Methods of modulating lipid absorption, compositions, and uses thereof

Abstract

The application provides a method for regulating lipid absorption, a composition and application thereof. The present inventors have been committed to the research on the correlation between diet and metabolism, and found in the previous research that compared with before diet restriction, re-feeding after diet restriction can significantly increase the body fat content. Further research shows that the composition of intestinal flora during normal diet after diet restriction changes significantly, and the proportion of lactobacillus increases significantly; high-protein diet after diet restriction can significantly inhibit the increase of the proportion of small intestinal lactobacillus, and the diversity of flora is significantly increased. Clearing the intestinal flora can effectively inhibit the increase of body fat caused by re-feeding after diet restriction. The present inventors have also found that a five-compound combination of a specific lactobacillus strain can significantly increase lipid absorption and fatty acid uptake of white adipose tissue.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological metabolism and food science, and more particularly, the present application relates to a method for regulating lipid absorption, a composition and application thereof. BACKGROUND

[0002] Since the mid-1970s, the number of obese people has increased significantly worldwide. Obesity threatens human health, and it significantly increases the risk of diseases such as type 2 diabetes, fatty liver, cardiovascular disease, cancer, and even increases the mortality rate of COVID-19 patients. Obesity is characterized by excessive accumulation of fat, and the root cause is an imbalance in energy homeostasis, i.e., too many calories are consumed and too few calories are expended.

[0003] Dietary restriction is considered an important method for improving lipid metabolism in normal weight or obese mammals and reducing fat content. In normal weight women and men, dieting for weight loss is increasingly popular. Restricting calorie intake and intermittent fasting as a commonly used dietary restriction method can significantly reduce the body weight of normal and mildly overweight adults, and the part of weight loss is mainly fat. However, normal weight women or men will experience weight rebound after dieting and returning to normal diet. 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.

[0004] On the other hand, for some people with malnutrition, frequent diarrhea or weight loss, it is necessary to improve lipid metabolism in order to promote lipid absorption.

[0005] Dietary factors are key determinants of the structure and function of the gut microbiota community, and nutrients can directly interact with microorganisms to promote or inhibit their growth, and the microbial community that takes more energy from specific dietary components has a good competitive advantage.

[0006] However, how does the composition of the host gut microbiota change during the re-feeding process after dietary restriction, and does the gut microbiota and its metabolites participate in the weight rebound after re-feeding after dietary restriction and the lipid metabolism of the small intestine and white adipose tissue during the re-feeding process? These issues remain to be elucidated in the art. SUMMARY

[0007] The purpose of the present application is to provide a method for regulating lipid absorption, a composition and application thereof.

[0008] In a first aspect of the application, there is provided a method of modulating lipid absorption or body weight, the method comprising: (a) modulating the level of a five-compound combination in the digestive tract (including the stomach or intestine), the five-compound combination being: a combination of DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA), 2-hydroxyisocaproic acid (HICA), and 2-hydroxy-3-methylbutyric acid (HMBA); or, (b) modulating the level of a Lactobacillus in the digestive tract (including the stomach or intestine), the Lactobacillus being a Lactobacillus having a 16S ribosomal RNA gene sequence as set forth in SEQ ID NO: 1.

[0009] In one or more embodiments, the modulating is increasing the level of the five-compound combination in the digestive tract, thereby increasing lipid absorption or body weight; or, the modulating is decreasing the level of the five-compound combination in the digestive tract, thereby decreasing lipid absorption or body weight.

[0010] In one or more embodiments, the modulating is increasing the level of the Lactobacillus in the digestive tract, thereby increasing lipid absorption or body weight; or, the modulating is decreasing the level of the Lactobacillus in the digestive tract, thereby decreasing lipid absorption or body weight.

[0011] In one or more embodiments, the lipid absorption is small intestinal lipid absorption.

[0012] In one or more embodiments, the lipid includes fatty acids of white adipose tissue.

[0013] In one or more embodiments, the increasing is a statistically significant increase, such as an increase of 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 100% or more.

[0014] In one or more embodiments, the decreasing is a statistically significant decrease, such as a decrease of 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 100% or more.

[0015] In one or more embodiments, the increasing lipid absorption includes inhibiting diarrhea or ameliorating malnutrition.

[0016] In one or more embodiments, the diarrhea is diarrhea resulting from increased secretion and / or decreased absorption.

[0017] In one or more embodiments, the increasing the level of the five-compound combination in the digestive tract includes ingesting an exogenous five-compound combination.

[0018] In one or more embodiments, the reducing the content of the five-compound combination in the digestive tract comprises reducing the amount of the lactic acid bacteria that metabolize the five-compound combination, preferably by antibiotics.

[0019] In one or more embodiments, the increasing the content of the lactic acid bacteria in the digestive tract comprises: first performing dietary restriction, and then resuming diet, and taking normal diet; or, taking exogenous lactic acid bacteria.

[0020] In one or more embodiments, the reducing the content of the lactic acid bacteria in the digestive tract comprises: first performing dietary restriction, and then resuming diet, and taking high-protein diet; or taking antibiotics.

[0021] In one or more embodiments, the lactic acid bacteria has a preservation number of CCTCC NO: M 20211687 in China Center for Type Culture Collection.

[0022] In another aspect of the present application, there is provided use of the five-compound combination or the lactic acid bacteria or the modulator thereof for preparing a composition for regulating lipid absorption or body weight; the five-compound combination is a combination of DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3-methylbutyric acid; or, the lactic acid bacteria is a lactic acid bacteria having a 16S ribosomal RNA gene sequence as shown in SEQ ID NO: 1.

[0023] In one or more embodiments, the antibiotics include (but are not limited to) vancomycin, ampicillin, neomycin, metronidazole, gentamicin, kanamycin, streptomycin, cefoperazone, erythromycin, tylosin, amoxicillin, penicillin, bacitracin, tetracycline, doxycycline or clindamycin, etc.

[0024] In one or more embodiments, the high protein diet (e.g., 1000 parts by weight total) includes: protein, carbohydrate, fat, fiber, minerals, and vitamins. In one or more embodiments, the protein content is 400-800 parts by weight (e.g., 450, 600, 650, or 800 parts by weight); preferably 500-700 parts by weight; the carbohydrate content is 150-350 parts by weight (e.g., 160, 180, 190, 200, 220, 230, 240, or 280 parts by weight); preferably 180-300 parts by weight; the fat content is 50-90 parts by weight (e.g., 55, 65, 75, or 85 parts by weight); preferably 60-80 parts by weight; the fiber content is 30-70 parts by weight (e.g., 35, 45, 55, or 65 parts by weight); preferably 40-60 parts by weight; the mineral content is 15-55 parts by weight (e.g., 28, 32, 38, or 42 parts by weight); preferably 25-45 parts by weight; and the vitamin content is 8-18 parts by weight (e.g., 9, 12, 14, 16, or 18 parts by weight); preferably 10-16 parts by weight.

[0025] In one or more embodiments, the protein includes a protein selected from the group consisting of: casein, cysteine, whey protein, and soy protein.

[0026] In one or more embodiments, the ratio of casein to cysteine in the high protein diet is 50-80:1, preferably 55-75:1, and more preferably 60-70:1.

[0027] In one or more embodiments, the carbohydrate is corn starch and / or maltodextrin; preferably, the ratio of corn starch to maltodextrin is 8-10:12-15.

[0028] 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.

[0029] In one or more embodiments, the dietary restriction includes, but is not limited to, regular dieting, intermittent dieting, time-restricted dieting, low-energy dieting mimicking dieting, gradient increasing or gradient decreasing dieting.

[0030] In one or more embodiments, the time period for which the dietary restriction is used is the time period required for a significant reduction in body weight and lipid. For example, this time period is at least 2 days (e.g., 2-100 days), at least 3, 4, 5, 6, 7, 8, or more days, e.g., 9, 10, 15, 20, 30, 45, 60, 80, 100, or more days.

[0031] In one or more embodiments, the dietary restriction is a gradient increasing or gradient decreasing dieting (such as providing 10%, 25%, 65% of food amount respectively within three days, providing 65%, 25%, 10% of food amount respectively within three days, and the like).

[0032] In one or more embodiments, the time for resuming diet, or taking normal diet, is the time required for significant increase of lipid, such as 1-100 days (more specifically such as 2, 3, 4, 5, 6, 8, 10, 15, 20, 30, 50, 70, 80, 90 days).

[0033] In one or more embodiments, the time for resuming diet, or taking high-protein diet, is the time required for significant increase of lipid, such as 1-100 days (more specifically such as 2, 3, 4, 5, 6, 8, 10, 15, 20, 30, 50, 70, 80, 90 days).

[0034] In one or more embodiments, the method for regulating lipid absorption or body weight is a non-diagnostic and therapeutic method.

[0035] In one or more embodiments, the method for dietary restriction and re-feeding is a non-diagnostic and therapeutic method.

[0036] In one or more embodiments, in the combination of five compounds, the weight ratio (or weight / volume ratio) of DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid, and 2-hydroxy-3-methylbutyric acid is 20-40:10-20:6-10:30-50:15-25.

[0037] In another aspect of the present application, a composition for regulating (increasing) lipid absorption or body weight is provided, comprising: DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid, and 2-hydroxy-3-methylbutyric acid, in a weight ratio (or weight / volume ratio) of 20-40:10-20:6-10:30-50:15-25; or, Lactobacillus, wherein the Lactobacillus is a Lactobacillus having a 16S ribosomal RNA gene sequence as shown in SEQ ID NO: 1; preferably, the Lactobacillus is a Lactobacillus having a preservation number of CCTCC NO: M 20211687 in China Center for Type Culture Collection, a metabolite, culture, or cell lysate thereof.

[0038] In one or more embodiments, the weight ratio of DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid, and 2-hydroxy-3-methylbutyric acid is 25-35:12-18:7-9:35-45:17-23.

[0039] 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 in a ratio of 30 ± 3: 15 ± 1.5: 8 ± 0.8: 40 ± 4: 20 ± 2 by weight.

[0040] In one or more embodiments, the DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid, 2-hydroxy-3-methylbutyric acid are in a ratio of 30: 15: 8: 40: 20 by weight.

[0041] In one or more embodiments, the five-compound combination is dissolved in water or an aqueous solvent.

[0042] In one or more embodiments, the five-compound combination is mixed with a food-acceptable carrier or an industry-acceptable carrier.

[0043] In one or more embodiments, the composition is a solid, semi-solid, or liquid preparation.

[0044] In one or more embodiments, the composition is a food composition.

[0045] In one or more embodiments, the method, use, or composition is for use in an animal; preferably in a mammal; more preferably including: a rodent (such as a mouse), a primate (including a human and a non-human primate, such as an ape, a monkey, a chimpanzee), a domestic animal (such as a pig, a dog, a chicken, a duck, a rabbit, etc.).

[0046] In one or more embodiments, the Lactobacillus is obtained by a method comprising: isolating a Lactobacillus having a 16S ribosomal RNA gene sequence as set forth in SEQ ID NO: 1 from intestinal microorganisms; and preferably further comprising: proliferating the isolated Lactobacillus.

[0047] In one or more embodiments, the Lactobacillus is identified by primers having sequences as set forth in SEQ ID NO: 2 and SEQ ID NO: 3.

[0048] In one or more embodiments, the Lactobacillus is identified by primers having sequences as set forth in SEQ ID NO: 4 and SEQ ID NO: 5.

[0049] In another aspect of the present application, an isolated Lactobacillus, a metabolite, a culture, or a cell lysate thereof is provided, wherein the Lactobacillus has a preservation number of CCTCC NO: M20211687 at the China Center for Type Culture Collection.

[0050] Other aspects of the application will be apparent to those of ordinary skill in the art in view of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 , dietary restriction followed by re-feeding alters gut microbiota, which in turn induces fat accumulation, enhances lipid absorption in the small intestine, and fatty acid uptake in white adipose tissue.

[0052] (A) Principal coordinate analysis of the cecal fecal microbiota of mice. Analysis was calculated based on Bray-Curtis distance. AL, indicates ad libitum feeding; NP, indicates normal protein diet; HP, indicates high protein diet; DR, indicates dietary restriction by providing mice with 10%, 25%, and 65% of their food intake for 3 days, respectively; AL-NP(D0), indicates normal feeding before dietary restriction; DR-NP(D4) and DR-HP(D4), indicates 1 day of re-feeding with normal protein chow or high protein chow, respectively, after 3 days of dietary restriction; DR-NP(D6) and DR-HP(D6), indicates 3 days of re-feeding with normal protein chow or high protein chow, respectively, after 3 days of dietary restriction. Each dot represents an individual mouse. PC1, PC2, and PC3 indicate the percent variance explained by each coordinate. N = 3-6 mice / group.

[0053] (B) Alpha diversity of the gut microbiota from mice in (A). Alpha diversity is represented by the Shannon index.

[0054] (C and D) Proportional abundance of class-level (C) and family-level (D) of the gut microbiota from mice in (A).

[0055] (E) Proportional abundance of Lactobacillus genus of the gut microbiota from mice in (A).

[0056] (F) Re-feeding stage treatment with antibiotics significantly attenuates fat accumulation in mice. DR, indicates dietary restriction by providing mice with 10%, 25%, and 65% of their food intake for 3 days, respectively, followed by normal diet. Gray shaded portion indicates the re-feeding stage, during which experimental mice were treated with antibiotics and control mice were not treated with antibiotics. ABX, indicates antibiotics. N = 9 mice / group.

[0057] (G and H) Representative fluorescent images of fresh small intestinal tissue (G) and their villus cryosections (H) of mice. DR, indicates dietary restriction by providing mice with 10%, 25%, and 65% of their food intake for 3 days, respectively, followed by normal diet. Experimental mice were treated with antibiotics during the re-feeding stage. Mice were gavaged with a mixture of BODIPY (boron-dipyrromethene) fluorescently labeled fatty acid analogs and olive oil on day 5, and tissues were harvested 2 hours post-gavage.

[0058] (BODIPY relative concentration in the small intestine (I) and serum (J) of mice, mice from (G) figure, N=7-8 mice / group.

[0059] (K and L) Representative fluorescence pictures of fresh inguinal and epididymal white adipose tissues (K) and their frozen sections (L) of mice, mice from (G) figure.

[0060] (M) BODIPY relative concentration in the inguinal and epididymal white adipose tissues of mice, mice from (G) figure, N=7-8 mice / group.

[0061] a or *, p < 0.05; b, p < 0.01; c or ***, p < 0.001; NS, not significant.

[0062] Figure 2 Lactobacillus sp. Lam-1 enhances intestinal lipid absorption and white adipose tissue fatty acid uptake and promotes body fat accumulation in mice.

[0063] (A) Phylogenetic tree of the isolated Lactobacillus sp. Lam-1 (L. murinus) and its relatives based on 16S ribosomal RNA gene sequences. Bar statistics represent sequence differences.

[0064] (B) BODIPY relative concentration in the feces of mice treated with control or Lam-1 strain. Feces produced by mice 10 min to 2 h after gavage with a mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil, N=8 mice / group.

[0065] (C and D) Representative fluorescence pictures of fresh small intestine tissues (C) and their villi frozen sections (D) of mice, mice from (B) figure, tissues were collected 2 h after gavage.

[0066] (E and F) BODIPY relative concentration in the small intestine (E) and serum (F) of mice, mice from (B) figure, N=8 mice / group.

[0067] (G and H) Representative fluorescence pictures of fresh inguinal and epididymal white adipose (G) and their frozen sections (H) of mice, mice from (B) figure, tissues were collected 2 h after gavage.

[0068] (I) BODIPY relative concentration in the inguinal and epididymal white adipose of mice, mice from (B) figure, N=8 mice / group.

[0069] (J) Body fat content of mice after gavage with control or Lam-1 strain. D0 indicates before gavage, D5 and D10 indicate 5 and 10 days of consecutive gavage, respectively, N = 8-9 mice / group.

[0070] (K) Body fat percentage content relative to body weight of mice after gavage with control or Lam-1 strain. D0 indicates before gavage, D5 and D10 indicate 5 and 10 days of consecutive gavage, respectively, N = 8-9 mice / group.

[0071] (L and M) Daily food intake (L) and water intake (M) of mice during consecutive gavage with control or Lam-1 strain.

[0072] (N) Body temperature of mice after gavage with control or Lam-1 strain. D0 indicates before gavage, D5 and D10 indicate 5 and 10 days of consecutive gavage, respectively, N = 8-9 mice / group.

[0073] * p < 0.05; ** p < 0.01; NS, not significant.

[0074] Figure 3 , Metabolites of the gut microbiota enhance lipid absorption in the small intestine and fatty acid uptake in white adipose tissue during the re-feeding phase after dietary restriction.

[0075] (A) Metabolite composition heat map of the small intestine of mice before dietary restriction and after re-feeding with normal or high-protein diet. DR, indicates that mice were subjected to dietary restriction by providing 10%, 25%, 65% of the food amount for three days, respectively, N = 3-6 mice / group.

[0076] (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 in the cecal feces of mice, N = 4 mice / group.

[0077] (G) Metabolite concentrations in the cecal feces of mice after gavage with control or Lam-1 bacteria, N = 9 mice / group.

[0078] (H) Food intake of mice after gavage with water or a mixture solution containing 5 compounds, PLA, HPLA, ILA, HICA, and HMBA, for 24 hours, N = 10 mice / group.

[0079] (I) Relative BODIPY concentration in the feces of mice after gavage with water or a mixture solution of 5 compounds. Feces produced by mice 10 min to 2 hours after gavage with a mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil were collected, N = 10 mice / group.

[0080] (J and K) Representative fluorescent pictures of fresh small intestine tissue (J) and their villi frozen sections (K) of mice from (I) figure, tissues were collected 2 hours after gavage.

[0081] (L) BODIPY relative concentration of mouse small intestine, mice from (I) figure, N=10 mice / group.

[0082] (M and N) Representative fluorescent pictures of fresh inguinal and epididymal white adipose tissue (M) and their frozen sections (N) of mice from (I) figure, tissues were collected 2 hours after gavage.

[0083] (O) BODIPY relative concentration of mouse inguinal and epididymal white adipose tissue, mice from (I) figure, N=10 mice / group.

[0084] (P) Overall flow chart of the experiment. Re-feeding after dietary restriction induces increase of lactobacilli and their metabolites, which in turn promotes small intestine lipid absorption and white adipose tissue fatty acid uptake, finally resulting in increased body weight. High protein diet intervention or antibiotic treatment during the re-feeding phase can alleviate the increased body weight after dietary restriction.

[0085] * or #, p<0.05; ** or ##, p<0.01; NS, not significant.

[0086] Figure 4 , antibiotic treatment during the re-feeding phase after dietary restriction can inhibit the increase of food intake and body fat percentage and the decrease of lean body mass percentage.

[0087] (A) Food intake of mice without antibiotic and with antibiotic treatment during the re-feeding phase after dietary restriction. DR, represents the dietary restriction by providing mice with 10%, 25%, 65% of food amount respectively for three days. The gray shaded part represents the re-feeding phase, mice in experimental groups were treated with antibiotics during the re-feeding phase, mice in control groups were not treated with antibiotics during the re-feeding phase. ABX, represents antibiotics, N=9 mice / group.

[0088] (B and C) Body fat percentage (B) and lean body mass percentage (C) of mice from (A), N=9 mice / group.

[0089] a, p<0.05; b, p<0.01; c, p<0.001.

[0090] Figure 5 , PLA, HPLA and ILA gavage alone and mixed gavage have no significant effect on small intestine lipid absorption and white adipose tissue fatty acid uptake.

[0091] (A) Food intake of mice 24 hours after gavage with water, DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), or indolelactic acid (ILA), N=6 mice / group.

[0092] (B) Relative BODIPY concentration in feces of mice. Feces produced by mice 10 minutes to 2 hours after gavage with a mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil were collected, N=6 mice / group.

[0093] (C) Representative fluorescence pictures of fresh small intestine tissue of mice from (B), tissue was collected 2 hours after gavage.

[0094] (D) Relative BODIPY concentration in small intestine of mice from (B), N=6 mice / group.

[0095] (E) Representative fluorescence pictures of fresh inguinal and epididymal white adipose tissue of mice from (B), tissue was collected 2 hours after gavage.

[0096] (F) Relative BODIPY concentration in inguinal and epididymal white adipose of mice from (B), N=6 mice / group.

[0097] (G) Food intake of mice 24 hours after gavage with water or a solution containing PLA, HPLA, and ILA, N=7 mice / group.

[0098] (H) Relative BODIPY concentration in feces of mice. Feces produced by mice 10 minutes to 2 hours after gavage with a mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil were collected, N=6 mice / group.

[0099] (I) Representative fluorescence pictures of fresh small intestine tissue of mice from (H), tissue was collected 2 hours after gavage.

[0100] (J) Relative BODIPY concentration in small intestine of mice from (H), N=7 mice / group.

[0101] (K) Representative fluorescence pictures of fresh inguinal and epididymal white adipose of mice from (H), tissue was collected 2 hours after gavage.

[0102] (L) Relative BODIPY concentration in inguinal and epididymal white adipose of mice from (H), N=7 mice / group.

[0103] NS, no significant.

[0104] Figure 6 , HICA, or HMBA had no significant effect on small intestine lipid absorption and fatty acid uptake in white adipose tissue.

[0105] (A and G) Food intake of mice after 24 hours of gavage with 2-hydroxyisocaproic acid (HICA) (A) or 2-hydroxy-3-methylbutyric acid (HMBA) (G), N = 7-8 mice / group.

[0106] (B and H) Relative BODIPY concentration in feces of mice. Feces produced from 10 min to 2 hours after gavage with a mixture of BODIPY fluorescently labeled fatty acid analogs and olive oil were collected, N = 7-8 mice / group.

[0107] (C and I) Representative fluorescence pictures of fresh small intestine tissue of mice. Mice were from (B) and (H) respectively, tissue was collected 2 hours after gavage.

[0108] (D and J) Relative BODIPY concentration in small intestine of mice. Mice were from (B) and (H) respectively, N = 6 mice / group.

[0109] (E and K) Representative fluorescence pictures of fresh inguinal and epididymal white adipose tissue of mice. Mice were from (B) and (H) respectively, tissue was collected 2 hours after gavage.

[0110] (F and L) Relative BODIPY concentration in inguinal and epididymal white adipose of mice. Mice were from (B) and (H) respectively, N = 6 mice / group.

[0111] NS, not significant. DETAILED DESCRIPTION

[0112] The present inventors have dedicated to study the correlation between diet and metabolism. In previous studies, the present inventors found that re-feeding after dietary restriction significantly increased body fat content compared to before dietary restriction. Further studies showed that the composition of gut microbiota during normal diet after dietary restriction was significantly changed, in which the proportion of Lactobacillus was significantly increased. High-protein diet after dietary restriction significantly inhibited the increase of Lactobacillus proportion in small intestine, while the diversity of gut microbiota was significantly increased. Elimination of gut microbiota could effectively inhibit the increase of body fat caused by re-feeding after dietary restriction. The present inventors also found that a specific strain of Lactobacillus and a five-compound combination could significantly increase lipid absorption and fatty acid uptake of white adipose tissue.

[0113] TERMINOLOGY

[0114] As used herein, the terms “dietary (food or foodstuff) restriction”, “dietary (food or foodstuff) control”, “intake restriction”, “intake control” and “dieting” are used interchangeably.

[0115] As used herein, the term "dietary restriction" refers to a specific period of time during which the subject's food intake is significantly less than the "normal diet." The "normal diet" generally refers to the subject's daily or natural food intake without the dietary restriction or prior to the dietary restriction.

[0116] As used herein, the term "resuming diet, normal diet" refers to a specific period of time after the "dietary restriction" period, during which the subject resumes the natural state of food supply and consumes a regular diet.

[0117] As used herein, the term "resuming diet, high protein diet" refers to a specific period of time after the "dietary restriction" period, during which the subject resumes the natural state of food supply but consumes a high protein diet.

[0118] As used herein, the term "increasing the amount of the five-compound combination / lactobacilli in the digestive tract" includes increasing the amount in the digestive tract by ingesting the five-compound combination / lactobacilli, for example, by adding the five-compound combination / lactobacilli to food or by taking the five-compound combination / lactobacilli alone.

[0119] As used herein, the term "composition of the present invention" includes food, health supplement, etc., as long as the components are adjusted as described in the present invention.

[0120] As used herein, the terms "comprising" or "including," or "having" include "consisting of," "consisting essentially of," and "consisting." "Consisting essentially of" means that in the composition, in addition to the essential ingredients or components, a small amount of an auxiliary ingredient or component, and / or an impurity can be contained, which does not affect the essential ingredients. For example, a sweetener or a flavoring agent can be contained to improve the taste, an antioxidant to prevent oxidation, and other additives commonly used in the art.

[0121] As used herein, the term "nutritionally acceptable" or "dietetically acceptable" ingredient is a substance that is suitable for use in humans and / or animals without excessive adverse side effects (e.g., toxicity, irritation, and allergic response) - i.e., the benefits outweigh the risks, on a reasonable benefit / risk basis.

[0122] As used herein, the term "effective amount" refers to an amount that is functional or active for humans and / or animals and is acceptable to humans and / or animals.

[0123] As used herein, "parts by weight" or "parts by weight" are used interchangeably, and the parts by weight can be any one fixed weight expressed in micrograms, milligrams, grams or kilograms (such as 1 μg, 1 mg, 1 g, 2 g, 5 g, 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 a composition consisting of 1 g of component a + 9 g of component b, or 10 g of component a + 90 g of component b, etc. In the composition, the percentage content of a certain component = (the parts by weight of the component / the sum of the parts by weight of all components) x 100%. Therefore, in a composition consisting of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and component b is 90%.

[0124] As used herein, the term "unit dosage form" or "unit dosage form" refers to the preparation of the composition of the present application into a dosage form required for single administration for the convenience of taking, including but not limited to various solid preparations (such as tablets), liquid preparations. The unit dosage form contains the composition of the present application suitable for single, single day or unit time administration.

[0125] Lipid metabolism after re-uptake after dietary restriction

[0126] The present inventors found that the body fat content of animals after dietary restriction and re-feeding was significantly increased compared to before dietary restriction, and the composition of the intestinal flora of animals during normal feeding after dietary restriction was significantly changed, in which the proportion of lactobacillus was significantly increased to about 60%, and the flora diversity was reduced. The present inventors proved through experiments that high-protein diet after dietary restriction can effectively inhibit the accumulation of animal body fat content. The results of the present application show that compared to animals after dietary restriction and normal feeding, high-protein diet after dietary restriction can significantly inhibit the increase of the proportion of lactobacillus in the small intestine, and the flora diversity is significantly increased. Treating animals with antibiotics to eliminate intestinal flora can effectively inhibit the increase of body fat caused by re-feeding after dietary restriction. Further, the present inventors found that the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue of animals treated with antibiotics were significantly weakened. After spreading the cecal feces of animals during normal feeding after dietary restriction on a plate and picking single clone strains for isolation and culture, the present inventors obtained a kind of lactobacillus with the 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1 (referred to as Lam-1 strain in the examples), which can significantly increase the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue when administered to animals. At the same time, continuous gavage of the lactobacillus to animals can significantly increase the body fat content but does not affect the food intake of animals. To further explore the mechanism of the increase in weight caused by re-feeding after dietary restriction involving intestinal flora, the present inventors analyzed the flora metabolite composition of the cecal feces of animals, and found that compared to animals before dietary restriction, the flora metabolite composition of animals after dietary restriction and re-feeding normal diet was significantly changed, in which the concentrations of five metabolites, DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA), 2-hydroxyisocaproic acid (HICA) and 2-hydroxy-3-methylbutyric acid (HMBA), were significantly increased, and the increase in the concentration of the five metabolites can be significantly inhibited by high-protein diet. In addition, the concentration of the five metabolites in the cecal feces of animals after gavage of Lam-1 strain was also significantly increased, indicating that the five metabolites were produced by lactobacillus. Further research found that the five compounds can significantly increase the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue. In summary, the research results of the present inventors show that targeting intestinal lactobacillus by high-protein diet or antibiotic treatment and then inhibiting the lipid absorption of the small intestine can be used as an effective method to prevent obesity after dietary restriction.

[0127] Although a series of antibiotics are preferably listed in the embodiments of the present application, however, in addition to vancomycin, ampicillin, neomycin, metronidazole, there are other antibiotics with similar functions, such as but not limited to gentamicin, kanamycin, streptomycin, cefoperazone, erythromycin, tylosin, amoxicillin, penicillin, bacitracin, tetracycline, doxycycline or clindamycin, etc., which can also be applied in the present application.

[0128] Based on the new findings of the present inventor, a method for regulating lipid absorption or body weight is provided, comprising: (a) adjusting the content of a five-compound combination in the digestive tract (including stomach or intestine), the five-compound combination being: a combination of DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA), 2-hydroxyisocaproic acid (HICA), and 2-hydroxy-3-methylbutyric acid (HMBA); or (b) adjusting the content of Lactobacillus in the digestive tract (including stomach or intestine), the Lactobacillus being Lactobacillus having a 16S ribosomal RNA gene sequence as shown in SEQ ID NO: 1.

[0129] In the present application, the Lactobacillus has a specific 16S ribosomal RNA gene sequence, and the specific sequence is also disclosed in the present application, so that those skilled in the art can isolate such strains from intestinal metabolites under the disclosure of the present application, and thus apply them to regulate lipid metabolism.

[0130] In the present application, the five compounds are all known or commercially available compounds in the art.

[0131] In the method of the present application, food intake or dietary control is performed in advance, which can be performed by various approaches, including but not limited to: regular dieting, intermittent dieting, time-limited dieting, low-energy dieting simulating dieting, gradient-increasing or gradient-decreasing dieting. Food intake or dietary control is performed according to the needs or planning of the subject; for example, for humans, the planning can be performed for a long period of time (e.g. 3-6 months or longer) or a medium length of time (e.g. 1-3 months) or a short period of time (e.g. 3-30 days). This process is usually tested by a significant decrease in body weight, which is also measured according to the needs or planning of the subject, for example, a significant decrease of 2-40% of body weight; more specifically, for example, 3%, 5%, 8%, 10%, 15%, 20%, 30%, 35%, etc.

[0132] In the present application, during the "dietary restriction" process, there is no specific restriction on the types of food to be taken in, which can be regular food but with controlled intake amount (significantly reduced intake amount) or low-energy food. However, as a preferred mode of the present application, the "dietary restriction" is a gradient-increasing or gradient-decreasing restriction scheme, for example, the daily intake amount is planned according to the planned dietary restriction time, and is regularly, rhythmically or in a wave pattern. Preferably, even if there is a period of increasing to a higher point, the higher point is lower than the level of "normal diet".

[0133] In the "restoration of food intake" stage, regular diet or food with adjusted protein content can be taken in order to achieve the purpose of regulating lipid absorption.

[0134] Lactobacillus

[0135] In the present application, a new type of Lactobacillus is isolated, which is a Lactobacillus having a 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1; preferably, the Lactobacillus has a preservation number of CCTCC NO: M 20211687 in China Center for Type Culture Collection, and also includes similar strains with the same function (also having a 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1).

[0136] The strain of the present application is a living cell, once the strain of the present application is obtained, the strain of the present application can be obtained in large quantities by means of inoculation, subculture, regeneration, etc. This is usually to inoculate it into a solid plate culture medium or a liquid culture medium to obtain the living cell of the present application by expanding the culture of the strain. The obtained living cell can be further subjected to laboratory domestication, genetic breeding and molecular genetic operation, etc. to obtain mutants and transformants. In addition, the strain of the present application can also be used as a biological engineering host cell for heterologous expression.

[0137] Further, the Lactobacillus of the present application can be used as a starting strain, and further improved by means of laboratory domestication, genetic breeding, molecular genetic operation, etc. to obtain a derivative strain with higher yield or more optimized enzyme system. The strain obtained by further screening and optimization using the Lactobacillus of the present application as a starting strain should also be included in the overall scope of the present application.

[0138] Methods familiar to those skilled in the art can be used to mutagenize the living strain of the present application, and cause changes (optimization) in the genetic code, biological properties and morphology of the living cell. These methods include physical methods such as rays, particles, lasers, ultraviolet light, chemical mutagenesis methods such as alkylating agents, base analogs, hydroxylamine, acridine pigments, etc. The mutagenesis can be one method or multiple generations of mutagenesis of multiple methods, and is not limited to these methods. Based on the strain provided by the present application, further breeding by physical and chemical methods can be carried out, and other regulatory genes can be introduced, and the obtained mutants and transformants can be used to obtain strains with further improved performance, and the breeding method is one or more of the above.

[0139] Methods familiar to those skilled in the art can be used to construct expression constructs (vectors) and further modify the strain of the present application. For example, the signal pathway, signal pathway and proteins involved in the signal pathway related to lipid absorption production found or newly found in the strain are further improved (for example, the expression of beneficial factors is increased, and the expression of harmful factors is reduced).

[0140] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known in the art. The steps used are well known in the art.

[0141] Based on the lactic acid bacteria obtained, the present application also provides a cell culture, a cell metabolite, a cell culture supernatant or a cell lysate of the lactic acid bacteria, which also has the function of regulating lipid absorption or body weight.

[0142] After obtaining the strain of the present application, a person skilled in the art can conveniently obtain its culture, for example, by referring to some of the culture media or culture processes provided in the specific embodiments of the present application, or by using culture media or culture processes that are appropriately changed from those in the embodiments of the present application but can also obtain cultures, so as to obtain a cell culture. The cell culture contains active strains, thereby playing a role in regulating lipid absorption or body weight.

[0143] The cell metabolite is a kind of substance produced or secreted by the strain of the present application during culture, which can be directly secreted by the cell into the culture medium, or separated from the cell after a certain treatment. The cell product can be separated, purified or concentrated.

[0144] The cell culture supernatant is the remaining culture solution after removing the cells and solid impurities during or after the culture of the strain of the present application, which can be unconcentrated or concentrated. Generally, the cells and solid impurities can be removed by methods such as centrifugation, filtration, etc.

[0145] The cell lysate is a mixture formed by lysing the cells using a lysing agent after the culture of the strain of the present application or at the end of the culture. The cell lysate can be a product after removing the solid impurities after lysing. According to the need, it can be a purified or concentrated product.

[0146] Composition

[0147] Based on the new findings of the present inventors, the present application 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 the weight ratio (or weight volume ratio) of 20-40:10-20:6-10:30-50:15-25.

[0148] The present application also provides a composition for regulating (increasing) lipid absorption or body weight, comprising lactic acid bacteria, wherein the lactic acid bacteria is lactic acid bacteria having the 16S ribosomal RNA gene sequence shown in SEQ ID NO: 1.

[0149] The compositions can be food compositions, and in some embodiments, the compositions can further comprise a food or nutraceutical acceptable carrier.

[0150] The ranges of formulations shown herein are intended to be exemplary and are not intended to limit the scope of the application. It is understood that the effective amount of each component can vary depending on the actual application when used to develop a food or composition. For example, the effective amount can vary depending on whether the food or composition is to be prepared in concentrated or diluted form, and such variations are intended to be included within the scope of the application.

[0151] In some preferred embodiments of the application, the compositions are in unit dosage form. When the compositions are prepared in unit dosage form, the compositions are administered in 2 to 6 unit doses per day, such as 2, 3, or 4 unit doses per day, depending on the dietary regimen. The unit dosage forms can be added to food for consumption, for example, in accordance with the mode of action of the compositions of the application.

[0152] In some embodiments of the application, intake regimens for animals such as mice are provided. Conversion of the intake amounts for animals such as mice to amounts suitable for humans is readily performed by one of skill in the art, for example, using the Meeh-Rubner formula: A = k x (W2 / 3) / 10,000. In this formula, A is the body surface area in m2, W is the body weight in g, and k is a constant that varies with the species of animal, typically 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It is understood that the conversion of intake amounts can vary depending on the food and time of day, and can be determined by one of skill in the art based on experience.

[0153] Applications

[0154] The inventors' results show that the proportion of lactobacilli increases to about 60% in animals after dietary restriction followed by re-feeding, and that the concentration of their corresponding metabolites also significantly increases, while this process is inhibited when the animals are re-fed a high protein diet. The inventors' results demonstrate that the administration of the lactobacilli and their metabolites disclosed herein to animals significantly increases the absorption of lipids in the small intestine and the uptake of fatty acids by white adipose tissue.

[0155] Diarrhea is generally due to increased secretion, decreased absorption, or both, and the inventors' results suggest that the lactobacilli or the combination of five compounds metabolized thereby can also be used as probiotics for the treatment of diarrhea.

[0156] The inventors' results also suggest that, in the case of over-nutrition, it can be more important to develop interventions specifically targeting the small intestinal microbiota, either by decreasing the proportion or activity of certain microorganisms that can promote fat absorption, or by increasing the proportion or abundance of microorganisms that can inhibit fat absorption.

[0157] For malnutrition, the abundance and activity of the microorganisms associated with fat absorption can also be increased or decreased to treat. For example, in the case of intestinal failure (e.g., small bowel resection or Crohn's disease) or other environmental intestinal diseases, methods targeting the intestinal flora can be developed to promote more efficient digestion and absorption of nutrients.

[0158] In addition, the results of the inventors' studies show that targeting the inhibition of lactobacilli by special diets such as high-protein diets or antibiotic treatment can be an effective method for reducing small intestinal fat absorption and resisting weight gain after dietary restriction.

[0159] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out according to the conventional conditions, such as the conditions described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Science Press, or according to the conditions recommended by the manufacturer.

[0160] Abbreviation explanation

[0161] In the present application, the abbreviations involved and their full names are as shown in Table 1.

[0162] Table 1

[0163] Abbreviations Full names AL Ad libitum DR Dietary restriction NP Normal protein HP High protein D0 Day 0 D4 Day 4 D5 Day 5 D6 Day 6 D10 Day 10 ABX Antibiotics BODIPY Dipyrromethene Boron Difluoride L. murinus Lactobacillus murinus CFU Colony forming unit PLA DL-3-phenyllactic acid HPLA 4-hydroxyphenyllactic acid ILA Indole-lactic acid HICA 2-hydroxyisocaproic acid HMBA 2-hydroxy-3-methylbutyric acid Zeitgeber time Figure 1

[0164] Animals and feed

[0165] The mice used in the experiments were all C57BL / 6J strain mice purchased from Shanghai Slac Laboratory Animal Co., Ltd. The normal standard feed (Chow) for mice was provided by the animal house and purchased from Shanghai Slac Laboratory Animal Co., Ltd. The 20% normal protein (NP) and 60% high protein (HP) feed were purchased from Shanghai Fanpo Biotechnology Co., Ltd. The 20% normal protein feed was prepared according to the AIN-93G rodent diet formula (D10012G, Research Diets Inc.), with the difference that it did not contain the antioxidant tBHQ and cornstarch was used to replace sucrose, containing 20% casein, 0.3% cysteine and 49.7% cornstarch. The high protein (HP) feed was prepared on the basis of the 20% normal protein feed, containing 60% casein, 0.9% cysteine and 9.1% cornstarch. The specific food composition is shown in Table 2 below.

[0166] Table 2, NP and HP diet ingredients

[0167]

[0168]

[0169] Reagents

[0170] BODIPY 500 / 510 C1, C12 fatty acid (4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3- lauric acid) was purchased from Molecular Probes; OCT compound was purchased from Sakura; Nonidet P-40 was purchased from Sangon Biotech; Chloral hydrate, methanol, acetonitrile, chloroform were purchased from Sinopharm Chemical; MRS medium was purchased from Hybio; Vancomycin was purchased from Meilunbio; Ampicillin, Neomycin, Metronidazole were purchased from Shengong Bioengineering; DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA) were purchased from TCI; 2-hydroxyisocaproic acid (HICA), 2-hydroxy-3-methylbutyric acid (HMBA) were purchased from Sigma-Aldrich.

[0171] Dietary restriction experiment

[0172] All mouse experiments were performed according to the guidelines of the Animal Care and Use Committee of Shanghai Nutrition and Health Institute. 8-week-old C57BL6 / J strain mice were purchased and acclimated in the experimental mouse house for 3-5 days. Then the mice were single-caged and acclimated for 5 days, and the following dietary restriction and re-feeding experimental mice were also single-caged. During the single-caged acclimation period before dietary restriction, the mice were given the feed used during dietary restriction. The average food intake of the mice for the three days before dietary restriction was used as a reference for the following experiment. Dietary restriction was performed by adding the specified amount of food to the mice at ZT12 (19:00). All animal experiments were performed on male mice.

[0173] Body weight and body composition measurement

[0174] Mouse body weight and body composition were measured at ZT8 (15:00). Fat and lean body mass content was determined using an EchoMRI-100H body composition analyzer (EchoMRI).

[0175] Body temperature measurement

[0176] Mouse body temperature was measured by using a RET-3 rectal probe (Physitemp) connected to a BAT-12 thermometer (Physitemp), and the measurement time was at ZT3 (10:00).

[0177] Tissue and fecal collection

[0178] Mice were anesthetized with 6% chloral hydrate at the indicated time points, and the tissues of interest, such as small intestine, inguinal white adipose tissue, and epididymal white adipose tissue, were isolated and snap-frozen in liquid nitrogen and stored at -80°C. Blood was collected from the apex of the heart using a 1-ml syringe, and the serum was collected by centrifugation at 1000 g for 30 min at 4°C and stored at -80°C. Mouse feces were collected and stored at -80°C. Mouse cecal contents were collected, snap-frozen in liquid nitrogen, and stored at -80°C.

[0179] Lipid and fatty acid absorption experiments

[0180] Mice were gavaged with a mixture of BODIPY 500 / 510 C1, C12 fatty acid (0.5 pg / g body weight) and olive oil (10 pl / g body weight) at ZT12 (19:00). After gavage, mice were provided with water ad libitum but no food. Feces were collected from mice at 10 min to 2 h after gavage, then freeze-dried and ground with a mortar and pestle, and stored at -20°C. Two hours after gavage with BODIPY-labeled fatty acid analog, mice were anesthetized with 6% chloral hydrate, and tissue and blood samples of interest were collected. The everted proximal jejunum, inguinal white adipose tissue, or epididymal white adipose tissue was either directly observed under a fluorescence microscope or embedded in OCT compound and sectioned. For the detection of BODIPY fluorescence intensity, the proximal jejunum, inguinal white adipose tissue, or epididymal white adipose tissue was first homogenized in RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholic acid sodium, 0.1% SDS), and the supernatant was read for fluorescence signal after centrifugation. The fluorescence signal of the extracted tissue samples or serum samples was measured by a microplate reader (Varioskan Flash, Thermo Scientific), with an excitation wavelength of 492 nm and an emission wavelength of 520 nm. The dried and ground fecal samples were treated with a mixture of water and chloroform (1:2, by volume), and the organic phase was read for fluorescence signal after centrifugation.

[0181] 16S ribosomal RNA gene sequencing analysis

[0182] Genomic DNA of bacteria was extracted from mouse cecal feces, followed by 10 ng of purified DNA for PCR amplification. The V3 and V4 regions of the 16S ribosomal RNA gene were amplified using universal primers for bacteria, forward primer sequence: 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: GGACTACNNGGGTATCTAAT (N represents A or G or C or T) (SEQ ID NO: 3). The PCR products were then mixed in equimolar amounts. Sequencing was performed using the Novaseq 6000 platform (Illumina) next, resulting in 2 x 250 base pair dual-end reads. High-quality filtered reads were obtained by QIIME 2 (version 2019.4) software and R packages, followed by searching against the GreenGenes (version 13.8) reference database. Beta diversity analysis was performed based on Bray-Curtis distances to explore the clustering differences of gut microbiota among samples, and visualized by principal coordinate analysis. Alpha diversity of the microbiota was calculated based on the gut microbiota gene profiles of each sample and based on the Shannon index, using Kruskal Wallis and dunn tests. The proportional abundance of the microbiota was assessed at the class, family, and genus taxonomic levels, respectively.

[0183] Antibiotic treatment

[0184] After the mice were fed with 10%, 25%, and 65% of the food amount for three days, respectively, the mice were provided with sufficient food, and at the same time, the mice were gavaged with high concentrations of antibiotics for 5 consecutive days. The high concentrations of antibiotics were 10 mg of vancomycin, 10 mg of ampicillin, 10 mg of neomycin, and 10 mg of metronidazole suspended in 0.2 ml of water. Thereafter, the mice were gavaged with low concentrations of antibiotics every day. The low concentrations of antibiotics were 2 mg of vancomycin, 4 mg of ampicillin, 4 mg of neomycin, and 4 mg of metronidazole suspended in 0.2 ml of water. During this period, the body fat, lean body mass, and food intake of the mice were detected to study the effect of antibiotic treatment on the weight gain induced by re-feeding after dietary restriction. To study the effect of antibiotic treatment on intestinal lipid absorption and fatty acid uptake of white adipose tissue, after the mice were fed with 10%, 25%, and 65% of the food amount for three days, respectively, the mice were provided with sufficient food, and at the same time, the mice were gavaged with high concentrations of antibiotics. Then, 18 hours later, the mice were gavaged with high concentrations of antibiotics again. Six hours later, the mice were gavaged with a mixture of BODIPY fluorescently labeled fatty acid analogs (0.5 μg / g body weight) and olive oil (10 μl / g body weight). Thereafter, blood and tissue samples were collected at the designated time points.

[0185] Isolation and identification of lactobacillus murinus

[0186] Cecal fecal samples were collected from mice after a day of normal protein diet following dietary restriction, and then diluted with sterile anaerobic PBS at a 1:10 dilution. 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. Individual colonies were randomly picked and re-cultured in MRS broth for 24 hours. The full-length 16S ribosomal RNA gene of the monoclonal strain was amplified using universal primers 27F (5'- AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 4)) and 1492R (5'- CTACGGCTACCTTGTTACGA-3' (SEQ ID NO: 5)) followed by sequencing. The isolated Lactobacillus murinus (strain Lam-1) was stored in 20% or 6% glycerol and kept in a -80°C freezer until further use. Reference sequences of model strains similar to the Lam-1 strain were obtained from the GenBank database by Blast, and multiple sequences were combined and aligned with ClustalW software. A phylogenetic tree was constructed using the "neighbor-joining" algorithm of MEGA 7.0 software.

[0187] Full-length 16S ribosomal RNA gene sequence (SEQ ID NO: 1):

[0188]

[0189] Gavage treatment with Lam-1

[0190] For the lipid absorption experiment in mice gavaged with Lam-1, mice were first gavaged with 0.2 ml of PBS containing 6% glycerol (control) or Lam-1 strain dissolved in PBS containing 6% glycerol (10 10 CFU), where the Lam-1 strain was pre-warmed in a 37°C water bath for 5-10 min at the time of gavage. 24 hours later, mice were again gavaged with the same PBS or Lam-1 strain, and mice were gavaged 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 at the indicated time points. For the phenotype detection experiment in mice gavaged with Lam-1, mice were gavaged with 0.2 ml of PBS containing 6% glycerol (control) or Lam-1 strain dissolved in PBS containing 6% glycerol (10 10 CFU) at 17:00 every day, and food and water intake were measured every day. Body temperature, body weight, body fat, and other indicators were measured before gavage and on days 5 and 10 after gavage.

[0191] Non-targeted metabolomics and data analysis

[0192] About 80 mg of caecal faeces was added with 0.2 ml of water and 0.8 ml of methanol / acetonitrile (1 :1 v / v), followed by homogenization, vortexing, ultrasonication on ice for 30 minutes. Subsequently, it was left at -20°C for 1 hour, then centrifuged at 14000 g for 20 minutes. The supernatant containing the metabolites was vacuum freeze-dried and dissolved in 0.1 ml of acetonitrile / water (1 :1 v / v) and determined using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The mass spectrometer was run in negative and positive ionization mode with a capillary voltage set at 5.5 kV. The raw mass spectrometry data were extracted and peaks were determined, isotopes and adducts were annotated using the R package CAMERA. Compounds were identified by comparing the accurate m / z values and spectra with a database of authentic standards available. Regarding the classification of metabolites, the metabolites that were significantly increased in both DR-NP (D4) and DR-HP (D4) groups compared to the AL-NP (D0) group (excluding the metabolites that were significantly decreased in the DR-HP (D4) group compared to the DR-NP (D4) group), were classified as "unblockable increase"; the metabolites that were significantly decreased in both DR-NP (D4) and DR-HP (D4) groups compared to the AL-NP (D0) group (excluding the metabolites that were significantly increased in the DR-HP (D4) group compared to the DR-NP (D4) group), were classified as "unblockable decrease"; the metabolites that were significantly decreased in the DR-NP (D4) group compared to the AL-NP (D0) group and significantly increased in the DR-HP (D4) group compared to the DR-NP (D4) group or not significantly different compared to the AL-NP (D0) group, were classified as "blockable decrease"; the metabolites that were significantly increased in the DR-NP (D4) group compared to the AL-NP (D0) group and significantly decreased in the DR-HP (D4) group compared to the DR-NP (D4) group or not significantly different compared to the AL-NP (D0) group, were classified as "blockable increase".

[0193] Targeted quantitative analysis of PLA, HPLA, ILA, HICA and HMBA

[0194] About 60 mg of cecal feces was added with 0.2 ml of water and 0.8 ml of methanol and 1 μl of formic acid, followed by homogenization, vortexing, and ultrasonication on ice for 30 minutes. Subsequently, it was left at -20°C for 3 hours, and then centrifuged at 14000 g for 20 minutes. The supernatant was filtered with a 0.22 μm PTFE hydrophilic filter, and then used for the next step of detection. The processed sample and different concentrations of DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), indolelactic acid (ILA), 2-hydroxyisocaproic acid (HICA), and 2-hydroxy-3-methylbutyric acid (HMBA) were used as standard compounds, and were subjected to liquid chromatography-tandem mass spectrometry (LC-MS / MS) measurement. A calibration curve was plotted based on the mass spectrometry detection results of each standard compound, and the concentrations of the corresponding five compounds in the sample were calculated.

[0195] Metabolite gavage treatment

[0196] Mice were gavaged with water, 60 mg / ml of PLA, 30 mg / ml of HPLA, 15 mg / ml of ILA, a mixture of the three compounds (including 60 mg / ml of PLA, 30 mg / ml of HPLA, and 15 mg / ml of ILA), or a mixture of five compounds (including 30 mg / ml of PLA, 15 mg / ml of HPLA, 8 mg / ml of ILA, 40 mg / ml of HICA, and 20 mg / ml of HMBA), and the pH of each solution was adjusted to 7.0 with sodium hydroxide. The gavage volume was 0.15 ml. After 24 hours, the mice were again gavaged with the same amount of water or the specified compounds, and were simultaneously gavaged with a mixture of BODIPY fluorescently labeled fatty acid analogues (0.5 μg / g body weight) and olive oil (10 μl / g body weight). The feces and tissues such as the small intestine, inguinal, and epididymal white adipose tissues of the mice were collected at the specified time points.

[0197] Data analysis and statistics

[0198] The statistical analysis of the data was performed using Excel software, and all numerical calculation results were displayed in the form of mean ± standard deviation (SD). All graphs were plotted using GraphPad Prism software. The significant differences between different groups were analyzed by two-tailed Student’s t-test. A p-value less than 0.05 was considered to be a statistically significant difference.

[0199] Storage of sequencing data

[0200] The raw data of 16S ribosomal RNA gene high-throughput sequencing of mouse cecal feces has been stored in the NCBI website (https: / / www.ncbi.nlm.nih.gov / sra) with the accession number PRJNA757842. The complete sequence of 16S ribosomal RNA gene of the isolated Lactobacillus Lam-1 has been stored in GenBank with the number MZ955456 (SEQ ID NO: 1).

[0201] Example 1, Gut microbiota regulates fat accumulation, small intestinal lipid absorption and fatty acid uptake of white adipose tissue during re-feeding after dietary restriction

[0202] Diet affects the structure and function of gut microbiota, which can affect the lipid absorption of small intestine and the lipid metabolism of adipose tissue. In previous experiments, the inventors have proved that re-feeding after dietary restriction can promote the lipid absorption of small intestine and the fatty acid uptake of white adipose tissue and make mice gain weight. To study whether gut microorganisms participate in the enhanced small intestinal absorption and weight gain induced by re-feeding after dietary restriction, the inventors first restricted the diet (DR) of mice, i.e. from the first day to the third day, the mice were fed with 10%, 25% and 65% of the food amount, respectively, and then from the fourth day, the mice were re-fed with sufficient normal protein diet (NP) or high protein diet (HP). The mice were given ad libitum diet (AL) before dietary restriction (D0). The cecal feces samples of mice were collected before dietary restriction (i.e. D0) and one day (i.e. D4) and three days (i.e. D6) after re-feeding with normal protein diet or high protein diet, and then the composition of gut microbiota was analyzed by 16S ribosomal RNA gene sequencing. In order to evaluate how re-feeding after dietary restriction affects the structure of gut microbial community, the inventors studied the alpha and beta diversity of gut microbiota of each sample to compare the diversity of gut microbiota within the sample and between the samples. For beta diversity, the results of principal coordinate analysis showed that there were obvious clustering differences between the gut microbiota of mice from before dietary restriction, after dietary restriction with normal protein diet and after dietary restriction with high protein diet Figure 1 A). In addition, the results of alpha diversity of gut microbiota calculated by Shannon index evaluation showed that compared with mice before dietary restriction, the alpha diversity of gut microbiota of mice after dietary restriction decreased significantly after being re-fed with normal protein diet for one day, while the corresponding high protein diet could significantly change this effect Figure 1 B). Next, the inventors analyzed the proportional abundance of gut microbiota at different classification levels of each group of samples. The inventors found that compared with mice before dietary restriction, the proportions of Bacilli, Lactobacillaceae and Lactobacillus of mice after dietary restriction increased extremely significantly after being re-fed with normal protein diet for one day, reaching about 60% Figure 1 C- Figure 1E) However, compared to mice that were refed a normal protein diet for one day after dietary restriction, mice that were refed a high protein diet for one day after dietary restriction had significantly lower abundances of the class of bacteria, the family of Lactobacillaceae, the genus of Lactobacillus, etc. Figure 1 C- Figure 1 E) These data indicate that refeeding after dietary restriction significantly alters the composition of the gut microbiota, leading to a significant enrichment of the genus of Lactobacillus.

[0203] To investigate the role of the gut microbiota in the refeeding-induced fattening after dietary restriction, the inventors treated mice with antibiotics to eliminate intestinal microbes during the refeeding period after dietary restriction. After mice were fed 10%, 25%, and 65% of the food amount for 3 consecutive days, respectively, refeeding significantly increased the body fat content of mice and led to the fattening of mice, while the treatment of mice with antibiotics during refeeding significantly inhibited the accumulation of body fat Figure 4 F), while the treatment of mice with antibiotics significantly inhibited the increase in food intake and the percentage of body fat and the decrease in the percentage of lean body mass Figure 4 A- Figure 1 C).

[0204] Further, the inventors gavaged mice with BODIPY (boron-difluoride-dipyrromethene) fluorescently labeled fatty acid analogs, and collected blood and tissue samples from mice after 2 hours. Fresh proximal jejunum tissue was directly observed under a fluorescence microscope. The inventors found that the fluorescence intensity of tissue from mice treated with antibiotics during the refeeding period after dietary restriction was lower than that from mice refed after dietary restriction Figure 1 G).

[0205] Subsequently, the inventors performed frozen sectioning of the proximal jejunum tissue, and found that the fluorescence intensity of the proximal jejunum villi of mice treated with antibiotics during the refeeding period was lower than that of mice refed after dietary restriction Figure 1 H).

[0206] Then, the inventors treated the proximal jejunum tissue with RIPA lysis buffer, and then centrifuged to obtain the supernatant for detection of fluorescence intensity, and found that the relative concentration of BODIPY in the proximal jejunum tissue of mice treated with antibiotics during the refeeding period was significantly lower than that of mice refed after dietary restriction Figure 1 I). Meanwhile, the relative concentration of BODIPY in the serum of mice treated with antibiotics during the refeeding period was also significantly lower than that of mice refed after dietary restriction Figure 1 J). The detection of the relative fluorescence intensity of BODIPY in the small intestine and serum proved that the treatment of mice with antibiotics during the refeeding period after dietary restriction inhibited the lipid absorption of the small intestine.

[0207] In addition, fresh inguinal and epididymal white adipose tissue was directly observed under a fluorescence microscope, and the inventors found that the fluorescence intensity of tissue from mice treated with antibiotics was lower than that from mice refed after dietary restriction Figure 1K) Subsequently, the inventors sectioned the inguinal and epididymal white adipose tissues and found that the fluorescence intensity of the tissues of the antibiotic-treated mice was also reduced Figure 1 L) Next, the inventors treated the inguinal and epididymal white adipose tissues with RIPA lysis buffer and then centrifuged the tissues to obtain the supernatant for fluorescence intensity detection. The results showed that the fluorescence intensity of the tissues of the antibiotic-treated mice was significantly reduced Figure 2 M) The BODIPY relative fluorescence intensity detection results of the white adipose tissues proved that the antibiotic treatment during the re-feeding stage after dietary restriction inhibited the fatty acid uptake of the white adipose tissues.

[0208] In summary, these data proved that the re-feeding after dietary restriction induced changes in the intestinal microbiota, which in turn increased the fat content, enhanced the lipid absorption of the small intestine and the fatty acid uptake of the white adipose tissues.

[0209] Example 2, Lactobacillus Lam-1 can enhance the lipid absorption of the small intestine and the fatty acid uptake of the white adipose tissues and promote the accumulation of body fat in mice

[0210] Since the proportion of Lactobacillus increased to about 60% during the re-feeding stage after dietary restriction, to explore whether this dominant bacterium led to the enhancement of the lipid absorption of the small intestine and the fatty acid uptake of the white adipose tissues, the inventors isolated 8 monoclonal strains from the cecal feces of mice fed with 10%, 25% and 65% of the food amount for 3 days and then normally fed with protein diet for one day (DR-NP(D4) group) and performed sequencing analysis. The results showed that these monoclonal strains had the same 16S ribosomal RNA gene sequence, indicating that they were the same strain, and the strain could be obtained based on the 16S ribosomal RNA gene sequence (e.g., isolating the strain from the intestinal microbiota and identifying the Lactobacillus with the sequence).

[0211] 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)) Figure 2 A) One of the 8 monoclonal strains was randomly selected and named Lam-1.

[0212] To determine whether Lactobacillus Lam-1 would lead to the enhancement of the lipid absorption of the small intestine and the fatty acid uptake of the white adipose tissues, the inventors gavaged the mice with control or 10 10 CFU (colony-forming unit) of Lam-1 bacteria, with a 24-hour interval, and at the same time, gavaged the mice with BODIPY fluorescently labeled fatty acid analogs. As shown in Figure 2As shown in B, the relative BODIPY level in the feces of mice gavaged with Lam-1 bacteria was significantly reduced, which suggested that the mice had enhanced lipid absorption capacity. Further, the inventors found that the fluorescence intensity of fresh proximal jejunum tissues and their villus frozen sections of mice gavaged with Lam-1 strain was increased (C and 2D), while the relative concentration of BODIPY in the proximal jejunum tissues and serum was significantly increased (E and 2F) compared with that of control mice. Then the inventors analyzed the fatty acid uptake of white adipose tissues, and found that the fluorescence intensity of fresh inguinal and epididymal white adipose tissues and their frozen sections of mice gavaged with Lam-1 strain was increased (G and 2H), while the relative concentration of BODIPY in the corresponding tissues was significantly increased (I) compared with that of control mice. Figure 2 Figure 2 Further, the inventors found that after continuous gavage with Lam-1 strain for 5 days and 10 days, the body fat content and the percentage of body fat relative to body weight of mice were significantly increased (J and 2K), while the food intake and water intake of mice did not change significantly (L and 2M) during the gavage process. In addition, the body temperature of mice did not change significantly (N) after gavage with Lam-1, which suggested that the energy consumption of mice did not change significantly. Based on the above results, it can be concluded that the Lam-1 strain can promote the lipid absorption of mice and thus lead to obesity. The 16S ribosomal RNA gene sequences of the 8 monoclonal strains are the same, which suggests that other monoclonal strains in the 8 monoclonal strains have the same function as Lam-1. Figure 2 Figure 2 Further, the inventors found that after continuous gavage with Lam-1 strain for 5 days and 10 days, the body fat content and the percentage of body fat relative to body weight of mice were significantly increased (J and 2K), while the food intake and water intake of mice did not change significantly (L and 2M) during the gavage process. In addition, the body temperature of mice did not change significantly (N) after gavage with Lam-1, which suggested that the energy consumption of mice did not change significantly. Based on the above results, it can be concluded that the Lam-1 strain can promote the lipid absorption of mice and thus lead to obesity. The 16S ribosomal RNA gene sequences of the 8 monoclonal strains are the same, which suggests that other monoclonal strains in the 8 monoclonal strains have the same function as Lam-1.

[0213] Further, the inventors found that after continuous gavage with Lam-1 strain for 5 days and 10 days, the body fat content and the percentage of body fat relative to body weight of mice were significantly increased (J and 2K), while the food intake and water intake of mice did not change significantly (L and 2M) during the gavage process. In addition, the body temperature of mice did not change significantly (N) after gavage with Lam-1, which suggested that the energy consumption of mice did not change significantly. Based on the above results, it can be concluded that the Lam-1 strain can promote the lipid absorption of mice and thus lead to obesity. The 16S ribosomal RNA gene sequences of the 8 monoclonal strains are the same, which suggests that other monoclonal strains in the 8 monoclonal strains have the same function as Lam-1. Figure 2 Figure 2 Further, the inventors found that after continuous gavage with Lam-1 strain for 5 days and 10 days, the body fat content and the percentage of body fat relative to body weight of mice were significantly increased (J and 2K), while the food intake and water intake of mice did not change significantly (L and 2M) during the gavage process. In addition, the body temperature of mice did not change significantly (N) after gavage with Lam-1, which suggested that the energy consumption of mice did not change significantly. Based on the above results, it can be concluded that the Lam-1 strain can promote the lipid absorption of mice and thus lead to obesity. The 16S ribosomal RNA gene sequences of the 8 monoclonal strains are the same, which suggests that other monoclonal strains in the 8 monoclonal strains have the same function as Lam-1. Figure 3 The above research results prove that the enriched lactobacillus during the re-feeding process after dietary restriction can enhance the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue, and thus promote lipid accumulation to cause obesity in mice.

[0214] Example 3, Metabolites produced by lactobacillus up-regulate the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue

[0215] In view of the above experimental results of using antibiotics to eliminate intestinal flora and gavaging Lam-1 bacteria, the inventors further explored how the intestinal flora regulates the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue of mice during re-feeding after dietary restriction. To this end, the inventors collected the cecal feces of mice before dietary restriction and mice fed with 10%, 25%, and 65% of food within three days, and then re-fed with normal protein or high protein diet, and detected and analyzed the intestinal flora metabolites in the feces by non-targeted metabolomics.

[0216]

[0217] ​​​​As expected, the gut microbiota metabolite composition of the cecal feces of mice fed a normal protein diet after dietary restriction was significantly changed compared to mice before dietary restriction, and the changes of some metabolites could be blocked by a high protein diet Figure 3 A).

[0218] In the group of "blockable increase", compared to mice before dietary restriction, the concentrations of 5 metabolites in the cecal feces of mice fed a normal protein diet for one day after dietary restriction (DR-NP(D4) group) were significantly increased, which were DL-3-phenyllactic acid (PLA), 4-hydroxyphenyllactic acid (HPLA), 2-hydroxyisocaproic acid (HICA), 2-hydroxy-3-methylbutyric acid (HMBA) and indolelactic acid (ILA) respectively Figure 3 A), and the increase of the concentrations of these 5 metabolites could be blocked to some extent by a high protein diet. At the same time, by using 5 purchased compounds as standards, the concentrations of the 5 metabolites in the samples were detected and analyzed by targeted quantitative detection and analysis by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the results further confirmed the changes in the concentrations of the 5 metabolites between different groups Figure 3 B-3F). In addition, the inventors found that the concentrations of the 5 metabolites in the corresponding cecal feces of mice were significantly increased after being gavaged with Lactobacillus Lam-1 Figure 5 G), which indicated that the 5 metabolites were produced by Lactobacillus.

[0219] The above data prove that the re-feeding after dietary restriction induces changes in the composition of cecal fecal metabolites, and the changes of some metabolites can be blocked by a high protein diet, such as PLA, HPLA, HICA, HMBA and ILA, and the concentrations of the 5 metabolites can be increased by Lactobacillus.

[0220] To explore whether the changes of these metabolites would lead to enhanced small intestinal lipid absorption and white adipose fatty acid uptake, the inventors planned to use the most significantly changed metabolites to treat mice. Among the 5 most significantly changed metabolites in the group of "blockable increase", PLA, HPLA and ILA can be produced by intestinal bacteria by mobilizing phenylalanine, tyrosine and tryptophan in the diet, respectively, and using the same enzyme for reduction reaction. Therefore, the inventors first studied the effects of these three compounds on metabolism, and the inventors found that neither PLA, HPLA and ILA treated alone nor the three compounds together had a significant effect on food intake, small intestinal lipid absorption and white adipose fatty acid uptake of mice Figure 6 A-5L). In addition, the inventors found that there was also no significant effect after HICA or HMBA treatment Figure 3However, when mice were gavaged with a mixture solution of 5 compounds, PLA, HPLA, ILA, HICA and HMBA, and at the same time, the mice were gavaged with BODIPY fluorescently labeled fatty acid analogs, the inventors found that the fecal BODIPY relative concentration of mice was significantly reduced but the food intake was not significantly changed Figure 3 H and 3I), which suggests that the ability of mice to absorb lipids is enhanced.

[0221] Further, the inventors found that compared with the control group of mice gavaged with water, the proximal jejunum tissue and their villus frozen sections of mice gavaged with 5 compounds had increased fluorescence intensity Figure 3 J and 3K), while the corresponding proximal jejunum BODIPY relative concentration was significantly increased Figure 3 L).

[0222] At the same time, the inventors also analyzed the fatty acid uptake of white adipose tissue. Compared with the control group of mice gavaged with water, the fluorescence intensity of fresh inguinal and epididymal white adipose tissue and their frozen sections of mice gavaged with 5 compounds was increased Figure 3 M and 3N), while the corresponding tissue BODIPY relative concentration was significantly increased Figure 3 O).

[0223] The above experimental results show that some intestinal bacteria, such as Lactobacillus, produce 5 metabolites, PLA, HPLA, ILA, HICA and HMBA, which can up-regulate the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue.

[0224] In the present application, the inventors found that re-feeding after dietary restriction promotes the enrichment of Lactobacillus in the small intestine and its metabolites, while high-protein diet or antibiotic treatment can inhibit this enrichment. The increased Lactobacillus and its metabolites can enhance the lipid absorption of the small intestine and the fatty acid uptake of white adipose tissue, and ultimately make it obese ​ P).

[0225] Preservation of biological materials

[0226] The strain (Lactobacillus murinus Lam-1) of the present application has been preserved in the China Center for Type Culture Collection (Wuhan, China Wuhan University), the preservation date is December 29, 2021, and its preservation number is CCTCC NO: M20211687.

[0227] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but should not be understood as limiting the scope of the patent of the present application. 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 application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. Meanwhile, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a reference. SEQUENCE LIST <110> SHANGHAI INSTITUTE OF NUTRITION AND HEALTH, CHINESE ACADEMY OF SCIENCES <120> METHODS OF MODULATING LIPID ABSORPTION, COMPOSITIONS AND USES THEREOF <130> 218319 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1455 <212> DNA <213> L. murinus <400> 1 tgctatacat gcaagtcgaa cgaaacttct ttatcaccga gtgcttgcac tcaccgataa 60 agagttgagt ggcgaacggg tgagtaacac gtgggcaacc tgcccaaaag agggggataa 120 cacttggaaa caggtgctaa taccgcataa ccatagttac cgcatggtaa ctatgtaaaa 180 ggtggctatg ctaccgcttt tggatgggcc cgcggcgcat tagctagttg gtggggtaaa 240 ggcttaccaa ggcaatgatg cgtagccgaa ctgagaggtt gatcggccac attgggactg 300 agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa tgggcgaaag 360 cctgatggag caacgccgcg tgggtgaaga aggtcttcgg atcgtaaaac cctgttgtta 420 gagaagaaag tgcgtgagag taactgttca cgtttcgacg gtatctaacc agaaagccac 480 ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttat ccggatttat 540 tgggcgtaaa gggaacgcag gcggtctttt aagtctgatg tgaaagcctt cggcttaacc 600 ggagtagtgc attggaaact gggagacttg agtgcagaag aggagagtgg aactccatgt 660 gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaagcgg ctctctggtc 720 tgtaactgac gctgaggttc gaaagcgtgg gtagcaaaca ggattagata ccctggtagt 780 ccacgccgta aacgatgaat gctaagtgtt ggagggtttc cgcccttcag tgctgcagct 840 aacgcaataa gcattccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga 900 cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta 960 ccaggtcttg acatcttttg ccaatcctag agataggact ttcccttcgg ggacaaaatg 1020 acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 1080 GAGCGCAACCC TTATTGTTAG TTGCCAGCAT TAAGTTGGGC ACTCTAGCAA GACTGCCGG 1140 TGACAAACCG GAGGAAGGTG GGGATGACGT CAAATCATCA TGCCCCTTAT GACCTGGGCT 1200 ACACACGTGC TACAATGGAC GGTACAACGA GTCGCAAGAC Cgcgaggttt agcaaatctc 1260 Ttaaagccgt TCTCAGTTCG GATTGTAGG CTGCAACTCG CCtacatgaa GTCGGAATCG C 1320 TAGTAATCGC GGATCAGCAT GCCGCGGTGA TACGTTCCCG GGCCttgtac acccgccc 1380 GTCACACCAT GAGAGTTTGT AACACCCAAA GCCGgtgggg Taaccttttg GAGCCAGCCG 1440 TCTAAGGTGG ACAGA 1455 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <400> 2 CCTAYGGGRB GCASCAG 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 GGACTACNNG GTATCTAAT 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 AGAGTTTGAT CCTGGCTCAG 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 ctacggctac cttgttacga 20

Claims

1. Use of a combination of five compounds for preparing a composition for increasing lipid absorption or body weight after dietary restriction and re-intake; The five-compound combination is: DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indole lactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3-methylbutyric acid; wherein the weight ratio of DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indole lactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3-methylbutyric acid is: 20-40: 10-20: 6-10: 30-50: 15-25.

2. The use according to claim 1, characterized in that The dietary restrictions include: conventional dieting, intermittent dieting, time-restricted dieting, low-energy diet that simulates dieting, and gradient-increase or gradient-decrease dieting.

3. The use according to claim 1, wherein In the five-compound combination, the weight ratios of DL-3-phenyllactic acid, 4-hydroxyphenyllactic acid, indolelactic acid, 2-hydroxyisocaproic acid and 2-hydroxy-3-methylbutyric acid are: 30±3: 15±1.5: 8±0.8: 40±4: 20±2.

4. The use according to claim 1, wherein The increased lipid absorption includes inhibiting diarrhea or improving malnutrition.

5. The use according to claim 4, characterized in that The diarrhea is caused by increased secretion and / or decreased absorption.

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