Composite microbial agent and its application

Abstract

The present invention discloses a composite bacterial agent and its application. The composite bacterial agent provided by the present invention comprises Lactobacillus plantarum, Leuconostoc mesenteroides and Weissella cibaria. The present invention also provides a method for preparing the composite bacterial agent, a method for preparing kimchi, kimchi and the application of the composite bacterial agent in preparing kimchi. When the composite bacterial agent provided by the present invention is used to prepare kimchi, it has the advantages of simple use, short fermentation time, good kimchi flavor, good taste, low nitrite content and stable quality, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biological fermentation, and specifically relates to a composite bacterial agent and application thereof. Background Art

[0002] Kimchi is one of the typical representatives of my country's traditional fermented foods. It is made mainly from vegetables as raw materials, and then goes through processes such as adding auxiliary materials, soaking and fermenting in salt water, and killing microorganisms, ultimately forming a type of fermented food with unique flavor and crispy taste.

[0003] However, due to the complexity of the microorganisms carried, the fermentation process is complicated and the fermentation time is long, which cannot bring stable and good kimchi quality. The starter prepared by directly using a fixed amount of bacteria in artificial fermentation can directly form a fermentation system once activated, which has a wide control range and is conducive to forming a standardized fermentation process. The starter can be preserved for a long time, which has a positive impact on the industrialization and marketization of kimchi products and the improvement of kimchi quality.

[0004] The selection and ratio of starter cultures determine the differences in the flavor and quality of kimchi products. Therefore, starter cultures with good fermentation characteristics, rapid fermentation, and suitability for kimchi systems are the focus of current research. At present, the research on kimchi starter cultures at home and abroad is still mainly focused on the screening and optimization of single-bacteria fermentation, and there is little research on the impact of multi-bacteria composite fermentation on kimchi flavor. Summary of the invention

[0005] The problem to be solved by the present invention is to provide a composite bacterial agent to solve the defects of the prior art in that the fermentation process of kimchi is complicated, the fermentation time is long, and stable and good kimchi quality cannot be achieved. The composite bacterial agent provided by the present invention has the advantages of simple use, short fermentation time, good kimchi flavor, good taste, low nitrite content, and stable quality when used to prepare kimchi, and has broad application prospects.

[0006] The present invention mainly solves the above technical problems through the following technical solutions.

[0007] A first aspect of the present invention provides a composite bacterial agent, comprising Lactobacillus plantarum, Leuconostoc mesenteroides and Weissella cibaria.

[0008] In some embodiments of the present invention, the ratio of Lactobacillus plantarum, Leuconostoc mesenteroides and Weissella sinusovorans in the composite bacterial agent is 80-200:0.5-10:0.5-10.

[0009] In some preferred embodiments of the present invention, the compounding ratio of the Lactobacillus plantarum, the Leuconostoc mesenteroides and the Weissella sinusoidalis is 100:1:1.

[0010] In some embodiments of the present invention, the Lactobacillus plantarum is CICC 21836; the Leuconostoc mesenteroides is CICC 24385; and / or the Weissella sinusoidalis is LMG 21843.

[0011] In some embodiments of the present invention, the dosage form of the composite bacterial agent includes liquid, powder, granule and immobilized bacterial agent.

[0012] A second aspect of the present invention provides a method for preparing a composite bacterial agent, wherein the composite bacterial agent comprises Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Weissella cibaria.

[0013] In some embodiments of the present invention, the Lactobacillus plantarum is CICC 21836, the Leuconostoc mesenteroides is CICC 24385, and / or the Weissella sinusoidalis is LMG 21843.

[0014] In some embodiments of the present invention, the compounding ratio of the Lactobacillus plantarum, the Leuconostoc mesenteroides and the Weissella sinusoidalis is 80-200:0.5-10:0.5-10.

[0015] In some preferred embodiments of the present invention, the compounding ratio of the Lactobacillus plantarum, the Leuconostoc mesenteroides and the Weissella sinusoidalis is 100:1:1.

[0016] The third aspect of the present invention provides a method for preparing kimchi, comprising the step of contacting the composite bacterial agent as described in the first aspect with raw materials for fermentation; the composite bacterial agent comprises Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Weissella cibaria.

[0017] In some embodiments of the present invention, the Lactobacillus plantarum is CICC 21836, the Leuconostoc mesenteroides is CICC 24385, and / or the Weissella sinusoidalis is LMG 21843.

[0018] In some embodiments of the present invention, the inoculation amount of the composite bacterial agent is 0.8×10 9 ~2×109 CFU / mL, the Leuconostoc mesenteroides and the Weissella sinusitis were 0.5×10 7 ~1×10 8 CFU / mL; and / or, the fermentation temperature is 10-35°C.

[0019] In some embodiments of the present invention, the inoculation amount of the composite bacterial agent is 1×10 9 CFU / mL, the Leuconostoc mesenteroides and the Weissella sinusitis were 1×10 7 CFU / mL.

[0020] In some embodiments of the present invention, the raw material is vegetables;

[0021] In some embodiments of the present invention, the method further comprises the step of adding auxiliary materials; the auxiliary materials include seasonings, spices, water; and / or, the method further comprises the step of adding calcium salts, such as calcium chloride, calcium sulfate and / or calcium carbonate, and the final calcium ion concentration is 500-900 mg / kg.

[0022] In some embodiments of the present invention, the seasoning includes salt and / or granulated sugar; the spice includes ginger, star anise, cinnamon and / or garlic.

[0023] A fourth aspect of the present invention provides kimchi, characterized in that the kimchi is prepared using the composite bacterial agent as described in the first aspect or the method as described in the third aspect.

[0024] A fifth aspect of the present invention provides a use of the composite bacterial agent as described in the first aspect in preparing kimchi.

[0025] When the composite bacterial agent provided by the present invention is used for preparing pickles, it has the advantages of simple use, short fermentation time, good pickle flavor, good taste, low nitrite content and stable quality, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Changes of pH and total acid content during fermentation of white radish with different Lactobacillus plantarum.

[0027] Figure 2 The total acid content and pH value changes during the fermentation of white radish by Leuconostoc mesenteroides.

[0028] Figure 3 It is the total acid content and pH value of pickled radish.

[0029] Figure 4 It is the free amino acid content of pickled radish.

[0030] Figure 5The nitrite content of pickled radish.

[0031] Figure 6 Analysis of volatile components of pickled radish.

[0032] Figure 7 Sensory evaluation of pickled radish.

[0033] Figure 8 The effect of calcium ion content on the hardness of pickled radish.

[0034] 1-Calcium 420: Calcium ion concentration is 420 mg / kg;

[0035] 2-Calcium 502: Calcium ion concentration is 502mg / kg;

[0036] 3-Calcium 890: Calcium ion concentration is 890mg / kg;

[0037] 4-Calcium 1300: Calcium ion concentration is 1300mg / kg. DETAILED DESCRIPTION

[0038] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and are not limited to the scope of the present invention. The implementation conditions adopted in the examples can be further adjusted according to the conditions of the specific manufacturer, and the unspecified implementation conditions are usually the conditions in conventional experiments.

[0039] Introduction and Overview

[0040] Various aspects of the present invention will be described below. It will be apparent to those skilled in the art that the present invention may be implemented according to only some or all aspects of the present invention. For purposes of illustration, specific numbering, materials, and configurations are provided herein to enable a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention can be implemented without specific details. In other examples, well-known features have been omitted or simplified in order not to obscure the present invention.

[0041] Various operations are described herein as multiple discrete steps, in sequence, and are explained in a manner that is most helpful in understanding the invention; however, the sequential description should not be construed as implying that these operations are necessarily order dependent.

[0042] Various embodiments will be described based on the reactants of typical types. It will be apparent to those skilled in the art that the present invention can be implemented using any number of different types of reactants, not just those provided here for illustrative purposes. In addition, it will also be apparent that the present invention is not limited to any specific mixing example.

[0043] In this article, the dilution ratios not specifically specified are all volume ratios. The strains used in this article are all publicly available commercial strains.

[0044] Data Statistical Analysis:

[0045] One-way ANOVA analysis was performed using the statistical software SPSS (version: 18.0; SPSS Inc., Chicago, IL, USA) (P < 0.05 was considered significant). Origin 2018 software was used for drawing. The experimental indicators were measured three times in parallel, and the samples were measured three times in parallel. The results were expressed as mean ± standard deviation.

[0046] Example 1 Obtaining the composite bacterial agent

[0047] 1. Screening of Lactobacillus plantarum

[0048] Twelve strains of Lactobacillus plantarum with good fermentation flavor were selected for screening. During the fermentation process of pickled radish, when the pH of the pickled radish was less than 4 and the total acid mass fraction was greater than 0.3%, the pickled radish was generally considered to be fermented mature. Figure 1 It can be seen that Lactobacillus plantarum 2 has a higher total acid content and a lower pH value. Lactobacillus plantarum 2 has a good acid production ability during the fermentation of pickled radish, which can greatly shorten the fermentation period of pickled radish. Therefore, Lactobacillus plantarum 2 has a better potential for fermenting pickled cabbage than other Lactobacillus plantarum.

[0049] The 12 strains of Lactobacillus plantarum used for screening are shown in Table 2, all of which are publicly available commercial strains.

[0050] Table 2 Lactobacillus plantarum used

[0051] serial number strain serial number strain 1 CICC 22201 7 ATCC 8014 2 CICC 21836 8 CICC 20279 3 CICC 20022 9 CICC 20022 4 ATCC 14917 10 CICC 20242 5 CICC 20261 11 CICC 21796 6 CICC 20267 12 CICC 20766

[0052] 2. Screening of Leuconostoc mesenteroides

[0053] Depend on Figure 2 It can be seen that on the 10th day of fermentation, the total acid content of the pickled radish inoculated with Enterobacterium mesenteroides 3 was higher than that of other Enterobacterium mesenteroides. Although the pH value was higher than that of the pickled radish fermented with Enterobacterium mesenteroides 4, 6, 7 and 8, the total acid was a more important screening index than the pH value. Taking all factors into consideration, Enterobacterium mesenteroides-3 has a strong acid production ability, which will shorten the kimchi fermentation cycle, so Enterobacterium mesenteroides 3 was selected for the subsequent compound bacterial fermentation test.

[0054] The 14 strains of Leuconostoc mesenteroides used for screening are shown in Table 2, all of which are publicly available commercial strains.

[0055] Table 3. Leuconostoc mesenteroides used

[0056]

[0057]

[0058] 3. Obtaining compound bacterial agents

[0059] Table 4 Screening of composite bacterial agents

[0060] Total acid pH Example 3.74±0.02 2.91±0.004 Comparative Example 1 2.62±0.18 3.72±0.004 Comparative Example 2 3.22±0.06 3.20±0.01 Comparative Example 3 2.96±0.10 3.42±0.008

[0061] Among them, the examples are Lactobacillus plantarum CICC 21836, Leuconostoc mesenteroides CICC 24385 and Weissella sinusoids LMG 21843 (the inoculation amount of Lactobacillus plantarum is 1×10 9 CFU / mL, the inoculation amount of Leuconostoc mesenteroides and Weissella sinusitis was 1×10 7 CFU / mL); Comparative Example 1 is Lactobacillus plantarum ATCC 8014 and Leuconostoc mesenteroides CICC 22179 (the inoculum size is 1×10 9 CFU / g); Comparative Example 2 was Lactobacillus plantarum CICC 22201, Leuconostoc mesenteroides CICC24388, Weissella vegetativeis LMG 21843 and Lactobacillus fermentum CICC 21800 (the inoculation amount was 1×10 9 CFU / g); Comparative Example 3 was Lactobacillus plantarum CICC 20022, Leuconostoc mesenteroides CICC 22180 and Weissella vegetativeis LMG 21843 (the inoculation amount was 1×10 9 CFU / g).

[0062] Example 2 Quality Analysis of Kimchi Fermented with Composite Bacterial Agent

[0063] Taking white radish as an example, wash and peel the fresh white radish, cut it into a rectangular shape of 2.5 cm in length, 2.5 cm in width and 2 cm in thickness, and put it into a jar with a ratio of white radish to water = 1:2 and seal it for fermentation at a constant temperature of 25℃.

[0064] There are three fermentation methods for fermenting white radish:

[0065] Experimental group: Inoculated with the selected composite bacteria, the inoculation amount of Lactobacillus plantarum CICC 21836 was 1×10 9 CFU / mL, the inoculum size of Leuconostoc mesenteroides CICC 24385 and Weissella sinusoidalis LMG 21843 was 1×10 7 CFU / mL.

[0066] Positive control group: commercial kimchi starter was added, and pickled radish was fermented according to the instructions: 1g of starter was fermented with 1000g of white radish. The starter contained the following bacteria: Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus rhamnosus, and the inoculation amount was 1×10 9 CFU / g.

[0067] Negative control group: no additional microorganisms were inoculated and natural fermentation was allowed.

[0068] 1 Total Acid and pH

[0069] Determination of total acid: refer to the acid-base titration method in GB 12456-2021 "Determination of total acid in food".

[0070] Determination of pH value: Calibrate the cleaned pH meter, that is, rinse the electrode head with ultrapure water and wipe it clean with filter paper, and place it in standard buffer solutions with pH values ​​of 4.0, 6.86 and 9.18 respectively. After calibration, the slope should be maintained at 95%-100%. When three calibration points appear on the display, the calibration is completed. Take 25g of pickled radish and mix it with an equal amount of CO2-free water, place it in a tissue grinder to make a homogenate, transfer it to a conical flask, wait for the homogenate to cool to 25℃, and measure the pH value with a pH meter.

[0071] like Figure 3 As shown, the total acid content of the experimental group was significantly higher than that of the positive control and the negative control (P < 0.05), reaching (3.74 ± 0.02) g / Kg. The pickled radish in the experimental group reached maturity (when the pH of the pickled cabbage is < 4 and the total acid mass fraction is > 0.3%, it can be considered mature).

[0072] 2 Types and contents of free amino acids

[0073] The types and contents of free amino acids have a great influence on the flavor and quality of pickled radish. The taste characteristics of free amino acids can be divided into three categories: umami (aspartic acid, glutamic acid and glutamine), sweet (serine, glycine, threonine, alanine and proline) and bitter amino acids (histidine, arginine, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine and lysine). The above 17 amino acids were tested.

[0074] Detection method:

[0075] Sample pretreatment: Take 10g of pickled radish and dry it at 80℃, put it in a mortar, grind it with quartz sand, transfer it all to a conical flask, add 25mL of ultrapure water, ultrasonically treat it for 30min, then centrifuge and take the supernatant for derivatization treatment.

[0076] Chromatographic conditions: C18 amino acid special analytical column (250mm×4.6mm, 5μm); mobile phase A: acetonitrile-water (4:1, V / V), mobile phase B: 0.1mol / L sodium acetate buffer solution (adjusted to pH 6.5 with acetic acid)-acetonitrile (97:3, V / V), flow rate 1.0mL / min, detection wavelength 254nm, column temperature 36℃, injection volume 20μL.

[0077] Elution conditions: 0~11min, 0%~1.5% A, 100%~98.5% B; 11~21.7min, 1.5%~7.6% A, 98.5%~92.4% B; 21.7~23.9min, 7.6%~11% A, 92.4%~89% B; 23.9~39min, 11%~30% A, 89%~70% B; 39~42min, 30%~70% A, 70%~30% B; 42~45min, 70%~100% A, 30%~0% B; 45~52min, 100% A, 0% B; 52~55min, 100%~0% A, 0% ~ 100% B; 55 ~ 70min, 0% A, 100% B.

[0078] Test results:

[0079] The results are as follows Figure 4 As shown, the content of sweet amino acids in the pickled radish of the experimental group was higher than that of other control groups and the blank group, and the content of bitter amino acids was significantly lower than that of the control group and the blank group, indicating that the flavor quality of kimchi fermented with the composite bacterial agent is better.

[0080] 3. Nitrite content

[0081] Determination shall be carried out according to the spectrophotometric method in GB 5009.33-2016 “Determination of nitrite and nitrate in food”.

[0082] The national limit standard stipulates that the nitrite content in pickled radish cannot exceed 20 mg / kg.

[0083] like Figure 5 As shown, the nitrite content in the pickled radish of the experimental group was significantly lower than that of the control group and the blank group.

[0084] 4. Analysis of aroma components

[0085] 5 g of pickled radish was weighed and made into homogenate, and the volatile flavor substances in the pickled radish inoculated with composite bacteria, commercial fermentation culture and natural fermentation were analyzed by HS-SPME-GC-MS.

[0086] Test results such as Figure 6As shown in the figure. Esters are one of the main sources of pickled radish flavor. In the middle and late stages of fermentation, alcohols in pickled radish react with organic acids to form rich esters, among which (E)-4-(methylthio)-3-butenyl isothiocyanate and trierucic acid glyceride have high contents. Most of these aroma substances come from the enzymatic hydrolysis of thioglucosides in pickled radish itself. Isothiocyanates are considered to be unique flavor substances of cruciferous plants and their fermented products. They have a strong aroma, making pickled radish spicy and have antibacterial and anti-cancer effects. Ethyl acetate is an important flavor substance in kimchi. The content of ethyl acetate in the experimental group was significantly higher than that in the control group and the blank group. The pickled radish fermented by the composite starter in the experimental group had more aroma components given by esters. Alcohols also have an important influence on the flavor of pickled radish. Ethanol, propanol and butanol give pickled radish a sweet and mellow aroma. 2,3-Butanediol and phenylethanol also have a pleasant aroma. Under the three fermentation methods, the experimental group produced more alcohol substances than the positive control and natural fermentation, such as 2-ethylhexanol, 2-propyl-1-pentanol and 2,3-butanediol, providing more mellow aroma. In addition, the experimental group had a better ethanol content than the control group and the blank group. Acid substances are produced in large quantities through the lactic acid fermentation pathway due to the vigorous metabolism of Lactobacillus plantarum. With the growth and reproduction of microorganisms, the content of acid substances increases, which not only increases the sour taste of pickled radish, but also contributes to the formation of ester substances. Both the positive control group and the experimental group produced a certain amount of acetic acid, among which the experimental group had the highest content, reaching 0.89μg / kg. A large amount of acetic acid can improve the flavor of kimchi, give pickled radish a soft sour taste, and have a benign improvement on the flavor of pickled radish. Terpenes were only found in the experimental group, which were dextrorotatory terpenes, and the relative content was high, reaching 0.55μg / kg. Dextrorotatory terpenoids are not only the main flavor component of old kimchi water fermentation kimchi, but also one of the main flavor components in natural fermentation. They have a light aroma like flowers and make a certain contribution to the aroma of kimchi.

[0087] Example 3 Sensory Evaluation of Pickle Quality

[0088] A sensory evaluation team composed of 16 sensory evaluators evaluated the fermentation taste, sweetness, sourness, saltiness, astringency, viscosity, crispness and other characteristics of kimchi in the experimental group, control group and blank group, with 10 points as the full score. The scores were statistically analyzed and the results were as follows: Figure 7 As shown in the figure, the comprehensive sensory evaluation of the experimental group was significantly better than that of the control group and the blank group.

[0089] Example 4 Effect of calcium ion content on kimchi hardness

[0090] In the process of further optimizing the taste of kimchi, the inventors found that calcium ions can increase the hardness of pickled radish, but the higher the calcium ion concentration, the better. Figure 8As shown, kimchi has an advantage in taste when the calcium ion content is in the range of 500-900 mg / kg.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent is Lactobacillus plantarum ( Lactobacillus plantarum )、Leuconostoc mesenteroides( Leuconostoc mesenteroides ) and Weissella sinusitis ( Weissella Cibaria ); The ratio of the plant lactobacillus, the mesenteroides and the Weissella sinusitis in the composite bacterial agent is 100:1:1; The plant lactobacillus is CICC 21836; The Leuconostoc mesenteroides is CICC 24385; The Weissella sinensis strain is LMG 21843.

2. The composite bacterial agent according to claim 1, characterized in that The dosage forms of the composite bacterial agent include liquid, powder, granule and immobilized bacterial agent.

3. A method for preparing a composite bacterial agent, characterized in that: The composite bacterial agent is Lactobacillus plantarum ( Lactiplantibacillus plantarum )、Leuconostoc mesenteroides( Leuconostoc mesenteroides ) and Weissella sinusitis ( Weissella cibaria ); The Lactobacillus plantarum is CICC 21836, the Leuconostoc mesenteroides is CICC 24385, and the Weissella sinusoidalis is LMG 21843; The compounding ratio of the plant lactobacillus, the mesenteroides and the Weissella sinusitis is 100:1:

1.

4. A method for preparing kimchi, characterized in that: The method comprises the step of contacting the composite bacterial agent as claimed in claim 1 or 2 with raw materials for fermentation.

5. The method according to claim 4, characterized in that The inoculation amount of the composite bacterial agent is 0.8×10 9 ~2×10 9 CFU / mL, the Leuconostoc mesenteroides and the Weissella sinusitis were 0.5×10 7 ~1×10 8 CFU / mL.

6. The method according to claim 4, characterized in that The fermentation temperature is 10-35°C.

7. The method according to claim 4, characterized in that The raw materials are vegetables.

8. The method according to claim 4, characterized in that The method further comprises the step of adding auxiliary materials; the auxiliary materials comprise spices and water.

9. The method according to claim 4, characterized in that The method further comprises the step of adding seasoning.

10. The method according to claim 4, characterized in that The method further comprises the step of adding calcium salt; the calcium salt comprises calcium chloride, calcium sulfate and / or calcium carbonate, and the final concentration of calcium ions is 500-900 mg / kg.

11. Use of the composite bacterial agent according to claim 1 or 2 in preparing kimchi.

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