Salt-tolerant zygosaccharomyces rouxii with high ethanol yield and application thereof in preparation of soy sauce

Abstract

The invention discloses a salt-tolerant zygosaccharomyces rouxii strain with high ethanol yield and application thereof in preparation of soy sauce, and belongs to the technical field of microorganisms, the salt-tolerant zygosaccharomyces rouxii strain SS-y178 is preserved in the China Center for Type Culture Collection on July 15, 2024, the preservation number is CCTCC NO: M 20241563, the preservation address is Wuhan University, Wuhan, China, the salt-tolerant zygosaccharomyces rouxii strain is applied to fermentation production of soy sauce, and the salt-tolerant zygosaccharomyces rouxii strain can be used for preparing the soy sauce. The method has the advantages of high yield of ethanol, promotion of fermentation fat production and increase of the types of lipid substances, has good effects in promotion of fat increase and aroma enhancement of soy sauce, and has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a salt-tolerant Zygosaccharomyces rouxii with high ethanol production and application thereof in preparing soy sauce. Background Art

[0002] Soy sauce is a condiment made from soybeans, wheat and other raw materials, which is fermented by microorganisms to produce flavor and color. Flavor is one of the most important quality indicators of soy sauce, and it is also one of the issues that consumers and producers are most concerned about.

[0003] Flavor is composed of smell and taste. It is the impression left by food on the human sense organs such as taste, smell and touch. It is an intricate sensory system, and its formation is also relatively complex. Volatile esters make a very important contribution to the flavor of soy sauce. They are mainly synthesized by yeast with alcohols and fatty acids as substrates. Among them, ethyl esters account for a relatively large proportion of volatile esters in soy sauce, which is related to the higher concentration of ethanol in soy sauce.

[0004] At present, Lu's yeast is used as the main fermentation yeast in soy sauce fermentation. For example, CN118272242 A discloses a Lu's yeast ZB441 and its application. The strain focuses on the performance of producing succinic acid and small molecule peptides, thereby effectively improving the aroma and taste of the fermented product; however, it does not pay attention to the ethanol production performance of the strain; currently, there is no report on Lu's yeast with high ethanol production. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the shortcomings of the prior art and breed a high-ethanol-producing salt-tolerant Zygosaccharomyces rouxiiSS-y178, which is classified and named Zygosaccharomyces rouxiiSS-y178, and deposited in the China Center for Type Culture Collection on July 15, 2024, with a deposit number of CCTCC NO: M20241563, and the deposit address is Wuhan University, Wuhan, China.

[0008] As a preferred embodiment of the Zygosaccharomyces rouxii of the present invention, the Zygosaccharomyces rouxii grows in a 20% NaCl w / w environment and has a strong stress resistance.

[0009] Another object of the present invention is to overcome the deficiencies in the prior art and provide a use of Zygosaccharomyces rouxii in preparing soy sauce.

[0010] As a preferred embodiment of the application of the present invention, the Zygosaccharomyces rouxii has a high ethanol production capacity.

[0011] As a preferred embodiment of the application of the present invention, the salt-tolerant Zygosaccharomyces rouxii improves the flavor of soy sauce during soy sauce fermentation and soy sauce brewing.

[0012] Beneficial effects of the present invention:

[0013] The invention provides a salt-tolerant Zygosaccharomyces rouxii SS-y178 with high ethanol production, which is classified and named as Zygosaccharomyces rouxii SS-y178, deposited in China Center for Type Culture Collection on July 15, 2024, with a deposit number of CCTCC NO:M 20241563, and a deposit address of Wuhan University, Wuhan, China. The strain has a fast growth rate and a high ethanol production, can grow in an environment of 20% NaCl (w / w), is applied to soy sauce fermentation production, has a high production of alcohol and a high production of unsaturated fatty acids and their ester derivatives with fat aroma, promotes fermentation and fat production, has good effects in promoting the fat increase, flavor enhancement and quality enhancement of soy sauce, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0015] Figure 1 This is a mortality curve of the starting strain of Zygosaccharomyces rouxii at different irradiation times in the embodiment of the present invention.

[0016] Figure 2 This is a color diagram of some mutagenized strains of Zygosaccharomyces rouxii on a TTC plate in the examples of the present invention.

[0017] Figure 3 This is a data diagram of flavor substances in soy sauce fermentation using Zygosaccharomyces rouxii in an embodiment of the present invention.

[0018] Figure 4This is a sensory evaluation diagram of Zygosaccharomyces rouxii used in soy sauce fermentation in an example of the present invention.

[0019] Figure 5 This is a diagram showing the aroma evaluation results of two soy sauces, SS-C8 and SS-y178, using the QDA method in an embodiment of the present invention. DETAILED DESCRIPTION

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0021] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0022] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0023] The high-ethanol-producing salt-tolerant Zygosaccharomyces rouxii SS-y178 of the present invention is classified and named Zygosaccharomyces rouxii SS-y178, which was deposited in the China Center for Type Culture Collection on July 15, 2024, with a deposit number of CCTCC NO: M 20241563, and the deposit address is Wuhan University, Wuhan, China.

[0024] Example 1: Mutagenesis and screening of Zygosaccharomyces rouxii with high alcohol production

[0025] Transfer the seed culture solution to YPD medium at a ratio of 2% and culture to the logarithmic phase. Centrifuge the bacterial solution and discard the supernatant. Wash the bacterial sludge with sterile water, resuspend the cells with sterile saline containing 5% glycerol, and adjust the bacterial concentration OD600 to between 0.6-0.8.

[0026] Wipe the mutagenesis chamber with 75% ethanol and irradiate with ultraviolet light for 30 minutes. In a sterile environment, take 10μL of bacterial solution on a sterilized slide and place the slide in the well. The mutagenesis time is 0, 30, 90, 120 and 150s respectively. After mutagenesis, put the slide into a centrifuge tube containing 990μL sterile saline. Spread 100μL of gradient diluted bacterial solution on a YPD plate and culture at 30℃. Record the number of single colonies on the plate and calculate the lethality at the corresponding irradiation time according to formula 1-1.

[0027]

[0028] The lethality curve was drawn with the mutagenesis time as the horizontal axis and the mutagenesis lethality as the vertical axis ( Figure 1 ). Select a mutagenesis time with a lethality of about 90%, and apply the mutagenesis to five groups of strains in parallel, then spread them on YPD plates. Place the plates upside down and culture them in an incubator at 30°C.

[0029] After mutagenesis, the strains cultured on the plate were screened on TTC plates (upper layer: 5g glucose, 15g agar, 0.5g TTC, 1000mL water; lower layer: 10g glucose, 2g peptone, 1.5g yeast, 0.4g magnesium sulfate heptahydrate, 1.1g potassium hydrogen phosphate trihydrate, 20g agar, 1000mL water, pH 5.6-5.8). Single colonies with large diameter and vigorous growth on the plate were selected for liquid culture. After 48 hours, 5μL of bacterial liquid was inoculated on the lower layer of TTC medium. The plate was inverted and cultured in a 30℃ incubator. After 48 hours, the upper layer of TTC medium was poured into the plate, and the colony color was observed after standing for 2 hours away from light ( Figure 2 ) and select colonies with darker red color for rescreening.

[0030] Single colonies were picked from the plate and transferred to YPD medium for ethanol fermentation rescreening. Single colonies were picked from the plate and transferred to YPD medium, cultured at 30°C with shaking for 48 h, and then centrifuged. The supernatant was taken to determine the ethanol content.

[0031] Add 1mL of the test solution and 2mL of 5% potassium dichromate solution to the colorimetric tube, and make up to 10mL with distilled water. Boil in a water bath for 10 minutes, then cool, and then measure the absorbance at 600nm to calculate the crude ethanol content. Take a 10mL colorimetric tube, add 1.5mL DNS solution, 50μL of the test solution, and make up to 10mL with distilled water. Boil in a water bath for 5 minutes, then cool to room temperature with running water, measure the absorbance at a wavelength of 540nm, and calculate the mass fraction of glucose.

[0032] Calculate the ethanol content according to formula 1-2:

[0033]

[0034] After mutagenesis, the ethanol production of the strain increased from 12.38 mg / mL to 13.76 mg / mL.

[0035] Strains with high ethanol production were selected for genetic stability testing. The rescreened strains y129 and y178 were continuously cultured in liquid culture for 7 generations, and the ethanol fermentation concentration was measured.

[0036] It was found that the ethanol production of the strain numbered y178 was stable at about 14 mg / mL during the 7th generation (Table 1). The strain was deposited in the China Center for Type Culture Collection on July 15, 2024, and was classified and named Zygosaccharomyces rouxiiSS-y178. The deposit number is CCTCC NO:M 20241563, and the deposit address is Wuhan University, Wuhan, China.

[0037] Table 1 Genetic stability of mutant strains

[0038]

[0039] Example 2: Determination of stress resistance of Zygosaccharomyces rouxii

[0040] Pour 2 mL of bacterial solution frozen at -80°C into 100 mL YPD liquid culture medium (2% soy peptone, 2% glucose, 1% yeast powder, adjust pH = 7), and culture at 30°C with a shaker at 120 rpm for 48 h. Then, inoculate the cultured bacterial solution into 100 mL of fresh YPD liquid at a ratio of 2%, and culture at 30°C with a shaker at 120 rpm until OD 600 = 0.7, which is considered to be activated.

[0041] The activated strain was inoculated into YPD liquid medium containing 0%, 4%, 8%, 12%, 16%, and 20% NaCl (w / w) in batches at a 2% inoculum. The culture was cultured at 30°C for 120 hours, the culture solution was centrifuged to collect the cells, and dried in an oven at 55°C for 24 hours to constant weight. The dry weight was used to reflect the biomass.

[0042] The results of the test are as follows Figure 3 , Considering that Zygosaccharomyces rouxii needs to play a practical role in soy sauce fermentation, the biomass greater than 0.01 g / L was selected as the threshold to evaluate the stress resistance of Zygosaccharomyces rouxii.

[0043] The NaCl tolerance limit threshold was found to be 20% (w / w).

[0044] Example 3: Application of Zygosaccharomyces rouxii in the flavor enhancement of brewing peanut meal soy sauce

[0045] Take 30g bran and 33g water in a conical flask. After high-pressure sterilization, place it at room temperature, inoculate Aspergillus oryzae 3.042, and culture it at 32℃ for about 12h. After shaking the flask, culture it flat at 30℃. After about 7h, shake the flask again, and observe whether the appearance of the koji material is dark green at about 72h. If so, dry it in an oven at 55℃, and obtain the seed koji after complete drying.

[0046] Soak the soybeans for 4 hours and sterilize them with an equal amount of peanut meal. Keep steaming for 10 minutes and then keep the high pressure (0.8MPa) for 20 minutes. After the temperature drops to 37°C, add 0.3% seed koji to the raw materials and culture at 30°C. Turn the koji at 8h and 12h, and make Daqu after 36h. Mix Daqu and 18% (w / w) NaCl solution in a mass ratio of 1:2.5 and start salt water fermentation. Ferment at 15°C for 30 days, then raise the temperature to 30°C for 30 days.

[0047] The starting strain SS-C8 and the mutagenic strain SS-y178 stored in the -80℃ ultra-low temperature refrigerator were taken out from the strain bank and placed in the clean bench for operation. 20μL of the bacteria were inoculated into the sterilized 20mL YPD liquid culture medium for initial activation, and the prepared solution was placed in a shaker for shaking. After 12 hours, the growth of the strains was observed. Generally, the initial activation of the strains was completed in 15-24 hours.

[0048] In the clean bench, add the bacterial solution that has been activated initially to a new 20 mL YPD liquid culture medium for secondary activation. The amount of bacterial solution added should make the OD600 of the culture medium 0.1. Put the prepared solution into a shaker for shaking. After the secondary activation is completed, the OD600 of the bacterial solution is detected and recorded. Generally, the OD600 should be around 1.

[0049] The fermentation raw materials were supplemented with 0.5% by mass of activated starting strain SS-C8 (the induced starting strain, which was Saccharomyces rouxii preserved in the laboratory) and induced strain SS-y178 respectively. Samples were taken after 150 days of warm fermentation, and the basic physicochemical properties of the soy sauce were determined.

[0050] As shown in Table 1, the total acid, amino acid nitrogen and soluble salt-free solids of soy sauce were between 0.95-1.02 g / 100 mL, 0.91-0.92 g / 100 mL and 14.60-14.81 g / 100 mL, respectively, and there was no significant difference among the samples (p>0.05).

[0051] The reducing sugar content in the soy sauce inoculated with the starting strain SS-C8 was the highest, at 27.84 mg / mL, and the ethanol content was the lowest, at 72.7 mg / mL. The ethanol content of sample SS-y178 was 83.42 mg / mL, which was significantly higher than that of the starting strain.

[0052] Table 1 Physicochemical properties of peanut meal soy sauce inoculated with different yeasts

[0053]

[0054]

[0055] The volatile flavor substances in the experimental group and the control group were detected by headspace solid phase microextraction and gas chromatography-mass spectrometry. 5 mL of sample was taken into the headspace bottle, and 0.9 g of NaCl and 210 μL of 2-octanol solution (20 ppm) were added. Equilibrate at 60 °C for 15 min. The sample was extracted with a 50 / 30 μm DVB / CAR / PDMS fiber (Supelco, Bellefonte, PA, USA) for 30 min. GC-MS and Rtx-5 MS column (30 m×0.25 mm×0.25 μm, Restek, Bellefonte, PA, USA) were used to analyze the volatile compounds. Helium was used as the carrier gas at 1.0 mL / min, and the split ratio was 10:1. The column temperature program was: keep at 40 °C for 3 min, increase to 150 °C at a rate of 4 °C / min for 1 min, and finally increase to 250 °C at a rate of 8 °C / min for 6 min. MS parameters: ionization source (EI, electron ionization), injection temperature (250°C), ion source temperature (220°C), scan range (35-500 m / z) and scan rate (3.00 scans / s). Compounds were identified using mass spectrometry and RI values. The concentrations of key odor-active compounds were calculated using the standard curve of the corresponding standard substances. Quantification was performed using the internal standard method using 2-octanol as the internal standard substance.

[0056] The types and relative contents of volatile compounds in soy sauce Figure 4 As shown. A total of 82 volatile compounds were identified in soy sauce, including 27 esters, 13 alcohols, 11 acids, 15 aldehydes, 7 ketones, 3 phenols, 2 pyrazines, and 4 alkanes. 71 and 84 volatile compounds were detected in SS-C8 and SS-y178 soy sauces, respectively. SS-C8 contained 22 esters, 17 alcohols, 5 acids, 14 aldehydes, 5 ketones, 2 phenols, 2 pyrazines, and 2 alkanes. Compared with SS-C8, SS-y178 reduced 4 alcohols and 2 pyrazines, and increased 5 esters, 6 acids, 1 aldehyde, 2 ketones, 1 phenol, and 2 alkanes. The most abundant species in soy sauce are esters, alcohols, and aldehydes. It can be seen that the mutated Lu's yeast reduced the types of alcohols produced in soy sauce fermentation and increased the types of esters.

[0057] Alcohols are the substances with the highest relative content in soy sauce. The one with the highest relative content of alcohols is SS-y178. The relative content of alcohols in SS-y178 is 64.96%, which is 17.55% higher than that of SS-C8. Compared with SS-C8, SS-y178 contains more acids and ketones.

[0058] According to different odor characteristics, the volatile flavor substances in soy sauce are divided into 10 categories, including sweet, fruity, floral, roasted nut, mellow, malty, creamy, caramel, and smoky. The specific OAV values ​​are shown in Table 2. Compared with SS-C8, the fruity, mellow, malty, creamy, and smoky aromas of SS-y178 increased by 18.39%, 443.8%, 125.8%, 33.2%, and 204.1%, respectively, and the total OAV values ​​of sweet, floral, and caramel aroma substances decreased by 17.1%, 23.2%, and 16.6%, respectively. Compared with SS-S1, the total OAV values ​​of sweet, fruity, mellow, malty, caramel and smoky aroma substances in SS-y178 increased by 62.9%, 37.6%, 90.0%, 29.7%, 43.9% and 232.2%, respectively, and the total OAV values ​​of floral and creamy aroma substances decreased by 13.5% and 35.8%, respectively.

[0059] Table 2 OAV values ​​of main flavor substances in soy sauce

[0060]

[0061]

[0062] A sensory scoring panel was formed by 10 professionally trained personnel (5 males and 5 females). The color, aroma, taste, and body of the soy sauce were scored with a full score of 100. The specific scoring criteria are shown in Table 3.

[0063] Table 3 Soy sauce sensory evaluation table

[0064]

[0065] The aroma of the samples was analyzed using a quantitative descriptive analysis method to evaluate the flavor of the soy sauce samples. A total of seven reagents were used to characterize the sensory properties: ethanol (mellow aroma), 4-ethylguaiacol (smoky aroma), 3-methylbutyraldehyde (malt aroma), HEMF (caramel aroma), soybean paste (soy sauce aroma), 2-phenylethanol (floral aroma), and ethyl acetate (fruity aroma). Each sample was coded with three random numbers. The flavor intensity of the samples was scored from 1 (lowest intensity) to 9 (highest intensity).

[0066] The sensory evaluation team used the QDA method to evaluate the aroma of SS-C8 and SS-y178 soy sauces. The specific results are as follows: Figure 5SS-y178 has stronger floral, fruity, mellow, smoky, soy sauce and malt aromas than SS-C8. The overall flavor of SS-y178 is better than that of SS-C8 fermented by the starting strain, which is similar to the OAV analysis results of soy sauce.

[0067] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A high-ethanol-producing, salt-tolerant Zygosaccharomyces rouxi SS-y178 was deposited in the China Center for Type Culture Collection on July 15, 2024, with the deposit number CCTCC NO: M20241563, and the deposit address is Wuhan University, Wuhan, China.

2. The Zygosaccharomyces rouxii of claim 1, wherein: The Zygosaccharomyces rouxii grows in a 20% NaCl w / w environment and has a strong stress resistance ability.

3. Use of the Zygosaccharomyces rouxii described in claim 1 or 2 in preparing soy sauce.

4. The use according to claim 3, characterized in that: The Zygosaccharomyces rouxii has high ethanol production.

5. The use according to claim 4, characterized in that: The salt-tolerant Zygosaccharomyces rouxii can improve the flavor of soy sauce during soy sauce fermentation and soy sauce brewing.

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