Growth-promoting probiotic and use thereof

Abstract

A growth-promoting probiotic and the use thereof, and a Lactobacillus plantarum and the use thereof. The Lactobacillus plantarum has good tolerability and safety, and can be used in the preparation of food raw materials, probiotic powder, food additives, feed additives, drugs, liquid drinks, food products and health care products. The Lactobacillus plantarum or a formulation containing the Lactobacillus plantarum can promote the growth of a subject.

Description

Growth promoting probiotics and uses TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and food, in particular to a probiotic and its application in promoting growth. BACKGROUND

[0002] In recent years, the role of intestinal microbes in human health has attracted widespread attention. Studies have found that intestinal microbes play a crucial role in the development of the host's immune system, nutrient absorption and the maintenance of metabolic homeostasis.

[0003] Children with growth retardation usually show growth retardation, developmental delay and high morbidity. Some studies have shown that children born by cesarean section or fed with milk powder have low levels of intestinal flora due to the lack of maternal genetic flora, which leads to a decrease in intestinal flora diversity, low immunity and growth retardation. Intestinal flora disorder is related to growth retardation in children. Transplanting the flora of malnourished children into germ-free mice can cause weight loss and metabolic disorders in germ-free mice. The above results prove the causal relationship between microorganisms and malnutrition, and also show that adjusting the composition of intestinal flora has a positive effect on human metabolism and growth conditions.

[0004] Probiotics are a class of active microorganisms that can produce beneficial effects on the host when sufficient amounts are ingested. Many scholars have used probiotics to promote the growth and development of animals. Some probiotics such as Lactobacillus acidophilus, Enterococcus faecium and Escherichia coli strains have been independently proven to promote growth, increase body weight, prevent pathogen colonization and inhibit muscle atrophy in different animals. Studies have found that probiotics can enhance the metabolic utilization of nutrients and improve feed efficiency by producing digestive enzymes such as amylase or B vitamins. Studies have also shown that probiotics and their cell wall components can improve the growth hormone sensitivity of peripheral tissues and increase the circulating levels of IGF-1, thereby promoting the growth of chronically malnourished mice and fruit flies. Probiotic supplementation is one of the strong candidates for promoting host growth.

[0005] Child growth is closely related to intestinal flora health. Meta-analysis has found that some clinical experimental studies have shown that the addition of probiotics can improve the weight and height of normal children aged 0-59 months, and studies in non-clinical environments have also shown that probiotics can promote the growth of malnourished children. Most current probiotic treatments focus on the growth of sick children, and there are few studies on the impact of probiotics on the growth and development of normal children.

[0006] In addition, due to the influence of geographical environment, genetic genes and dietary habits, the composition of intestinal flora of human beings in different countries in the world is significantly different. It is found that the diversity of intestinal flora of Chinese population is significantly higher than that of western population, and there is also a significant difference in the composition of intestinal dominant flora between the two. This makes the imported probiotic strains not completely adapt to the intestinal environment of Chinese people, and may have low colonization efficiency or even no beneficial effect.

[0007] Therefore, it is of important practical significance to develop a probiotic strain that can promote the growth and development of normal children and is more suitable for the intestinal flora of Chinese people.

[0008] SUMMARY

[0009] Due to the significant difference in the composition of intestinal flora of people in different regions, the present technology aims to screen new growth-promoting probiotic strains that meet the normal diet or nutritional background in China; by detecting the growth-promoting effect of the strain in different model organisms (fruit flies, zebrafish) larvae, the influence of the strain on the growth and development of the host under normal nutritional background is explored, filling the technical gap in the domestic and foreign markets.

[0010] In addition, the present application compares the effect of probiotics under different nutritional backgrounds (normal protein level, low protein level), providing a theoretical basis for the rational application of probiotics and the development of downstream products.

[0011] The present application aims to evaluate the growth-promoting effect of a probiotic strain in different animal models and explore its mechanism, including the following specific contents:

[0012] 1. Isolation and identification of probiotics and safety evaluation

[0013] The present application isolates a strain of Lactiplantibacillus plantarum from fermented food. Phenotypic identification, molecular biological identification and phylogenetic analysis determine that the strain is Lactiplantibacillus plantarum. Before the probiotic bacteria exert their probiotic function, they need to colonize through the gastrointestinal tract. The acid and bile salt tolerance of the strain is detected, and the drug resistance of the probiotic bacteria is detected by using hemolysis plate and various antibiotics to determine its safety.

[0014] 2. Growth-promoting effect of probiotics on different animal models

[0015] The present application explores the influence of probiotics on the growth performance of different model animals under normal nutritional background, detects the larval size, pupation time and eclosion time of fruit flies, and the body weight, body length and weight gain rate of zebrafish.

[0016] 3. Influence of probiotics on host growth under different nutritional backgrounds

[0017] The effect of probiotics may be different under different nutritional backgrounds. The present application sets two different nutritional backgrounds of normal protein level and low protein level to compare the effect of probiotics under different nutritional backgrounds.

[0018] In a first aspect, the present application provides Lactiplantibacillus plantarum Hi188, which has a preservation number of CGMCC No. 30250.

[0019] The present application also provides a preparation comprising Lactiplantibacillus plantarum Hi188.

[0020] The preparation comprising Lactiplantibacillus plantarum Hi188 is one or several of a food raw material, a probiotic bacterial powder, a food additive, a feed additive, a medicine, a liquid drink, a food, or a health product.

[0021] The preparation comprising Lactiplantibacillus plantarum Hi188 is a solid or liquid preparation; when the preparation is a liquid preparation, it comprises 1.0 x 10 5 CFU to 1.0 x 10 12 CFU / ml of Lactiplantibacillus plantarum Hi188; when the preparation is in a solid form, it comprises 1.0 x 10 5 CFU to 1.0 x 10 12 CFU / g of Lactiplantibacillus plantarum Hi188.

[0022] The preparation comprising Lactiplantibacillus plantarum Hi188 further comprises an excipient, which includes one or several of inulin, fructooligosaccharide, skimmed milk powder, desalted whey powder, lactoferrin, casein phosphopeptide, or hydrolyzed egg yolk powder.

[0023] In another aspect, the present application provides use of Lactiplantibacillus plantarum Hi188 in the preparation of a preparation or kit for promoting the growth of a subject.

[0024] In embodiments, the amount of Lactiplantibacillus plantarum Hi188 in the preparation or kit for promoting the growth of a subject is 1.0 x 10 5 CFU to 1.0 x 10 12 CFU / ml or 1.0 x 10 5 CFU to 1.0 x 10 12 CFU / g.

[0025] In a specific embodiment of the present application, the Lactobacillus plantarum Hi188 or the preparation comprising the Lactobacillus plantarum Hi188 can be present as an active ingredient in a kit; the kit refers to any product form (such as a health product box, a medicine box, etc.) comprising the active ingredient, which optionally further comprises a containing device, an outer package and / or an instruction manual. Among them, the instruction manual can be used to provide the administration information of the active ingredient, such as the administration amount, the administration frequency, or to indicate the use of the active ingredient, such as for promoting the body length or height increase of a subject, promoting the body weight increase of a subject, or promoting the development of a subject.

[0026] The present application also provides the use of a preparation comprising the Lactobacillus plantarum Hi188 in promoting the growth of a subject.

[0027] In embodiments, promoting the growth of a subject includes one or more of promoting the body length increase of a subject, promoting the body weight increase of a subject, or promoting the development of a subject.

[0028] In embodiments, the subject is a fruit fly, a zebrafish or a mammal.

[0029] In embodiments, the mammal is a mouse or a human.

[0030] In the present application, the normal protein refers to the protein level of the prior art conventionally taken for maintaining the normal growth and development of an organism; and the low protein refers to a protein level that significantly affects the normal growth and development of an organism, such as a protein level of 20%-60% lower than the normal protein.

[0031] The development and application of probiotics in the era of functional foods are the current research hotspots, which meet the needs of people for functional and healthy foods. Among them, functional products based on probiotics are welcomed by the public. The beneficial effects of the present application are that the present application not only focuses on the growth-promoting effect of probiotics on young organisms under normal nutrition, but also focuses on the effect of probiotics under different nutrition, which expands the range of reasonable application of probiotics, provides important reference value for the development of growth-promoting probiotics, and provides a solution for improving the height problem of children with growth retardation, and provides more choices for normal children with higher expected height requirements. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 shows the colony morphology of Lactobacillus plantarum Hi188, wherein A is an overall top view; and B is a partial view.

[0033] Figure 2 shows the chromosome genome circle map of Lactobacillus plantarum Hi188.

[0034] Figure 3 shows the phylogenetic tree of Lactobacillus plantarum Hi188.

[0035] Figure 4 shows the growth curve of Lactiplantibacillus plantarum Hi188.

[0036] Figure 5 shows the hemolytic test of Lactiplantibacillus plantarum Hi188.

[0037] Figure 6 shows the effect of Lactiplantibacillus plantarum Hi188 on the growth of Drosophila melanogaster larvae under normal nutrition conditions, wherein A is the comparison of the 5th day photos of Drosophila melanogaster larvae of the normal nutrition group and the normal nutrition Hi188 addition group; B is the comparison of the body length statistics of Drosophila melanogaster larvae of the normal nutrition group and the normal nutrition Hi188 addition group on the 5th day; wherein CD is the normal nutrition group, CD+Hi188 is the normal nutrition added with 10 9 CFU Lactiplantibacillus plantarum Hi188 group, ** indicates P<0.01.

[0038] Figure 7 shows the comparison of the effects of Lactiplantibacillus plantarum Hi188 and the comparative strains LP299v, WCFS1 and Drosophila melanogaster larvae growth under normal nutrition conditions, wherein A is the comparison of the 5th day photos of Drosophila melanogaster larvae of CON, LP299v, Hi188, WCFS1; B is the comparison of the body length statistics of Drosophila melanogaster larvae of CON, LP299v, Hi188, WCFS1 on the 5th day; wherein CON is the normal nutrition group; LP299v is the normal nutrition added with 10 9 CFU Lactiplantibacillus plantarum LP299v group, Hi188 is the normal nutrition added with 10 9 CFU Lactiplantibacillus plantarum Hi188 group, WCFS1 is the normal nutrition added with 10 9 CFU Lactiplantibacillus plantarum WCFS1 group.

[0039] Figure 8 shows the effect of Lactiplantibacillus plantarum Hi188 on the growth of Drosophila melanogaster larvae under low nutrition conditions, wherein A is the comparison of the 5th day photos of Drosophila melanogaster larvae of the normal nutrition group, the low nutrition group and the low nutrition Hi188 addition group; B is the comparison of the body length statistics of Drosophila melanogaster larvae of the normal nutrition group, the low nutrition group and the low nutrition Hi188 addition group on the 3rd to 5th day; wherein CD is the normal nutrition group; LD is the low nutrition group; LD+Hi188 is the low nutrition added with 10 9 CFU Lactiplantibacillus plantarum Hi188 group.

[0040] Figure 9 shows the effect of Lactiplantibacillus plantarum Hi188 on the growth of zebrafish larvae under normal nutrition conditions, wherein A is the comparison of the 4th week photos of zebrafish larvae of the normal nutrition group and the normal nutrition Hi188 addition group; B is the comparison of the body length statistics of zebrafish larvae of the normal nutrition group and the normal nutrition Hi188 addition group on the 4th week; wherein CD is the normal nutrition group; CD+Hi188 is the normal nutrition added with 10 9 CFU Lactiplantibacillus plantarum Hi188 group, **** indicates P<0.0001.

[0041] Figure 10 shows the effect of Lactiplantibacillus plantarum Hi188 on the growth of low-nutrition zebrafish larvae, wherein A is the comparison of the 4th week of zebrafish larvae in the normal nutrition group, low nutrition group and low nutrition Hi188 addition group; B is the comparison of the body length of zebrafish larvae in the normal nutrition group, low nutrition group and low nutrition Hi188 addition group from 1 to 4 weeks; wherein CD is the normal nutrition group; LD is the low nutrition group; LD+Hi188 is the low nutrition group added with 10 9 CFU Lactiplantibacillus plantarum Hi188 group.

[0042] Figure 11 is the comparison of the body weight and body length of three-week-old mice, wherein A is the comparison of the body weight of three-week-old mice in the normal control group (CON), Hi188 group and WJL group; B is the comparison of the body length of three-week-old mice in the normal control group (CON), Hi188 group and WJL group.

[0043] Figure 12 is the comparison of the body weight and body length of four-week-old mice, wherein A is the comparison of the body weight of four-week-old mice in the normal control group (CON), Hi188 group and WJL group; B is the comparison of the body length of four-week-old mice in the normal control group (CON), Hi188 group and WJL group; C is the body weight gain of four-week-old mice in the normal control group (CON), Hi188 group and WJL group; D is the body length growth of four-week-old mice in the normal control group (CON), Hi188 group and WJL group; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0044] Figure 13 is the comparison of the femur and tibia length of four-week-old mice, wherein A is the comparison of the femur length of four-week-old mice in the normal control group (CON), Hi188 group and WJL group; B is the comparison of the tibia length of four-week-old mice in the normal control group (CON), Hi188 group and WJL group; ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0045] Figure 14 is the photograph of the femur and tibia of four-week-old mice, wherein A is the comparison of the femur of four-week-old mice in the normal control group (CON), Hi188 group and WJL group; B is the comparison of the tibia of four-week-old mice in the normal control group (CON), Hi188 group and WJL group.

[0046] Deposit Description

[0047] Taxonomy: Lactiplantibacillus plantarum

[0048] Name: Hi188

[0049] Latin name: Lactiplantibacillus plantarum

[0050] Depositary: China General Microbiological Culture Collection Center

[0051] Abbreviation of the depositary: CGMCC

[0052] Address: No. 1, Xibaixili, Chaoyang District, Beijing

[0053] Date of deposit: April 2, 2024

[0054] Registration number of the depositary center: CGMCC No. 30250 DETAILED DESCRIPTION

[0055] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to specific examples and with reference to the accompanying drawings.

[0056] The methods used in the following examples are all conventional methods unless otherwise specified, and the reagents used are all commercially available reagents unless otherwise specified.

[0057] In the examples of the present application, both the fruit fly and the zebrafish are conventional model organisms in the prior art, and both can be commercially available, for example, wild type Canton S strain of Drosophila melanogaster is purchased from the National Fruit Fly Resource Center (China, Shanghai), and zebrafish AB strain is purchased from the National Zebrafish Resource Center (China, Wuhan).

[0058] Example 1 Isolation and identification of Lactobacillus plantarum Hi188

[0059] 1) Isolation and purification

[0060] Take 2.5 mL of fermented bean curd liquid (Wu Jing Town, Minhang District, Shanghai) and add it to a 10 ml test tube, then add 2.5 ml of 1x PBS buffer, centrifuge at 3000 r / min for 5 min, take 100 μL of the supernatant and add it to 40 ml of MRS broth (purchased from Hangzhou Baisi Biological Technology Co., Ltd.), incubate at 30°C with shaking at 200 r / min overnight. Dilute the bacterial solution 10 times with sterile 1x PBS buffer, take 100 μL and spread it on MRS solid medium (add 1.5% agar to MRS broth) using a spreader, invert culture at 30°C, and obtain single colonies of lactic acid bacteria. Pick single colonies with different morphologies from the plate, purify them by repeated streaking, then place them in a constant temperature incubator at 30°C and incubate continuously for 48 h, then repeat the above steps until pure colonies are obtained. 4

[0061] 2) Passage and freezing

[0062] ​The colonies on the MRS solid medium were scraped with a inoculating loop and inoculated into 10 ml of MRS broth medium, which was incubated at 30°C for 12 h at 200 r / min. Then, 60% sterilized glycerol was mixed with the bacterial solution at a volume ratio of 1:3 to obtain a final glycerol concentration of 15%, and the mixture was shaken and stored at -80°C.

[0063] 3) Strain identification and phylogenetic analysis

[0064] A small amount of the frozen bacterial strain was used to perform plate streaking on MRS solid medium using the three-zone streaking method, and the single colony morphology was observed. On the general MRS solid medium, Lactiplantibacillus plantarum Hi188 presented white, round, uniform, slightly raised, smooth surface, and neat edge colonies (Fig. 1).

[0065] Fresh single-strain bacterial solution was used as a template for PCR amplification in the PCR amplification system. The bacterial 16S rRNA gene primer sequences used were: forward primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1); reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2). The sequencing results were subjected to homologous sequence alignment using the BLAST software in the NCBI database, and a phylogenetic tree was established using the MEGA software. The isolated and identified strain was named Lactiplantibacillus plantarum Hi188. The 16S rRNA gene sequence obtained by 16S rRNA gene sequencing is as follows:

[0066] The whole-genome sequencing and analysis results of the strain showed that Hi188 was composed of one chromosomal DNA and ten plasmid DNAs, with a chromosomal sequence length of 3,108,079 bp and a GC content of 44.75 (Fig. 2). The phylogenetic tree of Hi188 is shown in Fig. 3. Hi188 strain was clustered with four other Lactiplantibacillus plantarum strains. Hi188 was identified as Lactiplantibacillus plantarum and belonged to the strains allowed for food use in the List of Strains Allowed for Food Use.

[0067] Example 2 Characteristics of Lactiplantibacillus plantarum Hi188

[0068] 1) Growth curve determination

[0069] The growth curve of the strain was observed in MRS broth medium at 37°C for 24h. A single colony was inoculated in a centrifuge tube containing 40mL MRS broth medium and cultured at 37°C, 200r / min. Every 2h, 200μL of the mixed bacterial suspension was taken and the absorbance value at 600nm was measured with blank medium as control. Three parallel tests were set up and the growth curve was drawn. The strain Hi188 entered the logarithmic phase at 6h and the stationary phase at about 22h (Figure 4). 600nm

[0070] 2) Cholatesalt tolerance test

[0071] The concentration of cholatesalt in intestinal juice is 0.03%-0.3%. Therefore, the cholatesalt tolerance of Hi188 at 0.3% cholatesalt was tested.

[0072] Firstly, MRS broth and bovine cholatesalt (purchased from Beijing Solabio Science and Technology Co., Ltd.) were used to prepare MRS broth medium containing 0.3% cholatesalt, which was sterilized in a high-pressure steam sterilizer at 121°C for 15min and then cooled for use. After the strain was cultured in MRS broth for 8h, it was inoculated in 40ml MRS broth medium or MRS broth medium containing 0.3% cholatesalt at an inoculation amount of 1%. The MRS broth containing 0.3% cholatesalt with bacteria was used as the treatment group, the MRS broth containing 0.3% cholatesalt without bacteria was used as the negative background group of the treatment group, the MRS broth with bacteria but without cholatesalt was used as the positive control, and the MRS broth without bacteria and cholatesalt was used as the negative background group of the positive control. Each treatment and control was repeated three times. The culture was carried out at 37°C, 200r / min for 3h, and the OD value of the bacterial solution was detected at 0h, 1h, 2h and 3h. The survival rate was calculated according to the formula:

[0073] Survival rate (%) = (OD value of treatment group - OD value of negative background of treatment group) / (OD value of positive control - OD value of negative background of positive control group) x 100%

[0074] The survival rates of the strain at 1h, 2h and 3h are shown in Table 1 below. The results show that the plant lactobacillus Hi188 has good cholatesalt tolerance.

[0075] Table 1 Survival rate of plant lactobacillus Hi188 to cholatesalt

[0076] 3) Artificial gastric juice and artificial intestinal juice tolerance test

[0077] ​Before the probiotics exert their probiotic functions, they need to colonize the gastrointestinal tract. In this study, the tolerance of Hi188 to artificial gastric juice (pH = 3, purchased from Shanghai Creative Technology Co., Ltd.) and artificial small intestinal juice (pH = 6.8, purchased from Shanghai Creative Technology Co., Ltd.) was detected. After Hi188 strain was cultured for 24 h, 1 ml of bacterial solution was inoculated into 9 ml of artificial gastric juice or small intestinal juice. The artificial gastric juice or small intestinal juice with inoculation was used as the treatment group, and the blank artificial gastric juice or small intestinal juice without bacterial inoculation was used as the negative control. Each treatment and control was repeated 3 times. The culture was incubated at 37°C for 3 h on a 200 r / min shaking table. The viable cell count at 0 h, 1 h, 2 h and 3 h was counted, and the survival rate was calculated according to the following formula:

[0078] Survival rate (%) = viable cell count of treatment group / viable cell count of 0 h treatment group x 100%

[0079] Table 2 Survival rate of Lactobacillus plantarum Hi188 in artificial gastric juice and small intestinal juice

[0080] The results are shown in Table 2. The survival rate of Lactobacillus plantarum Hi188 in artificial gastric juice and artificial small intestinal juice after 3 h treatment was more than 50%, indicating that Lactobacillus plantarum Hi188 has strong survival ability in artificial gastric juice and small intestinal juice.

[0081] Example 3 Safety identification of Lactobacillus plantarum Hi188

[0082] 1) Hemolytic test

[0083] The isolated Hi188 strain was inoculated on a blood plate (purchased from Anhui Wuhu Oukai Biotechnology Co., Ltd.). The plate was incubated in a 37°C incubator for 36 h, and the hemolysis phenomenon was observed. As shown in Figure 5, Lactobacillus plantarum Hi188 was a white smooth colony, and no hemolysis phenomenon was observed on the blood plate, indicating that the target strain had no obvious pathogenicity and was highly safe.

[0084] 2) MIC value detection

[0085] The experiment used microdilution method to determine the drug resistance of the strain to antibiotics ampicillin (Amp), gentamicin (Gen), kanamycin (Kan), erythromycin (Ery), clindamycin (Cli), tetracycline (Tet) and chloramphenicol (Chl), according to the standard method in ISO 10932 / IDF223. The drug resistance of the strain was expressed by the minimum inhibitory concentration (MIC).

[0086] The detection range of mass concentration was set to 0.5-256 μg / mL, and antibiotic storage solutions were prepared using various antibiotics. Ampicillin sodium, kanamycin sulfate and chloramphenicol were purchased from Yixing Biotechnology Co., Ltd. (Shanghai), gentamicin and tetracycline hydrochloride were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., erythromycin was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd., and clindamycin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The antibiotics were accurately weighed according to the titer of the reagent used, dissolved in the appropriate solvent, and shaken to mix.

[0087] The LSM liquid medium was composed of 90% IST liquid medium (purchased from Shandong Top Bioengineering Co., Ltd.) and 10% MRS broth (purchased from Hangzhou Baisi Biological Technology Co., Ltd.). This medium was used for MIC value determination. The prepared high-concentration antibiotic storage solution was diluted with LSM liquid medium to 2 times the highest concentration for determination.

[0088] The OD of the activated strain was measured 600nm The corresponding dilution was diluted with LSM medium, and 1% inoculation amount was inoculated into the antibiotic diluent with different concentration gradients, so that the number of viable bacteria was 3×10 5 CFU / ml. After incubation at 37°C for 24 h in a 96-well enzyme-labeled plate, the OD 600nm The minimum antibiotic concentration without bacterial growth was taken as the MIC value of the strain. L. plantarum LP WCFS1 ( purchased from China General Microbiological Culture Collection Center) as a control strain, and the broth with bacteria was used as a positive control well, and the broth without bacteria was used as a negative control well, all with 3 replicates. When the results of the parallel groups were inconsistent, repeated experiments were performed.

[0089] The MIC detection results are shown in Table 3, where - indicates that the antibiotic concentration inhibits bacteria, + indicates that the antibiotic concentration does not inhibit bacteria, Hi188 and LP WCFS1The results of the resistance to various antibiotics were similar. Hi188 was more sensitive to kanamycin, tetracycline, and chloramphenicol than LP. WCFS1 .

[0090] Table 3 Comparison of the drug resistance of Lactobacillus plantarum Hi188 and LP WCFS1

[0091] Note: The critical value is based on the EFSA standard. If the MIC value is greater than the critical value, it is resistant (R), and if the MIC value is less than or equal to the critical value, it is sensitive (S).

[0092] Example 4 Growth-promoting effect of Lactobacillus plantarum Hi188 on fruit flies

[0093] Adult fruit flies were mated to produce eggs, which were transferred to sterile normal protein medium within 8 hours of egg production. Five parallel culture tubes were set up for each treatment. The medium was yeast-corn flour medium. The medium was boiled for 5 min and sterilized at 121°C for 15 min. 10 9 CFU of Lactobacillus plantarum Hi188 was resuspended in 200 μL of sterile water and evenly inoculated on the medium. The control group was added with the same amount of sterile water, and then the eggs were placed in a 25°C incubator for culture, with a 12 / 12 hour dark / light cycle and a humidity of about 40%. On days 3-5, fruit fly larvae growing in the control group and the Hi188 group were randomly collected every day, and the fruit fly larvae were photographed and body length was measured using Image J software under a stereomicroscope.

[0094] Two different nutritional backgrounds, normal protein level and low protein level, were set up to compare the effects of probiotics under different nutritional backgrounds. In the experiment with normal protein background, the yeast-corn flour medium formula was 1 L of feed containing 20 g of white sugar, 20 g of brown sugar, 80 g of corn flour, 40 g of yeast, 10 g of agar powder, and 1.5-2% propionic acid, with a yeast content of 40 g / L. In the experiment with low protein background, the yeast content in the medium was adjusted to 10 g / L, and the other ingredients were the same as in the normal protein medium.

[0095] The results showed that under normal protein background, the body length of fruit fly larvae in the Hi188 addition group was significantly longer than that in the control group, indicating that Lactobacillus plantarum Hi188 can promote the growth of fruit fly larvae (Figure 6). Lactobacillus plantarum LP299v (isolated from healthy human intestinal mucosa, purchased from the Swedish probiotic production company Probi), WCFS1 (isolated from human oral saliva, purchased from the China Center for Type Culture Collection) were used as control strains. After comparing the growth effects, Lactobacillus plantarum Hi188 had a significantly better growth-promoting effect on fruit fly larvae body length than the control strains Lactobacillus plantarum LP299v, and the growth-promoting effect of Hi188 was also significantly better than that of the other control strain WCFS1 (Figure 7).​

[0096] Under the low-protein nutrition background, it was found according to the photograph on the 5th day that the body length of the Drosophila larvae in the low-nutrition group was obviously lower than that in the normal-nutrition group, and the growth was underdeveloped (Fig. 8A). Meanwhile, it was observed that the pupation time and the eclosion time of the Drosophila in the Hi188 addition group were earlier than those in the low-nutrition group, that is, the adverse effects of the low-nutrition developmental retardation were alleviated. In combination with the statistical results of the body length on the 3rd to 5th days, the addition of 10 9 CFU of Hi188 obviously promoted the growth and development of the Drosophila larvae and alleviated the adverse effects of the low nutrition (Fig. 8).

[0097] Further, the present embodiment also verified that the addition of 1.0 x 10 5 CFU and 1.0 x 10 12 CFU of the Lactobacillus plantarum Hi188 had similar effects.

[0098] Example 5: Growth-promoting effect of Lactobacillus plantarum Hi188 on zebrafish

[0099] The zebrafish parent fish were allowed to mate for 4-6 h, and then the fish eggs were collected in salt water (sea salt 1.5 mL / L) containing 9 μL / L of methylene blue, and the fish eggs were grouped and placed in an incubator for culture in salt water at 27 ± 1°C under a 14 / 10 h dark / light cycle. Three parallel groups were set for the control group and the treatment group. The fish eggs were fed with egg yolk twice a day for the first two days after hatching. Subsequently, the fish were fed with feed twice a day, and at the same time, the Hi188 bacterial solution resuspended in sterile water was added to the water body to make the final concentration of Hi188 in the water body of the Hi188 addition group 10 8 CFU / mL, and the control group was added with an equal amount of sterile water. The zebrafish larvae were photographed and the body length was measured under a body microscope using Image J software every week for 1-4 weeks.

[0100] Two different nutrition backgrounds of normal protein level and low protein level were set to compare the effects of probiotics under different nutrition backgrounds. In the experiment of normal protein background, the feed formula was 1 L of feed containing 500 g of fish meal, 300 g of starch, 20 g of fish oil, 15 g of multi-vitamin, 15 g of multi-mineral, 4 g of sodium carboxymethyl cellulose, 144.38 g of cellulose, 0.5 g of choline chloride, 0.02 g of butylated hydroxytoluene, 0.1 g of dimethyl-β-thionine propionate, 1 g of calcium hydrogen phosphate, and the protein content was 36% determined by Kjeldahl determination. In the experiment of low protein background, 1 L of feed was adjusted to contain 300 g of fish meal, 40 g of fish oil, and 324.38 g of cellulose, and other ingredients were the same as those in the normal protein feed, and the protein content was 21% determined by Kjeldahl determination.

[0101] After 4 weeks, compared with the normal nutrition group and the normal nutrition Hi188 addition group, the results showed that in the normal protein background, the growth and development of zebrafish larvae in the Hi188 addition group were significantly improved, the eye diameter was widened, the body length was increased, and the body area was increased, which proved the promoting effect of Lactobacillus plantarum Hi188 on the growth of zebrafish larvae (Figure 9).

[0102] Compared with the normal nutrition background, the body length of zebrafish larvae in the low protein nutrition background was significantly smaller, and the development was significantly delayed. Compared with the low nutrition group, the body length of zebrafish larvae in the low nutrition addition group with a final concentration of 10 8 CFU / ml Hi188 was significantly improved, the body length was increased, the eye diameter was widened, and the body area was increased, which to some extent alleviated the adverse effects of low protein nutrition (Figure 10), which proved the promoting effect of Hi188 on the growth of zebrafish larvae in the low protein nutrition background.

[0103] Example 6 Promoting effect of Lactobacillus plantarum Hi188 on mice

[0104] Three-week-old male C57BL / 6J mice (Shanghai Slac Laboratory Animal Co., Ltd.) were used as experimental objects, and the experimental period was one week. The three-week-old mice in the normal control group (CON) were gavaged with sterile saline, and the mice in the Hi188 group and the WJL group were gavaged with an equal amount of Lactobacillus plantarum Hi188 or Lactobacillus plantarum WJL (disclosed in Martin Schwarzer et al., (2023) Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice. Science Doi: 10.1126 / science.ade9767), respectively, and the gavage dose was 1×10 9 CFU / mouse, and the gavage volume was 0.2 ml / mouse. There were 10 mice in the normal control group and the Hi188 group (n = 10), and 5 mice in the WJL group (n = 5).

[0105] At the age of 3 weeks, there was no significant difference in body weight and body length between the mice in the Hi188 group and the mice in the normal control group (Figure 11), but at the age of 4 weeks, the body weight and body length of the mice in the Hi188 group were significantly higher than those of the mice in the normal control group (Figure 12), which indicated that the Hi188 strain could promote the increase of body weight and the linear growth of body length of mice.

[0106] The femur and tibia length of the Hi188 group mice at 3 weeks of age was not significantly different from that of the normal control group of young mice, but the femur and tibia length of the Hi188 group mice at 4 weeks of age was significantly greater than that of the normal control group of young mice (Figures 13 and 14), which indicates that the Hi188 strain can promote the growth of the femur and tibia of mice.

[0107] The above results demonstrate the growth-promoting effect of the Hi188 strain on mice.

[0108] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, substitutions and variations to the above-described embodiments within the scope of the present application.

Claims

1. Lactiplantibacillus plantarum Hi188, with the accession number of CGMCC No. 30250.

2. A preparation comprising the Lactiplantibacillus plantarum Hi188 of claim 1.

3. The preparation of claim 2, which is one or more of a food raw material, a probiotic bacterial powder, a food additive, a feed additive, a medicine, a liquid drink, a food, or a health product.

4. The preparation of claim 2 or 3, which is a solid or liquid preparation; wherein the preparation comprises Lactobacillus plantarum Hi 188 in an amount of 1.0 x 10 5 CFU / mL or 1.0 x 10 12 CFU / mL or 1.0 x 10 5 CFU / mL or 1.0 x 10 12 CFU / g.

5. The preparation of claim 2 or 3, further comprising an excipient, which comprises one or more of inulin, fructooligosaccharide, skim milk powder, desalted whey powder, lactoferrin, casein phosphopeptide, or hydrolyzed egg yolk powder.

6. Use of the Lactiplantibacillus plantarum Hi188 of claim 1 in the preparation of a preparation or a kit for promoting growth in a subject.

7. The use of claim 6, wherein, The amount of the plant Lactobacillus Hi188 is 1.0 x 10 5 CFU / ml or 1.0 x 10 12 CFU / ml or 1.0 x 10 5 CFU / ml or 1.0 x 10 12 CFU / g.

8. The use of claim 6 or 7, wherein promoting growth in the subject comprises: one or more of promoting an increase in body length or height, promoting an increase in body weight, or promoting development in a subject.

9. The use of claim 6 or 7, wherein the subject is a fruit fly, a zebrafish, or a mammal.

10. The use of claim 9, wherein the mammal is a mouse or a human.

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