CN116855395A - Salt-tolerant zygosaccharomyces succinogenes M03 capable of producing succinic acid in high yield and application thereof - Google Patents

Salt-tolerant zygosaccharomyces succinogenes M03 capable of producing succinic acid in high yield and application thereof Download PDF

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CN116855395A
CN116855395A CN202310880715.XA CN202310880715A CN116855395A CN 116855395 A CN116855395 A CN 116855395A CN 202310880715 A CN202310880715 A CN 202310880715A CN 116855395 A CN116855395 A CN 116855395A
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salt
soy sauce
zygosaccharomyces
strain
tolerant
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滑欢欢
梁亮
梁小玲
扈圆舒
符姜燕
黄敏斯
伍加文
潘丽娟
赵红娟
张晓辉
尹何南
梁翠婷
王哲哲
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Guangdong Meiweixian Flavoring Foods Co Ltd
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Guangdong Meiweixian Flavoring Foods Co Ltd
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/50—Soya sauce
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Abstract

The invention discloses a salt-tolerant zygosaccharomyces M03 with high succinic acid yield, and the preservation number is GDMCC No. 63586. Also discloses a microbial agent containing the zygosaccharomyces M03. The application of the zygosaccharomyces M03 or the microbial preparation containing the salt-tolerant zygosaccharomyces M03 in soy sauce fermentation, soy sauce brewing or improving soy sauce flavor. The invention takes saccharomycete M02 separated from fermented soy sauce mash as an initial strain, adopts an ARTP mutagenesis method to carry out mutagenesis on the initial strain to obtain a mutant bacterial colony, adds a high-salt wort culture medium containing calcium carbonate for primary screening, adds the high-salt wort culture medium into the initial soy sauce mash for soy sauce brewing, takes physicochemical indexes such as amino acid nitrogen, succinic acid, formic acid, acetic acid and the like in crude oil as judgment bases, takes sensory evaluation such as delicate flavor, sour flavor, ester flavor and the like as auxiliary indexes, and finally screens out salt-tolerant zygote saccharomycete M03 strain which not only produces succinic acid in high yield, but also produces formic acid and acetic acid in low yield or does not produce formic acid and total acid.

Description

Salt-tolerant zygosaccharomyces succinogenes M03 capable of producing succinic acid in high yield and application thereof
Technical Field
The invention belongs to the technical field of soy sauce brewing, and particularly relates to salt-tolerant zygosaccharomyces succinogenes M03 for high yield of succinic acid and application thereof.
Background
Soy sauce is a traditional fermented condiment originating in china and has a long history of development. Soy sauce is a liquid seasoning with special color, fragrance and taste, which is formed by taking soybean and wheat as main raw materials and fermenting for a long time under the combined action of a plurality of microorganisms.
Flavor is one of the most important quality indicators of soy sauce and one of the most interesting problems for consumers and producers. The flavor consists of smell and taste, is the impression of food on sense organs such as taste, smell, touch and the like, is an intricate and complex sensing system, and is relatively complex to form. The flavor formation of soy sauce mainly depends on the metabolism and decomposition of protein, starch, fat and other substances by various microorganisms and enzymes produced by the microorganisms, and amino acids, small peptides, sugar, glycerol, organic acids, various volatile substances and the like are produced.
In the fermentation process of the high-salt diluted soy sauce mash, salt-tolerant aroma-producing saccharomycetes are generally added for improving the flavor of soy sauce, so that the single and unsaturated flavor of aspergillus oryzae for soy sauce brewing is improved, and the flavor and grade of the soy sauce are improved. The addition of the aroma-enhancing yeasts in the fermentation process improves the aroma to a certain extent, but the flavor is relatively single, and aroma substances generated by the aroma-enhancing yeasts are mainly ester aroma and mellow aroma, and the excellent aroma-enhancing yeasts, especially the yeasts for improving the delicate flavor of soy sauce are relatively lacking.
The salt-tolerant yeasts directly related to the flavor of the soy sauce mainly comprise zygosaccharomyces rouxii, torulopsis mutans yeast and the like, are microorganisms which are often utilized in the soy sauce fermentation process, mainly perform alcoholic fermentation, can also participate in the production of flavor substances such as esters, furans, ketones and succinic acid in the soy sauce fermentation process, and are main fermentation yeasts in the soy sauce fermentation process. The research shows that the volatile matters in the high-salt diluted state fermented soy sauce sample are acids and alcohols, and the main volatile matters account for 47% and 14% of the total content respectively, and the research results show that the content of organic acid matters in the high-salt diluted state fermented soy sauce sample is closely related to the flavor of the organic acid matters.
The organic acid in the soy sauce is mainly lactic acid and acetic acid, the aroma of the soy sauce mainly comes from the content of the organic acid, the organic acid can adjust the uncomfortable taste brought by high salt content in the soy sauce fermented by a high-salt diluted state process, the skeleton system forming the flavor of the soy sauce is alcohols, esters and phenols in all the flavor substances, the proportion of the esters is the largest, and the organic acid is a precursor substance of the esters.
In the organic acid of the soy sauce, lactic acid can endow the soy sauce with mellow and long-lasting flavor, and the soy sauce with high lactic acid content generally has good flavor; if the oxalic acid content is too large, the soy sauce is astringent, so that the soy sauce is not expected to contain too much oxalic acid; formic acid and acetic acid are volatile acids, and are undesirably contained in soy sauce in too large an amount; succinic acid or its salt has special taste of shellfish, and is the only organic acid capable of improving soy sauce flavor.
Sodium succinate has obvious seafood and shellfish taste, has the coordinated freshness-increasing effect when being mixed with monosodium glutamate and I+G, has better stability to heat and better solubility, and can improve one component of soy sauce delicacies; the soy sauce can be used alone or together with sodium glutamate, and can play a role in enhancing the freshness of soy sauce. At present, microorganisms capable of producing succinic acid in the soy sauce fermentation process are mainly saccharomycetes, and meanwhile, the saccharomycetes also produce organic acids such as oxalic acid, formic acid and acetic acid which have poor flavor for soy sauce.
In the prior art, no saccharomycete is used for improving the flavor of the soy sauce, succinic acid is produced at high yield, acetic acid is produced at low yield and formic acid is produced at low yield, so that the invention hopes to provide a salt-tolerant saccharomycete which can produce succinic acid at high yield, does not produce acetic acid or produce acetic acid and formic acid at low yield through screening, and the saccharomycete is added in the soy sauce fermentation stage, so that the content of succinic acid in the soy sauce can be obviously improved, thereby improving the delicate flavor of the soy sauce, reducing the bad pungent smell in the soy sauce and improving the flavor quality of the soy sauce.
Disclosure of Invention
The invention aims to provide salt-tolerant zygosaccharomyces M03 capable of producing succinic acid at high yield and a microbial preparation containing the zygosaccharomyces M03, wherein the salt-tolerant zygosaccharomyces M03 or the microbial preparation can produce succinic acid at high yield, produce no or low yield of volatile organic acids such as formic acid and acetic acid, can further improve the delicate flavor of soy sauce, and effectively solve the problem of insufficient natural delicate flavor of soy sauce.
The invention also aims to provide the salt-tolerant zygosaccharomyces M03 for producing the succinic acid with high yield or the application of the zygosaccharomyces M03-containing microorganism in soy sauce brewing or improving the flavor of the soy sauce.
The first object of the present invention can be achieved by the following technical means: a salt-tolerant zygosaccharomyces sp M03 with high yield of succinic acid, wherein the storage number of the zygosaccharomyces sp M03 is GDMCC No. 63586, and the storage date is 2023, 06 and 26.
Culturing the zygosaccharomyces M03 in a malt juice culture medium with high salt content (salt concentration 10 wt%) at 32.5 ℃ for 2d, wherein the thallus is turbid, is sterile and free of film production, has clove smell, has special shellfish smell, and has no bad pungent smell; when the thallus is observed under an optical microscope, the yeast cells are elliptic, and buds can be clearly seen.
The zygosaccharomyces M03 is cultured on a high-salt (salt concentration 10 wt%) wort agar plate at 32.5 ℃ for 3 days, wherein the colony diameter is about 1mm, the colony diameter is about 0.5mm, the surface is smooth, the surface is milk cheese-like, the surface is smooth, the surface is opaque, the reflection is not generated, the edge is neat, and the shape of a circle is round.
The invention relates to a mutagenesis breeding method of salt-tolerant zygosaccharomyces succinogenes M03 for high yield of succinic acid, which comprises the following steps: firstly, an initial strain is obtained from sauce mash primary screening, then the initial strain is screened again, the strain M02 with the best performance is selected and subjected to mutagenesis by adopting an ARTP technology to obtain a mutant strain, and sauce mash fermentation and screening again are carried out to obtain salt-tolerant zygosaccharomyces succinogenes M03 with high yield of succinic acid.
The initial strains obtained from the sauce mash primary screening comprise: and (3) coating and separating a sample containing saccharomycetes by adopting a high-salt wort culture medium flat plate, and selecting saccharomycetes with smaller transparent circles to obtain the initial strain.
Rescreening the starting strain comprises: selecting a plurality of single bacterial colonies with small transparent rings, putting the single bacterial colonies into a liquid high-salt wort culture medium to obtain a culture bacterial liquid, adding the culture bacterial liquid into an initial soy sauce mash, fermenting at constant temperature to obtain raw soy sauce, detecting physical and chemical indexes of amino acid nitrogen, succinic acid, formic acid, acetic acid, total acid and salt in the raw soy sauce, performing sensory evaluation on the fresh taste, the sour taste and the ester taste, and selecting a strain M02 with the best performance as a basic strain to perform mutation breeding.
The conditions for mutagenesis using the art technique are: inoculating saccharomycete M02 into a liquid high-salt wort culture medium to obtain a culture solution, uniformly coating the culture solution onto an ARTP slide, setting the power to be 120W and the air quantity to be 10SLM, selecting the action time to be 10s, 20s, 30s, 40s, 60s and 80s, transferring each mutagenesis slide into an EP tube containing 10mL of wort after mutagenesis is finished, performing vortex elution for 1.5min, coating 100 microliters of eluent onto high-salt (10%) wort containing 3 permillage calcium carbonate, culturing for 72h in a 32.5 ℃ incubator to obtain a primary screening plate, selecting 10 single colonies with small transparent circles, inoculating into the high-salt (10%) wort liquid culture medium, and culturing for 72h at 32.5 ℃ to obtain the culture solution.
The soy sauce mash fermentation re-screening comprises: adding 10 strains of culture solution of yeast strain obtained by primary screening into the initial stage of sauce mash (adding during mash preparation) according to a ratio of 6% 6 Fermenting at 30 ℃ for 120 days, stirring once per week, ensuring normal proliferation and fermentation of saccharomycetes, filtering to obtain crude oil, checking physicochemical indexes such as succinic acid, acetic acid, formic acid, amino acid nitrogen, total acid and the like in the crude oil, performing sensory evaluation on the crude oil in flavor, sourness and ester taste, selecting the strain with the best performance as a starting strain for mutation breeding, and comprehensively obtaining the best salt-tolerant zygosaccharomyces M03 by taking a reference sample as a reference strain without adding saccharomycetes culture solution and adding 10% of equal amount of salt water.
As a preferred embodiment of the invention, the mutation breeding method of the salt-tolerant zygosaccharomyces M03 strain is as follows:
1. isolation and screening of the starting Strain M02:
(1) initial screening of starting strain
The applicant finds that the raw soy sauce fermented for 90 days in one pot has a full fresh flavor, a soft flavor and no pungent sour taste, and the amino acid nitrogen content reaches 1.1g/100mL; coating and separating by using a high-salt wort culture medium added with 0.3% of calcium carbonate, and selecting 10 yeasts with smaller transparent rings, wherein the yeasts are named as SY01, SY02, SY03, SY04, SY05, SY06, SY07, SY08, SY09 and SY10;
(2) compound sieve for starting strain
Taking 2 kg of mature Daqu, dividing into 10 parts, respectively mixing with 600g of salt water with the concentration of 20 Baume to prepare soy sauce mash, carrying out constant-temperature fermentation for 120 days at the temperature of 30 ℃ according to 6 wt% (the yeast strain is added when the yeast strain is added to the initial soy sauce mash preparation), filtering to obtain raw soy sauce, carrying out sensory evaluation on the physicochemical indexes of fresh taste, sour taste and ester taste of the raw soy sauce, selecting the strain M02 (namely SY 07) with the best performance as a basic strain, and carrying out mutation breeding.
2. ARTP mutagenesis by the starting Strain M02
The method comprises the steps of using saccharomycete M02 of high-yield succinic acid obtained by separation in soy sauce mash as a basic strain, adopting an ARTP technology to carry out mutagenesis on a starting strain, using the mortality rate and the like as indexes, and using the mutagenesis time as a variable to determine mutagenesis parameters so as to obtain a mutant strain; screening was then performed using high salt wort plating medium supplemented with 0.3wt% calcium carbonate. Namely, a colony with a small transparent circle compared with the original strain M02 is picked up in a high-salt wort flat-plate medium added with 0.3wt% of calcium carbonate, and is subjected to primary screening to obtain 10 positive mutant strains named SY11, SY12, SY13, SY14, SY15, SY16, SY17, SY18, SY19 and SY20, and then sauce mash fermentation re-screening is performed; the yield of the organic acid of the obtained saccharomycete M03 (namely SY 16) is far higher than that of the original strain, the total acid is lower than that of the original strain, the volatile acids such as formic acid, acetic acid and the like are not detected, and the obtained product has rich fresh flavor, unique shellfish flavor and soft taste, does not stimulate sour taste, and can obviously improve the flavor of fermented seasonings such as soy sauce, fermented sauce and the like.
The primary screening method comprises the following steps: the samples containing yeasts were subjected to coating separation using a plate supplemented with 3wt% of high salt (10 wt%) wort medium, and the size of the transparent circles of the individual colonies on the plate was observed, indicating a lower total acid production as the transparent circles were smaller.
The re-screening method comprises the steps of picking single colony with small transparent ring, and obtaining culture bacterial liquid in a wort culture medium with high salt content (10wt%); adding the culture bacterial liquid into the initial soy sauce mash according to the proportion of 6 wt%o, fermenting at the constant temperature of 30 ℃ for 120d to obtain crude oil, detecting physicochemical indexes such as succinic acid, acetic acid, formic acid, amino acid nitrogen and the like in the raw soy sauce according to a conventional method, and performing sensory evaluation on the fresh taste, the sour taste and the ester taste.
3. Research on genetic stability of mutant strain M03
Continuously passaging microzyme M03 in a liquid high-salt (10wt%) wort culture medium for 10 times, adding culture solutions of the 1 st generation, 5 th generation and 10 th generation strains into the initial soy sauce mash, fermenting at a constant temperature of 30 ℃ for 60d, filtering to obtain crude oil, detecting that physicochemical indexes such as succinic acid, acetic acid, formic acid, amino acid nitrogen and the like in the crude oil are stable among the generations, wherein microzyme M03 does not show reversion mutation until at least the 10 th generation, and the genetic stability is good.
The invention also provides a microbial agent containing the salt-tolerant saccharomycete M03 for producing the succinic acid at high yield.
The second object of the present invention can be achieved by the following technical means: the application of the salt-tolerant zygosaccharomyces succinogenes M03 for producing the succinic acid in soy sauce fermentation, soy sauce brewing or improving soy sauce flavor.
The salt-resistant joint saccharomycete M03 is added in the soy sauce fermentation process, so that the fermented crude oil has better and delicious flavor, soft and palatable comprehensive taste, outstanding delicate flavor and good flavor harmony, and is suitable for producing high-grade soy sauce and other products in seasoning enterprises.
The invention also provides application of the microbial agent containing the salt-tolerant zygosaccharomyces succinogenes M03 for high yield of the succinic acid in soy sauce fermentation, soy sauce brewing or soy sauce flavor improvement.
The salt-tolerant zygosaccharomyces M03 is subjected to expansion culture by adopting a saccharomycete expansion culture system and a soy sauce culture medium to obtain a culture solution, the culture solution is added into the initial soy sauce mash according to the proportion of 6 wt%and fermented for 180 days to obtain crude oil, the amino acid nitrogen content in the crude oil is equivalent to that of a control sample (without adding saccharomycete M03), the succinic acid content is improved by 2 times, the acetic acid and formic acid are greatly reduced, the delicate flavor is obviously improved, the taste is soft, the non-irritating sour taste and the ester taste are obvious, and the soy sauce flavor can be greatly improved and the quality of the soy sauce is further improved after the culture solution is repeatedly applied to soy sauce brewing.
Preferably, the salt tolerant zygosaccharomyces sp.m 03 or the microbial agent containing the salt tolerant zygosaccharomyces sp.m 03 is added at the time of initial fermentation of the moromi, i.e. at the time of 0d of fermentation of the moromi.
The salt-tolerant zygosaccharomyces M03 provided by the invention is applied to the production of fermented seasonings such as soy sauce or fermented sauce, can obviously improve the delicate flavor of the fermented seasonings such as soy sauce, shortens the fermentation period, and solves the problem of single flavor of modern soy sauce. Meanwhile, the cost of saccharomycetes is low, the operation of the fermentation process is simple, the method is suitable for large-scale industrial production and high-end soy sauce brewing, and the application prospect is wide.
The invention has the following beneficial effects:
(1) The invention takes saccharomycete M02 separated from fermented soy sauce mash with complete delicate flavor, soft flavor and no stimulating sour taste, the amino acid nitrogen content reaches 1.1g/100mL as an original strain, adopts an ARTP mutagenesis method to mutagenize the original strain to obtain a mutant bacterial colony, adds 3 permillage of high-salt (10wt%) wort culture medium of calcium carbonate for primary screening, adds the high-salt wort culture medium into the initial soy sauce mash for soy sauce brewing, takes physicochemical indexes of amino acid nitrogen, succinic acid, formic acid, acetic acid and the like in crude oil as judgment basis, takes sensory evaluation of the delicate flavor, the sour taste, the ester taste and the like as auxiliary indexes, and finally screens salt-tolerant zygomycete M03 strain which not only produces the succinic acid in high yield, does not produce the formic acid and the acetic acid and has low total acid;
(2) The salt-tolerant zygosaccharomyces M03 with high yield of succinic acid, low yield or no yield of volatile acids such as acetic acid, formic acid and the like is obtained, the strain is high-salt tolerant, and is added into the initial soy sauce mash to brew soy sauce, crude oil obtained by fermentation has low total acid, outstanding delicate flavor, no pungent sour taste and high content of succinic acid, and gives special fragrance to soy sauce, so that the flavor of the soy sauce is improved, and the quality of the soy sauce is further improved;
(3) The salt-tolerant zygosaccharomyces M03 strain obtained by the invention can be used as a fermentation functional bacterium to be applied to traditional fermentation industries such as soy sauce, fermented sauce and the like and food industry, so that the product has more outstanding delicate flavor, soft flavor, no-irritation sour taste and outstanding ester flavor, the product quality is obviously improved, good operability and economic benefit are achieved, the fermentation process and the freshness-enhancing process are integrated, and the labor cost and material cost of enterprises are obviously reduced.
Drawings
FIG. 1 shows growth conditions of the base strain M02 after mutagenesis in a wort agar plate containing 10wt% of common salt and 0.3wt% of calcium carbonate at 32.5℃for 3 days, and shows the size of transparent circles on the front and back sides of the plate containing calcium carbonate;
FIG. 2 shows a culture broth obtained by inoculating yeast M03 in example 2 to a high-salt (10 wt%) malt extract liquid medium and culturing at 32.5℃for 2 d;
FIG. 3 is a diagram showing the morphology of a cell obtained by inoculating a yeast M03 to a high-salt (10 wt%) malt extract liquid medium in example 2 and culturing the cell at 32.5℃for 2d under a common optical microscope;
FIG. 4 is a graph showing growth of the yeast M03 obtained in example 2 on a high-salt (10 wt%) wort agar plate at 32.5℃for 3 days.
Detailed Description
The following examples are only for illustration of the invention, and the scope of the invention is not limited to the following examples. The object of the present invention can be achieved by those skilled in the art based on the above disclosure of the present invention and the ranges taken by the parameters.
The test reagents, methods and apparatus described in the examples below, unless otherwise indicated, are all those conventional in the art; the specific conditions are not noted in the examples and are carried out under conventional conditions or under conditions recommended by the manufacturer; the kit biomaterial, unless otherwise specified, is commercially available.
Example 1: isolation and screening of base strains
1. Initial screening of starting strain
The inventor separates the strain with high organic acid content, high succinic acid content and low total acid from the sauce mash of Guangdong delicious fresh flavoring food Co-Ltd, and observes the cell morphology and the plate colony morphology through an optical microscope, and the strain is identified as saccharomycete and named as M02.
In actual production, the inventor finds that the sauce mash flavor of part of the fermentation tanks is obviously superior to other fermentation tanks in sensory evaluation, and the sauce mash flavor is mainly characterized by strong fresh flavor, soft sour flavor, coordinated mouthfeel and obvious seafood flavor. The inventor collects high quality soy sauce mash, screens it and mutagenizes it.
The specific scheme is as follows:
a jar of fermented soy sauce mash (the material is called fermented soy sauce mash after the Daqu is mixed with brine and pumped into a fermentation tank for fermentation) is selected, and when the soy sauce mash is fermented for 90 days, the fresh flavor is sufficient, the flavor is soft, no sour taste is stimulated, and the amino acid nitrogen content reaches 1.1g/100mL.
50g of soy sauce mash is taken and added into 450g of sterile physiological saline and mixed into bacterial suspension.
The bacterial suspension is subjected to 10-time gradient dilution with sterile physiological saline for 5-7 gradients, 0.1mL and 0.2mL of the dilutions are respectively taken and coated on a high-salt (the salt content is 10%) wort culture medium added with 0.3% of calcium carbonate for coating separation, and the culture is carried out for 3d at 32.5 ℃. 10 single colonies with smaller transparent rings in the plates are picked up and named as SY01, SY02, SY03, SY04, SY05, SY06, SY07, SY08, SY09 and SY10; after each single colony was streaked and separated 3 times, the single colony was transferred to a liquid wort medium (sterilization condition, sterilization at 121 ℃ C. For 30 min) having a salt content of 10%, and cultured at a constant temperature of 32.5 ℃ C. For 48 hours at 180r/min, so that the concentration of the bacterial cells reached 1.0X10 8 And (3) obtaining culture solution with the concentration of more than one mL, and carrying out rescreening and preservation operation.
2. Basic strain re-screening
Taking soybeans and wheat as raw materials, soaking and cooking the soybeans, roasting the wheat, uniformly mixing the raw materials according to the mass ratio of 1:0.3, and preparing the yeast according to a conventional method; taking 2 kg of yeast, dividing into 10 parts, mixing 200g of yeast with 600g of 20 Baume salt water to obtain soy sauce mash.
Adding 10 strains of culture solution of yeast strain obtained by primary screening into the initial stage of soy sauce mash (adding during mash preparation) according to a proportion of 6wt% 6 Fermenting at 30deg.C for 120 days, stirring once per week to ensure normal proliferation and fermentation of yeastFiltering to obtain crude oil, checking physical and chemical indexes of amino acid nitrogen, succinic acid, formic acid, acetic acid, total acid and the like in the crude oil, performing sensory evaluation on the crude oil with delicate flavor, sour flavor and ester flavor, selecting the strain with the best performance as a basic strain for mutation breeding, and supplementing 10% of saline water with the same amount by using a control sample without adding saccharomycetes culture solution.
TABLE 1 physicochemical results analysis of crude oil fermented by different bacteria (detection result unit: g/100 mL)
Sample of Amino acid nitrogen Succinic acid Formic acid Acetic acid Total acid Salt content
Control sample 0.95 0.08 0.25 0.61 2.4 16.5
SY01 0.98 0.15 0.21 0.52 2.2 16.3
SY02 0.96 0.14 0.15 0.45 2.1 16.4
SY03 0.95 0.12 0.14 0.50 1.9 16.3
SY04 0.97 0.10 0.23 0.41 2.0 16.5
SY05 1.0 0.08 0.16 0.35 1.7 16.2
SY06 0.98 0.15 0.10 0.32 1.8 16.4
SY07 0.95 0.2 0.08 0.22 1.7 16.6
SY08 0.94 0.1 0.14 0.32 2.2 16.5
SY09 0.96 0.17 0.12 0.41 2.1 16.3
SY10 0.97 0.09 0.11 0.37 2.2 16.2
As shown in the results of Table 1, the inventor has separated and purified 90d soy sauce mash of a fermenter with strong fresh flavor, soft sour flavor, coordinated taste and obvious seafood flavor, the obtained saccharomycete strain is added into crude oil obtained at the initial stage of soy sauce mash, after adding saccharomycete SY07, the succinic acid content in the crude oil is highest, the volatile acid formic acid and acetic acid are obviously reduced, the total acid is also greatly reduced, and the effect of the strain SY07 is optimal as seen from physicochemical indexes; the flavor is obviously superior to other samples, and is mainly characterized by strong fresh flavor, soft sour flavor, coordinated taste and obvious seafood flavor
On the basis, the test sample is subjected to sensory evaluation by an evaluator in the internal organization of the company, each index is divided into 10 minutes at most, and the evaluation result is shown in Table 2
TABLE 2 sensory evaluation scoring results for crude oil fermented by adding yeast
As can be seen from the sensory results in table 2, the higher the score, the more prominent the index, the higher the flavor, ester taste and aroma scores, and the better the crude oil flavor; the sour taste and the pungency are opposite, and the higher the score, the worse the flavor of the crude oil, and the more the sour taste is, the more the pungency of the crude oil is dispersed. The result shows that after the yeast SY07 is added, the comprehensive result of the appearance and the evaluation of the fermented crude oil is optimal.
According to the results of tables 1 and 2, strain SY07 was added to the moromi in the early stage for soy sauce fermentation and the effect of improving the flavor of crude oil was better than that of the control strain and other strains selected by screening, and this strain was designated as yeast M02 and subjected to ARTP mutagenesis breeding as a starting strain.
Example 2: mutagenesis and identification of the basic Strain M02
1. ARTP mutagenesis of base Strain M02
Inoculating yeast M02 into liquid high-salt (10%) wort culture medium (sterilizing condition, sterilizing at 121deg.C for 30 min), culturing in glycerol storage tube inoculated with yeast M02 at 32.5deg.C for 48 hr to obtain thallus concentration of 1.0X10% 8 Above the volume/mL, a culture broth was obtained, and mutagenesis was prepared.
The mutagenesis mode is as follows: 10 microliter of culture solution is uniformly coated on an ARTP slide, the power is set to 120W, the air quantity is 10SLM, and the action time is selected from 10s, 20s, 30s, 40s, 60s and 80s. After mutagenesis, each mutagenic slide was transferred to an EP tube containing 10mL wort and vortexed for 1.5min.
100. Mu.l of the eluate was applied to a high salt (10 wt%) wort containing 3% calcium carbonate and incubated for 72h at 32.5℃in an incubator to give a preliminary screening plate as shown in FIG. 1. 10 single colonies with small transparent circles (with the action time of 60 s) are selected and inoculated into a high-salt (10wt%) malt juice liquid medium and cultured for 72 hours at the temperature of 32.5 ℃ to obtain a culture solution.
2. Mutant strain rescreening
The soybean and the wheat are used as raw materials, the soybean is soaked and steamed, the wheat is roasted, and the raw materials are uniformly mixed according to the mass ratio of 1:0.3, and the yeast is prepared according to a conventional method.
Taking 2 kg of yeast, dividing into 10 parts, mixing 200g of yeast with 600g of 20 Baume salt water to obtain soy sauce mash.
Adding 10 strains of culture solution of yeast strain obtained by primary screening into the initial stage of soy sauce mash (adding during mash preparation) according to a proportion of 6wt% 6 Fermenting at 30deg.C for 120 days, stirring once per week to ensure normal proliferation and fermentation of yeast, and filtering to obtain crude oil.
Checking physicochemical indexes such as amino acid nitrogen, succinic acid, formic acid, acetic acid, total acid, salt and the like in crude oil, performing sensory evaluation on the crude oil such as delicate flavor, sour flavor and ester flavor, selecting the strain with the best performance as a starting strain for mutation breeding, and supplementing 10% of salt water with the same amount if a control sample is a yeast culture solution without adding yeast; the 5 batches were repeatedly validated and the results averaged and the results are shown in table 3 below.
TABLE 3 physicochemical results analysis of crude oil fermented by the addition of different mutant strains (detection result units: g/100 mL)
Sample of Amino acid nitrogen Succinic acid Formic acid Acetic acid Total acid Salt content
Starting strain 0.95 0.2 0.08 0.22 1.7 16.4
SY11 0.95 0.19 0.07 0.28 1.8 16.3
SY12 0.96 0.15 0.12 0.32 2.1 16.2
SY13 0.97 0.17 0.14 0.29 1.9 16.1
SY14 0.97 0.21 0.06 0.24 2.0 16.2
SY15 1.0 0.18 0.07 0.35 1.7 16.2
SY16 0.97 0.29 0 0 1.5 16.3
SY17 0.95 0.20 0.10 0.12 1.8 16.4
SY18 0.96 0.17 0.04 0.22 2.0 16.2
SY19 0.98 0.23 0.05 0.21 2.1 16.3
SY20 0.97 0.20 0.12 0.17 1.9 16.2
As shown in the results of Table 3, the inventors carried out ARTP mutagenesis by taking M02 as a basic strain as a starting strain, and obtained 10 mutant strains by primary screening, the obtained mutant strains were added into crude oil obtained by fermentation at the early stage of soy sauce mash, after the mutant strain SY16 is added, the succinic acid content in the crude oil is improved by 45% compared with the starting strain, formic acid and acetic acid of volatile acid are not detected, the total acid is also greatly reduced, and compared with the starting strain, the total acid is reduced by 11.7%, and the effect of the strain SY16 is optimal as can be seen from physicochemical indexes; the flavor is obviously superior to other samples, and the flavor is mainly characterized by strong fresh flavor, soft sour flavor, coordinated taste, no pungent sour flavor and obvious seafood flavor.
On the basis, sensory evaluation is carried out on test samples obtained by fermenting mutant strains by using tissue evaluation personnel in the company, each index is divided into 10 minutes at most, and the evaluation results are shown in table 4.
TABLE 4 sensory evaluation scoring results for crude oil fermented by adding yeast
As can be seen from the sensory results in Table 4, the higher the score, the more prominent the index, the higher the flavor, ester taste and aroma scores, and the better the crude flavor; the sour taste and the pungency are opposite, and the higher the score, the worse the flavor of the crude oil, and the more sour, the crude oil has a pungent taste to be spread. The result shows that the comprehensive result of the appearance and the evaluation of the fermented crude oil is optimal after the mutant strain SY16 is added.
According to the results of tables 3 and 4, the mutant strain SY16 was added to the soy sauce at the early stage of the moromi fermentation to improve the flavor of the crude oil, and the best strain SY16 was selected and designated as yeast M03.
Culturing in malt juice medium containing 10% salt at 32.5deg.C for 2d, wherein the thallus is turbid, sterile, free of film, and has shellfish flavor, ester flavor and no unpleasant smell, as shown in figure 2; when the cells were observed under a common optical microscope, the yeast cells were spherical or oval, and obvious buds were observed, as shown in FIG. 3.
The resulting yeast M03 was cultured on a high-salt (salt content: 10 wt%) wort agar plate at 32.5℃for 3 days, and the colony was cheese-like, milky white, smooth in surface, opaque, non-reflective, clean in edge, round, and about 1mm in colony diameter, and the growth of the yeast M03 on the wort agar plate (salt content: 10 wt%) was shown in FIG. 4.
The strain is preserved in the microorganism strain collection of Guangdong province at 2023, 06 and 26 days, and the preservation number is: GDMCC No. 63586, deposit address: building 59, post code of national institute No. 5, mitrex, guangdong province: 510075. its taxonomic name was identified as zygosaccharomyces sp.
Example 3: genetic stability detection of yeast M03 strain
Inoculating the salt-tolerant zygosaccharomyces sp M03 screened in the example 2 on a high-salt wort solid slant culture medium containing 10wt% of salt, continuously carrying out passage 10, observing the growth condition of each generation of strains, fermenting and culturing the 1 st generation, 5 th generation and 10 th generation slant strains according to the method in the example 2, and checking physicochemical indexes such as succinic acid, acetic acid and formic acid in crude oil after fermentation; meanwhile, inoculating the 1 st generation, 5 th generation and 10 th generation inclined plane strains to a malt juice culture medium containing 10% of salt, culturing for 48 hours at 32.5 ℃ to obtain a culture solution, checking the alcoholic strength, total acid, succinic acid, formic acid and acetic acid indexes of the culture solution, judging the genetic stability of the culture solution, and if the physicochemical errors of the ten-generation strain culture solution and the fermentation crude oil are within the error range of 10%, indicating that the genetic stability of the strain is good, wherein the specific data are shown in Table 5.
TABLE 5 Yeast strain passage stability test results (test results Unit: g/100 mL)
As shown in the results of Table 5, the yeast M03 has good genetic stability from the physical and chemical results of the crude fermentation oil and the physical and chemical results of the culture medium.
Example 4: acid resistance test of Yeast M03 Strain
In the soy sauce fermentation process, the pH of the fermented soy sauce mash is acidic, and gradually becomes lower as the fermentation time increases until the fermentation is completed, and the pH of the soy sauce mash is about 4.8, so that yeast added to the soy sauce mash is required to have stronger acid resistance.
The pH of the malt juice culture medium with high salt content (10 wt% of salt) is regulated to 4.0-6.5, the pH is increased gradually by 0.5 gradient, 3 parallel gradient steps are carried out on each gradient, the inclined solid strain of M03 is inoculated, the culture is carried out for 48 hours under the conditions of 32.5 ℃ and 180r/min, the indexes such as alcohol content, succinic acid, acetic acid, formic acid, total acid and the like in the culture solution are measured, the growth condition of the strain is judged according to the content of the strain, and the acid resistance result of the strain is shown in table 6.
TABLE 6 acid resistance results for Yeast Strain M03 (test results Unit: g/100 mL)
As can be seen from the results in Table 6, the yeast strain M03 was still able to grow normally at pH4.0, and as the pH increased, the yeast strain was still able to grow well in an acidic environment, and therefore, the yeast strain M03 had good acid resistance.
Example 5: salt tolerance test of Yeast M03 Strain
The yeast added into the soy sauce mash has higher salt tolerance because of higher concentration of the salt in the soy sauce fermentation process, so that the yeast can grow and propagate in the high-salt soy sauce mash.
The salt concentration of the malt juice liquid culture medium is regulated to 8% -20%, the gradient increment is carried out by 2% -3%, 3 parallel are carried out on each gradient, the slant solid strain of the yeast strain M03 is inoculated, the culture is carried out for 48 hours under the condition of 180r/min at the temperature of 32.5 ℃, the indexes such as alcohol degree, succinic acid, acetic acid, formic acid, total acid and the like in the culture solution are measured, the growth condition of the strain is judged according to the content of the indexes, and the salt resistance performance result of the strain is shown in the table 7.
TABLE 7 results of salt tolerance of Yeast strain M03 (test results Unit: g/100 mL)
As can be seen from the results in Table 7, the salt tolerant zygosaccharomyces strain M03 was still able to grow normally at a common salt concentration of between 8% and 20%, and the yeast strain was able to grow well in an acidic environment with an increase in the common salt concentration, and therefore, the salt tolerant zygosaccharomyces strain M03 had good common salt tolerance.
Example 6: ethanol tolerance test of salt tolerant zygosaccharomyces M03 Strain
During the fermentation process of soy sauce, after yeast is added, ethanol is generated in the fermented mash, the concentration of ethanol can inhibit the growth and propagation of yeast, and the yeast added into the soy sauce mash needs to have higher ethanol resistance to grow and propagate in the fermented soy sauce mash.
The ethanol concentration of the malt juice culture medium with high salt content (salt concentration 10%) is regulated to 0% -4% (v/v), 1% gradient increment is adopted, 3 parallel gradient steps are carried out on each gradient, the slant solid strain of the yeast strain M03 is inoculated, the culture is carried out for 48 hours under the conditions of 32.5 ℃ and 180r/min, indexes such as succinic acid, acetic acid, formic acid and total acid in the culture solution are measured, the growth condition of the strain is judged according to the content of the succinic acid, acetic acid and formic acid, and the ethanol resistance of the strain is shown in Table 8.
TABLE 8 results of ethanol tolerance of Yeast Strain M03 (test results Unit: g/100 mL)
As can be seen from the results in Table 8, the salt tolerant zygosaccharomyces sp M03 was still able to grow normally at an ethanol concentration of between 0% and 4%, and the yeast sp was able to grow well in an acidic environment with an increase in the ethanol concentration, and therefore, the yeast sp M03 had good ethanol tolerance.
Example 7: test of optimal addition timing of Yeast M03 Strain culture solution to beer
Inoculating the salt-tolerant zygosaccharomyces sp M03 selected in example 2 to malt juice medium containing 10% salt, culturing at 32.5deg.C under 180r/min for 48 hr to obtain thallus concentration of 1.0X10 8 And (3) obtaining culture solution with the volume of more than one per mL for later use.
Taking soybeans and wheat as raw materials, soaking and cooking the soybeans, roasting the wheat, uniformly mixing the raw materials according to the mass ratio of 1:0.3, and preparing the yeast according to a conventional method; taking 2 kg of yeast, dividing into 10 parts, mixing 200g of yeast with 600g of 20 Baume salt water to obtain soy sauce mash.
Respectively adding culture solution of saccharomycete M03 into sauce mash for fermentation for 0d, 15d, 30d and 45d to ensure that saccharomycete content in the sauce mash is more than or equal to 10 6 Fermenting at 30 deg.c for 120 days, stirring once a week to ensure the normal proliferation and fermentation of saccharomycetes, filtering to obtain crude oil, checking physical and chemical indexes of succinic acid, acetic acid, formic acid, total acid, etc. in the crude oil, and determining the best adding time of the strain according to the content of the crude oil, and the result is shown in Table 9.
TABLE 9 physicochemical results of crude oil fermented at different addition times of Yeast (detection result units: g/100 mL)
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As can be seen from the results in Table 9, the yeast strain M03 was added at the time of moromi fermentation for 0d, and the crude fermentation oil results were optimal, so that the optimal timing of adding the salt tolerant zygosaccharomyces strain M03 was moromi fermentation for 0d.
Example 8: application of salt-tolerant zygosaccharomyces M03 strain in soy sauce brewing
Culturing the bacterial cells: the preserved salt-tolerant zygosaccharomyces M03 is respectively used with 10% of foodActivating the malt extract agar medium, inoculating a loop of thallus into sterilized 300mL malt extract medium containing 10wt% salt (sterilizing condition, 121 deg.C for 30 min), culturing at 32.5deg.C for 48 hr/min, and culturing until thallus concentration reaches 1.0X10 8 The culture solution is obtained by gradually expanding culture with a saccharomycete expanding culture system with the concentration of thalli reaching 1.0x10 8 And (3) the concentration of the active component is more than one/mL.
Application in soy sauce fermentation:
(1) the soybean and the wheat are taken as raw materials according to the proportion of 1:0.3 and are prepared into yeast according to the conventional method;
(2) adding yeast into 2.2 times of salt water to obtain soy sauce mash, wherein the salt water concentration is 20 Baume, and adding salt-tolerant zygosaccharomyces M03 culture solution for culturing to obtain soy sauce mash with thallus content of more than or equal to 10 6 And (5) fermenting the soy sauce mash according to a conventional fermentation mode. The control sample is not added with saccharomycete culture solution, and is supplemented with an equal amount of saline (the salt concentration is 10 percent);
(3) after fermentation, crude oil in the mature soy sauce mash is extracted, physical and chemical indexes such as amino acid nitrogen, succinic acid, formic acid, acetic acid, salt and the like in the crude oil are detected, sensory evaluation of delicate flavor, sour flavor and ester flavor is carried out, and analysis results are shown in tables 10 and 11.
TABLE 10 comparison of physicochemical results of crude oil fermented by the starting strain M02 and the mutagenized strain M03 (detection result unit: g/100 mL)
Remarks: the control sample is a crude oil sample obtained by adding the original strain M02 for fermentation, and the test sample is a crude oil sample obtained by adding the mutagenesis strain M03 for fermentation.
The results in Table 10 show that the crude oil obtained by fermenting salt-tolerant zygosaccharomyces sp M03 has slightly higher amino acid nitrogen content than the original strain, and has far higher succinic acid content than the original strain, and particularly low acetic acid and formic acid yields, so that the application of the yeast strain M03 to soy sauce brewing can obviously improve the flavor of soy sauce, improve the quality of soy sauce and reduce the soy sauce brewing cost.
The invention also sumps 20 appraisers with abundant experience to carry out sensory evaluation, wherein the evaluation indexes of the sensory evaluation comprise fresh taste, sour taste, aroma and irritation, the score is 0-5 minutes, the higher the evaluation score is, the better the index is, the comprehensive evaluation refers to the comprehensive sense of a sample and the sense of the whole appraiser, and the evaluation result is shown in table 11.
Table 11 sensory evaluation scoring results for crude oil fermented by adding yeast
Sample of Fresh flavor Sour taste Taste of esters Irritation (irritation) Fragrance of fragrance Comprehensive evaluation
Starting strain M02 3.7 4.1 2.8 3.8 2.6 3.3
MutagenesisStrain M03 4.2 3.5 4.1 2.7 4.1 4.2
As can be seen from the sensory results in Table 11, the higher the score, the more prominent the index, the higher the flavor, ester taste and aroma scores, and the better the crude flavor; the sour taste and the pungency are opposite, and the higher the score, the worse the flavor of the crude oil, and the more sour, the crude oil has a pungent taste to be spread. The result shows that the comprehensive result of the appearance and the evaluation of the fermented crude oil is optimal after the mutagenesis microzyme M03 is added.
In conclusion, the salt-tolerant zygosaccharomyces M03 is added to ferment the soy sauce, so that the taste and flavor of the soy sauce can be obviously improved, the quality of the soy sauce is comprehensively improved, and the application prospect is wide.
The present invention may be summarized in other specific forms without departing from the spirit or essential characteristics thereof. The above-described embodiments of the present invention are to be considered in all respects only as illustrative and not restrictive. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments according to the essential technology of the present invention fall within the scope of the present invention.

Claims (6)

1. A salt-tolerant zygosaccharomyces (Zygosaccharomyces sp) M03 for high succinic acid production, which is characterized in that: the preservation number of the salt-tolerant zygosaccharomyces M03 is GDMCC No. 63586, and the preservation date is 2023, 06 and 26.
2. The mutation breeding method of the salt-tolerant zygosaccharomyces M03 for high yield of succinic acid according to claim 1, which is characterized by comprising the following steps: firstly, an initial strain is obtained from sauce mash primary screening, then the initial strain is screened again, the strain M02 with the best performance is selected and subjected to mutagenesis by adopting an ARTP technology to obtain a mutant strain, and sauce mash fermentation and screening are carried out again to obtain salt-tolerant zygosaccharomyces M03 with high succinic acid yield.
3. A microbial agent comprising the high-succinic acid-producing salt-tolerant zygosaccharomyces sp M03 according to claim 1.
4. Use of the salt tolerant zygosaccharomyces M03 of high yield succinic acid according to claim 1 in soy fermentation, soy brewing or improving soy flavor.
5. Use of a microbial agent comprising the high-succinic acid-producing salt-tolerant zygosaccharomyces M03 according to claim 1 in soy fermentation, soy brewing or improving soy flavor.
6. Use according to claim 4 or 5, characterized in that: the salt tolerant zygosaccharomyces strain M03 or the microbial agent containing the salt tolerant zygosaccharomyces strain M03 is added when the sauce mash is initially fermented, namely, when the sauce mash is fermented for 0d.
CN202310880715.XA 2023-07-18 2023-07-18 Salt-tolerant zygosaccharomyces succinogenes M03 capable of producing succinic acid in high yield and application thereof Pending CN116855395A (en)

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