Saccharomycetes HAU2501 and application thereof in preparation of fermented vegetables

Abstract

The invention provides yeast HAU2501 and application thereof in preparation of fermented vegetables, and belongs to the technical field of fermentation microorganisms. The invention discloses saccharomycetes HAU2501, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is July 21, 2025, and the preservation number is CGMCC No.35320. The saccharomycetes HAU2501 has the advantages that the saccharomycetes HAU2501 can be used for preparing the saccharomycetes HAU2501; the saccharomycetes HAU2501 can be used as an excellent aroma-producing leavening agent, can effectively improve the flavor quality of the fermented vegetables through synergistic fermentation with lactic acid bacteria, and provides a theoretical basis and a technical reference for flavor regulation and control of traditional fermented vegetables and development of functional leavening agents.

Description

Technical Field

[0001] This invention relates to the field of fermentation microbial technology, and in particular to a yeast strain HAU2501 and its application in the preparation of fermented vegetables. Background Technology

[0002] Fermented vegetables are a typical representative of traditional fermented foods in my country, widely popular for their tender, crisp texture, unique flavor, and pleasant aroma. They are also believed to have dietary functions such as relieving greasiness, stimulating appetite, and aiding digestion. The flavor and texture of traditionally fermented vegetables mainly rely on the synergistic action of microorganisms such as bacteria, yeast, and molds. These microorganisms participate in the conversion of carbohydrates, proteins, and other components in the vegetable matrix through enzyme production and other metabolic activities, thereby regulating the color, texture, and flavor characteristics of the product. In addition, fermentation not only gives vegetables their unique sensory qualities but also significantly enhances their nutritional and health value. During fermentation, microbial metabolism produces a variety of bioactive substances such as phenols, organic acids, and vitamins. Studies have confirmed that these components have physiological functions such as antioxidation, anti-inflammation, and assisting in regulating blood pressure.

[0003] During the natural fermentation of vegetables, lactic acid bacteria attached to the surface of the vegetables become the dominant bacteria in the fermentation process by utilizing their metabolic products and the resulting low-oxygen environment. As the main functional microorganisms in fermented vegetables, lactic acid bacteria produce organic acids, mainly lactic acid, and other bioactive components during metabolism, giving the product unique flavor and nutritional characteristics. However, traditional vegetable fermentation is essentially a synergistic process involving multiple types of microorganisms. Recent studies have gradually revealed that yeast, as an important member of the fermentation system, also plays an indispensable role. The participation of appropriate amounts of yeast can not only enrich the flavor layers of fermented vegetables, but also further regulate the fermentation process and product distribution through synergistic or competitive relationships with lactic acid bacteria. For example, Yan Yuge et al. used aroma-producing yeast and *Lactobacillus plantarum* to ferment chili sauce and found that the variety of flavor substances increased significantly, especially the content of esters, alcohols, and ketones, giving the product a richer fruity and floral aroma. Li Jiaxin et al.'s research showed that *Saccharomyces cerevisiae* (Wickham yeast)... Wickerhamomyces anomalus ), Pichia kudriozwij ( Pichia kudriavzevii Co-fermenting sauerkraut with *Lactobacillus plantarum* and *Leuconostoc mesenteroides* not only inhibits excessive acid production by lactic acid bacteria and reduces peak nitrite levels, but also improves the firmness and total ester content of the sauerkraut. Currently, while the synergistic effect of yeast and lactic acid bacteria has shown initial potential to improve the flavor and quality of fermented vegetables, related research is mostly focused on model strains or single fermentation systems. For chili peppers, a specific substrate, research on the impact of compound fermentation on the quality of chili pepper fermentation is relatively lacking, and the development of specialized compound fermentation agents adapted to this substrate has not yet formed a systematic research system.

[0004] To improve the quality of fermented chili peppers, this invention uses naturally fermented chopped chili peppers and mustard greens as the source of microbial strains, and selectively screens yeast strains that are resistant to acid, salt, and ethanol, and possess excellent aroma and ester production capabilities. Based on this, the screened yeast strains are combined with high-quality lactic acid bacteria obtained in the laboratory to construct a yeast-lactic acid bacteria composite fermentation system. This system is then applied to chili pepper fermentation verification experiments, aiming to develop a dedicated composite fermentation agent suitable for chili pepper fermentation, thereby providing microbial resources and technological basis for producing high-quality fermented chili peppers with harmonious flavor and rich nutrition. Summary of the Invention

[0005] The purpose of this invention is to provide a yeast strain HAU2501 and its application in the preparation of fermented vegetables.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a yeast strain (Maudiozyma humilis) HAU2501, which is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of July 21, 2025 and accession number CGMCC No. 35320.

[0007] This invention provides a compound fermentation agent comprising the aforementioned yeast HAU2501 and lactic acid bacteria.

[0008] Preferably, the lactic acid bacteria are selected from one or more of Lactobacillus plantarum H3D, Lactobacillus pentosus 6d-16, Pediococcus pentosus 206, Pediococcus pentosus 6-12, Weissella esculenta T244, and Lactobacillus brevis T216.

[0009] Preferably, the lactic acid bacteria are a combination of *Westernella esculenta* T244, *Lactobacillus brevis* T216, *Lactobacillus plantarum* H3D, and *Pediococcus pentosaceus* 206, wherein the mass ratio of *Westernella esculenta* T244, *Lactobacillus brevis* T216, *Lactobacillus plantarum* H3D, and *Pediococcus pentosaceus* 206 is 1~3:1~3:1~3:1~3, and the bacterial content of each is 1×10⁻⁶. 11 ~1.5×10 11 CFU / g.

[0010] Preferably, the lactic acid bacteria are a combination of *Lactobacillus pentosaccharide* 6d-16, *Pediococcus pentosaccharide* 6-12, and *Lactobacillus plantarum* H3D, wherein the mass ratio of *Lactobacillus pentosaccharide* 6d-16, *Pediococcus pentosaccharide* 6-12, and *Lactobacillus plantarum* H3D is 1~3:1~3:1~3, and the bacterial content of each is 1×10⁻⁶. 11 ~1.5×10 11 CFU / g.

[0011] Preferably, the mass ratio of yeast HAU2501 to lactic acid bacteria is 1~3:1~3, and the bacterial content of each is 1×10⁻⁶. 11 ~1.5×10 11 CFU / g.

[0012] This invention provides the application of the yeast HAU2501 or the compound fermentation agent in the preparation of fermented vegetables.

[0013] Preferably, the vegetable is a chili pepper.

[0014] This invention provides a method for increasing the aroma content in fermented vegetables, wherein the yeast HAU2501 or the compound fermentation agent is mixed with vegetables for fermentation to obtain fermented vegetables with increased aroma content.

[0015] Preferably, the amount of yeast HAU2501 added is 0.08~0.12wt% or the amount of compound fermentation agent added is 0.15~0.25wt%.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention screened a superior aroma-producing yeast strain from naturally fermented mustard greens and chopped chili pepper samples. Maudiozyma humilis The yeast strain HAU2501 possesses high ester-producing activity, rapid fermentation capacity, and good tolerance to multiple stresses. When this strain was combined with lactic acid bacteria for the fermentation of chopped chili peppers, the results showed that the total acid, total ester, and amino acid nitrogen content of the mixed fermentation group were significantly higher than those of the pure lactic acid bacteria fermentation group, and the sensory scores were also significantly improved. Furthermore, its flavor profile was significantly different from both the pure lactic acid bacteria fermentation group and the naturally fermented group. Therefore, yeast HAU2501 can be used as an excellent aroma-producing fermenting agent. Co-fermentation with lactic acid bacteria can effectively improve the flavor quality of fermented vegetables, providing a theoretical basis and technical reference for flavor regulation of traditional fermented vegetables and the development of functional fermenting agents. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 The figure shows the acid resistance test results of different strains in Example 1. Different lowercase letters between groups indicate significant differences (P<0.05). Figure 2 The figure shows the salt tolerance test results of different strains in Example 1. Different lowercase letters between groups indicate significant differences (P<0.05). Figure 3 The figure shows the results of ethanol tolerance tests on different strains in Example 1. Different lowercase letters between groups indicate significant differences (P<0.05). Figure 4 The colony (a), cell (b), and phylogenetic tree (c) of strain X1 in Example 1 are shown. Figure 5 The results of multivariate statistical analysis of volatile compounds in chopped chili peppers fermented using different strain combinations in Example 2 are shown, where a is the OPLS-DA plot, b is the permutation test plot, and c is the VIP plot.

[0019] Preservation Instructions

[0020] The yeast strain (Maudiozyma humilis) HAU2501 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 21, 2025, and the accession number is CGMCC No. 35320. Detailed Implementation

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. The materials, reagents, instruments and equipment used in the following embodiments are as follows: Materials and Reagents: Chopped Chili Peppers: Chili peppers were purchased from a farmers' market and naturally fermented in the laboratory to make chopped chili peppers. Fermented Mustard Greens: A gift from Guotai Food Co., Ltd. Anhydrous Ethanol, Glycerin, Agar, Phenolphthalein, Sodium Chloride, Sodium Hydroxide, Lactic Acid (all analytical grade): Sinopharm Chemical Reagent Co., Ltd.; YPD Culture Medium: Haibo Biotechnology Co., Ltd. Instruments and Equipment: SQ810C Automatic High-Pressure Steam Sterilizer: Chongqing Yamato Technology Co., Ltd.; HCB-1300V Clean Bench: Qingdao Haier Biomedical Co., Ltd.; UV-1801 Ultraviolet / Visible Spectrophotometer: Beijing Beifen Ruili Analytical Instrument Co., Ltd.; SPL250 Biochemical Incubator: Tianjin Laiboteri Instrument Equipment Co., Ltd.; CX31 Optical Microscope: Olympus (China) Co., Ltd.; Bante920 pH Meter: Shanghai Bante Instrument Manufacturing Co., Ltd.; SW-2FD Vortex Shaker: Shanghai Medical University Scientific Instrument Factory; Agilent 8890 Gas Chromatograph, Agilent 7250 Gas Chromatograph / Quadrupole Time-of-Flight Mass Spectrometer: Agilent Technologies.

[0022] Data Processing and Analysis: All experiments were independently repeated three times, and data are expressed as mean ± standard deviation. Excel 2016 was used for data processing and preliminary calculations, and Origin 2024 was used for chart creation. Phylogenetic trees were constructed based on specific gene sequences using MEGA 6.0 software. Partial least squares discriminant analysis (OPLS-DA) was performed using SIMCA software, and one-way ANOVA and other statistical tests were conducted using IBM SPSS Statistics 26 software. P < 0.05 was considered statistically significant.

[0023] Example 1

[0024] 1. Isolation and purification of yeast

[0025] 10 g of fermented mustard greens and chopped chili peppers were weighed separately, added to sterile physiological saline, and eluted thoroughly by shaking to obtain bacterial suspensions. The bacterial suspensions were serially diluted and plated on YPD plates, and incubated at 28°C for 3 days. Typical yeast colonies were picked, and pure strains were obtained through multiple streak purification processes and preserved in glycerol tubes at -80°C.

[0026] Results: A total of 21 bacterial strains were isolated and purified from fermented chopped chili peppers and fermented mustard greens. Colony morphology observation showed that most strains were milky white or pale yellow, with smooth or slightly wrinkled surfaces and regular edges; some strains formed distinct colony protrusions. Microscopic examination revealed that their cell morphology was mostly round or oval, consistent with typical yeast cell characteristics.

[0027] 2. Yeast screening

[0028] Aroma production capacity determination: Yeast was inoculated onto YPD solid medium by the spread plating method and cultured at 28℃ for 3 days before sensory evaluation. The aroma production capacity of the yeast was preliminarily determined by the olfactory method, and the evaluation criteria are shown in Table 1.

[0029] Table 1 Sensory evaluation criteria for screening yeast strains

[0030] Ester production performance was determined in accordance with GB / T10345—2007 "Analytical Methods for Baijiu".

[0031] Gas production performance determination: The Durham tube fermentation method was adopted. 100 μL of yeast culture stored in glycerol at -80℃ was inoculated into a test tube containing 10 mL of YPD liquid medium and activated at 28℃ for 24 h. The culture was then inoculated into YPD medium containing Durham tubes at a volume ratio of 1% and cultured at 28℃ for 48 h. The gas production of Durham tubes in the medium was carefully observed and recorded at 24 h and 48 h to compare the fermentation capacity of each strain.

[0032] The results of the aroma-producing capacity test are shown in Table 2. It can be seen that all strains can produce volatile aromas, but their aroma intensities vary significantly. Strains X1, X5, X8, and X12 produce rich sweet wine, ester, or alcohol aromas with good aroma persistence; while strains C6 and C8 have relatively weaker aroma intensities. Based on these results, seven strains with outstanding aroma-producing capacity (X1, X5, X8, X12, C6, C8, and B2) were selected for subsequent experiments.

[0033] Table 2. Results of aroma production capacity determination for different strains

[0034] The gas production and ester production performance are shown in Table 3. Esters are key components in the formation of fermentation flavor. Most aroma-producing yeasts can synthesize ester compounds with fruity aroma characteristics during metabolism, which have an important impact on the aroma composition and quality of the product. The results of the total ester content determination in the fermentation broth of each strain by saponification neutralization titration showed that X1, B2, and C6 had better ester production capacity, with total ester contents of 1.48 g / L, 1.14 g / L, and 1.03 g / L, respectively. The gas production capacity in the Durham tube can be used as an indicator to evaluate the fermentation rate of yeast strains. As shown in Table 3, there are significant differences in gas production capacity among different strains: X1, X8, and X12 filled the Durham tube with gas within 48 h, indicating a high fermentation rate; X5 and B2 produced about 3 / 4 of the tube volume, indicating a moderate fermentation rate; while C6 and C8 produced about 1 / 2 of the tube volume, indicating a slower fermentation rate.

[0035] Table 3. Results of gas production and ester production capacity determination for different strains

[0036] Note: "+, ++, +++, ++++" indicate that gas production reaches 1 / 4, 1 / 2, 3 / 4, and the entire volume of the Durbin tubule, respectively. Different lowercase letters between groups in the table indicate significant differences (P<0.05) (the same applies below).

[0037] 3. Yeast tolerance study

[0038] Acid tolerance test: Activated yeast culture was inoculated at a rate of 1% into YPD liquid medium at pH 3.0, 3.5, 4.0, 4.5, and 5.0, respectively, and incubated at 28℃ for 48 h. The OD of the fermentation broth was then measured. 600 value.

[0039] Salt tolerance test: Activated yeast culture was inoculated at a rate of 1% into YPD liquid medium with NaCl concentrations of 2%, 4%, 6%, 8%, and 10%, respectively, and cultured at 28℃ for 48 h. The OD of the fermentation broth was then measured. 600 value.

[0040] Ethanol tolerance test: Activated yeast culture was inoculated at a 1% inoculum into YPD liquid medium containing 2%, 4%, 6%, 8%, and 10% ethanol (v / v), respectively, and incubated at 28℃ for 48 h. The OD of the fermentation broth was then measured. 600 value.

[0041] Acid tolerance is crucial for the survival and function of yeast in fermentation systems. During the lactic acid fermentation of vegetables such as chili peppers, lactic acid bacteria, as the dominant flora, produce large amounts of lactic acid, causing the system pH to drop to 3.2-3.6. This acidic environment significantly inhibits yeast growth, thus affecting its fermentation capacity. Therefore, screening for yeast strains with excellent acid tolerance is key to ensuring the smooth progress of the fermentation process. Figure 1 As shown, under strongly acidic conditions of pH 3.0-3.5, the growth of all strains was significantly inhibited, with X1 exhibiting relatively strong tolerance. When pH ≥ 4.0, all strains grew well, and X1 showed the highest OD value. 600 The value was consistently higher than that of other strains, indicating that it had the best acid resistance among the tested strains.

[0042] During vegetable fermentation, yeast often faces high salt stress. The salt tolerance of yeast directly affects its fermentation performance; strains with poor salt tolerance are prone to slow fermentation initiation or even premature termination of the fermentation process. Figure 2 As shown, under low-salt conditions of 2% NaCl, the OD values ​​of strains X5 and X8 were... 600 A value less than 0.5 indicates poor growth; while the OD values ​​of the other five strains, including X1, are lower. 600 The OD value was around 1.5, indicating relatively good growth. As the NaCl concentration increased, the overall activity of each strain showed a decreasing trend, but X1's OD value remained relatively high. 600 The value was consistently higher than that of other strains, indicating superior salt tolerance.

[0043] During fermentation, the continuous accumulation of ethanol can have a toxic effect on yeast cells, inhibiting their normal growth and ultimately leading to the termination of fermentation. Figure 3 As shown, when the ethanol volume fraction was 2%, all strains except X5 grew well. With increasing ethanol concentration, the growth of all strains was inhibited to varying degrees, but X1 showed the highest OD value. 600 Its value was consistently higher than that of other strains, demonstrating optimal ethanol tolerance.

[0044] 4. Yeast identification

[0045] As shown above, strain X1 exhibits superior aroma, ester, and gas production capabilities, as well as strong tolerance to acid, salt, and ethanol. Therefore, strain X1 was identified. The purified yeast strain was streaked onto YPD plates and cultured at 28℃ for 2 days. Colony morphology was observed and recorded. The plate cultures were then sent to Wuhan Natural Power Biotechnology Co., Ltd. for 26S rDNA sequencing. The obtained sequences were compared in the NCBI GenBank database, and a phylogenetic tree was constructed using MEGA 6.0 software to analyze strain homology and determine its taxonomic position.

[0046] Colony and cell morphology characteristics of strain X1 are as follows: Figure 4 As shown. The colonies are round, milky white, with a raised center and a smooth, moist surface. Figure 4 (a) Microscopic examination showed that its cells were oval-shaped and reproduced through budding, consistent with the morphological characteristics of typical yeast. Figure 4 (b). Phylogenetic analysis results based on 26S rDNA sequences ( Figure 4 (c) further indicates that strain X1 and Maudiozyma humilis The homology is higher than 99%, indicating that strain X1 belongs to yeast (Maudiozyma humilis).

[0047] Therefore, strain X1 was named yeast (Maudiozyma humilis) HAU2501, deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on July 21, 2025, with accession number CGMCC No. 35320.

[0048] Example 2

[0049] 1. Analysis of antagonism between yeast and lactic acid bacteria

[0050] Whether there is antagonistic interaction between yeast and lactic acid bacteria directly affects the construction of mixed bacterial systems and their subsequent fermentation performance. Therefore, clarifying the compatibility between mixed strains is of great research significance. The antagonistic effect between yeast X1 and each lactic acid bacteria strain in lactic acid bacteria combination Q (T244;T216;H3D;206) and lactic acid bacteria combination Y (6d-16;6-12;H3D) was analyzed using the agar diffusion method (Oxford cup method). Yeast was added as an indicator bacteria to the culture medium, followed by 200 μL of lactic acid bacteria fermentation broth. After culturing for 24 h, the presence or absence of inhibition zones was observed to determine the antagonistic effect.

[0051] H3D is Lactobacillus plantarum ( Lactobacillus plantarum ); 6d-16 is Lactobacillus pentosolicus ( Lactobacillus pentosus ); 206 and 6-12 are Pediococcus pentosaceus ( Pediococcus pentosaceusT244 is *Westernella esculenta* (…). Weissella cibaria T216 is Lactobacillus brevis ( ); Lactobacillus brevis Lactic acid bacteria combination Q has been published in Qin Shuangxia, Zhao Lingyan, Deng Fangming. Construction of mixed fermentation microbial community of lactic acid bacteria in fermented chili peppers [J]. China Brewing, 2024, 43(07):117-125., and lactic acid bacteria combination Y has been published in Yang Jian. Selection and aroma-producing microorganisms of traditional fermented chili peppers and study on aroma-producing mechanism [D]. Hunan Agricultural University, 2020. DOI:10.27136 / d.cnki.ghunu.2020.000666. The bacterial count of each of the above lactic acid bacteria is 1×10⁻⁶. 11 CFU / g, the mass ratio of each lactic acid bacteria in lactic acid bacteria combination Q is 1:1:1:1, and the mass ratio of each lactic acid bacteria in lactic acid bacteria combination Y is 1:1:1.

[0052] The results are shown in Table 4. In all test combinations, regardless of the type of lactic acid bacteria used as the test bacteria, no obvious inhibition zone was observed around the Oxford cup, nor was any inhibition or sparse colony growth observed. These results indicate that there is no antagonistic reaction between yeast X1 and any of the tested lactic acid bacteria, suggesting that it is suitable for compound use in terms of biocompatibility and can be used as a base strain for the subsequent construction of compound fermentation systems.

[0053] Table 4. Antagonism between yeast X1 and lactic acid bacteria

[0054] Note: "–" indicates no antagonism.

[0055] 2. Screening of fermentation strain combinations

[0056] To investigate the effect of yeast-lactic acid bacteria combination on enhancing the flavor and quality of chopped chili peppers and to screen the optimal fermentation combination, the following 7 experimental groups were set up (the bacterial count of each strain is 1×10⁻⁶). 11 CFU / g): A: The salt content is 8% (w / w), and it is naturally fermented without inoculating any microorganisms; B: Salt addition is 8% (w / w), and lactic acid bacteria combination Y is inoculated for fermentation at an inoculation amount of 0.2% (w / w). C: Salt addition is 8% (w / w), and lactic acid bacteria combination Q fermentation is inoculated at an inoculation amount of 0.2% (w / w). D: Salt content is 8% (w / w), and fermentation is carried out by inoculating yeast X1 (0.1%, w / w) + lactic acid bacteria combination Y (0.1%, w / w); E: Salt addition is 8% (w / w), fermentation is carried out with yeast X1 (0.1%, w / w) + lactic acid bacteria combination Q (0.1%, w / w); F: Salt addition is 8% (w / w), fermentation is performed using commercially available yeast A (0.1%, w / w) + lactic acid bacteria combination Y (0.1%, w / w); the commercially available yeast A is brewer's yeast (Saccharomyces cerevisiae). Saccharomyces cerevisiae CGMCC 2.1, purchased from the China General Microbiological Culture Collection Center; G: Salt addition is 8% (w / w), fermented with commercially available yeast B (0.1%, w / w) + lactic acid bacteria combination Y (0.1%, w / w); the commercially available yeast B is Pichia pastoris (… Pichia mandshurica CGMCC 2.1026, purchased from the China General Microbiological Culture Collection Center.

[0057] All samples were fermented at room temperature for 7 days. After fermentation, physicochemical indicators, sensory properties and volatile flavor compounds were analyzed, and the optimal combination of compound fermentation was determined through comprehensive evaluation.

[0058] Physicochemical index determination: Total acid content was determined according to GB 12456—2021 "National Food Safety Standard - Determination of Total Acidity in Food". Esterification capacity was determined according to GB / T10345—2007 "Analytical Methods for Baijiu". Amino acid nitrogen content was determined according to GB 5009.235—2016 "National Food Safety Standard - Determination of Amino Acid Nitrogen in Food".

[0059] Volatile component determination: Volatile components were determined using a two-dimensional gas chromatography-mass spectrometry (GC-MS) system, with slight modifications to the method described in Liu, Yuan, et al. Sensory score prediction and key aroma compounds characterization in fermented chopped pepper. Food Chemistry:X,102743(2025). The first-dimensional column was an HP-5ms UltraInert capillary column (15.0 m × 0.25 mm × 0.25 μm), and the second-dimensional column was an HP-5ms-15m+SV+2D capillary column (21.97 m × 250 μm × 0.25 μm). High-purity helium (99.99%) was used as the carrier gas at a constant flow rate of 1.0 mL / min. The injection port temperature was 250℃, and a split injection mode was employed. The column oven temperature program was as follows: initial temperature 50℃, increased to 80℃ at 4℃ / min, held for 1 min, then increased to 240℃ at 3℃ / min, held for 4 min. Mass spectrometry conditions were as follows: ion source temperature 200℃, electron ionization energy 70 eV, mass scan range set to m / z 45-350. Quantitative analysis was performed using the internal standard method, with 2-octanol as the internal standard. The content (μg / g) of the target aroma component was calculated using the following formula: Aroma component content = peak area of ​​aroma component × internal standard content / peak area of ​​internal standard.

[0060] Sensory evaluation: The sensory evaluation panel consisted of 12 evaluators with experience in sensory analysis. To minimize subjective bias, all samples were aliquoted into transparent disposable evaluation cups and blind-evaluated using three-digit random numbers. Based on the sensory evaluation criteria shown in Table 5, the samples were comprehensively evaluated from five dimensions: color, texture, crispness, taste, and aroma.

[0061] Table 5 Sensory Evaluation Criteria for Fermented Chili Peppers

[0062] Overall Product Quality Evaluation: The overall quality of the product is evaluated using the weighted comprehensive evaluation method. The specific calculation formula is as follows:

[0063] The results of total acid, total ester, amino acid nitrogen content, sensory scores and comprehensive scores for different treatment groups are shown in Table 6, and the results of volatile substances and their contents are shown in Table 7.

[0064] As shown in Table 6, the total acid content of chili peppers fermented after combining yeast X1 with lactic acid bacteria combination Y and combination Q was 0.86 g / 100 g and 0.72 g / 100 g, respectively; the total ester content was as high as 21.69 mg / g and 17.08 mg / g, respectively; and the amino acid nitrogen content was as high as 0.231 g / 100 g and 0.186 g / 100 g, respectively. The sensory scores were 85.73 and 82.63, respectively. All indicators were significantly better than other treatment groups, indicating that the combined fermentation of chili peppers with yeast X1 and lactic acid bacteria (especially combination Y) can bring unexpected effects of significantly enhancing the aroma and flavor. Other combinations of yeast and lactic acid bacteria did not produce similar effects.

[0065] Table 7 shows that a total of 130 aroma compounds were identified in the chopped chili fermented with different microbial combinations, including 37 esters, 27 alcohols, 25 alkenes, 24 alkanes, 3 aldehydes, 3 acids, 5 ketones, 5 phenols, and 1 other substance. Among them, the yeast X1 and lactic acid bacteria mixed fermentation groups (groups D and E) detected 67 and 63 volatile compounds, respectively, and the number of aroma compounds in these groups was higher than that in the natural fermentation group (group A), the pure lactic acid bacteria fermentation group (groups B and C), and the mixed fermentation group of commercially available yeast and lactic acid bacteria (groups F and G). The total volatile aroma compounds (TVCs) in the yeast X1 and lactic acid bacteria mixed fermentation groups (groups D and E) were 658.39±56.67 μg / g and 337.79±32.05 μg / g, respectively. These TVC contents were higher than those in the naturally fermented group (group A 59.62±6.74 μg / g), the pure lactic acid bacteria fermentation group (group B 123.67±11.78 μg / g, group C 75.85±10.97 μg / g), and the commercially available yeast and lactic acid bacteria mixed fermentation group (group F 40.38±10.56 μg / g, group G 30.13±6.67 μg / g). This indicates that the mixed fermentation of vegetables with yeast X1 and lactic acid bacteria can significantly enrich the variety of TVCs and increase their total TVC content.

[0066] Furthermore, the co-fermentation of vegetables with yeast X1 and lactic acid bacteria (especially lactic acid bacteria combination Y) produces a large number of new volatile aroma compounds: ethyl undecanoate 10.38±3.55μg / g, linaloate 57.17±21.24μg / g, methyl acetate 0.98±0.31μg / g, behenyl acetate 1.37±0.33μg / g, isoamyl 8-methyl-6-nonenoate 2.11±0.99μg / g, and (S)-2-hexanol 1.70± 0.12 μg / g, cis-geraniol 3.13±1.44 μg / g, geraniyllinalool 2.39±0.58 μg / g, cis-6-pentadecan-1-ol 3.13±1.26 μg / g, 4,4-dimethyl-3-(3-methyl-3-butenylidene)-2-methylenebicyclo[4.1.0]heptane 4.48±2.18 μg / g, 2-isopropyl-3-vinylethylene oxide 2.96±1.36 μg / g, 3,7-dimethyl-1,3,6-octtriene 1.06±0.15 μg / g, (E)-10-methyl-4-undecene 1.14 ±0.13 μg / g, (E)-7-tetradecene 1.64±0.73 μg / g, 4,6,8-trimethyl-1-nonene 1.24±0.41 μg / g, (Z)-14-methyl-8-hexadecenal 1.98±1.06 μg / g, glycolic acid 3.92±1.88 μg / g, 3-hydroxy-2-butanone 2.41±1.22 μg / g, 1,2-dimethylcyclohexane 60.12±20.43 μg / g, 3-methyl-5-propylnonane 1.49±0.35 μg / g, 2,6-dimethyloctadecane 1.69±0.86 μg / g.

[0067] Table 6. Effects of different strain combinations on fermented chili peppers

[0068] Table 7. Volatile substances and their contents in fermentation of different strain combinations

[0069] Note: ND indicates not detected.

[0070] Further, orthogonal partial least squares discriminant analysis (OPLS-DA) was used to distinguish the flavor profiles of chili pepper samples from different fermentation methods. The results ( Figure 5Figure a) shows that the sample points of the naturally fermented group (A) and the lactic acid bacteria single-strain fermentation group (B, C) are highly clustered, indicating that there is no significant difference in the composition of volatile flavor compounds among these three groups, suggesting that lactic acid bacteria Y and Q have similar effects on flavor regulation as single fermenting agents; while the compound fermentation group (D, E) with added yeast X1 is significantly separated from the above three groups, indicating that the participation of yeast is the key factor leading to a significant change in the flavor profile of chili peppers. The permutation test results ( Figure 5 (b) further confirms that the OPLS-DA model has good reliability and stability, providing a statistical basis for the differential analysis of volatile flavor compounds under different fermentation methods.

[0071] Using a VIP value > 1 as the screening criterion, differential aroma compounds that significantly contribute to distinguishing different fermentation methods were extracted from the model. VIP diagram ( Figure 5 As shown in c), linalool (floral and fruity aroma), methyl salicylate (wintergreen aroma), isoamyl alcohol (brandy aroma), 2-methoxy-3-isobutylpyrazine (green bell pepper and green pea aroma), isoamyl acetate (banana flavor), and phenethyl acetate (rose aroma) are the core volatile compounds that distinguish the flavor profiles of each group. These compounds exhibit significantly different distribution characteristics among the different fermentation groups, contributing the most to the differentiation of flavor profiles and being the main reason for the differentiated flavor characteristics resulting from different fermentation methods.

[0072] As can be seen from the above embodiments, the present invention has screened an excellent aroma-producing yeast strain from naturally fermented mustard greens and chopped chili pepper samples. Maudiozyma humilis (Strain X1) possesses high ester-producing activity, rapid fermentation capacity, and good tolerance to multiple stresses. When this strain was combined with lactic acid bacteria for the fermentation of chopped chili peppers, the results showed that the total acid, total ester, and amino acid nitrogen content of the mixed fermentation group were significantly higher than those of the pure lactic acid bacteria fermentation group, and the sensory scores were also significantly improved. Furthermore, its flavor profile was significantly different from both the pure lactic acid bacteria fermentation group and the naturally fermented group. Among them, the mixed fermentation group D exhibited the best quality, with significantly increased levels of key aroma components such as phenethyl acetate and phenylethanol, forming a unique flavor profile. In conclusion, strain X1 can serve as an excellent aroma-producing fermenting agent. Co-fermentation with lactic acid bacteria can effectively improve the flavor quality of chopped chili peppers, providing a theoretical basis and technical reference for flavor control of traditional fermented vegetables and the development of functional fermenting agents.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A yeast strain (Maudiozyma humilis) HAU2501, deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on July 21, 2025, with accession number CGMCC No. 35320.

2. A compound fermentation agent, characterized in that, Includes the yeast HAU2501 and lactic acid bacteria as described in claim 1.

3. The compound fermentation agent according to claim 2, characterized in that, The lactic acid bacteria are selected from one or more of the following: Lactobacillus plantarum H3D, Lactobacillus pentosus 6d-16, Pediococcus pentosus 206, Pediococcus pentosus 6-12, Weissella esculenta T244, and Lactobacillus brevis T216.

4. The compound fermentation agent according to claim 3, characterized in that, The lactic acid bacteria are a combination of *Westernella tamariscina* T244, *Lactobacillus brevis* T216, *Lactobacillus plantarum* H3D, and *Pediococcus pentosaceus* 206, with a mass ratio of 1~3:1~3:1~3:1~3, and a bacterial content of 1×10⁻⁶ for each. 11 ~1.5×10 11 CFU / g.

5. The compound fermentation agent according to claim 3, characterized in that, The lactic acid bacteria are a combination of *Lactobacillus pentosaccharide* 6d-16, *Pediococcus pentosaccharide* 6-12, and *Lactobacillus plantarum* H3D, with a mass ratio of 1~3:1~3:1~3, and a bacterial content of 1×10⁻⁶ for each strain. 11 ~1.5×10 11 CFU / g.

6. The compound fermentation agent according to claim 2, characterized in that, The mass ratio of yeast HAU2501 to lactic acid bacteria is 1~3:1~3, and the bacterial content of each is 1×10⁻⁶. 11 ~1.5×10 11 CFU / g.

7. The application of the yeast HAU2501 according to claim 1 or the compound fermentation agent according to any one of claims 2 to 6 in the preparation of fermented vegetables.

8. The application according to claim 7, characterized in that, The vegetable in question is a chili pepper.

9. A method for increasing the aroma substance content in fermented vegetables, characterized in that, The yeast HAU2501 described in claim 1 or the compound fermenting agent described in any one of claims 2 to 6 is mixed with vegetables for fermentation to obtain fermented vegetables with increased aroma content.

10. The method according to claim 9, characterized in that, The amount of yeast HAU2501 added is 0.08~0.12wt% or the amount of compound fermentation agent added is 0.15~0.25wt%.

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