A compound microbial inoculant for reducing salt, enhancing flavor and aroma, and protecting crispness and color of mustard and a fermentation method thereof

Abstract

This invention provides a compound microbial agent and fermentation method for reducing salt content, enhancing aroma and flavor, and preserving crispness and color in mustard greens, belonging to the field of pickled foods. The compound microbial agent includes *Lactobacillus plantarum* H3D, *Pediococcus pentosaceus* B244, and *Lactobacillus plantarum* BZ1-6, with a live bacteria ratio of 1-3:0.5-2:0.5-2. This compound microbial agent achieves five effects: high acid production, high aroma production, high freshness production, high antibacterial activity, and preservation of crispness and color, providing a foundation for constructing a high-performance fermentation agent system. Fermented mustard greens prepared using this compound microbial agent and fermentation method exhibit a richer variety and higher content of volatile flavor compounds, a more intense aroma, and a more complex flavor. Furthermore, the fermentation cycle of the fermented mustard greens produced by this application is significantly shortened, sensory scores are significantly increased, and flavor layers are more harmonious and prominent.

Description

Technical Field

[0001] This invention belongs to the field of pickled foods, and particularly relates to a compound microbial agent and fermentation method for reducing salt, enhancing aroma and flavor, and preserving crispness and color of mustard greens. Background Technology

[0002] mustard( Brassica juncea Mustard greens (Brassica oleracea var. chinensis) are biennial or annual herbaceous plants belonging to the Brassicaceae family and the Brassicaceae genus, with a long history of cultivation. They are an important economic vegetable. Their leaves are wrinkled, pungent, and crisp, making them suitable for both fresh consumption and widespread use in pickling and fermentation, and they are very popular with consumers. Based on their morphological characteristics and edible uses, mustard greens can be mainly divided into leaf-type, stem-type, root-type, and seed-type. Leaf-type and stem-type mustard greens are commonly used for pickling and fermentation. The mustard green industry is large-scale and economically beneficial, especially in traditional pickled foods such as sauerkraut, pickled mustard tuber, and preserved vegetables, where it occupies a core position.

[0003] Huarong large-leaf mustard greens ( Brassica juncea var.longepetiolata (L.)Czern.et Coss.) is a specialty of Huarong County, Yueyang City, Hunan Province, and is one of my country's famous local specialty vegetable varieties. It belongs to the Brassicaceae family, Brassica genus, and Brassica rapa species. Brassica juncea This variety is renowned for its large, thick, crisp leaves, low fiber content, and rich aroma. It is suitable for making fermented products such as pickled mustard greens, sauerkraut, and preserved vegetables, and is particularly well-suited for low-salt lactic acid fermentation, making it a crucial raw material for traditional fermented mustard greens. With a long history of cultivation and strong adaptability, it is widely planted in autumn, resulting in a concentrated harvest period. The annual planting area reaches hundreds of thousands of acres, making it a vital pillar for increasing local farmers' income and developing local industries. Its high water content and moderate dry matter content in fresh mustard greens are ideal for the growth and reproduction of lactic acid bacteria during fermentation, contributing to the formation of a unique fermented flavor.

[0004] Fermented mustard greens are a traditional fermented food made primarily from mustard greens. Through natural or artificially controlled microbial fermentation, a series of biochemical reactions occur, resulting in a unique flavor, texture, and higher nutritional value. Lactic acid bacteria dominate the fermentation process, producing acid and inhibiting bacterial growth while promoting the formation of various flavor compounds and beneficial metabolites. This gives it a pleasantly sour and fragrant taste, a long shelf life, and safety and hygiene, making it one of the representative categories of traditional fermented vegetables in my country.

[0005] Fermentation of mustard greens not only preserves their original nutrients but also generates various flavor compounds during the process, resulting in a richer and more unique taste. Lactic acid fermentation through natural or artificial inoculation can significantly improve the flavor profile and safety of mustard greens, as well as enhance their antioxidant and probiotic properties. Traditional fermented mustard greens rely on open, natural fermentation, which is greatly affected by climate, environment, and microbial fluctuations, leading to inconsistent product flavor and quality. Further research is needed. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a compound microbial agent and fermentation method for reducing salt content, enhancing aroma and flavor, and preserving crispness and color in mustard greens.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a compound microbial agent for reducing salt content, enhancing aroma and flavor, and maintaining crispness and color in mustard greens. The compound microbial agent includes Lactobacillus plantarum H3D, Pediococcus pentosaceus B244, and Lactobacillus plantarum BZ1-6. The ratio of viable bacteria of *Lactobacillus plantarum* H3D, *Pediococcus pentosaceus* B244 and *Lactobacillus plantarum* BZ1-6 is 1~3:0.5~2:0.5~2; The preservation number of the *Lactobacillus plantarum* H3D is CGMCC NO.23495; The preservation number of the Pediococcus pentosaceus B244 is CGMCC No. 39361; The preservation number of the Lactobacillus plantarum BZ1-6 is CGMCC No. 39362.

[0008] This invention provides the application of the aforementioned compound microbial agent in the processing of pickled vegetables.

[0009] This invention provides a fermentation method for reducing salt content, enhancing aroma and flavor, and preserving crispness and color in mustard greens, comprising the following steps: After mixing the pretreated mustard greens, pickling agent, and the compound microbial agent, ferment for 14-18 days. The amount of the compound microbial agent added is 0.05% to 0.2% of the mass of the pretreated mustard greens.

[0010] Preferably, the pickling preparation contains the following components in the following mass ratio: Salt: Ethanol: Sodium metabisulfite: Lactose = 50~60: 10~30: 0.05~0.15: 20~40.

[0011] Preferably, the amount of the pickling agent added is 9% to 12% of the mass of the pretreated mustard greens.

[0012] Preferably, the viable count of the compound microbial agent is ≥1×10⁻⁶. 11 CFU / g.

[0013] Preferably, the pretreatment includes soaking the mustard greens in salt water and then dehydrating them; The mass concentration of the saline solution is 3% to 5%; Soaking time is 1-3 hours; The dehydration process involves dehydrating the mustard greens until their moisture content is 75% to 90%.

[0014] Preferably, the pretreatment temperature is 16~28℃.

[0015] Preferably, the fermentation temperature is 16~28℃.

[0016] This invention provides mustard greens prepared by the fermentation method described above.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a compound microbial agent and fermentation method for reducing salt content, enhancing aroma and flavor, and preserving crispness and color in mustard greens. This invention isolates multiple strains of lactic acid bacteria from naturally fermented vegetable samples and uses acid production, amino acid production, aroma production, and antibacterial activity as the main evaluation indicators. Ultimately, three superior strains, H3D, B244, and BZ1-6, were selected to form a compound fermentation system, exhibiting good symbiotic relationship. Through inoculation fermentation experiments with different mixing ratios, the optimal ratio of H3D, B244, and BZ1-6 was determined to be 2:1:1, achieving high acid production (total acid up to 1.71 g / kg), high aroma production (acetoin 39.52 mg / L), high freshness production (amino acid nitrogen 0.132 g / 100 mL), high antibacterial activity (inhibition diameter greater than 20 mm against Staphylococcus aureus and Escherichia coli), and preservation of crispness and color (crispness: 672.49 g / L). : 48.62, a -0.91, b The five-fold effect (40.72%) provides a foundation for constructing a high-performance fermentation agent system. Furthermore, this invention optimizes the formulation of the pickling agent. When the addition amounts of salt, edible ethanol (98%), lactose, and sodium metabisulfite are 5.59%, 1.97%, 2.95%, and 0.1093‰, respectively, the sensory score of the fermented mustard greens reaches 89 points, which is basically consistent with the model prediction result of 88.09. The amino acid nitrogen content is 0.26 g / 100 g, and the nitrite content is only 0.03 mg / kg, far below the national standard limit. Electronic nose measurement and principal component analysis revealed that the response values ​​of the optimized fermentation formulation group (TG) on multiple sensors were significantly higher than those of the natural fermentation control group (CK), indicating that it has a richer variety and higher content of volatile flavor compounds. PCA analysis further showed that the flavor characteristics of the two groups of samples differed significantly, with the optimized group having a richer aroma and more complex flavor, significantly improving the flavor of the fermented mustard greens. The fermentation cycle of the fermented mustard greens of this invention is shortened from 20 days in natural fermentation to 16 days, and the total acid content is 1.53 g / kg. In terms of quality improvement, the amino acid nitrogen content of the inoculated fermented mustard greens is increased from 0.213 g / 100g in natural fermentation to 0.248 g / 100g, the crispness is increased from 578.37 g in natural fermentation to 672.49 g, and the nitrite residue is reduced from 0.04 mg / kg in natural fermentation to 0.02 mg / kg. The sensory score is improved from 81 points in natural fermentation to 90 points, and the number of flavor compounds is increased from 49 to 64, especially showing a significant advantage in the accumulation of alcohols and esters, resulting in a more harmonious and prominent flavor profile. Attached Figure Description

[0018] Figure 1 The effect of salt addition on the sensory score and amino acid nitrogen content of fermented mustard greens; Figure 2 The effect of ethanol addition on the sensory score and amino acid nitrogen concentration of fermented mustard greens; Figure 3 The effect of citric acid addition on sensory scores, amino acid nitrogen concentration, and crispness of fermented mustard greens; Figure 4 The effect of sodium metabisulfite addition on sensory score, amino acid nitrogen concentration and crispness of fermented mustard greens; Figure 5 The effect of lactose addition on sensory scores, amino acid nitrogen concentration, and crispness of fermented mustard greens; Figure 6 The response surface and contour lines show the effect of the interaction between various factors on the sensory score of fermented mustard. Figure 7 The results of electronic nose detection of fermented mustard in the experimental and control groups are shown. Figure 8 The results of principal component analysis of mustard greens under different fermentation methods were obtained based on electronic nose detection. Figure 9 The pH value after 24 hours of bacterial culture; Figure 10 The amino acid nitrogen content of the strain after 24 hours of culture; Figure 11 The acetoin content of the strain after 24 hours of culture; Figure 12 The antibacterial effect of BZ1-6 against Escherichia coli (A) and Staphylococcus aureus (B) is shown in the figure. Figure 13 Growth curves of strains H3D, B244, BZ1-6 and mixed cultures; Figure 14 The changes in total acid content during natural fermentation and inoculated fermentation; Figure 15 The changes in amino acid nitrogen content during natural fermentation and inoculated fermentation; Figure 16 The changes in glucosinolate content during inoculation fermentation and natural fermentation; Figure 17 The changes in the content of volatile flavor compounds during inoculation and fermentation; Figure 18 This represents the change in the content of volatile flavor compounds during natural fermentation.

[0019] Certificate of Preservation of Microbial Strains

[0020] Lactobacillus plantarum ( Lactobacillus plantarum H3D, accession number: CGMCC NO.23495, deposited at China General Microbiological Culture Collection Center, deposited on September 28, 2021, at No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0021] The Pediococcus pentosaceus ( Pediococcus pentosaceus B244 has the accession number CGMCCNo.39361, is deposited at the China General Microbiological Culture Collection Center, was deposited on June 22, 2026, and is located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The plant lactobacillus ( Lactibacillus plantarum The accession number of BZ1-6 is CGMCCNo.39362, the depositary institution is the China General Microbiological Culture Collection Center, the deposit date is June 22, 2026, and the deposit address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing. Lactobacillus fermentum ( Lactobacillus fermentumThe accession number of 664 is CGMCC NO.25609, the depositary institution is the China General Microbiological Culture Collection Center, the deposit date is August 29, 2022, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing (hereinafter referred to as 664). Lactobacillus plantarum ( Lactobacillus plantarum The accession number of 729 is CGMCC NO.25610, the depositary institution is the China General Microbiological Culture Collection Center, the deposit date is August 29, 2022, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing (hereinafter referred to as 729). Lactobacillus fermentum ( Lactobacillus fermentum The accession number of 755 is CGMCC NO.23494, the depositary institution is the China General Microbiological Culture Collection Center, the deposit date is September 28, 2021, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing (hereinafter referred to as 755). ( Lactobacillus nenjiangensis The accession number of 577 is CGMCC NO.25612, the depositary institution is the China General Microbiological Culture Collection Center, the deposit date is August 29, 2022, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing (hereinafter referred to as 577). ( Lactiplantibacillus plantarum The accession number of w3-2 is CGMCC NO.25611, the depositary institution is the China General Microbiological Culture Collection Center, the deposit date is August 29, 2022, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing (hereinafter referred to as 3-2). Pediococcus pentosaceus ( Pediococcus pentosaceus The accession number of 6d-16 is CGMC NO.23498, the accession date is September 28, 2021, and the accession address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (hereinafter referred to as 6d-16). Pediococcus pentosaceus ( Pediococcus pentosaceus The accession number of 6-12 is CGMCC NO.23496, the accession date is September 28, 2021, and the accession address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing (hereinafter referred to as 6-12). Detailed Implementation

[0022] This invention provides a compound microbial agent for reducing salt content, enhancing aroma and flavor, and maintaining crispness and color in mustard greens. The compound microbial agent includes Lactobacillus plantarum H3D, Pediococcus pentosaceus B244, and Lactobacillus plantarum BZ1-6. The preservation number of the *Lactobacillus plantarum* H3D is CGMCC NO.23495; The preservation number of the Pediococcus pentosaceus B244 is CGMCC No. 39361; The preservation number of the Lactobacillus plantarum BZ1-6 is CGMCC No. 39362; In this invention, the ratio of viable bacteria of *Lactobacillus plantarum* H3D, *Pediococcus pentosaceus* B244 and *Lactobacillus plantarum* BZ1-6 is 1~3:0.5~2:0.5~2, preferably 1.5~2.5:0.7~1.5:0.7~1.5, and more preferably 2:1:1.

[0023] This invention provides the application of the aforementioned compound microbial agent in the processing of pickled vegetables.

[0024] This invention provides a compound microbial agent for reducing salt content, enhancing aroma and flavor, and preserving crispness and color in mustard greens, along with a fermentation method, comprising the following steps: The pretreated mustard greens, pickling agent, and compound microbial agent are mixed and then fermented. In this invention, the pretreatment preferably includes soaking the mustard greens in salt water and then dehydrating them; the mass concentration of the salt water is preferably 3%~5%, more preferably 3.5%~4.5%, and even more preferably 4%; the soaking time is preferably 1~3 h, more preferably 1.5~2.5 h, and even more preferably 2 h; the dehydration is preferably to dehydrate the mustard greens to a moisture content of 75%~90%, more preferably 80%~86%, and even more preferably 83.3%; the dehydration method is preferably air drying; and the pretreatment temperature is preferably 16~28℃, more preferably 20~26℃, and even more preferably 24℃.

[0025] In this invention, the preferred mass ratio of salt, ethanol, sodium metabisulfite, and lactose in the pickling agent is 50-60:10-30:0.05-0.15:20-40, more preferably 52-58:15-25:0.07-0.12:25-35, and even more preferably 55:9:19.7:0.1093:29.5. The preferred amount of the pickling agent added is 9%-12% of the mass of the pretreated mustard greens, more preferably 10%-11%, and even more preferably 10.52%.

[0026] In this invention, the preferred amount of the compound microbial agent is 0.05% to 0.2% of the mass of the pretreated mustard greens, more preferably 0.07% to 0.15%, and even more preferably 0.1%. The viable count of the compound microbial agent is preferably ≥1×10⁻⁶. 11 CFU / g.

[0027] In this invention, pretreated mustard greens, pickling agent and the compound microbial agent are mixed and fermented. The fermentation time is preferably 14-18 days, more preferably 15-17 days, and even more preferably 16 days. The fermentation temperature is preferably 16-28℃, more preferably 20-26℃, and even more preferably 24℃.

[0028] This invention provides mustard greens prepared by the fermentation method described above.

[0029] 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.

[0030] Example 1

[0031] (1.1) Test materials

[0032] Huarong large-leaf mustard greens: Harvested in early April 2024 in Hucheng Township, Huarong County, and immediately transported back to the laboratory.

[0033] Test reagents: Sodium hydroxide, phenolphthalein reagent, potassium hydrogen phthalate, formaldehyde, concentrated hydrochloric acid, p-aminobenzenesulfonic acid, and naphthylethylenediamine hydrochloride, all analytical grade, Sinopharm Chemical Reagent Co., Ltd.; Plate counting agar and lauryl sulfate peptone broth, Sinopharm Chemical Reagent Co., Ltd.; Sodium nitrite, sodium tetraborate, potassium ferrocyanide, and zinc acetate, all analytical grade, Fuchen (Tianjin) Chemical Reagent Co., Ltd.

[0034] Instruments and equipment: Electronic nose purchased from AIRSENSE GmbH, Germany; HCB-1600H ultra-clean workbench purchased from Haier Biomedical Co., Ltd.; Forma900 ultra-low temperature freezer purchased from Thermo Fisher Scientific Ltd.; Leici PHS-2F pH meter purchased from Shanghai Instrument & Electronics Scientific Instrument Co., Ltd.; AR1124CN precision electronic balance purchased from Shanghai Kunquan Biotechnology Co., Ltd.; HH-S6 constant temperature water bath purchased from Zhengzhou Yarong Instrument Co., Ltd.

[0035] (1.2) Processing technology flow

[0036] Raw material selection → washing → cutting → marinating → bottling.

[0037] (1.3) Detection

[0038] Single-factor experiments combined with Box-Behnken response surface methodology were used to study the effects of salt (4%, 6%, 8%, 10%, 12%), lactose (1%, 2%, 3%, 4%, 5%), ethanol (0%, 1%, 2%, 3%, 4%, 5%), citric acid (0%, 0.5%, 1%, 1.5%, 2%, 2.5%), and sodium metabisulfite (0‰, 0.1‰, 0.2‰, 0.3‰, 0.4‰, 0.5‰) on the quality of fermented mustard greens (the percentages are by mass, and the ‰ is by mass per thousand). The optimal formula for natural low-salt fermentation of Huarong large-leaf mustard greens was determined. Moisture content: determined according to Method 1 of GB5009.3—2016 "Determination of Moisture in Food"; pH value: determined using a pH meter; Total acid content: determined according to the acid-base titration method of GB / T12456—2008 "Determination of Total Acid in Food"; Amino acid nitrogen content: determined according to the acidity titration method of GB5009.235—2016 "Determination of Amino Acids in Food"; Nitrite content: determined according to the ultraviolet spectrophotometry method of GB / T5009.33—2016 "Determination of Nitrite and Nitrate in Food"; Escherichia coli content: determined according to the plate count method of GB 4789.3-2016 "Microbiological Examination of Food - Coliform Count".

[0039] Texture determination of fermented mustard greens: Mustard greens fermented for 25 days were trimmed into 2 cm × 3 cm strips for texture profile analysis (TPA). A P / 2 probe was used, with the following parameters: pre-test velocity 1.00 mm / s, test velocity 0.5 mm / s, post-test upward velocity 1.0 mm / s, 5 s interval between two compressions, 60% deformation, and trigger force 0.1 N. The measured parameters included hardness, elasticity, breaking force, adhesion, cohesion, and chewiness. Six measurements were performed on each sample.

[0040] Sensory evaluation

[0041] Fermented mustard greens for 25 days were collected, and 10 trained evaluators conducted sensory evaluations according to the sensory evaluation criteria for fermented mustard greens (maximum score 100 points). During the evaluation process, each evaluator independently scored the samples based on their own perception of the sensory characteristics. The arithmetic mean of the scores given by the 10 evaluators was taken as the sensory evaluation score of the fermented mustard green sample, thereby quantifying the sensory quality of the fermented mustard greens. The sensory evaluation criteria for fermented mustard greens are shown in Table 1.

[0042] Table 1 Sensory Evaluation Criteria for Fermented Mustard Greens

[0043] (1.4) Results of single-factor experiments

[0044] The effects of salt addition on the sensory score and amino acid nitrogen content of fermented mustard greens are shown in the figure. Figure 1 As the amount of salt added increased, the sensory score of fermented mustard greens showed a trend of first increasing and then decreasing. Specifically, when the salt addition was 6%, the sensory score of fermented mustard greens reached its peak at 88 points. At this point, the amino acid nitrogen content of fermented mustard greens was 0.394 g / 100 g, which was significantly different from other groups (P<0.05). Considering both sensory score and key indicators such as amino acid nitrogen content, the optimal salt addition was finally determined to be 6%.

[0045] The effects of ethanol addition on the sensory score and amino acid nitrogen concentration of fermented mustard greens are shown in the figure. Figure 2 The results showed that with increasing ethanol concentration, the sensory score and amino acid nitrogen content of fermented mustard greens initially increased and then decreased. The sensory score reached its highest value of 90 points when the ethanol concentration was 2%. At this point, the amino acid nitrogen content of the fermented mustard greens was 0.359 g / 100 g. Therefore, 2% was ultimately determined to be the optimal ethanol concentration for the fermentation of mustard greens.

[0046] The effects of citric acid addition on the sensory score, amino acid nitrogen concentration, and crispness of fermented mustard greens are shown in the figure. Figure 3 As the concentration of citric acid increased, the sensory quality of the fermented mustard greens did not change significantly in the early stage. Further increases in concentration led to a decrease in the sensory score. Therefore, citric acid did not have a significant effect on improving the quality of mustard greens, and the amount of citric acid added was not considered as a factor in the response surface methodology.

[0047] The effects of sodium metabisulfite addition on the sensory score, amino acid nitrogen concentration, and crispness of fermented mustard greens are shown in the figure. Figure 4 The results showed that as the concentration of sodium metabisulfite increased, the sensory score of fermented mustard greens showed a slight increase followed by a decrease. When the addition amount was 0.1‰, the sensory score of fermented mustard greens reached the highest value of 85 points, at which point the amino acid nitrogen content of fermented mustard greens was 0.368g / 100g.

[0048] The effects of lactose addition on the sensory score, amino acid nitrogen concentration, and crispness of fermented mustard greens are shown in the figure. Figure 5 The results showed that the sensory score of fermented mustard greens first increased and then decreased with increasing lactose concentration. The highest sensory score (90 points) was achieved when the lactose content was 3%. Simultaneously, at this lactose concentration, the amino acid nitrogen content was 0.327 g / 100 g. Therefore, at a lactose content of 3%, fermented mustard greens achieved a good balance in terms of flavor and nutrition.

[0049] (1.5) Optimization of natural low-salt fermentation of Huarong large-leaf mustard greens using response surface methodology

[0050] To further improve the sensory quality and flavor of fermented mustard greens, based on single-factor experiments, four factors were selected: salt (A), ethanol (B), sodium metabisulfite (C), and lactose (D). Sensory scores (Y) were used as the response values. A four-factor, three-level experiment was designed using Design-Expert 13.0 software and the Box-Behnken central composite design principle to optimize the fermentation conditions of the fermented mustard greens. The factors and levels of the optimized fermented mustard greens experiment are shown in Table 2.

[0051] Table 2 Factors and levels in the response surface methodology for optimizing mustard fermentation conditions.

[0052] The optimal response surface methodology for fermented mustard conditions and the results are shown in Table 3.

[0053] Table 3. Response surface methodology and results for optimizing fermented mustard conditions

[0054] Statistical analysis of the results obtained using Design-Expert 13 software yielded the following quadratic multinomial regression equation for the sensory score of fermented mustard greens: Sensory score = 91.2 - 2.33 A-0.5833 BC-1.25 D=0.5 AB+3.25 AC+1.25 AD-1.25 BC-1.5 BD-0.5 CD-5.14 A2-5.02 B2-5.39 C2-5.27 D2, the regression equation for the sensory scores of fermented mustard greens was analyzed by variance, and the results are shown in Table 4.

[0055] Table 4. Analysis of Variance for Regression Models

[0056] Note:" "Indicates a significant impact on the results (p < 0.05);" "" indicates that the effect on the result is extremely significant (p < 0.01)

[0057] Table 4 shows that the F-value of the sensory rating model is 19.08, p < 0.001, indicating that the model is highly significant; the F-value of the lack-of-fit term is 4.2, p > 0.05, indicating that it is not significant; the model's coefficient of determination R0 is...2 =0.9502, the corrected coefficient of determination is R 2 =0.9004, coefficient of variation 1.84%, signal-to-noise ratio 13.8558, indicating that the model has a high degree of fit, the experimental method is reliable, and it can be used for analysis and prediction. Based on the F-value, the influence of each response factor on the sensory score of fermented mustard is A>D>C>B, i.e., salt>lactose>sodium metabisulfite>ethanol. In the regression model of sensory scores, the first-order terms C and D have a significant impact on the sensory score of fermented mustard (p<0.05), while the first-order term A, the interaction term AC, and the quadratic term A... 2 B 2 C 2 D 2 The effect on the sensory score of fermented mustard was extremely significant (p < 0.01), while the other items were not significant (p > 0.05).

[0058] Response surface methodology and contour plots were obtained using DesignExpert 13.0 software. These were used to generate response surface methodology and contour plots showing the impact of interactions among various influencing factors on the sensory scores of fermented mustard greens. Figure 6 The optimal fermentation conditions for fermented mustard greens were found to be: 5.593% salt, 1.967% ethanol, 0.1093‰ metabisulfite, and 2.952% lactose. Under these optimized conditions, the predicted sensory score for fermented mustard greens was 88.069. Based on actual operational experience, the optimal fermentation formula for mustard greens was adjusted to 5.593% salt, 1.967% ethanol, 0.1093‰ metabisulfite, and 2.952% lactose. Three parallel verification experiments were conducted under these conditions, and the actual sensory score was 89, which is basically consistent with the model prediction, indicating that the optimization results are reliable and the process is feasible.

[0059] To systematically compare the differences in volatile flavor compounds of fermented mustard under optimized fermentation conditions (TG) and unoptimized conditions (CK), electronic nose technology was used to analyze the odor characteristics of samples from different treatments. Headspace extraction was used for electronic nose analysis. 5g of fermented mustard sample was weighed into a centrifuge tube, leveled at the bottom, and allowed to stand for 30 min before injection. Three replicates were set for each sample. Sampling time: 1 s / group; sensor self-cleaning time: 60 s; sensor zeroing time: 10 s; sample preparation time: 5 s; injection flow rate: 400 mm / min; analysis sampling time: 70 s. Results are as follows: Figure 7As shown: Specifically, the response values ​​of the three sensors W5S (sensitive to nitrogen oxides), W1W (sensitive to inorganic sulfides), and W2W (sensitive to organic sulfides and aromatic components) were significantly higher, with W1W showing the highest response value, followed by W2W, W1S (sensitive to short-chain alkanes such as methane), and W2S (sensitive to volatile substances such as alcohols, ethers, aldehydes, and ketones). This result indicates that the main volatile components in fermented mustard greens are inorganic sulfides, organic sulfides, and alcohols and aldehydes. Further analysis revealed that, except for the sensors W1C (aromatic components, toluene), W6S (mainly selective for hydrogen), and W3S (sensitive to long-chain alkanes), the optimal formulation group showed higher response values ​​on all other sensors than the control group. This indicates that the optimized formulation not only promoted the formation of aromatic compounds, sulfides, and aldehydes and ketones, but also improved the overall richness and concentration of volatile flavor compounds. Principal component analysis (PCA) was performed on the two samples, and the results are as follows: Figure 8 As shown, the variance contribution rates of PC1 and PC2 were 72.5% and 27.27%, respectively, with a cumulative variance contribution rate of 99.77% (>85%). This reflects the main informational characteristics of volatile substances in mustard greens fermented using different methods. The greater the distance between different samples, the greater the difference in aroma. Significant differences in flavor were observed between the two groups of fermented mustard greens, with the optimized group showing an increased proportion of substances and a clear advantage over the other group. Therefore, principal component analysis effectively distinguished mustard green samples fermented using different methods.

[0060] The nitrite content was determined by ultraviolet spectrophotometry according to GB / T5009.33—2016 "Determination of Nitrite and Nitrate in Food". The results showed that the nitrite content was only 0.03 mg / kg (far below the national standard) and there was no E. coli contamination, indicating high safety.

[0061] Example 2

[0062] (2.1) Materials and reagents

[0063] MRS broth, MRS medium, and LB broth were purchased from Guangdong Huankai Microbial Technology Co., Ltd. The DreamTaq Green PCR Master Mix was purchased from Thermo Fisher Scientific. Acetonitrile standard and nitrite standard were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Bacterial genomic DNA extraction kit and agarose were purchased from Beijing Solarbio Science & Technology Co., Ltd. Sodium hydroxide, creatine, naphthol, phenolphthalein, zinc acetate, borax, potassium ferrocyanide, agar powder, naphthylethylenediamine hydrochloride, and p-aminobenzenesulfonic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. The non-iodized salt was purchased from Xuetian Salt Industry Group Co., Ltd.

[0064] (2.2) Isolation and screening of superior lactic acid bacteria

[0065] After thawing, the strain was inoculated into MRS broth medium at a 1% inoculum size, thoroughly mixed, and then placed in an anaerobic incubator at 37°C for 18 h to obtain the first-generation activated bacterial solution. To ensure the stability of the strain's activity, the above activation steps were repeated until the third-generation activated bacterial solution was obtained for later use. The pH value of the fermentation broth after 24 h of culture was used as a quantitative indicator of acid production capacity; the acetoin content was used as a key characterization parameter for aroma production performance; and the amino acid nitrogen content was used to accurately assess the strain's ability to decompose and metabolize proteins and its efficiency in synthesizing umami substances, thereby comprehensively analyzing the functional characteristics and application potential of the lactic acid bacteria strain during the fermentation process.

[0066] Since fermentation agents are often used in vegetables with high salt content, the salt tolerance of the bacterial strains is crucial. The strains used for screening were further tested for salt tolerance. The activated strains were inoculated at a 1% inoculum into MRS broth containing 2%, 4%, 6%, 8%, and 10% iodine-free salt, and incubated at 37°C for 48 h. The acid production of the strains was measured to determine their activity at different salt concentrations. Strains with higher acid production at 6% salt concentration were selected. Acid production was determined using the acid-base titration method in GB / T12456—2008 "Determination of Total Acid in Food". The pH value of the culture medium after 48 h of strain incubation is shown in [reference needed]. Figure 9 The results showed the pH changes of each strain after fermentation. The lower the pH, the stronger its acid-producing ability. Most strains had pH values ​​concentrated between 3.7 and 4.3 after cultivation. Among them, the pH value of H3D was significantly lower than that of other strains, reflecting its strong ability to rapidly produce acid and reduce the pH of the system during cultivation, making it a promising fermentation strain.

[0067] H3D and B244 can be preliminarily identified as dominant strains with strong acid-producing capabilities. During subsequent fermentation, they are expected to effectively inhibit the growth of harmful microorganisms by rapidly lowering the pH, thereby improving the safety and flavor quality of the fermentation substrate. Therefore, these strains have significant application value in the selection of strains and development of starter cultures for fermented mustard greens. Further multi-dimensional evaluation can be conducted, combining indicators such as aroma production capacity, amino acid nitrogen yield, and antibacterial ability, to screen for the lactic acid bacteria strain with the best overall performance.

[0068] Amino acid nitrogen is one of the important indicators for determining the degree of fermentation of fermented products. Higher amino acid content indicates better umami flavor and quality in fermented mustard greens. During the fermentation of vegetables, lactic acid bacteria convert glucose, lactose, and other substances into amino acids and other metabolites. Different types of lactic acid bacteria may produce different types of amino acids. The amino acid nitrogen production capacity of various lactic acid bacteria is shown in [link to relevant data]. Figure 10 .

[0069] according to Figure 10 The results of the amino acid nitrogen production capacity measurements of various strains shown in the figure clearly demonstrate the significant differences in performance among different strains in this indicator. Amino acid nitrogen is one of the important indicators reflecting the degree of protein decomposition and the ability to generate flavor substances during fermentation, and its content directly affects the nutritional value and umami level of the fermented product. The results show that strains BZ1-6, 6-12, 577, and H3D performed excellently in this indicator, with significantly higher amino acid nitrogen content than other strains. This indicates that these strains have strong protein hydrolysis capacity and high protease activity, enabling them to more fully decompose the proteins in the raw materials during fermentation and release abundant free amino acids.

[0070] Among them, strain 6-12 stood out, with the highest amino acid nitrogen production among all strains, indicating its strongest potential fermentation capacity and the potential to become a dominant fermentation strain. BZ1-6, 577, and H3D also had high levels of amino acid nitrogen, indicating that they play a positive role in improving the flavor, umami, and nutritional quality of fermented mustard greens.

[0071] Comprehensive analysis reveals that strains such as BZ1-6, 6-12, 577, and H3D, due to their strong ability to produce amino acid nitrogen, exhibit significant advantages in the formation of flavor compounds and the improvement of nutritional quality in fermented mustard greens. They can serve as core candidate strains for further development of starter cultures and formulation construction, providing theoretical basis and technical support for optimizing fermentation processes and improving product quality. Future research can further investigate their symbiotic ability with other functional strains and their stability and adaptability under different process conditions to achieve efficient, stable, and safe fermentation production.

[0072] Aroma-producing lactic acid bacteria generate special aroma compounds during fermentation, imparting a unique fragrance to fermented products. Therefore, screening aroma-producing lactic acid bacteria and selecting strains with good aroma can improve the taste and flavor of products. Acetaminophen is a metabolic product of microorganisms and plants, possessing a pleasant aroma. Studies have shown that acetoin is related to the transformation of flavor substances such as diacetyl, acetaldehyde, 2,3-butanediol, tetramethylpyrazine, pyruvic acid, and diacetone. Therefore, the acetoin content reflects, to some extent, the metabolism and accumulation of other flavor substances. The acetoin content produced by various lactic acid bacteria after cultivation in MRS broth medium with added salt is as follows: Figure 11As shown, the strains with better acetoin production ability are H3D, B244, and 664.

[0073] (2.3) Determination of the inhibitory ability of lactic acid bacteria against foodborne pathogens

[0074] Fermented vegetable products are susceptible to various potential contaminants that can directly harm human health through the food chain. Foodborne pathogens are a significant source of contamination. Therefore, screening for strains that inhibit spoilage and pathogenic bacteria is crucial for fermentation agents. Salmonella, Escherichia coli, Staphylococcus aureus, and Listeria are common foodborne pathogens. Lactic acid bacteria, on the other hand, can secrete antibacterial substances such as organic acids, hydrogen peroxide, proteins, and small peptides to inhibit the growth of pathogens.

[0075] The antibacterial activity of lactic acid bacteria strains was determined using the Oxford cup plate diffusion method. Indicator bacteria were *Escherichia coli* and *Staphylococcus aureus*. The cultured indicator bacteria were mixed with sterile LB agar medium cooled to approximately 45°C. The final concentration of indicator bacteria was 1.0 × 10⁻⁶. 6 After CFU / mL concentration, the solution was poured into plates and allowed to cool and solidify. 200 μL of the test strain suspension was then added to each well of the Oxford cup, with MRS broth medium used as a blank control. The plates were placed in a 4°C refrigerator for diffusion for 2 hours, then transferred to a 37°C incubator for 24 hours. The diameter of the inhibition zone around the Oxford cup was measured using calipers.

[0076] Table 5 shows the inhibition diameters of various lactic acid bacteria strains against Escherichia coli and Staphylococcus aureus. The table indicates that strains 3-2, BZ1-6, and B244 have good inhibitory effects on these two foodborne pathogens.

[0077] Table 5. Results of antibacterial effects of each strain.

[0078] Note: "+" indicates that the inhibition diameter is less than 10mm, "++" indicates that the inhibition diameter is between 11mm and 20mm, and "+++" indicates that the inhibition diameter is greater than 21mm.

[0079] (2.4) Analysis of antagonism and symbiosis of lactic acid bacteria strains

[0080] Antagonism: Activated lactic acid bacteria strains were inoculated into MRS solid medium and incubated statically at 37°C for 24 h. Antagonism was then tested using the Oxford cup plate diffusion method. Results showed no inhibition zones on the outside of the Oxford cup, nor did the colonies become sparser closer to the cup, indicating no antagonistic reaction between any of the 10 strains, making them suitable as a combination strain. Considering the differences in species and characteristics, and combined with 16S rDNA identification results, *Lactobacillus plantarum* H3D, BZ1-6, and *Pediococcus pentosaceus* B244 were selected as the combination strains for further testing.

[0081] Symbiotic relationship: Activated lactic acid bacteria strains were inoculated into MRS broth medium. For single-strain culture, the inoculum size was 2%. For mixed culture, each strain was inoculated at the same proportion, with a total inoculum size of 2%. The cultures were incubated statically at 37°C for 24 h. After incubation, 2 mL of both the single-strain and mixed-strain cultures were used to determine the OD (octane rating). 600 Value; after individual culture, take 1 mL of the culture broth from each lactic acid bacteria culture and mix them, then take 2 mL of the mixed culture broth to measure the OD value. 600 Values. The OD600nm values ​​of the bacterial cultures after each combination are shown in Table 6.

[0082] Table 6 Results of symbiotic tests for each strain

[0083] Table 6 shows that the OD values ​​of combinations of 2 or 3 lactic acid bacteria strains were... 600 The values ​​were all higher than or equal to the bacterial density of the three lactic acid bacteria strains cultured individually and then mixed (p<0.05), indicating a significant symbiotic relationship between the three lactic acid bacteria strains, further suggesting that the four lactic acid bacteria strains are suitable as a compound strain. Comparison of the three culture methods shows that the culture method of mixing the three strains after inoculation individually yields better results. 16S rDNA identification of H3D and BZ1-6 showed a strong symbiotic relationship. Lactobacillus plantarum The homology was 100%, and it was identified as *Lactobacillus plantarum*, B244 and... Pediococcus pentosaceus The homology was 100%, and it was identified as Pediococcus pentosaceus.

[0084] (2.5) Determination of growth curves of lactic acid bacteria strains

[0085] The growth curves of the strains were plotted using a turbidimetric method. Activated lactic acid bacteria strains were inoculated into MRS broth at a 1% inoculum. For mixed culture, each strain was inoculated into the MRS broth at the same proportion, with a total inoculum of 1%. The cultures were incubated statically at 37°C, and OD values ​​were measured every 2 hours. 600 Values, with cultivation time (x) as the x-axis, OD 600 Plot the growth curve with the value (y) as the ordinate. The growth curve is shown below. Figure 13 As shown, the growth curves of the four lactic acid bacteria and the mixed strains all exhibit a typical "S" shape, with three distinct phases. The first 0-2 hours of culture is the lag phase, during which the lactic acid bacteria grow relatively slowly. Strains H3D, B244, BZ1-6, and the mixed strains show rapid cell density growth from 2-10 hours, entering the logarithmic growth phase. The final 10-32 hours is the stationary phase, with OD... 600The value reached its highest. The trend of change in the mixed culture of the three lactic acid bacteria strains was not significantly different from that in the individual cultures, indicating that there was no antagonistic effect among the three lactic acid bacteria strains, further suggesting that these three strains are suitable as compound strains.

[0086] (2.6) Determination of the proportion of fermentation strains

[0087] Based on three dominant strains—H3D, B244, and BZ1-6—mixed fermentation experiments were conducted with different mass ratios (including 1:1:1, 2:1:1, 2:2:1, 1:2:1, 1:1:2, and 1:2:2). By measuring key indicators such as amino acid nitrogen, acetoin content, and inhibition zone diameter, the performance of each combination in terms of flavor enhancement, product safety, and antibacterial activity was comprehensively evaluated. The aim was to screen for the optimal strain combination, providing a theoretical basis and practical foundation for subsequent fermentation process optimization. The relevant results are shown in Table 7.

[0088] Table 7 Results of fermentation ratios of H3D, B244, and BZ1-6

[0089] Note: "+" indicates that the inhibition diameter is less than 10 mm, "++" indicates that the inhibition diameter is between 11 mm and 20 mm, "++++" indicates that the inhibition diameter is greater than 21 mm, and "-" indicates no inhibitory effect.

[0090] Amino acid nitrogen is an important indicator reflecting the degree of protein degradation and flavor enhancement in fermentation products. As shown in the table, the amino acid nitrogen content of the 2:1:1 ratio is 0.151 g / 100mL, which is higher than that of single-strain fermentation and other mixed fermentation combinations. This indicates that this combination can more effectively promote protein degradation, increase the accumulation of small-molecule nitrogen sources, and thus enhance the richness of fermentation flavor compounds.

[0091] Acetoin is a metabolic byproduct of certain bacterial strains during fermentation, and its content is related to the type and metabolic characteristics of the fermenting microbial community to some extent. Although BZ1-6 produced the lowest acetoin content during fermentation alone (22.65 mg / L), the 2:1:1 mixed fermentation combination produced 39.52 mg / L of acetoin, which was slightly higher than other combinations and comparable to that of H3D single strain (39.41 mg / L). This indicates that this ratio improved nitrogen source utilization while maintaining excellent flavor characteristics of the fermentation product.

[0092] In terms of antibacterial activity, the 2:1:1 combination showed a large inhibition zone diameter against both Escherichia coli and Staphylococcus aureus, indicating that it has good antibacterial ability and safety, which is beneficial to improving the preservation performance of fermented products.

[0093] In summary, the 2:1:1 ratio of mixed fermentation strains has comprehensive advantages in promoting protein degradation, maintaining better flavor characteristics, and enhancing antibacterial properties, and can be used as an optimal combination for optimizing the quality of fermented mustard greens.

[0094] Example 3

[0095] (3.1) Materials and Methods

[0096] (3.1.1) Reagents

[0097] DNA Extraction Kit, Omega; Agarose, Biowest; AxyPrep DNAGel Extraction Kit, Axygen; Q5 high-fidelity DNApolymerase, NEB; AxyPrep Gel Recovery Kit, Omega.

[0098] (3.1.2) Preparation and sampling of fermented mustard greens

[0099] Fresh, high-quality, disease-free Huarong large-leaf mustard greens are selected as raw materials, and the following steps are performed in sequence: raw material selection → washing → cutting → pretreatment → pickling → bottling and fermentation.

[0100] In the pretreatment stage, after harvesting, the mustard greens were soaked in a 4% salt solution for 2 hours and then dehydrated appropriately by natural sun drying (corresponding to a moisture content of approximately 83.3%, TG8 treatment method). Subsequently, 5.593% salt, 1.967% ethanol, 0.1093‰ sodium metabisulfite, and 2.952% lactose were added according to the optimized additive combination, stirred evenly, and then bottled.

[0101] The experimental group was inoculated with 0.1% of a compound microbial agent (strain ratio of H3D:B244:BZ1-6 = 2:1:1) before bottling as the inoculation fermentation group, while the control group did not add any fermentation agent and relied solely on natural microorganisms for fermentation as the natural fermentation group. All samples were subjected to low-salt anaerobic fermentation under sealed conditions.

[0102] To ensure comparative analysis between the naturally fermented group and the inoculated fermentation group at similar fermentation stages, this study primarily used the similarity of total acid content during the fermentation process of the two groups to determine the sampling time points, thereby guaranteeing the scientific rigor and comparability of subsequent quality index comparisons. Based on the preliminary experimental results, samples were taken from the naturally fermented group (ZR) at days 0, 5, 10, 15, and 20 of fermentation, designated ZR1, ZR2, ZR3, ZR4, and ZR5; samples were taken from the inoculated fermentation group (JZ) at days 0, 4, 8, 12, and 16, designated JZ1, JZ2, JZ3, JZ4, and JZ5. These time points correspond to relatively matching stages of total acid content during the fermentation process of the two groups, providing a reliable basis for subsequent comparative analysis of physicochemical indicators, flavor components, and sensory qualities.

[0103] The trends of total acid content during natural fermentation and inoculated fermentation are as follows: Figure 14 As shown in the figure. Preliminary experimental results combined with sensory evaluation analysis indicate that the product exhibits the best flavor and significantly improved sensory scores when the total acid content of the mustard greens approaches 1.5 g / kg. Therefore, a total acid content of 1.5 g / kg was used as the key point for determining fermentation maturity. The total acid content of the inoculated fermented mustard greens reached 1.5 g / kg on day 16, while natural fermentation required day 20 to reach the same level. At this point, the sensory scores of the two groups were 90 points (inoculated fermentation group) and 81 points (natural fermentation group), respectively. Furthermore, the nitrite content in the inoculated group was only 0.02 mg / kg, significantly lower than the 0.04 mg / kg in the natural fermentation group, demonstrating its advantages in improving both sensory quality and safety. The total acid content of the naturally fermented samples on days 0, 5, 10, 15, and 20 was 0.094, 0.25, 0.45, 1.45, and 1.53 g / kg, respectively. The total acid content of the inoculated fermentation samples on days 0, 4, 8, 12, and 16 corresponded substantially to these values, indicating that the two groups were at similar fermentation stages at these time points. Therefore, these time points were selected as the corresponding sampling times to ensure the comparability of subsequent quality indicators. In summary, inoculated fermentation completes acid accumulation in a shorter time, indicating a faster acidification rate, stronger acid production capacity, and higher fermentation efficiency and quality control capabilities.

[0104] The changes in amino acid nitrogen content of mustard greens during natural fermentation and inoculated fermentation are as follows: Figure 15As shown in the figure, during inoculation fermentation, the amino acid nitrogen content increased significantly in the early stage and then stabilized. The amino acid nitrogen content was 0.208 g / 100g on day 15 of natural fermentation and 0.258 g / 100g on day 12. To compare the two fermentation methods, inoculation fermentation allows mustard greens to accumulate more amino acid nitrogen in a shorter time. This indicates that inoculation with specific microorganisms can accelerate the fermentation process and improve fermentation efficiency. This may be because the dominant microbial community inoculated with the inoculated microorganisms can metabolize the substrate more rapidly, generating fermentation products such as amino acid nitrogen. In the later stage of inoculation fermentation, the amino acid nitrogen content of the inoculated mustard greens decreased slightly. This may be because, in the later stages, some fermenting microorganisms continue to metabolize free amino acids as carbon or nitrogen sources, leading to a decrease in amino acid nitrogen content.

[0105] Changes in glucosinolates during natural fermentation and inoculated fermentation, such as... Figure 16 As shown, regardless of whether it was inoculated fermentation or natural fermentation, the glucosinolate content in mustard greens continuously decreased with prolonged fermentation time. The initial glucosinolate content of fresh mustard greens was 9.41 mg / g. During inoculated fermentation, the glucosinolate content at days 4, 8, 12, and 16 was 6 mg / g, 4.19 mg / g, 3.02 mg / g, and 3.02 mg / g, respectively. During natural fermentation, the glucosinolate content at days 5, 10, 15, and 20 was 8.62 mg / g, 7.34 mg / g, 5.91 mg / g, and 3.26 mg / g, respectively. Inoculated fermentation can more efficiently promote the degradation of glucosinolates, especially showing a significant advantage in the early and middle stages of fermentation. The main reason is that the inoculated functional strains may have stronger glucosinolate enzyme activity, which can rapidly destroy the glucosinolate structure and accelerate its metabolic decomposition; at the same time, the inoculated strains quickly occupy the dominant ecological niche, inhibiting competition from natural microorganisms and ensuring the stable and efficient function of the functional bacteria. In addition, the organic acids produced by the strain's metabolism can also lower the pH of the system, providing a more suitable environment for enzymatic reactions.

[0106] The content of volatile flavor compounds, such as Figure 17 and 18 As shown, 49 major flavor compounds were clearly detected during natural fermentation, while the number was even higher, reaching 64, during inoculated fermentation. At the level of alcohols, the two systems exhibited distinct differences. In the inoculated fermentation system, some alcohols, such as leaf alcohol and n-butanol, showed a rapid increase in content in the early stages of fermentation and maintained a high level throughout the entire fermentation process. This is likely due to the stronger alcohol-producing capacity of the specific inoculated microorganisms, which can more efficiently synthesize alcohols through specific metabolic pathways using sugars and other substances in the fermentation substrate. In contrast, during natural fermentation, the changes in alcohol content were relatively gradual, the growth trend was not prominent, and the peak value was significantly lower.

[0107] Esters, key components that impart unique aromas to fermented products, exhibit significant differences between the two fermentation methods. During inoculation fermentation, esters show significantly greater diversity and accumulation compared to natural fermentation. This is likely because the inoculated microorganisms secrete highly active esterases and related enzymes, greatly promoting esterification reactions between alcohols and acids, thereby accelerating ester synthesis. In contrast, during natural fermentation, ester synthesis is slower, with limited increases in both diversity and concentration.

[0108] Furthermore, the changes in aldehydes and ketones are also noteworthy. In the early stages of fermentation, the contents of aldehydes and ketones increased to some extent in both fermentation methods. However, as fermentation progressed, the trends diverged. In inoculated fermentation, the contents of some aldehydes declined rapidly after the middle stage, while the contents of ketones remained relatively stable, or even increased slightly. During natural fermentation, the fluctuations in the contents of aldehydes and ketones were smaller, and the overall trend differed significantly from that of inoculated fermentation. These differences fully reflect the different metabolic pathways and metabolic intensities of different microbial communities. In summary, inoculated fermentation has significant advantages in terms of the diversity of flavor compounds and the generation and accumulation of key flavor compounds, providing a strong basis for a deeper understanding of the flavor formation mechanism during fermentation and guiding the precise control of the flavor quality of fermented products in actual production.

[0109] 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 compound microbial agent for reducing salt content, enhancing aroma and flavor, and preserving crispness and color in mustard greens, characterized in that, The compound microbial agent includes Lactobacillus plantarum H3D, Pediococcus pentosaceus B244 and Lactobacillus plantarum BZ1-6; The ratio of viable bacteria of *Lactobacillus plantarum* H3D, *Pediococcus pentosaceus* B244 and *Lactobacillus plantarum* BZ1-6 is 1~3:0.5~2:0.5~2; The preservation number of the *Lactobacillus plantarum* H3D is CGMCC NO.23495; The preservation number of the Pediococcus pentosaceus B244 is CGMCC No. 39361; The preservation number of the Lactobacillus plantarum BZ1-6 is CGMCC No. 39362.

2. The application of the compound microbial agent according to claim 1 in fermented vegetable processing.

3. A fermentation method for reducing salt, enhancing aroma and flavor, and preserving crispness and color in mustard greens, characterized in that... Includes the following steps: The pretreated mustard greens, pickling agent, and compound microbial agent as described in claim 1 are mixed and fermented for 14-18 days. The amount of the compound microbial agent described in claim 1 is 0.05% to 0.2% of the mass of the pretreated mustard greens.

4. The fermentation method according to claim 3, characterized in that, The pickling preparation contains the following components in the following mass ratio: Salt: Ethanol: Sodium metabisulfite: Lactose = 50~60: 10~30: 0.05~0.15: 20~40.

5. The fermentation method according to claim 4, characterized in that, The amount of the compound microbial agent added is 9% to 12% of the mass of the pretreated mustard greens.

6. The fermentation method according to claim 3, characterized in that, The viable count of the compound microbial agent is ≥1×10⁻⁶. 11 CFU / g.

7. The fermentation method according to claim 3, characterized in that, The pretreatment includes soaking the mustard greens in salt water and then dehydrating them; The mass concentration of the saline solution is 3% to 5%; Soaking time is 1-3 hours; The dehydration process involves dehydrating the mustard greens until their moisture content is 75% to 90%.

8. The fermentation method according to claim 7, characterized in that, The pretreatment temperature is 16~28℃.

9. The fermentation method according to claim 3, characterized in that, The fermentation temperature is 16~28℃.

10. Mustard greens prepared by the fermentation method according to any one of claims 3 to 9.

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