A method for the fermentative production of beta-glucosidase by kluyveromyces

Abstract

The application discloses a method for fermenting and producing beta-glucosidase by Kluyveromyces, and comprises the following steps: preparing an enzyme production medium: weighing a carbon source and a filler, adding distilled water to the carbon source and the filler, sterilizing at 121 DEG C for 40-50 min to obtain the enzyme production medium; solid fermentation: inoculating Kluyveromyces into the enzyme production medium and adding bran koji, uniformly stirring and culturing, wherein the concentration of the Kluyveromyces is 1x10 8 CFU; the bran koji contains Rhizopus Q303; preparing an enzyme solution: after the fermentation is completed, adding an appropriate amount of acetic acid buffer into the enzyme production medium, uniformly mixing, leaching for 2 h, and then filtering to obtain the enzyme solution; the Kluyveromyces and the Rhizopus Q303 are co-cultured to ferment and produce beta-glucosidase, and the culture conditions are optimized, so that the purpose of efficiently improving the enzyme production level of beta-glucosidase is achieved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a method for producing β-glucosidase by fermentation of Kluyveromyces. BACKGROUND

[0002] β-glucosidase is also known as β-glucosidase hydrolase or β-D-glucoside hydrolase. In 1973, Wohler and Liebig first discovered β-glucosidase in bitter almond juice, and since then, researches on the enzymatic properties and other aspects of β-glucosidase have been reported. β-glucosidase not only plays an important role in the sugar metabolic pathway of some organisms, but also plays an important role in various fields related to human life. For example, the improvement of β-glucosidase in food, the improvement of the flavor and aroma of beverages such as mango, apple and fruit wine, etc. The enzyme can hydrolyze glycosides in fruit juice into terpene alcohols and aromatic aldehyde oxides, which are the main components of aroma, and have a good effect on the aroma of beverages. Li Ping et al. found that the aroma of lemon juice and apple juice samples treated by β-glucosidase was more full and soft, and the inherent aroma components were more prominent. In addition, β-glucosidase has important application value in the fields of energy, agriculture, medicine, etc.

[0003] β-glucosidase is widely present in many plants, insects, and microorganisms such as yeast, Trichoderma and bacteria. Microbial β-glucosidase has more extensive biochemical properties, substrate properties and stability than β-glucosidase from other sources. In addition, microbial β-glucosidase has high yield and high activity, so it has attracted widespread attention from researchers in recent years. At present, the research on microbial sources of β-glucosidase at home and abroad mainly includes bacteria, molds and yeasts. The mold source of β-glucosidase is studied earlier, and the reported molds include Aspergillus oryzae, Aspergillus niger, Trichoderma viride, Penicillium, etc. The yeasts mainly include the heat-resistant yeast Pichia pastoris, Kluyveromyces marxianus, Kluyveromyces lactis and Hansenula, etc. Among them, Kluyveromyces has become a research hotspot in the food industry due to its safety. Through the existing research, it is found that different strains and fermentation methods make the yield of β-glucosidase different. For example, the enzyme activity of β-glucosidase produced by molds is 28.15 U / mL ～140U / mL. Fang Shangling et al. can reach 93.48U / mL by optimizing the bacterial fermentation process, and the research on the production of β-glucosidase by Kluyveromyces is mostly liquid fermentation, and the enzyme activity is low. For example, β-glucosidase produced by Kluyveromyces liquid fermentation by Park Chunhong et al. can obtain 80mg of single product per liter of fermentation broth; and the enzyme activity is increased by 1.81 times by Xu Yan'er et al. through the fermentation of a strain of Kluyveromyces isolated from grape wine after mutagenesis. How to further improve the enzyme production level of β-glucosidase is a big problem that needs to be overcome by those skilled in the art. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a method for Kluyveromyces fermentation to produce β-glucosidase, so as to at least achieve the purpose of efficiently improving the enzyme production level of β-glucosidase.

[0005] The purpose of the present application is achieved by the following technical scheme: a method for Kluyveromyces fermentation to produce β-glucosidase, comprising the following steps:

[0006] S1, preparation of enzyme production medium: weigh the carbon source and filler, and add distilled water, then sterilize at 121℃ for 40-50min to obtain the enzyme production medium;

[0007] S2, solid fermentation: inoculate Kluyveromyces into the enzyme production medium and add bran koji, stir uniformly and culture, the concentration of the Kluyveromyces is 1×10 8 CFU; the bran koji contains Rhizopus Q303;

[0008] S3, enzyme solution preparation: after fermentation, add an appropriate amount of acetic acid buffer to the enzyme production medium, mix uniformly, extract for 2h, and then filter to obtain the enzyme solution.

[0009] The present application utilizes the Kluyveromyces fermentation to produce β-glucosidase, but the Kluyveromyces cannot directly utilize the carbon source (buckwheat flour, etc.) in the enzyme production medium, therefore, the present application adopts the method of co-culturing with Rhizopus Q303, so as to provide sufficient nutrients for the Kluyveromyces and ensure the growth of the bacterial cells. Rhizopus Q303 is an excellent strain selected from Xiaoqu by Guizhou Light Industry Research Institute, which has strong saccharification ability, fast growth rate and stable performance. Co-culturing with Kluyveromyces effectively ensures the nutrient supply for the growth of yeast cells. Rhizopus Q303 has been used in China for a long time for food fermentation, and is a recognized food safety strain, therefore, the present application adopts the co-culturing of Kluyveromyces and Rhizopus Q303 for fermentation to produce β-glucosidase, which ensures the biological safety of the enzyme.

[0010] Further, before adding the Kluyveromyces, the carbon source and other raw materials are treated at 30 DEG C for 6 hours by adding bran koji; or the raw materials are pretreated at 60 DEG C for 6 hours by adding saccharifying enzyme; or the bran koji or the saccharifying enzyme is added into the enzyme production medium together with the Kluyveromyces to perform enzyme production fermentation.

[0011] Further, the carbon source in the enzyme production medium is any one of buckwheat flour, wheat, corn and rice, and the filler is any one or several of buckwheat hull and bran; the addition ratio of the buckwheat hull and the bran in the filler is 1:0 or 0:1 or 1:1 or 1:4 or 4:1; and the total amount of the filler is 20% or 30% or 40% or 50% or 60%.

[0012] Further, the nitrogen source in the enzyme production medium is any one of peptone, yeast extract, corn steep liquor dry powder and ammonium sulfate; and the addition amount of the nitrogen source is 0.5% or 1.0% or 1.5% or 2.0% or 2.5% or 3.0% or 4.0%.

[0013] Further, the water content in the enzyme production medium is 30% or 50% or 70% or 90% or 110% or 130%.

[0014] Further, the inoculation amount of the Kluyveromyces is 5% or 10% or 20% or 30% or 40% or 50% or 60%.

[0015] Further, the addition amount of the bran koji is 0.4% or 0.6% or 0.8% or 1.0%.

[0016] Further, in the step S2, the fermentation temperature is 20 DEG C or 25 DEG C or 30 DEG C or 35 DEG C or 40 DEG C; and the fermentation time is 24 hours or 36 hours or 48 hours or 72 hours or 96 hours.

[0017] Further, the carbon source is buckwheat flour, the filler is buckwheat hull, the filling ratio of the filler is 50%, the addition amount of the bran koji is 0.8%, the nitrogen source is corn steep liquor, the water content in the medium is 90%, the inoculation amount in the fermentation is 10%, the fermentation temperature is 25 DEG C, and the fermentation time is 72 hours.

[0018] Further, the enzyme production level of the Kluyveromyces fermentation method for producing beta-glucosidase is 192.6 U / g.

[0019] The beneficial effects of the present application are:

[0020] (1) The application is derived from traditional folk fermented food, which has been eaten by people for a long time and has good safety, and is more conducive to the production of beta-glucosidase in food and pharmaceutical industries, expands the application of Kluyveromyces in food, pharmaceutical and other industries, and has higher enzyme activity in the fermentation production of beta-glucosidase. The method disclosed in the application is used for fermentation, and the bacteria grow vigorously, and the enzyme production is high and the activity is high.

[0021] (2) The application co-cultures Kluyveromyces and Rhizopus Q303 to ferment and produce beta-glucosidase. Rhizopus Q303 first liquefies and saccharifies buckwheat powder, a starchy raw material, to solve the problem that yeast cannot directly utilize macromolecular substances such as starch, so that Kluyveromyces marxianus can make more full use of raw materials, the bacteria grow vigorously, and the production of beta-glucosidase is more conducive. The growth rate of Kluyveromyces is fast, the fermentation time is short, and Rhizopus Q303 is a strain that has been used for food fermentation for a long time and has biological safety, which ensures the biological safety of beta-glucosidase.

[0022] (3) The application optimizes the fermentation conditions, and the optimal conditions are as follows: the optimal carbon source is buckwheat powder, the optimal filler is buckwheat shell, the filling ratio of the filler is 50%, the addition amount of bran is 0.8%, the optimal nitrogen source is corn syrup, the addition amount is 1.5%, the water content of the culture medium is 90%, the inoculation amount is 10%, the fermentation temperature is 25°C, and the fermentation time is 72h. After optimization, the enzyme production level is increased from the initial seed liquid enzyme production of 0.6U / g, the initial solid-state culture medium of 12.9U / g to 192.6U / g, which is about 15 times higher than the initial solid-state culture medium before optimization. The effect is obvious. The process flow of the method is simple, the obtained beta-glucosidase has high enzyme activity, and the method is a good production method of beta-glucosidase.

[0023] (4) The application effectively improves the yield of beta-glucosidase by using solid-state fermentation, and the solid-state fermentation has the following advantages: extensive processing and production can be implemented; buckwheat shells are used as fillers for solid-state materials, which effectively utilizes the by-products of agricultural products; through solid-state fermentation and bacterial culture, the bacterial protein content of solid-state materials is effectively improved, which provides a new idea for the production of bacterial protein feed; buckwheat powder and buckwheat shells are used as raw materials, buckwheat has multiple high-quality production areas in the country, and the use of buckwheat shells as fillers for fermentation culture medium makes the by-products of agricultural products more effectively utilized, laying a foundation for the high-value production of agricultural by-products. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The influence of fermentation methods (a) and treatment methods (b) on enzyme production;

[0025] Figure 2 Effect of carbon source type on enzyme production

[0026] Figure 3 Effect of filler type (a) and filling ratio (b) on enzyme production

[0027] Figure 4 Effect of bran koji addition amount on enzyme production

[0028] Figure 5 Effect of nitrogen source type (a) and nitrogen source addition amount (b) on enzyme production

[0029] Figure 6 Effect of initial water content of fermentation medium on enzyme production

[0030] Figure 7 Effect of fermentation inoculation amount on enzyme production

[0031] Figure 8 Effect of fermentation temperature on enzyme production

[0032] Figure 9 Effect of fermentation time on enzyme production DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be described in further detail below in conjunction with the accompanying drawings, but the protection scope of the present application is not limited to the following description.

[0034] EMBODIMENT

[0035] A method for fermentatively producing β-glucosidase by Kluyveromyces, comprising the following steps:

[0036] S1, preparation of enzyme production medium: weigh the carbon source and filler, and add distilled water to it, then sterilize at 121℃ for 40-50min to obtain the enzyme production medium; the carbon source in the enzyme production medium is any one of buckwheat flour, wheat, corn and rice, and the filler is any one or several of buckwheat hull and bran; the addition ratio of buckwheat hull and bran in the filler is 1:0 or 0:1 or 1:1 or 1:4 or 4:1; the total amount of the filler is 20% or 30% or 40% or 50% or 60%; the bran is pretreated before adding, first add the bran to the carbon source and other raw materials at 30℃ for 6h; or add glucoamylase to pretreat the raw materials at 60℃ for 6h; or add the bran or glucoamylase together with kluyveromyces in the enzyme production medium for enzyme production fermentation; the nitrogen source of the enzyme production medium is any one of peptone, yeast extract, corn syrup dry powder and ammonium sulfate; the addition amount of the nitrogen source is 0.5% or 1.0% or 1.5% or 2.0% or 2.5% or 3.0% or 4.0%; the water content of the enzyme production medium is 30% or 50% or 70% or 90% or 110% or 130%; the inoculation amount of the kluyveromyces is 5% or 10% or 20% or 30% or 40% or 50% or 60%; the addition amount of the bran is 0.4% or 0.6% or 0.8% or 1.0%;

[0037] S2, solid state fermentation: inoculate the kluyveromyces into the enzyme production medium and add bran, stir uniformly and then culture, the concentration of the kluyveromyces is 1×10 8 CFU; the bran contains rhizopus Q303; the fermentation temperature is 20℃ or 25℃ or 30℃ or 35℃ or 40℃; the fermentation time is 24h or 36h or 48h or 72h or 96h;

[0038] S3, preparation of enzyme solution: after the fermentation is completed, add an appropriate amount of acetic acid buffer solution to the enzyme production medium, mix uniformly, soak for 2h, then filter with gauze to obtain the enzyme solution.

[0039] The enzyme activity of the β-glucosidase produced in the example is determined

[0040] Take 0.1mL of the crude enzyme solution in step S3 in a test tube, add 0.2mL of p-nitrophenyl-β-D-glucopyranoside solution and 0.7mL of acetic acid buffer solution with pH=5, react in a 45℃ water bath for 30min, then add 2mL of sodium carbonate solution to terminate the reaction, and then add 9mL of distilled water to mix uniformly, and take the heated inactivated crude enzyme solution as a blank control, and measure the absorbance value at 410nm.

[0041] Enzyme activity definition: the enzyme activity unit (U) is defined as: the amount of enzyme required to hydrolyze 1μmol of p-nitrophenol per minute under the reaction conditions of pH 5.0 and 30℃, and the detection results are shown inFigures 1-9 The effect of solid-state fermentation and liquid-state fermentation on the production of glucose oxidase, lipase and alpha-amylase, and the significance analysis of the effect of solid-state fermentation on the enzyme content of bran koji are shown in Table 1.

[0042] Table 1:

[0043] Group Glucose oxidase Lipase Alpha-amylase Beta-glucosidase A ND ND ND 0.482±0.103b B 14.44±0.412a 37.485±1.971 53.091±0.291a 178.945±17.497a C ND ND 23.379±0.089c 3.125±0.597b

[0044] Note: In the figure, the group letters respectively represent A: Kluyveromyces liquid-state fermentation, enzyme activity unit is U / mL; B: Rhizopus 303 and Kluyveromyces co-culture solid-state fermentation, enzyme activity unit is U / g dry matter; C: bran koji (Rhizopus Q303), enzyme activity unit is U / g; ND: not detected.

[0045] From Figures 1-9 As can be seen from Table 1 and Table 1, the optimal carbon source of the present application is buckwheat flour, the optimal filler is buckwheat shell, the filling ratio of the filler is 50%, the addition amount of bran koji is 0.8%, the optimal nitrogen source is corn syrup, the water content of the culture medium is 90%, the fermentation inoculation amount is 10%, the fermentation temperature is 25℃, and the fermentation time is 72h. After optimization, the enzyme production level is from the initial seed liquid enzyme production amount of 0.6U / g, the initial solid-state culture medium of 12.9U / g, to 192.6U / g, which is about 15 times higher than that before optimization, and the optimization effect is obvious.

[0046] In order to explore whether solid-state fermentation has an advantage in improving enzyme activity, in addition to the determination of beta-glucosidase, the present application also compares and determines the enzyme production of other glucose oxidase, lipase, alpha-amylase and the like in liquid-state fermentation and solid-state fermentation, and determines the enzyme activity of the bran koji containing the enzymes. The specific method is as follows:

[0047] (1) Sample preparation: the Kluyveromyces liquid-state fermentation sample is the seed liquid of YPD culture medium liquid culture, which is recorded as sample A; the Kluyveromyces and Rhizopus co-culture solid-state fermentation sample is the solid-state fermentation culture medium obtained in the step, which is sampled, extracted and other enzyme activity detection samples are obtained in the same way, which is recorded as sample B; a certain mass of bran koji for fermentation is added with a certain proportion of buffer solution for extraction for 2h, and the filtrate is obtained as sample C; the enzyme activity of the above three groups of samples is determined respectively.

[0048] (2) Enzyme activity determination method and enzyme activity definition:

[0049] Determination of glucose oxidase:

[0050] The required reagents are: ① 2% glucose phosphate buffer, ② 0.1mol / L sodium hydroxide solution, ③ phenolphthalein indicator, and ④ 0.1mol / L hydrochloric acid.

[0051] Detection method: take 2% glucose phosphate buffer 5 mL in 250 mL conical flask, on the constant temperature shaker 30 ℃, 150 rpm preheating 3 minutes, accurate to add 1 mL of the determination solution, immediately placed on constant temperature shaker, 30 ℃, 150 rpm oscillation reaction 60 minutes (accurate timing) take out; Immediately accurate to add 20 ml of 0.1 mol / L sodium hydroxide solution, stop the reaction; Add phenolphthalein indicator 1 drop, titrate the remaining sodium hydroxide with 0.1 mol / L hydrochloric acid, record the consumption of hydrochloric acid milliliter A. Blank control for taking 2% glucose phosphate buffer 5 mL in 250 mL conical flask, accurate to add 20.00 ml of 0.1 mol / L sodium hydroxide solution, shake, then accurate to add 1 mL of enzyme solution, phenolphthalein indicator 1 drop, titrate the remaining sodium hydroxide with 0.1 mol / L hydrochloric acid, record the consumption of hydrochloric acid milliliter B. Enzyme activity is calculated as follows:

[0052] Glucose oxidase activity unit (GOD / g or mL) = (B-A) x N x f x 1000 / 60

[0053] In the formula:

[0054] A----reaction after the consumption of hydrochloric acid (mL);

[0055] B----reaction before the consumption of hydrochloric acid (mL);

[0056] N----hydrochloric acid molar concentration (mol / L);

[0057] f-----dilution multiple

[0058] 60----for 60 minutes of reaction, in minutes;

[0059] (3) lipase determination:

[0060] The required reagents are: ① 1 mmol / L p-NP standard solution, ② 0.5 mol / L 1 mL trichloroacetic acid, ③ 1 mol / L Na2CO3 solution, ④ 0.5 mol / L NaOH solution, ⑤ 10 mmol / L p-nitrophenyl palmitate, ⑥ Tris-HCl buffer

[0061] Standard curve: prepare 1 mmol / L p-nitrophenol standard solution; The p-NP standard solution is diluted with 1 mol / L Na2CO3 solution to 100-600 μmol / L 6 gradients, and then 1 mL of each is added to 1 mL of 0.5 mol / L trichloroacetic acid and 3 mL of NaOH solution. Mix well and measure the absorbance at 410 nm with a spectrophotometer. Draw the standard curve of p-nitrophenol. Reagent addition as shown in Table 2:

[0062] Table 2:

[0063] 1 mmol / 1 p-np 0 0.1 0.2 0.3 0.4 0.5 0.6 Na2CO3 1 0.9 0.8 0.7 0.6 0.5 0.4 Trichloroacetic acid 1 1 1 1 1 1 1 NaOH 3 3 3 3 3 3 3

[0064] Detection method: 10 mmol / L of p-nitrophenyl palmitate (p-NPP) was prepared, 20 μL of p-NPP substrate solution was taken, 780 μL of Tris-HCl buffer was added, 200 μL of enzyme solution was added after preheating at 37°C for 5 min, 1 mL of 0.5 mol / L trichloroacetic acid was added after reaction for 10 min, and the reaction was terminated after mixing and standing for 5 min, then 3 mL of 0.5 mol / L NaOH was added to adjust the pH, and the absorbance was measured at 410 nm. The blank control was obtained by replacing 200 μL of enzyme solution with 200 μL of deionized water. The enzyme activity was obtained according to the standard curve.

[0065] The lipase enzyme activity was calculated as follows:

[0066]

[0067] In the formula, X is the lipase activity, U / mL; c is the p-NP concentration, μmol / L; V is the final volume of the reaction solution after adjusting the pH, mL; V' is the amount of enzyme solution, mL; and t is the reaction time, min.

[0068] (4) Determination of α-amylase:

[0069] Required reagents: ① Original iodine solution: weigh 11.0 g of iodine and 22.0 g of potassium iodide, dissolve the iodine completely with a small amount of water, and make up to 500 mL, store in a brown bottle. Dilute iodine solution: take 2.00 mL of original iodine solution, add 20.0 g of potassium iodide dissolved in water and make up to 500 mL, store in a brown bottle. ② Soluble starch solution (20 g / L): weigh 2.000 g (accurate to 0.001 g) of soluble starch (based on absolute dryness) in a beaker, add a small amount of water to make a slurry, slowly add 70 mL of boiling water while stirring, then rinse the starch-containing beaker with water, pour the wash into it, stir and heat until completely transparent, cool and make up to 100 mL of solution. ③ Phosphate buffer (pH = 6.0): weigh 45.23 g of disodium hydrogen phosphate and 8.07 g of citric acid, dissolve in water and make up to 1000 mL, and use after correction with a pH meter. ④ Acid solution [c(HCl) = 0.1 mol / L]: prepared according to GB / T601.

[0070] Detection method: take 20.0 mL of soluble starch solution in a test tube, add 5.00 mL of phosphate buffer, shake well, and preheat in a 60°C ± 0.2°C (thermostable α-amylase preparation is placed in a 70°C ± 0.2°C) constant temperature water bath for 8 min; add 1.00 mL of diluted enzyme solution, immediately start timing, shake well, and accurately react for 5 min.

[0071] Take 1.00 mL of the reaction solution and add it to a test tube containing 0.5 mL of hydrochloric acid solution and 5.00 mL of dilute iodine solution, shake well, and use 0.5 mL of hydrochloric acid solution and 5.00 mL of dilute iodine solution as a blank, and quickly measure the absorbance at a wavelength of 660 nm with a 10 mm cuvette. According to the absorbance in Appendix A of the national standard, the concentration of the test enzyme solution is obtained.

[0072] The detection results show that the determination results of the above three enzymes find that the solid-state fermentation method in the application has a significant effect on the improvement of the enzyme production of these enzymes, especially through the optimization of the fermentation conditions, the production of β-glucosidase is obviously improved.

[0073] The above description is only the preferred embodiments of the present application, and it should be understood that the present application is not limited to the forms disclosed herein, and should not be considered as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concepts described herein by the above teachings or related art or knowledge. Any modification and change made by those skilled in the art without departing from the spirit and scope of the present application shall be within the protection scope of the claims of the present application.

Claims

1. A method for the fermentative production of β-glucosidase by Kluyveromyces, characterized in that, It comprises the following steps: S1, preparation of enzyme production medium: weigh the carbon source and filler, and add distilled water to it, then sterilize at 121℃ for 40-50min to obtain the enzyme production medium; the carbon source is any one of buckwheat flour, wheat, corn and rice, and the filler is any one or several of buckwheat shell and bran; S2, solid fermentation: inoculating the enzyme-producing medium with Kluyveromyces and adding bran koji, stirring uniformly and then culturing, the concentration of the Kluyveromyces being 1 x 10 8 CFU; the bran koji containing Rhizopus Q303; the Kluyveromyces being Kluyveromyces marxianus; S3, enzyme solution preparation: after fermentation, add an appropriate amount of acetic acid buffer to the enzyme production medium, mix uniformly, then extract for 2h, and then filter to obtain the enzyme solution.

2. A method for the production of β-glucosidase by fermentation of a Kluyveromyces according to claim 1, characterized in that: Before adding Kluyveromyces fermentation, first add bran koji to treat the carbon source raw material at 30℃ for 6h; or add bran koji and Kluyveromyces to the enzyme production medium together for enzyme production fermentation.

3. The method of claim 1, wherein the Kluyveromyces cell is a Kluyveromyces lactis cell. In the filler, the addition ratio of buckwheat shell and bran is 1:0 or 0:1 or 1:1 or 1:4 or 4:1; the total amount of the filler is 20% or 30% or 40% or 50% or 60%.

4. A method of fermentative production of β-glucosidase by Kluyveromyces according to claim 3, characterized by: The nitrogen source of the enzyme production medium is any one of peptone, yeast extract, corn steep powder and ammonium sulfate; the addition amount of the nitrogen source is 0.5% or 1.0% or 1.5% or 2.0% or 2.5% or 3.0% or 4.0%.

5. A method of fermentative production of β-glucosidase by Kluyveromyces according to claim 4, characterized by: The water content of the enzyme production medium is 30% or 50% or 70% or 90%.

6. The method of claim 1, wherein the Kluyveromyces cell is a Kluyveromyces lactis cell. The inoculation amount of the Kluyveromyces is 5% or 10% or 20% or 30% or 40% or 50% or 60%.

7. The method of producing β-glucosidase by fermentation of Kluyveromyces according to claim 1, characterized by: The addition amount of bran koji is 0.4% or 0.6% or 0.8% or 1.0%.

8. The method of claim 1, wherein the Kluyveromyces cell is a Kluyveromyces lactis cell. In step S2, the fermentation temperature is 20℃ or 25℃ or 30℃ or 35℃ or 40℃; the fermentation time is 24h or 36h or 48h or 72h or 96h.

9. The method of producing β-glucosidase by fermentation of Kluyveromyces according to any one of claims 1 to 8, characterized in that: The carbon source is buckwheat flour, the filler is buckwheat shell, the filling ratio of the filler is 50%, the addition amount of bran koji is 0.8%, the nitrogen source is corn steep liquor, the water content of the medium is 90%, the inoculation amount for fermentation is 10%, the fermentation temperature is 25℃, and the fermentation time is 72h.

10. The method of producing β-glucosidase by fermentation of Kluyveromyces according to claim 9, characterized by: The enzyme production level of the method for producing β-glucosidase by Kluyveromyces fermentation is 192.6U / g.

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