Pediococcus pentosaceus, fermented product of Pediococcus pentosaceus and corn stigma and application of Pediococcus pentosaceus and

Abstract

The invention discloses pediococcus pentosaceus as well as a fermented product of the pediococcus pentosaceus and corn stigma and application of the pediococcus pentosaceus and the corn stigma to The invention discloses a pediococcus pentosaceus and corn stigma co-fermentation product. The pediococcus pentosaceus and corn stigma co-fermentation product is obtained by co-fermentation of pediococcus pentosaceus and corn stigma. Researches find that a fermentation product obtained by co-fermentation of the pediococcus pentosaceus and the corn stigma has the effects of reducing blood sugar, improving liver functions and relieving liver fatty degeneration, and the effect is better than that of fermentation liquor obtained by independent fermentation of the pediococcus pentosaceus and the corn stigma. In addition, a strain of pediococcus pentosaceus is obtained through separation and is named as BCST003. The pediococcus pentosaceus BCST003 is obtained by screening from corn stigma, and is safe and reliable. And experiments prove that compared with the existing pediococcus pentosaceus strain, the pediococcus pentosaceus BCST003 has the advantage that the effect of reducing blood sugar and blood fat of fermentation liquor obtained by co-fermenting the pediococcus pentosaceus BCST003 and the corn stigma is better. The invention provides a new thought and direction for further development and utilization of corn stigma and fermented products thereof, and has good application prospect and value.

Description

Technical Field

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

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

[0003] Corn silk, as a byproduct of corn plants, has been regarded by traditional medicine since ancient times as a natural herbal medicine with diuretic, detumescent, and blood sugar lowering effects, and it is also low in cost. Modern research shows that corn silk is rich in polysaccharides, flavonoids, organic acids, and a variety of trace elements, which show good biological activity in regulating blood sugar, blood lipids, and anti-oxidation. However, corn silk products that are directly eaten or simply extracted by soaking in water have very limited blood sugar lowering effects and are difficult to meet demand.

[0004] Pediococcus pentosaceus is 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. The prior art also reports the application of Pediococcus pentosaceus strains in the preparation of fat-reducing and blood sugar-lowering products, such as Chinese patent CN117815272A discloses that Pediococcus pentosaceus strain A21358 has the ability to reduce the content of glucose and fat particles in the body of nematode models. However, the effect of Pediococcus pentosaceus in fat-reducing and blood sugar-lowering is not satisfactory. Summary of the invention

[0005] The present invention aims to provide a Pediococcus pentosaceus BCST003 and application thereof in reducing blood sugar and lipids.

[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 having blood sugar lowering and blood 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 use of the above-mentioned Pediococcus pentosaceus BCST003 or the above-mentioned product.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] Fermentation, as a biotransformation process, can utilize the enzyme system of microorganisms to change the chemical composition of raw materials and generate new substances with higher biological activity. The present invention has obtained a strain of Pediococcus pentosaceus BCST003. The research proves that the fermentation liquid after co-fermentation of Pediococcus pentosaceus BCST003 and corn silk has the effects of lowering blood sugar, improving liver function and alleviating liver fatty degeneration.

[0012] The invention provides a Pediococcus pentosaceus and corn silk co-fermentation product, which is obtained by co-fermenting the Pediococcus pentosaceus and corn silk.

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

[0014] The fermentation liquid refers to the fermentation liquid obtained by co-fermentation and cultivation of Pediococcus pentosaceus and corn silk.

[0015] The fermentation product refers to the product obtained after removing the bacteria from the fermentation liquid.

[0016] Preferably, the Pediococcus pentosaceus is Pediococcus pentosaceus BCST003 or Pediococcus pentosaceus 22227, the Pediococcus pentosaceus BCST003 has been deposited in Guangdong Provincial Microbiological Culture Collection Center on September 14, 2024, and its preservation number is GDMCC No: 65140.

[0017] Preferably, the fermentation condition is 35-39° C. for more than 24 hours.

[0018] As an optional embodiment, the fermentation condition is fermentation at 37° C. for 24 to 48 hours.

[0019] As an optional implementation scheme, the bacterial suspension of Pediococcus pentosaceus BCST003 is inoculated into a corn silk solution for fermentation.

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

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

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

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

[0024] As an optional 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 optional embodiment, the preparation method of the corn silk solution is to crush 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 has been deposited in Guangdong Provincial Microbiological Culture Collection Center on September 14, 2024, and its preservation number is GDMCC No: 65140.

[0027] The present invention provides a product, which contains the co-fermentation product of Pediococcus pentosaceus and corn silk, or contains the Pediococcus pentosaceus BCST003.

[0028] The present invention has found that the fermentation liquid after co-fermentation of Pediococcus pentosaceus and corn silk has the effects of lowering blood sugar, improving liver function and reducing liver fatty degeneration, and the effect is better than the fermentation liquid of the two fermentations alone. Therefore, the present invention claims to protect the following application scheme:

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

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

[0031] Use of the above-mentioned Pediococcus pentosaceus and corn silk co-fermentation product, the above-mentioned Pediococcus pentosaceus BCST003 or the above-mentioned product in the preparation of a product for improving liver function or mild hepatic fatty degeneration.

[0032] Specifically, the improvement of liver function is manifested in reducing the levels of alanine aminotransferase and aspartate aminotransferase in the blood.

[0033] The use of the co-fermentation product of Pediococcus pentosaceus and corn silk, the Pediococcus pentosaceus BCST003 or the product in the preparation of a drug for treating fatty liver.

[0034] The use of the above-mentioned Pediococcus pentosaceus and corn silk co-fermentation product, the above-mentioned Pediococcus pentosaceus BCST003 or the above-mentioned product in the preparation of drugs for treating diabetes.

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

[0036] The present invention has found that the fermentation liquid after co-fermentation of Pediococcus pentosaceus and corn silk has the effects of lowering blood sugar, improving liver function and alleviating liver fatty degeneration, and the effect is better than the fermentation liquid fermented by the two alone. In addition, the present invention has isolated a strain of Pediococcus pentosaceus, named BCST003. The Pediococcus pentosaceus BCST003 of the present invention is screened and obtained from corn silk, which is safe and reliable. And experiments have shown that the Pediococcus pentosaceus BCST003 is better than other existing Pediococcus pentosaceus strains in the effect of reducing blood sugar and lipids in the fermentation liquid after co-fermentation with corn silk. The present invention provides new ideas and directions for further development and utilization of corn silk and its fermented products, and has good application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0038] Figure 2 The Gram staining results of BCST003 strain.

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

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

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

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

[0043] Figure 7The results are the analysis of compounds whose contents changed significantly after fermentation with different treatments.

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

[0045] Fig. 9 This is the result of the effect of co-fermentation of Pediococcus pentosaceus BCST003 and corn silk on flavonoids in the fermentation broth.

[0046] Fig.10 This is the effect of co-fermentation of Pediococcus pentosaceus BCST003 and corn silk on total phenolics in the fermentation broth.

[0047] Fig.11 This is the result of the effect of co-fermentation of Pediococcus pentosaceus BCST003 and corn silk on the total reducing sugar in the fermentation broth.

[0048] Fig.12 The results of fasting blood glucose concentration changes in mice treated with different methods.

[0049] Fig.13 The results of oral glucose tolerance test of mice treated with different methods (different letters indicate significant differences, P<0.05; same letters indicate no significant differences, P>0.05).

[0050] Fig.14 The results of insulin tolerance test of mice treated with different methods (different letters indicate significant differences, P<0.05; same letters indicate no significant differences, P>0.05).

[0051] Fig.15 The results of alanine aminotransferase detection in the serum of mice treated with different methods (different letters indicate significant differences, P<0.05; the same letters indicate no significant differences, P>0.05).

[0052] Fig.16 These are the results of the detection of aspartate aminotransferase levels in the serum of mice treated with different methods (different letters indicate significant differences, P<0.05; the same letters indicate no significant differences, P>0.05).

[0053] Fig.17 The whole liver, HE staining, PAS staining and Oil Red staining images of mice treated differently. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples 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 art.

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

[0056] MRS medium: 10.0 g of casein digest, 10.0 g of beef extract powder, 4.0 g of yeast extract powder, 2.0 g of triammonium 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, and 1.5 g of Tween-80. The above ingredients were dissolved in 1 L of single distilled water, and the final pH value was adjusted to 5.7±0.2.

[0057] The Bergey's Manual for Identification of Bacteria in the following examples is "Berger's Manual for Identification of Bacteria. 8th Edition [M]. Science Press, Buchanan, Gibbons. 1984.".

[0058] Pediococcus pentosaceus The 22227 strain was commercially available.

[0059] Corn silk was purchased from Guangzhou Zhixin Pharmaceutical Co., Ltd.

[0060] Example 1 Obtaining Pediococcus pentosaceus BCST003 strain

[0061] 1. Experimental Methods

[0062] (1) Strain screening and identification

[0063] The corn silk and water mixture was put into a juicer at a ratio of 1:4 for natural fermentation. The fermentation liquid was obtained by continuous fermentation for 9 days. The fermentation liquid was continuously diluted with deionized water in a 10-fold dilution gradient. 100 μL of the dilution gradient was taken out respectively. -3 , 10 -4 , 10 -5 The diluted fermentation broth was evenly spread on MRS medium (added with 3% CaCO3) and cultured in a 37°C constant temperature incubator for 48 hours. The strain with obvious transparent zone was selected, and single colonies were isolated by plate streaking method. One of the single colonies was selected and recorded as BCST003 strain for subsequent identification.

[0064] (2) 16S rDNA sequencing and identification

[0065] The genomic DNA of the BCST003 strain was extracted as a sample, and the quality of the genomic DNA was detected by agarose gel electrophoresis.

[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] The paired-end sequencing data was filtered 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, the results showed that the BCST003 strain has multiple 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 the strain was named Pediococcus pentosaceus BCST003. It was deposited in the Guangdong Provincial Microbiological Culture Collection on September 14, 2024, with the collection number GDMCC No: 65140, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0086] Example 3 Changes in chemical composition of Pediococcus pentosaceus and corn silk co-fermentation

[0087] 1. Experimental Methods

[0088] 1. Set up three treatment groups, namely:

[0089] (1) BCST003 group,

[0090] (2) Corn silk group,

[0091] (3) Pediococcus pentosaceus BCST003 and corn silk co-fermentation group (referred to as co-fermentation group).

[0092] The specific processing is:

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

[0094] Corn silk group: Wash and dry the dried corn silk, crush it into powder with an ultra-high speed crusher, sterilize it under high pressure, and obtain corn silk powder for later use. Mix the corn silk powder with water in a ratio of 1:10 and ferment at 37°C, and measure the relevant indicators at 0h, 24h, and 48h.

[0095] Pediococcus pentosaceus BCST003 and corn silk co-fermentation group: 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 in PBS (p=7.4) to obtain 1×10 9 cfu / mL of Pediococcus pentosaceus BCST003 strain suspension. Wash and dry the dried corn silk, crush the corn silk into powder with an ultra-high speed crusher, sterilize it under high pressure, and obtain corn silk powder for use. Mix the corn silk powder with water in a ratio of 1:10, inoculate the BCST003 strain suspension into the mixed solution at a ratio of 1:100 V / V, ferment at 37°C, and measure the relevant indicators at 0h, 24h, and 48h.

[0096] 2. LC-MS detection of chemical composition changes

[0097] The sample was taken out of the -80℃ freezer, thawed on ice, and vortexed for 10 seconds after thawing to mix. Take 50μL of the sample into an EP tube and add 150μL of pre-cooled ice methanol (containing 1μg / mL 2-chlorophenylalanine as an internal standard). Vortex for 3min, centrifuge at 12000g at 4℃ for 10min. Pipette the supernatant into another new EP tube. Centrifuge the supernatant at 12000g at 4℃ for 5min, and take the supernatant into the liner tube of the injection bottle for UPLC-MS / MS analysis.

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

[0099] The mass spectrometry conditions mainly include: electrospray ionization (ESI) temperature 500°C, mass spectrometry voltage 5500V (positive), -4500V (negative), ion source gas I (GS I) 55psi, gas II (GS II) 60psi, curtain gas (CUR) 25psi, collision-activated dissociation (CAD) parameters set to 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-high performance liquid chromatography (UPLC) (Shim-pack UFLC SHIMADZU CBM30A) and tandem mass spectrometry (MS / MS)

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

[0102] 2. Experimental Results

[0103] After 24 hours of fermentation, the chemical components of the fermentation broth of the BCST003 group, corn silk group, and co-fermentation group were tested in detail using liquid chromatography-mass spectrometry (LC-MS), and the chemical composition data after co-fermentation were analyzed.

[0104] PCA analysis results of chemical components after fermentation with different treatments Figure 5 The heat map of chemical component contents after fermentation under different treatments is shown in Figure 6 The analysis results of compounds with significant changes in content after fermentation under different treatments are shown in Figure 7 As shown in the figure, the results showed that the chemical composition of corn silk changed significantly after fermentation by P. pentosaceus. Among all the detected compounds, the following compounds changed most significantly: zeaxanthin, formononetin, protocatechuic acid, chlorogenic acid, luteolin, caffeic acid, rutin, acetic acid, methyl butyrate, and citric acid. These compounds showed significant changes in abundance in the fermented samples, indicating that the metabolic activity of P. 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 its stability and improved bioavailability 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 an increase in the levels of caffeic acid, acetic acid, methyl butyrate, and citric acid in the fermented samples. These compounds have potential applications in food preservation, flavor formation, and health benefits.

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

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

[0109] 1. Experimental Methods

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

[0111] (1) Determination of total sugar content

[0112] Preparation of standard curve: 1 mL of glucose standard solution with 0, 10, 20, 30, 40, 60, 80 μg / mL is prepared, 4 mL of anthrone reagent is added, and the solution is quickly immersed in an ice water bath to cool. After adding each tube, immerse them in a boiling water bath together, and cover the tube mouth to prevent evaporation. Boil again in the water bath, boil for 10 minutes, 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. Draw a standard curve with the standard glucose content (μg) as the horizontal coordinate and the absorbance value as the vertical coordinate. Dilute the test solution 10 times and operate according to the above method to calculate the total sugar content.

[0113] (2) Determination of polyphenol content

[0114] The polyphenol content was determined using the Folin Ciocalteu method. Specifically, 0.2 mL of appropriately diluted sample was mixed 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, the absorbance was read at 765 nm using a UV-visible spectrophotometer. The polyphenol content was calculated.

[0115] (3) Determination of flavonoid content

[0116] Preparation of rutin standard curve: accurately weigh 10 mg of rutin standard, dissolve it in 60% ethanol solution to 50 mL, and obtain a rutin standard solution with a concentration of 0.2 mg / mL. Take 0.4, 0.8, 1.2, 1.6, and 2.0 mg / mL rutin standard solutions respectively, add ethanol solution to 2 mL, add 1 mL of sodium nitrite solution, shake well, let stand for 6 minutes, add 1.5 mL of aluminum nitrate solution, shake well, let stand for 6 minutes, add 4 mL of 40% sodium hydroxide solution, shake well, and let stand for 15 minutes. The ethanol solution was diluted to 10 mL to obtain 0.008, 0.016, 0.024, 0.032, and 0.04 mg / mL rutin standard solutions, respectively. The sodium nitrite-aluminum nitrate colorimetric method was used to determine the absorbance of the extract solution. Finally, the least squares method was used for linear regression 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] For sample testing, aspirate 0.5 mL of sample, dilute the test solution 10 times, and operate according to the above method.

[0118] (4) Determination of reducing sugar content

[0119] Dilute the fermentation liquid to be tested 10 times, take 2 mL of the dilution and add 1.5 mL of DNS solution, mix well, react in a boiling water bath for 10 min, cool, dilute to 25 mL with distilled water, and shake well. Use the absorbance corresponding to the glucose standard solution (0-0.028 mg / mL) at 540 nm to draw a standard curve (y=0.0073x+0.0328, R 2 =0.9986), and calculate the reducing sugar content in the fermentation broth.

[0120] 2. Experimental Results

[0121] The experimental results showed that compared with the corn silk group, the co-fermentation group showed significant differences in many aspects.

[0122] (1) In terms of pH changes, the pH value of the co-fermentation group dropped significantly to 3.8 within 48 hours, while the pH value of the corn silk group alone did not change significantly ( Figure 8 This result may be related to the acidic metabolites produced by Pediococcus pentosaceus during the fermentation process, which can reduce the pH value of the environment and thus affect the pH value 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) ( Fig. 9 ), which indicated that Pediococcus pentosaceus promoted the release of flavonoids from corn silk during fermentation.

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

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

[0126] Example 5 Detection of hypoglycemic and lipid-lowering effects

[0127] 1. Experimental Methods

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

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

[0130] (1) Establishment of 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). Free drinking water was provided to the blank control group, and the model group mice were fed with ordinary feed. After 4 weeks of high-sugar and high-fat diet (HFD), the mice were intraperitoneally injected with 40 mg / kg of streptozotocin (STZ) freshly prepared in citric acid buffer (0.1 M, pH 4.5) for 5 consecutive days in the 5th week; the blank control group was injected with an equal volume of citric acid buffer. On the 3rd and 7th days after the first injection of STZ, the blood glucose level in the tail vein blood was measured, and mice with blood glucose concentration greater than 11.1 mmol / L were confirmed as hyperglycemic mice.

[0132] (2) Experimental groups and drug administration

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

[0134] At the same time, a blank control group of mice was set up.

[0135] The mice in the blank control group (CON) and the model group were intragastrically administered with normal saline, and the mice in the CS group, the Pediococcus pentosaceus BCST003 group, the CS-Pediococcus pentosaceus BCST003 group, and the CS-Pediococcus pentosaceus Group 22227 was treated with medication for 8 consecutive weeks. The specific medication methods for each group are as follows:

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

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

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

[0139] CS-Pediococcus pentosaceus Group 22227: Oral administration of corn silk and Pediococcus pentosaceus 22227 co-fermentation liquid, once a day, oral gavage dose 0.2mL / 10g.

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

[0141] (2) Oral glucose tolerance test (OGTT)

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

[0143] (3) Insulin tolerance test (ITT)

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

[0145] (4) Biochemical index detection

[0146] All mice were fasted and allowed to drink water freely for 12 h before being killed. The eyeballs of the mice were removed to collect blood. The blood was collected into a 1.5 mL sterile, pyrogen-free EP tube and centrifuged at 300 g for 10 min. The transparent supernatant was aspirated into a new EP tube. After repeated centrifugation, the transparent supernatant was collected again and stored in a -80 ° C refrigerator. The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were detected using an automatic biochemical analyzer.

[0147] (5) HE staining

[0148] 1) Sampling: Fresh mouse liver tissue and skeletal muscle were fixed in 4% paraformaldehyde solution at room temperature for more than 24 hours. The tissue was removed from 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 in the dehydrator, and dehydrate according to the gradient alcohol, specifically: treat in 75% alcohol, 85% alcohol, 90% alcohol, 95% alcohol, anhydrous ethanol, benzene, xylene I, xylene II, paraffin I, paraffin II, and paraffin III in sequence. The treatment time in each solution is 4h, 2h, 1.5h, 1.5h, 0.5h, 10-20min, 10-20min, 10-20min, 1.5h, 1.5h, and 1h, respectively.

[0150] 3) Embedding: Place the wax-impregnated tissue in an embedding machine for embedding. First, place 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, and attach the corresponding label. Cool it in a freezer at 20°C. After the wax solidifies, take out the wax block from the embedding frame and trim it.

[0151] 4) Slice: Use a paraffin slicer to slice the trimmed wax block, with a slice thickness of 4 μm. Float the slices in 40°C warm water on the slicer, flatten the tissue, pick it up with a slide, and bake it in a 60°C oven. After the water is dried and the wax is melted, take it out and store it at room temperature for later use.

[0152] 5) Dewaxing of paraffin sections: Place the sections in xylene I and xylene II for 20 minutes each. After taking them out, place them in anhydrous ethanol I, anhydrous ethanol II, and 75% alcohol for 5 minutes each. Finally, wash them with distilled water.

[0153] 6) Hematoxylin staining: Stain the sections in hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution (1% hydrochloric acid alcohol) for a few seconds, rinse with tap water, blue with bluing solution (0.6% ammonia water), and rinse with running water.

[0154] 7) Eosin staining: After the sections were dehydrated in 85% alcohol for 5 minutes, they were taken out and dehydrated in 95% alcohol for 5 minutes, and finally stained in eosin solution for 5 minutes.

[0155] 8) Dehydration and sealing: Put the slices into anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, xylene I, and xylene II in turn for 5 minutes each. After the slices become transparent in xylene, take them out and dry them slightly, and then seal them with neutral gum.

[0156] 9) Use a microscope to collect and analyze images.

[0157] (6) Oil Red O staining

[0158] 1) Preparation of liver frozen sections: Cut an appropriate amount of mouse liver, embed the tissue block in OCT frozen section embedding medium, transfer it to a pre-cooled cryostat, wait for it to completely solidify and the tissue sample to be fixed in the cryostat, set the section thickness to 5 μm, take an adhesive slide and use the front side to attach the cut sample and mark it.

[0159] 2) Dry the slices: Dry the frozen slices at room temperature.

[0160] 3) Oil red staining: Place the sections in Oil Red O working solution and stain for 7-10 minutes in the dark.

[0161] 4) Color adjustment of sections: Place the sections in differentiation solution (60% ethanol) for 5-10 seconds until the stroma is clear. The infiltration time can be appropriately changed to control the color under the microscope until the fat 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) Hematoxylin staining of cell nuclei: Place the slices in hematoxylin staining solution for 2-4 minutes to stain the nuclei on the slices; rinse the slices twice with distilled water to wash away the stain that is not bound to the slices; then use differentiation solution (1% hydrochloric acid alcohol) to rapidly differentiate for 2-3 seconds to remove the dye adsorbed in the cytoplasm and the excessive dye bound to the cell nucleus. After rinsing the slices with distilled water, use hematoxylin blueing solution (0.6% ammonia water) to blue for 2-3 seconds, and then wash with tap water.

[0164] Glycerol gelatin sealing: first use absorbent paper to absorb the moisture around the slice, and then seal the slice with an aqueous sealing medium (glycerol gelatin, etc.). When sealing, if bubbles are found, do not press the coverslip to avoid displacement of lipid droplets, or temporarily do not seal the slice and observe directly.

[0165] (7) PAS staining

[0166] 1) Sampling: Fresh mouse liver tissue and skeletal muscle were fixed in 4% paraformaldehyde solution at room temperature for more than 24 hours. The tissues were removed from the fixative, trimmed flat in a fume hood, and placed in a dehydration box.

[0167] 2) Dehydration: Place the dehydration box into the hanging basket in the dehydrator, and dehydrate according to the gradient alcohol, specifically: treat in 75% alcohol, 85% alcohol, 90% alcohol, 95% alcohol, anhydrous ethanol, benzene, xylene I, xylene II, paraffin I, paraffin II, and paraffin III in sequence, and the treatment time is 4h, 2h, 1.5h, 1.5h, 0.5h, 10-20min, 10-20min, 10-20min, 1.5h, 1.5h, and 1h, 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, put it in the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Place it in a freezer at 20°C to cool. After the wax solidifies, take out the wax block from the embedding frame and trim it.

[0169] 4) Sectioning: Use a paraffin slicer to slice the trimmed wax block into 4 μm thick slices. Float the slices in 40°C warm water on the slide spreader. Flatten the tissue and pick it up with a glass slide. Place the slices in a 60°C oven and bake until the water is dried and the wax is melted. Remove the slices and store at room temperature for later use.

[0170] 5) Dewaxing of paraffin sections: Place the sections in xylene I and xylene II for 20 minutes each. After taking them out, place them in anhydrous ethanol I, anhydrous ethanol II, and 75% alcohol for 5 minutes each. Finally, wash with distilled water.

[0171] 6) Periodic acid staining: After dewaxing and water washing, immerse the slices in the periodic acid staining solution in the microwave repair box for 15 minutes, then take out the slices and wash them with distilled water for 2-3 times.

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

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

[0174] 9) Dehydration and sealing: The slices were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min. After the slices became transparent in xylene, they were taken out of xylene, slightly dried, and sealed with neutral gum.

[0175] 10) Microscope examination, image acquisition and analysis.

[0176] 2. Experimental Results

[0177] The results of fasting blood glucose concentration changes in mice treated with different methods are shown in Fig.12 The results of oral glucose tolerance test and insulin tolerance test of mice treated with different methods are shown in Fig.13 and Fig.14 As shown, the results showed that compared with the model group, the CS-Pentocereus BCST003 group and the CS-Pentocereus The blood glucose level of mice treated with group 22227 was significantly reduced. The fermented broth of corn silk fermented by 22227 strains can reduce the blood sugar content of mice, among which BCST003 strain has a significantly better blood sugar-lowering effect than 22227 strain( Fig.12 ).

[0178] The results of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice treated with different methods were as follows: Fig.15 and Fig.16 As shown, the results showed that compared with the model group, the CS-Pentocereus BCST003 group and the CS-Pentocereus The levels of ALT and AST in the mice treated with 22227 group were significantly reduced, and the levels of ALT and AST in the serum of the mice treated with CS-Pentosaceae BCST003 group were higher than those of the mice treated with CS-Pentosaceae The 22227 group was lower. This indicates that the co-fermentation product of BCST003 strain and corn silk has the effect of improving liver function.

[0179] The whole liver, HE staining, PAS staining and oil red staining of mice treated with different methods are shown in the figure. Fig.17 As shown, the results show that:

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

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

[0182] The liver tissue of each group of mice was stained with PAS to detect the content of liver glycogen. The results of PAS staining showed that compared with the CON group, the liver cell structure of the MOD group was damaged and the glycogen storage was lower. After treatment with the co-fermentation liquid of corn silk and Pediococcus pentosaceus BCST003, the liver cell structure was significantly restored and the glycogen storage in the liver cells increased significantly.

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

[0184] In summary, the results of this experiment show that the co-fermentation product of Pediococcus pentosaceus BCST003 and corn silk has significant effects in lowering blood sugar, improving liver function and alleviating liver fatty degeneration, etc. 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 implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A co-fermentation product of Pediococcus pentosaceus and corn silk, characterized in that: Obtained by co-fermentation of Pediococcus pentosaceus and corn silk.

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

3. The co-fermentation product of Pediococcus pentosaceus and corn silk according to claim 1 or 2, characterized in that: The Pediococcus pentosaceus is Pediococcus pentosaceus BCST003 or Pediococcus pentosaceus 22227, the Pediococcus pentosaceus BCST003 has been deposited in Guangdong Provincial Microbiological Culture Collection Center on September 14, 2024, and its preservation number is GDMCC No: 65140.

4. 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.

5. A product, characterized in that The invention comprises the co-fermentation product of Pediococcus pentosaceus and corn silk as described in any one of 1-3, or the Pediococcus pentosaceus BCST003 as described in claim 4.

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

7. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to any one of claims 1 to 3, the Pediococcus pentosaceus BCST003 according to claim 4, or the product according to claim 5 in the preparation of lipid-lowering products.

8. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to any one of claims 1 to 3, Pediococcus pentosaceus BCST003 according to claim 4, or the product according to claim 5 in the preparation of a product for improving liver function or mild hepatic fatty degeneration.

9. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to any one of claims 1 to 3, the Pediococcus pentosaceus BCST003 according to claim 4, or the product according to claim 5 in the preparation of a drug for treating fatty liver.

10. Use of the co-fermentation product of Pediococcus pentosaceus and corn silk according to any one of claims 1 to 3, Pediococcus pentosaceus BCST003 according to claim 4, or the product according to claim 5 in the preparation of a drug for treating diabetes.

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