NL2026360B1 - Uses of Bifidobacterium animalis A12 in the Control of Diabetes or Hyperlipidemia, Especially Weight Gain or Obesity - Google Patents
Uses of Bifidobacterium animalis A12 in the Control of Diabetes or Hyperlipidemia, Especially Weight Gain or Obesity Download PDFInfo
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- NL2026360B1 NL2026360B1 NL2026360A NL2026360A NL2026360B1 NL 2026360 B1 NL2026360 B1 NL 2026360B1 NL 2026360 A NL2026360 A NL 2026360A NL 2026360 A NL2026360 A NL 2026360A NL 2026360 B1 NL2026360 B1 NL 2026360B1
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- 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
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
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- 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
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
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- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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Abstract
The present invention relates to the field of microbiology, specifically relates the uses of Bifidobaclerium animalis A12 in the control of diabetes or hyperlipidemia, especially 5 weight gain or obesity. Bifidobaclerium animalis A12 is particularly outstanding in inhibiting d-glucosidase activity, inhibiting glucose transport, increasing glucose tolerance level in the body, alleviating insulin resistance and leptin resistance, increasing the secretion level of glucagon-like peptide-l, or increasing the size and quantity of islet ß - cells and so on. Bifidobaclerium animalis A12 can prevent and/or treat diabetes or 10 hyperlipidemia, and especially, it may control body weight gain and obesity. 15
Description
DESCRPTION Uses of Bifidobacterium animalis A12 in the Control of Diabetes or Hyperlipidemia, Especially Weight Gain or Obesity
FIELD The present invention relates to the field of microbiology, specifically relates the uses of Bifidobacterium animalis A12 in the control of diabetes or hyperlipidemia, especially weight gain or obesity.
BACKGROUND With development of economy and improvement of people's living level, people begin to pay attention to obesity, insulin resistance and impaired glucose tolerance and etc. caused by high-calorie and poorly structured diet (high fat, high protein, and low carbohydrate). Long-term poor dietary habit will eventually lead to diabetes. In recent years, diabetes has become popular all over the world as the third chronic disease that seriously endangers human health after tumors and cardiovascular diseases while China has the second largest number of diabetes patients in the world, secondary only to India. Therefore, it is urgent to develop uses as preventive measures to improve blood glucose level.
In terms of functional foods, the effect of probiotics for improvement of blood glucose level has been proved. However, there are very few relevant uses at present. The mechanism of action is not definite, and many strains, starter cultures and key technologies for functional probiotics are monopolized by foreign countries. Some studies have shown that foreign probiotics are not necessarily suitable for the Chinese physical characteristics and physique Therefore, it is important to study the probiotics suitable for the specific physiological constitution of Chinese people.
SUMMARY The purpose of present invention is to provide uses of Bifidobacterium animalis A12 in the control of diabetes or hyperlipidemia, especially weight gain or obesity based on probiotics.
To attain the above object, in a first aspect, the present invention provides uses of Bifidobacterium animalis in preparation of products that inhibit g-glucosidase activity, inhibit glucose transport, increase glucose tolerance level in the body, alleviate insulin resistance and leptin resistance, increase the secretion level of glucagon-like peptide-1, or increase the size and quantity of islet B — cells; wherein the Bifidobacterium animalis has a Preservation Number of CGMCC No. 17308.
In a second aspect, the present invention provides uses of Bifidobacterium animalis in preparation of products used for the prevention and/or treatment of diabetes; wherein the Bifidobacterium animalis has a Preservation Number of CGMCC No. 17308.
In a third aspect, the present invention provides uses of Bifidobacterium animalis in 40 preparation of products used for the prevention and/or treatment of weight gain or obesity. wherein the Bifidobacterium animalis has a Preservation Number of CGMCC No. 17308.
1
In a fourth aspect, the present invention provides a food or drug containing substance from Bifidobacterium animalis cells and/or metabolites of Bifidobacterium animalis, wherein the metabolites are provided in culture solution of the Bifidobacterium animalis; wherein the Bifidobacterium animalis has a Preservation Number of CGMCC No. 17308.
The present invention can achieve the following beneficial effects: 1) The present invention proposes the uses of Bifidobacterium animalis in inhibiting a-glucosidase activity, inhibiting glucose transport, increasing glucose tolerance level in the body, alleviating insulin resistance and leptin resistance, increasing the secretion level of glucagon-like peptide-1, or increasing the size and quantity of islet B — cells and so on.
2) The present invention provides a specific strain of Bifidobacterium animalis, Preservation Number: CGMCC No. 17308, which is particularly outstanding in inhibiting a-glucosidase activity, inhibiting glucose transport, increasing glucose tolerance level in the body, alleviating insulin resistance and leptin resistance, increasing the secretion level of glucagon-like peptide-1, or increasing the size and quantity of islet B - cells and so on.
3) The Bifidobacterium animalis CGMCC No. 17308 provided by present invention can prevent and/or treat diabetes or hyperlipidemia, and especially, it may control body weight gain and obesity; 4) The Bifidobacterium animalis CGMCC No. 17308 provided by present invention is isolated from the excrement of breastfed infants in China, and so, it is suitable for the specific physiological constitution of Chinese people. It is very important for us to expand the probiotics library with independent intellectual property rights of China and enhance the functionality of fermented foods.
Other features and advantages of the present invention will be further detailed in the embodiments hereunder.
Biological Preservation Bifidobacterium animalis The Bifidobacterium animalis disclosed in present invention was preserved in China General Microbiological Culture Collection Center (CGMCC) (address: Institute of Microbiology under Chinese Academy of Sciences, Building 3, No.1 Beichen West Road, Chaoyang District, Beijing, postal code: 100101) on March 6, 2019 and numbered as CGMCC No. 17308, called for short as A12.
BRIEF DESCRITION OF THE DRAWINGS The accompanying drawings are provided here to facilitate further understanding on the 40 present disclosure, and constitute a part of this document. They are used in conjunction with the following examples to explain the present disclosure, but shall not be comprehended as constituting any limitation to the present disclosure.
Figure 1 is the transepithelial electrical resistance (TEER) change curve of Caco-2 cells during the culture process.
45 Figure 2 shows the micrographs of inverted microscope (A: *100) and scanning electron microscope (B: x7,000; C: x10,000) on the 21st day for culture of Caco-2 cells.
2
Figure 3 shows the effect of the treatment on glucose transport capacity in the Transwell model for all groups. Figure 4A shows the tolerance evaluation for each strain in simulated gastric juice. Figure 4B shows the tolerance evaluation for each strain in simulated intestinal fluid. Figure 5A shows the change in body weight of mice with time for each group.
Figure 5B shows the serum insulin levels of mice treated in each group.
Figure 5C shows the serum leptin levels of mice treated in each group. Figure 6 shows the immunofluorescence staining results for pancreatic tissues of mice in each group, including nucleus (blue), insulin (green) and glucagon (red). Figure 7 shows the ratio of B-cells to a-cells in the islet tissues of mice treated in each group.
Figure 8 shows the change in blood glucose level with time of mice given glucose solution by gavage in each group.
Figure 9 shows the AUC for glucose tolerance peak within 120min of mice given glucose solution by gavage in each group. Figure 10 shows the serum GLP-1 content of mice treated in each group.
Figure 11 shows relative transcription levels of S-1, SGLT-1, Glut-2, GCG and PC3 in the small intestine of mice treated in each group.
DETAILED DESCRPTION Hereunder some embodiments of the present invention will be detailed. It should be understood that the embodiments described herein are only provided to describe and explain the present invention rather than constitute any limitation to the present invention.
Introduction on Bifidobacterium animalis CGMCC No. 17308 (Bifidobacterium animalis A12) The Bifidobacterium animalis mentioned above is isolated from the excrement of breastfed infants.
Liquid culture of Bifidobacterium animalis mentioned above may be produced large amount of viable Bifidobacterium animalis. There are no special requirements for the culture method. The method is acceptable as long as it enables the proliferation of Bifidobacterium animalis. For example, viable Bifidobacterium animalis may be inoculated in 107 CFU/mL inoculum size into a Lactobacillus culture solution, and cultured under an anaerobic or aerobic condition at 15-38°C temperature for 8-72h to obtain a culture solution. the lactobacillus culture solution may be selected from kinds of media suitable for lactobacillus culture well known in the field. For example, the lactobacillus culture solution may be milk and/or the lactic acid bacteria (MRS) broth described in "Lactic Acid Bacteria - Biological Basis and Application" (Yang Jiebin, Light Industry Press, 1996).
40 The inventor of present invention found that Bifidobacterium animalis A12 was more advantageous for growth in the modified MRS broth, and the substance from Bifidobacterium animalis A12 cells or metabolite cultured thereby was better in above effect. The formula for modified MRS broth 1s as follows: K2PO:: 1-3g/L, anhydrous sodium acetate: 4-6 g/L, malt extract powder: 4-6 g/L, magnesium sulfate: 0.4-0.6 g/L, beef extract: 8-12 g/L, ammonium 45 citrate: 1-3 g /L, maltose :15-25 g /L, soy peptone: 4-6 g /L, manganese sulfate monohydrate: 3
02-03 g/L, Twean-80: 0.5-1.5mL /L, cysteine hydrochloride: 0.4-0.6 g/L, inulin: 4-6 g/L. The viable Bifidobacterium animalis in above medium may be further separated. There are no special requirements for the separation. The method is acceptable as long as it may enrich the Bifidobacterium animalis from culture solution. For example, the separation may be implemented by centrifugation and/or filtration, and the conditions of the centrifugation and filtration may be well-known conditions. That aspect will not be further detailed here.
The o-glucosidase inhibitor is an oral hypoglycemic agent that may treat diabetes through delaying the absorption of carbohydrates in the intestine. It slows down the breakdown of carbohydrates into glucose through competitively inhibiting various a-glucosidases at the small intestine, thus slowing the absorption and transport of glucose in the intestine, reducing postprandial hyperglycemia, and improving glucose tolerance. The inventor of present invention found in a study that Bifidobacterium animalis A12 can be used as a a-glucosidase inhibitor.
L cells in the intestine may secrete glucagon-like peptide-1 (GLP-1), which can promote the production of insulin by islet - cells and inhibit the production of glucagon by islet a - cells, thus regulating the body's blood glucose balance and improving glucose tolerance. The inventor of present invention found that Bifidobacterium animalis A12 can increase the secretion of glucagon-like peptidin-1 (GLP-1).
In addition, the inventor of present invention also found in a study that Bifidobacterium animalis A12 could increase the size of islet cells and the quantity of islet B - cells, as well as alleviate insulin resistance and leptin resistance, thus further improving the glucose tolerance. Based on above findings, in a first aspect, the present invention provides uses of Bifidobacterium animalis CGMCC No. 17308 in preparation of products that inhibit a-glucosidase activity, inhibit glucose transport, increase glucose tolerance level in the body, alleviate insulin resistance and leptin resistance, increase the secretion level of glucagon-like peptide-1, or increase the size and quantity of islet B - cells.
In a second aspect, the present invention provides uses of Bifidobacterium animalis CGMCC No. 17308 in the preparation of products used for the prevention and/or treatment of diabetes.
In a third aspect, the present invention also provides uses of Bifidobacterium animalis CGMCC No. 17308 in preparation of products used for the prevention and/or treatment of weight gain or obesity.
As compared with other kinds of Bifidobacterium animalis, the Bifidobacterium animalis CGMCC No. 17308 provided in the present invention is more outstanding in above effects.
The inventor of present invention further found during the study that both the substance from Bifidobacterium animalis A12 cells and metabolites of Bifidobacterium animalis A12 have 40 above functions. Thus, during above uses, the substance from Bifidobacterium animalis cells of Bifidobacterium animalis A12 (live bacteria of Bifidobacterium animalis CGMCC No. 17308) and/or its metabolites may be used.
Therefore, above products may contain live bacteria of Bifidobacterium animalis CGMCC No. 17308, metabolites of Bifidobacterium animalis CGMCC No. 17308, or both.
45 As known, the metabolites generally exist in the culture solution of the bacteria, and so, the metabolites can be obtained by obtaining a clear liquid from a solid-liquid separation process for culture solution of the bacteria. The culture solution of the bacteria can be obtained as follows: cultivate Bifidobacterium animalis CGMCC No. 17308 provided by the invention at a 4 temperature of 35-37°C under anaerobic conditions for 12-72 hours. The solid-liquid separation method, for example, may be centrifugation method and/or filtration method. In a fourth aspect, the present invention provides a food or drug containing the substance from Bifidobacterium animalis cells and/or metabolites of Bifidobacterium animalis CGMCC No. 17308; wherein the metabolites are provided in culture solution of the Bifidobacterium animalis. According to the present invention, preferably, 10-70 parts (more preferably 30-50 parts) of metabolites of the Bifidobacterium animalis contained relative to 100 parts of the food or drug by weight when the food or drug containing the metabolites of the Bifidobacterium animalis, 103-10! CFU (more preferably 107-10° CFU) substance from Bifidobacterium animalis A12 cells relative to lg of the food or drug by weight when the food or drug containing the substance from Bifidobacterium animalis A12 cells. In above preferred cases, the effect of the food or drug on prevention and treatment of hyperlipidemia is more significant. Wherein, the content of the metabolites is measured with the clear liquid obtained through solid-liquid separation of the culture solution.
CFU (Colony-Forming Units) refers to the number of viable bacteria. For viable culture count, a colony formed through growth and propagation of a single bacterium or a group of bacteria on a solid medium is called a colony forming unit, which represent the number of viable bacteria. The cultured medium can be diluted to a suitable level and coated on a plate for incubation.
According to the present invention, the foods may be any type of foods, such as fruit juice products, dairy products, soy products, and etc. which contain a food as product substrate. The foods may vary dependent on different consumers. The foods may also contain conventional additives, such as flavors, minerals, vitamins, stabilizers, thickeners, and preservatives, which are acceptable as auxiliary materials for foods.
In addition, the foods that meet above requirements may contain the culture solution of the Bifidobacterium animalis (for example, fermented dairy products produced through fermentation of the Bifidobacterium animalis), isolated live bacteria of the Bifidobacterium animalis, and etc.
According to the invention, the drugs may be prepared into any types, such as tablets, emulsions, capsules, suppositories, and etc., and may vary dependent on the users. The drugs may also contain pharmaceutically acceptable adjuvants as product substrate, e.g. excipients, lubricants, flavoring agents, stabilizers, thickeners, preservatives, and etc, which may be selected according to specific dosage forms. That is well known to those skilled in the art. According to present invention, the foods or drugs may be prepared through adding 40 substance from Bifidobacterium animalis A12 cells and/or metabolites of Bifidobacterium animalis A12 to the product substrate.
According to present invention, there are no special restrictions on the method of adding the substance from Bifidobacterium animalis A12 cells and/or metabolites of Bifidobacterium animalis A12 to the product substrate. For example, the substance from Bifidobacterium 45 animalis A12 cells and/or metabolites of Bifidobacterium animalis A12 can be directly mixed with the product substrate in a predetermined proportion before preparation of corresponding products.
Hereunder present invention will be further detailed in preparation examples, embodiments and comparisons, but present invention is not limited to those embodiments. 5
In the following preparation examples, embodiments and comparisons: Experimental strain: the Bifidobacterium animalis disclosed in present invention was preserved in China General Microbiological Culture Collection Center (CGMCC) (address: Institute of Microbiology under Chinese Academy of Sciences, Building 3, No.1 Beichen West Road, Chaoyang District, Beijing, postal code: 100101) on March 6, 2019 and numbered as CGMCC No. 17308, called for short as A12. Reference strain: Bifidobacterium animalis BB12, called for short as BB12. Modified MRS broth formula: (K2PO4: 2 g/L, anhydrous sodium acetate: 5 g/L, malt extract powder: 5 g/L, magnesium sulfate: 0.5 g /L, beef extract: 10 g /L, ammonium citrate: 2 g /L, maltose: 20 g /L, soy peptone: 5 g/L, manganese sulfate monohydrate: 0.25 g /L, Twean-80: 1 ml/L, cysteine hydrochloride: 0.5 g /L, inulin: 5 g /L., supplemented with distilled water to 1000 ml (solid medium added with 15 g agar), pH6.5, sterilized at 121 °C for 15 min, and then stored before use. Above reagents were purchased from Biomics Biotech; Caco - 2 cells (human colon carcinoma cell lines) were purchased from Procell Life Science &Technology Co, Ltd, Wuhan, Article number: CL - 0050, derived from human colon carcinoma cells. In the Transwell chamber, Caco - 2 cells can form dense monolayer tissues, differentiate into cellular villous surface similar to that of the small intestine cells (apical, equivalent to the side of the enteric cavity) and basal surface (basolateral, equivalent to the side of inner bowel wall), as well as kinds of enzymes, including a-glucosidase. The Transwell model constructed by Caco-2 cells has been widely used in biochemistry, toxicology and nutrient uptake and transport studies. Preparation Example Preparation of bacteria solution
1. Inoculate Generation 2 of activated Bifidobacterium animalis A12 into the modified MRS broth, and perform an anaerobic culture at 37°C for 12h. After the culture, take 8000 g of the culture for centrifugation at 4 °C for 10 min, collect the supernatant, filter through a 0.22 um membrane, adjust the pH value to 6.5. The fermented supernatant was thus obtained.
2. Inoculate Generation 2 of activated Bifidobacterium animalis A12 and Bifidobacterium animalis BB12 into the modified MRS broth, and perform an anaerobic culture at 37°C for 12h. After the culture, take 8000 g of the culture for centrifugation at 4 °C for 10 min, and collect the bacteria. Wash with sterile PBS buffer (pH 7.4) for 3 times and resuspend it in PBS solution. Adjust the number of cells to 10°%/mL. TheA12 and BB12 viable bacterial suspensions were thus obtained. Example 1: 40 This example was used to illustrate the establishment of a Transwell cell model and the inhibitory effect of Bifidobacterium animalis A12 fermented supernatant on glucose transport.
1. Inoculate Caco-2 cells to a 12-well Transwell chamber (membrane aperture: 0.4um, area for growth: 1.12 em?), add 500 pL of 2x 10° cells /mL suspension to the enteric cavity-side of transport tank (upper compartment, AP side), and 1.5 mL of cell thallus solution to the 45 basal side (lower compartment, BL side). Change the medium once a day. After 21 days of cell growth and differentiation when the transepithelial electrical resistance (TEER) was more than 500Q-cm’, the cell fusion was complete and the Transwell cell model could be used in the transport experiment. 6
The results were as shown in Fig. 1. TEER increased rapidly in the first 11 day, reached the maximum in 12 days (1300 +/ - 110.82 Q cm?), indicating dense monolayer structure formed by Caco - 2 cells. TEER increased slightly 12-21 days after cell inoculation, and then the number of cells tended to be stable, indicating that the cell proliferation stopped and cell differentiation began. The closely connected structures and microvilli structure of the cells on day 21 were observed with inverted microscope and scanning electron microscope (Fig. 2). Monolayer Caco-2 cells (Fig. 2A: cellular morphology under optical inverted microscope (x100), 2B: under scanning electron microscope (x7,000)) and differentiated monolayer apical microvilli of Caco-2 cells (Fig. 2C: under scanning electron microscope (x 10,000)) were observed.
2. Transwell chamber was rinsed with PBS twice to remove glucose. Add 475 pL of 20 mmol/L maltose (after filtration sterilization) as the substrate, 25 uL of Bifidobacterium animalis A12 fermented supernatant (CFS - A12 group) prepared as above or 25 uL of modified MRS broth as negative control (blank control group) (total 0.5 mL of substrate and samples together) to the mucous membrane layer, and then add 1.5 mL of PBS to the lower layer. Incubate at 37 °C for 2 h, and test the content of glucose in the upper and lower chambers, based on which the inhibition rate (%) of glucose transport was calculated according to the following formula. Inhibition rate (%) = [1-(A/B)]* 100 Where, A =the lower chamber (test group) / [the upper chamber (test group) the lower chamber (test group)], B = the lower chamber (blank group) / [the upper chamber (test group) + the lower chamber (blank group)]. The calculated inhibition rate of glucose transport by Bifidobacterium animalis A12 was
25.93% 0.003% while Bifidobacterium animalis BB12 had no inhibitory effect on glucose transport. Therefore, the supernatant of Bifidobacterium animalis A12 showed excellent inhibitory activity and potential anti-diabetes ability. The results were as shown in Fig. 3. In the upper and lower chamber of Transwell, the glucose concentration of Bifidobacterium animalis A12 group was significantly higher than that of the blank control group (0.611+£0.002,0.140+0.001; 0.091+0.002,0.141+0.004, p <0.01). Example 2: This example was used to demonstrate the effect of Bifidobacterium animalis on a-glucosidase in vitro and on the simulated alimentary canal. 1) Preparation of cellular contents Inoculate the strains (A12 and BB12) in 2 v/v % inoculum size into the modified MRS broth, and perform an anaerobic culture at 37°C for 12h. Subculture to the third generation and store 40 before use. Centrifuge the activated bacteria solution at 6 000xg, 4 °C for 10 min, collect the supernatant, adjust the pH value to 6.0, filter through a 0.22 um membrane, and collect the filtrate as fermented supernatant. Wash the bacterial precipitation with sterile PBS buffer (pH7.4) for 3 times and resuspend it in PBS solution. Adjust the concentration of bacteria to about 1x10° CFU/mL. Then crush with Beadbeater for 30s, centrifuge at 12,000 xg for 10 min, 45 and the collected supernatant are the cellular contents.
2) Determination of a-glucosidase In the 210 pL reaction system of 96-well plate, add in sequence 50 pL of 0.1 mol/L PBS solution(pH6.8), 50 uL of 20mmol/L PNPG solution (substrate of a-glucosidase), 30 pL of 7 sample solution (A12 cellular content or BB12 cellular content), or 30 uL of PBS solution (0.1mol/L, pH6.8) as blank control, and incubate at 37°C for 10 min.
Then add 30 uL of 0.4 U/mL o-glucosidase solution or the same volume of PBS solution (0. 1mol/L, pH6.8) as blank control.
Continue the reaction at 37 °C for 30 min, and finally, add 50 uL of Imol/L Na:COs solution to stop the reaction.
After the reaction, measure the absorbance value at 405 nm with a microplate reader in triplicate for each group, and calculate the a-glucosidase inhibition rate (%) with the following formula. a-glucosidase inhibition rate (%) =[1-(A—B)/(C—D)]x 100% In the formula: A represents the sample group containing sample solution and a-glucosidase solution; B represents the blank group containing sample solution, but no a-glucosidase solution (including PBS solution of the same volume as a-glucosidase solution). C represents the control group containing a-glucosidase solution, but no sample solution (including PBS solution of the same volume as sample solution) . D represents the blank group containing no sample solution (including PBS solution of the same volume as sample solution) and no a-glucosidase solution (including PBS solution of the same volume as a-glucosidase solution). Where the o-glucosidase inhibition rate of A12 cell content is 18.57+1.90, while BB12 cell content showed no a-glucosidase inhibitory effect. 3) Evaluation on tolerance of simulated gastrointestinal tract Inoculate the strains (A12 and BB12) in 2 v/v % inoculum size into artificial gastric juice of pH 3 (pepsin 1:10,000, Sigma, USA, final concentration: 3 g /L) and artificial intestinal juice of pH 6.98 (trypsin 1:250, Sigma, USA, final concentration: 1 g /L) and incubate at 37°C.
Perform gradient dilution at 0 h, 1 h, 2 h, 3 h and 4 h of incubation, and select an appropriate gradient for plate count in triplicate for each group.
The tolerance in artificial gastric juice was as shown in Fig. 4A, and the tolerance in artificial intestinal juice was as shown in Fig. 4B.
According to Figs. 4A and 4B, A12 showed a gastrointestinal tolerance better than that of BBI2. Example 3: This example was used to illustrate the effect of bifidobacteria on the body weight in obese mice Test mice: 60 healthy male C57BL / 6J mice aged 3-5 weeks.
The mice were fed in standard cages (3 mice per cage) for free access to water and food.
For the animal house for test, the temperature was maintained at 22-25 °C, and the humidity was maintained at 40-60%, with strict 12h light /12h dark cycle.
The mice were fed a basal diet for seven days to adapt to the 40 new environment before the study began.
The mice were randomized into 4 groups (n = 15 for each group), as follows: (1) LFD group (basal diet +PBS 0.2ml); (2) HFD group (high-fat diet + PBS 0.2ml); (3) CFS-A12 group (high-fat diet and 0.2ml of Bifidobacterium animalis A12 fermented 45 supernatant), 8
(4) Al2 viable bacteria group (high-fat diet and 0.2ml of viable Bifidobacterium animalis A12 (10° CFU/ml); Feed for 10 weeks. Measure the body weight and daily food intake of the rats with a digital scale weekly. As shown in Fig. SA, high-fat diet led to the development of adverse conditions in mice, which was mainly manifested as weight gain. At the 10th week, the body weight in LFD group and CFS-A12 group decreased significantly (LFD: 25.40+1.42, CFS-A12: 26.35+1.31; HFD:
29.85+1.01, P <0.05). Obviously, in CFS-A 12 group, the adverse conditions caused by high-fat diet were significantly alleviated, which was mainly manifested as weight loss (P <0.05). The viable A12 group had little effect on weight loss in the early stage (0-7 days), but showed a good weight loss effect (27.61+1.53) in the 10th week. Example 4: This example was used to illustrate the effect of Bifidobacterium animalis on insulin resistance and leptin resistance in obese mice Test mice: 75 healthy male C57BL / 6J mice aged 3-5 weeks. The mice were fed in standard cages (3 mice per cage) for free access to water and food. For the animal house for test, the temperature was maintained at 22-25 °C, and the humidity was maintained at 40-60%, with strict 12h light /12h dark cycle. The mice were fed a basal diet for seven days to adapt to the new environment before the study began. The mice were randomized into 5 groups (n = 15 for each group), as follows: (1) LFD group (basal diet +PBS 0.2ml); (2) HFD group (high-fat diet + PBS 0.2ml); (3) CFS-A12 group (high-fat diet and 0.2ml of Bifidobacterium animalis A12 fermented supernatant), (4) A12 viable bacteria group (high-fat diet and 0.2ml of viable Bifidobacterium animalis A12 (10° CFU/ml)); (5) BBI2 viable bacteria group (high-fat diet and 0.2ml of viable Bifidobacterium animalis A12 (10° CFU/ml) ); After 10 weeks of feeding, all the mice fasted for 12h, but during the process, water was given. Then the mice were given glucose solution by gavage at 2.5g/kg body weight. After 2 hours, the mice were sacrificed, and the fresh blood samples were taken and placed at room temperature for 30min. The samples were centrifuged at 3000g and 4°C for Smin, and the serum was stored at -80°C until determination. The serum insulin and leptin levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit. The results were as shown in Figs.5B and SC. As compared with the LFD group, the intake of 40 high-fat diet resulted in development of adverse conditions, which was mainly manifested as increased insulin and leptin levels as well as insulin resistance and leptin resistance. As compared with the HFD group, the serum insulin levels of other groups decreased significantly, and the A12 group and CFS-A 12 group had an effect better than the BB12 group. These results suggested that daily intake of Bifidobacterium animalis A12 effectively improved metabolic 45 disorders in mice of poor glucose tolerance, and also alleviated insulin resistance and leptin resistance. 9
Example 5: This example was used to illustrate the effect of Bifidobacterium animalis on islet cell size and quantity of islet B — cells. Test mice: 60 healthy male C57BL / 6J mice aged 3-5 weeks. The mice were fed in standard cages (3 mice per cage) for free access to water and food. For the animal house for test, the temperature was maintained at 22-25 °C, and the humidity was maintained at 40-60%, with strict 12h light /12h dark cycle. The mice were fed a basal diet for seven days to adapt to the new environment before the study began.
The mice were randomized into 4 groups (n = 15 for each group), as follows: (1) LFD group (basal diet +PBS 0.2ml); (2) HFD group (high-fat diet + PBS 0.2ml); (3) A12 viable bacteria group (high-fat diet and 0.2ml of viable Bifidobacterium animalis A12 (10° CFU/mD)); (4) BBI2 viable bacteria group (high-fat diet and 0.2ml of viable Bifidobacterium animalis A12 (10° CFU/ml)); After 10 weeks of feeding, all the mice fasted for 12h, but during the process, water was given. Then the mice were given glucose solution by gavage at 2.5g/kg body weight. After 2 hours, the mice were sacrificed. The fresh pancreatic tissue was taken and the pancreas was sectioned (n=3 for each group). Antibodies to insulin and glucagon were used for immunostaining, and the nuclei were labeled with DAPI. For automatic image acquisition, the emission filter of FITC (536/40), Texas Red (624/40) and DAPI (447/60) was used to capture the whole pancreas section. The images were observed with a fluorescence microscope, as shown in Fig. 6 (nucleus: blue, insulin: green, with quantity of islet B — cells reflected, glucagon: red, with quantity of islet a — cells reflected). The ratio of B — cells to a -cells was as shown in Fig. 7. The results showed that both viable A12 and viable BB12 increased the islet B - cells. In addition, the ratio of islet B - cells to islet a - cells in the HFD group decreased significantly as compared with that in the LFD group (HFD: 1.01+0.85, LFD: 2.74+0.57, P <0.05). The ratios of B — cells to a -cells in the viable A12 group and viable BB12 group were higher than in the HFD group (A12 viable bacteria group: 3.65+0.94, BB12 viable bacteria group: 2.33+0.83, p >0.05). The results showed that Bifidobacterium animalis A12 could protect the islet and increase the quantity of islet B - cells.
Example 6: This example was used to illustrate the effects of Bifidobacterium animalis on glucose tolerance level in obese mice Test mice: 60 healthy male C57BL / 6] mice aged 3-5 weeks. The mice were fed in standard 40 cages (3 mice per cage) for free access to water and food. For the animal house for test, the temperature was maintained at 22-25 °C, and the humidity was maintained at 40-60%, with strict 12h light /12h dark cycle. The mice were fed a basal diet for seven days to adapt to the new environment before the study began.
The mice were randomized into 4 groups (n = 15 for each group), as follows: 45 (1) LFD group (basal diet +PBS 0.2ml}); (2) HFD group (high-fat diet + PBS 0.2ml), 10
(3) CFS-A12 group (high-fat diet and 0.2ml of Bifidobacterium animalis A12 fermented supernatant); (4) Al2 viable bacteria group (high-fat diet and 0.2ml of viable Bifidobacterium animalis A12 (10° CFU/ml)); After 10 weeks of feeding, all the mice fasted for 12h, but during the process, water was given.
Then the mice were given glucose solution by gavage at 2.5g/kg body weight. The blood glucose level of the tail vein among the mice was measured by a glycemeter at Omin, 30min, 60min and 120min (as shown in Fig. 8). The curve of blood glucose change was drawn, and the Area Under Curve (AUC) for glucose tolerance was calculated (as shown in Fig. 9).
The results showed that the blood glucose of mice in each group had an increasing trend within 30 minutes after gavage of glucose. The normal group of mice showed normal glucose tolerance. The blood glucose of mice in the normal group increased slowly within 30 minutes, decreased gradually after 30 minutes, and returned to the fasting level after 2 hours. The AUC in HFD group was significantly higher than in LFD group, CFS-A12 group and A12 viable bacteria group (P < 0.05), indicating that only high-fat diet increased the AUC level of mice, and the modeling was successful. Significant alleviation of the conditions in CFS-A12 group and A12 viable bacteria group indicated that Bifidobacterium animalis A12 effectively improved the hyperglycemia and glucose tolerance level, which was mainly reflected in the decreased AUC level.
Example 7: This example was used to illustrate the effect of Bifidobacterium animalis on GLP-1 secretion in obese mice.
The serum samples prepared in Embodiment 6 were taken out for detection of GLP-1 secretion by ELISA kit, so as to further explore the regulation effect of Bifidobacterium animalis A12 on GLP-1 secretion.
The results were as shown in Fig. 10. As compared with LFD group and HFD group, the GLP-1 level of mice in CFS-A12 group and A12 viable bacteria group increased significantly. There was no significant change in GLP-1 level in LFD group and HFD group. The results showed that Bifidobacterium animalis A12 played a hypoglycemic role through increasing the GLP-1 secretion.
Example 8: This example was used to illustrate the effect of Bifidobacterium animalis on glucosidase expression and synthesis in obese mice.
After completion of Example 6, the relative transcription levels of S-1 (a-glucosidase), SGLT-1 (sodium and glucosecotransport carrier L), GLUT-2 (glucose assisted diffusion 40 transporter 2), GCG (proglucagon), and PC3 (prohormone convertase 3) in each test group were analyzed by RT-PCR. According to the description, total RNA was extracted from pancreatic tissue using Trizol (Ambion, USA). The concentration and purity of each sample were measured on NanoDrop Lite (Thermo Scienti Fi C, USA). cDNA was obtained through reverse transcription using PrimeScript TM IT 1st Strand cDNA Synthesis Kit (TaKaRa, Japan).
45 Primer sequences were shown in Table 1. The circulating conditions were as follows: 95°C for 5min.95°C, 10s, 58°C, 30s and 72°C, 30s, for 40 cycles. The mRNA multiple was calculated with the 2-AACt method with B-actin as the control (internal reference gene). The results were as shown in Fig. 11.
1
Table1 Gene for SEQ ID Gene ID | primers sequences amplification NO. forward | TGAGAGGGAAATCGTGCGTGAC 1 primer NM-007393.3 B-actin reverse | GCTCGTTGCCAATAGTGATGACC 2 primer forward | GCCGCTGATTGGGAAGGTT 3 primer 14387 S-1 reverse
GCCTCTAACGAAGTTGGACGGT primer forward GTTTGCCTATGGAACTGGGAGC 5 primer 20537 SGLT-1 reverse GTCTGGAATGGGCTTGGTGAG 6 primer forward TGCCATCTTCCTCTTTGTCAGTTT 7 primer 20526 GLUT-2 reverse
GAAGCAGAGGGCGATGACAA EB primer forward GCTTATAATGCTGGTGCAAGG 9 primer 14526 GCG reverse CTGGGAAGCTGGGAATGAT 10 primer forward TGGAGTTGCATATAATTCCAAAGTT 11 primer 18548 PC3 reverse AGCCTCAATGGCATCAGTTAC 12 primer Figure 11A showed that the expression of S-1 mRNA in the small intestine of mice was significantly reduced in the CFS group (p<0.05), indicating that the fermented supernatant of Bifidobacterium animalis A12 reduced the expression of o-glucosidase in the small intestine of mice, and the decomposition into glucose from blocked carbohydrates reduced the glucose absorbed in the small intestine. According to Figs. 11B and 11C, the expression of glucose absorption and transport related genes (SGLT-1 and Glut-2) was significantly decreased in the 12
Al2 viable bacteria group as compared with HFD group (P <0.05), indicating that the viable Bifidobacterium animalis A12 played a hypoglycemia role through blocking the glucose absorption and transport. As shown in Figs. 11D and 11E, as compared with HFD group, all the other groups had the GCG mRNA levels significantly increased (P <0.05), while for the PC3 mRNA level, there was no significant change in the viable CFS-A12 group and A12 viable bacteria group as compared with HFD group. The results showed that Bifidobacterium animalis A12 could stimulate the synthesis of GLP-1, thus playing a hypoglycemic effect. The above content describes in detail the preferred embodiments of the present disclosure, but the present disclosure is not limited thereto. A variety of simple modifications can be made in regard to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, including a combination of individual technical features in any other suitable manner, such simple modifications and combinations thereof shall also be regarded as the content disclosed by the present disclosure, each of them falls into the protection scope of the present disclosure.
13
Sequence Listing ORE-2026360-L0602 - NLO ref.nr. P6096329NL
SEQUENCE LISTING <110> Beijing University of Agriculture <120> Uses of Bifidobacterium animalis A12 in the Control of Diabetes or Hyperlipidemia, Especially Weight Gain or Obesity <130> INL64234NXY-59083 <160> 12 <170> PatentIn version 3.3 <21e> 1 <211> 22 <212> DNA <213> |Â-actin forward primer <400> 1 tgagagggaa atcgtgcgtg ac 22 <2105 2 <211> 23 <212> DNA <213> |Â-actin reverse primer <400> 2 gctcgttgcc aatagtgatg acc 23 <2105 3 <211> 19 <212> DNA <213> S-I forward primer <400> 3 gccgctgatt gggaaggtt 19 <2105 4 <211> 22 <212> DNA <213> S-I reverse primer <400> 4 gcctctaacg aagttggacg gt 22 <216> 5 <211> 22 Pagina 1
Sequence Listing ORE-2026360-L0602 - NLO ref.nr.
P6096329NL <212> DNA <213> SGLT-1 forward primer <400> 5 gtttgcctat ggaactggga gc 22 <210> 6 <211> 21 <212> DNA <213> SGLT-1 reverse primer <400> 6 gtctggaatg ggcttggtga g 21 <210> 7 <211> 24 <212> DNA <213> GLUT-2 forward primer <400> 7 tgccatcttc ctctttgtca gttt 24 <210> 8 <211> 20 <212> DNA <213> GLUT-2 reverse primer <400> 8 gaagcagagg gcgatgacaa 20 <210> 9 <211> 21 <212> DNA <213> GCG forward primer <400> 9 gcttataatg ctggtgcaag g 21 <210> 10 <211> 19 <212> DNA <213> GCG reverse primer <400> 10 ctgggaagct gggaatgat 19 Pagina 2
Sequence Listing ORE-2026360-L0602 - NLO ref.nr.
P6096329NL <21e> 11 <211> 25 <212> DNA <213> PC3 forward primer <400> 11 tggagttgca tataattcca aagtt 25 <21e> 12 <211> 21 <212> DNA <213> PC3 reverse primer <400> 12 agcctcaatg gcatcagtta c 21 Pagina 3
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