Trulopsis variabilis and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses torulopsis variabilis and application thereof. Wild torulopsis variabilis is separated from soy sauce mash, a strain of torulopsis variabilis with high yield of aspartic acid, threonine, glutamic acid, valine, methionine, isoleucine, leucine and lysine is obtained through ARTP mutagenesis, the collection number of the torulopsis variabilis ZB444 is GDMCC No: 65240, the classification name of the Latin name of the torulopsis variabilis is Wickhamiella versitis, the collection number of the torulopsis variabilis ZB444 is CGMCC No: 65240, and the collection number of the torulopsis variabilis ZB444 is CGMCC No: 65240. The strain is preserved in Guangdong Microbial Culture Collection Center on October 12, 2024. The strain is applied to soy sauce fermentation, the content of various amino acids and the content of total amino acids in soy sauce can be increased, the nutritional value of the soy sauce can be increased, and meanwhile the flavor of the soy sauce is enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to Torulopsis heterotrophica and applications thereof. Background Art

[0002] Soybeans have high nutritional value. The vegetable protein, fat, carotene, riboflavin, niacin and other ingredients they contain are similar to those of pork, beef, mutton, milk and other foods. However, when soybeans are eaten directly, the digestion and absorption rate of nutrients in them is low. Under normal circumstances, the digestion and absorption rate of dry fried beans is below 50%, and the digestion and absorption rate of cooked beans is about 65%, which results in a waste of nutrients in soybeans. The soy sauce production process utilizes the effects of beneficial bacteria such as Aspergillus and yeast to decompose and convert soybeans into various amino acid compounds, esters, sugars and vitamins, which can make the nutrients in soybeans easier to be absorbed by the human body. Among them, amino acids are the decomposition products of proteins and are also important indicators for evaluating the nutritional value of food. The nutritional value of food can be reflected in the amino acid composition.

[0003] Amino acids are the basic building blocks of proteins, and proteins are one of the three major nutrients for human life activities. Since the human body cannot synthesize them or the synthesis speed cannot match the body's needs, the eight amino acids, lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, and valine, must be obtained from food, and soy sauce contains these eight amino acids. At the same time, free amino acids also give soy sauce sweetness, umami, bitterness and other flavors, making soy sauce rich in flavor. At present, research on amino acids in soy sauce focuses on increasing the content of umami amino acids in soy sauce, thereby improving the taste and flavor of soy sauce. Few people pay attention to increasing the content of various amino acids in soy sauce to improve the nutritional value of soy sauce.

[0004] Yeast is an important microorganism in soy sauce fermentation, which has a great influence on the quality of soy sauce. It can further decompose raw materials and provide various flavor substances and nutrients for soy sauce. In the prior art, the research on the role of yeast in soy sauce fermentation focuses on the improvement of the flavor of soy sauce by the aroma substances produced by yeast. Therefore, breeding yeast with high amino acid production is of great significance in improving the flavor of soy sauce and improving the nutritional value of soy sauce. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a Torulopsis heterotrophica and its application.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a Torulopsis versatilis, wherein the Torulopsis versatilis is Wickerhamiella versatilis ZB444, and its deposit number is GDMCC No: 65240.

[0008] The invention discloses a wild-type Torulopsis heteromutans isolated from soy sauce mash, which is obtained by ARTP mutagenesis, and the mutagenesis conditions are as follows: power 110-120W, gas volume 10SLM, and mutagenesis time 15-30s. A mutant strain ZB444 with high yield of multiple amino acids is selected from the mutants, and has the characteristics of high yield of aspartic acid, threonine, glutamic acid, valine, methionine, isoleucine, leucine and lysine. The strain is applied to soy sauce fermentation to increase the content of multiple amino acids and the total amino acid content in the soy sauce, thereby improving the nutritional value of the soy sauce and enhancing the flavor of the soy sauce.

[0009] The Wickerhamiella versatilis ZB444 of the present invention, whose Latin name is classified as Wickerhamiella versatilis, was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 12, 2024, with a deposit number of GDMCC No: 65240, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the contact number is 020-87137633 37656629.

[0010] In a second aspect, the present invention provides the bacterial body, fermentation supernatant and / or intracellular material of the Torulopsis heterotrophica.

[0011] In a third aspect, the present invention applies the Torulopsis heteromutans in soy sauce fermentation.

[0012] The invention inoculates the Wickerhamiella versatilis ZB444 into soy sauce crude oil for fermentation, and can increase aspartic acid, threonine, glutamic acid, valine, methionine, isoleucine, leucine and lysine of the crude oil by 9%, 17%, 18%, 13%, 993%, 11%, 11% and 13% respectively, and increase the total amino acid content of the crude oil by 9%, thereby improving the nutritional value of the soy sauce. At the same time, the flavor of the soy sauce can be enhanced after the inoculation and fermentation of the Wickerhamiella versatilis ZB444.

[0013] In a fourth aspect, the present invention uses the Torulopsis heterotrophica to increase the amino acid content of soy sauce.

[0014] As a preferred embodiment of the application of the present invention, the Torulopsis heterotrophica can increase the content of total amino acids in soy sauce. The amino acids include aspartic acid, threonine, glutamic acid, valine, methionine, isoleucine, leucine and lysine.

[0015] In a fifth aspect, the present invention uses the Torulopsis heterotrophica to enhance the flavor of soy sauce.

[0016] In a sixth aspect, the present invention provides a soy sauce produced by fermenting the Torulopsis heterotrophica.

[0017] In a seventh aspect, the present invention provides a method for preparing soy sauce, comprising inoculating the Torulopsis heterotrophica into crude soy sauce oil and subjecting the soy sauce to fermentation.

[0018] As a preferred embodiment of the preparation method of the present invention, the inoculation amount of Torulopsis heterotrophica is 1% to 5%, so that the yeast content in the crude oil reaches 1×10 5 ~1×10 7 cfu / mL.

[0019] As a preferred embodiment of the preparation method of the present invention, the fermentation time is 15 to 30 days, during which aeration and stirring are performed for 15 to 40 minutes every 1 to 3 days.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention discloses a wild type Torulopsis heterovaria separated from soy sauce mash, which is obtained by ARTP mutagenesis, and a mutant ZB444 with high yield of multiple amino acids is bred from the mutants. The strain is applied to soy sauce fermentation to increase the contents of multiple amino acids and the total amino acid content in the soy sauce, and can increase the aspartic acid, threonine, glutamic acid, valine, methionine, isoleucine, leucine and lysine of crude oil by 9%, 17%, 18%, 13%, 993%, 11%, 11% and 13% respectively, and the total amino acid content of crude oil by 9%, thereby improving the nutritional value of the soy sauce. At the same time, the flavor of the soy sauce can be enhanced after the Torulopsis heterovaria is inoculated and fermented. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the colony morphology of yeast strain ZB444;

[0023] Figure 2 This is a phylogenetic tree analysis diagram;

[0024] Figure 3 This is a radar chart of the sensory evaluation results of fermented crude oil. DETAILED DESCRIPTION

[0025] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0027] Example 1: Screening of yeast strain ZB444

[0028] 1. Strain isolation and purification

[0029] Take 10g of traditional soy sauce mash sample and add it to 90mL of sterile saline containing 10% sodium chloride, shake it in a shaker at 200rpm for 30min to mix thoroughly. Pipette 10mL of supernatant onto folded filter paper and filter to obtain bacterial solution. After diluting the bacterial solution 10, 100, and 1000 times, take 100μL of each multiple bacterial solution and spread it on the solid culture medium plate, culture it at 28℃~32℃ for 72~120 hours, and observe the colonies growing on the plate. Pick some colonies with morphology that meet the characteristics of yeast and make slides for observation under a microscope. After confirming that it is yeast by morphological identification with reference to "YEAST", streak the single colony onto a new solid culture medium plate for separation and purification.

[0030] The solid culture medium formula (w / v) is: 0.5% to 1.5% yeast extract, 1% to 2% peptone, 2% to 4% glucose, 0.1% to 0.2% chloramphenicol, 1.5% to 2.5% agar, and the rest is water.

[0031] 2. Screening of starting strains by shake flask fermentation

[0032] A single colony is picked from the strain separation and purification plate and inoculated into a triangular flask containing 50 mL of seed culture medium, and cultured in a shaker at 28°C to 32°C at 180rpm to 220rpm for 24 to 36 hours to obtain a seed solution. Subsequently, the seed solution is inoculated into 200 mL of liquid fermentation medium at a 1% inoculation amount, and cultured at 28°C to 32°C for 10 to 20 days. The fermentation liquid is taken to detect the residual glucose content. If the residual glucose content is less than 0.5%, the amino acid nitrogen content is further detected. According to the test results, the strain with the highest amino acid nitrogen content is selected as the starting strain.

[0033] The seed culture medium formula (w / v) includes: 0.5% to 1% yeast extract, 0.5% to 1% peptone, 1% to 2% glucose, 0.1% to 0.2% chloramphenicol, and the rest is water.

[0034] Liquid fermentation medium formula (w / v): 15% to 30% molasses, 0.5% to 1.5% yeast extract, 1% to 3% peptone, 0.05% to 0.1% magnesium sulfate, 0.05% to 0.1% potassium dihydrogen phosphate, 10% to 15% sodium chloride, and the rest is water.

[0035] The glucose content is tested in accordance with the method of GB5009.8-2023 "National Food Safety Standard for Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Foods"; the amino acid nitrogen content is tested in accordance with the method of GB5009.239-2016 "National Food Safety Standard for Determination of Amino Acid Nitrogen in Foods".

[0036] 3. Mutagenesis of starting strain

[0037] The selected starting strains were induced to mutate using atmospheric pressure room temperature plasma (ARTP) mutagenesis technology.

[0038] Mutagenesis equipment: ARTP-ⅢS, Wuxi Yuanqing Tianmu Biotechnology Co., Ltd., operated normally according to the equipment instruction manual.

[0039] Experimental operation: Pick a single colony from the selected starting strain plate and inoculate it into a triangular flask containing 50mL seed culture medium, and culture it in a shaker at 28℃~32℃ at 180rpm~220rpm for 18~30 hours to obtain logarithmic phase bacterial solution. Take 1mL of logarithmic phase bacterial solution and centrifuge to collect the bacteria, wash it 3 times with 0.9% saline, and resuspend it with 0.9% saline to OD 600 At 0.9-1.1, 10 μL of OD 600 The bacterial suspension of 0.9 to 1.1 is evenly applied on the ARTP metal slide, and then the slide is transferred to the ARTP carrier. Set the power to 110W to 120W, the gas volume to 10SLM, the mutagenesis time to 15s to 30s on the control panel, and click "Start" to process the sample. After the sample is processed, transfer the metal slide to a centrifuge tube containing 1mL of 0.9% saline, and shake it thoroughly to form a new bacterial suspension.

[0040] The seed culture medium formula (w / v) includes: 0.5% to 1% yeast extract, 0.5% to 1% peptone, 1% to 2% glucose, 0.1% to 0.2% chloramphenicol, and the rest is water.

[0041] 4. Screening of high-yield amino acid mutants

[0042] The new bacterial suspension obtained after mutagenesis was diluted 1000 times and spread on the growth plate. Select the colonies with good growth from the growth plate, dip a small amount of bacteria with a toothpick, and inoculate them in a 96-well plate with 100 μL liquid screening medium in each well. Each colony was inoculated in 1 well, and 2 wells were left on each plate to inoculate the starting strain. After the inoculated well plate was sealed with a ventilated sealing film, it was placed in a well plate shaker and cultured at 28°C to 32°C and 250rpm to 300rpm for 3d to 5d. Subsequently, the total free amino acid content of each well culture solution was detected by the well plate system, and the mutant strains with a total free amino acid content higher than the starting strain and in the top 20 were selected, and further shake flask liquid fermentation screening was carried out. Among them, a yeast strain ZB444 had the highest total free amino acid content, which was 14% higher than the control.

[0043] The growth plate formula (w / v) includes: 0.5% to 1% yeast extract, 0.5% to 1% peptone, 1% to 2% glucose, 0.1% to 0.2% chloramphenicol, 12% to 16% sodium chloride, 2% to 2.5% agar powder, and the rest is water.

[0044] Liquid screening medium (w / v): 10% to 15% glucose, 0.5% to 1.5% yeast extract, 1% to 3% peptone, 0.05% to 0.1% magnesium sulfate, 0.05% to 0.1% potassium dihydrogen phosphate, 10% to 15% sodium chloride, and the rest is water.

[0045] The formula of the shake flask liquid fermentation medium (w / v) is: 15% to 30% molasses, 0.5% to 1.5% yeast extract, 1% to 3% peptone, 0.05% to 0.1% magnesium sulfate, 0.05% to 0.1% potassium dihydrogen phosphate, 10% to 15% sodium chloride, and the rest is water.

[0046] The method for detecting the total free amino acid content was based on the “Ninhydrin Method for Determination of Amino Acid Content in Honey and Fructose-Glucose Syrup” by Yang Yuanfan et al. (Journal of Chinese Institute of Food Science and Technology, 2013(2):6).

[0047] Example 2: Identification of yeast strain ZB444

[0048] The genome of yeast strain ZB444 was extracted using the OMEGA column yeast genomic DNA extraction kit according to the instructions and sent to Guangdong Meige Gene Technology Co., Ltd. for 18S rDNA fragment sequencing. The obtained sequence is shown in SEQ ID No: 1.

[0049] The sequencing sequence of the 18S rDNA fragment (SEQ ID No: 1) is:

[0050]

[0051] The results of strain identification and sequencing were imported into the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) for nucleic acid sequence BLAST. Strains with a similarity of more than 97% were selected based on the comparison results, and their fasta format sequence files were downloaded. The downloaded sequence files were imported into Mega 11 software for sequence comparison, and a phylogenetic tree was constructed based on the comparison results. Figure 2 The comparison results showed that the homology between the strain and Torulopsis heterovariata was 99%.

[0052] Combined with the observation of biological characteristics and sequencing analysis, the yeast strain ZB444 is a Torulopsis versatilis, and was named Wickerhamiella versatilis ZB444. The classification name of its Latin name is Wickerhamiella versatilis. It was deposited in the Guangdong Provincial Microbiological Culture Collection on October 12, 2024, with the deposit number GDMCC No: 65240. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the contact number is 020-8713763337656629.

[0053] Example 3: Application of yeast strain ZB444 in soy sauce fermentation

[0054] The soy sauce crude oil fermented to maturity according to the conventional high-salt dilute fermentation process described in "Soy Sauce Science and Brewing Technology" compiled by Bao Qi'an was used to set up a control group and an experimental group. The experimental group was inoculated with Torulopsis variegata ZB444 to make the yeast content in the crude oil reach 1×10 5 ~1×10 7 cfu / mL, aerate and stir for 30 minutes on the day of inoculation to mix the materials, then aerate and stir for 30 minutes every other day, and ferment for 20 days in total. Except for not inoculating yeast, the other operations of control group 1 were the same as those of the experimental group. Control group 2 was inoculated with the starting strain, and the other operations and inoculation bacteria counts were the same as those of the experimental group.

[0055] The amino acid nitrogen content is tested in accordance with the method of GB5009.239-2016 "National Food Safety Standard Determination of Amino Acid Nitrogen in Foods"; the content of various amino acids is tested in accordance with the method of GB 5009.124-2016 "National Food Safety Standard Determination of Amino Acids in Foods".

[0056] The comparison of various indicators of crude oil after fermentation is shown in Table 1.

[0057] Table 1 Comparison of fermentation-expired crude oil indicators between the control group and the experimental group

[0058]

[0059]

[0060] Note: The relative content is based on the control group content as 1, and the other values ​​are the corresponding multiples of the control group content.

[0061] By inoculating Torulopsis mutans ZB444 into crude soy sauce material for fermentation, the aspartic acid, threonine, glutamic acid, valine, methionine, isoleucine, leucine and lysine content of the crude oil can be increased by 9%, 17%, 18%, 13%, 993%, 11%, 11% and 13% respectively, and the total amino acid content of the crude oil can be increased by 9%, thereby improving the nutritional value of soy sauce.

[0062] Example 4: Effect of yeast strain ZB444 on soy sauce flavor

[0063] Twelve experienced appraisers were recruited to conduct a comprehensive sensory evaluation of the three crude soy sauce products obtained in Example 3, and the crude soy sauce products were scored for their shape, color, aroma, and umami taste. The individual scores were 0-4 points. The higher the score, the better the index. The evaluation index was averaged. The results are shown in Table 2 and Figure 3 shown.

[0064] Table 2 Sensory evaluation results of fermented crude oil

[0065] Group posture Color aroma Umami Overall taste score Control group 1 3.6 3.3 3.0 3.2 3.2 Control group 2 3.6 3.2 3.4 3.2 3.3 Experimental Group 3.6 3.2 3.6 3.6 3.7

[0066] The results showed that the soy sauce obtained by fermentation with Torulopsis mutans ZB444 had better aroma, more umami taste and better overall taste than the soy sauce not inoculated with yeast and the soy sauce fermented with the starting strain.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. 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 solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A Torulopsis heterotrophica, characterized in that The Torulopsis versatilis is Wickerhamiella versatilis ZB444, and its deposit number is GDMCC No: 65240.

2. The bacterial cell, fermentation supernatant and / or intracellular material of Torulopsis heterotrophica according to claim 1.

3. Use of the Torulopsis heterotrophica according to claim 1 in soy sauce fermentation.

4. Use of the Torulopsis heterotrophica according to claim 1 in increasing the amino acid content of soy sauce.

5. The use according to claim 4, characterized in that: The amino acids include aspartic acid, threonine, glutamic acid, valine, methionine, isoleucine, leucine and lysine.

6. Use of the Torulopsis heterotrophica described in claim 1 in enhancing the flavor of soy sauce.

7. A soy sauce, characterized in that The method is prepared by fermenting the Torulopsis heterotrophica described in claim 1.

8. A method for preparing soy sauce, characterized in that: The soy sauce crude oil is inoculated with the Torulopsis heterotrophica of claim 1 and fermented.

9. The preparation method according to claim 8, characterized in that: The inoculation amount of the Torulopsis heterotrophica is 1% to 5%, so that the yeast content in the crude oil reaches 1×10 5 ~1×10 7 cfu / mL.

10. The preparation method according to claim 8, characterized in that: The fermentation time is 15 to 30 days, during which ventilation and stirring are performed for 15 to 40 minutes every 1 to 3 days.

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