United States Patent 5,290,571
Bounous , et al. March 1, 1994
Biologically active whey protein concentrate
The present invention is concerned with a whey protein composition comprising a
suitable concentration of whey protein concentrate wherein the whey protein
concentrate contains proteins which are present in an essentially undenatured
state and wherein the biological activity of the whey protein concentrate is
dependent on the overall amino acid and small peptides pattern resulting from
the contribution of all its protein components and a method of producing said
whey protein composition. The invention also relates to several applications of
Inventors: Bounous; Gustavo (Montreal, CA), Gold; Phil (Westmount, CA),
Kongshavn; Patricia A. L. (St. Lambert, CA)
Assignee:Immunotec Research Corporation, Ltd. (Quebec, CA)
[*] Notice: The portion of the term of this patent subsequent to July 27,
2010 has been disclaimed.
Appl. No.: 07/417,246
Filed: October 4, 1989
Related U.S. Patent Documents
Application NumberFiling DatePatent NumberIssue Date
Current U.S. Class:424/535 ; 426/72; 514/2; 514/21; 514/251; 514/276;
514/885; 530/365; 530/833
Current International Class: A23L 1/305 (20060101); A61K 35/20 (20060101);
A61K 38/17 (20060101); A61K 035/20 ()
Field of Search: 514/2,21,251,276,885 530/365,833 424/535 426/72
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Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Witz; Jean C.
Attorney, Agent or Firm: White; John P.
Parent Case Text
This application is a continuation-in-part of U.S. application Ser. No. 289,971
filed Dec. 23, 1988, now abandoned, and a continuation of application U.S.
application Ser. No. 188,271, filed Apr. 28, 1988, now abandoned.
The embodiments of the invention in which an exclusive property of privilege is
claimed are defined as follows:
1. A whey protein composition comprising an undenatured whey protein concentrate
obtained from raw bovine, goat or sheep milk which contains substantially all
the heat labile whey protein present in the raw milk, the whey protein being
present in an amount sufficient to provide an amount of from about 18 to about
28 grams of whey protein per 100 grams of composition, Vitamin B.sub.1 in an
amount of at least 1.5 mg per 100 grams of composition and Vitamin B.sub.2 in an
amount of at least 1.5 mg per 100 grams of composition.
2. The composition as in claim 1, wherein the amount of Vitamin B.sub.1 is in
the range of from 1.5 mg to 2.0 mg per 100 g of composition.
3. The composition as in claim 1, wherein the amount of Vitamin B.sub.2 is in
the range of from 1.5 mg to 2.0 mg per 100 g of composition.
4. The composition as in claim 1, wherein the composition is free of lactose.
5. The composition as in claim 1 having immunoenhancing properties that are heat
labile, insensitive to pancreatic digestion and dependent upon the undenatured
6. A method of improving the immune response in mammals as measured by sheep red
blood cell injection comprising administering orally to the mammal an
undenatured whey protein concentrate obtained from bovine, goat or sheep milk
containing substantially all the heat labile whey protein present in the raw
milk, the whey protein being present in an amount sufficient to provide an
amount of from about 18 g to about 28 g of whey protein per 100 g of
composition, Vitamin B.sub.1 in an amount of at least 1.5 mg per 100 grams of
composition and Vitamin B.sub.2 in an amount of at least 1.5 mg per 100 grams of
composition, the Vitamin B.sub.1 and Vitamin B.sub.2 being administered in
amounts in excess of minimum daily requirements.
7. The method as in claim 6, wherein the amount of Vitamin B.sub.1 is an amount
of from 1.5 mg to 2.0 mg per 100 g of composition.
8. The method as in claim 6, wherein the amount of Vitamin B.sub.2 is an amount
of from 1.5 mg to 2.0 mg per 100 g of composition.
9. The method as in claim 6, wherein the minimum daily requirement of Vitamin
B.sub.1 is 0.5-0.6 mg per 1000 calorie.
10. The method as in claim 6, wherein the minimum daily requirement of Vitamin
B.sub.2 is 1.2-1.5 mg per day.
11. The method as in claim 6, wherein the minimum daily requirement of Vitamin
B.sub.1 is 0.5 milligrams per 1000 calorie and the minimum daily requirement for
Vitamin B.sub.2 is 0.5-0.6 milligrams per 1000 calorie or 1.2-1.5 milligrams per
12. A method of improving the immune response in mammals as measured by sheep
red blood cell injection comprising the step of administering to a mammal a
composition comprising Vitamin B.sub.2 in an amount in excess of minimum daily
requirements, and an undenatured whey protein concentrate containing
substantially all the heat labile whey protein present in the raw milk in an
amount sufficient substantially to satisfy the daily requirements of protein of
13. A method of improving the immune response in mammals as measured by sheep
red blood cell injection comprising the step of administering to the mammal an
undenatured whey protein concentrate containing substantially all the heat
labile whey protein present in the raw milk, said whey protein concentrate
having immunoenhancing properties that are heat labile, insensitive to
pancreatic digestion and dependent upon the undenatured state and being
administered in an amount substantially sufficient to satisfy requirements of
protein of said mammal.
BACKGROUND OF THE INVENTION
Throughout this application, various publications are referred to by Arabic
numerals within parenthesis. Full citations for these references may be found at
the end of the specification, immediately preceding the claims. The disclosures
of these publications are hereby incorporated by reference in order to more
fully describe the state of the art at the time of the invention described and
The present invention is based on the surprising discovery that undenatured whey
protein concentrate has an enhanced immunological effect. More specifically, the
invention relates to the effect of the oral administration of whey protein
concentrate in undenatured conformation on the immune response to sheep red
blood cells, host resistance to pneumococcal infections, development of
chemically induced colon carcinoma and tissue glutathione.
The present invention shows the correlation between the undenatured conformation
of whey protein concentrate (w.p.c.) and host immunoenhancement whereby chemical
indices of denaturation are given and the demonstration that the same crucial
role of molecular conformation (undenatured state) applies to glutathione GSH
promotion, which is the other major biological activity of w.p.c. Equally
important is the demonstration that another protein source such as egg white,
with the same high cysteine content as w.p.c. does not enhance GSH synthesis,
further demonstrating the specificity of w.p.c. with respect to the described
Whey and whey protein have been utilized from time immemorable for nutritional
purposes. In addition, whey was recommended in folk and ancient medicine for the
treatment of various diseases .sup.(1,2) and, in one instance, lifetime feeding
of hamsters with a whey protein diet has been shown to promote longevity with no
Dairy products are widely used as a good source of nutrition. In addition,
claims have been made to the effect that fermented whole milk (yogurt) is
beneficial in the management of some types of intestinal infections. Certain
dietary regimes based on ill defined natural or cultured dairy products are said
to be associated with long life expectancy in some regions of the U.S.S.R., for
Since time immemorial, serum lactis, which is latin for milk serum or whey, has
been administered to the sick for the treatment of numerous ailments. In 1603,
Baricelli reported on the therapeutic use of cow or goat milk serum sometimes
mixed with honey or herbs. The spectrum of illnesses treated with whey include
jaundice, infected lesions of skin, those of the genito-urinary tract with
purulent secretions, gonorrhea, epilepsy, quartan fever and other febrile states
of different origins. Indeed, the common denominator of most of these illnesses
appears to be a septic condition. Although physicians of both ancient times and
of the middle ages agreed that whey treatment should be carried out over a
period of several days, a difference of opinion appear to exist concerning the
daily amount prescribed. Thus, Galen, Hippocrates and Dioscoride insisted on a
minimum daily amount of two 12 ounce latin libras, and up to five libras a day
according to gastric tolerance. This would represent between one to two liters
of whey a day. Baricelli on the other hand, reflecting the trend of his time,
limited the amount prescribed to one libra a day, given in fractionated doses on
an empty stomach.
Since then, numerous articles published in Europe through the 17th, 18th and
19th centuries have advocated the therapeutic use of whey. In an Italian
textbook published in the middle of the 19th century.sup.(15), at the dawn of
scientific medicine, an interesting distinction is made between whole milk and
milk serum. Milk is recommended firstly as a nutrient especially in patients
with strictures of the gastro intestinal track. In this respect the author
emphasises that the benefits of the then popular "milk therapy" of cachexia and
tuberculosis are due only to the nutritional property of milk. Secondly, the
milk was prescribed in the treatment of poisoning because milk components would
presumably neutralize ingested toxic material. Thirdly, milk therapy was
suggested for the alleged capacity of this fluid to coat and soothe ulcers of
the gastrointestinal track. Milk serum, on the other hand, was advocated in the
treatment of pneumonitis, acute inflammatory diseases of the intestines and
urogenital track, in spite of its recognized lower nutritional quality. Finally,
the author emphasized the ineffectiveness of whey in the treatment of disorders
of the nervous system.
The prime difference between whey (serum lactis) and whole milk is the near
absence in the former of the caseins, the casein-bound calcium and phosphate,
most of the fat and the fat soluble vitamins. The actual concentration in whey
of "whey proteins" is usually similar to that in milk. Hence quantitative
differences between whey and milk could not be construed to represent a key
factor in the alleged therapeutic effect of whey treatment because, if any, they
imply the lack, in whey, of some important nutrients. Some previously collected
data .sup.(5-10) of the present inventors provide a scientific background to the
presumed benefit of intensive treatment with "serum lactis". The importance of
the characteristic amino acid and peptide profile of whey protein concentrate in
the immune enhancing effect of the whey protein concentrate (WPC) has been
shown. The caseins represent 80% of the total protein content of cows milk while
WPC is only 20%. Hence, it is conceivable that it is the separation of WPC from
the caseins in whey which represents the crucial qualitative change, since this
would render the amino acid profile and associated small peptides patterns of
whey proteins unaltered by that of the caseins, once the digestive process has
released free amino acids from all ingested proteins.
The search for the possible mechanism of immunoenhancement by whey protein
feeding has revealed to us the provocative possibility that whey protein
concentrate may contribute to a broader biological effect of a protective nature
involving susceptibility to cancer and general detoxification of environmental
agents. All these conditions appear to be somehow related to changes in
glutathione which is a ubiquitous element exerting a protective effect against
superoxide radicals and other toxic agents.
Glutathione is a tripeptide thiol (L-gamma-glutamyl-L-cysteinylglycine) with a
broad range of vital functions that include detoxification of xenobiotics and
protection of cells against oxygen intermediates and free radicals, by-products
of oxygen-requiring metabolism.sup.(42-45). Modulation of intracellular
glutathione affects the proliferative immune response of lymphocytes which may
be inhibited by oxidative injury.sup.(46-48). Glutathione protect the cells
against radiation and alkylating agents.sup.(49-50). Age-related or
experimentally induced glutathione depletion in the lens is associated with
cataract formation.sup.(51,52). Oxidative DNA damage is rapidly and effectively
repaired. The human body is continually repairing oxidized DNA. A small fraction
of unrepaired lesions, however, could cause permanent changes in DNA and might
be a major contributor to old age diseases and cancer.sup.(53). Indeed, several
age associated diseases may be induced by free radicals.sup.(54). It appears
that whereas data on age-related changes in tissue vitamin E and other
antioxidants are, at best, contradictory.sup.(55), the tissue glutathione levels
are more consistently reported to decline with old age in laboratory
animals.sup.(56,57) and man.sup.(58-61).
For these reasons there has been interest in the factors that influence
intracellular glutathione synthesis and especially in ways of increasing
cellular levels of glutathione.
Glutathione is composed of three amino acids: glutamic acid, glycine and
cysteine. Availability of cysteine is a limiting factor in the synthesis of
glutathione.sup.(62,63). Cysteine is derived from dietary protein and by
trans-sulfuration from methionine in the liver. Various methods have been tried
in order to increase cellular levels of glutathione. Administration of free
cysteine is not an ideal method because this amino acid is rapidly oxidized,
toxio.sup.(64) and may actually cause glutathione depletion.sup.(65). Similar
problems have been encountered with i.p. injection of N-acetyl cysteine to rats,
although oral administration of this compound appeared to prevent
paracetamol-induced glutathione depletion.sup.(65). Administration of compounds
that are transported and converted intracellularly into cysteine, such as
L-2-oxothiazolidine-4-carboxylate are useful in increasing cellular
glutathione(66) acting as an intracellular delivery system for cysteine. Hepatic
glutathione doubled four hours after injection, returned to normal 8 hours later
but was below normal after 16 hours.sup.(66). Another approach for increasing
tissue glutathione levels was found in s.c. injection of gamma
glutamylcyst(e)ine in mice: glutathione increased in the kidney by about 55%,
40-60 minutes after injection, returning to near control values 2 hours
later.sup.(67). The administered compound is transported intact and serves as a
substrate for glutathione synthetase. It was also reported that about 2 hours
after i.p. administration of gamma-glutamyl cysteinyl-glycyl monomethyl (or
monoethyl) ester to mice, the liver and kidney glutathione levels were doubled,
with return to normal values after 8 hours.sup.(68). Similar increases in
glutathione tissue levels were attained by Meister by administering an alkyl
monoester of glutathione (U.S. Pat. No. 4,784,685, Nov. 15th, 1988), to mice.
Such esters are transported into tissue cells, and are deesterified within the
cells, thus leading to increased cellular levels of glutathione. The kinetics of
tissue glutathione increments attained with this method are similar to those
described following i.p. injection of methyl or ethyl esters of
glutathione.sup.(68). The effectiveness of these methods has been clearly
demonstrated in acute experiments.sup.(68,69). (U.S. Pat. No. 4,784,685); in
mice treated with L-2-oxothiazolidine-4-carboxylate the expected drop in
glutathione tissue level subsequent to acetaminophen injection, was replaced by
an actual increase in tissue glutathione values and survival. Other methods to
increase tissue glutathione levels are based on the "overshoot" of glutathione
concentration, following depletion by diethylmaleate or BSO. These studies were
done in vitro on murine cell lines.sup.(70). Also pre-exposure of rats to
hypoxia was found to increase lung glutathione.sup.(71).
The administration of glutathione itself is of little consequence on tissue
glutathione levels, because it apparently cannot be transported intact across
the cell membrane.sup.(68).
Some of said methods of increasing intracellular levels of glutathione
concentration are either toxic or dangerous owing to the risks related to the
initial phase of glutathione depletion. The methods involving the use of
gamma-glutamyloyst(e)ine, athiazolidine or glutathione esters (U.S. Pat. No.
4,784,685) offer an interesting possibility for short term intervention.
However, their long term effectiveness in producing sustained elevation of
cellular glutathione has not been shown, nor has the possible toxicity of their
long term use been disproved. Indeed, glutathione and glutathione disulfide were
found to be positive in the most commonly used short term tests for
carcinogenicity and mutagenicity. Relevant to our invention are recent data
indicating specifically that a lack of the GSH precursor, cysteine, rather than
a decrease in biosynthetic enzyme activities is responsible for the deficiency
of GSH noted in aging animals.sup.(73). Similarly, the fall in cytosolic GSH in
the liver of chronic ethanol fed rats does not appear to be caused by a
limitation in the capacity of gamma-glutamylcysteine synthetase
Our studies have shown that the observed enhancement of the immune response is
associated with greater production of splenic glutathione in immunized mice fed
whey protein concentrate in comparison to mice fed casein, cysteine enriched
casein or egg white protein in similar dietary concentration. The efficiency of
dietary cysteine in inducing supernormal glutathione levels is greater when it
is delivered in the whey protein than as free cysteine or within the egg white
protein. Glutathione was found at higher levels in the heart and liver of whey
protein fed old mice in comparison to mice fed the corresponding casein diet,
the egg white protein or Purina Mouse Chow.
The use of mice as biological test subjects in research is commonly practiced
world-wide. It is generally accepted that if a biological phenomenon occurs in
two different mammalian species, it can be applied to other mammalian species
including man. Our studies carried out in several unrelated strains of mice of
both sexes therefore are of great benefit in gauging the biological activity of
whey protein concentrate which appears to be independent of specific genetic or
hormonal influences. Perhaps most importantly human milk has by far the highest
whey protein/casein ratio than any other mammal. (See in this regard
"Evolutionary Traits in Human Milk Proteins", Bounous et al, Medical Hypotheses
(1988) 27, 133-140). Presumably nature has prepared humans, through the only
obligatory form of nutrition, to handle undenatured whey proteins for their best
metabolic advantage. In fact, one would anticipate that the favourable
biological activity of whey protein concentrate in rodents might be more
pronounced in the human host.
(a) Whey Protein:
Whey proteins are the group of milk proteins that remain soluble in "milk serum"
or whey after precipitation of caseins at pH 4.6 and 20.degree. C. The major
whey proteins in cow's milk are beta-lactoglobulin (.beta. L), alpha-lactalbumin
(.alpha. L), immunoglobulin and serum albumin (SA) in order of decreasing
The product of industrial separation of this protein mixture from whey is called
"whey protein concentrate" (WPC) or isolate The WPC used in most of our
experiments is from bovine milk (Lacprodan-80 from "Danmark Protein A.S."). Use
in its undenatured state is indicated as U- Lacp, and in its denatured state is
indicated as D-Lacp. Lactalbumin (L) is the term traditionally used to define
(c) SRBC=Sheep red blood cells;
(d) PFC=Plaque forming cells (spleen): enumeration of PFC in spleen is used to
assess the humoral immune response to SRBC injection;
(e) GSH=Glutathione (L-gamma-glutamyl-L-cysteinylglycine);
(g) The defined formula diets tested varied only in the type of protein.
(h) Whey of bovine milk contains approximately 6 g per liter protein, most of
the lactose, mineral and water soluble vitamins.
A suitable source of whey protein concentrate is the material known by the trade
mark PROMOD, which is a protein supplement provided in powder form by Ross
Laboratories, a Division of Abbott Laboratories, U.S.A. This is a concentrated
source of high quality protein which is useful for providing extra protein to
persons having increased protein needs, or those who are unable to meet their
protein needs with their normal diet. It contains whey protein concentrate and
soy lecithin. It has the following nutrients:
______________________________________ PER 5 G PROTEIN (ONE NUTRIENTS SCOOP)
______________________________________ Protein 5.0 g Fat Does not exceed 0.60 g
Carbohydrate Does not exceed 0.67 g Water Does not exceed 0.60 g Calcium Does
not exceed 23 mg (1.15 mEq) Sodium Does not exceed 13 mg (0.57 mEq) Potassium
Does not exceed 65 mg (1.66 mEq) Phosphorus Does not exceed 22 mg Calories 28
It has the following typical amino acid composition per 100 g protein. 100 g
PROMOD protein yields approximately 105 g of amino acids.
TYPICAL AMINO ACID COMPOSITION per 100 g Protein
Essential Amino Acids:
Histidine, 1.9 g;
Isoleucine, 6.2 g;
Leucine, 10.8 g;
Lysine, 9.3 g;
Methionine, 2.2 g;
Phenylalanine, 3.6 g;
Threonine, 7.3 g;
Tryptophan, 1.9 g;
Valine, 6.0 g.
Non-Essential Amino Acids:
Alanine, 5.3 g;
Arginine, 2.6 g;
Aspartic Acid, 11.2 g;
Cysteine, 2.6 g;
Glutamic Acid, 18.2 g;
Glycine, 2.1 g;
Proline, 6.5 g;
Serine, 5.6 g;
Tyrosine, 3.4 g
Diets used in our studies
Diets are prepared in the following way: 20 g of selected pure protein, 56 g of
product 80056 protein free diet powder containing corn syrup, corn oil, tapioca
starch, vitamins and minerals (Mead-Johnson Co. Inc., U.S.A.), 18 g cornstarch,
2 g wheat bran; 0.05 g Nutramigen vit-iron premix (Bristol-Myers, Ontario,
Canada), 2.65 g KCl; 0.84 g NaCl. The carbohydrate and lipid components of our
formula diets were the same. The only variable in the various purified diets was
the type of protein (20 g protein/100 g diet). At this concentration in diet all
the different proteins tested provided the daily requirements of essential amino
acids for the growing mouse.sup.(12). Vitamins and minerals were the same in
each set of experiments and were added in the amount necessary to provide daily
requirements for the growing mouse.sup.(13,14). Table 1, below, indicates the
variation in suggested vitamin requirements for mouse diets and their contents
in some of our formulations. Therefore all the formula diets used in our
experiments were designed to provide adequate nutrition as demonstrated by
normal body growth, serum protein and by the absence of hair loss, dermatitis,
cataract, ataxia, fatty liver etc. The latter symptoms were of course present in
very old mice and were related to the aging process.
VITAMIN AND MINERAL CONTENT OF TEST DIETS (amount/100 g diet) JACKSON (1) (range
of amount recommended in TEST Jackson labora- DIETS tories diets)
VITAMINS: AIN 76 (2) Vitamin A, IU 1295.sup. 1800 24-550 400 Vitamin D, IU
260.sup. 360 14-506 100 Vitamin E, IU 11.6.sup. 18 1-2.7 5.0 Vitamin K, mg
0.06.sup. 0.09 -- 0.005 Thiamine(Vitamin B1), mg 0.34.sup. 0.63 0.22-0.99 0.60
Riboflavin(Vitamin B2), mg 0.38.sup. 0.69 0.24-1.1 0.60 Vitamin B6, mg 0.26.sup.
0.36 0.1-0.55 0.70 Vitamin B12, mg 0.0012.sup. 0.054 0.0039-0.0055 0.001 Niacin,
mg 5.1.sup. 9.2 2.6-14.3 3.0 Folic acid, mg 0.063.sup. 0.12 .05-.27 0.2
Pantothenic acid, mg 1.93.sup. 3.38 1-5.5 1.6 Biotin, mg 0.031.sup. 0.058
0.019-0.165 0.02 Vitamin C, mg 53.3.sup. 65 -- -- Choline, mg 44.sup. 76 49-145
100 Inositol, mg 19.8.sup. 19.8 -- -- MINERALS: AIN 76 Calcium, mg 430 # 520
Phosphorus, mg 260 # 400 Magnesium, mg 63.2 # 50 Iron, mg 7.9.sup. 3.5 Zinc, mg
3.57 # 3.0 Copper, mg 0.47 # 0.60 Iodine, mg 0.023.sup. 0.02 Sodium, mg 232.sup.
100 Potassium, mg 997.sup. 360
after minerals analysis (1) Hoag W. G., Dickie M. M. "Nutrition: in Green E. L.
(Ed) Biology of the laboratory mouse McGrawHill NY 1966 pp 39-43. Jackson was
our supplier. (2) The mouse in biomedical research, vol III Eds Foster H. L.,
Seall J. D., Fox J. B., Academic press 1983, NY pp 57-58
Immunization for Plaque Assays
The diet-fed mice were immunized by an intravenous injection of 5.times.10.sup.6
washed sheep red blood cells obtained weekly from Institut Armand-Frappier,
Laval des Rapides, Quebec, Canada.
Plaque Forming Cell (PFC) Assay
The method used for assaying IgM plaque forming cells was essentially the one
described by Cunningham and Szenberg.sup.(101), with certain minor
modifications. Spleen cell suspensions were prepared by gently tamping the
spleen through a 50-mesh stainless steel screen, and collecting the cells in
balanced salt solution (BSS) supplemented with 10% heat-inactivated calf serum
(Grand Island Biological Company, Montreal, Quebec, Canada). The spleen cells
were washed and made up to 15 ml with BSS. Sheep red blood cells were washed
twice and made up to a 20% concentration. Guinea pig serum (Grand Island
Biological Company, Montreal, Quebec, Canada) as a source of complement was
diluted 1/15 with BSS All stock solutions were kept on ice water until used The
test consisted of mixing 0.05 ml of spleen cells, 0.15 ml of sheep red blood
cells and 0.75 ml of the complement solution in a test tube at 37.degree. C. The
whole mixture was immediately withdrawn and put into slide chambers, sealed with
warm paraffin wax, and incubated at 37.degree. C. for 45 to 60 min. The number
of plaque forming cells was counted and their total number per spleen estimated
by multiplying the number of plaque forming cells in each sample (0.05 ml spleen
cells) by 300. The values are expressed per total organ rather than per 10.sup.6
spleen cells, since this appears to reflect more accurately the functional
status of the spleen per se.
Mice were assayed for the plaque forming cell response to sheep red blood cells
normally on the fifth day after immunization when the response was shown to peak
or, in the kinetic study, on days 3, 4, 5 and 6 post-immunization.
The mean plaque forming cell values were compared among the dietary groups using
either Student's test, when two groups were being compared, or the of variances
(ANOVA) for more than two groups. Because of the heterogeneity of variances
among groups, the adjustment given by Brown and Forsythe was used
Spleen Glutathione Content
Ninety milligrams of mouse spleen were weighed using a Mettler PM-300 balance
and samples varied from 90 mg by less than 5 mg (5%). The samples were then
homogenized in 5-sulfosalicylic acid (5% w/v) Homogenates were centrifuged for 5
min in a microfuge at 10,000.times.g. The assay was carried out using the
supernatants on the same day according to the methods of Anderson.sup.(72).
Values are expressed as .mu.mol per g/wet tissue.
Buthionine Sulfoximine Experiments
In some experiments, following three weeks of whey protein feeding and one day
prior to immunization with sheep red blood cells, mice were injected i.p. with
450 mg/kg of buthionine sulfoximine (BSO) (S-[n-butyl] homocysteine
sulfoximine), a specific inhibitor of gamma-glutamyloysteine synthetase At the
same time 20 mM of BSO was added to the drinking water
Description of the Prior Art
An imposing number of publications deal with the association of nutritional
deficiencies, including protein energy malnutrition, and infection in the human
and animal host.sup.(16). For example, mice fed with insufficient amounts of
protein, exhibit less growth or
even weight loss and increase the susceptibility to infection by Staphylococcus
French Patent Publication 2,296,428 relates to the dietetic and therapeutic use
of lactoserum protein compositions for the treatment for the treatment of
malnutrition and diarrhea of, diarrhea, in infants and adults. This reference,
however, does not establish the biological activity (immunoenhancement) of the
whey protein diet unrelated to its nutritional quality. The improvement shown by
the subjects treated with these whey protein compositions appeared to result
from the increased nourishment from the protein compositions particularly in
studies relating to malnourished infants.
British Patent Specification 1,495,940 relates to an anti-cancer active whey
fraction. A whey fraction having the molecular weight of from 6000 to 20,000 is
utilized (I.P. injection) in the treatment of cancer and leukemia. The
particular mechanism of the effective fractions of whey against cancer has not
been shown. This includes irradiated whey.
PCT/U.S 87/00036 (W087/04050) relates to an immunologically active whey fraction
and recovery process. Disease resistance and growth rates in animals including
humans is enhanced by oral administration of the whey fraction. This reference
discloses a method for concentrating from whey, a product containing
immunologically active (antigen binding) immunoglobulin [Ig]]that, when fed to
new born calves at a very high concentration of 7% of total solids, provides a
substantial transfer of natural passive immunity as evidence by blood Ig levels
and increased resistance to infections. This reference does not appreciate nor
prove a cause-effect relationship between passive immunity and the development
of active immunity.
Dietary protein deficiency has been found to reduce the incidence of
spontaneous.sup.(80) or transplanted.sup.(80,81) tumors. Most of the definitive
studies concerning protein and cancer have utilized protein underfeeding.
Although some evidence indicates that the higher the protein intake, the greater
the tumor incidence.sup.(82,83), data concerning the effect of raising protein
intake on carcinogenesis and tumor development are not definitive.sup.(84).
Studies have focused on the quantity of protein and its amino acid supply rather
than its source.sup.(84). Only a few data are available on the effect of protein
type in nutritionally adequate and similar diets on the development of tumors.
Jacquet et al.sup.(85) reported that feeding milk retarded on the average by a
factor of 0.4 tumor growth in rats implanted with epithelioma T8. This is
consistent with some epidemiological studies showing that consumption of milk or
dairy products may reduce the risk of cancer.sup.(86,87). In mice inoculated
with Ehrlich ascites tumor cells, feeding yogurt reduced the number of tumor
cells by a factor of 0.2-0.28.sup.(88). It was also reported that mice fed a
milk protein formula diet, exhibited inhibition of tumor volume by a factor of
0.2 to 0.7, following s.c. injection of DMH-induced colon tumor cells in
comparison to mice fed other types of protein.sup.(89). A comparable degree of
tumor inhibition was noted in milk protein fed mice injected s.c. with herpes
virus transformed cells.sup.(90). However, in another article, submitted several
months later, the same group of authors reported results ". . . different from
those expected in light of our previous findings". Milk protein feeding did not
inhibit tumor growth in the same strain of mice injected with DMH.sup.(91). The
previously reported anti-cancer biological property of dietary milk proteins was
absent, in spite of the preservation of their good nutritional quality.sup.(91).
The authors provide no explanation for the apparent contradiction.
DMH-induced colon tumors appear to be similar to those found in humans as far as
type of lesions and chemotherapeutic response characteristics are
In light of our findings on the lability cf the biological property of whey
protein concentrate, it is conceivable that the whey protein fraction of the
milk protein mixture, used in the later experiments, was partially or totally
denatured. Various types of cheeses and yogurt were recently found to suppress
the growth of several experimental tumors in mice in proportion to the duration
of feeding. The tumor size was reduced by a factor of 0.17 to 0.70 depending on
the type of tumor.sup.(92). In spite of variations in the type of tumor and in
the control diets used in all these studies it is apparent that the level of
tumor inhibition reported with dairy product feeding is comparable to that which
we obtained with a formula diet containing casein as protein source.
These previous uses of whey protein in various forms and the treatment of
various diseases do not appreciate the enhancement of the immunological effect
of the whey protein concentrate when in undenatured conformation and in many
cases improvement of the patient is a result of the nutritional benefit of whey.
Further, this biological activity is dependent on the combined effect of all the
protein components of the whey protein concentrate and cannot be obtained using
whey protein fractions. Should a presumed biologically active material form a
part of a particular protein component, it is apparent that its effective
bioavailability is strongly influenced by the co-existence of the other protein
components of WPC through digestive-absorptive process. The activity is not
specifically related to the nutritional efficiency of the whey protein
concentrate. Denaturation abolishes the described biological activity without
affecting the nutritional quality of the whey protein concentrate.
Accordingly, the principle object of the present invention is to provide a
method for improving the humoral immune response in mammals by the oral
administration of undenatured whey protein concentrate.
A further object of the invention is to provide a method for increasing the
concentration level of glutathione in the organs of mammals through the use of
undenatured whey protein concentrate through its oral administration.
A further object of the invention is a process for enhancing the resistance to
bacterial infection, particularly pneumococcal infection, enhanced resistance to
slow growing carcinoma such as colon carcinoma through the utilization of whey
protein concentrate in an undenatured state.
SUMMARY OF THE INVENTION
The present invention relates to a biologically active whey protein composition
comprising a suitable concentration of whey protein concentrate wherein the whey
protein concentrate contains the proteins which are present in an undenatured
state and wherein the biological activity of the undenatured whey protein
concentrate is based on the overall amino acid and associated small peptides
patterns resulting from the contribution of all its protein components.
The invention further relates to the inclusion of Vitamin B.sub.1 and B.sub.2 in
the biologically active whey protein at above the minimum recommended daily
requirements resulting in a composition having a further increase in biological
The invention still further relates to a method for producing a whey protein
concentrate composition comprising the steps of: a) immediately after milking,
cooling the milk to a temperature in the range of 2.degree. C. to 10.degree. C.
and removing impurities, b) after another cleaning of the milk, precipitation of
the curd by reducing the pH to about 4.6 with lactic acid initially at
20.degree. C. c) addition of rennet and raising the temperature to about
30.degree. C. for 20 minutes to promote expulsion of whey from the curd and
followed by agitation to resolve at low speed, d) thermal treatment of the
pasteurization type of the remaining product in the vat and agitation at high
speed for cheese production, e) irradiation and separation of the whey, and f)
ultrafiltration of whey using a membrane having a molecular weight cut off of
substantially 10,000 or less, said method being characterized in that the
fraction of whey to be used for subsequent production of whey protein
concentrate is not heated and the material from which it is derived is slowly
agitated to minimize protein denaturation, said ultrafiltration being carried
out in a production line comprising up to 20 frame-type modules holding a large
number of membranes achieving a final undenatured protein concentrate in dry
matter, wherein said ultrafiltration is carried out at a temperature in the
range of 4.degree. C. to 20.degree. C.
The invention still further relates to a method for improving the humoral immune
response in mammals, the method comprising the steps of administering orally to
a mammal, a therapeutically or prophylactically effective amount of undenatured
whey protein concentrate having biological activity wherein the biological
activity is based on the overall amino acid and associated small peptides
pattern resulting from the contribution of all its protein components.
Enhancement of the humoral immune response results in enhanced resistance to
bacterial infection, particularly pneumococcal infection; enhance resistance to
colon carcinoma, particularly chemically induced colon carcinoma; delayed or
decreased mortality or a combination of the above.
The invention yet further relates to increasing the rate of synthesis, rate of
replenishment and concentration levels of glutathione in animal organs through
the step of administering to an animal a therapeutically or a prophylactically
effective amount of undenatured whey protein concentrate having biological
activity, the biological activity being based on the overall amino acid and
associated small peptides pattern resulting from the contribution of all its
The invention also relates to various food supplements, drugs and the like
containing the biologically active whey protein composition alone or in
combination with Vitamins B.sub.1 and B.sub.2.
The above, and other objects, features and advantages of the present invention,
will become apparent from the following detailed description of preferred
embodiments to be read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings which form part of this specification:
FIG. 1 shows plaque forming cells/spleen (PFC) on the day showing peak
production of PFC following immunization with 10.sup.6 SRBC. Effect of two weeks
of dietary treatment with 20 g/100 g diet of either lactalbumin (L) i.e. whey
protein concentrate, casein (C), Spirulina maxima protein (Sp), soy protein (S),
wheat protein (W), Scenedesmus protein (So), corn (Co) protein, egg white
protein (E), beef protein (B), fish protein (F), Purina Mouse Chow (P), or 20
g/100 g diet of a mixture containing 50% L and 50% S (L/S), or 80% L and 20% C,
or 20% L and 80% C (L/C). Each value represents the mean.+-.SD.
FIG. 2 shows plaque forming cells/spleen (PFC) on the day showing peak
production of PFC following immunization with 10.sup.6 SRBC. Effect of 3 weeks
of dietary treatment with 20 g/100 g diet of either whey protein concentrate
(WPC), casein (C), whey protein concentrate hydrolysate, casein hydrolysate,
beta-lactoglobulin (.beta.L), alpha-lactalbumin (.alpha. L), gamma-globulin
(.gamma. G) or bovine serum albumin (SA). Each value represents the mean.+-.SD.
FIG. 3 and related Table 3 and 4 illustrate the effect of various sources of
whey protein concentrate and casein (20 g/100 g diet) on spleen PFC response to
5.times.10.sup.6 SRBC in mice.
FIG. 4 and related Table 5 illustrates the effect of heat denaturation on the
immunoenhancing property of whey protein concentrate.
FIG. 5 illustrates plaque forming cells/spleen (PFC) on the day (day 5) showing
peak production of PFC following immunization of C3H/HeN mice with
FIG. 6 illustrates spleen glutathione as percent of values of unimmunized
C3H/HeN male mice fed with the corresponding diet for three weeks.
FIG. 7a illustrates plaque forming cells/spleen (PFC) on day 5, and FIG. 7b
shows spleen glutathione levels on day 4, following immunization with 10.sup.6
sheep red blood cells (SRBC).
FIG. 8 illustrates the liver glutathione content in male mice C57BL/6NIA fed
undenatured whey protein (U-Lacp), denatured whey protein (D-Lacp), casein, egg
white protein or purina diet-fed counterparts at age 10 weeks, 17, 20 and 21
FIG. 9 illustrates the heart glutathione content of male mice C57BL/6NIA fed
undenatured whey protein (U-Lacp), denatured whey protein (D-Lacp), casein, egg
white protein or purina diet-fed counterparts at age 10 weeks, 17, 20 and 21
FIG. 10 illustrates the survival curves of 2 month old male C57/BL/6NIA mice fed
casein, Purina Mouse Chow and whey protein.
FIG. 11 illustrates the effect of 26 days dietary treatment on PFC response to
DETAILED DESCRIPTION OF THE INVENTION
An assessment has been made of the effect on the immune response Of different
types of proteins in nutritionally adequate and similar diets. Mice fed formula
diets containing 20% or 28% whey protein pancreatic hydrolysate (LAD, Nestle)
were found to produce more plaque forming cells to sheep red blood cells than
mice fed Purina mouse chow containing about 22% protein from various sources and
of similar nutritional efficiency. The immunoenhancing effect of LAD was maximal
at 20% concentration.sup.(5). A 20 g net protein/100 g diet provides a good
method to assess the effect of protein type on the immune system. At this level
most protein supplies the minimum daily requirement of all indispensible amino
acids for the growing mouse.sup.(12-14) and this is important because the amino
acid adequacy is not the variable under investigation.
In subsequent studies, a comparison was made regarding the effect of dietary
whey protein concentrate (WPC) to that of other purified proteins in formula
diets of similar nutritional efficiency. The effect of graded amounts of dietary
WPC, casein (C), soy (S), wheat (W), protein and Purina rodent chow (stock diet)
on the immune responsiveness of C3H/HeN mice has been investigated by measuring
the specific humoral immune response to sheep red blood cells (SRBC), and horse
red blood cells (HRBC). The nutritional efficiency of these diets was normal and
similar. The immune response of mice fed the WPC diets, was found to be almost
five times higher than that of mice fed the corresponding C diets. The humoral
immune response of mice fed C, S, and W diets was substantially lower than that
of mice fed stock diet, whereas that of mice fed L (WPC) diet was higher. The
above-described immune effect of all tested proteins was obtained at 20 g/100 g
concentration with no further increments with 30- and 40 g/100 g protein in the
Because the whey protein concentrate was tested in comparison to a limited
number of proteins, we could not ascertain at that time whether the enhancement
of the humoral immune response observed in five (5) unrelated strains of mice
fed a whey protein diet, was due to a real immunoenhancement, in absolute terms,
by whey protein feeding or immuno-depression by the other food proteins tested.
Indeed, it can now be stated that these few purified food proteins (casein, soy
and wheat) used as "control" for the whey protein mixture were immunosuppressive
when compared to all of the other purified food proteins subsequently tested,
though nutritionally adequate and similar at 20% concentration in diet.
In fact, subsequent testing of whey protein against most commercially available
purified food proteins (casein, soy, wheat, corn, egg white, beef, fish protein,
gamma globulin, beta-lactoglobulin, alpha-lactalbumin, serum albumin, spirulina
maxima or scenedesmus algae protein) established that indeed mice fed whey
protein concentrate exhibit the highest immune response to foreign antigen
(SRBC).sup.(31) (FIG. 1). These proteins are nutritionally similar and adequate
at the 20 g/100 g diet concentration (Table 2, below).
As indicated in FIG. 1, mice fed the lactalbumin (w.p.c.) diet for 2 weeks
exhibit a plaque forming cell response to sheep red blood cells which is higher
than that of mice fed any other protein type or Purina mouse chow. The mean
number of plaque forming cells per spleen 5 days after i.v. injection with
5.times.10.sup.6 sheep red blood cells; in the lactalbumin diet-fed mice was
487%, 494%, 736%, 927%, 309%, 284%, 230%, 214%, and 177% of that noted in
casein, Spirulina, soy protein, wheat protein, Scenedesmus, corn protein, egg
albumin, beef or fish protein diet-fed mice respectively, and 168% of that of
Purina-fed mice. These differences are all statistically significant (P=0.004).
The number of plaque forming cells per spleen in Purina-fed mice was 170% of
that in corn protein diet-fed mice (P=0.0005) and the value of the latter group
was 171% of that noted in casein-fed mice (P=0.0005). No significant difference
was observed between fish protein diet-fed, beef protein diet-fed and Purina-fed
The addition of lactalbumin (w.p.c.) to either soy protein or casein produced a
significant increment in the humoral immune response of the host. In a 50:50
mixture with soy protein, lactalbumin induced a 4-fold increment in the immune
response in comparison to a purely soy protein diet. In an 80: 20 mixture with
casein, lactalbumin induced a 3-fold increment and, in a 20:80 mixture with this
protein, a 2-fold increase in the immune response was seen in comparison to a
purely casein diet. It was found that mice fed a lactalbumin diet for at least 2
weeks exhibit a sustained enhancement of the humoral immune response to sheep
red blood cells in comparison to mice fed most of the commercially available
edible animal or plant proteins in formula diets of comparable nutritional
efficiency. This effect persists as long as dietary treatment is continued (up
to 2 months has been tested). It is clear that despite great differences in
immune response to SRBC, no difference is seen in food consumption, final
weight, and serum proteins among mice fed the various purified proteins at 20
g/100 g diet concentration (see Table 2, below).
Thus, it can now be concluded that the newly discovered immune enhancing
biological activity of whey protein concentrate is not related to the already
known nutritional quality of this protein which is primarily based on
digestibility and amino acid content. In fact, the nutritional property of whey
protein concentrate at 20 g protein per 100 g diet concentration as used in
experimentation is similar to that of the other proteins tested.
Effect of 19 days dietary regimen on food consumption, body growth, total serum
protein and edevelopment of spleen.sup.h Avg Consumption Initial Weight Final
Weight Serum Protein Average Spleen Protein type (g/mouse/day .+-. SEM).sup.a
(g).sup.b (% initial wt.).sup.c (g/100 ml).sup.d Wt. (mg).sup.3 cells 10.sup.6
Lactalbumin.sup.j 2.8 .+-. 0.1 22.6 .+-. 0.6 118.0 .+-. 3.2 5.8 .+-. 0.2 117
.+-. 2.1 194 .+-. 4.0 Casein 2.9 .+-. 0.2 23.0 .+-. 0.8 117.8 .+-. 4.6 6.1 .+-.
0.3 113 .+-. 3.6 150 .+-. 4.1 Spirulina maxima 2.9 .+-. 0.3 19.8 .+-. 0.9 121.0
.+-. 1.8 5.4 .+-. 0.5 104 .+-. 3.4 138 .+-. 6.0 protein Soy protein 3.1 .+-. 0.2
21.2 .+-. 0.3 114.1 .+-. 1.3 6.0 .+-. 0.4 107 .+-. 3.8 144 .+-. 4.3 Wheat
protein 2.9 .+-. 0.2 20.0 .+-. 0.3 115.0 .+-. 2.2 5.9 .+-. 0.3 109 .+-. 2.6 139
.+-. 8.0 Scenedesmus 3.1 .+-. 0.4 23.0 .+-. 0.3 113.0 .+-. 3.0 6.1 .+-. 0.1 107
.+-. 4.0 152 .+-. 10.0 protein Corn protein 3.1 .+-. 0.2 22.8 .+-. 1.1 115.5
.+-. 5.4 5.6 .+-. 0.2 118 .+-. 3.2 160 .+-. 2.0 Egg albumin 3.0 .+-. 0.1 20.7
.+-. 0.6 116.0 .+-. 2.9 5.8 .+-. 0.3 114 .+-. 3.0 157 .+-. 6.0 Fish protein 2.8
.+-. 0.4 20.9 .+-. 0.3 117.1 .+-. 1.3 5.5 .+-. 0.1 105 .+-. 2.4 152 .+-. 5.0
Beef protein 2.9 .+-. 0.4 22.0 .+-. 0.3 113.0 .+-. 1.9 5.7 .+-. 0.3 109 .+-. 1.8
150 .+-. 5.0 Lactalbumin/Soy 2.9 .+-. 0.3 20.7 .+-. 0.5 121.0 .+-. 4.7 5.8 .+-.
0.5 110 .+-. 8.0 180 .+-. 7.0 (50:50) Lactalbumin/Casein 2.7 .+-. 0.4 23.6 .+-.
0.4 121.0 .+-. 2.0 5.6 .+-. 0.4 112 .+-. 4.0 148 .+-. 4.9 (80:20)
Lactalbumin/Casein 3.0 .+-. 0.2 23.4 .+-. 0.5 116.0 .+-. 2.0 6.0 .+-. 0.3 118
.+-. 4.0 145 .+-. 5.0 (20:80) Nonpurified diet.sup.g 3.2 .+-. 0.3 21.1 .+-. 0.5
114.7 .+-. 1.8 5.8 .+-. 0.2 114 .+-. 1.9 189
6.0 .sup.a The average food consumption over the 18 days feeding period was not
considered different by ANOVA .sup.b,c,d,e,f The average initial body weight
(b), increase in body weight (c), total serum protein (d) and spleen weight (e)
were not considered different by ANOVA. The numbers of cells per spleen (f) in
lactalbumin and Purina fed groups were higher by ANOVA (p: 0.0001) than the
corresponding values in the casein, wheat, soy and fish protein groups. .sup.g
Purina mouse chow, Ralston Purina Company, St. Louis, MO., (estimated 22 g
protein from various sources per 100 g diet). .sup.h Mice received 5 .times.
10.sup.6 SRBC on day 14. .sup.j Lactalbumin = Whey Protein Concentrate.
FIG. 2 shows plaque forming cells/spleen
(PFC) on the day showing peak production of PFC following immunization with
10.sup.6 SRBC. Effect of 3 weeks of dietary treatment with 20 g/100 g diet of
either whey protein concentrate (WPC), casein (C), whey protein concentrate
hydrolysate, casein hydrolysate, beta-lactoglobulin (.beta. L),
alpha-lactalbumin (.alpha. L), gamma-globulin (G) or bovine serum albumin (SA).
Each value represents the Means.+-.SD When protein hydrolysate was given, the
plaque forming cell response in mice fed the whey protein diet was found to be
504% of that noted in the casein diet-fed mice (p=0.0004) (FIG. 2). When free
amino acid mixture was given, the plaque forming cell response in mice fed the
whey protein amino acid diet was found to be 332% of that of the casein amino
acid diet-fed counterpart (p=0.0001) (FIG. 2). Our results (FIG. 2) indicate
that animals fed diets containing 20 g/100 g diet of any one of the four major
components of whey protein (.beta. L, .alpha. L, .gamma. G, SA) developed a
plaque forming cell response to sheep red blood cells inferior to (p.+-.0.0002)
that of mice fed a diet containing 20 g whey protein/100 g diet.
PREPARATION OF UNDENATURED WHEY PROTEIN CONCENTRATE
Immediately after milking, the milk is cooled to 4.degree. C. and kept in a
cooling tank for delivery to the cheese factory. The precipitation of the curd
is obtained by reducing the pH to about 4.6 with lactic acid initially at
20.degree. C. Following the addition of rennet (normally three ounces/1000
pounds of milk), the temperature is raised to about 30.degree. C. for 20 minutes
to promote expulsion of whey from the curd, allowing the agitation in the vat to
resolve at low speed.
When sufficient quantity of whey is obtained, the product remaining in the vat
is pasteurized in the standard fashion to obtain reduction of bacteria and
agitated at high speed for cheese production. The whey is then irradiated with a
source of gamma-irradiation. The radiation dose will vary from 5 to 15 kGy
according to bacteria content of the whey, to reach equivalent antibacterial
effect of standard pasteurization with minimal protein denaturation (measured by
changes in soluble protein, i.e. protein concentration in whey before and after
To obtain primarily undenatured whey to be used for subsequent production of
whey protein concentrate, the whey is not heated and the material from which it
is derived is slowly agitated to minimize protein denaturation. The prevention
of denaturation by maintaining high solubility avoids co-precipitation of whey
proteins with the caseins, whey protein loss is minimized, thus increasing the
protein content of whey. The whey is then cooled to 6.degree. C.
For the production of undenatured whey protein concentrate, the whey is
separated and concentrated through ultrafiltration, which allows for selective
separation of protein from lactose, salts and water under mild conditions of
temperature and pH. This is a physicochemical separation technique in which a
pressurized solution flows over a porous membrane. The membrane allows the
passage of only relatively small molecules.
To prevent excessive microbial growth during residence time and protein
denaturation, the plant is operated below 10.degree. C. most of the time. A thin
layer membrane of polymeric material (polysulphone) with a cut off value of
approximately 10,000 is used, so that protein components of MW.gtoreq.15,000 and
more are retained To speed up filtration, the liquid is fed on the membrane at a
pressure of 5 bar (Kg/cm.sup.2).
A frame type module is constructed to hold a large number of these membranes.
The production line consists of 18 such modules. In the last 10 modules,
demineralized water is added and then removed through the membranes carrying
lactose and minerals with it. To maintain velocities adequate to minimize
concentration polarization and fouling, recirculation pumps are used in each
A final protein concentrate with 80% protein (undenatured) in dry matter can be
FACTORS RESPONSIBLE FOR THE IMMUNOENHANCING EFFECT OF WHEY PROTEIN CONCENTRATE
(a) Whey Protein Mixture
Our studies show that the immunoenhancing effect of WPC in comparison to C is
maintained when these two proteins are replaced in formula diets by a pancreatic
hydrolysate (20% free amino and 80% oligo peptides with MW less than 1000) (see
FIG. 2).sup.(32). Our results also indicate that mice fed diets containing any
one of the four major protein components of the WPC mixture developed a PFC
response to SRBC inferior to that of mice fed the corresponding whey protein
mixture. We can thus conclude that the observed immunoenhancing effect of WPC is
dependent upon the contribution of all its protein components. For these reasons
we can assume that this phenomenon is not related to milk protein allergy or
some other manifestation of oral immunization.
(b) Undenatured Conformation of the Whey Protein Concentrate
Recent observations have revealed to us that the described biological activity
of the whey protein concentrate, already shown to be unrelated to its
nutritional quality, is actually dependent on the undenatured conformation of
the proteins. This discovery was made accidentally when a batch of whey protein
concentrate that was sent to us by the usual supplier failed to exhibit the
immunoenhancing effect previously described while exhibiting the same
nutritional efficiency. Upon analysis it appeared that this preparation was less
soluble and exhibited all the characteristic indirect signs of denaturation
(D-Lacp), quite different indeed from the previous samples of undenatured whey
protein (U-Lacp) exhibiting strong biological activity. Data on FIG. 3 (i.e.
Table 3, below) indicate the relationship between the degree of denaturation of
whey protein concentrate and the PFC immune response of the host.
INDIRECT INDICES OF DENATURATION OF WHEY PROTEIN CONCENTRATES SIGMA D-LACP.
DENATURED PROMOD SAPRO U-LACP U-LAD
SOLUBILITY: 82.8% 0% 93.7% 91.6% 94.5% -- (3%P) pH: 6.4 pH: 4.8 pH: 5.9 pH: 6.2
pH: 6.5 -- LIGHT TRANSMITTANCE: 49.7% 19.3% 68.6% 63.6% 79.0% -- (750 NM,
0.15%P) SOLUBILITY INDEX: 72.8% 0% 84.7% 83.8% 92.0% -- (pH 4.6, 3.0%P)
The related Table 4, below, further indicates the lack of correlation between
nutritional efficiency and denaturation of protein. In the natural state, the
milk whey proteins have a definite conformation which, when exposed to heat
above a certain critical level, is disrupted. In contrast to caseins, the whey
proteins are rapidly denatured by heating. Denaturation of whey proteins causes
unfolding of their globular structure to form a random coil conformation. In
addition to heating, other processing treatment, e.g. pumping, mixing, aeration,
vacuum evaporation and drying further promote protein denaturation.sup.(33). The
half cystine residues, frequent in some of the whey proteins.sup.(11), are
connected by intramolecular disulfide bonds which contribute to the spatial
configuration of the molecule and partly block unfolding of the
molecule.sup.(34). The free sulphydryl content of whey increases on heating due
to an unfolding and subsequent exposure of buried sulphydryl groups, with
rupture of the disulfide bonds in different whey proteins.sup.(35,36). Heat
denaturation unfolds and exposes the poorly soluble hydrophobic amino acid
residues to water. The denaturation of whey protein is pH sensitive.sup.(36).
Hence, the extent of denaturation is normally assessed by loss of solubility at
"natural" (intrinsic pH of an aqueous solution of the specific protein powder)
pH.sup.(37) or at pH 4.5.sup.(34,36,37), and decrease in light transmission of
the solution.sup.(37). In our studies we evaluated whey protein concentrate
denaturation by the following methods:
Solubility measurements: After dispersion of a 3% protein solution in distilled
water at room temperature and, in some cases, pH adjustment, the solution was
stirred and then centrifuged for 20 minutes at 40,000.times.g. The protein
content of the supernatant was determined by the Lowry method. Percent
solubility was computed as the portion of total protein recovered in the
Light transmittance: The initial 3% protein solution was diluted to 0.15% in
distilled H.sub.2 O. The light transmittance of blank (distilled H.sub.2 O) and
sample was measured at 750 nm with the spectrophotometer immediately after
CASEIN D-LACP DENATURED PROMOD SAPRO U-LACP U-LAD
Initial Weight (g): 19.7 .+-. 0.2 19.4 .+-. 0.4 20.0 .+-. 0.5 23.6 .+-. 0.3 22.2
.+-. 0.2 18.8 .+-. 0.4 20.1 .+-. 0.3 Final Weight (as % 124% .+-. 2 121.8 .+-.
0.8 122% .+-. 2 120% .+-. 2 121% .+-. 2 122% .+-. 1 121.6% .+-. 1.8 3 of initial
wt.): Serum Protein (mg/dl): 5.4 .+-. 0.1 5.7 .+-. 0.1 5.1 .+-. 0.1 5.3 .+-. 0.1
5.5 .+-. 0.1 5.8 .+-. 0.2 5.7 .+-.
D-Lacp = Denatured whey protein concentrate, Lacprodan80 by "Danmark Protein",
Denmark. ULacp = Undenatured whey protein concentrate, Lacprodan80 by "Danmark
Protein", Denmark. ULad = Pancreatic hydrolysate of undenatured whey protein
concentrate by Nestle, Switzerland. Promod = Whey protein concentrate by Ross
Laboratories, Montreal. Sapro = Whey protein concentrate by Saputo Ltd.,
It is apparent from FIG. 3 that a positive relation exists between the
undenatured state of whey protein concentrate in the diet and the intensity of
the humoral immune response to SRBC. The level of immune response is not related
to the nutritional efficiency of the whey protein concentrate but to its
undenatured conformation (FIG. 3, and associated Table 3 and 4, above). Hence,
the independence of the biological activity (immunoenhancement) from the
nutritional aspect of the whey protein concentrate, shown in our previous short
term experiments (FIGS. 1 and 2, Table 2, above) is confirmed. Further evidence
of the inhibitory effect of heat denaturation on the immunoenhancing property of
whey protein concentrate was obtained by heating a partially denatured whey
protein concentrate (Promod). This procedure produced a significant drop in the
immunoenhancing property of the diet without change in its nutritional
efficiency (FIG. 4 and associated Table 5).
Effect of three weeks of dietary treatment CASEIN PROMOD C3H/HeN Mice Non-heated
Heated Non-heated Heated
Initial weight (g): 20.4 .+-. 0.2 23.8 .+-. 0.3 24.2 .+-. 0.3 21.8 .+-. 0.3
Final weight 130% .+-. 2 120% .+-. 2 119% .+-. 2 127% .+-. 4 (% of initial wt.):
Spleen weight (mg): 92 .+-. 4 107 .+-. 5 104 .+-. 3 131 .+-. 7 Protein (mg/dl):
5.4 .+-. 0.1 5.5 .+-. 0.1 5.3 .+-. 0.1 5.6 .+-. 0.1
Promod nonheated vs. promod heated: p < .01 (90.degree. C. for 10 minutes)
Promod nonheated vs. casein nonheated: p < .01 Mean .+-. S.E.M. (N = 10)
Preliminary heat treatment of the concentrated whey protein solution will not
improve its overall digestibility; hence the whey protein concentrate used in
the preparation of the pancreatic hydrolysate LAD was undenatured. The absence
of cysteine in the free amino acid fraction of LAD is consistent with the
knowledge that pancreatic trypsin does not hydrolyse the disulfide
cross-linkage.sup.(38) characteristic of the native whey protein which is
instead split in the process of denaturation.sup.(34-36,39-41).
LAD is composed of small peptides (approx 80%) and of free acid amines (approx.
20%). The molecular weights of peptides varies between 450 and 1000. A large
percentage of essential or nutritionally important acid amines are present in
free form: Lys (63%), Arg (39%), His (18%), Met (59%), Ile (22%), Leu (32%), Tyr
(80%), Phe (56%) and Trp (99%). LAD is an experimental product which should not
be used for clinical treatment of humans.
DIETARY WHEY PROTEIN AND PNEUMOCOCCAL INFECTION
Because our studies had shown that dietary protein type influences the humoral
immune response, we then proceeded to investigate the effect of U-Lacp in diet
on the resistance of mice to pneumococcal infection. Pneumococci represent the
group of encapsulated high virulence organisms against which the body employs a
humoral immune response. C3H/HeJ mice fed a diet
containing 20 g U-Lacp/100 g diet showed improved survival after i.v. infection
with Streptococcus pneumoniae type 3 as compared to similarly infected mice fed
a 20 g C/100 g diet of similar nutritional efficiency.sup.(10) (Table 6 below).
On the basis of our various studies, it was shown that the enhanced resistance
of mice fed the whey protein diet to infection with Streptococcus pneumoniae
type 3 was independent of the weight of the animal at the time of infection and
the weight gained before infection (animals were fed the diets for 2 weeks prior
TABLE 6 ______________________________________ SUSCEPTIBILITY TO TYPE 3 S.
PNEUMONIAE OF THREE SERIES OF MICE FED DIETS OF VARIOUS PROTEIN TYPES.sup.1
Ratio of alive:dead mice Days Experiment 1 Experiment 2 Experiment 3
Post-Infection.sup.2 C L C L C L ______________________________________ 0
(10.sup.2) 8:0 8:0 10:0 10:0 10:0 10:0 2 8:0 8:0 10:0 10:0 10:0 10:0 3 7:1 8:0
10:0 10:0 10:0 10:0 4 7:1 8:0 9:1 10:0 9:1 10:0 9 (10.sup.3) 7:1 8:0 9:1 10:0
9:1 10:0 11 7:1 8:0 9:1 10:0 9:1 10:0 12 7:1 8:0 5:5 9:1 8.2 10:0 13 6:2 8:0 4:6
9:1 8:2 10:0 14 5:3 8:0 4:6 9:1 7:3 9:1 40 5:3 8:0 4:6 9:1 7:3 9:1
______________________________________ .sup.1 Mice were infected after 2 wk
treatment with casein diet (C) (20 g casein/100 g diet), or lactalbumin diet (L)
(20 g/100 g). .sup.2 Injected i. v. in 1% FCSRinger; 9 days after infection with
10.sup.2 pneumococci the surviving mice were infected with a dose of 10.sup.3
pneumococci. C = Casein L = Lactalbumin = Whey Protein Concentrate. Overall
mortality is 36% in the C fed groups and this is significantly higher (P =
0.002) than that of the L fed mice which is 7.1%.
MECHANISM RESPONSIBLE FOR THE IMMUNOENHANCING EFFECT OF WHEY PROTEIN CONCENTRATE
Over the past few years we have attempted to identify the changes induced by
dietary protein type which might directly or indirectly affect the humoral
immune responsiveness. In mice not challenged with an immunogenic stimulus, the
type of protein in the diet was found to have little or no effect on a variety
of parameters examined. Thus, body growth, food consumption, serum protein,
minerals and trace metals, circulating leukocytes and more specifically, the
genesis of bone marrow B lymphocytes were all within normal
limits.sup.(5-10,31). These findings confirm that at 20 g/100 g diet
concentration, the proteins provide an adequate daily supply of essential amino
acids for the growing mice. The only significant effect of protein type was
found to be a change in plasma amino acid profile, which essentially conformed
to the amino acid composition of the ingested protein, with the notable
exception of cysteine (Tables 7 and 8, below).
We were particularly intrigued by the finding that, in spite of an 8- fold
higher cysteine content in WPC, the plasma level of cysteine in WPC diet-fed
mice was not different from that in their C diet-fed counterparts. The fate of
the excess cysteine was a matter of interest. Dietary cysteine is a rate
limiting substrate for the synthesis of glutathione (GSH) which is necessary for
lymphocyte proliferation. GSH is dependent upon the supply of cysteine which is
derived from dietary protein. The redox state of the lymphocyte can modulate the
intracellular concentration of cyclic GMP, which is known to be intimately
involved in lymphocyte proliferation.
Our studies have shown that the observed enhancement of the immune response is
associated with greater production of splenic glutathione in immunized mice fed
whey protein in comparison to mice fed a casein or cysteine enriched casein
diet. The efficiency of dietary cysteine in inducing supernormal glutathione
levels is greater when it is delivered in the whey protein than as free
TABLE 7 ______________________________________ AMINO ACID COMPOSITION OF TEST
PROTEIN.sup.(a) (g/100 g protein) WHEY PROTEIN AMINO ACID CASEIN CONCENTRATE
______________________________________ Phenylalanine 5.3 .+-. 0.2 3.4 .+-. 0.3
Tryptophan 1.4 .+-. 0.2 2.1 .+-. 0.0 Glycine 2.0 .+-. 0.1 2.0 .+-. 0.2 Serine
6.2 .+-. 0.5 5.2 .+-. 0.4 Leucine 10.0 .+-. 0.4 10.4 .+-. 0.7 Isoleucine 6.0
.+-. 0.6 6.1 .+-. 0.8 Valine 7.1 .+-. 0.3 5.8 .+-. 0.8 Methionine 2.9 .+-. 0.2
2.1 .+-. 0.3 Cysteine 0.3 .+-. 0.1 2.3 .+-. 0.3 Aspartic acid 7.3 .+-. 0.1 10.7
.+-. 0.7 Glutamic acid 22.9 .+-. 0.2 18.8 .+-. 0.7 Histidine 3.0 .+-. 0.1 2.0
.+-. 0.2 Tyrosine 6.0 .+-. 0.1 3.0 .+-. 0.4 Proline 11.6 .+-. 0.4 6.1 .+-. 0.7
Arginine 4.0 .+-. 0.1 2.8 .+-. 0.3 Alanine 3.1 .+-. 0.3 4.9 .+-. 0.4 Lysine 8.2
.+-. 0.1 9.2 .+-. 0.5 Threonine 4.6 .+-. 0.3 6.8 .+-. 1.3
______________________________________ .sup.(a) Value expressed as Mean .+-.
S.D. of data from reliable sources.
TABLE 8 ______________________________________ EFFECT OF DIETARY PROTEIN TYPE ON
PLASMA AMINO ACID PATTERNS Lactalbumin 20 g % (whey protein nmol/ml Amino Acid
concentrate) Casein 20 g % P-value ______________________________________
Isoleucine 90 .+-. 5 95 .+-. 8 -- Leucine 125 .+-. 5 113 .+-. 4 -- Valine 232
.+-. 10 278 .+-. 13 0.025 Methionine 72 .+-. 3 92 .+-. 6 0.025 Cystine 37 .+-. 3
37 .+-. 3 -- Phenylalanine 51 .+-. 1 75 .+-. 4 0.0005 Tyrosine 55 .+-. 2 83 .+-.
5 0.005 Threonine 310 .+-. 7 223 .+-. 2 0.0005 Tryptophan -- -- -- Lysine 301
.+-. 6 323 .+-. 7 -- Histidine 50 .+-. 1 64 .+-. 4 0.005 Arginine 61 .+-. 4 92
.+-. 6 0.005 Glycine 142 .+-. 7 144 .+-. 7 -- Serine 120 .+-. 8 132 .+-. 4 --
Alanine 437 .+-. 18 382 .+-. 19 0.05 Proline 52 .+-. 5 117 .+-. 10 0.0005
Aspartic Acid 24 .+-. 2 16 .+-. 1 0.005 Glutamic Acid 65 .+-. 2 44 .+-. 4 0.005
______________________________________ Mean .+-. SD.
METHOD TO INCREASE TISSUE GLUTATHIONE
We further explored the interaction of dietary protein, GSH and the host immune
response We investigated whether a different protein source such as egg white,
with the same high level of cysteine as whey protein concentrate (Table 9,
below), had a similar effect in promoting higher GSH tissue content We already
knew that an egg white protein diet does not enhance the host immune response
above average. Whereas the static GSH level in spleen was found unaltered by
U-Lacp feeding for three weeks, our studies in young adult C3H mice showed that
enhancement of spleen cell immune response to SRBC (FIG. 5) is associated with
sustained elevation of splenic GSH during the antigen-driven clonal expansion of
the lymphocytes in U-Lacp (undenatured whey protein)-fed mice in comparison to a
pattern of decline observed in spleen GSH levels in mice fed either of the
nutritionally equivalent D-Lacp (denatured whey protein), casein, cysteine
enriched casein, or egg white protein diets (FIG. 6). The latter four groups
also exhibited a lower immune response (FIG. 5). Administration of S-(n-butyl)
homocyteine sulfoximine, which reduces the splenic glutathione level by half,
produces a marked drop in the humoral immune response of whey protein (U-Lacp)
diet-fed mice. This is further evidence of the important role of glutathione in
the immunoenhancing effect of dietary whey protein (FIG. 7).
TABLE 9 ______________________________________ AMINO ACID COMPOSITION (g/100 g
protein) Whey Protein Egg White Amino Acid Concentrate* Protein**
______________________________________ Aspartic acid 11.3 7.9 Threonine 7.2 4.4
Serine 6.1 7.9 Glutamic acid 20.1 14.1 Proline 6.6 3.8 Glycine 2.0 3.7 Alanine
5.4 7.6 Valine 6.5 7.8 Isoleucine 6.7 6.5 Leucine 11.2 8.8 Tyrosine 2.9 4.2
Phenylalanine 3.1 6.4 Lysine 9.5 6.0 Histidine 1.9 2.2 Arginine 2.7 5.9
Methionine 2.2 3.9 Cysteine 2.4 2.4 Tryptophan 1.7 1.5
______________________________________ *Lacprodan-80 from Danmark Protein A/S,
Copenhagen, Denmark, 1986; used i our experiments. **Values calculated from
"Amino Acid Content of Foods", U.S.D.A., 1957. Values from cysteine analyzed by
Sigma on samples used = 2.38 g/100 g protein and in our laboratory = 2.4 g/100 g
Tissue Glutathione Assay
Ninety milligrams of mouse heart or layer were homogenized in 5-sulfosalicylic
acid (5% w/v). Homogenates are centrifuged for 5 minutes in a microfuge at
10,000.times.g. The assay is carried out using the supernatants on the same day
according to the method of Anderson.sup.(72). Values are expressed as .mu.mol/g
wet tissue (FIGS. 8 and 9).
After three months on either diet initiated at age 17 months, GSH content was
found to be higher in the liver and heart of U-Lacp (undenatured whey protein)
fed mice compared to the D-Lacp (denatured whey protein), casein, egg white
protein or Purina diet-fed counterparts (FIGS. 8 and 9). The GSH values in heart
and liver of mice fed Purina laboratory chow was similar at age 10 weeks, 17,
20, 21 months The U-Lacp diet appears to enhance the GSH content of heart and
liver above "normal" values after 3 and 4 months of continuous feeding (FIGS. 8
In conclusion, after three weeks on the U-Lacp diet, spleen GSH content is
increased during the antigen driven clonal expansion of the lymphocytes in young
adult C3H/HeN mice as compared to a decline in controls fed D-Lacp, casein or
egg white protein diets (FIG. 6). In old C57BL/6N1A mice, long term feeding of
U-Lacp diet results in a moderate but sustained increase in liver and heart GSH
levels (FIGS. 8 and 9). The GSH enhancing activity of WPC is restricted to its
undenatured form (U-Lacp). This property is not solely due to the high cysteine
content of WPC because another protein source with similar cysteine content (egg
white) (see Table 9) does not exhibit this biological activity. This property of
U-Lacp does not depend specifically on its nutritional efficiency as evaluated
by body weight, serum proteins, and food consumption, but appears to depend on
the primary, secondary and tertiary structure of the protein in its native form.
Some of the previously discussed methods of increasing intracellular levels of
glutathione concentration are either toxic.sup.(64) or dangerous owing to the
risks related to the initial phase of glutathione depletion.sup.(70,71). The
methods involving the use of gamma-glutamyloyst(e)ine.sup.(67),
athiazolidine.sup.(69) or glutathione esters.sup.(68) (U.S. Pat. No. 4,784,685)
offer an interesting possibility for short term intervention. However, their
long term effectiveness in producing sustained elevation of cellular glutathione
has not been shown, nor has the possible toxicity of their long term use been
disproved. Indeed, glutathione and glutathione disulfide were found to be
positive in the most commonly used short term tests for carcinogenicity and
mutagenicity.sup.(64). Relevant to our invention are recent data indicating
specifically that a lack of the GSH precursor, cysteine, rather than a decrease
in biosynthetic enzyme activities is responsible for the deficiency of GSH noted
in aging animals.sup.(73). Similarly, the fall in cytosolic GSH in the liver of
chronic ethanol fed rats does not appear to be caused by a limitation in the
capacity of gamma-glutamyloysteine synthetase activity.sup.(74).
Data in FIGS. 8 and 9 show that the concentration of liver and heart glutathione
in control Purina fed mice remains very constant over time. On the other hand a
moderate but sustained elevation of tissue GSH was noted in mice fed the
nutritionally equivalent whey protein (U-Lacp) diet. Only minuscule quantities
of glutathione and no breakdown products that can be readily attributed to
glutathione are excreted in urine.sup.(75). The magnitude of change in cellular
glutathione concentration that can be achieved may be quite limited, perhaps
reflecting the critical importance of this molecule and the attendant tight
regulatory control. Glutathione itself serves as a negative feedback on the GSH
synthetic enzymes, which obviously limits cellular capacity to increase GSH
concentration.sup.(42). Glutathione reductase maintains GSH in its predominant
reduced form (.gtoreq.90%). This serves both to maintain this functional state
and also to control cellular concentration since reduced glutathione (GSH)
cannot cross the membrane, whereas the oxidized form (GSSG) can and does afflux,
resulting in decreased total glutathione. Besides these enzymes, gamma
glutamyltranspeptidase (GGT) is important in GSH metabolism. GGT serves as a
salvage pathway for glutamyl moieties at the cell membrane level, passing them
back into the cytosol to be used in GSH synthesis. Increased activity of this
enzyme has been associated with elevated GSH concentration in a number of cell
lines and malignant tissues.sup.(76,77).
The effects of a small increment in cellular GSH may be greater than expected.
For example, there are many reports of human and murine tumor cell lines
selected in vitro for resistance to a variety of chemotherapeutic agents. In a
number of these cell lines cellular GSH is increased consistently by 2-fold
compared to the drug sensitive parental cell line, despite the fact that the
level of drug resistance is often much greater, e.g. as much as
30-fold.sup.(77-79). In these cell lines, depletion of cellular GSH by selective
inhibition of synthesis restores drug sensitivity to the resistant cells. This
is effective only if the GSH depletion is maintained throughout the
Given the fact that cellular GSH is very tightly regulated, that a 2- fold
increase may be maximal, and that the effect of small increments in GSH may be
amplified by a variety Of GSH-utilizing enzymes (e.g. glutathione peroxidase,
glutathione-S-transferase), the reproducible change in GSH concentration
observed in animals fed the whey-rich diet is likely to have biological
importance. The chronic nature of this augmentation may contribute significantly
to this effect.
A METHOD TO INHIBIT THE GROWTH OF CHEMICALLY INDUCED COLON CANCER
Our findings show that in mice fed a casein diet the number and size of DMH
induced colon carcinoma were reduced by a factor of 0.3 and 0.4 respectively in
comparison to Purina fed controls (Table 10, below). However, in mice fed the
whey protein diet with similar nutritional efficiency the number and size of
DMH-induced colon carcinoma were reduced four fold in comparison to the Purina
fed controls (Table 10, below). DMH- induced colon tumors appear to be similar
to those found in humans as far as type of lesions and chemotherapeutic response
characteristics are concerned.sup.(93,94). The superiority of the anti-cancer
effect of whey protein in comparison to casein has been reported in our previous
study. About 80% of the proteins in bovine milk are caseins and the remaining
20% are whey proteins.sup.(95,96). In addition, using the traditional process of
preparing casein, the amount of whey protein co-precipitated along with the
casein varies from about 40 to 60% of the total amount of whey protein present
in the milk.sup.(97). Therefore it is conceivable that the minor anti-cancer
effect seen with casein could be due to the relatively (to caseins) small amount
of whey protein co-precipitated with it. It is apparent from the above described
studies that the antitumor activity of the dairy products is in the protein
fraction and more specifically, as our invention demonstrates, in the whey
protein component of milk.
Effect of dietary milk protein on animal growth and tumour development in A/J
mice treated with the carcinogen 1,2-Dimethylhydrazine. Whey Protein Casein
Purina Pur/Whey Pur/Cas 28 Weeks.sup.a 28 Weeks.sup.a 28 Weeks.sup.a 20/8
Weeks.sup.b 20/8 Weeks.sup.b
Initial 21.7 .+-. 0.5 21.5 .+-. 0.7 21.9 .+-. 0.8 21.9 .+-. 0.4 22.0 .+-. 0.7
Weight.sup.c (g) Final 21.5 .+-. 0.3 21.8 .+-. 0.4 19.7 .+-. 0.7 21.3 .+-. 1.0
21.0 .+-. 0.6 Weight.sup.c (g) Number of 8.4 .+-. 1.5 24.7 .+-. 3.0 35.9 .+-.
2.6 15.1 .+-. 3.2 21.7 .+-. 4.3 Tumours.sup.c Tumour Area.sup.c 38.8 .+-. 6.4
90.9 .+-. 10.6 160.0 .+-. 11.4 47.9 .+-. 10.4 77.7 .+-. 10.9
ANOVA: solid line(s) connect those means not significantly different (p < 0.05).
Group Whey Pur/Whey Pur/Casein Casein Purina Number of Tumours ##STR1## Tumour
.sup.a Mice treated with DMH for 24 weeks, and then sacrificed 4 weeks later.
.sup.b Mice treated with DMH for 24 weeks, and then sacrificed 4 weeks later.
They were maintained on Purina Mouse Chow for 20 weeks and then switched to
either Whey Protein or Casein diet for the remaining 8 weeks. .sup.c Mean .+-.
SURVIVAL STUDIES: THE BIOLOGICAL ACTIVITY IS DEPENDENT ON THE UNDENATURED
CONFORMATION OF WPC
(a) Survival of Old Mice During a Limited Time Period
Our study shows that the mean survival time, over a limited observation period
of 6-7 months ending when 55% of male C57BL/6N1A mice were dead, is increased by
about 30% in mice commenced on the undenatured whey protein (U-Lacp) diet at the
onset of senescence (age 21 months) in comparison with "controls" fed the
nutritionally equivalent Purina mouse chow. The survival curve of Purina fed
mice was very similar to that of casein diet-fed mice (FIG 10). However, in the
subsequent four months, mice on undenatured whey protein diet were switched to a
denatured whey protein concentrate (D-Lacp) diet. During this period, the time
of death of the remaining whey protein diet-fed mice became similar to that of
their casein diet or Purina-fed counterparts. Throughout the study repeat
bioassays of PFC formation confirmed the correlation between host
immunoenhancement and undenatured state of WPC in diet as indicated in FIG. 3.
In the second part of the study, when the difference between survival curves
began to narrow, the immunoenhancing property of WPC was absent although its
nutritional quality was preserved (D-Lacp). Throughout the entire study no
significant intergroup difference was seen in calorie intake, and body weight.
Since longevity is dependent primarily upon the genome of the individual it is
unlikely that delayed mortality over a limited period of time would have
influenced overall longevity. However, at least in terms of the immunoenhancing
effect of the diet, this study could be regarded as a single direction
cross-over from test (U-Lacp) to control (D-Lacp) diets, showing that the
biological activity of WPC on survival of old mice is dependent upon its
undenatured state and correlating directly with the PFC assay used in our study
(as illustrated in FIG. 3).
(b) Short and Long Term Survival of Mice with DMH-Induced Colon Cancer
In DMH treated mice we noticed a difference between mortality by the 28 weeks
end point and the survival time to the end of the experiment in relation to
dietary protein type. During the first seven months of study, the mice fed
undenatured whey protein (U-Lacp) had no death as compared to a 33% mortality
observed towards the end of this period in the casein and Purina groups. In the
subsequent four months mice on whey protein were fed denatured whey protein
(D-Lacp). During this latter period the D-Lacp diet appeared to have no
favourable effect on survival in comparison to the casein diet (Table 11,
below). Throughout the study repeat bioassays of spleen PFC were done to
document the physiologic effects of the diets on immune function as reported
previously and the stability of these effects. The immunoenhancing effect of the
U-Lacp diet was consistently confirmed for the first 7 months of the study;
however, in the following four months (D-Lacp), the immunoenhancing effect
previously observed in mice fed the U-Lacp diet was absent. The values of PFC
response in relation to either the U-Lacp diet or the D-Lacp diet were
consistent with those presented in FIG. 3. This study therefore confirms the
hypothesis that the biological activity of WPC on survival of tumor bearing mice
is dependent upon its undenatured state correlating directly with the PFC assay
used in our study.
TABLE 11 ______________________________________ Effect of dietary milk protein
on short and long term survival in A/J mice treated with the carcinogen 1,2-
Dimethylhydrazine for 24 weeks. DIETARY GROUP.sup.b Whey Protein.sup.d Casein
Purina ______________________________________ Mortality.sup.a at 28 weeks 0% 33%
33% Survival time.sup.c in weeks. 40 41 30
______________________________________ .sup.a Significance by Chi Square
analysis: Whey Protein vs. Purina vs. Casein p < 0.05. .sup.b Originally 12 mice
per group. .sup.c Survival time in weeks from the first dose of carcinogen. Whey
protein and Casein differ significantly from Purina, MantelCox test p < 0.01.
.sup.d Undenatured Whey Protein used from weeks 3 to 28. Denatured Whey Protein
used from week 28 until end.
Synergistic Role of Vit. B.sub.2, B.sub.1 in the Immunoenhancing Effect of
Dietary Whey Protein Concentrate
While whey protein represent an optimal source of cysteine, the rate limiting
substrate for the biosyntheses of GSH, Vit. B.sub.2 and B.sub.1 are important
elements in the function of the GSH redox cycle.
Glutathione (GSH) status in tissues is maintained mainly in the reduced state
(GSH:GSSG, 250), which is achieved by the efficient GSH peroxidase and reductase
system coupled to the NADP+/NADPH redox pair. Endogenous toxic H.sub.2 O.sub.2
is reduced to H.sub.2 O through the oxidation of GSH to GSSG catalyzed by GSH
peroxidase At the expense of cellular NADPH, GSSG is effectively reduced back to
GSH by NADPH:GSSG reductase, thus maintaining thiol balance. As a result, GSSG
reductase has a great capacity to protect cells against oxygen toxicity from
endogenous active oxygen species.
Vit. B.sub.1 (thiamin) is involved in the transketolase reaction of the pentose
phosphate shunt yielding NADPH and pentose.
Vit. B.sub.2 (riboflavin): The coenzyme derivatives of riboflavin, flavin
mononucleotide (FMN) sequentially from riboflavin. Vit B.sub.2 deficient animals
exhibit marked decreases in activities of FMN and FAD-requiring enzymes such as
In this sense, it is conceivable that all these water soluble vitamins naturally
present in whey, play an essential role for optimal function of the GSH redox
cycle particularly when whey protein intake, as shown in our experiments, has
produced higher level of GSH synthesis and storage in the tissues.
The present studies (FIG. 11) show that dietary levels of Vit. B.sub.1, B.sub.2
slightly above recommended allowance (Table 12, diets 5, 6; below) contribute to
the immunoenhancing effect of dietary whey protein concentrate. Whey protein, by
providing optimal bioavailability of the limiting substrate (cysteine) enhances
the synthesis and storage of GSH. On the other hand, higher than normal intakes
of Vit. B.sub.1 and B.sub.2 appears to be necessary to maintain the GSH redox
cycle at a level higher than normal, thus allowing the development of a better
than normal, immune response to SRBC. Individually the effect of each of the
vitamins in whey protein fed mice is limited; however, their synergistic effect
on the immune response of whey protein fed mice is apparent (FIG. 11, diets 5, 6
and diet 1). The same vitamins are ineffective on the immune response of casein
diet-fed mice. Although all these water-soluble vitamins are present in whey, it
is interesting to note that the main natural source of the single most effective
vitamin, riboflavin, is whey to which Vit. B.sub.2 gives its characteristic
VITAMIN CONTENT OF TEST DIETS VITAMINS (mg/100 g Diet) (Diet 1) Diet 3 Diet 4
Diet 5 Diet 6 Diet 7 Diet 8
B1 0.34 1.42 0.9 2.7 1.0 VIT. B2 0.38 1.47 0.9 2.7 0.6 VIT. B6 0.26 0.7 AC.
FOLIC 0.063 0.1 VIT. C 53.3 118.3
In conclusion, dietary intake of Vit B.sub.1 and particularly B.sub.2 above
recommended daily allowance contribute to the development of enhanced immune
response in whey protein fed animals: Vitamin B.sub.2 +B.sub.1 appears to
produce the strongest effect. When intake of these vitamins is at or slightly
below these levels, growth and animal appearance are normal, but the response to
immune challenge is below the maximum potential of whey protein fed mice. The
whey protein composition according to the invention comprises in combination
said WPC together with vitamins B.sub.1 and B.sub.2 in amount of 1.5 to 2.0 mg
B.sub.1 and 1.5 mg to 2.0 mg B.sub.2 per 100 g diet.
As reported in Nutrition Reviews' PRESENT KNOWLEDGE IN NUTRITION, The Nutrition
Foundation, Inc. (1984), the current U.S. recommended dietary allowance (RDA)
for Vitamin B.sub.1 (thiamin) is 0.5 mg per 1000 kcal. This amount is based on
assessments of varying levels of dietary thiamin on clinical signs of
deficiency, on excretion of thiamin and its metabolites, and on ETKA and TPP
effects. The present RDA for thiamin is 0.5 mg per 1000 kcal.
The allowance for Vitamin B.sub.2 (riboflavin) in males 11 to 51 plus years in
age ranges from 1.2 to 1.5 mg per day and for women from 1.2 to 1.3 mg,
according to estimates of the Food and Nutrition Board of the National Academy
of Sciences. Levels are to be increased by 0.3 mg during pregnancy, by 0.5 mg
during lactation and possibly should be related to energy expenditure. As
reported in The Commonwealth Bureau of Animal Nutrition's NUTRITION ABSTRACTS
AND REVIEWS, Volume 28, No. 2 (1958), the RDA for riboflavin is about 0.6 mg per
1000 Cal for women and about 0.5 mg per 1000 Cal for men.
In the stomach, whey is separated from milk by the action of gastric juice. It
is conceivable that the transit and absorption of the water-soluble vitamins and
proteins of whey occur faster than those of the protein (casein) and vitamin
constituents of the milk coagulum (curd). Hence the whey protein and vitamins
including the vitamins B.sub.1 and B.sub.2 could enter the systemic circulation
at a different rate than that of other milk constituents and express their
synergistic effect on the immune system and the GSH redox cycle.
The immunoenhancing and the other specific biological properties of dietary whey
protein described in this application, are heat labile and dependant upon the
undenatured (native) state of the protein (which can also be affected by
vigorous shaking, solvents, extreme ph changes, etc.) and are independent of its
nutritional quality which is unaltered by the process of denaturation.
Unlike most other commercially available when protein which are denatured, the
whey protein used in Our experiments, produced in Denmark (Lacprodan - 80) is
90% undenatured (U.D. in FIG. 11). This protein displays the greatest tendency
to denature under heat thus exposing its free sulfhydryl group. When experiments
were done using a batch of w.p.c. received after a long surface transport from
Denmark through the U.S. in exceptionally hot and humid weather (summer 1988),
the immunoenhancing property of w.p.c was lost (FIG. 11, 2d-8d). These
experiments, while indicating the synergistic role of vit. B.sub.1 and B.sub.2,
in the immunoenhancing effect of the diet, also show the negative effect of a
presumably partially denatured whey protein. Previous studies have shown that
the immunoenhancing property of dietary whey protein is probably related to an
optimal intracellular transport and availability of the cysteine which is a
limiting precursor for glutathione synthesis. It is conceivable that partial
denaturation of this protein had brought about the loss of its specific
biological property by altering GSH synthesis, without an effect on its
Although specific preferred embodiments of the invention have been described
above with reference to the accompanying drawings, it will be apparent that the
invention is not limited to those precise embodiments, and that many
modifications and variations could be effected therein by one statement in the
art without departing from the spirit or scope of the invention as defined in
the appended claims.
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