Saccharomyces cerevisiae Y-7 strain, complex starter culture prepared from the strain and application of the starter culture in low-salt fermented sausages

Abstract

This invention relates to a strain of brewing yeast ( Saccharomyces cerevisiae This invention discloses a yeast strain Y-7 and a compound starter culture comprising *Lactobacillus plantarum* Z43 and *Saccharomyces cerevisiae* Y-7, wherein the effective viable cell ratio of *Lactobacillus plantarum* Z43 to *Saccharomyces cerevisiae* Y-7 is 10:1 or 20:1. The *Saccharomyces cerevisiae* Y-7 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25587. This invention also provides the application of the above-mentioned compound starter culture in low-salt fermented sausages, which has the advantages of a short fermentation cycle, high safety, and high sensory evaluation.

Description

Technical Field

[0001] This invention relates to the field of microbial and meat fermentation, specifically to Saccharomyces cerevisiae Y-7 and a compound fermenting agent made from this strain, and the application of this fermenting agent in low-salt fermented sausages. Background Technology

[0002] Fermented sausages are a traditional fermented meat product, widely loved by consumers. With increasing awareness of health and food safety, low-salt fermented sausages are gaining popularity. However, reducing salt content can significantly impact product flavor and safety. Lactic acid bacteria can lower pH levels by producing acid, inhibiting the growth of spoilage microorganisms, improving product safety and quality stability, and ensuring product quality. This is a key microbial strain developed for meat fermentation. In existing technologies, Chinese patent application CN202110659527.5, "A strain of Debaryo myces hansenii with good fermentation performance and aroma-producing function and its screening method," applies Debaryo myces SH4 to fermented sausages. Chinese patent application CN201910480536.0, "A fermented sausage containing a compound of Saccharomyces ruvidii and Lactobacillus plantarum," uses a compound of Saccharomyces ruvidii and Lactobacillus plantarum for fermentation. However, existing fermented sausages are produced at relatively low temperatures, with long processing times, and generally high salt content. With increasing health awareness and a greater pursuit of flavor, existing technologies, such as those mentioned above, have issues with high salt content, long fermentation cycles, and safety in the fermentation process of fermented sausages, which need further improvement. Summary of the Invention

[0003] To overcome the shortcomings of the prior art, the present invention provides a brewing yeast strain Y-7 and a compound fermenting agent ZY made from the strain, which can improve the flavor of fermented sausages and reduce the salt content of fermented sausages, as well as the application of the fermenting agent in low-salt fermented sausages. The fermentation cycle is short, the safety is high, and the sensory score is high.

[0004] The technical solution adopted in this invention is: This invention discloses a strain of Saccharomyces cerevisiae Y-7, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25587.

[0005] This invention discloses a compound fermentation agent comprising *Lactobacillus plantarum* Z43 and *Saccharomyces cerevisiae* Y-7; the *Lactobacillus plantarum* Z43 is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 23962. Further, the effective viable cell ratio of the *Lactobacillus plantarum* Z43 and *Saccharomyces cerevisiae* Y-7 is 10:1 or 20:1. Further, the preparation method includes the following steps: (1) The activated Lactobacillus plantarum Z43 strain was inoculated into MRS liquid medium and cultured at 37℃ for 12h to obtain Lactobacillus plantarum Z43 fermentation broth; Saccharomyces cerevisiae Y-7 was inoculated into PDA liquid medium and cultured at 37℃ for 24h at 150r / min to obtain Saccharomyces cerevisiae Y-7 fermentation broth. (2) Centrifuge the fermentation broth obtained in step (1) at 8000 r / min for 5 min at 4℃, and collect the precipitated bacterial cells. After washing three times with physiological saline, resuspend in physiological saline and adjust the bacterial concentration to 10. 9 CFU / mL, respectively obtained Lactobacillus plantarum Z43 bacterial culture and Y-7 brewer's yeast bacterial culture; (3) Mix the Lactobacillus plantarum Z43 bacterial solution and Saccharomyces cerevisiae Y-7 bacterial solution obtained in step (2) in a certain proportion.

[0006] Further, in step (1), the concentration of the *Lactobacillus plantarum* Z43 strain inoculated into the MRS liquid medium is 10. 7 cfu / mL; the concentration of the Saccharomyces cerevisiae Y-7 inoculated into PDA liquid medium was 10. 7 CFU / mL.

[0007] Further, in step (1), the MRS liquid culture medium comprises the following components at the following concentrations: peptone 10.0 g / L, beef meal 8.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, Tween 80 1.0 g / L, sodium acetate 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, and manganese sulfate 0.05 g / L. The pH of the MRS liquid culture medium is 6.2 ± 0.1, and it is sterilized at 121°C for 20 min. The PDA liquid culture medium comprises the following components at the following concentrations: potato extract powder 3.0 g / L and glucose 20.0 g / L. The pH of the PDA liquid culture medium is 5.6 ± 0.2, and it is sterilized at 121°C for 20 min.

[0008] Furthermore, in step (2), when washing with physiological saline, each time an equal volume of physiological saline is used to wash the precipitated bacteria.

[0009] The present invention also provides the application of the above-mentioned compound fermentation agent in low-salt fermented sausage.

[0010] Furthermore, in the low-sodium fermented sausage, the amount of low-sodium salt added is 1.5%. The sodium content of the low-sodium salt is 29505 mg / 100g.

[0011] Furthermore, the compound fermentation agent is mixed with the meat and inoculated with the compound fermentation agent before sausage stuffing. At the time of inoculation, the amount of *Lactobacillus plantarum* Z43 added is 10... 7 The effective viable cell ratio of Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7 was 10:1 or 20:1; fermentation was carried out at 37°C for 3 days after inoculation.

[0012] The beneficial effects of this invention are: (1) The *Lactobacillus plantarum* Z43 of this invention was screened from air-dried Cantonese sausage, which is derived from fermented meat products, has high safety, and is adapted to the sausage fermentation environment. The *Saccharomyces cerevisiae* Y-7 of this invention was isolated from traditional sourdough, and its optimal growth temperature is 37°C.

[0013] (2) Inoculation with Lactobacillus plantarum Z43 of the present invention can grow rapidly, produce antibacterial substances rapidly, produce acid rapidly to reduce the pH value of the product, inhibit the growth of spoilage microorganisms, and ensure the safety of fermented sausage products.

[0014] (3) The low-salt fermented sausage made with the compound fermentation agent ZY of the present invention has high safety and high sensory score.

[0015] (4) This invention combines plant lactus Z43 with brewing yeast Y-7 to develop a high-quality meat product fermentation agent for the production of low-salt fermented sausages, which makes up for the defects in flavor of low-salt fermented sausage products, increases consumer acceptance, and has broad application prospects in the fermentation of low-salt meat products.

[0016] (5) The present invention effectively shortens the production cycle of fermented sausage, and fermentation can be completed in 3 days.

[0017] (6) The fermented sausage products using the present invention have a lower salt content, with a low sodium salt addition of only 1.5%. Attached Figure Description

[0018] Figure 1 The effect of different temperatures on the growth of strain Y-7; Figure 2 The effect of different pH values ​​on the growth of strain Y-7; Figure 3 Effects of different NaCl concentrations on the growth of strain Y-7; Figure 4 Morphology of strain Y-7; Figure 5 Sensory evaluation results of fermented sausage samples; Figure 6 pH values ​​of fermented sausage samples; Figure 7 Results of volatile basic nitrogen determination in fermented sausage samples. Detailed Implementation

[0019] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0020] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit this application or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps described in these embodiments do not limit the scope of this application. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0023] The Saccharomyces cerevisiae Y-7 strain provided by this invention was deposited on August 24, 2022, at the China General Microbiological Culture Collection Center (CGMCC No. 25587; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postcode: 100101), with the accession number CGMCC NO. Saccharomyces cerevisiae Y-7, i.e., Saccharomyces cerevisiae Y-7.

[0024] The *Lactobacillus plantarum* strain Z43 provided by this invention was deposited on November 24, 2021, at the China General Microbiological Culture Collection Center (CGMCC No. 23962; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postcode: 100101), with the accession number CGMCC NO. Lactobacillus plantarum Z43.

[0025] The present invention also provides a compound fermentation agent formed by combining the above two strains, including Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7, wherein the effective live cell ratio of Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7 is 10:1 or 20:1.

[0026] The combination of two strains of bacteria is a low-salt fermented sausage starter with great development potential. Low-salt fermented sausages made using the compound starter ZY of this invention have a pure flavor, a moderate saltiness, firm and elastic filling, a rose-red cut surface, and are safe and healthy.

[0027] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. Pork hind leg meat: purchased from Beiguo Supermarket Yizhuang Store; casings (Yigeng Yiyun Salted Pork Casings, Hebei Heqing Casing Co., Ltd.); salt (Haijing Balanced Low Sodium Salt, sodium content 29505mg / 100g, Huanghua Tongbao Special Salt Co., Ltd.); white sugar (Qi Shen Brand Grade 1 White Sugar, Jilin Qi Shen Food Co., Ltd.); monosodium glutamate (Lotus Health Monosodium Glutamate, Lotus Health Industry Group Co., Ltd.); meat product color protectant (Kangquan High-Efficiency Meat Product Color Protectant, Dalian Guotai Food Co., Ltd.). (Liability Company), Thirteen Spices (Wang Shouyi Thirteen Spices Seasoning Group Co., Ltd., Zhumadian City), Starch Powder (Haoshihui Edible Corn Starch, Beijing Haoshihui Seasoning Technology Co., Ltd.). In the quantitative experiments described below, three replicates were conducted, and the average value of the results was taken.

[0028] Example 1: Screening of yeast Y-7 I. Strain Screening 1. Weigh 25g of traditional sourdough and add it to 225mL of sterile physiological saline. Dilute it 10 times with sterile physiological saline and then spread 200μL of the diluted solution onto PDA medium plates. Incubate at 28℃ for 48h.

[0029] 2. After completing step 1, pick a single colony that matches the morphology of yeast colonies from the PDA medium plate, and repeatedly isolate and purify it until pure colonies are obtained.

[0030] 3. The strains purified in step 2 were screened, and colonies with a wine-like aroma and fast growth were selected. Further growth characteristic experiments were conducted to screen a strain that could adapt to the fermentation conditions of Lactobacillus plantarum Z43 in sausages, and it was named strain Y-7. 4. Growth characteristics of strain Y-7 4.1 Strains Y-7 were inoculated into PDA liquid medium and cultured at 150 r / min for 24 h at different temperatures. OD600 was measured to investigate the effect of different temperatures on the growth of this strain. Figure 1 This strain grows well in environments ranging from 27°C to 37°C, with optimal growth occurring at 37°C.

[0031] 4.2 Strains Y-7 were inoculated into PDA liquid medium with different pH values ​​and cultured at 37℃ and 150 r / min for 24 h. OD600 was measured to investigate the effect of different pH values ​​on the growth of this strain. Figure 2 .Depend on Figure 2 It can be seen that different pH levels have little effect on the growth of strain Y-7, and it can grow well under culture conditions of pH 4-8.

[0032] 4.3 Strains Y-7 were inoculated into PDA liquid medium with different NaCl concentrations and cultured at 37℃ and 150 r / min for 24 h. OD600 was measured to investigate the effect of different NaCl concentrations on the growth of this strain. Figure 3 .Depend on Figure 3 It can be seen that low NaCl concentration has little effect on the growth of strain Y-7, while the growth of strain Y-7 is significantly affected as the NaCl concentration increases.

[0033] The results above show that the optimal growth temperature for this strain is 37℃, which coincides with the optimal growth temperature of lactic acid bacteria, especially *Lactobacillus plantarum* Z43, facilitating the production of fermented sausages using the two strains. Furthermore, strain Y-7 can adapt to the slightly acidic environment required for lactic acid bacteria growth, and its growth is not affected by the pH decrease caused by acid production. Although the growth of this strain is somewhat affected by high NaCl concentrations, low concentrations of salt have a relatively small impact on its growth, making it suitable for low-salt fermented sausage production.

[0034] II. Morphological and Molecular Identification of Strain Y-7 1. Identification of the strain: Individual morphology: oval, lemon-shaped, see Figure 4 .

[0035] Colony morphology: Round, white, moist colonies, opaque, smooth surface, with a weak aroma, and a colony diameter of about 2 mm.

[0036] Molecular biological identification: Total DNA was extracted from strain Y-7 using a yeast genomic DNA extraction kit (TIANGEN). The 26S rDNA sequence of strain Y-7 was amplified using universal primers NL1 and NL4 and sequenced. The sequencing results are shown in Sequence 1 of the sequence listing. BLAST alignment results showed that the 26S rDNA of strain Y-7 had more than 99% similarity to multiple Saccharomyces cerevisiae strains in the NCBI database.

[0037] Based on the above identification, strain Y-7 can be determined to belong to Saccharomyces cerevisiae, therefore it has been renamed Saccharomyces cerevisiae Y-7.

[0038] Example 2: Preparation of a flavorful, low-salt fermented sausage 1. Preparation of compound fermentation agent ZY Lactobacillus plantarum Z43 was inoculated into MRS liquid medium (final concentration 10). 7 CFU / mL, inoculation ratio is Lactobacillus plantarum Z43 fermentation broth was obtained by incubating at 37℃ for 12 h with 1% (1%). Saccharomyces cerevisiae Y-7 was inoculated into PDA liquid medium (final concentration 10%). 7 CFU / mL (inoculation ratio of 1%), cultured at 150 r / min at 37℃ for 24 h to obtain Saccharomyces cerevisiae Y-7 fermentation broth; the fermentation broths of Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7 were centrifuged at 8000 r / min for 5 min at 4℃. The precipitated cells were washed with an equal volume of physiological saline to the corresponding fermentation broth each time, that is, an equal volume of physiological saline to the fermentation broth obtained in step (1) each time, for a total of 3 washes, resuspended with physiological saline, and the bacterial concentration was adjusted to 10. 9CFU / mL were used to obtain Lactobacillus plantarum Z43 bacterial suspension and Saccharomyces cerevisiae Y-7 bacterial suspension, respectively. The Lactobacillus plantarum Z43 bacterial suspension and Saccharomyces cerevisiae Y-7 bacterial suspension were then mixed in different proportions to obtain the compound fermentation agent ZY.

[0039] Percentage of effective live bacteria: Compound fermentation agent ZY-1, Lactobacillus plantarum Z43: Saccharomyces cerevisiae Y-7 = 5:1; Compound fermentation agent ZY-2, Lactobacillus plantarum Z43: Saccharomyces cerevisiae Y-7 = 10:1; The compound fermentation agent ZY-3 contains Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7 in a ratio of 20:1.

[0040] 2. Sausage making recipe Selected pork hind leg meat, with 70% lean meat and 30% fat. Seasonings: 1.5% white sugar, 1.5% Haijing balanced low-sodium salt, 0.3% MSG, 0.4% starch, 0.2% meat product color protectant, and 0.25% thirteen-spice powder.

[0041] 3. Fermented sausage preparation steps Raw meat → minced fat and lean meat → seasonings added and mixed evenly → fermentation agent added and mixed evenly → sausage stuffing → fermentation (37℃±1℃, 72h) → finished product; 4. Quality evaluation of fermented sausages (1) Sensory evaluation of fermented sausage samples Sensory evaluation was conducted by a sensory evaluation team of 10 food professionals (5 men and 5 women), whose members were trained according to GB / T 22210-2008 "Sensory Evaluation Standard for Meat and Meat Products". There were five groups of samples, numbered ZY-1, ZY-2, ZY-3, CK1, and CK2 respectively. Sample ZY-1 came from the fermented sausage group inoculated with compound starter culture ZY-1; sample ZY-2 came from the fermented sausage group inoculated with compound starter culture ZY-2; sample ZY-3 came from the fermented sausage group inoculated with compound starter culture ZY-3; sample CK1 came from the control group without starter culture; and sample CK2 came from the fermented sausage group inoculated with *Lactobacillus plantarum* Z43 (at a concentration of 10 μL). 7 CFU / g).

[0042] (In ZY-1, ZY-2, and ZY-3, the amount of Lactobacillus plantarum Z43 added during inoculation was 10.) 7 CFU / g).

[0043] Samples were randomly numbered. Evaluation panel members were required to score all samples. When evaluating different samples, members were instructed to rinse their mouths with warm water to minimize the influence of different samples, and evaluations were conducted without communication between members. A double-blind method was used for testing. The main evaluation criteria were color, odor, texture, and taste. The sensory evaluation standards are shown in Table 1. The sensory evaluation results of the fermented sausage samples are shown below. Figure 5 .

[0044] Table 1 Sensory Evaluation Criteria

[0045] like Figure 5 As shown in the sensory evaluation results, considering all sensory indicators of the fermented sausage, the 10 evaluators... The researchers gave higher ratings to the groups inoculated with compound fermentation agents ZY-2 and ZY-3, believing that the overall acceptability of the ZY-2 and ZY-3 groups was better than the other three groups. Among them, the ZY-3 group had the best sensory evaluation, with the fermented sausages having a brighter color, a denser texture, better slicing properties, a purer taste, and a fuller aftertaste.

[0046] Inoculation with ZY-2 and ZY-3 compound fermentation agents increased the fermentation aroma and reduced the sourness, resulting in a richer aroma profile and showcasing the unique flavor profile of the mellow fermented sausage. The compound fermentation agents ZY-2 and ZY-3 demonstrated excellent quality as aroma-producing fermentation agents.

[0047] (2) Determination of pH value of fermented sausage samples The pH was measured by directly inserting a calibrated pH meter into the fermented sausage. The results are as follows: Figure 6 As shown in the figure, after one day of fermentation, the pH values ​​of all groups decreased significantly. The pH values ​​of the fermented sausage groups inoculated with compound fermentation agents ZY-2 and ZY-3 decreased to 4.90±0.80 and 4.90±0.20, respectively, reaching the safety limit. This indicates that the addition of the compound fermentation agent ZY of this invention can rapidly reduce the pH value of the fermented sausage product, thereby inhibiting the growth of harmful microorganisms and ensuring the safety and quality of the fermented sausage. During the fermentation process, inoculation with compound fermentation agents ZY-2 and ZY-3 can rapidly reduce the pH value of the fermented sausage, which not only helps to inhibit the growth of spoilage microorganisms and improve the safety of the fermented sausage product, but also promotes the formation of a good sausage flavor.

[0048] (3) Determination of volatile basic nitrogen content in fermented sausage samples Volatile basic nitrogen was determined according to GB 5009.228-2016, "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food". The results are as follows: Figure 7As shown in the figure, the volatile basic nitrogen values ​​of all groups increased during fermentation. However, the volatile basic nitrogen content of the fermented sausage groups inoculated with compound starter cultures ZY-1, ZY-2, and ZY-3 was significantly lower than that of the blank control group (CK1 group) and the fermented sausage group inoculated with Lactobacillus plantarum Z43 (CK2 group). This indicates that the addition of compound starter culture ZY, composed of Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7, can significantly slow down the decomposition rate of proteins in fermented sausages, effectively inhibit the production of volatile basic nitrogen in the product, improve product safety, and extend shelf life.

[0049] (4) Determination of Enterobacter count in fermented sausage samples Enterobacteriaceae were counted in fermented sausage samples according to the national standard GB / T 4789.41-2016 "Food Microbiology Examination: Enterobacteriaceae Examination".

[0050] Table 2. Changes in the number of Enterobacteriaceae in fermented sausages of different experimental groups during fermentation.

[0051] Note: Lowercase letters represent differences in Enterobacter count at different fermentation times within the same group, while uppercase letters represent differences in Enterobacter count between different experimental groups at the same fermentation time (P<0.05). Table 2 shows that the number of Enterobacteriaceae decreased with increasing fermentation time. The fermentation strains inhibited or killed harmful bacteria in the fermented sausage by lowering the pH through acid production or by producing antibacterial metabolites. Table 2 also shows that the Enterobacteriaceae count in the sausage group inoculated with compound starter culture ZY was significantly lower than that in groups CK1 and CK2 (P < 0.05). At 3 days of fermentation, the Enterobacteriaceae count in the sausage group inoculated with compound starter culture ZY was 0, ensuring the safety of the fermented sausage, while the Enterobacteriaceae count in the CK1 group remained at (1.0 ± 0.5) × 10⁻⁶. 4 CFU / g. Therefore, the addition of compound fermentation agent ZY plays an important role in improving the safety of low-salt fermented sausages.

[0052] (5) Determination of volatile flavor compounds in fermented sausage samples Volatile flavor compounds in fermented sausages were determined using a Flavor Spec analyzer. 2g of sausage was placed in a 20mL headspace vial and incubated at 60℃ for 20 minutes before injection. Gas chromatography-ion mobility spectrometry (GC-IMS) conditions were: analysis time 30 min; column type: MXT-WAX, column length 30 m, inner diameter 0.53 mm, film thickness 1.0 μm (RESTEK, USA); column temperature 60℃; carrier gas N2; IMS temperature 45℃. Automatic headspace sampling (AHS) conditions were: injection volume 200 μL; incubation time 15 min; incubation temperature 60℃; injection needle temperature 85℃; incubation speed 500 rpm. The results are shown in Table 3. The results indicate that compared to groups CK1 and CK2, the inoculated compound fermentation agent ZY exhibited a richer variety of volatile flavor compounds, particularly alcohols and esters, which is closely related to the fermentation of *Saccharomyces cerevisiae* Y-7 in the compound fermentation agent. Compared with the blank control group, the fermented sausage groups inoculated with compound starter culture ZY showed significantly increased levels of 3-methyl-1-butanol dimer, 3-methyl-1-butanol monomer, ethanol, 1-pentanol dimer, 1-pentanol monomer, 1-penten-3-ol, 2-methyl-1-propanol, 2-methyl-2-propanol, and 2-propanol monomer. The levels of alcohols in the fermented sausages in all groups inoculated with compound starter culture ZY were significantly higher than those in the CK2 group inoculated with *Lactobacillus plantarum* Z43. The types and contents of esters in the fermented sausages inoculated with compound starter culture ZY, such as butyl acetate, ethyl butyrate, 3-methylbutyrate dimer, 3-methylbutyrate monomer, ethyl acetate monomer, ethyl hexanoate dimer, ethyl hexanoate monomer, ethyl propionate, propyl acetate, ethyl lactate dimer, ethyl lactate monomer, isoamyl acetate dimer, isoamyl acetate monomer, and 2-methyl-ethyl propionate, were significantly higher than those in the CK1 and CK2 groups. These esters are an important basis for the flavor of fermented sausages, which shows that inoculation with the compound fermentation agent ZY plays an important role in the formation of good flavor.

[0053] Table 3 Comparison of volatile compounds in fermented sausage samples from different experimental groups using GC-IMS

[0054]

[0055] The *Lactobacillus plantarum* Z43 strain of this invention was screened from dried Cantonese sausage, derived from fermented meat products, and is highly safe and adaptable. Sausage fermentation environment. It not only meets the basic standards for meat fermentation agents but also produces antibacterial substances, inhibiting the growth of *Escherichia coli*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Proteus*. *Saccharomyces cerevisiae* Y-7, isolated from fermented sourdough, can adapt to acidic environments, with an optimal growth temperature of 37℃, and can tolerate certain salt concentrations. The compound fermentation agent ZY, prepared by combining *Saccharomyces cerevisiae* Y-7 and *Lactobacillus plantarum* Z43, exhibits excellent sausage fermentation capabilities. When applied to low-salt fermented sausages, it can improve the flavor of the fermented sausages.

[0056] This invention uses the compound fermentation agent ZY to ferment sausages, which reduces the salt content of the product, shortens the production cycle, improves the flavor and quality of fermented sausages, effectively inhibits the production of volatile basic nitrogen, and can inhibit or kill harmful microorganisms such as Enterobacteriaceae, thereby improving product safety.

[0057] Using the compound fermentation agents ZY-3 and ZY-2 of this invention, low-salt fermented sausages (Haijing Balanced Low Sodium Salt 1.5%) are produced, which have better flavor and sensory quality, and the production cycle is short (fermentation cycle 3 days), with stable and safe quality.

[0058] The preparation of fermented sausages using the compound fermentation agents ZY-3 and ZY-2 of this invention has broad application prospects.

[0059] This invention introduces yeast and lactic acid bacteria to co-produce fermented sausages, exploring a new fermentation process and developing a high-grade, flavorful, low-salt fermented sausage that is safe, healthy, nutritious, and delicious. The product quality is stable and has a pleasant aroma. With a unique flavor, high nutritional value, and no seasonal limitations, this low-salt fermented sausage boasts a rich, aromatic flavor and a rose-red appearance. It has a firm texture, rich flavor, and a mellow, pleasant taste.

[0060] This invention utilizes yeast, which has similar growth conditions to lactic acid bacteria, for co-fermentation. On the one hand, yeast can provide the necessary growth factors for the growth of lactic acid bacteria, promoting their growth and acid production. On the other hand, the ethanol produced by yeast fermentation can react with the lactic acid and fatty acids produced by lactic acid bacteria fermentation to generate aromatic esters, compensating for the flavor defects caused by low salt content. Therefore, applying lactic acid bacteria-yeast to fermented sausage production can not only improve the fermentation performance of fermented sausages but also enhance the aroma of fermented sausage products.

[0061] Chinese patent application CN201910480536.0, entitled "A Fermented Sausage Containing a Compound of Saccharomyces ruvidii and Lactobacillus plantarum," describes a fermented sausage made from a compound of Saccharomyces ruvidii and Lactobacillus plantarum. In the aforementioned patent, the fermentation temperature is relatively low (22-25℃), the production time is relatively long (15 days), and the salt content is relatively high (2%). According to existing technology, using this compound cannot overcome the technical problem of low salt content in fermented sausages. Because it cannot effectively inhibit the growth of other microorganisms, the subsequent fermentation process is difficult to control, affecting product quality and safety. The two fermenting bacteria, after being combined, can only be used in sausages with high salt content. This invention creatively uses a compound of Saccharomyces ruvidii and a specific Lactobacillus plantarum Z43. The yeast strain used is different from that of this invention. The compound fermenting agent can be directly used in the production of low-salt fermented sausages, and the fermentation time is short. A flavorful, low-salt fermented sausage can be produced in 3 days under 37℃ fermentation conditions using this compound fermenting agent. The resulting low-salt fermented sausage has a sodium content of only 1.5%, which has significant promotional value.

[0062] This invention introduces yeast for co-fermentation based on lactic acid bacteria fermentation. *Lactobacillus plantarum* Z43, with antibacterial properties, is isolated from air-dried sausage and is adapted to the sausage fermentation environment. It grows rapidly in the fermented sausage environment, quickly inhibiting the growth of putrefactive microorganisms by producing acids and antibacterial substances, and generating aroma components by decomposing proteins and fats. Using *Lactobacillus plantarum* Z43 as a starter culture effectively ensures safety and quality while improving the product's flavor. *Saccharomyces cerevisiae* Y-7, isolated from fermented sourdough, is adapted to acidic environments and can grow and produce alcohols at 37°C. Combining it with *Lactobacillus plantarum* Z43 in low-salt fermented sausages fully leverages their synergistic effect, improving the flavor of the fermented sausage. This invention explores a new fermentation process, shortens fermentation time, and develops a high-quality, safe, and delicious low-salt fermented sausage with a rich, mellow flavor.

[0063] Currently, the technical solution of this invention has undergone pilot testing, which is a small-scale experiment before the product is mass-produced. After the pilot testing was completed, a user survey was conducted on a small scale, and the survey results showed that user satisfaction was high. Now, preparations are underway for the formal production and industrialization of the product (including intellectual property risk warning surveys).

[0064] The embodiments described above are preferred embodiments of the present invention, and not an exhaustive list of all feasible implementations of the present invention. For those skilled in the art, various improvements made without departing from the spirit and essence of the present invention should be considered to be included within the scope of protection of the present invention.

Claims

1. A compound fermentation agent, characterized in that, It is composed of Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7; Lactobacillus plantarum Z43 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 23962; Saccharomyces cerevisiae Y-7 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 25587; The effective live bacteria ratio of Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7 is 10:1 or 20:

1.

2. The compound fermentation agent according to claim 1, characterized in that, The preparation method includes the following steps: (1) The activated Lactobacillus plantarum Z43 strain was inoculated into MRS liquid medium and cultured at 37℃ for 12h to obtain Lactobacillus plantarum Z43 fermentation broth; Saccharomyces cerevisiae Y-7 was inoculated into PDA liquid medium and cultured at 37℃ for 24h at 150r / min to obtain Saccharomyces cerevisiae Y-7 fermentation broth. (2) Centrifuge the fermentation broth obtained in step (1) at 8000 r / min for 5 min at 4℃, take the precipitated cells, wash them 3 times with physiological saline, resuspend them with physiological saline, and adjust the cell concentration to 10. 9 CFU / mL, respectively, were used to obtain Lactobacillus plantarum Z43 bacterial suspension and Saccharomyces cerevisiae Y-7 bacterial suspension; (3) Mix the Lactobacillus plantarum Z43 bacterial solution and Saccharomyces cerevisiae Y-7 bacterial solution obtained in step (2) in a certain proportion.

3. The compound fermentation agent according to claim 2, characterized in that, In step (1), the concentration of the *Lactobacillus plantarum* Z43 strain inoculated into the MRS liquid medium is 10. 7 cfu / mL; the concentration of the Saccharomyces cerevisiae Y-7 inoculated into PDA liquid medium was 10. 7 CFU / mL.

4. The compound fermentation agent according to claim 2, characterized in that, In step (1), the MRS liquid culture medium comprises the following components at the following concentrations: peptone 10.0 g / L, beef meal 8.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, Tween 80 1.0 g / L, sodium acetate 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, magnesium sulfate 0.2 g / L, and manganese sulfate 0.05 g / L. The pH of the MRS liquid culture medium is 6.2±0.1, and it is sterilized at 121℃ for 20 min. The PDA liquid culture medium comprises the following components at the following concentrations: potato extract powder 3.0 g / L and glucose 20.0 g / L. The pH of the PDA liquid culture medium is 5.6±0.2, and it is sterilized at 121℃ for 20 min.

5. The compound fermentation agent according to claim 2, characterized in that, In step (2), when washing with physiological saline, use physiological saline of the same volume as the fermentation broth corresponding to the precipitated bacteria each time.

6. The application of the compound fermenting agent according to any one of claims 1-5 in low-salt fermented sausage.

7. The application according to claim 6, characterized in that, The low-sodium salt content in the low-salt fermented sausage is 1.5%.

8. The application according to claim 6, characterized in that, The compound fermentation agent is mixed with the meat and inoculated before sausage stuffing. At the time of inoculation, the amount of *Lactobacillus plantarum* Z43 added is 10. 7 The effective viable cell ratio of Lactobacillus plantarum Z43 and Saccharomyces cerevisiae Y-7 was 10:1 or 20:1; fermentation was carried out at 37°C for 3 days after inoculation.

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