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WO2025246169A1 - Lactobacillus gasseri bn9 pour améliorer la capacité de perception de la sucrosité, aliment fermenté associé à ceci et utilisation associée - Google Patents

Lactobacillus gasseri bn9 pour améliorer la capacité de perception de la sucrosité, aliment fermenté associé à ceci et utilisation associée

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Publication number
WO2025246169A1
WO2025246169A1 PCT/CN2024/128053 CN2024128053W WO2025246169A1 WO 2025246169 A1 WO2025246169 A1 WO 2025246169A1 CN 2024128053 W CN2024128053 W CN 2024128053W WO 2025246169 A1 WO2025246169 A1 WO 2025246169A1
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WO
WIPO (PCT)
Prior art keywords
lactobacillus gasseri
preparation
product
obese
products
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Pending
Application number
PCT/CN2024/128053
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English (en)
Chinese (zh)
Inventor
李莉
赵伟
杨政超
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Jiangnan University
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Jiangnan University
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Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • This invention relates to Lactobacillus gasseri BN9, which enhances the perception of sweetness, its fermented foods, and their applications, and belongs to the field of microbial technology.
  • the gut microbiota often referred to as the "internalized external environment," participates in various physiological activities of the host, including digestion, metabolism, and immunity.
  • the gut-brain axis is a bidirectional communication system between the gut microbiota and the brain; the gut microbiota can influence host brain function and behavior through this axis.
  • gut microbiota can regulate the host's sensory decision-making, influencing responses to odors and food choices; it can also affect the host's perception of sweetness. Therefore, regulating the gut microbiota through probiotics, thereby modulating an individual's sweetness perception and reducing active sugar intake, represents a novel and potential approach.
  • probiotics are mainly to regulate intestinal function; no research has reported that any probiotic can improve individual.
  • the technical problem to be solved by this invention is to provide a probiotic that can effectively improve an individual's ability to perceive sweetness and its application.
  • this invention provides Lactobacillus gasseri BN9, which was deposited on January 28, 2024, at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No:64320. The deposit address is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
  • the viable count of Lactobacillus gasseri BN9 in the microbial preparation is not less than 1 ⁇ 108 CFU/mL or 1 ⁇ 108 CFU/g.
  • the present invention also provides a product containing the above-mentioned Lactobacillus gasseri BN9 or the above-mentioned microbial preparation.
  • the product includes food, medicine, or health products.
  • the food includes fermented food or feed additive; the fermented food includes dairy products, soy products, fruit and vegetable products or other fermented foods containing Lactobacillus gasseri BN9.
  • the viable count of Lactobacillus gasseri BN9 in the product is not less than 1 ⁇ 108 CFU/mL or 1 ⁇ 108 CFU/g.
  • the fruit and vegetable products include one or more products made from cucumbers, carrots, beets, celery, cabbage, and other edible fruits and vegetables.
  • the fermented food further contains additives, which are selected from one or more combinations of spices, fruit and vegetable juices, flower tea juices, colorants, acidity regulators, preservatives, antioxidants, thickeners, and sweeteners.
  • the fermented food is processed into solid food, liquid food, or semi-solid food.
  • the present invention also provides the use of the above-mentioned Lactobacillus gasseri BN9 or the above-mentioned microbial preparation in the preparation of drugs that enhance the ability to perceive sweetness.
  • the present invention also provides the use of the above-mentioned Lactobacillus gasseri BN9 or the above-mentioned microbial preparation in the preparation of pharmaceuticals or health products that help maintain healthy blood sugar levels.
  • the present invention also provides the application of the above-mentioned Lactobacillus gasseri BN9 or the above-mentioned microbial preparation in the preparation of products that reduce the weight of obese individuals.
  • the Lactobacillus gasseri BN9 provided by this invention has excellent intestinal colonization ability, can significantly improve the sweet taste perception ability of healthy individuals, obese individuals, and diabetic individuals, and has the effect of lowering blood sugar.
  • Lactobacillus gasseri BN9 of the present invention can significantly improve the body's ability to perceive sweetness, significantly increase the content of amino acids such as glutamic acid (7.99 times), glycine (6.02 times), and lysine (6.74 times) in the intestine, and significantly increase the content of amino acids such as glutamine (6.68 times), serine (4.81 times), and glycine (7.02 times) in the blood;
  • Lactobacillus gasseri BN9 of this invention significantly improves the body's sweet taste perception, reduces the weight of obese mice, and improves blood glucose homeostasis.
  • Feeding Lactobacillus gasseri BN9 can increase the preference of obese and diabetic mice for low-concentration sucrose solutions (10 mmol/L and 15 mmol/L sucrose solutions) to normal levels, reduce the weight of obese mice by approximately 21%, reduce peak blood glucose levels by approximately 23.75%, and reduce the area under the oral glucose tolerance test curve by approximately 29.40%; and reduce peak blood glucose levels in diabetic mice by approximately 26.87% and reduce the area under the oral glucose tolerance test curve by approximately 31.58%.
  • the Lactobacillus gasseri BN9 described in this invention can improve an individual's sweet taste perception ability and can be used to prepare related functional foods, health products and pharmaceuticals to reduce an individual's sugar intake. It has a very broad application prospect in regulating taste impairment caused by diseases such as obesity and diabetes and controlling an individual's blood sugar and weight.
  • Lactobacillus gasseri BN9 taxonomically named Lactobacillus gasseri
  • GDMCC No.: 64320 located at the Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
  • Figure 1 shows the effect of Lactobacillus gasseri BN9 on the sweet taste perception ability of individual bumblebees. Lactobacillus gasseri BN9 significantly improved the sweet taste perception ability of individuals; while the control strains Lactobacillus gasseri CN3 and Lactobacillus rhamnosus did not affect the sweet taste perception ability of individuals; **P ⁇ 0.01.
  • Figure 2 shows the effects of feeding Lactobacillus gasseri BN9 on amino acids in the gut and hemolymph of bumblebees.
  • A Differences in hindgut amino acids
  • B Differences in hemolymph amino acids.
  • Figure 3 shows the effect of feeding Lactobacillus gasseri BN9 on sucrose preference in obese mice; *P ⁇ 0.05.
  • Figure 4 shows the effects of feeding Lactobacillus gasseri BN9 on body weight and glucose tolerance in obese mice; (A) body weight; (B) glucose tolerance test; **P ⁇ 0.01.
  • Figure 5 shows the effect of feeding Lactobacillus gasseri BN9 on sucrose preference in diabetic mice; *P ⁇ 0.05.
  • Figure 6 shows the effect of feeding Lactobacillus gasseri BN9 on glucose tolerance in diabetic mice; **P ⁇ 0.01.
  • MRS solid medium tryptone 10.00 g/L, yeast extract 5.00 g/L, beef extract 10.00 g/L, glucose 20.00 g/L, triammonium citrate 2.00 g/L, anhydrous sodium acetate 5.00 g/L, magnesium sulfate 0.10 g/L, manganese sulfate 0.05 g/L, dipotassium hydrogen phosphate 2.00 g/L, Tween 80 1.00 mL/L, agar powder 15.00 g/L, pH adjusted to 6.5.
  • MRS liquid culture medium tryptone 10.00 g/L, yeast extract 5.00 g/L, beef extract 10.00 g/L, glucose 20.00 g/L, triammonium citrate 2.00 g/L, anhydrous sodium acetate 5.00 g/L, magnesium sulfate 0.10 g/L, manganese sulfate 0.05 g/L, dipotassium hydrogen phosphate 2.00 g/L, Tween 80 1.00 mL/L, pH adjusted to 6.5.
  • the ordinary feed involved in this invention was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., product number XTCON50J; the high-fat feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., product number XTHF60.
  • the blood glucose meter and test strips involved in this invention were purchased from Roche Pharmaceuticals Ltd., model ACCU-CHEK Guide.
  • Lactobacillus gasseri CN3 and Lactobacillus rhamnosus involved in the following examples are self-selected strains.
  • the obtained PCR product was sent to a professional sequencing company for sequencing.
  • the sequencing results were searched and compared for similarity in the GeneBank database using BLAST.
  • the sequencing results identified the strain as belonging to Lactobacillus gasseri and named it Lactobacillus gasseri BN9.
  • the strain was stored at -80°C for later use.
  • Example 2 Effects of Lactobacillus gasseri BN9 on the sweet taste perception ability of individual bumblebees
  • Sweet taste perception tests were conducted on bumblebees at 12 days of age. The specific methods and results are as follows: Individual bumblebees that had been fasted for 12 hours were placed in a tester and given 2 ⁇ l of water (0%) and sucrose solutions of 0.1%, 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, and 40% concentrations, respectively. The feeding behavior of the bumblebees was observed and recorded to assess their sweet taste perception ability. Feeding on sucrose solution indicated that the bumblebees responded to the corresponding concentration of sugar solution.
  • Example 3 Effects of Lactobacillus gasseri BN9 on host amino acid metabolism
  • the content of glutamic acid increased from 0.15 mg/g to 1.26 mg/g, an increase of 7.99 times; the content of lysine increased from 0.14 mg/g to 0.96 mg/g, an increase of 6.74 times; and the content of glycine increased from 0.16 mg/g to 0.98 mg/g, an increase of 6.02 times.
  • feeding with *Lactobacillus gasseri* BN9 significantly increased the levels of amino acids such as glutamine, serine, and glycine in bumblebee hemolymph.
  • Glutamine levels increased from 0.21 mg/g to 1.44 mg/g, a 6.68-fold increase; serine levels increased from 0.28 mg/g to 1.38 mg/g, a 4.81-fold increase; and glycine levels increased from 0.18 mg/g to 1.26 mg/g, a 7.02-fold increase.
  • Glycine and lysine are associated with maintaining brain function and regulating blood sugar; serine is associated with blood sugar regulation and weight control; glutamine and glutamate can be interconverted in the body, and glutamate plays an important role in the transmission and regulation of taste information in taste receptors. Therefore, *Lactobacillus gasseri* BN9 may enhance the sweet taste perception ability of individual bumblebees by regulating the amino acid content in the hindgut and hemolymph.
  • Example 4 Lactobacillus gasseri BN9 can improve the sweet taste perception ability of obese mice.
  • the normal control group was fed a standard diet for 12 weeks; the obese and obese + Lactobacillus gasseri BN9 groups were fed a high-fat diet for 12 weeks.
  • the normal control and obese groups received an equal volume of physiological saline by gavage daily for 4 weeks. The weight of all mice was recorded every two weeks. From week 13, all three groups were fed a standard diet, and subsequent tests were conducted.
  • Example 5 Lactobacillus gasseri BN9 can improve individual obesity symptoms.
  • Example 4 the body weight and oral glucose tolerance test (OGTT) of the three groups of mice were compared. Body weight was recorded every two weeks, and OGTT was measured at week 16 after the glucose preference test. Eight weeks of high-fat diet resulted in significantly higher body weight in the mice compared to the control group. Supplementation with Lactobacillus gasseri BN9 was then initiated at week 9. Compared to the obese group, the obese mice fed with Lactobacillus gasseri BN9 showed an average body weight reduction of 2.03 g and 8.97 g at weeks 10 and 12, respectively. One month of Lactobacillus gasseri BN9 feeding significantly reduced the body weight of obese mice by 21.34% (Figure 4A).
  • mice were fasted for 14 hours but allowed free access to water. Then, each mouse was administered sterile glucose solution by gavage at a dose of 2 g glucose/kg body weight. Blood was collected from the tail of a lancet, and blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes after gavage (0 min represents the fasting blood glucose level before gavage). OGTT curves were plotted, and the area under the curve (AUC) was calculated. Obese mice exhibited impaired glucose tolerance, while supplementation with *Lactobacillus gasseri* BN9 improved glucose tolerance in obese mice.
  • AUC area under the curve
  • Lactobacillus gasseri BN9 can improve the sweet taste perception ability of diabetic mice.
  • the normal control group mice were fed a standard diet for 12 weeks.
  • a high-fat diet was fed for 12 weeks, followed by an 8-week high-fat diet.
  • Mice fed a high-fat diet were then given a single intraperitoneal injection of streptozotocin (STZ, 50 mg/kg) to induce diabetes and obtain diabetic mice.
  • STZ streptozotocin
  • mice in each group Two identical water bottles were placed in each cage, one containing a sucrose solution with a concentration set to [missing information]. Two solutions were tested: one containing 1, 5, 10, 15, 20, 25, and 50 mmol/L, and the other containing deionized water, ensuring consistent solution volume in both solutions. To avoid errors caused by positional bias, the positions of the two tubes were swapped every 24 hours, and each sugar solution concentration was tested for 48 hours. During testing, the solution concentration was increased sequentially from low to high. Results showed that diabetic mice exhibited a decreased preference for lower concentrations of sucrose solution, while feeding Lactobacillus gasseri BN9 significantly improved their preference for lower concentrations, restoring it to a level comparable to the control group.
  • the preference rate was 57.96% in diabetic mice fed with Lactobacillus gasseri BN9, compared to 60.32% in the control group and 52.9% in diabetic mice; for a 15 mmol/L sucrose solution, the preference rate was 60.21% in mice fed with Lactobacillus gasseri BN9, compared to 62.53% in the control group and 54.86% in diabetic mice. It is evident that diabetic mice have a decreased ability to perceive sweetness, while Lactobacillus gasseri BN9 can improve the ability to perceive sweetness in diabetic mice (Figure 5).
  • Activated Lactobacillus gasseri BN9 was cultured anaerobically at 37°C for 48 hours in MRS liquid medium. 30 mL of the Lactobacillus gasseri BN9 culture was placed in a centrifuge tube and centrifuged at 4°C and 4000 rpm for 10 min. The supernatant was discarded, and the bacterial sludge was collected. After washing 2-3 times with strictly sterilized physiological saline, an appropriate amount of cryoprotectant was added and mixed thoroughly. The mixture was pre-cooled at -80°C for 4 hours and then freeze-dried under the following conditions: cold trap temperature -70°C, drying time 24 hours, and vacuum degree 1 Pa. After freeze-drying, the bacterial powder was immediately collected into aseptic packaging bags and sealed to obtain Lactobacillus gasseri BN9 bacterial agent.
  • Fresh milk and white sugar were mixed and homogenized (5:1), sterilized at 140°C for 2 seconds, cooled to 35°C, and then inoculated with the Lactobacillus gasseri BN9 inoculum prepared in this invention.
  • the mixture was sealed and fermented at 35°C for 4 hours, and then transferred to a refrigerator at 4°C for 12 hours for post-ripening to obtain the final fermented product (bacterial concentration above 108 CFU/mL).
  • This invention enables the production and preparation of other fermented foods using Lactobacillus gasseri BN9 fermentation.
  • These fermented foods include solid foods, liquid foods, and semi-solid foods.
  • the fermented foods include dairy products, soy products, and fruit and vegetable products.
  • Products include milk, sour cream, and cheese; the fruit and vegetable products include cucumber, carrot, beet, celery, and cabbage products.

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Abstract

Sont divulgués Lactobacillus gasseri BN9 pour améliorer la capacité de perception de la sucrosité, un aliment fermenté associé à ceci et une utilisation associée. La présente invention concerne le domaine technique des micro-organismes. La prise du Lactobacillus gasseri BN9 peut améliorer significativement la capacité de perception de la sucrosité d'organismes sains et réguler le métabolisme des acides aminés intestinaux. De plus, la prise du Lactobacillus gasseri BN9 peut améliorer significativement la capacité de perception de la sucrosité des organismes obèses et diabétiques, réduire le poids corporel des souris obèses et améliorer l'homéostasie glycémique d'individus obèses et diabétiques. En conclusion, le Lactobacillus gasseri BN9 peut être utilisé dans la préparation d'aliments fonctionnels, de produits de soins de santé et de médicaments pour améliorer et augmenter la capacité de perception de la sucrosité d'individus et présente de très larges perspectives d'application dans la régulation d'une déficience du goût provoquée par des maladies telles que l'obésité et le diabète, la régulation du glucose sanguin chez un individu et d'autres aspects.
PCT/CN2024/128053 2024-05-29 2024-10-29 Lactobacillus gasseri bn9 pour améliorer la capacité de perception de la sucrosité, aliment fermenté associé à ceci et utilisation associée Pending WO2025246169A1 (fr)

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CN202410677525.2A CN118703356A (zh) 2024-05-29 2024-05-29 提高甜味感知能力的格氏乳杆菌bn9、其发酵食品及其应用

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