A strain of brewer's yeast and its application in chili pepper fermentation

Abstract

This invention discloses a strain of brewing yeast and its application in chili pepper fermentation, relating to the fields of fermentation engineering and biotechnology. The taxonomic name of the brewing yeast YMZ002 is... Saccharomyces cerevisiae The strain, *Saccharomyces cerevisiae* YMZ002, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 2, 2025, with accession number CGMCC No. 35084. YMZ002 exhibits high salt tolerance, maintaining high metabolic activity even at a 10% salt concentration. Fermentation of chili peppers using this strain yielded chili peppers with suitable ethanol concentrations, increased content of esters such as ethyl acetate and ethyl lactate, and increased content of terpenoids and aromatic compounds such as linalool, while significantly reducing the content of fusel oils such as isoamyl alcohol and isobutanol, thus improving the quality of the fermented chili peppers. Furthermore, compared to natural fermentation, the fermentation cycle can be shortened by more than 35%.

Description

Technical Field

[0001] This invention relates to the fields of fermentation engineering and biotechnology, and in particular to a strain of brewing yeast and its application in chili fermentation. Background Technology

[0002] Traditional chili fermentation relies heavily on wild microorganisms in the natural environment, which has drawbacks such as long fermentation cycles, unstable product quality, and susceptibility to contamination by other bacteria (such as coliforms and molds).

[0003] To address the issue of long natural fermentation cycles, existing technologies have attempted to introduce artificially cultivated yeast for fermentation. For example, patent document CN106722565A discloses a method for chili fermentation using brewing yeast combined with compound microbial powder. Although this shortens the fermentation time, conventional brewing yeast suffers severe stress on cell metabolic activity in the high-salt environment (usually around 10%) during the later stages of chili fermentation, leading to a significant decrease in aroma production.

[0004] Furthermore, yeast easily metabolizes during fermentation to produce fusel alcohols such as isobutanol and isoamyl alcohol. Fusel alcohols not only contribute to the rough taste of the product, but excessive intake can also cause dizziness and other adverse reactions. Patent document CN113604372A discloses a brewing yeast with low fusel alcohol production and high ester production, but it primarily targets conventional low-salt alcohol fermentation systems such as fruit wine and rice wine, and does not solve the technical challenge of these strains failing to survive or experiencing metabolic inhibition in a 10% high-salt substrate. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a brewing yeast strain and its application in chili fermentation. The brewing yeast can maintain vigorous metabolic activity in a high-salt environment and can increase esters while reducing fusel alcohols.

[0006] In order to achieve the objective of this invention, the following solution is proposed: A new strain of *Saccharomyces cerevisiae*, YMZ002, was collected and screened from homemade fermented soybean paste produced by farmers in Meishan City, Sichuan Province over a long period of natural fermentation. Its taxonomic name is [omitted]. Saccharomycescerevisiae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 2, 2025, with accession number CGMCC No. 35084.

[0007] The 26S rDNA of Saccharomyces cerevisiae YMZ002 was sequenced, and its sequence is shown in SEQ ID NO.1.

[0008] Saccharomyces cerevisiae YMZ002 can be used for chili fermentation under high-salt conditions of 10% NaCl. The ethanol content in the fermentation broth was determined by gas chromatography, and the content of volatile flavor compounds was determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry. The results showed that the ethanol content was 0.3%–1.5% (V / V); ethyl acetate was 30 mg / kg–100 mg / kg; ethyl lactate was 20 mg / kg–60 mg / kg; linalool was 1 mg / kg–5 mg / kg; phenethyl alcohol was 5 mg / kg–15 mg / kg; isoamyl alcohol was less than 15 mg / kg; and isobutanol was less than 10 mg / kg.

[0009] The beneficial effects of this invention are as follows: 1. Saccharomyces cerevisiae YMZ002 can maintain high metabolic activity even at a high salt concentration of 10%; Saccharomyces cerevisiae YMZ002 can quickly become the dominant bacteria, inhibit the growth of miscellaneous bacteria, and shorten the fermentation cycle by more than 35% compared with natural fermentation. 2. In chili peppers fermented with brewer's yeast YMZ002, the ethanol concentration was appropriate, which increased the content of esters such as ethyl acetate and ethyl lactate, as well as terpenoids such as linalool, and significantly reduced the content of fusel alcohols such as isoamyl alcohol and isobutanol. Attached Figure Description

[0010] Figure 1 A microscope image of Saccharomyces cerevisiae YMZ002 is shown. Figure 2 The colony diagram of Saccharomyces cerevisiae YMZ002 is shown; Figure 3 The bar chart shows the ethanol content in simulated chili pepper juice inoculated with various strains; Figure 4 The bar chart shows the contents of ethyl acetate, ethyl lactate, linalool, and phenylethanol in the fermented chili pepper juice of various strains. Figure 5 The bar chart shows the content of isoamyl alcohol and isobutanol in the simulated chili juice inoculated with various strains. Detailed Implementation

[0011] Example 1 This embodiment provides the screening and identification process of Saccharomyces cerevisiae YMZ002, as detailed below: Homemade fermented broad bean paste (doubanjiang) was collected from farmers in Meishan City, Sichuan Province, and fermented naturally over a long period. 10g of the doubanjiang sample was added to 90mL of sterile physiological saline and mixed thoroughly. Then, it was serially diluted to prepare 10... -3 10 -4 10 -5Three gradient bacterial suspensions. Spread 100 μL of the bacterial suspension onto YPD solid medium supplemented with 10% NaCl and incubate at 30°C for 24 h. Pick... Figure 2 The single colony shown was smooth, milky white, and had neat edges. It was then subjected to multiple streak purifications to obtain a primary screening strain with salt tolerance.

[0012] Chili pepper simulated juice (double-screened fermentation substrate): Fresh Erjingtiao chili peppers are juiced, filtered to remove residue, and 10% NaCl and 3% glucose are added by weight-volume ratio. After sterilization, it is ready for use.

[0013] The above-mentioned pre-screened strains were inoculated into YPD liquid medium and activated by shaking culture at 30°C for 24 hours. At an inoculation rate of 5% (V / V), each activated bacterial solution was inoculated into a triangular flask containing 100 mL of chili pepper simulated juice, equipped with a one-way vent valve, and allowed to ferment statically for 15 days.

[0014] Fermentation broths from each strain were collected, and the ethanol content was determined by gas chromatography. Headspace solid-phase microextraction-gas chromatography-mass spectrometry (HSP-GC-MS) was used to determine the volatile flavor compounds. Strain YMZ002 exhibited the best metabolic characteristics in 10% NaCl chili pepper simulated juice. like Figure 3 As shown, the ethanol content is 0.8% (V / V). like Figure 4 As shown, ethyl acetate reached 82 mg / kg, ethyl lactate reached 45 mg / kg, linalool 3.2 mg / kg, and phenylethanol 8.5 mg / kg; like Figure 5 As shown, the content of fusel alcohols is low, with isoamyl alcohol at only 12 mg / kg and isobutanol at only 7 mg / kg.

[0015] The YMZ002 strain was identified as the final screening strain, i.e., the strain of this invention.

[0016] Microscopic observation results of Saccharomyces cerevisiae YMZ002 are as follows: Figure 1 As shown, the cells are oval or ovate in shape.

[0017] 26S rDNA sequencing was performed using universal yeast primers NL1 and NL4 on *Saccharomyces cerevisiae* YMZ002, and the sequence is shown in SEQ ID NO. 1. It was ultimately identified as *Saccharomyces cerevisiae*. Saccharomycescerevisiae ).

[0018] Example 2 This embodiment provides the application process of brewing yeast YMZ002 in chili fermentation, as detailed below: Raw material processing: Take fresh Erjingtiao chili peppers, remove the stems, wash and drain the surface water, and coarsely crush them into chili pepper pieces of 0.5cm~1.0cm.

[0019] Salting and inoculation: Add 10% salt by weight and mix well. After activating the *Saccharomyces cerevisiae* YMZ002 in YPD liquid medium, collect the cells by centrifugation, resuspend in sterile physiological saline, and inoculate at 2.0 × 10⁻⁶. 6 Add the final concentration of CFU / g to the chili flakes and mix well.

[0020] Fermentation: The mixture is placed in a fermentation tank, sealed with water, and fermented at 25°C for 18 days to obtain fermented chili peppers.

[0021] Example 3 Fermented chili peppers were centrifuged or pressed and filtered to obtain the fermentation broth. The ethanol and flavor compounds in the fermentation broth were then analyzed and compared, as detailed below: Example 2: Inoculation with Saccharomyces cerevisiae YMZ002.

[0022] Comparative Example 1: No strains were inoculated, only 10% salt was added.

[0023] Comparative Example 2: Inoculated with commercially available Angel high-activity dry brewing yeast, with the same inoculation amount and conditions as in Example 2.

[0024] The ethanol content in each group of fermentation broth was determined by gas chromatography, and the content of volatile flavor compounds in each group of fermentation broth was determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry. The results are as follows: The fermentation broth of Example 2 contained: 1.1% ethanol (v / v); 88 mg / kg ethyl acetate, 51 mg / kg ethyl lactate, 4.2 mg / kg linalool, 10.5 mg / kg phenylethanol; 13.5 mg / kg isoamyl alcohol, and 8.2 mg / kg isobutanol. No coliform bacteria or pathogenic bacteria were detected in the fermentation system.

[0025] The fermentation broth of Comparative Example 1 contained 0.2% ethanol (V / V), 14 mg / kg ethyl acetate, 6 mg / kg ethyl lactate, and no linalool or phenylethanol was detected. The fermentation period lasted up to 28 days, with slight growth of mold and other microorganisms in the early stages.

[0026] The fermentation broth of Comparative Example 2 contained 1.8% ethanol (V / V), 29 mg / kg ethyl acetate, 21 mg / kg ethyl lactate, and trace amounts of linalool. Under high-salt conditions, its fusel alcohol metabolism was abnormal, with isoamyl alcohol reaching 42 mg / kg and isobutanol reaching 28 mg / kg.

[0027] In summary, using brewing yeast YMZ002 can not only shorten the fermentation cycle, but also significantly increase the content of esters and terpenoids in floral aroma compounds. At the same time, its isoamyl alcohol and isobutanol content is reduced by more than 50% compared with ordinary yeast, showing remarkable effects.

[0028] Example 4 This embodiment provides a salt tolerance test for brewer's yeast YMZ002, as detailed below: YMZ002 and commercially available Angel high-activity dry Saccharomyces cerevisiae were inoculated into salt-free YPD liquid medium, respectively. They were cultured in a shaker at 30℃ and 200 rpm for 12–16 h to reach the logarithmic growth phase. Subsequently, the concentration of both seed cultures was standardized using sterile physiological saline and adjusted to the initial absorbance OD. 600 =1.0.

[0029] Prepare two sets of 250mL Erlenmeyer flasks, one containing 100mL of basic YPD liquid medium (salt-free group) and the other containing YPD liquid medium supplemented with 10% NaCl (high-salt group). Inoculate the standardized *Saccharomyces cerevisiae* YMZ002 seed culture and Angel Yeast high-activity dry *Saccharomyces cerevisiae* seed culture into these two media at an inoculation rate of 5% (v / v). Place in a constant-temperature shaker and culture continuously at 30℃ and 200 rpm for 24 hours.

[0030] After 24 hours of incubation, the culture medium of each group was collected, and the absorbance (OD) value was measured at a wavelength of 600 nm using a UV-Vis spectrophotometer. 600 The calculation formula is used to determine the OD of the high-salt group. 600 / Salt-free group OD 600 ×100% yields the relative growth and survival rate.

[0031] Measurements showed that under salt-free conditions, the final growth concentrations of *Saccharomyces cerevisiae* YMZ002 and Angel Yeast high-activity dry *Saccharomyces cerevisiae* were basically the same; however, under high-salt conditions of 10% NaCl, the growth of Angel Yeast high-activity dry *Saccharomyces cerevisiae* was severely inhibited, and its OD... 600 The OD value was only about 15% of that of the salt-free group; however, the brewer's yeast YMZ002 of this invention still maintained vigorous proliferation ability, and its OD value was only about 15% of that of the salt-free group. 600 The value reached 85% of its own salt-free group.

[0032] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be unique or to limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from its scope are all within the scope of protection of the present invention.

Claims

1. A strain of brewing yeast YMZ002, characterized in that, Its taxonomic name is Saccharomyces cerevisiae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 2, 2025, with accession number CGMCC No. 35084.

2. The application of the brewing yeast according to claim 1, characterized in that, Used for chili pepper fermentation.

3. The application according to claim 2, characterized in that, The fermentation system contains 10% NaCl.

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