U.S. patent application number 12/845485 was filed with the patent office on 2011-01-20 for bioactive complex compositions and methods of use thereof.
This patent application is currently assigned to Texas A&M University System. Invention is credited to Michael H. Gurin, Adela Mora-Gutierrez.
Application Number | 20110014279 12/845485 |
Document ID | / |
Family ID | 38228971 |
Filed Date | 2011-01-20 |
United States Patent
Application |
20110014279 |
Kind Code |
A1 |
Mora-Gutierrez; Adela ; et
al. |
January 20, 2011 |
BIOACTIVE COMPLEX COMPOSITIONS AND METHODS OF USE THEREOF
Abstract
A bioactive complex composition having enhanced oxidative
stability, emulsion stability, mineral rich transparent beverages
and a wide range of functional health benefits. The composition may
include as a base composition individual ingredients or a
synergistic blend of mineral salts, Omega-3 rich oils,
phospholipids, chitosan, and alpha-casein, beta-casein,
kappa-casein or protein fragments, glycopeptides, phosphopeptides.
The composition may optionally be further utilized for the
prevention of hypercholesterolemia, bone (and teeth) mineral loss,
treatment of mental health diseases, heart health, additional
nutritional supplementation, and treatment of additional medical
conditions.
Inventors: |
Mora-Gutierrez; Adela;
(Houston, TX) ; Gurin; Michael H.; (Glenview,
IL) |
Correspondence
Address: |
ROSENBAUM & SILVERT, P.C.
1480 TECHNY ROAD
NORTHBROOK
IL
60062
US
|
Assignee: |
Texas A&M University
System
College Station
TX
|
Family ID: |
38228971 |
Appl. No.: |
12/845485 |
Filed: |
July 28, 2010 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11530635 |
Sep 11, 2006 |
7780873 |
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12845485 |
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10784842 |
Feb 23, 2004 |
7118688 |
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11530635 |
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11306582 |
Jan 3, 2006 |
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10784842 |
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Current U.S.
Class: |
424/451 ;
424/94.1; 426/541; 426/602; 426/648; 514/1.1; 514/1.9; 514/15.7;
514/16.9; 514/7.4; 530/350; 530/395 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61K 2800/56 20130101; A61K 31/05 20130101; A61P 9/10 20180101;
A61K 47/61 20170801; A61P 9/12 20180101; A61K 2800/522 20130101;
A61K 38/40 20130101; A61Q 11/00 20130101; A61P 3/02 20180101; A61K
8/64 20130101; A61P 25/00 20180101; A61P 19/10 20180101; A61K
31/6615 20130101; A61P 3/06 20180101; A61P 39/06 20180101; A61K
36/87 20130101; A61K 8/738 20130101 |
Class at
Publication: |
424/451 ;
424/94.1; 514/1.1; 514/1.9; 514/7.4; 514/15.7; 514/16.9; 530/350;
530/395; 426/648; 426/602; 426/541 |
International
Class: |
A61K 9/48 20060101
A61K009/48; A61K 38/43 20060101 A61K038/43; A61K 38/02 20060101
A61K038/02; C07K 14/00 20060101 C07K014/00; A61P 3/06 20060101
A61P003/06; A61P 25/00 20060101 A61P025/00; A61P 9/12 20060101
A61P009/12; A61P 9/10 20060101 A61P009/10; A61P 39/06 20060101
A61P039/06; A61P 3/02 20060101 A61P003/02; A61P 19/10 20060101
A61P019/10; A23L 1/305 20060101 A23L001/305; A23D 7/005 20060101
A23D007/005; A23L 1/30 20060101 A23L001/30 |
Claims
1. A bioactive composition comprising a polycationic protein or
protein fragment complex whereby the complex is operable as an
electron transfer bridge.
2. The composition according to claim 1, whereby the complex and
electron transfer bridge are within an oil and water emulsion with
a water phase and whereby the oil phase is comprised of at least
one selected from the group consisting of reducing sugar including
glucose, cyclodextrin, or combinations thereof, fat soluble
vitamins, Omega-3 rich oils, fat-soluble nutraceutical or
pharmaceutical actives, oils rich in ferulic acid including olive,
rice bran, corn, or oat oils, ionic liquids, ionic emulsifiers,
monoglycerides, diglycerides, or combinations thereof.
3. The composition according to claim 2, whereby the oil phase is
further comprised of chelating agents including EDTA, lactoferrin,
phytic acid, cyclodextrin, permeation enhancers, or combinations
thereof.
4. The composition according to claim 2, whereby the water phase is
comprised of at least one selected from the group consisting of
lactoferrin, phytic acid, trehalose, acetyl L-carnitine, permeation
enhancers, or combinations thereof.
5. The composition according to claim 3, whereby the oil to
cyclodextrin ratio is at least 10:1.
6. The composition according to claim 1, whereby the electron
transfer bridge pH is adjusted to a pH between 5.5 and 6.5 by means
including potassium hydroxide, calcium hydroxide, magnesium
hydroxide, or combinations thereof.
7. The composition according to claim 1, whereby the complex is
further comprised of at least one selected from the group
consisting of electron donor transfer, nucleotides including
pyrimidines, nucleosides, ribose, cyclodextrin, antioxidants,
Omega-3 rich oils, beta-glucan, or combinations thereof.
8. The composition according to claim 1, whereby the electron
transfer bridge is comprised of at least on selected from the group
consisting of canola protein isolate, canola protein fragment,
keratin, keratin, or combinations thereof complexed with at least
one from the group consisting of chitosan, caseinosphosphopeptide,
or combinations thereof.
9. The composition according to claim 1, whereby the electron
transfer bridge is within an oil in water emulsion that is further
comprised of a polycationic chitosan void of iron.
10. The composition according to claim 2, whereby the composition
is further comprised of electron transfer mediators including
potassium salts, lactic acid salts, derivatives of potassium salts,
derivatives of lactic acid salts, and combinations thereof.
11. The composition according to claim 1, whereby the composition
is an efficacious means for hypercholesterolemia prevention
products, bone mineral loss prevention products, enhancing
cognitive performance, reducing disorders of mental health,
reducing triglyceride levels, reducing blood pressure levels,
reducing arterial plaque, increasing free electron flow in an
aqueous room temperature electride solution or combinations
thereof.
12. A bioactive composition having reduced susceptibility to
oxidation comprised of an oil phase having a low electrical
resistance pathway from the oil to the antioxidant whereby free
radicals seek an electron from the antioxidant rather than damaging
the lipid molecules within the oil phase.
13. The composition according to claim 12, whereby the composition
is further comprised of at least one selected from the group
consisting of ionic emulsifiers, absorbed carbon dioxide,
polyphenols, lactic acid, lactic acid derivatives, gallic acid,
gallic acid derivatives, carnosic acid, carnosic acid derivatives,
green tea extracts, chelators including EDTA, synthetic
antioxidants including BHA, BHT, and TBHQ, oils having Omega-3,
Omega-6, and Omega-9 fractions, oils rich in ferulic acid, oils
rich in phytic acid, lactoferrin, antioxidants, galactolipids,
cyclodextrin, or combinations thereof.
14. The composition according to claim 12, whereby the cyclodextrin
encapsulates at least one selected from the group consisting of
coenzyme Q10, mixed tocopherols, mixed tocotrienols, oil soluble
antioxidants, or combinations thereof.
15. The composition according to claim 12, whereby the composition
is further comprised of non-ionic surface active agents within the
oil and water interface.
16. A bioactive composition comprised of at least one complex
selected from the group consisting of proteins, peptides, amino
acids, or combinations thereof salted-in with at least one selected
from the group consisting of lactic acid mineral salts, lactic acid
derivatives, potassium salts, potassium derivatives, or
combinations thereof.
17. The composition according to claim 16, wherein the bioactive
complex is further comprised of mineral salts salted in for means
including enhanced solubilization, reduced bitterness, controlled
saltiness, or combinations thereof.
18. The composition according to claim 16, whereby the composition
is further comprised of at least one selected from the group
consisting of polycationic chitosan, phosphate buffering agent,
citric acid, phytic acid, gallic acid, lactic acid, or combinations
thereof.
19. The composition according to claim 16, whereby the composition
is further complexed with chitosan as a means to reduce
bitterness.
20. A bioactive composition operable to reduce triglycerides and
total cholesterol comprised of at least one selected from the group
consisting of vanillin, o-vanillin, fat-soluble vanillyl
acylamides, or combinations thereof, and gallic acid, and at least
one selected from the group consisting of EDTA, cyclodextrin, CoQ10
encapsulated by cyclodextrin, lactoferrin or thermally stabilized
lactoferrin, mixed tocopherols, mixed tocotrienols, or combinations
thereof.
Description
CROSS REFERENCE TO RELATE APPLICATIONS
[0001] The present application is a divisional application of U.S.
patent application Ser. No. 11/530,635, filed Sep. 11, 2006, which
is a continuation-in-part of U.S. patent application Ser. No.
10/784,842, filed on the Feb. 23, 2004 and is now U.S. Pat. No.
7,118,688, and a continuation-in-part of U.S. patent application
Ser. No. 11/306,582, filed on the Jan. 3, 2006, all of which are
hereby incorporated by reference in their entirety with priority
claims therein.
TECHNICAL FIELD OF THE DISCLOSURE
[0002] The present invention relates to bioactive complex
compositions, particularly compositions formed from natural
ingredients, and methods for using said compositions to enhance
efficacy and bioavailability of actives. The invention comprised of
a polycationic complexation system further provides enhanced
bioavailability of phospholipids, Omega-3 rich oils, oil soluble
actives, and minerals for applications ranging from memory
enhancers to oral care.
BACKGROUND
[0003] It is known that whatever their kind and origin,
nutraceuticals and pharmaceuticals, that are not readily water
soluble, have relatively limited bioavailability. Many factors are
recognized in the art as limiting bioavailability including
relatively limited membrane fluidity, solubility, unstable
dispersions or emulsions. Additionally the presence of competing
non-actives for the same enzymatic functionality, such as Omega-6
versus Omega-3. Mammals cannot interconvert the Omega-3 and Omega-6
fatty acids and their metabolism requires the same desaturation
enzymes.
[0004] Numerous other competing interactions take place in vivo,
including calcium and magnesium absorption, minerals required for
enzymatic activation including zinc, acid intake and blood pH being
too acidic, gastrointestinal and/or brain barrier permeation to
name a few.
[0005] The presence of Omega-3 oils in food is of great importance
since they cannot be synthesized by human and animal tissues and
should thereby be provided with the diet. In tissues these
essential fatty acids are converted to longer and more unsaturated
fatty acids of the Omega-6 and Omega-3 families, such as
arachidonic acid (AA), eicosapentaenoic (EPA), and docosahexaenoic
(DHA), which are present in marine oils (fish, microalgae) in
relatively high amounts. The health benefits of linoleic acid,
alpha-linolenic acid, AA, EPA and DHA are well documented in the
literature. These benefits include hypolipidemic, anti-thrombotic,
and anti-inflammatory properties. They are also essential fats for
growth, brain function, and visual acuity, especially for infants.
Omega-3's are further recognized for their positive impact on
psychiatric, brain, and neurologic conditions.
[0006] Many products ranging from functional foods and
confectioneries to nutraceuticals and pharmaceuticals are emulsions
or may be made into emulsions. An emulsion is a colloidal
dispersion of two immiscible liquids, such as oil and water, in the
form of droplets. If oil droplets are finely dispersed in water,
then this is an oil-in-water or O/W emulsion. When water droplets
are finely dispersed in oil, then this is a water-in-oil or W/O
emulsion. O/W and W/O emulsions play a prominent role in the
preparation of a wide range of products including foods,
pharmaceutical products and cosmetics. It would be thus desirable
to provide enhanced bioavailability compositions formed from
natural ingredients and methods to effectively increase efficacy
within highly polyunsaturated oils in O/W and W/O emulsions.
[0007] Numerous other products ranging from functional foods and
beverages may incorporate powder actives. The powder's solubility
and bioadhesion characteristics in vivo are of critical importance
to the bioavailability of the functional active. It is also highly
desirable for the powder to be delivered within a transparent
beverage, or at other times having an opacifier impact. Regardless
of the delivery system, it is always desirable for the delivery
system to have a pleasing taste absent of bitterness. At other
times, the powder's taste is desired to be salty, sweet, or even
creamy thus adding taste functionality beyond the health and
nutrition functionality.
[0008] Another significant area addressing the inclusion of
functional actives centers around oxidation stability. One such
prior art method is the utilization of a class of surface active
agents or emulsifiers, as noted in the art U.S. Pat. No. 5,079,016
by Todd, Jr. on Jan. 7, 1992 entitled "Color-stabilized carotenoid
pigment compositions and foods colored therewith having increased
resistance to oxidative color fading" includes surface active
agents or emulsifiers with strong stabilizing and synergistic
properties with natural antioxidants: sorbitan esters, such as mono
and tri oleates and stearates, lactic acid esters of monoglycerides
and diglycerides. Surface active agents or emulsifiers with strong
stabilizing and modest synergistic properties include polyglycerol
esters of fatty acids, such as octaglycerol monooleate,
decaglycerol capric-caprylate, and decaglycerol tetraoleate,
mono-diglycerides of fatty acids, such as glycerol mono oleate,
acetylated monoglycerides, citric acid esters of mono-diglycerides,
lecithin, and propylene glycol esters of fatty acids. The prior art
notes an unexpected synergism exhibited between rosemary and
sorbitan trioleate, as a preferred representative of the class of
non-ionic surface active agents. This example shows that
tocopherols significantly interact with these stabilizers to
further improve the stability of the carotenoid pigments. This
prior art identifies the importance of very specific and unique
synergistic combinations.
SUMMARY
[0009] The present invention relates to compositions and methods
for enhancing the bioavailability and efficacy of a range of
Omega-3 rich oils, minerals, and delivery systems thereof.
[0010] In addition, the compositions may offer nutritional and
pharmacological benefits including: (1) enhanced membrane fluidity,
having improved brain functions (e.g., memory, reduced ADD and
ADHD, etc.) activity in animal bodies; (2) enhanced soluble
complexes with calcium and chitosan for bioadhesion, providing
remineralization of teeth and bone in animal bodies, (3) enhanced
delivery of actives including the families of recognized
fat-soluble antioxidants (tocopherols, tocotrienols, beta-carotene,
coenzyme Q.sub.10), choline, carnitine, and essential fatty acids
(DHA, EPA).
[0011] Specific embodiments of the present invention are further
described in the following detailed description.
DETAILED DESCRIPTION
[0012] The term "electron transfer", hereinafter also referred to
as "ET", is the process by which an electron moves from one atom or
molecule to another atom or molecule. ET is a mechanistic
description of the thermodynamic concept of redox, wherein the
formal oxidation states of both reaction partners change. Numerous
essential processes in biology employ ET reactions, including:
oxygen binding/transport, photosynthesis/respiration, metabolic
syntheses, and detoxification of reactive species. Additionally,
the process of energy transfer can be formalized as a two electron
exchange (two concurrent ET events in opposite directions). ET
reactions commonly involve transition metal complexes, but there
are now many examples of ET in organic molecules. The term
"electron transfer mediator", which is interchangeably used with
electron transport mediator, is defined as means of increasing the
effective mobilization of electrons including the tunneling or
bridging across molecular scale interfaces. Without being bound by
theory, an electron transfer mediator provides a low resistance
path for electron mobility. The term "electron transfer bridge" is
the coupling mechanism for intramolecular electron transfer between
donor and acceptor.
[0013] The term "alkalide" is defined as a class of ionic compounds
where the cations are of the Type I group (Alkali) elements Na, K,
Rb, Cs (no known `Lithide` exists). The cation is an alkali cation
complexed by a large organic complexant. The resulting chemical
form is A+[Complexant]B-, where the complexant is a Cryptand, Crown
Ether, or Aza-Crown.
[0014] The term "electride" is defined as being just like alkalides
except that the anion is presumed to be simply an electron that is
localized to a region of the crystal between the complexed
cations.
[0015] The term "Omega-3" includes all enzymatically altered forms
of Alpha-linolenic including Stearidonic acid, Eicosapentaenoic
acid "EPA", and Docosahexaenoic acid "DHA".
[0016] The term "oil rich in Omega-3" includes all oils having
greater than 20% Omega-3 content.
[0017] The term ubiquinone
50,2,3-dimethoxy-5-methyl6-pentacontdacaenyl-benzoquinone is also
hereinafter referred to as coenzyme Q.sub.10 or CoQ10.
[0018] The present invention includes compositions and methods for
enhancing bioavailability and efficacy of actives. The compositions
have an impact in multiple categories including: antioxidation,
mental health, immunity, bone health, triglyceride and total
cholesterol reduction, and the modulation of metabolic pathways by
influencing electron flux.
[0019] The compositions may enhance delivery of highly
polyunsaturated lipids. They may include non-reducing sugars, sugar
polyols, medium-chain triglycerides, sulfated polysaccharides,
caseinophosphopeptides, phospholipids, chitosan and polyphenols.
These compositions may be used in O/W or W/O emulsions or further
subjected to post processing to yield free flowing powders as
recognized in the art (e.g., spray drying, freeze drying,
absorption plating, etc.).
[0020] Selected embodiments contain sulfated polysaccharides. These
may include compounds containing at least one polymeric sugar
moiety covalently attached to a sulfate group. One example of a
sulfated polysaccharide is the carrageenan class of compounds.
Other examples of sulfated polysaccharides include chondroitin
sulfate, sulfated cyclodextrins, dextran sulfate and heparin
sulfate.
[0021] The compositions may also include ingredients selected from
the group of non-reducing sugars, sugar polyols, medium-chain
triglycerides, polysaccharides, alpha-casein, beta-casein,
kappa-casein or protein fragments, glycopeptides, phosphopeptides,
alpha, beta, gamma or delta tocopherols, alpha, beta, gamma or
delta tocotrienols, tocopherols, tocotrienols, beta-carotene,
phospholipids and chitosan, or combinations thereof.
[0022] The compositions may also include pH modifiers including
lactic acid, gallic acid, citric acid, ascorbic acid, gluconic
acid, and chelating agents including citric acid, choline citrate
or combinations thereof.
[0023] In selected embodiments, the compositions may include food,
beverage, and confectionery ingredients including: non-reducing
sugars, sugar polyols, or combinations thereof; modified starches;
polysaccharides; glycerides selected from enzymatically modified
oils, fats, and fatty acids of mono-, di-, and tri-glycerides;
glycerides selected from lipolyzed modified oils, fats, and fatty
acids of mono-, di-, and tri-glycerides; cocoa powder; Sucralose;
and combinations thereof.
[0024] The first embodiment of the composition is as a synergistic
active as a means to provide superior protection of lipids (and
other oxidative unstable products) against oxidation.
[0025] Antioxidation
[0026] A free radical is an active atom or molecule that has an
unpaired electron in its outer shell. The free radical seeks to
become stable by stealing an electron from another molecule.
Unfortunately, while this results in the original molecule becoming
stable, the second molecule becomes a free radical instead. The
generation of free radicals can set off a chain reaction of free
radical formation, as molecules seek to stabilize by grabbing
electrons from the molecules surrounding them, and leaving new free
radicals in their place. Antioxidants are molecules that can give
up an electron to a free radical but do not become free radicals
themselves.
[0027] The preferred embodiment is the presence of an electron
transfer bridge, hereinafter referred to as ETB, across the
interface of the O/W emulsion. The particularly preferred electron
transfer bridge has an iron-sulfur cluster. The specifically
preferred ETB is a chitosan-caprine CPP complex in which the
enzymatically-modified caprine casein referred to as
caseinophosphopeptide (CPP) is high in the sulfur-containing amino
acid cysteine and the basic amino acid lysine (caprine CPP contains
around 0.5% cysteine and 5% lysine whereas bovine CPP contains 0%
cysteine and around 3% lysine). The iron content of the chitosan
preparation should be between 50 and 400 ppm, particularly
preferred at less than 200 ppm. The particularly preferred
embodiment of the caprine CPP-chitosan complex is further comprised
of a reducing sugar (e.g., glucose) within the oil phase, without
being bound by theory attributed to thermal decomposition of
reducing sugars and nitrogen-based compounds present in Omega-3
oil-based formulations previously stabilized with Vitamin E (mixed
tocopherols or mixed tocotrienols). The thermal decomposition of
reducing sugar (glucose)-amino acid (lysine)-glucosamine (chitosan)
compounds are referred to as the Maillard browning reaction. The
specifically preferred source of glucose molecules is a
gamma-cyclodextrin-coenzyme Q10 inclusion complex from Wacker
Chemical Corporation (Adrian, Mo., USA) under the commercial name
of Cavamax CoQ10.TM.. (75% glucose and 25% coenzyme Q10 on w/w %).
It is the synergistic combination of glucose, cysteine, lysine,
iron-containing chitosan and coenzyme Q10 that regenerates the
tocopheryl free radicals into antioxidative tocopherol molecules).
The Maillard browning reaction is initiated by condensation of a
carbonyl group on the reducing sugar with free amino groups of
amino acids, proteins, and peptides (caprine CPP), and amine groups
of glucosamines (chitosan) thereby enhancing specific antioxidant
activity in oil-in-water and water-in-oil emulsions.
[0028] The Cavamax CoQ10.TM. can be dissolved either in the oil or
in the water phase. In the presence of strong chelating agents such
as EDTA, lactoferrin and phytic acid, Cavamax CoQ10.TM. exhibits
superior "protective" effect against oxidation of Omega-3 oils when
added to the oil phase. Furthermore, the amount of Cavamax
CoQ10.TM. can be reduced to levels below 0.5 wt. % when added
directly to the oil phase. However, in the presence of weak
chelating agents such as grape seed and grape pomace extract higher
levels of Cavamax CoQ10.TM. are required. Cyclodextrins, which are
ring shaped sugar compounds, are recognized in the art as a means
to encapsulate Omega-3 oils to minimize oxidation of Omega-3 oil
droplets by air, but the inclusion of cyclodextrins within the oil
phase at a ratio of cyclodextrin to oil at less than 10 wt. % is
unique (i.e., a ratio of cyclodextrin to oil of at least 10:1. The
preferred level of cyclodextrin on a w/w % basis to the oil of less
than 5 wt. %, particularly preferred is less than 1% on a w/w %
basis to the oil. The specifically preferred cyclodextrin is a
complex of gamma-cyclodextrin and coenzyme Q10. It is anticipated
that fat soluble antioxidants, vitamins, or electron donor
compounds complexed/encapsulated in gamma-cyclodextrin and then
subsequently incorporated into the lipid/oil to provide superior
protection against lipid oxidation. The preferred process method is
to add Cavamax CoQ10.TM. to the oil phase followed by
emulsification with egg yolk phospholipids to avoid encapsulating
the caprine CPP-chitosan complex that contains added iron (chitosan
lactate with added iron).
[0029] The key step of caprine CPP-chitosan complex preparation is
adjusting the pH of the caprine CPP-chitosan complex solution to a
range of pH 5.5-6.5, though the preferred embodiment has a pH 6.0
preferably with either potassium hydroxide or calcium hydroxide.
Potassium hydroxide is the preferred pH adjuster except in
situations resulting in too bitter taste in which calcium hydroxide
achieves good results. Another alternative is the combination of
both potassium hydroxide and calcium hydroxide with the ratio being
determined by taste impact.
[0030] Lipid oxidation of O/W emulsions is promoted by endogenous
transition metals that are naturally present in the oil,
surfactant, and/or water. Iron is known to be more soluble at low
pHs. Therefore, we anticipate lipid oxidation rates to be higher at
pH 3.0 than 6.0. In the particular case of caprine CPP-chitosan
complex, we are dealing with cationic oil-in-water emulsion
droplets that oxidize more slowly than anionic emulsion droplets
(at pH 3.0, chitosan exhibits a polycationic charge), without being
bound by theory, because of their ability to repel cationic metals,
thus decreasing interfacial iron concentrations and lipid oxidation
rates. Water-soluble chelators inhibit lipid oxidation by binding
aqueous-phase iron. Metal chelators however exhibit differences in
iron binding potential. A recognized in the art superior metal
chelator is EDTA. Iron bound to citrate can be more catalytically
active than free iron. Therefore, citric acid is the least
effective chelator at inhibiting lipid oxidation of the chelators
tested (i.e., EDTA, malic acid, citric acid, fumaric acid, etc.) in
the presence of iron. When citric and/or malic acid are used, the
usage levels should be preferably less than 0.01% wt. (based on the
weight of the emulsion) to chelate iron (or copper) and further
preferably in applications stored at temperatures below 30.degree.
C. The specifically preferred organic acid is lactic acid (or it's
derivative) due to the combined electron donor capacity and lack of
catalytic impact.
[0031] Preferential Food & Beverage Specifications--Avoid
[0032] Avoid the fructose or fructose syrups in combination with
Omega-3 oils and caprine CPP-chitosan complex. High fructose
syrups, scientifically linked to the obesity pandemia, create
thermal decomposition products of fructose-lysine (caprine CPP is
`rich` in the amino acid lysine), thus exhibiting more
"pro-oxidant" and "genotoxic" activity as compared to the thermal
decomposition products of glucose-lysine.
[0033] Avoid the combination of ascorbic acid (and ascorbyl
palmitate) in the presence of iron, particularly at low pH
conditions (pH<6.0) and especially when the phospholipids have
iron within the oil/water interface such as egg yolk phospholipids.
The result when used in combination with an Omega-3 rich oil is
particularly pro-oxidant with ascorbic acid, which without being
bound by theory the ascorbic acid keeps iron (III) in the reduced
form of iron (II) that is highly prooxidant. Iron is less
"prooxidant" at pH.gtoreq.6.0.
[0034] Sodium chloride (NaCl) content should be as low as possible.
Sodium chloride acts as pro-oxidant or antioxidant depending on the
nature of the system involved.
[0035] Avoid using "plain" pectin, as the higher the content of
methoxy groups present on the pectin molecule (less negative
charges), the lower the interaction with the chitosan molecule
(positively charged polymer at low pH).
[0036] Preferential Food & Beverage Specifications--Include
[0037] Antioxidants recognized in the art including vanillin,
vanillin derivatives, bee propolis, grape seed extract, grape
pomace extract, quercitin, tetrahydrocurcuminoids CG, ginger,
turmeric, capsaicin, spirulina, Trolox (water soluble tocopherol by
Hoffman Roche) and green tea (known to contain Superoxide Dismutase
"SOD"), clove (according to USDA is nature's richest known source
phytonutrient called eugenol, which enhances the metabolism of DHA,
inactivates free radicals, and promotes nerve cell health),
rosemary extract (especially high carnosic acid) and camosic acid
derivatives, and walnut extracts. The addition of grape seed
extract or grape pomace extract to O/W Menhaden oil-based emulsions
treated with Cavamax CoQ10.TM. (0.91 wt. %) dispersed in the water
phase (in the presence of the caprine CPP-chitosan complex of the
present invention) exhibits high antioxidant activity after 21-days
storage at 60.degree. C. These grape-derived polyphenols exhibit
both radical scavenging activity and mineral chelating
activity.
[0038] Corn oil is the only plant oil high in ubiquinone (coenzyme
Q) (200 ppm). Canola and soybean oil are high in gamma-tocopherol,
recognized as having the highest antioxidant activity. Palm oil is
high in mixed tocopherols and tocotrienols.
[0039] Ferulic acid is naturally present in extra-virgin olive oil,
rice bran oil, corn, and oat oil. Curcumin is very similar to
ferulic acid. The phytochemical ferulic acid is also found in the
leaves and seeds of many plants, but especially in cereals such as
brown rice, whole wheat and oats. Ferulic acid is also present in
coffee, apple, artichoke, peanut, orange and pineapple. Ferulic
acid is an antioxidant that neutralizes free radicals (superoxide,
nitric oxide and hydroxyl radical) that could cause oxidative
damage of cell membranes and DNA. Ferulic acid helps to prevent
damage to our cells caused by ultraviolet light. Exposure to
ultraviolet light actually increases the antioxidant potency of
ferulic acid. Ferulic acid is often added as ingredient of
anti-aging supplements. Studies have shown that ferulic acid can
decrease blood glucose levels and can be of help to diabetes
patients. Like many other antioxidants, ferulic acid reduces the
level of cholesterol and triglyceride, thereby reducing the risk of
hearth disease.
[0040] Fruit concentrate sweetener as humectant that comprises a
blend of hydrolyzed starch having a dextrose equivalent (D.E.) of
up to approximately 25; fruit juice or fruit syrup concentrate of
at least approximately 40 wt. % soluble solids and approximately 0
wt. % insoluble solids thereby forming a liquor having a dry weight
composition of approximately 40 to approximately 65 wt. % complex
carbohydrates; and approximately 35 to approximately 55 wt. %
simple sugars from the fruit juice or fruit syrup concentrate; and
approximately 0 to approximately 5 wt. % nutritional components
occurring naturally in the fruit juice or fruit syrup
concentrate
[0041] Galactolipids, such as from oat oil, also provide numerous
benefits within the preferred embodiment. Galactolipids as
recognized in the art protect O/W emulsions such that the lipids
are not enzymatically degraded until the small intestine. The
preferred embodiment is further comprised of galactolipids as a
means to enable fat soluble nutraceutical and pharmaceutical
actives incorporated into Omega-3 rich oils provide superior
efficacy and bioavailability of both Omega-3 and their inclusive
actives.
[0042] Gallic acid is preferably added to O/W marine oil-based
emulsions at concentrations.ltoreq.50 ppm (0.005 wt. %, based on
the weight of the emulsion). Gallic acid, found in plant materials
such as blackberry bark, henna, tea, and as a component of
hydrolyzable tannins, combats the browning effects that result from
food processing. The antioxidant action of gallic acid and gallates
is similar to that of other catechins, which behave as antioxidants
by electron donation to free radical oxidants in aqueous
solution.
[0043] Another implementation of the inventive technology is
keratin (protein hydrolyzate/peptide obtained from wool (Cynatine
FLX.TM., Keratec Corporation, New Zealand) complexed with the
caprine CPP-chitosan of the present invention, or separately
complexed with chitosan. Keratin can also be utilized as an
alternative to caprine CPP, as keratin is also a sulfur-bearing
protein.
[0044] Lactic acid is preferably included in the preparation of O/W
emulsions, in particular at low pH, because lactic acid is an
"electron donor" compound.
[0045] Utilize lactoferrin, preferably thermally-stabilized
lactoferrin as comprised of proprietary ingredients of TAMUS 1408,
and preferably further comprised of oils rich in ferulic acid. The
inclusion of 1 mM lactoferrin or 5 mM phytic acid was included in
an O/W emulsion comprised of Menhaden oil (OmegaPure.TM., Omega
Protein Corporation, Houston, Tex., USA) blended with olive oil.
Phytic acid is added at levels not higher than 0.05 wt. % (based on
the weight of the emulsion) in the absence or presence of
lactoferrin. It is important to isolate iron-containing proteins
(e.g., lactoferrin) from the cyclodextrin, thus the inclusion of
cyclodextrin is within the oil phase whereas lactoferrin is present
in the water phase. As recognized in the art, the addition of
bicarbonate improves the heat stability of iron-lactoferrin
complexes thereby enhancing the oxidative stability of oil-in-water
emulsions. Thus the infusion of carbon dioxide, at least during
product storage, is anticipated to enhance shelf-life. The further
inclusion of a cocktail of thermal stabilizing-compounds (i.e.,
TAMUS 1408) prevents the iron-lactoferrin complexes from undergoing
denaturation (unfolding) during pasteurization at 90.degree. C. for
3 minutes. Finally, chelating agents are known to stabilize
lactoferrin, in particular EDTA (U.S. Pat. No. 7,034,126 and U.S.
Pat. No. 7,026,295). Lactoferrin in combination with EDTA and
caprine CPP-chitosan complex significantly inhibits lipid oxidation
of O/W Menhaden-oil emulsions blended with Smart Balance Omega.TM.
oil (U.S. Pat. No. 5,578,334). Transition metals decrease the
oxidative stability of food emulsions through their ability to
decompose lipid peroxides into free radicals. In food emulsions,
free radicals are usually generated in the aqueous phase, and these
radicals have important implications for the oxidation of
emulsified oils.
[0046] Phospholipids "rich" in phosphatidylcholine (PC) such as
those present in egg yolk phospholipids is the emulsifier of choice
to increase "bilayer" adhesion between "negatively" charged
phospholipids "rich" in PC and "positively" charged chitosan-CPP
complex. Oil-in-water or water-in-oil emulsions containing oil
droplets coated with phospholipids-caprine CPP-chitosan exhibit
enhanced oxidative stability. Phosphatidyl serine (SerineAid
50P.TM.) effectively inhibits iron-induced lipid peroxidation of
egg yolk PC in emulsion droplets coated by anionic egg yolk PC and
cationic caprine CPP-chitosan, indicating that incorporation of
phosphoserine group into the phospholipid-cationic bilayers is
helpful to enhance the oxidative stability of food lipids such as
DHA and EPA. A water-soluble emulsifier GlycerolPhosphoCholine
Hydrate 85% (GPC 85.TM.) can also be added to the oil-in-water or
water-in-oil emulsions to inhibit iron-induced peroxidation of egg
yolk PC. The combination of SerineAid 50P.TM., GPC 85.TM. and egg
yolk PC-caprine CPP-chitosan results in synergistic inhibition of
lipid oxidation, because multicomponent antioxidant systems can
inhibit oxidation at many different phases of oxidation.
SerineAid.TM. and GPC 85.TM. decrease the number of free radicals
generated in a system by inhibiting metal-catalyzed oxidation.
[0047] The further inclusion of phytic acid enhances the oxidative
stability. Phytic acid is a powerful inhibitor of iron-driven
radical (--OH) formation because of its ability to form a unique
iron chelate that becomes catalytically inactive. Unlike most other
iron chelates, Fe.sup.3+-phytate does not retain a reactive
coordination site, thus it does not support --OH generation.
[0048] The polyphenols found in tea are more commonly known as
flavanols or catechins and comprise 30-40 percent of the
extractable solids of dried green tea leaves. The main catechins in
green tea extract are epicatechin, epicatechin-3-gallate,
epigallocatechin, and epigallocatechin-3-gallate (EGCG), with the
latter being the highest in concentration. Green tea leave oils
have demonstrated significant antioxidant, anticarcinogenic,
anti-inflammatory, thermogenic, probiotic, and anti-microbial
properties in numerous human, animal, and in-vitro studies. Green
tea extracts are a natural source of gallic acid, which is a
preferred antioxidant playing a particularly synergistic role with
Cavamax CoQ10.TM.. Coenzyme Q10 with d-limonene contains smaller,
nano-sized coenzyme Q10 particles that enhance this absorption into
other oils/fats. Cavamax CoQ10.TM. dissolved at 0.91 wt. % in the
"water" phase of the O/W Menhaden oil-based emulsion "regenerates"
the tocopheryl radicals (prooxidant activity) into antioxidative
tocopherol molecules (antioxidant activity) when the caprine
CPP-chitosan complex is used with polyphenols, in particular with
grape seed extract. Electron transfer mediators (i.e., potassium
hydroxide, calcium hydroxide, magnesium hydroxide) are essential to
"activate" the aromatic rings, such as those present in the
polyphenol(s) and coenzyme Q10 molecules. The gas chromatography
(GC) data of O/W Menhaden oil-based emulsions treated with caprine
CPP-chitosan complex, polyphenols (grape seed extract, grape pomace
extract, bee propolis), Cavamax CoQ10.TM. (dissolved in the water
phase at a level of 0.91 wt. % based on the weight of emulsion),
and the preferred electron transfer mediator potassium hydroxide
(used to adjust the pH of the O/W Menhaden-oil based emulsions to
6.0) after 28 days storage at 60.degree. C. are as follows: Control
(formulation only): 100% (decomposition into hydroperoxides), Grape
seed extract: 4.09%; Grape Pomace Extract: 23.19%; and Bee
Propolis: 33.92%. The number of GC peaks (oxidation volatile
products) present in the treated samples is decreased (4 or 3
peaks) as compared to the control sample that has a total of 20
peaks. Each individual peak is associated with an aldehyde compound
(decomposition product of hydroperoxides). One preferred embodiment
is the polyphenols derived from the fruit of Solanum melongena.
[0049] Potassium salt, preferably monobasic potassium phosphate, is
the preferred buffering agent because potassium phosphate is used
in the food and pharmaceutical industries to "sequester"
deleterious trace minerals (iron, copper), to provide potassium
ions to the formulation, and to play an active role in the electron
transport process. The major mineral "activator" in vivo within
cells is potassium, which is present in healthy cells at levels 20
times those of sodium. In other words, potassium is an "activator
of metabolic functions" in vivo, in addition to the "in-food" role
of electron transport. Thus, the potassium salt serves a
multifunctional role of buffering agent, chelating agent,
electrolyte, and electron transport mediator.
[0050] Sodium oleate is recognized in the art as a means to reduce
micelle size within emulsions.
[0051] Sterols in oat oil are recognized in the art as a mean to
protect oils against deterioration at frying temperatures.
[0052] Transglutaminase (TG) is known to interact with
alpha.sub.s1-casein and form the basis of milk-protein-based edible
films and coatings.
[0053] Utilize trehalose, a naturally occurring osmolyte, to
stabilize the caprine CPP-chitosan complex and help retain the
activity of caprine CPP (and lactoferrin) in solution as well as in
the freeze-dried state. The preferential hydration of the caprine
CPP-chitosan complex by trehalose, without being bound by theory,
enables the electron tunneling or mean free path to be increased as
a means to enhance electron transfer into the water phase.
[0054] The inventive composition further includes methods to
stabilize lipids that are not prepared as emulsions.
[0055] Stabilized Oil (as Compared to Emulsion)
[0056] It is recognized that electron transfer within the
non-conductive oil phase is poor. This in combination with the oil
being susceptible to oxidation through the presence of dissolved
oxygen, pro-catalysts such as iron, and peroxides, the generation
of free radicals (even in the presence of antioxidants) creates
oxidation by-products. The failure to create a high-surface area
"pathway" for the electrons to transfer or tunnel limits the range
of the free radical electron distance to the mean free path. In the
event that the antioxidant is not present within this range, the
free radical creates oxidative by-products. Without being bound by
theory, the presence of components within the oil phase that
provide the free radical with a low electrical resistance pathway
to the antioxidant will enable the free radical to seek an electron
from the antioxidant rather than damaging the lipid molecules
within the oil phase.
[0057] The preferred embodiment is a protein-chitosan or
peptide-chitosan complex whereby the chitosan provides binding
capacity with the host oil. The particularly preferred protein or
peptide is a sulfur bearing-protein. The particularly preferred
chitosan has iron present, such that the
caseinophosphopeptide-chitosan complex creates an iron-sulfur
cluster. The protein or peptide is preferably further comprised of
divalent mineral salts such as inorganic calcium, magnesium, and
zinc salts (i.e., calcium chloride, magnesium chloride, zinc
chloride) or organic calcium, magnesium, and zinc salts (i.e.,
calcium lactate, magnesium lactate, zinc lactate). Milk mineral
(TruCal.TM., Glanbia Foods, Twin Falls, Id., USA) exhibits a
synergistic antioxidant effect with the caprine CPP-chitosan
complex in O/W Menhaden oil-based emulsions. The specifically
preferred caprine CPP-chitosan complex is further complexed with at
least one selected from the group consisting of trehalose, ribose,
glucose, or cyclodextrins. The preferred gamma-cyclodextrin
encapsulates at least one selected from the group consisting of
coenzyme Q10, mixed tocopherols, mixed tocotrienols, or additional
oil soluble antioxidants.
[0058] The preferred manufacturing process incorporates polyols, or
preferably trehalose or ribose prior to drying by means known in
the art including spray drying and freeze drying.
[0059] The preferred oil is further comprised of at least one
selected from the group consisting of ionic liquids, ionic
emulsifiers, absorbed carbon dioxide, polyphenols, gallic acid,
green tea extracts, chelators including EDTA, synthetic
antioxidants including BHA, BHT, and TBHQ.
[0060] Other Functional Roles
[0061] The oxidative stability testing with lactoferrin was
conducted under experimental conditions (i.e., pH, ionic strength)
that favor "electrostatic" and "hydrophobic" interactions.
Electrostatic and hydrophobic interactions are crucial for the
biological activity of lactoferrin (i.e., antimicrobial activity,
anticancer activity). Protein-lipid interactions, possible
electrostatic, involving lipid-binding-induced structural changes
to lactoferrin, are inferred by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and analytical
ultracentrifugation (AU). The incorporation of oils having Omega-3,
Omega-6, and Omega 9 induces structural changes to lactoferrin
through lipid-binding, which have a significant role on the
biological activity of lactoferrin. The preferred oil has the
presence of ferulic acid, with the particularly preferred oil being
selected from the group consisting of rice bran oil or virgin olive
oil. The specifically preferred oil is the Smart Balance Omega.TM.
oil, which is a commercial oil blend containing canola, soybean and
olive oils (Heart Beat Foods, Cresskill, N.J., USA) designed to
increase the HDL concentration and the HDL/LDL ratio (U.S. Pat. No.
5,578,334).
[0062] A preferred embodiment as a means to reduce triglycerides
and total cholesterol is the combination of vanillin (or
o-vanillin, fat-soluble vanillyl acylamides) and gallic acid and at
least one selected from the group consisting of EDTA, coenzyme Q10
encapsulated by gamma-cyclodextrin, thermally-stabilized
lactoferrin (or EDTA), mixed tocopherols, mixed tocotrienols, or
combinations thereof. A particularly preferred embodiment is the
combination of vanillin, gallic acid or green tea extracts, EDTA or
thermally-stabilized lactoferrin, mixed tocopherols and
tocotrienols, and coenzyme Q10 encapsulated by gamma-cyclodextrin
all within an oil rich in Omega-3 further encapsulated by an oil
rich in ferulic acid. Vanillin reacts preferentially with protein
lysine residues. Thus the specifically preferred embodiment is
further comprised of caprine CPP-chitosan complex. The prior art of
U.S. Pat. No. 6,599,522 by Mokshagundam et. al., on Jul. 29, 2003
titled "Triglyceride reducing agent" utilizes ascorbic acid and
EDTA. Ascorbic acid functionality is limited by the pH sensitivity
of the host environment, whereas gallic acid is not and further
gallic acid in combination with Cavamax CoQ10.TM. is critical to
the regeneration of tocopheryl free radicals (prooxidant activity)
into antioxidative tocopherol molecules (antioxidant activity)
within an Omega-3 rich oil. EDTA is a synthetic
chelator/antioxidant having known toxicity, whereas lactoferrin is
a natural bioactive milk protein exhibiting both iron-chelating
activity and numerous recognized benefits including enhanced
immunity.
[0063] The preparation of the caprine CPP-chitosan complex is one
of the most critical steps in achieving the many functional
benefits. One exemplary method to prepare the CPP is as
follows.
[0064] CPP Preparation
[0065] Four versions of HEC chitosan (M-40L, M80L, FM-40L, FM-80)
were prepared in which iron (.ltoreq.200 ppm), lactic acid, and
sufficient calcium, potassium, or magnesium hydroxide to adjust pH
to 6.0 were added followed by freeze-drying. The freeze-dried
chitosan preparations were subsequently used to prepare the caprine
CPP-chitosan complex (Meyenber's goat milk) again followed by
freeze-drying. The HEC chitosans (M-40L, M80L, FM-40L, and FM-80)
complexed with caprine CPP exhibit `good` antioxidant activity in
O/W Menhaden oil-based emulsions stored at 40.degree. C. for 7
days. Since the HEC chitosans are "free" of protein, the thermal
decomposition of reducing sugar-amino-amine compounds (Maillard
reaction products) are expected to be smaller than those of Orcas'
and Cargill's chitosans. This means `lower` antioxidant activity of
HEC chitosans than those of Orcas' and Cargill's chitosans. In the
case of chitosan lactate manufactured by Pronova Biopolymers
Corporation, the formation of caprine CPP-chitosan complex was
achieved by adjusting the pH to 6.0 with around 5 ml of 0.1 N
potassium hydroxide. In the case of Orcas' chitosan or Cargill's
chitosan around 50 ml of 0.1 N potassium hydroxide was added
resulting in chitosan-CPP preparation excessively bitter and
hygroscopic. Therefore, the preparation of Orcas' chitosan or
Cargill's chitosan must use calcium hydroxide, magnesium hydroxide,
or mineral milk TruCal.TM. (Glanbia Foods, Twin Falls, Id., USA) to
avoid these detrimental effects on flavor and storage stability of
caprine CPP-chitosan preparations.
[0066] Additional testing has been performed using a variety of
chitosan forms, resulting in the preferred chitosan that have trace
amounts of iron. Alternatively, a pure form of chitosan can be
complexed with iron with the resulting chitosan-iron complex being
used to achieve comparable iron levels as present in the Pronova's
chitosan (i.e., less than 200 ppm). The more preferred chitosan is
a non-shellfish source chitosan such as resulting from fermentation
process of the fungi Aspergillus niger as available from Cargill
Food & Pharma Specialties (Cedar Rapids, Ia., USA). The
specifically preferred chitosan salt is chitosan lactate. Chitosan
as chitosan-alpha lipoic acid complex is also a particularly potent
component of the antioxidant and/or nanoemulsion compositions.
Thiolated chitosan is additionally a multifunctional chitosan form
that has the secondary benefit of being a superior bioadhesive and
mucoadhesive. The superior calcium absorption, without being bound
by theory, may be attributed to the increased mucoadhesive
properties within the gastrointestinal tract yielding controlled
release of the caprine CPP. A thiolated chitosan includes
chitosan-4-thio-butyl-amidine (a.k.a. chitosan-TBA). The further
addition of glutathione, most notably reduced glutathione, yields
superior bioadhesive properties.
[0067] It is important to note that chitosan that is void of iron
also serves a critical role, which is enhancing the emulsion
stability. Iron, which is a known catalyst for oxidation, cannot be
in an excessive amount without moving into a prooxidant
scenario.
[0068] The preparation of the emulsion is also another critical
step, with the following method being an exemplary method.
[0069] Emulsion Preparation
[0070] The resulting caprine CPP-chitosan complexes were each added
to O/W Menhaden oil-based emulsions blended with Smart Balance
Omega.TM. oil (12.5% Menhaden and 12.5% Smart Balance Omega.TM.
oil) in the absence and presence of caprine CPP-chitosan complex at
pH 6.0. All samples were further comprised of Cavamax CoQ10.TM. and
egg yolk phospholipids (Omega 6-PL-85.TM.). After 7 days storage at
40.degree. C. the p-anisidine value for each treatment is as
follows: Treatment 1 (control) (4.25); treatment 2 (M-40L and CPP)
(3.60); treatment 3 (M80L and CPP) (3.54); treatment 4 (FM-40 and
CPP) (3.61); and treatment 5 (FM-80 and CPP) (3.59). Food-Grade
EDTA (30 ppm w/w, based on the weight of the oil) was used as a
chelating agent in treatments T2 thru T5.
[0071] In other embodiments, the compositions may be made into
products including: hypercholesterolemia prevention products in a
mammal comprised of calcium and magnesium salts; bone mineral loss
prevention products in a mammal comprised of calcium and magnesium
salts; oils rich in Omega-3 products comprised of calcium and
magnesium salts; oil-soluble flavor products; oil-soluble vitamin,
nutraceutical, or pharmaceutical products; products having
vegetable oils including rice bran oil, flax, chia, hemp, castor,
soybean, lesquerella, dehydrated castor oil, rich in Omega-3, or
conjugated linoleic acid, animal oils including fish, egg, poultry,
and beef oils rich in Omega-3, or conjugated linoleic acid, or
combinations thereof; microalgae oils rich in Omega-3, or
conjugated linoleic acid, or combinations thereof; beverage
products being transparent comprised of calcium and magnesium
salts; cocoa products having improved creaminess, reduced
bitterness, and reduced oxidation; protein rich products, comprised
of high-methoxyl pectins or pectin alginates or combinations
thereof having reduced protein settling and sedimentation; protein
rich products having reduced protein settling and sedimentation;
oil-in-water micro- and nano-emulsions having increased emulsion
and oxidation stability; or water-in-oil micro- and nano-emulsions
having increased emulsion and oxidation stability.
[0072] The present invention may function as an antioxidant in a
variety of ways. For instance, sucrose has demonstrated its
potential as a fat-solubilizing agent for natural vitamins such as
provitamin A (beta-carotene) and vitamin E (tocopherol) as well as
polyphenolic compounds and caprine CPP and as an antioxidant agent
(invert sugar) in fat emulsions.
[0073] The compositions may also include polysaccharides such as
sulfated polysaccharides. Sulfated polysaccharides may include
iota-, kappa-, or lambda-carrageenan, or combinations thereof.
[0074] Compositions of the present invention may also include
alpha-casein, beta-casein, kappa-casein or protein fragments,
glycopeptides, phosphopeptides and combinations thereof.
Phosphopeptides may include phosphopeptides high in
alpha.sub.s2-casein and medium-chain triglycerides such as
caseinophosphopeptides (CPP). CPP may be isolated from caprine
(goat) milk. CPP have a particularly potent ability to form soluble
complexes with calcium. The increased solubility of the
calcium-bound caprine CPP-chitosan complex may further enhance the
mineral absorption to remineralize teeth, especially through the
chitosan adhesion within the oral cavity.
[0075] Compositions may further include alpha, beta, gamma or delta
tocopherols, alpha, beta, gamma or delta tocotrienols, tocopherols,
tocotrienols, beta-carotene, phospholipids, chitosan or
combinations thereof. A preferred mix of tocotrienols and
tocopherols is extracted from palm sources.
[0076] The compositions may also include polyphenols. A preferred
polyphenol is derived from the fruit of Solanum melongena.
Additional preferred polyphenols are derived from apple, cocoa,
grapes, pomegranate, and tea.
[0077] Fat emulsion particles containing sucrose or sorbitol
increase the solubility (and therefore, dispersion) of tocopherol
(vitamin E) and beta-carotene (provitamin A) present in flaxseed
oil. Fat particles containing sucrose or sorbitol will also
increase the solubility (dispersion) of cocoa (polyphenolic
compounds), eggplant-carrageenan complex (polyphenolic compounds)
and caprine caseinophosphopeptide-chitosan complex. The enhanced
antioxidant activity observed in O/W emulsions containing Canadian
flaxseed oil stems from the cooperation among tocopherols,
beta-carotene, phospholipids, sorbitol, proprietary cocoa mix, and
selected antioxidant compositions of the present invention. The
chitosan further enhances bioadhesion, which for oral consumption
of actives further improves the efficacy of the active by
encouraging sublingual and buccal administration thus avoiding
gastrointestinal deterioration.
[0078] Phospholipids used in embodiments of the invention may
include phospholipids from the group of egg yolk, soybean
phospholipids, or combinations thereof. TBA studies confirm the
synergistic antioxidant effects among soybean phospholipids
(lecithin), beta-carotene (provitamin A), tocopherol (vitamin E),
and sorbitol (sugar alcohol) or sucrose (non-reducing sugar) in
flaxseed oil-based nanoemulsions. The resulting flaxseed oil-based
nanoemulsions and the further use of soybean phospholipids,
sorbitol or sucrose along with homogenization minimize the lipid
oxidation of Omega-3, Omega-6, and Omega-9 fatty acids. The shelf
life of these essential polyunsaturated fatty acids (Omega-3,
Omega-6, Omega-9) in O/W nanoemulsions are therefore greatly
extended by some antioxidant compositions of the present invention.
Identical benefits are realized with a proprietary cocoa mix and
subsequent high-pressure homogenization.
[0079] Lecithin is widely used in lipid-based food products as an
antioxidant synergist. The structure of phospholipid molecules
enables lecithin to establish a protective coating on the surface
of the oil droplet, thereby retarding lipid oxidation. The process
of homogenization entraps not only the phospholipid molecules but
also the tocopherol and beta-carotene molecules in the oil droplets
that result in enhanced protection against lipid oxidation. The
production of low-fat products is further improved by the method of
incorporating selected antioxidant compositions of the invention
and egg yolk phospholipids to impart a rich and creamy mouthfeel
characteristic in low-fat products. Lecithin also has importance in
increasing efficacy due to the choline present.
[0080] Phosphatidylcholine is a phospholipid that is a major
constituent of cell membranes. Choline comprises about 15 wt. % of
the weight of phosphatidylcholine. Phosphatidylcholine is also
known as PtdCho or lecithin. Lecithins containing
phosphatidylcholine (PC) are produced from vegetable, animal and
microbial sources, but mainly from vegetable sources. Soybean,
sunflower and rapeseed are the major plant sources of commercial
lecithin. Soybean is the most common source. Eggs themselves
naturally contain from 68 to 72 wt. % phosphatidylcholine, while
soya contains from 20 to 22 wt. % phosphatidylcholine.
Phosphatidylcholine is important for normal cellular membrane
composition and repair. Phosphatidylcholine is also the major
delivery form of the essential nutrient choline.
[0081] Choline itself is a precursor in the synthesis of the
neurotransmitter acetylcholine, the methyl donor betaine and
phospholipids, including phosphatidylcholine and sphingomyelin
among others. Phosphatidylcholine's role in the maintenance of
cell-membrane integrity is vital to all of the basic biological
processes. These are: information flow that occurs within cells
from DNA to RNA to proteins; the formation of cellular energy and
intracellular communication or signal transduction. Choline is an
essential component of phospholipids, and is the building block for
acetylcholine, a major neurotransmitter of the central nervous
system. When a declining choline level becomes a limiting factor in
the synthesis of acetylcholine, which can occur during exercise and
other stressful activities, peak physical and mental performance
can be affected. Choline has also been shown to potentiate the
secretion of human growth hormone (hGH), a master hormone that in
part regulates basal metabolism and hence body composition. Since
the intrinsic release of hGH declines significantly after
adolescence, manifestations are seen as diminished resistance to
illness, vitality, and recovery, losses in muscle mass, increases
in fat mass, and negative changes in sleep patterns.
[0082] Phosphatidylcholine, particularly phosphatidylcholine rich
in polyunsaturated fatty acids, has a marked fluidizing effect on
cellular membranes. Decreased cell-membrane fluidization and
breakdown of cell-membrane integrity, as well as impairment of
cell-membrane repair mechanisms, are associated with a number of
disorders, including liver disease, neurological diseases, various
cancers and cell death. A particular preferred phospholipid rich in
phosphatidyl choline is derived from egg yolks (Omega-6-PL-85.TM.)
manufactured by Belovo Incorporated (Pinehurst, N.C., USA).
[0083] The further inclusion of additional sources of choline and
phosphatidyl containing compounds is anticipated as yielding
enhanced efficacy and benefits themselves as recognized in the art
for a wide range of psychiatric, brain, and other functional
benefits. Exemplary compounds include phosphatidylserine,
alpha-glyceryl phosphoryl choline, and choline citrate. Scientific
research and clinical investigations have shown that
phosphatidylserine, for example, plays a critical role in
maintaining optimal mental performance. A particular preferred
phospholipid rich in phosphatidylserine is SerineAid 50 P.TM..
(Chemi Nutraceuticals, White Bear Lake, Minn., USA). Another
particularly preferred phospholipid is alpha-glyceryl phosphoryl
choline (GPC 85.TM.) from Science & Ingredients Corporation
(Carlsbad, Calif., USA). (GPC 85.TM.) is comprised of 15 wt. %
water and 85 wt. % GPC).
[0084] The further addition of pH modifiers including lactic acid,
gallic acid, sodium acid sulfate, citric acid, ascorbic acid,
gluconic acid or combinations thereof may improve the oxidative
stability. The yet further addition of chelating agents including
citric acid may also enhance the oxidative stability. Although
citric acid controls the conversion of sucrose to invert sugar,
accelerated storage conditions (i.e., a temperature of 60.degree.
C. for more than 7 days) can lead to the formation of invert sugar
(a mixture of glucose and fructose). Enhanced solubility may reduce
the chalkiness associated with precipitated compounds, an important
criteria in the inclusion of the nanoemulsions in functional foods,
confectioneries, and beverages for superior texture and
mouthfeel.
[0085] In a specific embodiment, the invention includes a
composition having ingredients selected from the group of:
non-reducing sugars, sugar polyols, or combinations thereof;
modified starches; polysaccharides; glycerides selected from
enzymatically modified oils, fats, and fatty acids of mono-, di-,
and tri-glycerides; glycerides selected from lipolyzed modified
oils, fats, and fatty acids of mono-, di-, and tri-glycerides;
fruit concentrate sweetener as humectant that comprises a blend of
hydrolyzed starch having a dextrose equivalent (D.E.) of up to
approximately 25; fruit juice or fruit syrup concentrate of at
least approximately 40 wt. % soluble solids and approximately 0 wt.
% insoluble solids thereby forming a liquor having a dry weight
composition of approximately 40 to approximately 65 wt. % complex
carbohydrates; and approximately 35 to approximately 55 wt. %
simple sugars from the fruit juice or fruit syrup concentrate; and
approximately 0 to approximately 5 wt. % nutritional components
occurring naturally in the fruit juice or fruit syrup concentrate;
cocoa powder; Sucralose; or combinations thereof.
[0086] Cocoa powder contains around 20 wt. % raw protein. Maillard
reactions are initiated by a condensation between the free amino
group of amino acid, peptide, or protein and the carbonyl group of
a reducing sugar to give a N-substituted glycosyl-amino compound
followed by the reversible formation of the Schiff base, which
cyclizes to the NB substituted glycosylamine and its then converted
into the Amadori compound. The Amadori rearrangement is catalyzed
by weak acids and is considered the key step of the Maillard
reaction. Amadori compounds formed during the early stage of the
Maillard reaction are responsible for the loss of nutritional value
of amino acids and proteins, because their biological activity is
reduced by the formation of Amadori compounds. Cocoa powder also
contains around 10 wt. % polyphenols, which have antioxidative
effects (Dreosti I. E. in Nutrition 16, 692-694 (2000)). The
ability of cocoa powder to inhibit lipid oxidation in O/W emulsion
systems with added sucrose (pH 6.6) is influenced by heat
treatments. An extensive acid hydrolysis of sucrose, by heat, is
detrimental to the antioxidant capacity of cocoa powder. However,
for formulated O/W emulsions that have sorbitol (pH 6.6), cocoa
powder shows enhanced oxidative stability upon storage at
60.degree. C. for 28 days.
[0087] A wide range of products may be manufactured by inclusion of
the compositions of the invention including: hypercholesterolemia
prevention products in a mammal including salts selected from the
group of calcium and magnesium salts; bone mineral loss prevention
products in a mammal including salts selected from the group of
calcium and magnesium salts; oils rich in Omega-3 products, further
comprised of salts selected from the group of calcium and magnesium
salts; oil-soluble flavor products; oil-soluble vitamin,
nutraceutical, or pharmaceutical products; products having
vegetable oils including rice bran oil, flax, chia, hemp, castor,
soybean, lesquerella, dehydrated castor oil, rich in Omega-3, or
conjugated linoleic acid, animal oils including fish, egg, poultry,
and beef oils rich in Omega-3, or conjugated linoleic acid, or
combinations thereof; microalgae oils, rich in Omega-3, or
conjugated linoleic acid, or combinations thereof; beverage
products being transparent including salts selected from the group
of calcium and magnesium salts; cocoa products having improved
creaminess, reduced bitterness, and reduced oxidation; protein rich
products including high-methoxyl pectins or pectin alginates or
combinations thereof having reduced protein settling and
sedimentation; protein rich products having reduced protein
settling and sedimentation; oil-in-water micro- and nano-emulsions
having increased emulsion and oxidation stability; or water-in-oil
micro- and nano-emulsions having increased emulsion and oxidation
stability.
[0088] Additional preferred actives include actives that further
enhance transport through cellular membranes/mitochondria. One
exemplary is Acetyl L-carnitine, an amino acid-like compound
related to choline. It is a brain support supplement that may
assist in the conversion of choline into acetylcholine, one of the
body's key neurotransmitters. Additional L-carnitine components
include components selected from the group consisting of free
L-carnitine, L-carnitine L-tartrate, L-carnitine magnesium citrate
and acetyl-L-carnitine.
[0089] The acetyl group that is part of acetyl-L-carnitine
contributes to the production of the neurotransmitter
acetylcholine, which is required for mental function. Several
double-blind clinical trials suggest that acetyl-L-carnitine delays
the progression of Alzheimer's disease and enhances overall
performance in some people with Alzheimer's disease. Alzheimer's
research has been done with the acetyl-L-carnitine form, rather
than the L-carnitine form, of this nutrient. L-carnitine is
traditionally made in the body from the amino acids lysine and
methionine, and is needed to release energy from fat. It transports
fatty acids into mitochondria, though the body requires adequate
lysine, methionine, vitamin C, iron, niacin, and vitamin B6 to
produce carnitine.
[0090] Additional preferred actives include actives that suppress
delta-5 desaturase enzyme. A more preferred active is sesame
lignans, an extract from sesame seeds. A series of specific actives
are found in sesame lignans including sesamin and sesamol. Sesame
lignans suppress the enzyme (delta-5 desaturase) that converts DGLA
into arachidonic acid. By blocking the undesirable enzyme (delta-5
desaturase), more DGLA is available for conversion into beneficial
prostaglandin.
[0091] Yet further additional actives include permeation enhancers,
with exemplaries including acylcarnitine, phosphatidylcholine,
fatty acids (eicosapentaenoic acid (EPA), docosahexaenoic acid
(DHA), oleic acid, capric acid, linoleic acid, and their
monoglycerides), bile salts (cholate, taurocholate and
derivatives), salicylates (3- or 5-methoxy-salycilate, salicylate),
homovanilate, surfactants (sodium dodecyl sulfate (SDS), Triton
X-100, Brij), chelating agents (ethylenediamine tetraacetic acid
(EDTA), citric acid, phytic acid, enamine derivatives), and
aprotinin.
[0092] The inclusion of such permeation enhancers, in combination
with the small size of the nanoemulsion composition enables
permeation through various administration routes including the
skin, blood capillary or membrane barriers. Therefore, the
nanoemulsion composition is suitable for dermal, peroral, enteral,
parenteral, ocular, pulmonary, and transmucosal administration
routes. The term "permeation enhancer" includes all actives and
methods that promote permeation through the cellular membranes,
such as mitochondria of cells including, though not limited to,
gastrointestinal, and brain cells.
[0093] Additional functionality includes enhancing cognitive
performance, and reducing disorders of mental health. The use of
specific actives such as cholines and serines is recognized in the
art for the treatment of a wide range of cognitive and mental
health disorders. These include as one exemplary, though not
limited, enhancing memory functions in Alzheimer patients. The
present invention uniquely achieves a synergistic impact on such
cognitive and mental health conditions by enhancing the delivery of
such actives and concurrently the delivery of Omega-3. The realized
benefits are greater than each individual component.
[0094] The range of products include, but are not limited to,
confectionery, baked goods, spreads, dressings, salad dressings,
nutraceutical supplements, functional foods products, ice cream,
seed milks, dairy products, pharmaceutical tablets, syrups, and
medicines, functional confectionery products, mineral-enriched
drinks, and oral care products. The specific complexation of
caseinophosphopeptide (CPP) with chitosan further leverages the
bioadhesive properties of chitosan, thus the complex may provide
both superior solubility for the calcium and adhesion to the teeth
both of which are required conditions for teeth
remineralization.
[0095] Compositions of the present invention may include O/W and
W/O emulsions prepared with vegetable and animal oils that contain
a significant amount of highly polyunsaturated fatty acids such as
rice bran oil, flaxseed oil, chia oil, hemp oil, soybean oil,
lesquerella oil, castor oil, dehydrated castor oil, menhaden oil,
sardine oil, herring oil, salmon oil, anchovy oil, and other oils
rich in Omega-3, or conjugated linoleic acid. The oil content of
the O/W and W/O emulsions may vary according to the oil species
component used and other components but may be within the range of
0.1-95 w/v %, preferably 1-85 w/v %. Embodiments of the present
invention also may be effective when applied to oil flavors such as
fruit and herb flavored oils, cheese flavored oils, butter flavored
oils, and oil soluble vitamin, nutraceutical or pharmaceutical
products.
[0096] Oil-in-water (O/W) emulsions that include small lipid
droplets dispersed in an aqueous medium form the basis of many
kinds of foods, e.g., milk, cream, beverages, dressings, dips,
sauces, batters and deserts. Emulsions are thermodynamically
unstable systems because of the unfavorable contact between oil and
water phases, and because the oil and water phases have different
densities, hence they will always breakdown over time. Use of
emulsifiers, which are surface-active ingredients that absorb to
the surface of freshly formed lipid droplets during homogenization,
usually retards emulsion breakdown. Once absorbed, they facilitate
further droplet disruption by lowering the interfacial tension,
thereby reducing the size of the droplets produced during
homogenization. Emulsifiers also reduce the tendency for droplets
to aggregate by forming protective membranes and/or generating
repulsive forces between the droplets. A good emulsifier should
rapidly adsorb to the surface of the lipid droplets formed during
homogenization, rapidly lower the interfacial tension by a
significant amount and protect the droplets against aggregation
during emulsion processing, storage and utilization.
[0097] Emulsions prepared with egg yolk phospholipids and the
nanoemulsion compositions of the present invention have improved
stability against phase separation and particle aggregation. Recent
studies for the purpose of enhancing flavor release have shown that
the release of non-polar flavors from O/W emulsions during
mastication is controlled by encapsulating the oil droplets within
biopolymer particles (Malone et al. in Flavor Release, ACS
Symposium Series, American Chemical Society, pp. 212-217 (2000)).
Biopolymer particles are created by the caprine
caseinophosphopeptide-chitosan complex and eggplant-carrageenan
complex that are embodiments of the inventive antioxidant
compositions.
[0098] Different emulsifiers are categorized as being ionic or
non-ionic. Ionic compounds may be cationic, anionic or amphoteric.
Ionic emulsifiers have a problem; however, they can react with
various ions to form complexes that adversely affect performance.
Non-ionic emulsifiers tend not to react with ions and are used most
extensively in the food industry. Without being bound by theory,
the presence of a non-ionic surface active agent limits the
creation of adverse complexes with the polycationic CPP-chitosan
complex. The utilization of non-ionic components is desired within
the oil and water interface. Whereas, the preferred oil soluble
components within the oil phase are ionic as a means to increase
the electrical conductivity and/or electron transport such that
electron transport can be enhanced from antioxidant and/or electron
donor within the oil phase and/or through the interface (preferably
through CPP-chitosan emulating channel proteins) such that water
soluble antioxidants become the preferred electron donor (as the
water phase has higher electrical conductivity and/or electron
transport). The particularly preferred embodiment is where the
"channel protein" is a phosphopeptide. Without being bound by
theory, the negative charge of the phosphate group enhances the
electron transfer across the liquid-liquid interface. It is
recognized in the art that prior binding of ubiquinone (i.e.,
CoQ10) to protein followed by the subsequent binding of
phospholipids is a means to reactive enzymatic reactions. The
preferred embodiment is further comprised of whey protein--lactic
acid--CoQ10 complex to further encapsulate the Omega-3 rich oil
emulsion.
[0099] One exemplary non-ionic surface active agent is
polyglycerol. One method known in the art to solubilize organic
carboxylic acids is through the utilization of diglycerides. The
inventive preferred embodiment is comprised of carboxylic acids
selected from the group consisting of lactic acid, gallic acid, or
combinations thereof. The particularly preferred is the combination
of lactic acid and gallic acid, without being bound by theory, is
attributed to lactic acid's and gallic acid's superior electron
donor capacity.
[0100] One exemplary ionic emulsifier, which is preferentially oil
soluble, is lecithin (or phosphatidyl choline). Lecithin is unique
in that it is both able to be dissolved in oil and carry a charge
(critical for electron transport). The conductivity of the host oil
can be modified using ionic emulsifiers, as noted in the art, such
as lecithin and charged compounds with a hydrophobic end such as
alcohols. The preferred embodiment only utilizes charged compounds
with a hydrophobic end, such as alcohol, which is limited to
solutions absent of proteins such as the particularly preferred
embodiment, as it cause protein precipitation (i.e.,
CPP-chitosan).
[0101] The caseinophosphopeptide (CPP) employed as nanoemulsion
compositions of the present invention may include
alpha.sub.s2-casein as isolated from caprine (goat) milk. Caseins
and caseinophosphopeptides exhibit different degrees of
phosphorylation, and a direct relationship between the degree of
phosphorylation and mineral chelating activity has been described
(Kitts, D. D. in Can. J. Physiol. Pharmacol. 72, 423-434 (1994)).
Accordingly based on phosphorylation, alpha.
sub.s2-casein>alpha.sub.s1-casein>beta-casein>kappa-casein.
Caseinophosphopeptide isolated from caprine (goat) milk high in
alpha.sub.s2-casein (alpha.sub.s2-casein=29.2% of total casein) has
more mineral chelating activity than a caseinophosphopeptide
isolated from bovine (cow) milk (alpha.sub.s2-casein=12.1% of total
casein). The phosphoric group of phosphoserine and carboxic groups
of acidic amino acids present in the caseinophosphopeptide isolated
from caprine (goat) milk high in alpha.sub.s2-casein, without being
bound by theory, likely complexes with metal ions such as iron and
copper. Complexation with other critical nutritional minerals, such
as selenium, zinc, and magnesium may further increase their
bioavailability.
[0102] It would also be understood to one skilled in the art that
other milk high in alpha. sub.s2-casein may be suitable for the
present invention. Choice of milk may be influenced, inter alia, by
economic factors and availability of particular milk. The selection
of milk containing high levels of alpha.sub.s2-casein, which is low
in alpha.sub.s1-casein, may be carried out by reversed-phase high
performance liquid chromatography (RP-HPLC) (Mora-Gutierrez et al.
in J. Dairy Sci. 74, 3303-3307 (1991)). The casein composition of
the caprine caseinophosphopeptide is normally as follows:
alpha.sub.s2-casein content=29.2 wt. %, alpha.sub.s1-casein
content=5.9 wt. %; beta-casein content=50.5 wt. % and kappa-casein
content=14.4 wt. %.
[0103] The fat in caprine (goat) milk is also rich in medium-chain
triglycerides (MCT) (C6:0 Caproic, C8:0 Caprylic and C10:0 Capric)
which are absorbed in the proximal intestine and do not require
bile salts to be absorbed (Vanderhoof et al. in J. Parenter.
Enteral Nutr. 8, 685-689 (1984)). These MCT have become of
considerable interest to the medical profession because of their
unique benefits in many metabolic diseases of humans (Babayan V. K.
in J. Amer. Oil Chem. 59, 49A-51A (1981)). The bone (femur and
sternum) is the preferential organ for the deposit of magnesium in
animals fed a caprine (goat) milk diet, which has been ascribed to
its special characteristics concerning lipid composition (rich in
MCT) (Lopez-Aliaga et al. in J. Dairy Sci. 86, 2958-2966 (2003)).
Lipids are associated with proteins (caseins) in milk and their
content in bound lipid fractions is high (Cerbulis J. in J. Agric.
Food Chem. 15, 784-786 (1967)). The MCT content of the caprine
caseinophosphopeptide used in this inventive antioxidant
composition is high because this caprine caseinophosphopeptide is
produced from caprine (goat) milk with a fat content of 1 wt. % by
enzymatic hydrolysis and acid precipitation with chitosan.
Chitosan, which assumes a polycationic character at acidic pH,
exhibits a high fat-binding capacity (No et al. in J. Food Sci. 65,
1134-1137 (2000)).
[0104] In an exemplary embodiment of the invention, caprine (goat)
milk (1% fat content) characterized by a high alpha. sub.s2-casein
content is used as the starting material in a method of the present
invention: (a) digesting the casein present in caprine (goat) milk
high in alpha.sub.s2-casein with 0.01% (w/v) trypsin (enzymatically
modified proteins through trypsin digestion) at a substantially
neutral pH to produce a crude caseinophosphopeptide, (b) reducing
the pH to 4.5 with 2% (w/v) chitosan (SEACURE L 110 with 70 wt. %
deacetylation; Pronova Biopolymer, Inc., Oslo, Norway) dissolved in
10% citric acid (w/v), (c) removing the unreacted casein from the
supernatant by centrifugation, (d) permitting the supernatant to
stand for 20 hours at 4.degree. C., (e) adjusting the pH of the
supernatant to about 6.0, then adding calcium chloride (0.2% w/v)
and ethanol (40% v/v), to precipitate a calcium-bound
caseinophosphopeptide, which is recovered by centrifugation. This
calcium-bound caseinophosphopeptide may be washed with deionized
water and dried by lyophilization. The composition of the
lyophilized product is provided in Table 1.
TABLE-US-00001 TABLE 1 Caprine caseinophosphopeptide composition
Per 100 grams Kjeldahl N 6.49 Calcium 8.61 Phosphorus 2.76
Medium-chain triglycerides 9.71
[0105] A food grade acidulant may be added to the fat emulsion
before adding the acid-soluble caprine caseinophosphopeptide. The
acid-soluble caprine caseinophosphopeptide may be added to an
acidic environment ranging from approximately pH 2.0 to 5.7. The
food grade acidulant may be citric acid, ascorbic acid, gluconic
acid, and mixtures thereof. The acidulant in the fat emulsion may
be mostly citric acid. Citric acid sequesters deleterious trace
metals, particularly copper and iron, which hasten deterioration of
color, flavor and vitamin A content.
[0106] As used herein, the term LBJ refers to a mixture of sugars
and soluble fiber derived from eggplant (Solanum melongena). To
produce LBJ in one example, whole eggplant is slurried with water
to which citric acid and iota-carrageenan are added. This mixture
is reacted at elevated temperature under controlled conditions for
a specific period of time. The resulting slurry of sugars/soluble
fiber (LBJ) is subsequently treated with an adsorptive resin
functional to remove from the sugars/soluble fiber (LBJ) bitter
taste components, color and odor components. The treated
sugars/soluble fiber (LBJ) solution may be concentrated and dried
if desired to powder form. The further addition of polyphenols,
specifically the polyphenols derived from the fruit of Solanum
melongena is possible.
[0107] More specifically, in an exemplary embodiment, an aqueous
solution containing 0.50 wt. % citric acid and 0.25 wt. %
iota-carrageenan is heated at 45.degree. C. for 6 hours with
continuous stirring. Eggplant samples may be obtained from local
food stores or any other source and stored under refrigeration at
approximately 4.degree. C. until use if necessary. About one hour
prior to use, the eggplant samples are removed from refrigeration
and equilibrated at room temperature at about 22.degree. C. The
eggplants (0.7 kg) are rinsed with water, peeled and then sliced
into 4-5 mm thick slices. These are immediately immersed in a
treatment bath containing the mixed-acid solution of citric acid
and iota-carrageenan. The treatment bath with the sliced eggplants
and mixed-acid solution of citric acid and iota-carrageenan is then
heated to a temperature that may be in the range 70.degree. C. to
80.degree. C., typically 75.degree. C. This elevated temperature
may be maintained for at least 2 hours but possibly held at such
elevated temperature for longer, e.g., about 4 hours, and then
cooled to between 0.degree. C. and 50.degree. C., in a particular
embodiment about 4.degree. C., for a period of time, typically
about 12 hours. Finally, the mixture is decanted through Whatman
No. 4 filter paper or similar filtration medium.
[0108] In an exemplary embodiment, the aqueous slurry/solution
(LBJ) is passed through a column of an adsorptive resin. The
adsorptive resin may be a polymeric resin, which functions to
remove bitterness, odor and color from the aqueous slurry/solution
(LBJ). One suitable class of adsorptive resins for use are
polymeric cross-linked resins composed of styrene and
divinylbenzene such as, for example, the Amberlite series of
resins, e.g., Amberlite XAD-2, Amberlite XAD-4 and Amberlite
XAD-16, which are available commercially from Supelco of
Bellefonte, Pa. Other polymeric crosslinked styrene and
divinylbenzene adsorptive resins suitable for use according to the
invention are XFS-4257, XFS-4022, XUS-40323 and XUS-40322
manufactured by Dow Chemical Company of Midland, Mich., and other
similar resins.
[0109] Treatment of the aqueous slurry/solution (LBJ) in accordance
with this invention may be conducted in various manners such as by
a batch treatment or by passing the aqueous slurry/solution (LBJ)
through a column containing the adsorptive resin. The column size
selected depends upon the sample size and the concentration of the
aqueous slurry/solution (LBJ).
[0110] More specifically, in an exemplary embodiment, a batch of
approximately 100 g of Amberlite XAD-2 is slurried in water and
poured into an open glass chromatography column (2.times.30 cm)
fitted with a Teflon stopcock. The column is then prepared for use
by washing it with two liters of twice-distilled water, two liters
of distilled methanol (reagent grade), and finally two liters of
distilled water. The aqueous slurry/solution (LBJ) treated in the
column may preferably be free of insoluble material so as to not
plug the column or impede flow. Generally, the concentration of
eggplant undergoing treatment may be in the range of about 50 to
70% by weight. The pH of the slurry/solution (LBJ) may be in the
range of pH 3 to 4. The flow rate of the aqueous slurry/solution
(LBJ) through the column may preferably be slow enough to allow
sufficient time for the undesired bitterness, color and odor to be
adsorbed in the adsorptive resin. Column flow rates between one to
five bed volumes/hour are generally satisfactory.
[0111] One aqueous slurry/solution (LBJ) according to the present
invention contains a fructose portion of 3.7 wt. % and a sucrose
portion of 1.5 wt. % as determined by high-performance liquid
chromatography (HPLC). Thus, this natural composition exhibits a
high hygroscopic property. Saccharide polymers may be used as
spray-drying aids in the manufacture of this natural composition.
The composition may include between around 5 and 10% by weight
maltodextrin. The maltodextrin may have a low DE, generally not
exceeding about 10. The aqueous slurry/solution (LBJ) is mixed with
maltodextrin DE=10 at a concentration of 6% (by weight) after the
aqueous slurry/solution (LBJ) is passed through a column of the
adsorptive resin. Then, the aqueous slurry/solution (LBJ 10) is
dried by spray drying or the like to provide a product that is well
suited for use as a natural antioxidant ingredient for fat
emulsions. The composition of this product is provided in Table
2.
TABLE-US-00002 TABLE 2 LBJ 10 physicochemical composition Per 100
grams carbohydrate portion 92.21 nitrogen content 0.71 fat portion
0.16 ash portion 2.33 dietary fiber portion 0.41 soluble fiber
portion 0.41 fructose portion 3.72 glucose portion 4.26 sucrose
portion 1.48 maltose portion 2.19 sugar portion 11.65
[0112] The numerical values for carbohydrate, crude protein, fat
portion, ash portion, dietary fiber portion, soluble fiber portion,
and sugar portion are those according to a general analysis.
[0113] Carrageenans exhibit thickening or viscosity-increasing
effect. The viscosity of the LBJ 10 composition of Table 2, which
has 0.25 wt. % iota-carrageenan, is rather low, i.e., about 11 cps
(1%, 22.degree. C.), and it tastes slightly sweet and is odorless.
Carrageenans such as kappa-carrageenan and lambda-carrageenan can
also be used in the preparation of LBJ 10. Carrageenans are known
to interact with casein (and derived phosphopeptides) to modify
food texture by improving water holding capacity (Mora-Gutierrez et
al. in J. Agric. Food Chem. 46, 4987-4996 (1998)). In some
embodiments of the invention, the combination of egg yolk
phospholipids, caprine caseinophosphopeptide and LBJ 10 impart
richness, lubricity and creaminess to fat-reduced emulsions.
Because antioxidant activities are correlated with the phenolic
contents of foods, the total phenolic content of LBJ 10 was
determined using methods described by Singlenton et al., Analysis
of Total Phenols and Other Oxidation Substrates and Antioxidants by
Means of Folin-Ciocalteu Reagent, Methods in Enzymology, Oxidants
and Antioxidants, 1998, pp. 152-178. The total phenolic content of
LBJ 10 was 45.mu.mol gallic acid equivalents/g of LBJ 10.
[0114] The present invention includes compositions of natural
antioxidants including tocopherols, beta-carotene, egg yolk or
soybean phospholipids, sucrose or sorbitol, caprine
caseinophosphopeptide-chitosan complex, eggplant (LBJ 10), and
citric acid. Specific antioxidant ingredients of the present
invention may include from about 0.01 to about 0.03% by lipid
content of tocopherols, from about 0.01 to about 0.03% by lipid
content of beta-carotene, from about 0.05 to about 0.5% by weight
of emulsion of egg yolk or soybean phospholipids, from about 2 to
about 20% by weight of emulsion of sucrose or sorbitol, from about
0.01 to about 0.05% by weight of emulsion of caprine
caseinophosphopeptide-chitosan complex, from about 0.01 to about
0.2% by weight of emulsion of eggplant (LBJ 10), and from about
0.01 to about 0.5% by weight of emulsion of citric acid.
[0115] One specific composition includes about 0.01% tocopherols,
0.01% beta-carotene, 0.1% egg yolk or soybean phospholipids, 10%
sorbitol, about 0.05% caprine caseinophosphopeptide-chitosan
complex, about 0.01% eggplant (LBJ 10), and about 0.01% citric
acid, all by weight of emulsion.
[0116] Unrefined Canadian flaxseed oil is rich in tocopherols and
beta-carotene. A specific embodiment of the composition of the
present invention, especially effective for O/W emulsions prepared
with Canadian flaxseed oil, is as follows: 0.05% caprine
caseinophosphopeptide-chitosan complex, 0.01% eggplant (LBJ 10),
and 0.01% citric acid by weight of emulsion.
[0117] The fat emulsion may be produced by conventional technology.
An exemplary production process includes adding egg yolk or soybean
phospholipids in suitable amounts to a predetermined amount of the
oil component, homogenizing the mixture, adding sorbitol, caprine
caseinophosphopeptide-chitosan complex, eggplant (LBJ 10), and
citric acid in suitable amounts to a predetermined amount of the
water component, and emulsifying the entire mixture with a
homogenizing machine such as the conventional homo-mixer,
homogenizer, ultrasonic homogenizer, or pressure homogenizer. The
mixture may preferably be finely dispersed by homogenization to
ensure a homogeneous equal dispersion of the natural antioxidant
composition in all the oil particles. The average particle diameter
of the fat emulsion particles is within the range of 5-50 nm. The
emulsified mixture may be pasteurized using conventional
methods.
[0118] Some natural antioxidant compositions of the present
invention may exhibit antioxidant activity superior to prior
compositions or synthetic antioxidants. Some natural antioxidant
compositions of the present invention may also offer a number of
health benefits, including helping to promote bone health by
boosting calcium and magnesium absorption, and a healthy
cardiovascular system by lowering blood serum cholesterol levels.
Thus in certain embodiments, the amount of caprine
caseinophosphopeptide and eggplant (LBJ 10) may range from the
minimum amount which will stabilize the oil against oxidation, or
effectiveness, to at least that amount which will promote bone
health and protect against heart disease in animal or human bodies.
In general, the amount of caprine caseinophosphopeptide-chitosan
complex and eggplant (LBJ 10) used may range from 0.01 to 0.05% by
weight for caprine caseinophosphopeptide-chitosan complex and 0.01%
to 0.1% by weight for eggplant (LBJ 10).
[0119] Exemplary Data
[0120] The procedures utilized for all of the below examples are
identical except where explicitly noted. Therefore, only the
differences are noted from one example to the next example as
indicated in square brackets [ . . . ]. Further, ascorbyl palmitate
(0.03% w/w, based on the weight of the oil, is added to oil blend
followed by homogenization is only added to pH 6.0 and not pH 3.0
(as it is pro-oxidant at the lower pH values). The ionic
emulsifier, egg yolk phospholipids (.omega.6-PL-85.TM.), was used
in the present oxidative stability studies of O/W Menhaden
oil-based emulsions `blended` with Smart Balance Omega.TM. oil
(Examples 1 thru 9). All results are within Table 3.
[0121] The following examples are included to demonstrate specific
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention. However, those of skill in
the art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
[0122] Examples 1 through 9 demonstrate Omega-3 emulsions with a
range of synergistic actives.
[0123] The health benefits of some embodiments of the present
invention are explained in detail in Examples 10 through 12.
Example 1
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 6.0) Containing
Caprine CPP-Chitosan Complex and Food-Grade EDTA
(ethylenediaminetetraacetate)
[0124] A=Control
[0125] Menhaden oil/Smart Blend Omega oil at 1:1 ratio (25%) is
homogenized with egg yolk phospholipids "rich" in PC (0.3% w/w,
based on the weight of the oil) followed by homogenization, then
Cavamax CoQ10.TM. (0.3% w/w, based on the weight of the emulsion)
is added to this oil blend followed by homogenization. An aqueous
solution (75% of double deionized water) containing potassium
phosphate monobasic (0.01% w/w, based on the weight of the
emulsion), mannitol (4% w/w, based on the weight of the emulsion)
and trehalose (2% w/w, based on the weight of the emulsion) is
stirred. [The pH of this aqueous solution (water phase) is adjusted
to 6.0 with 0.1 N potassium hydroxide (KOH)]. The emulsified oil
blend (25%) is added to the aqueous solution (75%) followed by
homogenization.
[0126] B=Treatment
[0127] Menhaden oil/Smart Blend Omega oil at 1:1 ratio (25%) is
homogenized egg yolk phospholipids "rich" in PC (0.3% w/w, based on
the weight of the oil), then Cavamax CoQ10.TM. (0.3% w/w, based on
the weight of the oil) is added to this oil blend followed by
homogenization. [Ascorbyl palmitate (0.03% w/w, based on the weight
of the oil, is added to oil blend followed by homogenization.] An
aqueous solution (75% of double deionized water) containing
potassium phosphate monobasic (0.01% w/w, based on the weight of
the emulsion), mannitol (4% w/w, based on the weight of the
emulsion) and trehalose (2% w/w, based on the weight of the
emulsion) is stirred. Gallic acid (0.002% w/w, based on the weight
of the emulsion) and [Ca-EDTA (0.0070% w/w, based on the weight of
the oil) are added and stirred. The pH of this aqueous solution
(water phase) is adjusted to 6.0 with 0.1 N potassium hydroxide
(KOH)]. Caprine CPP-chitosan complex (0.04% w/w, based on the
weight of the emulsion) is added to this aqueous solution and
homogenized. The emulsified oil (negatively charged oil phase)
(25%) is added to this aqueous solution (positively charged water
phase) (75%) followed by homogenization.
[0128] Samples were stored in 50 ml capped, sterile brown glass
bottles in an incubator for 28 days at 40.degree. C., and, samples
were taken at 14 and 28 days. Samples were analyzed for the
peroxide value (hydroperoxides) and p-anisidine value (aldehydes).
The antioxidant activity of the composition according to an
embodiment of the present invention is demonstrated below.
Example 2
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 6.0) Containing
Caprine CPP-Chitosan Complex and Phytic Acid
[0129] B=Treatment
[0130] [and phytic acid (0.3% w/w, based on the weight of the
emulsion) are added and stirred. The pH of this aqueous solution
(water phase) is adjusted to 6.0 with 0.1 N potassium hydroxide
(KOH)].
Example 3
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 3.0) Containing
Caprine CPP-Chitosan Complex and Food-Grade EDTA
(ethylenediaminetetraacetate)
[0131] A=Control
[0132] [The pH of this aqueous solution (water phase) is adjusted
to 3.0 with 10 mM lactic acid.]
[0133] B=Treatment
[0134] No ascorbyl palmitate. [The pH of this aqueous solution
(water phase) is adjusted to 3.0 with 10 mM lactic acid. Gallic
acid (0.002% w/w, based on the weight of the emulsion) and Ca-EDTA
(0.007% w/w, based on the weight of the oil) are added and stirred.
Gallic acid is acidic. Thus, the pH of this aqueous solution (water
phase) should be re-adjusted to 3.0 with 0.1 N potassium hydroxide
(KOH)].
Example 4
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 3.0) Containing
Caprine CPP-Chitosan Complex and Phytic Acid
[0135] A=Control
[0136] [The pH of this aqueous solution (water phase) is adjusted
to 3.0 with 10 mM lactic acid.]
[0137] B=Treatment
[0138] No ascorbyl palmitate. [The pH of this aqueous solution
(water phase) is adjusted to 3.0 with 10 mM lactic acid. Gallic
acid (0.002% w/w, based on the weight of the emulsion) and phytic
acid (0.3% w/w, based on the weight of the emulsion) are added and
stirred. Gallic acid is acidic. Thus, the pH of this aqueous
solution (water phase) should be re-adjusted to 3.0 with 0.1 N
potassium hydroxide (KOH)].
Example 5
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 3.0) Containing
Caprine CPP-Chitosan Complex and Lactoferrin
[0139] A=Control
[0140] [The pH of this aqueous solution (water phase) is adjusted
to 3.0 with 10 mM lactic acid.]
[0141] B=Treatment
[0142] No ascorbyl palmitate. [The pH of this aqueous solution
(water phase) is adjusted to 3.0 with 10 mM lactic acid. Gallic
acid (0.002% w/w, based on the weight of the emulsion) and
lactoferrin (0.01% w/w, based on the weight of the emulsion) are
added and stirred. Gallic acid is acidic. Thus, the pH of this
aqueous solution (water phase) should be re-adjusted to 3.0 with
0.1 N potassium hydroxide (KOH)].
Example 6
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 3.0) Containing
Caprine CPP-Chitosan Complex, Lactoferrin and Grape Seed
Extract
[0143] A=Control
[0144] [The pH of this aqueous solution (water phase) is adjusted
to 3.0 with 10 mM lactic acid.]
[0145] B=Treatment
[0146] No ascorbyl palmitate. [The pH of this aqueous solution
(water phase) is adjusted to 3.0 with 10 mM lactic acid. Grape seed
extract (0.01% w/w, based on the weight of the emulsion) and
lactoferrin (0.01% w/w, based on the weight of the emulsion) are
added and stirred].
Example 7
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 3.0) Containing
Caprine CPP-Chitosan Complex, Lactoferrin and Resveratrol
[0147] A=Control
[0148] [The pH of this aqueous solution (water phase) is adjusted
to 3.0 with 10 mM lactic acid.]
[0149] B=Treatment
[0150] No ascorbyl palmitate. [The pH of this aqueous solution
(water phase) is adjusted to 3.0 with 10 mM lactic acid.
Resveratrol (0.01% w/w, based on the weight of the emulsion) and
lactoferrin (0.01% w/w, based on the weight of the emulsion) are
added and stirred].
Example 8
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 6.0) Containing
Caprine CPP-Chitosan Complex, Food-Grade EDTA
(ethylenediaminetetraacetate) and New Zealand Wool-Derived Keratine
(Cynatine FLX.TM.)
[0151] A=Control
[0152] B=Treatment
[0153] [New Zealand wool-derived keratine (Cynatine FLX.TM.) are
added to this aqueous solution (pH 6.0) and homogenized.]
Example 9
25% O/W Menhaden/Smart Blend Omega Emulsions (pH 3.0) Containing
Caprine CPP-Chitosan Complex, Food-Grade EDTA
(ethylenediaminetetraacetate) and New Zealand Wool-Derived Keratine
(Cynatine FLX.TM.)
[0154] A=Control
[0155] [The pH of this aqueous solution (water phase) is adjusted
to 3.0 with 10 mM lactic acid.]
[0156] B=Treatment
[0157] No ascorbyl palmitate. [The pH of this aqueous solution
(water phase) is adjusted to 3.0 with 10 mM lactic acid. Gallic
acid (0.002% w/w, based on the weight of the emulsion) and Ca-EDTA
(0.007% w/w, based on the weight of the oil) are added and
stirred.] [New Zealand wool-derived keratine (Cynatine FLX.TM.) are
added to this aqueous solution (pH 3.0) and homogenized.]
TABLE-US-00003 TABLE 3 14 days peroxide 14 days anisidine 28 days
peroxide 28 days anisidine Sample value value value value 1A 4.8
mequiv/kg 10.7 9.2 mequiv/kg 16.5 1B 1.5 mequiv/kg 4.3 3.3
mequiv/kg 7.1 2A 4.9 mequiv/kg 10.5 9.0 mequiv/kg 16.1 2B 3.0
mequiv/kg 6.7 5.7 mequiv/kg 10.3 3A 3.5 mequiv/kg 6.9 8.1 mequiv/kg
12.6 3B 0.9 mequiv/kg 3.9 1.5 mequiv/kg 5.8 4A 3.7 mequiv/kg 6.9
8.0 mequiv/kg 12.7 4B 2.4 mequiv/kg 4.7 5.1 mequiv/kg 8.3 5A 3.9
mequiv/kg 7.0 8.3 mequiv/kg 12.3 5B 2.7 mequiv/kg 4.8 5.8 mequiv/kg
8.6 6A 3.9 mequiv/kg 6.9 8.3 mequiv/kg 12.3 6B 2.6 mequiv/kg 4.8
5.6 mequiv/kg 8.5 7A 3.9 mequiv/kg 7.0 8.3 mequiv/kg 12.3 7B 2.7
mequiv/kg 4.9 5.5 mequiv/kg 8.6 8A 4.8 mequiv/kg 10.7 9.2 mequiv/kg
16.5 8B 1.3 mequiv/kg 4.1 3.2 mequiv/kg 7.0 9A 3.5 mequiv/kg 6.9
8.1 mequiv/kg 12.6 9B 1.1 mequiv/kg 4.0 1.4 mequiv/kg 5.5
Example 10
Cholesterol-Lowering Activity in Rats
[0158] Rats (Sprague-Dawley type, 7 weeks of age, male) were fed a
diet low in calcium and high in animal fat. These rats were divided
into three groups each being formed of 12 rats having a similar
mean body weight of 200-205 grams, then three kinds of
heat-sterilized O/W nanoemulsions i.e., an O/W nanoemulsion of
0.05% (w/v) caprine caseinophosphopeptide-chitosan complex and
0.01% (w/v) eggplant (LBJ 10) supplemented with calcium (300 ppm),
an O/W nanoemulsion supplemented with calcium (300 ppm), and an O/W
nanoemulsion non-supplemented with calcium were respectively given
from feeding bottles to the rats as drinking water. Composition of
these O/W nanoemulsions was identical in terms of flaxseed oil (1
g/L), soybean phospholipids (0.1 g/L), sucrose (4 g/L), and citric
acid (5.0 g/L) content. O/W nanoemulsions were supplemented with
calcium gluconate (3 g/L).
[0159] The three groups of rats were free to take the feed and
water in, during the treatment period of 21 days. At the end of the
21-day, rats were deprived of food overnight and anesthetized by
intraperitoneal injection of sodium pentobarbital (40 mg/kg body
weight). Blood collection was carried out from cardiac puncture.
With respect to analysis, measurements were carried out using a
DU-530 Spectrophotometer made by Beckman by means of a colorimetric
method.
[0160] Results of the measurement for blood serum total cholesterol
are shown in Table 4.
TABLE-US-00004 TABLE 4 Group Cholesterol, mg/dL Control
(non-supplemented) 84.92 .+-. 7 Control (supplemented) 78.36 .+-. 5
Natural antioxidant composition 67.30 .+-. 4 (supplemented)
[0161] According to the above results, it has been proved that the
increase in serum cholesterol of male Sprague-Dawley rats fed a low
calcium and high animal fat diet has been lowered by the addition
of an antioxidant composition according to an embodiment of the
present invention (caprine caseinophosphopeptide-chitosan complex
combined with eggplant (LBJ 10) and citric acid at levels of 0.05%
(w/v), 0.01% (w/v), and 0.5% (w/v), respectively) to a
calcium-supplemented O/W emulsion.
[0162] This natural antioxidant composition, therefore, can be
applied to O/W nanoemulsions as physiologically functional
factor.
Example 11
Calcium and Magnesium Bioavailability in Rats
[0163] Rats (Sprague-Dawley type, 7 weeks of age, male) were fed an
egg white-diet low in calcium. Chromic oxide (Cr.sub.2O.sub.3, 0.5
g/kg diet), an insoluble and unabsorbed marker, was added to the
egg white-diet to allow estimation of apparent Ca and Mg absorption
by determining the ratio of Ca:Cr and Mg:Cr in the diet and feces.
These rats were divided into four groups each being formed of 12
rats and having a similar mean body weight of 200-205 grams, then
three kinds of heat-sterilized O/W nanoemulsions i.e., an O/W
nanoemulsion of 0.05% (w/v) caprine caseinophosphopeptide-chitosan
complex and 0.01% (w/v) eggplant (LBJ 10) supplemented with calcium
(300 ppm), an O/W nanoemulsion supplemented with calcium (300 ppm),
and an O/W nanoemulsion non-supplemented with calcium were
respectively given from feeding bottles to the rats as drinking
water. Composition of these O/W nanoemulsions was identical in
terms of flaxseed oil (1 g/L), soybean phospholipids (0.1 g/L),
sucrose (4 g/L), and citric acid (5.0 g/L) content. O/W
nanoemulsions were supplemented with calcium gluconate (3 g/L).
[0164] The three groups of rats were free to take the feed and
water in, during the treatment period of 21 days. Food intake was
measured every day. Feces were collected during the last 3 days and
freeze-dried. At the end of the 21-day, rats were deprived of food
overnight and anesthetized by intraperitoneal injection of sodium
pentobarbital (40 mg/kg body weight). The right femurs were excised
for measurement of Ca, and Mg content. The amounts of Ca, Mg, and
Cr in the diets and feces were quantified by atomic absorption
spectrometry (Varian Analytical Instruments, Walnut Creek, Calif.,
USA) after wet-washing with an acid mixture (16 mol/L HNO3:9 mol/L
HClO4=3:1). The right femurs were treated with 1N HNO3 and ashed at
550 degrees C. Ca and Mg content were determined in the same manner
as in the case of the diets and feces. Apparent Ca absorption was
calculated by the following formula: Apparent Ca absorption
(%)=100[(Ca intake/Cr intake)-(Ca in the feces/Cr in the
feces)]/(Ca intake/Cr intake). Apparent Mg absorption was
calculated in a similar manner.
[0165] The apparent Ca and Mg absorption, and femoral bone Ca and
Mg content of rats fed the three different O/W nanoemulsions are
shown in Table 5.
TABLE-US-00005 TABLE 5 Apparent Ca Apparent Mg Bone Ca content Bone
Mg content Group absorption (%) absorption (%) (mg/femur)
(mg/femur) Control (non- 49 .+-. 5.7 51 .+-. 4.2 89.63 .+-. 0.27
4.47 .+-. 0.13 supplemented Control 54 .+-. 6.0 49 .+-. 5.1 97.08
.+-. 0.19 4.31 .+-. 0.27 (supplemented) Antioxidant 59 .+-. 5.0 61
.+-. 5.9 103.20 .+-. 0.14 5.62 .+-. 0.11 Composition
(supplemented)
[0166] The data show enhanced Ca and Mg bioavailability from the
O/W nanoemulsion containing an antioxidant composition according to
an embodiment of the present invention.
Example 12
Bone Metabolism and Dynamic Strength of Bone in Rats
[0167] Rats (Sprague-Dawley type, 7 weeks of age, male) were fed a
diet low in calcium. These rats were divided into four groups each
being formed of 12 rats and having a similar mean body weight of
200-205 grams, then three kinds of heat-sterilized O/W
nanoemulsions i.e., an O/W nanoemulsion of 0.05% (w/v) caprine
caseinophosphopeptide-chitosan complex and 0.01% (w/v) eggplant
(LBJ 10) supplemented with calcium (300 ppm), an O/W nanoemulsion
supplemented with calcium (300 ppm), and an O/W nanoemulsion
non-supplemented with calcium were respectively given from feeding
bottles to the rats as drinking water. Composition of these O/W
nanoemulsions was identical in terms of flaxseed oil (1 g/L),
soybean phospholipids (0.1 g/L), sucrose (4 g/L), and citric acid
(5.0 g/L) content. O/W nanoemulsions were supplemented with calcium
gluconate (3 g/L).
[0168] The three groups of rats were free to take the feed and
water in, during the treatment period of 21 days. At the end of the
21-day, rats were deprived of food overnight and anesthetized by
intraperitoneal injection of sodium pentobarbital (40 mg/kg body
weight). The left femurs were collected from the animals and soft
tissue was removed. The left femur from each animal was subjected
to bone mineral content (BMC), bone mineral density (BMD), and bone
mechanical strength (BMS) measurements using dual-energy X-ray
absorptiometry (DEXA), which is a typical method used to study the
status of bone growth. Table 6 shows the beneficial effects of an
antioxidant composition according to an embodiment of the present
invention on bone metabolism and dynamic strength of bone in
rats.
TABLE-US-00006 TABLE 6 Group BMC (g) BMD (g/cm2) BMS (kg force)
Control (non-supplemented) 0.1912 .+-. 0.012 0.1346 .+-. 0.004
8.402 .+-. 0.321 Control (supplemented) 0.2041 .+-. 0.012 0.1432
.+-. 0.004 8.591 .+-. 0.298 Antioxidant composition 0.2134 .+-.
0.012 0.1518 .+-. 0.004 9.567 .+-. 0.298 (supplemented)
[0169] The data clearly indicate that the O/W nanoemulsion
containing an antioxidant composition according to an embodiment of
the present invention strengthens the femur bones in rats by
enhancing the amount of magnesium retained in bone (Example 11),
and that this results from increased apparent magnesium absorption
(Example 11).
[0170] The caprine caseinophosphopeptide-chitosan-MCT bound
complexes, which are present in the above antioxidant composition
according to an embodiment of the present invention, are thermally
stable and deliver large amount of magnesium to the proximal
intestine, the site for magnesium absorption. Thus the complexes
per se can provide physiological activity of magnesium to low-pH,
protein-based beverages and transparent beverages processed by heat
treatment. The complexes prevent protein sedimentation in low pH
(3.5-4.2) beverages when used in combination with high-methoxyl
pectins or pectin alginates.
Example 13
Transparent Low-pH (3.0-4.2) Beverages Containing Caprine
Caseinophosphopeptide
[0171] A big factor in the drop in calcium and magnesium
consumption in the United States is the fact that soft drinks have
replaced milk in the American diet. Milk is an excellent source of
calcium (1,310 mg/L) and also contains magnesium (120 mg/L). A
Consumer Beverage Consumption study conducted in late 2000,
surveyed a total of 1,379 participants in two age groups-adults
(19-64; 320 males/358 females) and teens (12-18; 326 boys/375
girls). Adults reported that their favorite beverage is "cold,
refreshing, and satisfying" whereas teens prefer their drinks to be
"cold, refreshing, and delicious". In this survey, teens and
adults, milk drinkers and non-milk drinkers expressed comments
regarding their concern with health issues, additives, chemicals,
handling and spoilage.
[0172] A growing body of research now shows that the more soft
drinks teenagers consume, the higher their risk of broken bones
and, in later life, osteoporosis. Since 1970 Americans have more
than doubled their soft drink consumption while drinking less milk.
Consumers want a cold, refreshing, satisfying, portable, and
healthy beverage. Caprine caseinophosphopeptide-chitosan complex
can be used in transparent low-pH (3.0-4.2) beverages fortified
with calcium and magnesium to prevent the loss of these minerals
from bone and therefore, lowering the risk of bone fractures.
[0173] Caprine caseinophosphopeptide-chitosan complex can also form
the building stones for mineral-fortified, low-pH beverages
tailored for individuals with lactase non-persistence, a reduced
capacity to metabolize lactose. The presence of lactose in milk is
detrimental for those individuals that suffer from lactose
intolerance. The ingestion of one to two glasses of milk can lead
to abdominal discomfort and diarrhea in such individuals. Many
studies have noted racial differences in the incidence of lactose
intolerance. In the United States is estimated that only 10-15% of
adult Caucasians react adversely to lactose, whereas 70% of adult
Afro-Americans are lactose intolerance. The incidence of lactose
intolerance in adult Asians is 95%. The beverage food industry
could formulate a calcium- and magnesium-fortified beverage
containing caprine caseinophosphopeptide-chitosan complex to export
to the Far East.
Example 14
Coated Nuts
[0174] Long shunned by dieters for their fat content, nuts have
made a big-time dietary come back. Recent epidemiological studies
suggest that frequent nut consumption may be protective against
heart disease and other chronic diseases. As mentioned earlier, the
level of fat in the diet influences magnesium absorption because
fatty acids have a greater tendency to form soaps with calcium than
magnesium (Van Dokkun et al. in Ann. Nutr. Metab. 27, 361-367
(1983)).
[0175] Recent research studies have shown that increased lipid
proportion of the diet improves the digestive utilization of
magnesium in clinical cases of malabsorption syndrome (Alferez et
al. in J. Dairy Res. 68, 451-461 (2001)). Increased proportions of
protein in the diet also favor magnesium absorption (Pallares et
al. in J. Agric. Food Chem. 44, 1816-1820 (1996)). Nuts are rich in
fat, protein, and magnesium. The inventive antioxidant composition
promotes a significant increase of magnesium absorption, which is
reflected in the greater quantity of this mineral stored in femoral
bone. Magnesium is associated with strong bones. People who crunch
on nuts coated with the inventive antioxidant composition can lower
the risk of bone fractures.
[0176] The further addition of coenzyme Q10 also enhances the
antioxidative stability of lipids, including within the inventive
nanoemulsions. The more preferred coenzyme Q10 is infused into the
oil phase prior to emulsion. The particularly preferred coenzyme
Q10 is modified through means known in the art, such as
"encapsulation" into gamma-cyclodextrin provided by Wacker in their
Cavamax CoQ10.TM. product, to be solubilized in the oil phase of
the emulsion. The amount of coenzyme Q10 within the oil phase can
range from 0.25% to 5% of the oil on a w/w basis. The preferred
amount of coenzyme Q10 is within the range of 0.25% to 1.25%. The
more preferred amount of the coenzyme Q10 is within the range of
0.25% to 1.0%. The presence of coenzyme Q10 in essentially any
amount, including trace amounts, yields superior antioxidative
stability of the lipid phase. The further ability of to regenerate
oxidized forms of Vitamin E and glutathione by coenzyme Q10 is a
further synergistic benefit of the combination of glutathione and
coenzyme Q10.
[0177] The resulting testing has both expanded the range of
acceptable pH modifiers (and the range of suitable pH from 2.8 to
6.6) and in fact has lead to the more preferred pH modifiers now
including gallic acid, fumaric acid, pantothenic acid, and choline
citrate. The combination of the preferred pH modifiers has a
synergistic effect in terms of oxidative stability with the further
inclusion of electron transfer mediators such as potassium
hydroxide, calcium hydroxide, and magnesium hydroxide. The role of
said electron transfer mediators has been further elucidated as a
superior understanding of the theory explaining the significant
gains in antioxidative stability being largely attributed to the
creation of an electron transfer bridge at the interface of the oil
and water phases of emulsions in general and particularly in the
more susceptible nanoemulsions.
[0178] Additional more preferred polyols now include trehalose. The
combinations of trehalose with the noted electron transfer
mediators yield superior oxidative stability. Trehalose has the
secondary advantage of protecting proteins (and derived peptides),
an important benefit in providing enhanced antioxidative stability
when the protein and/or peptide is exposed to elevated temperatures
and/or when the emulsion is dried. In addition to the protein
stabilizer trehalose, polyethylene glycol (PEG) can be added to the
emulsion prior to drying. PEG provides surface modification of the
protein and/or peptide. Thus the utilization, without being bound
by theory, of trehalose and/or PEG provides further encapsulation
of the lipid phase within the micelles.
[0179] Additional more preferred phospholipids includes
phospholipids rich in phosphatidylcholine (PC). Rich is
contextually defined as being of greater than 40% on a weight
basis. Thus the preferred formulation is the inclusion of PC rich
phospholipids into the oil phase during emulsification. An
important integral component of the inventive bioactive complex is
the electrostatic interactions of the polycationic milk
peptide-chitosan complex with negatively charged bilayers, such as
PC rich phospholipids. The combination markedly reduces the fluid
spacing between the negatively charged lipid bilayers. These
bridges are stabilized by increased adhesion arising from increased
van der Waals interactions between the opposing bilayers,
electrostatic interactions between the pi electrons in the phenol
ring (e.g., tocopherols) and the --(N.sup.+CH.sub.3).sub.3 groups
on the PC headgroups, decreased hydration repulsion between
bilayers, and hydrogen bonds between the H-bond-donating moieties
on the polyphenols, particularly tocopherols, and H-bond-accepting
groups n the bilayer. The increased bilayer adhesion is essential
for enhanced oxidative stability of emulsions.
[0180] One exemplary formulation procedure is the homogenization of
Omega Pure.TM. Menhaden fish oil (25%) for 4 minutes with egg yolk
phospholipids "rich" in PC at 0.2% w/w (Sigma Chemical 60% PC)
based on the weight of the fish oil, then 0.25% gamma-cyclodextrin
encapsulated coenzyme Q10 (w/w, based on the weight of the fish
oil) is added with subsequent homogenization for 4 minutes. An
aqueous solution (75% of double deionized water) containing
mannitol (4% w/w/, based on the weight of the emulsion) and
trehalose (2% w/w, based on the weight of the emulsion) is
magnetically stirred for 4 minutes. Gallic acid (0.002% w/w, based
on the weight of the emulsion) is subsequently added and
magnetically stirred for 2 minutes. The pH of this aqueous solution
(water phase) is adjusted to pH 6.0 with the preferred electron
transfer mediator of potassium hydroxide 0.1 N
(alternatively/preferably a combination of KOH and choline citrate
is used). The earlier prepared caprine casein
phosphopeptide-chitosan complex (0.04% w/w, based on the weight of
the emulsion) is added to this aqueous solution and homogenized for
1 minute. The oil phase and aqueous phase are combined and
homogenized for 4 minutes (production methods would include
multiple pass high pressure homogenization) resulting into an oil
in water emulsion. Without being bound by theory, the resulting
micelles at the interface between the oil and water phase contain
an iron-sulfur cluster as a means to provide an electron transfer
bridge across the two phases.
[0181] Additional more preferred casein phosphopeptides are
complexed with additional minerals including, though not limited
to, magnesium, manganese, selenium, zinc, and iron. The
particularly preferred mineral is in the lactate form. And the
specifically preferred mineral is zinc lactate. Zinc has secondary
advantages associated with protection of RNA and DNA, while calcium
has secondary advantages associated with the many health benefits
as known in the art for calcium including bone building, and
cholesterol reduction. Additional preferred proteins include
keratin and canola, and fragments thereof of keratin and canola
protein isolates. More preferred proteins include phosphopeptides
of keratin and canola proteins. A supplier of keratin includes
Keratec of New Zealand with the preferred product being their
Keratec IFP.TM., a purified protein fraction isolated from pure New
Zealand wool. The Keratec Pep product has a high proportion of
cystine derived amino acids as part of the peptide backbone. The
particularly preferred Keratec product is Cynatine FLX.TM.. A
supplier of canola protein isolates includes Burcon of Canada in
the form of Puratein.TM. and Supertein.TM..
[0182] Additional emulsifiers include the utilization of non-ionic
emulsifiers such that the polycationic charge created by the
chitosan is not "bound" by the emulsifier.
[0183] Additional antioxidants include vanillin, bee propolis,
grape seed extract, grape pomace extract, quercitin, carotenoids,
and lactoferrin. A preferred antioxidant has reducing sugars
eliminated from the antioxidant. Numerous methods exist as known in
the art, including the enzymatic elimination of sugars such as
glucoseoxidase. Lactoferrin, a recognized powerful antioxidant,
provides a synergistic impact of binding otherwise pro-oxidant
iron, thus without being bound by theory effectively removes the
catalytic iron while providing the inherent antioxidant
benefits.
[0184] Transparent Beverage
[0185] Chitosan is recognized in the art as a means to enhance the
intestinal absorption of calcium (Ca) in mammals. Magnesium (Mg) is
also recognized as a means to increase bone strength in mammals
whereas calcium is known to increase bone density in mammals.
However, high dietary Ca/Mg ratios interfere with Mg absorption,
because Ca and Mg share common intestinal absorption pathways
(Thebault et al. in Adv. Chronic Kidney Dis. 13, 110-117 (2006)).
When Ca is elevated with respect to Mg, Ca out-competes Mg for the
absorption pathways and hypomagnesaemia (low magnesium in the
blood) results. With respect to calcium absorption in humans, it
has been shown that bovine CPP administration (87.5 mg) leads to
significantly better absorption of co-ingested calcium (250 mg) in
normal post-menopausal women with low calcium absorption values
(n=17), as determined using an intrinsic Ca label in the calcium
source, at a CPP:Ca ratio of 0.35 (Heaney et al. in Bone Miner.
Met. 12, 77-81, (1994)). These findings suggest that CPP
supplementation is particularly useful for persons with a low basal
absorptive performance. This profile of action is fortunate in that
it is precisely in such persons with poor absorption that
enhancement is needed. Note: 87.5 mg CPP/250 mg Ca=0.35 (CPP/Ca
ratio).
[0186] In order to overcome this competing scenario between Ca and
Mg, the preferred embodiment is comprised of caprine CPP, which is
"rich" in medium chain triglycerides "MCT". MCT favors the
absorption of magnesium. The more preferred embodiment is comprised
of both chitosan and caprine CPP as the synergistic combination
achieves results greater than the individual components. Without
being bound by theory, the chitosan provides both superior
bioadhesion within the GI tract and protects the caprine CPP for a
longer period of time against enzymatic degradation of the peptide.
The result is superior absorption of both Ca and Mg. The lipid
composition rich in MCT and vitamin D both favor the absorption and
deposit of magnesium in the femur. Caprine CPP is also high in
amino acid lysine that is partially responsible for higher
absorption of divalent cations in laboratory animals as compared to
bovine milk. Similar results are anticipated from bovine sources of
casein that are all characterized by high levels of alphas1-casein
(bovine casein is characterized by low or intermediate amounts of
alphas2-casein but not high alphas2-casein). Another embodiment is
the synergistic combination of chitosan and GPC, which without
being bound by theory binds minerals, in particular calcium ions,
because of the combined phosphate groups in GPC and bioadhesion and
chelating properties of chitosan. Yet another embodiment is caprine
CPP-chitosan complex with egg yolk phospholipids (negatively
charged) having phosphate groups.
[0187] The dietary deficiency of minerals within the normal
consumption of food and beverage products is placing new demands on
creating new methods for efficiently delivering meaningful minerals
supplementation. However, meaningful levels often create taste,
clarity, and bioavailability constraints. One such exemplary is the
demand for transparent beverage systems meeting the requirement of
"excellent source" as defined by the FDA (i.e., 20% of RDA per
serving).
[0188] One embodiment to achieve a transparent beverage is achieved
by the acid precipitation of casein into small peptides (CPP) with
the addition of acids such as HCl, citric acid, phytic acid, lactic
acid, malic acid, etc. One exemplary preparation is achieved by
mixing 2% (w/v) chitosan with 10% (w/v) citric acid prior to the
addition into goat's milk. The selection of caprine milk vs. bovine
milk achieves superior transparency. Virtually any protein is a
candidate for salting-in in the presence of high concentrations of
mineral salts, though milk proteins (e.g., casein, whey) and their
derived peptides are superior. Several amino acids also bind
mineral salts, though to a lower extent than protein hydrolyzates.
The salting-in process of caprine CPP with TruCal.TM. can be
achieved without the inclusion of chitosan. The preferred
embodiment is further comprised of a phosphate buffering agent,
such as monobasic potassium phosphate. The preferred acids are
comprised of at least one acid selected from the group consisting
of citric acid, phytic acid, gallic acid and lactic acid. Citric
acid is of particular significance because of both its lack of
contributing to an in vivo acidic environment, and the recognized
within the art interaction with phytic acid as a means to reduce
arterial plaque. Phytic acid is of particular significance because
of the prior mentioned interaction with citric acid, it's
recognized in the art debittering of potassium, and it's excellent
chelating power. The novel combination of citric acid, phytic acid,
and mineral salts selected from the group of potassium, calcium,
magnesium, and zinc with the method of salting-in yields a highly
bioavailable, good tasting, and transparent beverage. Gallic acid
is of particular significance because of its antioxidant capacity,
which is of particular importance as a means to reduce the oxidized
cholesterol responsible for the plaque build up. Lactic acid is of
particular significance because of the electron donor capacity,
which has particular importance when the beverage is comprised of
oxidation sensitive oils including Omega-3 rich oils, borage oil,
etc. Prior art of an oral chelator (U.S. Pat. No. 7,009,067 by
Coppolino on Mar. 7, 2006 titled "Hexa-citrated phytate and process
of preparation thereof") claims to dissolve artery plaque and
removes excess copper, zinc, and iron deposits in the brain tissue
to treat age-related degenerative disorders, such as Alzheimer's
disease. The Coppolino complex is one of hexa-citrated phytate
based on the steps of slowly adding sufficient amount of calcium
carbonate in increments to aqueous phytic acid. Coppolino's
invention is inferior to the inventive preferred embodiment of a
calcium or magnesium salt (excluding carbonate, and preferably
lactate) due in part to lower solubility and bioavailability of
calcium carbonate, antioxidant capacity, poor taste, and lack of
transparency. A particularly preferred embodiment is a complex
comprised of gallic acid (or green tea extract), magnesium lactate,
phytic acid, and monobasic potassium phosphate. A more specifically
preferred complex is further comprised of at least one selected
from the group consisting of chitosan, GPC 85.TM., egg yolk PC
(Omega 6-PL-85.TM.), caprine CPP, trehalose and ribose. Without
being bound by theory, the chitosan provides superior bioadhesion
and chelating, the gallic acid provides superior antioxidant
capacity, the magnesium lactate provides both a source of magnesium
and lactic acid for electron donor capacity, the phytic acid is an
excellent chelating agent, and monobasic potassium phosphate
provides both the electrolyte capacity of potassium, and the
necessary phosphates. GPC 85.TM. and egg yolk PC ((Omega
6-PL-85.TM.), are both permeation enhancers and orthomolecular.
Caprine CPP enhances mineral transport and bioavailability. And
trehalose and ribose enhance hydration of the resulting complex.
The yet further inclusion of potassium salts in combination with
the phytic acid concurrently enhances the electrolyte content, the
debittering of the potassium, and the synergistic impact in
achieving a highly functional transparent beverage. The superior
process method further includes the salting-in of said minerals and
acids.
[0189] The significant deficiency of magnesium deficiency in the
diet is recognized in the art as a means to prevent calcium from
being deposited in bones, impair kidney, adrenal, heart, brain,
muscle and digestive function, compromises nerve transmissions,
restrict carbohydrate metabolism, inhibit the activities of "B"
vitamins, retard new cell growth, slow the production of DNA, and
so on. One of magnesium's most important duties is the formation of
ATP, which is the molecule that provides the energy for virtually
everything that occurs within the cells. When we lack magnesium,
ATP becomes scarce, metabolism slows, homeostasis becomes more
difficult to maintain, stress takes ever greater tolls, and fatigue
sets in. An oral chelator comprised of at least one magnesium salt
is superior to an oral chelator void of magnesium, as arterial
plaque is a function of numerous dietary and health deficiencies.
Many of these deficiencies result from blood that is too acid, and
the epidemic proportions of diabetes and obesity, all of which
magnesium and potassium will provide health benefits. The further
combination with phytic acid, which is recognized in U.S. Pat. No.
4,952,568 to Sawai et al. directed to methods for treating type II
diabetes by administering phytic acid salts in amounts sufficient
to moderate blood glucose levels to foods may be used to delay
starch digestion and glycemic response, is more synergistic with
magnesium as compared to calcium having the opposite effect. By
virtue of their polyoxy nature, many sugar alcohols (i.e.,
mannitol) form interesting although chemically weak complexes with
several polyvalent cations. For various physiologic and nutritional
purposes the complexes with calcium, iron, copper and possibly
several trace elements in general are important (Knuuttila et al.
in Bone & Mineral 6:25-31 (1989)). Trehalose obtained from
Cargill Food & Pharma Specialties is known to enhance the
absorption of calcium perhaps related to the same mechanism
ascribed to the polyols (Yoshizane et al. in U.S. Pat. No.
6,440,446 (2002)). Trehalose is an osmolyte (polyols also exhibit
an "osmotically" active nature). Non-reducing sugars such as
sucrose, sorbitol and mannitol are known to "chelate" minerals.
Ribose is recognized as a key building block to DNA and RNA.
[0190] Another embodiment of producing a transparent beverage is
comprised of sequential steps of adding a buffering agent, followed
by the addition of an acid, then addition of protein complex, then
addition of incremental amounts of minerals in combination with
further addition of acid until the earlier of failure to eliminate
cloudiness or achieving desired level of mineral inclusion. The
preferred buffering agent is monobasic potassium phosphate, which
has the secondary benefit of being a potassium source. The
preferred protein is a hydrolyzed protein (peptide) having enhanced
solubility. The particularly preferred protein is further complexed
with at least one selected from the group of chitosan, egg yolk PC
(Omega 6-PL-85.TM.), and GPC 85.TM.. The specifically preferred
peptide complex is caprine caseinophosphopeptide (CPP) complexed
with chitosan (CPP-chitosan complex). Without being bound by
theory, the selection of caprine CPP chitosan complex provides
superior bioavailability of minerals, specifically zinc and
magnesium in addition to calcium. The preferred acid includes acids
selected from the group consisting of gallic acid, malic acid,
citric acid, lactic acid, glutamic acid, gluconic acid, and phytic
acid. The preferred minerals are comprised of calcium salts,
magnesium salts, zinc salts and potassium salts. Particularly
preferred minerals are obtained from dairy milk that contains the
natural balance of calcium, phosphorous, magnesium and additional
trace minerals. The inventive transparent beverage utilizes
TruCal.TM. obtained from Glanbia Foods (Twin Falls, Id., USA). It
is fundamentally important to recognize in the formulation of any
food system that calcium loss can take place from too much acid in
the diet, which then demands means to increase blood alkanity. A
preferred pH adjuster for transparent beverages is therefore citric
acid (i.e., calcium citrate) due to its in vivo acid neutralizing
impact. This preference for citric acid, however, does not exist
when high levels of emulsified oils are present within the
beverage, as citric acid induces phase separation.
[0191] An important and recognized means to reduce in vivo acidity
is through the inclusion of potassium. A preferred embodiment of
reducing the bitterness and metallic notes associated with
potassium salts (and also zinc salts) respectively is the
sequential addition of zinc or potassium salts preceding the
subsequent addition of other salts including calcium and magnesium
salts. The selection of zinc including zinc sulfate has significant
health and nutritional benefits, which when complexed with caprine
CPP-chitosan yields superior bioavailability and enzymatic
inhibition protecting the peptide in vivo, without being bound by
theory, for sustained transport of minerals across the
gastrointestinal barrier. Preferred potassium salts are potassium
phytate, recognized as a means to reduce the metallic notes of
potassium.
[0192] The above sequential steps required for salting-in the
minerals is a process significantly more time consuming and complex
as compared to the traditional adding then mixing in of ingredients
for commercial beverage production. The further inclusion of at
least one ingredient selected from soluble fibers, bulk sweeteners,
amino acids, peptides, and soluble proteins enables the resulting
highly solubilized/transparent mineral complex (i.e., enhanced
solubilization) to be transformed into a powder utilizing methods
known in the art (including spray drying and freeze drying). The
resulting dry powder, which may include excipients known in the art
to maintain free-flowing powders (e.g., fumed silica), is readily
added and solubilized within the traditional commercial beverage
production methods. The preferred formulation includes trehalose
providing a synergistic impact on both protein (and amino acids,
peptides) protection against thermal degradation/denaturing and
superior bone health. Trehalose, as well as other polyols, is a low
glycemic sweetener thus promoting health in terms of obesity and
diabetes. A preferred solubilized fiber FiberSol-2.TM. (ADM,
Decatur, Ill., USA) is readily soluble up to 70% w/w % solids in
water/juices etc.
[0193] Natural Opacifier
[0194] The inventive complex further includes embodiments that
result in providing a natural source of opacity to a beverage while
concurrently providing health and nutrition benefits. One preferred
embodiment is comprised of the enzymatically modified caprine
casein resulting in the caprine caseinophosphopeptide (CPP). The
specifically high content of MCT and calcium increases the opacity
of beverage formulations, as the CPP that is high in beta-casein
content, has a high affinity for iron. The highly soluble complexes
comprised of TruCal.TM. and caprine CPP formed in the presence of
chitosan having embedded iron (or alternatively iron-added)
preferably as a chitosan lactate derived from microbial
fermentation (Cargill, USA), yield a very "milky" solution. These
findings have application in the food, pharmaceutical, and cosmetic
industries as an all natural "opacifying" agent (and for whitening
aqueous food compositions). Exemplary health benefits of iron-added
chitosan complexed with TruCal.TM. include enhanced calcium,
magnesium, and iron absorption for healthy and strong bones. The
preferred embodiment is far superior to the prior art of calcium
citrate in terms of promoting bone density and bone mechanical
strength. The presence of iron is recognized in the literature as
being a vital component for bone mineral density (Harris et al. in
J. Nutr. 133:3598-3602 (2003)).
[0195] Transparent Beverage Preparation
[0196] For a calcium-fortified beverage (480 mg Ca/240 mL) with a
serving size of 8 oz (240 mL) the amount of each ingredient would
be as follows: calcium carbonate 1.2 grams (0.5% w/v), acid-soluble
caprine CPP-chitosan complex: 0.96 grams (0.4% w/v), and citric
acid 0.72 grams (0.3% w/v). For a calcium-fortified beverage (960
mg Ca/240 mL) with a serving size of 8 oz (240 mL) the amount of
each ingredient would be as follows: calcium carbonate 2.4 grams
(1.0% w/v), acid-soluble caprine CPP-chitosan complex: 1.92 grams
(0.8% w/v), and citric acid 1.44 grams (0.6% w/v).
[0197] The preferred embodiment of the transparent beverage
utilizes the acid-soluble CPP-chitosan complex as prepared by a
two-step precipitation procedure. The two-step precipitation
procedure is conducted only when using high amounts of the
acid-soluble caprine CPP-chitosan complex (0.5%-1% w/v) for
incorporation into carbonated transparent beverages containing
calcium carbonate as the calcium source. For incorporation into
highly transparent calcium fortified beverages containing calcium
lactate or calcium gluconate as the calcium source, you can use the
acid-soluble caprine CPP-chitosan complex prepared by the one-step
precipitation procedure, but the level of the acid-soluble caprine
CPP-chitosan complex cannot be higher than 0.05% (w/v).
[0198] A particularly preferred embodiment utilizes both a calcium
and magnesium fortified beverage containing from about a 2:1 ratio
to a 1:1 ratio of acid-soluble caprine CPP-chitosan complex
precipitated with Ca and Mg respectively.
Example 15
Calcium-Fortified Apple Juice Containing Caprine CPP-Chitosan
Complexed with TruCal.TM.
[0199] Step 1: To 1000 ml of 100% apple juice Mott's.TM. with no
added sugar (ingredients: apple juice, water, and apple juice
concentrate) was added 0.14% (w/v) high methoxy pectin (HMP) #7050
(Cargill Food & Pharma Specialties, Cedar Rapids, Ia., USA)
(1.40 grams) followed by homogenization with a hand-held
homogenizer.
[0200] Step 2: 3.33 grams of calcium gluconate (9.48% elemental
calcium) and 3.83 grams of calcium lactate (13.95% elemental
calcium) were added to the apple juice under constant stirring.
Each 8 Oz (240 ml) of apple juice contains 75.76 mg elemental
calcium from calcium gluconate and 128.23 mg elemental calcium from
calcium lactate (approximately 200 mg elemental calcium per
serving).
[0201] Step 3: 0.04% (w/v) caprine CPP-chitosan complexed with
TruCal.TM. (0.40 grams) is then added to the calcium-fortified
apple juice followed by homogeneous mixing.
[0202] Step 4: The calcium-fortified apple juice containing the
caprine CPP-chitosan complexed with TruCal.TM. is transferred to a
bottle and capped. Heat sterilization is conducted for 20 minutes
at 83.degree. C.
[0203] Samples of caprine CPP-chitosan "complexed" with TruCal.TM.
were used to prepare either fruit beverages (i.e., apple juice) or
transparent beverages (with added malic acid and citric acid) are
as follows: Product "A" (TruCal.TM. with milk peptide, calcium
lactate, and chitosan lactate with added iron derived from
microbial fermentation (Cargill Food & Pharma Specialties,
Cedar Rapids, Ia., USA; Product "B" (TruCal.TM. with milk peptide,
calcium lactate, chitosan lactate with added iron derived from crab
shells (Orcas International Corporation, Flanders, N.J., USA));
Product "C" (TruCal.TM. with milk peptide, calcium lactate,
chitosan citrate derived from microbial fermentation (Cargill, USA;
and Product "D" (TruCal.TM. with milk peptide, calcium lactate,
chitosan citrate derived from crab shells (Orcas International
Corporation, USA. Another set of calcium-enriched beverages were
prepared as above but replacing calcium lactate by either calcium
chloride or calcium gluconate. The taste of the calcium-enriched
formulations containing calcium lactate was superior and achieved a
transparent beverage w/300 mg of elemental calcium per 8 Oz serving
size.
[0204] Preferred means to fully hydrate the peptides include
trehalose, sucrose or corn syrup solids (i.e., glucose). This is
particularly important for higher solubility or dispersability in
the presence of high amounts of otherwise insoluble mineral salts
as present in TruCal.TM.. A preparation containing 13% of Daily
Value for carbohydrates, based on a serving size of 240 ml, of corn
syrup solids enabled 31.25% RDI of calcium per 500 ml serving size
(TruCal.TM. contains 28% elemental Ca) to be achieved in a
transparent water beverage. The addition of a high methoxy pectin
(HMP #7050) (Cargill, USA) significantly reduces cloudiness. The
further addition of a highly soluble mineral salt (e.g., calcium
chloride) induces the salting-in effect.
[0205] 500 ppm (0.5 mg/ml) of caprine CPP-chitosan complex will
prevent the precipitation of 100 ppm (0.10 mg/ml) of elemental
calcium (100% of calcium-solubilizing power). The bone
mineralization studies detailed in the original patent application
were conducted with only 0.05% (w/v) caprine caseinophosphopeptide
(CPP) supplemented with calcium (300 ppm). The calcium source
utilized in the study was calcium gluconate, recognized as having
high solubility in water.
Example 16
Solubility Enhancement of TruCal.TM. for Beverage Applications
[0206] Product "A" (TruCal.TM. with milk peptide, calcium lactate,
and chitosan lactate with added iron derived from microbial
fermentation (Cargill, USA; Product "B" (TruCal.TM. with milk
peptide, calcium lactate, chitosan lactate with added iron derived
from crab shells (Orcas International Corporation, USA)); Product
"C" (TruCal.TM. with milk peptide, calcium lactate, chitosan
citrate derived from microbial fermentation (Cargill, USA; and
Product "D" (TruCal.TM. with milk peptide, calcium lactate,
chitosan citrate derived from crab shells (Orcas International
Corporation, USA.
[0207] Corn syrup solids (15% w/v), high methoxy pectin (0.35%
w/v), TruCal.TM. milk peptide complex (0.04% w/v), citric acid
(0.10% w/v), malic acid (0.35%), calcium chloride dihydrate (0.44%
w/v), trehalose (2% w/v), and double deionized water (81.72% w/v).
Dissolve the corn syrup solids, high methoxy pectin, citric acid,
malic acid and calcium chloride dihydrate in double deionized water
with the use of a hand-homogenizer for 3 minutes. The
TruCal.TM.-milk peptide complex is added to this aqueous solution
followed by homogenization for another 3 minutes. 1.8 grams of
TruCal.TM. in a 500 ml serving size will give 50% RDI of calcium.
The TruCal.TM.-milk peptide complexes (Product A, Product B,
Product C, and Product D) contain on average 8 mg elemental calcium
as TruCal.TM.. The calcium chloride dihydrate provides 60% RDI of
calcium in a 500 ml serving size. The total amount of elemental
calcium provided by the transparent beverage is 1,108 mg. The
samples are poured in a clear glass bottle and heated at 85.degree.
C. for 15 minutes. The process of pasteurization enhances the
solubility of the TruCal.TM.-milk peptide complex (enhanced
transparency of the calcium-fortified beverage). These
calcium-fortified beverages remain transparent at room temperature
for at least 30 days.
TABLE-US-00007 TABLE 7 Product Content Amount in 500 ml A Product A
plus TruCal .TM. 0.20 g plus 1.80 g B Product B plus TruCal .TM.
0.20 g plus 1.80 g C Product C plus TruCal .TM. 0.20 g plus 1.80 g
D Product D plus TruCal .TM. 0.20 g plus 1.80 g E TruCal .TM. 1.8
g
[0208] In all above samples tested chalkiness, cloudiness, and
sedimentation are not observed, and the acidic taste is acceptable
for beverage formulation.
[0209] During the development of the antioxidant and transparent
beverage applications, it became readily apparent that the
inventive complexation approach was a foundation for flavor
platforms. The specific benefits, realized as a result of
controlling ingredients (i.e., mineral salts, acids, enzymatically
modified proteins, chitosan, etc.) and process steps resulted in
powders having sweetness, creaminess, controlled saltiness, and
reduced bitterness.
[0210] Low Sodium Salt Replacement
[0211] An embodiment of the complex has been created with the
combination of TruCal.TM. "complexed" with caprine CPP-chitosan,
whereby the chitosan is a lactate salt with added iron. The
chitosan (manufactured by Cargill) has a very good taste profile
due to the bitterness being almost entirely masked by TruCal.TM.
and the chitosan lactate. The normal chalkiness associated with
calcium mineral salts is no longer perceived. The degree of
saltiness is controlled by both the amount of salted-in mineral
salts and the pH level. As noted earlier in the sequential
salting-in process, a preferred embodiment contains a level of
sodium reduced by at least 50% by the final mineral salting-in
being NaCl. Numerous food applications are such that the perception
of salt is a surface phenomenon, thus a presence of NaCl towards
the periphery of the salted-in peptide will represent a higher
perceived level vs. either stand alone NaCl crystals or salted-in
peptides comprised entirely of NaCl. Furthermore, the caprine CPP
effectively binds cations and keep them soluble in the digestive
tract yielding higher bioavailability for the less inherently
soluble minerals including iron.
[0212] Peptide and Hydrolysate Debittering
[0213] The preparation of the preferred peptide as complexed with a
chitosan salt (preferably with chitosan lactate) has resulted in
debittered peptides (anticipated for amino acids and protein
hydrolysates as well). The combination further protects the peptide
against degradation thus enhancing and prolonging the mineral
transport capabilities leading to superior bioavailability of a
wide range of minerals. Virtually any protein (e.g., soy, canola,
pea, etc.) is a candidate for enzymatic modification by protease
(e.g., trypsin) to split the protein molecule into small protein
peptides. The preferred chitosan salt is chitosan lactate to bring
the pH down to 3.0-4.0, resulting in a peptide having very low
bitterness and saltiness. The key to mask the bitterness of caprine
CPP is to neutralize the caprine CPP-chitosan preparation with an
alkali (to a range of pH 5.5-6.5, though the preferred embodiment
has a pH 6.0).
[0214] Mental Health
[0215] The individual benefits associated with Omega-3 rich oils
and phospholipids including phosphatidylcholine,
phosphatidylserine, and glycerolphosphocholine are widely
recognized in the broad area of mental health. However, significant
benefits are achieved beyond their individual benefits due to the
high bioavailability of nutraceutical and pharmaceutical actives
contained within an oil-in-water emulsion. The preferred combined
formulation utilizes an Omega-3 rich oil as a carrier oil for said
nutraceutical and pharmaceutical actives. Exemplary nutraceutical
actives include plant sterols, vitamins (e.g., Vitamin A, D, and
K), and coenzyme Q10. Preferred pharmaceutical actives are selected
from the group consisting of fat soluble actives, peptides, DNA,
and enzymes.
[0216] The preferred embodiment utilizes a low concentration of GPC
85 (0.01%-0.04% wt., based on the weight of the emulsion,
respectively) in combination with the caprine CPP-chitosan complex
(0.04% wt., based on the weight of the emulsion) in the aqueous
phase. The chitosan assumes a polycationic character at acidic pH,
therefore maximum interaction between the negatively charged GPC 85
and CPP-chitosan complex is observed at acidic pH. At pH 6.0, the
caprine CPP-chitosan complex exhibits less positive charge
(interaction with GPC 85 is at a minimum), yet still a net positive
charge.
[0217] Another embodiment is further comprised of DNA. The caprine
CPP-chitosan complex exhibit a strong net positive charge (and
caprine CPP contains high amounts of a basic amino acid: lysine).
Caprine CPP also contains high amounts of beta-casein. The ideal pH
for chitosan (and milk proteins or peptides) to bind to DNA is
around 6.0-6.5. A more preferred embodiment further includes the
presence of an "electron donor transfer" likely to further enhance
the bioavailability of DNA for gene therapy and other nucleotides
(i.e., pyrimidines such as citodine or uridine). A more
specifically preferred embodiment further includes ribose, which is
a sugar present in the DNA molecule and other nucleotides. A
complex of ribose, caprine CPP-chitosan, gamma-cyclodextrin
(CAVAMAX CoQ10.TM.), and polyphenols have the ability to stabilize
Omega-3 rich oils in vitro, with the significant in vivo benefits
attributed to the synergistic impact of the combined formulation.
The Maillard Reaction Products (MRPs) formed due to interaction of
the free amino group of the amino acid lysine present in the
caprine CPP molecule and the free amine group of D-glucosamine
present in the chitosan molecule and a reducing sugar (glucose
units present in Cavamax CoQ10.TM.) exhibit "antioxidant" activity.
The preferred embodiment is anticipated to provide protection to
the range of nucleosides, nucleotides, proteins, protein
hydrolysates, peptides, amino acids, and enzymes.
[0218] The preferred embodiment inherently incorporates chitosan's
recognized properties as noted in U.S. Pat. No. 6,184,037 by
Rolland, et al. on Feb. 6, 2001 titled "Chitosan related
compositions and methods for delivery of nucleic acids and
oligonucleotides into a cell" being useful in complexing and
condensing nucleic acids or complexing oligonucletides. DNA, which
is a polyanionic nucleic acid has a high net negative charge due to
the presence of two phosphate moieties on each base pair. Rolland
further teaches the conclusion that "DNA is an excellent candidate
for complexation with chitosan and chitosan oligomers for non-viral
gene delivery". Neutralization of the negative charge of DNA by the
amine groups of chitosan and chitosan oligomers results in
condensation of DNA into a compact particle which protects the DNA
from nuclease degradation and delivers the DNA, either specifically
or non-specifically, to target cells". Rolland does not anticipate
the benefits of incorporating Omega-3 rich oils as a further means
of increasing the efficacy and bioavailability associated with the
preferred embodiment.
[0219] Yet another embodiment includes a range of individual
actives recognized for immune system enhancement. One such active
is beta-glucan being recognized as having an immunomodulatory
action. The beta-glucan has multiple negatives associated with the
stand-alone active, which include non-desirable taste, low
solubility, and low bioavailability. A preferred
chitosan-beta-glucan complex has reduced bitterness, high
bioadhesion, and higher solubility, all of which contribute to
superior bioavailability. The more preferred complex is further
comprised of caprine caseinophosphopeptides (CPP). And the
particularly preferred complex has zinc lactate salted-in as a
means of providing high bioavailability of zinc (also recognized
for immune system enhancement). The selection of caprine
caseinophosphopeptides (CPP) further enhances the bioavailability
of magnesium. Another preferred modified caprine CPP-chitosan is a
further complex of caprine caseinophosphopeptide, chitosan and
beta-glucan. The resulting complex, without being bound by theory,
is an especially powerful immunity enhancer as the combined complex
performs better than each individual component due to the
synergistic effects of high zinc binding, the bioadhesive and
protein protection by the chitosan, the higher chelation
capabilities as compared to animal chitosan by the combined
chitosan and beta-glucan, and superior electron transfer due to the
iron presence in the chitosan creating an iron-sulfur cluster due
to the complex of caprine caseinophosphopeptide and chitosan. The
specifically preferred caprine
caseinophosphopeptide-zinc-beta-glucan-chitosan is further combined
with EDTA as a further means to prevent the "leakage" under high
acidic pH (i.e., .ltoreq.4.0) of ferrous ions `bound` to the
chitosan salt leading to catalytic oxidation of lipids. Another
benefit of the synergistic combination is the reduced bitterness of
the zinc and beta-glucan making the product more suitable for
functional food and beverage products. Zinc and magnesium are
especially noted for their direct effect on osteoblastic activity.
Another preferred delivery includes the further addition of omega-3
oils as a means to inhibit inflammation. A more preferred delivery
further includes Vitamin D within the Omega-3 to provide high
efficacy delivery of Vitamin D (and/or additional fat soluble
Vitamins, nutraceuticals, and pharmaceuticals). The further
inclusion of a thermally-stabilized lactoferrin, as provided by
TAMUS 1408 has both an "in-food" and "in-vivo" impact, which are
reduced lipid oxidation and enhanced immunity, respectively.
[0220] Numerous observations of magnesium have been cited by
Guosong Liu in "Magnesium for Memory" Prepared Foods Newsletter
Dec. 6, 2004. Liu further states that magnesium in the American
diet has declined since the Industrial Revolution and that the high
fat content of the modern diet prevents magnesium from being
absorbed. In his experiment, he fed lab rats the equivalent of a
human dose of 400 mg of magnesium a day. The result, he said, was
that the mineral increased the activity of receptors that control
learning and memory. Specifically, it enhanced the activity at the
synapse, the gap between two neurons, or brain cells. However,
numerous other factors not cited by Liu adversely impact magnesium
bioavailability. These include competitive interaction with calcium
salts. Therefore the preferred embodiment of caprine
caseinophosphopeptides (CPP) further enhances the bioavailability
of magnesium ions in part due to the high
medium-chain-triglycerides (MCT) content. The particularly
preferred embodiment comprises salted-in magnesium lactate. Certain
foods, due to the high acidity taste of lactate salts, can utilize
potassium hydroxide to neutralize the lactic acid present in the
chitosan preparation (i.e., lactate) during the preparation of
caprine CPP. Magnesium hydroxide and calcium hydroxide can also be
utilized in combination of instead of potassium hydroxide as a
means to reduce the peptide "bitterness". The preferred method
being a sequential salting-in process of lactic acid salts followed
by the hydroxide salts. In addition to the mental health gains
associated with magnesium, numerous additional benefits including
energy metabolism and protein synthesis are recognized.
[0221] The combination of phospholipids with chitosan has multiple
synergistic impact including permeation enhancers. A preferred
embodiment includes phosphatidylserine (PS), which is an acidic
phospholipid that in combination phosphatidylcholine (PC),
significantly decreases the iron-induced oxidation of egg yolk PC
when utilized as an emulsifier in Omega-3 rich oils. Phospholipids
`rich` in PC are utilized to increase "bilayer" adhesion whereby
the increased bilayer adhesion between the "negatively" charged
phospholipids PC and "positively" charged caprine CPP-chitosan
complex to stop Omega-3 degradation in O/W emulsions. Phospholipids
`rich` in phosphatidylserine (PS) (i.e., SerineAid 50P.TM.) are
necessary to chelate iron present in the egg yolk phospholipids, in
particular in acidic emulsions (pH<5.0). Oil-in-water emulsions
prepared with egg yolk PC (Omega 6-PL-85.TM.) and alpha-glyceryl
phosphoryl choline (GPC 85.TM.) exhibit enhanced oxidative
stability because GPC 85.TM. is also an effective iron chelator.
Clearly the preferred embodiment introduces synergistic benefits
when at least one emulsifier is selected from the group of
phospholipids consisting of PC, PS (i.e., SerineAid 50P.TM.) and
alpha-glyceryl phosphoryl choline (GPC 85.TM.). GPC 85.TM. is
particularly preferred when complexed with either the caprine
CPP-chitosan complex and/or caprine CP alone. Formulations that
require additional phospholipids beyond the required levels for
complexation preferably are comprised of GPC 85.TM. within the
water phase of the emulsion.
[0222] Literature supports that mucoadhesive polymers enhance the
peroral peptide drug delivery. Thiolated polycarbophil and
thiolated chitosan in combination with reduced glutathione (GSH)
are potent enhancers of peptide transport across intestinal mucosae
by increasing the parecellular permeability due to opening of
intercellular junctions (Bernkop-Schn urch et al. in J. Control
Release 93, 95-103 (2003)). Furthermore calcium plays an important
role in maintaining the thermodynamic stability of several serine
proteases (e.g., trypsin), which is the basis of their resistance
against autoprotolysis. Additionally, chitosan improves transport
by increasing the paracellular permeability of the intestinal
epithelium (Sharma et al. in Pharmazie 61, 495-504 (2006)).
Mucoadhesive polymers as platforms for peroral peptide delivery
comprised of chitosan-EDTA conjugates are very useful drug-carrier
matrixes in overcoming the enzymatic barrier to orally administered
peptide and protein drugs (Bernkop-Schn a{umlaut over (r)}ch, A.
and Pasta, M. in J. Pharmaceutical Sciences 87, 430-434, 1998). The
preferred embodiment comprised of both caprine CPP-chitosan and
EDTA, though the EDTA is contained within the water phase as a
means of protecting the Omega-3 oil, is anticipated to have
comparable performance to the chitosan-EDTA conjugates.
[0223] Muranishi et al. in Chem Phys Lipids 28: 269-279 (1981)
further supports that fatty acids including oleic, capric, and
linoleic acids have a strong and rapid action on permeability of
lipid bilayer, while chelating agents such as EDTA, citric acid,
phytic acid have strong to moderate activity. Thus it is
reasonable, without being bound by theory that the preferred
caprine CPP-chitosan complex combined with the preferred presence
of capric MCT, oleic fatty acids, EDTA, chitosan, and oils rich in
phytic acid would further improve the permeability. And the
superior binding of calcium, magnesium, and zinc to the preferred
caprine caseinophosphopeptide (CPP) further enhances the
bioavailability of said minerals.
[0224] A yet further embodiment of the invention is the
"encapsulation" of the Omega-3 rich oil in olive oil. The
combination of olive oil, which is rich in ferulic acid, and
lactoferrin is a synergistic antioxidant combination to protect
Omega-3 rich oils from oxidation. Clark et al. as cited in
http://www.algatech.com/bio.htm compared lycopene and astaxanthin
absorption from corn oil and olive oil emulsions in rats.
Absorption of lycopene and astaxanthin from both oils increased
with the amount infused into the rat's duodenum. The average
recovery of astaxanthin in the lymph from the olive oil emulsion
was 20%, but decreased to 13% from emulsions containing corn oil.
Lycopene was not as well absorbed as astaxanthin. The average
recovery of LYC was 6% from olive oil emulsions but only 2.5% when
infused with corn oil. They concluded that the type of oil with
which a carotenoid is consumed can substantially influence its
absorption. Astaxanthin is a powerful antioxidant and can serve as
a potent free-radical scavenger. Moreover, astaxanthin has been
found to provide many essential biological functions, including
protection against lipid-membrane peroxidation of essential
polyunsaturated fatty acids and proteins, DNA damage and UV light
effects; it also plays an important role in immunological defense.
Astaxanthin is capable of crossing the blood-brain barrier in
mammals. The preferred embodiment of Omega-3 rich oil, which is an
orthomolecule, encapsulated by olive oil, which is rich in ferulic
acid, in combination with thermally-stabilized lactoferrin, green
tea extracts (or gallic acid) and coenzyme Q10 encapsulated by
gamma-cyclodextrin all within micelles comprised of at least one of
the group consisting of chitosan, caprine CPP-chitosan, caprine
CPP, GPC 85.TM., Omega 6-PL-85.TM., and SerineAid 50P.TM. has
superior bioavailability and free radical protection both in vitro
and in vivo.
[0225] Another embodiment further includes trehalose. Trehalose is
recognized as a means to protect proteins from unfolding, thus it
is obvious that trehalose will protect the caprine
caseinophosphopeptide (CPP) of the present invention. Trehalose has
also been identified as being effective against protein
agglomeration in mice having the Huntington protein. Without being
bound by theory, the further inclusion of trehalose providing
superior oxidation protection to Omega-3 rich oils (i.e., by
encapsulating the Omega-3 rich oil during the process of spray
drying) has the further potential to enhance mental health by the
synergistic combination of Omega-3 and trehalose. It is recognized
that the biosynthesis of trehalose phosphate can occur by either
one of two reactions:
UDP-glucose+glucose-6-P>>trehalose-P+UDP. It is further
hypothesized that in vivo trehalose has a role within mental health
in the production of uridine, citodine, and/or choline within the
brain. Thus the particularly preferred embodiment is comprised of a
trehalose to Omega-3 ratio ranging from a low of 5% w/w of water
phase, 5% w/w of total oil phase in emulsion to a 1:1 ratio of
trehalose to Omega-3 fraction of Omega-3 rich oil.
[0226] Electron Transfer
[0227] Without being bound by theory, the creation of iron sulfur
clusters is a fundamental component in the electron transfer from
the oil phase into the water phase of the emulsion. Thus,
effectively an electron transfer bridge is created across the
interface of between the oil and water phase. The existence of this
electron transport bridge is critical to the realization of
significantly superior oxidative stability of lipids. The irony of
this result is such that, iron which is otherwise a procatalyst,
must be present in at least trace amounts as a means of creating
the electron transfer bridge. Such electron transport bridge
includes thialoto-bridged complexes. Electron-rich thiolato groups
have a great affinity for various metal ions. This includes
metal-bound thiolato sulfur centers. Broadly, the incorporation of
thiolated complexes, metalloproteins and/or protein complex having
an iron-sulfur cluster within the emulsion interface, without being
bound by theory, enhances electron transfer between the phases.
[0228] The further inclusion of an electron transfer mediator
within the antioxidant and nanoemulsion compositions, without being
bound by theory, enhances electron transfer out of the lipid phase
of the emulsion into the water phase. The preferred electron
transfer mediator is potassium hydroxide, an acceptable food
ingredient. The potassium hydroxide serves a significant secondary
role of providing potassium mineral supplementation, a noted
deficient mineral especially in the American diet.
[0229] Without being bound by theory, the inventive combination of
an electron transfer mediator, a molecular electron transfer
bridge, and an iron-sulfur cluster (or metalloproteins) creates an
aqueous, room temperature electride solution as indicated by the
presence of a sapphire blue solution indicative of free electrons
(increased free electron flow). A wide range of applications are
anticipated for stable room temperature electride solutions,
including nutraceutical, pharmaceutical, energy transfer, and
oxidative stability applications.
[0230] U.S. Pat. No. 7,045,339 by Sorenson, Jr., et al. on May 16,
2006 titled "Electron donors for chlorinated solvent source area
bioremediation" notes that lactic acid or salts of lactic acid, or
mixtures thereof are illustrative electron donors including oleyl
lactylic acid, linoleyl lactylic acid, linolenoyl lactylic acid,
stearoyl lactylic acid, palmitoyl lactylic acid, myristoyl lactylic
acid, lauroyl lactylic acid, caproyl lactylic acid, mixtures
thereof, mixtures with fatty acids or salts thereof, mixtures with
lactic acid or salts thereof, mixtures with fatty acids and lactic
acid and salts thereof, and the like. In a specific embodiment of
the invention, the electron donor is a member selected from the
group consisting of lactic acid, salts thereof, lactate esters, and
mixtures thereof. Illustrative salts of lactic acid include sodium
lactate, potassium lactate, lithium lactate, ammonium lactate,
calcium lactate, magnesium lactate, manganese lactate, zinc
lactate, ferrous lactate, aluminum lactate, and mixtures thereof,
wherein sodium lactate is especially illustrative. In another
specific embodiment of the invention, the electron donor is a
member selected from the group consisting of oleyl lactylic acid,
oleic acid or salts thereof, and lactic acid or salts thereof. The
preferred embodiment of the present invention for chitosan is a
chitosan lactate. A more preferred embodiment includes lactate
salts selected from the group of potassium, calcium, zinc, and
magnesium due to their multifunctional role within the applied
food, nutraceutical or pharmaceutical system. Potassium is a
superior electrolyte as compared to sodium. Zinc and magnesium play
vital health roles in bone, mental, and immune system health.
Caprine CPP-chitosan complexes comprised of zinc lactate
demonstrated superior antioxidant performance to the other lactate
salts.
[0231] Without being bound to theory, electron transfer reactions
(most notably within oil and water emulsions) depend upon multiple
factors such as electrolyte concentration (potassium is the mineral
activator within cells), lipids (phosphatidylcholine and
phosphatidylserine), divalent cations (calcium, magnesium), and
iron-sulfur clusters (chitosan lactate with added iron; caprine CPP
contains cysteine). The specific protection of Omega-3 oils within
oil-in-water or water-in-oil emulsions is highly dependent on
electron transfer across the micelle interface. A more preferred
embodiment further includes whey protein (concentrates and
isolates) that are rich in cysteine. Cysteine, which is recognized
as a means to maintain a healthy immune system through glutathione
synthesis yields glutathione, a potent antioxidant within in vivo
cellular structures. The particularly preferred embodiment of the
caprine CPP-chitosan complex has sufficient iron within the matrix
to create an iron-sulfur cluster. Cysteine is a sulfur-containing
amino acid, which is most likely the critical amino acid in
creating the iron-sulfur cluster. Therefore, the superior
protection against lipid oxidation mimics the in vivo cellular
structure that is an interface layer comprised of phospholipids,
channel proteins, and catalytic proteins. The inventive caprine
CPP-chitosan complex (in the preferred embodiment) plays the role
of the channel protein by enabling efficient electron transport
across the oil-water interface. The further inclusion of a lactic
protein furthers the electron transport mechanism, as lactic acid
is an electron donor. The further inclusion of lactoferrin mimics a
catalytic protein. The preferred protein is whey protein with
lactic acid. The particularly preferred protein is a whey protein
hydrolysate isolate with lactic acid. And the further inclusion of
coenzyme Q10 encapsulated by gamma-cyclodextrin provides for the
production of Maillard Reaction Products with antioxidant
properties through the glucose present in gamma-cyclodextrin during
food processing (i.e., pasteurization, baking). Without being bound
by theory, the mechanisms taking place are as follows: (1)
Plant-derived polyphenols are metal-chelating agents and
free-radical scavenging agents; (2) Coenzyme Q10 regenerates
tocopheryl free radicals (prooxidant activity) into antioxidative
tocopherol molecules (antioxidant activity); (3) Gamma-cyclodextrin
(CAVAMAX.TM.) is comprised of glucose which enables the formation
of thermally-derived compounds known as Maillard Reaction Products
with specific antioxidant activity; (4) Tocopherols and
tocotrienols are highly effective antioxidants in oil-in-water and
water-in-oil emulsions at levels greater than 500 ppm; (5)
Potassium hydroxide, which is the preferred electron transfer
mediator, is used to adjust the pH of the oil-in-water or
water-in-oil emulsions to 6.0, and to inject electrons to chemical
structures containing an aromatic ring (i.e., tocopherols,
tocotrienols, grape seed extract, grape pomace extract, bee
propolis, green tea, coenzyme Q10, BHA, BHT, and TBHQ.
[0232] Modulation of Metabolic Pathways by Influencing Electron
Flux
[0233] The utilization of N-acetyl cysteine, which is a well-known
scavenger of reactive oxygen species (ROS) is anticipated as a
means of facilitating electron transport in addition to the
recognized role of protecting the liver from injury. The liver is
the organ that detoxifies our body from harmful substances such as
alcohol, nitrates present in cured meats, pesticides present in
conventional grown fruits and vegetables, etc. The U.S. patent
application by McCleary "Metabolic Uncoupling Therapy"
(20040043013) focuses on the importance of electron transport
reactions within the human cell though fails to recognize the
importance of in vitro protection of Omega-3 rich oils in order to
provide oxidative stability. The preferred embodiment of the
present invention is superior and substantially novel by the
realization of 1) coenzyme Q10 being encapsulated by cyclodextrin,
preferably gamma-cyclodextrin, 2) encapsulated coenzyme Q10 being
within the oil phase, 3) same but for alpha lipoic acid as in #2
and #1, 4) the presence of amino acids in the form of a complex
having an iron-sulfur cluster, and 5) The U.S. Pat. No. 6,579,866
by McCleary on Jun. 17, 2003 titled "Composition and method for
modulating nutrient partitioning" though explicitly requires the
utilization of hydroxycitric acid, whereas the inventive preferred
embodiment recognizes the significant detriment of citric acid to
the emulsion and oxidative stability of the Omega-3 rich oils.
[0234] Additional antioxidants including eugenol, which is
recognized as enhancing the metabolism of DHA and inactivating free
radicals, grape seed extract, and ginger, turmeric, spirulina, and
green tea which are known to contain Superoxide Dismutase "SOD" (a
potent in vivo natural antioxidant). A particularly preferred
antioxidant is or contains gallic acid, such as green tea. In the
present invention gallic acid is also used as a buffering agent at
concentrations.ltoreq.50 ppm (0.0005 wt. %) to adjust the pH of the
oil-in-water emulsions to 6.0 in combination with the preferred
electron transfer mediator potassium hydroxide. Gallic acid is an
antibrowning agent and a potent antioxidant. Gallic acid scavenges
superoxide anions that are generated enzymatically. The oxidation
of unsaturated fatty acids in biological membranes decreases
membrane fluidity and disrupts membrane structure and function
(Machlin, L. and Benedic, A. in FASEB J. 1, 441-445 (1987); Slater,
T. F. and Cheeseman, K. H. in Proceedings of the Nutrition Society
46, 1-12 (1987). Cellular damage due to lipid peroxidation causes
serious disturbance that can result in ischemia-reperfusion injury
(Sugawara et al. in J. Clin. Exp. Med. 163, 237-238 (1992)),
coronary arteriosclerosis (Kok et al. in Atherosclerosis 86, 85-90
(1991)), or diabetes mellitus (Sugawara et al. in J. Clin. Exp.
Med. 163, 237-238 (1992); it also is linked to aging and
carcinogenesis (Yagi, K. in Antioxidants and Disease Prevention;
CRC Press: Boca Raton, Fla., USA (1997); Garewal, H. S. in
Antioxidants and Disease Prevention; CRC Press: Boca Raton, Fla.,
USA (1997)). Inhibition of membrane peroxidation has a protective
effect on the initiation and promotion of certain cancers. In
general, various reactive oxygen molecules link damage to the
initiation and development of cancer. The primary role of
antioxidants is to protect against such damage. Therefore, gallic
acid or green tea, which is a rich source of gallic acid, can be
considered even as cancer-fighting ingredients or chemopreventive
agents. A research report by Sakagami et al. in Anticancer Res. 17,
377-380 (1997) indicates that gallic acid induced apoptotic cell
death in human promyelocytic leukemia HL-60 cells. Because
superoxide anions reportedly enhanced the oxidation rate of
L-tyrosine to L-DOPA by tyrosinase (Wood et al. in Biochem.
Biophys. Acta 1074, 378-385 (1991)), the scavenging activity of
gallic acid and green tea is of considerable benefit.
[0235] A surprising feature of the bioactive complex of the present
invention is the creation of a sapphire blue color within an
aqueous solution. It is hypothesized that the sapphire blue color,
which without being bound by theory, is indicative of free
electrons such as electrides. The blue color only is evident in
caprine CPP-chitosan formulations containing chitosan lactate with
embedded iron. The blue "solution" is obtained with chitosan
lactate manufactured by Pronova Biopolymer Corporation (Portsmouth,
N.H., USA) and caprinecaseinophosphopeptide (CPP) at about 400 ppm
(caprine CPP-chitosan complex), in combination with gallic acid at
about 20 ppm. Increasing the levels of gallic acid increased the
deepness of the blue color. The pH of the solution is at least 6.0
(can also be greater, but not less). This finding is particularly
unique in that electrides to date have not been present at either
room temperature or in the presence of water (let alone within an
aqueous solution). Applications of such an electride solution range
from energy conversion applications to energy transfer (e.g., fuel
cells, solar to electricity conversion, heat transfer, etc.).
[0236] The preferred caprine CPP preparation contains cysteine
(bovine CPP does not contain cysteine, another reason for enhanced
effectiveness of the caprine CPP-chitosan complex vs. a bovine
CPP-chitosan complex). The --SH groups of L-cysteine decide the
conformation of proteins (and derived peptides) and the catalytic
activity of enzymes. Without being bound by theory, the thiol
(--SH) groups of cysteine forms "iron-sulfur centers" (iron atoms
paired with an equal number of acid-labile sulfur atoms) that are
essential for electron transfer. Recent experimental studies have
provided evidence that the --SH groups of L-cysteine enhance the
electron transfer ability between protein molecules and
chitosan-stabilized gold nanoparticles (Feng et al. in Anal.
Biochem. 342, 280-286 (2005)). These active --SH groups (i.e.,
L-cysteine) of proteins (and peptides) have also been shown to
facilitate electron transfer and electrocatalize the redox of
phenolic compounds when dissolved in aqueous solution (Zhao et al.
in J. Environmental Science and Health 41, 447-456 (2006)). The
"high" affinity of the beta-casein component present in high
amounts in caprine CPP (>50%) for iron as compared with bovine
CPP (<30%) is one major factor proving the efficient electron
transfer turnover-rate of the caprine CPP-chitosan complex. Thus,
the caprine CPP molecule "strongly" interacts with the iron
"embedded" in the chitosan molecule. In the event that the source
of chitosan is naturally (or due to production processes) iron
deficient, the chitosan can be embedded with iron during the
creation of the chitosan complex. Chitosan complexes have ranged
with 20 ppm of iron to 700 ppm. In the event that the chitosan
complex is being utilized to provide antioxidant protection for an
"encapsulated" oil/lipid, it is essential to maintain the caprine
CPP-chitosan with the embedded iron to be less than 500 ppm of the
total emulsion weight. It is anticipated that both additional
levels of chitosan and/or caprine CPP-chitosan can be utilized only
when said chitosan is void of iron, especially under high acid pH
conditions. Under high acid conditions, the embedded iron is
"leaked" from the matrix and becomes the recognized pro-catalyst.
Thus one of the novel features of the preferred embodiment is the
superior antioxidant performance during the presence of an
otherwise traditional pro-oxidant.
[0237] In living organisms, iron-sulfur centers (clusters) are
associated with an enzyme, succinate NADH dehydrogenase, which is
involved in electron transport reactions. By undergoing Fe (II)-Fe
(III) cycles, the prostetic groups of this succinate NADH
dehydrogenase transfer reducing equivalents to the next electron
carrier ubiquinone (coenzyme Q). The complex of succinate NADH
dehydrogenase with the iron-sulfur proteins contains two kinds of
electron-carrying structures. Real life is emulated in a preferred
embodiment containing the caprine CPP-chitosan complex of the
present invention. The caprine CPP molecule `per se` is a
`bioactive` milk protein fraction or peptide whereas an enzyme is a
protein specialized to catalyze a specific metabolic reaction. This
in vivo process is emulated in vitro in a test tube that contains
an aqueous solution of caprine CPP-chitosan complex (0.05% w/v)
where the iron-sulfur centers (clusters) are associated with a
casein protein (or preferably a casein peptide, or particularly
preferred a caprine CPP), and the chitosan molecule having iron
present. By undergoing Fe (II)-Fe (III) cycles, the caprine
CPP-chitosan complex (iron-sulfur complex) acts as an electron
carrier structure transporting electrons to or from ubiquinone
(coenzyme Q10). Electron transfer reactions between the iron-sulfur
clusters of the CPP-chitosan complex and coenzyme Q10 play an
important role in stabilizing Omega-3 oils against oxidative
degradation in vitro and very likely in vivo (the living cell).
[0238] Moreover, iron (II) complexes might be formed with the
further inclusion of gallic acid (Fenton-type reactions) (Strlic et
al. in J. Agric. Food Chem. 50, 6313-6317 (2002)). Gallic acid is
known to be readily oxidized, chemically or electronically, in
acidic and alkaline solutions. The o-quinone products from gallic
acid are formed via the semiquinone radical and are susceptible to
secondary reactions at pH>7. It should be noted here that we
have conducted our electron-transfer studies at pH 6.0; therefore,
gallic acid does not fully oxidize. Furthermore, studies conducted
by other scientists have shown that at pH<7.0 iron (II) does not
complex with gallic acid. Our studies by gas chromatography (GC)
with O/W Menhaden oil-based emulsions emulsified with egg yolk PC
(Omega 6-PL-85.TM.) confirm research by Strlic et al. (2002) in
which the addition of Fe (II) or Fe (III) to an aqueous solution of
gallic acid has a prooxidative effect. Therefore, the main
mechanism taking place in our studies conducted with caprine
CPP-chitosan complex (500 ppm), gallic acid (10 ppm-50 ppm), and
coenzyme Q10 (0.25%-1.0%) is due to electron transport reactions
between the CPP-chitosan complex (iron-sulfur complex) and coenzyme
Q10. Indeed, control samples containing gallic acid, alone or in
combination with coenzyme Q10, exhibit prooxidative properties, and
it promotes the production of hydroxyl radicals due to the presence
of iron from egg yolk PC (Omega 6-PL-85.TM.). The further inclusion
of trehalose at concentrations of 1% to 2% (w/v) is essential to
stabilize the CPP-chitosan complex at incubation
temperatures.gtoreq.40.degree. C. Gallic acid is used in our
studies as a "buffer" to acidify the aqueous media; subsequent
addition of the preferred electron transfer mediator (0.1 N KOH) is
preferred to bring pH of the aqueous solution to 6.0.
[0239] It is recognized in the art, that thionins is a family of
peptides found solely in higher plants. Specifically as cited in
"Biochemical fuel cells" by Eugenii Katz et al., of Institute of
Chemistry and The Farkas Center for Light-Induced Processes, The
Hebrew University of Jerusalem in Jerusalem, Israel, thionin
consists of 45-48 amino acid residues. 6-8 of these are cysteine
forming 3-4 disulfide bonds. Microbial cells have also been grown
in the presence of various nutritional substrates. For example,
Proteus vulgaris bacteria were grown using glucose, galactose,
maltose, trehalose and sucrose as primary electron donors and used
in a biofuel cell with thionine as a diffusional electron transfer
mediator. We, the applicants of this invention, note that the
toxicity of thionin precludes the use for food ingredient
applications, though the potential for both non-edible and
pharmaceutical applications remains as a means to achieve excellent
electron transport. Additional means to achieve electron transport
reactions include ferredoxin and flavodoxin. As expected, electron
transport reactions are only favored in the aqueous phase rather
than in the oil phase. The higher the oil content of the emulsion
(.gtoreq.30%), the lower the conductivity of electrons. This means
relatively "poor" oxidative stability protection of Omega-3 oils
when the caprine CPP-chitosan complex of the present invention is
contained within the oil phase.
[0240] Electron transfer plays a vital role in numerous
applications including the performance of antimicrobials. The
inventive caprine CPP-chitosan complex potentiates antimicrobials,
antivirals, etc. known in the art and/or serves as a primary
antimicrobials or antivirals.
[0241] Although only exemplary embodiments of the invention are
specifically described above, it will be appreciated that
modifications and variations of these examples are possible without
departing from the spirit and intended scope of the invention.
* * * * *
References