CN110257285B

CN110257285B - Lactobacillus plantarum with functions of efficiently degrading nitrite and strongly producing acid and application thereof

Info

Publication number: CN110257285B
Application number: CN201910531157.XA
Authority: CN
Inventors: 陈加平; 苏容容; 熊蝶; 袁岚玉; 冯武
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2021-12-14
Anticipated expiration: 2039-06-19
Also published as: CN110257285A

Abstract

The invention provides lactobacillus plantarum with functions of efficiently degrading nitrite and strongly producing acid, which is named as lactobacillus plantarum P42 with the preservation number as follows: CCTCC NO. M2019369. The invention also provides the application of the lactobacillus plantarum in fermenting pickles; inoculating the strain to an MRS culture medium containing 200mg/L of nitrite, culturing at 37 ℃ for 24 hours, wherein the degradation rate of the nitrite is 97.28 +/-0.64%, and the pH of a culture solution is changed from 5.83 +/-0.01 to 3.45 +/-0.02; after culturing for 48 hours at 37 ℃, the degradation rate of nitrite reaches 99.66 +/-0.29 percent. The lactobacillus plantarum P42 is inoculated and fermented to obtain the pickle, the maximum nitrite content is only 1.85 +/-0.03 mg/kg after the next day, and the prepared fermented pickle is superior to the naturally fermented pickle in color, texture, taste, smell and overall acceptability.

Description

Lactobacillus plantarum with functions of efficiently degrading nitrite and strongly producing acid and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to lactobacillus plantarum with functions of efficiently degrading nitrite and strongly producing acid and application thereof.

Background

Lactic acid bacteria are a general term for a group of bacteria that can utilize fermentable sugars and produce large amounts of lactic acid. Lactic acid bacteria are the dominant bacteria in the pickle and play an important role in the fermentation process of the pickle. It can not only shorten the fermentation time of the pickle, but also generate various substances such as lactic acid, acetic acid, amino acid and the like in the fermentation process, thereby further improving the nutritive value of the vegetables.

The pickle is one of the traditional Chinese fermented vegetables, is characterized by being sour, tasty and pleasant in fragrance, and is widely eaten in many areas of China, especially in the north of China. The pickles are popular among many people because of their good taste and high nutritive value. The home-made kimchi is generally prepared by mixing fresh or salted vegetables (e.g., cabbage, radish, carrot, cowpea, lotus root) as a raw material, soaking in a flavored (e.g., ginger, pepper, zanthoxylum, garlic) brine, and then subjecting to anaerobic fermentation at room temperature. However, nitrate existing in vegetable tissues is easily converted into nitrite during fermentation of kimchi, and accumulation of nitrite in kimchi becomes a common problem.

Nitrite (Nitrite), NIT for short, is often added into fermented meat products, can well keep bright red color of the meat products, can also enhance the sensory quality of the meat products, and also has an antiseptic effect. However, excessive consumption of food containing nitrite can result in excessive intake of nitrite in human body, and may cause methemoglobinemia. Meanwhile, nitrite can be combined with secondary amine which is a protein decomposition product in food to generate N-nitroso compound with strong carcinogenicity, and the risk of carcinogenesis is caused. Therefore, reducing the nitrite content is very important to maintain food safety.

The existing methods for degrading nitrous acid mainly comprise three methods, namely a chemical method, a biological method and a biological enzymolysis method. The chemical method has strong capability of degrading nitrite, but the antioxidants VC and VE are easy to be oxidized, and are not beneficial to being applied to a complex food system. The biological enzyme method degrades nitrite, mainly uses microbe to generate nitrite reductase to decompose nitrite. However, since the nitrite reductase found in the current research is basically intracellular, it is difficult to widely apply it to the food industry production. The application of biological methods is a safer and less costly method. One of the simplest, safe and effective methods is to use lactobacillus strains to control the nitrite content in the pickled product and pickle fermentation process. Many researches show that the lactic acid bacteria separated from the pickled vegetables can degrade nitrite in the pickled vegetables, but it is very difficult to screen a lactic acid bacteria strain which has both nitrite degradation and strong acid production capacity.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides lactobacillus plantarum with functions of efficiently degrading nitrite and strongly producing acid and application thereof. The strain has extremely strong acid production capability and acid resistance capability and also has extremely strong capability of degrading nitrite, is identified as lactobacillus plantarum, is inoculated into pickle products obtained by pickle, and has good sensory evaluation: the texture analysis and the sensory analysis show that the pickled vegetables fermented by the lactobacillus plantarum are better than the naturally fermented pickled vegetables in color, texture, mouthfeel, smell and overall acceptability.

One of the purposes of the invention is to provide a Lactobacillus plantarum with functions of efficiently degrading nitrite and strongly producing acid, which is named Lactobacillus plantarum P42(Lactobacillus plantarum P42) and China Center for Type Culture Collection (CCTCC) NO. M2019369.

The lactobacillus plantarum P42 is a bacterial strain separated and screened from home-made pickle in Chongqing by the inventor, and is found to have extremely strong acid production capacity and acid resistance and also have extremely strong capability of degrading nitrite, specifically: the bacterial strain is inoculated to an MRS culture medium containing nitrite, the degradation rate of the bacterial strain to the nitrite is 97.28 +/-0.64 percent after the bacterial strain is cultured for 24 hours at 37 ℃, and the pH of a culture solution is changed from 5.83 +/-0.01 to 3.45 +/-0.02; after culturing for 48h at 37 ℃, the degradation rate of nitrite reaches 99.66 +/-0.29%, and the pH of the culture solution is changed from 5.83 +/-0.01 to 3.49 +/-0.02.

The strain is identified as Lactobacillus plantarum (Lactobacillus plantarum) by colony morphology, biochemistry and 16S rRNA sequencing analysis, and is named as Lactobacillus plantarum P42.

The second purpose of the invention is to provide the application of the lactobacillus plantarum with the functions of efficiently degrading nitrite and strongly producing acid in the fermentation of pickled vegetables.

The invention also aims to provide a fermentation inoculant which comprises fermentation liquor obtained by fermenting the lactobacillus plantarum P42, or dry powder inoculant obtained by spray drying the fermentation liquor.

The fourth object of the present invention is to provide a method for preparing fermented kimchi by adding the bacterial suspension or dry powder microbial inoculum prepared from lactobacillus plantarum P42 according to claim 1, Chinese cabbage, salt solution and seasonings into a kimchi jar for fermentation at the beginning of the fermentation of kimchi.

The bacterial liquid prepared by the lactobacillus plantarum P42 is mixed by 1 × 10⁸CFU/mL～1×10¹¹The inoculation amount of CFU/mL is inoculated into the pickle (and corresponding auxiliary materials are added), the bacterial suspension is added at the beginning of fermentation, the inoculation amount of the lactobacillus plantarum P42 is 4%, and the unit of the inoculation amount is mL/L.

The fifth purpose of the invention is to provide the pickle product prepared by the method. The maximum nitrite content of the pickle products obtained by inoculating the lactic acid bacteria P42 in the pickle fermentation process reaches 1.85 plus or minus 0.03mg/kg next day, which is far lower than 20mg/kg specified by the national standard (GB 15198). And the pH value of the brine is changed from 6.78 +/-0.04 to 3.54 +/-0.3 in the fermentation process of the pickle.

The invention has the beneficial effects that:

1. the inventor separates and screens a plurality of strains from home-made pickles in Chongqing, Sichuan, Shaanxi and Hubei provinces, and finally screens to obtain a strain, wherein the strain has extremely strong acid production capability and acid resistance capability and also has extremely strong capability of degrading nitrite, and specifically comprises the following steps: inoculating the Lactobacillus plantarum P42 to MRS culture medium containing 200mg/L nitrite, culturing at 37 deg.C for 24h, wherein the degradation rate of nitrite is 97.28 + -0.64%, and the pH of the culture solution is changed from 5.83 + -0.01 to 3.45 + -0.02; after culturing for 48h at 37 ℃, the degradation rate of nitrite reaches 99.66 +/-0.29%, and the pH of the culture solution is changed from 5.83 +/-0.01 to 3.49 +/-0.02. The identified strain is lactobacillus plantarum named lactobacillus plantarum P42.

2. The inventor inoculates lactobacillus plantarum P42 to pickle obtained pickle products, and tests show that the maximum nitrite content reached the next day of fermentation is only 1.85 plus or minus 0.02mg/kg, which is far lower than 20mg/kg specified by the national standard (GB 15198). And after fermenting for 3 days, the pH of the brine in the pickle fermentation process is changed from 6.78 +/-0.04 to 3.51 +/-0.25, and the brine in the natural fermentation is changed from 6.72 +/-0.07 to 5.47 +/-0.01 in the third day.

The lactobacillus plantarum P42 can accelerate the ripening of the pickle and the fermented pickle has good sensory evaluation when being applied to the pickle fermentation process: the texture analysis and the sensory analysis show that the pickled vegetables fermented by the lactobacillus plantarum P42 are better than the naturally fermented pickled vegetables in terms of color, texture, mouthfeel, smell and overall acceptability.

The preservation date of the strain is 5 months and 17 days in 2019, and the preservation number is CCTCC NO. M2019369. The Lactobacillus plantarum is classified and named as Lactobacillus plantarum P42(Lactobacillus plantarum P42), the name of the preservation unit is the China center for type culture Collection, and the address is Wuhan university in Wuhan City, Hubei province, China, zip code: 430072.

drawings

FIG. 1 is a morphological photograph of gram-stained lactic acid bacterium P42 under a microscope;

FIG. 2 is a phylogenetic tree analysis of strain P42.

Detailed Description

Example 1 directed enrichment isolation and identification of Strain P42

1. Directional enrichment and isolation of Strain P42

Collecting samples from home-made sauerkraut of Chongqing, Sichuan, Shaanxi and Hubei provinces, adding 90mL Lactobacillus plantarum liquid culture medium (LPB) with pH4.0, performing enrichment culture at 37 deg.C for 12 hr, and gradually diluting with 10 times gradient of sterile water (10 times of gradient)^-1～10^-5) Coating 200 μ L of the mixture on a coating containing 0.5% CaCO₃Carrying out anaerobic culture in a lactobacillus plantarum solid medium (LPA) flat plate at 37 ℃ for 48h, selecting a bacterial colony with a large calcium melting ring, carrying out streaking separation and purification for multiple times, and screening multiple strains of microorganisms with large calcium melting rings.

Respectively inoculating the microbial colonies with larger calcium melting rings to an MRS liquid culture medium, standing and culturing at 37 ℃ to a stable period, inoculating the microbial colonies to an MRS liquid culture solution containing 200mg/L according to the inoculation amount of 4% for culturing, and measuring the NaNO in the MRS liquid by adopting a Grignard colorimetry after culturing for 24-48h₂The cell density was measured by spectrophotometry, and the pH of the bacterial solution was measured by a pH meter.

The result shows that the bacterial strain P42 from home-made pickle in Hubei province has the degradation rate of 97.28 +/-0.64 percent after being cultured for 24 hours at 37 ℃, and the pH of the culture solution is changed from 5.83 +/-0.01 to 3.45 +/-0.02; after culturing for 48h at 37 ℃, the degradation rate of nitrite reaches 99.66 +/-0.29%, and the pH of the culture solution is changed from 5.83 +/-0.01 to 3.49 +/-0.02.

2. Identification of Strain P42

(1) Thallus and colony morphological characteristics of strain P42

The bacterial strain P42 preserved in glycerol in a refrigerator at the temperature of-80 ℃ is unfrozen on ice, a circulant is taken by using an inoculating loop to perform lineation on an MRS agar plate, a culture dish is placed in a constant temperature incubator upside down to be cultured for about 48 hours at the temperature of 37 ℃, and the colony morphology is observed. Single colonies growing on MRS agar plates were stained with gram stain, and the morphology of the cells was observed under a microscope, as shown in FIG. 1.

The observed colony color is milky white and opaque, the colony shape is round, small and convex, the edge is neat, the surface is smooth and moist, the picking is easy, and the fermentation sourness is light. The gram staining result shows that the cells are bluish purple after being stained, gram positive bacteria exist in short rod shape and exist singly or in pairs.

(2) Genetic characterization

The activated strain P42 was identified by 16SrDNA gene sequence analysis. Firstly, extracting DNA of a strain by adopting an SK8255-Ezup column type bacterial genome DNA extraction kit, then taking the genomic DNA of the strain as an amplification template of PCR, and carrying out PCR amplification by taking a PCR reaction primer as a universal primer of bacteria. And finally, carrying out agarose gel electrophoresis on the product after PCR amplification, and sending the product meeting the sequencing requirement to a company Limited in biological engineering (Shanghai) for sequencing after electrophoresis.

The sequencing results were compared with the Blast search program for gene sequences from the database (NCBI) and phylogenetic trees were constructed (fig. 2). The homology of the P42 strain and model strains, namely Lactobacillus plantarum strain BCH-2, Lactobacillus plantarum L.P 205, Lactobacillus plantarum strain CSI9 and Lactobacillus plantarum strain CSI3, is found to reach 99%. The strain P42 is determined to be Lactobacillus plantarum by combining physiological and biochemical characteristics, and is named as Lactobacillus plantarum P42(Lactobacillus plantarum P42). And the culture is preserved in the China center for type culture Collection in 2019, 5 and 17 months, with the preservation number of CCTCC NO. M2019369.

2, the capability of lactobacillus plantarum P42 in degrading nitrite and the capability of strongly producing acid are measured

1. Preparation of Lactobacillus plantarum P42 bacterial suspension

Taking the slant of Lactobacillus plantarum P42 stored in a refrigerator at 4 ℃, streaking with an inoculating loop, inoculating on MRS agar medium, and culturing at 37 ℃ in a constant temperature incubator for 24 h. Inoculating a single colony in an MRS liquid culture medium, and culturing for 16h at 37 ℃ in a constant temperature incubator. Transferring turbid MRS liquid culture medium into a sterile centrifuge tube in a super clean bench for centrifugation, discarding supernatant, washing the bacteria twice with 0.85% sterile normal saline, adding 0.85% sterile normal saline, mixing well, and making into 1 × 10⁸CFU/mL of bacterial suspension is ready for use.

2. Inoculating lactobacillus plantarum P42 bacterial suspension

5mL of sterilized 2000. mu.g/mL NaNO₂The solution was added to 45mL of MRS broth to obtain a solution containing 200mg/L of NaNO₂The culture broth of (4), and inoculating a bacterial suspension of Lactobacillus plantarum P42 to a culture broth containing 200mg/L of NaNO₂In the broth medium of (1). Wherein the inoculation amount is 2% (v/v), and the fermentation temperature is 37 ℃.

Simultaneously inoculating bacterial suspension of lactobacillus plantarum P42 to NaNO containing 300mg/L₂In the broth medium of (1). Wherein the inoculation amount is 2% (v/v), and the fermentation temperature is 37 ℃.

3. Sampling and measuring the content of nitrite and the change of pH value

And centrifuging the fermentation liquor (4 ℃, 5min, 10000r/min) at the time points of 0h, 4h, 8h, 12h, 24h, 36h and 48h of fermentation, measuring the content of the nitrite in the obtained supernatant by using a colorimetric nitrite method, and calculating the degradation rate. Inoculating to NaNO containing 200mg/L₂The results in the broth culture are shown in Table 1, inoculated with 300mg/L NaNO₂The results in the broth culture are shown in Table 2.

TABLE 1

As can be seen from Table 1, the sample contains 200mg/L of NaNO₂Under the condition of the MRS culture medium, the degradation rate of the lactobacillus plantarum P42 to nitrite reaches 97.28 +/-0.64% after 24 hours, and the pH is changed from 5.83 +/-0.01 to 3.45 +/-0.02, which shows that the lactobacillus plantarum has extremely strong acid production capability and acid resistance capability and also has extremely strong capability of degrading nitrite.

TABLE 2

As can be seen from Table 2, the sample contains 300mg/L of NaNO₂Under the condition of the MRS culture medium, the degradation rate of the lactobacillus plantarum P42 on nitrite reaches 96.01 +/-0.93 percent after 24 hours, and the pH is changed from 5.83 +/-0.01 to 3.79 +/-0.03, which shows that the lactobacillus plantarum contains 300mg/L of high-concentration NaNO₂Under the condition, the acid-proof agent still has extremely strong acid-producing capability and acid-proof capability and also has extremely strong capability of degrading nitrite.

Example 3 inoculation of Lactobacillus plantarum P42 for kimchi fermentation experiment

1. Preparation of pickled vegetable

The formula of the pickle is as follows: 400g of Chinese cabbage, 30g of garlic, 30g of ginger, 5g of pepper, 5g of dry red pepper, 40g of salt and 1L of cold boiled water. Cleaning Chinese cabbage, cutting, draining for later use, slicing ginger, peeling garlic for later use, putting raw materials and seasonings into a pickle jar, adding salt water, and performing an experimental group: the lactobacillus plantarum P42 bacterial suspension is inoculated as a starter for fermentation, and the inoculation amount is 40 mL. The fermentation temperature of the pickle is 25 ℃, the fermentation time is 7d, the control group is naturally fermented, and the lactobacillus plantarum P42 bacterial suspension is not inoculated.

2. Nitrite content in pickled vegetable and pH measurement of brine

Table 3 shows the nitrite content and the pH of the brine in the kimchi measured from the kimchi and the kimchi brine.

TABLE 3

As can be seen from Table 3, the maximum nitrite content of 33.14. + -. 19.63mg/kg is reached the next day in the natural fermentation process, and the maximum nitrite content of 1.85. + -. 0.03mg/kg is reached the next day by the lactobacillus plantarum P42 inoculation fermentation. The nitrite content of the pickled vegetable fermented by inoculating the lactobacillus plantarum P42 is always at a very low level and is always lower than that generated in the natural fermentation process. The fermented pickle inoculated with the lactobacillus plantarum P42 can greatly reduce the nitrite content in the pickle. The pH of the brine of the fermented pickle inoculated with the lactobacillus plantarum P42 is quickly reduced from 6.78 +/-0.04 to 4.06 +/-0.28 in the first day and is basically stabilized to 3.51 +/-0.25 in the third day, and the change of the naturally fermented brine is slowly changed from 6.72 +/-0.07 to 5.47 +/-0.01 in the first day. The fermentation time can be greatly shortened by inoculating the P42 fermented pickle.

3. Performing texture analysis and sensory evaluation on sauerkraut

The texture analysis and sensory evaluation of kimchi after 7 days of kimchi fermentation are shown in tables 4 and 5.

TABLE 4 texture Change of naturally fermented kimchi and fermented kimchi inoculated with Lactobacillus plantarum P42

As can be seen from table 4, the texture (hardness, stickiness, chewiness, elasticity and resilience) of the kimchi fermented naturally at the same temperature was similar to that of the kimchi fermented by lactobacillus plantarum P42 (P > 0.05). This indicates that the inoculation of lactobacillus plantarum P42 did not change the texture of kimchi.

TABLE 5 sensory evaluation of naturally fermented kimchi and fermented kimchi inoculated with Lactobacillus plantarum P42

As can be seen from table 5, the lactobacillus plantarum P42 inoculated fermented kimchi was superior to the naturally fermented kimchi in terms of color, texture, taste, smell, and overall acceptability. It is demonstrated that the fermented kimchi inoculated with lactobacillus plantarum P42 can improve the sensory quality of kimchi. The taste of the high-quality pickled vegetable is not fresh Chinese cabbage taste or too heavy sour taste, and the sour taste is within the acceptable degree of consumers; the pH of the brine of the pickle inoculated with the lactobacillus plantarum P42 is 3.51 +/-0.25, and the pickle has good sour taste.

The above description is only an example of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. Lactobacillus plantarum with functions of efficiently degrading nitrite and strongly producing acid is characterized in that Lactobacillus plantarum P42 is named, and the preservation number is as follows: CCTCC NO. M2019369.

2. The use of lactobacillus plantarum with the functions of efficiently degrading nitrite and strongly producing acid according to claim 1 in the fermentation of kimchi.

3. A zymophyte agent is characterized in that the zymophyte agent is: a fermentation broth obtained by fermenting Lactobacillus plantarum P42 according to claim 1, or a dry powder microbial inoculum obtained by spray-drying a fermentation broth obtained by fermenting Lactobacillus plantarum P42 according to claim 1.

4. A method for preparing fermented sauerkraut, which comprises adding the bacterial suspension or dry powder bacterial agent of Lactobacillus plantarum P42 of claim 1, Chinese cabbage, salt solution and flavoring agent into a sauerkraut jar for fermentation at the beginning of sauerkraut fermentation.

5. The method of preparing fermented kimchi according to claim 4, wherein the concentration of the bacterial suspension is 1 x 10⁸CFU/mL-1×10¹¹CFU/mL, and the bacterial suspension was added at the very beginning of the fermentation, the inoculum size of Lactobacillus plantarum P42 was 4%, the unit of said inoculum size being mL/L.