CN116004479A - Lactobacillus plantarum and application thereof - Google Patents

Abstract

The invention discloses a lactobacillus plantarum and application thereof, belonging to the technical field of microbial fermentationLactiplantibacillus plantarum) The sun-15 is preserved in China Center for Type Culture Collection (CCTCC) with a preservation date of 2022, 10 months and 19 days and a preservation number of CCTCC NO: m20221595. The invention takes lactobacillus plantarum sun-15 and lactobacillus acidophilus KLDS1.0901 as fermentation strains, and the optimal fermentation process conditions are as follows: the ratio of lactobacillus (Lactobacillus plantarum: lactobacillus acidophilus) is 2:1, the inoculation amount of lactobacillus in 250g of Chinese cabbage is 450 μl, and the addition amount of corn juice is 7.5g. The pickled Chinese cabbage without salt is mature in 5 days, and the finished pickled Chinese cabbage without salt has good quality characteristics and better stability in 60 days of storage period.

Description

Lactobacillus plantarum and application thereof

Technical Field

The invention relates to lactobacillus plantarum and application thereof, and belongs to the technical field of microbial fermentation.

Background

Fermented foods have been an important component of the human diet worldwide, and the diversity of the fermented product categories reflects the diversity of microorganisms in driving the fermentation process.

Northeast pickled Chinese cabbage is a popular traditional fermented food in Chinese pickled Chinese cabbage, the traditional fermentation period depending on natural microorganisms can reach tens of days, and the complex microbial activities can also make the fermentation process difficult to control. Current developments in the northeast sauerkraut industry have made it difficult to meet consumer demand.

Pickled Chinese cabbage is widely favored by consumers due to its own nutritional value (rich in vitamins, organic acids, amino acids, etc.) and unique flavor. In order to meet the higher demands on the yield and quality of pickled Chinese cabbage, continuous researches on the pickled Chinese cabbage fermentation process and fermentation system are carried out. At present, the fermentation of pickled Chinese cabbage by adding a large amount of salt and inoculating lactobacillus is the most effective means for controlling the fermentation process and optimizing the product quality. In general, the growth of spoilage bacteria and pathogenic bacteria is inhibited by adding high-concentration salt into traditional pickled Chinese cabbage, so that the growth of lactic acid bacteria with poor salt tolerance is inhibited, the growth of harmful bacteria cannot be effectively controlled, if harmful bacteria such as escherichia coli cannot be inhibited from propagating wantonly, the report of food-borne pathogenic bacteria in high-hydrochloric acid foods is more in recent years, the high-salt intake also increases the health risk, causes certain diseases, inhibits the growth of lactic acid bacteria, prolongs the fermentation period, and therefore, the rise of low-salt fermentation products becomes hot. However, natural fermentation under low-salt or salt-free conditions often does not guarantee the quality of the product.

Under salt-free conditions, research to ensure stable fermentation and ultimately obtain high quality pickled Chinese cabbage remains of little interest. Therefore, the research of developing the non-hydrochloride pickled Chinese cabbage is of great significance.

Disclosure of Invention

The invention aims to solve the technical problem of providing a lactobacillus plantarum strain for salt-free fermentation of pickled Chinese cabbageLactiplantibacillus plantarum）suan-15。

Meanwhile, the invention provides a mixed starter, which is prepared by compounding excellent strains screened from northeast pickled Chinese cabbage with lactobacillus acidophilus KLDS1.0901 and corn juice.

Meanwhile, the invention provides a preparation method of the mixed starter, which utilizes the excellent strain screened from the northeast pickled Chinese cabbage to find an optimal fermentation condition capable of improving the product quality in a salt-free environment, improves the pickled Chinese cabbage quality, reduces the high-salt hazard, and simultaneously can avoid the adverse effect caused by secondary fermentation of the traditional fermented pickled Chinese cabbage in the daily production and storage processes.

Meanwhile, the invention provides the non-hydrochloric pickled Chinese cabbage and the preparation method thereof, and provides a certain theoretical guidance and technical support for developing the non-hydrochloric pickled Chinese cabbage.

Meanwhile, the invention provides a lactobacillus plantarum strainLactiplantibacillus plantarum) Use of suan-15 in fermenting non-hydrochloric acid pickle.

Meanwhile, the invention provides application of the mixed starter in fermenting the non-hydrochloric pickled Chinese cabbage.

In order to solve the technical problems, the invention adopts the following technical scheme:

lactobacillus plantarum strain Lactiplantibacillus plantarum) And (3) suan-15, wherein the lactobacillus plantarum is preserved in China center for type culture collection (China center for type culture collection), the preservation date is 2022, 10 and 19 days, and the preservation number is CCTCC NO: m20221595.

Comprises a plant lactobacillus strainLactiplantibacillus plantarum) A mixed starter of suan-15 comprising lactic acid bacteria and corn juice; the lactobacillus comprises lactobacillus plantarum suan-15 and lactobacillus acidophilusLactobacillus acidophilus) KLDS1.0901; the viable count of the lactobacillus plantarum suan-15 and the lactobacillus acidophilus KLDS1.0901 is 6 multiplied by 10 ⁹ CFU/mL; the volume ratio of the lactobacillus plantarum suan-15 to the lactobacillus acidophilus KLDS1.0901 is 2:1;250g of cabbage has a lactic acid bacteria inoculation amount of 450 mu L and a corn juice addition amount of 7.5g.

A method for preparing a mixed starter, comprising the steps of: the viable count is 6 multiplied by 10 ⁹ CFU/mL lactobacillus plantarum span-15 and lactobacillus acidophilus KLDS1.0901 are respectively activated in MRS liquid culture medium for two generations to obtain bacterial liquid, the bacterial liquid is diluted by saturated NaCl solution for 10 times to obtain diluted liquid, 450 mu L of lactobacillus plantarum span-15 diluted liquid and lactobacillus acidophilus KLDS1.0901 diluted liquid are mixed according to the proportion of 2:1, the supernatant is removed by centrifugation, bacterial sludge is obtained, the bacterial sludge is washed twice by saturated NaCl solution, and 15.0g of sterile fermentation culture liquid consisting of 7.5g of corn juice and distilled water with the same mass is added.

The preparation method of the corn juice comprises the following steps: pulping fruit corn and water according to a mass ratio of 1:10, filtering with four layers of gauze, collecting filtrate, and sterilizing at 121deg.C for 20min.

The centrifugation process is 8000r/min for 3min.

A preparation method of a non-hydrochloric pickled Chinese cabbage adopting a mixed starter comprises the following steps:

s1, preprocessing cabbage: removing roots and old leaves of Chinese cabbage, cleaning, scalding for 3-5 seconds by boiling water, draining, shredding, weighing 250g each time, and filling into a polyethylene film bag;

s2, adding the mixed starter into the polyethylene film bag in the step S1, fully mixing, and sealing by using a vacuum fresh-keeping machine;

s3, fermenting: fermenting at 30deg.C for 5 days to obtain mature non-hydrochloride pickled Chinese cabbage;

s4, storing: stored at 4℃for 60 days.

Lactobacillus plantarum strainLactiplantibacillus plantarum) Use of suan-15 in fermenting non-hydrochloric acid pickle.

An application of mixed starter in fermenting non-hydrochloric pickled Chinese cabbage is provided.

A method for preparing a non-hydrochloride pickled Chinese cabbage.

Compared with single lactobacillus fermentation, the invention has important contribution to improving the quality of fermentation products by mixed fermentation. The mixed fermentation can make the number of enterobacteriaceae lower than the detection limit in a short time. In addition, strains of mixed ferments may produce metabolites that mutually stimulate or inhibit growth, which further complicate the dynamic changes in the structure of the flora.

The invention screens out a plant lactobacillus from 9 plants separated from northeast pickled Chinese cabbage, has the best fermentation performance and the strongest antibacterial capability, and combines the plant lactobacillus with lactobacillus acidophilus, and after optimizing the fermentation condition, the process is used for fermenting the pickled Chinese cabbage without salt; then, measuring a plurality of indexes including pH value, total acid content, nitrite content, reducing sugar content, vitamin C content and organic acid content in the fermentation process, and analyzing the sensory characteristics of the finished product through an electronic nose and an electronic tongue; finally, the stability of the non-hydrochloric pickled vegetable in storage at 4℃for 60 days was evaluated.

Through the research, the invention has the following beneficial effects:

(1) 2 strains of lactobacillus, namely lactobacillus plantarum span-15 and lactobacillus acidophilus KLDS1.0901, are screened out by taking the growth activity, the acid production capacity and the antibacterial capacity as screening indexes, and the optimal growth temperature of lactobacillus plantarum is 30 ℃ as the fermentation temperature.

(2) The sensory evaluation score is used as an index, and the optimal fermentation process condition is determined through a single factor test and a response surface test: the ratio of lactobacillus (lactobacillus plantarum suan-15: lactobacillus acidophilus KLDS 1.0901) is 2:1, the inoculation amount of lactobacillus in 250g of Chinese cabbage is 450 mu L, and the addition amount of corn juice is 7.5g.

(3) The non-hydrochloride pickled Chinese cabbage is mature in 5 days, and the detection of some indexes in the fermentation process shows that: the pH value, the reducing sugar content, the vitamin C content and the nitrite content are obviously reducedp<0.05 Total acid content and total organic acid level are obviously increasedp<0.05 A) is provided; the fermentation degree of the non-hydrochloric pickled Chinese cabbage is more thoroughThe residual sugar amount is less, the organic acid content is obviously increased, the nitrite content is extremely low and reaches 0.23+/-0.10 mg/kg, and the finished product of the non-hydrochloride pickled Chinese cabbage has good quality characteristics.

(4) The pH value and the total acid content of the non-hydrochloride pickled Chinese cabbage are stable at 4 ℃, and the nitrite content is continuously reduced, so that the stability of the non-hydrochloride pickled Chinese cabbage in a storage period of 60 days is proved to be better.

Drawings

FIG. 1 is a schematic diagram of a test technique of the present invention;

FIG. 2 shows growth curves of 9 Lactobacillus plantarum strains at different temperatures, wherein a) 22 ℃; b) 26 ℃; c) 30 ℃; d) 34 ℃; e) 38 ℃;

FIG. 3 shows the pH of 9 Lactobacillus plantarum fermentation broths at different temperatures, wherein a) 22 ℃; b) 26 ℃; c) 30 ℃; d) 34 ℃; e) 38 ℃;

FIG. 4 shows the acid yield of 9 Lactobacillus plantarum strains at different temperatures, wherein a) 22 ℃; b) 26 ℃; c) 30 ℃; d) 34 ℃; e) 38 ℃;

FIG. 5 shows the bacteriostatic effect of various Lactobacillus plantarum on E.coli ATCC 25922;

FIG. 6 shows fermentation characteristics of Lactobacillus acidophilus KLDS1.0901 and Lactobacillus acidophilus KLDS1.0902 at 30 ℃; wherein a) a growth curve; b) Fermentation broth pH and strain acid yield;

FIG. 7 shows the pH and total acid content of different proportions of lactic acid bacteria fermented non-hydrochloric acid pickle; wherein a) the pH value changes; b) Total acid content variation;

FIG. 8 shows the variation of nitrite content in different proportions of lactic acid bacteria fermented non-hydrochloric acid pickled vegetable;

FIG. 9 is a plot of the sensory scores of different proportions of lactic acid bacteria fermented non-hydrochloric acid vegetables;

FIG. 10 shows the pH and total acid content changes in fermented non-hydrochloric acid vegetables at different lactic acid bacteria inoculum sizes; wherein a) the pH value changes; b) Total acid content variation;

FIG. 11 shows the variation of nitrite content in fermented non-hydrochloric acid vegetable under different lactic acid bacteria inoculation;

FIG. 12 is a plot of the sensory scores of fermented non-hydrochloric acid vegetables at different lactic acid bacteria inoculum sizes;

FIG. 13 shows the pH and total acid content of fermented non-hydrochloric acid vegetables at various corn juice additions; wherein a) the pH value; b) Variation of total acid content;

FIG. 14 shows the change in nitrite content of fermented non-hydrochloric acid vegetable at various corn juice addition levels;

FIG. 15 is a plot of the sensory scores of fermented non-hydrochloric acid vegetables at different corn juice additions;

FIG. 16 is response surface test interaction;

FIG. 17 shows the pH and total acid content changes during fermentation;

FIG. 18 is a nitrite standard curve and changes in nitrite content during fermentation; wherein a) a nitrite standard curve; b) The change of nitrite content in the fermentation process;

FIG. 19 shows the change in reducing sugar and vitamin C content during fermentation;

FIG. 20 is a radar chart of an electronic nose sensor versus a sample;

FIG. 21 is a PCA analysis of samples from different fermentation periods by the electronic nose;

FIG. 22 is an LDA analysis of samples from different fermentation periods by the electronic nose;

FIG. 23 is a radar chart of an electronic tongue sensor versus a sample;

FIG. 24 is a graph showing the effect of 4℃on pH, total acid content and nitrite content of a non-salted vegetable;

figure 25 is the effect of 4 ℃ storage on the no-hydrochloride pickled cabbage sensory score.

Detailed Description

The invention will be further described with reference to the accompanying drawings.

In the invention, the Latin name of the lactobacillus plantarum sun-15 isLactiplantibacillus plantarumAnd (3) suan-15 is preserved in China center for type culture collection (CCTCC NO), wherein the preservation date is 2022, 10 months and 19 days: m20221595. Latin name of Lactobacillus acidophilus KLDS1.0901 Lactobacillus acidophilusKLDS1.0901, which is preserved in the northeast agricultural university dairy emphasis laboratory and isolated from Xinjiang traditional fermented dairy products, the lactobacillus acidophilus KLDS1.0901 in the present invention is obtained from the prior art sources: lactobacillus acidophilus KLDS1.0901 fermentation characteristics for yogurtInfluence of antioxidant Activity [ J]Food research and development, 2019, 22.

1 materials and methods

1.1 materials and instruments

1.1.1 test strains

The test was performed using lactic acid bacteria isolated from northeast sauerkraut and deposited in the major laboratory of dairy education department of northeast agricultural university. The specific strain information is as follows: the bacterial strain sources are lactobacillus plantarum span-1, lactobacillus plantarum span-3, lactobacillus plantarum span-5, lactobacillus plantarum span-9, lactobacillus plantarum span-12, lactobacillus plantarum span-14, lactobacillus plantarum span-15, lactobacillus plantarum span-16, lactobacillus plantarum span-17 and lactobacillus acidophilus KLDS1.0901, lactobacillus acidophilus KLDS1.0902 and escherichia coli ATCC25922 preserved in a laboratory.

1.1.2 Test materials

Fresh cabbages used in the test are purchased from the Biyou supermarket in the International conference center of Harbin city of Heilongjiang province.

1.1.3 Culture medium

MRS liquid Medium/L: 5g of peptone, 10g of tryptone, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 20g of glucose, 5g of yeast powder, 0.25g of manganese sulfate, 0.58g of magnesium sulfate, 2g of dipotassium hydrogen citrate, 5g of beef extract, 1g of tween-80 and the balance of distilled water.

Corn juice fermentation medium: pulping fruit corn and water according to a mass ratio of 1:10, filtering with four layers of gauze, collecting filtrate, and sterilizing at 121deg.C for 20min.

1.2 Test method

1.2.1 Screening of lactic acid bacteria

1.2.1.1 Measurement of growth Properties of Lactobacillus plantarum at different temperatures

Activating the strain in MRS liquid culture medium for two generations to reach thallus concentration of 6×10 ⁹ CFU/mL was inoculated into MRS liquid medium at an inoculum size of 2%, cultured at 22℃at 26℃at 30℃at 34℃at 38℃for 24 hours, and OD was measured at 0, 4, 8, 12, 16, 20 and 24 hours, respectively ₆₀₀ And the pH value of the culture solution is repeated three times, the average value is taken, and the growth curve of the strain is drawn.

1.2.1.2 Determination of acid production amount of lactobacillus plantarum at different temperatures

Activating the strain in MRS liquid culture medium for two generations to reach thallus concentration of 6×10 ⁹ CFU/mL is inoculated into MRS liquid culture medium with 2% inoculum size, cultured for 24h at 22 ℃, 26 ℃, 30 ℃, 34 ℃ and 38 ℃ respectively, 1mL of fermentation broth is measured and placed into a 150mL conical flask, 50mL of distilled water is used for dilution, phenolphthalein is used as an indicator, 0.1mol/L NaOH standard solution is used for titration until the solution is reddish, the volume of the NaOH standard solution is recorded, the process is repeated three times, and the average value is obtained.

Acid yield (%) = C×V×0.09×100/V ₀

And (3) injection:C-NaOH standard solution concentration (mol/L);V-consume the volume (mL) of NaOH standard solution;V ₀ -volume of sample solution (mL).

1.2.1.3 Determination of bacteriostasis of different Lactobacillus plantarum: adopts oxford cup quantitative diffusion method.

1.2.1.4 Screening of Lactobacillus acidophilus with optimal fermentation Performance at selected temperature

Activating the strain in MRS liquid culture medium for two generations to reach thallus concentration of 6×10 ⁹ CFU/mL was inoculated into MRS liquid medium at an inoculum size of 2%, cultured at the optimum growth temperature for Lactobacillus acidophilus with the best fermentation performance for 24h, and OD was measured at 0, 4, 8, 12, 16, 20 and 24h, respectively ₆₀₀ And the pH value of the culture solution is repeated three times, the average value is taken, and the growth curve of the strain is drawn. The method for measuring the acid yield of the strain is the same as 1.2.1.2.

1.2.2 Optimization of production process of pickled Chinese cabbage without salt

1.2.2.1 The fermentation process of the non-hydrochloric pickled Chinese cabbage comprises the following steps: removing roots and old leaves of commercially available cabbages, cleaning, scalding for 3-5 seconds by boiling water, draining, shredding, weighing 250g of the cabbages each time, filling the cabbages into a polyethylene film bag, inoculating lactobacillus plantarum suan-15 obtained by screening and lactobacillus acidophilus KLDS1.0901 according to a certain proportion, fully mixing the cabbages with a certain amount of corn juice culture medium, sealing the cabbages by a vacuum fresh-keeping machine, and selecting a proper temperature for fermentation.

1.2.2.2 Fermentation process optimization

(1) Lactic acid bacteria compounding ratio is excellentAnd (3) chemical treatment: the number of living bacteria is 6×10 ⁹ CFU/mL lactobacillus plantarum span-15 and lactobacillus acidophilus KLDS1.0901 are respectively activated in MRS liquid culture medium for two generations to obtain bacterial liquid, the bacterial liquid is diluted 10 times by normal saline to obtain diluted bacterial liquid, 450 mu L of lactobacillus plantarum span-15 diluted bacterial liquid and lactobacillus acidophilus KLDS1.0901 diluted bacterial liquid are placed in an EP tube according to the proportion of 3:1, 2:1, 1:1, 1:2 and 1:3, centrifugation is carried out for 3min at 8000rpm, supernatant is removed to obtain bacterial mud, the bacterial mud is washed twice by the normal saline, and the bacterial mud is added into 15.0g of sterile fermentation culture liquid consisting of 7.5g of corn juice and distilled water with the same mass. And determining the optimal compounding proportion by taking the sensory evaluation score as an index.

(2) Optimizing the inoculation amount of the strain: the viable count is 6 multiplied by 10 ⁹ CFU/mL of Lactobacillus plantarum span-15 and Lactobacillus acidophilus KLDS1.0901 were activated in MRS broth for two generations, the broth was diluted 10-fold with physiological saline, 150. Mu.L, 300. Mu.L, 450. Mu.L, 600. Mu.L and 750. Mu.L of Lactobacillus plantarum span-15 and Lactobacillus acidophilus KLDS1.0901 were placed in EP tubes at 1:1, centrifuged at 8000rpm for 3min, the supernatant was removed, the puree washed twice with physiological saline, and added to 15.0g of sterile fermentation broth consisting of 7.5g of corn juice and the same mass of distilled water. And determining the optimal strain adding amount by taking the sensory evaluation score as an index.

(3) Optimizing the adding amount of corn juice: the viable count is 6 multiplied by 10 ⁹ CFU/mL of Lactobacillus plantarum span-15 and Lactobacillus acidophilus KLDS1.0901 were activated in MRS broth for two generations, the broth was diluted 10-fold with physiological saline, 450. Mu.L of Lactobacillus plantarum span-15 and Lactobacillus acidophilus KLDS1.0901 were placed in EP tube at 1:1, centrifuged at 8000rpm for 3min, the supernatant was removed, the bacterial sludge was washed twice with physiological saline, and 15.0g of sterile fermentation broth prepared from 2.5g, 5.0g, 7.5g, 10.0g and 12.5g of corn juice and distilled water was added. And determining the optimal corn juice adding amount by taking the sensory evaluation score as an index.

(4) Fermentation process parameter response surface test: according to the result of the single factor test, the Design-expert13.0 software is adopted to carry out test Design, the sensory evaluation score is selected as a response value, 3 independent variable factors of the lactobacillus compound proportion (A), the strain inoculation amount (B) and the corn juice addition amount (C) are optimized, and the specific test factor level codes are shown in table 1.

TABLE 1 Box-Benhnken center test factor level coding

1.2.2.3 Physical and chemical index and chemical component detection method

(1) Determination of pH: 5.00g of pickled Chinese cabbage is mixed with the same amount of water for homogenate, and measured by a pH meter.

(2) Determination of total acids: reference is made to the method in GB 12456-2021 determination of total acids in food.

(3) Determination of nitrite content: reference is made to GB5009.33-2016 "determination of nitrite and nitrate in food safety national Standard food".

(4) Determination of reducing sugar: reference is made to the direct titration method in GB5009.7-2016 "determination of reducing sugars in food safety national standard food".

(5) Determination of vitamin C: reference is made to the titration method of 2, 6-dichloroindophenol in GB5009.86-2016 "determination of anti-Cycloblood acid in food safety national Standard food".

(6) Determination of organic acids: reference is made to the method in GB5009.157-2016 "determination of organic acids in food".

(7) Electronic nose analysis: the electronic nose analysis is carried out by a DM6 electronic nose device, and comprises sensors W1C, W5S, W3C, W6S, W5C, W1S, W1W, W2S, W W and W3S, wherein the main volatile gas types are benzene, oxynitride, ammonia and aromatic compounds, hydride, short-chain alkane, methyl, aldolone, terpene, organic sulfide and long-chain alkane. Briefly, 2g of the sauerkraut sample was placed in a 20mL headspace bottle, capped with a polytetrafluoroethylene membrane, and equilibrated at room temperature prior to testing. Headspace gas was injected at a constant flow rate of 300mL/min for 60s, and sensor signals were recorded every second during the test. After each test was completed, the air after filtration was used for rinsing for 30s. Setting 3 times of parallel tests, taking an average value for subsequent statistical analysis.

(8) Electronic tongue analysis: electronic tongue analysis was performed using an SA402B electronic tongue apparatus, comprising five taste sensors, AAE, CT0, CA0, AE1, and C00, for testing umami, salty, sour, sweet, and bitter tastes, respectively. Briefly, 5g of a pickled Chinese cabbage sample was homogenized by adding 100mL of ultrapure water, sonicated for 15min, and centrifuged at 10000 rpm for 10min, and the supernatant was subjected to loading analysis. Setting 3 times of parallel tests, taking an average value for subsequent statistical analysis.

(9) Sensory evaluation: 10 persons are selected to carry out sensory evaluation on the finished pickled Chinese cabbage, and the color of vegetables, the color of a pickling solution, the hardness and softness of the pickled Chinese cabbage, package and smell are taken as evaluation items, and specific scoring criteria are shown in the following table.

TABLE 2 sensory evaluation criteria for the Pickle-free finished products

1.2.3 Storage stability test at 4 ℃): and (3) preserving the finished product fermented by the optimal fermentation condition for 60 days at the temperature of 4 ℃, detecting the pH value, the total acid content and the nitrite content of the pickled Chinese cabbage every 5 days, and evaluating the stability of the non-pickled Chinese cabbage under the conventional refrigeration condition by comprehensive sensory evaluation.

1.2.4 And (3) data processing: all experiments were set up in three replicates, data were shown as "mean ± standard deviation", and data were subjected to ANOVA statistical analysis using SPSS 20.0 software for level of significant differences p<0.05. The plots were made using the OriginPro 9.1 software.

2 results and analysis

2.1 Screening of fermentation strains

2.1.1 Growth activity of Lactobacillus plantarum at different temperatures

The growth activity of the lactobacillus plantarum at different temperatures is shown in figure 2, and the pH value is shown in figure 3. As shown in the figure, 9 lactobacillus plantarum are in a slow growth period within 0-4 hours, and in order to adapt to a new inoculation environment, the strain grows slowly and has an OD (optical density) at the stage ₆₀₀ The value is slowly increased, and the pH value of the fermentation liquor is slowly decreased. As the strain grows into log phase, the strain grows vigorously and OD ₆₀₀ The pH value is rapidly increased, the pH value is rapidly decreased, and the duration is 4-20 hours. After that, the growth enters a stable stage, the proliferation speed of the strain is reduced, and the OD ₆₀₀ The value is kept relatively stable, and the pH value of the fermentation liquor is stabilized between 3.65 and 4.10. In the comprehensive view, 9 lactobacillus plantarum can grow well in the temperature range of 22-38 ℃. Wherein, at 30 ℃, lactobacillus plantarum span-15 shows better growth activity, and the pH of the fermentation liquor drops fastest and drops from 5.83+/-0.02 to 3.70+/-0.01 above other culture temperatures. This result reflects the good growth characteristics and acid producing ability of the strain.

2.1.2 Acid producing ability of lactobacillus plantarum at different temperatures

The acidogenic capacity of Lactobacillus plantarum at different temperatures is shown in FIG. 4. As shown in the figure, the pH value of the fermentation liquid is in negative correlation with the acid production amount of the thalli, the acid production amount of 9 lactobacillus plantarum is slowly increased within 0-4 hours, the acid production amount is rapidly increased in the logarithmic growth stage of the thalli, and then the trend is gentle during 20-24 hours. At 20 ℃ and 26 ℃, the acid production difference of different lactobacillus plantarum is larger, but the acid production is lower than the higher temperature. At 30 ℃, the acid yield of the lactobacillus plantarum suan-15 is increased most within 24 hours, and the final acid yield can reach 1.821+/-0.02%, which is consistent with the research result of the strain growth activity. Therefore, lactobacillus plantarum span-15 with the best fermentation performance is selected as a subsequent fermentation strain, and the optimal culture temperature of the lactobacillus plantarum span-15 is selected as the fermentation temperature.

2.1.3 Inhibition ability of different Lactobacillus plantarum to Escherichia coli

Pathogenic escherichia coli is a common food-borne pathogen, and traditional fermented pickled Chinese cabbage is easily polluted by pathogenic bacteria and spoilage bacteria, especially escherichia coli, so that the quality of products is influenced and the human health is endangered. The inhibitory activity of 9 Lactobacillus plantarum on escherichia coli ATCC25922 is shown in figure 5, and the strong bacteriostatic ability is that the bacteriostatic activity of Lactobacillus plantarum span-15 and Lactobacillus plantarum span-9, 2 Lactobacillus plantarum has no significant difference p>0.05). The diameter of the inhibition zone ranges from 10mm to 17mm, and particularly the lactobacillus plantarum span-15 shows the strongest antibacterial activity, and the diameter of the inhibition zone reaches 16.40+/-0.30 mm. By using the strainThe safety of the fermented pickled Chinese cabbage can be ensured due to the strong antibacterial property.

2.1.4 Fermentation performance of Lactobacillus acidophilus at 30deg.C

The fermentation characteristics of Lactobacillus acidophilus KLDS1.0901 and Lactobacillus acidophilus KLDS1.0902 are shown in FIG. 6.2 Lactobacillus acidophilus has slow growth and OD within 0-4 hours ₆₀₀ And the acid yield of the strain is slowly increased, the pH value of the fermentation liquor is slowly reduced, and no obvious difference exists. At the log phase of strain growth, lactobacillus acidophilus KLDS1.0901 grew more vigorously, proliferated faster, and produced more acid than lactobacillus acidophilus KLDS 1.0902. The acid yield can reach 1.27% after 24 hours. Lactobacillus acidophilus KLDS1.0901 has more excellent fermentation properties at 30 ℃. Therefore, lactobacillus acidophilus KLDS1.0901 was selected as the fermenting strain for the subsequent compounding of the starter. Lactobacillus plantarum span-15 and lactobacillus acidophilus KLDS1.0901 are selected as strains with optimized fermentation process parameters, and are hereinafter respectively called lactobacillus plantarum and lactobacillus acidophilus for short.

2.2 Optimization of fermentation process parameters

2.2.1 Optimization of lactic acid bacteria compounding proportion

2.2.1.1 pH and Total acid content

The non-hydrochloric acid pickled Chinese cabbage inoculated with Lactobacillus plantarum and Lactobacillus acidophilus in different proportions has pH and total acid content as shown in figure 7 during fermentation. The lactobacillus compounding proportion has obvious influence on the pH value and the total acid content in the fermentation process of the non-hydrochloride pickled Chinese cabbage. The initial pH value of the sample is between 6.0 and 6.2, the pH value of all sample groups is rapidly reduced on the 1 st day in the fermentation process, then gradually reduced, finally stabilized, and the total acid content is slowly increased from rapid increase until fermentation is mature. The inoculated lactobacillus is better adapted to anaerobic environment, thereby utilizing energy source substance metabolism to produce acid and proliferation in large quantity. The pH value and the total acid content of all the sample groups tend to be stable after 5 days, and the acidic environment caused by flora metabolism has a certain inhibition effect on the growth of lactobacillus. At this time, the pH value of the sample group is lower than 3.4, and the total acid content is 0.53-0.65 g/100g, so that the fermentation is considered to be mature.

At the time of fermentation maturation, the ratio of lactobacillus (lactobacillus plantarum: lactobacillus acidophilus) is 2:1The pH value of the mixed fermentation group with the ratio of 1:1 is obviously lower than that of other sample groups; the corresponding total acid is accumulated to the maximum of 0.61-0.64 g/100g, and no obvious difference exists between the two groups p>0.05). The pH value of the mixed fermentation group of lactobacillus (lactobacillus plantarum: lactobacillus acidophilus) with the ratio of 1:3 is the highest and is 3.35+/-0.01; the total acid content is obviously lower than that of other sample groupsp<0.05 0.54.+ -. 0.03g/100 g).

2.2.1.2 Nitrite content

The non-hydrochloride pickled Chinese cabbage inoculated with lactic acid bacteria in different proportions has nitrite content in the fermentation process as shown in figure 8. The nitrite content in all sample groups showed a continuous decreasing trend throughout the fermentation process, no nitrite peak appeared, and was always below the highest limit value of 20mg/kg, decreasing to the lowest level after 5 days. At the initial stage of fermentation (0-1 day), the proportion of lactobacillus (lactobacillus plantarum: lactobacillus acidophilus) is similar to the decrease trend of the nitrite content of a 1:2 sample group and a 1:3 sample group, and the proportion is similar to the decrease trend of a 3:1 sample group, a 2:1 sample group and a 1:1 sample group.

When the ratio of the inoculated lactobacillus (lactobacillus plantarum: lactobacillus acidophilus) is 2:1, the nitrite content reaches the same group minimum level of 0.23+/-0.10 mg/kg at the time of fermentation maturation. When the ratio of lactobacillus (lactobacillus plantarum: lactobacillus acidophilus) is 1:3, the nitrite content reaches the highest level of the same group, which is 1.31+/-0.10 mg/kg. These results show that the nitrite concentration of the fermented pickled Chinese cabbage can be effectively controlled by inoculating the two strains of lactic acid bacteria for mixed fermentation.

2.2.1.3 sensory evaluation

Sensory evaluation of lactic acid bacteria fermented non-hydrochloric acid dish in different proportions is shown in fig. 9. The lactobacillus (lactobacillus plantarum: lactobacillus acidophilus) has a ratio of 2:1, and the sensory indexes of the lactobacillus plantarum/lactobacillus plantarum sample are superior to those of other sample groups except the tissue morphology, and the comprehensive score is 42.36+/-0.10. Overall, no bag expansion occurred in all sample groups, and the comprehensive scoring condition was 2:1 sample groups>1:1 sample group>3:1 sample group>1:3 sample set>A 1:2 sample group, wherein the scores of the 3:1 sample group, the 1:3 sample group and the 1:2 sample group are not significantly differentp>0.05). For this index of tissue morphology, lactic acid bacteria (Lactobacillus plantarum: lactobacillus acidophilus)Bacteria) ratio of 2:1, which may have a significant relationship with its lower pH. And (3) integrating the results of all indexes, wherein the sensory evaluation score of the pickled Chinese cabbage product is highest when the mixed bacteria ratio is 2:1, and the pickled Chinese cabbage product is regarded as the most suitable compound ratio for fermentation.

2.2.2 Optimization of lactic acid bacteria inoculum size

2.2.2.1 pH and Total acid content

The pH and total acid content of the non-hydrochloric acid pickled Chinese cabbage with different lactobacillus inoculation amounts are shown in figure 10. Within the first 1 day, the pH value of all the pickled vegetable samples rapidly drops from about 6.0 to about 3.2-3.3; the change of the total acid content is inversely related to the pH value, the total acid content increases with time in all samples, and after fermentation for 5 days, the total acid content is stabilized within the range of 0.54-0.62 g/100g, and the total acid content reaches the matured pickled Chinese cabbage standard in DBS 22/025-2014 local standard pickled Chinese cabbage. In the early fermentation period (0-3 days), the pH value is reduced along with the increase of the inoculation amount of the lactic acid bacteria, and the total acid content is increased along with the increase of the inoculation amount of the lactic acid bacteria; subsequently, the inoculation amount of the lactic acid bacteria is 450 mu L of sample group, and the index change of the sample group is not significantly different from that of the sample group of 600 mu L and the sample group of 750 mu L p>0.05）。

When the inoculation amount of the lactic acid bacteria is 450 mu L, the pH value at the time of fermentation maturation is the lowest, the total acid content accumulated after 3.24+/-0.01,5 days is the highest, and the total acid content reaches 0.61+/-0.01 g/100g. The mixed inoculum size showed a more pronounced acidification rate at 750 μl, corresponding to pH. In the research, the pH value at the early stage of fermentation is quickly reduced to below 4.0, and meanwhile, the total acid content is quickly increased, so that the added mixed bacteria can be ensured to be dominant bacteria, the fermentation is obviously accelerated, and the production of pickled Chinese cabbage is accelerated.

2.2.2.2 Nitrite content

The change of nitrite content in the fermented non-hydrochloric acid dish with different lactobacillus inoculum is shown in figure 11. The nitrite content of all the sauerkraut samples was continuously reduced during fermentation and reduced to a minimum level on day 5. In the early fermentation period (0-3 days), the nitrite content is reduced along with the increase of the lactobacillus inoculation amount, and the lactobacillus inoculation amount is no obvious difference between a 450 mu L sample group and 600 mu L sample group and 750 mu L sample groupp>0.05). When the inoculation amount of the lactic acid bacteria is 450-750 mu L, the nitrite content of the sample group is always lower than that of other sample groups, and the minimum nitrite content of the fermentation on the 5 th day is 0.65+/-0.21 mg/kg. The lactobacillus inoculation amount shows the highest nitrite content at 150 mu L, which is 1.31+/-0.10 mg/kg. Furthermore, the nitrite content in all the fermented samples was lower than the initial content, and these results indicate that the nitrite content in the non-hydrochloride pickled Chinese cabbage was effectively controlled.

2.2.2.3 Sensory evaluation

Sensory evaluation of different lactobacillus inoculation amounts of fermented non-hydrochloric acid vegetables is shown in figure 12. The sample group with the inoculation amount of the lactobacillus of 450 mu L has better sensory indexes in terms of color, tissue morphology and smell of the stain than other sample groups, and the comprehensive score is 42.2+/-0.12. In the whole, the bag expansion phenomenon does not occur in all the sample groups, and the comprehensive scoring condition is that the lactobacillus inoculation amount is 450 mu L of the sample group, and the sample group is more than 600 mu L of the sample group, more than 300 mu L of the sample group, more than 150 mu L of the sample group, and more than 750 mu L of the sample group. And (3) integrating the results of all indexes, wherein the sensory evaluation score of the pickled Chinese cabbage product with the lactobacillus inoculation amount of 450 mu L is highest, and the pickled Chinese cabbage product is regarded as the lactobacillus inoculation amount most suitable for fermentation.

2.2.3 Optimization of corn juice addition

2.2.3.1 pH and Total acid content

The pH and total acid content of the fermented non-hydrochloric acid dish were varied at various corn juice additions as shown in FIG. 13. After fermentation for 1 day, lactobacillus proliferates rapidly in the fermentation system, the pH value of the acid accumulated by metabolism reduces rapidly to about 3.5, and then the too low acid environment inhibits the growth of lactobacillus, so that the pH value of the sample group reduces slowly, and the total acid content increases slowly until the sample group is stable. It is obviously observed that in the early fermentation period (0-2 days), the pH value is reduced along with the increase of the adding amount of the corn juice, the total acid content is increased along with the increase of the adding amount of the corn juice, then the adding amount of the corn juice is in the range of 2.5-7.5 g, and the lower the pH value is, the higher the total acid content is; when the addition amount is within the range of 7.5-12.5 g, the difference between the pH value and the total acid content is not obvious along with the increase of the addition amount p>0.05). Therefore, the acid level of the sample group with the addition amount of 7.5-12.5 g is most obvious, and the lowest pH value of the mature sample is3.24+ -0.01, corresponding to a total acid content of up to 0.62+ -0.01 g/100g.

2.2.3.2 Nitrite content

The results of the study on the change of the nitrite content of the fermented non-hydrochloric acid pickled vegetable under different corn juice addition amounts are shown in figure 14. All sample groups showed a trend of continuously decreasing nitrite content, and consistently below the initial content. In the fermentation process, when the adding amount of corn juice is 7.5-12.5 g, the nitrite content of a sample group is always lower than that of other sample groups, the lowest nitrite content is 0.65+/-0.21 mg/kg on the 5 th day of fermentation, and at the moment, the nitrite content difference of the sample group with the adding amount of corn juice of 7.5-12.5 g is not obviousp>0.05). The corn juice shows the highest nitrite content of 1.55+/-0.10 mg/kg when the adding amount of the corn juice is 2.5g, and the nitrite content reduction rate is obviously lower than that of a sample group of 7.5-12.5 gp<0.05）。

2.2.3.3 Sensory evaluation

The sensory evaluation results of the fermented non-hydrochloric acid pickle with different corn juice addition amounts are shown in figure 15.

The five indexes of vegetable color, stain color, tissue morphology, packaging and smell are combined, and obvious differences exist between sample groups p<0.05). The corn juice added at 7.5g showed the best vegetable color and smell, corresponding to the highest total acid content of the sample group, probably due to the high acidity improving the quality of the pickled cabbage. And combining the sensory scores in all aspects, wherein the total score of the fermented non-hydrochloric acid pickled Chinese cabbage is higher than that of other sample groups and is 41.48+/-0.35 when the adding amount of the corn juice is 7.5g, and the total score of the fermented non-hydrochloric acid pickled Chinese cabbage is regarded as the optimal adding amount of the fermented corn juice.

2.2.4 Optimization of optimal fermentation parameters

On the basis of a single-factor experiment, a proper horizontal range is selected, 3 factors including a lactobacillus compounding proportion (A), a strain inoculation amount (B) and a corn juice adding amount (C) are used as independent variables, a sensory score (Y) is used as a response value, a non-hydrochloric pickled vegetable fermentation process is optimized according to a Box-Behnken center combination design principle, and a sensory score of 17 groups of experiments is shown in table 3.

TABLE 3 response surface test design and results

According to regression model analysis of variance, a second polynomial regression equation of sensory evaluation score to independent variable lactobacillus compound proportion (A), strain inoculum size (B) and corn juice additive amount (C) is obtained as follows:

Y=45.63+0.72A+1.92B+1.74C-2.17AB+1.48AC+0.68BC-1.71A ² -4.68B ² -3.93C ²

TABLE 4 regression model analysis of variance

Note that: * Representing remarkable%p<0.05 A) is provided; * Representing extremely remarkable% p<0.01 A) is provided; -representing insignificant.

As can be seen from Table 4, the response surface model established in the present study is extremely remarkablep<0.01 The model mismatch term has an F value of 1.45,pa value of 0.3543, not significantp>0.05 The regression equation is shown to have better fit to the test. The determination coefficient R of the model ² = 0.9761, correct the determination coefficient R ² _Adj = 0.9454, illustrating that the model can interpret changes in 94.54% response values. Quadratic terms AC, BC, A in the simulation equation ² 、B ² And C ² All have obvious influence on the results, which indicates that the compounding proportion of the lactic acid bacteria, the inoculation amount of the strain and the addition amount of the corn juice have important influence on the sensory quality of the fermented non-hydrochloric pickled Chinese cabbage.

The response surface test interaction is shown in figure 16, the sensory evaluation of the fermented non-hydrochloric acid pickle is greatly influenced by the addition amount of the strain, and the related response surface is more curved; the interaction between the inoculation amount of the lactic acid bacteria and the ratio of the lactic acid bacteria is more obvious, the contour is elliptical, and the figure color changes more quickly. When the inoculation amount of the lactic acid bacteria is fixed, the interaction of the compounding proportion of the lactic acid bacteria and the addition amount of the corn juice has small influence on sensory scores, the response curved surface is gentle, the figure color change is slow, and the response curve is consistent with the analysis of variance.

The maximum estimated value of Y is 46.11 according to the simulation equation, the inoculation amount of the lactic acid bacteria is 476.13 mu L, the compounding ratio of the lactic acid bacteria (lactobacillus plantarum: lactobacillus acidophilus) is 2.22:1, and the adding amount of corn juice is 8.19g. The optimal conditions for fermenting the non-hydrochloric pickled Chinese cabbage are as follows: the inoculation amount of the lactobacillus is 450 mu L, the compounding ratio of the lactobacillus (lactobacillus plantarum: lactobacillus acidophilus) is 2:1, and the adding amount of the corn juice is 7.5g.

2.3 physical and chemical index changes during fermentation

2.3.1 changes in pH, total acid content: in order to evaluate the quality of the fermented non-hydrochloric pickled Chinese cabbage under the optimal parameters, the pH value and the total acid content in the whole fermentation process are monitored, and the changes of the pH value and the total acid content of the pickled Chinese cabbage within 5 days of fermentation are shown in figure 17. The initial pH value of the fermentation is found to be 6.11+/-0.04, the pH value is rapidly reduced to 3.55+/-0.02 on the 1 st day of the fermentation, then gradually reduced to 3.24+/-0.01 in 2-5 days, the total acid content is increased along with the fermentation time, and the total acid content reaches 0.64+/-0.01 g/100g on the 5 th day. The non-hydrochloride pickled Chinese cabbage has higher acidification speed than traditional fermented pickled Chinese cabbage, and greatly shortens the production time.

2.3.2 variation of nitrite content: as shown in fig. 18, nitrite is considered as an important index for evaluating food safety. With the progress of fermentation, lactic acid bacteria proliferate rapidly, acid production is rapid, nitrate reducing bacteria are inhibited, nitrite is decomposed by lactic acid bacteria, therefore, obvious reduction of nitrite content is observed, and finally, 0.23+/-0.10 mg/kg is reached. Compared with the traditional fermented pickled Chinese cabbage, the nitrite content in the pickled Chinese cabbage is lower, and no nitrite peak is observed in the whole fermentation process. The results prove that the safety of pickled Chinese cabbage can be effectively improved by fermenting mixed lactobacillus (lactobacillus plantarum suan-15 and lactobacillus acidophilus KLDS 1.0901) under the salt-free condition.

2.3.3 variation of reducing sugar and vitamin C: the metabolism of lactic acid bacteria mainly uses reducing sugar as a substrate, and the content of the reducing sugar can reflect the degree of fermentation. The change of reducing sugar and vitamin C in the fermentation process is shown in figure 19, and the reducing sugar content is obviously reduced in the whole fermentation processp<0.05 The content of reducing sugar in the fresh Chinese cabbage is 5.01+/-0.17 g/100g,on day 5, the consumption rate is reduced to 0.57+/-0.01 g/100g and reaches 88.62 percent. The reducing sugar content reducing rate is obviously higher than that of 1-5 days in the fermentation period of 0-1 daysp<0.05 During the fermentation, the pH value is also the fastest in the descending speed, and the total acid accumulation speed is the fastest, which shows that the lactic acid bacteria can utilize the reducing sugar more efficiently within 0-1 day of fermentation, and meanwhile, the acid substances are accumulated more rapidly. With the continuous deepening of the acid level, the gradual consumption of nutrients gradually increases the growth restriction of lactic acid bacteria, so that the utilization rate of reducing sugar is reduced within 1-5 days of fermentation.

From a nutritional point of view, pickled Chinese cabbage is a good source of bioactive compounds for people, and in particular ascorbic acid in the product has anticancer and anti-aging properties. The change of the vitamin C content in the fermentation process of the non-hydrochloric pickled Chinese cabbage is shown in figure 19, and the change trend of the vitamin C content and the pH value is obviously observed to be consistent, the vitamin C content is rapidly reduced in the early stage of fermentation, then is slowly reduced, and reaches 1.34+/-0.01 mg/100g when the fermentation is mature. Compared with 0 day, the vitamin C content in the fermentation maturation is reduced by more than 90%.

2.3.4 variation of organic acids: the dynamic change of the organic acid content during the fermentation of the non-hydrochloric acid pickled Chinese cabbage is shown in Table 5. The content changes of five organic acids of lactic acid, succinic acid, oxalic acid, citric acid and acetic acid were monitored altogether, wherein lactic acid and acetic acid were not detected at day 0, indicating that these two organic acids were not present in the cabbage matrix, whereas succinic acid, oxalic acid and citric acid were present in small amounts in the cabbage matrix. Among the 5 organic acids, lactic acid and oxalic acid are the main acid products in the fermentation process of the non-hydrochloric acid pickled Chinese cabbage, and compared with fresh cabbage, the lactic acid and oxalic acid contents of the mature non-hydrochloric acid pickled Chinese cabbage are increased by 26.5 times and 12.3 times respectively. With the continuous accumulation of organic acid in the fermentation process, lactic acid is the most important metabolite in the fermentation process of pickled Chinese cabbage, the content increase is most remarkable in the whole fermentation process, and the maximum value 26.523 +/-0.170 mg/mL is reached at the time of fermentation maturity on the 5 th day, which is 2.4 times of the total amount of other 4 organic acids. Oxalic acid, acetic acid and citric acid increased significantly after 1 day of fermentationp<0.05 And slowly increases during subsequent fermentation. In contrast, succinic acid content is in fermentationAfter 1 day, an increase of 0.434mg/mL followed by a slight decrease in 1-5 days.

TABLE 5 variation of organic acids during fermentation

Note that: representing no detection in the sample.

2.3.5 Electronic nose analysis

2.3.5.1 Signal response of electronic nose to pickled Chinese cabbage with different fermentation time

Electronic noses can recognize a range of different odors by mimicking the human olfactory organ. The electronic nose is sensitive to the smell information of the acquired sample and small changes in volatile compounds can lead to differences between sensor responses. The radar chart of the electronic nose sensor on the pickled Chinese cabbage samples in different fermentation periods is shown as 20. For the 4 samples, the strongly responsive sensors were W1W and W5S (W1W sensor is sensitive to organosulfides and W5S sensor is sensitive to oxynitrides), indicating that oxynitrides and organosulfides may have a greater effect on the flavor of the sample. Of all samples, the response values for W3C, W S and W5C (W3C sensor is sensitive to ammonia, aromatic molecules, W6S sensor is sensitive to hydrides, and W5C sensor is sensitive to olefins, aromatic, polar molecules) were the lowest. The radar patterns of the samples are similar in shape, indicating that the fragrance characteristics of the 4 samples are similar, but the volatilization intensity of each gas is greatly different. In addition, the response value of the sample fermented for 0 day is obviously lower than that of the sample fermented for 5 days, which indicates that the aroma characteristics of the ripe non-hydrochloride pickled Chinese cabbage are greatly different from those of the pickled Chinese cabbage.

2.3.5.2 Principal Component Analysis (PCA) results

In order to more directly observe and more intuitively interpret the relationship between the samples and the sensor, regularity and variability between samples are evaluated by principal component analysis. PCA analysis of samples from different fermentation periods by the electronic nose is shown in FIG. 21. The contribution rates of PC1 (X axis) and PC2 (Y axis) are respectively 99.85% and 0.11%, and the total contribution rate is more than 95%, which shows that 4 samples have very different odors, so that pickled Chinese cabbages in different fermentation periods can be distinguished. In addition, the analytical data points of the 5-day and 3-day fermented samples are slightly coincident, and the other sample data points are not coincident, which indicates that the change of the flavor compound in the initial stage of fermentation is significantly larger than that in the later stage of fermentation, which is consistent with the previous results of the acid level change study.

2.3.5.3 Linear Discriminant Analysis (LDA) results

The results of LDA analysis of samples from different fermentation periods by the electronic nose are shown in FIG. 22. The contribution rates of the discrimination type LD1 (X axis) and LD2 (Y axis) are 93.00% and 6.73%, respectively, and the total contribution rate is 99.73%. While no PCA analysis is large in scale, LDA plots can also show some degree of near-far differences in fragrance between different samples. The 5-day and 3-day samples were closely spaced, indicating similar odors, consistent with the results of the PCA analysis.

2.3.6 Electronic tongue analysis

The results of the electronic tongue analysis in the samples of mature sauerkraut and cabbage are shown in FIG. 23. In general, the sour taste and the salty taste are important sensory indexes of the pickled Chinese cabbage, and as no salt is added in the study, no obvious difference between salty parameter indexes in the study results is caused in two samplesp>0.05). In addition, it can be obviously observed that the sour taste and the delicate flavour parameter indexes are obviously different in the two samplesp<0.05 And these organoleptic characteristics are more intense in pickled cabbage, indicating that the fermentation effectively improves the organoleptic quality of the product, which is also consistent with the lower pH results.

2.4 Evaluation of storage stability at 4 ℃

2.4.1 Variation of basic physicochemical index

Dynamic changes in pH, total acid content and nitrite content of the non-hydrochloric acid dish stored at 4℃for 60 days are shown in FIG. 24. The pH value is not changed obviously in the whole in 60 days of storage periodp>0.05 The corresponding total acid content also has no obvious change trend. The pH value is always about 3.23-3.25 during storage, and the total acid content is about 0.60-0.64 g/100 g. Therefore, under the condition of 4 ℃ refrigeration, putrefying bacteria in the pickled Chinese cabbage cannot be greatly proliferated to consume organic acid, and the product still effectively maintains a relatively stable acidic system.

The change of the nitrite content in the pickled Chinese cabbage is in a trend of overall reduction in the storage period of 60 days, and the nitrite content is obviously reduced after 60 daysp<0.05 Almost undetectable in the sample, far below the maximum limit of 20mg/kg for nitrite content in pickled vegetables specified by the national standard GB 2762-2017. The research results show that the non-hydrochloride pickled Chinese cabbage can better keep the good quality characteristics of the non-hydrochloride pickled Chinese cabbage at the low temperature of 4 ℃, and greatly reduces the health risk caused by high salt and high nitrite in the traditional pickled Chinese cabbage to human bodies.

2.4.2 Sensory evaluation

The sensory scores of the no-hydrochloride sauerkraut stored at 4deg.C for 60 days are shown in FIG. 25. The clear observation shows that the sensory quality of the non-salted vegetables is always kept in a good state at the temperature of 4 ℃, which is consistent with no obvious change of the pH value and the total acid content of the non-salted vegetables in 2.4.1 research, and the sensory total score is between 41.0 and 42.3. The vegetable color and odor scores decreased slightly with increasing storage time, but also maintained good vegetable color and unique sour odor at all times.

3. Discussion of the invention

3.1 selection of fermentation strains

Generally, the fermentation of pickled Chinese cabbage can be mainly divided into two modes of spontaneous fermentation and inoculated starter fermentation, but the spontaneous fermentation process is complex and uncontrolled, so that the inoculated starter fermentation is more and more extensive. With the increasing diversity of pickled Chinese cabbage starter cultures, the selection of lactic acid bacteria strains has become the focus of research.

Compared with exogenous isolated strains, the strains isolated from the self-fermentation have higher metabolic capacity, can effectively improve the nutrition and sensory properties of the product and prolong the shelf life.

The strain inoculated to the fermented pickled Chinese cabbage is required to have higher growth activity and acid production capacity so as to shorten the fermentation time and enhance the flavor characteristics of the strain. The traditional fermented pickled Chinese cabbage is fermented at 15-25 ℃, but the low temperature condition can limit the rapid proliferation of lactobacillus in a short period, so that the research mainly selects 9 lactobacillus plantarum separated from northeast pickled Chinese cabbage to perform the research of fermentation characteristic and bacteriostasis characteristic at 22-38 ℃. Test results show that the lactobacillus plantarum span-15 has the best growth activity and the highest acid production at 30 ℃; lactobacillus plantarum suan-15 has remarkable antibacterial activity, so that the strain is selected as a fermentation strain for subsequent experiments, and the optimal growth temperature of the strain is determined as the subsequent fermentation temperature at 30 ℃.

Even though fermentation using a single strain may significantly improve the quality of pickled cabbage, efficient control of stable progression of fermentation is challenging due to the utilization of the strain of material in the white cabbage substrate and other instability factors. Therefore, 2 strains of lactobacillus acidophilus with stronger viability in an acidic environment are selected, the growth activity and the acid production capacity at 30 ℃ are measured, and finally lactobacillus acidophilus KLDS1.0901 is selected as a mixed strain for subsequent fermentation so as to ensure the stability of fermentation.

3.2 optimization of fermentation Process

The mixed fermentation requires selection of optimal fermentation conditions to promote optimal effect of lactic acid bacteria on the food substrate. In general, the main parameter conditions include the type of the probiotics, the strain inoculation amount of the probiotics, the inoculation proportion of the probiotics, the salt concentration, the fermentation temperature and the like.

The sweet corn called fruit corn contains 3% -4% higher sugar content than common corn, especially oligosaccharide and reducing sugar, can provide required carbon source for lactobacillus in the initial growth stage, and the rich amino acid can be used as nitrogen source for lactobacillus in the initial growth stage, so that the growth delay of lactobacillus is shortened, and the purpose of rapid proliferation is achieved. In addition, the corn juice is rich in various vitamins (such as B vitamins and the like), and can be used as a growth promoting factor for promoting the growth and reproduction of lactobacillus. Lactic acid bacteria make full use of these nutrients to metabolize acid, thereby promoting a rapid rise in acid levels. In the study, it can be obviously observed that the pH value of the sample can be rapidly reduced to about 3.5-4.0 within 1 day after corn juice is added, and a large amount of generated acid is accumulated. This is also probably due to the fact that under salt-free conditions, the inoculated lactic acid bacteria are not inhibited by the high salt environment, the fermentation environment tends to be stable, and the maturation standard is reached on day 5. The more corn juice is added, the more soluble sugar is available for lactic acid bacteria in the initial stage The more the contents of the components and the growth promoting factors are, so that the larger the total acid content is along with the increase of the addition amount in the early fermentation stage, but no obvious difference exists when the addition amount is 7.5-12.5 gp>0.05）。

In order to ensure the stability of the fermentation process, the prior art generally adopts 10 ⁷ ~10 ⁹ CFU/mL was used as the initial inoculum size. In addition, a certain amount of salt is added at the beginning of fermentation, so that the activity of spoilage bacteria and pathogenic bacteria is reduced, the moisture in vegetables is reduced, and a certain flavor is provided. The mature non-salted vegetable can ensure the acid level in the product even under the non-salted condition, thereby ensuring the quality and the sense of the product. In the study, the smaller the pH value is along with the increase of the inoculation amount of the lactic acid bacteria in the early fermentation period, the larger the total acid content is along with the increase of the inoculation amount of the lactic acid bacteria; then, when the inoculation amount of the lactic acid bacteria is 450-750 mu L, no obvious difference existsp>0.05）。

The invention utilizes the synergistic or antagonistic action among the mixed bacterial strains to achieve the aim of effectively multiplying probiotics and inhibiting the growth of harmful bacteria. The present study gave the result of having optimal fermentation quality at a lactic acid bacteria ratio of 2:1. Meanwhile, when lactobacillus acidophilus is more, the pH value of the sample is raised, the total acid content is reduced, and the nitrite content is increased.

3.3 Material Change condition during fermentation and quality evaluation

In order to more comprehensively evaluate the quality characteristics of the non-hydrochloride pickled Chinese cabbage, the research carries out quantitative analysis of 5 main organic acids and determination of reducing sugar and vitamin C content in the fermentation process. As is evident from the data, the reducing sugar content is significantly reduced during fermentation and the overall organic acid level is significantly increased, indicating that lactic acid bacteria metabolically convert carbohydrates to organic acids. Succinic acid is present in the cabbage matrix and can be metabolized to other compounds such as malic acid, which accounts for the decrease in succinic acid content during fermentation. The increased lactic acid in a short period of time inhibits the growth of, for example, nitrate-reducing bacteria, and plays a role in inhibiting the excessive production of nitrite. In addition, the antioxidation of vitamin C in Chinese cabbage inhibits nitrate from being oxidized into nitrite to a certain extent. The decrease in vitamin C content with fermentation in this study was likely due to the reduction of nitrite to NO by vitamin C, and thus was effective in reducing nitrite content in the fermentation system. Vitamin C, in addition to having antioxidant capacity, can also be involved in enzymatic activities of the glycolytic pathway such as hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, which have a significant impact on the significant accumulation of lactate in the study. The fermentation process of pickled Chinese cabbage is very complex, and the content of lactic acid which is a main chemical component depends on the sugar content in raw materials and the number of lactic acid bacteria, so that the quality of the product cannot be judged according to the content of the chemical component only. The utilization rate of the reducing sugar can indirectly indicate the fermentation degree, and the research shows that the utilization rate of the reducing sugar reaches 88.62 percent, which indicates that the fermentation is more thorough.

Storage Performance evaluation at 3.44 ℃

Since lactic acid bacteria produce metabolites such as organic acids which inhibit the growth of spoilage bacteria during fermentation, the mature sauerkraut product can be stored periodically. Storage at high temperature can cause excessive proliferation of microorganisms in the pickled Chinese cabbage, and reduce shelf life of the product. The proliferation of various microorganisms in a fermentation system can be limited at 4 ℃, and the color and taste of the pickled Chinese cabbage can be better maintained.

The pH value, the total acid content and the nitrite content of the pickled Chinese cabbage during storage are key indexes taking the quality characteristics thereof into consideration. The pH value and the total acid content of the pickled Chinese cabbage in the study do not change significantly during the storage periodp>0.05 The nitrite content is reduced to 0mg/kg after 10 days, and the fact that the vacuum packaging technology is used for fermentation and storage is proved, so that a stable fermentation system is more controllable, secondary fermentation during storage is avoided, and further the storage stability of pickled Chinese cabbage is affected.

The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (9)

1. PlantLactobacillus plantarumLactiplantibacillus plantarum) The suan-15 is characterized in that the lactobacillus plantarum is preserved in China center for type culture collection, wuhan, with a preservation date of 2022, 10 months and 19 days, and a preservation number of CCTCC NO: m20221595.

2. Comprising a strain of Lactobacillus plantarum according to claim 1Lactiplantibacillus plantarum) A mixed starter culture of suan-15, which is characterized by comprising lactobacillus and corn juice; the lactobacillus comprises lactobacillus plantarum suan-15 and lactobacillus acidophilusLactobacillus acidophilus) KLDS1.0901; the viable count of the lactobacillus plantarum suan-15 and the lactobacillus acidophilus KLDS1.0901 is 6 multiplied by 10 ⁹ CFU/mL; the volume ratio of the lactobacillus plantarum suan-15 to the lactobacillus acidophilus KLDS1.0901 is 2:1;250g of cabbage has a lactic acid bacteria inoculation amount of 450 mu L and a corn juice addition amount of 7.5g.

3. The mixed starter according to claim 2, wherein the method for producing the mixed starter comprises the steps of: the viable count is 6 multiplied by 10 ⁹ CFU/mL lactobacillus plantarum span-15 and lactobacillus acidophilus KLDS1.0901 are respectively activated in MRS liquid culture medium for two generations to obtain bacterial liquid, the bacterial liquid is diluted by saturated NaCl solution for 10 times to obtain diluted liquid, 450 mu L of lactobacillus plantarum span-15 diluted liquid and lactobacillus acidophilus KLDS1.0901 diluted liquid are mixed according to the proportion of 2:1, the supernatant is removed by centrifugation, bacterial sludge is obtained, the bacterial sludge is washed twice by saturated NaCl solution, and 15.0g of sterile fermentation culture liquid consisting of 7.5g of corn juice and distilled water with the same mass is added.

4. A mixed starter according to claim 3, wherein the corn juice is produced by a process comprising: pulping fruit corn and water according to a mass ratio of 1:10, filtering with four layers of gauze, collecting filtrate, and sterilizing at 121deg.C for 20min.

5. A mixed starter according to claim 3, wherein the centrifugation process is 8000r/min for 3min.

6. A method for preparing a non-hydrochloric acid dish using the mixed starter according to claim 2, comprising the steps of:

s1, preprocessing cabbage: removing roots and old leaves of Chinese cabbage, cleaning, scalding for 3-5 seconds by boiling water, draining, shredding, weighing 250g each time, and filling into a polyethylene film bag;

s2, adding the mixed starter into the polyethylene film bag in the step S1, fully mixing, and sealing by using a vacuum fresh-keeping machine;

s3, fermenting: fermenting at 30deg.C for 5 days to obtain mature non-hydrochloride pickled Chinese cabbage;

s4, storing: stored at 4℃for 60 days.

7. A strain of lactobacillus plantarum according to claim 1Lactiplantibacillus plantarum) Use of suan-15 in fermenting non-hydrochloric acid pickle.

8. Use of the mixed starter according to claim 2 for fermenting pickled cabbage.

9. A method of making a non-salted vegetable as claimed in claim 6.

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