U.S. patent application number 17/406537 was filed with the patent office on 2022-09-08 for gastro-resistant microencapsulates, and uses thereof to stimulate in-vivo ileal glp-1 release in a mammal.
This patent application is currently assigned to NUABIOME LIMITED. The applicant listed for this patent is NUABIOME LIMITED. Invention is credited to Sinead BLEIEL.
Application Number | 20220280600 17/406537 |
Document ID | / |
Family ID | 1000006549529 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220280600 |
Kind Code |
A2 |
BLEIEL; Sinead |
September 8, 2022 |
GASTRO-RESISTANT MICROENCAPSULATES, AND USES THEREOF TO STIMULATE
IN-VIVO ILEAL GLP-1 RELEASE IN A MAMMAL
Abstract
A cold-gelated mono-nuclear microencapsulate comprises a unitary
liquid core encapsulated within a gastro-resistant,
ileal-sensitive, polymerized denatured protein membrane shell,
wherein the liquid core comprises a GLP-1 release stimulating agent
in a substantially solubilised form. The GLP-1 release stimulating
agent is a native protein selected from native dairy protein,
native vegetable protein or native egg protein.
Inventors: |
BLEIEL; Sinead; (Dublin,
IE) |
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Applicant: |
Name |
City |
State |
Country |
Type |
NUABIOME LIMITED |
Dublin |
|
IE |
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Assignee: |
NUABIOME LIMITED
Dublin
IE
|
Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20220040258 A1 |
February 10, 2022 |
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Family ID: |
1000006549529 |
Appl. No.: |
17/406537 |
Filed: |
August 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15536298 |
Jun 15, 2017 |
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PCT/EP2015/079905 |
Dec 15, 2015 |
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17406537 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/168 20130101;
A61K 35/20 20130101; A61K 9/5089 20130101; A61K 9/5052 20130101;
A23P 10/30 20160801; A23L 33/10 20160801; A61K 36/48 20130101; A61K
31/7016 20130101; A23L 2/52 20130101; A23L 33/19 20160801; A23V
2002/00 20130101; A61K 9/0053 20130101; A23L 33/185 20160801; A61K
38/1709 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 9/50 20060101 A61K009/50; A23P 10/30 20060101
A23P010/30; A61K 35/20 20060101 A61K035/20; A61K 36/48 20060101
A61K036/48; A61K 38/16 20060101 A61K038/16; A23L 33/185 20060101
A23L033/185; A23L 33/19 20060101 A23L033/19; A23L 33/10 20060101
A23L033/10; A23L 2/52 20060101 A23L002/52; A61K 9/00 20060101
A61K009/00; A61K 31/7016 20060101 A61K031/7016 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 15, 2014 |
GB |
1422259.0 |
Claims
1. A mono-nuclear microencapsulate comprising a core material
encapsulated within a gastro-resistant, ileal-sensitive,
polymerized protein membrane shell, wherein the core material
comprises a GLP-1 release stimulating agent selected from the group
consisting of native dairy protein, native vegetable protein,
native egg protein, disaccharide, or a mixture thereof, in a
substantially solubilised form.
2. (canceled)
3. The mono-nuclear microencapsulate as claimed in claim 1 in which
the GLP-1 release stimulating agent is native pea protein.
4. The mono-nuclear microencapsulate as claimed in claim 1 in which
the core material has a GLP-1 release stimulating agent
concentration of 6-8% (w/v).
5. The mono-nuclear microencapsulate as claimed in claim 1 in which
the protein of the membrane shell is selected from the group
consisting of whey protein isolate, whey protein concentrate, milk
protein concentrate, or pea protein isolate.
6. The mono-nuclear microencapsulate as claimed in claim 1 in which
the core material forms at least 50% of the microencapsulate
(v/w).
7. The mono-nuclear microencapsulate as claimed in claim 1 in which
the core material forms 70-95% of the microencapsulate (v/w).
8. (canceled)
9. The mono-nuclear microencapsulate as claimed in claim 1 in which
the core material comprises 7-9% native protein (w/v).
10. The mono-nuclear microencapsulate as claimed in claim 1 in
which the core material comprises disaccharide.
11. A composition suitable for oral administration to a mammal
comprising a multiplicity of mono-nuclear microencapsulates
according to claim 1.
12-16. (canceled)
17. A method of inducing satiety in a mammal comprising a step of
orally administering to the mammal a mono-nuclear microencapsulate
of claim 1.
18. A method of inducing or promoting weight loss in a mammal
comprising a step of orally administering to the mammal a
mono-nuclear microencapsulate of claim 1.
19. A method of glycaemic management, promoting insulin secretion,
reducing blood sugar levels, or treating or preventing obesity, in
a mammal, comprising a step of orally administering to the mammal a
mono-nuclear microencapsulate of claim 1.
20-21. (canceled)
22. A method of making a microencapsulate having a unitary liquid
core encapsulated within a gastro-resistant polymerized protein
membrane shell, which method employs a double nozzle extruder
comprising an outer nozzle concentrically formed around an inner
nozzle, the method comprising the steps of: co-extruding a
core-forming solution comprising a GLP-1 release stimulating agent
through the inner nozzle of a double nozzle extruder and a protein
solution through the outer nozzle of the double nozzle extruder to
form microdroplets; and curing the microdroplets.
23. The method as claimed in claim 14 in which the core forming
solution comprises a GLP-1 release stimulating agent selected from
a native dairy protein, a native vegetable protein, a disaccharide,
or any mixture thereof, in a substantially solubilised form.
24. The method as claimed in claim 15 in which the native vegetable
protein is native pea protein.
25. The method as claimed in claim 15 in which the protein solution
is selected from whey protein isolate or whey protein concentrate
at a concentration of 10-12% (w/v), milk protein concentrate at a
concentration of 4-6% (w/v), or pea protein isolate at a
concentration of 7-9% (w/v).
26-27. (canceled)
28. The mono-nuclear microencapsulate as claimed in claim 1 in
which the polymerized protein is a polymerized denatured
protein.
29. The method as claimed in claim 15 in which the protein solution
comprises a denatured protein solution.
30. The method as claimed in claim 15 further comprising the step
of drying the cured microdroplets.
31. The method as claimed in claim 19 wherein the drying step
comprises vacuum/drum drying the cured microdroplets.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation application of U.S.
application Ser. No. 15/536,298 filed Jun. 15, 2017, which is a 35
U.S.C. .sctn. 371 National Phase Entry Application of International
Application No. PCT/EP2015/079905 filed Dec. 15, 2015, which
designates the U.S. and claims benefit under 35 U.S.C. .sctn.
119(a) of European Provisional Application No. 1422259.0 filed Dec.
15, 2014, the contents of which are incorporated herein by
reference in their entireties.
BACKGROUND TO THE INVENTION
[0002] The worldwide, rapidly increasing prevalence of overweight
and obesity has triggered research into food or food products that
have therapeutic potential in the management of overweight,
obesity, and associated diseases. For example, so-called functional
foods containing nutrients that cause larger reductions in food
intake than would be expected on the basis of their caloric
contents alone. These functional foods may have a role in dieting
plans to improve compliance by reducing between-meal hunger,
postponing subsequent meal consumption and reducing caloric intake.
Recent studies have shown that under normal physiological
situations undigested nutrients can reach the ileum, and induce
activation of the so-called "ileal brake", a combination of effects
influencing digestive process and ingestive behaviour. The
relevance of the ileal brake as a potential target for weight
management is based on several findings: First, activation of the
ileal brake has been shown to reduce food intake and increase
satiety levels. Second, surgical procedures that increase exposure
of the ileum to nutrients produce weight loss and improved
glycaemic control. Third, the appetite-reducing effect of chronic
ileal brake activation appears to be maintained over time.
Together, this evidence suggests that activation of the ileal brake
is an excellent long-term target to achieve sustainable reductions
in food intake.
[0003] Given this background, obesity represents 21.sup.st Century
problem--perhaps like never before--the thin line between
maintaining both energy intake and a healthy lifestyle. Much has
been written in both media and science literature about the health
consequences of obesity and inactivity. Despite the attention that
this public health phenomenon has received, obesity has replaced
more traditional problems such as under-nutrition and infectious
diseases as a significant cause of ill-health. Acknowledging that
there is no a clear treatment for obesity and that no single
intervention provides answers for all patients, this presented
encapsulation invention tackles this issue with significant health
benefits via activation of the ileal brake system.
[0004] Activation of the ileal brake is associated with secretion
of gut peptides such as peptide YY (PYY) and Glucagon-Like
Peptide-1 (GLP-1). GLP-1 is known to reduce food intake and hunger
feelings in humans and is assumed to be an important mediator of
ileal brake activation. Activation of the ileal brake is associated
with secretion of gut peptides such as PYY and GLP-1. GLP-1 is
known to reduce food intake and hunger feelings in humans and is
assumed to be an important mediator of ileal brake activation.
Furthermore, GLP-1 is an incretin derived from the transcription
product of the proglucagon gene that contributes to glucose
homeostasis. GLP-1 mimetics are currently being used in the
treatment of Type 2 diabetes. Recent clinical trials have shown
that these treatments not only improve glucose homeostasis but also
succeed in inducing weight loss. Increasing endogenous GLP-1
secretion by functional foods can be expected to mimic these
effects in obese and overweight subjects, because the pathways
involved in GLP-1 secretion and incretin effects are preserved in
obese subjects and in patients with Type 2 diabetes.
[0005] As outlined, GLP-1, is a hormone that delays gastric
emptying and promotes a feeling of satiety. To date, research has
demonstrated that treatment with GLP-1 can potentially enhance
endogenous secretion of insulin after a meal, resulting in improved
glucose homoeostasis and suppressed appetite. As a result,
GLP-1--related drugs have gained credibility from food formulators
with much fanfare and anticipated potential for the treatment of
obesity and Type 2 Diabetes. However, several major obstacles
hinder the oral delivery of GLP-1 due to sensitivity to stomach
acid and enzymes. Furthermore, GLP-1 production is low in obese and
dieting individuals. In other words, GLP-1 requires added
protection to succeed as a credible treatment for Type II Diabetes
or obesity. Although, GLP-1 treatments are currently available for
the treatment for Diabetes Type II; treatments require subcutaneous
injection twice daily, which can cause severe nausea in some
patients, especially when treatment is initiated. The invasive
nature of subcutaneous administration (i.e. injection) can cause
patient discomfort and reduced treatment compliance; hence, an oral
GLP-1 format would significantly improve patient comfort, reduce
primary healthcare costs and reduce the requirement for primary
intervention to treat the follow-on diseases of diabetes.
[0006] To-date, the vast majority of oral satiety ingredients
tested, utilize peptide doses at least 80.times. greater than
equivalent injectable doses. The higher dose quantity being tested
in oral formats clearly compensates for significant losses of
peptides experienced during oral delivery, which makes viable
commercial applications more problematic. This invention seeks to
stimulate the natural release of GLP-1 in the gut in reaction to
the presence of native dietary protein in the upper intestine. In
this way, the costly delivery of satiety ingredients in excessive
doses can be avoided.
[0007] Lipids contain essential fatty acids, and the addition of
lipids to food products in general increases palatability. These
properties make lipids an attractive target to develop functional,
caloric intake--reducing foods. For lipids to influence hunger and
food intake, digestion to fatty acids is an essential step. Several
studies in humans have shown that the effect of lipids on food
intake can be augmented by altering the type of fatty acids in
triacylglycerols: by increasing fatty acid chain length or by
increasing the proportion of unsaturated fatty acids within
triacylglycerols.
[0008] Another approach to increase the effects of lipids on
satiety and food intake is by delaying lipolysis. This results in
the exposure of more distal parts of the small intestine to fat and
fatty acids. Exposure of the ileum to specific nutrients, including
lipids activates the so-called "Ileal brake". This distal ileal
feedback mechanism was initially discovered as an inhibition in
small intestinal motility and transit after ileal fat exposure.
Activation of the ileal brake also has profound effects on satiety
and food intake. After ingestion of a regular meal, only a small
proportion of the ingested nutrients will reach the ileum.
Therefore, the extent to which the ileal brake has a role in
regulation of satiety and food intake under physiologic conditions
is uncertain. The magnitude of the effect of ileal brake activation
on food intake has been most convincingly shown in animal studies
using the ileal transposition technique. In this procedure, a small
segment of distal ileum is re-sected with preservation of
innervation and vasculature. This segment is transpositioned more
proximal with anastomoses between duodenum and proximal jejunum.
Regular feeding in this model profoundly activates the ileal brake.
The ileal transposition procedure results in marked reductions in
food intake and body weight. In humans, studies using
catheter-assisted ileal fat infusions have also reported reductions
in hunger and food intake after ileal fat administration. A dose of
3 g fat, delivered into the ileum, already significantly reduces
between-meal hunger. The data from human and animal experiments
indicate that foods or food constituents that enable exposure of
the ileum to an increased amount of fatty acids have great
potential in the regulation of body weight in obese and overweight
patients.
[0009] Most of the articles published to date describe the delivery
of macronutrients to the ileum by "ileal infusion" using a
naso-ileal catheter for the studies. For example, Shin et al.
(2013) (Lipids, CHOs, proteins: can all macronutrients put a
`brake` on eating?, Shin H S, Ingram J R, McGill A T, et al.,
Physiological Behaviour 2013 Aug. 15: 114-23.) gives a very
comprehensive review on the mechanism and mediators of the
activation of the ileal brake.
[0010] There is a predominance of evidence for an ileal brake on
eating that comes from lipid studies, where direct lipid infusion
into the ileum suppresses both hunger and food intake. Outcomes
from oral feeding studies are less conclusive with no evidence that
`protected` lipids have been successfully delivered into the ileum
in order to trigger the brake. An example of oral feeding studies
are the ones related to `Fabuless` (Olibra).RTM. a "protected"
lipid emulsion. According to these studies the effects were
attributed to the arrival of the emulsion into the distal ileum and
the subsequent stimulation of the ileal brake (Burns A A,
Livingstone M B E, Welch R W, Dunne A, Reid C A, Rowland I R
(2001). The effects of yoghurt containing a novel fat emulsion on
energy and macronutrient intakes in non-overweight, overweight and
obese subjects. Int J Obes 25, 1487-1496); (Burns A A, Livingstone
M B E, Welch R W, Dunne A, Robson P J, Lindmark L et al. (2000).
Short-term effects of yoghurt containing a novel fat emulsion on
energy and macronutrient intakes in non-obese subjects. Int J Obes
24, 1419-1425.); (Burns A A, Livingstone M B E, Welch R W, Dunne A,
Rowland I R (2002). Dose-response effects of a novel fat emulsion
(Olibra) on energy and macronutrient intakes up to 36 h
post-consumption. Eur J Clin Nutr 56, 368-377); (Diepvens K,
Steijns J, Zuurendonk P, Westerterp-Plantinga M S (2008).
Short-term effects of a novel fat emulsion on appetite and food
intake. Physiological Behaviour 95, 114-117). However, these oral
delivery studies neither demonstrated that the lipid emulsions were
protected from absorption in the duodenum or jejunum nor that they
were indeed delivered into the ileum. Dobson et al. (2002) (The
effect of ileal brake activators on the oral bioavailability of
atenolol in man, International Journal of Pharmaceutics, Clair L.
Dobson' Stanley S. Davis' Sushil Chauhan' Robert A. Sparrow' Ian R.
Wilding, Volume 248, Issues 1-2, 6 Nov. 2002, Pages 61-70) describe
the complexities of exploiting natural gastrointestinal processes
to enhance the oral bioavailability of drugs. For the study they
used atenolol as model drug, and oleic acid and the monoglyceride
DGM-04 were formulated into modified release capsules (starch or
hard gel) that were targeted to the small intestine. Their
conclusion was that ileal brake activators can sometimes influence
drug behaviour in the gastrointestinal tract (GI) but the
exploitation of a natural process to enhance the bioavailability of
drugs will not be straightforward.
[0011] For regulation of satiety and food intake, sensing and
signaling from the gastrointestinal tract is crucial. Human
intubation studies and surgical models in animal studies have shown
the potential of ileal brake activation in weight management and in
treating diabetes. Under physiologic conditions, only a small
amount of dietary fat reaches the ileum. Postponing lipolysis and
fat absorption is a well-sought-after target in the development of
functional foods. Fabuless (DSM Food Specialities, Delft,
Netherlands), a vegetable oil emulsion consisting of palm oil and
oat oil, has shown some promise in this respect. Diepvens et al.,
(2007,2008) (Diepvens K, Steijns J, Zuurendonk P,
Westerterp-Plantinga M S (2008). Short-term effects of a novel fat
emulsion on appetite and food intake. Physiological Behaviour 95,
114-117); (Diepvens K, Soenen S, Steijns J, Arnold M,
Westerterp-Plantenga M. Long-term effects of consumption of a novel
fat emulsion in relation to body-weight management. International
Journal of Obesity 2007; 31:942-9) showed that weight management
after initial weight loss improved significantly after ingestion of
a yogurt containing Fabuless twice daily compared with placebo.
However, the mechanism underlying the effect of Fabuless was
previously unknown. Knutson et al., (2010) (Knutson L, Koenders D J
P C, Fridblom H, Viberg A, Sein A, Lennernas H. Gastrointestinal
metabolism of a vegetable-oil emulsion in healthy subjects. Am J
Clinical Nutrition 2010; 92:515-24) reported new data on the
gastrointestinal behavior of the Fabuless emulsion. During a human
intervention study, the authors compared intragastric infusion of
yogurt with either the Fabuless emulsion or milk fats on lipid
digestion in a crossover design. An inflated balloon prevented
passage of luminal contents beyond the proximal jejunum and allowed
sampling at regular intervals. The authors observed that the
treatment containing the test product yielded significantly higher
amounts of fatty acids in the jejunum compared with the control
treatment. This was attributed to the formation of needle-shaped
fatty acid crystals after the Fabuless treatment, in which the
galactolipids from oat oil seem to play a crucial role.
Galactolipids have also been shown to delay and reduce lipolysis by
sterically hindering the absorption and penetration of pancreatic
colipase and lipase into the oil-water interphase in the duodenum.
The mechanism proposed by Knutson et al (2010) is that these
crystals function as a "slow-release capsule," gradually dissolving
while traversing the gastrointestinal tract. This may result in
increased exposure of the ileum to fatty acids, thereby activating
the ileal brake. The concept that Fabuless indeed activates the
ileal brake needs confirmation in human studies. In the study by
Diepvens et al 2010, however, the increase in GLP-1 secretion as a
result of the Fabuless treatment was only observed at 180 min after
ingestion of the test product. One may argue whether this small
increase in GLP-1 secretion can be expected to improve glucose
homeostasis and induce weight loss. Knutson et al., 2010 also
points to intriguing mechanisms involving gradual release of free
fatty acids from lipid crystals, which are formed through the
action of galactolipids.
[0012] Little data exists on whether carbohydrates or protein may
induce the ileal brake and suppress food intake, although there is
a lot of evidence that both clearly have GI mediated effects (e.g.
Groger et al., 1997--Ileal carbohydrates inhibit cholinergically
stimulated exocrine pancreatic secretion in humans. Int J
Pancreatol. 22: 23-9; Karhunen et al., 2008--Effect of protein,
fat, carbohydrate and fibre on gastrointestinal peptide release in
humans. Regul Pept. 149(1-3):70-8; Majaars et al., 2008--Ileal
brake: a sensible food target for appetite control. A review.
Physiol Behay. 95: 271-81; Geraedts et al., 2011a, Mol Nutr Food
Res. 55 (3):476-84. 2011b PLoS One; 6: e24878.). All of them use
catheters for the macronutrient delivery. Some studies state that
proteins have been shown to be more satiating than carbohydrates,
which in turn are more satiating than fats (Westerterp-Plantenga et
al., 2003 (Westerterp-Plantenga et al., 2003, High protein intake
sustains weight maintenance after body weight loss in humans);
(Westerterp-Plantenga et al., 2004), (MS. Westerterp-Plantenga, M.
P. Lejeune, L Nijs, M van Ooijen, E. M. Kovacs, Journal Obesity
Relation Metabolism Disorder., 28 (2004), pp. 57-64), Maliaars et
al, 2008 (Maljaars J, Symersky T, Kee B C, Haddeman E, Peters H P,
et al. (2008) Effect of ileal fat perfusion on satiety and hormone
release in healthy volunteers. International Journal of Obesity 32:
1633-1639).
[0013] It has been confirmed that glucose sensors are present in
both the proximal and the distal GI tract with a feedback loop to
inhibition of gastric emptying when glucose was delivered to the
ileum. The response was related to the length of the SI exposed to
the nutrient (Lin et al. 1989). But according to Shin et al. (2013)
there are no clinical studies which have investigated the effect of
ileal infusion of CHO on appetite related outcomes. In relation to
proteins, although there is growing evidence that is the most
satiating of the macronutrients and may have a role to play in
weight control (Poppitt et al., 1998; Anderson et al., 2004; Weigle
et al., 2005).
[0014] There are no animal models or clinical settings assessing
the role of protein-induced ileal brake on appetite and food
intake. The article by Van Avesaat et al. (2014), (Ileal brake
activation: macronutrient-specific effects on eating behavior? van
Avesaat M, Troost F J., Ripken D., Hendriks H F, Masclee A A,
International Journal of Obesity, 2014) seems to be one of the few
(according to them, the only one) with human data on effects of
ileal exposure to carbohydrates and proteins on food intake and
satiety. Still, this study has been done delivering macronutrients
through a catheter.
[0015] Van Avesaat et al. (2014) demonstrate that with respect to
satiety feelings, only infusion of high-dose protein resulted in a
significant decrease in hunger. Infusions of lipids or high-dose
carbohydrates did not significantly affect feelings of hunger and
satiety. Scientists observed an increase in CKK and GLP-1 plasma
levels after protein infusions. And they also observed increase in
PYY secretion following lipid and carbohydrate infusion.
Apparently, they are the first to demonstrate that ileal infusion
of all three macronutrients induces a decrease in food intake and
that this effect is dose dependent. It is concluded that an ileal
brake-satiating effect leads to a decrease in food intake obtained
with small amounts of lipid, protein and carbohydrates. Ileal
infusion of equicaloric amounts of these macronutrients modulates
food intake, GI peptide release (CCK, GLP-1 or PYY) and feelings of
hunger.
[0016] To summarise prior art to date, extensive literature exists
on the physiology of the ileal brake mechanism: it's activation
(dietary macronutrients), it's effects (delayed gastric emptying,
decreased peristaltic pressure waves in the intestine, etc) and
it's mediators (GI peptides like GLP-1 and PYY). Most of the
clinical studies deliver the macronutrient with a catheter, and the
ones that use oral feeding are inconclusive due to stomach
breakdown of the macronutrients.
[0017] WO2009/053487 (Universiteit Maastricht) describes methods
for treatment or prevention of obesity, or inducement of satiety,
that involve oral delivery of intact pea or wheat protein in a
delivery vehicle that is resistant to hydrolysis. Enteric coated
capsules and microparticles are described as suitable delivery
vehicles. The microparticles are made using 20 g sugar nonpareil
particles that are coated with a thin film of intact pea protein (1
g), which is then dried and further coated with an acid-resistant
polymer such as EUDRAGIT (7 g). Thus, only about 2-5% (w/w) of the
resultant microparticles is intact pea protein, which necessitates
the use of a high dosage of microparticles to achieve a clinically
effective satiety effect.
[0018] It is an object of the invention to overcome at least one of
the above-referenced problems.
STATEMENTS OF INVENTION
[0019] The invention addresses the problems of the prior art,
especially oral delivery of native proteins to the proximal ileum
to stimulate ileal GLP-1 release by means of the ileal brake
mechanism. The invention addresses these problems by providing
cold-gelated mono-nuclear microencapsulates having a liquid core of
GLP-1 release stimulating agent encapsulated within a
gastro-resistant, ileal-sensitive, denatured protein membrane. The
membrane protects the core material (i.e. native protein or
disaccharide) during transit through the acidic environment of the
stomach, preventing digestion of the active agent contained within
the core, and releases the core material when it reaches the ileal
environment. In addition, the use of a mono-nuclear core-shell type
of encapsulate allows for a greater payload of core material (up to
92% of microencapsulate by weight) compared with the nonpareils of
WO2009/053487 that deliver less than 5% of intact protein. In
addition, as the microencapsulates of the invention are formed by
cold gelation, food grade proteins of dairy or vegetable origin may
be employed to generate the gastro-resistant, ileal-sensitive,
membrane shell, thus obviating the need for specialized synthetic
excipients such as EUDRAGIT. Data is provided below demonstrating
that the microencapsules of the invention survive transit through
the stomach, release their contents in the ileum, and deliver a
high payload of GLP-1 release stimulating agents to the proximal
ileum in an active form.
[0020] In a first aspect, the invention provides a mono-nuclear
microencapsulate comprising a core material encapsulated within a
gastro-resistant, ileal sensitive, polymerized denatured protein
membrane shell. Typically, the microencapsulate is cold-gelated.
Typically the core material is liquid. Typically the core material
comprises a GLP-1 release-stimulating agent. Typically the core
material is selected from a dairy protein, egg protein, vegetable
protein, or disaccharide. Typically, the denatured protein
comprises dairy protein or vegetable protein.
[0021] In a further aspect, the invention provides a typically
cold-gelated, mono-nuclear, microencapsulate comprising a liquid
core encapsulated within a gastro-resistant, ileal sensitive,
polymerized denatured protein membrane shell, wherein the liquid
core comprises a native protein, wherein the polymerized denatured
protein membrane shell optionally comprises denatured pea protein
or denatured whey-containing dairy protein.
[0022] In a further aspect, the invention provides a typically
cold-gelated, mono-nuclear, microencapsulate comprising a liquid
core encapsulated within a gastro-resistant, ileal sensitive,
polymerized denatured protein membrane shell, wherein the liquid
core comprises a native protein selected from native protein of
dairy or vegetable origin, wherein the polymerized denatured
protein membrane shell optionally comprises denatured pea protein
or denatured whey-containing dairy protein.
[0023] In a further aspect, the invention provides a typically
cold-gelated, mono-nuclear, microencapsulate comprising a liquid
core encapsulated within a gastro-resistant, ileal sensitive,
polymerized denatured protein membrane shell, wherein the liquid
core comprises a native dairy protein, native pea protein,
disaccharide, or any mixture thereof, and wherein the polymerized
denatured protein membrane shell optionally comprises denatured pea
protein or denatured whey-containing dairy protein.
[0024] In a further aspect, the invention provides a typically
cold-gelated, mono-nuclear, microencapsulate comprising a liquid
core encapsulated within a gastro-resistant, ileal sensitive,
polymerized denatured protein membrane shell, wherein the liquid
core comprises a 6-8% solution of native whey protein, native
casein, native milk protein, native pea protein, disaccharide, or
any mixture thereof, and wherein the polymerized denatured protein
membrane shell comprises denatured pea protein.
[0025] In a further aspect, the invention provides a typically
cold-gelated, mono-nuclear, microencapsulate comprising a liquid
core encapsulated within a gastro-resistant, ileal sensitive,
polymerized denatured protein membrane shell, wherein the liquid
core comprises a 6-8% solution of native pea protein, sucrose, or
any mixture thereof, and wherein the polymerized denatured protein
membrane shell comprises denatured pea protein.
[0026] In one embodiment, at least 50% of the microencapsulate
comprises the liquid core (w/w). In one embodiment, at least 60% of
the microencapsulate comprises the liquid core (w/w). In one
embodiment, at least 70% of the microencapsulate comprises the
liquid core (w/w). In one embodiment, about 70-95% of the
microencapsulate comprises the liquid core (w/w).
[0027] Preferably, the liquid core comprises a GLP-1 release
stimulating agent. In one embodiment, the GLP-1 release stimulating
agent is provided in a substantially solubilised form.
[0028] Preferably, the GLP-1 stimulating agent is a native protein.
In one embodiment, the native protein is selected from native dairy
protein, native vegetable protein, disaccharide, or a mixture
thereof. Data is provided below demonstrating that native dairy and
vegetable protein, and disaccharide, delivered to the proximal
ileum by means of the microencapsulates of the invention, stimulate
the release of GLP-1.
[0029] Typically, the native dairy protein is selected from casein,
whey or a mixture thereof.
[0030] Typically, the native vegetable protein is selected from pea
protein, wheat protein or rice protein, or any mixture thereof.
[0031] Typically, the disaccharide is selected from sucrose or
maltose.
[0032] Preferably, the unitary liquid core has a GLP-1 stimulating
agent concentration of 5-10% (w/v).
[0033] Preferably, the unitary liquid core has a GLP-1 stimulating
agent concentration of 6-8% (w/v).
[0034] Preferably, the protein of the gastro-resistant membrane
shell is selected from whey-containing dairy protein or vegetable
protein.
[0035] Typically, the protein of the gastro-resistant membrane
shell is selected from whey protein isolate, whey protein
concentrate, milk protein concentrate, or pea protein isolate.
[0036] The invention also relates to a composition suitable for
oral administration to a mammal comprising a multiplicity of
microencapsulates of the invention.
[0037] Typically, the composition is selected from a food product,
a beverage, a food ingredient, a nutritional supplement, or oral
dosage pharmaceutical.
[0038] The invention also relates to a microencapsulate of the
invention, or a composition of the invention, for use in a method
of inducing satiety in a mammal, in which the microencapsulate or
composition is administered orally.
[0039] The invention also relates to a microencapsulate of the
invention, or a composition of the invention, for use in a method
of promoting weight loss a mammal, in which the microencapsulate or
composition is administered orally.
[0040] The invention also relates to a microencapsulate of the
invention, or a composition of the invention, for use in a method
of treating or preventing obesity a mammal, in which the
microencapsulate or composition is administered orally.
[0041] The invention also relates to a microencapsulate of the
invention, or a composition of the invention, for use in a method
of glycaemic management in a mammal, in which the microencapsulate
or composition is administered orally.
[0042] The invention also relates to a microencapsulate of the
invention, or a composition of the invention, for use in a method
of promoting insulin secretion in a mammal, in which the
microencapsulate or composition is administered orally.
[0043] The invention also relates to a microencapsulate of the
invention, or a composition of the invention, for use in a method
of reducing blood sugar levels in a mammal, in which the
microencapsulate or composition is administered orally.
[0044] The invention also relates to a method of making a
mono-nuclear microencapsulate having a liquid core encapsulated
within a gastro-resistant polymerized denatured protein membrane
shell, which method employs a double nozzle extruder comprising an
outer nozzle concentrically formed around an inner nozzle, the
method comprising the steps of:
[0045] co-extruding a core-forming solution through the inner
nozzle of the double nozzle extruder and a denatured protein
solution through the outer nozzle of the double nozzle extruder to
form mono-nuclear microdroplets; and curing the mono-nuclear
microdroplets in an acidic gelling bath.
[0046] Preferably, the core forming solution comprises a GLP-1
release stimulating agent. In one embodiment, the GLP-1 release
stimulating agent is provided in a substantially solubilised form.
In one embodiment, the GLP-1 release stimulating agent comprises
native protein or disaccharide. In one embodiment, the GLP-1
release stimulating agent comprises native dairy protein. In one
embodiment, the GLP-1 release stimulating agent comprises native
egg protein. In one embodiment, the GLP-1 release stimulating agent
comprises native vegetable protein. In one embodiment, the native
protein in the core-forming solution is solubilized by physical
means (i.e. sonication) or chemical means (pH). In one embodiment,
the native protein is pea protein, and the solution has a pH of at
least 10. In one embodiment, the native protein is milk protein,
and the solution has a pH of 7-8. In one embodiment, the solution
of native protein has a protein concentration of 6-8% (w/v).
[0047] In one embodiment, the native dairy protein is selected from
casein, whey or a mixture thereof.
[0048] In one embodiment, the native vegetable protein is selected
from pea protein, wheat protein or rice protein, or any mixture
thereof.
[0049] In one embodiment, the disaccharide is selected from sucrose
or maltose.
[0050] In one embodiment, the core forming solution has a GLP-1
stimulating agent concentration of 5-10% (w/v).
[0051] In one embodiment, the core forming solution has a GLP-1
stimulating agent concentration of 6-8% (w/v).
[0052] In one embodiment, the core forming solution comprises
surfactant. In one embodiment, the core forming solution comprises
0.001 to 0.01% surfactant (v/v).
[0053] In one embodiment, the denatured protein solution comprises
whey-containing dairy protein or vegetable protein.
[0054] In one embodiment, the denatured protein solution comprises
whey protein isolate, whey protein concentrate, milk protein
concentrate, or pea protein isolate.
[0055] In one embodiment, the denatured protein solution has a
protein concentration of 4-12% (w/v). When the protein is pea
protein, the protein concentration is suitably 7-9%, preferably
about 8% (w/v). When the protein is whey protein, the protein
concentration is suitably 10-12%, preferably about 11% (w/v). When
the protein is milk protein protein, the protein concentration is
suitably 4-6%, preferably about 5% (w/v).
[0056] Preferably, the denatured protein solution is prepared by
heat denaturation at a temperature of 70-90.degree. C. for a period
of 30-60 minutes. Preferably, the denatured protein solution is
fully denatured.
[0057] Typically, the denatured protein solution is rapidly cooled
immediately after heat denaturation to prevent immediate gelation
of the solution.
[0058] Preferably the core-forming solution is treated to remove
soluble matter.
[0059] Preferably the denatured protein solution is treated to
remove soluble matter.
[0060] In one embodiment, the core forming solution and denatured
protein solution are heated prior to and/or during extrusion. In
one embodiment, the solutions are heated to 30-40.degree. C.
BRIEF DESCRIPTION OF THE FIGURES
[0061] FIGS. 1A and 1B. Light microscopy illustration of
mononuclear microencapsulates (FIGS. 1A and 1B) generated using a
concentric nozzle for protection of macronutrients.
[0062] FIGS. 2A and 2B. Mononuclear microencapsulates after
vacuum/drum drying and membrane thinning process. FIG. 2A
represents a bar of 100 microns and FIG. 2B illustrates a bar of 40
microns.
[0063] FIG. 3. Identification and characterization of in vivo
enzymatic action
[0064] FIG. 4. Effect of in vivo stomach incubation on the tensile
strength of microencapsulate with encapsulated pea protein (Black
column); casein (dark grey column) and sucrose (light grey column).
Data is a average of 12 independent triplicate testings. Image
illustrates the integrity maintained of micro-encapsulates after in
vivo stomach incubation.
[0065] FIG. 5. Microscope images of intact microencapsulates in the
human stomach (A and B) and duodenum (C and D) 35 minutes after
oral ingestion.
[0066] FIG. 6. Microscopic image showing progressive
microencapsulate degradation in the human ileum 90 minutes after
oral ingestion of encapsulated macro-nutrients. Bars (white)
represent 100 microns and (black) 20 microns, respectively.
[0067] FIG. 7. Confocal imagery of digested microencapsulates in
the human ileum 90 minutes after ingestion
[0068] FIG. 8. Intact native protein (black lines) and peptide
release (blue lines) as measured by size exclusion HPLC within the
ileum. Trace amounts of peptides identified in the intestinal
digesta at T=10 min are represented by the red baseline.
[0069] FIG. 9. Data Biacore analysis for detection of sucrose in
the jejunum, (black line), in the proximal ileum 90 minutes (blue
line) and 120 minutes (red line) after ingestion of
microencapsulated sucrose doses. The dose response in the ileum is
represented by the blue arrow.
[0070] FIG. 10a-10c. Absorption of native protein (casein or pea
protein isolate) was significantly increased and controlled as a
result of the microencapsulate encapsulation technique (left
column; FIG. 10c) compared to standard microbead extrusion
encapsulation (right column; FIG. 10c). The right column represents
protein encapsulated in protein microbeads relative to protein
encapsulated in microencapsulates. Column represents relative
absorption in the proximal ileum. Bar represents 20 microns.
DETAILED DESCRIPTION OF THE INVENTION
[0071] The present invention utilizes cost-efficient, clean-label,
food-grade materials to generate micron-sized capsules for
controlled delivery of native protein and/or disaccharide (sucrose)
to the proximal ileum for stimulation of the ileal break mechanism
and insulin regulation.
[0072] This invention outlines the generation of microcapsules with
a membrane formulated from a thermal-treated protein source.
Depending on the protein source, the protein can be partially or
fully denatured. This protein can be sourced from dairy (whey or
casein) or vegetable (pea, rice or wheat) ingredients.
[0073] In one embodiment, the core of the capsule will contain a
GLP-1 release stimulating agent, for example a native protein with
vegetable or dairy origin i.e. pea protein, egg protein, whey or
casein. It can also contain a disaccharide such sucrose or maltose.
Single ingredients or combinations of the aforementioned
ingredients (native protein and disaccharides) can also be
encapsulated as the core material.
[0074] This protein membrane (which is made from thermally treated
protein) has proven protection against harsh stomach acid and
challenging proteolytic enzymes in the upper intestine. This unique
delivery model generates micro-capsules with a gastro-resistant
outer membrane that reacts to intestinal conditions and releases
the core ingredient(s) at the proximal ileum, the systemic target
site.
[0075] Data has demonstrated the release of native protein (pea
protein or casein) and/or disaccharides (sucrose) at the human
proximal ileum, resulting in the production of GLP-1.
[0076] The generation of GLP-1 as a result of native protein and/or
disaccharide delivery to the proximal ileum, stimulated the ileal
break mechanism.
[0077] Evidence exists to demonstrate that secreted GLP-1 further
triggers the secretion of insulin in pancreatic .beta.-cells.
Definitions
[0078] "Cold-gelated": means formed by cold-gelation, in which
liquid microdroplets are extruded or sprayed into a gelling bath
and immediately cured in a gelling bath due to polymerization of
the denatured protein surface film. The bath may be heated or sold.
Examples of cold-gelation are described in the literature, for
example PCT/EP2010/054846 and PCT/EP2014/062154.
[0079] "Mono-nuclear": as applied to the microencapsulate means
that the core material is provided as a single core or nucleus
surrounded by a membrane shell, and is different to the microbeads
described in the prior art, for example PCT/EP2010/054846 and
PCT/EP2014/062154, in which the encapsulated material is provided
as a multiplicity of discrete droplets distributed throughout a
continuous matrix of encapsulating material. The use of
mono-nuclear microencapsulates allows greater amounts of core
material to be encapsulated compared to single nozzle microbead
formation.
[0080] "Microencapsulate": means a mononuclear core/shell type
structure having an average dimension in the range of 30-150
microns, preferably 80-120 microns as determined using a method of
laser diffractometery (Mastersizer 2000, Stable Micro Systems,
Surrey, UK). This method is determines the diameter, mean size
distribution and D (v, 0.9) (size at which the cumulative volume
reaches 90% of the total volume), of micro-encapsulates with
diameters in the range of 0.2-2000 .mu.m. For microencapsulate size
analysis, micro-encapsulate batches were re-suspended in Milli-Q
water and size distribution is calculated based on the light
intensity distribution data of scattered light. Measurement of
microencapsulate size is performed at 25.degree. C. and six runs
are performed for each replicate batch (Doherty et al., 20111)
(Development and characterisation of whey protein micro-beads as
potential matrices for probiotic protection, S. B. Doherty, V. L.
Gee, R. P. Ross, C. Stanton, G. F. Fitzgerald, A. Brodkorb, Food
Hydrocolloids Volume 25, Issue 6, August 2011, Pages 1604-1617).
Preferably, the microencapsulate is substantially spherical as
shown in the attached figures.
[0081] "Gastro-resistant": means that the microencapsulates can
survive intact for at least 60 minutes in the simulated stomach
digestion model described in Minekus et al., 1999 and 2014 (A
computer-controlled system to simulate conditions of the large
intestine with peristaltic mixing, water absorption and absorption
of fermentation product, Minekus, M., Smeets-Peeters M, Bernalier
A, Marol-Bonnin S, Havenaar R, Marteau P, Alric M, Fonty G, Huis
in't Veld J H, Applied Microbiology Biotechnology. 1999 December;
53 (1):108-14) and (Minekus et al., 2014, A standardised static in
vitro digestion method suitable for food--an international
consensus, Minekus, A. et al., Food Function, 2014, 5, 1113).
[0082] "Ileal-sensitive": means that the microencapsulates are
capable of releasing their contents in vivo in the ileum of a
mammal.
[0083] "GLP-1 release stimulating agent" means an agent that is
capable of stimulating STC cells to release GLP-1 in an in vitro
cell model described below. Preferably, the GLP-1 release
stimulating agent is selected from a native protein and a
disaccharide. Preferably, the GLP-1 release stimulating agent is
selected from a native protein of dairy or vegetable origin.
Preferably, the GLP-1 release stimulating agent is pea protein, egg
protein, casein, whey protein, disaccharide, or a mixture
thereof.
[0084] "Native" as applied to protein means that the protein is not
denatured, i.e. typically at least 90% and preferably all of the
protein by weight is in its native, non-denatured, form. In one
embodiment, the native protein is slightly hydrolysed, e.g. up to
20% hydrolysis, by suitable means, e.g. a suitable hydrolyzing
enzyme, such that it still functions as a GLP-1 release stimulating
agent.
[0085] "Native protein of dairy origin": means native whey protein,
native casein protein, native milk protein, or a mixture thereof,
in any form for example whey protein isolate, whey protein
concentrate, caseinate, milk protein concentrate or the like.
[0086] "Native protein of vegetable origin": means native pea
protein, native wheat protein, native rice protein, in any forms
for example as a concentrate or isolate, or proteins derived from
other vegetable sources. Preferably, the term means native pea,
wheat or rice protein.
[0087] "Dairy protein" as applied to the core means casein, whey,
or combinations thereof. Typically, the dairy protein is a bovine
dairy protein, preferably a dairy protein isolate or concentrate.
In one embodiment, the dairy protein is selected from milk protein
concentrate, whey protein concentrate, whey protein isolate, and a
caseinate, for example sodium caseinate. Typically, the liquid core
comprises 6-8% dairy protein, ideally 6.6-7.5% (w/v). Typically the
solvent for the dairy protein has a pH of 7-8, ideally about
7.5.
[0088] "Vegetable protein": typically means a protein derived from
a vegetable or plant, for example pea, wheat or rice, or any
combination thereof. The protein may be in the form of a
concentrate or an isolate.
[0089] "Pea protein" should be understood to mean protein obtained
from pea, typically total pea protein. Preferably the pea protein
is pea protein isolate (PPI), pea protein concentrate (PPC), or a
combination of either. Typically, the liquid core comprises 6-8%
pea protein, ideally 6.6-7.5% (w/v). Typically the solvent for the
pea protein has a pH of greater than 10 or 10.5. Ideally, the pea
protein is solubilised in an alkali solvent.
[0090] "Alkali solvent" means an aqueous solution of a suitable
base for example NaOH or KOH. Preferably, the alkali solvent
comprises an aqueous solution of 0.05-0.2M base, more preferably
0.05-0.15. Ideally, the alkali solvent comprises an aqueous
solution of 0.075-0.125 M base. Typically, the alkali solvent is an
aqueous solution of NaOH, for example 0.05-0.2M NaOH. Preferably,
the alkali solvent comprises an aqueous solution of 0.05-0.2M NaOH
or KOH, more preferably 0.05-0.15 NaOH or KOH. Ideally, the alkali
solvent comprises an aqueous solution of 0.075-0.125 M NaOH or
KOH.
[0091] "pH of at least 10" means a pH of greater than 10, typically
a pH of 10-13 or 10-12. Ideally, the pH of the pea protein solution
is 10.5 to 11.
[0092] "Disaccharide" means a sugar molecule comprising two linked
saccharide units, for example sucrose, maltose, trehalose or the
like. Preferably, the disaccharide is sucrose or maltose.
[0093] "Polymerised": as applied to the protein of the membrane
shell means that the protein is crosslinked as a result of
cold-gelation in a gelling bath. Preferably, the polymerized
protein forms a water impermeable shell. Typically, the gelling
bath is acidic
[0094] "Denatured": means partially or fully denatured. Preferably
at least 90%, 95% or 99% of the protein is denatured. A method of
determining the % of denatured protein is provided below.
[0095] "Whey-containing dairy protein" means a whey protein (i.e.
whey protein isolate or concentrate) or a milk protein that
contains whey (i.e. milk protein concentrate). When the protein is
whey, the denatured whey protein solution typically comprises at
least 50%, 60% or 70% denatured whey protein. When the protein is
milk protein, the denatured milk protein solution typically
comprises 4-6%, preferably 5-5.5% denatured milk protein.
Preferably, the milk protein is milk protein concentrate.
[0096] "Pea protein solution" means a liquid pea protein
composition comprising soluble pea protein and optionally insoluble
pea protein. The methods of the invention provide for pea protein
solutions comprising high levels of soluble pea protein, typically
greater than 80%, or 90% (for example, 85-95% soluble pea protein).
When the pea protein is mixed with alkali solvent, the amount of
soluble pea protein will gradually increase during the resting step
until high levels of the pea protein is solubilised in the alkali
solvent, at which point the pea protein solution is heat-denatured.
This results in a solution of denatured pea protein having very
high levels of denatured pea protein present in the form of soluble
denatured pea protein aggregates.
[0097] The term "soluble" or "solubilised" or "substantially
solubilized" as applied to protein, especially protein in the
liquid core, should be understood to mean that the protein is
present as soluble pea protein aggregates. Typically, the terms
mean that the soluble aggregates will not come out of solution upon
centrifugation at 10,000.times.g for 30 minutes at 4.degree. C.
[0098] "Resting the native protein solution" means leaving the
native protein solution rest for a period of time to allow the
native protein to solubilise in the solvent. Generally, the native
protein solution is allowed to rest for at least 20, 25, 30, 35,
40, or 45 minutes. Typically, the native protein solution is rested
at room temperature. Typically, the native protein solution is
rested for a period of time until at least 90% of the native
protein has been solubilised.
[0099] "Conditions sufficient to heat-denature the protein without
causing gelation of the protein solution" means a temperature and
time treatment that denatures at least 95% or 99% of the protein
present in the solution while maintaining the solution in a form
suitable for extrusion (i.e. readily flowable). The temperature and
times employed may be varied depending on the concentration of the
pea protein solution. Thus, for example, when an 8% pea protein
solution (w/v) is used, the solution may be treated at a
temperature of 80-90.degree. C. for 20-30 minutes (or preferably
85.degree. C. for 25 minutes). However, it will be appreciated that
higher temperatures and shorter times may also be employed.
[0100] "Rapidly cooled" means actively cooling the solution to
accelerate cooling compared with simply allowing the solution to
cool at room temperature which the Applicant has discovered causes
the solution to gel. Rapid cooling can be achieved by placing the
solution in a fridge or freezer, or on slushed ice, until the
temperature of the solution has been reduced to at least room
temperature.
[0101] "Treated to remove soluble matter" means a separation or
clarification step to remove soluble matter such as insoluble
protein from the protein solution. In the specific embodiments
described herein, centrifugation is employed (10,000.times.g for 30
minutes at 4.degree. C.) is employed, but other methods will be
apparent to the skilled person such as, for example, filtration or
the like.
[0102] "Solution of denatured protein" means a solution of protein
in which at least 90%, 95% or 99% of the total protein is
denatured. A method of determining the % of denatured protein in a
protein solution is provided below.
[0103] "Immediately gelling the droplets in an acidic gelling bath
to form microbeads" means that the droplets gel instantaneously
upon immersion in the acidic bath. This is important as it ensures
that the droplets have a spherical shape and homogenous size
distribution. Surprisingly, instantaneous gelation is achieved by
employing an acidic bath having a pH less to the pI of the pea
protein, for example a pH of 3.8 to 4.2.
[0104] "Acidic gelling bath" means a bath having an acidic pH that
is capable of instantaneously gelling the droplets. Typically, the
acidic gelling bath has a pH of less than 5, for example 3.5 to
4.2, 3.7 to 4.2, or 3.8 to 4.2. The acidic gelling bath is
generally formed from an organic acid. Ideally, the acid is citric
acid. Typically, the acidic gelling bath has an acid concentration
of 0.1M to 1.0M, preferably 0.3M to 0.7M, and more preferably 0.4M
to 0.6M. Typically, the acidic gelling bath has a citric acid
concentration of 0.1M to 1.0M, preferably 0.3M to 0.7M, and more
preferably 0.4M to 0.6M. Preferably, the acidic gelling bath
comprises 0.4 to 0.6M citric acid and has a pH of less than 4.3,
typically 3.8 to 4.2.
[0105] "Double nozzle extruder" means an apparatus comprising an
outer nozzle concentrically arranged around an inner nozzle, and in
which the denatured protein solution is extruded through the outer
nozzle and the core-forming solution is extruded through the inner
nozzle to form microdroplets which are gelled in the gelling
bath.
[0106] Examples of double nozzle extruders include instrumentation
provided by BUCHI Labortechnik (www.buchi.com) and GEA NIRO
(www.niro.com).
[0107] "Cured mono-nuclear microdroplets in the acidic gelling
bath" means that the microdroplets are allowed remain in the
gelling bath for a period of time sufficient to cure (harden) the
microbeads. The period of time varies depending on the
microdroplets, but typically a curing time of at least 10, 20, 30,
40 or 50 minutes is employed.
Experimental A: Manufacture of Microcapsules
[0108] A: Preparation of Native Protein (Loading Material)
[0109] Materials
[0110] The following materials have been tested as loading
materials in microcapsules: [0111] Whey protein isolate (WPI)
[0112] Whey protein concentrate (WPC) [0113] Milk Protein
concentrate (MPC) [0114] Sodium caseinate (NaCa) [0115] Pea Protein
isolate (PPI) [0116] Sucrose
[0117] The core/loading material can be a native protein with
vegetable or dairy origin. Disaccharides have also been tested and
sucrose appears to be the best candidate for loading.
[0118] Method
[0119] Prepare a protein dispersion i.e. Suspend 7.0% (w/w),
protein basis) in distilled water and disperse under agitation at
4.degree. C. for 24 hours using an overhead stirrer (Heidolph RZR
1, Schwabach, Germany) Prepare a disaccharide dispersion i.e. 7.0%
(w/w) in distilled water and disperse under agitation at ambient
temperature for 24 hours using an overhead stirrer. When using
dairy or vegetable protein sources, HPLC analysis must be performed
initially in order to validate the protein and calcium
concentration i.e. protein & calcium content will be
significantly different between concentrates and isolates. When
using milk based proteins (WPI, WPC, MPC or NaCa), adjust solution
to pH 7.5 (using IN/4N NaOH) and add 0.003% Tween 20 in order to
encourage the dissolution. When dispersing pea protein (PPI) adjust
to pH 10.5 (using IN/4N NaOH) and add 0.004% tween-80 to enhance
protein solubility.
[0120] Store solutions at ambient temperature in order to permit
full protein hydration.
[0121] Centrifuge at 2000.times.g for 20 minutes at room
temperature to remove any undesirable protein agglomerates present
form the powder processing. All protein solutions are filtered
through 0.45 .mu.m HVLP membranes (Millipore USA) under a pressure
of 4 bar using a stainless steel dead-end filtration device. All
milk-based protein solutions (WPI, WPC, MPC or NaCa), are sonicated
for 90 seconds to remove air pockets formed during filtration. Pea
Protein (PPI) is placed under vacuum to remove dissolved air
droplets. This process avoids i) blockage of protein in the
concentric nozzle and ii) flow discrepancies during encapsulation
process which would effect encapsulation efficiency.
[0122] B: Preparation of Capsule Material
[0123] Materials [0124] Whey protein isolate (WPI) [0125] Whey
protein concentrate (WPC) [0126] Milk Protein concentrate (MPC)
[0127] Pea Protein isolate (PPI)
[0128] Method
[0129] Heat-treat the pea protein solution (8.0% w/w) under
agitation (200 rpm) at 85.degree. C. and maintain that temperature
for a duration of 25 minutes. For MPI, protein concentration must
be diluted to 5.2% (w/w,) on a protein basis using phosphate
buffered saline (PBS) prior to heat treatment at 78.degree. C. for
a duration of 45 minutes. The presence of calcium requires a lower
MPI protein concentration to avoid polymerization during heating
phase. MPI comprises of .beta.-lactoglobulin and .beta.-casein;
hence a more transparent protein dispersion will be generated for
use in subsequent encapsulation steps. Heat-treatment of whey
protein solutions (WPI, WPC) is performed using the original
prepared concentration (11% protein solution, w/w) under agitation
(150 rpm) at 78.degree. C. for 45 minutes. Upon completion of the
heat treatment step, transfer the protein solutions to crushed ice
for immediate cooling. Continue agitation (200 rpm) for 2 hours
(room temperature) to prevent further polymerisation of the protein
agglomeration. The protein solution in stored overnight (min. 8
hours) at refrigeration temperature. Equilibrate the solution at
ambient temperature.
[0130] C: Encapsulation Procedure
[0131] Mono-nuclear microcapsules were prepared using the
co-extrusion laminar jet break-up technique. The encapsulator was
fitted with one of two different sized concentric nozzles (internal
and external). Heat-treated protein (pea or milk sources) was
prepared as outlined above. Heat treated protein dispersions are
supplied to the external nozzle using an air pressure regulation
system which enabled flow rates of 5-6.6 L/min to be generated
using a maximum head pressure of 0.85-1.1 bar. The desired flow
rate is set using a pressure reduction valve. The internal phase
(native protein, non-heat treated or sucrose) is supplied using a
precision syringe pump connected to the inner nozzle to supply the
inner phase at flow rates of between 9 and 17.3 L/min. Hence the
native material (to be encapsulated; the encapsulant) i.e. casein
and/or sucrose is incorporated into the inner core. They can be
delivered as a sole protein source or disaccharide source--or they
can be blended into a mixture. Spherical microcapsules are obtained
by the application of a set vibrational frequency, with defined
amplitude, to the co-extruded liquid jet consisting of outer layer
of heat-treated protein (pea or milk) material and inner core
consisting of native casein and/or sucrose
[0132] The material in the inner and outer nozzle are both heated
to 35.degree. C. in order to allow for better flowability in
commercial operations. The resulting concentric jet breaks up into
microcapsules, which fall into a magnetically stirred gelling bath
20 cm below the nozzle. The gelling bath consisted of 36 g/l citric
acid, 10 mM MOPS, pH 4.0. Tween-80 is added (0.1-0.2% (v/v)) to
reduce the surface tension of the gelation solution. To prevent
coalescence of the microcapsules during jet break-up and/or when
entering the gelling bath, a high negative charge was induced onto
their surface using an electrostatic voltage system which applied
an electrical potential of 0-2.15 kV between the nozzle and an
electrode, placed directly underneath the nozzle As microcapsules
fall through the electrode, they were deflected from their vertical
position resulting in their impact occurring over a larger area in
the gelation solution Microcapsules were allowed to harden for at
least 45 minutes to ensure complete gelation and were then washed
and filtered using a porous mesh to remove any un-reacted
components.
Experimental B: Characterisation of Microcapules and In-Vitro,
Ex-Vivo and In-Vivo Testing
Experimental Methods
[0133] Light Microscopy--Bright-field light microscopy measurements
were also carried out using a BX51 light microscope (Olympus,
Essex, UK). Samples were deposited on glass slides and analysed on
the same day.
[0134] Atomic Force Microscopy (AFM)--Atomic Force Microscopy (AFM)
images were obtained using Asylum Research MFP-3D-AFM (Asylum
Research UK Ltd. Oxford, UK) in AC-mode. Prior to imaging, all
samples were diluted (.times.50, .times.100) in MilliQ H.sub.2O and
10 .mu.L aliquots were deposited onto freshly cleaved mica surfaces
and subsequently dried in a desiccator. An aluminum reflex coated
cantilever with a tetrahedral tip (AC 240), spring constant of 1.8
N/m (Olympus Optical Co. Ltd, Tokyo Japan), working frequency of
50-- 90 kHz, and scan rate at 1 Hz was used for air-dried samples.
The radius of curvature of the tetrahedral tip was 10 (.+-.3)
nm.
[0135] Confocal Scanning Laser Microscopy (CSLM)--Fluorescent
microscopy was performed using a Leica TCS SP5 confocal scanning
laser microscope (CSLM) (Leica Microsystems, Wetzler, Germany)
Micro-encapsulates were stained using fast green or Thiazole orange
(TO) dye for fluorescence of the protein micro-encapsulates.
Samples were analysed using .times.63 magnification objective with
a numerical aperture of 1.4. Confocal illumination was provided by
an argon laser (488 nm laser excitation) and red-green-blue images
(24 bit), 512.times.512 pixels, were acquired using a zoom factor
of 2.0, giving a final pixel resolution of 0.2 .mu.m/pixel.
[0136] Mechanical Strength--The mechanical strength of
micro-encapsulates were examined using a texture analyzer (TA-XT2i,
Stable Micro Systems, Godalming, UK) as a function of stomach
incubation time (0-180 minutes). Briefly, a specific force was
applied to a micro-encapsulates monolayer and the quantity of
rupture of the micro-encapsulates was assigned as a measure of
mechanical stability. A procedure was developed for measurement of
mechanical strength and physical integrity of empty and
macronutrient-loaded micro-encapsulates with necessary compression
conditions acquired from the manufacturer. Strength assays were
performed using a 20 mm diameter cylindrical aluminum probe at a
mobile speed of 0.3 mm/s in compression mode. A rupture distance of
95% was applied and the peak force (expressed in gram force)
exerted by the probe on the micro-encapsulate mono-layer was
recorded as a function of compression distance leading to a force
vs. incubation time relation. Analysis was conducted on 15
monolayer samples per batch and a total of 10 replicate batches
were analysed at each time point to obtain statistically relevant
data.
[0137] HPLC analysis--Size exclusion chromatography was carried out
on FPLC system (AKTA purifier, GE Healthcare) equipped with a
Superose 12 10/300 GL column (GE Healthcare Bio-Sciences, Uppsala,
Sweden). Pea and Milk protein isolates (100 mg) were dissolved in 1
ml borate buffer (0.1 M sodium borate, 0.2 M sodium chloride, pH
8.3). The proteins were eluted at a flow rate of 0.4 ml per min.
The aforementioned buffer was used as mobile phase/eluent. The
eluate was continuously monitored at 280 nm. Molecular weight
standard kits for gel filtration chromatography (Sigma Aldrich, St.
Louis, Mo., USA) were used for calibration.
[0138] Capsule Surface hydrophobicity (SH)--SH of whey
microencapsulates were determined using the SDS binding method
outlined by Kato et al., 1984 (Kato, A., Matsuda, T., Matsudomi,
N., & Kobayashi, K (1984). Determination of protein
hydrophobicity using sodium dodecyl sulfate binding. Journal of
Agricultural and Food Chemistry, 32, 284-288) with particular
adjustment for milk and/or pea protein profiles. Protein
micro-encapsulates were suspended in sodium dihydrogen phosphate
dihydrate buffer (0.02 M; pH 6.0), while SDS reagent (w/v=40.37 mg
L.sup.-1) and methylene blue (w/v=24.0 mg L-.sup.1) were prepared
separately in fresh buffer solutions. Individual micro-encapsulate
batches were mixed with SDS reagent (1:2 ratio), incubated for 30
minutes at 20.degree. C. under slight agitation and subsequently
dialyzed against the phosphate buffer (v/v, ratio 1:25) for 24 h at
20.degree. C. Mixtures of 0.5 mL of dialysate, 2.5 mL of methylene
blue, and 10 mL of chloroform were centrifuged at 2,500.times.g for
5 minutes. The extinction co-efficient (.epsilon.) of the
chloroform phase was assessed at a wavelength of .lamda.=655 nm
(according to Hiller and Lorenzen, 2000) (Hiller, B., &
Lorenzen, P. C. (2008), Surface hydrophobicity of physicochemically
and enzymatically treated milk proteins in relation to techno
functional properties, Journal of Agricultural and Food Chemistry,
56 (2), 461-468). Measurements were performed in triplicate and SH
of fresh microencapsulate batches were assessed relative to batches
procured as a function of gastric and intestinal incubation time.
Native and heat-treated milk and pea proteins represented positive
and negative controls, respectively, and all treatments contained
equivalent protein concentration.
[0139] SDS-PAGE--The average molecular weights (AMW) of peptides
procured during micro-encapsulate digestion in intestinal media
were estimated by SDS-PAGE under reducing conditions according to
the method described by Laemmli, 1970 (Laemmli, U. K, 1970,
Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature, 227 (5259), 680-685). Treated samples
were loaded onto a stacking gel 12% acrylamide and a 4% stacking
gel, both containing 0.1% SDS. The running buffer used was free
from .beta.-mercaptoethanol due the disassociating effect it has on
the protein. This caused the break-up of protein aggregates by
reducing intra- and intermolecular disulphide bonds. The
electrophoresis was performed at a constant voltage of 180 V in a
mini Protean II system (Bio-Rad Alpha Technologies, Dublin,
Ireland) and gels were stained in 0.5% Coomassie brilliant blue
R-250, 25% iso-propanol, 10% acetic acid solution. The AMW of the
protein bands of electrophoretically separated matrix components
were estimated by comparison of their mobility to those of standard
proteins (Precision Plus Protein.TM. Standards, Bio-Rad Alpha
Technologies).
[0140] Size Distribution Analysis--Mean size distribution and D (v,
0.9) (size at which the cumulative volume reaches 90% of the total
volume), of micro-encapsulates were determined using a laser
diffractometer (Mastersizer 2000, Stable Micro Systems, Surrey, UK)
with a range of 0.2-2000 .mu.m. For particle size analysis,
micro-encapsulates batches were resuspended Milli-Q water and size
distribution was calculated based on the light intensity
distribution data of scattered light. Measurement of
micro-encapsulate size was performed at 25.degree. C. and three
runs were performed for each replicate batch. Micro-encapsulate
diameter and size distribution were determined as a function of
incubation time, acetate concentration and pH in addition to GI
sample analysis.
[0141] Micro-encapsulate Digestion--The Degree of hydrolysis (DH)
of micro-encapsulates was investigated directly by quantification
of cleaved peptide bonds via the o-phthaldialdehyde (OPA)
spectrophotometric assay, which involved using N-acetyl-1-cysteine
(NAC) as the thiol reagent. To assay proteolysis, 100 .mu.l of each
GI sample was added to an equal volume of 24% (w/v) trichloroacetic
acid (TCA). Analysis was performed in triplicate for each
micro-encapsulate batch obtained. Adler-Nissen, 1979 (Determination
of the degree of hydrolysis of food protein hydrolysates by
trinitrobenzenesulfonic acid, Adler-Nissen, J., Journal of
Agricultural. Food Chemistry, 1979, 27 (6), 1256-1262).
[0142] Free Amino Acid Analysis--Samples procured from digestion
studies were deproteinised by mixing the sample with equal volumes
of 24% (w/v) TCA and allowed to stand for 10 min before
centrifugation at 14,400.times.g for 10 minute (Microcentaur, MSE,
London, UK). Supernatants were removed and diluted with 0.2M sodium
citrate buffer, pH 2.2 to give a final concentration of 125 nM/ml
Amino acids were quantified using a Jeol JLC-500/V amino acid
analyzer (Jeol (UK) Ltd., Garden city, Herts, UK) fitted with a
Jeol Na.sup.+ high performance cation exchange column Amino acid
analysis was performed in triplicate on all GI sample.
[0143] Cell Culture--STC-1 cells are maintained in Dulbecco's
Modified Eagles Medium (Sigma) with 10% fetal bovine serum (Sigma),
100 units/mL penicillin, and 100 mg/mL streptomycin as additional
supplements, at 37.degree. C. in 5% CO.sub.2/air humidity. All
studies were performed on cells with passage number 30-35.
[0144] Digestion/Delivery Testing
[0145] In Vitro Studies
[0146] In vitro digestion modeling was performed to elucidate the
stability and subsequent digestibility of microencapsulates during
gut transit. The procedure consists of subjecting (encapsulated and
control) treatments to a two-stage digestive process: gastric
incubation and intestinal incubation. During in vitro analysis,
various factors like digestive enzymes, bile salts, pH, etc. were
integrated to simulate transit and digestion of encapsulation
systems along the gastrointestinal tract i.e. USP formulation.
During the gastric phase, microencapsulates are acidified and a
porcine pepsin suspension added under agitation. During the
intestinal phase, the pH is neutralised and the mixture incubated
at 37.degree. C. in the presence of intestinal enzymes such as
trypsin and chymotrypsin under controlled temperature and agitation
conditions., Minekus et al., 2014 (A standardised static in vitro
digestion method suitable for food--an international consensus,
Minekus, A. et al., Food Function, 2014, 5, 1113).
[0147] Ex Vivo Studies
[0148] Gastric and intestinal contents from pigs were collected and
pooled within 2 hour of slaughter. The starved animals (12 hour
prior slaughter) were not prescribed any medicated feed prior to/at
the time of collection, gastric and intestinal juices were subject
to centrifugation and filtration, and the final suspensions were
checked for sterility on brain heart infusion agar (Oxoid Ltd.).
Preliminary tests confirmed the absence of indigenous gut
microflora within gastric contents; and intestinal contents were
screened for relevant background micorflora. Standard enzyme assays
were performed to validate the enzyme activity and action.
[0149] In Vivo Studies (Porcine)
[0150] Transit time of microencapsulates along the porcine GI tract
was investigated during an in vivo porcine study. Feeding studies
were compliant with European Union Council Directive 91/630/EEC
(outlines minimum standards for the protection of pigs) and
European Union Council Directive 98/58/EC (concerns the protection
of animals kept for farming purposes). Two weeks-post weaning, nine
male pigs (Large White.times.Landrace) were blocked by weight (mean
weight of 15.2.+-.0.45 kg) and housed individually in pens designed
to provide reasonable space for free movement and normal activity,
thereby assuring normal GI motility. All pens equipped with a
single feeder and nipple drinker were located in light-controlled
(0600 to 1730 h) rooms with temperatures maintained at 28-30 Degree
C. throughout the trial using a thermostatically controlled space
heater. Day -7 to day 0 represented the acclimatisation period,
during which animals were fed a non-medicated commercial diet (free
of antimicrobials, performance enhancers, and sweeteners) twice
daily at 0730 and 1530 h (350 g/serving) with ad libitum access to
fresh water. Pigs were randomly assigned to three groups (n=3), all
of which were fasted for 16 h prior to capsule administration
microencapsulates, using protein-free milk permeate (MP; Kerry
Ingredients, Co. Kerry, Ireland) as the delivery medium. Feeding
was staggered by 15 min and as a replacement for their morning
feed. Animal variation was kept to a minimum since 1) the
relationship between feeding and porcine gastric emptying is
influenced by many factors and 2) the rate of emptying can be
related to the metabolic requirement of the body. Previous marker
transit studies in pigs showed that the majority of ingested feed
would have transited to the small intestine within 2 h; however
sequential intestinal recovery of microencapsulates may surpass
these expectations due to the nature of the delivery system. Hence,
sampling was conducted 1 h (n=3), 2 h (n=3) and 3 h (n=3) after
administration of microencapsulates Upon ingestion of the capsules,
pigs were subsequently sacrificed by captive-bolt stunning followed
by exsanguination, in the same order as they were fed. Segments of
porcine stomach and intestine (mucosa, duodenum, jejunum, ileum,
colonic fluid & tissue) were analysed to verify the
absence/presence of micro encapsulates.
[0151] In Vivo Studies (Human)
[0152] A human study was designed whereby four participants were
intubated with a 145 cm nasoduodenal catheter. The catheter was
introduced into the stomach and the tip was positioned in the
intestine under radiological guidance and verification. Following
overnight fasting, participants were instructed to consume the
encapsulated prototype within 5 minutes (40 mL volume+approx. 120
mL water). After 180-220 min the nasoduodenal catheter was removed
and subjects were allowed to eat ad liteum. Position of the
catheter is shown on the Table 1.
[0153] Results
[0154] Encapsulation Efficiency
[0155] Encapsulation of native macronutrients i.e. casein, native
pea protein, sucrose were performed according to the aforemetioned
method using a concentric nozzle to create a defined core and outer
membrane for protection of the encapsulated GLP-1 stimulating
ingredient. FIG. 1 illustrates the homogenous mono-nuclear nature
of micro-encapsulate batches produced using the presented
invention.
[0156] Size Distribution & Drying Effects
[0157] According to light microscopy, micro-beads demonstrated
diameters of approx. 200 .mu.m with a narrow range size
distribution (.+-.1.2 .mu.m) as shown in FIG. 2. Laser
diffractometry was also incorporated and confirmed a D(v, 0.9)
values for micro-encapsulates, revealing a diameter of
201.7.+-.0.90 .mu.m and 183.42.+-.0.90 .mu.m, pre- and post-drying
respectively. FIG. 2B also visualises the effect of membrane
thinning post drying. The strength of micro-encapsulates
significantly increases as a function of drying.
[0158] Stomach Incubation & Strength of Micro-Encapsulates
[0159] Strength of micro-beads was analyzed as a function of
gastric incubation time in vivo (pH 1.2-1.4; 37.degree. C.). No
difference in micro-bead strength was reported for stomach
incubation and enzyme-activated stomach conditions did not
significantly (p, 0.001) weakened micro-bead strength. Tensile
strength of micro-encapsulated remained unchanged with no reported
leakage or loss of encapsulated casein, pea protein or sucrose.
After 180 min gastric incubation, encapsulated casein, pea protein
and sucrose microencapsulates maintained high tensile strength
52.03.+-.1.27 nN, 60.31.+-.0.27 nN and 58.23.+-.0.12 nN,
respectively. Hence, microencapsulates were capable of surviving
stomach transit to achieve intestinal delivery.
[0160] Light microscopy (FIG. 4) validated robust micro-bead
integrity after 180 min gastric incubation and did not reveal
contractile membranes on the micro-bead periphery after 180 min; a
penetrating effect only recognized in peptic-activated capsules.
Chromatography (SEC) confirmed the absence of peptides in gastric
media after 180 min, and microencapsulates expressed negligible DH;
hence, proteolysis was averted during enzyme-activated gastric
incubation. Table 1 and FIG. 3 show the identification and
characterization of in vivo enzymatic action.
TABLE-US-00001 TABLE 1 Identification and characterisation of
in-vivo enzymatic action .mu.mole Protein Enzyme Assay Tyrosine
Content Activity Substrate equivalent GI Section (n = 4) (n = 4) (n
= 4) (n = 4) Duodenal Time 10 min Trypsin Azo- 21.14 Contents 0.014
mg/mL casein (.+-.1.87) (.+-.0.00873) Time 55 min Chymotrypsin
319.75 0.0098 mg/mL (.+-.21.982) (.+-.0.00119) Proximal Time 35 min
Trypsin Azo- 2.38 jejunum/IIeum 2.23 mg/mL casein (.+-.0.0321)
(.+-.0.00981) Time 120 min Chymotrypsin 89.75 11.76 mg/mL
(.+-.11.027) (.+-.0.1382)
[0161] Intestinal Incubation
[0162] Micro-encapsulates were subsequently tested for intestinal
delivery during in vivo transit trials. FIG. 5 illustrates the
maintenance of micro-encapsulate integrity in the duodenum 35
minutes after oral ingestion of micro-encapsulates and degradation
was not evident.
[0163] Ileum Degradation
[0164] Micro-encapsulate degradation evolved according to
expectations during intestinal conditions (in vivo), since protein
matrices demonstrated reciprocal sensitivity to pH and enzymatic
proteolysis, an imperative pre-requisite for an ileal physiological
carrier medium. FIG. 6 illustrates the degradation of
microencapsulates as a function of ileum incubation time. As time
progressed, the capsulate membrane gradually degrades to release
the mononuclear core material.
[0165] Liberation of Core Material
[0166] The release of core, GLP-1 stimulating material is
identified using methods such as chromatography (FIG. 8), Bradford
assay, Surface Plasmon Resonance (FIG. 9) and High pH Anion
Exchange Chromatography with Pulsed Amperometric Detection
(HPAEC-PAD) to measure sucrose and protein.
[0167] Choice of Encapsulation Technology
[0168] FIG. 10 illustrate the novelty with regard to the
microencapsulates with a mononuclear core that can control core
release at the ileum. On the contrary, microbeads (FIG. 10B)
represent a weak delivery vehicle for native macronutrients, due to
the lack of segregation and compartmentalisation of the native
component within the encapsulation structure. FIG. 10A, however,
illustrates encapsulates with a defined mononuclear core to enable
protection of native macronutrients.
* * * * *