U.S. patent application number 14/769877 was filed with the patent office on 2016-01-07 for bioactive compositions from fruit and methods for their production.
The applicant listed for this patent is AKZO NOBEL CHEMICALS INTERNATIONAL B.V.. Invention is credited to Artyom Duev, Olga V. Dueva-Koganov, Michael Koganov, Steven Micceri.
Application Number | 20160000848 14/769877 |
Document ID | / |
Family ID | 50241441 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160000848 |
Kind Code |
A1 |
Koganov; Michael ; et
al. |
January 7, 2016 |
Bioactive Compositions From Fruit and Methods for Their
Production
Abstract
The present invention relates to bioactive fractions
(ingredients) derived from fruit juices. The bioactive fractions
are either free of or substantially free of patulin and protein.
Further, the bioactive fractions have antioxidant, free radical
scavenging, moisturizing and buffering properties. The present
invention also relates to a method for isolating bioactive
fractions that are derived from fruit juices and are free or
substantially free of protein and patulin. The present invention
also relates to a method for preparing a bioactive fractions
derived from fruit juices that are stabilized and are either free
of or substantially free of patulin and protein.
Inventors: |
Koganov; Michael; (White
Plains, NY) ; Dueva-Koganov; Olga V.; (White Plains,
NY) ; Duev; Artyom; (White Plains, NY) ;
Micceri; Steven; (Milford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AKZO NOBEL CHEMICALS INTERNATIONAL B.V. |
Amersfoort |
|
NL |
|
|
Family ID: |
50241441 |
Appl. No.: |
14/769877 |
Filed: |
March 12, 2014 |
PCT Filed: |
March 12, 2014 |
PCT NO: |
PCT/EP2014/054758 |
371 Date: |
August 24, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61792709 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
424/765 ;
424/777 |
Current CPC
Class: |
A61K 36/736 20130101;
A61K 2236/37 20130101; A61P 13/02 20180101; A61K 2236/31 20130101;
A61K 36/88 20130101; A61P 39/06 20180101; A61P 1/04 20180101; A61K
36/73 20130101; A61K 36/185 20130101; A61P 1/16 20180101; A61K
36/45 20130101; A61K 36/81 20130101; A61K 36/752 20130101 |
International
Class: |
A61K 36/73 20060101
A61K036/73 |
Claims
1. A bioactive fraction derived from fruit, wherein said fraction
is substantially free of patulin and protein.
2. The bioactive fraction of claim 1 wherein said fruit is selected
from peaches, pears, bananas, plums, apples, pineapples, apricots,
cherries, grapes, blueberries, raspberries, blackberries,
cranberries, tangerines, prickly pears, nectarines, pomegranates,
oranges, grapefruit, tomatoes, and combinations or mixtures
thereof.
3. The bioactive fraction of claim 1 wherein said fraction
comprises less than 25 microgram/kg patulin and less than 0.1%
total protein content.
4. The bioactive fraction of claim 1 wherein said fraction is
derived from apple.
5. A composition comprising the bioactive fraction of claim 1
wherein the composition is a leave-on product selected from the
group consisting of cream, dressing, gel, lotion, ointment, liquid,
a spray applicator, and combinations thereof, or a wash-off product
selected from the group consisting of hand dishwashing detergent,
liquid hand soap, bar soap, body wash, shampoo, general purpose
cleanser, and combinations thereof.
6. A method for isolating a bioactive fraction that is derived from
fruit juice of an Apple (Pyrus malus), said method comprising
providing fresh fruit of Apple (Pyrus malus); processing the fruit
under conditions effective to yield a fruit juice least
substantially free of protein and patulin, said processing the
fresh fruit comprising: (i) separating the fresh fruit into the
initial fruit juice and a fiber enriched precipitate; and (ii)
subjecting the initial fruit juice to pH adjustment to yield a
first fruit juice supernatant and a first precipitate; subjecting
said first juice supernatant to microwave treatment to yield a
second fruit juice supernatant and a second precipitate and
subjecting said second fruit juice supernatant to centrifugation
and filtration steps to yield a third fruit juice supernatant and a
third precipitate, wherein said third fruit juice supernatant is
substantially free of patulin and protein.
7. The method of claim 6 wherein said third fruit juice supernatant
comprises less than 25 microgram/kg patulin and less than 0.1%
total protein content.
8. A method for isolating the bioactive fraction of claim 1 from
fruit which comprises processing said fruit to obtain a fruit
juice, and subjecting said fruit juice to the following process
steps: (1) subjecting said fruit juice to a membrane fraction
destabilization step yielding a destabilized fruit juice; (2)
subjecting said destabilized fruit juice to a membrane fraction
separation step to yield a membrane fraction and a fruit juice
supernatant, and (3) subjected said fruit juice supernatant to the
stabilizing step to yield stabilized bioactive fraction derived
from fruit juice.
9. The method of claim 8 wherein said membrane fraction
destabilization step comprises subjecting said fruit juice to
electromagnetic waves of from 2.45 GHz up to about 7.0 for a time
effective to achieve destabilization of the membrane fraction.
10. The method of claim 9 wherein said fruit juice is subjected to
electromagnetic waves of from 3.0 GHz up to about 6.0 GHz for a
time effective to achieve destabilization of the membrane
fraction.
11. The method of claim 9 wherein the temperature of said fruit
juice during said membrane destabilizing step is maintained below
about 40.degree. C.
12. The method of claim 9 wherein the temperature of said fruit
juice during said membrane destabilizing step is maintained below
about 35.degree. C.
13. The method of claim 9 wherein the temperature of said fruit
juice during said membrane destabilizing step is maintained below
about 30.degree. C.
14. The method of claim 9 wherein the temperature of said fruit
juice during said membrane destabilizing step is maintained below
about 20.degree. C.
15. The method of claim 9 wherein the electromagnetic waves
necessary to destabilize the fruit juice are generated by a
magnetron, power grid tube, klystron, klystrode, crossed-field
amplifier, travelling wave tube, and/or a gyrotron.
16. The method of claim 8 wherein said stabilizing step comprises
incubating the bioactive fraction in a mixture of at least one
preservative, at least one antioxidant, at least one stabilizer or
mixtures or combinations thereof to yield a stabilized bioactive
serum fraction.
17. The method of claim 16 wherein said preservatives, antioxidant
and/or stabilizers are selected from potassium sorbate, sodium
benzoate, sodium metabisulfite, pentylene glycol or mixtures or
combinations thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to bioactive fractions
(ingredients) that include isolated bioactive fractions derived
from fruit juices that are either free of or substantially free of
patulin and protein. Further, the bioactive fractions have
antioxidant, free radical scavenging, moisturizing and buffering
activities and properties.
BACKGROUND OF THE INVENTION
[0002] Apples are a part in all food diets and their therapeutic
value is well known for different illnesses (determines the
absorption of gastric secretions, the elimination of toxins, has
diuretic effect). Firmness and sugar content are important quality
attributes that directly influence consumers on purchasing fresh
apple fruit. Organic acids are an important component of fruit
flavor and, together with soluble sugars and aromas, contribute to
the overall organoleptic quality of fresh apple fruits. Malic acid
is the predominant organic acid in apple fruits [Campeanu, G., G.
Neata and G. Darjanschi (2009). Chemical composition of the fruits
of several apple cultivars growth as biological crop. Notulae
Botanicae Horti Agrobotanici Cluj-Napoca 37(2):161-164].
[0003] Malic acid is the major component of apple that is founding
to maintain the liver in a healthy condition and it help in
digestion process. The content of organic acids might be also of
interest in that certain acids may lead to a lowering of the
postprandial blood glucose and insulin responses [Suni, M., M.
Nyman, N.-A. Eriksson, L. Bjork and I. Bjorck (2000). Carbohydrate
composition and content of organic acids in fresh and stored
apples. J. Sci. Food Agric. Journal of the Science of Food and
Agriculture 80:1538-1544]. Apples, and especially apple peels, have
been found to have a potent antioxidant activity and can greatly
inhibit the growth of liver cancer and colon cancer cells. The
total antioxidant activity of apples with the peel was
approximately 83 .mu.mol vitamin C equivalents, which means that
the antioxidant activity of 100 g apples (about one serving of
apple) is equivalent to about 1500 mg of vitamin C However, the
amount of vitamin C in 100 g of apples is only about 5.7 mg.
Vitamin C in apples contributed less than 0.4% of total antioxidant
activity [Boyer, J. and R. H. Liu (2004).
[0004] Apple phytochemicals and their health benefits are well
known. Nutrition Journal 3(5):
http://www.nutritionj.com/content/3/1/5]. (Boyer and Liu, 2004).
Lee et al. [Lee, K, Y. Kim, D. Kim, H. Lee and C. Lee (2003). Major
phenolics in apple and their contribution to the total antioxidant
capacity. Journal of Agriculture and Food Chemistry 51:6516-6520]
found that the average concentration of ascorbic acid among six
apple cultivars was 12.8 mg/100 g fruit. Apples contain a large
concentration of flavonoids, as well as a variety of other
phytochemicals, and the concentration of these phytochemicals may
depend on many factors, such as cultivar, harvest, storage and
processing of the apples. Consumers are becoming more interested in
the content of the health-promoting compounds in fruit because of
their antioxidant activity [Robards, K., P. D. Prenzler, G. Tucker,
P. Swatsitang and W. Glover (1999).]
[0005] Phenolic compounds and their role in oxidative processes in
fruits is also well known. Food Chem. 66:401-436]. Fruit juice is a
fluid expressed from various fruits by crushing and pressing. It
can be clear, cloudy or pulpy. Term apple cider in the United
States is often used to mean cloudy, unfermented, unpreserved apple
juice; it contains intracellular and extracellular water,
carbohydrates (sugars, pectin, hemicellulose, cellulose, starch),
protein, lipids, organic acids (malic, citric, tartaric, acetic,
ascorbic), tannins, phenolic acids and complex phenols, vitamins,
minerals, fiber, carotenoids, anthocyanins, traces of chlorophyll.
These solids are categorized as soluble, which is readily expressed
in the juice and insoluble, consisting primarily of the press
residue. Despite the small amounts of some compounds, they can
influence dramatically the appeal, stability, or health value of
the fruit. Many intrinsic (fruit-specific) and extrinsic
(process-dependent) factors influence juice composition. If the
juice is processed or clarified, it is then called apple juice
[Principles and practices of small and medium-scale fruit juice
processing by R. P. Bates, J. R. Morris and P. G. Crandall FAO
(2001) Agricultural Services Bulletin].
[0006] Malic acid and/or sodium malate that are present in apple
juice are being used in cosmetic products and food for pH
adjustment and effective buffering purposes, as well as flavor
additive. Malic acid is an alpha-hydroxy acid (AHA) which may have
some exfoliating action, and there are products which claim it in
that capacity. Sodium malate might have humectant and moisturizing
properties. Malic acid is naturally and predominantly found in
apples; it acts as gentle exfoliator while providing nutrients to
the skin/scalp as component of tricarboxylic acid cycle (TCA cycle)
or the Krebs cycle.
[0007] Thus, apple's composition, known health benefits of apple
and its purported ability to keep the doctor away, suggests that
this fruit is expected to have a long life in drink, skin, hair and
oral care products and applications. However, there are certain
undesirable components that are present in apple fruit and in apple
juice, namely patulin and protein.
[0008] Patulin or 4-Hydroxy-4H-furo[3,2-c] pyran-2(6H)-one is a
mycotoxin produced by certain species of the genera Aspegillus and
Penicillium. Apples used for processing into juice production could
be inadvertently damaged during transportation. The certain
percentage of fruit with varying degrees of damage (rot) can
contain patulin. It is common in apples and other fruit that is
damaged prior to processing for juice production. Patulin is
produced by various molds, which primarily infect the moldy part of
apples. Removing the moldy and damaged parts of the fruit may not
eliminate all the patulin because some of it may migrate into sound
parts of the flesh. Also, patulin can be produced within the fruit,
even though it may not be visibly moldy. If moldy apples are used
to produce apple juice, the patulin goes into the juice. It is not
destroyed by heat treatments such as the pasteurization process.
Patulin is a natural human toxin and therefore can have genetic
affects within cells, including a developing fetus, the immune
system and the nervous system. The recommended advisory level is 50
microgram of patulin/kg in apple juice [Guidance on the control of
patulin in directly pressed apple juice,
http://www.newark-sherwooddc.gov.uk/ppimageupload].
[0009] Penicillium expansum, is one such fungus and it is
responsible for decomposition of apples and other fruit. The United
Kingdom Ministry of Agriculture, Fisheries and Food in its Food
Surveillance Paper No. 36 (1993) "Mycotoxins "Third Report"
indicates that Penicillium expansum which produces patulin is a
common problem in a diverse range of product (e.g. apples, peaches,
pears, bananas, pineapples, apricots, cherries and grapes). They
indicate that for apple juices patulin levels are generally higher
in cloudy juices than in clear juices (highest levels in their data
showed as 434 microgram/kg and 118 microgram/kg respectively).
Mycotoxins are undesirable in the ingredients from apple or other
fruit because of their toxicity to animals and potential toxicity
to human beings. The toxic activity of patulin, its teratogenicity,
carcinogenicity and mutagenicity is known and is of concern.
[0010] The Codex Alimentarius Commission as part of the United
Nations joint FAO/WHO Food Standards Programme in their 28th
Session (June 1997) in respect of patulin indicates a PMTDI
(Provisional Maximum Tolerable Intake) of 0.4 micrograms per
kilogram body weight per day (i.e. 0.4 micrograms/kg.bw/day). It
was reported that apple juice can occasionally be heavily
contaminated notwithstanding that apple juice generally
(particularly single strength apple juice eg; 11.5.degree. Brix)
has patulin levels of below 50 micrograms per liter. The lower
recommendations (eg; to below 25 micrograms of patulin per liter or
even lower) are now being considered and recommended. It is known
that in some apple juice samples (where there is a significant use
of windfall and/or rotting fruit) to be as high as 1500
microgram/l. However apple juice more commonly contains patulin up
to 200 microgram/l.
[0011] U.S. Pat. No. 6,248,382 Miller et al. describes process for
reducing the patulin concentration in fruit juices which includes
presenting the juice to a resin material having in abundance
micropores of less than 20 .ANG. minimum pore width and at least a
pore surface capable of retaining patulin by the forces of
chemisorption. Preferably the resin has weak base functionality and
is substantially devoid of mesopores and macropores. The resin
preferably has a surface area of greater than 900 m.sup.2 /g (BET)
and the resin has been hypercross-linked whilst in the swollen
state. Regeneration involves the conversion of the resin held
patulin to a more easily flushed out derivative using ammonia or a
volatile base, preferably generated in situ from a high pH
solution.
[0012] S. Drusch et al. investigated the stability of patulin in an
aqueous juice-like model system. At acidic pH, the presence of
ascorbic acid reduced the stability of patulin. After 34 days,
patulin was reduced to 30% of its initial concentration in the
presence of ascorbic acid compared to 68-71% in samples without
ascorbic acid. Conditions during storage (presence of light, oxygen
and/or metal ions) influenced the stability of patulin.
Furthermore, it was possible to induce degradation of patulin by
either generating hydroxyl radicals or by adding the rather stable
radical diphenyl-1-picrylhydrazyl (DPPH). Data this study indicate
that patulin is decomposed by free radicals generated by oxidation
of ascorbic acid to dehydroascorbic acid. Rapid oxidation of
ascorbic acid in the presence of oxygen, catalysed by free metal
ions, resulted in a decrease of patulin. After complete oxidation
of ascorbic acid, no further patulin degradation was observed. In
contrast, slow oxidation of ascorbic acid in the presence of
metal-chelators induced a continuous, slow oxidation of patulin.
Due to low oxygen content in the headspace of a food package,
addition of ascorbic acid to products such as apple juice, prior to
filling, cannot be considered as an effective decontamination
strategy [Stability of patulin in a juice-like aqueous model system
in the presence of ascorbic acid S. Drusch, S. Kopkaa and J.
Kaedinga Food Chemistry Volume 100, Issue 1, 2007, Pages
192-197].
[0013] Proteins, including those in fruit juices, can cause protein
contact dermatitis in sensitive individuals. Shortly after contact
with the causative proteinacous material, such individuals can
experience symptoms such as acute urticarial or vesicular eruption
on the skin, often accompanied by pruritus, burning, and/or
stinging [V. Janssens, et al., "Protein contact dermatitis: myth or
reality?", British Journal of Dermatology 1995; 132: 1-6].
[0014] Thus, it is highly desirable to obtain and use apple juice
that contains as little patulin and as little protein as possible:
and a significant task exists in simultaneous removing or
substantially reducing the concentrations of patulin and protein in
apple juice.
SUMMARY OF THE INVENTION
[0015] The present invention also relates to a method for isolating
bioactive fractions that are derived from fruit juices and are free
or substantially free of protein and patulin. The present invention
also relates to a method for preparing a bioactive fractions
derived from fruit juices that are stabilized and are either free
of or substantially free of patulin and protein.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention provides a commercially viable process
for removing or substantially reducing the patulin and protein
content in fruit juices. The primary objective of this invention is
patulin and protein removal or substantiate reduction of their
content in bioactive ingredients (fractions) obtained from various
apple cultivars (Granny Smith, Golden Delicious, Gala, Cameo,
McIntosh, Braeburn, etc.); however, there is no reason to expect
that patulin and protein content will not be reduced in bioactive
fractions (ingredients) obtained from other fruit juices (e.g.
peaches, pears, bananas, plums, apples, pineapples, apricots,
cherries, grapes, blueberries, raspberries, blackberries,
cranberries, tangerines, prickly pears, nectarines, pomegranates,
oranges, grapefruit, tomatoes, and combinations or mixtures
thereof) with the use of described process.
[0017] As used herein, "substantially free of patulin" means less
than 25 microgram/kg content that is determined by the official
method of analysis of the association of Official Analytical
Chemists (AOAC) 995.10, Patulin in Apple Juice.
[0018] As used herein, "substantially free of proteins" means less
than 0.1% total protein content determined by hydrolyzed and
un-hydrolyzed amino acid analysis conducted on Hitachi L-8900 amino
acid analyzer.
[0019] The formulations containing the bioactive fractions
(ingredients) of the present invention may be prepared using
methodologies that is well known by an artisan of ordinary
skill.
[0020] As used herein, "topical application" generally refers to
techniques relating to directly laying on or spreading the
bioactive ingredients of the present invention or formulations
containing these bioactive ingredients onto the outer skin using,
e.g., by use of the hands or an applicator such as a wipe. The
bioactive ingredients of the present invention are "cosmetically
acceptable."
[0021] As used herein, the term "cosmetically acceptable" refers to
bioactive ingredients, formulations, cosmetically active agents, or
inert ingredients that are suitable for use in contact with
mammalian tissues (e.g., the skin of humans) without undue
toxicity, incompatibility, instability, irritation, allergic
response, and the like, commensurate with a reasonable benefit/risk
ratio.
[0022] It should be noted that, depending on conditions of fruit
(e.g. apple) cultivation, year of growth, and particular harvest,
the dry matter content in fruit can vary and it may impact the
consistency of fruit juice properties and thus reproducibility of
fruit juice derived bioactive fractions (ingredients).
[0023] The present invention allows for the standardization of
initial fruit juice properties to improve reproducibility of
bioactive (fractions) ingredients. The standardization of initial
fruit juice properties can be improved by exploring uniform
conditions for apple cultivation and harvesting.
[0024] The isolated bioactive fractions (ingredients) are combined
with a stabilizing agent. Particularly suitable stabilizing agents
can include, without limitation, a preservative, a stabilizer
and/or mixtures thereof. The isolated bioactive ingredients can be
further concentrated and then stabilized for further utilization in
skin care for topical, oral and functional drink applications. The
bioactive ingredients of the present invention can further be
included in delivery systems that are commonly used in the art.
[0025] An exemplary method of preparing the isolated bioactive
fractions derived from fruit juices that are either free of or
substantially free of patulin and protein involves harvesting,
collecting, and washing of the fresh fruit. Suitable steps to
follow for preparing the fresh fruit biomass include, for example,
the following: (1) preservation of the inherent moisture content of
the fruit; (2) preservation of fruit integrity during harvesting;
(3) minimization of environmental impact and time factors of
biological degradation of the fruit biomass; and (5) cleaning of
the fruit biomass prior to processing (e.g., prior to grinding and
maceration). Each of these steps is discussed below.
Preservation of Inherent Moisture Content:
[0026] The harvest should be done to avoid wilting due to moisture
loss. Optimal conditions are those where natural moisture content
is maintained and preserved.
Preservation of Fruit Integrity During Harvesting:
[0027] Harvesting of the fruit biomass is conducted in a manner
that avoids or minimizes the chopping, mashing, crushing, or other
type of injury of the fruit. For large-scale industrial harvesting,
where it may not be possible to avoid chopping due to the type of
equipment required, care is taken to minimize injury that could
lead to microbial growth, moisture loss, intensification of
oxidation, polymerization, isomerization, and hydrolysis processes
(i.e., unwanted catabolic processes) in collected fruit. Further,
particular attention is made to minimize injury during and after
harvest.
Minimization of Environmental Impact and Time Factors of
Degradation:
[0028] Delivery time of the fruit material to the processing
facility and exposure of biomass to sun, high temperature, and
other negative environmental factors, should be minimized to
prevent the impact of unwanted degradation processes as described
above. For example, in one embodiment of the present invention, the
delivery time for the fruit for further processing does not exceed
30 minutes from the time of harvest. In another embodiment, fruits
that undergo long distance transport are treated to a post-harvest
procedure involving immediately placing the fruit biomass into
Styrofoam coolers containing bags of frozen gel packs to help
maintain freshness and natural moisture content during overnight
delivery to the processing facility. As a nonlimiting example, for
many fruit species it is beneficial to not only minimize delivery
time for processing, but to also keep the fruit material cool, by
refrigeration if necessary, to prevent and/or minimize unwanted
degradation prior to and/or during processing.
Cleaning Step Prior to Grinding and Maceration:
[0029] A washing step to remove debris from the fruit prior to
further processing is performed once the fruit is harvested. The
washing is achieved using a low-pressure rinse for a short duration
under conditions to prevent the initiation of the release of the
juice from the fruit, to cause injury, or to remove valuable
components. For example, in one embodiment of the present
invention, the washing of the fruit biomass was accomplished in
less than or equal to 5 minutes with a water pressure of less than
or equal to 1 kg/cm.sup.2. Residual water wash did not contain any
green or yellow pigments, which indicates the absence of subsequent
injury. The excess water is removed from washed fruit biomass in
order to keep the dry matter content close to natural level.
[0030] After the fruit is harvested, as described above, further
processing of the fruit is performed to yield fruit juice. In one
embodiment, the fruit biomass is subjected to grinding, maceration,
and pressing to separate the intracellular content, i.e., the fruit
juice, and to separate it from the fiber-enriched press-cake
containing predominantly cell walls.
[0031] An example of a suitable processing protocol involves the
steps described below. A hammer mill may be used to grind the fruit
to yield fruit tissue particles of a small size in a short time and
without significant increase of biomass temperature. In one
embodiment, a modified hammer mill is used to produce the maximum
size of macerated fruit particles less than or equal to 0.5
centimeters during less than or equal to 10 seconds of treatment,
where the increase of biomass temperature is less than or equal to
5.degree. C.
[0032] Exposure of ground and macerated fruit biomass is minimized
to prevent the impact of unwanted catabolic processes, as described
above. The separation of fruit juice from fiber-enriched material
(or press-cake) is commenced as soon as possible after grinding and
maceration of the fruit biomass. The fruit biomass is processed in
a short time and without significant increase in temperature. In
one embodiment, immediately after grinding and maceration, the
fruit biomass is pressed using a horizontal, continuous screw press
(Compact Press "CP-6", Vincent Corporation, FL). The pressure on
the cone is maintained at level 24 kg/cm.sup.2, screw speed is at
12 rpm, and biomass temperature increase is less than or equal to
5.degree. C.
[0033] The initial fruit juice usually contains small fiber
particles, starch, pectic substances, hemicellulose and cellulose
particles which can absorb valuable fruit juice components and also
block the hoses and pumps. The above particles should be removed by
filtration or low-speed centrifugation. For example, the initial
fruit juices produced after the pressing step are filtered through
four layers of nylon fabric prior to using the fruit cell juice in
the methods of the present invention.
[0034] Once fruit juice is separated, the fruit cell juice is
relatively stable colloidal dispersion in which organelles
represent the dispersed phase and cytoplasm represents the
continuous phase. Fruit juice is then treated to a processes
involving (1) triggering destabilization of above colloidal
dispersion performing a "initiation of membrane fraction
aggregation step" to yield a destabilized fruit juice and (2)
performing a "membrane fraction separation step" on destabilized
cell juice mixture to yield a membrane fraction (containing
nucleous, mitochondria, or combination of thereof) and a fruit
juice supernatant. In one embodiment, initiation of membrane
fraction destabilization is accomplished by subjecting said cell
juice to electromagnetic waves at a frequency of 2.45 GHz. In
another embodiment the frequency employed is greater than 2.45 GHz
up to 7.0 GHz. In another embodiment, the frequency employed is any
frequency between 2.45 and 7.0 GHz effective to achieve
destabilization of the membrane fraction. After destabilization is
achieved, a membrane fraction separation step is performed. This
step includes, for example, separating of destabilized fruit juice
into the membrane fraction and the fruit juice supernatant using
separating techniques including filtration, or centrifugation, or
combination of thereof.
[0035] A variety of instruments can be employed in the process of
the invention in order to generate the electromagnetic waves
necessary to destabilize the fruit juice including, but not limited
to magnetrons, power grid tubes, klystrons, klystrodes,
crossed-field amplifier, travelling wave tubes, and gyrotrons. One
such instrument includes, but is not limited to high power
magnetron. Conventional and industrial magnetrons operate at a
frequency of 915 MHz and 2.45 GHz and can be employed. However at
those frequencies undesirable heat is can be generated that can
denature the cell juice composition. It is therefore advantageous
to use electromagnetic waves operating at frequencies that are
substantial higher than the frequencies of conventional or
industrial magnetrons, which allows for destabilization of the
fruit juice without undesirable denaturing due to heat generation.
This frequency is typically above the frequency of conventional
microwave magnetrons, i.e., above 2.45 GHz, in another embodiment
greater than 2.45 GHz and less than about 7 GHz; in another
embodiment from about 3 to about 6 GHz, and in yet another
embodiment, any frequency between 2.45 and 7.0 GHz effective to
achieve destabilization of the fruit juice. During the
destabilizing step of the invention the temperature of the fruit
juice is beneficially maintained below 40.degree. C., in another
embodiment below about 35.degree. C., in another embodiment below
about 30.degree. C., in another embodiment below about 25.degree.
C., in another embodiment below about 20.degree. C.
[0036] The freshly obtained membrane fraction is a paste having
color and specific odor that is raw material source specific. The
membrane fraction is represented predominantly by hydrophobic
compounds present in the fruit. The composition of the membrane
fraction includes predominantly phospholipids, membrane proteins,
chromoplasts, traces of chloroplasts, traces of chlorophyll,
nucleus, mitochondria and carotenoids.
[0037] After the bioactive fractions derived from fruit juices that
are either free of or substantially free of patulin and protein is
produced, it is then subjected to the stabilizing step to yield
stabilized fruit juice. In one embodiment, the stabilizing step
involves incubating the bioactive fraction in a mixture of at least
one preservative, at least one antioxidant and at least one
stabilizer to yield a stabilized bioactive serum fraction. Suitable
preservatives, antioxidant and stabilizers for use in the present
invention include, for example, potassium sorbate, sodium benzoate,
sodium metabisulfite and pentylene glycol.
[0038] The present invention also relates to a bioactive topical
formulation suitable for topical application to mammalian skin
and/or hair. The composition can be a leave-on product such as, for
example, a cream, dressing, gel, lotion, ointment, liquid, a spray
applicator, and combinations thereof, or a wash-off product such as
for example a hand dishwashing detergent, liquid hand soap, bar
soap, body wash, shampoo, general purpose cleanser, and
combinations thereof.
[0039] In one embodiment, the bioactive topical formulation
includes a topically effective amount of the bioactive composition
of the present invention. The bioactive topical formulation can
further include a topically acceptable carrier. Suitable topically
acceptable carriers can include, without limitation, a hydrophilic
cream base, a hydrophilic lotion base, a hydrophilic surfactant
base, a hydrophilic gel base, a hydrophilic solution base, a
hydrophobic cream base, a hydrophobic lotion base, a hydrophobic
surfactant base, a hydrophobic gel base, and/or a hydrophobic
solution base. In one embodiment, the bioactive composition can be
present in an amount ranging from between about 0.001 percent and
about 90 percent of the total weight of the bioactive topical
formulation.
[0040] The following examples are intended to illustrate particular
embodiments of the present invention, but are by no means intended
to limit the scope of the present invention.
EXAMPLE 1
Preparation of Bioactive Fractions (Ingredients) from Apple Fruit
(Granny Smith Cultivar)
[0041] Below is a description of relevant aspects of one embodiment
of the method of the present invention.
[0042] The apple whole fruits are collected directly from the trees
at the apple farm. Inspection and removal of unsound fruits is
conducted. After that the apples are thoroughly washed in filtered
city water before processing. The whole fruits are then grinded
(macerated), pressed and filtered to produce initial fruit juice,
the initial source of cosmetic ingredient Recentia.RTM. PM [INCI:
Pyrus Malus (Apple) Juice], CAS RN 1310712-55-0 or functional drink
ingredient Purecentia.TM. PM.
[0043] The yield of initial apple fruit juice from apple fruits
after grinding, pressing and filtration is about 63 to 69%
weight/weight.
[0044] The initial fruit juice is then subjected to various
treatments including pH adjustments, focused microwave irradiations
and separations by centrifugations and filtrations. The pH of
initial juice and interim supernatants are adjusted with dilute
hydrochloric acid (HCl) solution (5N) or sodium hydroxide (NaOH)
25% solution prior to focused microwave processing steps. The pH of
initial apple juice that is about 3.2 to 3.6 is adjusted using a
titration method utilizing 5 N Hydrochloric Acid (HCl) to decrease
the pH of the cell juice to about 3.0. Then the pH adjusted fruit
juice is immediately exposed to microwave irradiation using
specially designed continuous flow system having 2.45 GHz frequency
and 3,200 Watt output power (Microwave Research & Applications,
Inc., Laurel, Md.). This system is equipped with constant speed
stirrer BDC 1850 (Caframo Ltd., Wiarton, Ontario, Canada) and a
temperature control probe. This treatment continued until the
temperature of the fruit juice in microwave chamber reached about
92 to 96.degree. C. and then the treated fruit juice is immediately
pumped through a continuous flow centrifuge device which is
connected with a 1 HP recirculating chiller (Model 6106 P,
Polyscience Corporation, Niles, Ill.) to yield a supernatant 1 and
precipitate 1. The pH of supernatant 1 is then adjusted using a
titration method utilizing 25% solution of Sodium Hydroxide (NaOH)
to increase the pH of the supernatant 1 from about 3.0 to about
8.0. Then the pH adjusted supernatant 1 is immediately exposed to
microwave irradiation using specially designed continuous flow
system having 2.45 GHz frequency and 3,200 Watt output power
(Microwave Research & Applications, Inc., Laurel, Md.). This
system is equipped with constant speed stirrer BDC 1850 (Caframo
Ltd., Wiarton, Ontario, Canada) and a temperature control probe.
This treatment continued until the temperature of the supernatant 1
in microwave chamber reached about 92 to 96.degree. C. and then the
treated supernatant 1 is immediately pumped through a continuous
flow device which was connected with a 1 HP recirculating chiller
(Model 6106 P, Polyscience Corporation, Niles, Ill.) to produce the
supernatant 2 and precipitate 2.
[0045] The pH of supernatant 2 is then adjusted using a titration
method utilizing 5 N Hydrochloric Acid (HCl) to decrease the pH of
the supernatant 2 to from pH about 8.0 to pH about 3.0 and
centrifuged again to yield the supernatant 3 and precipitate 3.
Appropriate preservatives and stabilizers are then added to
supernatant 3 to yield Recentia.RTM. PM or Purecentia.TM. PM.
[0046] Dose rate of microwave treatments were about 1380
joules/sec. During focused microwave irradiation steps there is no
concentration of juice supernatants due to the evaporation. The
system that is used is completely sealed and prevents any loses of
intracellular water due to the evaporation.
[0047] The temperatures during the process are regulated, monitored
and controlled throughout the process and are kept below 30.degree.
C. with the exception of the microwave treatments where temperature
is increased to about 92 to 96.degree. C. at which point it is
immediately and rapidly cooled back to below 30.degree. C. It was
unexpectedly found that the biological constituents remaining in
cosmetic ingredient Recentia.RTM. PM or functional drink ingredient
Purecentia.TM. PM are water soluble components of the initial apple
juice that are free or substantially free from patulin and
protein.
Example 2
Characteristics and Properties of Bioactive Ingredient
Recentia.RTM. PM [INCI: Pyrus Malus (Apple) Juice]
[0048] Bioactive Ingredient Recentia.RTM. PM [INCI: Pyrus Malus
(Apple) Juice] was prepared according to the process described in
Example 1.
[0049] Analyses of Bioactive Ingredient Recentia.RTM. PM were
conducted to determine its various physico-chemical and microbial
characteristics.
[0050] The selected physico-chemical characteristics of
Recentia.RTM. PM [INCI: Pyrus Malus (Apple) Juice] and test methods
are presented below in Table 1.
TABLE-US-00001 TABLE 1 The selected physico-chemical
characteristics of Recentia .RTM. PM [INCI: Pyrus Malus (Apple)
Juice] and test methods. Property Minimum Maximum Test Method Units
Appearance Clear to Yellow Clear to Yellow Determined
organoleptically. N/A Liquid Liquid Odor Characteristic
Characteristic Determined organoleptically. N/A Color 0.5 7.0
Determined on Lovibond Comparator 3000 Gardner Scale. Gardner Turn
on comparator lamp. Measure 8 mL of sample into scale sample tube.
Insert tube into comparator. Rotate the knobs until two color
standards nearest in color to the sample have been located. Record
the value of the sample color accordingly. If the color of the
sample is substantially similar to both, rather than a single
standard, then record it as a value between the values of the two
standards. Dry Matter 9.0 14.7 Dry matter is determined by
comparing the weights of % liquid sample with residual dry matter
after water has been evaporated. Procedure is based on standard
laboratory practices commensurate with available equipment. Take
three disposable aluminum weighing dishes (VWR 25433- 016) and
distinctly number them with a permanent marker on the outside.
Allow marker ink to dry. Turn on the Ohaus Explorer E00640 balance
(Ohaus Corporation) and allow it to start up. Zero the weight.
Place a dish on the balance and record the weight for that dish as
`tare`. Without removing the dish from the balance, add
approximately 4 mL of liquid sample and record the weight for that
dish as `tare + wet`. Set the dish with sample aside. Repeat the
above with two remaining dishes. Leave the dishes for 24 hours in
the ThermoScientific "Lindberg Blue M" Gravity oven at 105 degrees
Celsius. Remove the dishes and allow them to cool for approximately
5 minutes at room temperature. Weigh each dish with dried residue
individually and record the weight as `tare + dry`. Dry matter
percentage is calculated as (`tare + dry` - `tare`)/(`tare + wet` -
`tare`) * 100. Record dry matter percentage for the sample as mean
of dry matter percentage for the three dishes. Refractive 1.350
1.360 Determined by measuring on Reichert Arias 500 nD index
refractometer with temperature regulation provided by Cole- Parmer
Polystat temperature controller, model number 12108-10. Procedure
is based on the instruction manual for Arias 500 refractometer,
sections 6.0, 4.1 and 4.4-4.5. Turn on the temperature controller,
set temperature to 20 degrees Celsius. Turn on refractometer.
Ensure that Automatic Reading Method is enabled. Deposit
approximately 0.5 mL of deionized water on the surface of the lower
measuring prism. Close the cell, taking care to avoid bubble
formation. Turn shadowline adjustment knob to bring the shadow line
within the crosshairs. Wait for temperature at refractometer
measuring cell to completely stabilize, then push Read button.
Retry above steps until refractive index of deionized water is
determined as 1.333 at least three times in a row. Rinse the lower
and upper surfaces of the measuring cell with deionized water and
blot dry with lint-free wipe. Deposit approximately 0.5 mL of
sample on the surface of the lower measuring prism. Close the cell
and turn the shadowline adjustment knob to bring the shadowline
within the crosshairs. Wait for temperature at refractometer
measuring cell to completely stabilize, then push Read button and
note the reading. Rinse the lower and upper surfaces of the
measuring cell with deionized water and blot dry with lint-free
wipe. Repeat above steps until stable readings have been obtained
for sample material at least three times in a row. Record the value
of these stable readings as refractive index of the sample
material. Density 1.03 1.08 Determined with Densito 30PX
densitometer from Mettler g/cm.sup.3 Toledo. Procedure is based on
Operating Instructions for Densito 30PX, sections 4 and 6. Push and
hold the power button on the densitometer for a second to turn it
on. Once the instrument starts up, ensure that the display is set
to g/cm3 units. Depress the plunger button fully, submerge the
sampling tube in deionized water and slowly push the fill trigger
to fill the sample loops with 4 cm3 of deionized water. Avoid
bubble intake or formation. Note the reading. If it deviates by
more than 0.05% from expected density of water at the ambient
temperature, recalibrate the densitometer as per Operating
Instructions. After ensuring that the unit display and calibration
for densitometer are proper, eject the water into a waste
receptacle by depressing the plunger button fully. Submerge the
sampling tube in liquid sample and push the fill trigger to fill
the sample loops with 4 cm3 of sample. Avoid bubble intake or
formation. Note the reading once it stabilizes. Eject the sample
from densitometer into a waste receptacle and repeat steps above
for additional readings, until receiving three matching readings in
a row. Record that as the value of density (specific gravity) for
the sample. pH 3.0 5.0 Determined by measuring on a pH meter such
as Denver N/A Instrument Model 250 pH/ISE/conductivity meter with
pH/ATC electrode number 300729.1. Procedure is based on Denver
Instrument Company 301127.1 Rev. D manual, pages ii and 9 through
12. Turn on the meter and wait for the software to start up. Push
Cal Data button, select pH channel and press Enter. Check that the
calibration is current and slope within acceptable parameters -
manual recommends between 90% and 105%. For product specification
determination, used values between 95% and 100%. If calibration is
out of date or slope outside acceptable boundaries, open the window
on the pH electrode and rinse the electrode head in deionized
water. Then push the Standardize button, select pH channel and
press Enter. Select Manual Buffer Entry and press Enter. Type in
the pH value of a standardization buffer and press Enter. Submerge
the head of the pH electrode into a small beaker of the buffer,
stirred on a magnetic stirrer and press Enter. Wait for the reading
to stabilize. Repeat the above steps with two additional buffers to
complete three-point calibration. If calibration slope is outside
acceptable parameters, repeat. The pH buffers used to calibrate the
pH meter during the product specification determination were
acquired from Thermo Electron Company. In particular, Orion
Application Solution number 910107 was used as pH 7.00 standard,
Orion Application Solution number 910104 was used as pH 4.01
standard, and Orion Application Solution number 910110 was used as
pH 10.01 standard. After ensuring that pH meter calibration is
current and within acceptable bounds, rinse the pH electrode head
with deionized water and blot it lightly with a lint-free wipe.
Pour approximately 20 mL of a sample to be tested into a small
beaker. Add a magnetic stir bar and set it to be stirred at a low
speed on a magnetic stirrer. Ensure that the window at the base of
pH electrode is open, submerge the electrode head in liquid sample
and wait for pH reading to stabilize. Record the stable reading as
the pH value for the sample. UV max 260 265 Determined on Ultrospec
9000 UV/Visible dual-beam nm spectrophotometer (GE Healthcare Life
Sciences) with 5- position thermostatted cell changer. The
procedure is based on chapter 10 section 3 of GE Healthcare
Ultrospec 7000/8000/9000 UV/Visible Spectrophotometers User Manual
and chapter 4 section 3 of GE Healthcare Life Sciences Datrys Life
Science Manual version 2.2.0.0. Instrument control is provided by
Datrys Life Science software version 2.2.0.0 (GE Healthcare) on the
attached computer and/or built-in application suite of the device.
Temperature regulation is provided by CB20 Mini Circulator (Torrey
Pines Scientific). Turn on the circulator and set the temperature
to 20 degrees Celsius and pump speed to 50%. Turn on the
spectrophotometer and/or the attached control computer and wait for
the startup procedures to finish. Take two quartz spectrophotometer
cuvettes with 1 cm path length and volume of at least 3 ml.
Dispense 3 mL deionized water into the cuvette which will be used
as the blank, and 2.998 mL deionized water into the cuvette which
will hold the sample. Add 2 microliters of liquid sample to the
sample cuvette with micropipettor, for effective 1 in 1500 v/v
dilution. For materials that absorb UV light especially strongly or
especially weakly, a different dilution may be required. Close the
two cuvettes with cuvette caps and mix by repeated inversion while
avoiding bubble formation. Open spectrophotometer sample
compartment cover. Place the blank cuvette into the reference cell
holder and the sample cuvette into the position number 1 in the
cell changer. Make sure that clear sides of the cuvettes are the
ones which will be facing the beam. Close the cover and wait to
allow the cuvette temperature to equilibrate at 20 degrees Celsius.
Perform a scan and analysis using the following parameters: start
wavelength 200.0 nm, end wavelength 400.0 nm, step 0.5 nm,
bandwidth 1 nm, fast scan speed, lamp mode set as pulse, reading
mode set as absorbance, feature type set as "Peaks", detection set
as "Sensitive". Report the wavelength of characteristic peak as
.lamda. max. In case of extremely broad and shallow peaks,
detection may be set as "Custom" with appropriate minimum peak
height. In case of no identifiable peaks being present in the plot,
.lamda. max is the wavelength of maximum absorbance. These tasks
may be accomplished in the "Wavescan" application in built-in
instrument control software, or "Wavelength Scan" application in
Datrys Life Science version 2.2.0.0 software running on attached
control computer. Total Plate <10 <10 Determined as per US
Pharmacopoeia XXX, NF25, <61>, CFU/gm Count Microbiological
Limit Tests Mold/Yeast <10 <10 Determined as per US
Pharmacopoeia XXX, NF25, <61>, CFU/gm Microbiological Limit
Tests E. coli Negative/10 gm Negative/10 gm Determined as per US
Pharmacopoeia XXX, NF25, <61>, CFU/gm Microbiological Limit
Tests Salmonella sp. Negative/10 gm Negative/10 gm Determined as
per US Pharmacopoeia XXX, NF25, <61>, CFU/gm Microbiological
Limit Tests Staphylococcus aureus Negative/10 gm Negative/10 gm
Determined as per US Pharmacopoeia XXX, NF25, <61>, CFU/gm
Microbiological Limit Tests Pseudomonas sp. Negative/10 gm
Negative/10 gm Determined as per US Pharmacopoeia XXX, NF25,
<61>, CFU/gm Microbiological Limit Tests Patulin Not detected
<25 By the official method of analysis of the association of
Microgram/kg (Below the method Official Analytical Chemists (AOAC),
Patulin in Apple detection limit of Juice. 1 microgram/kg (or 0.001
ppm) Protein <0.03 <0.1 Amino acid analysis conducted on
Hitachi L-8900 amino % acid analyzer.
[0051] Recentia.RTM. PM and Purecentia.TM. PM are readily soluble
in water in any proportion and are biodegradable ingredients.
Recentia.RTM. PM satisfies skin care industry requirements
regarding total plate count, mold and yeast count, and absence of
pathogens.
[0052] Recentia.RTM. PM and Purecentia.TM. PM have an effective
system of preservatives.
[0053] Recentia.RTM. PM and Purecentia.TM. PM were determined to be
stable (i.e., maintaining physical and chemical integrity) for at
least 12-18 months while stored at a temperature of between 4 and
25 degree. C. in a closed container protected from light.
[0054] Table 2 includes the data related to free radical scavenging
activity of Recentia.RTM. PM.
TABLE-US-00002 TABLE 2 DPPH (2,2-Diphenyl-1-Picrylhydrazyl) free
radical scavenging activity of Recentia .RTM. PM DM % mg DPPH/g
sample Material CAS RN # w/w (as supplied) Recentia .RTM. PM
1310712-55-0 13.33 6.02 Lot A30510MSS-0551
Method
[0055] DPPH (2,2-Diphenyl-1-Picrylhydrazyl) free radical scavenging
activity was determined by a kinetic colorimetric assay adapted for
use with glass-coated polypropylene 96-well microtiter plates
(catalog number 400 062) from SUN-SRi (Rockwood, Tenn.) and Synergy
2 microplate reader from BioTek Instruments Inc (Winooski, Vt.).
Absorbance was measured at 515 nm wavelength. Reaction volume in
each microplate well was 200 .mu.l, with initial concentration of
DPPH equal to 114 .mu.M. L-ascorbic acid was used as positive
control. DPPH (Sigma D9132) and USP L-ascorbic acid (Sigma A-2218)
were obtained from Sigma-Aldrich (St. Louis, Mo.).
[0056] Stoichiometry of the reaction was calculated and expressed
as units weight test article necessary to quench 1 unit weight
DPPH. This can easily be recalculated as DPPH equivalent per sample
weight. This method was adapted from procedure described in "Use of
a free radical method to evaluate antioxidant activity" by W.
Brand-Williams et al, published in LWT--Food Science and
Technology, Volume 28, Issue 1, 1995, pp 25-30.
* * * * *
References