A microbial inoculant for secondary fermentation of pickled vegetables in jars on an industrial scale

Abstract

The present application is a kind of microbial inoculant for the secondary fermentation of pickled vegetables in jars on an industrial scale.The microbial inoculant is a solid composite inoculant and contains Lactobacillus plantarum, Weissella confusa and Kazachstania exigua, and the viable cell count of each bacterium in the inoculant is ≥1‑5×10 9 CFU / g.The mass ratio of Lactobacillus plantarum bacterial powder, Weissella confusa bacterial powder and Kazachstania exigua bacterial powder is 6‑8:1‑2:1‑2.The microbial inoculant is used to reduce the content of nitrite and biogenic amines in the secondary fermentation process in jars, and to prevent the growth of coliform bacteria, bacillus, pseudomonas and mold in the secondary fermentation process in jars.

Description

Technical Field

[0001] This invention belongs to the field of industrial-scale salted vegetable processing technology, specifically a microbial agent for secondary fermentation of industrial-scale salted vegetables in jars. Background Technology

[0002] Salted vegetables are a traditional Chinese fermented food with a long history and rich cultural significance. They are widely used as side dishes, appetizers, and condiments. Their crispness and tangy flavor are highly favored by consumers, and demand is increasing year by year. This has led to a shift in production methods from small workshops to large-scale industrial processing. Currently, the common process for industrial-scale salted vegetables involves using fresh vegetables as raw materials, pickling them in a high concentration of salt (over 12%), followed by washing, desalting, dehydration, seasoning, bottling, and sterilization. Microorganisms grow and metabolize slowly under the high-salt conditions of industrial-scale salting, and the complex processes cause a loss of fermentation flavor. Companies can only compensate for this loss by adding acids and spices. Therefore, industrial-scale single-fermentation salted vegetables rely heavily on post-processing seasoning and have lost the flavor of traditional pickled vegetables. Currently, most companies have begun to use a secondary fermentation process to give salted vegetables the flavor of traditional pickled vegetables, thereby improving their quality and market competitiveness.

[0003] The secondary fermentation of industrial-scale salted vegetables refers to the process of placing salted vegetables (salted embryos) that have been fermenting in brine tanks for a period of time (1-6 months) into earthenware jars for fermentation again after simple processes such as washing and desalination. However, currently, the secondary fermentation of industrial-scale salted vegetables mainly relies on natural fermentation, which has a long fermentation cycle, often exceeding one month. This leads to difficulties in the turnover of earthenware jars, low utilization efficiency, and hinders the production of salted vegetables and the profitability of enterprises. Furthermore, the washing and desalination processes before secondary fermentation reduce the number of microorganisms, resulting in weak secondary fermentation and low acid production, making it difficult to achieve the flavor of "old-fashioned pickled vegetables." Moreover, the multiple processing steps involved in salting vegetables may contaminate environmental microorganisms, leading to spoilage such as film growth and softening, and higher levels of nitrites and biogenic amines, seriously affecting the safety of salted vegetables for consumption. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the prior art by providing a microbial inoculant for the secondary fermentation of salted vegetables on an industrial scale. This inoculant is a compound of *Lactobacillus plantarum*, *Weissella confusa*, and *Kazachstania exigua* in a specific ratio. *Weissella confusa* in this inoculant has strong salt tolerance, enabling rapid initiation of fermentation in industrial-scale salted vegetables. It assists *Lactobacillus plantarum* in its growth and acid production, shortening the maturation period of industrial-scale salted vegetables during secondary fermentation. Simultaneously, *Weissella confusa* also promotes the production of appropriate amounts of alcohols by *Kazachstania exigua*. This microbial inoculant can degrade undesirable flavors such as butyric acid and formic acid in salted vegetables, inhibit the growth of spoilage microorganisms, reduce biogenic amine content, and improve the food safety of salted vegetables.

[0005] To achieve the above-mentioned objectives, the specific technical solution of this invention is as follows:

[0006] A microbial agent for secondary fermentation of salted vegetables on an industrial scale, comprising a solid compound microbial agent containing *Lactobacillus plantarum*, *Weissella confusa*, and *Kazachstania exigua*, with each microorganism having a viable count ≥1-5 × 10⁻⁶. 9 CFU / g.

[0007] Furthermore, the mass ratio of Lactobacillus plantarum powder, Weissella confusa powder, and Kazachstania exigua powder was 6-8:1-2:1-2.

[0008] As a preferred option, this microbial agent is used in the secondary fermentation process of salted vegetables on an industrial scale.

[0009] Preferably, the amount of the microbial agent added is 0.1-0.3% of the total mass of the raw materials.

[0010] The aforementioned secondary fermentation process refers to the process in which, depending on the type of vegetable, after the vegetables have fermented in a brine pool for 1-6 months, the salted vegetables are washed, desalted, and then placed in earthenware jars for further fermentation.

[0011] The process of using microbial inoculants for secondary fermentation in a fermentation jar includes the following steps:

[0012] 1) Take out the vegetables that have been fermenting in the brine tank for 1-6 months, wash them, desalinate them, and dry their surface moisture;

[0013] 2) Place the salted vegetables, after drying the surface moisture, into a ceramic jar. Mix the cooled boiled water with the microbial inoculant and add it to the ceramic jar. Ferment at room temperature for 5-7 days under sealed conditions.

[0014] Furthermore, this microbial agent is used to reduce the content of nitrite and biogenic amines in the secondary fermentation process.

[0015] Furthermore, this microbial agent is used to inhibit the growth of coliform bacteria, Bacillus, Pseudomonas, and mold during the secondary fermentation process.

[0016] Compared with the prior art, the positive effects of the present invention are reflected in:

[0017] (i) The microbial agent of the present invention, Weissella confusa, has strong salt tolerance and can quickly start fermentation in industrial-scale salted vegetables, assist Lactobacillus plantarum in growth and produce a large amount of acid, shorten the maturation cycle of industrial-scale salted vegetables in secondary fermentation, and improve the efficiency of fermentation containers such as ceramic jars.

[0018] (ii) The microbial agent of the present invention contains Weissella confusa which is beneficial to Kazachstania exigua in producing an appropriate amount of alcohols. Furthermore, the microbial agent can degrade unpleasant flavors such as butyric acid and formic acid in salted vegetables, giving the "old-fashioned pickled vegetables" flavor to industrial-scale salting ponds. The vegetables can be directly used for cooking and seasoning after being removed from the jar, and have high consumer acceptance.

[0019] (III) The microbial agent of the present invention can reduce the content of nitrite and biogenic amines, improve the food safety of salted vegetables, prevent the growth of putrefactive microorganisms such as coliform bacteria, Bacillus, Pseudomonas, and mold in salted vegetables, prevent spoilage and deterioration, and make them more suitable for storage. Attached Figure Description

[0020] Figure 1 This is a graph showing the pH change of salted radishes during secondary fermentation in the example.

[0021] Figure 2 This is a graph showing the change in total acid concentration of salted radishes during secondary fermentation in the example.

[0022] Figure 3 This is a bar chart showing the content of organic acids and alcohols in samples 1, 2, and 8 during fermentation maturation in the examples.

[0023] Figure 4 The image shows the evaluation results of the color, odor, taste, and texture of samples 1, 2, and 8 at the fermentation maturity stage in the examples. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the invention to the following embodiments. Various substitutions and modifications made based on ordinary technical knowledge and common practice in the art without departing from the above-described technical concept of this invention should be included within the scope of this invention.

[0025] Unless otherwise specified, % in this application document refers to wt%; unless otherwise specified, all raw materials used are commercially available products.

[0026] The Lactobacillus plantarum mentioned in this application is prior art, with accession number CGMCC No. 14931.

[0027] The *Weissella confusa* fusion strain mentioned in this application is prior art, with accession number CGMCC No. 23918.

[0028] The Kazachstania exigua mentioned in this application is prior art, with accession number CGMCC No. 12791.

[0029] The preparation method of the compound microbial agent includes the following steps:

[0030] (1) Take 100uL of Lactobacillus plantarum and Weissella confusa strain preservation solution respectively and incubate them in 10mL of MRS broth at 37℃ for 12-18h.

[0031] Take 100 μL of the Kazachstania exigua strain preservation solution and incubate it in 10 mL of YPD liquid medium at 28 °C for 20–24 h.

[0032] (2) The bacterial cultures were inoculated into expansion culture media at 3% of their total volume and cultured for further growth. The expansion culture medium for *Lactobacillus plantarum* consisted of: 2.5-3.5% glucose, 1-1.5% peptone, 0.5-1% yeast extract, and 0.5-1.0% beef extract. The culture conditions were: initial pH 6.0-6.5, culture temperature 30-37℃, shaking speed 80-100 r / min, and culture time 12-16 h. The expansion culture medium for *Weissella confusa* consisted of: 1.5-2.5% glucose, 0.5-1.0% peptone, 0.5-1.0% yeast extract, and 0.5-1.0% beef extract. The culture conditions were: initial pH 6.0-6.5, culture temperature 30-37℃, shaking speed 80-100 r / min, and culture time 10-14 h. The expansion medium for Kazachstania exigua consisted of 1.0-1.5% peptone, 0.5-1.0% yeast extract, and 2-3% sucrose. The culture conditions were: initial pH 4.5-5.0, culture temperature 28-32℃, shaker speed 80-100 r / min, and culture time 15-18 h.

[0033] (3) The culture medium after expansion was centrifuged at 4℃ and 4000 r / min for 20 min to obtain bacterial sludge. A protectant was added, with the ratio of protectant to bacterial sludge being 1:2-3 by weight. The mixture was then freeze-dried under vacuum to obtain bacterial powder. The protectant consisted of the following raw materials in parts by weight: 9 parts skim milk powder, 2 parts trehalose, 0.6 parts glycerol, 2.5 parts sorbitol, and 1.2 parts maltodextrin. The drying temperature was -60 to -50℃, the vacuum degree was 1 Pa, and the time was 30-35 h. After freeze-drying, the bacterial powders were prepared as follows: the bacterial powder obtained from Lactobacillus plantarum was designated as A, the bacterial powder obtained from Weissella confusa was designated as B, and the bacterial powder obtained from Kazachstania exigua was designated as C. The viable count in each bacterial powder was approximately 1-5 × 10⁻⁵. 9 CFU / g.

[0034] (4) Mix bacterial powders A, B and C in a mass ratio of 6-8:1-2:1-2 to form a compound bacterial agent.

[0035] Example 1:

[0036] A microbial agent for secondary fermentation of salted vegetables on an industrial scale, comprising a solid compound microbial agent containing *Lactobacillus plantarum*, *Weissella confusa*, and *Kazachstania exigua*, with each microorganism having a viable count ≥ 2 × 10⁻⁶. 9 CFU / g.

[0037] The preparation method of this compound microbial agent includes the following steps:

[0038] (1) Take 100uL of Lactobacillus plantarum and Weissella confusa strain preservation solution respectively and incubate them in 10mL of MRS broth at 37℃ for 12h.

[0039] Take 100 μL of the Kazachstania exigua strain preservation solution and incubate it in 10 mL of YPD liquid medium at 28 °C for 20 h.

[0040] (2) The bacterial cultures were inoculated into expansion culture medium at 3% of the total volume and cultured for a further period. The expansion culture medium for Lactobacillus plantarum consisted of 2.5% glucose, 1.0% peptone, 0.5% yeast extract, and 0.5% beef extract. The culture conditions were: initial pH of the medium 6.5, culture temperature 35℃, shaking speed 100 r / min, and culture time 16 h.

[0041] The expansion medium for Weissella confusa consisted of 1.5% glucose, 0.5% peptone, 1.0% yeast extract, and 0.5% beef extract. The culture conditions were: initial pH 6.5, culture temperature 30℃, shaker speed 100 r / min, and culture time 14 h.

[0042] The expansion medium for Kazachstania exigua consisted of 1.5% peptone, 1.0% yeast extract, and 3.0% sucrose. The culture conditions were: initial pH 5.0, culture temperature 28℃, shaker speed 100 rpm, and culture time 18 h.

[0043] (3) The culture medium after expansion was centrifuged at 4℃ and 4000 r / min for 20 min to obtain bacterial sludge. A protectant was added, with a mass ratio of protectant to bacterial sludge of 1:3. The mixture was then freeze-dried under vacuum to obtain bacterial powder. The protectant consisted of the following raw materials in parts by weight: 9 parts skim milk powder, 2 parts trehalose, 0.6 parts glycerol, 2.5 parts sorbitol, and 1.2 parts maltodextrin. The drying temperature was -60℃, the vacuum degree was 1 Pa, and the time was 32 h. After freeze-drying, the bacterial powders were prepared as follows: the bacterial powder obtained from Lactobacillus plantarum was designated A, the bacterial powder obtained from Weissella confusa was designated B, and the bacterial powder obtained from Kazachstania exigua was designated C. The viable count of each bacterial powder was approximately 1 × 10⁻⁶. 9 CFU / g or higher.

[0044] (4) Mix bacterial powders A, B and C in a mass ratio of 8:1:1 to obtain compound bacterial agent 1#.

[0045] Example 2:

[0046] A microbial agent for secondary fermentation of salted vegetables on an industrial scale, comprising a solid compound microbial agent containing *Lactobacillus plantarum*, *Weissella confusa*, and *Kazachstania exigua*, with each microorganism having a viable count ≥1×10⁻⁶. 9 CFU / g.

[0047] The preparation method of the microbial inoculant is the same as in Example 1. Microbial powders A, B, and C are mixed in a mass ratio of 6:2:2 to obtain compound microbial inoculant #2.

[0048] Example 3:

[0049] A microbial agent for secondary fermentation of salted vegetables on an industrial scale, comprising a solid compound microbial agent containing *Lactobacillus plantarum*, *Weissella confusa*, and *Kazachstania exigua*, with each microorganism having a viable count ≥1×10⁻⁶. 9 CFU / g.

[0050] The preparation method of the microbial inoculant is the same as in Example 1. Microbial powders A, B, and C are mixed in a mass ratio of 5:3:2 to obtain compound microbial inoculant #3.

[0051] Example 4:

[0052] A microbial agent for secondary fermentation of salted vegetables on an industrial scale, comprising a solid compound microbial agent containing only *Lactobacillus plantarum* and *Weissella confusa*, with each microorganism having a viable count ≥1×10⁻⁶. 9 CFU / g. The preparation method of the microbial inoculant is the same as in Example 1. Mix bacterial powders A and B at a mass ratio of 7:3 to obtain compound inoculant #4.

[0053] Example 5:

[0054] A microbial agent for secondary fermentation of salted vegetables on an industrial scale, comprising a solid compound microbial agent containing only *Lactobacillus plantarum* and *Kazachstania exigua*, with each microorganism having a viable count ≥1×10⁻⁶. 9 CFU / g. The preparation method of the microbial inoculant is the same as in Example 1. Mix bacterial powders A and C at a mass ratio of 7:3 to obtain compound inoculant #5.

[0055] Example 6:

[0056] A microbial agent for secondary fermentation of salted vegetables on an industrial scale, comprising a solid compound microbial agent containing only *Weissella confusa* and *Kazachstania exigua*, with each microorganism having a viable count ≥1×10⁻⁶. 9 CFU / g. The preparation method of the microbial inoculant is the same as in Example 1. Mix bacterial powders B and C at a mass ratio of 7:3 to obtain compound inoculant 6#.

[0057] Comparative Example 1:

[0058] Lactobacillus plantarum CICC 22195 was purchased from the China Industrial Microbial Culture Collection Center (CICC), and a compound microbial agent was prepared by fusing Weissella confusa CICC 24453 and Kazachstaniaexigua CICC 33206 with it. The preparation followed the steps in Example 1.

[0059] Example 7:

[0060] (1) Select radishes that have been fermented in the salt tank of the enterprise for about a month, cut them into small pieces, wash them with clean water to remove salt three times, dry them and weigh 67kg for secondary fermentation in the jar, and weigh 1kg for basic index determination.

[0061] (2) Put the salted radish processed in step (1) into a 100L earthenware jar, add 200g of microbial agent to 32.8kg of cooled boiled water, mix well, add to the earthenware jar and seal, and place at room temperature for 7 days to ferment.

[0062] (3) Industrial-scale salted radishes fermented with the microbial agents of Examples 1-6 are samples 1-6, industrial-scale salted radishes fermented with Comparative Example 1 are sample 7, and industrial-scale salted radishes fermented without adding microbial agents are sample 8.

[0063] 1. Basic indicators for salted radishes

[0064] Salted radishes from step 1 of Example 7, before secondary fermentation, were selected. Their salinity, pH, total acid, and the number of microorganisms such as lactic acid bacteria and yeast were measured and compared with fresh radishes. The results are shown in Table 1. The salted radishes before secondary fermentation had higher salinity and lower lactic acid bacteria counts, which are the main reasons for the difficulty and long maturation period of secondary fermentation. Therefore, it is necessary to use microbial agents to regulate fermentation.

[0065] Table 1. Physicochemical and microbiological indicators of industrial-scale salted radishes before secondary fermentation.

[0066]

[0067] 2. Comparison of fermentation effects

[0068] According to national standard methods, the pH value and total acid concentration of samples 1, 2, 3, 5, 7, and 8 were determined during the fermentation process. The results are as follows: Figure 1 and Figure 2 As shown in the diagram, Samples 1 and 2 exhibited the fastest decrease in pH and the fastest increase in total acid concentration, reaching 0.8 g / 100 g by day 7 of fermentation, meeting the acidity requirements for removal from the fermentation tank. Samples 7 and 8 showed a slower increase in total acid (and a slower decrease in pH), indicating that the microbial agent of this patent can be effectively used for industrial salted vegetable fermentation. Sample 5 showed a slow increase in total acid, with a concentration of 0.53 g / 100 g by day 7 of fermentation, indicating that *Weissella confusa* has good salt tolerance, can quickly initiate fermentation, and assists *Lactobacillus plantarum* in growth and large-scale acid production. The fermentation effect of Sample 3 was not as good as that of Samples 1 and 2, indicating that the fermentation effect was best when the ratio of *Lactobacillus plantarum*, *Weissella confusa*, and *Kazachstania exigua* was 6-8:1-2:1-2.

[0069] 2. Comparison of flavor substance content

[0070] According to the national standard method, the concentrations of lactic acid, acetic acid, and ethanol in samples 1, 2, 4, 5, 6, and 8 at fermentation maturity were determined by high performance liquid chromatography (HPLC). The results are as follows: Figure 3 As shown in the diagram, Samples 1 and 2 have significantly higher lactic acid and ethanol contents than Sample 8, indicating that the microbial agent of this patent can enhance the flavor of industrial-scale salted vegetables. While Samples 4 and 5 have similar lactic acid and acetic acid contents to Sample 8, their ethanol concentrations are extremely low, indicating that *Kazachstania exigua* can form a certain amount of ethanol, and *Weissella confusa* can promote the formation of higher concentrations of ethanol from *Kazachstania exigua*. Sample 6 has a higher ethanol concentration but a lower lactic acid content. Therefore, the microbial agent of this invention can produce a large amount of organic acids and a suitable amount of alcohol in industrial-scale salted vegetables, and the absence of any one of these bacteria will adversely affect the flavor of the product.

[0071] 3. Sensory comparison

[0072] Ten professional sensory experts were randomly selected to score the color, aroma, taste, and texture of samples 1, 2, and 8 at the point of fermentation. The scoring criteria are shown in Table 2. Commercially available pickled vegetables were used as a control sample. The results are as follows: Figure 4 As shown, the sensory scores of Sample 1 and Sample 2 were significantly better than those of Sample 8, and they also had a better taste, a richer aroma, and were not much different from the control sample. This indicates that the secondary fermentation of salted vegetables by microbial agents in Examples 1 and 2 was effective on an industrial scale and imparted a "traditional pickled vegetable" flavor.

[0073] Table 2 shows the sensory evaluation criteria.

[0074]

[0075] 4. Comparison of unpleasant taste substances and food safety

[0076] According to national standard methods, the contents of formic acid, propionic acid, butyric acid, nitrite, and biogenic amines, as well as the quantities of coliform bacteria, Pseudomonas, and mold in samples 1, 2, and 8 were determined, and the results are shown in Table 1. Industrial-scale salted vegetables often accumulate unpleasant flavor substances such as formic acid, propionic acid, and butyric acid during the fermentation stage due to high salt concentration and poor sealing, severely affecting the product's flavor. Samples 1 and 2, which used the microbial agent of this invention, did not show any unpleasant flavor substances, while sample 8 had a very high content. This indicates that the microbial agent of this invention can effectively degrade formic acid, propionic acid, and butyric acid, improving the product's flavor. Regarding safety, the nitrite and biogenic amine contents of samples 1 and 2 were much lower than those of sample 8, and samples 1 and 2 did not show any microorganisms such as coliform bacteria, Pseudomonas, Bacillus, or mold, while sample 8 showed Pseudomonas, Bacillus, and mold. Therefore, industrial-scale salted vegetables fermented a second time using this patented microbial agent have higher food safety, are less prone to spoilage, and are more suitable for storage.

[0077] Table 3. Determination of unpleasant taste substances and safety-related indicators.

[0078]

[0079]

[0080] The examples described above are merely preferred embodiments of this patent, but the scope of protection of this patent is not limited thereto. It should be noted that, for those skilled in the art, without departing from the principles of this patent, based on the technical solution and patent concept of this patent, several improvements and modifications can be made, and these improvements and modifications should also be considered within the scope of protection of this patent.

Claims

1. The application of a microbial inoculant in the secondary fermentation of salted vegetables on an industrial scale, characterized in that... This microbial agent is a solid compound microbial agent, and contains... Lactobacillus plantarum , Weissella confusa and Kazachstania exigua The viable count of each type of bacteria in the inoculant is ≥1-5×10⁻⁵. 9 CFU / g; In this application, the microbial agent is used to reduce the content of nitrite and biogenic amines in the secondary fermentation process, and at the same time inhibit the growth of coliform bacteria, Bacillus, Pseudomonas and mold in the secondary fermentation process. Among them, Lactobacillus plantarum has the accession number CGMCC No. 14931; Weissella confusa has the accession number CGMCC No. 23918; and Kazachstania exigua has the accession number CGMCC No. 12791.

2. The application as described in claim 1, characterized in that: Lactobacillus plantarum Bacterial powder, Weissella confusa Bacterial powder and Kazachstania exigua The mass ratio of the bacterial powder is 6-8:1-2:1-2.

3. The application of the microbial agent as described in claim 1 or 2, characterized in that: The amount of this microbial agent added is 0.1-0.3% of the total mass of the raw materials.

4. The application of the microbial agent as described in claim 3, characterized in that, The aforementioned secondary fermentation process refers to the process in which, depending on the type of vegetable, after the vegetables have fermented in a brine pool for 1-6 months, the salted vegetables are washed, desalted, and then placed in earthenware jars for further fermentation.

5. The application of the microbial agent as described in claim 4, characterized in that, The specific application steps are as follows: 1) Take out the vegetables that have been fermenting in the brine tank for 1-6 months, wash them, desalinate them, and dry their surface moisture; 2) Place the salted vegetables, after drying the surface moisture, into a ceramic jar. Mix the cooled boiled water with the microbial inoculant and add it to the ceramic jar. Ferment at room temperature for 5-7 days under sealed conditions.

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