Lactobacillus plantarum Lrld-22 and application thereof

Abstract

The invention relates to the technical field of food microorganisms, in particular to lactobacillus plantarum Lrld-22 and application thereof. According to the invention, the strain Lrld-22 is separated from Qinghai yak fermented milk, the strain Lrld-22 is used for fermenting hickory nut and torreya grandis fermented milk for domestication, and the new lactobacillus plantarum is separated and identified from the hickory nut and torreya grandis fermented milk. Research tests show that the lactobacillus plantarum has the functions of reducing uric acid, relieving gout symptoms and removing helicobacter pylori at the same time, and a good basis is provided for enriching the application of the types of probiotics.

Description

Technical Field

[0001] The invention relates to the technical field of food microorganisms, and in particular to a strain of Lactobacillus plantarum Lrl d-22 and an application thereof. Background Art

[0002] Probiotics have a positive impact on human and / or animal health, and most probiotics are composed of lactic acid bacteria. Lactobacillus is a major group of lactic acid bacteria, and there are many differences in their physiological and genetic levels. Unlike the distribution of Lactobacillus casei and Lactobacillus delbrueckii, Lactobacillus can not only be isolated in some specific ecological environments, but Lactobacillus plantarum strains are widely present in meat, dairy products, vegetables, wine, silage and the gastrointestinal tract. The ubiquitous nature of Lactobacillus plantarum is a reflection of its strong metabolic pathways and evolutionary adaptability, so it can improve the nutritional value, flavor and food preservation of fermented foods, and play a vital role in the production of fermented vegetables, beverages, kefir, cheese, and fermented meat products.

[0003] Although lactic acid bacteria are safe and often used in food fermentation and biotechnology industries, as the application scope of probiotic lactic acid bacteria expands, safety issues are increasingly concerned. They may also have adverse reactions such as serious infections and drug-resistant gene transfer. In vitro testing is still the main method for evaluating laboratory properties, but it still cannot meet the needs of risk assessment of a large number of strains. Whole genome sequencing of lactic acid bacteria strains is a high-throughput and effective technical means to evaluate the safety of lactic acid bacteria. Lactic acid bacteria are regarded as GRAS (Generally Recognized as Safe) by the United States Department of Agriculture (USDA) and QPS (Qualified Preferred Scheme) by the European Food Safety Authority (EFSA). Therefore, screening and verifying lactic acid bacteria by genomic and metabolomics analysis is a very important task.

[0004] With the development of social economy, changes in lifestyle and dietary structure, the incidence of hyperuricemia has increased year by year, becoming the second largest metabolic disease after diabetes. In addition to causing gout, elevated blood uric acid levels are also related to the occurrence and development of kidney, endocrine metabolism, and cardiovascular diseases. Therefore, the prevention and treatment of uric acid has become a widely discussed issue for researchers.

[0005] Helicobacter pylori (H.pylori) is a Gram-negative, spiral-shaped, microaerophilic bacterium that can produce urease, an enzyme that can decompose urea in the stomach into carbon dioxide and ammonia, thereby helping the bacteria survive in the gastric acid environment. Helicobacter pylori infection can almost always cause active inflammation of the gastric mucosa, and on this basis, some patients may also develop a series of diseases such as peptic ulcers and gastric cancer. It is the only bacterium discovered so far that can survive in the highly acidic environment of the human stomach and is considered to be one of the main pathogens of gastric ulcers and gastric cancer. The bismuth quadruple regimen is currently commonly used for the treatment of Helicobacter pylori infection, combining proton pump inhibitors, bismuth agents and two antibiotics to achieve the purpose of treatment. However, more effective and less side-effect-prone regimens are needed for the prevention and treatment of Helicobacter pylori to improve compliance with prevention and treatment. Summary of the invention

[0006] In the face of the general demand for providing more and safer lactic acid bacteria strains, the present invention has discovered a new plant lactobacillus through long-term and extensive screening and genomic and metabolomics research, and verified that it has the functions of lowering uric acid, relieving gout symptoms and eliminating Helicobacter pylori, thereby completing the present invention.

[0007] The present invention provides a plant lactobacillus strain Lrld-22, which can be provided by Anqing Vocational and Technical College and is numbered Lrld-22-AQVTC-312. The strain has been disclosed in the paper "Effects of Carya cathayensis Sarg. and Torreya grandis nuts on the physicochemical properties and lactic acid bacteria of yogurt", Dong Liu, et al, Italian Journal of Food Science, Vol. 37, No. 1, pp. 319-332, DOI 10.15586 / ijfs.v37i1.2688.

[0008] In one aspect of the present invention, the present invention provides a bacterial agent, which comprises the Lactobacillus plantarum Lrld-22; in one embodiment, the Lactobacillus plantarum bacterial agent is prepared by fermenting the Lactobacillus plantarum Lrld-22 and then freeze-drying it.

[0009] In one aspect of the present invention, the present invention discloses the use of Lactobacillus plantarum Lrld-22 in the preparation of a composition for reducing or controlling uric acid.

[0010] In one aspect of the present invention, the present invention discloses the use of Lactobacillus plantarum Lrld-22 in preparing a composition for treating Helicobacter pylori infection.

[0011] In one embodiment, the composition is food, medicine, or health food. The food or health food further contains excipients acceptable to food or health food; the medicine further contains pharmaceutically acceptable excipients.

[0012] In one embodiment, the food, health food, and medicine further contain other probiotics and / or effective ingredients.

[0013] In one embodiment, the food or health food dosage form is a dairy product, capsule, tablet, candy, powder, drop, or oral liquid preparation added with Lactobacillus plantarum Lrld-22.

[0014] In one embodiment, the pharmaceutical dosage form is a tablet, capsule, powder, or liquid preparation to which Lactobacillus plantarum Lrld-22 is added.

[0015] In one embodiment of the present invention, the plant lactobacillus of the present invention can be used in combination with other probiotics of the genus Lactobacillus or Bifidobacterium, or can be used in combination with one or more functional components including celery seed extract, salvia miltiorrhiza extract, sophora japonica extract, turmeric extract, inulin, vitamins, carotene, grape seed extract, kudzu root extract, mulberry extract, lotus leaf extract, Poria extract, dendrobium extract, rattan tea extract, and blueberry extract to enhance its function. The probiotics of the genus Lactobacillus include one or more of Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Streptococcus thermophilus, Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus salivarius, and Lactobacillus helveticus; the probiotics of the genus Bifidobacterium include one or more of Bifidobacterium adolescentis, Bifidobacterium animalis subspecies, Bifidobacterium animalis lactis subspecies, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subspecies, and Bifidobacterium longum infantis subspecies.

[0016] The plant lactobacillus Lrld-22 provided by the present invention is isolated from Qinghai yaks and domesticated nut fermented milk. By sequencing and analyzing the whole genome of Lrld-22, the Lrld-22 genome consists of a single circular chromosome of 3,246,150 bp in length, carrying 3284 genes, 99 non-coding RNAs and some repeated sequences, and the GC content of the genome accounts for 44.58%, and no prophage is found; at the same time, two plasmid sequences of 81471 bp and 41833 bp in size are detected in the bacteria. The genome is annotated in terms of functions such as carbohydrate utilization and fatty acid metabolism, and it is found that plant lactobacillus Lrld-22 has a good utilization ability for carbohydrates and fats. After research and testing, it was found that plant lactobacillus Lrld-22 has the effect of reducing uric acid content, relieving the symptoms of gout, and also has the effect of clearing Helicobacter pylori.

[0017] Beneficial Effects

[0018] The present invention discovered a new strain of Lactobacillus plantarum Lrld-22, and verified that it has the functions of lowering uric acid, relieving gout symptoms and clearing Helicobacter pylori, providing a good basis for enriching the application of the probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The colony characteristic diagram and bacterial morphology diagram of Lactobacillus plantarum Lrld-22 of the present invention;

[0020] Figure 2 This is a schematic diagram of the genome circle of Lactobacillus plantarum Lrld-22 of the present invention;

[0021] Figure 3 A schematic diagram of a plasmid circle of Lactobacillus plantarum Lrld-22 of the present invention;

[0022] Figure 4 It is another schematic diagram of the plasmid circle of Lactobacillus plantarum Lrld-22 of the present invention;

[0023] Figure 5 This is a schematic diagram of the percentage of functional gene annotations of Lactobacillus plantarum Lrld-22 of the present invention in the COG database;

[0024] Figure 6 This is a bar graph of CAZy functional gene annotation analysis of Lactobacillus plantarum Lrld-22 of the present invention;

[0025] Figure 7 for Figure 6 This is a fan-shaped proportion diagram of the annotation analysis of CAZy functional genes of Lactobacillus plantarum Lrld-22 of the present invention;

[0026] Figure 8This is a schematic diagram of reducing uric acid content by Lactobacillus plantarum Lrld-22 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The equipment and reagents used in the embodiments and test examples can be obtained from commercial sources unless otherwise specified. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] Example 1: Isolation of strain Lrld-22

[0029] A method for isolating a strain comprises the following steps:

[0030] Step 1: Select materials, which are fermented milk from Qinghai yaks and fermented milk from domesticated nuts;

[0031] Step 2: strain separation, dilute 10 mL of sample with 90 mL of sterile water, homogenize with high-speed vortex for 1 minute, prepare serial ten-fold dilutions with sterile water, pipette 100 μL of each dilution into sterile culture dishes and culture on MRS agar medium at 37°C for 24-48 h;

[0032] Step 3: Strain culture: pick a single colony for liquid pure culture, and collect the strain after centrifugation at 5000×g / min for 6 minutes.

[0033] refer to Figure 1 The results showed that the strain was isolated from Qinghai yak fermented milk, and was used to ferment pecan and Torreya grandis fermented milk for domestication. Then 22 strains were isolated and identified from pecan and Torreya grandis fermented milk. Lrld-22 with better growth performance was taken for further study. After cultivation, it was found that the colony was round, milky white, with a smooth and convex surface, neat edges, and opaque. The colony diameter was about 3-5mm. Gram staining results showed that it was positive and rod-shaped.

[0034] Example 2 Identification of strain Lrld-22

[0035] The strain identification was carried out by combining morphological and molecular biological techniques. The morphological and biochemical reactions were further identified by full-length nucleotide sequencing of the 16S rDNA gene. The primers used were bacterial universal primers 27F and 1492R. The PCR amplification conditions were carried out according to the conditions provided by the kit. The sequence results were compared with those of the National Center for Biotechnology Information in the United States, and the phylogenetic tree was constructed using the tools provided by the platform.

[0036] The results showed that the 16S rRNA sequence amplified a sequence length of 1440bp, and the sequence of the isolated strain had a homology of more than 99% with the sequences of multiple strains of Lactobacillus plantarum. The phylogenetic tree showed that the strain Lrld-22 had the highest homology with Lactobacillus plantarum. Therefore, the strain Lrld-22 was identified as Lactobacillus plantarum based on the comprehensive colony characteristics and morphological results, and was named Lactobacillus plantarum Lrld-22. At present, the bacteria is provided by Anqing Vocational and Technical College and is numbered Lrld-22-AQVTC-312. The bacteria has been published in the paper "Effects of Carya cathayensis Sarg.and Torreya grandis nuts on the physicochemical properties and lactic acid bacteria of yogurt", Dong Liu, et al, Italian Journal of Food Science, Vol. 37, No. 1, pp. 319-332, DOI 10.15586 / ijfs.v37i1.2688.

[0037] Example 3 Whole genome sequencing of Lactobacillus plantarum Lrld-22

[0038] Genomic DNA was extracted according to the instructions of the DNA extraction kit, and the DNA concentration and A 260 / A 280 and A 260 / A 230 The integrity and purity of DNA were evaluated by 1.5% agarose gel electrophoresis. After the quality reached the requirements for library construction, whole genome sequencing was performed using the BGISEQ platform and Nanopore platform of BGI.

[0039] See Table 1 and Figure 2 The results show that the present invention performs data evaluation and sequence assembly on the sequencing sample Lactobacillus plantarum Lrld-22, obtains the genome assembly results and basic genome characteristics (see Table 1); and splices the assembled sequences into a genome circle map (see Figure 2 ); The results showed that the genome sequence of Lactobacillus plantarum Lrld-22 had 1 circular chromosome and 2 plasmids after assembly (see the circle diagram Figure 3-Figure 4 ); the total genome length is 3369454bp, of which the ring chromosome size is 3246150bp, the GC content (%) is 45.48%, the average length is 859.12bp, the longest is 313638, and the total number of reads is 3284.

[0040] Table 1 Assembly results and genome characteristics of Lactobacillus plantarum Lrld-22

[0041]

[0042]

[0043] Example 4: Prediction and annotation of functional genome of Lactobacillus plantarum Lrld-22:

[0044] The functions of the assembled Lactobacillus plantarum Lrld-22 genome were annotated. (1) The COG database can divide protein functions into 26 categories, each of which is composed of orthologous sequences. By aligning protein sequences, they are annotated to a certain COG classification to infer the function of the sequence. L. plantarum Q180 is one of the most intensively studied Lactobacillus plantarum and has a high reference value. It also has a strong bile salt tolerance, can survive and colonize in the human intestine, and has a high probiotic potential. Therefore, using L. plantarum Q180 as a reference strain can deepen our understanding of Lactobacillus plantarum Lrld-22 to a certain extent. See Figure 5 , Lactobacillus plantarum Lrld-22 obtained 2653 functional gene annotations in the COG database, accounting for 80.79% of CDS. These gene annotations mainly involve three aspects: cells, information transfer and metabolism. The annotation results show that there are 271 genes involved in the transfer and metabolism of carbohydrates; 222 genes involved in the metabolism and transfer of amino acids; 152 genes related to the formation of cell walls, cell membranes, and cell envelopes; 67 genes related to defense mechanisms; and of course, there are some genes involved in protein transcription, translation, modification, phospholipid transfer and metabolism, and energy generation and transfer.

[0045] (2) CAZy functional gene annotation analysis: CAZy is a well-known professional database related to carbohydrate enzymes in the art. Figure 6-7 ), Lactobacillus plantarum Lrld-22 received the highest annotation amount in glycoside hydrolases (GHs), accounting for 54%, followed by glycosyltransferases (GTs) and carbohydrate-related modules (CBMs), which accounted for 29% and 14%, respectively. Carbohydrate esterases (CEs) also received 2% of annotations, which can catalyze the deesterification of various carbohydrate substrates, and 1% of auxiliary activity enzymes were also annotated. Combining the COG and CAZy annotation results, it shows that Lactobacillus plantarum Lrld-22 has a strong carbohydrate metabolism ability and has good prebiotic potential.

[0046] Example 5 Evaluation of the effect of plant lactobacillus Lrld-22 in reducing uric acid:

[0047] Evaluation of the effect of reducing uric acid: The uric acid concentration in the culture medium was detected by high performance liquid chromatography. A standard curve was drawn with the uric acid peak area and uric acid concentration (mg / L), and the uric acid peak area detected in the sample to be tested was used to obtain the concentration using the standard curve and calculate the degradation rate;

[0048] The results of uric acid test in rats are as follows Figure 8 As shown in the figure, compared with the blank control group (CK), the uric acid concentration of the model group (M) increased to 212 μmol / L. After 30 days of administration, the blood uric acid content of the Lactobacillus plantarum Lrld-22 treatment group decreased to varying degrees compared with the model group (M). The uric acid concentration of the low-dose group (LT) decreased to 182 μmol / L. The high-dose group (LH) was fed with 1 mL 3x10 9 The probiotic Lactobacillus plantarum Lrld-22 reduced uric acid to an average of 117 μmol / L, which was significantly lower than that of the hyperuricemia model group (P<0.01), indicating that Lactobacillus plantarum Lrld-22 has a good therapeutic effect on hyperuricemia symptoms. This is strongly related to oxidative stress and metabolic syndrome in its biological process. Probiotics can affect oxidative stress and uric acid levels by improving insulin sensitivity, and the conversion of purine nucleotides in the biosynthesis process is closely related to uric acid metabolism.

[0049] Example 6 Effect and mechanism of plant lactobacillus Lrld-22 in eliminating Helicobacter pylori

[0050] The experimental animals were randomly divided into a control group (CK), a model group (Model), and six Lactobacillus plantarum Lrld-22 intervention groups (LT, HT, TCKLT, TCKHT, TLT, and THT). LT was a low-dose (3x10 7 ) Lactobacillus plantarum Lrld-22 treatment group, HT was high dose (3x10 9 ) Lactobacillus plantarum Lrld-22 treatment group, TCKLT was a low dose (3x10 7 ) Lactobacillus plantarum Lrld-22 treatment group, TCKHT was a high dose (3x10 9 ) Lactobacillus plantarum Lrld-22 treatment group, TLT is a low dose (3x10 7 ) Lactobacillus plantarum Lrld-22 treatment group, THT was a high dose (3x10 9) Lactobacillus plantarum Lrld-22 treated group. The animal model of Helicobacter pylori infection was established. After fasting for 24 hours, 5% NaHCO3 was given by gavage. 30 minutes later, 10 9 CFU / ml Helicobacter pylori 0.2ml, 4 times in a row, each time interval of 48h. The control group was provided with an equal amount of normal saline. After 30 days of treatment, gastric tissue was taken, washed with DEPC water and the rapid urease (RUT) test was used to determine whether Helicobacter pylori was eliminated.

[0051] The results are shown in Table 2. The Helicobacter pylori clearance rates of the LT group and the HT group were 19.6% and 62.2%, respectively, and those of the TCKLT group and the TCKHT group were 16.3% and 63.5%, respectively. The clearance effect was significant compared with the group with the same dose in which the polysaccharide gene exoY was successfully knocked out. In addition, the polysaccharide content of Lactobacillus plantarum Lrld-22 with the polysaccharide gene exoY knocked out was significantly reduced, indicating that polysaccharides are the key component for its clearance of Helicobacter pylori. In addition, Helicobacter pylori infection can cause an imbalance in the number of lactic acid bacteria in the intestines of mice. Supplementation of Lactobacillus plantarum Lrld-22 can significantly (P < 0.01) improve the intestinal flora of mice, especially the group without knocking out the gene exoY. The effect is more obvious, which also shows that the exoY gene and the polysaccharides regulated by it have a good promoting effect on the colonization of the intestine by Lactobacillus plantarum Lrld-22.

[0052] Table 2

[0053]

[0054] It should be noted that: ** indicates that the clearance rate is significantly different compared with the model group (Model) at P < 0.05 and P < 0.01 (same row);

[0055] a, A indicates significant difference compared with the normal group (CK) at P < 0.05 and P < 0.01 (same row);

[0056] b, B indicates significant difference compared with the model group (Model) at P < 0.05 and P < 0.01 (same row).

[0057] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Application of Lactobacillus plantarum Lrld-22 in the preparation of a composition for reducing or controlling uric acid.

2. Application of Lactobacillus plantarum Lrld-22 in the preparation of a composition for treating Helicobacter pylori infection.

3. The use according to claim 1 or 2, characterized in that: The composition is food, medicine or health food.

4. The use according to claim 3, characterized in that: The food and health food also contain excipients acceptable to food and health food; the medicine also contains pharmaceutically acceptable excipients.

5. The use according to claim 3, characterized in that: The food, health food and medicine also contain other probiotics and / or functional ingredients.

6. The use according to claim 3, characterized in that: The food and health food dosage forms are dairy products, capsules, tablets, candies, powders, drops, and oral liquid preparations added with Lactobacillus plantarum Lrld-22.

7. The use according to claim 3, characterized in that: The drug dosage form is tablets, capsules, powders and liquid preparations added with Lactobacillus plantarum Lrld-22.

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