CN115369054B

CN115369054B - Bean pulp hydrolysate and preparation and application thereof

Info

Publication number: CN115369054B
Application number: CN202210671202.3A
Authority: CN
Inventors: 赵海锋; 罗杨; 易沄昕; 程超; 张志超
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2023-08-22
Anticipated expiration: 2042-06-15
Also published as: CN115369054A

Abstract

The invention discloses a soybean meal hydrolysate and preparation and application thereof, and the method comprises the following steps: pulverizing soybean meal into soybean meal powder, preparing soybean meal feed liquid, regulating pH to 5.5-8.5, and then steaming the soybean meal feed liquid at 60-100 ℃ for 10-30 min; after the temperature is reduced to 40-60 ℃, adding protease compound for enzymolysis; and after the enzymolysis is finished, inactivating enzyme in a boiling water bath for 10-15 min, and then freeze-drying the hydrolysate to obtain the soybean meal hydrolysate. The soybean meal hydrolysate prepared by the invention can be used as a nitrogen source of a culture medium for producing vitamin K2 (MK-7) by fermenting bacillus subtilis, the soybean meal hydrolysate is used for replacing soybean peptone as the nitrogen source to optimize a fermentation culture medium, the cost of the optimized culture medium is obviously reduced, the yield of vitamin K2 (MK-7) is improved, and the production cost of the optimized culture medium unit is 45.14% of that of the original culture medium by cost accounting.

Description

Bean pulp hydrolysate and preparation and application thereof

Technical Field

The invention relates to the field of microbial fermentation, in particular to a soybean meal hydrolysate and preparation and application thereof.

Background

Vitamin K2 is a fat-soluble vitamin with 2-methyl-1, 4-naphthoquinone parent nucleus, and is named as MK-n according to the difference of the number of isoprene units carried on a C3 side chain, wherein n is the number of isoprene units, such as MK-4, MK-5, MK-6, MK-7 and the like. Research shows that MK-7 has long side chain, long half life, easy accumulation in blood, obvious physiological effect compared with other vitamin K2, important effect in human body, such as preventing osteoporosis, cardiac and cerebral vascular diseases, treating thrombus, etc. and is one of essential important vitamins.

Although vitamin K2 (MK-7) has many important physiological functions, human cells cannot synthesize, and only bacteria in the intestinal tract can synthesize a small amount of vitamin K2 (MK-7) to be absorbed and utilized by human bodies, so that the daily required vitamin K2 (MK-7) mainly comes from meat, dairy products and fermented foods. Industrial production of MK-7 has been carried out by three production processes, namely extraction, chemical synthesis and microbial fermentation, from the 90 th century of 20 th. In recent years, microbial fermentation has become a hotspot for vitamin K2 (MK-7) research and production, but the lower yield of bacillus subtilis becomes a main factor for restricting the development of industry.

The current patent for producing vitamin K2 (MK-7) by using bacillus subtilis mainly focuses on strain mutagenesis, shortening of fermentation period, product purification and the like, but the nutrition components in a fermentation medium are not studied in detail. The nitrogen source is used as an important nutrient substance for synthesizing cell substances (amino acids, proteins, nucleic acids and the like) of bacteria and nitrogenous metabolites, has an important effect on microbial growth and product synthesis, but is also a main component of a culture medium, is critical to cost control, and is difficult to apply to industrial production due to high price, and the soybean peptone has good strain growth and high MK-7 yield when being used as the nitrogen source.

Therefore, it is significant to select bacillus subtilis with high yield of vitamin K2 (MK-7) and stable passage, and then to research nitrogen source substitutes to reduce the cost of the culture medium.

Disclosure of Invention

In view of the above-mentioned drawbacks and shortcomings of the prior art, a primary object of the present invention is to provide a method for preparing a hydrolysate of soybean meal.

Another object of the present invention is to provide a soybean meal hydrolysate prepared by the above method.

It is still another object of the present invention to provide the use of the above-mentioned soybean meal hydrolysate.

The aim of the invention is achieved by the following technical scheme:

a method for preparing a soybean meal hydrolysate, comprising the following steps:

(1) Fully pulverizing commercial soybean meal into soybean meal powder;

(2) Preparing soybean meal powder and distilled water into soybean meal feed liquid, regulating the pH to 5.5-8.5, and then steaming the feed liquid at 60-100 ℃ for 10-30 min;

(3) After the temperature is reduced to 40-60 ℃, adding the protease compound, stirring at constant temperature, and simultaneously utilizing ultrasonic waves to assist enzymolysis;

(4) And after the enzymolysis is finished, inactivating enzyme in a boiling water bath for 10-15 min, and then freeze-drying the hydrolysate to obtain the soybean meal hydrolysate.

Further, the crude protein content of the soybean meal in the step (1) is more than 30.0wt%.

Further, the grain size of the soybean meal powder in the step (1) is 50-150 mu m.

Further, the feed liquid ratio of the soybean meal feed liquid in the step (2) is 1 (4-8).

Further, the feed liquid ratio of the soybean meal feed liquid in the step (2) is 1:5.

Further, the protease complex in the step (3) is a complex of at least two enzymes selected from neutral protease, pepsin, trypsin, papain, endoprotease and alkaline protease.

Further, the protease complex in the step (3) is a complex obtained by mixing neutral protease and pepsin in a mass ratio of (1-5): 1, preferably in a mass ratio of 2:1.

Further, the addition amount of the protease complex in the step (3) is 0.5-2.0% of the mass of the soybean meal in the step (2).

Further, the protease complex in the step (3) is added in an amount of 1% by mass of the soybean meal in the step (2).

Further, the enzymolysis time in the step (3) is 4-8 hours, and the technical parameters of the ultrasonic-assisted enzymolysis are as follows: the ultrasonic power is 150-600W, the pulse working time is 4-10 s, and the pulse interval time is 0-4 s.

Furthermore, the enzymolysis time in the step (3) is 6 hours, and the technical parameters of the ultrasonic-assisted enzymolysis are as follows: ultrasonic power 150W, pulse on time 4s, pulse interval time 4s.

The soybean meal hydrolysate prepared by the invention can be used as a nitrogen source of a culture medium for producing vitamin K2 (MK-7) by fermenting bacillus subtilis.

Further, the culture medium comprises 60-150 g/L of soybean meal hydrolysate, 50-70 g/L of glycerol, 5-10 g/L of yeast extract, 0.1-0.5 g/L of calcium chloride, 0.1-0.5 g/L of anhydrous dipotassium hydrogen phosphate, 0.1-0.5 g/L of magnesium sulfate and the balance of distilled water.

Further, the culture medium comprises 60g/L of soybean meal hydrolysate, 70g/L of glycerol, 10g/L of yeast extract, 0.1g/L of calcium chloride, 0.1g/L of anhydrous dipotassium hydrogen phosphate, 0.1g/L of magnesium sulfate and the balance of distilled water.

The invention also provides a high-quality bacillus subtilis, which is named as bacillus subtilis (Bacillus subtilis) HG-1 and is stored in the Guangdong province microorganism strain collection center, wherein the storage address is the No. 100 laboratory building 5 building of Mitrex in the View district of Guangzhou, and the storage number is GDMCC No:1.3412, the preservation date is 2022, 6 and 9.

The invention has the following benefits:

(1) The bacillus subtilis HG-1 strain provided by the invention has high yield and short fermentation period, the yield of vitamin K2 (MK-7) is 88.06mg/L when the strain is fermented for 60 hours, meanwhile, the strain is genetically stable, and the yield of vitamin K2 (MK-7) is not changed obviously after continuous passage.

(2) According to the invention, soybean meal hydrolysate is used for replacing soybean peptone as a nitrogen source to optimize the fermentation medium, so that the cost of the optimized medium is obviously reduced, the yield of vitamin K2 (MK-7) is improved, and the unit production cost of the optimized medium is 45.14% of that of the original medium through cost accounting.

Drawings

FIG. 1 shows colony morphology of the original strain Y-7 on agar.

FIG. 2 shows the morphology of the cells of the original strain Y-7 under an optical microscope.

FIG. 3 shows the 16s rRNA gene sequencing results of the original strain Y-7.

FIG. 4 shows the relationship between ARTP mutagenesis treatment time and mortality of original strain Y-7.

FIG. 5 is a comparison of vitamin K2 (MK-7) production by HG-1 strain and other strains.

FIG. 6 is a study of HG-1 strain fermentation cycle.

FIG. 7 is a study of genetic stability of HG-1 strain.

FIG. 8 is a comparison of yield and production costs for the original and optimized media.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. The raw materials related to the invention can be directly purchased from the market. For process parameters not specifically noted, reference may be made to conventional techniques.

The soybean peptone used in the examples was purchased from Qingdao Gaokou Haibo biotechnology Co., ltd., the yeast extract was purchased from Angel Yeast Co., ltd., the commercial natto was Japanese mountain big North sea duct delicious natto, the soybean meal (crude protein content: more than 30.0 wt%) was purchased from Shijia Jiujiu Wu Muye Co., ltd., and the neutral protease and pepsin were purchased from Shanghai leaf biotechnology Co., ltd.

Example 1: isolation and identification of original strains

(1) Medium composition

Seed medium composition: 20g/L of soybean peptone, 30g/L of glycerol, 10g/L of yeast extract, 0.1g/L of calcium chloride, 0.1g/L of anhydrous dipotassium hydrogen phosphate, 0.1g/L of magnesium sulfate and the balance of distilled water.

Fermentation medium composition: 50g/L of soybean peptone, 70g/L of glycerol, 10g/L of yeast extract, 0.1g/L of calcium chloride, 0.1g/L of anhydrous dipotassium hydrogen phosphate, 0.1g/L of magnesium sulfate and the balance of distilled water.

(2) Isolation and screening of original strains

Weighing 10g of commercial natto, completely crushing, adding into 10mL of sterile physiological saline, swirling for 5min to fully dissolve out the strain, and centrifuging for 5min at 8000rpm to obtain bacterial liquid; the bacterial liquid is treated by water bath at 80 ℃ for 30min to kill the mixed bacteria, and is cooled to room temperature, and the bacterial liquid is diluted to 10 of the original concentration ^-8 After doubling, the cells are spread on a solid medium (containing 1, 4-dihydroxy-2-naphthoic acid serving as a structural analogue) and are cultured at 37 ℃ for 18 hours until obvious colonies are formed, and then the colonies with large diameters and good growth vigor are selected for fermentation re-screening.

Culturing 90 strains in seed culture medium at 37deg.C and 150rpm for 8 hr; then, the resulting mixture was inoculated into a fermentation medium having a liquid loading amount of 30mL/250mL at an inoculum size of 2wt%, and fermented at 37℃and 150rpm for 3 days, followed by collecting the fermentation broth.

(3) Vitamin K2 (MK-7) extraction and determination

Vitamin K2 (MK-7) extraction: adding an organic reagent (the volume ratio of the organic reagent to the fermentation liquor is 6:1, the volume ratio of the organic reagent to the fermentation liquor is 2:1, and the normal hexane and the isopropanol) into the collected fermentation liquor, sufficiently oscillating for 30min, completely collecting an upper organic phase after extraction, concentrating under reduced pressure at 45 ℃ to obtain a light yellow oily substance, adding 1mL of methanol for redissolution after blowing nitrogen, passing through a 0.22 mu m filter membrane, and transferring into a 1.5mL brown sample bottle for storage.

Determination of vitamin K2 (MK-7) content: MK-7 content was determined by High Performance Liquid Chromatography (HPLC). The column was Agilent TC-C18 (4.6 nm. Times.250.0 mm,5.0 μm). The detection parameters are as follows: 100% methanol (chromatographic grade), absorption wavelength: 270nm, flow rate: 1mL/min, sample injection amount: column temperature 10 μl: 50 ℃. MK-7 yields of 90 different original strains are shown in Table 1.

TABLE 1 production of different original strains MK-7

(4) Identification of original strains

Through re-screening, a strain Y-7 with higher vitamin K2 (MK-7) yield (21.44 mg/L) is selected for morphological observation, physiological and biochemical identification and 16s rRNA strain identification. The identification result shows that the strain is bacillus subtilis, and finally, the Y-7 is taken as an original strain for subsequent experiments. The specific identification results are as follows:

morphological observation: the colonies were spread on agar medium and cultured at 37℃for 18 hours until distinct colonies were formed, and the colonies were observed to be gray-white in morphology, nearly circular in shape, dry in surface, opaque, wrinkled, matted, not smooth in edges in the shape of split leaves, and darker in central color than the edge portions (see FIGS. 1 and 2).

And (3) physiological and biochemical identification: several physiological and biochemical experiments were performed on the isolated original strain Y-7, including gram staining, contact enzyme, V-P assay, V-P culture, pH assay, gelatin liquefaction, starch hydrolysis, D-glucose fermentation, D-xylose fermentation, D-mannitol fermentation, naCl salt-tolerant growth, nitrate reduction, etc., and further determined to be Bacillus subtilis.

Identification of 16s rRNA strain: the whole genome of the strain was extracted by means of a genome kit, and 16s rRNA amplification was performed using universal primers (27F and 1492R), and the sequencing results were compared at NCBI, confirming that it belongs to the genus Bacillus. Primer information is shown in Table 2, and the 16s rRNA sequencing results are shown in FIG. 3.

TABLE 2 primer information

Example 2: ARTP mutagenesis and screening of original strains

The seed medium and fermentation medium used in this example were the same as in example 1.

(1) Preparation of bacterial suspension

Inoculating Y-7 into seed culture medium, culturing at 37deg.C and 150rpm for 16 hr, washing strain with sterile physiological saline for several times and diluting appropriately until the bacterial suspension OD ₆₀₀ The value was at 0.6.

(2) ARTP mutagenesis treatment

Uniformly coating 10 mu L of bacterial suspension on a sterile slide, transferring the slide to an ARTP operation bin, placing the slide to a corresponding hole site by forceps, enabling the slide to be positioned right below an airflow port, and setting instrument parameters as follows: the power is 110W, the air flow is 10SLM, and the mutagenesis time is 150s. The relationship between ARTP mutagenesis treatment time and mortality is shown in figure 4, and the graph shows that the mortality gradually rises along with the prolongation of the mutagenesis time, when the mutagenesis time is 220s, the mortality reaches 81.3%, and the mutagenesis time is continued to be prolonged after 220s, so that the mortality has no obvious change, and the ARTP mutagenesis can directly act on bacterial genetic materials without causing great damage and influence on bacterial morphology. The final ARTP mutagenesis time was 220s.

After mutagenesis, transfer slide glass to 1mL sterile physiological saline, shake elution to form new bacterial suspension.

(3) Resistance screening of structural analogues

Diluting the new bacterial suspension to the original concentration of 10 ^-8 After doubling, 100. Mu.L of the strain is coated on a solid medium (containing 1, 4-dihydroxyl-2-naphthoic acid serving as a structural analogue), and after culturing for 18 hours at 37 ℃,90 single colonies with good growth vigor are selected for fermentation and re-screening, the mutant strain is represented by A-n, A represents a mutation mode ARTP, and n represents a strain number.

(4) Fermentation double screen

Picking colonies into a seed culture medium, and culturing for 8 hours at 37 ℃ and 150 rpm; then, the resulting mixture was inoculated into a fermentation medium having a liquid loading amount of 30mL/250mL at an inoculum size of 2wt%, and fermented at 37℃and 150rpm for 3 days, followed by collecting the fermentation broth.

(5) Vitamin K2 (MK-7) extraction and determination

Adding an organic reagent (the volume ratio of the organic reagent to the fermentation liquor is 6:1, the volume ratio of the organic reagent to the fermentation liquor is 2:1, and the normal hexane and the isopropanol) into the collected fermentation liquor, sufficiently oscillating for 30min, completely collecting an upper organic phase after extraction, concentrating under reduced pressure at 45 ℃ to obtain a light yellow oily substance, adding 1mL of methanol for redissolution after blowing nitrogen to dry, passing through a 0.22 mu m filter membrane, transferring into a 1.5mL brown sample bottle, and preserving for subsequent yield measurement.

The vitamin K2 (MK-7) yields of 90 different mutant strains are shown in Table 3. Wherein strain 43 gives the highest yield, up to 78.40mg/L, of about 366% of the original self-screening strain Y-7; and is also significantly higher than other bacillus subtilis CICC2637 (yield 10.26mg/L, deposited in China center for type culture collection), CICC10263 (yield 19.15mg/L, deposited in China center for type culture collection), and CIMI1.784 (yield 17.54mg/L, obtained from Guangdong province microorganism) deposited by the inventors, the yield pairs are shown in FIG. 5. The mutant strain with the number of 43 is named as bacillus subtilis Bacillus subtilis HG-1 and is preserved in the microorganism strain collection center in Guangdong province, the preservation address is the building 5 of the experiment building of the Mitsui No. 100 of the Va Xiuzhou area of Guangdong province, and the preservation number is GDMCC No:1.3412, the preservation date is 2022, 6 and 9.

TABLE 3 production of different mutant strains MK-7

Example 3: research on fermentation period and genetic stability of mutagenized strain HG-1

The fermentation medium composition used in this example was the same as in example 1.

(1) Fermentation period

HG-1 was continuously fermented for 144 hours, sampled every 12 hours, and changes in the amount of cells and the yield of vitamin K2 (MK-7) during the fermentation were observed. As shown in FIG. 6, the strain grows rapidly in the early stage of fermentation, and the dry weight of the strain reaches a peak value at 48 hours; when fermentation is carried out for 60 hours, the yield of vitamin K2 (MK-7) reaches a peak value of 78.40mg/L, which shows that HG-1 has vigorous activity, high growth speed and short fermentation period.

(2) Genetic stability

HG-1 was serially passaged for 12 generations, and MK-7 production was measured for evaluation of genetic stability. As shown in FIG. 7, the effect of passage number on MK-7 production has no obvious effect on HG-1 production, and the productivity is basically stable and maintained at the same high level, indicating that the strain HG-1 has better genetic stability.

Example 4: research on high-quality nitrogen sources

1. Preparation of bean pulp hydrolysate

(1) Pretreatment of soybean meal: fully crushing commercial soybean meal into soybean meal powder with the particle size of 50-150 mu m.

(2) Enzymolysis of soybean meal powder: adding a proper amount of distilled water into 5g of soybean meal powder, preparing soybean meal feed liquid according to a feed liquid ratio of 1:5, adjusting the pH value to 6.0, and then steaming the soybean meal feed liquid at 80 ℃ for 10min.

(3) After the soybean meal feed liquid is cooled to 40 ℃, adding a compound obtained by mixing neutral protease and pepsin at a ratio of 2:1 (W/W), wherein the addition amount of the protease compound is 1.0 percent (namely, the addition amount of the neutral protease is 0.033g and the addition amount of the pepsin is 0.017 g.) of the soybean meal powder, stirring and hydrolyzing for 6 hours at constant temperature, and adopting ultrasonic auxiliary enzymolysis during which the ultrasonic auxiliary enzymolysis technical parameters are that the ultrasonic power is 150W, the pulse working time is 4s and the pulse interval time is 4s.

(4) And after the enzymolysis is finished, inactivating enzymes in a boiling water bath for 15min, and then performing freeze-drying treatment on the hydrolysate to obtain 4.2g of soybean meal hydrolysate which is in a light yellow granule shape.

2. Determination of the concentration of a Nitrogen Source

(1) Preparing culture mediums with different nitrogen source concentrations

The culture medium with different nitrogen source concentrations is prepared by taking soybean meal hydrolysate and soybean peptone with different concentrations (50 g/L,60g/L,70g/L,80g/L,90g/L,100g/L, 120g/L and 150 g/L) as nitrogen sources, and mixing with glycerol with the concentration of 70g/L, 10g/L yeast extract, 0.1g/L calcium chloride, 0.1g/L anhydrous dipotassium hydrogen phosphate, 0.1g/L magnesium sulfate and distilled water.

(2) Referring to example 2, steps (3) and (4), mutant strain HG-1 was used for fermentative production of MK-7, and the effect of different nitrogen source concentrations on MK-7 production was investigated, and the results are shown in Table 3. As shown in Table 4, MK-7 was found to be highest at a soybean meal hydrolysate concentration of 60g/L, at 88.06mg/L; MK-7 was produced at a concentration of 50g/L soytone at the highest yield of 78.40mg/L.

TABLE 4 MK-7 production corresponding to different concentration of Nitrogen sources

3. Cost accounting of culture Medium

By researching the nitrogen source of the culture medium, the cost of the culture medium is obviously reduced. Table 4 shows cost accounting tables for two fermentation media when soybean peptone (original medium, fermentation medium of the same composition as in example 1) and soybean meal hydrolysate (optimized medium, i.e., the above-mentioned medium with 60g/L soybean meal hydrolysate as nitrogen source) were used as nitrogen sources. As can be seen from Table 5, the costs of the original medium and the optimized medium were 13.747 yuan/L and 6.918 yuan/L, MK-7 production was 78.40mg/L and 88.06mg/L, respectively, and the unit production cost of the optimized medium was 0.079 yuan/mg, which was significantly lower than that of the original medium (0.175 yuan/mg), which was 45.14% of the previous.

TABLE 5 cost accounting for two fermentation media

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. The application of the soybean meal hydrolysate in improving the fermentation production of vitamin K2 by bacillus subtilis is characterized in that the preparation method of the soybean meal hydrolysate comprises the following steps:

(1) Pulverizing soybean meal into soybean meal powder;

(3) After the temperature is reduced to 40-60 ℃, adding a protease compound obtained by mixing neutral protease and pepsin in a mass ratio of 2:1, stirring at constant temperature, and simultaneously utilizing ultrasonic waves to assist enzymolysis;

(4) After enzymolysis, inactivating enzyme in boiling water bath for 10-15 min, and freeze drying the hydrolysate to obtain the soybean meal hydrolysate;

the deposit number of the bacillus subtilis is GDMCC No:1.3412;

the dosage of the bean pulp hydrolysate in the fermented culture medium component is 60-150 g/L.

2. Use of the soybean meal hydrolysate according to claim 1 for improving the fermentation production of vitamin K2 by bacillus subtilis, wherein the crude protein content of the soybean meal in step (1) is more than 30.0wt%.

3. The use of the soybean meal hydrolysate according to claim 1 for improving the fermentation production of vitamin K2 by bacillus subtilis, wherein the grain size of the soybean meal powder in the step (1) is 50-150 μm.

4. The use of the soybean meal hydrolysate in improving the fermentation production of vitamin K2 by bacillus subtilis according to claim 1, wherein the feed liquid ratio of the soybean meal feed liquid in the step (2) is 1 (4-8).

5. The use of the soybean meal hydrolysate according to claim 1 for improving the fermentation production of vitamin K2 by bacillus subtilis, wherein the protease compound in the step (3) is added in an amount of 0.5-2.0% of the mass of the soybean meal powder in the step (2).

6. The application of the soybean meal hydrolysate in improving the fermentation production of vitamin K2 by bacillus subtilis according to claim 1, wherein the enzymolysis time in the step (3) is 4-8 hours, and the technical parameters of the ultrasonic-assisted enzymolysis are as follows: the ultrasonic power is 150-600W, the pulse working time is 4-10 s, and the pulse interval time is 0-4 s.

7. The use of the soybean meal hydrolysate in improving the fermentation production of vitamin K2 by bacillus subtilis according to claim 1, wherein the components of the fermentation medium are 60-150 g/L of the soybean meal hydrolysate, 50-70 g/L of glycerol, 5-10 g/L of yeast extract, 0.1-0.5 g/L of calcium chloride, 0.1-0.5 g/L of anhydrous dipotassium hydrogen phosphate, 0.1-0.5 g/L of magnesium sulfate and the balance of distilled water.