A type of Indica sporozoan yeast and its application

Abstract

This invention provides a *Torulaspora indica* G2-2 strain and its application. *Torulaspora indica* G2-2 is deposited at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC M 2022937, and deposited on June 21, 2022. This invention also provides the application of *Torulaspora indica* G2-2 in white fermented soup. The preparation of the starter culture utilizes a co-culture method of *Torulaspora indica* and *Lactobacillus paracasei*, leveraging the synergistic fermentation and aroma-producing effects of the two strains during the fermentation of white fermented soup.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an Indica sporozoan yeast and its applications. Background Technology

[0002] Sour soup, a traditional food of the Miao and Dong ethnic groups, is popular in Guizhou Province, China. It mainly includes "white sour soup" made from glutinous rice or rice and "red sour soup" made from tomatoes and red peppers. Sour soup, fermented from grains, is not only refreshing and delicious, but also has anti-fatigue, anti-aging, cardiovascular disease prevention, and immune regulation functions. These health benefits mainly come from the metabolism of probiotics during the fermentation process of sour soup.

[0003] Non-Saccharomyces yeasts (aromatic yeasts) mainly include *Hanamitratus*, *Kluyveromyces*, *Mercuriae*, *Pichia pastoris*, *Rhodotorula*, *Cryptospira*, *Schizosaccharomyces*, and *Cytomyces*. These yeasts produce a wide variety of volatile substances in high amounts, resulting in prominent aromas, and are widely present in brewing raw materials and fermentation environments. The aroma components in white sour soup mainly originate from the co-fermentation of lactic acid bacteria and yeast. During fermentation, aroma precursors are hydrolyzed and mostly exist in the form of glycosides. These glycosides are further decomposed by enzymes produced by microorganisms to form free aroma components.

[0004] Currently, the fermentation of white sour soup remains a traditional, small-scale, natural fermentation process. This process is lengthy and susceptible to the influence of raw material quality, bacterial contamination, and environmental factors. Furthermore, the lack of sterilization processes leads to batch-to-batch inconsistencies in quality and flavor. While some companies can produce white sour soup industrially, most of these productions involve adding organic acids, resulting in strong acidity and a lack of aroma. Therefore, selecting suitable dominant microbial strains for white sour soup fermentation offers significant economic and social benefits. Summary of the Invention

[0005] To address the problems in the existing technology, this invention provides Indica spore-forming yeast and its application, which uses Indica spore-forming yeast and Lactobacillus paracasei for synergistic fermentation. The process is simple and preserves the special flavor of naturally fermented sour soup to the greatest extent.

[0006] The technical problem solved by this invention is achieved by the following technical solution:

[0007] The first objective of this invention is to provide a Torulaspora indica, characterized in that the Torulaspora indica G2-2 is deposited at the China Center for Type Culture Collection, located at Luojia Mountain, Wuchang, Wuhan, Hubei Province, China, with accession number CCTCC M 2022937 and deposit date of June 21, 2022.

[0008] Furthermore, the sequence number of Torulaspora indica G2-2 is shown in SEQ ID NO.1.

[0009] Furthermore, the screening method for Torulaspora indica G2-2 includes the following steps:

[0010] S1. Weigh a sample of white sour soup from Southwest China and place it in a test tube containing sterile deionized water. Shake thoroughly to form a mother suspension. Prepare the mother suspension into 10... -1 10 -2 10 -3 10 -4 10 -5 Deionized water gradient dilution was prepared for later use; each of the gradient dilutions was spread onto YEPD solid medium, incubated at 35°C for 48 h, and then isolated and purified to obtain the isolated strain;

[0011] S2. The isolated strain obtained in S1 was inoculated into YEPD liquid medium and cultured at 30℃ and 150 rpm for 24 h to obtain seed culture.

[0012] S3. The seed culture obtained in S2 was inoculated into YNB liquid medium at an inoculation rate of 1%, and cultured at 30℃ and 150rpm for 6h. Then it was transferred to YNB auxotrophic medium and cultured in a constant temperature incubator at 30℃ for 24-48h to obtain Torulaspora indica G2-2.

[0013] Furthermore, the YNB nutrient deficiency culture medium comprises 0.67% YNB, 0.5% cellobiose, and 2% agar.

[0014] The second objective of this invention is to provide an application of Torulaspora indica G2-2 in the fermentation of white sour soup.

[0015] Furthermore, the application of Torulaspora indica G2-2 combined with Lactobacillus paracasei in the fermentation of white sour soup.

[0016] Furthermore, the method for preparing the white sour soup is to ferment the glutinous rice hydrolysate with Torulasporaindica G2-2 and Lactobacillus paracasei as inoculum.

[0017] Furthermore, the inoculum size of Torulaspora indica G2-2 was 0.1%, and the inoculum size of Lactobacillus paracasei was 8%.

[0018] Furthermore, the preparation method of the glutinous rice enzymatic hydrolysate is as follows: glutinous rice is crushed using a high-speed grinder and sieved through an 80-mesh sieve to obtain glutinous rice flour. Pure water is boiled, and 1% by mass of glutinous rice flour is added while stirring. Then, the mixture is transferred to a 60°C water bath for gelatinization for 30 minutes, with continuous stirring. After gelatinization, 1% by mass of α-amylase is added, and after liquefaction for 30 minutes, the mixture is placed in a 95°C water bath for 20 minutes. Then, it is cooled to about 36°C to obtain the glutinous rice enzymatic hydrolysate.

[0019] Furthermore, the inoculation density of the Torulaspora indica G2-2 was 10-1. 7 cfu / mL.

[0020] Furthermore, after inoculation with Torulaspora indica G2-2, the fermentation temperature of the glutinous rice hydrolysate is 30-35℃.

[0021] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0022] This invention is based on the characteristics of Guizhou-style white sour soup and is developed using Torulaspora indica G2-2, a microorganism that plays a key role in the fermentation process of white sour soup, thus preserving the unique flavor of naturally fermented sour soup to the greatest extent. The invention employs a combined culture method of Torulaspora indica and Lactobacillus paracasei in the preparation of the starter culture, utilizing the synergistic fermentation, acid production, and aroma production effects of the two strains during the white sour soup fermentation process. No organic acids such as citric acid, tartaric acid, or acetic acid are added during the preparation of the white sour soup. The above-mentioned method for preparing ripening and acid-producing white sour soup is simple, accelerates the ripening of white sour soup, shortens the fermentation cycle, produces highly stable products, has low equipment requirements, is easy to control, has high production efficiency, and low production costs.

[0023] The white sour soup used in this invention is made by farmers in Guizhou.

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0025] Figure 1 The colony morphology of Torulaspora indica G2-2 in this invention on YEPD solid medium. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0027] Furthermore, unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention can be obtained commercially or prepared using existing methods. In each embodiment of this invention, the quantitative experiments were performed in triplicate, and the results were averaged.

[0028] Example 1: Isolation, screening and identification of Torulaspora indica G2-2

[0029] 1.1 Isolation of strains

[0030] The isolation of bacterial strains involves three steps: sampling, enrichment, and initial and secondary screening, as detailed below:

[0031] 1. Sampling

[0032] Samples of homemade white sour soup were collected from farmers in Guizhou using ice boxes, placed in clean sampling bags, labeled, and transported back to the laboratory for storage at 4°C as samples.

[0033] 2. Enrichment and initial screening

[0034] In a sterile laminar flow hood, 10 mL of homemade white sour soup sample was weighed and placed into a test tube containing 90 mL of sterile deionized water. The mixture was then vortexed thoroughly to prepare a mother suspension. Then, using deionized water, the mother suspension was further refined into 10... -1 10 -2 10 -3 10 -4 10 -5 Gradual dilutions of deionized water were prepared, with 100 μL of each dilution spread onto YEPD solid medium. Different morphologies of the bacteria were streaked and purified to obtain isolated strains, which were named G2-1, G2-2, G2-3, and G2-4, respectively. The strains were stored at -80°C with 50% glycerol for later use. The YEPD solid medium consisted of 20 g glucose, 10 g yeast extract, 20 g peptone, and 1000 mL deionized water.

[0035] 3. Secondary screening

[0036] The glycosidase activity of the isolated strains obtained from the initial screening was determined using the following method: Strains G2-1, G2-2, G2-3, and G2-4 were inoculated into YEPD liquid medium and cultured at 30℃ and 150 rpm for 24 h to prepare seed solutions. These seed solutions were then inoculated at a rate of 1% into 250 mL Erlenmeyer flasks containing 100 mL of carbon-free YNB medium and cultured at 30℃ and 150 rpm for 6 h. After the excess sugar was consumed, 5 μL was transferred to YNB auxotrophic medium and cultured in a 30℃ incubator for 24-48 h. Growth was observed; better growth indicated stronger glycosidase activity. The experimental results are shown in Table 1.

[0037] The growth of the strains showed that the G2-2 strain of this invention exhibited good growth, possessed a certain content of glycosidase, and had the potential to degrade aroma precursors and enhance the aroma of white sour soup. The YNB auxotrophic medium consisted of 0.67% YNB, 0.5% cellobiose, and 2% agar. 20g of agar was added to the YNB solid medium. The YNB auxotrophic medium was sterilized at its natural pH and 115℃ for 20 minutes.

[0038] Table 1 Comparison of glycosidase activities

[0039]

[0040] 1.2 Strain Identification

[0041] A series of physiological and biochemical identifications were performed on the purified Torulaspora indica G2-2 culture obtained above, and the 26S rDNA D1 / D2 region sequence was identified using the PCR method. The identification results are as follows:

[0042] 26S rDNA D1 / D2 region amplification and sequencing were performed, and the sequencing results are shown in SEQ ID NO.1.

[0043] The *Torulaspora indica* G2-2 strain, after homology comparison, showed a 99.5% high similarity to *Torulaspora indica* strain YFA121867 (GenBank accession no. MG214672). Based on its morphological and physiological-biochemical characteristics, the strain was named *Torulaspora indica* G2-2. The *Torulaspora indica* G2-2 isolated and screened in this example was cultured on YEPD solid medium, and its colony morphology was observed. The results are shown in the attached figure. Figure 1As shown. See appendix. Figure 1 It can be seen that the Torulaspora indica G2-2 of this invention forms milky white, round, regular-edged, smooth and glossy colonies on YEPD solid medium.

[0044] The Torulaspora indica G2-2 obtained by the above method is deposited at the China Center for Type Culture Collection, located at Luojia Mountain, Wuchang, Wuhan, Hubei Province, China, with accession number CCTCC M 2022937 and deposit date of June 21, 2022.

[0045] Example 2: Preparation of white sour soup using Torulaspora indica G2-2 and Lactobacillus paracasei.

[0046] The screened *Saccharomyces cerevisiae* G2-2 was co-fermented with *Lactobacillus paracasei* to prepare white sour soup. The specific method is as follows:

[0047] S1. *Saccharomyces cerevisiae* G2-2 stored at -80℃ was activated and passaged on YEPD solid and liquid media. The activated *Saccharomyces cerevisiae* G2-2 was inoculated into 100 mL of YEPD liquid media and cultured at 30℃ and 150 rpm for 24 h on a shaker. The cells were then collected by centrifugation at 8℃ and 4000 rpm for 5 min, and resuspended in sterile water to a concentration of 10. 7 cfu / mL; *Lactobacillus paracasei* stored at -80℃ was activated and passaged in MRS solid and liquid media. The activated *Lactobacillus paracasei* was inoculated into 100 mL of MRS liquid media and cultured at 35℃ and 80 rpm for 24 h on a shaker. The cells were then collected by centrifugation at 8℃ and 4000 rpm for 5 min, and resuspended in sterile water to a concentration of 10. 7 cfu / mL.

[0048] S2. Glutinous rice is crushed using a high-speed grinder and sieved through an 80-mesh sieve to obtain glutinous rice flour. Pure water is boiled, and 1% of the glutinous rice flour is added while stirring. The mixture is then transferred to a 60℃ water bath for gelatinization for 30 minutes, with continuous stirring. After gelatinization, 1% α-amylase is added, and the mixture is liquefied for 30 minutes. It is then placed in a 95℃ water bath for 20 minutes, followed by cooling to approximately 36℃ to obtain glutinous rice enzymatic hydrolysate. *Saccharomyces cerevisiae* G2-2 is inoculated at a rate of 0.1%, and *Lactobacillus paracasei* is inoculated at a rate of 8% into the glutinous rice enzymatic hydrolysate. Fermentation is carried out at 30-35℃ for 5 days to obtain white sour soup.

[0049] Two samples were used as control experiments: one fermented with Lactobacillus paracasei alone, and the other without the addition of Indica spore-forming yeast G2-2 and Lactobacillus paracasei, while the glutinous rice hydrolysate was naturally fermented.

[0050] The physicochemical properties of the white sour soup co-fermented with *Saccharomyces cerevisiae* G2-2 and *Lactobacillus paracasei* obtained in this invention were compared with those of the control experiments of naturally fermented white sour soup and white sour soup fermented with *Lactobacillus paracasei*. The comparison results are shown in Table 2. It can be seen that the co-fermentation of *Saccharomyces cerevisiae* G2-2 and *Lactobacillus paracasei* can rapidly increase the total acid content and decrease the total sugar and reducing sugar content in the white sour soup system, thereby improving the fermentation flavor and enhancing the product quality. While the white sour soup obtained by adding only *Lactobacillus paracasei* has a better flavor than the naturally fermented white sour soup, its total acid content is low, and its total sugar content, especially the reducing sugar content, is high, thus making it difficult to achieve the unique flavor of naturally fermented sour soup. In summary, the differences in fermentation methods have different effects on the production and preparation of white sour soup. Multi-strain enhanced fermentation can achieve the quality of natural fermentation and has a faster acid production rate. It also exhibits strong fermentation characteristics in terms of physicochemical quality. The white sour soup prepared by co-fermentation of Saccharomyces Indica and Lactobacillus paracasei of this invention has superior quality in terms of pH, total acid content, and reducing sugar consumption rate. As can be seen from Table 2, each physicochemical index is very close to that of natural farmhouse white sour soup.

[0051] Table 2 Physicochemical index analysis of fermented white sour soup

[0052]

[0053] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0054] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A Saccharomyces indicans characterized by: The Torulaspora indica G2-2 is preserved in China Center for Type Culture Collection, Wuhan, Hubei, China, and the preservation number is CCTCC M 2022937, and the preservation time is June 21, 2022.

2. The Yarrowia lipolytica of claim 1, wherein: The Indica Saccharomyces sp. Torulaspora indica The nucleotide sequence of G2-2 is shown as SEQ ID NO.

1.

3. The I. indica according to claim 1, wherein, The screening method of the Indica Saccharomycopsis ( Torulaspora indica ) G2-2 includes the following steps: S1, the white acid soup sample in the southwest region is weighed and placed into a test tube containing sterile deionized water, and a mother suspension is formed after being shaken thoroughly; the mother suspension is prepared into 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 gradient dilutions of deionized water for standby; each of the gradient dilutions is coated on YEPD solid culture medium, and after being cultured at 35°C for 48 h, isolated strains are obtained through separation and purification; S2, inoculating the isolated strain obtained in S1 into YEPD liquid culture medium, and culturing at 30 DEG C and 150 rpm for 24 h to obtain a seed liquid; S3, inoculate the seed liquid obtained in S2 into YNB liquid culture medium at an inoculation amount of 1%, culture at 30°C and 150 rpm for 6 h, then transfer into YNB nutrient-deficient culture medium, and place in a 30°C constant temperature incubator to culture for 24-48 h to obtain the Indica spore round yeast (Saccharomyces boulardii) G2-2. Torulaspora indica ) G2-2.

4. A Saccharomyces indicanus (S. indicans) strain according to any one of claims 1-3, wherein the strain is a Saccharomyces indicanus (S. indicans) strain deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 14023. Torulaspora indica ) G2-2 in combination with Lactobacillus paracasei in the fermentation of white vinegar.

5. The use according to claim 4, wherein: The preparation method of the white acid soup is to ferment the Indica Saccharomyces boulardii (G2-2) and Lactobacillus paracasei as a strain to the waxy rice enzymatic hydrolysate. Torulaspora indica ) G2-2 and Lactobacillus paracasei as a strain to the waxy rice enzymatic hydrolysate.

6. The use according to claim 5, characterized in that: The said Indica Saccharomyces Torulaspora indica The inoculation amount of G2-2 was 0.1%, and the inoculation amount of Lactobacillus paracasei was 8%.

7. The use according to claim 5, wherein: The preparation method of the waxy rice enzymatic hydrolysate is as follows: waxy rice is broken by a high-speed crusher, sieved through an 80-mesh screen to obtain waxy rice powder, pure water is boiled, 1% waxy rice powder by mass fraction is added while stirring, then the mixture is moved into a 60 DEG C water bath for gelatinization for 30 min, the mixture is continuously stirred during the gelatinization, 1% alpha-amylase by mass fraction is added after the gelatinization, the mixture is placed in a 95 DEG C water bath for 20 min after being liquefied for 30 min, then the mixture is cooled to about 36 DEG C to obtain the waxy rice enzymatic hydrolysate.

8. Use according to any one of claims 4 to 7, wherein: S. inudicatum Torulaspora indica G2-2 inoculation density of 10 7 cfu / mL.

9. The use according to claim 7, characterized in that: The said Indica Saccharomycopsis Torulaspora indica After G2-2 inoculation, the fermentation temperature of the waxy rice enzymatic hydrolysate is 30-35℃.

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