Bacillus amyloliquefaciens strain yp2 and application thereof

Abstract

The application discloses a bacillus amyloliquefaciens YP2 and application thereof, and relates to the technical field of microbial food fermentation. The bacillus amyloliquefaciens YP2 is preserved in the China General Microbiological Culture Collection Center, 1, Xibei Road, Beichen, Chaoyang District, Beijing, and the preservation time is November 27, 2023, the preservation number is CGMCC No. 29157, and the classification and naming is bacillus amyloliquefaciens. The 16S rDNA sequence is shown as SEQ ID NO. 1. The application adopts the bacillus amyloliquefaciens YP2 and application thereof, and the total phenol content of the obtained fermentation liquor is increased, the fermentation liquor has the functions of reducing blood sugar and antioxidant activity, and can be widely applied to the preparation of blood sugar reducing drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial food fermentation, in particular to a strain of Bacillus amyloliquefaciens YP2 and application thereof. BACKGROUND

[0002] Diabetes is one of the major chronic diseases endangering human health. Current studies have confirmed that long-term postprandial hyperglycemia is one of the important factors inducing type 2 diabetes (Esposito, K.; et al. Regression of carotid atherosclerosis by control of postprandial hyperglycemia in type 2 diabetes mellitus. Circulation, 2004, 110, 214-219. Dong, H.Q.; et al. Inhibitory potential of trilobatin from lithocarpus polystachyus rehd against α-glucosidase and α-amylase linked to type 2 diabetes. Food Chem, 2012, 130, 261-266.).

[0003] For the characteristics of type II diabetes, the therapeutic drugs include insulin sensitizers, insulin secretion promoters and a-glucosidase inhibitors, etc. Among them, a-glucosidase inhibitors can reduce the generation of glucose by inhibiting the activity of carbohydrate hydrolase, thereby avoiding the increase of blood glucose. It has been reported that oral a-GI can delay starch digestion, improve hyperglycemia and diabetic complications (Elbendary, A. A.; et al. Isolation of antimicrobial producing actinobacteria from soil samples. Saudi J. Biol. Sci, 2018, 25, 44-46). For Chinese patients whose dietary structure is mainly based on grains, taking a-glucosidase inhibitors will be effective in treating diabetes in Chinese patients. At present, the a-glucosidase inhibitors on the market mainly include acarbose, miglitol, voglibose, etc. However, patients taking these drugs will have adverse reactions such as bloating and vomiting. In recent years, more and more scholars have paid attention to the ability of natural ingredients in blood glucose regulation, such as alkaloids, polysaccharides, polyphenols, flavonoids, polypeptides, etc. The sources of these natural ingredients are very rich, including plants, animals, chemical synthesis and microbial fermentation. Compared with the other three sources, microorganisms are more suitable for large-scale production of a-glucosidase inhibitors due to their short growth cycle, easy cultivation and low cost. Therefore, it is particularly important to further explore strains with a-glucosidase inhibitory capacity and more efficient a-glucosidase inhibitors.

[0004] Bacillus amyloliquefaciens belongs to the genus Bacillus and the family Bacillaceae, and can produce various active substances such as lipopeptides, cyclic dipeptides, exopolysaccharides, vitamins, purine nucleosides and poly-gamma-glutamic acid. The primary and secondary metabolites of Bacillus amyloliquefaciens can be used as a new type of substance in food and medicine. These products have multiple biological activities such as blood glucose lowering, antiviral, antitumor, anticancer, immunomodulation and antioxidant. Therefore, Bacillus amyloliquefaciens as a probiotic has more value in food fermentation processing, and it is of great significance to explore microbial strains that can produce blood glucose lowering functional substances. However, the industrialization processing of Bacillus amyloliquefaciens is mostly limited to the production of soy products such as tempeh, fermented bean curd and miso, and there is less research on the processing of Bacillus amyloliquefaciens fermented fruit and vegetable juice, so further exploration is needed.

[0005] Carrots are a kind of fruits and vegetables with rich nutritional value, and have excellent processing characteristics and various physiological regulation functions. Studies have found that carrots have the effects of invigorating the spleen and stomach, nourishing the liver and eyesight, reducing phlegm and relieving cough, clearing heat and detoxifying, and enhancing immunity. Therefore, the processing of carrots is very promising. SUMMARY

[0006] The application aims to provide a bacillus amyloliquefaciens YP2 and application thereof, a fermentation liquor obtained by the bacillus amyloliquefaciens YP2 has increased total phenol content, has hypoglycemic and antioxidant activities, and can be widely applied to preparation of hypoglycemic drugs.

[0007] To achieve the above-mentioned purpose, the application provides a bacillus amyloliquefaciens YP2, which is preserved in the China General Microbiological Culture Collection Center, 1, Beichen West Road, Chaoyang District, Beijing, and has a preservation time of November 27, 2023, a preservation number of CGMCC No. 29157, and a classification name of bacillus amyloliquefaciens.

[0008] Further, the 16S rDNA sequence of the bacillus amyloliquefaciens YP2 is shown in SEQ ID NO. 1.

[0009] The application also provides a microbial fermentation liquor with alpha-glucosidase inhibition capacity, which is obtained by fermentation of the bacillus amyloliquefaciens YP2.

[0010] Further, the optimal fermentation conditions are that the addition amount of lactose is 25 g / L, the initial pH is 5, the inoculation amount is 2%, and the culture temperature is 34 DEG C.

[0011] Further, the effective components of the microbial fermentation liquor include 1-deoxynojirimycin, cyclic dipeptide and polypeptide, and exopolysaccharide with alpha-glucosidase inhibition capacity.

[0012] Further, the alpha-glucosidase inhibition capacity of the microbial fermentation liquor is greater than 90%.

[0013] The application also provides application of the bacillus amyloliquefaciens YP2 in preparation of hypoglycemic drugs.

[0014] The application also provides application of the bacillus amyloliquefaciens YP2 in food fermentation.

[0015] The bacillus amyloliquefaciens YP2 and application thereof have the following advantages and positive effects:

[0016] 1. The bacillus amyloliquefaciens YP2 in the application is screened from fermented douchi, is suitable for production of hypoglycemic active substances, has alpha-glucosidase inhibition capacity, antioxidant activity and other excellent properties.

[0017] 2、The Bacillus amyloliquefaciens YP2 in the application has the largest proportion of genes in amino acid and carbohydrate metabolism in the genome, and the largest proportion (65.35%) of glycoside hydrolases and glycosyltransferases in the CAZy annotation results. The glycoside hydrolases can hydrolyze various sugar-containing compounds, and then participate in the synthesis of monosaccharides, oligosaccharides or sugar complexes. The glycosyltransferases can catalyze the connection of activated sugars and proteins, peptides, oligosaccharides and the like, thereby generating or enhancing the biological activity of the products. Therefore, the YP2 has good glycoside hydrolysis and transfer capacity, and thus has the potential to regulate blood glucose.

[0018] 3、The Bacillus amyloliquefaciens YP2 in the application has a gene cluster for synthesizing antibacterial peptides, so that the fermented carrot juice prepared by the Bacillus amyloliquefaciens YP2 has the potential of natural antibiosis, and thus the shelf life of the fermented carrot juice can be prolonged.

[0019] 4、The Bacillus amyloliquefaciens YP2 in the application can produce 1-deoxynojirimycin, and also has other alpha-glucosidase inhibitors, and can produce a variety of alpha-glucosidase inhibiting peptides by fermentation with bean dregs.

[0020] 5、The DNJ with a purity of >95% can be obtained by combining different types of resins to separate and purify the DNJ, which provides technical support for the standardized and industrialized production of high-purity DNJ.

[0021] 6、The pure Bacillus amyloliquefaciens is used for fermenting carrot juice in the application, so that no pathogenic bacteria such as mixed bacteria are contained, the production speed is improved, and the edible safety of the carrot juice is ensured; the carrot juice not only has blood glucose-lowering activity, but also has strong antioxidant activity; and the carrot juice is rich in total phenol content.

[0022] The technical solutions of the application will be further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The alpha-glucosidase inhibiting capacity of 9 strains of Bacillus from douchi (YP2, 3-3, 4-1, 4-3, 6-2, 7-1, 8-1, 10-1, 10-2) after 96h fermentation and the alpha-glucosidase inhibiting capacity of strain YP2 at different fermentation times are shown in the table, wherein A is the alpha-glucosidase inhibiting capacity of 9 strains of Bacillus from douchi after 96h fermentation, and B is the alpha-glucosidase inhibiting capacity of strain YP2 at different fermentation times.

[0024] Figure 2The colony morphology, growth curve, and pH and alpha-glucosidase inhibition ability changes during the growth process of Bacillus amyloliquefaciens YP2 in the embodiments of the present application, wherein A is the colony morphology of Bacillus amyloliquefaciens YP2, B is the growth curve of Bacillus amyloliquefaciens YP2, C is the pH change during the growth process of Bacillus amyloliquefaciens YP2, and D is the alpha-glucosidase inhibition ability change during the growth process of Bacillus amyloliquefaciens YP2;

[0025] Figure 3 The phylogenetic tree of Bacillus amyloliquefaciens YP2 based on 16S rRNA gene sequences in the embodiments of the present application;

[0026] Figure 4 The selection of components (carbon source and nitrogen source) of the fermentation medium based on DNJ synthesis of Bacillus amyloliquefaciens YP2 in the embodiments of the present application, wherein A is the selection of carbon source type, B is the selection of nitrogen source type, and C is the DNJ yield under different carbon sources and nitrogen sources;

[0027] Figure 5 The growth curve of Bacillus amyloliquefaciens YP2 and the pH, alpha-glucosidase inhibition ability, and DNJ yield changes during the growth process of Bacillus amyloliquefaciens YP2 in the lactose fermentation medium in the embodiments of the present application, wherein A is the pH change, B is the growth curve, C is the alpha-glucosidase inhibition ability, and D is the DNJ yield change;

[0028] Figure 6 The single-factor (initial pH, lactose addition amount, inoculation amount, and culture temperature) optimization results of the DNJ production conditions of Bacillus amyloliquefaciens YP2 in the lactose fermentation medium in the embodiments of the present application, wherein A is the initial pH, B is the lactose addition amount, C is the inoculation amount, and D is the culture temperature;

[0029] Figure 7 The extracellular polysaccharide and polypeptide results identified using the sucrose medium and the bean dregs medium in the embodiments of the present application, wherein A is the crude polysaccharide content, B is the alpha-glucosidase inhibition rate, C is the absorbance, and D is the polypeptide content of the crude extract;

[0030] Figure 8DNJ process in Bacillus amyloliquefaciens YP2 using cation exchange resin, macroporous resin, anion exchange resin, silica gel column chromatography, Sephadex LH-20 gel column chromatography to separate and purify lactose fermentation medium, wherein A is the pH and alpha-glucosidase inhibitory activity of the effluent after LXP-01 resin purification, B is the alpha-glucosidase inhibitory rate of the effluent after HP-20 resin purification, C is the alpha-glucosidase inhibitory rate of the effluent after D941 (weak base type) anion exchange resin purification, D is the alpha-glucosidase inhibitory rate of the effluent after silica gel column chromatography purification, E is the alpha-glucosidase inhibitory rate of the effluent after Sephadex LH-20 purification, and F is the liquid phase detection of DNJ purity;

[0031] Figure 9 Polypeptide process in Bacillus amyloliquefaciens YP2 using cation exchange resin, macroporous resin, and Sephadex LH-20 gel column chromatography to separate and purify bean dreg fermentation medium, wherein A is the pH and alpha-glucosidase inhibitory activity of the effluent after LXP-01 resin purification, B is the alpha-glucosidase inhibitory rate of the effluent after HP-20 resin purification, C is the alpha-glucosidase inhibitory rate of the effluent after Sephadex LH-20 purification, and D is the alpha-glucosidase inhibitory rate of the polypeptide under ethanol elution gradient;

[0032] Figure 10 Alpha-glucosidase inhibitory capacity of fermented fruit and vegetable juice prepared using different fruits and vegetables. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0034] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0035] The microbial fermentation broth with alpha-glucosidase inhibitory capacity is obtained by fermentation of Bacillus amyloliquefaciens YP2. The initial fermentation medium is LB liquid medium, and the fermentation conditions are temperature 37℃, rotation speed 180r / min, and culture time 96h.

[0036] The fermentation conditions for DNJ production are as follows: the fermentation medium is LB liquid medium containing 2.5% lactose, the fermentation conditions are temperature 34℃, rotation speed 180r / min-200r / min, and culture time 84h, and under these conditions, the DNJ content is 93μg / mL.

[0037] The fermentation conditions for producing exopolysaccharide are as follows: the fermentation medium is LB liquid medium containing 2.5% sucrose (pH 7.0), the fermentation conditions are: temperature 37℃, rotation speed 180r / min-200r / min, culture time 48h, and under the above conditions, the content of exopolysaccharide is 2.5mg / mL.

[0038] The fermentation conditions for producing cyclic dipeptide and polypeptide are as follows: the fermentation medium is LB liquid medium containing 2.5% lactose (pH 5.0), the fermentation conditions are: temperature 34℃, rotation speed 180r / min-200r / min, culture time 84h, and under the above conditions, the content of DNJ is 93μg / mL.

[0039] The separation and purification conditions of 1-deoxynojirimycin, cyclic dipeptide and polypeptide are as follows: after the fermentation broth is treated by ethanol precipitation, centrifugation is performed to obtain the supernatant, rotary evaporation is performed, the supernatant is loaded onto a cation exchange resin, ammonia is used for elution, the fraction eluted by 0.5M ammonia is collected, rotary evaporation is performed to remove ammonia, then the sample is loaded onto a macroporous resin, ethanol is used for elution to remove fat-soluble pigments and odor, rotary evaporation is performed to remove ethanol, after the pigments are removed by loading the sample onto an anion exchange resin, silica gel column chromatography is performed, different concentrations of chloroform-methanol-water (3:3:1) eluted fractions are collected, and then Sephadex LH-20 is used for elution. The obtained 1-deoxynojirimycin has a purity of >95%, and the cyclic dipeptide and polypeptide have a purity of >90%.

[0040] Deposit information

[0041] Bacillus amyloliquefaciens YP2 is deposited in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Beijing City, Chaoyang District, on November 27, 2023, with the deposit number CGMCC No. 29157, and is classified and named as Bacillus amyloliquefaciens. The 16S rDNA sequence of Bacillus amyloliquefaciens is shown in SEQ ID NO. 1.

[0042] Example 1: Isolation and screening of Bacillus amyloliquefaciens with hypoglycemic function

[0043] 1. Strain culture: 87 strains of bacteria were isolated from 56 samples of fermented soybeans collected nationwide (including Wanianhong fermented soybeans, Liyang fermented soybeans, delicious fermented soybeans, Anjun Yongchuan fermented soybeans, Yangjiang Qiaoyangjiang fermented soybeans, Guizhou self-made fermented soybeans, Jiangxi homemade fermented soybeans, Sanshi Yangjiang fermented soybeans, Taipingqiao fermented soybeans, Shandong Linqin Babao fermented soybeans, Zhenji fermented soy paste, Wuhan self-made fermented soybean dregs, Yunyang self-made fermented soybean dregs, Chongqing self-made fermented soybean dregs, fermented soybean dregs pills, fermented soybean dregs, etc.), and the supernatant was obtained by centrifugation after 96h culture in LB basic medium.

[0044] 2. Determination of α-glucosidase inhibition ability: Take 20 μL of fermentation broth supernatant, 20 μL of α-glucosidase and 60 μL of PBS (pH 8.0), mix them, incubate them in a 37°C incubator for 20 minutes, add 20 μL of PNPG substrate, continue incubating in a 37°C incubator for 60 minutes, add Na2CO3 solution to terminate the reaction, and measure the absorbance at 410 nm to calculate the enzyme activity inhibition rate of the fermentation broth supernatant on α-glucosidase. The results are as follows Figure 1 As shown, 9 strains with α-glucosidase inhibitory activity were obtained, with inhibition rates greater than 90%, among which Bacillus amyloliquefaciens YP2 performed better.

[0045] Example 2 Identification and Genomic Characterization Analysis of Bacillus amyloliquefaciens

[0046] 1. Strain characteristics: Inoculate Bacillus amyloliquefaciens YP2 into 5 mL of LB liquid medium and culture at 37°C, 180 rpm shaking for 24 hours. Collect the Bacillus amyloliquefaciens when the bacterial solution becomes noticeably turbid. Dilute the collected Bacillus amyloliquefaciens by streaking onto LB solid medium and culture in an inverted incubator at 37°C for 12 hours. Figure 2 A single colony was selected and re-inoculated into LB liquid medium and cultured at 37°C with a shaker at 180 rpm for 120 h. Samples were taken at 2 h, 4 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h. Changes in bacterial growth, pH, and enzyme inhibition ability of YP2 were measured over time. Figure 2 As shown in Figure B, YP2 grows fast, and its pH is neutral in the early growth stage (within 6 hours). As the growth time increases, the pH becomes alkaline; the pH remains basically unchanged after 48 hours ( Figure 2 YP2 did not inhibit α-glucosidase during the initial growth phase. However, after 24 h of fermentation, the fermentation broth was able to inhibit α-glucosidase activity. The inhibition rate gradually increased over time, which was contrary to the growth curve but consistent with the pH trend. This indicates that the substance in the fermentation broth that can inhibit α-glucosidase activity is a secondary metabolite of Bacillus amyloliquefaciens, and that this product is produced under alkaline conditions.

[0047] 2. Physiological and biochemical characteristics: The physiological and biochemical characteristics of YP2 were determined according to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems. The test results are shown in Table 1.

[0048] Table 1 Physical and chemical test results of strain YP2

[0049]

[0050] 3. Genetic characteristics: The DNA sequence of the strain YP2 of the present application was sequenced by Meiji Bio. The genomic sequence data of Bacillus amyloliquefaciens YP2 of the present application showed that the whole genome sequence was 4157426 bp, and the G+C content in the DNA was 45.96%. The genetic sequence of the strain YP2 of the present application was compared by gene (Genebank) sequence, and a phylogenetic tree was established by MEGA7.0, and the results are shown in Figure 3 Fig. 1. Through 16S rDNA sequence phylogenetic analysis, ANI and dDH value analysis, the strain YP2 was identified as Bacillus amyloliquefaciens.

[0051] 4. Gene annotation: The coding genes of Bacillus amyloliquefaciens YP2 of the present application were functionally annotated by COG (http: / / eggnog.embl.de / ) and KEGG (http: / / www.genome.jp / kegg / ) databases, and the carbohydrate active enzymes thereof were analyzed by CAZy database (http: / / www.cazy.org / ). The results of COG database annotation showed that amino acid transport metabolism (302 genes), transcription (287 genes) and carbohydrate transport metabolism (269 genes) were dominant in Bacillus amyloliquefaciens YP2. The results of KEGG database annotation showed that the most abundant in Bacillus amyloliquefaciens YP2 were carbohydrate metabolism (242 genes) and amino acid metabolism (208 genes), which were similar to the results of COG annotation. The results of CAZy annotation showed that glycoside hydrolases accounted for the largest proportion (33.07%) in carbohydrate active enzymes, followed by glycosyltransferases (32.28%). The above results showed that the genes involved in the carbohydrate and amino acid of Bacillus amyloliquefaciens YP2 accounted for a large proportion, and it was speculated that Bacillus amyloliquefaciens YP2 had good glycoside hydrolysis and transfer ability.

[0052] Example 3 Fermentation condition optimization of Bacillus amyloliquefaciens YP2 for DNJ production

[0053] 1. Optimization of medium components: LB was used as the basic medium (control), and 2.5% of carbon source components and nitrogen source components (pH 7.0) were added, respectively. The carbon source components were glucose, inulin, lactose, sorbitol, galactose, xylose, maltose, soluble starch, etc.; the nitrogen source components were acid hydrolysis casein, soybean meal, soybean protein, etc. After sterilization, 5% of activated bacteria liquid was inoculated, and after 96h of culture (37℃, 180rpm), the fermentation liquid was centrifuged to measure its inhibitory ability on a-glucosidase. The results are shown in Figure 4As shown in Figures A and B, after culturing with lactose as the carbon source or yeast extract or soy peptone as the nitrogen source, the fermentation broth has a very strong inhibitory ability against α-glucosidase (inhibition rate >70% after 16-fold dilution). Furthermore, fermentation broths with strong α-glucosidase inhibition ability were selected, and the presence of different levels of the hypoglycemic component 1-deoxynojirimycin (DNJ) in the fermentation broth was detected by high performance liquid chromatography, as shown in Figure 4. Figure 4 As shown in C. Obviously, lactose has a better DNJ yield. Therefore, the culture medium supplemented with lactose can promote the production of α-glucosidase inhibitors.

[0054] In order to understand the effects of these carbon sources on the growth of bacteria in more detail, we analyzed the effects of different lactose addition amounts (5, 15, 25, 35, 45 g / L) on the growth of YP2. Figure 5 As shown. Through the pH change curve ( Figure 5 A) It is not difficult to see that in the early stage of strain growth, the pH may drop due to the consumption of a large amount of carbon source, which produces a large amount of organic acid. As the fermentation progresses, the pH of the medium with less lactose added (5g / L) will rise first and reach alkaline. This may be because the ammonia substances produced during the metabolism of the strain continuously neutralize the organic acid and gradually become dominant. The medium with more lactose added can maintain the growth period for a longer time due to the continuous energy supply of lactose, and basically maintains the pH at around 7. By observing the growth of the bacteria ( Figure 5 In Figure B), YP2 reached its first growth peak at 24h in the culture media with different lactose addition amounts. After that, the cell density of the 5g / L lactose culture medium showed a slow decline, while the cell density of other culture media showed a fluctuation of decreasing cell density and then rose rapidly. This may be due to the complex metabolic process of the strain caused by lactose addition. In addition, higher concentrations of lactose addition (15g / L~45g / L) can maintain strain growth for a longer time, which is obviously more conducive to the production of α-glucosidase inhibitors. By measuring the α-glucosidase inhibitory ability ( Figure 5 C), a small amount of α-glucosidase inhibitors were produced at the 24th hour of fermentation. The changes in α-glucosidase inhibitory activity were observed with different lactose addition amounts. The addition of 35g / L lactose was beneficial to the production of α-glucosidase inhibitory substances, which was more conducive to DNJ synthesis ( Figure 5 D), and the DNJ production tended to be stable at the 96th hour, and the lactose addition amount of 35g / L was selected.

[0055] 2. Optimization of culture conditions: Single factor experiment combined with orthogonal test was used to analyze the effects of different initial pH (4, 5, 6, 7, 8, 9, 10); different lactose addition (5, 15, 25, 35, 45 g / L), different inoculation amount (1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%), and different temperature (31, 34, 37, 40 ° C) on DNJ production. Figure 6 and Table 2), the optimal fermentation conditions were finally determined to be: lactose addition of 25 g / L, initial pH of 5, inoculation of 2%, and culture temperature of 34°C. Under these conditions, the DNJ yield was approximately 93 μg / mL.

[0056] Table 2 Orthogonal experimental design results

[0057]

[0058]

[0059] Note: A is the amount of lactose added (g / L), B is the temperature (°C), C is the initial pH, D is the inoculation amount (%), and E is the error term.

[0060] Example 4 Analysis of exopolysaccharide production by Bacillus amyloliquefaciens YP2 fermentation

[0061] 1. Fermentation conditions: The fermentation medium was LB liquid medium (pH 7.0) containing 2.5% sucrose; the fermentation conditions were: temperature 37°C, rotation speed 180 rpm to 200 rpm, and incubation time 48 h.

[0062] 2. Fermentation broth treatment: The fermentation broth was centrifuged (8000 rpm, 5 min) to remove the bacteria, and triple ethanol was added to the supernatant. After alcohol precipitation for 12 h, the supernatant was centrifuged (8000 rpm, 5 min), and the precipitate was dissolved in deionized water.

[0063] 3. Determination of extracellular polysaccharide content: According to the phenol-sulfuric acid method, under the above fermentation conditions, the extracellular polysaccharide content of the crude extract was 2.5 g / L ( Figure 7 Middle A).

[0064] 4. α-glucosidase inhibition ability determination: After the crude polysaccharide was diluted eight times, it still had an α-glucosidase inhibition rate of more than 90% ( Figure 7 Middle B).

[0065] Example 5 Analysis of polypeptide production by fermentation of Bacillus amyloliquefaciens YP2

[0066] 1. Fermentation conditions: The fermentation medium is 4% soybean dregs medium; the fermentation conditions are: culture in a fermenter for 96 hours.

[0067] 2. Fermentation broth treatment: centrifugalize the broth (8000rpm, 5min) to remove the bacteria, then add equal volume of ethanol to the supernatant, and centrifugalize (8000rpm, 5min) after 12h of alcohol precipitation, and concentrate the crude extract by rotary evaporation.

[0068] 3. Polypeptide content determination: by UV full wavelength scanning, it is found that there is maximum UV absorption at 202nm, and weak absorption at 203-220nm. Figure 7 The polypeptide content of the crude extract under the above fermentation conditions accounts for 11% of the total composition (Medium C) according to the Coomassie Brilliant Blue method. Figure 7

[0069] 4. α-Glucosidase inhibition ability determination: the crude extract has an α-glucosidase inhibition rate of about 75% (Medium B). Figure 7

[0070] Example 6 Isolation and purification of Bacillus amyloliquefaciens α-glucosidase inhibitor DNJ

[0071] 1. Pretreatment: centrifugalize the lactose fermentation broth (8000rpm, 5min) to remove the bacteria, then add equal volume of ethanol to the supernatant, and centrifugalize (8000rpm, 5min) after 12h of alcohol precipitation to remove potential polysaccharides and proteins, and then concentrate the active substance crude extract by rotary evaporation.

[0072] 2. Strong acid cation exchange resin purification: ① Select LXP-01 resin for purification, first pretreat the column material to activate the column material, and finally use 4% hydrochloric acid to flush the column with 3 column volumes, and then use distilled water to rinse the column material layer until the effluent pH≥4, at which time the column material type is hydrogen type, ready for use.

[0073] ②Prepare the concentrated liquid sample for loading, after loading, first use distilled water to rinse 3 column volumes to remove uncharged and only anionic group-containing substances, and then use 0.5M ammonia water for elution, and detect the effluent pH and α-glucosidase inhibition activity during elution, such as Figure 8 Medium A, the main active substance in the effluent starts to be eluted before and after the pH becomes alkaline, collect all the active substances, then remove ammonia under reduced pressure, and freeze-dry for the next step of purification.

[0074] 3. Macroporous adsorption resin and anion exchange resin purification: ① Select HP-20 resin to remove weakly polar substances and some fat-soluble pigments. After pretreatment of the HP-20 macroporous resin, the sample concentrate is loaded and rinsed with water, and fat-soluble pigments and other substances are adsorbed by the macroporous resin, and the effluent is the active substance (Medium B), which is collected and concentrated by rotary evaporation for use. Figure 8 ​​​

[0075] 2. Further purification by D941 (weak base type) anion exchange resin to remove pigments and anion group containing substances, and to further enrich the active substances. After loading, directly water washing was performed, and the elution profile is shown in Fig. 1A, and the sample with α-glucosidase inhibitory activity ≥ 20% was collected, rotary evaporated and lyophilized for storage. Figure 8

[0076] 4. Purification by silica gel column chromatography: silica gel column chromatography was selected to separate the active substances with different polarities. The sample lyophilized powder was dissolved in methanol and mixed with silica gel at a ratio of 1:1.5, and then stirred thoroughly and rotary evaporated for storage. The silica gel was packed by wet method, and after the liquid level was stable, the silica gel powder containing the sample was added. After loading, purification was performed by gradually increasing the polarity of the eluent, and eluted with chloroform (100%), chloroform-methanol-water (20:6:0.5), chloroform-methanol-water (20:8:0.5), chloroform-methanol-water (20:10:1), chloroform-methanol-water (3:3:1), and finally methanol (100%) elution. Each gradient used 3 column volumes for elution. After elution, the α-glucosidase inhibitory activity of each tube was determined, and the active components were collected by TLC double verification, and the elution profile is shown in Fig. 1D (since pure chloroform itself contains 50%-60% unstable enzyme inhibitory activity, therefore there is a higher inhibitory activity when eluted with chloroform). Subsequently, we determined the DNJ content of different combined components according to the method of national standard GB / T40642-2021, and found that only the chloroform-methanol-water (3:3:1) elution component contained a large amount of DNJ. Figure 8

[0077] 5. Purification by Sephadex LH-20: Sephadex LH-20 was selected for further refinement of the sample. The lyophilized powder of the DNJ component was dissolved in a small amount of water, loaded, and the active components were collected, and the elution profile is shown in Fig. 1E. The DNJ was combined and lyophilized, and the purity was detected by liquid phase to be >95% (Fig. 1F). Figure 8 Figure 8

[0078] Example 7 Isolation and purification of Bacillus amyloliquefaciens α-glucosidase inhibitor polypeptide

[0079] 1. Pretreatment: The bean dregs fermentation broth was centrifuged (8000 rpm, 5 min) to remove the bacteria, and an equal amount of ethanol was added. After alcohol precipitation for 12 h, centrifugation (8000 rpm, 5 min) was performed to remove potential polysaccharides and proteins, and then rotary evaporation was performed to obtain the crude extract of active substances.

[0080] ​​​​2. Purification of strong acid type cation exchange resin: ① Select LXP-01 resin for purification, first pretreat the column material to activate the column material, and finally use 4% hydrochloric acid 3 times the column volume to flush the column, and use distilled water to rinse the column material layer until the effluent pH≥4, at this time the column material type is hydrogen type, ready for use.

[0081] ② Prepare the concentrated liquid sample for loading, after loading, first use distilled water to rinse 3 times the column volume to remove uncharged and only anionic group containing substances, after water washing, use 0.5M ammonia water for elution, detect the effluent pH and α-glucosidase inhibitory activity during elution, such as Figure 9 middle A, the main active substance in the effluent starts to be eluted before and after the pH becomes alkaline, collect all the active substances, then remove ammonia under reduced pressure, and freeze-dry for the next step of purification.

[0082] 3. Macroporous adsorption resin purification: select HP-20 resin to remove weakly polar substances and some fat-soluble pigments. After pretreatment of HP-20 macroporous resin, the sample concentrate is loaded and rinsed with water, and fat-soluble pigments and other substances are adsorbed by macroporous resin, and gradient elution is performed with different concentrations of ethanol, and the fraction with ethanol elution concentration>90% is collected Figure 9 middle B), collect and concentrate using a rotary evaporator for use.

[0083] 4. Sephadex LH-20 purification: select Sephadex LH-20 for further elution of the sample, the ethanol elution gradient is 50%, 70%, 90%, and 100%, and the 70% ethanol elution fraction Figure 9 middle C and D) is collected, and it is detected that 0.25mg / mL of polypeptide has an α-glucosidase inhibition rate of>90%.

[0084] Example 8 Application of Bacillus amyloliquefaciens YP2 in fermented fruit and vegetable juice

[0085] (1) Pretreatment: ① Pretreatment of carrots, corns and apples: select fresh fruits and vegetables, wash and peel the carrots, cut them into pieces, and weigh them, then add deionized water according to the material to liquid ratio of 1:2;

[0086] ② Pretreatment of black beans and soybeans: grind the black beans or soybeans in a cell disruptor until the powder is uniform and fine;

[0087] (2) Preparation of fruit and vegetable juice: ① Carrot, corn and apple juice: use a blender to mix evenly, filter the carrot, corn and apple homogenate with two to six layers of gauze or silk cloth, and prepare carrot juice, which is divided into 50mL bottles, and the conical flask has a capacity of 250mL;

[0088] (2) Fruit and vegetable juice: 50 mL of carrot juice, 50 mL of black bean juice, 50 mL of corn juice, 50 mL of tomato juice, 50 mL of apple juice, 50 mL of soybean juice, and 50 mL of watermelon juice were weighed and added with distilled water to prepare a medium with a ratio of 1% to 5% of the medium, and the conical flask had a capacity of 250 mL.

[0089] (3) Sterilization: the carrot, corn, apple juice, and tomato juice prepared in (2) were sterilized at 100°C for 10 min for standby;

[0090] (4) Fermentation:

[0091] A: Bacillus amyloliquefaciens was inoculated in LB solid medium for recovery for 12 h, and then a single colony was picked and inoculated in 50 mL of LB liquid medium for activation for 24 h;

[0092] B: Bacillus amyloliquefaciens YP2 in A was inoculated in the sterile fruit and vegetable juice obtained in (4) at an inoculation amount of 5%, and then cultured in a shaking bed for 24 h, and the culture conditions were 37°C and a shaking bed rotation speed of 180 rpm.

[0093] (5) Centrifugation: the fruit and vegetable juice fermented in (4) was centrifuged (4°C, 10,000 xg, 10 min), filtered with silk cloth, and the supernatant was stored in a refrigerator at 0°C to 4°C for standby.

[0094] (6) Analysis of α-glucosidase inhibiting capacity: the blood sugar reducing potential of the fermented carrot juice, black bean juice, and soybean juice was analyzed, and the α-glucosidase inhibiting rate was used as an index, and the results are shown in Table 2. The α-glucosidase inhibiting capacity of the carrot juice fermented by Bacillus amyloliquefaciens YP2 was as high as 94.72%, followed by the α-glucosidase inhibiting capacity of the black bean juice and the soybean juice, which were 54.05% and 46.91%, respectively. The tomato, corn, and apple did not have the α-glucosidase inhibiting capacity. Therefore, the fermented carrot juice has more application value. Figure 10

[0095] Further analysis of the total phenol content and the total sugar content found that, compared with the unfermented carrot juice (except for no inoculation, the same treatment was performed on the fermented sample), the total phenol content of the fermented carrot juice was increased by 4.04 times, and the total sugar content was 8.29 mg / mL (Table 3).

[0096] Further analysis of the total phenol content and the total sugar content found that, compared with the unfermented carrot juice (except for no inoculation, the same treatment was performed on the fermented sample), the total phenol content of the fermented carrot juice was increased by 4.04 times, and the total sugar content was 8.29 mg / mL (Table 3).

[0097] Table 3 Activity analysis of the fermented carrot juice

[0098] Unfermented sample Sample after fermentation DPPH inhibition rate (%) 70.33 95.04 Total phenol (μg / mL) 0.68 2.73 Total sugar (mg / mL) 9.10 8.29

[0099] Example 9 Mouse experiment​

[0100] 1. Experimental animals: adult animals were selected, female rats (180 ± 20 g) were selected.

[0101] 2. Reagents: tetraoxypyrimidine (C4H2N2O4·H2O, molecular weight 160.08) or streptozotocin, dexamethasone sodium phosphate injection, glucose or medical starch, blood glucose test paper or kit, insulin, triglyceride, total cholesterol determination kit

[0102] 3. High heat energy feed: lard 10%, sucrose 15%, egg yolk powder 15%, casein 5%, cholesterol 1.2%, sodium cholate 0.2%, calcium bicarbonate 0.6%, stone powder 0.4%, mouse maintenance feed 52.6%

[0103] 4. Instruments: blood glucose meter, full-automatic biochemical analyzer, visible spectrophotometer, enzyme marker, balance.

[0104] 5. Dose grouping and administration time of test sample: three dose groups (A, B, C), one model control group (M) and one blank control group (CK) were set up, A group was given fermented liquid at a dose of 10 mL, B group was given fermented liquid at a dose of 15 mL, C group was given fermented liquid at a dose of 20 mL, M group and CK group were given normal saline at a dose of 10 mL, and the gavage time was 30 days.

[0105] A, B, C, M groups were constructed according to the following modeling method: 25 animals were randomly selected after 3-5 days of adaptation, and the fasting blood glucose was measured as the baseline blood glucose value. Then the animals were fasted for 24 hours (free water), and tetraoxypyrimidine (freshly prepared before use) was injected for modeling, and the rats were injected with 50-80 mg / kg BW.iv or 120-160 mg / kg BW.ip. 5-7 days later, the animals were fasted for 3-5 hours, and the blood glucose was measured. The blood glucose value of 10-25 mmol / L was the successful animal of high blood glucose model.

[0106] High blood glucose model animals were randomly divided into 4 groups according to the blood glucose level of fasting 3-5 hours, A group, B group, C group, M group, CK group was blank control group, no treatment, A group, B group, C group were given different doses of fermented liquid, M group and CK group were given normal saline, for 30 days. The grouping and dose are shown in Table 4:

[0107] Table 4

[0108]

[0109] After 30 days, the fasting blood glucose value (fasting as before the experiment) was measured, and the blood glucose value and blood glucose reduction percentage of each group of animals were compared.

[0110] Blood glucose reduction rate % = (blood glucose value before experiment - blood glucose value after experiment) / blood glucose value before experiment * 100 %.

[0111] Results are shown in Table 5,

[0112] Table 5

[0113] Group Blood glucose value before experiment (mmol / L) Blood glucose value after experiment (mmol / L) Blood glucose reduction rate (%) Group A 17.5 10.9 37.7％ Group B 18.9 8.7 54.0％ Group C 19.2 7.8 59.4％ Group M 17.1 16.8 -0.6％ CK group 5.7 5.5 3.5％

[0114] As shown in Table 5, compared with the M group, the blood glucose values of the rats in the A group, the B group and the C group are all decreased, and the blood glucose value of the rats in the C group is decreased more obviously. Therefore, the fermentation liquor of Bacillus amyloliquefaciens in the present application is helpful to maintain the healthy level of blood glucose.

[0115] Therefore, the Bacillus amyloliquefaciens YP2 and the application thereof are adopted in the present application, the total phenol content of the obtained fermentation liquor is increased, the fermentation liquor has the activities of reducing blood glucose and resisting oxidation, and can be widely applied to the preparation of blood glucose-lowering drugs.

[0116] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application but not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that: the technical solutions of the present application can still be modified or replaced by the equivalent, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. Use of a Bacillus amyloliquefaciens strain YP2 in the preparation of a hypoglycemic drug, characterized in that: The microbial fermentation liquid obtained by fermenting Bacillus amyloliquefaciens YP2 has the ability to inhibit α-glucosidase, and the effective components of the microbial fermentation liquid include 1-deoxynojirimycin, cyclic dipeptide, polypeptide and exopolysaccharide having the ability to inhibit α-glucosidase; Bacillus amyloliquefaciens YP2 was deposited in the General Microbiology Center of China Culture Collection Administration Committee, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on November 27, 2023, with the deposit number CGMCC No. 29157, and was classified as Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ); The 16S rDNA sequence of Bacillus amyloliquefaciens YP2 is shown in SEQ ID NO.

1.

2. A microbial fermentation broth having α-glucosidase inhibitory ability, characterized in that: The invention is obtained by fermenting the Bacillus amyloliquefaciens YP2 as claimed in claim 1.

3. The microbial fermentation liquid according to claim 2, characterized in that The optimal fermentation conditions were: lactose addition of 25 g / L, initial pH of 5, inoculation of 2%, and culture temperature of 34 ℃.

4. The microbial fermentation liquid according to claim 2, characterized in that: The effective components of the microbial fermentation broth include 1-deoxynojirimycin, cyclic dipeptide, polypeptide and exopolysaccharide with α-glucosidase inhibitory ability.

5. Use of a Bacillus amyloliquefaciens strain YP2 in food fermentation, characterized by: Bacillus amyloliquefaciens YP2 was deposited in the General Microbiology Center of China Culture Collection Administration Committee, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on November 27, 2023, with the deposit number CGMCC No. 29157, and was classified as Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ); The 16S rDNA sequence of Bacillus amyloliquefaciens YP2 is shown in SEQ ID NO.1.

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