CN113667612A

CN113667612A - Yeast and application thereof

Info

Publication number: CN113667612A
Application number: CN202111091945.5A
Authority: CN
Inventors: 孙晓琪; 周其洋; 周斌; 黄磊; 李荔
Original assignee: Foshan Haitian Flavoring and Food Co Ltd; Foshan Haitian Gaoming Flavoring and Food Co Ltd
Current assignee: Foshan Haitian Flavoring and Food Co Ltd; Foshan Haitian Gaoming Flavoring and Food Co Ltd
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2021-11-19
Anticipated expiration: 2041-09-17
Also published as: CN113667612B

Abstract

The invention relates to the technical field of microbial fermentation. In particular, the invention relates to a yeast which is suitable for soy sauce fermentation production and can obviously improve the content of 4-ethylguaiacol, glutamic acid and/or beta-phenylethyl alcohol in soy sauce or sauce. The invention also relates to the use of said yeast in the production of soy sauce or sauce (e.g. low-salt or reduced-salt soy sauce or sauce). Furthermore, the present invention also relates to a method for preparing soy sauce or sauce (e.g., low-salt or reduced-salt soy sauce or sauce) comprising adding the yeast of the present invention during moromi fermentation.

Description

Yeast and application thereof

Technical Field

Background

Soy sauce is an indispensable seasoning at home, but about 19% of daily salt intake in the diet is derived from soy sauce. Because the intake of higher salt causes potential risks such as hypertension and cardiovascular diseases, the consumption demand of people for low-salt/reduced-salt soy sauce is increasing with the increase of health consciousness of consumers, and the eating of low-salt/reduced-salt fermented soy sauce is a trend.

At present, the low-salt/reduced-salt fermented soy sauce is obtained by adopting a low-salt solid state fermentation or high-salt dilute state fermentation salt reduction mode. The low-salt solid fermented soy sauce adopts 10 to 12 percent of saline water which is 1.1 to 1.2 times of the total mass of raw materials to be yellow, simultaneously keeps the fermentation temperature above 40 ℃, has the fermentation period within 1 month and has lower production cost, but because the fermentation temperature is higher and the fermentation time is shorter, microorganisms cannot effectively participate in the flavor fermentation of the soy sauce, the produced soy sauce has single taste, poor flavor, insufficient delicate flavor, slightly sour taste and poor flavor quality, and cannot meet the public demand. In the high-salt dilute soy sauce process, 18% -22% saline water which is 1.8-2.2 times of the total mass of the mixed raw materials is adopted for yellow falling and natural fermentation, but the high salt can greatly inhibit the activity of protein, so that the period of the high-salt dilute soy sauce is longer than that of low-salt fermentation, and is generally 2-6 months. Under the natural temperature fermentation environment, the main flora of the soy sauce fermentation, such as yeast and other microorganisms, are fully grown, so that the high-salt dilute soy sauce has mellow taste, delicious taste and rich fragrance, and is deeply popular. In order to obtain low-salt soy sauce with good taste and flavor, the development of low-salt soy sauce is carried out on the basis of a high-salt dilute soy sauce process at home and abroad.

The development of low-salt soy sauce is currently carried out around a high-salt dilute state process, and mainly develops from the following aspects: 1) desalting with electrodialysis equipment to obtain low-salt soy sauce at high cost, and removing part of flavor and fragrance substances of soy sauce by electrodialysis to slightly reduce quality; 2) concentrating the high-salt soy sauce by using normal-temperature concentration equipment to separate out the salt of the soy sauce in a saturated state, then removing the separated salt, and supplementing a certain amount of water to obtain the low-salt soy sauce, wherein the cost is high, the flavor of the soy sauce is greatly changed, and the quality is reduced; 3) the method has the advantages that the low-salt soy sauce is obtained by directly adding water into the high-salt soy sauce for dilution, the cost is lower, but the taste and the flavor of the soy sauce are obviously changed, and the quality is obviously reduced; 4) the high-salt dilute state process is improved, the salt content of the fermented soy sauce is reduced to 12% or less (hereinafter referred to as low-salt dilute state), and poor control of the process can cause the fermented low-salt soy sauce to have sour taste, thin flavor, weak aroma and insufficient delicate flavor. Therefore, in order to obtain high-quality low-salt fermented soy sauce, it is necessary to solve the problem of insufficient flavor, aroma and umami taste of low-salt soy sauce.

Disclosure of Invention

The inventor of the application obtains a product through a large amount of screeningWickerhamiella versatilisThe strain ZB435 can obviously improve the content of 4-ethylguaiacol in soy sauce or sauce, improve the proportion of pleasant aroma substances such as beta-phenethyl alcohol, isoamyl acetate, 3-methylthio propanol and the like, improve the content of glutamic acid in the soy sauce so as to improve the delicate flavor of the soy sauce, and has a certain effect of improving sour taste. In addition, the strain can increase the ethanol content in the soy sauce in the fermentation process, thereby improving the fragrance and the antiseptic power of the soy sauce, and improving the overall fragrance and the flavor of the soy sauce or the sauceHas good application effect. Based on this, the present inventors developed a novel strain for preparing soy sauce or paste and a method for preparing soy sauce or paste based on the strain.

Provided by the present applicationWickerhamiella versatilisThe strain ZB435 has wide salt tolerance range, has better growth capacity in an environment with 5% of low salt content, can normally grow in a high salt environment such as an environment with 20% of salt content, has certain growth capacity even in an environment with 25% of salt content, and is suitable for fermentation production of low-salt or reduced-salt soy sauce and conventional high-salt soy sauce. The ZB435 is added during soy sauce mash fermentation, so that the content of substances such as 4-ethylguaiacol, alcohols, glutamic acid and the like in soy sauce can be remarkably increased, the problems of low aroma, lack of delicate flavor and insufficient flavor of the soy sauce are solved, the ethanol content is increased, the soy sauce aroma can be improved, and the soy sauce preservative power is improved to a certain extent. In addition, the addition of ZB435 also has a significant effect on the enhancement of the umami taste of soy sauce and the alleviation of salty taste.

Accordingly, in one aspect, the present invention provides a method of making a semiconductor deviceWickerhamiella versatilisStrain ZB435, which was deposited at 29 months 6/2021 in the guangdong province collection of microorganisms with the deposit number GDMCC NO: 61755, the preservation address is No. 59 of the Zhou Dazhong 100 Jie, Guangzhou city.

In another aspect, there is provided the use of the strain of the present invention ZB435 in the preparation of soy sauce or sauce, and the use of the strain ZB435 for increasing the content of 4-ethylguaiacol, glutamic acid and/or β -phenylethyl alcohol in soy sauce or sauce.

In another aspect, there is provided the use of the strain of the invention ZB435 for the preparation of a low-salt or reduced-salt soy sauce or spread, and the use of the strain ZB435 for increasing the content of 4-ethylguaiacol, glutamic acid and/or β -phenylethyl alcohol in a low-salt or reduced-salt soy sauce or spread.

In another aspect, the present invention provides a method for increasing the content of 4-ethylguaiacol, glutamic acid and/or β -phenylethyl alcohol in soy sauce or paste, and a method for preparing soy sauce or paste. The method comprises adding the strain of the invention, ZB435, during moromi fermentation. In certain embodiments, the soy sauce or spread is a low-salt or reduced-salt soy sauce or spread.

In certain embodiments, the method is low salt solid state fermentation, high salt dilute state fermentation, or low salt dilute state fermentation.

In certain embodiments, the method comprises mixing the feedstock with brine to yellow off, fermenting moromi after the brine has yellow off, and adding the strain ZB435 to the moromi during fermentation.

In certain embodiments, the method is a low-salt dilute fermentation in which 1.8-2.2 times the total mass of the mixed raw materials (e.g., less than 18%, such as 12% -18%, 12% -15%, 15% -18%, 14% -16%, such as about 15%) of brine is employed to yellow and ferment, and the soy sauce obtained via the low-salt dilute fermentation has a salt content of no more than 12%.

In certain embodiments, the strain ZB435 is added to the moromi described above and fermentation is continued on days 20-40 (e.g., days 25-35, e.g., day 30) of moromi fermentation.

In certain embodiments, the moromi fermentation is continued for 80-100 days (e.g., 85-95 days, e.g., 90 days).

In certain embodiments, the strain ZB435 is added in an amount of 10⁴~10⁶Per g (e.g. 10)⁵Per g).

In certain embodiments, the low salt dilute state fermentation comprises: fermenting the moromi after the brine is yellowed at a first temperature, and then continuing the fermentation at a second temperature. In certain embodiments, the strain ZB435 is added during the fermentation at the second temperature.

In certain embodiments, the first temperature and the second temperature are selected from 25-40 ℃ (e.g., 25-35 ℃), and the second temperature is higher than the first temperature. In certain embodiments, the first temperature is 20-30 ℃ (e.g., 25 ℃). In certain embodiments, the second temperature is from 25 ℃ to 35 ℃ (e.g., 30 ℃).

In certain embodiments, fermentation is carried out at the first temperature for 10-30 days, such as 15-25 days, such as 20 days. In certain embodiments, fermentation at the second temperature is for 60-100 days, 60-90 days, 60-80 days, 65-75 days, e.g., 70 days.

In certain embodiments, the yellowing temperature is from 15 to 25 ℃ (e.g., 15 to 20 ℃ (e.g., 18 ℃).

In certain embodiments, the strain ZB435 is pre-cultured. In certain embodiments, the pre-culturing comprises the steps of: the strain ZB435 was inoculated into wort medium and cultured under aerobic conditions. In certain embodiments, the pre-culturing comprises the steps of: the strain ZB435 is inoculated into a wort medium containing 5% to 15% (e.g., 10%) NaCl and cultured at a temperature of 25 to 40 ℃ (e.g., 28 to 35 ℃ (e.g., 30 ℃) under aerobic conditions.

In certain embodiments, the method comprises one or more of the following steps:

pretreating bean raw materials;

pretreating starchy auxiliary materials;

mixing the pretreated bean raw materials and starchiness auxiliary materials, adding yeast seeds, and preparing into yeast;

mixing the koji with saline water to obtain soy sauce mash (i.e. yellow drop);

fermenting the sauce mash;

after the fermentation is finished, separating fermented sauce and/or sauce mash from the fermentation product;

blending, sterilizing and filling or any combination thereof the fermented sauce to obtain the sauce; or blending, sterilizing and filling or any combination thereof the fermented soy sauce to obtain the soy sauce.

In another aspect, the present invention provides a soy sauce or spread, which is prepared by the method as hereinbefore described. In certain embodiments, the soy sauce or spread is a low-salt or reduced-salt soy sauce or spread.

In another aspect, the present invention provides a method for culturing the strain of the present invention, ZB435, comprising the step of inoculating the strain ZB435 to a wort medium and culturing under aerobic conditions. In certain embodiments, the method comprises: the strain ZB435 is inoculated into a wort medium containing 5% to 15% (e.g., 10%) NaCl and cultured at a temperature of 25 to 40 ℃ (e.g., 28 to 35 ℃ (e.g., 30 ℃) under aerobic conditions.

The terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

Sauce mash: the koji is mixed with brine to form a semi-fluid mixture.

Soy sauce mash: the yeast is mixed with the salt water, and then is naturally layered to form a porous solid which is positioned on the upper layer.

Sauce juice: the finished koji is mixed with the saline water to form a liquid substance in the lower layer due to natural layering.

Low salt or reduced salt: the salt content is not higher than 12%.

Low salt thin fermentation (also referred to herein as low salt thin mash fermentation): adopts 1.8-2.2 times of the total mass of the mixed raw materials to make the saline water yellow and ferment. The salt content of the soy sauce obtained by the low-salt dilute state fermentation is not higher than 12%.

Advantageous effects of the invention

The strain ZB435 of the invention can obviously increase the content of 4-ethylguaiacol in soy sauce or sauce, increase the composition proportion of pleasant aroma substances such as beta-phenylethyl alcohol, reduce aldehyde pungent odor and improve sour taste, thereby improving the aroma and flavor of the soy sauce or the sauce, simultaneously not influencing the content of nutrient components such as total acid, amino acid nitrogen and the like in the soy sauce or the sauce, and obviously increasing the content of glutamic acid in the soy sauce or the sauce. Moreover, the strain ZB435 has a wide salt tolerance range, and the ethanol content in the soy sauce can be increased in the fermentation process, so that the fragrance and the preservative capability of the soy sauce are improved, and the current situation that the low-salt thin mash fermentation preservative capability is weak can be improved. Therefore, the strain is not only suitable for the fermentation production of the conventional high-salt soy sauce, but also suitable for the fermentation production of low-salt or reduced-salt soy sauce so as to obtain the low-salt or reduced-salt soy sauce or sauce with strong aroma, obvious sauce aroma and soft mouthfeel. In addition, the strain ZB435 of the invention is derived from natural soy sauce mash, and has good safety.

Sequence information

Table 1: information on the sequences to which the present application relates is described in the following table.

Drawings

FIG. 1 is a colony morphology of strain ZB435 (strain No. 7) on PDA-containing solid medium.

FIG. 2 shows the growth of strain ZB435 (strain No. 7) in media with different salt contents.

Description of biological Material preservation

The present invention relates to the following biological materials which have been deposited at the Guangdong province microbial culture Collection (Middledo 100, Vol. 59, Guangzhou city):

Wickerhamiella versatilisZB435 with accession number GDMCC NO: 61755 and the preservation date is 2021, 6 months and 29 days.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

The media and assay conditions used in the examples of the present invention are those conventional in the art unless otherwise specified. The reagents used in the examples of the present invention were all commercially available unless otherwise specified.

The formula of the culture medium is as follows:

PDA solid medium containing 8% NaCl: 30% of potato extract powder, 8% of NaCl, 2% of glucose, 1.5% of agar and 0.1% of chloramphenicol.

Maltose culture medium: 5% of maltose, 1% of casein, 0.2% of yeast extract and 0.2% of KH₂PO4，0.05% MgSO₄·7H₂O, 18% NaCl, 2% agar, pH 4.8.

TTC lower layer medium: 5% glucose, 1% tryptone, 0.8% yeast extract, 0.5% KH₂PO4，0.2% MgSO₄·7H₂O, 0.15% citric acid, 2% agar.

TTC upper medium: 0.05% TTC, 0.5% glucose, 2% agar.

Example 1 screening of target strains

1.1 salt tolerance screening of strains

Selecting sauce mash with special fragrance, and diluting the sauce mash to 10 degrees by grade with sterile normal saline^-1、10^-2、10^-3、10^-4And (5) concentration, and preparing the sauce mash diluent. And (3) uniformly coating 0.1 mL of sauce mash diluent on a PDA (potato dextrose agar) solid culture medium added with 8% NaCl, carrying out inverted culture at 30 ℃ for 48-72 h, and selecting single colonies growing on a flat plate and having different sizes, colors and forms, wherein the numbers of the single colonies are 1-8 in sequence. And (4) selecting 1-8 single colonies, and respectively continuously streaking on a PDA solid culture medium containing 8% NaCl until pure single colonies are obtained. The growth characteristics and colony morphology of strains No. 1-8 on PDA solid medium containing 8% NaCl are shown in Table 2. The colony morphology of the No. 7 strain on the PDA solid medium is shown in figure 1, and the colony is circular, neat in edge, smooth in surface, milky white and opaque.

TABLE 2 growth characteristics and colony morphology of the respective strains

1.2 growth characteristics screening of strains

1.2.1 maltose assimilation ability

And sequentially inoculating the single colonies 1-8 obtained by the previous step on a maltose culture medium (containing 18% of salt) in a point-to-point manner, carrying out inverted culture at 30 ℃ for 48-72 h, and selecting 5 strains in total of the single colonies 1, 3, 6, 7 and 8 which can grow on the maltose culture medium for subsequent screening.

The maltose assimilation ability of each of strains No. 1 to 8 is shown in Table 3.

TABLE 3 maltose Medium screening results

1.2.2 preliminary judgment of ability to produce wine by screening Yeast

Inoculating the 5 strains (No. 1, No. 3, No. 6, No. 7 and No. 8) into a TTC lower layer culture medium in a point-to-point manner, performing inverted culture at 30 ℃ for 48 h, pouring about 12 mL of TTC upper layer culture medium onto the TTC lower layer culture medium, performing light-shielding culture at 30 ℃ for 3-5 h after solidification, and observing the color of a bacterial colony. The discoloration of colonies of each strain is shown in Table 4.

TABLE 4 TTC Medium screening results

TTC (2,3, 5-triphenyltetrazolium chloride) is a color developing agent, and the activity of respiring enzyme in yeast, namely the alcohol production capacity of the yeast can be judged through the color developing agent, wherein the yeast with strong alcohol production capacity can show deep red and second pink. According to the TTC color-developing screening result, the screened yeasts have wine-producing capability, wherein the No. 1 and the No. 3 have stronger wine-producing capability in the screened yeasts, and the No. 7 and the No. 8 have weaker wine-producing capability than the No. 6.

1.3 Yeast morphology microscopy

1-2 drops of sterile normal saline is dripped in the center of a glass slide, a small yeast colony is picked by a sterile toothpick, the yeast colony is uniformly coated in the normal saline, a cover glass is covered on the bacteria colony, the microscopic examination is carried out, the magnification of an ocular lens of the microscopic examination is 10 x, and the objective lens is 20 x. The microscopic examination results are shown in table 5.

TABLE 5 microscopic examination of the morphology results

The microscopic examination result shows that the cell forms of 5 strains of bacteria in total, namely No. 1, No. 3, No. 6, No. 7 and No. 8, are different, wherein the cell forms of the 1 and No. 3 yeasts are relatively close, the cell forms of the 7 and No. 8 yeasts are relatively close, and the cell forms of the 7 and No. 8 yeasts are circular, but the individuals are relatively small.

Example 2 application and screening of strains in Soy sauce fermentation

2.1 Low-salt Dilute fermentation Process

The low-salt dilute state fermentation technology is adopted, the salt water with the concentration of 15% is used for yellowing, and the specific yellowing temperature and the early-stage fermentation temperature are shown in the table 6. In the low-salt dilute state fermentation process, the mixed bacteria such as lactic acid bacteria in the sauce mash easily and rapidly grow, so that the total acid in the sauce mash is too high, and the growth of microorganisms such as yeast is influenced. For this reason, the inventors examined the number of lactic acid bacteria and the total acid content change in the moromi mash during the fermentation process, and the results are shown in table 7.

TABLE 6 temperature control protocol for low-salt dilute fermentation process

TABLE 7 changes in the content of lactic acid bacteria and the total acid content of the moromi mash during the fermentation process

From the detection results in table 7, it can be seen that the temperature control scheme provided in table 6 can effectively inhibit the rapid proliferation of various bacteria such as acid-producing bacteria, thereby improving the phenomenon of rapid increase of total acid in the normal-temperature low-salt thin mash fermentation process, avoiding the significant decrease of the pH of the thick mash, and making various enzymes (such as neutral protease) slowly but still smoothly ferment in the early thick mash fermentation process, thereby laying a good foundation for the low-salt thin mash fermentation and improving the current situation of weak corrosion resistance of the low-salt thin mash fermentation.

2.2 Effect of the selected strains in Soy sauce fermentation

The yeast strains (No. 1, No. 3, No. 6, No. 7 and No. 8 strains) selected in the example 1 and the Torulopsis hunculensis Hubrew 2.14 are inoculated into soy sauce mash for fermentation, and the specific operation steps are as follows: the strains No. 1, No. 3, No. 6, No. 7 and No. 8 and Huniang 2.14 were cultured in the liquid medium of commercial wort culture medium (purchased from Kyork Microbiol. Co., Ltd., product No. 021120) supplemented with 10% NaCl. Inoculating according to 1% of inoculation amount, and performing shake culture at 30 ℃ and 150 rpm36-48 h, until the yeast concentration reaches 10⁸The fermented soybean is added into the soy sauce mash at each ml. The time for inoculating the soy sauce mash is 30 days of fermentation, and the inoculation amount of yeast liquid is 10⁵Per g, the fermentation protocol used the low salt, low temperature process protocol described in 2.1 above until the moromi fermentation matured. The physical and chemical indexes and aroma components of the fermented soy sauce are respectively measured by using a reducing sugar tester, a GC-MS (gas chromatography-Mass spectrometer) and other instruments, and the results show that the 4-ethylguaiacol content in the soy sauce of each test group is improved compared with that of a control group, wherein the flavor improvement effect of the No. 7 strain (named as ZB 435) fermented soy sauce is most obvious, and the application effect is better, and the specific data are shown in Table 8.

TABLE 8 relevant index of 4-ethylguaiacol in soy sauce fermented by yeasts

Note: the ratio of aroma is the ratio of the sum of peak area integral values of certain flavor substances to the sum of peak area integral values of total flavor substances.

The results in Table 8 show that the soy sauce fermented by the yeast strain ZB435 has a 4-ethylguaiacol content which is obviously superior to that of the non-yeast-added group and other screened yeasts, and the aroma ratio of the 4-ethylguaiacol is improved by about 6.8 times compared with that of the negative control group without the yeast. Moreover, compared with the Torulopsis torulosa Huniang 2.14 (purchased from Shanghai Dynasty brewing biological products Co., Ltd.) which is generally used in the industry at present, the Torulopsis torulosa has obvious advantages in increasing the content of 4-ethylguaiacol in soy sauce. Therefore, the yeast strain ZB435 is applied to soy sauce fermentation, so that the content of 4-ethylguaiacol in soy sauce can be effectively increased, and the flavor of the soy sauce is improved.

The No. 7 strain (ZB 435) of the invention is identified by 26S rDNA gene, and the gene sequence is shown in SEQ ID NO. 1. BLAST homologous sequence alignment of the above sequences on NCBI shows that the strain ZB435 belongs toWickerhamiella versatilis，Wickerhamiella versatilisAndTorulopsis versatilis、 Candida versatilisbelonging to synonyms.

This implementationExample No. 7 strain obtained by breeding was deposited in the Collection of microorganisms of Guangdong province at 29.06.2021 by the present inventors and namedWickerhamiella versatilisStrain ZB435, deposit NO GDMCC NO: 61755, the preservation address is No. 59 of the Zhou Dazhong 100 Jie, Guangzhou city.

2.3 salt tolerance test of the selected strains

Commercial wort liquid culture medium (purchased from Kyork microorganism science and technology Co., Ltd., product number: 021120) is used, different salt contents are prepared by adding different proportions of salt, and the cell number is detected by sampling at different culture time to verify the salt tolerance of the strain. The results are shown in FIG. 2.

The results in figure 2 show that the screened strain ZB435 has a wide adaptation range for salt concentration and strong salt tolerance, can grow normally under the condition that the salt concentration reaches 20%, and the yeast concentration can reach 10 after 36 hours⁸each/mL, obvious inhibition effect on growth of the yeast is achieved when the concentration is 25 percent, and the concentration of the yeast can reach 10 after the yeast is cultured for 60 hours⁸one/mL, and a certain survival and growth capacity is still obtained when the salt content is 28%. Therefore, the screened ZB435 has strong salt tolerance and wide application range to salt concentration, and not only can be applied to a low-salt moromi fermentation environment to obtain low-salt or salt-reduced soy sauce, but also can be applied to a conventional high-salt moromi fermentation environment.

Example 3 screening of influence of Strain on Soy sauce aroma component

Will be provided withWickerhamiella versatilisThe results of the measurements of the aroma and physicochemical indices of the fermented soy sauce, applied to the fermentation of low-salt moromi mash with the ZB435 strain according to the method described in example 22.2, are shown in tables 9-12.

TABLE 9 physical and chemical indexes of fermented soy sauce

As can be seen from table 9, the yeast ZB435 added to the low-salt soy sauce mash for fermentation did not significantly affect the basic physicochemical indexes of total acid, ammonia nitrogen, and the like of the soy sauce as a whole, but significantly increased the glutamic acid content in the soy sauce, the glutamic acid content in the ZB435 test group was increased by about 14% compared to the control group, the umami of the soy sauce mainly originated from glutamic acid in amino acids, and the higher the glutamic acid content, the better the umami of the soy sauce. Compared with the control group, the fermented soy sauce added with Shanghai brewing 2.14 has basically no change in physical and chemical indexes and no obvious effect of improving the content of glutamic acid.

TABLE 10 fragrance composition and ratio of fermented soy sauce

Note: the category ratio is calculated in such a way that the sum of peak area integral values of a certain category of flavor substances accounts for the sum of peak area integral values of the total flavor substances.

The alcohols, organic acids and esters contained in soy sauce are important components of soy sauce aroma. The results in table 10 show that when ZB435 yeast strains are added into soy sauce mash for fermentation, the substances with the most significant increase of aroma substances in soy sauce are alcohols, while aldehydes are significantly reduced, wherein ZB435 has a more significant increase effect on phenols and a small increase effect on esters. The overall improvement of Shanghai brewing 2.14 on soy sauce aroma was significantly inferior to ZB435, and the characteristic aroma of 4-ethylguaiacol was significantly lower than ZB 435.

TABLE 11 composition of fermented Soy sauce with different aroma classes

TABLE 12 variation of different characteristic aroma components in fermented Soy sauce

From tables 11 to 12, it can be seen that ZB435 can significantly increase the content of alcohol in soy sauce aroma compared to the control group without yeast fermentation, and the detected types of ZB435 are also increased from 12 types when no yeast is added to 15 types, wherein the most significant changes are ethanol and β -phenylethyl alcohol, and the content of ethanol is significantly increased, and the increase of ethanol content not only can improve soy sauce flavor, but also has a certain benefit for improving the preservative capability during the fermentation of low-salt thin mash. In addition, the aroma content of 3-methylthiopropanol in the ZB 435-added fermented soy sauce was increased by about 5.8 times. The 3-methylthiopropanol is present in volatile components of tomatoes, wine and soy sauce, and shows a sauce flavor and a meat flavor at a low concentration, and is also a typical aroma substance in soy sauce. Therefore, the effect of ZB435 addition on alcohols in soy sauce was shown overall to be an increase in higher alcohol species and abundance.

In addition, in the soy sauce fragrance fermented by adding ZB435, the ester abundance is increased by 3.7% compared with that of a control group, and the isoamyl acetate produced by fermentation is a flavor substance commonly found in fermented products, and the soy sauce fragrance is softer due to the existence of the isoamyl acetate. The esters can be used as basic fragrant substances to make key flavor compounds such as maltol, beta-phenylethyl alcohol, etc. have mellow smell and buffer salty taste of salt in soy sauce. The reduction of the aldehyde aroma content is mainly caused by the reduction of the 2-methyl butyraldehyde content, and the 2-methyl butyraldehyde has strong suffocating aroma and has unique cocoa and coffee aroma after being diluted. The ketone substances mainly have reduced species abundance, which is represented by the reduction of octanone, octanedione, butenone and other substances, and the butenone has an irritating fishy smell; the acetic acid content in the acid substances is obviously reduced, and the acid substances are reduced from 10 to 6.

From the aroma analysis data, after the ZB435 is added, the soy sauce aroma is adjusted to strong aroma, namely, the content of 4-ethylguaiacol in the soy sauce can be obviously increased, the aldehyde pungent smell is reduced, the sour taste is improved, the composition proportion of substances with pleasant aroma is increased, and the overall aroma is improved.

Example 4 sensory evaluation Effect of Yeast ZB435 fermented Soy sauce

20 appraisers with abundant experience are summoned to perform sensory appraisal on soy sauce fermented by adding yeast ZB435 respectively, the sensory appraisal method refers to GB 2717-2018, the appraisal indexes of the sensory appraisal include delicate flavor, thick taste, comprehensive mouthfeel, color and fragrance, the score is 0-5, and the higher the average score is, the better the index is. The sensory evaluation results are summarized in table 13.

TABLE 13 sensory evaluation of Soy sauce

The results in table 13 show that the umami taste of the yeast ZB435 fermented soy sauce was significantly improved in sensory evaluation compared to the control group, which may be correlated with the ability to increase the glutamic acid content in the soy sauce. Under the condition that the salt content index is not influenced, the soy sauce fermented by adding the ZB435 has lighter salty taste in the entrance, and an appraiser shows that the soy sauce has softer taste and better dense taste and improved sour taste compared with a control group, so that the ZB435 can obviously improve the content of 4-ethylguaiacol and the content of ester substances and has a certain effect of reducing acetic acid. Comprehensive taste evaluation shows that the soy sauce fermented by adding ZB435 has better improvement effect in taste, mainly reflected in reduction of salty taste, improvement of delicate flavor and improvement of sour taste.

In addition, the results in table 13 also show that the addition of ZB435 has a greater improvement in soy sauce aroma with a score significantly higher than soy sauce fermented without the addition of yeast. The appraiser shows that the soy sauce fermented by adding the yeast has strong fragrance, obvious sauce fragrance and lasting fragrance. In conclusion, the yeast ZB435 is added into the soy sauce mash for fermentation, so that the taste and aroma of the fermented soy sauce can be obviously improved.

Example 5 genetic stability assay for Yeast ZB435

The yeast ZB435 screened in the above examples is inoculated in YPD slant culture medium for continuous passage for 10 generations, the growth condition of strains of each generation is observed, the slant strains of the 1 st generation, the 5 th generation and the 10 th generation are inoculated in soy sauce mash for fermentation culture according to the method described in the example 22.2, and after the fermentation is finished, the content of 4-ethylguaiacol and glutamic acid in the fermentation liquor is measured by adopting high performance liquid chromatography to judge the genetic stability. If the content errors of the 4-ethylguaiacol and the glutamic acid measured in the fermentation liquor obtained by fermenting the 10 generations of strains are within the error range of 10 percent, the genetic stability of the strains is good, and the detection results are shown in Table 14. The results in table 14 show that strain ZB435 has good genetic stability.

TABLE 14 Yeast ZB435 passage stability test results

Remarking: the indexes of the 1 st generation of yeast ZB435 are 1.00, and the 5 th generation and the 10 th generation are respectively converted into corresponding ratios.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Sequence listing

<110> Hitachi (Gaoming) flavoring food Co., Ltd; haitian flavouring foods of Foshan City Ltd

<120> yeast and application thereof

<130> IDC210177

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 535

<212> DNA

<213> Artificial Sequence

<400> 1

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ccgggacgta tttgataaag ctataacaca tccgaagatg ccactttctt taccatttat 120

ccatccggca aaactgatgt tagccccttc cgaagaaggt gtgtagcttt aagcccttcc 180

acttcaacga tttcacgtgc tatttcactc tcttttcaaa gtgcttttca tctttccttt 240

acagtacttg ttcgctatcg gtctctcgcc aatatttagc cttagatgga atttaccacc 300

cacttagcgc tgcattccca aacaacgcga ctcgtagaaa gagtactaca cagacacagg 360

acctccgtgg cacggggcta tcaccctcca aggcgcggct ttccagccaa cttccacaga 420

gctgagtcca gcactccatc tccaaattac aactcctcaa aagaggattt caaatttggg 480

cttttgccgc ttcactcgcc gttactaggg caatccctgt tggtttcttt tcctc 535

Claims

1. A Wickerhamiella versatilis strain ZB435 which is deposited at 29 months 6 of 2021 in the Guangdong province collection of microorganisms with the deposition number gdmcc.no: 61755, the preservation address is No. 59 of the Zhou Dazhong 100 Jie, Guangzhou city.

2. A method for increasing the content of 4-ethylguaiacol, glutamic acid and/or β -phenylethyl alcohol in soy sauce or sauce, which comprises adding the strain ZB435 of claim 1 during moromi fermentation.

3. A method for preparing soy sauce or sauce, comprising adding the strain ZB435 of claim 1 during moromi fermentation.

4. The method of claim 2 or 3, wherein the method is low salt dilute state fermentation.

5. The method of claim 4, wherein the strain ZB435 is added to the moromi on days 20-40 of moromi fermentation.

6. The method of claim 4, wherein the moromi is fermented at a first temperature and subsequently at a second temperature; wherein the first temperature is 20-30 ℃ and the second temperature is 25-35 ℃.

7. The method of claim 2 or 3, wherein the soy sauce or spread is a low-salt or reduced-salt soy sauce or spread.

8. A soy sauce or paste obtained by the method of any one of claims 3 to 7.

9. A method of culturing the strain ZB435 of claim 1, comprising the step of inoculating the strain ZB435 in a wort medium and culturing under aerobic conditions.

10. The method of claim 9, wherein the method comprises: the strain ZB435 is inoculated in a wort medium containing 5% -15% NaCl and cultured at 25-40 ℃ under aerobic conditions.