WO2023134365A1 - Method for regulating lipid absorption, composition and application thereof - Google Patents

Abstract

A use of five compounds or a lactobacillus in the preparation of a composition for regulating lipid absorption or body weight, comprising: (a) a compound combination being a combination of DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indole lactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3-methylbutyric acid; or (b) a lactobacillus being lactobacillus having a 16S ribosomal RNA gene sequence represented by SEQ ID NO: 1, wherein the preservation number of the lactobacillus in the China Center for Type Culture Collection is CCTCC NO: M20211687. An isolated lactobacillus and metabolites, cultures or cell lysis products thereof, wherein the preservation number of the lactobacillus in the China Center for Type Culture Collection is CCTCC NO: M20211687.

Description

Methods, compositions and uses for modulating lipid uptake

The present invention claims the priority of the patent application number 202210049394.4 submitted to the State Intellectual Property Office of China on January 17, 2022, and the title of the invention is "Methods, Compositions and Applications for Regulating Lipid Absorption".

technical field

The invention belongs to the fields of biological metabolism and food science, and more specifically, the invention relates to a method, a composition and an application for regulating lipid absorption.

Background technique

Global obesity has increased significantly since the mid-1970s. Obesity threatens human health. It significantly increases the risk of diseases, such as type 2 diabetes, fatty liver, cardiovascular disease, cancer, etc., and even increases the mortality rate of patients with novel coronavirus pneumonia (COVID-19). Obesity is typically characterized by excessive accumulation of fat, the root cause of which is an imbalance in energy homeostasis, that is, too many calories consumed and too few calories burned.

Dietary restriction is considered an important method to improve lipid metabolism and reduce fat content in normal weight or obese mammals. Diets to lose weight are becoming increasingly popular among normal-weight women and men. Calorie restriction and intermittent fasting, commonly used dietary restriction methods, can significantly reduce body weight in normal and mildly overweight adults, and the weight loss is mainly fat. However, returning to a normal diet after a diet in a normal-weight woman or man can trigger weight regain. However, the mechanism of weight rebound after dietary restriction is still unclear, and effective interventions to prevent weight rebound after dietary restriction still need further research.

On the other hand, for some people with malnutrition, frequent diarrhea or emaciation, it is necessary to improve lipid metabolism in order to promote lipid absorption.

Dietary factors are key determinants of gut microbial community structure and function, nutrients can directly interact with microbes to promote or inhibit their growth, and microbiota that receive more energy from specific dietary components have a favorable competitive advantage.

However, how does the host gut microbiota composition change during refeeding after dietary restriction, and whether the gut microbiota and its metabolites are involved in refeeding-induced weight rebound after dietary restriction and small intestinal and white fat lipids during refeeding Metabolism, these issues have yet to be elucidated in the field.

Contents of the invention

The object of the present invention is to provide methods, compositions and applications for regulating lipid absorption.

In a first aspect of the present invention, there is provided a method of regulating lipid absorption or body weight, the method comprising: (a) regulating the content of a five-compound combination in the digestive tract (including the stomach or intestinal tract), the five-compound combination For: DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA), 2-hydroxyisocaproic acid (HICA) and 2-hydroxy-3-methylbutyric acid (HMBA) combination; or, (b) regulating the content of lactobacilli in the digestive tract (including stomach or intestinal tract), said lactobacillus is a lactobacillus having a 16S ribosomal RNA gene sequence shown in SEQ ID NO:1.

In one or more embodiments, the adjustment is to increase the content of the five-compound combination in the digestive tract, thereby increasing lipid absorption or body weight; or, the adjustment is to reduce the content of the five-compound combination in the digestive tract, thereby reducing lipid absorption or weight.

In one or more embodiments, the adjustment is to increase the content of Lactobacillus in the digestive tract, thereby increasing lipid absorption or body weight; or, the adjustment is to reduce the content of Lactobacillus in the digestive tract, thereby reducing lipid absorption or weight.

In one or more embodiments, the lipid absorption is intestinal lipid absorption.

In one or more embodiments, the lipids comprise fatty acids of white adipose tissue.

In one or more embodiments, the increase is a statistically significant increase, such as an increase of 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 100% or more.

In one or more embodiments, the reduction is a statistically significant reduction, such as a reduction of 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 100% or more.

In one or more embodiments, said increasing lipid absorption includes inhibiting diarrhea or improving malnutrition.

In one or more embodiments, the diarrhea is diarrhea caused by increased secretion and/or decreased absorption.

In one or more embodiments, said increasing the content of the five-compound combination in the digestive tract includes: intake of exogenous five-compound combination.

In one or more embodiments, reducing the content of the five-compound combination in the digestive tract includes: reducing the amount of lactobacilli that metabolize the five-compound, preferably using antibiotics to reduce the amount of lactobacilli.

In one or more embodiments, said increasing the content of Lactobacillus in the digestive tract includes: restricting the diet first, then restoring the diet, and taking a normal diet; or, taking in exogenous Lactobacillus.

In one or more embodiments, the reduction of the content of Lactobacillus in the digestive tract includes: restricting the diet first, then restoring the diet, taking a high-protein diet; or taking antibiotics.

In one or more embodiments, the preservation number of the lactobacillus in the China Center for Type Culture Collection is CCTCC NO: M 20211687.

In another aspect of the present invention, there is provided a five-compound combination or the use of lactobacilli or their regulators for the preparation of a composition for regulating lipid absorption or body weight; the five-compound combination is: DL-3-phenyllactic acid , 4-hydroxyphenyl lactic acid, indole lactic acid, 2-hydroxyisocaproic acid and 2-hydroxyl-3 methylbutyric acid combination; or, the lactobacillus has the 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1 Lactobacillus.

In one or more embodiments, the antibiotics include (but not limited to): vancomycin, ampicillin, neomycin, metronidazole, gentamicin, kanamycin, streptomycin, cephalosporin Piperidone, erythromycin, tylosin, amoxicillin, penicillin, bacitracin, tetracycline, doxycycline or clindamycin, etc.

In one or more embodiments, the high-protein diet (eg, 1000 parts by weight in total) includes: protein, carbohydrates, fat, cellulose, minerals and vitamins. Wherein, the protein content is 400-800 parts by weight (such as 450, 600, 650 or 800 parts by weight); preferably 500-700 parts by weight; wherein, the carbohydrate content is 150-350 parts by weight (such as 160, 180, 190, 200,220,230,240 or 280 parts by weight); preferably 180 to 300 parts by weight; wherein the fat content is 50 to 90 parts by weight (such as 55,65,75 or 85 parts by weight); preferably 60-80 parts by weight; wherein the cellulose content is 30-70 parts by weight (such as 35, 45, 55 or 65 parts by weight); preferably 40-60 parts by weight; wherein the mineral content is 15-55 parts by weight Parts (such as 28, 32, 38 or 42 parts by weight); preferably 25 to 45 parts by weight; wherein, the vitamin content is 8 to 18 parts by weight (such as 9, 12, 14, 16 or 18 parts by weight); relatively Preferably, it is 10 to 16 parts by weight.

In one or more embodiments, the protein comprises a protein selected from the group consisting of casein, cysteine, whey protein, soybean protein.

In one or more embodiments, in the high-protein food, the ratio of casein to cysteine is 50-80:1, preferably 55-75:1, more preferably 60-70:1 1.

In one or more embodiments, the carbohydrate is corn starch and/or maltodextrin; preferably, the ratio of corn starch and maltodextrin is 8-10:12-15.

In one or more embodiments, the vitamins include V10037 and choline bitartrate; preferably, the ratio of V10037 to choline bitartrate is 2-4:1; preferably 2.5-3.5:1.

In one or more embodiments, the dietary restrictions include (but are not limited to): conventional diet, intermittent diet, time-restricted diet, low-energy diet that simulates dieting, step-up or step-down diet.

In one or more embodiments, the period of dietary restriction is the time required for a significant reduction in body weight and lipids to occur. For example, this time is at least 2 days (such as 2～100 days), at least 3, 4, 5, 6, 7, 8 days or more days, such as 9, 10, 15, 20, 30, 45, 60, 80, 100 or more days.

In one or more embodiments, the dietary restriction is a step-up or step-down diet (such as providing 10%, 25%, 65% of the food amount in three days, providing 65%, 25%, 10% in three days, respectively. % of food, etc.).

In one or more embodiments, the time for resuming a diet and taking a normal diet is: the time required for a significant increase in lipids, such as 1 to 100 days (more specifically, 2, 3, 4, 5 , 6, 8, 10, 15, 20, 30, 50, 70, 80, 90 days).

In one or more embodiments, the time for resuming the diet and consuming a high-protein diet is: the time required for a significant increase in lipids, such as 1 to 100 days (more specifically, such as 2, 3, 4, 5, 6, 8, 10, 15, 20, 30, 50, 70, 80, 90 days).

In one or more embodiments, the method of modulating lipid absorption or body weight is a non-diagnostic and therapeutic method.

In one or more embodiments, the method of dietary restriction and reintake is non-diagnostic and therapeutic.

In one or more embodiments, in the five-compound combination, DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3-methylbutyric acid according to Parts by weight (or weight to volume ratio) are: 20-40:10-20:6-10:30-50:15-25.

In another aspect of the present invention, there is provided a composition for regulating (increasing) lipid absorption or body weight, comprising: DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisohexanoic acid Acid, 2-hydroxyl-3 methylbutyric acid, according to parts by weight (or weight to volume ratio): 20-40: 10-20: 6-10: 30-50: 15-25; or, lactobacillus, described The lactobacillus is a lactobacillus having a 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1; preferably, the lactobacillus is a lactobacillus whose preservation number is CCTCC NO: M 20211687 in the China Type Culture Collection Center, Its metabolites, culture or cell lysates.

In one or more embodiments, the DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indole lactic acid, 2-hydroxyisocaproic acid, and 2-hydroxy-3-methylbutyric acid are: 25 parts by weight -35:12-18:7-9:35-45:17-23.

In one or more embodiments, the DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3 methylbutyric acid are: 30 parts by weight ±3:15±1.5:8±0.8:40±4:20±2.

In one or more embodiments, the DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid, and 2-hydroxy-3-methylbutyric acid are: 30 parts by weight :15:8:40:20.

In one or more embodiments, the penta-compound combination is dissolved in water or an aqueous solvent.

In one or more embodiments, the five-compound combination is mixed with a food acceptable carrier or an industrially acceptable carrier.

In one or more embodiments, the composition is a solid, semi-solid or liquid formulation.

In one or more embodiments, the composition is a food composition.

In one or more embodiments, the method, application or composition can be used in animals; preferably in mammals; more preferably in rodents (such as mice), primates (including Humans and non-human primates such as apes, monkeys, orangutans), domestic animals (such as pigs, dogs, chickens, ducks, rabbits, etc.).

In one or more embodiments, the lactobacillus is a lactobacillus obtained by the following method: isolating a lactobacillus with a 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1 from intestinal microorganisms; preferably Alternatively, it also includes proliferating and culturing the isolated Lactobacillus.

In one or more embodiments, the genus of Lactobacillus of the 16S ribosomal RNA gene sequence is identified with primers of sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3.

In one or more embodiments, the Lactobacillus strain of the 16S ribosomal RNA gene sequence is identified with primers of sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5.

In another aspect of the present invention, isolated Lactobacillus, its metabolites, culture or cell lysate are provided, and the preservation number of the Lactobacillus in China Center for Type Culture Collection is CCTCC NO: M 20211687.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. Refeeding after dietary restriction alters the gut microbiota and induces fat accumulation, enhanced lipid absorption from the small intestine and fatty acid uptake from white fat.

(A) Principal coordinates analysis of fecal microbiota in mouse cecum. Analysis is based on Bray-Curtis distance calculations. AL, means free feeding; NP, means normal protein diet; HP, means high protein diet; DR, means to provide mice with 10%, 25%, 65% of the food in three days, and carry out dietary restriction; AL-NP (D0) means normal feeding before dietary restriction; DR-NP(D4) and DR-HP(D4), means refeeding 1 day of normal protein feed or high protein feed after 3 days of dietary restriction; DR-NP (D6) and DR-HP(D6), indicating that after 3 days of dietary restriction, refeed with normal protein feed or high protein feed for 3 days. Each dot represents an individual mouse. Principal coordinate 1, principal coordinate 2 and principal coordinate 3 indicate the percentage of variance explained by each coordinate, N=3-6 mice/group.

(B) Alpha diversity of the mouse gut microbiota from (A). Alpha diversity is represented by Shannon index calculation.

(C and D) Class-level (C) and family-level (D) proportional abundances of the mouse gut microbiota from (A).

(E) Proportional abundance of Lactobacillus spp. from the mouse gut flora in (A).

(F) Antibiotic treatment significantly attenuated body fat accumulation in mice during the refeeding period. DR means that the mice were fed with 10%, 25%, and 65% of the food amount respectively within three days, and then a normal diet was carried out. The gray shaded part indicates the refeeding period, during which the mice in the experimental group were treated with antibiotics, and the mice in the control group were not treated with antibiotics during the refeeding period. ABX means antibiotics, N=9 mice/group.

(G and H) Representative fluorescent pictures of fresh mouse intestinal tissues (G) and their cryosections (H). DR means that the mice were fed with 10%, 25%, and 65% of the food amount respectively within three days, and then a normal diet was carried out. Mice in the experimental group were treated with antibiotics during the refeeding phase. On the 5th day, the mice were fed with a mixture of BODIPY (boron fluoride dipyrrole) fluorescent-labeled fatty acid analogs and olive oil, and the tissues were collected 2 hours after the feeding.

(I and J) Relative concentration of BODIPY in small intestine (I) and serum (J) of mice from (G) panel, N=7-8 mice/group.

(K and L) Representative fluorescent pictures of fresh inguinal and epididymal white adipose tissue (K) and their cryosections (L) from mice from (G).

(M) The relative concentration of BODIPY in the white adipose tissue of the inguinal and epididymis of the mice, from the graph of (G), N=7-8 mice/group.

a or *, p<0.05; b, p<0.01; c or ***, p<0.001; NS, not significant.

Figure 2. Lactobacillus Lam-1 isolated and identified during the refeeding period after dietary restriction enhanced intestinal lipid absorption and fatty acid uptake in white adipose tissue and promoted body fat accumulation in mice.

(A) Phylogenetic tree of isolated Lactobacillus Lam-1 (L. murinus) and its relatives based on the sequence display of the 16S ribosomal RNA gene. Bar statistics represent differences in sequences.

(B) Relative concentration of BODIPY in feces of mice treated with control or Lam-1 strain. After intragastric administration of the mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil, the feces produced by the mice were collected from 10 minutes to 2 hours, N=8 mice/group.

(C and D) Representative fluorescent images of fresh small intestinal tissues (C) and their cryosections (D) from mice from (B), harvested 2 hours after gavage.

(E and F) Relative concentration of BODIPY in small intestine (E) and serum (F) of mice from panel (B), N=8 mice/group.

(G and H) Representative fluorescence images of fresh inguinal and epididymal white fat (G) and their cryosections (H) from mice from (B), tissues harvested 2 hours after gavage.

(I) The relative concentration of BODIPY in the white fat of the inguinal and epididymis of the mice, the mice are from the graph (B), N=8 mice/group.

(J) Body fat content of mice after continuous intragastric administration of control or Lam-1 strain. D0 means before gavage, D5 and D10 mean continuous gavage for 5 days and 10 days respectively, N=8-9 mice/group.

(K) Mouse body fat percentage relative to body weight after continuous gavage of control or Lam-1 strain. D0 means before gavage, D5 and D10 mean continuous gavage for 5 days and 10 days respectively, N=8-9 mice/group.

(L and M) Daily food intake (L) and water intake (M) of mice during continuous gavage of control or Lam-1 strain.

(N) Body temperature of mice after continuous gavage of control or Lam-1 strain. D0 means before gavage, D5 and D10 mean continuous gavage for 5 days and 10 days respectively, N=8-9 mice/group.

*, p<0.05; **, p<0.01; NS, not significant.

Figure 3. Microbiota metabolites during the refeeding phase after dietary restriction enhance lipid absorption in the small intestine and fatty acid uptake in white adipose tissue.

(A) Heat map of intestinal metabolite composition in mice re-fed normal protein or high protein diet before and after dietary restriction. DR means that mice were provided with 10%, 25%, and 65% of the food amount respectively within three days, and the diet was restricted, N=3-6 mice/group.

(B-F) DL-3-phenyllactic acid (PLA) (B), 4-hydroxyphenyllactic acid (HPLA) (C), indolelactic acid (ILA) (D), 2-hydroxyisocaproic acid ( HICA) (E) and 2-hydroxy-3-methylbutyric acid (HMBA) (F) concentrations, N=4 mice/group.

(G) Concentration of metabolites in cecal feces of mice after intragastric administration of control or Lam-1 bacteria, N=9 mice/group.

(H) Food intake of mice after intragastric administration of water or a mixed solution containing 5 compounds such as PLA, HPLA, ILA, HICA and HMBA for 24 hours, N=10 mice/group.

(1) The relative concentration of BODIPY in mouse feces in intragastric administration water or the mixed solution of 5 kinds of compounds. After intragastric administration of the mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil, the feces produced by the mice were collected from 10 minutes to 2 hours, N=10 mice/group.

(J and K) Representative fluorescence images of fresh small intestinal tissues (J) and their cryosections (K) from mice from (I), harvested 2 hours after gavage.

(L) Relative concentration of BODIPY in the small intestine of mice from (I) graph, N=10 mice/group.

(M and N) Representative fluorescent images of fresh inguinal and epididymal white fat (M) and their cryosections (N) from mice from (I), tissues harvested 2 hours after gavage.

(O) The relative concentration of BODIPY in the white fat of the inguinal and epididymis of mice, mice from (I) graph, N=10 mice/group.

(P) Overall flow chart of the experiment. Refeeding after dietary restriction induces an increase in Lactobacillus and its metabolites, which in turn promotes lipid absorption in the small intestine and fatty acid uptake in white adipose tissue, resulting in obesity, and high-protein diet intervention or antibiotic treatment during the refeeding period can alleviate Obesity following dietary restriction.

* or #, p<0.05; ** or ##, p<0.01; NS, not significant.

Figure 4. Antibiotic treatment during the refeeding phase after dietary restriction inhibits increases in food intake and percent body fat and decreases in percent lean body mass in mice.

(A) Food intake of antibiotic-free and antibiotic-treated mice during the refeeding phase after dietary restriction. DR means that 10%, 25%, and 65% of the food amount were provided to the mice for three days, and the diet was restricted. The gray shaded part indicates the refeeding period, during which the mice in the experimental group were treated with antibiotics, and the mice in the control group were not treated with antibiotics during the refeeding period. ABX means antibiotics, N=9 mice/group.

(B and C) Percent body fat (B) and percent lean body mass (C) from mice in (A), N=9 mice/group.

a, p<0.05; b, p<0.01; c, p<0.001.

Fig. 5. PLA, HPLA and ILA have no significant effect on small intestinal lipid absorption and fatty acid uptake in white adipose tissue by gavage alone or mixed gavage.

(A) Food intake of mice after intragastric administration of water, DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA) or indolelactic acid (ILA) for 24 hours, N=6 mice/group.

(B) Relative concentration of BODIPY in mouse feces. After intragastric administration of the mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil, the feces produced by the mice were collected from 10 minutes to 2 hours, N=6 mice/group.

(C) Representative fluorescent images of fresh small intestinal tissue from mice from (B), harvested 2 hours after gavage.

(D) Relative concentration of BODIPY in the small intestine of mice from (B), N=6 mice/group.

(E) Representative fluorescent images of fresh inguinal and epididymal white adipose tissue from mice from (B), harvested 2 hours after gavage.

(F) The relative concentration of BODIPY in the white fat of the inguinal and epididymis of the mice from (B), N=6 mice/group.

(G) Food intake of mice 24 hours after intragastric administration of water or a solution containing three compounds of PLA, HPLA and ILA, N=7 mice/group.

(H) Relative concentration of BODIPY in mouse feces. After intragastric administration of the mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil, the feces produced by the mice were collected from 10 minutes to 2 hours, N=6 mice/group.

(I) Representative fluorescent images of fresh small intestine tissue from mice from (H), harvested 2 hours after gavage.

(J) Relative concentration of BODIPY in the small intestine of mice from (H) panel, N=7 mice/group.

(K) Representative fluorescent images of fresh inguinal and epididymal white fat from mice from (H), tissue harvested 2 hours after gavage.

(L) The relative concentration of BODIPY in the white fat of the inguinal and epididymis of mice from the graph of (H), N=7 mice/group.

NS, not significant.

Figure 6. HICA or HMBA had no significant effect on small intestinal lipid absorption and fatty acid uptake in white adipose tissue.

(A and G) Food intake of mice after oral administration of 2-hydroxyisocaproic acid (HICA) (A) or 2-hydroxy-3-methylbutyric acid (HMBA) (G) for 24 hours, N=7-8 mice /Group.

(B and H) Relative concentrations of BODIPY in mouse feces. After intragastric administration of the mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil, the feces produced by the mice were collected within 10 minutes to 2 hours, N=7-8 mice/group.

(C and I) Representative fluorescent pictures of fresh small intestine tissue from mice. Mice are from (B) and (H) respectively, and tissues were harvested 2 hours after gavage.

(D and J) Relative concentrations of BODIPY in the small intestine of mice. Mice are from panels (B) and (H), respectively, N=6 mice/group.

(E and K) Representative fluorescent pictures of fresh inguinal and epididymal white adipose tissue from mice. Mice are from (B) and (H) respectively, and tissues were harvested 2 hours after gavage.

(F and L) Relative concentrations of BODIPY in the inguinal and epididymal white fat of mice. Mice are from panels (B) and (H), respectively, N=6 mice/group.

NS, not significant.

Detailed ways

The inventors are committed to studying the correlation between diet and metabolism. In previous studies, it was found that compared with before dietary restriction, re-intake after dietary restriction will significantly increase body fat content. Further studies have shown that the composition of the intestinal flora during the normal diet after dietary restriction changed significantly, and the proportion of Lactobacillus increased significantly; the high-protein diet after dietary restriction significantly inhibited the increase of the proportion of Lactobacillus in the small intestine, and the flora was diverse. Sex significantly increased. Elimination of gut microbiota effectively inhibits refeeding-induced body fat gain following dietary restriction. The present inventors also found that a five-compound combination of a specific class of Lactobacillus strains can significantly increase lipid absorption and fatty acid uptake in white adipose tissue.

the term

As used herein, the terms "diet (food or food) restriction", "diet (food or food) control", "intake restriction", "intake control" and "diet" are used interchangeably.

As used herein, the "dietary restriction" is a specific period, which is significantly different from the "normal diet"; during this "dietary restriction" period, the food intake of the test subject is significantly less on a "normal diet". The "normal diet (amount)" generally refers to the same subject's daily or natural food intake (amount) when no "diet restriction" or before "diet restriction".

As used herein, the "restoration of diet, intake of normal diet" is a specific stage, which is different from the "diet restriction" stage; Food supply status, the diet consumed is a regular diet/normal diet.

As used herein, the "recovery diet, intake of high-protein diet" refers to returning to the natural food supply state of the subject after the "diet restriction" period, but the food intake is a high-protein diet.

As used herein, "increasing the content of penta-combination/lactobacillus in the digestive tract" includes increasing the content in the digestive tract by ingesting penta-compound/lactobacillus, such as adding penta-compound/lactobacillus to food, or / Lactobacillus taken alone.

As used herein, the term "composition (composition) of the present invention" includes: food (composition), health product (composition), etc., as long as their component contents are regulated according to the present invention.

As used herein, the terms "comprising" or "comprising" include "comprising", "consisting essentially of", and "consisting of". "Consisting essentially of" means that in addition to essential components or essential components, the composition may also contain a small amount of secondary components and/or impurities that do not affect the active components. For example, sweeteners or flavoring agents to improve taste, antioxidants to prevent oxidation, and other additives commonly used in the art may be contained.

As used herein, the term "nutraceutical acceptable" or "food acceptable" is an ingredient that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic reactions), That is, a substance with a reasonable benefit/risk ratio.

As used herein, the term "effective amount" refers to an amount that can produce functions or activities on humans and/or animals and that can be accepted by humans and/or animals.

As used herein, "parts by weight" or "parts by weight" can be used interchangeably, and said parts by weight can be any fixed weight expressed in micrograms, milligrams, grams or kilograms (such as 1 μg, 1 mg, 1 g, 2g, 5g, or kg, etc.). For example, a composition consisting of 1 part by weight of component a and 9 parts by weight of component b can be 1 gram of component a+9 gram of component b, or 10 gram of component a+90 gram of component b etc. composition. In the composition, the percentage content of a certain component=(parts by weight of this component/sum of parts by weight of all components)×100%. Therefore, in a composition composed of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and the content of component b is 90%.

As used herein, the terms "unit dosage form" and "unit dosage form" refer to the preparation of the composition of the present invention into dosage forms required for single administration for the convenience of taking, including but not limited to various solid dosage forms (such as tablets), liquid agent. The unit dosage form contains the composition of the present invention in an amount suitable for single, single day or unit time administration.

Lipid metabolism in reintake after dietary restriction

In the research work of the present inventors, it was found that compared with before the dietary restriction, the body fat content of the animals re-fed after the dietary restriction was significantly increased, and the composition of the intestinal flora of the animal during the normal diet after the dietary restriction was significantly changed. Among them, Lactobacillus The ratio increased significantly to around 60%, while the diversity of the flora decreased. In the early stage, the present inventors proved through experiments that the high-protein diet after dietary restriction can effectively inhibit the accumulation of animal body fat content. The results of the present invention show that compared with animals fed normally after dietary restriction, the high-protein diet after dietary restriction can significantly Inhibit the increase in the proportion of Lactobacillus in the small intestine, and at the same time the diversity of the flora is significantly increased. Treating animals with antibiotics to clear intestinal flora can effectively inhibit the increase in body fat caused by refeeding after dietary restriction. Further, the inventors found that the intestinal lipid absorption and fatty acid uptake in white adipose tissue of animals treated with antibiotics were significantly weakened. Animal cecal feces in the normal diet process after dietary restriction is coated on a flat plate, and after picking a monoclonal strain to isolate and cultivate, the inventor has obtained a class of lactobacillus with the 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1 ( In the examples, it is called Lam-1 bacterial strain), and this bacterial strain can significantly increase the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue by gavage of this bacterial strain, meanwhile, the continuous gavage of the lactic acid bacteria strain can significantly increase the body fat content of the animal But it does not affect the food intake of animals. In order to further explore the mechanism of intestinal flora involved in refeeding after dietary restriction leading to obesity, the inventors analyzed the composition of bacterial metabolites in animal cecal feces and found that compared with animals before dietary restriction, refeeding after dietary restriction The metabolite composition of the flora of normal diet animals changed significantly, among them, DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA), 2-hydroxyisocaproic acid (HICA) and The concentration of five metabolites including 2-hydroxy-3-methylbutyric acid (HMBA) increased significantly, and the increase in the concentration of these five metabolites could be significantly inhibited by high-protein diet. In addition, the concentration of these five metabolites in the cecal feces of animals after gavage of Lam-1 strain also increased significantly, indicating that these five metabolites were produced by Lactobacillus. Further studies have found that these five compounds can significantly increase lipid absorption in the small intestine of animals and fatty acid uptake in white adipose tissue. In summary, the inventors' findings suggest that targeting intestinal Lactobacillus by high-protein diet or antibiotic treatment, thereby inhibiting lipid absorption in the small intestine, can be an effective method to prevent obesity after dietary restriction.

Although a series of antibiotics are preferably listed in the embodiments of the present invention, in addition to vancomycin, ampicillin, neomycin, and metronidazole, there are also other antibiotics that have similar functions to them, such as but not limited to gentamicin kanamycin, streptomycin, cefoperazone, erythromycin, tylosin, amoxicillin, penicillin, bacitracin, tetracycline, doxycycline or clindamycin, etc., they can also be used in the present invention.

Based on the inventor's new discovery, a method for regulating lipid absorption or body weight is provided, comprising: (a) regulating the content of a five-compound combination in the digestive tract (including the stomach or intestinal tract), the five-compound combination being: DL - a combination of 3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA), 2-hydroxyisocaproic acid (HICA) and 2-hydroxy-3-methylbutyric acid (HMBA); Or (b) regulating the content of lactobacillus in the digestive tract (including stomach or intestinal tract), the lactobacillus is a lactobacillus having the 16S ribosomal RNA gene sequence shown in SEQ ID NO:1.

In the present invention, the lactobacillus has a specific 16S ribosomal RNA gene sequence, and the present invention also discloses its specific sequence, so those skilled in the art can obtain it from intestinal metabolites under the disclosure of the present invention. Such strains are thus applied to the regulation of lipid metabolism.

In the present invention, the five compounds mentioned are all known in the art or commercially available.

In the method of the present invention, food intake reduction or diet control is carried out in the early stage, and this process can be carried out through a variety of ways, including but not limited to: conventional diet, intermittent diet, time-restricted diet, low-energy diet that simulates dieting, Gradient or descending gradient dieting. Food intake or diet control is carried out according to the needs or schedule of the subject; for example, for humans, the schedule can be at a longer (eg 3-6 months or longer) or intermediate length such as (1-3 months) or a shorter period (such as 3 to 30 days). This process usually takes a significant weight loss as the test standard, which is also measured according to the needs or plans of the subject, for example, a significant weight loss of 2-40%; more specifically, such as 3%, 5%, 8%, etc. %, 10%, 15%, 20%, 30%, 35%, etc.

In the present invention, during the "diet restriction" process, there is no specific restriction on the type of food ingested, and it can be conventional food, but the intake is controlled (the intake is significantly reduced), or it can be Eat low-calorie foods. However, as a preferred mode of the present invention, the "dietary restriction" is a gradient-increasing or gradient-decreasing restriction scheme, for example, according to the planned dietary restriction time, to plan the daily intake, regularity, rhythm or wave type to increment or decrement. Preferably, even if there is a period of escalation to a higher point, the higher point is below the level of a "normal diet".

In the phase of "restoring food intake", regular diet or food with regulated protein content can be taken in order to achieve the purpose of regulating lipid absorption.

Lactobacillus

In the present invention, a novel lactobacillus is isolated, and the lactobacillus is a lactobacillus with a 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1; The preservation number is CCTCC NO: M 20211687, which also includes similar strains with the same function (also having the 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1).

The bacterial strains of the present invention are living cells. Once the bacterial strains of the present invention are obtained, the bacterial strains of the present invention can be obtained in large quantities by means of inoculation, passage, regeneration and the like. Usually, the live cells of the present invention are obtained by inoculating them into solid plate culture medium or liquid culture medium to carry out expanded culture of strains. The obtained living cells can be further subjected to laboratory domestication, genetic breeding and molecular genetic manipulation to obtain mutants and transformants. In addition, the bacterial strain of the present invention can also be used as a bioengineering host cell for heterologous expression.

Furthermore, the Lactobacillus of the present invention can be used as a starting strain, which can be further improved through laboratory domestication, genetic breeding, molecular genetic manipulation and other means to obtain derivative strains with higher yield or more optimized enzyme system. Using the Lactobacillus of the present invention as the starting strain, the strains obtained through further screening and optimization by these artificial means should also be included in the overall scope of the present invention.

Methods well known to those skilled in the art can be used to mutagenize the living strains of the present invention, resulting in changes in gene coding, biological characteristics and morphological changes (optimization) of living cells. These methods include the use of physical methods such as rays, particles, lasers, and ultraviolet light, and chemical mutagenesis methods such as alkylating agents, base analogs, hydroxylamine, and acridine pigments. The mutagenesis may be multigenerational mutagenesis of one or more of the above methods, and is not limited to these methods. Based on the strains provided by the present invention, further breeding can be carried out in physical and chemical ways, and other regulatory genes can also be introduced, and the obtained mutants and transformants can be obtained together to obtain strains with further improved performance. The breeding method is one of the above-mentioned or a combination of more than one.

Methods well known to those skilled in the art can be used to construct expression constructs (vectors) and to further engineer the strains of the invention. For example, further improvement (such as increasing the expression of beneficial factors and reducing the expression of harmful factors) has been found or newly discovered in the strains related to lipid absorption and production of signaling pathways, signaling pathways and proteins involved therein.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. The procedures used are well known in the art.

On the basis of obtaining the lactobacillus, the present invention also provides the cell culture, cell metabolite, cell culture supernatant or cell lysate of the lactobacillus, which also has the function of regulating lipid absorption or body weight. Function.

After obtaining the bacterial strain of the present invention, those skilled in the art can obtain its culture conveniently, for example, can refer to some culture medium or culture process provided in the specific embodiment of the present invention, or utilize and exist in the embodiment of the present invention appropriate Cell cultures are obtained by altering the medium or the cultivation process but also obtaining the culture. The cell culture contains active strains, so as to regulate lipid absorption or body weight.

The cell metabolites are substances produced or secreted by the strain of the present invention during the culture process, which may be directly secreted by the cells into the culture medium, or separated from the cells after certain treatment. Said cellular product may be isolated, purified or concentrated.

The cell culture supernatant refers to the remaining culture solution after removing cells and solid impurities during or after the culture of the bacterial strain of the present invention, which may be unconcentrated or concentrated. Usually, cells and solid impurities can be removed by means such as centrifugation and filtration.

The cell lysate is a mixture formed by lysing cells with a cell lysing reagent during or after cultivating the bacterial strain of the present invention. The cell lysate may be a product after lysis with solid impurities removed. It may be a purified or concentrated product, as desired.

combination

Based on the inventor's new discovery, the present invention provides a composition for regulating (increasing) lipid absorption or body weight, comprising: DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2 -Hydroxyisocaproic acid, 2-hydroxy-3-methylbutyric acid, in parts by weight (or weight to volume ratio): 20-40: 10-20: 6-10: 30-50: 15-25.

The present invention also provides a composition for regulating (increasing) lipid absorption or body weight, including Lactobacillus, which is a Lactobacillus having a 16S ribosomal RNA gene sequence shown in SEQ ID NO:1.

The composition may be a food composition, and in some embodiments, the composition may also include a food or health product acceptable carrier.

The formulation range shown in the present invention can be used as a reference guide. However, it should be understood that when used in research and development to prepare foods or compositions, the effective dosage of each component may also vary with actual application conditions. For example, it is made into a concentrated form or a diluted form, etc., and these variations are also included in the present invention.

In some preferred modes of the present invention, the composition is in unit dosage form. When the composition is prepared into a unit dosage form, 2 to 6 doses of the composition in the unit dosage form are taken every day, such as 2, 3 or 4 doses according to dietary rules. According to the mode of action of the composition of the present invention, the unit dosage form can be taken with food, for example.

In the specific examples of the present invention, some intake schemes for animals such as mice are given. It is easy for those skilled in the art to convert the intake amount of animals such as rats into the intake amount suitable for humans. For example, it can be calculated according to the Meeh-Rubner formula: Meeh-Rubner formula: A=k×(W2/ 3)/10,000. In the formula, A is body surface area, calculated in m2; W is body weight, calculated in g; K is a constant, which varies with animal species. Generally speaking, mice and rats are 9.1, guinea pigs 9.8, rabbits 10.1, cats 9.9, dogs 11.2, monkey 11.8, human 10.6. It should be understood that intake conversions may vary according to food and time circumstances, as assessed by an experienced technician.

application

The inventor's research results show that the proportion of Lactobacillus in animals re-fed after dietary restriction increases to about 60%, and the concentration of its corresponding metabolites increases significantly at the same time, and when the animals after dietary restriction are re-fed high-protein diet can inhibit this a process. The inventor's experimental results prove that the Lactobacillus and its metabolites disclosed in the present invention can significantly increase the lipid absorption in the small intestine and the fatty acid intake of white fat by gavage to animals.

Diarrhea is generally due to increased secretion, decreased absorption, or both, and the results of the present invention suggest that the Lactobacillus or the five-compound combination metabolized by it may also be used as a probiotic for the treatment of diarrhea.

The inventors' findings also suggest that, for conditions of overnutrition, it may be more important to develop interventions that specifically target the small intestinal microbiota, either by reducing the proportion or activity of certain microbes that might promote fat absorption, or by increasing Proportional abundance of fat-absorbing microbes.

Malnutrition can also be treated by increasing or decreasing the abundance and activity of microorganisms associated with fat absorption. For example, approaches targeting the gut microbiota to promote more efficient nutrient digestion and absorption could be developed in cases of intestinal failure (such as small bowel resection or Crohn's disease) or other environmental enteropathy.

In addition, the research results of the present inventors show that targeted inhibition of Lactobacillus through special diets such as high-protein diets or antibiotic treatment can be used as an effective method to reduce intestinal lipid absorption and resist obesity after dietary restriction.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, generally follow the conditions described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, or according to the manufacturer's suggestion conditions of.

Acronyms

In the present invention, the abbreviations involved and their full names are shown in Table 1.

Table 1

Acronym	full name
AL	Free feeding (Ad libitum)
DR	Dietary restriction
NP	Normal protein diet (Normal protein)
HP	High protein diet
D0	Day 0 (Day 0)
D4	Day 4 (Day 4)
D5	Day 5 (Day 5)

D6	Day 6 (Day 6)
D10	Day 10 (Day 10)
ABX	Antibiotics
BODIPY	Dipyrromethene Boron Difluoride
L. murinus	Lactobacillus murinus Lactobacillus murinus
CFU	Colony forming unit
PLA	DL-3-phenyllactic acid (DL-3-phenyllactic acid)
HPLA	4-hydroxyphenyllactic acid (4-hydroxyphenyllactic acid)
ILA	Indole-lactic acid
HICA	2-hydroxyisocaproic acid
HMBA	2-hydroxy-3-methylbutyric acid (2-hydroxy-3-methylbutyric acid)
ZT	Ambient time (Zeitgeber time)

animals and feed

The mice used in the experiments were all C57BL/6J strain mice, which were purchased from Shanghai Slack Experimental Animal Co., Ltd. The normal standard feed (Chow) for mice was provided by the animal room and purchased from Shanghai Slack Experimental Animal Co., Ltd., and the 20% normal protein (NP) and 60% high protein (HP) feeds were purchased from Shanghai Fanbo Biotechnology Co., Ltd. The 20% normal protein feed is made according to the AIN-93G rodent diet formula (D10012G, Research Diets Inc.), the difference is that it does not contain the antioxidant tBHQ, and replaces sucrose with cornstarch, contains 20% casein, 0.3% cysteine and 49.7% corn starch. High protein (HP) feed was made on the basis of 20% normal protein feed, which contained 60% casein, 0.9% cysteine and 9.1% cornstarch. The specific food composition is shown in Table 2 below.

Table 2. NP and HP diet components

Reagent

BODIPY 500/510 C1, C12 fatty acid (4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-lauric acid), purchased from Molecular Probes ; OCT compound, purchased from Sakura; Nonidet P-40, purchased from Sangon Biotech; chloral hydrate, methanol, acetonitrile, chloroform, purchased from Shisheng Sebastian; MRS medium, purchased from Baihaibo Biotech; Lun Biology; ampicillin, neomycin, metronidazole, purchased from Sangon Bioengineering; DL-3-phenyl lactic acid (PLA), 4-hydroxyphenyl lactic acid (HPLA), indole lactic acid (ILA), purchased from TCI ; 2-Hydroxyisocaproic acid (HICA), 2-hydroxy-3 methylbutyric acid (HMBA), purchased from Sigma-Aldrich.

dietary restriction experiment

All mouse experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the Shanghai Institute of Nutrition and Health. Eight-week-old C57BL6/J strain mice were acclimatized in the experimental mouse room for 3-5 days after purchase. Then the mice were reared in a single cage and adapted for 5 days, and the subsequent diet restriction and re-feeding of the experimental mice were also reared in a single cage. During the stage of single-cage rearing before dietary restriction, the mice were fed with the feed used for dietary restriction. The average food intake of the mice in the first three days of dietary restriction was used as the dietary reference for the next experiment. Dietary restriction was performed by feeding mice the indicated amount of food at ZT12 (19:00). All animal experiments were performed on male mice.

Weight and Body Composition Measurements

Mouse body weight and body composition were measured at ZT8 (15:00). Fat and lean body mass were measured with EchoMRI-100H body composition analyzer (EchoMRI).

temperature measurement

Mouse body temperature was measured at ZT3 (10:00) using a RET-3 rectal probe (Physitemp) connected to a BAT-12 thermometer (Physitemp).

tissue and stool collection

Mice were anesthetized with 6% chloral hydrate at designated time points, and then the small intestine, inguinal and epididymal white adipose tissues of interest were isolated, snap-frozen in liquid nitrogen and stored in a -80°C refrigerator. The blood was collected by inserting a needle into the apex of the heart with a 1ml syringe, and the collected blood was centrifuged at 1000g for 30 minutes at 4°C, and then the serum was collected and stored in a -80°C refrigerator. The feces of the mice were collected and stored in a -80°C refrigerator. After the cecal contents of the mice were collected, they were snap-frozen in liquid nitrogen and stored in a -80°C refrigerator.

Lipid and Fatty Acid Absorption Assay

Mice were fed with BODIPY 500/510 C1, C12 fatty acid (4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-inda at ZT12 (19:00) A mixture of pro-3-lauric acid) (0.5 μg/g body weight) and olive oil (10 μl/g body weight). Mice were provided with sufficient water but no food after gavage. Mice feces were collected between 10 minutes and 2 hours after gavage, then freeze-dried and ground with a mortar and pestle before being stored in a -20°C freezer. After intragastric administration of BODIPY fluorescently labeled fatty acid analogs for 2 hours, the mice were anesthetized with 6% chloral hydrate, and then the tissue and blood samples of interest were collected. The isolated proximal jejunum, inguinal white adipose tissue, or epididymal white adipose tissue were directly observed under a fluorescence microscope, or embedded in OCT compound, and then sectioned. For the detection of fluorescence intensity of BODIPY, specifically, proximal jejunum, inguinal white adipose tissue or epididymal white adipose tissue were firstly dissolved in RIPA lysis buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Nonidet P- 40, 1% sodium deoxycholate, 0.1% SDS), and then centrifuged to get the supernatant to read the fluorescence signal. The fluorescence signal of the extracted tissue sample or serum sample is measured by a microplate reader (Varioskan Flash, purchased from Thermo Scientific), and the corresponding excitation wavelength is 492nm, and the emission wavelength is 520nm. The dried and ground feces samples were treated with a mixture of water and chloroform (volume ratio: 1:2), and then centrifuged to obtain the organic phase to measure the fluorescence signal.

16S ribosomal RNA gene sequencing analysis

Bacterial genomic DNA was extracted from mouse cecum feces, and then 10 ng of purified DNA was used for PCR amplification. Use bacterial universal primers to carry out PCR amplification of the V3 and V4 regions of the 16S ribosomal RNA gene. The forward primer sequence is: CCTAYGGGRBGCASCAG (Y represents C or T, R represents A or G, B represents G or C or T, S represents C or G) (SEQ ID NO: 2); Reverse primer sequence is: GGACTACNNGGGTATCTAAT (N represents A or G or C or T) (SEQ ID NO: 3). The PCR products were then mixed in equimolar amounts. Next, sequencing was performed using the Novaseq 6000 platform (Illumina), generating 2×250 base paired-end reads (reads). High-quality filtered reads were acquired with the QIIME 2 (version 2019.4) software and R package, and then searched against the GreenGenes (version 13.8) reference database. β-diversity analysis was performed based on Bray-Curtis distances to explore the clustering differences of intestinal flora among samples, and visualized by principal coordinate analysis. The alpha diversity of the microbiota was calculated based on the genetic profile of the gut microbiota for each sample, and based on the Shannon index, tested using the Kruskal Wallis and dunn tests. The proportional abundance of the bacterial community was assessed at the taxonomic level of class, family and genus, respectively.

antibiotic treatment

Re-feed the mice after feeding 10%, 25%, and 65% of the food amount in three days, that is, provide enough food for the mice, and at the same time give the mice intragastric administration of high-concentration antibiotics every day for 5 consecutive days, high-concentration antibiotics Antibiotics were prepared by suspending 10 mg of vancomycin, 10 mg of ampicillin, 10 mg of neomycin and 10 mg of metronidazole in 0.2 ml of water. Afterwards, the antibiotics of low concentration were given intragastrically to mice every day, and the antibiotics of low concentration were the vancomycin of 2mg, the ampicillin of 4mg, the neomycin of 4mg and the metronidazole of 4mg were suspended in the water preparation of 0.2ml. Body fat, lean body mass, and food intake of mice were measured during this period to study the effect of antibiotic treatment on refeeding-induced obesity after dietary restriction. To study the effect of antibiotic treatment on lipid absorption in the small intestine and fatty acid uptake in white adipose tissue, mice were fed with 10%, 25%, and 65% of the food volume for three days, and then provided enough food to the mice at the same time. Rats were gavaged with high concentrations of antibiotics. Then 18 hours later, the mice were given high-concentration antibiotics again. Then 6 hours later, the mice were fed with a mixture of BODIPY fluorescently labeled fatty acid analogs (0.5 μg/g body weight) and olive oil (10 μl/g body weight), and then blood and tissue samples were collected at designated time points.

Isolation and identification of Lactobacillus genus Lactobacillus murinus (L.murinus)

The cecal feces samples of mice re-fed with normal protein diet for one day after dietary restriction were collected, and then the samples were diluted with sterile anaerobic PBS at a dilution ratio of 1:10. A small portion of the dilution was spread on MRS agar plates and incubated in an anaerobic incubator (5% hydrogen, 5% carbon dioxide, 90% nitrogen) at 37°C for 48 hours. Single colonies were picked at random and cultured for an additional 24 hours in MRS broth. Amplification of full-length 16S ribosomal RNA from monoclonal strains using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 4)) and 1492R (5'-CTACGGCTACCTTGTTACGA-3' (SEQ ID NO: 5)) genes, followed by sequencing. Isolated Lactobacillus murine (strain Lam-1) was stored in 20% or 6% glycerol and kept in a -80°C freezer until further use. The reference sequence of type strains similar to Lam-1 strain was obtained from the GenBank database by Blast, and multiple sequences were combined and compared with ClustalW software. The phylogenetic tree was constructed using the "neighbor-joining" algorithm of MEGA 7.0 software.

Full-length 16S ribosomal RNA gene sequence (SEQ ID NO: 1):

Gastric administration of Lam-1

For the lipid absorption experiment of mice after gavage of Lam-1, the mice were first gavaged with 0.2 ml of PBS containing 6% glycerol (control) or the Lam-1 strain dissolved in PBS containing 6% glycerol (10 ¹⁰ CFU), wherein the Lam-1 strain was preheated in a 37°C water bath for 5-10min during gavage. After 24 hours, the mice were fed with the same PBS or Lam-1 strain again, and at the same time, the mice were fed with a mixture of BODIPY fluorescently labeled fatty acid analogs (0.5 μg/g body weight) and olive oil (10 μl/g body weight) . Feces, cecal contents, blood, and tissues such as small intestine, inguinal, and epididymal white fat were collected from the mice at the indicated time points. For the mouse phenotype detection experiment after gavage of Lam-1, the mice were gavaged with 0.2ml of PBS containing 6% glycerol (control) or Lam-1 dissolved in PBS containing 6% glycerol at 17:00 every afternoon. strain (10 ¹⁰ CFU), the food intake and water intake of the mice were detected every day, and the body temperature, body weight, body fat and other indicators of the mice were detected before gavage and on the 5th and 10th days after gavage.

Untargeted Metabolomics and Data Analysis

Take about 80 mg of cecal feces, add 0.2 ml of water and 0.8 ml of methanol/acetonitrile (volume ratio 1:1), then homogenize, vortex, and sonicate on ice for 30 minutes. This was followed by 1 hour at -20°C and centrifugation at 14000 g for 20 minutes. The supernatant containing metabolites was vacuum freeze-dried, dissolved in 0.1 ml of acetonitrile/water (volume ratio 1:1), and determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mass spectrometer was run in negative and positive ionization modes with the capillary voltage set at 5.5 kV. Raw mass spectrometry data were extracted and peaks identified, isotopes and adducts were annotated using the R package CAMERA. Compounds are identified by comparing accurate m/z values and spectra to an established database of available authentic standards. Regarding metabolite classification, compared with AL-NP(D0) group, both DR-NP(D4) group and DR-HP(D4) group had significantly increased metabolites (excluding DR-HP(D4) group with DR- NP(D4) group significantly decreased metabolites), which were classified as "unstoppable increase" category; compared with AL-NP(D0) group, DR-NP(D4) group and DR-HP(D4 ) groups (excluding metabolites that were significantly increased in the DR-HP(D4) group compared with the DR-NP(D4) group), were classified as "unstoppable reduction"; DR-NP (D4) group was significantly decreased compared with AL-NP(D0) group, while DR-HP(D4) group was significantly increased compared with DR-NP(D4) group or had no significant difference compared with AL-NP(D0) group , classified as "blockable reduction"; the DR-NP(D4) group significantly increased compared to the AL-NP(D0) group, while the DR-HP(D4) group compared to the DR-NP(D4) group Metabolites that were significantly reduced or not significantly different compared to the AL-NP(D0) group were classified as "blockable increases".

Targeted quantitative analysis of PLA, HPLA, ILA, HICA and HMBA

Take about 60 mg of cecal feces, add 0.2 ml of water, 0.8 ml of methanol and 1 μl of formic acid, then homogenize, vortex, and sonicate on ice for 30 minutes. This was followed by 3 hours at -20°C and centrifugation at 14000 g for 20 minutes. The supernatant was filtered with a 0.22 μm PTFE hydrophilic filter and used for the next step of detection. Treated samples along with DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA), 2-hydroxyisocaproic acid (HICA) and 2-hydroxy-3-methylbutyric acid (HMBA) and other compounds used as standards with different concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Draw a marked curve based on the mass spectrometry detection results of each standard compound, and use this to calculate the corresponding concentrations of the five compounds in the sample.

Metabolite gavage treatment

Mice were given water, 60mg/ml of PLA, 30mg/ml of HPLA, 15mg/ml of ILA, three compounds (including 60mg/ml of PLA, 30mg/ml of HPLA and 15mg/ml of ILA) or five A compound (comprising 30mg/ml of PLA, 15mg/ml of HPLA, 8mg/ml of ILA, 40mg/ml of HICA and 20mg/ml of HMBA) solution, each solution is adjusted to pH 7.0 with sodium hydroxide, poured The stomach volume was 0.15ml. After 24 hours, the mice were fed with the same amount of water or the specified compound again, and at the same time, the mice were fed with a mixture of BODIPY fluorescently labeled fatty acid analogs (0.5 μg/g body weight) and olive oil (10 μl/g body weight) liquid. Feces and tissues such as small intestine, inguinal and epididymal white fat were collected from the mice at the indicated time points.

Data Analysis and Statistics

Excel software was used for statistical analysis of data, and all numerical calculation results were presented in the form of mean ± standard deviation (SD). All graphs were drawn using GraphPad Prism software. Significant differences among different groups were analyzed by two-tailed Student's t-test. A p value of less than 0.05 was considered statistically significant.

Sequencing data storage

The raw data of high-throughput sequencing of 16S ribosomal RNA gene in mouse cecal feces has been deposited on the NCBI website (https://www.ncbi.nlm.nih.gov/sra), and the accession number is PRJNA757842. The complete sequence of the 16S ribosomal RNA gene of the isolated Lactobacillus Lam-1 has been deposited in GenBank, and its number is MZ955456 (SEQ ID NO: 1).

Example 1. Gut flora regulates fat accumulation, small intestinal lipid absorption, and fatty acid uptake in white adipose tissue during refeeding after dietary restriction

Diet affects the structure and function of gut microbiota, which can affect lipid absorption in the small intestine and lipid metabolism in adipose tissue. In the early stage, the inventors proved through experiments that refeeding after dietary restriction can promote lipid absorption in the small intestine and fatty acid uptake in white adipose tissue and make mice gain weight. Small intestine absorption and fattening, the present inventor carries out diet restriction (DR) to mouse at first, promptly feeds 10%, 25%, 65% food amount to mouse respectively from the first day to the 3rd day, then from the 3rd day Re-feeding began at four days, providing mice with adequate normal protein diet (NP) or high protein diet (HP). Mice before dietary restriction (D0) were given ad libitum diet (AL). The cecal fecal samples of the mice were collected before dietary restriction (ie D0) and after re-feeding a normal protein diet or a high protein diet for one day (ie D4) and three days (ie D6), and then the intestinal tract was analyzed by 16S ribosomal RNA gene sequencing. The bacterial composition was analyzed. To assess how refeeding after dietary restriction affects gut microbiome structure, the inventors studied the gut microbiota alpha and beta diversity of each sample to compare gut microbiota within samples and between samples diversity. For β-diversity, the results of principal coordinate analysis revealed significant clustering differences between the gut microbiota from mice fed a normal protein diet after dietary restriction, and mice fed a high-protein diet after dietary restriction (Fig. 1A). In addition, the results of gut microbiota α diversity calculated by Shannon index evaluation showed that compared with mice before dietary restriction, the gut microbiota α of mice subjected to dietary restriction were re-fed a normal protein diet for one day. Diversity was significantly reduced, whereas a corresponding high-protein diet significantly altered this effect (Fig. 1B). Next, the inventors analyzed the proportional abundance of intestinal flora at different taxonomic levels in each group of samples. The present inventors found that compared with mice before dietary restriction, the proportions of Bacillus class, Lactobacillus family, and Lactobacillus genus in mice subjected to dietary restriction were re-fed a normal protein diet for one day, and the proportions of the genus Lactobacillus all increased significantly, reaching Around 60% (Fig. 1C-Fig. 1E). However, compared with mice fed a normal protein diet for one day after dietary restriction, the abundance of Bacillus, Lactobacillus, and Lactobacillus genera in mice fed a high-protein diet for one day after dietary restriction were significantly reduced (Fig. 1C - Figure 1E). These data suggest that refeeding following dietary restriction significantly altered gut microbiota composition, resulting in a significant enrichment of Lactobacillus spp.

To investigate the role of gut microbiota in refeeding-induced obesity after dietary restriction, the inventors treated mice with antibiotics to clear gut microbes during the post-refeeding phase of dietary restriction. After feeding mice with 10%, 25%, and 65% food for 3 consecutive days, re-feeding can significantly increase the body fat content of mice and cause mice to gain weight, while re-feeding mice were given antibiotics After treatment, body fat accumulation was significantly inhibited (Figure 1F), while antibiotic treatment significantly inhibited the increase in food intake and percentage of body fat and the decrease in percentage of lean body mass in mice (Figure 4A-Figure 4C).

Furthermore, the present inventors administered BODIPY (boron dipyrrole fluoride) fluorescently labeled fatty acid analogs to mice, and collected blood and tissue samples from the mice 2 hours later. Fresh proximal jejunum tissues were taken for direct observation under a fluorescence microscope. The present inventors found that mice treated with antibiotics during the refeeding phase after dietary restriction had decreased tissue fluorescence intensity compared to mice refed after dietary restriction ( FIG. 1G ).

The inventors then performed frozen sections on the proximal jejunum tissue, and it can be seen that the fluorescence intensity of the proximal jejunal villi of the mice treated with antibiotics during the refeeding period decreased (Fig. 1H).

Then the inventor treated the proximal jejunum tissue with RIPA lysate, then centrifuged to get the supernatant to detect the fluorescence intensity, and found that the relative concentration of BODIPY in the proximal jejunum tissue of mice treated with antibiotics in the heavy feeding stage was significantly reduced (Fig. 1I). At the same time, the relative concentration of BODIPY in the serum of mice treated with antibiotics during the re-feeding period was also significantly reduced (Fig. 1J). BODIPY relative fluorescence intensity measurements in the small intestine and serum demonstrated that antibiotic treatment during the refeeding phase after dietary restriction inhibited lipid absorption in the small intestine.

In addition, fresh inguinal and epididymis white adipose tissue was directly observed under a fluorescence microscope, and the inventors found that the fluorescence intensity of the tissue in mice treated with antibiotics decreased ( FIG. 1K ). Subsequently, the inventors performed frozen sections on the inguinal and epididymal white adipose tissue, and it can also be seen that the fluorescence intensity of the mouse tissue treated with antibiotics decreased (Fig. 1L). Next, the inventors treated the inguinal and epididymis white adipose tissue with RIPA lysate, then centrifuged to take the supernatant to detect the fluorescence intensity, and found that the fluorescence intensity of the mouse tissue treated with antibiotics was significantly reduced (Figure 1M). BODIPY relative fluorescence intensity measurements of white adipose tissue demonstrate that antibiotic treatment during the refeeding phase after dietary restriction inhibits fatty acid uptake in white adipose tissue.

Taken together, these data demonstrate that refeeding after dietary restriction induces changes in the gut microbiota leading to increased fat content, small intestinal lipid absorption, and enhanced white fat fatty acid uptake.

Example 2, Lactobacillus Lam-1 can enhance the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue and promote the accumulation of body fat in mice

Since the proportion of Lactobacillus increased to about 60% during the refeeding period after dietary restriction, in order to explore whether this dominant bacterium led to the enhancement of intestinal lipid absorption and adipose tissue fatty acid uptake, the inventors fed 10% and 25% Lactobacillus respectively within 3 days. 8 monoclonal strains were isolated from the cecum feces of mice (DR-NP(D4) group) fed with a normal protein diet for one day after 65% of the food amount, and sequenced and analyzed, the results showed that these monoclonal strains had the same The 16S ribosomal RNA gene sequence indicates that they are the same bacterial strain, and the bacterial strain can be obtained based on the 16S ribosomal RNA gene sequence (such as isolating bacterial strains from intestinal flora and identifying lactobacilli with this sequence).

The phylogenetic tree analysis based on the 16S ribosomal RNA gene sequence showed that the isolated monoclonal strain was closest to Lactobacillus murinus (L. murinus (NR_112689)) (Fig. 2A). One of the eight monoclonal strains was randomly selected and named Lam-1.

In order to determine whether Lactobacillus Lam-1 would lead to enhanced lipid absorption in the small intestine and fatty acid uptake in white fat, the inventors administered control or 10 ¹⁰ CFU (colony forming units) of Lam-1 bacteria to mice at intervals of 24 hours The mice were fed with BODIPY fluorescent-labeled fatty acid analogs at the same time. As shown in Figure 2B, the relative level of BODIPY in feces of mice after oral administration of Lam-1 bacteria was significantly reduced, which suggested that the lipid absorption capacity of mice was enhanced. Further, the inventors found that compared with control mice, the fluorescence intensity of fresh proximal jejunal tissues and their villi cryosections of mice gavaged with Lam-1 increased (Figure 2C and 2D), while the proximal jejunum The relative concentration of BODIPY in tissue as well as in serum was significantly increased (Fig. 2E and 2F). Then the inventors analyzed the fatty acid uptake of white adipose tissue, and found that compared with control group mice, the fluorescence intensity of fresh groin and epididymis white adipose tissue and their cryosections of mice fed with Lam-1 bacterial strain increased ( Figures 2G and 2H), while the corresponding tissue relative concentration of BODIPY was significantly increased (Figure 2I).

Further, the inventors found that after continuous gavage of the Lam-1 strain for 5 days and 10 days, the body fat content of the mice and the percentage of body fat relative to the body weight increased significantly (Fig. 2J and 2K), and during the gavage The food intake and water intake of the mice did not change significantly (Fig. 2L and 2M). In addition, the body temperature of the mice did not change significantly after gavage of Lam-1 (Fig. 2N), which suggested that the energy expenditure of the mice did not change significantly. Analysis of the above results shows that the Lam-1 strain can promote lipid absorption in mice and lead to obesity in mice. The 16S ribosomal RNA gene sequences of the eight monoclonal strains were the same, suggesting that the other monoclonal strains in the eight monoclonal strains had the same function as Lam-1.

The above research results prove that the enriched Lactobacillus during refeeding after dietary restriction can enhance lipid absorption in the small intestine and fatty acid uptake in white fat, thereby promoting lipid accumulation and leading to obesity in mice.

Example 3. Metabolites Produced by Lactobacillus Upregulate Lipid Absorption in the Small Intestine and Fatty Acid Uptake in White Adipose Tissue

In view of the above experimental results of using antibiotics to clear intestinal flora and Lam-1 bacteria after gavage, the inventors want to further explore how intestinal flora regulates intestinal fat absorption and whitening of mice after diet restriction. Fatty acid uptake. To this end, the inventors collected the cecal feces of mice before dietary restriction and re-fed mice with normal protein or high-protein diet after three days of feeding with 10%, 25%, and 65% of the food amount, respectively, through non-targeted metabolomics To detect and analyze the metabolite components of the intestinal flora.

As expected, compared with mice before dietary restriction, the intestinal flora metabolite composition of cecal feces of mice re-fed with normal protein diet after dietary restriction was significantly changed, and some metabolite changes could be explained by high protein Diet blocking (Fig. 3A).

In the "blockable increase" group, compared with the mice before dietary restriction, the metabolites in the cecal feces of mice re-fed with a normal protein diet for one day after dietary restriction (DR-NP(D4) group), Five metabolites increased significantly, they were DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), 2-hydroxyisocaproic acid (HICA), 2-hydroxy-3 methylbutyric acid (HMBA ) and indole lactic acid (ILA) (Fig. 3A), the increase in the concentration of these five metabolites could be blocked to some extent by a high-protein diet. At the same time, 5 purchased compounds were used as standards, and the concentration of 5 metabolites in the sample was detected and analyzed by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results further confirmed the different Concentration changes of the 5 metabolites between groups (Fig. 3B-3F). In addition, the inventors found that the concentration of five metabolites in the corresponding cecum feces increased significantly after the mice were given Lactobacillus Lam-1 ( FIG. 3G ), which indicated that the five metabolites were produced by Lactobacillus.

The above data prove that refeeding after dietary restriction induces changes in the composition of cecal fecal metabolites, some of which can be blocked by a high-protein diet, such as PLA, HPLA, HICA, HMBA, and ILA, and these five metabolites The concentration of can be up-regulated by Lactobacillus.

To explore whether alterations in these metabolites lead to enhanced intestinal lipid absorption and fatty acid uptake in white fat, the inventors planned to treat mice with the most significantly altered metabolites. Among the five most significantly increased metabolites in the "blockable increase" group, the three metabolites PLA, HPLA and ILA can mobilize dietary phenylalanine and tyrosine respectively by gut bacteria and tryptophan, and are produced by reduction reactions using the same enzymes. Therefore, the inventors first studied the effects of these three compounds on metabolism, and the inventors found that PLA, HPLA and ILA were treated separately, or that the three compounds were treated together and would not affect the mice's food intake, intestinal lipid absorption and Fatty acid uptake by white fat had a significant effect (Fig. 5A-5L). Furthermore, the inventors found no significant effect after HICA or HMBA treatment (Fig. 6A-6L). However, when the mice were given a mixed solution of five compounds including PLA, HPLA, ILA, HICA and HMBA, and the mice were given BODIPY fluorescently labeled fatty acid analogues, the inventors found that the feces of the mice BODIPY was relatively Concentrations were significantly reduced without significant changes in food intake (Fig. 3H and 3I), suggesting enhanced lipid uptake in mice.

Further, the inventors found that compared with the control mice fed with water, the fluorescence intensity of the proximal jejunum tissue and their villi cryosections of the mice fed with the five compounds were increased (Fig. 3J and 3K), and the corresponding The relative concentration of BODIPY in the proximal jejunum was significantly increased (Fig. 3L).

At the same time, the inventors also analyzed the fatty acid uptake of white adipose tissue. Compared with the control group mice that were given intragastric water, the fresh inguinal and epididymal white adipose tissues and their frozen sections of mice that were given five compounds were The fluorescence intensity increased (Fig. 3M and 3N), and the corresponding tissue relative concentration of BODIPY increased significantly (Fig. 3O).

The above experimental results show that some intestinal bacteria, such as Lactobacillus, produce five metabolites including PLA, HPLA, ILA, HICA and HMBA, which can up-regulate lipid absorption in the small intestine and fatty acid uptake in white fat.

In the present invention, the inventors found that refeeding after dietary restriction promotes the enrichment of Lactobacillus and its metabolites in the small intestine, while high-protein diet or antibiotic treatment can inhibit this enrichment, and the increased Lactobacilli and their metabolites will Enhanced lipid absorption from the small intestine and fatty acid uptake in white fat, and ultimately hypertrophy (Fig. 3P).

Preservation of Biological Material

The strain of the present invention (Lactobacillus murinus Lam-1) has been preserved in the China Center for Type Culture Collection (Wuhan, China, Wuhan University), the preservation date: December 29, 2021, and its preservation number is CCTCC NO: M 20211687.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims. Also, all documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference.

Claims (12)

1. A method for regulating lipid absorption or body weight, characterized in that the method comprises:

   (a) Regulating the content of five compound combinations in the digestive tract, the five compound combinations are: DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indole lactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3 methylbutyl a combination of acids; or

   (b) regulating the content of lactobacillus in the digestive tract, said lactobacillus is a lactobacillus with the 16S ribosomal RNA gene sequence shown in SEQ ID NO:1.
2. The method according to claim 1, wherein the adjustment is to increase the content of the five-compound combination in the digestive tract, thereby increasing lipid absorption or body weight; or, the adjustment is to reduce the content of the five-compound combination in the digestive tract, thereby decreased lipid absorption or body weight; or

   The adjustment is to increase the content of Lactobacillus in the digestive tract, thereby increasing lipid absorption or body weight; or, the adjustment is to reduce the content of Lactobacillus in the digestive tract, thereby reducing lipid absorption or body weight.
3. The method according to claim 2, wherein said increasing lipid absorption includes inhibiting diarrhea or improving malnutrition; preferably, said diarrhea is caused by increased secretion and/or decreased absorption.
4. The method according to claim 2, wherein said increasing the content of the five-compound combination in the digestive tract comprises: intake of exogenous five-compound combination;

   The reduction of the content of the five-compound combination in the digestive tract includes: reducing the amount of lactobacillus that metabolizes the five-compound, preferably using antibiotics to reduce the amount of lactobacillus;

   The increase of the content of Lactobacillus in the digestive tract includes: first restricting the diet, then restoring the diet, and taking a normal diet; or, taking in the exogenous Lactobacillus;

   The reduction of the content of lactobacilli in the digestive tract includes: first restricting the diet, then restoring the diet, taking a high-protein diet; or taking antibiotics;

   Preferably, the antibiotics include: vancomycin, ampicillin, neomycin, metronidazole, gentamicin, kanamycin, streptomycin, cefoperazone, erythromycin, tylosin , amoxicillin, penicillin, bacitracin, tetracycline, doxycycline, or clindamycin.
5. The method according to claim 1, characterized in that, the preservation number of the lactobacillus in the China Center for Type Culture Collection is CCTCC NO: M 20211687.
6. Use of five compound combinations or lactobacilli or modulators that regulate them for the preparation of compositions for the regulation of lipid absorption or body weight;

   The five-compound combination is: DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indole lactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3 methylbutyric acid combination; or

   The lactobacillus is a lactobacillus having the 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1, its metabolites, culture or cell lysate; Lactobacillus whose preservation number is CCTCC NO: M 20211687.
7. The method according to any one of claims 1-5 or the application according to claim 6, wherein the high-protein diet includes: protein, carbohydrates, fat, cellulose, minerals and vitamins;

   Wherein, the protein content is 400-800 parts by weight; preferably 500-700 parts by weight;

   Wherein, the carbohydrate content is 150-350 parts by weight; preferably 180-300 parts by weight;

   Wherein, the fat content is 50-90 parts by weight; preferably 60-80 parts by weight;

   Wherein, the cellulose content is 30-70 parts by weight; preferably 40-60 parts by weight;

   Wherein, the mineral content is 15-55 parts by weight; preferably 25-45 parts by weight;

   Wherein, the vitamin content is 8-18 parts by weight; preferably 10-16 parts by weight.
8. The method or application according to claim 5, characterized in that, the dietary restrictions include: regular diet, intermittent diet, time-limited diet, low-energy diet that simulates dieting, and gradient-increasing or gradient-decreasing dieting.
9. The method according to claim 1 or the application according to claim 2, characterized in that, in the five compound combinations, DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid and 2-hydroxyl-3 methylbutanoic acid in parts by weight: 20-40: 10-20: 6-10: 30-50: 15-25.
10. A composition for regulating lipid absorption or body weight, characterized in that it comprises:

    DL-3-phenyl lactic acid, 4-hydroxyphenyl lactic acid, indole lactic acid, 2-hydroxyisocaproic acid, 2-hydroxy-3 methyl butyric acid, in parts by weight: 20-40: 10-20: 6-10: 30-50: 15-25; or

    Lactobacillus, said Lactobacillus is a Lactobacillus having a 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1, its metabolites, culture or cell lysate; preferably, said Lactobacillus is typically cultured in China The preservation number of CCTCC NO: M 20211687 is Lactobacillus.
11. The composition according to claim 10, wherein the lactobacillus is a lactobacillus obtained by the following method: isolating lactic acid having the 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1 from intestinal microorganisms Bacillus; preferably, also includes carrying out the proliferating culture of the isolated Lactobacillus.
12. Isolated Lactobacillus, its metabolites, culture or cell lysate, the preservation number of the Lactobacillus in China Center for Type Culture Collection is CCTCC NO: M 20211687.

Images