CN112063542A - Lactobacillus paracasei CICC6277 and application thereof in Xinjiang-style chili fermentation - Google Patents
Lactobacillus paracasei CICC6277 and application thereof in Xinjiang-style chili fermentation Download PDFInfo
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- CN112063542A CN112063542A CN202010034383.XA CN202010034383A CN112063542A CN 112063542 A CN112063542 A CN 112063542A CN 202010034383 A CN202010034383 A CN 202010034383A CN 112063542 A CN112063542 A CN 112063542A
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- nitrite
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
- A23L19/20—Products from fruits or vegetables; Preparation or treatment thereof by pickling, e.g. sauerkraut or pickles
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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Abstract
The invention relates to the field of food microorganisms, in particular to Lactobacillus paracasei (Lactobacillus paracasei) CICC6277 capable of efficiently and quickly degrading nitrite, generating biogenic amine at low level and inhibiting pathogenic microorganisms and application thereof in Xinjiang-style pepper fermentation. The strain is preserved in the China general microbiological culture Collection center, and the preservation numbers are as follows: CGMCC 17812. The strain is cultured in an improved MRS liquid culture medium for 24 hours, the degradation rate of nitrite can reach 94.82%, and the degradation rate can reach 100% after culturing for 36 hours; amino acid decarboxylase is negative; and is capable of inhibiting the growth of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella; the bacterial strain CICC6277 is applied to Xinjiang special pepper fermentation by adopting a microorganism strengthening technology, the content of nitrite is 0.81mg/kg, the content of biogenic amine is 4.22mg/kg, which are lower than those of a control group, and the edible safety of the fermented pepper is improved by strengthening the Lactobacillus paracasei (L.paracasei) CICC 6277.
Description
Technical Field
The invention relates to the field of food microorganisms, in particular to lactobacillus paracasei CICC6277 capable of rapidly degrading nitrite, generating biogenic amine at a low rate and inhibiting pathogenic microorganisms and application thereof in Xinjiang special pepper fermentation.
Background
Lactic acid bacteria are a general term for microorganisms which are gram-positive, non-motile or very little motile, anaerobic or facultative anaerobic, and have lactic acid as the main or only product of the fermentative metabolism. Lactic acid bacteria are widely distributed in nature and include strains of the genera Lactobacillus, lactococcus, Streptococcus, Pediococcus, and the like. Most of lactic acid bacteria have a probiotic function, can generate various organic acids in the fermentation process, have the characteristics of acid resistance, salt resistance, nitrite degradation capability, pathogenic microorganism inhibition, low generation of biogenic amine and the like, and are widely applied to the fermentation production of fruits and vegetables.
The fermentation of the fruits and vegetables is a mode of processing the fruits and vegetables by utilizing beneficial microorganisms and controlling production conditions, can keep the nutrition, color, aroma and taste of the vegetables to the maximum extent, has good sensory quality and is popular with consumers. The fruit and vegetable fermentation system is essentially a micro-ecological environment in which various microorganisms interact, wherein lactic acid bacteria play a crucial role. The lactobacillus paracasei CICC6277 obtained by screening can inhibit the growth of pathogenic microorganisms such as escherichia coli, staphylococcus aureus, listeria monocytogenes, salmonella and the like, can rapidly degrade nitrite, has the characteristic of low generation of biogenic amine, and can inhibit the growth of pathogenic microorganisms in fermented peppers, reduce the content of nitrite and biogenic amine in products and improve the edible safety of the fermented peppers by applying the strain to Xinjiang special pepper fermentation production through a microorganism strengthening technology.
Disclosure of Invention
The invention aims to provide a lactobacillus paracasei CICC6277 which is characterized by being capable of efficiently degrading nitrite, producing low biogenic amine and inhibiting the growth of pathogenic microorganisms.
Another object of the present invention is to provide a Xinjiang-style fermented chili pepper inoculated with the Lactobacillus paracasei CICC6277 and having a low content of nitrite and biogenic amine.
The Xinjiang-style fermented chili can overcome the defects of the prior art, can inhibit the growth of pathogenic microorganisms through inoculation, can quickly and efficiently degrade nitrite, and can produce Xinjiang-style fermented chili with low content of nitrite and biogenic amine by virtue of lactobacillus paracasei CICC6277 with low biogenic amine production.
The specific technical scheme of the invention is as follows:
lactobacillus paracasei CICC6277 is a Lactobacillus isolated from Xinjiang specialty pepper in laboratory, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC 17812, the preservation date of 2019, 5 and 15 days, the preservation unit address of No. 3 Hospital No. 1 North West Lu of the Yangtze district in Beijing city, and is named Lactobacillus paracasei by classification. The bacterial colony of the strain is milky white, round, smooth in surface, non-transparent and neat in edge. The thallus is in short rod shape, 0.5-0.6 μm × 1.2-2.6 μm, arranged singly or in pairs, gram-positive, and catalase-negative.
The lactobacillus paracasei CICC6277 has rapid nitrite degradation capability, the nitrite degradation rate can reach 94.82% after being cultured in an improved MRS liquid culture medium for 24 hours, and the degradation rate can reach 100% after being cultured for 36 hours. Lactobacillus paracasei CICC6277 amino acid decarboxylase is negative, which indicates that the strain has weak capability of producing biogenic amine in the process of growth and metabolism. In addition, lactobacillus paracasei CICC6277 has strong capability of inhibiting pathogenic microorganisms, and can inhibit the growth of four common pathogenic bacteria, namely escherichia coli, staphylococcus aureus, listeria monocytogenes and salmonella.
The lactobacillus paracasei CICC6277 is applied to the fermentation production process of Xinjiang specialty peppers, the obtained fermented peppers have the nitrite content of 0.81mg/kg, the biogenic amine content of 4.22mg/kg, nitrite and biogenic amine in the fermented peppers are reduced, and the problem of food safety brought by the nitrite and biogenic amine is solved to a certain extent. In addition, the total acid content of fermented pepper inoculated with the strain reached 2.54%.
Drawings
FIG. 1 shows the scanning electron microscope morphology of Lactobacillus paracasei CICC6277 of the invention
FIG. 2 shows the phylogenetic tree of the 16S rRNA gene of Lactobacillus paracasei CICC6277 according to the invention
FIG. 3 shows the nitrite degradation characteristics of Lactobacillus paracasei CICC6277 of the invention
Detailed description of the preferred embodiments
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
Example 1 identification of Strain CICC6277
The strain CICC6277 in the invention is obtained by separating and screening Xinjiang traditional fermented pepper, and is identified as lactobacillus paracasei from phenotype and genotype level by utilizing a multiphase identification technology.
1. Morphological identification
The morphological characteristics of the strain CICC6277 are identified by referring to the handbook of identifying common bacteria systems, and the strain is observed by a scanning electron microscope (figure 1). And (3) culturing the strain in an MRS culture medium at 37 ℃ for 24h to obtain the bacterial colony which is milky, round, smooth in surface, non-transparent and regular in edge. The thallus is in short rod shape, 0.5-0.6 μm × 1.2-2.6 μm, arranged singly or in pairs, gram-positive, and catalase-negative.
2. Physiological and biochemical characterization
The API 50 CHL identification system is used for physiological and biochemical characterization of the strain CICC 6277. The strain CICC6277 is identified as the cheese of Lactobacillus paracasei (Lactobacillus paracasei), the identification value is 99.1 percent, the T value is 0.99, and the identification result is credible. The strain can produce acid by using D-ribose, mannitol, esculin, salicin, sorbitol, D-cellobiose, D-maltose, D-sucrose, D-trehalose, D-gentiobiose, D-melezitose, D-raffinose, D-glucose, D-fructose, D-mannose, D-lactose, D-galactose and N-acetylglucosamine.
3. PCR amplification of 16S rRNA Gene
Extracting the genome of the lactic acid bacteria by using a bacterial genome DNA extraction kit. The 16S rRNA gene was amplified using primers 27f (5'-AGAGTTT GATCCTGGCTCAG-3') and 1492r (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction system is as follows: template 10 PCR buffer 5 u L, dNTP (2.5mmol/L)4 u L, template 2 u L, Taq DNA polymerase 1 u L, primer each 1 u L, make up deionized water to 50 u L. The reaction conditions are 94 ℃ for 5min, 94 ℃ for 50s, 52 ℃ for 50s, 72 ℃ for 50s, 33 cycles and 72 ℃ for 7 min.
4. Sequence analysis
Similarity comparison is carried out on the obtained 16S rRNA gene sequence and an NCBI database by using BLAST software, and a phylogenetic tree is constructed by using MEGA 5.0 software and a Neighbor Joining method. The strain CICC6277 is in the same branch with the lactobacillus paracasei (figure 2), and is identified as the lactobacillus paracasei by combining with phenotypic characteristics. The 16S rRNA gene sequence accession number is MN 315539.
EXAMPLE 2 characterization of Lactobacillus paracasei CICC6277
1. Nitrite degradation ability
According to GB 5009.33-2010: determination of nitrite and nitrite in food the nitrite degrading ability of Lactobacillus paracasei CICC6277 was determined. Inoculating with 1% inoculum size to a solution containing 0.125mg/mL NaNO2When the strain is cultured for 36 hours at 37 ℃ in the MRS culture medium, the degradation rate of nitrite can reach 100 percent (figure 3).
2. Amino acid decarboxylase test
In the microbial metabolism process, under the action of amino acid decarboxylase, biogenic amines are generated. The screened bacterial strain with negative amino acid decarboxylase as a leaven can reduce the content of biogenic amine in the fermentation production of fruits and vegetables. The test result of amino acid decarboxylase shows that the activities of the lactobacillus paracasei CICC6277 tryptophan decarboxylase, the lysine decarboxylase and the ornithine decarboxylase are all negative.
3. Bacteriostatic ability
The degree of inhibition of lactobacillus paracasei CICC6277 on salmonella enteritidis, escherichia coli, staphylococcus aureus and listeria monocytogenes was determined by a filter paper sheet method. The bacteriostatic diameter of the lactobacillus paracasei CICC6277 overnight culture on salmonella enteritidis reaches 22.3mm, the bacteriostatic diameter on escherichia coli reaches 13.5mm, the bacteriostatic diameter on staphylococcus aureus reaches 14.4mm, and the bacteriostatic diameter on listeria monocytogenes reaches 11.3 mm.
Example 3 application of Lactobacillus paracasei CICC6277 in Xinjiang specialty Pepper fermentation
Selecting fresh Xinjiang Capsici fructus, cleaning, crushing, adding 3% sucrose and 3% salt, inoculating 1% Lactobacillus paracasei CICC6277 (about 10)8CFU/mL), fermenting at constant temperature of 30 ℃ for 7 days to obtain the fermented pepper. The capsicum not inoculated with lactobacillus was used as a control. According to GB 5009.33-2010: the nitrite content of fermented pepper was determined in "determination of nitrite and nitrate in food". According to GB/T5009.208-2008: in the measurement of biogenic amine content in foods, the types and contents of biogenic amine in fermented capsicum and naturally fermented capsicum inoculated with lactobacillus paracasei cic 6277 were measured by HPLC method. According to GB/T12456-2008: acid-base titration method in determination of total acid in food determines total acid content of fermented Capsici fructus. The types and the contents of organic acids in the fermented pepper and the naturally fermented pepper inoculated with lactobacillus paracasei cic 6277 were measured by HPLC method by constructing an organic acid analysis model. The specific measurement results are shown in Table 1.
TABLE 1 fermented Pepper assay index
The lactobacillus paracasei CICC6277 is used for Xinjiang specialty pepper fermentation, and the nitrite content of the obtained fermented pepper is 0.81mg/kg, which is far lower than the nitrite content (2.63mg/kg) of a control group and is far lower than the national standard of 20 mg/kg. In the fermented pepper inoculated with the strain CICC6277, biogenic amine is mainly putrescine, the content of the biogenic amine is 4.22mg/kg, and compared with a control group, the biogenic amine content is reduced by 78.2 percent. Therefore, by inoculating lactobacillus paracasei CICC6277, the content of residual nitrite and biogenic amine in the fermented vegetables can be obviously reduced, and the food safety problem caused by nitrite and biogenic amine is solved. In addition, the total acid content of the fermented pepper inoculated with the lactobacillus paracasei CICC6277 is 2.54 percent higher than that of the naturally fermented pepper, and the content of lactic acid, succinic acid and acetic acid is obviously increased, which shows that the quality of the fermented pepper is improved by the reinforcement of the lactobacillus paracasei CICC 6277.
Claims (3)
1. Lactobacillus paracasei (I)Lactobacillus paracasei) CICC6277, the strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 17812.
2. Lactobacillus paracasei (L.paracasei) according to claim 1Lactobacillus paracasei) Application of CICC6277 in pepper fermentation production is provided.
3. The use as claimed in claim 2, wherein the capsicum is Xinjiang specialty capsicum.
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| CN113061550A (en) * | 2021-04-01 | 2021-07-02 | 新疆农业科学院微生物应用研究所(中国新疆—亚美尼亚生物工程研究开发中心) | Lactobacillus new strain Z6 and application thereof in food |
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| CN112940977A (en) * | 2021-03-05 | 2021-06-11 | 中国海洋大学 | Lactobacillus paracasei and application thereof |
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| CN116138429A (en) * | 2022-09-16 | 2023-05-23 | 新疆康美瑞农业发展有限公司 | Short Kazakhstan yeast XJ-65 and application thereof in pepper fermentation |
| CN116138429B (en) * | 2022-09-16 | 2024-04-02 | 新疆康美瑞农业发展有限公司 | Short Kazakhstan yeast XJ-65 and application thereof in pepper fermentation |
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