A strain of Pediococcus pentosaceus and its fermentation product with corn silk and its application in reducing blood sugar and lipid

Abstract

The present invention discloses a strain of Pediococcus pentosaceus, its fermentation product with corn silk, and its application in reducing blood sugar and lipid. The present invention discloses a co-ferment of Pediococcus pentosaceus and corn silk, which is obtained by co-fermenting Pediococcus pentosaceus and corn silk. The present invention finds through research that the fermented product after co-fermenting Pediococcus pentosaceus and corn silk has the effects of reducing blood sugar, improving liver function, and alleviating hepatic steatosis, and the effect is better than that of the fermentation broth of the two fermented separately. In addition, the present invention isolates and obtains a strain of Pediococcus pentosaceus, named BCST003. The Pediococcus pentosaceus BCST003 of the present invention is screened from corn silk and is safe and reliable. Moreover, experiments prove that compared with the existing Pediococcus pentosaceus strains, the fermentation broth after co-fermenting Pediococcus pentosaceus BCST003 with corn silk has a better effect in reducing blood sugar and lipid. The present invention provides new ideas and directions for the further development and utilization of corn silk and its fermentation products, and has good application prospects and value.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology. More specifically, it relates to a strain of Pediococcus pentosaceus and its fermentation product with corn silk and its application in reducing blood sugar and blood lipid. Background Art

[0002] In recent years, the number of people suffering from hypertension, hyperlipidemia, and hyperglycemia, commonly known as the "three highs" syndrome, has been increasing, seriously endangering people's health and safety. They are the three major risk factors for cardiovascular diseases, and they affect each other, jointly leading to the formation of arterial lipid plaques and arteriosclerosis, increasing the risk of diseases such as coronary heart disease, heart disease, and stroke. Poor long-term control will cause diabetes, peripheral vascular disease, coronary heart disease, myocardial infarction, hypertensive renal damage, heart failure, diabetic retinopathy, cerebral infarction, cerebral hemorrhage, pancreatitis, kidney disease, etc. Moreover, diabetic patients are also prone to secondary tuberculosis, pulmonary infections, etc. Once it develops severely to the point where drugs are needed, it is very difficult to control, and existing drugs also have many problems. For example, currently, drugs for treating hyperglycemia include insulin, metformin, etc., and drugs for treating hyperlipidemia include atorvastatin, rosuvastatin, ezetimibe, etc. These drugs may not only cause a series of side effects, but also long-term drug treatment will bring an economic burden to patients. Therefore, finding safe, effective, and economical natural substances for reducing blood sugar and blood lipid has become a current research hotspot, which can intervene in reducing blood sugar and blood lipid earlier, more safely, and at lower cost to control blood sugar and blood lipid levels.

[0003] Corn silk, as a by-product of corn plants, has been regarded as a natural herb with diuretic, detumescence, blood sugar-lowering and other effects in traditional medicine since ancient times, and the cost is also low. Modern research shows that corn silk contains rich polysaccharides, flavonoid compounds, organic acids and various trace elements, and these components show good biological activities in regulating blood sugar, blood lipid and antioxidant aspects. However, the blood sugar-lowering effect of directly eating or simply extracting corn silk products by soaking in water is very limited and difficult to meet the needs.

[0004] Pediococcus pentosaceus belongs to a kind of lactic acid bacteria and is widely used in agriculture, food, environmental protection and other fields. In recent years, the application of Pediococcus pentosaceus in probiotic fermentation products has gradually attracted more and more attention. There are also reports in the prior art on the application of Pediococcus pentosaceus strains in the preparation of fat-reducing and blood sugar-lowering products. For example, Chinese Patent CN117815272A discloses that Pediococcus pentosaceus strain A21358 has the effect of reducing the content of glucose and lipid globules in a nematode model in vivo. However, the effect of Pediococcus pentosaceus in reducing fat and blood sugar is not satisfactory. Summary of the Invention

[0005] The present invention aims to provide a strain of Pediococcus pentosaceus BCST003 and its application in reducing blood sugar and blood lipid.

[0006] The first object of the present invention is to provide a strain of Pediococcus pentosaceus BCST003.

[0007] The second object of the present invention is to provide a product with hypoglycemic and lipid-lowering effects.

[0008] The third object of the present invention is to provide a fermentation product of Pediococcus pentosaceus and corn silk.

[0009] The fourth object of the present invention is to provide the application of the above-mentioned Pediococcus pentosaceus BCST003 or the above-mentioned product.

[0010] The above objects of the present invention are achieved by the following technical solutions:

[0011] Fermentation, as a biological transformation process, can utilize the enzyme system of microorganisms to change the chemical composition of raw materials and generate new substances with higher biological activity. A strain of Pediococcus pentosaceus BCST003 was obtained in the research of the present invention. Through research, it was proved that the fermentation broth after co-fermentation of Pediococcus pentosaceus BCST003 and corn silk has the effects of reducing blood sugar, improving liver function and reducing hepatic steatosis.

[0012] The present invention provides a co-ferment of Pediococcus pentosaceus and corn silk, which is obtained by co-fermenting Pediococcus pentosaceus and corn silk.

[0013] Specifically, the co-ferment of Pediococcus pentosaceus and corn silk is a fermentation broth or a fermentation product obtained by co-fermenting Pediococcus pentosaceus and corn silk.

[0014] The fermentation broth refers to the fermentation broth obtained by co-fermenting Pediococcus pentosaceus and corn silk.

[0015] The fermentation product refers to the product obtained after removing the bacterial cells from the fermentation broth.

[0016] Preferably, the Pediococcus pentosaceus is Pediococcus pentosaceus BCST003 or Pediococcus pentosaceus 22227. The Pediococcus pentosaceus BCST003 was deposited in the Guangdong Provincial Culture Collection Center of Microorganisms on September 14, 2024, and its deposit number is GDMCC No: 65140.

[0017] Preferably, the fermentation conditions are fermentation at 35-39 °C for more than 24 hours.

[0018] As an alternative embodiment, the fermentation conditions are fermentation at 37 °C for 24-48 h.

[0019] As an alternative embodiment, the bacterial suspension of the above-mentioned Pediococcus pentosaceus BCST003 is inoculated into the corn silk solution for fermentation.

[0020] As an alternative embodiment, the concentration of the bacterial suspension is 1×10 8 ~1×10 10 cfu / mL.

[0021] As an alternative embodiment, the concentration of the bacterial suspension is 1×10 9 cfu / mL.

[0022] As an alternative embodiment, the volume ratio of the bacterial suspension to the corn silk solution is 1:(90 - 110).

[0023] As an alternative embodiment, the volume ratio of the bacterial suspension to the corn silk solution is 1:90 - 100.

[0024] As an alternative embodiment, the preparation method of the corn silk solution is to grind the corn silk into powder and then mix it with water.

[0025] As an alternative embodiment, the preparation method of the corn silk solution is to break the corn silk into powder, sterilize it to obtain corn silk powder, and then mix the corn silk powder with water in a ratio of 1:10 and ferment it at 37°C.

[0026] The present invention provides a strain of Pediococcus pentosaceus BCST003, which was deposited at the Guangdong Provincial Culture Collection Center of Microorganisms on September 14, 2024, and its deposit number is GDMCC No: 65140.

[0027] The present invention provides a product containing the co-ferment of the above-mentioned Pediococcus pentosaceus and corn silk, or containing the above-mentioned Pediococcus pentosaceus BCST003.

[0028] The present invention has found through research that the fermentation broth after the co-fermentation of Pediococcus pentosaceus and corn silk has the effects of reducing blood sugar, improving liver function, and alleviating hepatic steatosis, and the effect is better than that of the fermentation broth of the two fermented separately. Therefore, the present invention claims the following application schemes:

[0029] Use of the co-ferment of the above-mentioned Pediococcus pentosaceus and corn silk, the above-mentioned Pediococcus pentosaceus BCST003, or the above-mentioned product in the preparation of hypoglycemic products.

[0030] Use of the co-ferment of the above-mentioned Pediococcus pentosaceus and corn silk, the above-mentioned Pediococcus pentosaceus BCST003, or the above-mentioned product in the preparation of lipid-lowering products.

[0031] Use of the co-ferment of the above-mentioned Pediococcus pentosaceus and corn silk, the above-mentioned Pediococcus pentosaceus BCST003, or the above-mentioned product in the preparation of products for improving liver function or alleviating hepatic steatosis.

[0032] Specifically, the improvement of liver function is manifested as a reduction in the contents of alanine aminotransferase and aspartate aminotransferase in the blood.

[0033] Use of the above-mentioned Pediococcus pentosaceus and corn stigma fermentate, the above-mentioned Pediococcus pentosaceus BCST003, or the above-mentioned product in the preparation of a drug for treating fatty liver.

[0034] Use of the above-mentioned Pediococcus pentosaceus and corn stigma fermentate, the above-mentioned Pediococcus pentosaceus BCST003, or the above-mentioned product in the preparation of a drug for treating diabetes.

[0035] The present invention has the following beneficial effects:

[0036] The present invention has found through research that the fermentation broth after co-fermentation of Pediococcus pentosaceus and corn stigma has the effects of reducing blood sugar, improving liver function, and alleviating hepatic steatosis, and the effect is better than that of the fermentation broth of the two fermented alone. In addition, a strain of Pediococcus pentosaceus was isolated and obtained in the present invention, named BCST003. The Pediococcus pentosaceus BCST003 of the present invention was screened from corn stigma and is safe and reliable. And experiments have proved that compared with other existing Pediococcus pentosaceus strains, the fermentation broth after co-fermentation with corn stigma of Pediococcus pentosaceus BCST003 has better blood sugar and lipid-lowering effects. The present invention provides new ideas and directions for the further development and utilization of corn stigma and its fermentation products, and has good application prospects and value. Description of the Drawings

[0037] Figure 1 It is the 16S rDNA alignment result of BCST003 strain on NCBI.

[0038] Figure 2 It is the Gram staining result of BCST003 strain.

[0039] Figure 3 It is the whole genome circos map of BCST003 strain.

[0040] Figure 4 It is the KEGG gene function annotation map of BCST003 strain.

[0041] Figure 5 It is the PCA analysis result of chemical components after fermentation with different treatments (Group A is BCST003 group; Group B is corn stigma group; Group C is the co-fermentation group of Pediococcus pentosaceus BCST003 and corn stigma).

[0042] Figure 6 It is the heat map of chemical component contents after fermentation with different treatments (Group A is BCST003 group; Group B is corn stigma group; Group C is the co-fermentation group of Pediococcus pentosaceus BCST003 and corn stigma).

[0043] Figure 7Analysis results of compounds with significant changes in content after different treatments and fermentation.

[0044] Figure 8 Results of the effect of co-fermentation of Pediococcus pentosaceus BCST003 and corn silk on the pH of the fermentation broth.

[0045] Figure 9 Results of the effect of co-fermentation of Pediococcus pentosaceus BCST003 and corn silk on flavonoids in the fermentation broth.

[0046] Figure 10 Results of the effect of co-fermentation of Pediococcus pentosaceus BCST003 and corn silk on total phenols in the fermentation broth.

[0047] Figure 11 Results of the effect of co-fermentation of Pediococcus pentosaceus BCST003 and corn silk on total reducing sugars in the fermentation broth.

[0048] Figure 12 Results of the determination of fasting blood glucose concentration changes in mice with different treatments.

[0049] Figure 13 Results of the oral glucose tolerance test in mice with different treatments (different letters indicate significant differences, P < 0.05; the same letters indicate no significant differences, P > 0.05).

[0050] Figure 14 Results of the insulin tolerance test in mice with different treatments (different letters indicate significant differences, P < 0.05; the same letters indicate no significant differences, P > 0.05).

[0051] Figure 15 Results of the detection of alanine aminotransferase content in the serum of mice with different treatments (different letters indicate significant differences, P < 0.05; the same letters indicate no significant differences, P > 0.05).

[0052] Figure 16 Results of the detection of aspartate aminotransferase content in the serum of mice with different treatments (different letters indicate significant differences, P < 0.05; the same letters indicate no significant differences, P > 0.05).

[0053] Figure 17 Diagrams of the whole liver, HE staining, PAS staining, and oil red staining of mice with different treatments. Detailed implementation methods

[0054] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0055] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0056] MRS medium: 10.0 g of casein peptone, 10.0 g of beef extract powder, 4.0 g of yeast extract powder, 2.0 g of ammonium citrate, 5.0 g of sodium acetate, 0.02 g of magnesium sulfate heptahydrate (MgSO4·7H2O), 0.05 g of manganese sulfate tetrahydrate (MnSO4·4H2O), 2.0 g of dipotassium hydrogen phosphate, 20.0 g of glucose, 1.5 g of Tween-80. The above components are dissolved in 1 L of single-distilled water, and the final pH value is adjusted to 5.7 ± 0.2.

[0057] In the following examples, the Bergey's Manual of Determinative Bacteriology is "Bergey's Manual of Determinative Bacteriology. 8th Edition [M]. Science Press, Buchanan, Gibbons. 1984."

[0058] Pediococcus pentosaceus Strain 22227 is commercially available.

[0059] Corn silk is commercially available and purchased from Guangzhou Zhixin Pharmaceutical Co., Ltd.

[0060] Example 1 Obtaining of Pediococcus pentosaceus BCST003 Strain

[0061] I. Experimental Method

[0062] (1) Strain Screening and Identification

[0063] According to the ratio of corn silk to water of 1:4, put it into a juicer to extract the corn silk water mixture for natural fermentation, and continuously ferment for 9 days to obtain the fermentation broth. Continuously dilute the fermentation broth with deionized water at a 10-fold dilution gradient. Respectively pipette 100 μL of the diluted fermentation broth with dilution gradients of 10 -3 、10 -4 、10 -5 , evenly coat them on MRS medium (added with 3% CaCO3), and place them in a constant temperature incubator at 37°C for 48 h. Select the strains with obvious clear zones, and isolate single colonies by the method of streaking on a plate. Select one of the single colonies and record it as BCST003 strain, and conduct subsequent identification.

[0064] (2) 16S rDNA Sequencing Identification

[0065] Extract the genomic DNA of BCST003 strain as a sample, and use agarose gel electrophoresis to detect the quality of genomic DNA.

[0066] PCR amplification was performed using 16S rDNA specific detection primers (27F primer and 1492R primer) and genomic DNA of BCST003 strain as template. Each sample was repeated 3 times. The PCR products of the samples were mixed and detected by agarose gel electrophoresis. The PCR products were cut and recovered using the AxyPrepRNA gel recovery kit (AXYGEN). Referring to the preliminary quantitative results of electrophoresis, the PCR products were detected and quantified using the QuqntiFluorTM-ST blue fluorescence quantitative system (Promega). The samples were then sent to a sequencing company for sequencing, and the sequencing results were then compared with the BLAST in the GenBank database of NCBI.

[0067] The 27F primer sequence was (5′-3′): TACGGYTACCTTGTTACGACTT;

[0068] The 1492R primer sequence is (5'-3'): AGAGTTTGATCMTGGCTCAG.

[0069] 2. Experimental Results

[0070] (1) The 16S rDNA sequence was sequenced and then Blast compared on the NCBI website. The results are as follows: Figure 1 As shown, the results showed that the 16S rDNA sequencing sequence of the BCST003 strain had a similarity of up to 100% with that of Pediococcus pentosaceus, and the BCST003 strain was identified as Pediococcus pentosaceus.

[0071] At the same time, Gram staining was performed on the BCST003 strain, and the staining results were as follows Figure 2 As shown, the results showed that the strain was dark blue, indicating that the BCST003 strain was a Gram-positive bacterium. The BCST003 strain had a typical spherical or elliptical morphology and was relatively consistent in size, which was consistent with the typical characteristics of Pediococcus pentosaceus.

[0072] (2) Biochemical identification results of strains

[0073] The BCST003 strain was subjected to a series of microbial microbial biochemical identifications. The results of the strain biochemical identification are shown in Table 1. Combined with the data from the Bergey Bacterial Identification Manual, the results showed that the BCST003 strain was similar to Pediococcus pentosaceus in terms of physiological and biochemical characteristics. The characteristics of 22227 were basically the same, further proving that the BCST003 strain was Pediococcus pentosaceus.

[0074] Table 1 Biochemical identification results of strains

[0075]

[0076] Note: “+” represents a positive reaction, and “-” represents a negative reaction.

[0077] Example 2 Whole genome sequencing

[0078] 1. Whole genome sequencing

[0079] After extracting high-quality genomic DNA from the BCST003 strain, the purity, concentration, and integrity were checked using Nanodrop, Qubit, and 0.35% agarose gel electrophoresis. Large fragments of DNA were recovered using the BluePippin fully automated nucleic acid recovery system, followed by library construction (including DNA damage repair and end repair, adapter ligation, and Qubit library quantification). Finally, the prepared library was sequenced.

[0080] The sequencing results were first subjected to raw data quality control to filter out low-quality and short reads; the filtered reads were then assembled de novo, and the assembled draft genome was corrected; the genome components were then analyzed (including repetitive sequences, coding genes, non-coding RNA, prophages, gene islands, CRISPR, etc.); finally, functional annotation was performed, mainly including general databases such as Nr, Uniprot, COG, KEGG, and proprietary database annotations such as CAZyme, PHI, and CARD. In addition, genome map analysis was also performed (including genome circle maps, genome map display tools, and genome maps).

[0081] 2. Whole genome sequencing results

[0082] After filtering, the paired-end sequencing data was used to obtain high-quality data (clean reads) for comparison of the nt library. The whole genome circos diagram of the BCST003 strain is shown in the figure. Figure 3 As shown, the results showed that the nt library of strain BCST003 was most similar to that of Pediococcus pentosaceus, with a similarity of 96.2%, indicating that strain BCST003 was a new strain of Pediococcus pentosaceus.

[0083] The genome size of BCST003 strain is about 1.8Mb, with a GC content of 37.15%. After gene prediction and annotation, we found a total of 1836 protein-coding genes involved in a variety of biological processes and metabolic pathways. In addition, the genome of BCST003 strain also contains 3535bp of repetitive sequences, 57 tRNAs and 15 rRNAs.

[0084] By comparing known databases and literature, we have annotated the gene functions of the BCST003 strain in detail. The annotation results are as follows: Figure 4As shown in the figure, the results show that the BCST003 strain has a variety of potential biological functions and metabolic pathways, such as carbon metabolism, nitrogen metabolism, energy production and conversion, etc.

[0085] In summary, the BCST003 strain was identified as Pediococcus pentosaceus, and this strain was named: Pediococcus pentosaceus BCST003. It was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 14, 2024, with the deposit number GDMCC No: 65140, and the deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0086] Example 3 Chemical Composition Changes during Co-Fermentation of Pediococcus pentosaceus and Corn Silk

[0087] I. Experimental Methods

[0088] 1. Three treatment groups were set up, namely:

[0089] (1) BCST003 group,

[0090] (2) Corn silk group,

[0091] (3) Co-fermentation group of Pediococcus pentosaceus BCST003 and corn silk (abbreviated as co-fermentation group).

[0092] The specific treatments were as follows:

[0093] BCST003 group: Pediococcus pentosaceus BCST003 was inoculated into MRS medium and fermented and cultured at 37°C.

[0094] Corn silk group: The dry corn silk was washed, dried, and ground into powder with an ultra-high-speed crusher, and then sterilized under high pressure to obtain the corn silk powder for standby. The corn silk powder was mixed with water at a ratio of 1:10 and fermented at 37°C. Relevant indicators were measured at 0 h, 24 h, and 48 h respectively.

[0095] Co-fermentation group of Pediococcus pentosaceus BCST003 and corn silk: After the BCST003 strain was revived, it was cultured at 37°C for 24 hours and then centrifuged to obtain the precipitate. The precipitate was suspended with PBS (p = 7.4) to obtain a suspension of Pediococcus pentosaceus BCST003 strain at 1×10 9 cfu / mL. The dry corn silk was washed, dried, and ground into powder with an ultra-high-speed crusher, and then sterilized under high pressure to obtain the corn silk powder for standby. The corn silk powder was mixed with water at a ratio of 1:10, and the BCST003 strain suspension was inoculated into the mixture at a ratio of 1:100 V / V and fermented at 37°C. Relevant indicators were measured at 0 h, 24 h, and 48 h respectively.

[0096] 2. LC-MS Detection of Chemical Composition Changes

[0097] The sample was taken out from the -80 °C refrigerator and thawed on ice. After thawing, it was vortexed for 10 s to mix evenly. 50 μL of the sample was taken into an EP tube, and 150 μL of pre-cooled ice methanol (containing 2-chlorophenylalanine at 1 μg / mL as the internal standard) was added. It was vortexed for 3 min and centrifuged at 12000 g for 10 min at 4 °C. The supernatant was aspirated into another new EP tube. The supernatant was centrifuged again at 12000 g for 5 min at 4 °C, and the supernatant was taken into the liner of the injection vial for UPLC-MS / MS analysis.

[0098] The liquid phase conditions mainly include: chromatographic column: Waters ACQUITY UPLC HSS T3 C18 (1.8 μm, 2.1 mm * 100 mm); mobile phase: phase A is ultrapure water (0.04% acetic acid), phase B is acetonitrile (0.04% acetic acid); elution gradient: 0 min water / acetonitrile (95:5 V / V), 11.0 min is 5:95 V / V, 12.0 min is 5:95 V / V, 12.1 min is 95:5 V / V, 14.0 min is 95:5 V / V; flow rate 0.4 mL / min; column temperature 40 °C; injection volume 2 μL.

[0099] The mass spectrometry conditions mainly include: electrospray ionization (ESI) temperature 500 °C, mass spectrometry voltage 5500 V (positive), -4500 V (negative), ion source gas I (GS I) 55 psi, gas II (GS II) 60 psi, curtain gas (CUR) 25 psi, collision-activated dissociation (CAD) parameter settings are high. In the triple quadrupole (Qtrap), each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0100] The data acquisition instrument system mainly includes ultra-performance liquid chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A) and tandem mass spectrometry (MS / MS)

[0101] UPLC-MS / MS analysis was used to process the mass spectrometry data with Analyst 1.6.3. According to the information of Q1 (mass-to-charge ratio of precursor ions) in metabolites, the metabolites were co-identified by comparing with the existing self-built database. The MultiQuant software was used for the integration and calibration of chromatographic peaks. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to screen for differential variables. The screening conditions were VIP≥1, or metabolites with fold change≥2 and fold change≤0.5 were selected as target metabolites. If the difference in metabolites between the control group and the experimental group was more than 2-fold or less than 0.5-fold, the difference was considered significant.

[0102] II. Experimental Results

[0103] After 24 h of fermentation, the BCST003 group, the corn silk group, and the co-fermentation group used liquid chromatography-mass spectrometry (LC-MS) to conduct a detailed detection of the chemical components of the fermentation broth, and analyzed the chemical component data after co-fermentation.

[0104] The results of PCA analysis of the chemical components after fermentation with different treatments are as Figure 5 shown, the heat map of the chemical component contents after fermentation with different treatments is as Figure 6 shown, and the analysis results of the compounds with significant changes in content after fermentation with different treatments are as Figure 7 shown. The results indicate that after the fermentation by Pediococcus pentosaceus, the chemical components in corn silk have changed significantly. Among all the detected compounds, the following compounds have changed the most significantly: zeaxanthin, formononetin, protocatechuic acid, chlorogenic acid, luteolin, caffeic acid, rutin, acetic acid, methyl butyrate, and citric acid. These compounds showed significant abundance changes in the fermented samples, indicating that the metabolic activities of Pediococcus pentosaceus BCST003 may promote the generation or transformation of these bioactive substances.

[0105] Among them, the content of zeaxanthin increased significantly after fermentation (P<0.05), which may be related to the improved stability and bioavailability of zeaxanthin during the metabolism of Pediococcus pentosaceus BCST003. The contents of rutin, formononetin, protocatechuic acid, chlorogenic acid, and luteolin decreased significantly after fermentation.

[0106] In addition, we observed that the contents of caffeic acid, acetic acid, methyl butyrate, and citric acid in the fermented samples also increased. These compounds have potential application values in food preservation, flavor formation, and health benefits.

[0107] In summary, the co-fermentation of Pediococcus pentosaceus BCST003 significantly changed the key chemical components in corn silk, and these changes may be related to the improvement of its biological activity.

[0108] Changes in pH, flavonoids, polyphenols, and reducing sugars after co-fermentation in Example 4

[0109] I. Experimental method

[0110] Detect the changes in the content of pH, flavonoids, polyphenols, and reducing sugars during the fermentation process in the corn silk group, Pediococcus pentosaceus BCST003, and the co-fermentation group of corn silk in Example 3.

[0111] (1) Determination of the content of total sugar

[0112] Preparation of the standard curve: Prepare 1 mL of glucose standard solutions with concentrations of 0, 10, 20, 30, 40, 60, and 80 μg / mL. Add 4 mL of anthrone reagent, quickly immerse in an ice-water bath for cooling. After adding all the tubes, immerse them in a boiling water bath together, cover the tube mouths to prevent evaporation. Starting from when the water bath re-boils, boil for 10 min and then take out, cool to room temperature with an ice bath, and quickly measure the absorbance of the remaining tubes at a wavelength of 620 nm. Take the standard glucose content (μg) as the abscissa and the absorbance as the ordinate to draw the standard curve. Dilute the test solution by 10 times and operate according to the above method to calculate the content of total sugar.

[0113] (2) Determination of the content of polyphenols

[0114] Use the Folin Ciocalteu method to determine the polyphenol content. Specifically, mix 0.2 mL of appropriately diluted sample with 1.5 mL of 10-fold diluted Folin-Ciocalteu reagent and 1.5 mL of 7.5% (w / v) sodium carbonate. After standing at room temperature in the dark for 40 min, read the absorbance at 765 nm using a UV-visible spectrophotometer. Calculate the polyphenol content.

[0115] (3) Determination of the content of flavonoids

[0116] Preparation of the rutin standard curve: Accurately weigh 10 mg of rutin standard product, dissolve and make up the volume to 50 mL with 60% ethanol solution to obtain a rutin standard product solution with a concentration of 0.2 mg / mL. Respectively take 0.4, 0.8, 1.2, 1.6, 2.0 mg / mL rutin standard product solutions, supplement to 2 mL with ethanol solution, add 1 mL of sodium nitrite solution, shake well, let stand for 6 min, add 1.5 mL of aluminum nitrate solution, shake well, let stand for 6 min, add 4 mL of 40% sodium hydroxide solution, shake well, and let stand for 15 min. Dilute and make up the volume to 10 mL with ethanol solution to obtain rutin standard product solutions with concentrations of 0.008, 0.016, 0.024, 0.032, 0.04 mg / mL respectively. Use the sodium nitrite-aluminum nitrate colorimetric method to measure the absorbance of the extraction solution, and finally perform linear regression by the least squares method to obtain the regression equation between the rutin concentration C (mg / mL) and the absorbance A (y = 0.3083x + 0.0372, R 2= 0.9981).

[0117] Sample detection: Pipette 0.5 mL of the sample, dilute the test solution 10 times, and operate according to the above method.

[0118] (4) Determination of reducing sugar content

[0119] Dilute the test fermentation broth 10 times. Take 2 mL of the diluted solution and add 1.5 mL of DNS solution. Mix well. After reacting in a boiling water bath for 10 min, cool it, and make up the volume to 25 mL with distilled water. Shake well. Use the absorbance corresponding to the glucose standard solution (0 - 0.028 mg / mL) at 540 nm to plot the standard curve (y = 0.0073x + 0.0328, R 2 = 0.9986), and calculate the reducing sugar content in the fermentation broth.

[0120] II. Experimental results

[0121] The experimental results show that there are significant differences in multiple aspects between the co - fermentation group and the corn silk group.

[0122] (1) In terms of the change in pH value, the pH value of the co - fermentation group significantly decreased to 3.8 within 48 hours, while the pH value of the single corn silk group did not change significantly ( Figure 8 ). This result may be related to the acidic metabolites produced by Pediococcus pentosaceus during fermentation. These metabolites can reduce the pH value of the environment, thus affecting the acidity and alkalinity of the fermentation broth.

[0123] (2) In terms of flavonoid content, the co - fermentation group showed a significant increase (P value less than 0.01) ( Figure 9 ), indicating that Pediococcus pentosaceus promoted the release of flavonoid compounds in corn silk during fermentation.

[0124] (3) In terms of the change in total phenol content, compared with the corn silk group, the total phenol content of the co - fermentation group showed a certain upward trend ( Figure 10 ).

[0125] (4) In terms of reducing sugar content, the co - fermentation group also showed a significant decrease ( Figure 11 ). This may be because Pediococcus pentosaceus consumed a large amount of reducing sugar as an energy and carbon source during fermentation, resulting in a decrease in reducing sugar content.

[0126] Example 5 Detection of hypoglycemic and hypolipidemic effects

[0127] I. Experimental method

[0128] Using two different strains of Pediococcus pentosaceus (BCST003 and (22227) The corn silk was fermented, and a mouse model was established to evaluate the hypoglycemic and lipid-lowering effects of these fermentation products.

[0129] 1. Establishment, grouping and administration of the mouse model

[0130] (1) Establishment of the mouse model

[0131] After 1 week of adaptive feeding, Balb / c mice were randomly divided into a blank control group (CON, n = 5) and a high-sugar and high-fat model group (n = 25). They had free access to water. The blank control group was fed a normal diet, and the mice in the model group were fed a high-sugar and high-fat diet (HFD) for 4 weeks. In the 5th week, they were intraperitoneally injected with freshly prepared streptozotocin (STZ) at a dose of 40 mg / kg in citrate buffer (0.1 M, pH 4.5) for 5 consecutive days; the blank control group was injected with an equal volume of citrate buffer. On the 3rd and 7th days after the first injection of STZ, the blood glucose level in the tail vein blood was measured. Mice with a blood glucose concentration greater than 11.1 mmol / L were confirmed as hyperglycemic mice.

[0132] (2) Experimental grouping and administration

[0133] The mouse model established above was randomly divided into a model group (MOD), a corn silk group (CS group), a Pediococcus pentosaceus BCST003 group (pp003), a CS-Pediococcus pentosaceus BCST003 group (CS-pp003), and a CS-Pediococcus pentosaceus 22227 group (cs-pp22227), with 5 mice in each group.

[0134] At the same time, blank control group mice were set up.

[0135] The blank control group (CON) and the model group mice were gavaged with normal saline. The CS group, the Pediococcus pentosaceus BCST003 group, the CS-Pediococcus pentosaceus BCST003 group, and the CS-Pediococcus pentosaceus 22227 group were given drug treatment for 8 consecutive weeks. The specific drug administration methods for each group were as follows:

[0136] CS group: gavaged with the single fermentation broth of corn silk once a day, and the gavage dose was 0.2 mL / 10 g.

[0137] Pediococcus pentosaceus BCST003 group: gavaged with the single fermentation broth of Pediococcus pentosaceus BCST003 once a day, and the gavage dose was 0.2 mL / 10 g.

[0138] CS-Pediococcus pentosaceus BCST003 group: gavaged with the co-fermentation broth of corn silk and Pediococcus pentosaceus BCST003 once a day, and the gavage dose was 0.2 mL / 10 g.

[0139] CS-Pediococcus pentosaceus Group 22227: intragastric administration of corn silk and Pediococcus pentosaceus The co-fermentation broth of 22227 was administered intragastrically once a day at a dose of 0.2 mL / 10 g.

[0140] During the experiment, the mice in each group were given free access to water and food. The body weight (BW) and fasting blood glucose (FBG) concentration of the mice were measured every 2 weeks.

[0141] (2) Oral Glucose Tolerance Test (OGTT)

[0142] After 8 weeks of administration, the mice in each group were fasted overnight for 12 h, given free access to water, and intragastrically administered a 40% glucose solution at a dose of 2 g / kg. Tail vein blood was collected at 0 min, 30 min, 60 min, 90 min, and 120 min after intragastric administration of glucose, and the blood glucose value was measured and recorded using an ACCU-CHEK Performa (Roche vitality) glucose test strip method, and then the corresponding area under the curve (AUC) was calculated.

[0143] (3) Insulin Tolerance Test (ITT)

[0144] After 8 weeks of administration, the mice in each group were fasted for 6 h, given free access to water, and intraperitoneally injected with insulin (0.75 U / kg). Tail vein blood was collected at 0 min, 30 min, 60 min, 90 min, and 120 min after injection of insulin, and the blood glucose value was measured and recorded using an ACCU-CHEK Performa (Roche vitality) glucose test strip method, and then the corresponding area under the curve (AUC) was calculated.

[0145] (4) Detection of biochemical indexes

[0146] All mice were fasted and given free access to water for 12 h before sacrifice. The mice were anesthetized by removing the eyeballs to collect blood, and the blood was collected into a 1.5 mL sterile and pyrogen-free EP tube. After centrifugation at 300 g for 10 min, the clear supernatant was aspirated into a new EP tube. After repeating centrifugation once, the clear supernatant was collected again and stored in a -80 °C refrigerator. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were detected using an automatic biochemical analyzer.

[0147] (5) HE staining

[0148] 1) Specimen collection: Fresh mouse liver tissue and skeletal muscle were placed in 4% paraformaldehyde solution and fixed at room temperature for more than 24 h. The tissue was taken out of the fixative, trimmed flat in a fume hood, and placed in a dehydration box.

[0149] 2) Dehydration: Place the dehydration box into the hanging basket and put it into the dehydrator. Dehydrate according to gradient alcohol, specifically: Treat it successively in 75% alcohol, 85% alcohol, 90% alcohol, 95% alcohol, absolute ethanol, alcohol-benzene, xylene I, xylene II, paraffin I, paraffin II, and paraffin III. The treatment time in each solution is 4h, 2h, 1.5h, 1.5h, 0.5h, 10 - 20 min, 10 - 20 min, 10 - 20 min, 1.5h, 1.5h, and 1h respectively.

[0150] 3) Embedding: Place the tissue completed with wax infiltration into the embedding machine for embedding. First, put the melted wax into the embedding frame. Before the wax solidifies, take out the tissue from the dehydration box, place it into the embedding frame according to the requirements of the embedding surface, and attach the corresponding label. Place it on the freezing table at 20°C for cooling. After the wax solidifies, take out the wax block from the embedding frame and trim it.

[0151] 4) Sectioning: Use a paraffin microtome to section the trimmed wax block with a section thickness of 4μm. Float the section on the warm water at 40°C of the spreading machine. After flattening the tissue, pick it up with a glass slide and place it in an oven at 60°C for baking. After the water is baked dry and the wax melts, take it out and store it at room temperature for standby.

[0152] 5) Dewaxing the paraffin section to water: Place the section into xylene I and xylene II for 20 min each in sequence. After taking it out, then place it into absolute ethanol I, absolute ethanol II, and 75% alcohol for 5 min each in sequence. Finally, wash it with distilled water.

[0153] 6) Hematoxylin staining: Place the section into the hematoxylin staining solution for 3 - 5 min. After washing with tap water, differentiate it with the differentiating solution (1% hydrochloric acid alcohol) for several seconds, rinse with tap water, blue it with the bluing solution (0.6% ammonia water), and rinse with running water.

[0154] 7) Eosin staining: Place the section into 85% alcohol for dehydration for 5 min, then take it out and place it into 95% alcohol for dehydration for 5 min, and finally place it into the eosin staining solution for staining for 5 min.

[0155] 8) Dehydration and mounting: Place the section into absolute ethanol I, absolute ethanol II, absolute ethanol III, xylene I, and xylene II for 5 min each in sequence. After the section becomes transparent in xylene, take it out and let it dry slightly, and mount it with neutral balsam.

[0156] 9) Use a microscope for microscopic examination and perform image acquisition and analysis.

[0157] (6) Oil Red O staining

[0158] 1) Preparation of frozen liver sections: Appropriately cut the mouse liver, embed the tissue block in OCT frozen section embedding agent, transfer it to a pre-cooled cryostat. After it is completely solidified and the tissue sample is fixed in the cryostat, set the section thickness to 5 μm, take an adhesive glass slide and use the front side to attach the cut sample and make marks.

[0159] 2) Air-dry the sections: Air-dry the frozen sections at room temperature.

[0160] 3) Oil Red staining: Immerse the sections in Oil Red O working solution and stain them in the dark for 7 - 10 min.

[0161] 4) Section color adjustment: Immerse the sections in the differentiating solution (60% ethanol) for 5 - 10 s until the stroma is clear. Here, the infiltration time can be appropriately changed to control the color under the microscope so that the adipose tissue is bright red and the stroma is colorless.

[0162] 5) Wash the sections with water: Wash the sections 3 times with distilled water.

[0163] 6) Stain the cell nuclei with hematoxylin: Immerse the sections in hematoxylin staining solution and stain for 2 - 4 min until the nuclei on the sections are stained; wash the sections 2 times with distilled water to wash away the stain not bound to the sections; then quickly differentiate with the differentiating solution (1% hydrochloric acid alcohol) for 2 - 3 s to remove the dye adsorbed in the cytoplasm and the excessive dye bound in the cell nuclei. After washing the sections with distilled water, blue the sections with hematoxylin blueing solution (0.6% ammonia water) for 2 - 3 s and wash with tap water.

[0164] Mount with glycerol gelatin: First, blot the water around the sections with absorbent paper, and then mount the sections with an aqueous mounting medium (such as glycerol gelatin). When mounting, if air bubbles are found, do not press the cover glass to avoid lipid droplet displacement, or directly observe without mounting temporarily.

[0165] (7) PAS staining

[0166] 1) Specimen collection: Take fresh mouse liver tissue and skeletal muscle and fix them in 4% paraformaldehyde solution at room temperature for more than 24 h. Take out the tissue from the fixative, trim it flat in the fume hood and place it in a dehydration box.

[0167] 2) Dehydration: Put the dehydration box into the hanging basket in the dehydrator and dehydrate according to gradient alcohol, specifically: successively process in 75% alcohol, 85% alcohol, 90% alcohol, 95% alcohol, absolute ethanol, alcohol-benzene, xylene I, xylene II, paraffin I, paraffin II, paraffin III, and the treatment times are 4 h, 2 h, 1.5 h, 1.5 h, 0.5 h, 10 - 20 min, 10 - 20 min, 10 - 20 min, 1.5 h, 1.5 h, 1 h respectively.

[0168] 3) Embedding: Place the wax-impregnated tissue in an embedding machine for embedding. First, put the melted wax in the embedding frame. Before the wax solidifies, take out the tissue from the dehydration box, place it in the embedding frame according to the requirements of the embedding surface, attach the corresponding label, and place it on a freezing table at 20°C for cooling. After the wax solidifies, take out the wax block from the embedding frame and trim it.

[0169] 4) Sectioning: Use a paraffin slicing machine to slice the trimmed wax block with a thickness of 4 μm. Float the sections in warm water at 40°C on a spreading machine. After flattening the tissue, pick it up with a glass slide and bake it in an oven at 60°C until the water is dried and the wax is melted, then take it out and store it at room temperature for standby.

[0170] 5) Dewaxing of paraffin sections to water: Immerse the sections in xylene I and xylene II for 20 minutes each in sequence. After taking them out, then immerse them in absolute ethanol I, absolute ethanol II, and 75% alcohol for 5 minutes each in sequence, and finally wash with distilled water.

[0171] 6) Periodic acid staining: Immerse the sections dewaxed to water in periodic acid staining solution in a microwave repair box and stain for 15 minutes. Then take out the sections and wash them with distilled water 2 - 3 times.

[0172] 7) Schiff staining: Immerse the sections in Schiff staining solution in a microwave repair box and stain them in the dark for 30 minutes. Take out the sections and rinse them with running water for 5 minutes until the water in the staining cup turns red and then becomes colorless to stop the color development.

[0173] 8) Hematoxylin staining: Immerse the sections in hematoxylin staining solution and stain for 30 seconds, wash with tap water, differentiate with differentiating solution (1% hydrochloric acid alcohol) for several seconds, rinse with tap water, blue with blueing solution (0.6% ammonia water), and rinse with running water.

[0174] 9) Dehydration and mounting: Immerse the sections in absolute ethanol I for 5 minutes - absolute ethanol II for 5 minutes - absolute ethanol III for 5 minutes - xylene I for 5 minutes - xylene II for 5 minutes in sequence. After the sections become transparent in xylene, take out the sections from xylene, let them dry slightly, and mount them with neutral balsam.

[0175] 10) Microscopic examination and image acquisition and analysis.

[0176] II. Experimental results

[0177] The measured results of the fasting blood glucose concentration changes in mice with different treatments are as Figure 12 shown. The results of the oral glucose tolerance test and insulin tolerance test in mice with different treatments are respectively as Figure 13 and Figure 14 shown. The results show that compared with the model group, the CS-Pediococcus pentosaceus BCST003 group and CS-Pediococcus pentosaceus The blood glucose levels of the mice treated with the 22227 groups were significantly decreased. This indicates that the fermentation broths of the BCST003 strain and the 22227 strain after fermenting corn silk can both reduce the blood glucose levels of mice. Among them, the blood glucose-lowering effect of the BCST003 strain is significantly better than that of the 22227 strain ( Figure 12 ).

[0178] The detection results of the alanine aminotransferase (ALT) content and aspartate aminotransferase (AST) content in the sera of mice with different treatments are respectively Figure 15 and Figure 16 shown as follows. The results indicate that, compared with the model group, the ALT and AST contents of the mice treated with the CS-Pediococcus pentosaceus BCST003 group and the CS-Pediococcus pentosaceus 22227 group were significantly decreased, and the ALT and AST contents in the sera of the mice treated with the CS-Pediococcus pentosaceus BCST003 group were lower than those of the CS-Pediococcus pentosaceus 22227 group. This indicates that the co-fermentation product of the BCST003 strain and corn silk has the effect of improving liver function.

[0179] The overall liver, HE staining, PAS staining, and oil red staining diagrams of the mice with different treatments are as Figure 17 shown as follows. The results indicate that:

[0180] In terms of the liver appearance, the livers of the mice in the model group were yellowish in color, larger in size, and had a higher fat content. In contrast, the livers of the mice in the CS-Pediococcus pentosaceus BCST003 group were significantly redder, indicating a reduction in the degree of fatty degeneration.

[0181] By observing the liver tissue sections through hematoxylin-eosin (HE) staining, we found that the hepatocyte cords in the control group were arranged neatly, and no obvious necrosis or inflammatory cell infiltration was observed. In contrast, a large number of pathological changes such as hepatocyte fatty degeneration, cell swelling, and round vacuoles in the cytoplasm were visible in the liver tissue of the model group. However, the liver tissue of the mice in the CS-Pediococcus pentosaceus BCST003 group showed obvious improvement, with a reduction in vacuolization and inflammatory cell infiltration, and only a small amount of hepatocyte fatty degeneration, indicating that the co-fermentation product of the BCST003 strain and corn silk has the effect of reducing liver fatty degeneration.

[0182] By performing PAS staining on the liver tissues of each group of mice to detect the glycogen content. The PAS staining results indicate that, compared with the CON group, the liver cell structure of the mice in the MOD group was damaged and the glycogen storage was lower. After treatment with the co-fermentation broth of corn silk and Pediococcus pentosaceus BCST003, the liver cell structure was significantly restored, and the glycogen storage in the hepatocytes increased significantly.

[0183] After treating the liver with Oil Red O staining, we observed that the livers of the mice in the model group showed more obvious orange-red color, indicating increased lipid deposition, while the fat accumulation in the livers of the mice in the CS-Pediococcus pentosaceus BCST003 group was significantly reduced.

[0184] In summary, the results of this experiment indicate that the co-fermentation product of Pediococcus pentosaceus BCST003 strain and corn silk shows significant effects in reducing blood glucose, improving liver function, and alleviating hepatic steatosis. This provides new ideas and directions for the further development and utilization of corn silk and its fermentation products.

[0185] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A Pediococcus pentosaceus ( Pediococcus pentosaceus ) and corn silk co-fermentation, characterized in that, It is obtained by co-fermentation of Pediococcus pentosaceus and corn silk; the Pediococcus pentosaceus is Pediococcus pentosaceus BCST003, and the Pediococcus pentosaceus BCST003 has been preserved in the Guangdong Provincial Microbiological Culture Collection Center on September 14, 2024, and its preservation number is GDMCC No: 65140.

2. The co-fermentation product of Pediococcus pentosaceus and corn silk according to claim 1, characterized in that: It is a fermentation liquid or a fermentation product obtained by co-fermenting Pediococcus pentosaceus and corn silk. The fermentation product refers to a product obtained after removing the bacterial cells from the fermentation liquid.

3. A strain of Pediococcus pentosaceus BCST003, characterized in that: The Pediococcus pentosaceus BCST003 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 14, 2024, and its preservation number is GDMCC No: 65140.

4. A product, characterized in that Contains the co-fermentation product of Pediococcus pentosaceus and corn silk according to claim 1 or 2, or contains Pediococcus pentosaceus BCST003 according to claim 3.

5. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to claim 1 or 2, the Pediococcus pentosaceus BCST003 according to claim 3, or the product according to claim 4 in the preparation of hypoglycemic products.

6. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to claim 1 or 2, the Pediococcus pentosaceus BCST003 according to claim 3, or the product according to claim 4 in the preparation of lipid-lowering products.

7. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to claim 1 or 2, the Pediococcus pentosaceus BCST003 according to claim 3, or the product according to claim 4 in the preparation of a product for improving liver function or mild hepatic fatty degeneration.

8. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to claim 1 or 2, the Pediococcus pentosaceus BCST003 according to claim 3, or the product according to claim 4 in the preparation of a drug for treating fatty liver.

9. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to claim 1 or 2, the Pediococcus pentosaceus BCST003 according to claim 3, or the product according to claim 4 in the preparation of a drug for treating diabetes.

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