Lactobacillus paracasei LK10 and application thereof

Abstract

The invention discloses lactobacillus paracasei LK10 and application thereof, and relates to the technical field of microorganisms, the lactobacillus paracasei LK10 is obtained by being separated from yellow serofluid which is a by-product of bean curd processing, the name of the strain is Lacticasei paracasei LK10, and the preservation number of the strain is GDMCC No.64333. The lactobacillus paracasei LK10 is high in acid production speed and resistant to bile salt, metabolites can inhibit bacteria and resist oxidation, and the lactobacillus paracasei LK10 can be used for preparing the lactobacillus paracasei LK10. The method has a wide application prospect in food fermentation, preservation and health promotion.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus paracasei LK10 and an application thereof. Background Art

[0002] Lactobacillus paracasei, as a probiotic, is widely found in fermented dairy products and other fermented foods and has attracted much attention due to its various benefits to human health.

[0003] Traditional Lactobacillus paracasei strains have differences in acid production rate, bile tolerance and metabolite activity, which limits their efficiency and effectiveness in industrial applications. Therefore, screening Lactobacillus paracasei strains with excellent characteristics is of great significance for improving food fermentation quality, extending shelf life and developing new functional foods. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a Lactobacillus paracasei LK10 and an application thereof.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: a strain named Lacticaseibacillusparacasei LK10, which not only produces acid quickly, but also exhibits excellent bile salt tolerance. Its metabolites have significant antibacterial and antioxidant activities, providing new solutions for the fields of food fermentation, preservation and health promotion.

[0006] The Lacticaseibacillusparacasei LK10 described in the present invention is isolated from yellow pulp water, a byproduct of tofu processing, and its excellent properties are confirmed after a series of screening and identification. The strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No.64333 and a deposit date of January 29, 2024.

[0007] The Lacticaseibacillusparacasei LK10 of the present invention has the following characteristics:

[0008] (1) Rapid acid production: Under suitable fermentation conditions, the LK10 strain can rapidly produce organic acids such as lactic acid, effectively lowering the pH value of the fermentation environment, accelerating the fermentation process, and improving fermentation efficiency.

[0009] (2) Bile salt resistance: This strain has a high bile salt resistance and can grow stably and function under conditions that simulate the human intestinal environment, making it possible for its application in promoting intestinal health.

[0010] (3) Metabolite activity: The metabolites of the LK10 strain are rich in substances with antibacterial and antioxidant activities, which can effectively inhibit the growth of a variety of harmful microorganisms, while scavenging free radicals and protecting cells from oxidative damage, and have broad application potential.

[0011] In order to gain a deeper understanding of the genetic background and functional characteristics of the LK10 strain, the present invention conducted whole genome sequencing on it. The results showed that the whole genome of the LK10 strain consisted of 1 chromosome and 3 plasmids. The original sequencing data and assembly results have been uploaded to the NCBI database. The relevant information is as follows:

[0012] Bioproject: PRJNA1119716

[0013] Biosample: SAMN41663323

[0014] Compared with the existing technology, the beneficial effects of this scheme are as follows: based on the excellent characteristics of LK10 strains such as rapid acid production, bile salt tolerance and metabolite activity, it has broad application prospects in the fields of food fermentation, preservation and health promotion. For example, in the production of dairy products, kimchi, sour soup, tofu coagulant and other foods, LK10 strains can significantly improve fermentation efficiency and product quality; in terms of food preservation, its metabolites can be used as natural preservatives to extend the shelf life of food; in addition, LK10 strains can also be used as functional food raw materials for the development of health products with antibacterial and antioxidant effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the separation and identification of LK10 in the embodiment of the present invention, wherein Figure 1 A is a single colony, Figure 1 B is the purified colony. Figure 1 C is the Gram staining image of the strain;

[0016] Figure 2 is the growth characteristic curve of LK10 in the embodiment of the present invention, wherein Figure 2 A is the growth curve of LK10 in MRS liquid medium and the pH change curve of MRS, Figure 2 B is the growth curve of LK10 in MRS containing 0, 1.0%, and 3.0% ox bile salt;

[0017] Figure 3 The effect of LK10 inoculation amount and fermentation time on the quality of sour soup in the embodiment of the present invention is shown in FIG. Figure 3 A is the effect of different inoculation amounts on the total acid and sensory quality of sour soup. Figure 3 B shows the effect of different fermentation times on the total acid and sensory quality of sour soup. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0019] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0020] Example 1 Isolation and Identification of Lactobacillus paracasei LK10

[0021] The yellow slurry was diluted in gradients and spread on MRS agar plates supplemented with 2.0% calcium carbonate, cultured at 37°C for 36-48h, and single colonies with calcium-dissolving circles were picked with a sterile inoculation loop ( Figure 1 A) Perform streaking separation and, after multiple purifications, pick white or milky white, round, smooth colonies ( Figure 1 B) Gram staining showed that the bacteria were purple and rod-shaped, and were preliminarily identified as Gram-positive bacteria ( Figure 1 C); further 16S rDNA identification and NCBI blast revealed that the 16S rRNA of LK10 was 100% similar to that of Lactobacillus paracasei 2734 (Genebank Accession: MT611745.1), and it was identified as Lacticaseibacillusparacasei.

[0022] The 16S rRNA sequence of LK10 is shown as SEQ ID NO: 1, and its Genebank ID is PP859167 in NCBI.

[0023] Example 2 Whole genome sequencing of Lactobacillus paracasei LK10

[0024] LK10 was cultured in liquid culture until mid-logarithmic phase, and genomic DNA of LK10 was extracted. DNA integrity was confirmed by agarose electrophoresis, and its concentration and purity were evaluated by readings obtained by Drop 2000 micro-nucleic acid protein quantifier (Thermo Fisher, USA). PacBio library was constructed, and Illumina platform Pair End double-end sequencing and third-generation sequencing platform PacBioSMRT single-molecule sequencing were used. Fastp 0.20, SOAPdenovo 2.04 and GapCloser 1.12 software were used for quality control, assembly and gap region filling of the obtained data, respectively; bowtie22.5.1 and meryl software were used to perform GC-depth distribution analysis and K-mer frequency distribution analysis on the genome sequence obtained by preliminary assembly, respectively. The results of the genome assembly were used to predict chromosomes and plasmids using Prodigal 2.6.3 and GeneMarkS 4.3, and the coding sequences, tRNA, and rRNA distribution were predicted using Glimmer, tRNAscan-SE v2.0, and Barrnap 0.4.2 software. Gene annotation was performed by comparing with the six major databases of Non-Redundant protein database, Swiss-Prot database, Pfam database, Clusters of Orthologous Groups of proteins, Gene Ontology, and KEGG Database for functional annotation. The data was analyzed on the Majorbio cloud platform (www.majorbio.com). The raw data analysis data of the whole genome sequencing was uploaded to NCBI, and the upload results are as follows:

[0025] Bioproject: PRJNA1119716

[0026] Biosample:SAMN41663323

[0027] WGS accession:CP158067-CP158070

[0028] Sequence ReadArchive: SRR29409814, SRR29409813

[0029] Methylation data: SUPPF_0000005619

[0030] Example 3 Lactobacillus paracasei grows fast, produces acid quickly, and is resistant to bile salts

[0031] The activated strain was inoculated into MRS or MRS medium containing bile salts at a rate of 3.0% of the bacterial solution, and cultured in a 37°C incubator. The pH and OD were measured every 2 hours. 600 Value, do three replicates, with time as the horizontal axis and pH or OD as the horizontal axis 600nm The pH change curve and growth curve are plotted as the vertical axis ( Figure 2 ).

[0032] Example 4 Application of Lactobacillus paracasei LK10 in sour soup

[0033] Tomato, red pepper, ginger, garlic and other raw materials were inoculated with Lactobacillus paracasei LK10 and fermented at 37℃ for 8-14 days. The color difference, total acidity and sensory evaluation of sour soup were used as the evaluation indicators to study the effects of inoculation amount and fermentation time on the quality of sour soup. Figure 3 As shown in Tables 1 and 2, the prepared sour soup is red, has a mild sour taste and good flavor.

[0034] Table 1 Color difference of sour soup prepared with different inoculum amounts

[0035]

[0036] Table 2 Color difference of sour soup prepared at different fermentation times

[0037]

[0038] Example 5 Physiological activity of fermented sour soup of Lactobacillus paracasei LK10

[0039] The sour soup produced by fermentation of Lactobacillus paracasei LK10 was centrifuged at 4°C and 10000r / min for 10min, and the supernatant was taken for DPPH free radical scavenging ability and antibacterial test.

[0040] The DPPH free radical scavenging ability was determined according to the existing method. 2 mL of DPPH and 2 mL of acid soup were added to the ep tube and mixed. After standing for 20 minutes, the absorbance was measured at 517 nm. Anhydrous ethanol was used as the control group.

[0041] Calculation formula Where: A0 is the absorbance of ethanol, A1 is the absorbance of sour soup.

[0042] The antibacterial activity was determined by the antibacterial zone test. First, the filter paper was placed on the LB solid culture medium, and then 100uL of each component of the acid soup was dropped on the filter paper. After drying, the Escherichia coli was spread with a coating rod and sealed. The sealed culture dish was placed in an incubator for 24 hours, and the diameter of the antibacterial zone was measured.

[0043] As shown in Table 3, the scavenging rates of the sour soup stock solution and the centrifuged solution prepared by LK10 fermentation on DPPH were 58.53% and 47.51%, respectively, and the inhibition zones on Escherichia coli were 3.50 mm and 3.10 mm, respectively, indicating that the sour soup prepared by LK10 fermentation had good physiological activity.

[0044] Table 3 Physiological activity of sour soup

[0045]

[0046] In summary, the Lactobacillus paracasei LK10 of the present invention can be cultured and expanded by conventional microbial culture methods. In the process of food fermentation or health product preparation, parameters such as strain inoculation amount and fermentation conditions can be adjusted according to specific needs to give full play to its excellent characteristics. The Lactobacillus paracasei LK10 provided by the present invention and its application provide new technical means and solutions for the fields of food fermentation, preservation and health promotion, and have significant economic and social benefits.

[0047] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A Lactobacillus paracasei LK10, Its characteristics are: The strain is LacticaseibacillusparacaseiLK10, and its strain collection number is GDMCC No.64333.

2. a kind of Lactobacillus paracasei LK10 as claimed in claim 1, Its characteristics are: The whole genome of the strain consists of 1 chromosome and 3 plasmids.

3. Application of a Lactobacillus paracasei LK10 as claimed in claim 1 in food fermentation, preservation and health promotion, Its characteristics are: The Lactobacillus paracasei LK10 is separated from yellow pulp water, a by-product of tofu processing.

4. An application of Lactobacillus paracasei LK10 in food fermentation, preservation and health promotion.

5. A method of using Lactobacillus paracasei LK10 as claimed in claim 4 in food fermentation, preservation and health promotion, Its characteristics are: The Lactobacillus paracasei LK10 has a fast acid production speed and is resistant to bile salts, and its metabolites can inhibit bacteria and resist oxidation.

Images