CN111518734A

CN111518734A - High-yield guaiacol bacillus subtilis and application thereof

Info

Publication number: CN111518734A
Application number: CN202010528896.6A
Authority: CN
Inventors: 林斌; 陈申习; 杨强; 唐洁; 张龙; 宿智新
Original assignee: Jing Brand Co ltd
Current assignee: Jing Brand Co ltd
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2020-08-11

Abstract

The invention relates to a bacillus subtilis and application thereof, wherein the preservation number of the bacillus subtilis is CCTCCM 2020001, and the bacillus subtilis is obtained by separating and purifying distiller's yeast of Kyowa Ji Co. The bacillus subtilis can produce the guaiacol factor of 9.7mg/L, the content of the guaiacol in the fen-flavor liquor can be increased to 108%, and the bacillus subtilis can be used for producing the guaiacol factor in the fen-flavor liquor.

Description

High-yield guaiacol bacillus subtilis and application thereof

Technical Field

The invention relates to the field of microorganisms, and particularly relates to high-yield guaiacol bacillus subtilis and application thereof.

Background

Due to the unique production process, Chinese white spirit contains a large amount of volatile (phenols, acids, esters, terpenes, pyrazines, sulfur-containing substances and the like) and nonvolatile substances (volatile components such as amino acids, vitamins, mineral substances, polyhydric alcohols, cyclic ester active peptides and the like). They not only endow white spirit with color, flavor and taste, but also have various important physiological activities. The guaiacol substances (guaiacol, 4-methyl guaiacol, 4-ethyl guaiacol, 4-vinyl guaiacol and the like) are natural aroma compounds existing in liquor with sauce aroma, strong aroma, faint scent, sesame aroma and the like, and have important contribution to the aroma of the liquor. The gas mass spectrometry detection is carried out on guaiacol substances in 67 kinds of Chinese spirits, and the result shows that the content of 4-ethyl guaiacol is 1.3-167.5 micrograms/liter, and the content of 4-methyl guaiacol is 2.8-1709 micrograms/liter. The guaiacol substance has important contribution to the aroma of the white spirit as a white spirit flavor compound, has good active oxygen elimination function, can be used as a natural free radical scavenger, promotes microcirculation, enhances the immunity of a human body, can resist oxidation, prevents various diseases such as cardiovascular diseases and the like, and has the effects of preventing diseases, resisting aging and promoting the health of the human body. The biological transformation of microorganisms is an important source of guaiacol substances in Chinese white spirit, and as people seek health more and more, the fen-flavor white spirit which is one of main flavor types needs to increase health factors while improving flavor and quality. Screening functional strains producing health factors and improving the content of the health factors in white spirit become a current research hotspot. However, no report is found on the microbial strains for producing the guaiacol in the brewing process of the Xiaoqu fen-flavor liquor, so that the functional microbial strains for obtaining the high-yield guaiacol by screening from the brewing environment have extremely important effects on improving the flavor and the health connotation of the fen-flavor liquor.

Disclosure of Invention

The invention aims to solve the problems and provides a high-yield bacillus subtilis B257CCTCC NO: m2020001, deposit address: eight-way Lopa mountain China typical culture collection center in Wuchang district, Wuhan city, Hubei province, postcode: 430072.

the Bacillus subtilis B257 of the present invention is isolated from koji from Jinpai Co., Ltd, and its 16s rDNA is shown in Table 3. The bacteriological characteristics of this strain are as follows: the thallus is rod-shaped, has the length of 1.5-3 mu m and the width of 0.6-0.9 mu m, and has no capsule and mesogenic spores; the bacterial colony is milky white and opaque, the surface wrinkles are not smooth, and the edges of the bacterial colony are rough and diffuse; the physiological and biochemical characteristics are gram-positive, peroxidase-positive and V-P reaction-positive, can hydrolyze gelatin and starch, can utilize citrate to metabolize glucose to produce pyruvic acid, and can metabolize glucose, sucrose and maltose to produce acid.

The method for culturing the Bacillus subtilis B257, which is disclosed by the invention, comprises the following steps of: bacillus subtilis was inoculated into LB liquid medium containing 10g of sodium chloride, 10g of peptone, 5g of yeast extract powder, 1000ml of water and pH 7.0, and cultured at 37 ℃ and 150rpm for 72 hours.

The invention also discloses application of the Bacillus subtilis B257 in producing guaiacol.

The invention also discloses a microbial agent, which comprises the following components in percentage by weight: m2020001 Bacillus subtilis.

The invention also discloses a Xiaoqu fen-flavor liquor which is prepared from the following components in percentage by weight, wherein the preservation number is CCTCC NO: brewing with Bacillus subtilis M2020001.

The Bacillus subtilis strain provided by the invention is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 2 days in 2020, and the preservation number is CCTCC M2020001.

Compared with the prior art, the invention has the following beneficial effects: the bacillus subtilis with high guaiacol yield can produce 9.7mg/L of guaiacol and can improve the guaiacol content in fen-flavor liquor to 108%. Provides a new strain for guaiacol production and provides a valuable resource for fen-flavor liquor. The bacillus subtilis strain is separated from the distiller's yeast, can be used for producing white spirit, improves the content of the Xiaoqu fen-flavor white spirit, and provides a new way for enhancing the flavor of the spirit body and improving the health factors in the white spirit.

Drawings

FIG. 1 is a diagram showing the content of guaiacol health factor in laboratory test of functional bacteria B257 in accordance with the present invention;

FIG. 2 is a diagram showing the content of conventional flavor substances in a laboratory test of a functional bacteria B257 of the present invention;

FIG. 3 is a graph showing the effect of gram staining by Bacillus subtilis strain of the present invention (microscope 10X 100).

Detailed Description

The present invention is described in detail below with reference to the following embodiments and the attached drawings, it should be understood that the embodiments are only for illustrating the present invention and are not to be construed as limiting the present invention, and any modifications, equivalents and the like based on the present invention are within the scope of the present invention.

Example 1: guaiacol-producing bacillus screening

1. Screening of Bacillus in distiller's yeast of Hubei Jing brand Co Ltd

Materials: hubei Jinpai Co Ltd distiller's yeast

Culture medium:

LB culture medium: 10g of sodium chloride; 10g of peptone; 5g of yeast extract powder; 1000ml of water; and (5) bottling, placing in a sterilization pot at 121 ℃ for sterilization for 30 minutes, and cooling for later use, wherein the pH value is 7.0.

Separating and purifying bacillus:

adding 5g distiller's yeast sample into 95ml sterilized distilled water, placing into 150r/min shaking table for oscillating for 30min, placing the oscillated bacterial suspension in 75-80 deg.C water bath for 30min, performing gradient dilution, and collecting 200 μ L10 μ L^-3And 10^-4And adding the bacterial suspensions with the two gradients into an LB culture medium for coating, putting the LB culture medium into a 37 ℃ incubator for culture observation, observing and screening bacterial colonies on a flat plate, and picking the bacillus colonies for purification culture for next experimental screening. Ten bacillus B73, B79, B81, B99, B109, B156, B167, B172, B209, B213 and B257 with morphological differences are screened out.

2. Bacterial strain screening of bacillus for producing guaiacol factor

2.1 preliminary screening of guaiacol-producing strains

Experimental strains: bacillus species B73, B79, B81, B99, B109, B156, B167, B172, B209, B213 and B257 isolated from koji from Kyowa Kaltd.

Culture medium:

YGS medium: 2g of glucose, 1g of yeast extract powder, 1g of starch and 100mg of vanillic acid, adding 1000ml of water, heating and dissolving, bottling, sterilizing in a sterilization pot at 121 ℃ for 30 minutes, and cooling for later use.

The experimental method comprises the following steps:

(1) qualitative analysis of guaiacol production activity: the test strains were inoculated into YGS medium and cultured at 37 ℃ for 24 hours. 2mL of the bacterial solution was added to the reaction system (2mL of phosphate buffer solution + 20. mu.L (5U) of horseradish peroxidase (HRP) + 300. mu.L of 0.5% H₂O₂). And (3) standing the reaction solution at normal temperature for 5min, carrying out color comparison, and measuring the optical density value of OD 470. The capacity of each strain to metabolize glucose to generate guaiacol is judged according to the OD470 value.

(2) Quantitative analysis of guaiacol production activity: and (3) quantitatively analyzing the content of the guaiacol health factors in the fermentation liquor of the YGS culture medium of the bacillus by adopting GC-MS gas chromatography, and selecting the strain with high content of the health factors for re-screening.

The experimental results are as follows:

the ten selected bacillus strains B73, B79, B81, B92, B93, B99, B109, B156, B167, B172, B209, B213 and B257 are inoculated into a YGS culture medium for color reaction detection. The results show that (as shown in the table) ten total bacillus strains produce reddish brown substances in the reaction system, the absorbance value is between 0.366 and 1.851 at 470nm, and the ten microorganisms have the potential of producing guaiacol, wherein the absorbance values of B92 and B156 are both above 1.658, and the guaiacol producing activity is higher. And performing quantitative analysis on the content of the guaiacol health factors in the bacillus fermentation liquor by GC-MS gas chromatography in one step, wherein the result shows that: a total of five strains of Bacillus produced guaiacol at 16.89-21.88mg/L, with four strains (B73, B79, B81 and B257) having higher guaiacol content.

TABLE 1 Ten strains of Bacillus guaiacol Activity coloration and fermentation broth guaiacol content

Note that "-" indicates that the enzyme activity did not reach the detection value

2.2 rescreening of guaiacol-producing strains

Fermentation medium (bran culture medium) 2.5g of bran, adding 47.5mL of water, mixing, subpackaging in 250mL triangular flask, sterilizing at 121 deg.C for 30 min.

The experimental method comprises the following steps: inoculating the strains obtained by primary separation into fermentation medium at an inoculum size of 2% (v/m), and culturing at 37 deg.C and 160r/min for 6 days. Centrifuging the cultured bran fermentation liquor to obtain fermentation liquor supernatant, and detecting the fermentation liquor by using GC-MS. The results are shown in table 2 below:

TABLE 2 guaiacol content (mg/L) in liquid fermentation medium of four Bacillus bran strains

As can be seen from the table above, the content of guaiacol and 4-vinylguaiacol in the liquid fermentation medium of bran inoculated with the strain B257 is higher, 1.044mg/L and 9.7mg/L, which are respectively higher than those of the other three strains by 132-; the other two guaiacol factors are similar in content to B73, B79 and B257. Therefore, the strain B257 has better capability of producing guaiacol factors.

2.3 laboratory bench test of guaiacol-producing health factor strains

The experimental method comprises the following steps:

(1) preparation of experimental koji: adding the culture solution of the bacillus producing the health factor to be detected into the mixed distiller's yeast used for production according to a specific proportion, wherein 5.2g of the mixed distiller's yeast and 2ml of LB culture solution of the laboratory bacteria to be detected are needed for single sample, and the method is used for laboratory lab effect verification.

(2) Soaking grains: weighing the required experimental grain according to the amount of 500g of Australian sorghum of each sample, adding warm water at 60 ℃ (the water surface is higher than the grain surface), and soaking the grain in an oven at 60 ℃ for 24 hours.

(3) Steaming grains: firstly, putting the soaked grains into a sterilizing steamer for primary steaming at the temperature of 121 ℃ for 30 minutes. And after primary steaming is finished, adding 100 ℃ water to stew grains for 40 minutes, filtering the water, and putting the water into a sterilizing steamer to re-steam the grains for 30 minutes at 115 ℃.

(4) Mixing the grains and the yeast: and cooling the grains after re-steaming to 30 ℃, subpackaging in No. 12 self-sealing bags, each self-sealing bag is 1040g, and fully mixing with the experimental yeast.

(5) Saccharification: and (3) putting the mixed grain yeast into an incubator at 30 ℃ for saccharification for about 24 hours, keeping the seal of the self-sealing bag open, and sealing the opening with a wet towel to ensure the supply of oxygen and moisture in the saccharification process.

(6) And (3) sterilizing vinasse: weighing the vinasse used in the experiment according to the amount of 500g of a single sample, putting the vinasse into a sterilization steamer, and sterilizing at 121 ℃ for 30 minutes.

(7) Fermenting in a tank: mixing saccharified grain and sterilized distiller's grains according to the ratio of 1: 1, placing the mixture into a gas sampling bag, sealing, vacuumizing, placing the bag into an incubator at 30 ℃ for fermentation, and continuing for 7 days.

(8) Distilling wine: and 7 days later, filling fermented grains after fermentation into a special triangular flask for open fire distillation, taking 100ml of distilled wine sample, and sending the wine sample to a detection center for measuring wine body related gas phase index parameters and health factor indexes. The results are shown in FIGS. 1 and 2.

The gas phase data of the small test wine sample shows that in the aspect of guaiacol factors, the total content of the guaiacol factors in the white wine sample brewed by adding the healthy functional bacteria B257 distiller's yeast is 8.98mg/L, which is improved by 108 percent compared with the white wine sample without adding the B257 strain distiller's yeast. And the four guaiacol factors also show 11 to 237 percent increase compared with the strain without adding B257. Secondly, on the basis of conventional gas phase data of a wine sample, the distiller's yeast added with the health functional bacteria B257 has advantages in improving the alcohol content and the ethyl acetate, wherein the alcohol content is improved by 9.8 percent, and the ethyl acetate is improved by 8.1 percent; and in the fusel oil, the reduction is 5.5 percent. The visible health functional bacteria B257 can greatly improve the content of guaiacol on the basis of ensuring the wine yield and the quality of wine.

Example 2 identification of Bacillus strains

1. 16S rDNA sequence analysis

(1) DNA extraction

Collecting the thallus for use

The soil genome DNA extraction kit is used for DNA extraction, and the specific method refers to the instruction.

(2) Amplification of 16S rDNA fragment of Bacillus subtilis B257

And (3) taking the extracted Bacillus subtilis DNA as a template, and adopting bacterial 16S rDNA universal primers 27F and 1492R to amplify the 16S rDNA fragment. The amplification reaction system is 30 mu L, wherein the Premix Taq^TM(TaKaRa Taq^TMVersion2.0plus dye)15μL，ddH₂O13. mu.L, each of primers 27F and 1492R 0.5. mu.L, and DNA template 1. mu.L. And (3) amplification reaction conditions: 5min at 94 ℃; 30s at 94 ℃; at 55 ℃ for 50s and at 72 ℃ for 30s, and circulating for 32 s; extension at 72 ℃ for 10 mm and storage at 4 ℃.

(3)16S rDNA sequence analysis

The amplified 16S rDNA fragment was sent to the Wuhanhua large gene for sequencing to obtain the corresponding 16SrDNA amplification sequence results (shown in Table 3). And its species information (as shown in table 3) was obtained by comparison with the Blast search program in the national biotechnology information (NCBI).

TABLE 3 phylogenetic information of the strains and their 16S rDNA amplification sequence results

2. Morphological characteristics and biological characteristics identification of bacillus

The cells used in the following experiments were inoculated and activated in LB solid medium, and cultured at 37 ℃ for 1 to 3 days, and then used in the following experiments:

(1) microscopic observation of bacterial colony and thallus

Observing the colony morphology of the bacillus on the LB culture medium under the culture condition of 37 ℃; a small amount of the cells were picked up with an inoculating needle or a sterilized toothpick and placed on a slide glass, and microscopic observation of the cells was carried out under a microscope (10X 100).

The observations were as follows: the thallus is rod-shaped, has length of 1.5-3 μm and width of 0.6-0.9 μm, and has no capsule and mesogenic spore (as shown in figure 3); the bacterial colony is milky white and opaque, the surface wrinkles are not smooth, and the edges of the bacterial colony are rough and diffuse.

(2) Gram stain

Taking the newly activated thalli to perform smear fixation on a glass slide, staining the newly activated thalli for 1 minute by using ammonium oxalate crystal violet, and washing the newly activated thalli by purified water; adding iodine solution to cover the coated surface and dyeing for 1 minute; then washing with water, and absorbing water by using absorbent paper; adding a few drops of 95% alcohol, slightly shaking for decoloring, washing with water after 30 seconds, and absorbing water; and finally dyeing with safranin staining solution for 10 seconds, washing with purified water, drying and performing microscopic examination. The results are shown in Table 4.

(3) Catalase assay

When 3% hydrogen peroxide was directly added dropwise to the LB liquid medium of Bacillus which had been cultured for 3 days, it was immediately observed that the case where a large amount of bubbles were produced was positive (+) and the case where no bubbles were produced was negative (-). The results are shown in Table 4.

(4) Methyl Red test

The activated Bacillus bacteria are selected and inoculated into a general-purpose medium, and cultured at 36. + -. 1 ℃ or 30 ℃ preferably 30 ℃ for 3 to 5 days. From the next day, 1ml of the culture solution was taken every day, and 1-2 drops of methyl red indicator were added, whereby positive (+) showed bright red, weak positive showed pale red, and negative showed yellow (-). The result can be determined until positive is found or until the result is positive by day 5. The results are shown in Table 4.

(5) Starch hydrolysis test

Starch culture medium: 10g of soluble starch; 10g of peptone; 5g of glucose; 5g of sodium chloride; 5g of beef extract; 20g of agar; sterilizing at 121 deg.C for 20 min.

Inoculating activated Bacillus into starch agar plate, and culturing at 36 + -1 deg.C for 24-48 h. The iodine reagent is then directly dripped onto the surface of the medium plate. The results were immediately examined, and a positive reaction was a colorless transparent circle (+) formed around the colony or culture, and a negative reaction was a non-transparent circle (-). The results are shown in Table 4.

(6) Gelatin puncture test

Subpackaging peptone gelatin culture medium into test tubes, and sterilizing at 121 deg.C for 20 min. Activated bacillus was inoculated by puncture, after culturing at 25 ℃ for 21 days, the growth of the strain and the liquefaction of gelatin were observed, and the results are shown in table 4, where the gelatin liquefaction is positive (+) and the no liquefaction is negative (-).

(7) Citrate utilization test

Culture medium: 1g of sodium citrate; 1g of dipotassium phosphate; magnesium sulfate 0.2 g; 5g of sodium chloride; 1g of diammonium hydrogen phosphate; 20g of agar; sterilizing at 121 deg.C for 20min at pH of 6.8, and keeping.

Inoculating activated bacillus into a culture medium, culturing at 37 ℃ for 48 hours, adding 3 drops of bromothymol blue, and if the culture medium is changed from green to blue, determining the culture medium is positive (+); no color change, then it is negative (-). The results are shown in Table 4.

(8) V-P experiment

Inoculating activated bacillus into an LB culture medium, culturing for 48h at 37 ℃, taking 1ml of culture solution, adding 1ml of O' Meara reagent (40% sodium hydroxide aqueous solution added with 0.3% creatine), shaking for 1-2 min, standing in an incubator at 30 ℃ for 15min, and indicating that the reaction solution is eosin color, that is, the experimental result is positive (+), or else, the reaction solution is negative (-). The results are shown in Table 4.

(9) Sugar fermentation experiments

Preparing a bacillus culture medium: (NH)₄)₂HPO₄：1g，MgSO₄:0.2g, KCl 0.2g, yeast extract: 0.2g, sugar: 1% water-washed agar: 5-6g, distilled water: 1000mL, bromocresol purple: 0.4% ethanol solution: 2mL, pH 6.8-7.0. The indicator is added after the pH is adjusted. Subpackaging the above culture medium into test tubes, sterilizing at 115 deg.C for 20min to obtain culture medium with height of about 4-5cm, and changing color to yellow to positive (+) without changing to negative (-). Four kinds of saccharides such as sucrose, glucose, maltose and lactose were measured. The results are shown in Table 4.

TABLE 4 physiological and biochemical characteristics of the bacteria of the strains

The method comprises the following steps: "+" is positive and "+" is negative.

Claims

1. A high-yield guaiacol Bacillus subtilis B257 is characterized in that the preservation number is CCTCC NO: m2020001.

2. The use of the bacillus subtilis of claim 1 for increasing the content of guaiacol in white spirit.

3. The use according to claim 3, wherein the liquor is a Xiaoqu fen-flavor liquor.

4. A microbial inoculant comprising the collection number of claim 1 as CCTCC NO: m2020001 of Bacillus subtilis.

5. A Xiaoqu fen-flavor liquor, which is prepared from the liquor containing the preservation number of CCTCC NO: m2020001 is prepared by brewing the microbial agent of bacillus subtilis.