Low-salt compound lactic acid bacteria fermentation method for delicious flavor enhanced pickled vegetables

Abstract

The invention discloses a low-salt compound lactic acid bacteria fermentation method of delicious flavor enhanced pickled vegetables, and belongs to the technical field of pickled products. The method comprises the following steps: layering pretreated vegetables, adding salt, dehydrating, compacting, standing, turning over a vat, sub-packaging the vegetables into a fermentation container II after discharged juice is balanced, inoculating a prepared compound bacterium suspension of lactobacillus plantarum and saliva combined lactobacillus, sealing by adopting a water seal type sealing structure, and fermenting. According to the invention, the multi-dimensional quality improvement of the fermentation process is realized by reducing the salinity and adopting the scheme of optimized compounding of lactobacillus plantarum and saliva combined lactobacillus.

Description

Technical Field

[0001] The invention relates to the technical field of pickled products, and in particular to a low-salt composite lactic acid bacteria fermentation method for flavor-enhanced pickled vegetables. Background Art

[0002] Fermented vegetables are a traditional fermented food in my country. Fermentation enhances their flavor and extends their shelf life, making them a popular choice. Rich in vitamins, minerals, dietary fiber, and other important compounds, fermented vegetables offer a unique flavor, relieve greasiness, stimulate appetite, and promote digestion. They also boast numerous beneficial effects, including anti-aging, arteriosclerosis prevention, and cholesterol-lowering. Due to the availability of affordable raw materials and a simple production process, most fermented vegetables are currently produced through home-based, high-salt, natural fermentation. However, existing vegetable processing companies often suffer from small production scales, rudimentary equipment, outdated technology, poor sanitary conditions, and poor corporate management practices. The quality of pickled vegetables produced under these conditions is hampered by numerous factors, making it difficult to achieve standardized industrial production with consistent quality and control over fermentation conditions and processes. Furthermore, these products suffer from long fermentation cycles, low yields, inconsistent product quality, short storage times, and difficulty controlling harmful substances during the fermentation process. Furthermore, high-salt fermentation also leads to excessive salt and nitrite levels, posing a potential threat to consumer health. With the increasing demand for high-quality fermented vegetables and the growing emphasis on food safety, the traditional fermented food industry faces challenges in equipment automation, green manufacturing, quality control and safety in production.

[0003] Fermentation techniques utilizing wild fungi carried on raw vegetables and pickling ingredients are gradually replacing traditional methods and are widely used for vegetable fermentation. This fermentation creates a complex microecological environment, with lactic acid bacteria being the dominant microbial community in nearly all naturally fermented vegetable products. They contribute to the maturation and quality development of vegetables by enhancing nutritional value, improving food flavor, and inhibiting the growth of other bacteria. As a key component of traditional fermented vegetables, lactic acid bacteria play a crucial role in their quality and safety. Lactic acid bacteria metabolize and produce acid, which lowers the pH of the system, further inhibiting pathogens and promoting nitrite degradation. However, current lactic acid fermentation methods still suffer from unstable fermentation products from single strains and high nitrite content. Furthermore, improper fermentation control under high-salt conditions can lead to low nitrite degradation efficiency, suppressed strain activity, and increased risk of residual nitrite. Furthermore, the resulting pickled vegetables fail to meet both flavor quality and appearance requirements. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention provides a low-salt, composite lactic acid bacteria fermentation method for flavor-enhanced snow mustard greens. By reducing salinity and employing an optimized combination of Lactobacillus plantarum and Lactobacillus salivarius, the present invention achieves multi-dimensional quality improvements during the fermentation process.

[0005] The technical solutions of the present invention are as follows: The present invention provides a low-salt compound lactic acid bacteria fermentation method for preparing flavor-enhanced pickled vegetables, the low-salt compound lactic acid bacteria fermentation method comprising the following steps: S1: vegetable pretreatment; S2: Dehydration with salt: The pretreated vegetables are placed in fermentation container I in layers, with a salt layer between two adjacent layers of vegetables. A salt layer is placed on the surface of the top vegetable layer and then covered with a protective layer, which is then compacted under pressure and allowed to stand. S3: turning over the tank; S4: After the juice is balanced, the vegetables are divided into fermentation container II for later use; S5: Preparation of composite bacteria: Lactobacillus plantarum and Lactobacillus salivarius were activated and rejuvenated with culture medium, bacterial sludge was collected by centrifugation, washed with water, and then centrifuged again to collect bacterial sludge to obtain two bacterial suspensions for use; S6: mixing the two bacterial suspensions to obtain a composite bacterial suspension; inoculating the composite bacterial suspension into the vegetables obtained in step S4, sealing the vegetables with a water-sealed structure, and fermenting the mixture to obtain flavor-enhanced pickled vegetables.

[0006] Preferably, in step S2, the specific method of layered loading is: laying a layer of salt in the fermentation container I, and then alternatingly distributing a layer of vegetables and a layer of salt.

[0007] Preferably, in step S2, the protective layer is a plastic film layer; The pressure compaction is to place a 15-20 kg weight on the surface of the protective layer for compaction; The standing time is 2-3 days; The amount of salt added is 3-5% of the weight of the pretreated vegetables.

[0008] Preferably, in step S3, the turning over of the tank is to swap the position of the vegetables on the bottom layer of the fermentation container I with the vegetables on the surface layer, then cover with a protective layer, place a 15-20 kg weight on the surface of the protective layer to compact it, and let it stand until the exudate no longer increases.

[0009] Preferably, in step S5, the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum ) ZJ-23, deposit number: CGMCC No. 34276, deposit date: April 21, 2025; The saliva-associated lactobacillus is saliva-associated lactobacillus ( Lactobacillus salivarius )Ls01, the deposit number is: CGMCCNo.34275, and the deposit date is: April 21, 2025.

[0010] Preferably, in step S5, the concentration of the bacterial suspension is (0.5~1.5)*10 8 CFU / mL.

[0011] Preferably, in step S6, in the composite bacterial suspension, the volume ratio of the bacterial suspension of Lactobacillus plantarum to the bacterial suspension of Lactobacillus salivarius is 1:1 to 2:1.

[0012] Preferably, in step S6, the inoculation amount is 3-4.5%.

[0013] Preferably, in step S6, the water-sealed sealing is performed by completely covering the vegetables with a covering film, adding water to form a water seal layer on the surface of the covering film, and finally compacting the vegetables with a pressing block; The depth of the water layer is 6-8 cm; The mass of the briquette is 2-4 kg.

[0014] The second aspect of the present invention provides a flavor-enhanced pickled vegetable prepared by the method described in the first aspect.

[0015] The beneficial technical effects of the present invention are: The present invention reduces the effect of high salt on the activity of the strain by reducing salinity; the use of a compound of plant lactobacillus ZJ-23 and saliva-combined lactobacillus Ls01, combined with water-sealed anaerobic fermentation, not only reduces the salt content in pickled foods, ensuring food safety; it can also achieve the preparation of low-salt products and improve the color and flavor of the products. Specifically: the unique nitrite reductase system of the two strains and the low-salt environment achieve efficient degradation of nitrite; the optimized bacterial community actively synthesizes glutamic acid and other umami substances through the synergistic metabolism of the composite bacteria, thereby improving the flavor of the product. The flavor of the fermented product is significantly improved compared to natural fermentation; the water-sealed anaerobic fermentation system achieves precise control of the fermentation microenvironment, promotes beneficial fermentation dominated by lactic acid bacteria, and inhibits the growth of miscellaneous bacteria, solving the problem of abnormal product flavor due to insufficient oxygen control in traditional processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the effects of salinity on nitrite and sensory properties of fermented products.

[0017] In the figure: (A) shows the effect of salinity on nitrite in fermented products; (B) shows the effect of salinity on the sensory properties of fermented products.

[0018] Figure 2 This is a graph showing the effect of inoculation amount on nitrite and sensory properties of fermentation products.

[0019] In the figure: (A) shows the effect of inoculation amount on nitrite content of fermented products; (B) shows the effect of inoculation amount on sensory properties of fermented products.

[0020] Figure 3 Effect of inoculum size on nitrite and sensory properties of fermentation products.

[0021] In the figure: (A) shows the effect of bacterial strain ratio on nitrite content in fermented products; (B) shows the effect of bacterial strain ratio on sensory properties of fermented products.

[0022] Figure 4 It is the interactive hyperbolic surface and contour map in the response surface analysis of the fermentation process of the present invention.

[0023] In the figure: (A) interaction between salinity and inoculum size; (B) contour map of salinity and inoculum size; (C) interaction between salinity and compound ratio; (D) contour map of salinity and compound ratio; (E) interaction between inoculum size and compound ratio; (F) contour map of inoculum size and compound ratio.

[0024] Figure 5 This is the change in organic acid content during the fermentation process of Example 6 of the present invention.

[0025] In the figure: (A) changes in oxalic acid content; (B) changes in tartaric acid content; (C) changes in malic acid content; (D) changes in lactic acid content; (E) changes in acetic acid content; (F) changes in citric acid content; (G) changes in succinic acid content.

[0026] Figure 6 The changes in pH and total acid content of the pickled vegetables of Example 6 of the present invention and Comparative Example 1 during the fermentation process are shown.

[0027] In the figure: (A) pH change; (B) total acid content change.

[0028] Figure 7 Graph showing texture changes of pickled vegetables during fermentation according to Example 6 of the present invention and Comparative Example 1.

[0029] In the figure: (A) hardness change; (B) elasticity change; (C) chewiness change. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to the embodiments.

[0031] The present invention provides a low-salt pickled vegetable fermentation process based on composite lactic acid bacteria. By controlling the amount of salt added during the fermentation process, combined with the coordinated use of composite bacteria and water sealing treatment, the sensory quality of snow vegetables is improved, the nitrite content is reduced, and the color of the product is improved.

[0032] The low-salt composite lactic acid bacteria fermentation method for preparing flavor-enhanced pickled vegetables of the present invention comprises the following steps: S1: vegetable pretreatment; S2: Dehydration with salt: The pretreated vegetables are placed in fermentation container I in layers, with a salt layer between two adjacent layers of vegetables. A salt layer is placed on the surface of the top vegetable layer and then covered with a protective layer, which is then pressed and compacted, and allowed to stand. S3: turning over the tank; S4: After the juice is balanced, the vegetables are divided into fermentation container II for later use; S5: Preparation of composite bacteria: Lactobacillus plantarum and Lactobacillus salivarius were activated and rejuvenated with culture medium, bacterial sludge was collected by centrifugation, washed with water, and then centrifuged again to collect bacterial sludge to obtain two bacterial suspensions for use; S6: mixing the two bacterial suspensions to obtain a composite bacterial suspension; inoculating the composite bacterial suspension into the vegetables obtained in step S4, sealing the vegetables with a water-sealed structure, and fermenting the mixture to obtain flavor-enhanced pickled vegetables.

[0033] In some embodiments, in step S1, the vegetable is snow mustard greens.

[0034] In some embodiments, in step S1, the pretreatment includes: selecting fresh potherb mustard greens (Potherb mustard greens), manually removing leaves with insect spots and mechanical damage, and retaining intact green leaves. The trimmed potherb mustard greens are laid out flat in a ventilated, cool, and shady area to dry, allowing surface moisture to evaporate naturally and causing the leaves to slightly wilt and turn yellow.

[0035] In some embodiments, in step S2, the specific method of layered loading is: laying a layer of salt in the fermentation container I, and then alternatingly distributing a layer of vegetables and a layer of salt.

[0036] In some embodiments, in step S2, the protective layer is a plastic film layer; The pressurized compaction is to place a weight of 15 to 20 kg on the surface of the protective layer for compaction, including but not limited to 15 kg, 18 kg, and 20 kg.

[0037] The standing time is 2 to 3 days, including but not limited to 2 days and 3 days; The amount of salt added is 3-5% of the weight of the pretreated vegetables, including but not limited to 3%, 4%, and 5%.

[0038] In some embodiments, in step S3, the turning over of the tank is to swap the position of the vegetables on the bottom layer of the fermentation container I with the vegetables on the surface layer, then cover with a protective layer, and place a 15-20 kg weight on the surface of the protective layer to compact it, and let it stand until the exudate no longer increases.

[0039] In some embodiments, the strain Lactobacillus plantarum ( Lactobacillus plantarum ) ZJ-23 is deposited in the General Microbiology Center of China Culture Collection Administration Committee, with the deposit number: CGMCC No. 34276, the deposit date is April 21, 2025, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing); the saliva-combined Lactobacillus ( Lactobacillus salivarius ) Ls01 was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number: CGMCC No. 34275, the deposit date is April 21, 2025, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing).

[0040] The strain Lactobacillus plantarum ZJ-23 used in the present invention can be obtained by the following method: First, the strain is isolated from a snow vegetable sample; the snow vegetable sample from which the strain is obtained can be either commercially available snow vegetable or naturally fermented snow vegetable produced by farmers.

[0041] Second, it was identified as Lactobacillus plantarum through 16S rDNA of Sangon Biotech (Shanghai) Co., Ltd.

[0042] Third, the identified strains are preserved. The specific preservation information is as described above and will not be repeated here.

[0043] In some embodiments, the method for obtaining the saliva-associated Lactobacillus Ls01 is the same as that for obtaining Lactobacillus plantarum ZJ-23.

[0044] In some embodiments, in step S5, the concentration of the bacterial suspension is (0.5-1.5)*10 8 CFU / mL, including but not limited to 0.5*10 8 CFU / mL, 1.0*10 8 CFU / mL, 1.5*10 8 CFU / mL.

[0045] In some embodiments, in step S6, in the composite bacterial suspension, the volume ratio of the Lactobacillus plantarum ZJ-23 bacterial suspension to the Lactobacillus salivae Ls01 bacterial suspension is 1:1 to 2:1, including but not limited to 1:1, 1.5:1, and 2:1.

[0046] In some embodiments, in step S6, the inoculation amount is 3-4.5%, including but not limited to 3%, 4%, and 4.5%.

[0047] In some embodiments, in step S6, the water-sealing method comprises completely covering the vegetables with a covering film, adding water to form a water seal layer on the surface of the covering film, and finally compacting the water seal layer with a pressing block. The depth of the water layer is 6-8 cm, including but not limited to 6 cm, 7 cm, and 8 cm.

[0048] The mass of the briquette is 2-4 kg, including but not limited to 2 kg, 3 kg, and 4 kg.

[0049] In some embodiments, in step S6, the fermentation time is 25 days.

[0050] It is understandable that the combination of bacterial strains can significantly accelerate the acid production process in terms of fermentation kinetics, making the degradation of nitrite more thorough; in terms of flavor regulation, the synergistic effect of the bacterial community can promote the directional accumulation of umami amino acids (glutamate TAV>1) and beneficial organic acids (lactic acid, acetic acid), while effectively inhibiting the formation of bitter amino acids and oxalic acid, and significantly enriching volatile flavor substances such as alcohols and esters, and reducing the content of spicy ingredients such as allyl isothiocyanate; in terms of sensory quality, lactic acid bacteria can exhibit a series of enzyme activities, which can improve the sensory characteristics of fermented foods, including aroma, flavor and taste, optimize color, shorten ripening time, etc.; in terms of microbial ecology, it shows higher community richness and stability. The organic acids (lactic acid, acetic acid), bacteriocins, extracellular polysaccharides and other substances produced by lactic acid bacteria during the growth and metabolism process can improve the quality and safety of fermented foods by inhibiting the growth of pathogens or killing spoilage bacteria, and extend the storage period of fermented foods. In addition, the process method of the present invention breaks through the bottleneck of quality control of fermented vegetables under low-salt conditions, realizes the coordinated optimization of health attributes and flavor quality, and provides an innovative solution for the industrial upgrading of traditional fermented vegetables.

[0051] The second aspect of the present invention provides a flavor-enhanced pickled vegetable prepared by the method described in the first aspect.

[0052] In some embodiments, the pickled vegetable is snow mustard greens.

[0053] The following are specific embodiments.

[0054] Example 1 A pickling process for snow mustard greens comprises the following steps: (1) Raw material pretreatment We carefully select fresh potherb mustard greens (Potherb mustard greens), manually removing leaves with insect spots and mechanical damage, leaving the leaves intact and green. We then lay the potherb mustard greens flat in a well-ventilated, cool, and shady place to air dry for 24 hours, allowing the surface moisture to evaporate naturally and the leaves to wilt slightly and turn yellow.

[0055] (2) Low-salt dehydration treatment Weigh the snow vegetables after drying, and add salt according to 3% of the weight of the vegetables, and set aside; use a 50L food-grade plastic barrel as a fermentation container, and sprinkle a thin layer of salt on the bottom first; use a layered arrangement method to lay the snow vegetables in the fermentation container, specifically: for each layer of snow vegetables about 10cm thick, evenly sprinkle a layer of salt and compact each layer; stop filling when it is 10cm away from the barrel mouth, and sprinkle the remaining salt on the top layer; cover with food-grade PE plastic film (thickness 1mm), place a round vegetable pressing board with a diameter slightly smaller than the barrel mouth, and apply a weight of about 15kg on the board; dehydrate at room temperature (20±2℃) for 72 hours.

[0056] (3) Tank overturning Remove the pressing board, release the weight, and peel off the plastic film on the surface; turn the bottom layer of vegetables (about 1 / 3 of the amount) to the surface; re-layer and compact, restore the PE film covering, prevent the circular pressing board and apply a 20kg weight; continue dehydration until the juice is balanced (that is, the exudate no longer increases).

[0057] (4) Preparation for subpackaging Transfer the dehydrated snow vegetables and exudate into a 5L plastic bucket; each bucket should contain about 2kg, ensuring that the exudate completely submerges the vegetables.

[0058] (5) Preparation of composite strains Lactobacillus plantarum ZJ-23 and Lactobacillus salivarius Ls01 were removed from -80°C glycerol tubes and inoculated into 50 mL of MRS liquid medium. The culture was incubated at 37°C for 20 h to obtain the culture. The composition of the MRS liquid medium was as follows: peptone 10 g / L; beef powder 8 g / L; yeast powder 4 g / L; glucose 20 g / L; potassium dihydrogen phosphate 2 g / L; diammonium hydrogen citrate 2 g / L; sodium acetate 5 g / L; magnesium sulfate 0.2 g / L; manganese sulfate 0.04 g / L; Tween 80 1.0 g / L, with an initial pH of 6.2±0.2.

[0059] Transfer 1 mL of each culture to 100 mL of fresh MRS liquid medium, using the same medium as above, and incubate at 37°C for 18 h. Collect the bacterial sludge by centrifugation at 4500 rpm for 10 min under sterile conditions; wash twice with sterile saline and centrifuge again at 4500 rpm for 10 min to collect the bacterial sludge. Adjust the bacterial suspension concentration to OD 0. 600 =1.0 (approximately 10 8 CFU / mL). Two bacterial suspensions were obtained respectively, and the two bacterial suspensions were mixed at a volume ratio of 1:1 between saliva-associated Lactobacillus Ls01 and Lactobacillus plantarum ZJ-23 to obtain a composite bacterial suspension.

[0060] (6) Inoculation and fermentation Calculate the total weight of the snow cabbage (including exudate), and inoculate the composite bacterial suspension at a 3% inoculum volume. Evenly inoculate the cabbage and then apply water-sealing and stone-pressing. The specific method for water-sealing and stone-pressing is as follows: First, completely cover the cabbage with food-grade PE film (0.5 mm thick). Then, pour clean water onto the PE film to a depth of 6-8 cm to form a water seal. Then, place 3 kg of sterilized stones on top. Allow to ferment for 25 days. After fermentation, measure the pH.

[0061] (7) Post-processing The pickled vegetables were vacuum packed and pasteurized at 75℃ for 10 min.

[0062] Example 2 The method is basically the same as Example 1, except that the salinity in step (2) of low-salt dehydration is changed. In this embodiment, salt is weighed at 4% of the weight of the vegetables.

[0063] Example 3 The method is basically the same as Example 1, except that the salinity in step (2) of low-salt dehydration is changed. In this embodiment, salt is weighed at 5% of the weight of the vegetables.

[0064] Comparative Example 1 The method is basically the same as Example 1, except that no bacteria are added, and natural fermentation is used. That is, after step (4), the vegetable body is water-sealed and stone-pressed. The specific method of water-sealing and stone-pressing is as follows: first, the vegetable body is completely covered with food-grade PE film (0.5 mm), then clean water is injected into the surface of the PE film to form a water seal layer with a water depth of 6-8 cm, and then 3 kg of sterilized stones are pressed. The vegetable body is left to ferment for 25 days.

[0065] Comparative Example 2 The method is basically the same as Example 1, except that the salinity in step (2) of low-salt dehydration is changed. In this embodiment, salt is weighed at 6% of the weight of the vegetables.

[0066] The fermentation broths of Examples 1-3 and Comparative Examples 1-2 were fermented for 25 days and the nitrite content was determined. The results were as follows: Figure 1 As shown in Figure (A), in the inoculation and fermentation curve, 3% salinity corresponds to Example 1, 4% salinity corresponds to Example 2, 5% salinity corresponds to Example 4, and 6% salinity corresponds to Comparative Example 2. As can be seen from the figure, Example 3 has a lower nitrite content, while Comparative Example 1, which does not add bacteria, has a higher nitrite content at the same salt addition level.

[0067] At the same time, sensory evaluation was performed on the products of Examples 1-3 and Comparative Example 2 fermented for 25 days. The results are as follows: Figure 1 As shown in Figure (B), it can be seen from the figure that the sensory evaluation of the fermented product of Comparative Example 2 is significantly lower than that of the embodiment, indicating that controlling the amount of salt added can not only achieve low salt, but also improve the sensory performance of the product.

[0068] Example 4 The process is basically the same as Example 2, except that in step (6) of inoculation and fermentation, the inoculation amount of the strain is 4.5%.

[0069] Comparative Example 3 The process is basically the same as Example 2, except that in step (6) of inoculation and fermentation, the inoculation amount of the strain is 1.5%.

[0070] Comparative Example 4 The process is basically the same as Example 2, except that in step (6) of inoculation and fermentation, the inoculation amount of the strain is 6%.

[0071] Take the fermentation broth of Example 2, Example 4, Comparative Example 3 and Comparative Example 4 fermented for 25 days and measure the content of nitrite therein. The results are as follows: Figure 2 As shown in Figure (A), an inoculum size of 1.5% corresponds to Example 3, an inoculum size of 6% corresponds to Example 4, an inoculum size of 3% corresponds to Example 2, and an inoculum size of 4.5% corresponds to Example 4. As can be seen from the figure, the nitrite content of the inoculum size of 4.5% is relatively low, and the nitrite content corresponding to the inoculum size of 1.5% is the highest.

[0072] At the same time, sensory evaluation was performed on the products of Example 2, Example 4, Comparative Example 3 and Comparative Example 4 fermented for 25 days. The results are as follows: Figure 2 As shown in Figure (B), when the inoculation amount is 4.5%, the sensory performance is better. However, when the inoculation amount is increased to 6%, the sensory performance is significantly reduced.

[0073] Example 5 The method is basically the same as Example 2, except that in the preparation of the composite strain in step (5), the volume ratio of saliva-associated Lactobacillus Ls01 to plant Lactobacillus ZJ-23 is 1:2.

[0074] Comparative Example 5 The method is basically the same as Example 2, except that in the preparation of the composite strain in step (5), the volume ratio of saliva-associated Lactobacillus Ls01 to Lactobacillus plantarum ZJ-23 is 2:1.

[0075] Comparative Example 6 The method is basically the same as Example 2, except that in step (5), the composite bacteria are used. This comparative example only uses saliva combined with Lactobacillus Ls01. Step (5) is specifically as follows: Saliva-associated Lactobacillus salivarius Ls01 was removed from a -80°C glycerol tube and inoculated into 50 mL of MRS liquid medium and incubated at 37°C for 20 h. The composition of the MRS liquid medium was as follows: 10 g / L peptone, 8 g / L beef powder, 4 g / L yeast extract, 20 g / L glucose, 2 g / L potassium dihydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween 80, with an initial pH of 6.2 ± 0.2. One mL of the above culture was transferred to 100 mL of fresh MRS liquid medium and incubated at 37°C for 18 h. The bacterial sludge was collected by centrifugation at 4500 rpm for 10 min under sterile conditions. The sludge was washed twice with sterile saline and then centrifuged at 4500 rpm for 10 min to collect the bacterial sludge. The bacterial suspension concentration was adjusted to OD 0.00 with saline. 600 =1.0 (approximately 10 8 CFU / mL). Obtain bacterial suspension for later use.

[0076] Take the fermentation broth of Example 2, Example 5, Comparative Example 5 and Comparative Example 6 for 25 days and measure the content of nitrite therein. The results are as follows: Figure 3 As shown in Figure (A), the compounding ratio of 0 corresponds to Example 6, the compounding ratio of 0.5 corresponds to Example 5, the compounding ratio of 1 corresponds to Example 2, and the compounding ratio of 2 corresponds to Example 5. As can be seen from the figure, the nitrite content of using only one strain is relatively high.

[0077] At the same time, sensory scores were performed on the products of Example 2, Example 5, Comparative Example 5, and Comparative Example 6 fermented for 25 days. The results are shown in Figure 3 (B). It can be seen from the figure that when the compounding ratio is 0.5, the sensory performance is relatively better.

[0078] Through the above experiments, the present invention determined the optimal ranges of various parameters in the fermentation process. To further obtain the optimal process parameter combination, the present invention further performed a response surface design using a Box-Benken experiment to obtain the optimal parameter combination. The details are as follows: Based on the optimal factor levels from the above experiments, a three-factor, three-level Box-Behnken response surface design was conducted. Salinity was used as factor A, inoculum size as factor B, and the ratio of saliva-associated Lactobacillus Ls01 to Lactobacillus plantarum ZJ-23 as factor C. Sensory scores were used as responses. A response surface optimization experiment was conducted to determine the optimal combination. The factors and levels are shown in Table 1.

[0079] Table 1 Factor levels of Box-Benhnken design experiment

[0080] The experimental data were processed using Design-Expert 13.0 software, and the results are shown in Table 2-3.

[0081] Table 2 Response surface test results

[0082] Table 3 Analysis of significance results of response surface methodology

[0083] The experimental data were processed using Design-Expert 13.0 software. The model predicted that the sensory score of fermented snow vegetable reached the highest value of 92 when the salinity was 4.8%, the inoculation amount was 4.2%, and the compound ratio was 0.48.

[0084] Figure 4 The interaction hyperbolic surface and contour map corresponding to the corresponding surface method are given. It can be seen from the figure that when the compound ratio is fixed at the 0 level, the interaction effect of salinity A and inoculation amount B on the sensory score of fermented snow vegetable is obtained. Figure 4 As can be seen, the 3D image presents a steeply curved surface, with densely elliptical contour lines. This indicates that salinity A and inoculum size B have a significant interaction on the sensory score Y of fermented snow vegetable, which is consistent with the results in the table above. Similarly, the interaction between salinity A and compound ratio C is not significant, while the interaction between inoculum size B and compound ratio C is significant.

[0085] The pickling process of snow mustard greens under the above conditions was verified by specific experiments below, see Example 6 for details.

[0086] Example 6 A pickling process for snow mustard greens comprises the following steps: (1) Raw material pretreatment We carefully select fresh potherb mustard greens (Potherb mustard greens), manually removing leaves with insect spots and mechanical damage, leaving the leaves intact and green. We then lay the potherb mustard greens flat in a well-ventilated, cool, and shady place to air dry for 24 hours, allowing the surface moisture to evaporate naturally and the leaves to wilt slightly and turn yellow.

[0087] (2) Low-salt dehydration treatment Weigh the snow vegetables after drying, and add salt according to 4.8% of the weight of the vegetables, and set aside; use a 50L food-grade plastic barrel as a fermentation container, and sprinkle a thin layer of salt on the bottom first; use a layered arrangement method to lay the snow vegetables in the fermentation container, specifically: for each layer of snow vegetables about 10cm thick, evenly sprinkle a layer of salt and compact each layer; stop filling when it is 10cm away from the barrel mouth, and sprinkle the remaining salt on the top layer; cover with food-grade PE plastic film (thickness 1mm), place a round vegetable pressing board with a diameter slightly smaller than the barrel mouth, and apply a weight of about 15kg on the board; dehydrate at room temperature (20±2℃) for 72 hours.

[0088] (3) Tank overturning Remove the pressing board, release the weight, and peel off the plastic film on the surface; turn the bottom layer of vegetables (about 1 / 3 of the amount) to the surface; re-layer and compact, restore the PE film covering, prevent the circular pressing board and apply a 20kg weight; continue dehydration until the juice is balanced (that is, the exudate no longer increases).

[0089] (4) Preparation for subpackaging Transfer the dehydrated snow mustard greens and the exudate into a 5L plastic bucket (about 2kg per bucket), ensuring that the exudate completely submerges the vegetables. (5) Preparation of composite strains Lactobacillus plantarum ZJ-23 and Lactobacillus salivarius Ls01 were taken out from -80℃ glycerol tubes and inoculated into 50mL of MRS liquid medium respectively. The culture was incubated at 37℃ for 20 h. The composition of MRS liquid medium was as follows: peptone 10 g / L; beef powder 8 g / L; yeast powder 4 g / L; glucose 20 g / L; potassium dihydrogen phosphate 2 g / L; diammonium hydrogen citrate 2 g / L; sodium acetate 5 g / L; magnesium sulfate 0.2 g / L; manganese sulfate 0.04 g / L; Tween 80 1.0 g / L, with an initial pH of 6.2±0.2.

[0090] Transfer 1 mL of each culture to 100 mL of fresh MRS liquid medium and incubate at 37°C for 18 h. Aseptically centrifuge at 4500 rpm for 10 min to collect the bacterial sludge; wash twice with sterile saline and centrifuge again at 4500 rpm for 10 min to collect the bacterial sludge. Adjust the bacterial suspension concentration to OD 0. 600 =1.0 (approximately 10 8 CFU / mL). Two bacterial suspensions were obtained separately and mixed at a volume ratio of 1:0.48 to obtain a composite bacterial suspension.

[0091] (6) Inoculation and fermentation Calculate the total weight of the snow cabbage (including exudate); prepare a 4.2% inoculum of the composite bacterial suspension; evenly inoculate the cabbage with the composite bacterial suspension and then perform a water-sealing and stone-pressing process. The specific method for water-sealing and stone-pressing is as follows: First, completely cover the cabbage with food-grade PE film (0.5 mm thick). Then, pour clean water onto the PE film to a depth of 6-8 cm to form a water seal. Then, place 3 kg of sterilized stones on top. Allow the mixture to ferment for 25 days. After completion, test the pH of the fermentation liquid.

[0092] (7) Post-processing The fermented pickled vegetables were vacuum packed and pasteurized at 75°C for 10 min.

[0093] The total amount of total acid, the content of each organic acid, and the texture and color changes of the snow vegetable samples from the beginning of fermentation to 25 days of fermentation in the low-salt and composite bacteria fermentation of Example 6 (denoted as ZJ-Ls) and the low-salt natural fermentation group of Comparative Example 1 (denoted as NF) were respectively measured. At the same time, the free amino acid content of commercially available snow vegetable (denoted as MF) and the free amino acid content and nitrite content of the low-salt and composite bacteria fermentation of Example 6 (denoted as ZJ-Ls) and the low-salt natural fermentation group of Comparative Example 1 (denoted as NF) after 25 days of fermentation were also measured.

[0094] The test results showed that the total acid content of the product of Example 6 reached 79.8 g / L, the nitrite content was 0.129 mg / kg, and the total number of lactic acid bacteria was 4.39×10 7 The total acid content of the product of Comparative Example 1 was 64.2 g / L, the nitrite content was 0.454 mg / kg, and the total number of lactic acid bacteria was 3.38 × 10 7 CFU / mL.

[0095] The types and contents of free amino acids are shown in Table 4.

[0096] Table 4 Free amino acid content in fermented potherb mustard samples

[0097] Note: * indicates essential amino acids, ^ indicates semi-essential amino acids, and # indicates non-essential amino acids. TEAA indicates total essential amino acids, and TFAA indicates total free amino acids.

[0098] As can be seen from the table above, a total of 18 free amino acids were detected in the samples of the ZJ-Ls, NF, and MF groups, but the cysteine ​​content in the MF group and the serine content in the NF group were extremely low (less than 0.001 mg / mL). The proportion of total essential amino acids (TEAA) to total free amino acids (TFAA) in the samples of each group was 38.95%, 53.46%, and 44.81%, respectively. The main free amino acids in mature snow mustard greens samples from the ZJ-Ls group were glutamate (0.424 mg / mL), alanine (0.169 mg / mL), tryptophan (0.116 mg / mL), and lysine (0.097 mg / mL). The main free amino acids in samples from the NF group were alanine (0.420 mg / mL), tryptophan (0.347 mg / mL), proline (0.239 mg / mL), and valine (0.232 mg / mL). The MF group had the highest levels of alanine (0.703 mg / mL), leucine (0.344 mg / mL), and proline (0.307 mg / mL). Furthermore, at the end of fermentation, except for glutamate in the ZJ-Ls group, which had a taste activity value greater than 1, the TAV of each free amino acid in the other groups was less than 1, indicating that the ZJ-Ls group exhibited a stronger umami flavor than the other samples.

[0099] Types and contents of organic acids Figure 5As shown in the figure, (A) oxalic acid content changes; (B) tartaric acid content changes; (C) malic acid content changes; (D) lactic acid content changes; (E) acetic acid content changes; (F) citric acid content changes; and (G) succinic acid content changes. As can be seen, oxalic acid content in the ZJ-Ls and NF groups showed an overall downward trend over time, with the ZJ-Ls group consistently higher than the NF group. However, the ZJ-Ls group reached its lowest value of 0.274 mg / mL on day 20 of fermentation, and then saw a slight increase on day 25. Overall, the oxalic acid content in both groups decreased from 2.249 mg / mL and 1.040 mg / mL on day 1 to 0.818 mg / mL and 0.033 mg / mL on day 25. Tartaric acid content in the ZJ-Ls group initially increased and then decreased until stabilization in the middle and late stages of fermentation. The NF group showed the same trend as the ZJ-Ls group during the early stages of fermentation, but saw a slight rebound towards the end of fermentation. Both groups reached peak values ​​of 0.348 mg / mL and 0.236 mg / mL on day 5 of fermentation, respectively. Tartaric acid levels in the NF group reached their lowest value of 0.062 mg / mL on day 20. Tartaric acid levels in the ZJ-Ls group remained stable at 0.100-0.110 mg / mL from day 15 to day 25. Overall, tartaric acid levels in the ZJ-Ls group were higher than those in the NF group except on day 25, and the decline in tartaric acid levels in the fermentation group inoculated with the compound lactic acid bacteria was greater than that in the natural fermentation group. Changes in malic acid content differed between the two groups. In the ZJ-Ls group, the concentration first decreased in the early stage, from 1.999 mg / mL to 0.825 mg / mL, maintained a stable level in the middle stage of fermentation, and then decreased to a lower level in the late stage, reaching only 0.424 mg / mL when fermentation was mature. The change trend of the NF group in the early and middle stages of fermentation was the same as that of the ZJ-Ls group, but the decline was greater and the level was lower, from 1.872 mg / mL to 0.143 mg / mL. The content rebounded slightly in the late stage of fermentation, reaching 0.354 mg / mL on the 25th day.

[0100] Lactic acid and acetic acid were the two most important organic acids in the late fermentation stage, and their changing trends were basically the same in all groups. The content in the ZJ-Ls group first increased, then decreased, and then increased again at the end of fermentation; reaching peak values ​​of 11.014 mg / mL and 9.653 mg / mL on the 5th and 10th days of fermentation, respectively, and decreased to 9.079 mg / mL and 7.726 mg / mL at the end of fermentation; while the content in the NF group first increased and then decreased, reaching the highest levels of 8.507 mg / mL and 10.523 mg / mL on the 10th day, and slowly decreased to 1.351 mg / mL and 5.275 mg / mL at the end of fermentation. Citric acid was not detected in the fermented snow vegetable samples on the first day of fermentation. Its content then slowly increased in the ZJ-Ls group, reaching 0.107 mg / mL by the end of fermentation. In the NF group, it initially increased and then decreased, reaching a peak of 0.373 mg / mL on the 10th day and dropping to 0.130 mg / mL on the 25th day. Succinic acid content in the ZJ-Ls group decreased, eventually reaching a stable level of 1.881 mg / mL. In the NF group, however, it initially decreased rapidly, then rebounded to its initial level of 5.357 mg / mL on the 20th day, before dropping to a low of 1.170 mg / mL on the 25th day.

[0101] Compared with the potherb mustard greens of comparative example 1, the accumulation of organic acids in the potherb mustard greens of example 6 resulted in the characteristics of low pH and high acidity of the fermented potherb mustard greens (e.g. Figure 6 This helps preserve the snow vegetable.

[0102] The above analysis also shows that the total organic acid content of the potherb mustard greens in the ZJ-Ls group was higher than that in the other two groups. The oxalic acid content in the MF group was 4.649 mg / mL, significantly higher than in the other two groups. Excessive oxalic acid levels can damage the gastrointestinal mucosa and cause discomfort. It can also easily combine with calcium to form calcium oxalate crystals, increasing the risk of kidney stones and potentially causing arrhythmias. Therefore, samples with higher oxalic acid concentrations are not suitable for human consumption. The ZJ-Ls group contained higher levels of lactic acid and acetic acid. The pungent acetic acid had the highest flavor activity value, which may be a key characteristic of the fermented potherb mustard greens' unique flavor. The milder lactic acid partially neutralized the pungent sourness of acetic acid and further enhanced the sensory acceptability of the fermented potherb mustard greens. This lactic acid-fermented potherb mustard greens had a profound impact on the quality and flavor of the fermented potherb mustard greens. Overall, the fermented potherb mustard greens in the ZJ-Ls group exhibited a superior organic acid flavor.

[0103] The texture changes of snow mustard greens during the fermentation process of the embodiment and the comparative example were measured by a texture analyzer. The results are as follows: Figure 7As shown in the figure, the hardness of the ZJ-Ls group showed a downward trend from the first day to the fifth day, and was basically stable from the 10th to the 25th day, with a slight increase from the 15th to the 20th day. The overall change was not significant as time went on. The hardness at the time of fermentation maturity (the 25th day) was 1080.73±16.81g, which was not significantly different from the initial hardness (the 1st day). P >0.05). The hardness trend in the NF group was similar to that in the ZJ-Ls group, with a significant decrease from day 1 to day 15, reaching nearly the same level as the ZJ-Ls group on day 10. Later in fermentation, from days 15 to 25, hardness increased slightly, ultimately reaching 899.74 ± 130.05 g at the end of fermentation. Compared with the ZJ-Ls group, the NF group showed a more pronounced change in hardness, with the hardness lower than that of the ZJ-Ls group at the end of fermentation. In terms of elasticity, no significant changes were observed between the two groups. Chewability showed a similar trend to hardness, initially decreasing in the early stages of fermentation and then becoming relatively stable from the middle stages. Chewiness peaked in both groups on the first day of fermentation, with the NF group showing significantly higher chewiness than the ZJ-Ls group. This chewiness then decreased steadily from the first day to the tenth day, when it became the same in both groups and reached its lowest level during the fermentation process. At the end of fermentation, the chewiness of the fermented snow radish in the ZJ-Ls and NF groups was 218.88±18.01 g and 209.74±34.81 g, respectively. In summary, the texture of the snow radish fermented with the optimal process was not significantly different from that of the naturally fermented group.

[0104] The color changes of the potherb mustard greens during the fermentation process of the embodiment and the comparative example were tested by a colorimeter. The results are shown in Table 5.

[0105] Table 5 Color changes during fermentation of potherb mustard greens in Example 6

[0106] Table 6 Color changes of the potherb mustard greens during fermentation of Comparative Example 1

[0107] As can be seen from the table, as time changes, the L* values ​​of the two groups show a downward trend, the lightness L* becomes darker, and there is no obvious change trend in the red-green value a* value and the yellow-blue value b* value. Compared with the fermentation day 1 of the fermented mature (D25) snow vegetable sample, the L* value and the total color difference value ΔE of the ZJ-Ls group and the NF group are significantly reduced ( P< 0.05), L* values ​​decreased from 44.17 and 40.56 on the first day to 26.66 and 7.24 at the end of fermentation; the total color difference value ΔE decreased from 48.36 and 45.53 to 30.36 and 19.88, indicating that after fermentation, the brightness of fresh snow vegetable became significantly darker, the greenness decreased, and the yellowness faded but the change was not significant ( P >0.05).

[0108] Comparing the color changes of potherb mustard greens under different fermentation conditions showed that the rate of brightness change in the ZJ-Ls group was lower than that in the NF group, and the L* value at the end of fermentation was higher than that of the NF group, indicating that the inoculation of lactic acid bacteria allowed the fermented potherb mustard greens to better retain their color and slow down the decline in brightness. There were no significant differences in the red-green and yellow-blue values ​​between the two groups. Although there were significant fluctuations during the fermentation process, overall, there was no significant change between the beginning and end of fermentation. This shows that the inoculation of lactic acid bacteria has little effect on the color of the fermented potherb mustard greens, and the potherb mustard greens can still maintain a good color after fermentation.

[0109] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment. It is understood that other improvements and variations directly derived or imagined by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the scope of protection of the present invention.

Claims

1. A low-salt compound lactic acid bacteria fermentation method for flavor-enhanced pickled vegetables, characterized in that: The low-salt compound lactic acid bacteria fermentation method comprises the following steps: S1: vegetable pretreatment; S2: Dehydration with salt: The pretreated vegetables are placed in fermentation container I in layers, with a salt layer between two adjacent layers of vegetables. A salt layer is placed on the surface of the top vegetable layer and then covered with a protective layer, which is then pressed and compacted, and allowed to stand. S3: turning over the tank; S4: After the juice is balanced, the vegetables are divided into fermentation container II for later use; S5: activating and rejuvenating Lactobacillus plantarum and Lactobacillus salivarius respectively with culture medium, collecting bacterial sludge by centrifugation, washing with water, and collecting bacterial sludge by centrifugation again to obtain two bacterial suspensions respectively, which are set aside; S6: mixing the two bacterial suspensions to obtain a composite bacterial suspension; inoculating the composite bacterial suspension into the vegetables obtained in step S4, sealing the vegetables with a water-sealed structure, and fermenting the mixture to obtain flavor-enhanced pickled vegetables.

2. The low-salt composite lactic acid bacteria fermentation method according to claim 1, characterized in that: In step S2, the specific method of layered loading is: laying a layer of salt in the fermentation container I, and then arranging a layer of vegetables and a layer of salt alternately.

3. The low-salt composite lactic acid bacteria fermentation method according to claim 1, characterized in that: In step S2, the protective layer is a plastic film layer; The pressure compaction is to place a 15-20 kg weight on the surface of the protective layer for compaction; The standing time is 2 to 3 days; The amount of salt added is 3-5% of the weight of the pretreated vegetables.

4. The low-salt composite lactic acid bacteria fermentation method according to claim 1, characterized in that: In step S3, the turning over of the tank is to swap the position of the vegetables on the bottom layer of the fermentation container I with the vegetables on the surface layer, then cover with a protective layer, place a 15-20 kg weight on the surface of the protective layer to compact it, and let it stand until the exudate no longer increases.

5. The low-salt composite lactic acid bacteria fermentation method according to claim 1, characterized in that: In step S5, the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum ) ZJ-23, deposit number: CGMCC No. 34276; The saliva-associated lactobacillus is saliva-associated lactobacillus ( Lactobacillus salivarius )Ls01, the deposit number is: CGMCC No.34275.

6. The low-salt composite lactic acid bacteria fermentation method according to claim 1, characterized in that: In step S5, the concentration of the bacterial suspension is (0.5~1.5)*10 8 CFU / mL.

7. The low-salt composite lactic acid bacteria fermentation method according to claim 1, characterized in that: In step S6, in the composite bacterial suspension, the volume ratio of the bacterial suspension of Lactobacillus plantarum to the bacterial suspension of Lactobacillus salivarius is 1:1 to 2:

1.

8. The low-salt composite lactic acid bacteria fermentation method according to claim 1, characterized in that: In step S6, the inoculation amount is 3-4.5%.

9. The low-salt composite lactic acid bacteria fermentation method according to claim 1, characterized in that: In step S6, the water-sealed sealing is to completely cover the vegetables with a covering film, add water to form a water seal layer on the surface of the covering film, and finally compact it with a pressing block; The depth of the water layer is 6-8 cm; The mass of the briquette is 2-4 kg.

10. A pickled vegetable with enhanced umami flavor prepared by the method according to any one of claims 1 to 9.

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