Related application of fructus lycii fermented wine in regulating intestinal flora

Abstract

The invention discloses related application of fructus lycii fermented wine in regulating intestinal flora, and relates to the field of fructus lycii wine. It is found for the first time that the fructus lycii fermented wine can improve the relative abundance of ackermann bacteria, lactobacillus, bifidobacterium, flavonolysis bacteria and blue-green algae bacteria and meanwhile reduce the relative abundance of escherichia, shigella, proteusbacillus vulgaris, pseudomonas and klebsiella; the content of short-chain fatty acids in intestinal tracts can be remarkably increased, and the probiotic can be applied to the field of foods as prebiotics.

Description

Technical Field

[0001] The invention relates to the field of wolfberry wine, and in particular to the application of wolfberry fermented wine in regulating intestinal flora. Background Art

[0002] Lycium barbarum is one of the varieties that can be used as both medicine and food. It is rich in various types of functional substances such as wolfberry glycoprotein, polyphenols and their derivatives, carotenoids, etc. It has multiple pharmacological effects such as anti-aging, immune regulation, improving lipid metabolism, and regulating blood sugar.

[0003] Various wolfberry deep-processed products have appeared on the market. Among them, fermented wolfberry wine is a kind of fruit wine made from wolfberry through yeast fermentation. Since yeast can produce a large amount of aromatic esters and other ingredients during metabolism, its aroma is richer and stronger than that of soaked wine, and the variety of flavor compounds is also significantly increased. Fermented wolfberry wine has a low alcohol content and is suitable for a wide range of people. Its nutritional value and health benefits are welcomed by the market.

[0004] Currently, research on fermented wolfberry wine mainly focuses on the brewing process, and there is a lack of relevant research on its efficacy.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide related applications of wolfberry fermented wine in regulating intestinal flora.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides an application of wolfberry fermented wine in regulating intestinal flora or preparing a product for regulating intestinal flora, wherein the intestinal flora includes: a combination of any one or more of the genera Akkermansia, Lactobacillus, Bifidobacterium, Escherichia, Shigella, Proteus, Pseudomonas, Klebsiella, Flavonoids and Cyanobacteria.

[0009] In a second aspect, an embodiment of the present invention provides the use of wolfberry fermented wine in increasing the relative abundance of Proteobacteria and / or Bacteroidetes or in preparing a product that increases the relative abundance of Proteobacteria and / or Verrucomicrobia.

[0010] In a third aspect, an embodiment of the present invention provides the use of wolfberry fermented wine in reducing the relative abundance of Firmicutes and / or Verrucomicrobia or in preparing a product that reduces the relative abundance of Firmicutes and / or Verrucomicrobia.

[0011] In a fourth aspect, an embodiment of the present invention provides the use of wolfberry fermented wine in reducing the ratio of Firmicutes / Bacteroidetes or in preparing a product for reducing the ratio of Firmicutes / Bacteroidetes.

[0012] In a fifth aspect, an embodiment of the present invention provides the use of wolfberry fermented wine in increasing the content of short-chain fatty acids in the intestine or in preparing a product for increasing the content of short-chain fatty acids in the intestine.

[0013] The present invention has the following beneficial effects:

[0014] This application discovers for the first time that wolfberry fermented wine has prebiotic activity, which can increase the relative abundance of Akkermansia, Lactobacillus, Bifidobacterium, Flavonoids and Cyanobacteria, while reducing the relative abundance of Escherichia, Shigella, Proteus, Pseudomonas and Klebsiella. In addition, it can also significantly increase the content of short-chain fatty acids in the intestine, and can be used in the food field as a prebiotic. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 It is the liquid chromatogram of wolfberry wine glycoprotein LBFG-4;

[0017] Figure 2 This is the liquid chromatogram of wolfberry wine polyphenol LBFP-4;

[0018] Figure 3 This is a bar graph showing the distribution of intestinal flora at the family level after wolfberry wine and its component groups intervened in model rats;

[0019] Figure 4 This is a distribution bar graph showing the genus level analysis of intestinal flora in model rats after intervention with wolfberry wine and its component groups. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0021] On the one hand, an embodiment of the present invention provides the use of wolfberry fermented wine in regulating intestinal flora or preparing a product for regulating intestinal flora, wherein the intestinal flora includes: any one or more combinations of Akkermansia, Lactobacillus, Bifidobacterium, Escherichia, Shigella, Proteus, Pseudomonas, Klebsiella, Flavonifractor and Lachnoclostridium.

[0022] In some embodiments, regulating the intestinal flora comprises: upregulating the relative abundance of any one or more of the genera Akkermansia, Lactobacillus, Bifidobacterium, Flavonoids, and Cyanobacteria in the intestine.

[0023] Akkermansia, Lactobacillus and Bifidobacterium are beneficial bacteria in the intestinal flora. Upregulation of these bacteria can help enhance immunity, fight infection, aid digestion, and regulate gastrointestinal disorders.

[0024] In some embodiments, regulating the intestinal flora comprises: downregulating the relative abundance of any one or more of the genera Escherichia, Shigella, Proteus, Pseudomonas, and Klebsiella in the intestine.

[0025] Escherichia and Shigella are pathogenic bacteria.

[0026] Proteus is a conditional pathogen that can produce a strong toxin and is one of the main pathogens of diarrhea.

[0027] Pseudomonas is a gram-negative bacillus with strong drug resistance and ability to adapt to the environment. Under certain conditions, it can transform into a pathogenic bacterium. When the body's immunity is weakened or there is a primary disease, Pseudomonas can take the opportunity to invade and cause infection.

[0028] Klebsiella is a common Gram-negative bacillus in clinical practice. It is a conditional pathogen. Klebsiella infection mainly occurs in people with weakened immune function and underlying diseases, and can cause lung infection, urinary tract infection and nervous system infection.

[0029] On the other hand, an embodiment of the present invention provides the use of wolfberry fermented wine in increasing the relative abundance of Proteobacteria and / or Bacteroidetes or in preparing a product that increases the relative abundance of Proteobacteria and / or Verrucomicrobia.

[0030] On the other hand, an embodiment of the present invention provides the use of wolfberry fermented wine in reducing the relative abundance of Firmicutes and / or Verrucomicrobia or in preparing a product that reduces the relative abundance of Firmicutes and / or Verrucomicrobia.

[0031] On the other hand, an embodiment of the present invention further provides the use of wolfberry fermented wine in preparing a product for preventing, treating or assisting in treating intestinal flora imbalance.

[0032] In some embodiments, the intestinal flora imbalance includes: an imbalance of any one or more combinations of Akkermansia, Lactobacillus, Bifidobacterium, Escherichia, Shigella, Proteus, Pseudomonas, Klebsiella, Flavonoids and Cyanobacteria, or an imbalance of any one or more combinations of Proteobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia.

[0033] Optionally, the intestinal flora imbalance includes a decrease in any one or more of Akkermansia, Lactobacillus, Bifidobacterium, Flavonoids and Cyanobacteria. Optionally, the intestinal flora imbalance includes an increase in any one or more of Escherichia, Shigella, Proteus, Pseudomonas and Klebsiella. Optionally, the intestinal flora imbalance includes a decrease in Proteobacteria and / or Bacteroidetes. Optionally, the intestinal flora imbalance includes an increase in Firmicutes and / or Verrucomicrobia.

[0034] On the other hand, an embodiment of the present invention provides the use of wolfberry fermented wine in reducing the ratio of Firmicutes / Bacteroidetes or preparing a product for reducing the ratio of Firmicutes / Bacteroidetes.

[0035] On the other hand, an embodiment of the present invention provides the use of wolfberry fermented wine in preparing a product for reducing or inhibiting obesity.

[0036] On the other hand, an embodiment of the present invention provides the use of wolfberry fermented wine in increasing the content of short-chain fatty acids in the intestine or in preparing a product that increases the content of short-chain fatty acids in the intestine.

[0037] Short-chain fatty acids (SCFAs) are a type of saturated fatty acids composed of 1-6 carbon atoms as metabolites of intestinal microbial fermentation. SCFAs can be used as an energy source and undergo oxidative decomposition through the tricarboxylic acid cycle. Increasing the content of SCFAs can also speed up the rate of the tricarboxylic acid cycle and accelerate metabolism.

[0038] Studies have found that the levels of SCFAs in the feces of subjects who have lost weight have increased. SCFAs are beneficial to the host's metabolism. Acetate changes the expression of hypothalamic appetite-regulating neuropeptides by activating the tricarboxylic acid cycle, thereby reducing appetite. SCFAs can maintain healthy symbiotic microbiota, intestinal epithelial integrity, mucosal immunity, and improve metabolic diseases.

[0039] SCFAs are also closely related to type 2 diabetes. Experiments have confirmed that intestinal microbiota maintained by high dietary fiber can promote the production of butyrate and acetate, improve glucose tolerance, reduce glycated hemoglobin levels, and help alleviate type 2 diabetes. Butyrate is one of the main energy sources for intestinal epithelial cells. Elevated levels of butyrate can induce intestinal gluconeogenesis, reduce liver glucose production, reduce body weight, and improve intestinal wall function. SCFAs can also regulate glucose transporters through the adenylate-activated protein kinase signal transduction control system, increase the sensitivity of receptors to insulin, and further activate adenylate-activated protein kinase to promote the body's transport and consumption of glucose. Therefore, the balance of glucagon, insulin, and SCFAs is a key factor in maintaining blood sugar stability in the body.

[0040] Studies have found that SCFAs may delay the development of non-alcoholic fatty liver disease. After SCFAs enter the liver through the portal vein system, they can play a positive role in the development of non-alcoholic fatty liver disease by regulating the inflammatory response, lipid metabolism and glucose metabolism of liver tissue. SCFAs can change the intestinal microecology and protect the intestinal mucosal barrier, thereby delaying the progression of non-alcoholic fatty liver disease-related diseases.

[0041] In some embodiments, the short-chain fatty acids include any one or more combinations of acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid and isovaleric acid.

[0042] On the other hand, an embodiment of the present invention provides the use of wolfberry fermented wine in preparing a product for preventing or assisting in treating any one or more of obesity, diabetes, and non-alcoholic fatty liver disease.

[0043] "Treatment" as used herein includes preventing or alleviating a condition, reducing the rate at which a condition develops or progresses, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0044] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0045] In some embodiments, the product comprises: food and / or medicine.

[0046] In some embodiments, the method for preparing the wolfberry fermented wine includes: subjecting a mixture of wolfberry raw materials and fermentation bacteria to primary fermentation.

[0047] In some embodiments, the main fermentation conditions include: 15-30° C. The fermentation temperature can be any one of 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30° C. or a range between any two of them. When the residual sugar and specific gravity do not change for 1-3 days, the main fermentation is stopped, and the wine is allowed to stand and clarify for 2-3 days to obtain wolfberry original wine.

[0048] In some embodiments, the preparation of the wolfberry raw material includes: enzymatic hydrolysis of wolfberry pulp.

[0049] In some embodiments, the method for obtaining wolfberry pulp comprises: removing wolfberry seeds and / or wolfberry skins from wolfberries, and beating the wolfberries to obtain wolfberry pulp.

[0050] In some embodiments, the conditions of the enzymolysis include: 0-35°C, 1-15h. The enzymolysis temperature can be any one of 0, 5, 10, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35°C or a range between any two thereof. The enzymolysis time can be any one of 1, 2, 4, 6, 8, 10, 12, 14, 15h or a range between any two thereof.

[0051] In some embodiments, the enzyme used in the enzymolysis includes any one or more of pectinase and cellulase. Optionally, the amount of pectinase added is 0.1‰ to 5‰ of the mass of wolfberry jam, specifically 0.1‰, 0.5‰, 1‰, 1.5‰, 2‰, 3‰, 4‰, 5‰, any one or any two of them. Optionally, the amount of cellulase added is 0.1‰ to 5‰ of the mass of wolfberry jam, specifically 0.1‰, 0.5‰, 1‰, 1.5‰, 2‰, 3‰, 4‰, 5‰, any one or any two of them.

[0052] In some embodiments, after enzymatic hydrolysis and before main fermentation, the preparation of the wolfberry raw material further comprises: adjusting the sugar content of the enzymatic hydrolysis product to 15-25° Brix, and adjusting the pH to 3-4. Specifically, the sugar content is adjusted to any one of 15, 16, 18, 20, 21, 22, 24, and 25° Brix; and the pH is adjusted to any one of 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4.0, or a range between any two of them.

[0053] In some embodiments, the fermentation bacteria include: Saccharomyces cerevisiae.

[0054] The present invention has no special restrictions on brewer's yeast, and can be carried out using the fermentation bacteria disclosed in the prior art for wolfberry fermentation. In some embodiments, the brewer's yeast is brewer's yeast M-23-7-14 with a deposit number of CGMCC No. 23603.

[0055] In some embodiments, the method for preparing the wolfberry fermented wine further comprises: performing post-fermentation on the product after the main fermentation to obtain the wolfberry fermented wine.

[0056] In some embodiments, the post-fermentation conditions include: 15-30°C, 10-30 days. The temperature range can be any one of 15, 16, 18, 20, 22, 24, 26, 28, 30°C or a range between any two thereof; the time can be any one of 10, 12, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30 days or a range between any two thereof.

[0057] In some embodiments, the preparation method further comprises any one or more steps of clarifying, filtering and sterilizing the post-fermentation product after the post-fermentation is completed.

[0058] In some embodiments, the clarifying step includes: performing a sizing process using a bentonite-gelatin sizing material.

[0059] In some embodiments, the filtering step includes: filtering using a filter membrane.

[0060] In some embodiments, the sterilization includes high temperature sterilization, and the sterilization temperature can specifically be 75-77°C.

[0061] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0062] Example 1

[0063] A method for preparing wolfberry fermented wine comprises the following steps:

[0064] (1) Raw material sorting: remove the fruit stems, select fresh wolfberries without green fruit, rot, mildew, or bird bites, seal them, and quickly freeze them in a cold storage at -18°C for 3 days for future use;

[0065] (2) Pulping: Turn on the ultraviolet lamp and ozone generator to disinfect and sterilize the workshop environment for more than 1 hour. Use a double-channel pulping machine to pulp the fresh wolfberry preserved in step (1) to remove the wolfberry seed skin, obtain wolfberry pulp, and add 60 ppm of potassium metabisulfite.

[0066] (3) Enzymatic hydrolysis: adding 1‰ pectinase and 1‰ cellulase to the wolfberry pulp, stirring evenly and performing enzymatic hydrolysis at room temperature for 8 hours;

[0067] (4) Adjusting ingredients: adjusting the sugar content of wolfberry syrup to 21° Brix with sucrose and adjusting the pH to 3.6 with tartaric acid;

[0068] (5) Main fermentation: The wolfberry fermented mash was inoculated with activated yeast (wolfberry wine brewing yeast (Saccharomyces cerevisiae) M-23-7-14 (deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 23603 and a deposit date of October 14, 2021), and the fermentation temperature was controlled at 22°C. When the specific gravity was 1070 kg / m 3 When the pressure is above 1000 kg / m3, the circulation should be carried out every 4 hours, and the specific gravity should be between 1070 and 1030 kg / m3. 3 During the vigorous fermentation period, the wine is circulated every 2 hours. When there is no obvious sign of fermentation and the residual sugar and specific gravity do not change for two consecutive days, the fermentation is considered to be over, and the fermentation is terminated. The wine is allowed to stand and clarify for 2 to 3 days, and then squeezed and separated to obtain the original wolfberry wine.

[0069] (6) Finished product: The wolfberry wine enters the post-fermentation period, and the fermentation temperature is 20°C. After the post-fermentation is completed after 15 days, it is poured back, 800ppm bentonite and 100ppm gelatin are added for gelatinization, and after the gelatinization is completed, a 0.22μm sterile microporous filter is used to remove microorganisms. After bottling, a high pressure instantaneous machine is used for sterilization to obtain wolfberry fermented wine.

[0070] Example 2: Evaluation of the prebiotic efficacy of wolfberry fermented wine

[0071] 2.1 Preparation of wolfberry wine and its component groups

[0072] Take 100g of dried and crushed wolfberry powder, add 3000mL of water and reflux extraction at 90°C for 3h, repeat the extraction twice, filter, concentrate the filtrate at 60°C under reduced pressure to 100mL, add an equal volume of ethyl acetate and repeat the extraction 3 times, combine the ethyl acetate layers, concentrate under reduced pressure, add sterile saline to 20mL, which is the wolfberry carotenoid (LBD, content 50.56mg / 100g) component; add 4 times the amount of anhydrous ethanol to the water layer to an alcohol concentration of 80% , stand in a refrigerator at 4℃ for 12h, concentrate the supernatant under reduced pressure, add sterile saline to make up to 20mL, which is the wolfberry polyphenol (LBC, content 9.0mg / mL) component, filter the precipitate and dry at 50℃ to obtain the wolfberry glycoprotein (LBA, yield 8.56%) component; take an appropriate amount of LBA and add deionized water to dissolve, remove protein by Sevage method, and obtain the protein-free wolfberry polysaccharide (LBB, yield 3.68%) component after alcohol precipitation, washing and drying. Among them, the polyphenol content is determined by Folin phenol method, gallic acid is used as the standard, and the detection wavelength is 750nm; the carotenoid content is determined by ultraviolet spectrophotometry, and the detection wavelength is 450nm. Accurately weigh 200mg of each of LBA, LBB, and LBE (commercial wolfberry glycoprotein) and place them in a 20mL volumetric flask, add sterile saline to make up to the scale, and prepare a 10mg / mL sample solution. Accurately measure 2 mL of LBC and LBD each and place them in a 20 mL volumetric flask, add sterile saline to the scale to prepare a sample solution with a crude drug concentration of 0.5 g / mL.

[0073] Measure 300 mL of the wolfberry fermented wine sample prepared in Example 1, concentrate under reduced pressure to 16 mL, and obtain the total wolfberry wine sample LBF (with a glycoprotein content of 30.3 mg / mL and a polyphenol content of 12.6 mg / mL) for later use. Add 4 times the amount of anhydrous ethanol to LBF for alcohol precipitation, let it stand overnight in a refrigerator at 4°C, and filter it by suction. The mother liquor is wolfberry wine polyphenol LBFP-4. Test sample; the precipitate is washed with anhydrous ethanol and acetone in turn, dried at 60°C, and weighed, which is wolfberry wine glycoprotein LBFG-4.

[0074] The molecular weight and distribution of LBFG-4 samples were determined by HPSEC (see Figure 1 ), chromatographic conditions: LC-20AD liquid phase system; TSK G4000 PWXL chromatographic column (7.8mm ID×300mm); mobile phase pure water; flow rate 0.6mL·min-1; column temperature 30℃; RID-10A differential detector; injection volume 20μL. The characteristic peak composition of LBFP-4 sample was determined by HPLC ( Figure 2), which is basically the same as that of wolfberry polyphenols (LBC), the chromatographic conditions are: Agilent 1260 InfinityⅡ HPLC system; CosmosilC18 MS-Ⅱ (4.6mm ID.×250mm, 5μm) chromatographic column; 0.1% acetic acid water as mobile phase A, acetonitrile as mobile phase B, gradient elution (0-5min, 5% B; 5-20min, 5%-20% B; 20-30min, 20% B; flow rate 1.0mL / min; UV detection wavelength 320nm; column temperature 30℃; injection volume 20μL.

[0075] The monosaccharide composition and content of LBFG-4 were determined by PMP derivatization-HPLC, which showed that LBFG-4 contained mannose (0.928%, newly added to the wine sample after fermentation, derived from mannan produced by Saccharomyces cerevisiae), glucose (1.86%, derived from amylo-glucan), galactose (0.354%, derived from glycoprotein) and arabinose (0.127%, derived from glycoprotein). Further determination by visible spectrophotometry showed that LBFG-4 contained total neutral sugars (10.2%), uronic acid (3.5%) and protein (27.1%).

[0076] 2.2 Establishment of the model of antibiotic intervention on intestinal flora in rats and in vitro fermentation

[0077] Twelve SD male rats (weight 220-240g, Zhaoyan Suzhou New Drug Research Center Co., Ltd.) were randomly divided into two groups after 3 days of adaptive feeding in the SPF animal room, 6 rats in each model group and blank group. The model group (IR) (n=6) was given antibiotics by gavage with 50mg / kg imipenem-cilastatin sodium injection (5mg / mL); the blank group (BL) (n=6) was given the same dose of sterile saline. The antibiotic intervention period was 4 days in total, with regular gavage every day. After 4 days, the model group stopped antibiotic intervention. Fresh feces of male SD rats in the model group and the normal group were collected 24 hours after the cessation of antibiotic intervention, and mixed at a ratio of 1g feces plus 10mL sterile saline, vortexed to homogenize, and processed into a 10% (W / V) fecal suspension. After centrifugation at 800r / min for 10min, the fecal impurities were discarded to obtain the supernatant, which was the corresponding intestinal flora liquid. The intestinal flora liquid and the anaerobic culture liquid were mixed evenly at a ratio of 1:9 (V / V), placed in an anaerobic culture bag and cultured in a 37°C constant temperature incubator for 24 hours to obtain the corresponding incubation liquid for standby use.

[0078] The experiment was divided into a blank group, a model group, and an LBA-LBF administration group. The blank group was added with 1 mL of the BL group incubation solution and 1 mL of sterile saline; the model group was added with 1 mL of the IR group incubation solution and 1 mL of sterile saline; the administration group was added with 1 mL of the IR group incubation solution and 1 mL of each sample solution, and each group was operated in parallel for 5 times. The above test tubes were placed in an anaerobic culture bag and cultured in a constant temperature incubator at 37 ° C. One sample tube was taken out at 0 h and 24 h, respectively, and centrifuged at 12000 r / min for 10 min. The bottom sediment was the intestinal bacteria sample, which was stored in a -80 ° C refrigerator. The samples were entrusted to Hangzhou Lianchuan Biotechnology Co., Ltd. for high-throughput sequencing analysis of the 16S rRNA gene of the intestinal flora; 800 μL of the supernatant was taken, 80 μL of 50% sulfuric acid solution and 800 μL of ether were added, vortexed and mixed, centrifuged at 12000 r / min for 5 min, and the upper ether layer was taken for short-chain fatty acid determination.

[0079] 2.3 16S rRNA analysis of the prebiotic effects of wolfberry wine and its components

[0080] The 16S rRNA gene high-throughput analysis obtained the effective classification OTU values ​​of each group, which were 988 (BL group), 800 (IR group), 648 (LBA group), 700 (LBB group), 759 (LBC group), 690 (LBD group), 705 (LBE group), and 915 (LBF group), which can be used for subsequent statistical analysis. The Chao1 value α diversity analysis results showed that the richness of the model group decreased compared with the blank group, the LBF group could improve the richness of the intestinal flora of the model group rats, and the other drug groups had no significant regulatory effect. PCoA principal coordinate analysis showed that the distance between biological replicates in each group was close (R=0.9932) and the distance between groups was far, indicating that the repeatability of each group was good, the difference between groups was large, and the data was reliable. The distance between the model group and the blank group was far, indicating that the intestinal flora of the rats induced by antibiotics had changed significantly, and the model was successfully established. There was no crossover between the drug-treated groups and the model group, indicating that different components had different degrees of regulatory effects on the intestinal flora of the model group. Among them, the LBA (glycoprotein), LBB (polysaccharide) and LBE groups were relatively close, indicating that there were similarities between the three groups and their microbial compositions were similar; the LBC (polyphenol) and LBF (wolfberry wine) groups were relatively close, indicating that the microbial compositions of the two groups were similar.

[0081] The distribution of bacterial species in each group of samples can be classified and statistically analyzed at the level of phylum, family, genus, etc. Table 1 shows the changes in the relative abundance distribution of intestinal microorganisms in each group of samples at the phylum level. There are 19 bacterial phyla that can be annotated at the phylum level, among which Proteobacteria, Firmicutes, Bacteroidetes, Verrucomicrobia, and Actinobacteria have an abundance of more than 90% in each group, which are the most important flora in the intestine. Compared with the BL group, the model group treated with antibiotics significantly changed the abundance of intestinal flora. Compared with the model group, the abundance of Proteobacteria can be downregulated after treatment with Lycium barbarum glycoproteins LBA, LBB, and LBE, and LBB and LBE can reduce the ratio of Firmicutes to Bacteroidetes (F / B); Lycium barbarum polyphenols LBC and LBF groups both downregulated Proteobacteria and upregulated Firmicutes, and LBF increased the abundance of Bacteroidetes and reduced the abundance of Verrucomicrobia. The study found that the increase in Bacteroidetes helps to control weight, and the reduction in the F / B value of the wolfberry glycoprotein group has an inhibitory effect on obesity; Proteobacteria include pathogens such as Escherichia coli and Salmonella, and the wolfberry polyphenol group has a better function of inhibiting the growth of pathogenic bacteria.

[0082] Table 1 Relative abundance of the intestinal flora of wolfberry wine and its components at the main phylum level ( n=5)

[0083] Door Proteobacteria Firmicutes Bacteroidetes Verrucomicrobia BL 65.74±1.59 24.24±1.23 4.76±0.65 3.62±1.19 IR 54.42±1.65 28.46±1.92 11.21±0.96 4.72±1.08 LBA 36.13±1.85 42.84±2.27 11.88±1.04 7.30±3.07 LBB 47.93±4.09 29.83±1.70 12.98±3.20 7.74±2.65 LBC 32.11±2.35 47.16±1.40 8.81±1.34 6.17±2.99 LBD 71.74±6.25 15.87±5.40 6.68±1.05 5.03±3.18 LBE 42.41±4.37 24.30±2.57 16.70±1.11 14.44±5.74 LBF 31.03±0.95 51.74±2.69 13.53±1.35 2.79±1.29

[0084] Further statistical analysis results showed that the relative abundance of intestinal microorganisms at the genus level in each group of samples changed significantly. Figure 3-4Compared with the blank group, the relative abundance of pathogenic bacteria such as Proteus, Pseudomonas, and Klebsiella increased in the model group, while these genera in each medication group showed a significant downward trend, among which Pseudomonas returned to the level of the normal group. Compared with the model group, the LBA, LBB and LBE groups upregulated the relative abundance of Bacteroides, Enterococcus, Enterorhabdus, Akkermansia, Flavonifractor and Parabacteroides, and decreased the abundance of Klebsiella, Proteus, Clostridium, Pseudomonas, Eubacterium, Intestinimonas and Eisenbergiella; LBC and LBF increased the relative abundance of Clostridium, UBA1819 of Ruminococcus family and Eisenbergiella The relative abundance of Escherichia, Shigella, Klebsiella, Enterococcus, Proteus, Clostridium, Pseudomonas and Intestinimonas was downregulated by LBC. In addition, LBC could upregulate Akkermansia, Lactobacillus and Bifidobacterium, and downregulate Parabacteroides, Lachnoclostridium and Eubacterium. LBF had a more significant effect on upregulating Lachnoclostridium and Flavonifractor. LBD could increase the relative abundance of Klebsiella and Akkermansia, and decrease the relative abundance of Escherichia, Shigella, Bacteroides, Bifidobacterium and Lactobacillus. In general, the wolfberry treatment groups have different inhibitory effects on a variety of pathogenic bacteria, and can increase the abundance of beneficial bacteria such as Akkermansia, Bacteroides, Enterococcus, Lactobacillus and Bifidobacterium to varying degrees, suggesting that different nutrient components such as wolfberry glycoprotein, polyphenols, carotenoids, etc. have different degrees of prebiotic effects.

[0085] The above results suggest that wolfberry wine and its various nutrient components have the effect of regulating intestinal flora and have different selectivities.

[0086] 2.4 Determination of short-chain fatty acids

[0087] The composition and content of short-chain fatty acids in the samples were determined by gas chromatography. A mixed standard stock solution of 8.40 mg / mL acetic acid, 4.95 mg / mL propionic acid, 0.47 mg / mL isobutyric acid, 4.80 mg / mL n-butyric acid, 0.46 mg / mL isovaleric acid and 0.47 mg / mL n-valeric acid was prepared and stored at 4°C. Chromatographic conditions: Agilent HP 6890 gas chromatograph; Agilent DB-WAX gas chromatograph column (0.32 mm×60 m, 0.5 μm); the initial column temperature was 105°C and maintained for 0.5 min, then increased to 150°C at 5°C / min and maintained for 24 min. Flow rate 1.6mL / min; hydrogen 40mL / min; air 400mL / min; tail gas (nitrogen) 40mL / min; injection port temperature 220℃; detector is hydrogen flame ionization detector, detector temperature 230℃; injection volume 1μL, split ratio 50:1. Qualitative analysis is performed using peak retention time, and content is determined using peak area.

[0088] The results of the determination of the six common short-chain fatty acids in the intestinal flora are shown in Table 2, among which acetic acid, propionic acid and n-butyric acid are the main components, accounting for more than 90% of the total content. Compared with the blank group, the content of acetic acid, propionic acid, n-butyric acid and n-valeric acid in the model group decreased, the content of isobutyric acid and isovaleric acid increased, and the total content of short-chain fatty acids showed an overall downward trend. Compared with the model group, each drug-treated group can increase the content of each short-chain fatty acid to varying degrees, among which the acetic acid content significantly exceeded the level of the normal group, and the LBF group was the most significant.

[0089] Table 2 Effects of wolfberry wine and its components on the content of short-chain fatty acids in rat feces ( n=3)

[0090]

[0091] Short-chain fatty acids are volatile saturated fatty acids composed of 6 or fewer carbon atoms. They are produced by the fermentation of dietary fiber and other nutrients by anaerobic bacteria in the colon and play a key role in the establishment and maintenance of intestinal homeostasis. Lycium barbarum glycoprotein components LBA, LBB and LBE can significantly increase the content of acetic acid, propionic acid and butyric acid. These Lycium barbarum polysaccharide components can promote the production of acetic acid and the like by increasing the abundance of Lactobacillus and Bifidobacterium. Lycium barbarum polyphenol component LBC can greatly increase the abundance of Lactobacillus, Bifidobacterium and Clostridium, thereby significantly increasing the content of acetic acid and propionic acid. Carotenoid component LBD can also increase the content of various short-chain fatty acids. In particular, LBF showed a significant increase in all short-chain fatty acids. On the one hand, this is because Lycium barbarum wine is rich in Lycium barbarum polyphenols, glycoproteins and other nutrients. On the other hand, it may also be because some nutrients in Lycium barbarum wine can be converted into effective substances that are more likely to promote the reproduction of probiotics after fermentation, thereby significantly increasing the content of short-chain fatty acids. The above research results indicate that different effective ingredients of wolfberry can increase the production of short-chain fatty acids such as acetate, butyrate and propionate by regulating the abundance of related bacterial flora, which has a potential role in preventing the invasion of pro-inflammatory factors, thereby improving the intestinal barrier function, exerting prebiotic effects and improving the host's intestinal health.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of wolfberry fermented wine in regulating intestinal flora or preparing products regulating intestinal flora, characterized in that: The intestinal flora includes any one or more combinations of Akkermansia, Lactobacillus, Bifidobacterium, Escherichia, Shigella, Proteus, Pseudomonas, Klebsiella, Flavonoids and Cyanobacteria.

2. The use according to claim 1, characterized in that: The regulating of the intestinal flora comprises: upregulating the relative abundance of any one or more of the genera Akkermansia, Lactobacillus, Bifidobacterium, Flavonoids and Cyanobacteria.

3. The use according to claim 1, characterized in that: The regulating of the intestinal flora includes: downregulating the relative abundance of any one or more of the genera Escherichia, Shigella, Proteus, Pseudomonas, and Klebsiella.

4. Application of wolfberry fermented wine in increasing the relative abundance of Proteobacteria and / or Bacteroidetes or in preparing products that increase the relative abundance of Proteobacteria and / or Verrucomicrobia.

5. Use of wolfberry fermented wine in reducing the relative abundance of Firmicutes and / or Verrucomicrobia or in preparing products that reduce the relative abundance of Firmicutes and / or Verrucomicrobia.

6. Application of wolfberry fermented wine in reducing the ratio of Firmicutes / Bacteroidetes or in preparing products for reducing the ratio of Firmicutes / Bacteroidetes.

7. Use of wolfberry fermented wine in increasing the content of short-chain fatty acids in the intestine or in preparing products for increasing the content of short-chain fatty acids in the intestine.

8. The use according to claim 7, characterized in that: The short-chain fatty acids include any one or more combinations of acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid and isovaleric acid.

9. The use according to any one of claims 1 to 8, characterized in that: The preparation method of the wolfberry fermented wine comprises: subjecting a mixture of wolfberry raw materials and fermentation bacteria to primary fermentation.

10. The use according to claim 9, characterized in that: The preparation of the wolfberry raw material comprises: enzymolysis of wolfberry pulp; Optionally, the main fermentation conditions include: 15-30°C; Optionally, before the main fermentation, the preparation of the wolfberry raw material further comprises: adjusting the sugar content of the product after enzymatic hydrolysis to 15-25° Brix, and adjusting the pH to 3-4; Optionally, the fermentation bacteria include: Saccharomyces cerevisiae; Optionally, the cerevisiae yeast is cerevisiae yeast M-23-7-14 with a deposit number of CGMCC No. 23603; Optionally, the method for preparing the wolfberry fermented wine further comprises: performing post-fermentation on the product after the main fermentation to obtain the wolfberry fermented wine; Optionally, the post-fermentation conditions include: 15-30° C., 10-30 days.

Images