CN109797115B

CN109797115B - Bacillus amyloliquefaciens GZU05 for producing nattokinase and beta-glucoside and application method thereof

Info

Publication number: CN109797115B
Application number: CN201910025309.9A
Authority: CN
Inventors: 何腊平; 冯仑元; 李翠芹
Original assignee: Guizhou University
Current assignee: Change Innovation Wuhan Bio Tech Co ltd
Priority date: 2019-01-12
Filing date: 2019-01-12
Publication date: 2022-07-12
Anticipated expiration: 2039-01-12
Also published as: CN109797115A

Abstract

Bacillus amyloliquefaciens GZU05 capable of producing nattokinase and beta-glucoside, is classified and named as Bacillus amyloliquefaciens GZU05 according to microbiological classification, and has the following chemical name: bacillus amyloliquefaciens GZU05 which is preserved in China center for type culture collection (CCTCC M2018763) in 11-9.2018. It is collected from traditional fermented bean products of Guizhou, namely fermented soya beans. The application method comprises culturing the selected strain GZU05 with solid fermented natto culture medium to produce beta-glucosidase and nattokinase, and obtaining fermented soybean product rich in nattokinase. The GZU05 of the invention can be used for high-value conversion of ginkgo flavonoid glycoside and can produce nattokinase with high value, the enzyme activity of the nattokinase is high, the sensory property is good, and the invention is suitable for being popularized and applied to producing the nattokinase.

Description

Bacillus amyloliquefaciens GZU05 for producing nattokinase and beta-glucoside and application method thereof

Technical Field

The invention relates to a microorganism, an enzyme, bacillus amyloliquefaciens and an application method of the bacillus amyloliquefaciens.

Background

beta-Glucosidase (beta-D-Glucosidase, EC3.2.1.21) is a broad-source hydrolase. The characteristic is that the beta-D-glucose can be hydrolytically bonded to a terminal non-reducing beta-D-glucose glycosidic bond and release beta-glucose and corresponding ligands. The beta-glucosidase may be produced by yeast, aspergillus, trichoderma, bacteria, etc. According to the reports at home and abroad, the enzyme can be widely used for the biotransformation of flavonoids.

The production of enzymes by microbial fermentation is an effective way for biological enzyme sources. Domestic and foreign research shows that the microbial fermentation enzyme production can hydrolyze ginkgo flavone glycoside into ginkgo flavone aglycone, so that the aglycone activity is obviously higher than that of the ginkgo flavone glycoside. The ginkgo flavone is the best medicament for preventing and treating cardiovascular and cerebrovascular diseases, and currently, the ginkgo flavone is mainly extracted from ginkgo leaves, but the ginkgo flavone extracted from the ginkgo leaves mainly exists in the form of ginkgo flavone glycoside, while the biological activity of the ginkgo flavone glycoside is obviously less than that of the ginkgo flavone aglycone, and the enzymatic method is environment-friendly and has high selectivity, so that the ginkgo flavone glycoside is necessarily converted into the aglycone by utilizing beta-glucosidase.

At present, although the research results on beta-glucosidase are more, most microorganisms are from the nature and are from the traditional fermented food, and the research results are only reported. In addition, the traditional enzymes have poor substrate selectivity and cannot maximize the content of aglycone, and reports of hydrolyzing the ginkgo flavone glycoside into aglycone with high selectivity are few. Wuyi et al use fixed commercial beta-glucosidase to hydrolyze ginkgo flavone glycoside, and obtain better results. However, the hydrolysis of ginkgo flavonoid glycosides by non-commercial beta-glucosidase is rarely reported, and because commercial beta-glucosidase is expensive, the screening of beta-glucosidase strains producing hydrolyzed ginkgo flavonoid glycosides lays a foundation for the industrial production of ginkgo flavonoid aglycones, and the reduction of conversion cost is very important.

The human health is seriously affected by thrombotic diseases, according to statistics, at least 1200 million patients die of thrombotic diseases every year around the world, and about 260 million patients die of China. Therefore, the development of thrombolytic and antithrombotic drugs has been a hot point related to human health. Currently, Streptokinase (SK), Urokinase (UK) and tissue plasminogen activator (t-PA) are commonly used thrombolytic antithrombotic drugs in clinic, but these drugs have the disadvantages of pain in administration, large side effect, high price, short half-life and the like. And the nattokinase has the advantages of oral taking, safety, low price, strong fiber solubility and the like, and becomes a research hotspot of thrombolytic products in recent years.

In a Chinese patent database, a plurality of patents and application parts related to beta-glucosidase exist, for example, ZL2014106023384, namely a beta-glucosidase and beta-glucosidase mutant and application, ZL2015101756557, high-tolerance beta-glucosidase and application, 2017102790450, namely a strain of beta-glucosidase producing bacteria for hydrolyzing ginkgetin glycoside and a screening method thereof, and the like; however, no application related to Bacillus amyloliquefaciens GZU05 is available.

Disclosure of Invention

The invention aims to provide a bacillus amyloliquefaciens GZU05 capable of producing nattokinase and beta-glucoside enzymolysis, which has important effects on the beta-glucosidase capable of selectively hydrolyzing ginkgo flavonoid glycoside produced by fermented soya beans and the nattokinase for preventing and treating cardiovascular and cerebrovascular diseases.

The invention also aims to provide a method for applying the bacillus amyloliquefaciens to producing flavone aglycone and producing natto kinase rich fermented soybean.

The bacillus amyloliquefaciens GZU05 capable of producing nattokinase and beta-glucoside enzymolysis provided by the inventor is classified and named as bacillus amyloliquefaciens GZU05 and Latin literature name: bacillus amyloliquefaciens GZU05 which is preserved in the China center for type culture Collection in 11.9.2018 has the preservation number of CCTCC M2018763, the preservation unit address is Wuhan, Wuhan university, the postal code is 430072, and the telephone number is 027-.

The bacillus amyloliquefaciens GZU05 is collected from traditional fermented soybean product fermented soya beans in Guizhou, and has the performance of producing beta-glucosidase for selectively hydrolyzing ginkgo flavonoid glycoside and producing nattokinase.

The method for applying the bacillus amyloliquefaciens to producing flavonoid aglycone and producing fermented soybeans rich in nattokinase provided by the inventor comprises the following steps:

(1) Properly diluting a fermented bean product sample, coating the diluted fermented bean product sample on a gardenoside color development plate, culturing the diluted fermented bean product sample by using a gardenoside color development screening culture medium prepared in advance, screening out a strain with a large blue colony and a dark color, carrying out scribing, separation and purification on the strain, and carrying out slant preservation and glycerol preservation for later use after the strain is detected as a single strain by a gram staining microscope;

(2) converting ginkgo flavone by using strain fermentation liquor, and determining the enzyme activity of beta-glucosidase; simultaneously measuring the enzyme activity of the nattokinase, carrying out sensory evaluation on the natto product, and selecting a natto fermentation strain GZU05 which has high activity of beta-glucosidase and nattokinase and optimal comprehensive sensory evaluation;

(3) performing morphological analysis, physiological and biochemical determination and 16S rRNA sequence analysis on the strain GZU05 to determine that the strain belongs to the bacillus amyloliquefaciens;

(4) culturing the bacillus amyloliquefaciens GZU05 by using a solid fermented natto culture medium, producing nattokinase and beta-glucosidase by fermented natto, and fermenting for 48-72 hours; obtaining the fermented soybean product rich in nattokinase.

In the step (1) of the method, the formula of the gardenoside chromogenic screening culture medium is as follows: 5 parts of ginkgo leaf extract, 0.5 part of magnesium sulfate, 10 parts of monopotassium phosphate, 0.1 part of geniposide, 10 parts of sodium glutamate, 2 parts of ammonium sulfate and 20 parts of agar; the culture temperature is 37 ℃, and the culture time is 36-72 h.

In the step (2) of the method, the unit of enzyme activity is the amount of enzyme required by hydrolyzing the ginkgo flavone glycoside to generate 1 mu mol of reducing sugar on a glucose basis every 1min under the measuring condition, and is defined as an enzyme activity unit.

In the step (2) of the above method, the method for measuring β -glucosidase comprises: taking a 15mL test tube with a plug scale, adding 1.8mL of substrate, preheating at 50 ℃ for 3min, adding 0.2mL of glucosidase enzyme solution, bathing at 50 ℃ for 30min, adding 3mL of DNS reagent, bathing in boiling water for 10min, cooling and fixing the volume to 15 mL. Taking the inactivated enzyme solution as a blank control, measuring a light absorption value at 540nm, and measuring the content of the generated reducing sugar; the method for measuring the enzyme activity of the nattokinase comprises the following steps: taking 10 mu L of nattokinase solid state fermentation crude enzyme liquid to be spotted on a fibrin flat plate (four holes are parallel in one group), standing for 10min, moving into an incubator at 37 ℃, keeping the temperature for 16h, taking out, measuring the diameter of a lysis ring by using a vernier caliper, calculating the area of each lysis ring, and calculating the enzyme activity of the nattokinase according to a regression equation of a urokinase standard curve; the sensory evaluation method of the fermented soybean product rich in nattokinase comprises the following steps: after fermentation, the fermented product is scored by a weighting method, and sensory evaluation is carried out on 4 aspects of wire drawing (viscosity), color, smell and posture.

In the above step, the substrate is 1% of one of ginkgo flavone glycoside or amygdalin, geniposide, rutin, gentiobiose, maltotriose, arbutin, laminarin and trehalose.

In the step (4) of the above method, the medium is prepared by the following method: cleaning soybeans, removing mildewed, rotten, worm-eaten and malformed soybeans, removing impurities, washing the soybeans with deionized water, accurately weighing, adding 4 times of deionized water in which 1% of salt and 1% of glucose are dissolved by volume, completely immersing the soybeans at normal temperature for 18-24 hours, and thus obtaining the soybean milk.

In the step (4), the beta-glucosidase has high selectivity on the ginkgo flavonoid glycoside, the enzyme activity of hydrolyzing the ginkgo flavonoid glycoside is 4.2U/g, the enzyme can be stored for 2-4 days at room temperature and for 7-10 days at 4 ℃, the conversion capacity of the enzyme is kept above 45%, and the enzyme activity of the nattokinase produced by fermenting natto exceeds 6000U/g.

Compared with the prior art, the invention has the following advantages: the conversion rate of the biological compound enzyme prepared by the invention to the substrate ginkgetin can reach 88 percent, and the conversion rate of beta-glucosidase to the substrate ginkgetin can reach 45 percent; the fermentation method has simple process and raw materials, and reduces the production cost of the beta-glucosidase; and the environmental tolerance of the enzyme is obviously improved compared with the beta-glucosidase reported in the literature: the tolerance pH2.8-5.5, and the tolerance temperature 40-70 ℃; the conversion reaction can be continuously carried out, and the method is suitable for large-scale continuous production; ② the beta-glucosidase enzyme activity of the bacillus amyloliquefaciens GZU05 is 4.022U/g, the enzyme activity of the nattokinase is 5786.1 +/-510.9, and the bacillus amyloliquefaciens has the function of converting the ginkgo flavonoid glycoside into aglycone. The produced natto kinase has high enzyme activity and good sensory property, and is easy to be accepted by the public; the bacillus amyloliquefaciens GZU05 can be used for high-value transformation of ginkgo flavonoid glycoside and production of high-value nattokinase, and simultaneously provides a novel culture method for producing beta-glucosidase and nattokinase, and has high safety; because the soybeans are wide in source and moderate in price, the production cost of beta-glucosidase and nattokinase is reduced. Therefore, the bacillus amyloliquefaciens has good application prospect.

Drawings

FIG. 1 is a glucose concentration standard curve;

FIG. 2 is a urokinase standard curve;

FIG. 3 is a microscope (100X) photograph of strain GZU 05;

FIG. 4 is a phylogenetic tree diagram of strain GZU 05.

Detailed Description

The following examples serve to further illustrate the invention:

EXAMPLE 1 preparation of seed liquid culture Medium, Nattokinase and beta-glucosidase crude enzyme solution

1) Preparing a seed liquid culture medium (g/L), 10 parts of glucose, 5 parts of yeast extract, 10 parts of beef extract and 5 parts of NaCl, selecting 2-3 cyclomycelia from a slant, inoculating the mixture into the liquid seed culture medium, and culturing for 18 hours at 37 ℃ under the condition of 180 r/min.

2) The preparation method of the crude enzyme liquid of nattokinase and beta-glucosidase comprises the following steps: adding 10g of NaCl and 10g of glucose into each liter of deionized water according to the mass part ratio, heating to completely dissolve, cooling, and drying the beans: deionized water 1: 4 soaking the commercial non-transgenic soybeans completely in an immersion manner for 18 hours. Then the mixture is drained and filled into 50/250 triangular bottles for sterilization. Inoculating the seed liquid to the cooled solid fermentation culture medium according to the inoculation amount of 2-4%, culturing at 37 ℃ for 72h, and shaking once every 12h to form a fermented product. Extracting the fermented product with physiological saline at 4 deg.C for 24 hr, grinding with mortar, centrifuging at 4 deg.C under centrifugal force of 12000 × g for 12-18min, collecting supernatant, and breaking cells to obtain crude enzyme solution of beta-glucosidase and nattokinase;

Example 2: and (3) measuring the enzyme activity of the beta-glucosidase:

1) drawing a glucose standard curve: sucking 0, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of anhydrous glucose solution of 1mg/mL into a test tube with a plug scale, supplementing water to 2mL, adding 3mL of DNS reagent, mixing, boiling in boiling water for 10min, cooling, adding water to fix the volume to 15mL, and measuring the absorbance at the wavelength of 540nm of a spectrophotometer; taking the absorbance as the ordinate and the glucose amount as the abscissa, drawing a standard curve, and taking a linear regression equation as y ═

0.5841x +0.0044, R2 ═ 0.9991 (fig. 1);

(2) and (3) measuring the enzyme activity of the beta-glucosidase: detecting the enzyme activity of the beta-glucosidase of the strain by a DNS method, and selecting the strain with higher enzyme activity; taking a 15mL test tube with a plug scale, adding 1.8mL of a substrate (1% of one of ginkgo flavonoid glycoside or amygdalin, geniposide, rutin, gentiobiose, maltotriose, arbutin, laminarin and trehalose), preheating at 50 ℃ for 3min, adding 0.2mL of enzyme solution, carrying out water bath at 50 ℃ for 30min, adding 3mL of DNS reagent, carrying out boiling water bath for 10min, cooling and fixing the volume to 15 mL. The absorbance was measured at 540nm using the inactivated enzyme solution as a blank control. And calculating the enzyme activity of the beta-glucosidase in the sample according to a regression equation of a glucose concentration standard curve.

Definition of beta-glucosidase enzyme activity unit: under the measurement conditions, the amount of enzyme required to hydrolyze ginkgo flavone glycosides to produce 1. mu. mol of reducing sugars (in terms of glucose) per 1min was defined as 1 enzyme activity unit (U).

GZU05 the beta-glucosidase enzyme produced by fermented soya beans hydrolyzes ginkgo flavone glycosides with an enzyme activity of 3.696U/g, which is higher than that of enzyme hydrolyzing other substrates, and shows high selectivity to ginkgo flavone glycosides.

Example 3: nattokinase activity assay

The activity of the natto kinase enzyme is defined as follows: measuring the diameter of the lysis rings by using a vernier caliper, calculating the area of each lysis ring, and calculating the enzyme activity of the sample nattokinase according to a regression equation of a urokinase standard curve.

Preparation of urokinase standard curve: respectively preparing urokinase standard substances into 248IU/m L, 496IU/m L, 744IU/m L, 992IU/m L and 1240IU/m L, spotting 10 mu L of the urokinase standard substances into newly prepared fibrinogen flat plate holes (4 holes are parallel to 1 group), placing for 10min, culturing at 37 ℃ for 16h, taking out, measuring the diameter of a lysis ring, and calculating the area of each lysis ring; in terms of the area (x, mm) of the dissolving ring²) Urokinase standard curve is plotted with urokinase enzyme activity (y, IU/mL) as abscissa and urokinase enzyme activity (y, IU/mL) as ordinate. The linear regression equation is that y is 4.2016x +10.874, and R2 is 0.9994 (fig. 2). Then GZU05 is tested, and the nattokinase activity of the fermented natto is 6861U- g。

Example 4: precisely measuring the nattokinase by an ultraviolet spectrophotometry:

the ultraviolet spectrophotometer method is a measuring method which is established by the Japan nattokinase society and can accurately reflect the fibrinolytic activity of the nattokinase, and is also one of the world-recognized nattokinase activity measuring methods. The method has the advantages that the fibrinolytic activity content of the nattokinase can be objectively obtained, and various samples can be simultaneously detected. Because the measured value of the agarose-fibrinogen plate method is easy to change along with the change of the culture time and the thickness of the plate, the accuracy is poor and the sample measurement with high accuracy requirement is not suitable. The specific method comprises the following steps: 1.4mL of Tris-HCl (50mM, pH 7.8) buffer and 0.4mL of fibrinogen solution (7.2mg/mL) were added to the tube, incubated at 37 ℃ for 5min, then 0.1mL of thrombin (20U/mL) was added, further incubated at 37 ℃ for 10min to form an artificial thrombus, 0.1mL of a sample to be tested was added, incubated at 37 ℃ for 60min, 2mL of trichloroacetic acid (0.2moL/L) solution was added, the reaction was stopped by standing for 20min, centrifuged at 13000 r/min for 10min, and the supernatant was taken out and the absorbance was measured at a wavelength of 275 nm. Definition of enzyme activity: the amount of enzyme required for an increase in absorbance at 275nm of 0.01 per 1min was defined as 1 unit of fibrin-degrading enzyme activity. The nattokinase activity of the nattokinase obtained after GZU05 solid-state fermented natto is precisely measured for 36 hours by an ultraviolet spectrophotometry method reaches 116 FU/g.

Example 5: GZU05 solid state fermentation natto sensory evaluation and scoring condition

(1) GZU05 sensory evaluation of strain solid state fermentation: the fermented and cooked natto is scored by a weighting method, sensory evaluation is respectively carried out on four aspects of wire drawing (viscosity), color, smell and property and behavior, each index is divided into five grades, 10 professional persons respectively evaluate the indexes, and the average value, the scoring standard and the scoring weight are shown in table 1.

TABLE 1 sensory Scoring criteria for fermented products

(2) Sensory evaluation results of solid fermented natto

Good production strains are the basis for ensuring food fermentation, and selection of strains with good organoleptic properties is a prerequisite for subsequent fermentation. Inoculating GZU05 strain into solid fermentation culture medium containing semen glycines as main raw material, culturing under the same fermentation condition, recording the wire drawing length, color, smell and taste of each strain fermentation product, and calculating the comprehensive score of 4.2. The fermented natto is yellow to light yellow, and has long-term wiredrawing phenomenon when fermented soybeans are stirred, has light fragrance, coordinated fragrance and uniform posture, and the nattokinase activity in the fermented soybeans is higher than 6000U/g (wet soybeans).

Example 6: colony morphology identification, physiological and biochemical identification and 16SrRNA molecular biology identification

The strain is cultured on a screening medium plate for 48 hours, and the colony morphology and the colony color are observed and recorded.

Gram staining: selecting bacterial colonies on the flat plate for smear, fixation, crystal violet initial staining, mordant staining, decoloration, water washing, safranin counterstaining, drying and microscopic examination.

Observing the colony morphology characteristics of the strain, wherein the bacterial colony of the strain has an obvious blue bacteria ring, the bulge has wrinkles, and after gram staining, observing the cell morphology characteristics under an oil microscope of an optical microscope, and the result is shown in figure 3.

And (3) carrying out physiological and biochemical characteristic identification on the bacillus amyloliquefaciens GZU05, wherein the physiological and biochemical characteristics of the bacillus amyloliquefaciens GZU05, namely carbon source utilization, are shown in table 2, and the physiological and biochemical characteristics, enzyme activity and carbon source assimilation of the bacillus amyloliquefaciens GZU05 are shown in table 3.

The phylogenetic dendrogram of the bacillus amyloliquefaciens GZU05 is shown in fig. 4.

TABLE 2 Biochemical Properties of Strain GZU05 production of acid Using carbon Source

Note: +: positive, -: the negative result is negative, and the negative result is negative,

TABLE 3 physiological and biochemical characteristics of Strain GZU 05-enzyme Activity, carbon Source assimilation

Note: +: positive reaction; -: negative reaction; w: weak positive reaction

<110> Guizhou university

He Laping

<120> bacillus amyloliquefaciens GZU05 for producing nattokinase and beta-glucoside enzymolysis and application method thereof

<130> 11111111

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1449

<212> DNA

<213> Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)

<220>

<221> 16S rRNA

<222> (1)..(1449)

<400> 1

atggcgcgtg ctatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag 60

cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 120

accggggcta ataccggatg gttgtctgaa ccgcatggtt cagacataaa aggtggcttc 180

ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 240

aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 300

cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360

agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 420

caagtgccgt tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 540

aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 600

gtcattggaa actggggaac ttgagtgcag aagaggagag tggaattcca cgtgtagcgg 660

tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg gtctgtaact 720

gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc 780

gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca gctaacgcat 840

taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacgggggc 900

ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 960

ttgacatcct ctgacaatcc tagagatagg acgtcccctt cgggggcaga gtgacaggtg 1020

gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080

acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc cggtgacaaa 1140

ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1200

tgctacaatg gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc 1260

tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat 1320

cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380

cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tatggagcca gccgccgaaa 1440

ggggacccc 1449

Claims

1. Bacillus amyloliquefaciens (Latin academy name: Bacillus amyloliquefaciens) GZU05 capable of producing nattokinase and beta-glucoside enzymolysis starch is characterized in that the Bacillus amyloliquefaciens is preserved in the China center for type culture collection (CCTCC M2018763) in 2018, 11 and 9 months; the address of the preservation unit is Wuhan, Wuhan university, whose postcode is 430072 and telephone number is 027-one 68754952.