Lactic acid bacteria CF80 with high yield of amylase and application thereof

Abstract

The invention discloses lactic acid bacteria CF80 with high yield of amylase and application of the lactic acid bacteria CF80, and belongs to the technical field of bioengineering and food engineering. According to the invention, a strain of Lactobacillus sp. CF80 with high yield of amylase is obtained through screening, the Lactobacillus sp. CF80 is preserved in China Center for Type Culture Collection on January 20, 2025, and the preservation number is CCTCC NO: M 2025186. The strain is used for tartary buckwheat fermentation, the degradation rate of starch in tartary buckwheat can be remarkably increased, and the prepared tartary buckwheat protein peptide has high ACE inhibitory activity and has huge application potential in the field of food.

Description

Technical Field

[0001] The present invention relates to Lactobacillus CF80 with high amylase activity and its application, belonging to the technical fields of bioengineering and food engineering. Background Art

[0002] Lactic acid bacteria are a group of Gram-positive bacteria that can rapidly ferment carbohydrates and have lactic acid as the main metabolite. They are the most widely used probiotics and have important physiological and health functions such as regulating the balance of human intestinal flora, enhancing immunity, and delaying aging. They are widely used in industries such as food, medicine, and health products. In the field of fermented foods, lactic acid bacteria can improve the flavor and quality of foods and extend the shelf life of foods. Fermented foods with lactic acid bacteria are also considered functional foods. It is then pointed out that the vast majority of lactic acid bacteria cannot utilize starch, and their amylase activity is low or even non-existent, resulting in the inability to utilize starch and only being able to utilize the more expensive carbon source glucose.

[0003] Buckwheat is an extremely excellent cereal and contains various vitamins and trace elements essential for the human body. Buckwheat contains about 10% protein. Buckwheat protein peptides can effectively inhibit the activities of two key enzymes, renin and angiotensin-converting enzyme (ACE), in the renin-angiotensin-aldosterone system (RAAS), further blocking the conversion of angiotensinogen into angiotensin I (Angiotensin I, AngⅠ) and then into angiotensin II (Angiotensin II, AngⅡ), and then causing blood vessels to dilate and reducing vascular resistance, playing a role in lowering blood pressure. Buckwheat contains rich starch raw materials. These starch raw materials are not required when buckwheat protein peptides exert their blood pressure-lowering effect. Especially when preparing buckwheat protein peptides, these starch raw materials will be discarded or disposed of at a low price as waste, resulting in a waste of resources. If these starch raw materials can be used as a carbon source for microbial growth to develop probiotic products such as lactic acid bacteria, it will be possible to avoid waste of starch resources and greatly improve economic benefits.

[0004] However, lactic acid bacteria usually cannot utilize starch raw materials as a carbon source. For natural cereal raw materials that contain both starch and components such as protein, it is also a key technical problem that needs to be explored and solved for lactic acid bacteria to utilize starch as a carbon source without destroying and retaining other beneficial components. If the starch raw materials of buckwheat can be used as a carbon source for the growth of lactic acid bacteria strains without affecting the protein content of buckwheat, integrating the blood pressure-lowering activity of buckwheat protein peptides and probiotic properties, and developing lactic acid bacteria-fermented buckwheat foods, it has an important auxiliary effect on hypertensive patients in controlling blood pressure and enhancing body immunity.

[0005] However, due to the strict nutritional requirements of lactic acid bacteria and the fact that the vast majority of them cannot directly utilize starch, they grow slowly in tartary buckwheat and have a low utilization rate of starch, which has become a bottleneck restricting the development of fermented tartary buckwheat. Summary of the Invention

[0006] Technical Problem: The object of the present invention is to address the problems such as the slow growth of lactic acid bacteria in tartary buckwheat and the low utilization rate of tartary buckwheat starch. Four strains of lactic acid bacteria with high amylase production were screened, which can effectively degrade the starch raw materials in tartary buckwheat without affecting the protein content. The starch in fermented tartary buckwheat is more easily digested, and the ACE inhibitory rate of tartary buckwheat protein peptides remains unchanged or even increases, which has an important auxiliary effect on hypertensive patients in controlling blood pressure and enhancing the body's immunity.

[0007] The present invention provides a strain of lactic acid bacteria with high amylase production, which is Lactobacillus sp. CF80. It was deposited at the China Center for Type Culture Collection on January 20, 2025, with the deposit number CCTCC NO: M2025186, and the deposit address is Wuhan University, Wuhan, China.

[0008] In one embodiment, the Lactobacillus CF80 has the characteristic of high amylase production.

[0009] In one embodiment, the cell morphology of the Lactobacillus CF80 is rod-shaped, Gram-positive, and sporeless; the colony morphological characteristics are: the colony diameter is about 1 mm, round, white, slightly transparent, the colony edge is irregular, and the surface is rough; the physiological and biochemical characteristics are facultative anaerobic, non-motile, producing acid but not gas from glucose metabolism, capable of metabolizing starch, and the growth temperature is 37 °C; the full length of the 16S rDNA sequence of this strain is about 1104 bp, and its sequence is as shown in SEQ ID NO.1.

[0010] The present invention provides a microbial preparation containing the Lactobacillus CF80.

[0011] In one embodiment, the content of the Lactobacillus is not less than 1.0×10 7 cfu / mL or 1.0×10 7 cfu / g.

[0012] The present invention provides a method for producing amylase, which uses the Lactobacillus CF80 to culture in a medium at 30-37 °C for a period of time, and then collects the amylase.

[0013] In one embodiment, the medium includes but is not limited to MRS medium.

[0014] The present invention also provides a method for degrading starch in tartary buckwheat, which is to ferment using the Lactobacillus CF80.

[0015] In one embodiment, the fermentation uses tartary buckwheat as the raw material.

[0016] In one embodiment, the tartary buckwheat is tartary buckwheat granules or tartary buckwheat powder after being pulverized.

[0017] In one embodiment, the tartary buckwheat is pulverized and sieved through a 100-mesh sieve.

[0018] In one embodiment, the method is to inoculate Lactobacillus sp. CF80 into the fermentation system so that the cell concentration after inoculation is ≥ 1×10 8 cfu / mL or 1×10 8 cfu / g.

[0019] In one embodiment, the method is to ferment Lactobacillus sp. CF80 in a solid-state fermentation system containing tartary buckwheat granules at 35 - 40 °C for 96 - 144 h.

[0020] In one embodiment, the method is to ferment Lactobacillus sp. CF80 in a liquid fermentation system containing tartary buckwheat powder at 35 - 40 °C for 60 - 84 h.

[0021] The present invention also provides the application of Lactobacillus sp. CF80 in the food field.

[0022] In one embodiment, the application is to prepare tartary buckwheat protein or tartary buckwheat protein peptides.

[0023] In one embodiment, the application is to prepare a product containing tartary buckwheat protein and / or tartary buckwheat protein peptides.

[0024] Beneficial effects: The strain provided by the present invention has good subculture stability. After several subcultures, the production capacity of amylase can be stably maintained at a certain level. The enzyme activity of Lactobacillus sp. CF80 is 28.92 U / mL. Applying this strain to the fermentation of tartary buckwheat can reduce costs, protect the environment, and the fermented tartary buckwheat is more easily digested. The degradation rate of tartary buckwheat starch is significantly increased, reaching 78.36%, while the content of tartary buckwheat protein is not significantly affected, and the tartary buckwheat protein peptides still have strong ACE inhibitory activity, with a value of 91.84%. These data indicate that fermenting tartary buckwheat with lactic acid bacteria has important practical significance and application value.

[0025] Biological material preservation

[0026] Lactobacillus sp. CF80, classified and named as Lactobacillus sp.CF80, was deposited at the China Center for Type Culture Collection on January 20, 2025, with the deposit number CCTCC NO: M 2025186 and the deposit address being Wuhan University, Wuhan, China. Brief Description of the Drawings

[0027] Figure 1 Colony morphology diagrams of Lactobacillus pentosus CF55, Lactiplantibacillus plantarum CF60, L. plantarum CF66, and Lactobacillus sp. CF80. A: CF55; B: CF60; C: CF66; D: CF80.

[0028] Figure 2 Diagrams of starch content and starch degradation rate of tartary buckwheat fermented by L. pentosus CF55, L. plantarum CF60, L. plantarum CF66, and Lactobacillus sp. CF80. A: Total starch content; B: Tartary buckwheat starch degradation rate.

[0029] Figure 3 Diagrams of starch digestibility and relative contents of rapid, slow, and resistant starch of tartary buckwheat fermented by L. pentosus CF55, L. plantarum CF60, L. plantarum CF66, and Lactobacillus sp. CF80. A: Starch digestibility of fermented tartary buckwheat; B: Relative contents of rapid, slow, and resistant starch.

[0030] Figure 4 Diagrams of protein content and protein extraction amount of tartary buckwheat fermented by L. pentosus CF55, L. plantarum CF60, L. plantarum CF66, and Lactobacillus sp. CF80. A: Protein content; B: Protein extraction amount.

[0031] Figure 5 Diagram of ACE inhibitory activity of protein hydrolysates of tartary buckwheat fermented by L. pentosus CF55, L. plantarum CF60, L. plantarum CF66, and Lactobacillus sp. CF80. Detailed Embodiments

[0032] The technical content of the present invention will be further described below in conjunction with the embodiments: The following embodiments are illustrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Culture medium:

[0034] MRS medium: Glucose 20 g / L, peptone 10 g / L, beef extract powder 10 g / L, yeast powder 5 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.62 g / L, MgSO4·7H2O 0.58 g / L, MnSO4·H2O 0.19 g / L, Tween-80 1 mL, agar 20 g / L in solid medium, pH 6.2 ± 0.2, sterilized at 115 °C for 15 min.

[0035] SS-MRS medium: Soluble starch 10 g / L, peptone 10 g / L, beef extract powder 10 g / L, yeast powder 5 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.62 g / L, MgSO4·7H2O 0.58 g / L, MnSO4·H2O 0.19 g / L, Tween-80 1 mL, agar 20 g / L in solid medium, pH 6.2 ± 0.2, sterilized at 121 °C for 20 min.

[0036] TBS-MRS medium: Tartary buckwheat powder 15 g / L, peptone 10 g / L, beef extract powder 10 g / L, yeast powder 5 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4·3H2O 2.62 g / L, MgSO4·7H2O 0.58 g / L, MnSO4·H2O 0.19 g / L, Tween-80 1 mL, agar 20 g / L in solid medium, pH 6.2 ± 0.2, sterilized at 121 °C for 20 min.

[0037] Isolation, screening and identification of Lactobacillus amylolyticus in Example 1

[0038] Isolation of strains: Take a certain amount of samples from commercially available yogurt, cheese, fermented sausage, pickles, tartary buckwheat, self-made sourdough and soil, add an appropriate amount of sterile physiological saline and homogenize for 10 min. Take 1 mL of the homogenate and perform gradient dilution with sterile physiological saline, shake evenly, and screen for lactic acid bacteria. The medium used is MRS medium. The solid culture condition is static culture at 37 °C for 48 h, and the liquid culture condition is static culture at 37 °C for 24 h.

[0039] Screening of strains: The screening process of strains is divided into three steps. First, the typical strains obtained from the raw materials are inoculated on MRS agar medium and statically cultured at 37 °C for 48 h, and then separated and purified until pure cultures are obtained. Second, the typical strains obtained after separation and purification are inoculated on SS-MRS agar medium and statically cultured at 37 °C for 48 h. Then, Lugol's iodine solution is dropped around the strains, and Lactobacillus amylolyticus is screened according to the color-changing circle. Third, the strains that produce color-changing circles are inoculated into SS-MRS liquid medium and statically cultured at 37 °C for 24 h. Then, 1 mL of the bacterial liquid is centrifuged at 4500 r / min for 15 min, 40 μL of the supernatant is taken, 40 μL of 0.1 M PBS buffer is added, and after mixing, 100 μL of Lugol's iodine solution is added. After color development, it is diluted 10 times and 150 μL of the sample is transferred into a transparent flat-bottom 96-well microplate, and the absorbance value at 580 nm is measured by an enzyme-linked immunosorbent assay (ELISA) reader. According to the standard curve y = 0.128x + 0.0958 of the color reaction of different concentrations of soluble starch solution (prepared using 20 mg / mL soluble starch standard solution) and iodine solution, R 2 = 0.9993, the starch degradation rate of Lactobacillus amylolyticus is quantitatively calculated, and 4 strains with a starch degradation rate above 88% are obtained. Their colony morphologies are as Figure 1 shown.

[0040] Identification of strains: The DNA of the 4 strains with high starch degradation rate is extracted using FastPure Bacterial DNA Isolation Mini Kit - BOX 2 (Nanjing Novoprotein Scientific Co., Ltd., China). The upstream and downstream primers for PCR are universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), respectively. The thermal cycling parameters are pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s. After 35 cycles, extension at 72 °C for 10 min. The PCR products are sequenced by Suzhou Genewiz Biotechnology Co., Ltd. The gene sequences of the strains are aligned through the BLAST tool, and it is determined that the 4 strains are Lactobacillus pentosus L.pentosus CF55, Lactobacillus plantarum L.plantarum CF60, Lactobacillus plantarum L.plantarum CF66, and Lactobacillus sp.CF80, respectively.

[0041] Preparation of fermented tartary buckwheat samples in Example 2

[0042] Solid-state fermentation: It is used to determine the protein extraction amount in fermented tartary buckwheat. 20 g of tartary buckwheat granules are mixed with 40 mL of MRS medium without carbon source and sterilized. The strains screened in Example 1 are used at 5×10 8Inoculate them into the solid-state fermentation system at a concentration of cfu / g respectively, and ferment at 37 °C for 120 h. After fermentation, freeze-dry the tartary buckwheat granules and grind them through a 100-mesh sieve. The control group uses the same volume of sterile normal saline instead of the bacterial solution under the same conditions.

[0043] Liquid fermentation: Used to determine the total starch content, starch degradation rate in fermented tartary buckwheat, extract proteins and hydrolyze them into peptides to determine the ACE inhibitory activity. After grinding the tartary buckwheat granules, weigh 15 g and mix it with 300 mL of MRS medium without carbon source for sterilization. The strains screened in Example 1 were respectively inoculated at a concentration of 5×10 8 cfu / g and fermented at 37 °C for 72 h. The control group uses the same volume of sterile normal saline instead of the bacterial solution under the same conditions.

[0044] Determination of total starch content and starch degradation rate of fermented tartary buckwheat in Example 3

[0045] Weigh 0.1 g of the tartary buckwheat sample obtained after liquid fermentation and disperse it by shaking with 10 mL of sodium acetate solution (pH 5). Add 0.1 mL of α-amylase solution, transfer it to a 50 °C water bath for 10 min after boiling water bath for 30 min, then add 0.1 mL of amyloglucosidase solution, disperse it by shaking, and keep it in a 50 °C water bath for 30 min. Centrifuge the cooled sample at 10000 rpm for 5 min. Dilute the supernatant several times with sodium acetate solution, take 0.05 mL and mix it with 1.5 mL of glucose oxidase-peroxidase (GOPOD), and react at 50 °C for 30 min. Calculate the total starch content by recording the absorbance at 510 nm. After determination, the total starch contents of the tartary buckwheat samples fermented by 4 strains of bacteria, Lactobacillus pentosus CF55, Lactobacillus plantarum CF60, Lactobacillus plantarum CF66, and Lactobacillus sp. CF80, were 41.30%, 40.01%, 39.73%, and 31.30% respectively ( Figure 2 A).

[0046] According to the total starch content and the moisture content measured from the samples after liquid fermentation, calculate the remaining starch amount in the dry basis of the fermented tartary buckwheat samples, and then obtain the starch degradation rate of tartary buckwheat. After determination, the starch degradation rates of the tartary buckwheat samples fermented by Lactobacillus pentosus CF55, Lactobacillus plantarum CF60, Lactobacillus plantarum CF66, and Lactobacillus sp. CF80 were 43.68%, 50.62%, 47.46%, and 78.36% respectively ( Figure 2 B). It shows that these 4 strains of starch-degrading lactic acid bacteria can effectively degrade the starch raw materials in tartary buckwheat, and the effect of CF80 is the most significant.

[0047] Determination of the Digestibility of Fermented Tartary Buckwheat Starch in Example 4

[0048] Calculate and weigh a certain amount of fermented tartary buckwheat powder according to the total starch content measured in Example 3 (ensuring that the initial total starch content of each sample is 200 mg), add 10 mL of 0.2 mol / L sodium acetate buffer solution (pH 5.2), shake well, incubate in a constant temperature water bath shaker at 37 °C for 20 min to simulate oral digestion. Then add 14.5 mL of buffer solution. After taking out the 0 min sample, add the prepared complex enzyme solution of 0.6 μL of α-amylase (60.0 U) and glucoamylase (31.3 U), and supplement the buffer solution to make the total system reach 25 mL. Pipette 100 μL of the enzyme hydrolysis solution at 10, 20, 30, 45, 60, 90, 120, and 180 min respectively, and add 900 μL of absolute ethanol to terminate the reaction. After centrifugation, pipette 6 μL of the supernatant, add 174 μL of the assay reagent of the glucose kit, and incubate at 37 °C for 30 min. Measure the absorbance at 520 nm with an enzyme-labeled instrument and calculate the amount of glucose. Calculate the starch digestibility according to Formula 1.

[0049] Digestibility (%) = { (m s - m0) × 0.9 / [m × (100 - mc) × sc / 10000]} × 100 (Formula 1);

[0050] where, m s is the amount of glucose in the sample, m0 is the amount of glucose in the blank, 0.9 is the conversion coefficient between starch and glucose, m is the weight of the sample, mc is the moisture percentage of the sample, and sc is the starch percentage of the sample.

[0051] After determination, the digestibility of the fermented tartary buckwheat samples has increased ( Figure 3 A), the proportion of rapidly digestible starch has increased by 6.69% - 7.98%, the proportion of slowly digestible starch has not changed significantly (-3.18% - +4.10%), and the proportion of resistant starch has decreased by 3.63% - 12.08% ( Figure 3 B). It shows that adding lactic acid bacteria fermentation can effectively improve the digestive characteristics of tartary buckwheat.

[0052] Determination of the Protein Content and Protein Extraction Yield of Fermented Tartary Buckwheat in Example 5

[0053] The protein content was determined by the Kjeldahl method. After determination, the protein contents of the tartary buckwheat samples fermented by Lactobacillus pentosus CF55, Lactobacillus plantarum CF60, Lactobacillus plantarum CF66, and Lactobacillus sp. CF80 were 14.78%, 15.56%, 14.93%, and 14.68% respectively ( Figure 4 A).

[0054] The protein was extracted by the alkali-solution and acid-precipitation method, and then freeze-dried and weighed. After determination, the protein extraction amounts of the tartary buckwheat samples fermented by Lactobacillus pentosus CF55, Lactobacillus plantarum CF60, Lactobacillus plantarum CF66, and Lactobacillus sp. CF80 were 566.8, 564.8, 570.1, and 565.5 mg respectively ( Figure 4 B).

[0055] Therefore, the screened strains can effectively utilize the starchy raw materials in tartary buckwheat without affecting the protein content.

[0056] Example 6 Determination of ACE Inhibitory Activity

[0057] The protein extracted in Example 5 was hydrolyzed to obtain a hydrolysate. The specific steps were as follows: The protein extracted in Example 5 was used to prepare a tartary buckwheat protein dispersion with PBS buffer (pH 7.00), the pH was adjusted to 2.00, and pepsin (2500 U / mg, purchased from Sigma-Aldrich, catalog number 9001-75-6) at 4% of the protein mass was added, and hydrolysis was carried out at 37 °C for 2 h. Then the pH was adjusted to 7.00, and a certain amount of trypsin (1500 U / mg, purchased from Sigma-Aldrich, catalog number 9002-07-7) was added, and hydrolysis was carried out at 37 °C for several hours. The specific hydrolysis conditions were determined by single-factor and orthogonal experiments. After inactivating the enzyme by boiling, centrifugation was carried out at 10000 r / min for 15 min, and the supernatant was the crude tartary buckwheat protein hydrolysate.

[0058] 100 μL of 1 M N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) (dissolved in 50 mM Tris-HCl buffer containing 0.3 mM NaCl, pH 7.5), 50 μL of fermented tartary buckwheat protein hydrolysate, and 50 μL of ACE (dissolved in borate buffer, pH 8.3) were added successively to a 96-well plate and incubated at 37 °C for 30 min. The absorbance values before and after 30 min of reaction were measured at 340 nm. The blank was replaced with the buffer solution instead of the fermented tartary buckwheat protein hydrolysate. The ACE inhibitory activity of the fermented tartary buckwheat sample was calculated according to Equation 2.

[0059] ACE inhibitory activity (%) = [(ΔA 空白 - ΔA 样品 ) / ΔA 空白 × 100 (Equation 2);

[0060] where: ΔA 空白 is the decrease in absorbance of the blank group within 30 min, and ΔA 样品 is the decrease in absorbance of the sample group within 30 min.

[0061] It was determined that the ACE inhibitory activity of the peptides hydrolyzed from the proteins of the unfermented tartary buckwheat sample was 81.56%, and the ACE inhibitory activities of the protein hydrolysates of the fermented tartary buckwheat samples by Lactobacillus pentosus L.pentosus CF55, Lactobacillus plantarum L.plantarum CF60, Lactobacillus plantarum L.plantarum CF66, and Lactobacillus sp. CF80 were 77.14%, 87.52%, 82.15%, and 91.84% respectively ( Figure 5 ). Except for the fermented tartary buckwheat sample by CF55, the ACE inhibitory activities of the fermented tartary buckwheat samples by the other strains were all increased. Among them, the increase by CF80 was the largest.

[0062] Example 7 Determination of α-amylase activity

[0063] The strain CF80 obtained by screening in Example 1 was cultured in MRS liquid medium at 37 °C for 24 h, centrifuged at 8000 r / min for 10 min at 4 °C, and the fermentation supernatant was collected and used as the crude amylase extract. The α-amylase activity was determined according to the 3,5-dinitrosalicylic acid (DNS) method. 0.8 mL of 1% (W / V) soluble starch solution and 0.8 mL of phosphate buffer (0.1 M, pH 7.0) were added to a 20 mL stoppered test tube and preheated at 40 °C for 10 minutes. 0.4 mL of the crude enzyme solution was added and the reaction was carried out at 40 °C for 1 h. Then 2 mL of DNS reagent was added to terminate the reaction. The mixture was boiled for 5 minutes, and after the solution cooled, it was made up to 20 mL with distilled water. By recording the absorbance at 520 nm and using the maltose standard curve y = 0.5215x + 0.0677, R 2 = 0.999 (prepared using a 2 mg / mL maltose standard solution), the amount of reducing sugar released during starch hydrolysis was measured. The enzyme activity unit was defined as the amount of enzyme required to hydrolyze 1 mg of maltose in 1 h under the enzyme assay conditions. After determination, the enzyme activity of Lactobacillus sp. CF80 was 28.92 U / mL.

[0064] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Lactobacillus sp. CF80, which was deposited at the China Center for Type Culture Collection on January 20, 2025, with the deposit number CCTCC NO: M 2025186.

2. A microbial preparation containing the Lactobacillus CF80 described in claim 1.

3. The microbial agent according to claim 2, wherein The content of the Lactobacillus is not less than 1.0×10 7 cfu / mL or 1.0×10 7 cfu / g.

4. A method for producing amylase, characterized in that, Using the Lactobacillus CF80 in a culture medium, culturing at 30 - 37 °C for a period of time, and collecting amylase.

5. The method according to claim 4, characterized in that, The culture medium includes but is not limited to MRS medium.

6. A method for degrading starch in tartary buckwheat, characterized in that, Using tartary buckwheat as a raw material and fermenting with the Lactobacillus CF80 described in claim 1.

7. The method according to claim 6, wherein The tartary buckwheat is tartary buckwheat granules or tartary buckwheat powder after being pulverized.

8. The method according to claim 7, wherein The method is to inoculate the Lactobacillus CF80 into a fermentation system so that the cell concentration after inoculation is ≥ 1×10 8 cfu / mL or 1×10 8 cfu / g.

9. A method for preparing tartary buckwheat protein, characterized in that, Fermenting the Lactobacillus CF80 described in claim 1 or the microbial preparation described in any one of claims 2 - 3 in a culture medium containing tartary buckwheat granules or tartary buckwheat powder at 35 - 40 °C for at least 72 h.

10. The application of the Lactobacillus CF80 described in claim 1 or the method described in any one of claims 6 - 9 in the food field.

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