Application of lactobacillus casei or combined fungicide thereof in preparation of polypeptide through fermentation

Abstract

The invention discloses application of lactobacillus casei or a combined microbial agent thereof to preparation of polypeptide through fermentation. The application comprises the step of preparing the polypeptide through combined fermentation of lactobacillus casei GYX-1 and lactobacillus delbrueckii subsp. Bulgaricus GYX-2. The method specifically comprises the following steps: diluting fermentation liquor obtained by fermenting in a carya illinoensis cake meal fermentation culture medium, and then performing ultrafiltration. The obtained hypoglycemic peptide does not need to be subjected to liquid chromatography separation, the amino acid sequence of the hypoglycemic peptide is directly analyzed by LC-MS / MS, the hypoglycemic peptide is synthesized to further verify the alpha-glucosidase inhibitory activity of the hypoglycemic peptide, the sequence of the hypoglycemic peptide is determined, and the efficiency is improved; the hypoglycemic peptide derived from the carya illinoensis cake meal can be used for preparing a functional preparation, and has hypoglycemic activity by inhibiting alpha-glucosidase.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of a Lactobacillus casei strain or a combined bacterial agent thereof in the fermentation preparation of polypeptides. Background Art

[0002] Lactic acid bacteria are a type of Gram-positive, anaerobic or facultative anaerobic, acid-resistant cocci or rods that can ferment sugars into lactic acid. Lactic acid bacteria mainly include Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus rhamnosus. Lactic acid bacteria can utilize various nutrients in the raw materials in solid-state fermentation and hydrolyze proteins into polypeptides through their own enzymes. This fermentation method has low requirements for the mechanization of polypeptide extraction. Therefore, lactic acid bacteria biological fermentation has now developed into one of the hot spots of biological research.

[0003] Diabetes is a common disease worldwide, and about 90% of diabetic patients suffer from type 2 diabetes. Delaying the decomposition of carbohydrates by blocking the action of digestive enzymes that decompose carbohydrates is an effective strategy for preventing diabetes. Carbohydrates in food are mainly absorbed and utilized by the human body in the form of monosaccharides in the intestine. After the polysaccharides such as starch ingested by the human body are decomposed into oligosaccharides and disaccharides by saliva and pancreatic α-amylase, they need to be enzymatically hydrolyzed into glucose under the action of α-glucosidase before they can be absorbed and utilized. Therefore, α-glucosidase has become an effective target for inhibiting carbohydrate absorption. At present, the α-glucosidase-inhibited hypoglycemic drugs that have been clinically used are mainly acarbose and voglipose, but the treatment of these drugs will more or less cause adverse reactions or side effects. Studies have shown that polypeptides with α-glucosidase inhibitory activity are a class of inducible substances, which mainly compete with the binding between polysaccharides and enzymes through active polypeptide molecules, reduce the binding rate between enzymes and polysaccharides, and make a large number of polysaccharides unable to be decomposed into glucose due to the lack of corresponding enzyme catalysis.

[0004] Pecan nuts, also known as pecans, are native to North America and are now cultivated in Jiangsu, Zhejiang, Henan, Hebei and other provinces in my country. Pecan nuts are rich in nutrients and high in oil. As one of the important woody oil-bearing tree species, they are an important guarantee for national grain and oil security. In addition, pecan nuts are rich in protein, amino acids, and essential mineral elements such as potassium and calcium, which can meet the daily nutritional needs of most people. At present, most of the pecan nuts in my country are discarded as waste or used only as feed fertilizer, ignoring the reuse of the by-product of oil extraction, the pecan cake, and the research and development of related products. The protein content of the by-product pecan cake is as high as 30%. These proteins and peptides have a variety of physiological functions, including anti-oxidation, blood sugar regulation and immune regulation, and have important potential to become functional foods. However, screening peptides with good α-glucosidase inhibitory activity is still difficult and requires luck. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of a strain of Lactobacillus casei GYX-1 or a combined bacterial agent thereof in the fermentation preparation of polypeptides.

[0006] The technical problem that the present invention needs to solve is to provide a polypeptide prepared by the above method.

[0007] The technical problem that the present invention also solves is to provide a peptide with a molecular weight of ≤3kDa, and then quickly screen out peptides with good α-glucosidase inhibitory activity through computer-assisted screening and molecular docking methods, thereby obtaining peptides with blood sugar lowering function.

[0008] The final technical problem to be solved by the present invention is to provide the use of the polypeptide in the preparation of a drug or preparation for lowering blood sugar.

[0009] Technical solution: In order to solve the above technical problems, the present invention provides the use of Lactobacillus casei GYX-1 or its combined bacterial agent in fermentation preparation of polypeptides, wherein the Lactobacillus casei GYX-1 was deposited in the General Microbiological Center of China National Committee for the Preservation of Microbiological Cultures on June 19, 2018, with a deposit number of CGMCC No.15956.

[0010] Among them, the application includes combining Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 for fermentation to prepare polypeptides, and the Lactobacillus delbrueckii subsp bulgaricus GYX-2 was deposited in the General Microbiology Center of China National Culture Collection Administration on December 3, 2020, with the deposit number CGMCC No. 21287.

[0011] The combined fermentation step comprises: fermenting a starter prepared by combining Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 in a thin-shell pecan cake fermentation medium, diluting the fermented liquid, and then ultrafiltrating the resultant.

[0012] The present invention also provides a method for preparing thin-shell pecan polypeptides by solid-state fermentation of a lactic acid bacteria combination, the method comprising fermenting a starter prepared by a combination of Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 in a thin-shell pecan cake fermentation medium, diluting the fermented liquid and then ultrafiltrating the resultant, wherein the Lactobacillus casei GYX-1 was deposited in the General Microbiology Center of the China National Microbiological Culture Collection on June 19, 2018, with a deposit number of CGMCC No. 15956, and the Lactobacillus delbrueckii subsp bulgaricus GYX-2 was deposited in the General Microbiology Center of the China National Microbiological Culture Collection on December 3, 2020, with a deposit number of CGMCC No. 21287.

[0013] The preparation of the starter comprises: inoculating Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 into MRS liquid culture medium at a bacterial liquid inoculation amount of 1.50wt% to 3.00wt%, respectively, culturing and mixing them at a certain volume ratio.

[0014] Wherein, the volume ratio of Lactobacillus casei GYX-1 to Lactobacillus delbrueckii subsp bulgaricus GYX-2 is 0.3-5:1.

[0015] Preferably, the culture temperature of the Lactobacillus casei GYX-1 and the Lactobacillus delbrueckii subspecies bulgaricus GYX-2 is 37° C., the culture time is 24 hours, and they are inoculated into MRS culture medium at an inoculation rate of 2%.

[0016] The method for preparing the thin-shell pecan cake fermentation medium comprises: crushing the thin-shell pecan cake, drying, and sieving to obtain thin-shell pecan cake powder, and adding distilled water to the thin-shell pecan cake powder for sterilization. The thin-shell pecan cake-derived hypoglycemic peptide of the present invention is derived from the thin-shell pecan cake, which is a byproduct of oil extraction from the thin-shell pecan cake of an important woody oil crop.

[0017] The fermentation step comprises: heating the thin-shell pecan cake solid fermentation medium to 110-130° C., keeping warm for 10-30 minutes, cooling to room temperature, adding 12.00%-21.00wt% of a starter under aseptic operation conditions; placing the inoculated thin-shell pecan cake culture medium in a constant temperature incubator at 37° C. for fermentation for 6-16 hours.

[0018] Preferably, 1.5 mL of bacterial solution is added to the thin-shell pecan cake culture medium and fermented in a constant temperature incubator at 37° C. for 12 hours.

[0019] The fermentation broth is diluted according to a weight-to-volume ratio of the fermentation broth to water of 1:5-10 (g / mL), extracted by shaking at room temperature for 1-3 hours, centrifuged at 3000-5000×g for 5-20 minutes, and the supernatant is ultrafiltered.

[0020] Wherein, the ultrafiltration membrane pressure of the ultrafiltration is 0.1-0.5MPa.

[0021] Preferably, the ultrafiltration membrane pressure is 0.3 MPa.

[0022] The present invention also includes the polypeptide prepared by the method.

[0023] Wherein, the amino acid sequence of the polypeptide is FAGDDAPR or LAGNPDDEFRPQ.

[0024] The present invention also includes freeze-drying the ultrafiltrate to determine the α-glucosidase inhibitory activity.

[0025] The present invention also includes the analysis of the glucose-lowering peptide sequence: LC-MS / MS is used to analyze the amino acid sequence of the glucose-lowering peptide.

[0026] The present invention also includes molecular docking screening: using Peptide Ranker software to predict the potential biological activity of the identified peptides, 15 peptides with potential α-glucosidase inhibitory activity were screened. AutoDockVina was used to perform molecular docking on 10 peptides, and the structure file of α-glucosidase was obtained from the protein structure database. The ligand and H 2 O was docked with the peptides, and the peptides with the lowest binding energy after docking, FAGDDAPR and LAGNPDDEFRPQ, were screened out.

[0027] The present invention also includes external synthesis verification: solid phase synthesis is performed on the two screened polypeptides, and the α-glucosidase inhibitory activity thereof is collected and determined.

[0028] Preferably, the LC-MS / MS uses a liquid chromatography analysis column packing: Reprosil-Pur120C18-AQ (75 μm id×250 mm, 1.9 μm), and the instrument used is Easy nLC 1200 / OrbitrapExploris480.

[0029] Preferably, the structure file of the α-glucosidase is obtained from the Protein Data Bank (PDB) with the code 3WY1.

[0030] The present invention also includes the use of the polypeptide in the preparation of blood sugar lowering drugs or preparations.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) The hypoglycemic peptide derived from the thin-shell pecan cake of the present invention is derived from the by-product of oil pressing of the thin-shell pecan, has a high protein content, and has a high utilization value; (2) The method of the present invention adopts Lactobacillus casei and Lactobacillus delbrueckii subspecies bulgaricus for fermentation, which are listed in the "List of Bacteria that Can Be Used in Food" issued by the Ministry of Health, and not only have strong polypeptide production capabilities, but also have the advantages of high safety; (3) The present invention adopts a molecular docking method to screen peptides with potential hypoglycemic function, which has the characteristics of high efficiency and high accuracy; (4) The hypoglycemic peptide derived from the thin-shell pecan cake of the present invention can be used to prepare functional preparations, and the effect of lowering blood sugar is achieved by inhibiting the activity of α-glucosidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The effects of Lactobacillus casei and combined lactic acid bacteria on the production of peptides by fermentation;

[0033] Figure 2 is the peptide content of the lactic acid bacteria combined fermentation and unfermented samples;

[0034] Figure 3 The effect of different inoculation amounts on the production of peptides by fermentation;

[0035] Figure 4 The effect of different material-liquid ratios on the production of polypeptides by fermentation;

[0036] Figure 5 is the peptide content of different ultrafiltration fractions after fermentation;

[0037] Figure 6 It is the total ion current of components below 3 kDa after fermentation;

[0038] Figure 7 is the MS spectrum of peptide LAGNPDDEFRPQ;

[0039] Figure 8is the MS image of peptide FAGDDAPR;

[0040] Fig. 9 It is the molecular docking result of peptide LAGNPDDEFRPQ; Fig. 9 The left picture shows the overall docking situation after molecular docking. Fig. 9 The right picture is a partial enlargement;

[0041] Fig.10 It is the molecular docking result of peptide FAGDDAPR; Fig.10 The left picture shows the overall docking situation after molecular docking. Fig.10 The right picture is a partial enlargement;

[0042] Fig.11 The results are the α-glucosidase inhibitory activity results of the synthetic peptides LAGNPDDEFRPQ and FAGDDAPR. DETAILED DESCRIPTION

[0043] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0044] The experimental determination method used in the embodiments of the present invention is as follows:

[0045] 1. Determination of α-glucosidase inhibition rate

[0046] α-glucosidase and D-pyranoglucoside (PNPG) were prepared into 0.2U / mL and 5mg / mL solutions respectively using 0.1mol / L, pH 6.8 phosphate buffer (PBS). Pipette 20μL PBS, 20μL α-glucosidase solution and 20μL sample into a 96-well plate, incubate at 37°C for 5min, and mix thoroughly. Then add 20μL PNPG solution and react at 37°C for 30min. Finally, add 80μL Na 2 CO 3 The reaction was terminated, and the content of p-nitrophenol generated by the reaction system was detected at a wavelength of 405 nm and recorded. Three parallels were performed for each group to calculate the enzyme inhibition rate of the sample.

[0047]

[0048] OD blank: absorbance value of blank group, only PBS and Na 2 CO3 The absorbance of

[0049] OD negative: absorbance value of negative group, absorbance of sample replaced by an equal amount of buffer;

[0050] OD background: absorbance of the background group, the absorbance of an equal amount of buffer replacing α-glucosidase;

[0051] OD sample: absorbance value of the sample group, including the absorbance of the sample reaction system.

[0052] 2. Determination of polypeptide content: Biuret method was used to take an appropriate amount of glutathione and prepare 0 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.2 mg / mL, 1.6 mg / mL and 2 mg / mL with distilled water. 1 mL of standard solution of different concentrations was taken respectively, and 4 mL of biuret reagent was added. The mixture was fully mixed on a vortex mixer, allowed to stand for 10 min, and centrifuged at 2000 r / min for 10 min. The absorbance of the supernatant was measured at 540 nm. A standard curve was drawn based on the absorbance value (y) and the polypeptide content (x). The regression equation y = 0.1126x + 0.0031, R 2 =0.9929.

[0053] Mix the sample solution with 10% (w / v) trichloroacetic acid in equal volumes, let stand for 10 min, and centrifuge at 4000 r / min for 10 min. Take 1 mL of the supernatant and add 1 mL (0.1 mg / mL NaOH 0.75 mL, 0.01 mg / mL CuSO 4 0.25mL) biuret reagent, mix thoroughly, let stand for 30 minutes. Centrifuge at 2000r / min for 10 minutes, measure the absorbance of the supernatant at 540nm, and substitute the standard curve to calculate the absorbance.

[0054] The method for preparing thin-shell walnut polypeptide by fermenting a combination of Lactobacillus casei and Lactobacillus delbrueckii subspecies bulgaricus comprises the following steps:

[0055] Example 1 Fermentation of thin-shell pecan cake with Lactobacillus casei and lactic acid bacteria composition

[0056] (1) Preparation of starter: taking out glycerol-preserved Lactobacillus casei GYX-1 with a preservation number of CGMCC No. 15956 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 with a preservation number of CGMCC No. 21287 from a -80°C refrigerator, inoculating them into MRS liquid culture medium at an inoculum amount of 5% (v / v), and culturing them in an incubator at 37°C for 24 hours for activation; inoculating the activated Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 into MRS liquid culture medium at an inoculum amount of 2.00% under aseptic operation conditions, and culturing them to the logarithmic phase;

[0057] (2) Preparation of thin-shell pecan cake fermentation medium: Thin-shell pecan cake (provided by Jiangsu Academy of Agricultural Sciences) was crushed, dried at 55°C, and passed through a 40-mesh sieve to obtain thin-shell pecan cake powder. 5 g of thin-shell pecan cake powder was added to a 50 mL conical flask, 5 mL of distilled water was added, and sterilized at 121°C for 15 min.

[0058] (3) Inoculation and fermentation: Repeat steps (1) to (2) to prepare two more identical fermentation mediums, heat the above thin-shell pecan cake solid fermentation medium to 121°C, keep it warm for 20 minutes, cool it to room temperature, and under aseptic operation conditions, inoculate it with 1.5 mL of Lactobacillus casei GYX-1; add 1.5 mL of a combined bacterial solution of Lactobacillus casei GYX-1 and Lactobacillus delbrueckii bulgaricus GYX-2 in a volume ratio of 1:1. The above thin-shell pecan cake medium was placed in a constant temperature incubator at 37°C and fermented for 12 hours.

[0059] (4) Take out 2.00 g of fermented thin-shell pecan cake and dilute it with water at a ratio of 1:9 (g / mL), shake it at room temperature for 2 hours for extraction, centrifuge it at 4000×g for 10 minutes, take the supernatant to determine the polypeptide content and compare it with the unfermented sample. Figure 1 It can be seen that compared with uninoculated fermentation, Lactobacillus casei GYX-1 has better ability to produce polypeptides by fermentation. The polypeptide content of thin-shell pecan cake after fermentation reached 81.92±1.46mg / g, while the combined fermentation of Lactobacillus casei GYX-1 and Lactobacillus delbrueckii bulgaricus GYX-2 reached 86.17±1.94mg / g. Therefore, the combined strain fermentation was selected for subsequent process exploration.

[0060] Example 2 Effects of different fermentation times on walnut polypeptides

[0061] As in Example 1, steps (1) to (2) were repeated to prepare four identical fermentation mediums, except that only 1.5 mL of a combined bacterial solution of Lactobacillus casei GYX-1 and Lactobacillus delbrueckii bulgaricus GYX-2 in a volume ratio of 1:1 was added according to the method of step (3) and the polypeptide content was measured after fermentation for 0 h, 6 h, 12 h and 18 h to determine the optimal fermentation time. Figure 2 It can be seen that the combined fermentation of Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subspecies bulgaricus GYX-2 not only significantly increased the polypeptide content of thin-shell pecan cake, but also had the best effect when fermented for 12 hours, with the highest polypeptide content reaching 86.67±2.43 mg / g.

[0062] Example 2 Effect of inoculation amount on walnut polypeptide

[0063] As in Example 1, steps (1) to (2) were repeated to prepare three identical fermentation media, except that in step (3), 1.2 mL, 1.5 mL, 1.8 mL, and 2.1 mL of bacterial solution of Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subspecies bulgaricus GYX-2 were added at an inoculum amount of 12.00 wt%, 15.00 wt%, 18.00 wt%, and 21.00 wt%, respectively, in a volume ratio of 1:1. After fermentation for 12 h, the polypeptide content was determined. The results showed that ( Figure 3 ), when the inoculation amount was 15%, i.e., 1.5 mL of bacterial solution was added, the polypeptide content was significantly higher than that at other inoculation amounts, and the highest fermentation polypeptide content was 86.11±3.01 mg / g.

[0064] Example 3 Effect of material-liquid ratio on walnut polypeptide

[0065] As in Example 1, steps (1) to (2) were repeated to prepare three identical fermentation media, except that in step (2), 5 g of thin-shell pecan cake powder was added to distilled water at a material-liquid ratio of 1:1.4, 1:1, 1.4:1, and 1.2:1 (g / mL) for sterilization, and subsequent combined bacterial fermentation was performed for 12 hours. After fermentation, the polypeptide content was determined. The results are shown in FIG. Figure 4 As shown in the figure, different material-liquid ratios have a certain effect on the output of fermented polypeptides. When the material-liquid ratio is 1:1, the polypeptide output is the highest, which is 88.97±2.50 mg / g.

[0066] Therefore, the optimal fermentation conditions adopted were as follows: 5 g of thin-shell pecan cake powder was added with distilled water for sterilization in a solid-liquid ratio of 1:1 (g / mL), and 1.5 mL of bacterial solution of Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subspecies bulgaricus GYX-2 was added in a volume ratio of 1:1 for fermentation for 12 h.

[0067] Example 4

[0068] The supernatant of the combined fermentation of Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subspecies bulgaricus GYX-2 in Example 5 was ultrafiltered at an ultrafiltration membrane pressure of 0.3 MPa to determine the α-glucosidase inhibitory activity of different components; the results are as follows Figure 4 As shown, the component with a molecular weight of ≤3 kDa has a higher α-glucosidase inhibitory activity than other molecular weight components, and its α-glucosidase inhibitory activity reaches 34.72±4.03%. As a key enzyme related to type 2 diabetes, inhibiting α-glucosidase is one of the methods to control postprandial hyperglycemia. Therefore, the fermented thin-shell pecan cake crude polypeptide is expected to be used in the fields of lowering blood sugar and inhibiting obesity.

[0069] Example 5

[0070] The sequence analysis of the polypeptides of the thin-shell pecan cake meal comprises the following steps:

[0071] The peptide structure was identified by liquid chromatography-mass spectrometry, the chromatographic column was packing: Reprosil-Pur120C18-AQ (75 μm id×250 mm, inner diameter 1.9 μm), the mobile phase A was ultrapure water containing 0.1% formic acid, the mobile phase B was an aqueous solution containing 0.1% formic acid and 80% acetonitrile, and the specific chromatographic conditions were: 0-2 min, 8% B; 2-35 min, 8-28% B; 35-55 min, 28-40% B; 55-56 min, 40-95% B; 56-66, 95% B. The mass spectrometry conditions were as follows: the full scan range of the mass spectrometry was 100-1500 m / z, the primary mass spectrometry resolution was set to 120000, AGC was Standard, and MaximumIT was 20 ms; the secondary mass spectrometry resolution was set to 15000, AGC was Standard, and MaximumIT was 22 ms, and the peptide fragmentation collision energy was set to 30. The raw data of mass spectrometry detection were generated, and the total ion current was as shown in the figure. Figure 6 shown.

[0072] Example 6

[0073] The screening of the thin-shell pecan cake meal with potential hypoglycemic activity peptides comprises the following steps:

[0074] (1) Computer-assisted screening: Peptide Ranker software was used to predict the potential biological activity of the identified peptides, and 15 peptides with higher peptide ranks were screened out. Among them, two peptides were LAGNPDDEFRPQ and FAGDDAPR. The molecular weight of LAGNPDDEFRPQ was 1357.62, the length was 12, and the peptide ranker was 0.566446. The molecular weight of FAGDDAPR was 6847.3824, the length was 8, and the peptide ranker was 0.641361.

[0075] (2) Molecular docking screening: The LAGNPDDEFRPQ and FAGDDAPR obtained in step (3) were molecularly docked using the AutoDock Vina molecular docking software. The structure file of α-glucosidase was obtained from the Protein Data Bank (PDB) with the code 3WY1. The ligand and H 2 0 and then docked with the peptide. The docking results of LAGNPDDEFRPQ and FAGDDAPR are shown in Fig. 9 , Fig.10 As shown in the figure, the binding energy of LAGNPDDEFRPQ after docking is -7.5 kcal / mol, and the binding energy of FAGDDAPR is -7.6 kcal / mol.

[0076] Example 7 Synthesis and Verification

[0077] Based on the binding energy after docking, the score reflects the stability of the peptide after binding to the protein. The lower the binding energy, the stronger and tighter the binding between the peptide and the protein. Based on this, two peptides FAGDDAPR and LAGNPDDEFRPQ were selected from the peptides screened in Example 7 for solid phase synthesis (Nanjing Jiepeptide Biotechnology Co., Ltd. was commissioned to synthesize the synthetic peptide using solid phase chemical synthesis, and the obtained synthetic peptide was a polypeptide solid powder with a purity of >95%). Labeled as synthetic peptide FAGDDAPR and synthetic peptide LAGNPDDEFRPQ. Figure 8 is the MS spectrum of peptide LAGNPDDEFRPQ; Figure 7 The MS graph of polypeptide FAGDDAPR is shown in Figure 2. The synthetic peptides were prepared into 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, and 10 mg / mL concentration synthetic peptide solutions with deionized water, and the in vitro α-glucosidase inhibitory activity of the two synthetic peptides was measured. The results are shown in Figure 2. Fig.11 As shown, the IC of LAGNPDDEFRPQ 50 The IC value of FAGDDAPR is 4.431 mg / mL. 50The results showed that LAGNPDDEFRPQ and FAGDDAPR were active peptides with hypoglycemic function derived from walnut cake, which are expected to be used in the preparation of functional preparations for lowering blood sugar.

Claims

1. Application of Lactobacillus casei GYX-1 or its combined bacterial agent in fermentation to prepare polypeptides, wherein the Lactobacillus casei GYX-1 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on June 19, 2018, with a deposit number of CGMCC No.15956.

2. The use according to claim 1, characterized in that: The application includes fermenting Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 in combination to prepare polypeptides, wherein the Lactobacillus delbrueckii subsp bulgaricus GYX-2 was deposited in the General Microbiology Center of China National Culture Collection Administration on December 3, 2020, with the deposit number CGMCC No.21287.

3. The use according to claim 1 or 2, characterized in that: The combined fermentation step comprises: fermenting a starter prepared by combining Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 in a thin-shell walnut cake fermentation medium, diluting the fermented liquid, and then ultrafiltrating the resultant.

4. The use according to any one of claims 1 to 3, characterized in that: The preparation of the starter comprises: inoculating Lactobacillus casei GYX-1 and Lactobacillus delbrueckii subsp bulgaricus GYX-2 at a bacterial liquid inoculation amount of 1.50wt% to 3.00wt%, respectively, culturing, and mixing them at a certain volume ratio.

5. The use according to claim 4, characterized in that: The volume ratio of Lactobacillus casei GYX-1 to Lactobacillus delbrueckii subsp bulgaricus GYX-2 is 0.5-3:

1.

6. The use according to claim 3, characterized in that: The method for preparing the thin-shell pecan cake fermentation medium comprises: crushing the thin-shell pecan cake, drying, and sieving to obtain thin-shell pecan cake powder, and adding distilled water to the thin-shell pecan cake powder according to a material-liquid ratio of 1:1.4 to 1.2:1 (g / mL) for sterilization.

7. The use according to claim 3, characterized in that: The fermentation step comprises: heating the thin-shell pecan cake solid fermentation medium to 110-130° C., keeping the temperature for 10-30 minutes, cooling to room temperature, adding 12.00%-21.00wt% of a starter under aseptic operation conditions; placing the inoculated thin-shell pecan cake culture medium in a constant temperature incubator at 37° C. for fermentation for 6-16 hours.

8. The use according to claim 3, characterized in that: The fermentation broth is diluted according to a weight-to-volume ratio of the fermentation broth to water of 1:5-10 (g / mL), extracted by shaking at room temperature for 1-3 hours, centrifuged at 3000-5000×g for 5-20 minutes, and the supernatant is ultrafiltered. Preferably, the ultrafiltration membrane pressure of the ultrafiltration is 0.1-0.5 MPa.

9. The polypeptide prepared by the method according to any one of claims 1 to 7, characterized in that The amino acid sequence of the polypeptide is FAGDDAPR or LAGNPDDEFRPQ.

10. Use of the polypeptide according to claim 9 in the preparation of a drug or preparation for lowering blood sugar.

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