CN115404190A - High-salt-resistant Maotai-yeast-derived fusion Weissella JQ3 capable of degrading nitrite and application thereof - Google Patents

Abstract

The invention discloses a high-salt-resistant sauce yeast source fusion Weissella JQ3 capable of degrading nitrite and application thereof, and relates to the technical field of microorganisms. The preservation number of the Weissella confusa JQ3 provided by the invention is CGMCC No.21637, and the strain can utilize various saccharides; the yeast has the advantages of no beta hemolysis, no generation of biological amine substances, no generation of virulence factors, sensitivity to most antibiotics, higher safety, high salt resistance and low pH value, capability of degrading nitrite, capability of providing strain resources for the development of leavening agents of a plurality of fermented foods such as pickle, sour meat, sour fish and the like, and practical significance for the separation and application of functional microorganisms in the sauce koji.

Description

High-salt-resistant Maotai-yeast-derived fusion Weissella JQ3 capable of degrading nitrite and application thereof

Technical Field

The invention relates to a high-salt-resistant sauce yeast source fusion Weissella JQ3 capable of degrading nitrite and application thereof, belonging to the field of microorganisms.

Background

Nitrite can cause hypoxia, and forms N-nitrosamine compound with strong carcinogenic and teratogenic effects on human body with amine compound. Nitrite is widely present in fermented foods such as fermented vegetables, and intake of more than 80% of nitrate and nitrite in daily life is related to vegetables and fermented products thereof. In the food industry, nitrite, although useful as an additive in food processing, must be used in a carefully controlled amount. The nitrite specified in GB 2762-2017 is in vegetables and products (pickled vegetables) thereofNot more than 20 mg. Kg ^-1 . Therefore, how to control the accumulation of nitrite in fermented food is the key to ensure the safety and green production of the product.

Lactic acid bacteria can degrade nitrite by producing lactic acid and nitrite reductase, a process that is currently considered safer and faster than physical and chemical degradation. In addition, lactic acid bacteria are the dominant population in the vegetable fermentation process, and the generated organic acids such as lactic acid and the like can regulate the acidity, enrich the mouthfeel and inhibit the growth of part of harmful bacteria. However, the salt concentration of the fermented vegetables is usually increased to 8% or more during the pickling process, so that the shelf life of the vegetables is prolonged. The high salt environment can limit the survival and functioning of lactic acid bacteria. Therefore, the growth of lactic acid bacteria in a high-salt environment has an important influence on the exertion of the nitrite-reducing ability thereof. However, in the current patent, few lactic acid bacteria with salt tolerance and nitrite reduction are reported, and most of the lactic acid bacteria with nitrite reduction belong to the genus lactobacillus.

Weissella fusca (Weissella confusa) ((Weissella confusa) Is an important strain of Weissella in lactic acid bacteria and is a dominant flora in fermented foods such as pickle and the like. The Weissella confusa is a halotolerant bacterium which can grow under the salt concentration of 0-20 percent and is a potential food leavening agent for reducing nitrite and resisting salt. However, there is no report that Weissella confusa degrades nitrite so far.

Disclosure of Invention

The invention aims to provide Weissella confusa with soy sauce yeast source, which is high-salt resistant and can degrade nitriteWeissella confusa JQ3 and application thereof: the Weissella fuscaWeissella confusa JQ3 is preserved in China general microbiological culture collection center in 2021, 1 month and 15 days, with the preservation number of CGMCC No. 21637.

The Weissella fusion JQ3 bacterial colony is milky white and round, has a smooth surface and neat edges, and is convex in the middle; catalase negative, gelatin liquefaction negative, gram staining positive, and short rod-shaped thallus arranged in pairs or short chains; can utilize 0.5% galactose, 0.5% D-trehalose, 0.5% D-ribose, xylose, but cannot utilize esculin, salicin, raffinose, melibiose, cellobiose, arabinose.

The invention also provides a microbial preparation containing the Weissella confusa JQ3.

In one embodiment, the microbial preparation contains ≧ 1 × 10 ⁷ CFU/mL or 1X 10 ⁷ CFU/g Weissella fusiformis JQ3.

The invention also provides application of the Weissella confusa JQ3 in degrading nitrite.

In one embodiment, the application includes, but is not limited to, adding the weissella fusiformis JQ3 or the microbial preparation to a nitrite-containing or nitrite-prone environment.

In one embodiment, the application is the addition of the weissella fusiformis JQ3 to a high salt environment food or food ingredient to degrade nitrite.

In one embodiment, said application includes, but is not limited to, adding said weissella fusiformis JQ3 or said microbial preparation to the kimchi to be fermented or being fermented.

In one embodiment, the application is in particular: mixing the pickling liquid containing the Weissella confusa JQ3 with mustard, and fermenting at 25-30 ℃ for at least 4 days.

In one embodiment, the preparation method of the curing salt is: dissolving salt in water to obtain a NaCl solution, and mixing the NaCl solution with the Weissella confusa JQ3 bacterial suspension to obtain the pickling liquid.

The invention also provides application of the Weissella confusa JQ3 in producing low-nitrite food.

In one embodiment, the food product includes, but is not limited to, sour meat, sour fish.

Has the advantages that: the Weissella confusa provided by the invention is a dominant bacterium in a sauce koji fermentation process, has good tolerance to high-salt and low-pH environments in the fermentation process, and has good nitrite reducing property. Also can provide strain resources for the development of leaven of a plurality of fermented foods such as pickle, sour meat, sour fish and the like, and has practical significance for the separation and application of functional microorganisms in the sauce koji.

Biological material preservation

Weissella fusca (Weissella confusa)Weissella confusa) JQ3, classified and namedWeissella confusaIt has been preserved in China general microbiological culture Collection center (CGMCC) No.21637 at 1 month and 15 days 2021, and the preservation place is No. 3 Xilu No. 1 Beijing Hopkin, the area facing the sun, beijing.

Drawings

FIG. 1 is a colony morphology of Weissella fusiformis JQ3 of the present invention.

FIG. 2 is a gram stain of Weissella fusiformis JQ3 of the present invention.

FIG. 3 is a phylogenetic tree fusing Weissella JQ3 of the present invention.

FIG. 4 is a standard curve of nitrite content.

FIG. 5 is a graph showing the effect of the culture time of Weissella confusa JQ3 on the nitrite degradation rate.

FIG. 6 is a chart showing the results of hemolysis experiments with Weissella fusiformis JQ3 of the present invention.

Detailed Description

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1 isolation and characterization of the strains

Weighing 10 g Maotai koji sample, adding 90 mL sterile normal saline, shaking in a shaking table at 37 deg.C and 150 r/min for 30 min, mixing, and performing gradient dilution to 10 ^-5 And (4) sample liquid. Coating 100 mu L of the strain in an MRS screening culture medium containing natamycin 0.4 g/L and vancomycin 0.2 g/L, and culturing for 24-48 h at the constant temperature of a biochemical incubator at 37 ℃. After obvious bacterial colony grows out on the culture medium, selecting a single bacterial colony by using an inoculating loop under aseptic condition, and carrying out three-region streaking purification on an MRS solid culture medium for 3 times until the bacterial colony forms on the culture medium are consistent.

The physiological and biochemical identification is referred to the lactic acid bacteria classification identification and experimental method and is appropriately modified. The results are as follows:

bacterial colony, thallus morphology and physiological and biochemical characteristics: the Weissella confusa JQ3 bacterial colony is milky white and round, has a smooth surface and regular edges, and is convex in the middle; the thallus is in the shape of short rod and is arranged in pairs or short chains.

Catalase negative, gelatin liquefaction negative and gram staining positive; can utilize 0.5% galactose, 0.5% D-trehalose, 0.5% D-ribose, xylose, but cannot utilize esculin, salicin, raffinose, melibiose, cellobiose, arabinose.

Molecular biological identification: extracting DNA of fusion Weissella JQ3 and using the DNA as a template, performing PCR amplification by using a bacteria 16S rRNA universal primer (27F and 1492R), sequencing an amplification product, performing BZbiocloud sequence homology comparison on the measured 16S rRNA, and comparing a strain JQ3 with the strain JQ3Weissella confusaThe homology of the Weissella confusa is over 99 percent, and the Weissella confusa is judged to be constructed by a phylogenetic tree.

Example 2 investigation of salt tolerance of strains

Activating Weissella for three generations, and regulating OD _{600 nm} = 0.6. + -. 0.05, inoculating 2% strain to MRS liquid medium with salt content of 0%, 3%, 6%, 9%, 12%, 15%, 18%, 21%, 24%, 27%, 30%, 33%, 35%, culturing at 37 ℃ 24 h, determining OD _{600 nm} And (4) taking the absorbance value, coating the fermented bacterial liquid on an MRS solid culture medium, observing the number of viable bacteria and the growth condition of the strain, and determining the salt tolerance of the strain.

The growth rate of the strain at different salt concentrations was calculated by the formula:

growth rate (%) = (OD of culture solution at different NaCl concentrations) _{600 nm} Blank OD _{600 nm} ) /(OD in NaCl-free culture solution) _{600 nm} Blank OD _{600 nm} ）×100。

TABLE 1 Weissella fusiformis JQ3 salt tolerance experimental results

Salt concentrateDegree/%)	Growth rate/%)
		3	63.04±4.25
6	29.94±0.08
		9	11.10±0.49
12	10.38±0.24
		15	10.44±0.48
18	10.16±0.21
		21	11.59±0.25
24	11.00±0.74

The growth efficiency of Weissella confusa JQ3 at 3% salt concentration is 63.04 + -4.25%, the growth efficiency at 6% salt concentration is 29.94 + -0.08%, the growth efficiency at 9% salt concentration is 11.10 + -0.49%, the growth rate of Weissella confusa JQ3 is not greatly different when the salt concentration exceeds 12%, and Weissella confusa JQ3 can survive when the salt concentration approaches 35% of the salt solubility limit.

Example 3 Low pH environmental tolerance test of strains

Inoculating Weissella fusiformis JQ3 into an MRS liquid culture medium for overnight culture, inoculating the bacterial liquid into the MRS liquid culture medium with pH 3.5, pH 3 and pH 2.5 under aseptic conditions, standing and culturing at 37 ℃, sampling, gradient diluting and spreading on an MRS solid plate after respectively inoculating 0 h, 3 h, 8 h and 24 h, culturing at 37 ℃ for 24 h, calculating the survival rate, and repeating for three times.

Survival rate (%) = lgN _n /lgN _m X 100. In the formula N _m The viable count/(CFU/mL), N, of 0 h _n The viable count/(CFU/mL) of the fermentation liquor sampled at different times

The viable count of Weissella confusa JQ3 in 0 h is 2.41 multiplied by 10 ⁹ CFU/mL, viable count of 1.74X 10 after culturing 3 h in MRS liquid culture medium with pH of 3.5 ⁸ CFU/mL, the survival rate is 87.83 percent, and the viable count after 8 h is 1.29 multiplied by 10 ⁷ CFU/mL, survival rate of 75.79%, viable count after 24 h of 1.22 × 10 ⁶ CFU/mL, survival rate of 64.87%. The strain can survive in MRS liquid culture medium with pH of 3 for 3 h or more, and the viable count of 3 h is 1.73 multiplied by 10 ⁷ CFU/mL, survival rate of 77.15%. Can survive 3 h or more in MRS liquid culture medium with pH of 2.5, and the viable count of 3 h is 6.6 × 10 ⁵ CFU/mL, survival rate is 62.03%.

EXAMPLE 4 determination of nitrite degrading ability of Strain

Inoculating Weissella confusa JQ3 to sterilized mixture containing 60.17 mg/L NaNO ₂ Culturing in MRS liquid culture medium containing sodium nitrite, culturing 24 h, 48 h and 72 h at 37 deg.C, and measuring according to the operation method of GB 5009.33-2016 (determination of nitrite and nitrate in food).

(1) Determination of nitrite standard curve: accurately sucking sodium nitrite standard use solution (corresponding to 0.0 mu g, 0.1 mu g, 0.2 mu g, 0.3 mu g, 0.5 mu g, 0.8 mu g, 1.0 mu g, 2.0 mu g and 5.0 mu g of sodium nitrite) of 0.00 mL, 0.02 mL, 0.04 mL, 0.06 mL, 0.10 mL, 0.16 mL, 0.20 mL, 0.40 mL, 1.50 mL and 1.00 mL, respectively placing the mixture into 50 mL cuvettes with plugs, respectively adding 2 mL of 4 g/L sulfanilic acid solution into a standard tube and a sample tube, uniformly mixing, standing for 5 min, respectively adding 1 mL of 2 g/L naphthyl ethylenediamine hydrochloride solution, adding water to scale, uniformly mixing, standing for 30 min, adjusting the zero point by using a zero tube through a 2 cm cuvette, measuring the absorbance at a wavelength of 538 nm, and drawing a standard curve, wherein the standard curve is shown in figure 4.

(2) Sample treatment: accurately sucking 5.0 mL fermentation liquor, placing in a 250 mL triangular flask, adding 12.5 mL of 50 g/L saturated borax liquid, stirring uniformly, adding about 40 mL distilled water, placing the triangular flask in a boiling water bath, heating for 15 min, taking out, cooling to room temperature, adding 5 mL of 106 g/L potassium ferrocyanide solution, mixing uniformly, adding 5 mL of 220 g/L zinc acetate solution, mixing uniformly, and standing for about 30 min to precipitate protein. Transferring the liquid into a 100 mL volumetric flask, adding water to the scale, and mixing uniformly. And (4) carrying out suction filtration on the treatment solution by using a circulating water type vacuum pump, discarding the primary filtrate 30 mL, and collecting the residual filtrate for later use.

(3) And (3) sample determination: sucking 2.00 mL above treatment liquid into 50 mL volumetric flask, adding about 40 mL water, mixing, adding 2 mL 4 g/L sulfanilic acid solution, mixing, standing in the dark for 5 min, adding 1 mL 2 g/L naphthyl ethylenediamine hydrochloride solution, mixing, adding water to scale, standing in the dark for 15 min, measuring absorbance at 538 nm with 2 cm cuvette, replacing sample liquid with equal amount of distilled water, and performing blank control according to the same treatment method.

The calculation formula of the nitrite degradation rate is as follows: nitrite degradation rate = (initial nitrite concentration-nitrite concentration in culture solution after n hours of culture) ÷ initial nitrite concentration.

The results are shown in FIG. 5, where the degradation rate of nitrite was 80.77%,48 h was 83.57%, and the degradation rate of 72 h was 90.10% for 24 h.

Example 5 safety test

(1) Indole experiments

The Weissella fusiformis JQ3 is inoculated in peptone water culture medium, 24 h is cultured at the constant temperature of 37 ℃, 2~3 is dripped into the culture medium, and the color change is observed by slightly shaking a test tube.

The result of the Weissella JQ3 fusion indole experiment is negative, tryptophanase cannot be generated, and indole substances cannot be generated.

(2) Hemolysis test

Weissella fusiformis JQ3 was streaked on Columbia agar plates, and cultured at 37 ℃ for 24 h, and presence or absence of hemolysis and color were observed.

The hemolysis experiment result of Weissella confusa JQ3 is alpha hemolysis.

(3) Amino acid decarboxylase validation experiment

The tyramine production and histamine detection adopt a flat plate color development method. Respectively adding histidine and tyrosine into the culture medium, adjusting the final pH to 5.5 with the final concentration of 2%, sterilizing the culture medium, pouring into a flat plate, solidifying, streaking the activated Weissella fusiformis JQ3 on the culture medium flat plate containing amino acid, culturing at 37 ℃ for 3 d, and observing color change. The putrescine production and cadaverine detection adopt a biochemical identification tube method. Inoculating the activated Weissella confusa JQ3 bacterial suspension into an ornithine decarboxylase and lysine decarboxylase test tube, simultaneously inoculating into an amino acid decarboxylase control tube, covering the surface of the biochemical tube liquid with 5~8 drops of liquid paraffin, culturing at 37 ℃ for 24 h, and observing the color in the tube.

Weissella confusa JQ3 does not produce amino acid decarboxylase, and biogenic amines such as cadaverine, putrescine, histamine, tyramine and the like are not produced.

(4) Virulence gene detection

Amplification of primers disclosed in the reference Screening biological strains for safety of Evaluation of viral and antigenic activities of infectious from fungal chipscylA、asa1、ace、esp、gelE5363 and hyl, etc., to detect the virulence gene of the fusion Weissella JQ3.

Amplification conditions: pre-denaturation at 95 ℃ for 4 min;32 cycles (denaturation: 94 ℃,30 s; annealing: 56 ℃,1min; extension: 72 ℃ for 1 min); re-extension, 7 min at 72 ℃.

Reaction system: the total reaction system was 10. Mu.L. 2. mu.L Template, upstream and downstream primers 0.2. Mu.L, 5. Mu.L 2EasyTap ^@ PCR SuperMix，2.6 μL Nuclease-free Water。

Wessella fusiformis JQ3 is not producedesp、ace、gelE、hyl、cylAAnd (4) an isopoxicity factor.

(5) Susceptibility test

Diluting the activated bacterial liquid of two generations to 0.5 McLeod turbidity with sterile normal saline, coating on MRS plate, standing for 5 min, sticking corresponding drug sensitive paper sheets on the surface of the culture medium, sticking 3 tablets on each plate, setting 3 groups of each tablet in parallel, and culturing at 37 ℃ for 24 h. The diameter of the inhibition zone of each drug sensitive paper sheet is measured by a vernier caliper, and the sensitivity of the strain to antibiotics is evaluated according to the execution standard of antimicrobial drug sensitivity test.

TABLE 2 Weissella fusiformis JQ3 drug susceptibility test results

Tablet name	Diameter of bacteriostatic circle (mm)
		Ampicillin	25.43±0.75（S）
Cefotaxime	29.19±0.83（S）
		Kanamycin	10.95±0.90（R）
Erythromycin	24.70±0.69（S）
		Tetracycline derivatives	24.44±0.40（S）
Chloromycetin	25.12±1.25（S）
		Klineomyces spVegetable extract	29.41±0.56（S）
Vancomycin	6.35±0.00（R）
		Norfloxacin hydrochloride	15.82±0.91（I）

Note: s is sensitive, I is intermediate sensitive, and R is drug resistant.

The fusion Weissella JQ3 is sensitive to ampicillin, cefotaxime, erythromycin, tetracycline and clindamycin, drug-resistant to kanamycin and vancomycin and sensitive to norfloxacin mediation.

Example 6 application of the Strain in the production of kimchi

(1) Preparing a bacterial suspension of the zymophyte strain: preparing a Weissella fusion JQ3 bacterial solution: activating Weissella fusion JQ3 for three generations, and regulating OD _{600 nm} =0.6 ± 0.05, inoculating the strain into MRS broth culture medium at an inoculation amount of 2%, and performing static culture at 37 ℃ for 16-18 h to obtain bacterium liquid A; centrifuging the bacterial liquid A in 10000 r/min for 10 min, collecting thallus, diluting the thallus with 0.85% sodium chloride aqueous solution by mass fraction, and preparing the thallus with the concentration order of 1 × 10 ⁹ CFU/mL of bacterial suspension.

(2) Pretreatment of raw materials: selecting fresh leaf mustard, removing old leaves, cleaning, shaping, air drying, blanching at 10 s, and cooling.

(3) Preparing pickled vegetables: cleaning the container, and sterilizing with boiling water for 20 min to obtain the sterilized container. Preparing salt water with the concentration of 80 g/L; mixing the NaCl solution with the Weissella confusa JQ3 bacterial suspension prepared in the step (1) to prepare a pickling solution, wherein the final concentration of Weissella confusa JQ3 in the pickling solution is 1 x 10 ⁷ CFU/mL; placing the pickling liquid of 1500 mL and the mustard of 800 g step (2) in a sterilized container, and pickling at 30 ℃ for 4 days to obtain the fermented mustard.

Respectively taking natural fermentation and commercial strain Weissella confusa CICC24453 as a reference, and treating a natural fermentation group according to the methods in the steps (2) to (3) except that Weissella confusa JQ3 bacterial suspension is not inoculated; and (3) preparing bacterial suspension by using the Weissella confusa CICC24453 according to the step (1) and using the prepared bacterial suspension for preparing the fermented mustard according to the steps (2) - (3). And (4) measuring the content of the nitrite. The nitrite content of the pickled mustard after fermentation of 4 d is shown in table 3, and the texture analysis results of the pickled mustard are shown in table 4.

TABLE 3 nitrite content of pickled mustard

Fermentation mode	Nitrite content (mg/kg)
		Natural fermentation	14.05
Inoculation with commercial Weissella fusiformis CICC24453	6.69
		Inoculation of Weissella fusiformis JQ3	3.62

The content of nitrite in the fermented mustard prepared by fusing Weissella JQ3 is 3.62 mg/kg, is lower than that in a natural fermentation group (14.05 mg/kg), is lower than that in a commercial Weissella CICC24453 fermentation group (6.69 mg/kg), and is far lower than the national standard 20 mg/kg. Compared with natural fermentation, the degradation rate of the nitrite of the Weissella confusa JQ3 is 74.2%, and the degradation rate of the nitrite of the commercial Weissella confusa CICC24453 is 52.4%, which shows that the pickled mustard with the Weissella confusa JQ3 inoculated can greatly reduce the nitrite content in the pickled mustard, and the degradation effect is better than that of the commercial Weissella confusa CICC24453.

TABLE 4 texture analysis of pickled mustard

Fermentation mode	Degree of brittleness	Elasticity	Chewiness of the product
				Natural fermentation	112.03	0.66	44.06
Inoculation with commercial Weissella fusiformis CICC24453	59.73	0.59	20.79
				Inoculation of Weissella fusiformis JQ3	173.54	0.63	54.94

As can be seen from table 4, the texture (crispness, elasticity and chewiness) of the juncea inoculated with weissella JQ3 under the same conditions is similar to the elasticity of the naturally fermented and pickled juncea, and the crispness and chewiness are better; the crispness, elasticity and chewiness of the pickled mustard leaves inoculated with the commercial Weissella confusa CICC24453 are obviously lower than those of the pickled mustard leaves subjected to natural fermentation; the results show that the pickled mustard with the inoculated Weissella fusion JQ3 does not obviously change the original pickled mustard quality.

Comprehensive nitrite degradation data analysis shows that compared with Weissella confusa CICC24453 of the same species, the nitrite content in pickled mustard inoculated with Weissella confusa JQ3 is significantly reduced in fermented vegetables with 8% salt concentration, and the texture of the pickled mustard is not significantly changed by inoculating Weissella confusa JQ3.

Claims (10)

1. Weissella fusca (Weissella confusa)Weissella confusa) JQ3 is preserved in China general microbiological culture Collection center in 2021, 1 month and 15 days, with the preservation number of CGMCC No. 21637.

2. A microbial preparation comprising weissella fusiformis JQ3 as claimed in claim 1.

3. The microbial preparation of claim 2, wherein the microbial preparation comprises ≥ 1 x 10 ⁷ CFU/mL or 1X 10 ⁷ CFU/g Weissella fusiformis JQ3.

4. The use of Weissella confusa JQ3 as claimed in claim 1 for degrading nitrite.

5. The application according to claim 4, characterized in that it comprises: adding the Weissella confusa JQ3 of claim 1 or the microbial preparation of any one of claim 2~3 to an environment containing or susceptible to the production of nitrite.

6. The use according to claim 4, wherein the use is fermentation of the Weissella fusiformis JQ3 added to a high salt environment food or food material for a period of time.

7. Use according to claim 6, wherein the food product comprises kimchi.

8. A method for preparing low nitrate kimchi, characterized in that a pickling solution containing Weissella fusiformis JQ3 as defined in claim 1 is mixed with mustard and fermented at 25 to 30 ℃ for at least 4 days.

9. The method as claimed in claim 8, wherein the pickling solution is prepared by mixing the Weissella confusa JQ3 strain with an aqueous solution of salt.

10. Use of weissella fusiformis JQ3 as claimed in claim 1 for the production of low nitrite food products.

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