Plant lactobacillus LP.3 and application thereof

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a Lactiplantibacillus plantarum LP.3 and application thereof.The present application provides a Lactiplantibacillus plantarum LP.3, which has high yield of malic acid, is used for fermenting small leaf mustard, and significantly improves the content of malic acid in the product, while the content of citric acid and lactic acid is relatively low, which weakens the oligo-acid flavor, enhances the softness and coordination of the sour taste and richness, and improves the flavor and quality of the fermented small leaf mustard.The technical scheme provided by the present application adopts a direct-inoculation pure-strain fermentation technology, and exogenous Lactiplantibacillus plantarum LP.3 strains are added, which can rapidly multiply in the fermentation system, play a role in nutritional occupation, reduce the growth space of miscellaneous bacteria, inhibit the growth of miscellaneous bacteria, and show stable fermentation quality in large-scale fermentation jars, effectively solve the problem of unstable fermentation quality of small leaf mustard, improve the fermentation quality of small leaf mustard, reduce the risk of food safety, and help to realize large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a plant lactobacillus LP.3 and application thereof. BACKGROUND

[0002] Brassica juncea, a Brassica annual herb, is a famous characteristic vegetable in China and one of main raw materials for making pickles. Small leaf mustard is one of the most common leaf mustard, which is currently pickled by natural fermentation. However, the fermentation quality of batches is unstable, and the potential food safety risk is high. Traditional natural fermentation of small leaf mustard is easily contaminated by yeast in the environment, resulting in spoilage of fermented small leaf mustard and abnormal flavor. In addition, the natural fermented small leaf mustard has high contents of lactic acid and citric acid and low content of malic acid, resulting in single and uncoordinated sour taste, oligo-acid flavor, weak sour and weak jar aroma.

[0003] Patent CN 117814456 A discloses a strain of lactic acid bacteria for fermenting mustard, and the obtained mustard product has a flavor of alcohol and pleasant sourness. No food additives are added in the product, and the quality is stable. Patent CN 117683677 A discloses fermentation agents of Weissella hellena, Lactobacillus plantarum and Lactobacillus jensenii. The contents of 24 kinds of free amino acids and amino acid nitrogen and the types and contents of isothiocyanate compounds in fermented mustard are significantly improved, which effectively improves the flavor and quality of the product, and has good anti-inflammatory and antioxidant effects, which has obvious advantages compared with traditional fermentation agents. Patent CN 116376737 A discloses a strain of Lactobacillus paracasei ZF616, which can increase the content of malic acid in fermented soy sauce, increase the pleasant sour taste of soy sauce, and make the taste more sour and sweet, and the sweet and sour taste more full and coordinated, thereby improving the thickness of soy sauce.

[0004] Malic acid can be used in food, medicine, daily chemical industry and other industries, and has been widely used in various beverage and food industries, becoming the third largest food souring agent, only next to citric acid and lactic acid. Malic acid is an organic acid necessary for human body, and is an important organic acid affecting the rich, soft and coordinated sour taste of fermented small leaf mustard. The prior art optimizes mustard fermentation inoculum, but does not solve the technical problems of low malic acid content and single flavor. SUMMARY

[0005] Based on the above technical problems, the main purpose of the present application is to overcome the shortcomings of the background art. A Lactiplantibacillus plantarum LP.3 strain is screened, which has high yield of malic acid. The strain is used for fermentation of small leaf mustard, and the content of malic acid in the product is significantly increased, while the content of citric acid and lactic acid is relatively low, which weakens the oligo-acid flavor, enhances the softness and richness of the acid flavor. In addition, the LP.3 strain can rapidly proliferate in the early stage of fermentation, which plays a role in nutrient occupation, thereby inhibiting the growth of yeast to some extent. The problem of unstable fermentation quality of small leaf mustard is solved, the fermentation quality is improved, and the potential food safety risk is reduced.

[0006] To achieve the above-mentioned purpose, the inventors have conducted in-depth research and completed the present application scheme after repeated research and demonstration, as follows:

[0007] In a first aspect, the present application provides a Lactiplantibacillus plantarum LP.3, which has been deposited with the China General Microbiological Culture Collection Center on March 29, 2024, and the deposit number is CGMCC No. 30203.

[0008] The colony of the Lactiplantibacillus plantarum LP.3 strain in the MRS medium is milky white, with a neat and round edge, a smooth surface, and a lustrous wetness. After Gram staining, the bacterial body of the strain is observed under a microscope to be a typical bacillus shape, with a length of 0.5-0.8 μm and a width of 0.2-0.4 μm,

[0009] In a second aspect, the present application further provides a fermentation inoculant, which comprises the above-mentioned Lactiplantibacillus plantarum LP.3.

[0010] In a third aspect, the present application further provides the use of the above-mentioned Lactiplantibacillus plantarum or fermentation inoculant in mustard fermentation, preferably in the fermentation of small leaf mustard, especially for increasing the content of malic acid in fermented small leaf mustard, and improving the flavor and quality of fermented small leaf mustard.

[0011] The chemical name of malic acid is 2-hydroxy gamma-diacid. Due to the presence of an asymmetric carbon atom in the molecular structure, it exhibits optical rotation as right-handed (D-), left-handed (L-) and racemic (DL-). Malic acid is an essential organic acid for the human body and an ideal food additive with low calories. It has been widely used in various beverage and food industries and has become the third largest food acidulant, only next to citric acid and lactic acid. In terms of microbial fermentation method, the malic acid producing bacteria mainly include Aspergillus flavus, Aspergillus oryzae, Aspergillus parasiticus, Rhizopus chinensis and Pichia membranifaciens. Malic acid is applied in food, medicine, daily chemical industry and other industries, and can play an important role in human metabolism, including anti-fatigue, protection of liver, kidney and heart, treatment of anemia and liver dysfunction. At the same time, after normal physical labor and intensive labor, the intake of malic acid can effectively help the recovery of physical strength. Malic acid can enhance the absorption of drugs in the human body, and make the drugs taste fruity, which is more suitable for the elderly and children. In addition, malic acid can be used to accelerate the elimination of free radicals in the tissues of the elderly body. The existence of free radicals can damage the tissues and cells of the body, and then cause chronic diseases and aging effects. Malic acid can effectively control the tissue damage caused by the increase of free radicals. In recent years, malic acid and malate have also been applied to animal growth promoters.

[0012] In a fourth aspect, the present application further provides a fermentation method of small leaf mustard, which is a method of adding the above-mentioned Lactobacillus plantarum or fermentation inoculant to the small leaf mustard for fermentation.

[0013] Further, the inoculation time of the Lactobacillus plantarum or the fermentation inoculant is the early stage of fermentation.

[0014] Further, the inoculation amount of the Lactobacillus plantarum is 10 6 CFU / g

[0015] Further, the fermentation time is 40-50 days.

[0016] In a fifth aspect, the present application further provides a fermented small leaf mustard, which is prepared by the preparation method of the present application.

[0017] The one or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0018] 1. The Lactobacillus plantarum LP.3 provided by the present application has high malic acid yield, and the malic acid content of the fermented small leaf mustard is significantly increased, while the citric acid and lactic acid contents are relatively low, which weakens the oligo-acid flavor, enhances the softness and coordination of the acid flavor and richness, and improves the flavor and quality of the fermented small leaf mustard.

[0019] 2、The plant lactiplantibacillus LP.3 strain provided by the present application can ferment small leaf mustard, can show stable fermentation quality in large-scale fermentation jars, can effectively solve the problem of unstable fermentation quality of small leaf mustard, and is helpful to realize large-scale industrial production.

[0020] 3、The plant lactiplantibacillus LP.3 provided by the present application is derived from naturally fermented small leaf mustard, and has no safety risk.

[0021] 4、The technical scheme provided by the present application adopts a direct inoculation type pure strain fermentation technology, and adds an exogenous plant lactiplantibacillus LP.3 strain, which can rapidly reproduce in a fermentation system, plays a nutritional occupation role, reduces the growth space of miscellaneous bacteria, inhibits the growth of miscellaneous bacteria, improves the fermentation quality of small leaf mustard, and reduces the food safety risk. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a flavor profile of small leaf mustard with different treatments;

[0023] Figure 2 It is a colony morphology diagram of the plant lactiplantibacillus LP.3;

[0024] Figure 3 It is a cell morphology diagram of the plant lactiplantibacillus LP.3 under a microscope;

[0025] Figure 4 It is a microbial community change of small leaf mustard with different fermentation processes in a 350kg specification;

[0026] Figure 5 It is an area accumulation diagram of different organic acids in 600kg fermented small leaf mustard;

[0027] The plant lactiplantibacillus (Lactiplantibacillus plantarum) LP.3 provided by the present application has been preserved in the China General Microbiological Culture Collection Center on March 29, 2024, the address is No. 3, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 30203; the strain was received and registered by the preservation center on March 29, 2024, and was detected by the preservation center on March 29, 2024 as a living strain. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present application, the present application will be described more fully below. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0030] Unless otherwise indicated, the reagents, methods, and apparatuses employed in the present application are conventional food grade reagents, methods, and apparatuses in the art.

[0031] The terminology used in the specification of the present application herein is used only for the purpose of describing particular embodiments and is not intended to be limiting of the present application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0032] The terms and related determination methods involved in the present application are explained as follows:

[0033] 1. Lactic acid bacteria isolation determination method: refer to GB 4789.35-2023 National Food Safety Standard Food Microbiological Examination Lactic Acid Bacteria Examination.

[0034] 2. Yeast isolation determination method: refer to GB 4789.15-2016 National Food Safety Standard Food Microbiological Examination Mold and Yeast Count.

[0035] 3. Organic acid content determination method: refer to GB 5009.157-2016 National Food Safety Standard Determination of Organic Acids in Food.

[0036] 4. Sensory analysis method: refer to GB / T 10221-2021 / ISO 5492:2008 Sensory Analysis Terminology.

[0037] 5. Flavor profile analysis: refer to GB 12313-90 Sensory Analysis Method Flavor Profile Test.

[0038] Example 1 Screening of Plantaricaceae LP.3

[0039] 1. Treatment of raw materials:

[0040] Under sterile operation conditions, 25 g of small leaf mustard with good natural fermentation flavor was weighed and placed in 225 mL of sterile water, and a homogenizer was used for homogenization for 30 min. 10-fold gradient dilution was performed with sterile water to 10 -1 -10 -6 , 10 -4 , 10 -5 , 10 -6 , 3 dilution gradients, pour inoculation method was used to inoculate MRS and malt juice fixed culture medium, 1 mL / dish was inoculated for each dilution, and it was placed in a 30℃ constant temperature incubator for inverted anaerobic culture for 48 h.

[0041] Select the culture dish with 30-300 CFU / dish, mark the colony morphology and describe the morphological difference, and perform microscopic morphological examination on the colonies with different colony morphology and describe it. Select typical lactic acid bacteria for purification, and prepare glycerol freezing tubes after purification for 3 times, and store them in -80℃ medical refrigerator for standby. A total of 4 test microorganisms were obtained, named LP.3, LN.1, P.4, and CA.2.

[0042] 2. Primary screening of lactic acid bacteria performance

[0043] Take 200 μL of the glycerol freezing solution of each of the 4 strains of test bacteria selected above, and inoculate them into 100 mL of liquid medium (MRS medium), and incubate them at 37℃ for 48 h. Adjust the concentration of the bacterial solution to 1×10 8 CFU / mL, which is the test bacterial suspension. Determine the organic acid content of the test bacterial suspension, and record the results in Table 1.

[0044] Table 1 Determination of organic acid content of MRS culture of lactic acid bacteria

[0045]

[0046] According to the results in Table 1, the ability to produce malic acid is LP.3 > LN.1 > P.4 > CA.2, the ability to produce lactic acid is LN.1 > P.4 > LP.3 > CA.2, and the ability to produce citric acid is CA.2 > P.4 > LP.3 > LN.1. The content of malic acid produced by LP.3 is as high as 23.53 g / kg, and the contents of lactic acid and citric acid produced are low. The strains are applied to the fermentation of small leaf mustard to verify the performance of the strains in producing malic acid during the fermentation of small leaf mustard.

[0047] 3. Secondary screening of lactic acid bacteria

[0048] Take fresh small leaf mustard and wash it, and then soak it in 7°Be' salt water for 30 mins and take it out. Weigh it and neatly pack it into a 25 kg fermentation jar, and put the small leaf mustard to the mouth of the jar. Mix the test bacterial suspension evenly in the material water (3% (w / w) food-grade icing sugar, 3% (w / w) food-grade sodium chloride aqueous solution), and inoculate the lactic acid bacteria at a concentration of 1×10 6 CFU / g of the total system, add the material water to the mouth of the jar, seal the mouth of the jar with water, mark it, and place it in a dark place indoors for natural fermentation for 45 days. Maintain the mouth of the jar every 3-5 days, and try to avoid opening the jar during fermentation. The control group is the same as the test group except that it does not add the test strain.

[0049] Perform sensory analysis and flavor profile analysis on the fermented mature small leaf mustard, and determine the contents of L-malic acid, citric acid, and lactic acid, analyze the corresponding relationship of organic acids to taste-acid, and select the best fermentation strain according to the results of flavor profile analysis. The results are recorded in Tables 2, 3, and Figure 1 .

[0050] Table 2 Sensory flavor analysis of different processing small leaf mustard

[0051]

[0052]

[0053] Table 3 Determination of organic acid content of different processing small leaf mustard

[0054]

[0055] From Figure 1 The results shown in Table 2 and Table 2 show that the comprehensive impression LP.3 > LN.1 > CA.2 > P.4 > CK, LP.3 sensory flavor analysis, color, fragrance, taste and comprehensive evaluation effect is best, the sour taste is rich and harmonious, the jar aroma is thick. Compared with other groups, the small leaf mustard inoculated with LP.3 fermentation, the color of the heart and the color of the leaf are uniform golden yellow; the sour taste is slightly rich and soft, the sour taste is rich, there is no sterile smell, there is no fermentation pollution phenomenon, the jar aroma is thick, the sour aroma is thick, and the retention degree is long.

[0056] The results of the determination of organic acid content in Table 3 show that the L-malic acid content of the small leaf mustard fermented by the test lactic acid bacteria LN.1 and LP.3 is high, which is 2.16 g / kg and 3.77 g / kg respectively; the lactic acid content is low, which is 6.15 g / kg and 9.48 g / kg respectively; the citric acid content is low, which is 3.91 g / kg and 4.11 g / kg respectively. Combined with the results of sensory profile analysis, malic acid, citric acid and lactic acid are the main sour substances of small leaf mustard. The sour taste of biological source malic acid is rich and soft, the sour taste is sufficient, the sour taste is pure and the sour taste is harmonious. The sour taste of biological source citric acid and lactic acid is single, which presents oligo-acid flavor, and the sour taste is not harmonious. Therefore, the principle of selecting high-yield organic acid strain for fermented small leaf mustard is high-yield malic acid strain. Combined with the results of flavor profile analysis, sensory flavor analysis and organic acid content determination, LP.3 strain is selected as the excellent strain for small leaf mustard fermentation.

[0057] The colony morphology of the test LP.3 strain in pure culture in MRS medium is shown in Figure 2 The colony is milky white, the edge is neat and round, the surface is smooth, wet and shiny; the cell morphology observed under a microscope after Gram staining is shown in Figure 3 The strain is a typical bacillus morphology, the cell length is 0.5-0.8 μm, and the width is 0.2-0.4 μm, and the result is obtained by magnifying 1000 times. It is identified as Lactiplantibacillus plantarum by sequencing, and preserved in China General Microbiological Culture Collection Center on March 29, 2023, with the preservation number of CGMCC No.30203.

[0058] Example 2 Lactiplantibacillus plantarum LP.3 fermentation of small leaf mustard application test

[0059] Take 200 μL of Lactiplantibacillus plantarum LP.3 frozen bacteria solution and inoculate it into 100 mL of liquid medium (MRS medium) and incubate it at 37°C for 48 h to obtain the first-stage seed liquid. Inoculate the first-stage seed liquid into liquid medium (MRS medium) at a ratio of 1% and incubate it at 37°C for 24 h. After centrifugation, resuspend the bacterial cells in physiological saline and adjust the concentration of the bacterial liquid to 1 x 10 8 CFU / mL, which is the seed liquid.

[0060] Wash fresh small leaf mustard with a bubble washing machine, weigh it, and evenly pack it into 350 kg fermentation jars. Add Lactiplantibacillus plantarum LP.3 seed liquid to the brine (3% (w / w) food-grade icing sugar and 3% (w / w) food-grade sodium chloride aqueous solution), mix well, and inoculate the bacteria at a concentration of 1 x 10 6 CFU / g. Add the brine to the jar mouth, seal the jar mouth with water, mark it, and place it in a room indoors away from light for natural fermentation for 45 days. Perform jar mouth maintenance every 3-5 days and observe the yeast explosion. The yeast explosion is evaluated when the yeast film on the surface of the brine in the pickle jar exceeds 1 / 3 of the brine surface area. Avoid opening the jar during fermentation. The control group is the same as the test group except that it does not add Lactiplantibacillus plantarum LP.3. Take small leaf mustard fermented for 7 d, 14 d, 28 d, and 45 d for microbial flora analysis, and record the results in Table 3. Take small leaf mustard fermented for 45 d for sensory analysis, and record the results in Table 4. Figure 4

[0061] Table 4 Small leaf mustard 350 kg fermentation jar flavor profile test results

[0062]

[0063] Note: Scale A, evaluated with numbers; 0 = not present; 1 = just recognizable or trace; 2 = weak; 3 = moderate; 4 = strong; 5 = very strong.

[0064] According to the flavor profile analysis of small leaf mustard 350 kg in Table 4, Lactiplantibacillus plantarum LP.3 fermented small leaf mustard in a 350 kg jar fermentation system, and the flavor profile analysis score was 4.5. There was no yeast contamination during the entire fermentation process. The corresponding control group started to show yeast growth on the 18th day of fermentation, and on the 23rd day, there was a large explosion of yeast. The mature small leaf mustard had a yeast flavor, which affected the product flavor. Figure 4 ​The data of microbial groups in different processes of 350 kg size of small leaf mustard showed that the content of lactic acid bacteria group in LP.3 group was obviously higher than that in CK group during the whole fermentation process; the content of yeast was not obviously different at the beginning, and then obvious difference appeared on the 14th day of fermentation, and until the end of fermentation, the content of yeast in LP.3 group was significantly lower than that in CK group.

[0065] It can be seen that the small leaf mustard fermented by the exogenous addition of Lactobacillus plantarum LP.3 can increase the content of lactic acid bacteria group in the fermentation system of small leaf mustard, can play a dominant role in the early stage of small leaf mustard fermentation, can occupy the nutrients in advance, can inhibit the reproduction of yeast group, can play the advantage of good flavor of small leaf mustard fermented by exogenous pure strain group, can improve the flavor of fermented small leaf mustard, can ensure the stability of the fermentation quality of small leaf mustard, and can reduce the potential food safety risk.

[0066] Example 3 Fermentation of 600 kg of small leaf mustard by Lactobacillus plantarum LP.3

[0067] The Lactobacillus plantarum LP.3 seed liquid was prepared according to the method described in Example 2.

[0068] Fresh small leaf mustard was washed with a bubble washing machine, and then soaked in 7°Be' salt water for 30 mins and taken out. After weighing, it was evenly loaded into a 600 kg fermentation jar, and small leaf mustard was put into the jar mouth. The Lactobacillus plantarum LP.3 seed liquid was mixed uniformly in the material water (3% (w / w) food grade icing sugar, 3% (w / w) food grade sodium chloride aqueous solution), and the inoculation amount was 1×10 6 CFU / g, the material water was added to the jar mouth, the jar mouth was sealed with water, and the mark was made. It was placed in a dark place indoors for natural fermentation for 45 days. The jar mouth was maintained every 3-5 days, and the jar was opened as little as possible during fermentation. The control group was the same as the test group except that Lactobacillus plantarum LP.3 was not added. After fermentation, the small leaf mustard was subjected to organic acid determination and flavor profile analysis, and the results were recorded in Table 5 and Table 6. Figure 5

[0069] Table 5 Sensory analysis of 600 kg of small leaf mustard fermentation jar

[0070]

[0071] The results of sensory analysis of 600 kg of small leaf mustard fermentation jar in Table 5 showed that the small leaf mustard fermented by LP.3 had better sensory evaluation than the control group, the acid taste was rich, soft and coordinated, the acid taste was pure, the jar had good aroma, and the acid aroma was rich. The CK group had obvious oligo-acid taste, and the acid taste was single and uncoordinated.

[0072] Figure 5 ​The record results show that the malic acid content of the LP.3 group is 4.05 g / kg, and the malic acid content of the CK group is only 0.69 g / kg, further verifying that malic acid is the main substance of the rich and soft sour taste of small leaf mustard, and the yield of citric acid and lactic acid in the CK group is high, but the sour taste presents a few acid flavor, the sour taste is not coordinated, and the jar aroma is weak. Exogenous addition of plant lactobacillus LP.3 can promote the synthesis of malic acid in a 600 kg specification small leaf mustard fermentation system, and the malic acid content in the fermented mature mustard can be as high as 4.05 g / kg, proving that plant lactobacillus (Lactiplantibacillus plantarum) LP.3 has the advantage of high malic acid yield in the large-scale fermentation process of small leaf mustard, can significantly improve the flavor of fermented small leaf mustard, and improve the quality stability, thereby improving the fermentation quality of small leaf mustard and realizing large-scale industrial production.

[0073] The above-described embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent of the application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A strain of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum LP.3, characterized in that, The preservation number of the Lactiplantibacillus plantarum LP.3 is CGMCC No. 30203.

2. A fermentation inoculant characterized in that, The fermentation agent comprises the Lactiplantibacillus plantarum as claimed in claim 1.

3. Use of Lactobacillus plantarum as claimed in claim 1 or the fermentation inoculum as claimed in claim 2 in the fermentation of mustard, characterized in that, The mustard is small leaf mustard.

4. The use according to claim 3, wherein the compound is ###0002### The application is to increase the content of malic acid in fermented small leaf mustard.

5. A fermentation process of Brassica juncea characterized in that, The method is a method for adding the Lactiplantibacillus plantarum as claimed in claim 1 or the fermentation agent as claimed in claim 2 to small leaf mustard for fermentation.

6. The method of claim 5, wherein, The inoculation time of the Lactiplantibacillus plantarum or the fermentation agent is the initial stage of fermentation.

7. The method of any one of claims 5 or 6, wherein, The inoculation amount of the plant Lactobacillus is 10 6 CFU / g.

8. The method of claim 5, wherein, The fermentation time is 40-50 days.

9. A fermented leaf mustard, characterized by, The fermented small leaf mustard is prepared by the method as claimed in any one of claims 5-8.

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