Lactobacillus plantarum NSL0125 and application of lactobacillus plantarum NSL0125 in preparation of microbial preparation for promoting calcium supplement and growth and development

Abstract

The invention provides lactobacillus plantarum NSL0125 and application thereof in preparation of a microbial preparation for promoting calcium supplement and growth and development. The lactobacillus plantarum NSL0125 is classified and named as Lactobacillus plantarum and is preserved in the China Center for Type Culture Collection, and the preservation number of the lactobacillus plantarum NSL0125 is CCTCC (China Center for Type Culture Collection) NO: M 2024609. The lactobacillus plantarum NSL0125 is high in safety, free of pathogenicity and capable of fermenting various nutrient substances to supply energy for growth. Experiments prove that the lactobacillus plantarum NSL0125 can be colonized in intestinal tracts for a long time, and meanwhile, the lactobacillus plantarum NSL0125 has unique effects on the aspects of adsorption, transfer, absorption promotion and the like of calcium ions, so that calcium absorption, growth and development of organisms are effectively promoted. The lactobacillus plantarum NSL0125 is high in fermentation level and suitable for large-scale industrial production. A large number of experimental studies prove that the lactobacillus plantarum NSL0125 and the microbial preparation thereof have remarkable effects of promoting calcium supplement and growth and development, have good application value and have good market application prospects.

Description

Technical Field

[0001] The invention belongs to the field of microbial technology and biological preparations, and specifically relates to a plant lactobacillus NSL0125 and an application thereof in preparing a microbial preparation for promoting calcium supplementation and growth and development. Background Art

[0002] As people's health awareness continues to improve, the scientific supplementation and rational use of nutrients, especially calcium, have received unprecedented attention. As one of the indispensable minerals for the human body, calcium not only plays a core role in the formation and maintenance of bones and teeth, but also participates in many physiological processes such as nerve signal transmission, muscle contraction, and blood coagulation. Adequate calcium intake and efficient calcium utilization are of great significance for preventing osteoporosis, maintaining normal physiological functions, and ensuring the bone development of children and adolescents and the bone health of adults. However, in reality, there are widespread problems of insufficient calcium intake and low absorption and utilization, especially in children and adolescents in rapid growth periods and the elderly who are at increased risk of calcium loss. These problems are particularly prominent.

[0003] As a widely used probiotic, Lactobacillus plantarum has a strong ability to colonize the intestines, can form a stable flora in the intestines, help improve the balance of intestinal microecology, and create a favorable environment for calcium absorption. Although Lactobacillus plantarum has great potential in regulating calcium metabolism, there are still certain technical difficulties in how to effectively combine its functional characteristics with the need for calcium supplementation and develop biological preparations that have both efficient calcium supplementation and can promote growth and development. These difficulties include but are not limited to: how to screen Lactobacillus plantarum strains with excellent calcium metabolism regulation ability; how to optimize strain culture conditions to maximize their calcium metabolism-related functions; how to design a reasonable preparation formula to ensure that live bacteria effectively survive and function in the intestines. Therefore, the development of safe, effective and convenient Lactobacillus plantarum biological preparations has important theoretical significance and practical value for meeting market demand, improving calcium nutrition status, and promoting healthy growth and development. Summary of the invention

[0004] In view of the above technical problems, the object of the present invention is to provide a strain of Lactobacillus plantarum NSL0125 and its application in the preparation of a microbial preparation for promoting calcium supplementation and growth and development. The Lactobacillus plantarum NSL0125 is safe in source and non-pathogenic. Its microbial preparation uses Lactobacillus plantarum as an active ingredient and has the effects of calcium supplementation and promoting growth and development. The Lactobacillus plantarum NSL0125 and its microbial preparation provided by the present invention can not only efficiently supplement calcium but also help growth and development, meeting the current society's growing demand for effective calcium supplementation and rational utilization.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides a strain of Lactobacillus plantarum NSL0125, which is classified and named Lactobacillus plantarum , deposited in China Center for Type Culture Collection with the deposit number CCTCC M 2024609.

[0006] Furthermore, the 16S rRNA sequence of the Lactobacillus plantarum NSL0125 is shown as SEQ ID No.1.

[0007] The invention also provides a lactobacillus plantarum powder, which is obtained by mixing bacterial mud obtained by fermenting the lactobacillus plantarum NSL0125 with a freeze-drying protective agent and then subjecting the mixture to vacuum freeze-drying.

[0008] Furthermore, the mass ratio of the bacterial sludge of the Lactobacillus plantarum NSL0125 to the lyophilization protective agent is 1-3:1.

[0009] Furthermore, the formula and dosage of the fermentation medium of the plant lactobacillus NSL0125 are: in parts by weight, 30-45 parts of soluble starch, 20-40 parts of fish peptone, 2-6 parts of dipotassium hydrogen phosphate trihydrate, 4-9 parts of anhydrous sodium acetate, 4-9 parts of triammonium citrate, 1-2.5 parts of magnesium sulfate heptahydrate, 0.1-0.9 parts of manganese sulfate monohydrate, and 1.5-3 parts of Tween 80.

[0010] Preferably, the formula and dosage of the fermentation medium of Lactobacillus plantarum NSL0125 are: 40 parts of soluble starch, 30 parts of fish peptone, 2 parts of dipotassium hydrogen phosphate trihydrate, 9 parts of anhydrous sodium acetate, 5 parts of triammonium citrate, 0.5 parts of magnesium sulfate heptahydrate, 0.25 parts of manganese sulfate monohydrate, and 2 parts of Tween 80.

[0011] Furthermore, the lyoprotectant comprises the following components: 1-2 parts of trehalose, 1-2 parts of skim milk powder, 0.1-0.2 parts of sodium glutamate, 0.5-2 parts of Tween 80 and 0.5-2 parts of pure water, calculated in parts by weight.

[0012] Furthermore, the fermentation conditions of the Lactobacillus plantarum NSL0125 are as follows: pH 6.20-6.80 before fermentation, fermentation temperature 32-40° C., fermentation speed 100 rpm, and fermentation time 10-16 h.

[0013] Furthermore, the viable count of Lactobacillus plantarum NSL0125 in the Lactobacillus plantarum powder is not less than 4×10 11 CFU / ml.

[0014] The present invention also provides the use of the Lactobacillus plantarum NSL0125 or the Lactobacillus plantarum powder in the preparation of a microbial preparation and / or medicine for promoting calcium supplementation and / or growth and development.

[0015] Furthermore, the number of viable bacteria in the microbial preparation or drug is not less than 1×10 10 CFU / ml of Lactobacillus plantarum NSL0125.

[0016] The present invention also provides the use of the Lactobacillus plantarum NSL0125 or the Lactobacillus plantarum powder in the preparation of a microbial preparation for promoting calcium ion absorption.

[0017] Furthermore, the microbial preparation includes instant probiotic powder and probiotic active beverage.

[0018] The present invention mainly relates to instant probiotic powder.

[0019] Furthermore, the formula and dosage of the instant probiotic powder are: 2-5 parts of Lactobacillus plantarum powder, 10-15 parts of maltodextrin, 20-25 parts of skimmed milk powder, 5-8 parts of oligofructose, 5-10 parts of emulsified calcium, 1-4 parts of black bean powder, 1.5-2.5 parts of black date powder, 2.1-2.8 parts of hawthorn powder, and 3-6 parts of lemon powder, calculated by weight.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects: the present invention isolates a plant lactobacillus NSL0125, which has strong safety, no pathogenicity, no acute toxicity, and can ferment a variety of nutrients for energy growth. The plant lactobacillus NSL0125 is verified by experiments to be able to colonize in the intestinal tract for a long time, and it has a unique effect on the adsorption, transportation and absorption promotion of calcium ions, thereby effectively promoting the body's calcium absorption and growth and development. In addition, plant lactobacillus NSL0125 has excellent fermentation characteristics, a high fermentation level, and is suitable for large-scale industrial production. The present invention, through a large number of experimental studies, proves that plant lactobacillus NSL0125 and its biological preparation have a significant effect of promoting calcium supplementation and growth and development, and therefore has a good application value and a good market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a microscopic photograph of the bacteria of the Lactobacillus plantarum NSL0125. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments.

[0023] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0024] Example 1: Isolation and identification of Lactobacillus plantarum NSL0125 1. Separation and purification: The collected dairy products were added to 100 ml of MRS liquid shake flask medium for anaerobically cultured for 24 h, then streaked onto MRS solid medium and cultured anaerobically at 37°C for 48 h. The obtained single colony was inoculated into liquid medium MRS and cultured at 37°C for 24 h. Physiological saline was added to make a dilution gradient bacterial suspension to 10 -8 , select three appropriate gradient bacterial suspensions and take 1 mL each, inoculate into selective solid culture medium, and culture at 37℃ under anaerobic conditions for 48 h. Pick the typical colonies on the plate, repeat streaking separation for 3 generations, obtain the purified strains, perform Gram staining and observe under a microscope, select Gram-positive strains, and microscopically observe spherical, rod-shaped, and polymorphic strains. A rod-shaped bacterium was further selected from the multiple strains obtained, and a strain named NSL0125 was obtained.

[0025] 2. Morphological observation (1) Microscopic examination: Inoculate strain NSL0125 on a modified MRS solid plate and culture it anaerobically at 37°C for 48 h to observe the morphological characteristics of the colonies. Figure 1 As shown, the colony morphology is milky white, translucent, relatively round, smooth, with neat edges and obvious protrusions. The strain cells are round rod-shaped, medium-sized, non-spore-producing, and positive after Gram staining.

[0026] (2) Preservation of bacterial strains: The bacterial suspension was mixed with glycerol in a volume ratio of 7:3 and stored at -80°C ultra-low temperature to ensure its genetic stability and repeatability of subsequent experiments.

[0027] (3) 16S rRNA identification: DNA of strain NSL0125 was extracted as a template, and its 16S rRNA gene was amplified using universal primers. The DNA was then sent to a biological company for sequencing to obtain a complete 16S rRNA sequence. The sequencing result is shown in SEQ ID No. 1. After comparison with the NCBI database, its taxonomic status was confirmed to be Lactobacillus plantarum, and it had a high sequence similarity with strains that have the function of regulating calcium metabolism.

[0028] The screened Lactobacillus plantarum NSL0125 is preserved in the China Center for Type Culture Collection (CCTCC); address: Wuhan University, Wuhan, China; Lactobacillus plantarum NSL0125 Lactobacillus plantarum The deposit number is: CCTCC NO: M 2024609, and the deposit date is April 1, 2024.

[0029] Example 2: Safety evaluation of Lactobacillus plantarum NSL0125 1. Hemolytic test The blood agar plate method was used to detect whether the strain NSL0125 had hemolytic activity. The safety evaluation was performed based on the presence or absence of hemolysis to determine whether it had a destructive effect on red blood cells. Under sterile conditions, Lactobacillus plantarum was inoculated into Columbia blood agar plates with an inoculation loop and cultured at 37°C for 48 h. A blank test was performed at the same time to observe whether there was a hemolytic zone around the colonies. Hemolysis will form three characteristics on the blood plate: ①a hemolysis: a grass-green hemolytic zone will appear around the colonies, and this bacterium is generally pathogenic. ②β hemolysis: a wider transparent hemolytic zone appears around the colonies, which is generally highly pathogenic. ③γ hemolysis: no hemolytic zone appears around the colonies, and generally the strain is non-pathogenic. Using Staphylococcus aureus as a positive control, the experimental results showed that there was no hemolytic zone around each colony, so Lactobacillus plantarum NSL0125 was γ hemolytic and had no hemolytic activity, which also showed that Lactobacillus plantarum NSL0125 was non-pathogenic and safe.

[0030] 2. Animal oral acute toxicity test Twenty healthy adult SPF KM mice weighing 18-25 g were selected, half of which were male and half were female. The concentration of Lactobacillus plantarum NSL0125 in the growth phase was adjusted to 1×10 10 CFU / mL, fasting gavage once, each gavage volume of 0.2mL / 10g body weight. Immediately after gavage, the activity performance, body weight and physical condition of the animals were observed for 14 days.

[0031] The results showed that during the observation period, the mice had normal activity and appetite, and gained weight. After the experiment, the surviving mice were dissected and no abnormalities were found in their internal organs such as the heart, liver, spleen, lungs, kidneys, and intestines. This shows that Lactobacillus plantarum NSL0125 has no acute toxicity to mice and is safe.

[0032] Example 3: Preparation of freeze-dried powder of Lactobacillus plantarum NSL0125 The activated plant lactobacillus NSL0125 is streaked on an MRS solid culture medium, and anaerobic fermentation is carried out at 37°C for 48 h, and then the microbial suspension is collected, and the mixture is centrifuged at a speed of 8000 r / min for 10 min, and the centrifuged bacteria are collected to obtain bacterial mud; the bacterial mud is mixed with a freeze-drying protective agent solution, wherein the protective agent has a formula of 1-2 parts of trehalose, 1-2 parts of skim milk powder, 0.1-0.2 parts of sodium glutamate, 1 part of Tween 80, and 1 part of pure water, in parts by weight, and then mixed evenly according to the volume of the bacterial liquid before centrifugation to obtain a plant lactobacillus suspension; the probiotic suspension is pre-frozen at -60°C in a vacuum freeze dryer for 1 h, and after vacuuming to 20 Pa, the probiotic suspension is dried at a gradient temperature of -50°C, and freeze-dried into powder after 24 h, so as to obtain the probiotic freeze-dried powder with a high number of viable bacteria, which is stored in a -18°C cold storage after being packaged.

[0033] The freeze-dried powder was mixed with normal saline (0.85%), and the number of live bacteria was calculated by the live bacteria counting method. The survival rate was calculated by comparing the number of live bacteria before freeze-drying, and the freeze-dried powder containing the number of live bacteria not less than a specific standard (such as 1×10^10 CFU / ml in the "General Rules for Probiotics for Food").

[0034] Freeze-drying survival rate = (total number of viable bacteria per milliliter after freeze-drying and rehydration / total number of viable bacteria per milliliter of bacterial solution before freeze-drying) × 100%.

[0035] Example 4: Preparation of biological preparations Take 2-5 parts of probiotic dry powder, 10-15 parts of maltodextrin, 20-25 parts of skimmed milk powder, 5-8 parts of oligofructose, 5-10 parts of emulsified calcium, 1-4 parts of black bean powder, 1.5-2.5 parts of black date powder, 2.1-2.8 parts of hawthorn powder, and 3-6 parts of lemon powder, by weight.

[0036] Step 1: Evenly mix the prepared Lactobacillus plantarum NSL0125 freeze-dried powder with the above-mentioned auxiliary materials according to the formula ratio.

[0037] Step 2: Use wet granulation, fluidized bed drying and other processes to make biological preparations in the form of granules, tablets, capsules or powders.

[0038] Step 3: Dilute the lyophilized powder in the preparation using the gradient dilution method, and select the appropriate concentration to count the live bacteria using MRS agar medium.

[0039] The preparation must ensure that the number of viable bacteria is not less than the specified standard during the storage period, and have good taste, stability and convenience of taking, and the number of viable bacteria reaches 6×10 11 cfu / ml.

[0040] The Lactobacillus plantarum NSL0125 provided by the present invention can improve the bioavailability of calcium through the following mechanisms: 1. Lower the intestinal pH value: Lactobacillus plantarum lowers the pH value of the intestinal environment by producing organic acids such as lactic acid, increases the solubility of calcium ions, facilitates their release from food, and improves the absorbability of calcium.

[0041] 2. Release of calcium-regulated peptides: Lactobacillus plantarum can secrete some peptide substances, such as casein phosphopeptides (CPPs). These peptides directly or indirectly act on calcium channels, promote calcium ion transmembrane transport, and enhance calcium absorption efficiency.

[0042] 3. Regulate intestinal hormones and inflammatory responses: Lactobacillus plantarum indirectly affects the absorption and utilization of calcium by regulating the secretion of intestinal hormones (such as GLP-2, IGF-1, etc.) and inhibiting inflammatory responses.

[0043] In summary, the present invention provides a biological preparation with plant lactobacillus NSL0125 as a core component and its application, the preparation makes full use of the regulatory effect of plant lactobacillus on calcium metabolism, can effectively improve the absorption and utilization rate of calcium, promote the growth and development of human or animal, and has good safety. Through scientific preparation technology and effect verification, the present invention provides a novel product with strong practicality and broad market prospects for realizing efficient, safe and convenient calcium supplementation and growth promotion.

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A strain of Lactobacillus plantarum NSL0125, characterized in that: The classification of the Lactobacillus plantarum NSL0125 is named Lactobacillus plantarum , deposited in China Center for Type Culture Collection, with the deposit number: CCTCC NO: M2024609.

2. A plant lactobacillus powder, characterized in that: The Lactobacillus plantarum powder is obtained by mixing the bacterial mud of the Lactobacillus plantarum NSL0125 described in claim 1 with a freeze-drying protective agent and then subjecting the mixture to vacuum freeze-drying.

3. The plant lactobacillus powder according to claim 2, characterized in that The mass ratio of the bacterial mud of the Lactobacillus plantarum NSL0125 to the lyophilization protective agent is 1-3:

1.

4. The plant lactobacillus powder according to claim 2, characterized in that The lyophilization protective agent comprises the following components: 1-2 parts of trehalose, 1-2 parts of skim milk powder, 0.1-0.2 parts of sodium glutamate, 0.5-2 parts of Tween 80 and 0.5-2 parts of pure water, calculated by weight.

5. The plant lactobacillus powder according to claim 2, characterized in that The viable count of Lactobacillus plantarum NSL0125 in the Lactobacillus plantarum powder is not less than 4×10 11 CFU / ml.

6. Use of the plant lactobacillus NSL0125 described in claim 1 or the plant lactobacillus powder described in claim 2 in the preparation of a microbial preparation and / or a medicine for promoting calcium supplementation and / or growth and development.

7. The use according to claim 6, characterized in that: The number of viable bacteria in the microbial preparation or drug is not less than 1×10 10 CFU / ml of Lactobacillus plantarum NSL0125.

8. Use of the plant lactobacillus NSL0125 described in claim 1 or the plant lactobacillus powder described in claim 2 in the preparation of a microbial preparation for promoting calcium ion absorption.

9. The use according to claim 8, characterized in that: The microbial preparations include instant probiotic powders and probiotic active beverages.

10. The use according to claim 9, characterized in that: The instant probiotic powder comprises: 2-5 parts of plant lactobacillus powder, 10-15 parts of maltodextrin, 20-25 parts of skim milk powder, 5-8 parts of oligofructose, 5-10 parts of emulsified calcium, 1-4 parts of black bean powder, 1.5-2.5 parts of black date powder, 2.1-2.8 parts of hawthorn powder and 3-6 parts of lemon powder, calculated by weight.

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