CN116904377A - High-yield conjugated linoleic acid strain, lipid-lowering fermented milk and application - Google Patents

Abstract

The invention discloses a high-yield conjugated linoleic acid strain, a lipid-lowering fermented milk and application thereof, wherein the strain is named as lactobacillus fermentum L1, and the preservation number is CCTCC NO: m2023862, the preservation unit is China center for type culture Collection, and the preservation address is China university of Wuhan. The lactobacillus fermentum L1 can be used for producing the fermented milk with good flavor and texture and rich living bacteria number and conjugated linoleic acid. The fermented milk rich in conjugated linoleic acid can improve physiological and biochemical indexes, improve the abundance of bacteroides and bifidobacteria in intestinal flora, restore the balance of the intestinal flora, promote the secretion of primary bile acid, and convert the primary bile acid into secondary bile acid under the action of the bifidobacteria and the bacteroides, thereby realizing benign circulation of bile acid metabolism and achieving the effects of losing weight and reducing lipid.

Description

High-yield conjugated linoleic acid strain, lipid-lowering fermented milk and application

Technical Field

The invention relates to the field of microbial strains, in particular to a high-yield conjugated linoleic acid strain, lipid-lowering fermented milk and application.

Background

Conjugated linoleic acid is a group of conformational and positional isomers of linoleic acid, consisting of a series of geometrically heterogeneous octadecadienoic acids containing conjugated double bonds at the 9, 10, 11 carbon positions. Conjugated linoleic acid is known as a "novel nutrient of the twenty-first century", and is found mainly in dairy products and ruminant meat. Along with the improvement of the living standard of people and the increase of the demand for high-quality unsaturated fatty acid, conjugated linoleic acid gradually becomes a research hot spot due to the numerous physiological functions of resisting cancer, reducing lipid, resisting oxidation and the like. Conjugated linoleic acid has great research value in medicine research and development, nutrition and health care industries and the like.

Since 1966, the first strain, vibrio butyrate (kepler et al, 1966), which can produce conjugated linoleic acid, has been reported to date, and many CLA (conjugated linoleic acid) -producing strains have been discovered, such as bifidobacterium, lactobacillus fermentum, lactobacillus plantarum, lactobacillus reuteri, and the like. High purity CLA products are expensive because of the chemical production of CLA along with other byproducts; the synthesis of CLA using lactic acid bacteria is a very desirable route compared to chemical synthesis of CLA. The application obtains a lactobacillus strain with high conjugated linoleic acid yield through screening, and utilizes lactobacillus fermentation to produce the fermented milk rich in conjugated linoleic acid.

Disclosure of Invention

The application aims to provide a high-yield conjugated linoleic acid strain, and the mechanism of high-yield conjugated linoleic acid of Yunnan primary lactobacillus is analyzed based on Yunnan plateau characteristics and from theory around the demands of obese and overweight people on personalized nutrition; fermenting by lactic acid bacteria to produce fermented milk rich in conjugated linoleic acid and constructing a product characteristic component fingerprint; the product is combined with animal intervention experiments to clarify the lipid-lowering mechanism of the fermented milk rich in conjugated linoleic acid.

The technical scheme of the application is as follows: high-yield conjugated subunit Oleic acid strain named Lactobacillus fermentum L1%LimosilactobacillusfermentumL), the preservation number is CCTCC NO: m2023862, deposit unit: china center for type culture collection, with the preservation addresses: university of martial arts in chinese; preservation date: 2023, 5 and 30.

The invention also provides a microbial preparation containing the high-yield conjugated linoleic acid strain, and the bacterial concentration in each milliliter or gram of the microbial preparation is not less than 1 multiplied by 10 ⁹ CFU。

The invention also provides application of the high-yield conjugated linoleic acid strain or the microbial preparation in preparation of conjugated linoleic acid.

The invention also provides the lipid-lowering fermented milk, which is obtained by adopting the high-yield conjugated linoleic acid strain to ferment; or by fermentation with the microbial preparation.

Further, the preparation process of the lipid-lowering fermented milk comprises the following steps: dissolving skimmed milk with distilled water, adding safflower seed oil and acesulfame potassium, mixing, homogenizing at room temperature under 25-30MPa for 3-10min, and homogenizing repeatedly for 2-4 times; then maintaining the homogenized liquid at 75-85deg.C for 20-40min, cooling to 33-37deg.C, inoculating lactobacillus fermentum L1 into the homogenized liquid under aseptic condition, and fermenting at 33-36deg.C.

Further, after the lactobacillus fermentum L1 is activated, inoculating the lactobacillus fermentum L1 into MRS broth for culture until the third generation, and inoculating the lactobacillus fermentum L1 into homogenized liquid, wherein the inoculating amount is 0.8-1.5% (w/w).

Further, the fermented milk adopts lactobacillus fermentum L1, lactobacillus bulgaricus and streptococcus thermophilus as a starter together; the ratio of the lactobacillus fermentum L1 to the lactobacillus bulgaricus to the streptococcus thermophilus is 1.5-2.5:0.8-1.2:0.8-1.2.

Further, the addition amount of the skim milk is 8-15%, the addition amount of the safflower seed oil is 5-7%, and the addition amount of the acesulfame potassium is 0.008-0.015%.

The invention also provides application of the lipid-lowering fermented milk in lipid lowering and weight losing.

The invention screens and obtains the lactobacillus fermentum L1, and the safflower seed oil is taken as a substrate to produce conjugated linoleic acid with high yield. The skim milk containing safflower seed oil is fermented by lactobacillus fermentum L1, and is a fermented milk which is rich in conjugated linoleic acid, fine in texture and sweet and sour. The fermented milk rich in conjugated linoleic acid is different from the common yoghourt in characteristic active ingredients: mainly contains various characteristic components such as fatty acid, amino acid, organic acid, alkaloids, lignans and the like; the fatty acid is mainly myristic acid, palmitic acid, linoleic acid and conjugated linoleic acid, and the linoleic acid is at a core position of fatty acid metabolism; malic acid is a typical organic acid in the core position of organic acid metabolism, and gives good flavor to the product. The fermented milk rich in conjugated linoleic acid can improve the abundance of bacteroides and bifidobacteria in intestinal flora, restore the balance of the intestinal flora, promote the secretion of primary bile acid, and convert the primary bile acid into secondary bile acid under the action of the bifidobacteria and the bacteroides, thereby realizing benign circulation of bile acid metabolism and achieving the effects of losing weight and reducing lipid.

Compared with the prior art, the application has the following beneficial effects:

(1) According to the application, as the density of thalli and the intensity of signal molecules are increased, the S-adenosyl-L-methionine metabolic pathway is up-regulated, the luxS/AI-2 quorum sensing system is activated, the expression of key enzymes (eno, MCRA) is forward regulated, the efficiency of converting linoleic acid into CLA by thalli is improved, and the yield of CLA is further improved.

(2) The use of Lactobacillus fermentum L1 can produce fermented milk with good flavor and texture and high viable count and conjugated linoleic acid. The fermented milk product is characterized by comprising fatty acid, amino acid, organic acid, alkaloid and lignan. The content of fatty acid in all the characteristic components is highest, and linoleic acid is positioned in the center of fatty acid metabolism, so that the metabolism of arachidonic acid, the biosynthesis of unsaturated fatty acid, the metabolism of alpha-linolenic acid and the generation of conjugated linoleic acid are promoted; the typical organic acid is malic acid, which imparts a good flavor to the product. The product is fermented milk with rich active ingredients and high content of conjugated linoleic acid and probiotics.

(3) The fermented milk rich in conjugated linoleic acid can improve the abundance of bacteroides and bifidobacteria in the intestinal flora, so that the intestinal flora is balanced; up-regulating expression of genes related to bile acid metabolism, PPAR signal channels and primary bile acid biosynthesis pathways, and promoting secretion of primary bile acid; under the action of bifidobacteria and bacteroides, the primary bile acid is converted into the secondary bile acid, so that benign circulation of bile acid metabolism is realized, and the effects of losing weight and reducing lipid are achieved.

Drawings

FIG. 1 morphological features of Lactobacillus fermentum L1; the left graph shows colony morphology of lactobacillus fermentum L1 on an MRS agar plate, and the right graph shows gram staining microscopic examination of lactobacillus fermentum L1;

FIG. 2 CLA yield of Lactobacillus fermentum L1 in 4 fermentation substrates;

figure 3 comparison of CLA content of different dairy products, notes: l1 is the conjugated linoleic acid-enriched fermented milk; M1-M4 represent 4 different commercially available whole milk; Y1-Y4 represent 4 different commercially available yogurts;

FIG. 4 metabolite profiles of three fermented milks;

FIG. 5 is a fingerprint of a fermented milk enriched in conjugated linoleic acid;

FIG. 6 effect of fermented milk on food intake: the different letters within 11 to 13 weeks represent significant differences between groupsp＜0.05）；

Figure 7 effect of fermented milk on mouse body weight notes: the different letters within 11 to 13 weeks represent significant differences between groupsp＜0.05）；

Figure 8 mice of different groupings body type and abdominal fat size notes: a: different groups of mouse sizes; b: abdominal fat size of mice in different groups.

Fig. 9, organ weight notes for different groupings: a: abdomen fat weight; b: heart weight; c: liver weight; d: spleen weight; e: kidney weight;

FIG. 10 difference in the levels of intestinal microorganisms in mice; and (3) injection: the left side is a Bray-Curtis distance clustering tree structure, and the closer the sample clusters are, the shorter the branches are, and the more similar the composition of the representative grouping species is; on the right are species relative abundance profiles of each grouping at the portal level, the more occupied ratios

Large indicates higher abundance;

FIG. 11 expression levels of metabolites in bile acid metabolic pathways of different treatment groups;

FIG. 12 is a schematic of a lipid-lowering mechanism for conjugated linoleic acid enriched fermented milk.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the following description of the technical solution of the present invention is given by way of example and illustration only, and should not be construed as limiting the scope of the present invention in any way.

EXAMPLE 1 isolation and purification of seed

Taking 10mL of cow milk sample from Daxizhou, adding into 100mL of sterile physiological saline, shaking uniformly, gradient diluting, sucking 100uL, and coating on 1% CaCO ₃ -MRS solid medium, cultured at 37 ℃ for 48h. Selecting CaCO ₃ Colonies forming calcium-dissolving rings on MRS solid medium are repeatedly streaked on MRS solid medium for purification until the colony morphology of the strain is consistent. Inoculating purified lactobacillus to MRS slant culture, preserving at 4deg.C, inoculating the strain and 20% glycerol into freezing tube, mixing, and preserving at-80deg.C.

Mixing tween-80 and LA/MRS culture medium of 0.04% LA (linoleic acid) at a ratio of 1:1, adding distilled water, mixing with water-oil ratio (LA: water) of 1:1, and performing ultrasonic emulsification under ice water bath: power: 80w, single phacoemulsification time: 10s, intermittent time: 15s, total ultrasound time: and after the emulsification is finished, sterilizing for 5-8 min at 90-95 ℃.

Activating the strain obtained by screening to the third generation, performing microscopic examination, inoculating the strain into 15mL of MRS liquid culture medium added with 0.04% LA emulsion according to the inoculum size of 6% (v/v), and fermenting and culturing for 48h at 37 ℃ to obtain lactobacillus fermentum (lactobacillus fermentum L1) with highest CLA yield.

Identification of species

Morphological characterization of the species

As shown in fig. 1, the morphological characteristics of lactobacillus fermentum L1 are as follows: after the lactobacillus fermentum L1 is cultured in an MRS agar culture medium for 48 hours, the colony form is milky white, semitransparent, moist, smooth, and regular in edge, and has obvious bulges. The microscopic examination result of the lactobacillus fermentum L1 is as follows: no spores are produced and gram staining is a purple positive bacteria.

16SrDNA Gene sequencing

Bacterial genomes are extracted by using a TSINGKE plant DNA extraction kit (general purpose type), and bacterial general purpose primers are adopted by taking the genome of the extracted strain as a template: 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were subjected to PCR experiments on 16 SrDNA. And (3) sending the PCR product to Beijing engine biotechnology Co., ltd for sequencing, comparing and analyzing the sequencing result in NCBI database (www.ncbi.nlm.gov/BLAST /) by using BLAST tool with the existing sequences in GenBank database, analyzing the homology of the strain to be tested and the corresponding sequences of the known strain, and determining the strain species screened. The strain is subjected to 16SrDNA gene sequencing, as shown in SEQ ID NO.1, and the homology of the sequence and the 16SrDNA sequence of lactobacillus fermentum is over 99% as a result of gene sequencing, so that the screened lactobacillus is lactobacillus fermentum.

The strain selected was determined to be lactobacillus fermentum by morphology and 16SrDNA sequencing and was named lactobacillus fermentum L1 #Limosilactobacillusfermentum L), deposited in China center for type culture Collection, accession number: CCTCCNO: m2023862.

EXAMPLE 2 screening of conjugated linoleic acid production conditions by Strain

Effect of substrate on CLA production

The CLA yield of lactobacillus fermentum L1 in 4 fermentation substrates is explored by taking safflower seed oil, moringa seed oil, tea seed oil and walnut oil as fermentation substrates. The CLA yield profile of Lactobacillus fermentum L1 in 4 fermentation substrates is shown in FIG. 2.

As can be seen from FIG. 2, the CLA yields of Lactobacillus fermentum L1 in the 4 fermentation substrates were not identical. When the concentration of the fermentation substrate is in the range of 0% -12%, the CLA yield in the fermentation liquid with the moringa seed oil, the safflower seed oil and the tea seed oil as the substrates is in a trend of rising and then falling; when the fermentation lactobacillus L1 takes walnut oil as a fermentation substrate, the yield of CLA is in an ascending trend, and when the addition amount of the walnut oil is 12%, the CLA content in fermentation liquid is 314.86 mug/mL. When the addition amount of the moringa seed oil is 8%, the CLA content in the fermentation liquid is 858.8 mug/mL; when the adding amount of the tea seed oil is 10%, the CLA content in the fermentation liquor is 532.7 mug/mL; when the addition amount of safflower seeds is 6%, the CLA content in the fermentation liquor is 1025.26 mug/mL at the highest. Thus, compared with moringa seed oil, tea seed oil and walnut oil, safflower seed oil is more suitable for being used as a fermentation substrate of lactobacillus fermentum L1.

Effect of culture conditions on CLA yield

Because the culture time, temperature and initial pH value of the culture solution of the lactobacillus have influence on the growth metabolism of the lactobacillus, in order to ensure that the yield of CLA of the lactobacillus fermentum L1 in the safflower seed oil serving as a fermentation substrate is higher, the moringa seed oil is fermented respectively under different culture time, different culture temperature, different initial pH value of the culture solution and different inoculation amount, and the influence of different culture temperature, different time and different initial pH value of the culture solution on the number of living bacteria and the yield of CLA is explored; and the effect of different inoculum sizes on CLA production. Through single-factor and multi-factor analysis, the optimal culture temperature of the strain is finally determined to be 33-35 ℃, the culture time is 20-24h, and the initial pH value is 6.3-6.5.

Example 3 preparation of lipid-lowering fermented milk

The activated strain was inoculated into MRS broth at an inoculum size of 1% (37 ℃ C., 24 h) for cultivation to the third generation. Skim milk (10% w/w) was dissolved in hot distilled water and mixed with safflower seed oil (6% w/w) followed by the addition of 0.01% acesulfame potassium. Homogenizing at room temperature under 25-30MPa for 5min with high pressure homogenizer, and repeating homogenizing for 3 times. Then the homogeneous solution is heated in water bath under the condition of 80 ℃ for 30min, after the temperature is reduced to 35 ℃, the fermentation agent transferred to the third generation is inoculated into the homogeneous solution with the inoculum size of 1% (v/v) under the aseptic environment, and then the fermentation milk is obtained after the fermentation milk is cultured for 20-24h under the condition of 35 ℃.

When fermented milk production was performed, the control group (control) contained no safflower seed oil and fermented lactobacillus L1, SO (supplementsaf flowerseedoil) group contained safflower seed oil but not inoculated with fermented lactobacillus L1, the L1 group (inoculate fermented lactobacillus L1) contained safflower seed oil and fermented lactobacillus L1, except for the above-mentioned differences, the three treatment groups contained lactobacillus bulgaricus and streptococcus thermophilus (inoculum size was 1%), and the other conditions were unchanged, and each treatment group was repeated three times.

Example 4 Performance test of fermented milk

Process for fermenting milk rich in conjugated linoleic acid and quality analysis

The titrated acidity, pH and conjugated linoleic acid content of the different fermented milks during the different fermentation periods are shown in table 1. As is clear from Table 1, as the fermentation time was prolonged, both the TA (titrated acidity) and the CLA values of the 3 fermented milks increased, and the pH value was lowered, and when the fermentation time exceeded 20 hours, the titrated acidity of the three fermented milks exceeded 120℃T, and the pH value was lower than 4.2. The results of the sensory evaluation radar chart show that the overall score of the sensory scores of the L1 group is highest after fermentation for 20 hours, and the scores of the tissue state, the lubrication degree, the sweet-sour ratio and the aroma occupation are highest in all groups. Fermented milk has a good sweet-sour ratio that is more conducive to consumer acceptance. It has been shown that consumers are more receptive to fermented milk with titrating acidity at 120°t and pH between 3.9 and 4.2. The CLA content of the L1 group after fermentation for 20h is 814.26 mug/mL, which is significantly higher than that of the other groups, and the sensory score after fermentation for 24h is lower than 20h. In order to ensure that the flavor and the CLA content of the product are good, the sensory score and the CLA content are comprehensively considered, and the fermentation time of the fermented milk is fixed at 20 hours.

The texture and microbiological index of the different fermented milks after 20h fermentation (24 h after refrigeration) are shown in Table 2. As can be seen from the results in Table 2, the gel strength, viscosity and water holding capacity of the L1 group and the SO group are significantly higher than those of the control groupp<0.05 A) is provided; in addition, the number of viable bacteria of the three fermented milks exceeds 10 ⁸ CFU/mL, wherein the number of viable bacteria in the L1 group is the highest (10 ⁹ CFU/mL). The texture of dairy products is closely related to fatty acids, proteins are key nutritional components of fermented milk and are also important factors in the formation of colloidal systems. Whey protein in the fermented milk can be combined with casein through hydrogen bonds, disulfide bonds and the like to form a porous structure capable of maintaining small oil drops; in addition, the proteins on the surface of the porous structures can prevent the oil drops from being re-polymerized, so that the oil drops can reach a stable state in the gaps. With the addition of the fat, young's modulus (Young's modulus) of the fermented milk increases, which means that with the fermented milkThe water loss rate of the fermented milk is gradually reduced due to the increase of the content of medium oil. Therefore, the fermented milk added with safflower seed oil is more stable in structure and has better taste and texture than the defatted fermented milk (control group). The texture, flavor and mouthfeel of the fermented milk are related to the starter. The results show that after the lactobacillus fermentum L1 is added, the hardness, the consistency, the viscosity and other performances of the fermented milk are improved, and the taste and the texture of the L1 group are better than those of the SO group after the lactobacillus fermentum L1 is fermented.

TABLE 1 titrated acidity, pH and conjugated linoleic acid content of fermented milk at various fermentation times

Note that: the different lowercase letters in the table indicate that there are significant differences in the indices corresponding to the different fermentation time periods within the groupp< 0.05); the different capital letters represent that the corresponding indexes among groups have obvious difference in the same fermentation timep＜0.05）。

TABLE 2 texture and microbial indicators of fermented milk

Grouping

Gel strength

Viscosity of the mixture

Water holding capacity%

Number of viable bacteria

Mould fungus

Yeast

Control

9.51±0.90 b

5436.33±380.61 b

35.33±4.51 b

8.35±0.8 b

Not detected

Not detected

SO

26.19±1.22 a

8993.67±642.29 a

75.67±4.04 a

8.63±0.51 b

Not detected

Not detected

L1

27.67±0.85 a

7990.00±866.66 a

74.67±2.08 a

10.08±0.43 a

Not detected

Not detected

Note that: the different letters represent that significant differences exist between groupsp＜0.05）。

The results of comparing the CLA content of L1 with native 4-version cow milk and 4-version fermented milk are shown in FIG. 3. As can be seen from FIG. 3, L1 has a higher CLA content than the other 8 products. In sum, the skim milk containing safflower seed oil is fermented for 20 hours by utilizing the lactobacillus fermentum L1, the lactobacillus bulgaricus and the streptococcus thermophilus to obtain the fermented milk with good taste and texture and high conjugated linoleic acid and viable bacteria count.

Metabolite profile of fermented milk

The Principal Component Analysis (PCA) of the metabolites in the fermented milk was performed, and the results are shown in FIG. 4-A. From fig. 4-a, three samples were distributed in different intervals, and 3 samples from three treatment groups were more aggregated, indicating good reproducibility among the three sample groups, while there were differences among the groups. The metabolites (positive and negative ions combined) in the three samples were compared in pairs and the results are shown in FIGS. 4-B, C, D. As can be seen from FIG. 4-B, the L1 group identified 4241 different metabolites in total compared with the control group p< 0.05): there were 1970 relative upregulations, 2271 decreases; the SO group had 2045 metabolite levels increased and 2663 metabolite levels decreased compared to the control group (FIG. 4-C); the L1 group had 1038 up-regulated metabolites and 1149 down-regulated metabolites compared to the SO group (FIG. 4-D). This result demonstrates that there is a significant difference in the composition of fermented milk produced using different strainsp＜0.05）。

For the differencep< 0.05) metabolites were classified, all metabolites were classified into 35 categories except unidentifiable substances. The L1 and SO fermented milk has a large number of fatty acids, amino acids and derivatives thereof, organic acids, alkaloids, and lignans (more than 5). The fermented milk rich in conjugated linoleic acid contains 54 fatty acids, 43 amino acids and derivatives thereof, 12 organic acids, 11 alkaloids and 6 lignans. The characteristic active components of the L1 group and the SO group are similar in types, but the content is obviously differentp< 0.05). It follows that both the starter and the substrate affect the active ingredients of the fermented milk.

Characteristic active ingredient fingerprint

In order to analyze the characteristic components of the CLA-enriched fermented milk in detail, the metabolites with more kinds (more than 5 kinds) are further analyzed, and key metabolites are mined. Since the fermented milk has the largest variety of fatty acids, key fatty acids are searched by adopting KEGG metabolic pathway annotation, and the fatty acids are quantified so as to further confirm the composition ratio of the fatty acids; performing metabolic pathway annotation on the organic acid, searching for key organic acid, and analyzing the influence of the key organic acid on the flavor of the fermented milk; the amino acids and their derivatives, alkaloids, lignans are classified in detail to further understand the constitution of the characteristic components. Based on the key metabolites screened above, characteristics of fermented milk were constructed.

Fingerprint of active ingredient

Fatty acid

Fatty acids of both the L1 and SO groups are enriched in arachidonic acid metabolism (arasitinicosidmetabiolism), unsaturated fatty acid biosynthesis (biosynthesis furcatate fattyacids), alpha-linolenic acid metabolism (alpha-Linolentic biosystems), secondary metabolite biosynthesis (biosynthesis second farymethapsilosites). Upon integration of these metabolic pathways, changes in these metabolites were found to be related to the linoleic acid metabolic pathway, with linoleic acid (linoleic acid) being in a central metabolic position.

Amino acids

Amino acids and derivatives thereof are classified, and there are 12 amino acids which can be identified and classified in fermented milk. The amino acid content of the L1 fermented milk and the amino acid content of the SO fermented milk are differentpLess than 0.05), the variety of glycine, glutamic acid, threonine and methionine can be enriched through the fermentation of lactobacillus fermentum L1, and the amino acid spectrum of the fermented milk is enriched.

Organic acid

The organic acids of both the L1 group and the SO group are enriched in biosynthesis of secondary metabolites (biosynthesis) and in the citric acid cycle (TCA cycle), in glyoxylate and dicarboxylic acid metabolism (glyoxylate dicarboxylates), in biosynthesis of antibiotics (biosynthesis), in biosynthesis of saccharomyces cerevisiae (saccharomyces cerevisiae), and in oxycarboxylate metabolism (oxycarboxylate). Analysis of these metabolic pathways in combination found that L-malate was in a central metabolic position. The application detects 12 organic acids from the fermented milk rich in CLA, wherein the content of L-malic acid, cis-aconitic acid, ketoleucine, 4, 6-dioxoheptanoic acid and glutaric acid is relatively high. Studies have shown that cis-aconitic acid has a better effect in reducing ventilation (oliveiraet al, 2021) and bacteriostasis. The efficacy of ketoleucine, 4, 6-dioxoheptanoic acid and glutaric acid is rarely reported. In the application, the titrated acidity of the three fermented milks is the same as the variation trend of the malic acid content. When all samples are fermented for 20 hours, the malic acid content in the control group is the highest, and the second is the SO group; whereas the L1 group had the lowest malic acid and TA content. Fermented milk has a good sweet-sour ratio that is more conducive to consumer acceptance. The sensory evaluation score was highest after 20h fermentation of the L1 group fermented milk, which may also be related to malic acid. Notably, group L1 after addition of safflower seed oil and Lactobacillus fermentum L1, fat was metabolized by Lactobacillus fermentum L1. Malic acid is an important substrate of the TCA cycle, and ATP produced by the TCA cycle provides energy for biosynthesis of unsaturated fatty acids, biosynthesis of fatty acids, and linolenic acid metabolism of Lactobacillus fermentum L1. This means that in order to supply energy to other metabolic activities, malic acid of the L1 group is consumed in other metabolic pathways, so that malic acid of the L1 group is lower than the other two groups, and the content of malic acid gives a good sweet-sour ratio to the L1 group.

Other characteristic components

11 alkaloids are detected in the fermented milk, and the weight-losing effect of part of alkaloids is reported. Lignans are mostly found in plants and are of various kinds, and have functions of resisting cancer, resisting oxidation, regulating hormone metabolism level and the like (alessandral, 2018). In the application, 6 substances are detected in total by lignans: n-feruloyl-1, 4-butanediamine, 4-hydroxycinnamic acid, inter-o-coumaric acid, coumaric acid belonging to cinnamic acid and other lignans not identified (2), wherein 4-hydroxycinnamic acid, inter-o-coumaric acid, coumaric acid belonging to hydroxycinnamic acid can be classified as cinnamic acid.

Construction of finger print

The kind and content of characteristic components in the fermented milk and the functions reported in the literature are comprehensively considered, and the fingerprint spectrum established for the fermented milk rich in conjugated linoleic acid is shown in figure 5. The whole fingerprint is circular, and the high-yield CLA strain-lactobacillus fermentum L1 is positioned in the center of the fingerprint, which shows that thalli plays a dominant role in the production of the product; the outer ring is divided into 5 regions, and is composed of fatty acid, amino acid, organic acid, alkaloid and lignan, and the ratio of the outer ring to the inner ring is 42.86%, 34.13%, 9.52%, 8.73% and 4.76% respectively. Fatty acids are mainly composed of conjugated linoleic acid, palmitic acid and myristic acid. The amino acid is composed of 12 substances including glycine, glutamic acid, threonine, methionine, etc. The organic acid mainly comprises glutaric acid, L-malic acid, ketoleucine, cis-aconitic acid and 4,6 dioxoheptanoic acid. The alkaloid mainly comprises hypoxanthine, caffeine and betaine. The lignans mainly comprise N-feruloyl-1, 4-butanediamine and 4-hydroxycinnamic acid.

The characteristic active ingredients of the dairy product are different due to the changes of raw milk, fermentation process, starter and the like. By identifying the characteristic active ingredients of the product, the material composition and the nutritional characteristics of the product can be comprehensively known. More and more researches find that the synergistic effect of multiple nutrients is greater than the effect of single nutrient on human health or food characteristics, and the effect of the food and the effect of single nutrient should be equally valued in future researches. In general, the present application provides a fermented dairy product that is a CLA-enriched product, and that contains probiotics, as well as a variety of active ingredients.

In summary, the following conclusions can be drawn:

(1) The skim milk containing safflower seed oil is fermented by lactobacillus fermentum L1, so that the fermented milk with qualified organoleptic, physicochemical and microorganism indexes and good flavor and texture can be produced, and the conjugated linoleic acid content and viable bacteria number of the product are higher. Is a fermented milk rich in conjugated linoleic acid, fine in texture and sweet and sour.

(2) The fermented milk rich in conjugated linoleic acid is different from the common yoghourt in characteristic active ingredients: mainly contains various characteristic components such as fatty acid, amino acid, organic acid, alkaloids, lignans and the like; the fatty acid is mainly myristic acid, palmitic acid, linoleic acid and conjugated linoleic acid, and the linoleic acid is at a core position of fatty acid metabolism; malic acid is a typical organic acid in the core position of organic acid metabolism, and gives good flavor to the product.

(3) The characteristic component fingerprint of the fermented milk rich in conjugated linoleic acid consists of probiotics for high yield of conjugated linoleic acid, fatty acid (conjugated linoleic acid, palmitic acid, myristic acid), amino acid (glycine, glutamic acid, threonine, etc.), organic acid (L-malic acid, cis-aconitic acid, ketoleucine, glutaric acid, etc.), alkaloid (hypoxanthine, betaine, caffeine, etc.), and lignan (N-feruloyl-1, 4-butanediamine, cinnamic acid, etc.). Provides scientific basis for the identification of the fermented milk rich in conjugated linoleic acid and the construction of the fingerprint of the active ingredient characteristic of the fermented food.

EXAMPLE 5 study of lipid-lowering of fermented milk

Laboratory animals and groups

Male C57BL/6J mice of 3 weeks of age, grade APF, purchased from Hunan Stokes Lemonda laboratory animals Co., ltd., eligibility: SCXK (Hunan) 2019-0004. Mice were kept in 12/12h day and night period temperature controlled (22.+ -. 2 ℃) animal houses (university of agricultural food science, yunnan, academy of technology, animal houses) with 6 mice per cage, and were allowed to drink water freely. All mice were fed normal feed to the environment 1 week prior to intervention, and were gavaged daily during the intervention period, and fermented milk lyophilized powder was hydrated with distilled water, once a day and a night, each gavage volume was 0.4mL. The mice were randomly divided into 6 groups (18 mice per group) according to the guidelines for evaluation of health food function (2020 edition), each group: (1) normal group (NF): ordinary feed + distilled water; (2) model set (HF): high fat feed + distilled water; (3) group of plain yoghurt (HFY): high fat feed + medium dose normal yoghurt (0.67 g lyophilized powder/time); (4) high dose group (HFH): high fat feed + high dose conjugated linoleic acid enriched fermented milk (1 g lyophilized powder/time); dose group (HFM) in (5): high fat feed + medium dose conjugated linoleic acid enriched fermented milk (0.67 g lyophilized powder/time); (6) low dose group (HFL): high fat feed + low dose conjugated linoleic acid containing fermented milk (0.33 g lyophilized powder/time). The cages and pads were replaced every 3d during the acclimation period and the intervention period, with daily replacement of feed and water. The experimental unit uses license numbers: SYXK (Yunnan) K2015-0002, all experimental procedures were in accordance with animal welfare regulations.

Influence of fermented milk on physiological and biochemical indicators

Influence on the food intake and body weight of mice

Within 13 weeks of continuous intervention of fermented milk, the mice in each group had normal physical and mental conditions and no death phenomenon, and each group had normal drinking water and feeding, and the feeding and body weight of the mice within 14 weeks (including 1 week adaptation period) were shown in fig. 6 and 7, respectively.

As can be seen from the results of fig. 6, comparing the weekly feeding conditions between the different groups, the feeding amount of HFH group fed with 60% high fat feed was significantly higher than that of the other groups, and there was no significant difference in feeding amount of HFH, HFM, HFL, HFY group fed with high fat feed through fermented milk intervention, indicating that the appetite of mice was reduced through fermented milk intervention compared with the high fat group, but not related to the type of fermented milk and the gastric lavage dose. Compared with the food intake in the same group in different periods, the food intake of the mice in the later period of intervention is higher than that in the earlier period, and the mice tend to be stable after 9 weeks. This is related to the increase in age of the mice and their adaptation to the feed. Mice develop growth phase 4-8 weeks after birth, and 60-90d after birth is adulthood. As the age of mice increases, the feeding rate of mice increases and the feeding rate of mice after entering adulthood tends to stabilize. When the mice are transferred into a dry expectation (fed with high-fat feed) in the adaptation period (fed with common feed), the mice can not adapt to the feed in time due to the large difference of the raw material ratio of the high-fat feed and the common feed, so that the ingestion amount in the first week of intervention is low; in addition, the feed density and weight are different due to different proportions of the common feed and the high-fat feed, so that the ingestion rate of the HFH group is higher than that of other groups.

From the results of fig. 7, it can be seen that the body weight of the mice gradually increased during the adaptation period of week 1 and the intervention period of weeks 13 (weeks 2 to 14), and gradually became stable from week 9, and after week 9, the body weight of the HF group was significantly higher than that of the other groups, the body weight of the NF group was the lowest, and the HFH group and HFM group were next. In the later period of intervention (11-13 weeks of intervention), the average weight of the mice in the high-fat model group reaches 34.28g, which is significantly higher than that in the other groups (p < 0.05); the average body weight of the normal mice was 27.83g, significantly lower than the other mice (p < 0.05), with an average body weight of 29.92g for the HFH group, 31.15g for the HFM group, 32.22g for the HFL group and 32.29g for the HFY group. From the 14 week body weight profile, the body weight of the HFH group was closest to the NF group.

In summary, the mice had a reduced feeding rate after fermented milk intervention compared to the high-fat group, but the reduction in feeding rate was independent of the gastric lavage dose and the type of fermented milk. In addition, the weight of the high-fat mice can be obviously reduced through the intervention of the fermented milk rich in CLA, and particularly, the high-dose group has the best effect on reducing the weight of the high-fat mice.

Influence of fermented milk on body type, abdominal fat size and visceral index of mice

After 13 weeks of intervention, the body shape and abdominal fat size in a 1 x 1 square, and heart, liver, spleen, kidney, fat weights of each group of mice are shown in fig. 8, 9, respectively. As can be seen from fig. 8, NF group mice sizes were significantly smaller than the other groups, while HF group mice sizes were the largest among all groups. The mice body forms after the CLA-enriched fermented milk intervention show HFH groups, HFM groups < HFL groups, and the HFY and HFL groups have the similar body forms; as can be seen from the size of the abdominal fat in mice, the abdominal fat in the HF group was significantly greater than that in the HFH, HFM, HFL, NF group, with the NF group having the least abdominal fat, followed by the HFH group and HFM group.

From the results of fig. 9, it is seen that the NF group had the lowest fat weight, the HF group and the HFY group had the highest fat weight, and the HFH group had no significant difference in fat weight from the HFM group and the HFL group; there was no significant difference in cardiac weight in the HFH group compared to the NF group; the liver weights of NF group were lowest, HFY group were highest, while the liver weights of the other groups were not significantly different; there was no significant difference in spleen and kidney weights for each group of mice. In conclusion, compared with the normal group, the abdominal fat weight of the mice is obviously improved by feeding with the high-fat feed, so that the abdominal fat size and the body shape of the mice are larger; the intervention of the CLA-enriched fermented milk with different doses is helpful for returning the abdominal fat and body type of the mice to normal, and the abdominal fat size and weight of the mice show a certain dose dependency; particularly, through the intervention of the conjugated linoleic acid-rich fermented milk with high dosage, the abdominal fat of the high-fat mice can be obviously reduced, and the body shape of the high-fat mice is close to that of a normal group.

Influence of fermented milk on mouse blood lipid index

The changes in Triglyceride (TG), total Cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) in the serum of mice after intervention with the CLA-enriched fermented milk are shown in the results of Table 3. The high fat diet significantly increased TG, TC and LDL-C levels and the HDL-C levels were significantly reduced in the mouse serum. Compared with the model group, the content of TG, TC and LDL-C in the serum of the mice can be obviously reduced and the content of HDL-C can be improved after the high, medium and low dosages of fermented milk rich in conjugated linoleic acid and the ordinary yoghurt are subjected to dry prognosis. After the intervention of the high-dose conjugated linoleic acid-enriched fermented milk, the blood lipid index of the mice is not obviously different from that of the normal group, the blood lipid index of the mice in the low-dose group and that of the mice in the ordinary yogurt group are also not obviously different, and the blood lipid index of the mice and the stomach-filling dose show a certain dose dependency. TG containing LDL-C and HDL-C, TG, TC, LDL-C content too high indicates the possibility of hyperlipidemia; HDL-C is called-good cholesterol and has cardiovascular protecting effect (Suastikaet al, 2019). In conclusion, the blood lipid level of the high-fat mice can be regulated through the intervention of fermented milk. The weight and body type change conditions are combined, the weight and blood fat indexes between the common yoghourt group and the low-dose group are not obviously different, and the physiological and biochemical index change shows a certain dose dependency, so that the high-dose group, the medium-dose group, the common yoghourt group, the normal group and the model group are selected for subsequent experiments.

TABLE 3 influence of fermented milk on the blood lipid index of mice

Grouping	TG（mmol/L）	TC（mmol/L）	LDL-C（mmol/L）	HDL-C（mmol/L）
					HFH	1.02±0.36 b	3.95±0.31 c	2.55±0.36 c	1.84±0.36 ab
HFM	1.14±0.54 b	4.23±0.33 c	3.59±0.40 b	1.52±0.30 bc
					HFL	1.19±0.76 b	5.22±0.59 b	4.03±0.18 b	1.32±0.42 c
HFY	1.04±0.57 b	5.35±0.56 b	3.93±0.25 b	1.29±0.28 c
					NF	0.85±0.19 b	3.19±0.24 d	2.29±0.68 c	2.01±0.48 a
HF	2.36±0.37 a	6.40±0.73 a	5.06±0.39 a	0.70±0.25 d

Note that: the superscript letter differences represent significant differences between groups.

Effect of fermented milk on intestinal flora, serum metabolism and liver transcription

Effect of fermented milk on the intestinal flora of mice

To further explore the differences and similarities of intestinal microorganisms in mice of different treatment groups, the communities of relative abundance T30 at the genus level in the samples were clustered using Bray-Curtis (a distance index most commonly used in systematic clustering, mainly to describe the closeness between samples, the size of the distance being the main basis for sample classification) and the results are shown in fig. 10. From the results of fig. 10, it can be seen that at the generic level, the relative abundance of intestinal microorganisms of the 5 treatment groups was clustered into 4 classes, with the relative abundance of HF and HFY groups being most similar. Clostridium erysipelas of HFY, HFM, HFH, NF group compared with HF groupErysipelatoclostridium) The relative abundance is low. Notably, compared with the high-fat group, after intervention of the fermented milk rich in CLA, the bifidobacterium in the intestinal tract of the mice isBifidobacterium) Is increased. The above results show that the modification of intestinal flora structure caused by high-fat diet can be improved by the intervention of fermented milk rich in CLA, and harmful bacteria can be reduced ErysipelatoclostridiumIn aspects, the gastric lavage dose of the CLA-enriched fermented milk is inversely related; at the same time, the fermented milk rich in CLA is beneficial to improving the high-fat diet miceBifidobacteriumFlora abundance.

Effect of fermented milk on mouse liver Gene transcription

After comparing the HFH, HFM, HFY, NF group of HF,performing KEGG enrichment analysis on the differential expression genes between the two groups, and selectingpThe first 20 KEGG pathways with the smallest values draw a bubble map. The four comparison groups collectively annotated pathways were peroxisome proliferator activated receptor signaling pathway (PPARsignaling pathway) and bile secretion (bileSECRITION), which indicated that PPARsignaling pathway and bileSECRITION metabolism were significantly altered in either the normal group or the other three fermented milk intervention groups as compared to the HF groupp< 0.05). Peroxisome proliferator-activated receptors (PPARs) are receptors for fatty acids and derivatives thereof. PPARs have three subtypes (pparα, β and γ), exhibit different expression patterns in vertebrates, and PPAR plays a role in eliminating cellular lipids by regulating gene expression of liver and skeletal muscle lipid metabolism, pparβ being involved in lipid oxidation and cell proliferation; pparγ promotes adipocyte differentiation and enhances blood glucose uptake (Hauner, 2010). Bile is critical for digestion and absorption of fat and fat-soluble vitamins in the small intestine; and plays an important role in removing excessive cholesterol, medicines and toxic compounds. Secretion of bile depends on the function of the membrane transport system of hepatocytes and cholangiocytes, as well as the integrity of biliary tract structure and function (Kostersetal, 2008). Cholesterol is oxidized to bile acid in hepatocytes, then reacts with taurine or glycine to form primary bile acid, enters the intestinal tract, forms secondary bile acid through the actions of intestinal microorganisms and bile salt hydrolase and the like, and participates in emulsification and absorption of fat (klaasseen et al, 2010). Of the four comparison groups, the HFH group and NF group annotated the same metabolic pathway the most, for a total of 10: retinolmetanolism (retinol metabolism), PPAR signaling pathway, chemical carcinogenesis, bileretransmission (bile secretion), steroidogyne biosystems (steroid hormone biosynthesis), drug metanolism-other enzymes, ascobatin and hydrochloride metabolism, aralidogenic metanolism (Glycine), glycine, serine and threonine metabolism, poryindeand chlorophyllinetabilization. Furthermore, fermented milk intervention group (HFH, HFM, HFY) the common differential metabolic pathways contained compared to the HF group were mineral absorption, endocrinologically related factor-regulated calcium reabsorption, and Bilespeculation. Taken together, liver gene transcription in high-fat diet mice was indeed altered by fermented milk intervention, and these alterations were also in terms of pparsignalingapath (peroxisome proliferator-activated receptor signaling pathway) and bilerecryption (bile secretion) associated with fatty acid metabolism.

In order to further study the differential expression condition of liver gene transcription, focus on the main metabolic pathway and search for the change of the main pathway caused by fermentation milk intervention, the application carries out differential gene cluster thermogram analysis on differential genes related to PPARsignalinlingpathway (peroxisome proliferator activated receptor signal pathway), bileretion (bile secretion) and Primarybileacidiosynthesis (primary bile acid biosynthesis, belonging to bileretion pathway) in 5 treatment groups, and the gene expression of NF group and other 4 groups has obvious differencep＜0.05），

NF group most genes associated with bile acid metabolism were down-regulated relative to the other 4 supplemented high fat diet groups (HF, HFH, HFM, HFY); after intervention of fermented milk (HFH, HFM, HFY), there was a difference in the number of gene upregulations associated with bile acid metabolism, HFH > HFM > HFY. Notably, the expression of a number of genes CYP7A1, CYP27A1, CYP81, PPARα, PPARβ, PPARγ reported to be associated with bile acid metabolism was up-regulated in the HFH group compared to the NF group. In general, 5 treatment groups showed differences in liver gene transcription, and the expression of related genes in bile acid metabolic pathways of high-fat diet mice could be up-regulated by CLA-enriched fermented milk intervention.

Effect of fermented milk on serum metabolism

To further study the effect of fermented milk on serum metabolism in high-fat mice and to find key metabolic pathways, KEGG annotation was performed on the differential metabolites of the Normal (NF) and fermented milk intervention (HFH, HFM, HFY) groups in HF group, and the number of metabolic pathways enriched from HF group to the differential metabolites of the 4 treatment groups were analyzed in order from more to less: HFVSNF (25), HFVSHFY (9), HFVSHFM (9), HFVSHFH (8), HFVSNF annotated to 25 metabolic pathways altogether. The differential genes of the other 4 groups were annotated together to 4 metabolic pathways compared to the high fat diet group: pantothenate and coenzyme a biosynthesis (pantothenatecand abiosynthesis), pyrimidine metabolism (pyrimidemethabolism), biosynthesis of unsaturated fatty acids (biosystems) and primary bile acid biosynthesis (primarybileacbiosynthesis). The panthothenateand abiosynthesis are mainly cofactors and vitamin metabolism, the pyremidinesabolism is closely related to nucleotide metabolism, and the biosystems and primybileacbiosynthesis are also related to fatty acid and cholesterol metabolism. Overall, serum metabolic levels of high fat mice were significantly altered following fermented milk intervention.

Metabolic pathway annotation results for both the liver transcriptome and serum metabolome are directed to pathways associated with bile acid metabolism; the high-dose CLA-enriched fermented milk has the best intervention effect on high-fat mice, and the main component analysis results of the intestinal microbial diversity of the 16SrRNA, the liver gene transcription expression condition and the serum metabolite expression condition among different groups show that the HFH group is closest to the NF group. The present application selects the mouse serum sample with the same number as the liver transcriptome sample, and studies the expression level of the metabolites of the bile acid metabolism related pathway (PPARsignalingpathway, bilesecretion, primarybileacidbiosynthesis) of HFH group, NF and HF group, and the results are shown in FIG. 11. From fig. 11, it is clear that the metabolites in the bile acid metabolism pathway of the HFH group were significantly up-regulated, and it is noted that the content of chenodeoxycholic acid (Tauro-chenodeoxycholic acid) and ursodeoxycholic acid (ursodeoxycholic acid) in the HFH group was higher than those in the NF group and the HF group.

Effect of fermented milk on intestinal flora and bile acid metabolism

The result of the combination of the liver genome transcriptome and the serum metabolome of the mice shows that the pathways related to fatty acid metabolism and bile acid metabolism are changed after the intervention of fermented milk of the high-fat mice. Bile acid metabolism plays an important role in regulating fat metabolism; the application also discovers that the intestinal flora of the high-fat mice is obviously changed after the intervention of the fermented milk.

Liver gene transcription and bile acid metabolism

The high dose lavage group and high lipidic group (HFHVSHF) and normal group and high lipidic group (NFVSHF) bile acid metabolites were analyzed in association with genes related to bile acid metabolism. As a result, it was found that the differential metabolites show a certain correlation with genes in bile acid metabolism, in particular, metabolites in PPARα (Ppara), PPARβ (Ppar), aPPARγ (Pparg), cyp7a1, cyp27a1, cyp8b1 and PPAR signaling pathway (PPARSignalingapay), and chenodeoxycholic acid and ursodeoxycholic acid show a positive correlation. From the results of liver transcriptome, pparα, pparβ, pparγ, cyp7a1, cyp27a1, cyp8b1 were found to be up-regulated in HFH group, whereby it could be determined that genes (PAR α, pparβ, abpparγ) of pparignalingpay and genes (Cyp 7a1, cyp27a1, cyp8b 1) of primary bile acid biosynthesis (primariybileachosyntheie) pathway were up-regulated by intervention of conjugated linoleic fermented milk, and the content of chenodeoxycholic acid and ursodeoxycholic acid was increased. Peroxisome proliferator-activated receptors (PPARs) are receptors that regulate the expression of genes of interest, are ligand-activated transcription factors, and are capable of inducing the expression of hundreds of genes in almost every cell type. PPARs play a role in the clearance of cellular lipids by regulating the expression of genes for liver and skeletal muscle lipid metabolism. Each subtype of PPAR has different functions, pparα and pparβ are involved in lipid oxidation and cell proliferation, and pparγ promotes adipocyte differentiation and enhances blood glucose uptake. Pparα is the most widely and deeply studied PPAR family, and is closely related to lipid metabolism in the liver, and almost regulates all gene expression related to lipid metabolism in the liver, including fatty acid uptake, intracellular fatty acid activation and binding, fatty acid extension and desaturation, formation and breakdown of triglycerides and lipid droplets, plasma lipoprotein metabolism, and the like. In the process of regulating bile acid metabolism, PPAR family binds to Retinoid X Receptor (RXR) as heterodimer, recognizes bile acid metabolism target genes (CYP 7A1, CYP27A1, CYP81, etc.) and starts transcription of the target genes, promotes bile acid metabolism, and reduces blood lipid.

The classical bile acid synthesis pathway is initiated by cholesterol7 alpha-hydroxylase (CYP 7 A1), which is also an important pathway for bile acid synthesis in adults. CYP7A1 hydrolyzes cholesterol at position 7α to form 7α -hydroxycholesterol and is converted to 7α -hydroxy-4-cholest-3-one (designated C4) by 3β -hydroxy- Δ5-C27-steroid dehydrogenase (HSD 3B 7), C4 being a common precursor of Cholic Acid (CA) and chenodeoxycholic acid (CDCA), CA and CDCA being two major bile acids in the human liver (Gonz-lezetal, 2017). In mice and humans, the 7α -OH group in CDCA can be isomerized to 7β -OH to form ursodeoxycholic acid (UDCA). The CDCA content of the HFH group was up-regulated compared to the HF group. In conclusion, the high-fat diet mice can up-regulate the expression of partial metabolites of PPAR signal channels through the intervention of the fermented milk rich in CLA, up-regulate the expression of CYP7A1, CYP27A1 and CYP81 genes in bile acid metabolism, and promote the synthesis of primary bile acid.

Intestinal microorganisms and bile acid metabolism

The correlation analysis of bile acid metabolites of high dose lavage group and high lipid group (HFHVSHF) and normal group and high lipid group (NFVSHF) with intestinal microorganisms at genus level shows that FirmicutesAndErysipelatoclostridiumhas negative correlation with chenodeoxycholic acid and ursodeoxycholic acid; whileBacteroidalesAndBifidobacteriumpositive correlation with partial metabolites in PPAR signaling pathway, chenodeoxycholic acid and ursodeoxycholic acid. Intestinal microorganisms are closely related to bile acid metabolism, and researches show that primary bile acid generates secondary bile acid (deoxycholic acid, lithocholic acid and ketolithocholic acid) under the action of intestinal microorganisms through the actions of bile salt hydrolase and 7 alpha-hydroxysteroid dehydrogenase (7-HSDH) of the intestinal microorganisms, and the organism realizes the reutilization of the bile acid through intestinal liver circulation. Ridlon et al (2005) found that primary bile acids are converted in the gut to secondary bile acids and predominantly in the gutBacteroidalesAnd Clostridium bacteria [ ]Clostridium) In these bacteria, bile salt hydrolase (bilialysalarydrolyase, BSH) to form secondary bile acids, which are then partially utilized and partially circulated back to the liver through the intestinal liver for the next cycle. BSH has been found in bacteria which are primarily anaerobic, e.gBacteroidalesGenus Clostridium and LactobacillusLactobacillus) And colibacillus genus Escherichiacoli) The method comprises the steps of carrying out a first treatment on the surface of the In addition, BSH also helps the bacteria to build up tolerance to bile acids, thereby maintaining activity in secondary bile acid conversion and bile acid environments (Jonesetal, 2016). Liang et al (2020) found that bifidobacterium animalis subspecies had the efficacy of reducing blood lipid levels in hyperlipidemic mice, while bifidobacterium animalis subspecies upregulated liver LXR receptor transcription levels, and CYP7A1 protein expression, thereby regulating bile acid metabolism. Intestinal microorganisms play a key role in liver bile acid metabolism, and this interaction forms the intestinal-hepatic axis, and the homeostasis of intestinal micro-ecology is the basis of the virtuous circle of the intestinal-hepatic axis. Bile acids are not only closely related to fat digestion and metabolism, but also play an important role in transmission as signal molecules connecting intestinal microecology and liver bile acid metabolism by relying on negative feedback regulation mechanisms in the intestinal-hepatic circulation. Once the intestinal microecology is disturbed, bile acid circulation is affected, thereby causing lipid metabolism to be disturbed. In conclusion, through the intervention of the fermented milk rich in CLA, intestinal microorganisms of the high-fat mice can be regulated, the abundance of bifidobacterium and bacteroides is improved, the balance of the intestinal microorganisms is regulated, and meanwhile, the secretion of secondary bile acid is promoted, so that the emulsification and absorption of fat are realized, and the lipid-lowering effect is achieved.

Bile acid metabolism and lipid-lowering mechanism rich in conjugated linoleic acid

The comprehensive analysis of the results shows that the high-fat diet mice can up-regulate the expression of PPAR receptors in the liver through the intervention of the fermented milk rich in CLA, induce the expression of key genes (CYP 7A1, CYP27A1 and CYP 81) of bile acid metabolism in the liver, and further promote the secretion of primary bile acid (chenodeoxycholic acid); meanwhile, through the intervention of the fermented milk, the abundance of bifidobacterium and bacteroides in the intestinal tract of a mouse can be improved; the abundance of the firmicutes and the erysipelas clostridia is reduced, the ratio (F/B) of the firmicutes and the bacteroides is reduced, and the effect of restoring the intestinal microbial balance of the high-fat diet mice is achieved. Under the action of bifidobacterium and bacteroides, the primary bile acid is converted into secondary bile acid, and the secondary bile acid participates in emulsification and absorption of fat, so that the TC, TG, LDL-C content in serum of a high-fat mouse is reduced, the HDL-C content is increased, and the weight and body fat level of the mouse are reduced. In summary, the CLA-enriched fermented milk can achieve the effects of losing weight and reducing blood lipid by promoting bile acid metabolism and restoring intestinal microecological balance of high-fat mice through benign circulation of bile acid in the intestinal-hepatic axes (fig. 12).

In summary, (1) the CLA-enriched fermented milk improves physiological and biochemical index of high-fat mice: high fat mice were subjected to high doses of CLA

The fermented milk intervention can obviously reduce the food intake, the weight and the abdominal fat weight of the mice, so that the body type and the abdominal fat of the high-fat mice are similar to those of normal mice. Meanwhile, all blood lipid indexes of the high-fat mice tend to be normal through CLA fermented milk intervention;

(2) Upregulation of intestinal microorganisms, serum metabolism and liver transcription in high fat mice by CLA-enriched fermented milk: after the intervention of the fermented milk rich in CLA, the intestinal microorganism diversity of the high-fat mice is improved, the abundance of the bacteroides of the high-fat mice is improved at the portal level, and the F/B is reduced; at the genus level, the abundance of clostridium erysipelas is reduced, and the abundance of bifidobacteria is increased. After a dry prognosis, the liver gene transcription level of the high-fat mice is changed, the metabolic pathways of the differential genes are mainly concentrated in pathways related to bile acid metabolism, and key genes (Cyp 7a1, cyp27a1, cyp8b1, PPAR-alpha, PPAR-beta and PPAR-gamma) related to bile acid metabolism in the livers of the mice are up-regulated. Serum metabolites were changed in high-fat mice after CLA fermented milk intervention, and differential metabolites between different treatment groups and high-fat groups were enriched in primary bile acid biosynthesis (primary bile acid biosynthesis).

(3) Association analysis of differential metabolites with liver gene transcriptomes showed: the metabolites in CYP7A1, CYP27A1, CYP81, PPAR-alpha, PPAR-beta, PPAR-gamma genes and bile acid metabolism exhibit a positive correlation; the results of the correlation analysis of intestinal microorganisms and differential metabolites at the belonging level show that the bacteroides and bifidobacteria show a positive correlation with the metabolites in bile acid metabolism. In conclusion, after the intervention of the CLA-enriched fermented milk, the high-fat diet mice can up-regulate the expression of genes related to bile acid metabolism, promote the secretion of primary bile acid, restore the balance of intestinal microorganisms, convert the primary bile acid into secondary bile acid under the action of bifidobacteria and bacteroides, promote bile acid metabolism, and achieve the effects of losing weight and reducing lipid through the virtuous circle of intestinal-hepatic axes.

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the claims of the present invention.

Claims (9)

1. The high-yield conjugated linoleic acid strain is characterized in that the strain is named as lactobacillus fermentum L1, and the preservation number is CCTCC NO: m2023862, deposit unit: china center for type culture collection, with the preservation addresses: university of martial arts in chinese; preservation date: 2023, 5 and 30.

2. A microbial preparation comprising the strain of high-yield conjugated linoleic acid as claimed in claim 1, wherein the microbial concentration per milliliter or gram of the microbial preparation is not less than 1X 10 ⁹ CFU。

3. Use of the high-yield conjugated linoleic acid strain according to claim 1 or the microbial preparation according to claim 2 for the preparation of conjugated linoleic acid.

4. A lipid-lowering fermented milk, characterized in that the fermented milk is obtained by fermentation using the high-yield conjugated linoleic acid strain of claim 1; or by fermentation using the microbial preparation of claim 2.

5. The lipid-lowering fermented milk according to claim 4, wherein the lipid-lowering fermented milk is prepared by the following steps: dissolving skimmed milk with distilled water, adding safflower seed oil and acesulfame potassium, mixing, homogenizing at room temperature under 25-30MPa for 3-10min, and homogenizing repeatedly for 2-4 times; then maintaining the homogenized liquid at 75-85deg.C for 20-40min, cooling to 33-37deg.C, inoculating lactobacillus fermentum L1 into the homogenized liquid under aseptic condition, and fermenting at 33-36deg.C.

6. The lipid-lowering fermented milk according to claim 5, wherein the lactobacillus fermentum L1 is inoculated into MRS broth medium for cultivation to the third generation at an inoculum size of 0.8-1.5% after activation, and then inoculated into a homogenous liquid at an inoculum size of 0.5-3%.

7. The lipid-lowering fermented milk according to claim 6, wherein the fermented milk employs lactobacillus fermentum L1, lactobacillus bulgaricus and streptococcus thermophilus together as a starter; the ratio of the lactobacillus fermentum L1 to the lactobacillus bulgaricus to the streptococcus thermophilus is 1.5-2.5:0.8-1.2:0.8-1.2.

8. The lipid-lowering fermented milk according to claim 5, wherein the addition amount of the skim milk is 8-15%, the addition amount of the safflower seed oil is 5-7%, and the addition amount of the acesulfame potassium is 0.008-0.015%.

9. Use of a lipid-lowering fermented milk according to any of claims 4-8 for lowering lipid and reducing weight.