DESC:Field of the invention
The invention relates to nutritional compositions for enhanced absorption of proteins and useful for for body builders, athletes & sports persons for building body size and strength and by patients who are undergoing surgeries including convulsing patients for quick recovery.
Background of the invention
Whey protein contains all 9 essential amino acids and is low in lactose content. All Essential Benefits/Effects/Facts & Information of whey proteins especially for better muscle building are extensively discussed in a publication that can be accessed in https://examine.com/supplements/whey-protein/ .
Whey is generally used as a protein supplement as it is absorbed faster than other forms of protein and increases muscle protein synthesis, in addition to being vegetarian. Whey is known to deliver large amount of the amino acid L-cysteine, thereby alleviating deficiencies that occur during aging and diabetes, as well as other conditions. Whey also increases fat loss due to function of protein.
For muscle recovery and building the most important three amino acids are leucine, isoleucine and valine.
General requirement of protein is 1 gm per body weight. However during body building exercise the requirement is much more. When athletes take more than one scoop (24 gm) of protein daily they experience discomforts like bloating and constipation.
Enhancing the bioavailability of the amino acids from the whey proteins is of significance and it known that Bacillus species aid the process of enhancing bioavailability of the amino acids from whey.
However, when Bacillus species is added to the whey proteins and orally consumed in the form of supplementary foods, the bacteria get destroyed by the gastric juices produced by the stomach and are not available in a viable condition in sufficient numbers when they reach the intestine.
There has been an unmet need to provide formulations of whey proteins exhibiting high bioavailability of the desired amino acids from whey proteins when consumed in prescribed dosages.
The present invention addresses the unmet need in the market place and provides stable formulations of whey protein as food supplements that aid in enhanced bioavailability of the desired amino acids by the body that can be used by a range of subjects that include athletes, body builders and convulsing patients for quick recovery.
Summary of the invention
The present invention provides stable formulations of whey protein as food supplements that aid in enhanced bioavailability of the desired amino acids by the body that can be used by a range of subjects that include athletes, body builders and convulsing patients for quick recovery.
Another object of the invention is to provide formulations of whey proteins and encapsulated Bacillus coagulans so that the bacteria to survive the low pH when it comes in transits through the gut and cones in contact with gastric juices produced by the stomach and reach the intestines in a viable condition.
Another object of the invention is to provide the said formulations to subjects without them experiencing discomforts like bloating, constipation, flatulence, or diarrhea.
Another object of the invention is to provide the said formulations in which the Bacillus coagulans withstand the manufacturing processes, especially those required to make hot tea, coffee, cereals, and other foods.
Another object of the invention is to provide the said formulations and nutritional products for storage and transportation under tropical conditions wherein the Bacillus coagulans probiotics maintain viability.
Detailed description of the invention
It has surprisingly been found that a judicious combination of therapeutic amounts of lactobacilli probiotic bacteria in whey protein provide stable formulations to release proteases which helps to digest proteins into amino acids leading to enhanced liberation of amino acids from the protein and increasing the bio-absorption in the intestine to achieve increased efficacy.
The probiotic used in the recent invention is Bacillus Coagulans SNZ1969 which is encapsulated thereby providing stability in the acidic environment and the bile of the GI tract, and at temperatures of upto 100 deg centigrade and maintains viability at the site of action. Bacillus coagulans SNZ1969 strain has been deposited with the Microbial Type Culture Collection (MTCC) - assigned number MTCC 5724 and with Belgian Coordinated Collections of Microorganism (BCCM™ILGM) with the assigned number LMG S - 27484. Additionally, the partial gene sequencing of this strain can be found at the National Center for Biotechnology Information database (Swamy and Soman, 2013) as well as at DNA Data Bank of Japan (DDBJ). Additionally, Bacillus coagulans SNZ1969 spores preparation is exempt from the premarket approval requirement of the Federal Food, Drug, and Cosmetic Act because it has been determined to be Generally Recognized As Safe (GRAS), based on scientific procedures by the United States FDA (GRN000597).
Encapsulation of the Bacillus coagulans may be carried out using Cellulose acetate phthalate, Poly vinyl acetate phthalate, Shellac, Methacrylic acid copolymer, Sodium alginate, , Alginate, Chitosan, Carragenan, Zein, Gum arabic, Gum karaya, Mesquite gum Plant extracts, Galactomannans, Gluten Isolates (Pea, Soy), Caseins, Whey proteins, Gelatin, Fatty acids/alcohols, Glycerides, Waxes, Phospholipids, Xanthan, Gellan, Dextran and crosslinked CMC polymers namely: Methyl, Ethyl, Hydroxypropyl, Hydroxypropyl methyl, Ethyl methyl, Carboxy methyl and Ethyl hydroxyethyl.
In samples used in the present specification encapsulation of the Bacillus coagulans was done using Methacrylic acid copolymer plasticizers and opacifying agents. The dosage is 0.5 billion cfu/30gram serving of the formulation. However the probiotic dosage may vary from 0.2 billion – 20 billion per 30 gram serving.
In-vitro experiments were performed to test the release of amino acids from whey proteins by Bacillus coagulans versus whey protein without probiotic. 1% whey protein was dissolved in sucrose medium with and without Bacillus coagulans, and placed on a shaker at 37 0C with 160 rpm for 48 Hours. Following digestion of the protein the amino acid contents of the samples were quantified using Reverse Phase HPLC (The buffers used were Buffer A: 10mM Sodium acetate, pH6.4 with 6% acetic acid Buffer B: A buffer + Acetonitrile (40:60)). 1 ml of the given sample was taken and 4ml Methanol was added and incubated Over Night at -20?C. The sample was then centrifuged at 4?C, 13000 rpm for 15 min. The supernatant maintained at at 60?C (Under Nitrogen) in a heated dry bath. To this dried pellet, 50 microliter of PITC Reagent was added and incubates at 45?C for 1 hour in Thermomixer. The sample was then speed vacuumed to complete dryness. 200 microliter of A-Buffer was added, filtered using syringe filter and loaded 20UL into HPLC.
As a control, liberation of amino acids by Bacillus licheniformis which is well known to produce enhanced proteases and efficient for digesting whey protein was also tested using the protocol described above. The comparative results of these tests are given in table 1.

Table 1: Concentration of different amino acids in undigested whey protein control sample, whey protein sample digested with Bacillus coagulans and whey protein sample digested with Bacillus licheniformis.
Referring to table 1, it is clearly seen that the levels of 3 critical branched chain amino acids viz. valine, leucine and isoleucine go up several folds i.e. 18x, 4.8x and 3.4x respectively in formulations containing Bacillus coagulans as compared to those of the undigested control. These amino acids play a critical role in development of muscle mass. The level of another critical amino acid Arginine liberated following digestion goes up significantly following digestion by Bacillus coagulans. This amino acid plays a role in increasing blood circulation and thus helps is recovery following rigorous exercise.
The levels of free cysteine are increased 2.9 fold using formulations containing Bacillus coagulans . Cysteine helps to reduce aging and other complications. Further, the levels of all the amino acids with the exception of asparagine (which remains the same as the control sample) go up using formulations containing Bacillus coagulans. Eight out of nine essential amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) show enhancement when formulations containing Bacillus coagulans are used. Such enhancement of the amino acids is of significance as the human body cannot synthesize these amino acids and they must be consumed from foods.
Further table 1 demonstrates that Bacillus coagulans SNZ 1969 outperforms Bacillus licheniformis with respect to the liberation of free amino acids (with the exception of Proline) showing the remarkable synergy between the probiotic Bacillus coagulans and whey protein.
Typical compositions of the present invention comprise encapsulated Bacillus coagulans 0.2 billion – 20 billion per 30 gram serving, whey protein concentrate 75-95%, alkalised cocoa powder 2-20%, gum blend 0.1-1%, digestive enzymes 0.025-0.10%. The compositions optionally further comprise additional ingredients selected from the group consisting of alkali metals, sugars, carbohydrates, dietary fiber, vitamin A, vitamin C, calcium, iron, vitamin D, vitamin E, thiamin, riboflavin, niacin, vitamin B6, folate, vitamin B12, biotin, pantothenic acid, phosphorus, iodine, magnesium, zinc, selenium, copper, manganese, chromium, and molybdenum based on the targeted purpose for a targeted subject group.
In-vivo experiments to assess the impact of Whey Protein feeding along with Bacillus Coagulans on serum Amino Acid Levels and Muscle Growth have also been carried out. Following are the details of the experiments:
Male Adult Wistar Rats were divided in to 3 groups and fed with different diets mentioned bellow for a period of two weeks.
Normal Control Fed with normal chow diet
Whey Protein Fed with 20% Whey Protein diet
Whey Protein+ Bacillus Coagulans Fed with 20% whey protein along1.2 billion CFU Bacillus coagulans daily for a period of twenty eight days

Serum Amino Acid content and Muscle and Fat Pad contents were measured after twenty eight days of experimentation.
The results are given in figures 1-4.
The figures clearly demonstrate the efficacy of the present invention. Following trends are seen:
? Incremental trend in the Body Weights after twenty-eight days of Whey protein feeding
? Essential Amino Acid content in the serum increased in Whey Protein + BC Fed animals
? Skeletal Muscle content increased in the Whey protein + BC fed animals
? Peritoneal Fat content is reduced in Whey Protein + BC fed animals irrespective of their weight gain
Formulations containing Bacillus coagulans were also subjected to accelerated tests for performed by storing samples upto 37 º C and 70% humidity in their original packaging. 30 g samples (serving size) were taken out at monthly intervals and the viable cell count of Bacillus coagulans in the formulation, pathogens such as Escherichia coli, Salmonella species, Staphylococccus aureus, Pesudomonsa aeruginosa , including yeast and mould counts were monitored periodically upto 6 months of storage. The samples were also checked for their appearance retained their off white to brown color with characteristic odour at the end of six months of storage. No pathogens, yeast or mould were detected. Further the viable counts of the Bacillus coagulans in the initial sample reduced from 810 million spores to 582 million spores [samples stored at 37ºC)] and 650 million spores [samples stored at 25ºC)] which are well above the accepted limits of 500 million spores indicating stability.
,CLAIMS:We claim:
1. A stable formulation of therapeutic amounts of encapsulated bacilli probiotic bacteria, whey protein concentrate, alkalised cocoa powder, gum blend, digestive enzymes and optionally alkali metals, sugars, carbohydrates, dietary fiber, vitamin A, vitamin C, calcium, iron, vitamin D, vitamin E, thiamine, riboflavin, niacin, vitamin B6, folate, vitamin B12, biotin, pantothenic acid, vitamin k, phosphorus, iodide, magnesium, zinc, selenium, copper, chromium, and chloride, wherein the formulation is stable in acidic environment and the bile of the GI tract, and at temperatures of upto 100 °C and maintains viability at the site of action.

2. The probiotic as claimed in claim 1 is Bacillus Coagulans SNZ1969 strain deposited with the Microbial Type Culture Collection (MTCC) and assigned number MTCC 5724 and with Belgian Coordinated Collections of Microorganism (BCCM™ILGM) with the assigned number LMG S – 27484.

3. The encapsulation as claimed in claim 1 is carried out using Cellulose acetate phthalate, Poly vinyl acetate phthalate, Shellac, Methacrylic acid copolymer, Sodium alginate, Alginate, Chitosan, Carragenan, Zein, Gum arabic, Gum karaya, Mesquite gum Plant extracts, Galactomannans, Gluten Isolates (Pea, Soy), Caseins, Whey proteins, Gelatin, Fatty acids/alcohols, Glycerides, Waxes, Phospholipids, Xanthan, Gellan, Dextran and crosslinked CMC polymers namely: Methyl, Ethyl, Hydroxypropyl, Hydroxypropyl methyl, Ethyl methyl, Carboxy methyl and Ethyl hydroxyethyl.

4. A stable formulation is Bacillus coagulans SNZ1969 strain deposited with the Microbial Type Culture Collection (MTCC) and assigned number MTCC 5724 and with Belgian Coordinated Collections of Microorganism (BCCM™ILGM) with the assigned number LMG S – 27484 is 0.2 billion – 20 billion per 30 gram serving, whey protein concentrate 75-95%, alkalised cocoa powder 2-20%, gum blend 0.1-1%, and digestive enzymes 0.025-0.10% wherein the encapsulation is carried out using Methacrylic acid copolymer plasticizers and opacifying agents.

5. A stable formulation as claimed in any of the preceding claims when subjected to accelerated storage tests 37° C and 70% humidity for 6 months retained their off white to brown color with characteristic odour, with no pathogens, yeast or mould, wherein the viable counts of the Bacillus coagulans in the initial sample reduced from 810 million spores to 582 million spores [samples stored at 37ºC)] and 650 million spores [samples stored at 25ºC)] which are well above the accepted limits of 500 million spores.

6. The formulations as claimed in any of the preceding claims when used for invitro digestion of whey proteins when compared to undigested controls show
I. levels of amino acids viz. arginine valine, leucine and isoleucine go up several folds i.e. 18x, 4.8x and 3.4x respectively;
II. levels of free cysteine increase 2.9-fold;
III. levels of all the amino acids except for asparagine go up;
IV. eight out of nine essential amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) show enhancement.
7. The formulations as claimed in any of the preceding claims when used invivo, leads to incremental increase in body weight after two weeks, increase in essential amino acid content in the serum, increase in skeletal muscle content and reduction in peritoneal fat content irrespective of their weight gain.