Non-mycelium-producing rouxellia yeast capable of inhibiting mycelium-producing microorganisms, microbial inoculant and application thereof

Abstract

The application discloses a non-membrane-producing Rhodotorula conjugata capable of inhibiting membrane-producing microorganisms, a microbial inoculum and application, relates to the technical field of microorganisms and food fermentation, and the Rhodotorula conjugata (KDX-69) is preserved in the Guangdong Microbial Culture Collection Center on Nov. 14, 2025, and has a preservation number of GDMCC No: 67296. Zygosaccharomyces rouxii The new strain is obtained from a bean paste, does not produce membranes by itself, and can inhibit the growth of membrane-producing microorganisms; the new strain is applied to a low-salt fresh pepper bean paste fermentation process, can effectively inhibit membrane production, flower production and other spoilage and deterioration, reduce the content of biological amine in a product, improve product safety, improve product flavor, and has high application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganism and food fermentation, and particularly relates to a non-membrane-producing Zygosaccharomyces rouxii capable of inhibiting membrane-producing microorganisms, a microbial inoculum and application. BACKGROUND

[0002] Doubanjiang is a typical high-salt fermented condiment in China, and its salt content is usually between 15% and 22%. The high-osmotic environment formed by high salt is the most important means of preservation in the open fermentation process of doubanjiang, which can effectively inhibit the growth of miscellaneous bacteria and ensure the smooth progress of the fermentation process and the stability of the final product. However, when the natural "barrier" of low salt is weakened, the product is easily contaminated by spoilage microorganisms, especially the "flowering" deterioration caused by microorganisms such as Bacillus subtilis, Pichia membranifaciens and Pichia kudriavzevii is the most common and prominent. Membrane-producing microorganisms consume a large amount of nutrients such as sugars, organic acids and amino acids in the product during growth and metabolism, and form a layer of white, wrinkled bacterial membrane, which not only leads to a decrease in sensory quality, but also may produce an unpleasant odor, causing overall deterioration of the product, and even accompanied by the accumulation of harmful substances such as biogenic amines. At present, the control means of adding chemical preservatives has certain limitations, either because of the difficulty of implementation, or because it is contrary to the consumer trend of "clean label". And the existing biological control means often brings flavor interference, poor adaptability and safety risks due to the introduction of exogenous microorganisms. Therefore, there is an urgent need in the art for a new biological preservation strategy that is endogenous, adaptable to low-salt systems and safe and effective.

[0003] Zygosaccharomyces rouxii, Zygosaccharomyces rouxii as an important endogenous strain in the fermentation process of traditional fermented soy products, can be divided into membrane-producing Zygosaccharomyces rouxii and non-membrane-producing Zygosaccharomyces rouxii. Non-membrane-producing Zygosaccharomyces rouxii plays a role in flavoring during fermentation; while membrane-producing Zygosaccharomyces rouxii can form a white membrane on the surface of static sauce cake and produce a slight unpleasant odor. At present, Zygosaccharomyces rouxii is widely used in fermented foods as a flavor strain, but its application in the field of biological preservation has not been reported. Therefore, screening a new strain of non-membrane-producing Zygosaccharomyces rouxii with excellent antimicrobial and membrane-producing activity and applying it to low-salt fermented foods has important social and economic significance. SUMMARY

[0004] To solve the problems in the prior art, the present application provides a non-membrane-producing Zygosaccharomyces rouxii capable of inhibiting membrane-producing microorganisms, a microbial inoculum and application, which can isolate and screen a strain that does not form a bacterial membrane itself but can effectively inhibit membrane-producing spoilage microorganisms, thereby ensuring the smooth fermentation of condiments and improving the flavor of the product.

[0005] In order to achieve the purpose of the present application, the following scheme is adopted:

[0006] The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. Zygosaccharomyces rouxii The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism.

[0007] The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. Zygosaccharomyces rouxii The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. Zygosaccharomyces The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism.

[0008] The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. Zygosaccharomyces rouxii The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism.The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism.

[0009] The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. Zygosaccharomyces The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. rouxii The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism.

[0010] The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. Zygosaccharomyces rouxii The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism.

[0011] Specifically, the filamentous microorganism includes one or more of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus velezensis (Bacillus velezensis), Bacillus subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), Bacillus cereus (Bacillus cereus), Staphylococcus epidermidis (Staphylococcus epidermidis), Pichia membranifaciens (Pichia membranifaciens), Pichia kudriavzevii (Pichia kudriavzevii), Williopsis passalii (Williopsis passalii), Pichia farinosa (Pichia farinosa), Debaryomyces hansenii (Debaryomyces hansenii). Bacillus amyloliquefaciens Bacillus velezensis Bacillus subtilis Bacillus licheniformis Bacillus cereus Staphylococcus epidermidis Pichia membranifaciens Pichia kudriavzevii Wickerhamiella versatilis Pichia farinosa Debaryomyces hansenii The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism.

[0012] The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. Zygosaccharomyces rouxii The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism. The present application provides a non-filamentous Zygosaccharomyces rouxii (KDX-69) capable of inhibiting growth of a filamentous microorganism.

[0013] ​​​​​Specifically, for inhibiting the growth of film-producing microorganisms in low-salt fermented condiments.

[0014] Specifically, for inhibiting the growth of film-producing microorganisms in low-salt fermented condiments.

[0015] Specifically, for improving the quality of low-salt fermented condiments.

[0016] Specifically, the fermented condiments include one or more of soy sauce, bean paste, soybean paste, and douchi.

[0017] For example, in the case of bean paste, improving the quality means reducing the content of biogenic amines in low-salt fresh chili bean paste and improving the flavor of low-salt fresh chili bean paste. Specifically, it refers to reducing the content of tryptamine, phenethylamine, putrescine, histamine, tyramine, and other biogenic amines, and increasing the content of 4-ethylphenol, 4-ethylguaiacol, phenethyl alcohol, linalool, 9-octadecenoic acid ethyl ester, hexadecanoic acid methyl ester, tetradecanoic acid ethyl ester, linolenic acid methyl ester, linoleic acid ethyl ester, and ethyl palmitate in low-salt fresh chili bean paste.

[0018] The present application has the following advantages: a new strain that does not produce films itself and can inhibit the growth of film-producing microorganisms is screened from bean paste; the new strain is applied to the fermentation process of low-salt fresh chili bean paste, which can effectively inhibit the spoilage of film-producing microorganisms and other spoilage during the fermentation process of low-salt fresh chili bean paste, reduce the content of biogenic amines in the product, improve the safety of the product, and increase the content of 4-ethylphenol, 4-ethylguaiacol, phenethyl alcohol, linalool, 9-octadecenoic acid ethyl ester, hexadecanoic acid methyl ester, tetradecanoic acid ethyl ester, linolenic acid methyl ester, linoleic acid ethyl ester, and ethyl palmitate in low-salt fresh chili bean paste, thereby improving the flavor of the product, and thus having high application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The figure is the colony morphology of Zygosaccharomyces rouxii (Z. rouxii) KDX-69. Zygosaccharomyces rouxii The figure is the colony morphology of Zygosaccharomyces rouxii (Z. rouxii) KDX-69.

[0020] Figure 2 The figure is the colony morphology of Zygosaccharomyces rouxii (Z. rouxii) KDX-69. Zygosaccharomyces rouxii The figure is the colony morphology of Zygosaccharomyces rouxii (Z. rouxii) KDX-69.

[0021] Figure 3 The figure is the colony morphology of Zygosaccharomyces rouxii (Z. rouxii) KDX-69. Zygosaccharomyces rouxii The figure is the colony morphology of Zygosaccharomyces rouxii (Z. rouxii) KDX-69.

[0022] Figure 4 The figure is the colony morphology of Zygosaccharomyces rouxii (Z. rouxii) KDX-69.

[0023] Figure 5 The figure is the colony morphology of Zygosaccharomyces rouxii (Z. rouxii) KDX-69. Figure 5(A) is an ester, alcohol, phenol, aldehyde, acid, hydrocarbon, ketone compound content chart; Figure 5 (B) is a main volatile flavor substance composition chart.

[0024] Biological material preservation information: Zygosaccharomyces rouxii KDX-69 Zygosaccharomyces rouxii ) KDX-69, preserved in Guangdong Microbial Culture Collection Center, with a preservation date of November 14, 2025, and a preservation number of GDMCC No: 67296. DETAILED DESCRIPTION

[0025] Example 1

[0026] Zygosaccharomyces rouxii Zygosaccharomyces rouxii Screening of Zygosaccharomyces rouxii (KDX-69) is as follows:

[0027] 1.1 Isolation and purification

[0028] 10.0 g of soybean paste sample was weighed in 90 mL of sterile normal saline, and incubated at 30℃ with 120 r / min shaking for 2h to fully disperse the microbial cells; the supernatant was aspirated for serial gradient dilution, and 100µL of the dilution gradient was aspirated to 10 -3 ~10 -5 of the dilution liquid was spread on PDA medium; the spread plate was inverted in a 30℃±1℃ incubator for 48h; single colonies with typical yeast morphology were picked according to the colony morphology, and plate streaking purification was performed on PDA plates, which was repeated 2~3 times until a pure strain without impurities was obtained.

[0029] 1.2 Non-membrane-producing strain screening

[0030] (1) Preparation of legume simulation medium: after soaking and cooking, the cooked broad beans were pulverized into a paste with a pulverizer, and 5 times deionized water was added and mixed, and sterilized at 115℃ for 15min to prepare the legume simulation medium.

[0031] (2) Preparation of seed liquid: the isolated yeast was inoculated into 50mL of YPD medium, and incubated in a 30℃±1℃ shaking incubator at 120r / min for 48h to prepare the seed liquid.

[0032] (3) Test method: the prepared seed liquid was inoculated into 500mL of sterilized legume simulation medium, with an inoculation amount of 1×10 6 CFU / mL, and un-inoculated as a blank group; first, incubate in a 30℃±1℃ shaking incubator at 120r / min for 2d, then incubate in a general incubator at 30℃±1℃ for 5d; non-membrane-producing strains are screened by whether the simulation medium surface produces membrane or not.

[0033] 1.3 Screening of functional strains that inhibit the growth of biofilm-producing microorganisms

[0034] (1) Indicator bacteria: Bacillus amyloliquefaciens (BAM) Bacillus amyloliquefaciens SICC 1.740, Bacillus belyssus ( Bacillus velezensis SICC 1.618, Bacillus subtilis ( Bacillus subtilis SICC1.707, Bacillus licheniformis ( Bacillus licheniformis SICC 1.702, Bacillus cereus ( Bacillus cereus SICC 1.702, Staphylococcus epidermidis ( Staphylococcus epidermidis SIIA 141422, Pichia pastoris ( Pichia membranifaciens SICC 2.907, Pichia kudrica ( Pichia kudriavzevii SICC 2.991, Variable Variant Saccharomyces cerevisiae ( Wickerhamiella versatilis SICC 2.770, Pichia pastoris powder ( Pichia farinosa SICC 2.838, Hansenula polymorpha ( Debaryomyces hansenii SICC 2.926 is from the Sichuan Provincial Industrial Microbial Culture Collection Center (https: / / www.sc-sicc.org.cn / ).

[0035] (2) Preparation of indicator bacteria: Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Bacillus belesiensis ( Bacillus velezensis Bacillus subtilis ( Bacillus subtilis ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus cereus ( Bacillus cereus Staphylococcus epidermidis ( Staphylococcus epidermidis Indicator bacteria such as *Pichia pastoris* were inoculated into LB liquid medium and cultured at 37°C and 120 rpm for 12 h; *Pichia pastoris* ( Pichia membranifaciens ), Kudria zweipichia yeast ( Pichia kudriavzevii ), Variable Variant Saccharomyces cerevisiae ( Wickerhamiella versatilis Pichia pastoris (powdered) Pichia farinosa ), Hansenbali yeast ( Debaryomyces hansenii Bacillus amyloliquefaciens Bacillus velezensis Bacillus subtilis Bacillus licheniformis Bacillus cereus Staphylococcus epidermidis Pichia membranifaciens Pichia kudriavzevii Wickerhamiella versatilis Pichia farinosa Debaryomyces hansenii Bacillus amyloliquefaciens Bacillus velezensis Bacillus subtilis Bacillus licheniformis Bacillus cereus Staphylococcus epidermidis Pichia membranifaciens Pichia kudriavzevii Wickerhamiella versatilis Pichia farinosa Debaryomyces hansenii Bacillus amyloliquefaciens Bacillus velezensis Bacillus subtilis Bacillus licheniformis Bacillus cereus Staphylococcus epidermidis Pichia membranifaciens Pichia kudriavzevii Wickerhamiella versatilis Pichia farinosa Debaryomyces hansenii Bacillus amyloliquefaciens Bacillus velezensis Bacillus subtilis Bacillus licheniformis Bacillus cereus Staphylococcus epidermidis Pichia membranifaciens Pichia kudriavzevii Wickerhamiella versatilis Pichia farinosa Debaryomyces hansenii Bacillus amyloliquefaciens Bacillus velezensis Bacillus subtilis Bacillus licheniformis Bacillus cereus Staphylococcus epidermidis Pichia membranifaciens Pichia Inoculate into PD liquid medium and incubate at 30℃ and 120r / min for 24h.

[0036] (3) Seed culture preparation: The yeast strains that do not produce membranes themselves were screened above and inoculated into PD liquid culture medium and cultured at 30℃ and 120r / min for 24h.

[0037] (4) Test method: pour 10 mL of 2% agar into a flat plate and let it solidify; first place a sterile Oxford cup on it, then pour 15 mL of culture medium containing the indicator bacteria (the concentration of the indicator bacteria is 10 6 CFU / mL), remove the Oxford cup after it solidifies; add 100 μL of the seed liquid of the screened yeast strain that does not produce film itself into the well, place it at 4°C for 2 h, then place it in a 30°C incubator for 24 h, and compare the bacteriostatic ability by measuring the size of the inhibition zone.

[0038] 1.4 Experimental results

[0039] By investigating whether the screened strains produce film in the static culture in the simulated culture medium, 5 yeast strains that do not produce film themselves were screened, numbered as KDX-Y3, KDX-69, KDX-Y11, KDX-Y12 and KDX-Y15, and the inhibition ability of the 5 strains on film-producing microorganisms was further analyzed, and the results are shown in Table 1. The strain numbered KDX-69 has inhibition effect on 11 film-producing microorganisms, so the screening is successful, and the KDX-69 strain is regarded as the target strain.

[0040] Table 1: Inhibition of film-producing microorganisms

[0041]

[0042] Note: - indicates no inhibition zone; + indicates inhibition zone diameter 0 mm~4.9 mm; ++ indicates inhibition zone diameter 5.0 mm~9.9 mm; +++ indicates inhibition zone diameter 10.0 mm~22.0 mm.

[0043] Example 2

[0044] Zygozyma rouxii ( Zygosaccharomyces rouxii ) KDX-69 was identified as follows:

[0045] 2.1 Morphological identification

[0046] (1) Colony morphology observation: the strain KDX-69 purified in Example 1 was inoculated into YPD culture medium for activation, and after 48 h of shaking culture at 30°C, a small amount of bacterial liquid was taken with a disposable sterile inoculation loop and streaked on a PDA culture medium plate for incubation at 30°C for 48 h, and the colony morphology was observed.

[0047] (2) Cell morphology observation: a small amount of bacterial cells of a single colony on a YPD solid culture medium plate were evenly spread on a glass slide to make a temporary slide, and the bacterial cell morphology was observed under a 10x40 microscope.

[0048] 2.2 Molecular biology identification

[0049] The fresh bacterial liquid of the exponential growth phase of the strain KDX-69 purified in Example 1 was centrifuged to collect the bacterial cells, and the genomic DNA was extracted by using a fungal gene extraction kit; the 26S rDNA fragment was amplified by using the fungal universal primers NL1 (5'-TGCGTTGATTACGTCCCTGC) and NL4 (5'-GGTCCGTGTTTCAAGACGG); the polymerase chain reaction (PCR) amplification system (50 µL) was as follows: 2×PCR Mix 25 µL, DNA 1 µL, NL1 (100 µM) 0.1 µL, NL4 (100 µM) 0.1 µL, ddH2O 23.8 µL; the PCR reaction conditions were as follows: 94 ℃ pre-denaturation for 5 min, 94 ℃ denaturation for 30 s, 52 ℃ annealing for 45 s, 72 ℃ extension for 1 min, a total of 30 cycles, and 72 ℃ re-extension for 10 min; the amplified sample was sent to Beijing Qikexinye Biotechnology Co., Ltd. for sequencing; after sequencing, the basic local alignment search tool (BLAST) program in the national center for biotechnology information (NCBI) was used for splicing, and the data in the NCBI database were compared to determine the strain-species relationship.

[0050] 2.3 Experimental results

[0051] The colony morphology and microscopic morphological structure of the strain KDX-69 are shown in Figure 1 and Figure 2 As can be seen from the figures, the strain colony is slightly protruding, white and creamy, opaque, viscous in texture, and the surface is moist with a neat edge; the cell morphology is nearly spherical, with budding reproduction, and the diameter is 3.5 µm-8.5 µm, without the formation of pseudohyphae. The molecular biology identification results show that the full-length of the 26S rDNA gene sequence of the strain KDX-69 is 600 bp, and the gene sequence is shown in SEQ ID No: 1.

[0052] The strain KDX-69 is determined to belong to Zygoascus hermannii (Z. hermannii), Zygosaccharomyces and is named as Z. hermannii KDX-69.

[0053] Example 3

[0054] The physiological and biochemical characteristics of the Z. hermannii Zygosaccharomyces rouxii KDX-69 are as follows:

[0055] 3.1 Preparation of seed liquid

[0056] The Z. hermannii Zygosaccharomyces rouxii KDX-69 of Example 2 was inoculated into a 250 mL flask containing 50 mL of seed liquid medium, and the seed liquid was prepared by shaking culture at 28 ℃, 200 r / min for 24 h.Zygosaccharomyces rouxii KDX-69 was picked and inoculated into 50 mL of YPD medium and cultured in a shaking incubator at 30℃±1℃ and 120 r / min for 48 h to prepare seed culture.

[0057] 3.2 Experimental Methods

[0058] (1) Salt tolerance: The above seed liquid was subjected to 1×10 6 CFU / mL inoculum was inoculated into YPD liquid medium with NaCl concentrations of 0%, 4%, 8%, 12%, 16%, 18%, 20%, 22%, and 24%, respectively. The medium was incubated at 30℃ with shaking at 120 rpm for 48 h, and the optical density (OD) at 600 nm was measured. 600nm ).

[0059] (2) Temperature tolerance: The above seed liquid was subjected to a temperature of 1×10 6 CFU / mL inoculum was inoculated into YPD liquid medium and cultured at 20℃, 25℃, 30℃, 35℃, 40℃, and 45℃ with shaking at 120 rpm for 48 h. The optical density (OD) of the culture medium at 600 nm was measured. 600nm ).

[0060] (3) pH tolerance: The above seed solution was subjected to pH 1×10 6 CFU / mL inoculum was inoculated into YPD liquid medium at pH values ​​of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The medium was incubated at 30℃ with shaking at 120 rpm for 48 h, and the optical density (OD) at 600 nm was measured. 600nm ).

[0061] (4) Ethanol tolerance: The above seed liquid was treated with 1×10 6 CFU / mL inoculum was inoculated into YPD liquid medium with ethanol concentrations of 0%, 2%, 4%, 6%, 8%, and 10% (v / v), respectively. The medium was incubated at 30℃ with shaking at 120 rpm for 48 h, and the optical density (OD) at 600 nm was measured. 600nm ).

[0062] 3.3 Experimental Results

[0063] Luvian conjugated yeast ( Zygosaccharomyces rouxii The physiological and biochemical characteristics of KDX-69 are shown in Table 2. The growth temperature range of this bacterium is 25℃~40℃, the NaCl tolerance range is 0%~22%, the pH tolerance range is 3.5~8.0, and the ethanol tolerance range is 0%~6% (v / v).

[0064] Table 2 Statistical Table of Physiological and Biochemical Characteristics

[0065]

[0066] Example 4

[0067] Low-salt fermentation system of *Lourdesca leuciscus* ( Zygosaccharomyces rouxii The effects of KDX-69 on the growth of biofilm-producing microorganisms are as follows:

[0068] 4.1 Seed liquid preparation

[0069] (1) Luvian conjugated yeast ( Zygosaccharomyces rouxii KDX-69 seed culture: The KDX-69 seed culture from Example 2 was used to prepare the KDX-69 seed culture. Zygosaccharomyces rouxii KDX-69 was picked and inoculated into 50 mL of YPD medium and cultured in a shaking incubator at 30℃±1℃ and 120 r / min for 48 h to prepare seed culture.

[0070] (2) Seed culture of film-producing microorganisms: Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Bacillus belesiensis ( Bacillus velezensis Bacillus subtilis ( Bacillus subtilis ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus cereus ( Bacillus cereus Staphylococcus epidermidis ( Staphylococcus epidermidis Indicator bacteria such as *Pichia pastoris* were inoculated into LB liquid medium and cultured at 37°C and 120 rpm for 12 h; *Pichia pastoris* ( Pichia membranifaciens ), Kudria zweipichia yeast ( Pichia kudriavzevii ), Variable Variant Saccharomyces cerevisiae ( Wickerhamiella versatilis Pichia pastoris (powdered) Pichia farinosa ), Hansenbali yeast ( Debaryomyces hansenii Inoculate into PD liquid medium and incubate at 30℃ and 120r / min for 24h.

[0071] 4.2 Preparation of low-salt simulated culture medium

[0072] A fermented soybean paste sample was mixed with deionized water at a mass ratio of 1:2 after one month of fermentation. The mixture was boiled for 10 minutes, filtered through gauze, and the filtrate was collected. NaCl was added to the filtrate until the NaCl mass concentration was 10%. The mixture was then sterilized at 115°C for 15 minutes.

[0073] 4.3 Experimental Methods

[0074] Using Transwell chambers to conjugate Rhus fasciculata ( Zygosaccharomyces rouxiiKDX-69 and film-forming microorganisms were respectively separated and co-cultured, realizing the exchange between metabolites without physical contact between strains.

[0075] (1) Experimental group: The low-salt simulated medium was loaded into the inner and outer chambers of the Transwell chamber, and the activated Zygosaccharomyces rouxii (Z. rouxii) was inoculated into the inner chamber of the Transwell chamber at an inoculation amount of 2%, and the activated seed liquid of the film-forming microorganism was inoculated into the outer chamber of the Transwell chamber at an inoculation amount of 2%. After co-culturing at 30°C for 48 h, the traditional culture technique was used to count the film-forming microorganisms, and the influence of Z. rouxii (KDX-69) on the growth of the film-forming microorganisms was investigated. Zygosaccharomyces rouxii ) KDX-69 on the growth of the film-forming microorganisms, it can be seen that Z. rouxii (KDX-69) has a good inhibitory effect on the 11 strains of film-forming microorganisms in the co-culture, and the inhibitory effect on the film-forming bacteria is better than that on the film-forming yeasts. Compared with the control group, the co-culture of Z. rouxii (KDX-69) in the experimental group makes the number of film-forming microorganisms decrease by 29.63% to 81.37%, among which Bacillus amyloliquefaciens (B. amyloliquefaciens), Bacillus velezensis (B. velezensis), Bacillus subtilis (B. subtilis), Bacillus licheniformis (B. licheniformis), Bacillus cereus (B. cereus), Staphylococcus epidermidis (S. epidermidis), Pichia membranifaciens (P. membranifaciens), Pichia kudriavzevii (P. kudriavzevii), Wickerhamomyces variabilis (W. variabilis), Pichia powdery (P. powdery) and Hanseniaspora debaryi (H. debaryi) decrease by 72.4%, 56.1%, 66.48%, 56.7%, 72.76%, 81.37%, 29.63%, 43.69%, 28.38%, 60.36% and 41.39%, respectively. Zygosaccharomyces rouxii

[0076] (2) Control group: The same film-forming microorganism was inoculated into the inner chamber of the Transwell chamber as the outer chamber, and the other conditions were the same as those of the experimental group.

[0077] 4.4 Experimental results

[0078] The influence of Z. rouxii (KDX-69) on the growth of the film-forming microorganisms was investigated, and it can be seen that Z. rouxii (KDX-69) has a good inhibitory effect on the 11 strains of film-forming microorganisms in the co-culture, and the inhibitory effect on the film-forming bacteria is better than that on the film-forming yeasts. Compared with the control group, the co-culture of Z. rouxii (KDX-69) in the experimental group makes the number of film-forming microorganisms decrease by 29.63% to 81.37%, among which Bacillus amyloliquefaciens (B. amyloliquefaciens), Bacillus velezensis (B. velezensis), Bacillus subtilis (B. subtilis), Bacillus licheniformis (B. licheniformis), Bacillus cereus (B. cereus), Staphylococcus epidermidis (S. epidermidis), Pichia membranifaciens (P. membranifaciens), Pichia kudriavzevii (P. kudriavzevii), Wickerhamomyces variabilis (W. variabilis), Pichia powdery (P. powdery) and Hanseniaspora debaryi (H. debaryi) decrease by 72.4%, 56.1%, 66.48%, 56.7%, 72.76%, 81.37%, 29.63%, 43.69%, 28.38%, 60.36% and 41.39%, respectively. Zygosaccharomyces rouxii Figure 3 Zygosaccharomyces rouxii Zygosaccharomyces rouxii Bacillus amyloliquefaciens Bacillus velezensis Bacillus subtilis Bacillus licheniformis Bacillus cereus Staphylococcus epidermidis Pichia membranifaciens Pichia kudriavzevii Wickerhamiella versatilis Pichia farinosa Debaryomyces hansenii ​​​​​​​​​​​​​​​​

[0079] Example 5

[0080] Zygosaccharomyces rouxii Zygosaccharomyces rouxii The application of KDX-69 in low-salt fresh chili and broad bean sauce is as follows:

[0081] 5.1 Experimental method

[0082] (1) Preparation of broad bean sauce starter

[0083] After the broad beans were selected, impurities were removed, and soaked, they were blanched in boiling water for 3 min, then immediately cooled to 35-40°C, mixed with flour at a mass ratio of 8:2, inoculated with 0.03% Aspergillus oryzae spores (based on the total weight of broad beans and flour), and incubated at 30°C for 45 h, with timely turning of the starter every 12 h until the surface of the starter was covered with yellow-green mycelium, which was the broad bean sauce starter.

[0084] The inoculated Aspergillus oryzae in this example was KDX-66, with the taxonomic name Aspergillus oryzae , preserved in the Guangdong Microbial Culture Collection Center on November 14, 2025, with the preservation number GDMCC No: 67295. However, it should be noted that the Aspergillus oryzae spores inoculated during the preparation of the broad bean sauce starter are not the only limitation.

[0085] (2) Experimental group of low-salt fresh chili and broad bean sauce

[0086] First, fresh red chilies were washed, impurities were removed, and crushed, then mixed with the broad bean sauce starter at a mass ratio of 3:7, and 10% salt was added (based on the total mass of raw materials); then the mixed sauce mash was transferred to a clean pottery jar for natural fermentation, and the mash was stirred every 2-3 days during the fermentation process; finally, when the fermentation time was 20d, KDX-69 was inoculated at a concentration of 1x10 6 CFU / g sauce mash, and the fermentation was continued for about 90d to obtain low-salt fresh chili and broad bean sauce. Zygosaccharomyces rouxii

[0087] (3) Control group of low-salt fresh chili and broad bean sauce

[0088] The difference between the control group and the experimental group was that KDX-69 was not inoculated during the fermentation process, and the rest of the fermentation conditions were exactly the same. Zygosaccharomyces rouxii

[0089] 5.2 Index analysis

[0090] (1) Sensory quality analysis of low-salt fresh chili and broad bean sauce

[0091] ​​The sensory analysis of low-salt fresh chili pepper and bean sauce was performed by 12 professional sensory evaluation personnel on the aroma, flower aroma, fruit aroma, mellow flavor, spiciness, sourness and overall acceptance. The scoring was performed using a 5-point scale (0 indicating no sensation, 5 indicating very strong sensation), and the higher the score, the higher the preference. In addition, the production of film and flower during the fermentation process of low-salt fresh chili pepper and bean sauce was recorded.

[0092] (2) Volatile flavor substance detection

[0093] The volatile flavor components in low-salt fresh chili pepper and bean sauce were detected by headspace solid phase microextraction combined with gas chromatography-mass spectrometry. The specific operation is as follows:

[0094] Sample detection: accurately take 2.0 g of sample into a 15 mL headspace sample bottle, add 5 μL of internal standard (4-methyl-2-pentanol solution, concentration is 0.5 μg / mL), put the sample bottle into a 60 ℃ water bath for preheating for 2 min, then insert the aged SPME extraction head into the sample bottle, extract at 60 ℃ constant temperature for 50 min, then pull out the extraction head and insert it into the GC-MS gas chromatography inlet, analyze at 250 ℃ for 5 min, each sample is independently determined for 3 times.

[0095] Gas chromatography conditions: DB-WAX capillary column (60 m x 0.25 mm, 0.25 μm); carrier gas is helium, flow rate is 1 mL / min; inlet temperature is 250 ℃; no split injection; temperature program: initial temperature is 50 ℃, increase to 85 ℃ at 10 ℃ / min (hold for 1.5 min), then increase to 100 ℃ at 5 ℃ / min (hold for 1 min), increase to 175 ℃ at 2.5 ℃ / min (hold for 1.5 min), finally increase to 250 ℃ / min at 10 ℃ / min.

[0096] Mass spectrometry conditions: EI ion source, electron impact energy 70 eV; ion source temperature 230 ℃; interface 250 ℃; mass scan range 35 amu~350 amu; detector voltage 0.1 kv; tuning file stuneu; scan mode scan.

[0097] Qualitative and quantitative analysis: the chromatogram obtained by GC-MS is compared and searched in the standard spectrum library NIST11 by computer, the substances with similarity (SI)>80 (maximum value is 100) are selected for qualitative analysis, and each volatile component is accurately identified, at the same time, 4-methyl-2-pentanol (0.5 μg / mL) is used as internal standard for semi-quantitative analysis, and the mass concentration of each component is obtained.

[0098] (3) Biogenic amine detection

[0099] Sample extraction: Accurately weigh 10g of the ground sample into a 50mL centrifuge tube, add 1.25mL of 1.0mg / mL internal standard solution (1,7-diaminoheptane) and 15mL of 5% trichloroacetic acid solution, mix evenly with the sample, and extract by shaking at room temperature for 30min. Centrifuge at 8000r / min for 10min, and inject the supernatant into a 50mL volumetric flask. Perform a second extraction on the treated sample using the same method, combine the supernatants from the two extractions, and dilute to the mark with 5% trichloroacetic acid solution.

[0100] Sample derivatization: Take 1.0 mL of supernatant, add 200 µL of 2 mol / L sodium hydroxide, 300 µL of saturated sodium bicarbonate solution, and 2.0 mL of 10 mg / mL dansyl chloride solution in sequence, vortex to mix for 1 min, derivatize at 40 °C in the dark for 60 min, add 200 µL of 100 mg / mL proline solution, vortex for 1 min, and then place at room temperature in the dark for 15 min. Add 0.4 g of NaCl, vortex until the NaCl is completely dissolved, add 1 mL of diethyl ether, vortex for 30 s, allow to stand for separation, aspirate the upper organic phase, extract once more with 1 mL of diethyl ether, combine the two organic phases, evaporate to dryness in a 40 °C water bath, dissolve the residue with 1.0 mL of acetonitrile, filter through a 0.22 µm organic filter, and store the filtrate at 4 °C in the dark for later use.

[0101] Detection and analysis: The chromatographic column was a C18 column (250 mm × 4.6 mm, 5 μm), with a UV detection wavelength of 254 nm, an injection volume of 20 µL, and a column temperature of 35 ℃. Mobile phase A was 0.01 mol / L ammonium acetate solution (containing 0.1% acetic acid) - acetonitrile = (10:90, V / V), and mobile phase B was 0.01 mol / L ammonium acetate solution (containing 0.1% acetic acid) - acetonitrile = (90:10, V / V). The flow rate was 0.8 mL / min. The elution program was as follows: 0 min ~ 22 min, 60% ~ 85% mobile phase A; 22 min ~ 25 min, 85% ~ 100% mobile phase A; 25 min ~ 32 min, 100% mobile phase A; 32 min ~ 32.1 min, 100% ~ 60% mobile phase A; 32.1 min ~ 37 min, 60% mobile phase A.

[0102] 5.3 Experimental Results

[0103] Film formation during the fermentation of low-salt fresh chili broad beans, as follows Figure 4 As shown, the experimental group of low-salt fresh pepper broad beans was inoculated with Rhus zygosaccharidus ( Zygosaccharomyces rouxii After inoculation with KDX-69, no film formation or flowering occurred during fermentation; however, the control group showed severe film formation and flowering during fermentation. Sensory evaluation personnel conducted sensory evaluations on the low-salt fresh pepper broad beans of the experimental and control groups, and the results are shown in Table 3. Compared with the control group, the experimental group inoculated with KDX-69 showed significantly higher levels of film formation and flowering.Zygosaccharomyces rouxii After using KDX-69, the aroma of soy sauce, floral, fruity, and mellow flavors and overall acceptability of low-salt fresh chili bean paste are significantly improved, while the spiciness and sourness of the product are reduced.

[0104] Table 3. Sensory Score Statistics of Low-Salt Fresh Pepper Fermented Beans

[0105]

[0106] Furthermore, headspace solid-phase microextraction combined with gas chromatography-mass spectrometry was used to analyze the volatile flavor components of low-salt fresh chili bean paste. The results are as follows: Figure 5 As shown in Figure A, the experimental group was inoculated with *Gnaphalium rayum* (a type of yeast). Zygosaccharomyces rouxii KDX-69 significantly increased the content of volatile flavor compounds in low-salt fresh chili bean paste, especially ester compounds; followed by phenolic compounds and alcohol compounds. Figure 5 B shows the composition of the main volatile flavor compounds (≥1µg / g) in low-salt fresh chili bean paste. The main compounds, including 4-ethylphenol, 4-ethylguaiacol, phenethyl alcohol, linalool, ethyl 9-octadecenoate, methyl palmitate, ethyl myristate, methyl linoleate, ethyl linoleate, and ethyl palmitate, showed a significant increase in content, all exceeding 10µg / g. Furthermore, the total biogenic amine content in the experimental group (190.56mg / kg ± 3.53mg / kg) was significantly lower than that in the control group (528.03mg / kg ± 20.64mg / kg), especially tryptophan, phenylethylamine, putrescine, histamine, and tyramine, which decreased substantially (see Table 4).

[0107] Table 4. Statistical table of biogenic amine content in low-salt fresh chili broad beans

[0108]

[0109] In summary, *L. rouxii* (a type of yeast) Zygosaccharomyces rouxii KDX-69 enhanced fermentation low-salt fresh pepper bean paste can prevent the formation of film, deterioration and spoilage during fermentation, and improve the flavor and quality of the product.

[0110] The above embodiments are only used to illustrate the technical concept 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 protection scope of the present invention.

Claims

1. A non-filamentous Zygozyma sp. KDX-69 which can inhibit a filamentous microorganism, characterized in that, The taxonomic name is Zygosaccharomyces rouxii , and was preserved in Guangdong Microbial Culture Collection Center on November 14, 2025, with the preservation number GDMCC No: 67296.

2. A microbial inoculant, characterized in that, Zygosaccharomyces rouxii as claimed in claim 1.

3. Use of a Zygosaccharomyces rouxii according to claim 1, characterized in that, For inhibiting the growth of film-producing microorganisms, including one or more of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bacillus amyloliquefaciens ), Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ), Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ), Bacillus licheniformis (Bacillus licheniformis) Bacillus licheniformis ), Bacillus cereus (Bacillus cereus) Bacillus cereus ), Staphylococcus epidermidis (Staphylococcus epidermidis) Staphylococcus epidermidis ), Pichia membranifaciens (Pichia membranifaciens) Pichia membranifaciens ), Pichia kudriavzevii (Pichia kudriavzevii) Pichia kudriavzevii ), Wickerhamomyces anomalus (Wickerhamomyces anomalus) Wickerhamiella versatilis ), Pichia powdery (Pichia powdery) Pichia farinosa ), Debaryomyces hansenii (Debaryomyces hansenii) Debaryomyces hansenii ).

4. Use of the Zygosaccharomyces rouxii according to claim 1, characterized in that, For preparing low-salt fermented condiment.

5. Use of a Zygosaccharomyces rouxii according to claim 4, characterized in that, The fermented condiment includes one or more of soy sauce, bean sauce, soybean paste, and fermented soybean.

6. Use of a Zygosaccharomyces rouxii according to claim 4, characterized in that, For inhibiting film-forming and flower production during fermentation of low-salt fermented condiment.

7. Use of the Zygosaccharomyces rouxii according to claim 4, characterized in that, For reducing the content of biogenic amines in low-salt fermented condiment and increasing the content of one or more of 4-ethylphenol, 4-ethylguaiacol, phenylethanol, linalool, ethyl 9-octadecenoate, methyl hexadecanoate, ethyl tetradecanoate, methyl linolenate, ethyl linoleate, and ethyl palmitate.

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