WO2020204910A1 - Composition probiotique stable pour la gestion de l'intolérance au lactose - Google Patents

Composition probiotique stable pour la gestion de l'intolérance au lactose Download PDF

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Publication number
WO2020204910A1
WO2020204910A1 PCT/US2019/025296 US2019025296W WO2020204910A1 WO 2020204910 A1 WO2020204910 A1 WO 2020204910A1 US 2019025296 W US2019025296 W US 2019025296W WO 2020204910 A1 WO2020204910 A1 WO 2020204910A1
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WIPO (PCT)
Prior art keywords
lactose
bacillus coagulans
less
lactase
intolerance
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PCT/US2019/025296
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English (en)
Inventor
Muhammed Majeed
Kalyanam Nagabhushanam
Furqan Ali
Sivakumar Arumugam
Shaheen Majeed
Original Assignee
Muhammed Majeed
Kalyanam Nagabhushanam
Furqan Ali
Sivakumar Arumugam
Shaheen Majeed
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Application filed by Muhammed Majeed, Kalyanam Nagabhushanam, Furqan Ali, Sivakumar Arumugam, Shaheen Majeed filed Critical Muhammed Majeed
Priority to PCT/US2019/025296 priority Critical patent/WO2020204910A1/fr
Publication of WO2020204910A1 publication Critical patent/WO2020204910A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/54—Mixtures of enzymes or proenzymes covered by more than a single one of groups A61K38/44 - A61K38/46 or A61K38/51 - A61K38/53
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)

Definitions

  • the invention in general relates to probiotic compositions. More specifically, the present invention relates to a stable probiotic composition comprising Bacillus coagulans individually and/or in combination with multi-enzyme complex for the utilization of lactose and therapeutic management of lactose intolerance.
  • Lactose maldigestion is a common condition affecting up to 75 % of the world's population due to the decline in the activity of lactase enzyme from the normal range.
  • This enzyme is responsible for cleavage of lactose, a disaccharide carbohydrate consisting of glucose and galactose.
  • lactose a disaccharide carbohydrate consisting of glucose and galactose.
  • lactose maldigestion therefore, does not necessarily lead to lactose intolerance. Lactase is produced naturally in human body; however, sometime the production is affected by certain factors.
  • Lactose excess intake of Lactose may also be responsible for lactose intolerance.
  • probiotics gained the importance due to its various applications. They are live bacteria or yeast which supplements the gastrointestinal flora. Specifically strains belonging to Bifidobacterium and Lactobacillus are the most widely used probiotic bacteria for lactose intolerance as they are major groups of the gastrointestinal microbiota. Probiotics promote lactose digestion in lactose intolerance by increasing the overall hydrolytic capacity in the small intestine and increasing the colonic fermentation. Probiotics can decrease lactose concentration in fermented products, and increase active lactase enzyme entering the small intestine with the fermented products.
  • probiotics have been reported for the management of lactose intolerance it is well known in the scientific art that biological effects of probiotics or products thereof are strain specific and cannot be generalised among genera, species and strains (Probiotics: In
  • the present invention solves the above problem by disclosing the therapeutic potential of probiotic bacteria Bacillus coagulans for the management of lactose intolerance and increased utilization of lactose the invention also discloses a synergistic combination comprising probiotic bacteria Bacillus coagulans and multi-enzyme complex for increased lactose utilization.
  • Bacillus coagulans individually or in combination with multi-enzyme complex for the utilization of lactose.
  • Microbial Type Culture Collection & Gene Bank (MTCC), CSIR-Institute of Microbial
  • the present invention discloses the potential of probiotic bacteria Bacillus coagulans individually and/or in combination with multi-enzyme complex for the increased utilization of lactose.
  • the invention further discloses a method for the management of lactose intolerance using composition comprising Bacillus coagulans individually and/or in combination with multi-enzyme complex.
  • Fig. la and lb shows the graphical representation of utilization of lactose g/L (Fig. la) and percentage of reduction of lactose (Fig. lb) in MRS media supplemented with 2.5, 5.0,
  • Fig. 2 shows the graphical representation of utilization of lactose in dairy food
  • Fig. 3 shows the graphical representation of strain comparison of different strains of
  • Fig. 4 shows the graphical representation of effect of pH on the lactase (b- galactosidase) enzyme activity from Bacillus coagulans MTCC 5856
  • Fig. 5 shows the graphical representation of effect of temperature on the Lactase (b- galactosidase) enzyme activity from Bacillus coagulans MTCC 5856 was investigated at a temperature range from 10 to 70°C
  • Fig. 6 shows the graphical representation of effect of gastric acid on the stability of lactase (b-galactosidase) activity was evaluated from the range of pH 1.5 to 7.0 for different time intervals up to 180 min
  • Fig. 7 shows the graphical representation of thermo stability of Lactase enzyme was evaluated from the range of 37 to 70 °C
  • Fig. 8 shows the graphical representation of evaluation of utilization of Lactose by
  • FIG. 9 shows the graphical representation of evaluation of utilization of lactose by
  • present invention relates to a method of increasing lactose utilization said method comprising step of bringing into contact foods containing lactose with probiotic bacteria Bacillus coagulans to bring about the effect of increased lactose utilization.
  • Bacillus coagulans strain is specifically
  • the foods containing lactose is selected from the group comprising milk, sour cream and buttermilk, butter, yogurt, cheese, ice cream, bread and baked goods like waffles, pancakes, biscuits, cookies and breakfast mixes, breakfast cereals, instant potatoes, soups, potato chips and com chips and/or foodstuff wherein lactose is present.
  • invention in another most preferred embodiment, relates to a method of increasing lactose utilization, said method comprising step of bringing into contact foods containing lactose with probiotic bacteria Bacillus coagulans individually and/or in combination with multi-enzyme complex to bring about the effect of increased lactose utilization in synergistic manner.
  • Bacillus coagulans strain is specifically MTCC 5856.
  • the food containing lactose is selected from the group comprising milk, sour cream and buttermilk, butter, yogurt, cheese, ice cream, bread and baked goods like waffles, pancakes, biscuits, cookies and breakfast mixes, breakfast cereals, instant potatoes, soups, potato chips and com chips and/or foodstuff wherein lactose is present hi yet another related embodiment
  • the effective dose of Bacillus coagulans is 1x10 6 to 1x10 14 colony forming units (cfu) per unit dose. In further related embodiment, Bacillus coagulans is preferably 2x10 9 colony forming units (cfu) per unit dose.
  • the multi-enzyme complex comprises of a) a-amylase: not less than 24000 DU/g, b) cellulase: not less than 1100 CU/g, c) lipase: not less than 200 FlP/g, d) lactase: not less than 4000 ALU/g and e) neutral or acid protease: not less than 6000 PC/g.
  • the symptoms of lactose intolerance are selected from the group consisting of flatulence (wind), diarrhoea, bloated stomach, stomach cramps and pains, stomach rumbling, nausea, feeling sick, borborygmi, and vomiting.
  • lactose intolerance are selected from the group consisting of lactase deficiency, or hypolactasia, congenital alactasia, lactose ingestion, lactose malabsorption.
  • invention relates to a method for the therapeutic management of lactose intolerance in mammals, said method comprising step of administering a composition comprising probiotic bacteria Bacillus coagulans individually and/or in combination with multi-enzyme complex to mammals in need of such therapy.
  • mammal is human hi related embodiment
  • Bacillus coagulans strain is specifically MTCC 5856.
  • the effective dose of Bacillus coagulans is 1 x 10 6 to 1 x 10 14 colony forming units (cfu) per unit dose. In further related embodiment, Bacillus coagulans is preferably 2x 10 9 colony forming units (cfu) per unit dose.
  • the symptoms of lactose intolerance are selected from the group consisting of flatulence (wind), diarrhoea, bloated stomach, stomach cramps and pains, stomach rumbling, nausea, feeling sick, borborygmi, and vomiting.
  • lactose intolerance are selected from the group consisting of lactase deficiency, or hypolactasia, congenital alactasia, lactose ingestion, lactose malabsorption hi yet another related embodiment, the multi-enzyme complex comprises of a) a-amylase: not less than 24000 DU/g, b) cellulase: not less than 1100 CU/g, c) lipase: not less than 200 FlP/g, d) lactase: not less than
  • composition containing Bacillus coagulans and multi-enzyme complex is formulated with pharmaceutically/ nutraceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, antioxidants and preservatives and/or combined with other hepatoprotective compositions and administered orally in form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.
  • composition containing Bacillus coagulans and multi-enzyme complex is formulated with pharmaceutically/ nutraceutically acceptable excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, antioxidants and preservatives and/or combined with other hepatoprotective compositions and administered orally in form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.
  • Example 1 Utilization of Lactose by Bacillus coagulans MTCC 5856 in media
  • compositions 10 g/1
  • Lactose content was determined as per the 3, 5-Dinitrosalicylic acid (DNSA) method (Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426-428).
  • DNSA 3,5-Dinitrosalicylic acid
  • Example 2 Utilization of Lactose by Bacillus coagulans MTCC 5856 in Dairy
  • Bacillus coagulans MTCC 5856 The utilization of lactose in dairy foods by Bacillus coagulans MTCC 5856 was evaluated by growing Bacillus coagulans MTCC 5856 in skimmed milk media.
  • Bacillus coagulans MTCC 5856 was grown in media containing skimmed milk (20 g/L), soya peptone (5g/L), calcium carbonate (0.05g/L), Manganese sulfate (0.1 g/L), ammonium sulphate (1.46g/L) using 2.5 L fermenter (Bioengineering AG Sagenrainstrasse, Wald, Switzerland). The fermented broth was checked for enzyme activity at different intervals. Further, the supernatant was collected and carried out further for lactase enzyme assay. Lactase activity was determined as per standard method of Food Chemicals Codex (FCC) by using ortho-Nitrophenyl-b-galactoside
  • Lactose content was determined as per the 3, 5-Dinitrosalicylic acid (DNS A) method (Miller, G. L. (1959) by using lactose as standard. Use of dinitrosalicylic acid reagent for determination of reducing sugar.
  • Example 3 Lactase Enzyme assay (O-Nitrophenol beta-galactosidase enzyme assay)
  • O-nitrophenyl -b-Dgalactopyranoside (ONPG) enzyme assay was used for testing the lactase activity. 4 ml of 3.7 mg/ml ONPG in acetate buffer-(Dilute 5.8 ml of glacial acetic acid in 1 L
  • DM water adjust the pH to 4.5
  • 1 ml of the sample (supernatant of broth and cell free extract) was added and further incubated at 37 ° C for 15 min.
  • 1 ml of 1% Na 2 CO 3 was added and then 8 ml of DM water was added.
  • OD of samples was recorded at 420nm. Lactase activity is expressed in ALU/ml or
  • Example 5 Effect of temperature on the b-galactosidase activity from Bacillus coagulans MTCC 5856
  • FCC ortho-Nitrophenyl- b-galactoside
  • Example 6 Gastric acid and thermo-stability of the b-galactosidase activity from Bacillus coagulans MTCC 5856
  • Lactase activity was determined as per standard method (FCC) by using ONPG as substrate (Institute of Medicine. 2003. Food
  • lactase enzyme produced by the Bacillus coagulans MTCC 5856 was performed while incubating lactase enzyme in buffer at different temperature (50, 60 and 70°C).
  • Lactase activity was determined as per standard method of Food Chemicals Codex (FCC) by using ortho ⁇
  • Nitrophenyl-b-galactoside (ONPG) as substrate (Institute of Medicine. 2003. Food Chemicals.
  • Example 7 Combination study of Bacillus coagulans MTCC 5856 and multienzyme complex for the utilization of lactose in media and in dairy foods (skimmed milk)
  • lactose content was quantified by following DNSA method using lactose as standard.
  • One group without multi-enzyme complex was also taken in this experiment.
  • lactose content was quantified by following DNSA method using lactose as standard.
  • One group without multi-enzyme complex was also taken in this experiment.
  • DU Dextrinizing Unit
  • PU Protease Unit
  • CU Cellulase Unit
  • ALU Acid Lactase
  • Example 8 Formulations containing Bacillus coagulans and multi-enzyme complex for lactose intolerance
  • Bacillus coagulans and multi-enzyme complex is formulated with pharmaceutically/ nutraceutically acceptable compositions with excipients, adjuvants, bases, diluents, carriers, conditioning agents, bioavailability enhancers, antioxidants and preservatives and/or combined with other hepatoprotective compositions and administered orally in form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables and administered for treatment of alcohol induced and drug hepatotoxicity.
  • the following tables provide examples of different Bacillus coagulans and multi-enzyme complex compositions.
  • Tables 2-5 Provide illustrative examples of formulations containing Bacillus coagulans MTCC 5856 (LACTOSORE®) for the treatment/management of lactose intolerance.
  • Table 2 Bacillus coagulans tablet

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Abstract

La présente invention concerne une composition probiotique stable comprenant Bacillus coagulans individuellement et/ou en combinaison avec un complexe multienzymatique pour l'utilisation de lactose et la gestion thérapeutique de l'intolérance au lactose. L'invention concerne également un procédé de gestion de l'intolérance au lactose à l'aide d'une composition comprenant Bacillus coagulans individuellement et/ou en combinaison avec un complexe multienzymatique.
PCT/US2019/025296 2019-04-02 2019-04-02 Composition probiotique stable pour la gestion de l'intolérance au lactose WO2020204910A1 (fr)

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PCT/US2019/025296 WO2020204910A1 (fr) 2019-04-02 2019-04-02 Composition probiotique stable pour la gestion de l'intolérance au lactose

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PCT/US2019/025296 WO2020204910A1 (fr) 2019-04-02 2019-04-02 Composition probiotique stable pour la gestion de l'intolérance au lactose

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113341119A (zh) * 2021-05-13 2021-09-03 杭州海路医疗科技有限公司 间接检测乳糖不耐受的方法
CN114480175A (zh) * 2021-12-28 2022-05-13 广东海大集团股份有限公司 一种凝结芽孢杆菌菌剂及其制备方法与应用

Citations (2)

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Publication number Priority date Publication date Assignee Title
US20170035813A1 (en) * 2003-12-05 2017-02-09 Ganeden Biotech, Inc. Methods for the dietary management of irritable bowel syndrome and carbohydrate malabsorption
US20180117127A1 (en) * 2016-11-03 2018-05-03 Muhammed Majeed Enzyme composition for therapeutic management of muscle soreness

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170035813A1 (en) * 2003-12-05 2017-02-09 Ganeden Biotech, Inc. Methods for the dietary management of irritable bowel syndrome and carbohydrate malabsorption
US20180117127A1 (en) * 2016-11-03 2018-05-03 Muhammed Majeed Enzyme composition for therapeutic management of muscle soreness

Non-Patent Citations (1)

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Title
"A Superior Therapeutic Formulation Stable Room Temperature Probiotic", PREVENTIX NUTRITION, PROXYME-GI(TM, 25 April 2017 (2017-04-25), XP055746399, Retrieved from the Internet <URL:https://web.archive.Org/web/20170425160020/http://www.preventixnutrition.com/proxyme-gi.html> [retrieved on 20190509] *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113341119A (zh) * 2021-05-13 2021-09-03 杭州海路医疗科技有限公司 间接检测乳糖不耐受的方法
CN114480175A (zh) * 2021-12-28 2022-05-13 广东海大集团股份有限公司 一种凝结芽孢杆菌菌剂及其制备方法与应用

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