Lactobacillus acidophilus proliferation agent, lactobacillus acidophilus preparation and application of lactobacillus acidophilus proliferation agent

Abstract

The invention discloses a lactobacillus acidophilus proliferation agent, a lactobacillus acidophilus preparation and application of the lactobacillus acidophilus proliferation agent. Belongs to the technical field of probiotics. The lactobacillus acidophilus proliferation agent is dendrobium officinale ultra-fine powder, preferably, Yunnan dendrobium officinale ultra-fine powder, and the particle size of the lactobacillus acidophilus proliferation agent is smaller than or equal to 10 microns. According to the proliferation agent, the growth rate and the thallus number of the lactobacillus acidophilus in the culture process can be increased, the resistance of the lactobacillus acidophilus to cholate, gastric juice and intestinal juice is enhanced, and the survival rate of the lactobacillus acidophilus in gastrointestinal tracts is increased. The invention also provides a thallus preparation, which comprises lactobacillus acidophilus viable thallus and dendrobium officinale ultra-fine powder. The invention also relates to application of the product in preparation of food or drugs for improving intestinal microecology, and a method for promoting growth of lactobacillus acidophilus and improving gastrointestinal tract tolerance of lactobacillus acidophilus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of probiotics, in particular to a lactobacillus acidophilus proliferation agent, a bacterial preparation and applications thereof. BACKGROUND

[0002] With the pursuit of a healthy lifestyle, probiotic products are becoming increasingly popular in the market. Lactobacillus acidophilus, as an important probiotic, has multiple physiological activities such as regulating intestinal flora balance, enhancing immunity, and improving gastrointestinal function, and is widely used in functional foods and drugs. However, the survival rate of Lactobacillus acidophilus in the gastrointestinal tract is low, which seriously affects its probiotic effect.

[0003] Currently, the common Lactobacillus acidophilus preparations on the market have a large decrease in the number of live bacteria after the action of gastric acid and bile salts and other digestive fluids, making it difficult to effectively colonize in the intestinal tract. In order to improve the survival rate and proliferation capacity of Lactobacillus acidophilus, researchers have tried various methods, among which the addition of prebiotics is a common means. Traditional prebiotics mainly include oligosaccharides such as fructooligosaccharides and galactooligosaccharides, but these prebiotics have limited effects on promoting the proliferation of Lactobacillus acidophilus and are not ideal for improving its gastrointestinal tolerance.

[0004] Dendrobium officinale, as a precious traditional Chinese medicine, has the effects of nourishing yin and clearing heat, benefiting the stomach and generating saliva, and improving eyesight. In recent years, the application of Dendrobium officinale in the field of food and health products has been increasing. Studies have shown that Dendrobium officinale contains various active ingredients such as polysaccharides, alkaloids, and amino acids, which may have a regulatory effect on the growth of microorganisms.

[0005] In CN106822601A, "a kind of Dendrobium officinale enzyme superfine powder and its preparation method" is disclosed, which mentions that Dendrobium officinale is fermented by yeast and lactic acid bacteria to produce superfine powder. This product has good antioxidant properties. CN104940644A discloses "a lactic acid bacteria fermentation method of Dendrobium officinale, its fermentation product and application", which uses lactic acid bacteria to ferment Dendrobium officinale. On the basis of the original health care effect of Dendrobium officinale, active probiotics are introduced to improve the taste and flavor of Dendrobium officinale, and to improve its nutritional value and health care effect.

[0006] CN105707628B discloses "a composition of probiotic fermented Dendrobium officinale protocorm and its preparation method", which uses multiple strains of probiotics to batch ferment Dendrobium officinale protocorm, making its effective ingredients into active substances that are easily absorbed by the intestinal tract, thereby improving the utilization rate. CN108785536A discloses "a Dendrobium officinale probiotic herbal oral preparation and its preparation and application", which combines Dendrobium officinale powder with probiotic powder to effectively inhibit the reproduction of harmful bacteria in the oral cavity, throat, and intestinal tract, and balance the oral and intestinal microecology.

[0007] However, there is no research report on Dendrobium officinale super-fine powder as a Lactobacillus acidophilus proliferation agent in the prior art, and there is no related research on the ability of Dendrobium officinale super-fine powder to improve the tolerance of Lactobacillus acidophilus to gastric acid and bile salts. The existing probiotic preparations have low survival rate in the environment of gastric acid and bile salts, and the colonization effect is poor. Traditional prebiotics (such as oligosaccharides) have limited proliferation-promoting effect on Lactobacillus acidophilus, and it is difficult to meet the demand of improving the survival rate of Lactobacillus acidophilus in the gastrointestinal tract. Therefore, developing a proliferation agent that can effectively promote the growth of Lactobacillus acidophilus and improve its gastrointestinal tolerance is of great significance to improve the efficacy of probiotic preparations. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a new prebiotic application scheme capable of improving the proliferation ability and gastrointestinal tolerance of probiotics, aiming at the defects that the existing probiotic preparations have low survival rate in the environment of gastric acid and bile salts, and the colonization effect is poor, and the traditional prebiotics (such as oligosaccharides) have limited proliferation-promoting effect on Lactobacillus acidophilus.

[0009] The present application is realized by the following technical scheme: In a first aspect, the present application provides a Lactobacillus acidophilus proliferation agent, wherein the proliferation agent is Dendrobium officinale super-fine powder.

[0010] Preferably, the Dendrobium officinale super-fine powder is Dendrobium officinale super-fine powder produced in Yunnan, and the particle size is ≤ 10 μm.

[0011] Preferably, the Dendrobium officinale super-fine powder improves the growth rate and the number of bacterial cells during the culture of Lactobacillus acidophilus.

[0012] Preferably, the Dendrobium officinale super-fine powder can enhance the resistance of Lactobacillus acidophilus to bile salts, gastric juice and small intestinal juice, and improve its survival rate in the gastrointestinal tract.

[0013] Further, the mass concentration of the Dendrobium officinale super-fine powder for proliferating Lactobacillus acidophilus is 0.5-2.0 mg / mL, preferably 1.0 mg / mL.

[0014] Preferably, the preparation method of the Dendrobium officinale super-fine powder comprises the following steps: crushing the dried stems of Dendrobium officinale produced in Yunnan and passing through a No. 9 sieve to obtain super-fine powder with a particle size of ≤ 10 μm. The crushing process can promote the complete destruction of cells, increase the contact area between the drug and the bacterial cells, and thus better play the probiotic effect.

[0015] In a second aspect, the present application further provides a bacterial cell preparation, characterized in that it comprises Lactobacillus acidophilus live bacterial cells and Dendrobium officinale super-fine powder.

[0016] Preferably, the bacterial cell preparation can improve the survival rate of Lactobacillus acidophilus in the gastrointestinal tract.

[0017] In a third aspect, the present application also provides the use of the above-mentioned Lactobacillus acidophilus proliferation agent or bacterial body preparation in the preparation of a food or drug for improving the intestinal micro-ecology.

[0018] In a fourth aspect, the present application also provides a method for promoting the growth of Lactobacillus acidophilus and improving its gastrointestinal tolerance, characterized in that the Dendrobium officinale super-fine powder is added as a prebiotic to the Lactobacillus acidophilus culture system or bacterial body preparation.

[0019] The present application has the following beneficial effects: 1. The Dendrobium officinale super-fine powder can significantly promote the growth and proliferation of Lactobacillus acidophilus, and the proliferation effect is significantly higher than that of the Dendrobium officinale polysaccharide group and other concentrations (0.5 / 1.5 / 2.0 mg / mL) in the same group. Through comparative experiments, it is proved that the effect of the Dendrobium officinale super-fine powder as a prebiotic is better than that of traditional prebiotics, which provides a new choice for the development of probiotic preparations.

[0020] 2. In the in-vitro simulation of bile salt, artificial gastric juice (pH 3.0) and artificial small intestinal fluid environment, the viable count of Lactobacillus acidophilus with the addition of Dendrobium officinale super-fine powder is significantly higher than that of the blank group and the inulin group, indicating that the Dendrobium officinale super-fine powder can effectively improve the tolerance of Lactobacillus acidophilus to the harsh environment of the gastrointestinal tract. This result suggests that Dendrobium officinale may protect the bacterial body by maintaining membrane stability or inhibiting trypsin activity, thereby solving the technical problem of low survival rate of existing probiotic preparations in the gastrointestinal tract.

[0021] 3. The Dendrobium officinale super-fine powder can improve the colonization ability of Lactobacillus acidophilus in the gastrointestinal tract to a certain extent, making it better to play a probiotic role, which provides a new technical means for improving the intestinal micro-ecology. Compared with the prior art, the application of the Dendrobium officinale super-fine powder as a prebiotic not only promotes the growth of Lactobacillus acidophilus, but also improves its survival rate in the gastrointestinal tract, thereby more effectively exerting the health function of probiotics. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 for the proliferation effect of Dendrobium officinale super-fine powder and Dendrobium officinale polysaccharide on Lactobacillus acidophilus; Figure 2 for the effect of Dendrobium officinale on the growth rate of Lactobacillus acidophilus; Figure 3 for the effect of Dendrobium officinale on the growth of Lactobacillus acidophilus in bovine bile salt; Figure 4 for the effect of Dendrobium officinale on the growth of Lactobacillus acidophilus in artificial gastric juice; Figure 5 for the effect of Dendrobium officinale on the growth of Lactobacillus acidophilus in artificial small intestinal fluid. DETAILED DESCRIPTION

[0023] The application will be further described in detail below with reference to the accompanying drawings and examples, but in no way limits the application, any transformation or improvement based on the teaching of the application falls into the protection scope of the application.

[0024] The process, conditions, reagents, experimental methods, etc. for implementing the application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the application has no special limitations. The experimental methods not specified in the specific conditions in each example are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer.

[0025] Unless otherwise specified, the meanings of all professional terms and scientific terms used in the specification are the same as the meanings generally understood by the person skilled in the art to which the application belongs. However, if there is a conflict, the specification including the definition shall prevail.

[0026] Lactobacillus acidophilus, strain number 1878, from the Chinese Academy of Sciences Institute of Microbiology. Dian production of Dendrobium candidum from Yunnan Tianbao Huabio Biological Resources Development Company.

[0027] Example 1 The present example provides a Lactobacillus acidophilus proliferation agent, which is a Dian production of Dendrobium candidum ultrafine powder.

[0028] Specifically, the Lactobacillus acidophilus proliferation agent is prepared from Dian production of Dendrobium candidum, and is treated by ultrafine grinding process so that the particle size of the ultrafine powder is not more than 10 μm. The particle size of the Dendrobium candidum ultrafine powder is controlled to be below 10 μm, which can significantly improve the bioavailability and release efficiency of active ingredients, thereby more effectively promoting the proliferation of Lactobacillus acidophilus.

[0029] In the preparation process, the freshly picked Dian production of Dendrobium candidum is first washed and dried, and then ground to an ultrafine powder state with a particle size of not more than 10 μm by low-temperature ultrafine grinding technology.

[0030] Example 2 Proliferation effect of Dendrobium candidum on Lactobacillus acidophilus (1) The basic culture medium for Lactobacillus acidophilus: add distilled water to 1000 mL of MRS medium (see Table 1 for MRS medium composition) to completely dissolve, and sterilize at 121 ℃ for 20 min. The experimental medium replaces glucose with a certain concentration of Dian production of Dendrobium candidum ultrafine powder and Dendrobium candidum polysaccharide sample.

[0031] Table 1 MRS medium composition

[0032] (2) Strain activation and seed liquid preparation: Lactobacillus acidophilus 1878 glycerol-preserved strain was thawed in an ice bath, then streaked on MRS solid medium and cultured anaerobically at 37°C for 48 hours; a single colony was picked and inoculated in 5 mL of MRS liquid medium, and cultured anaerobically at 37°C and 180 rpm until the OD 600 value reached 0.8-1.0 to obtain the logarithmic phase seed liquid.

[0033] (3) Optimal addition concentration screening: MRS base medium without glucose was prepared, and Dian-produced Dendrobium officinale and Dendrobium officinale polysaccharide were added to a final concentration of 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, and 2.0 mg / mL, respectively; MRS base medium without the addition of Dian-produced Dendrobium officinale and Dendrobium officinale polysaccharide was used as a negative control; each experimental group was inoculated with 1% (v / v) seed liquid, and cultured anaerobically at 37°C for 48 hours; the OD 600 value was measured, and each group had 3 replicates; The results, as shown in Figure 1 , showed that Dian-produced Dendrobium officinale ultra-fine powder and Dendrobium officinale polysaccharide both had a certain promoting effect on the growth of Lactobacillus acidophilus, and the promoting effect on the growth of Lactobacillus acidophilus was different at different concentrations of the added samples in the culture medium. The OD 600 value of Lactobacillus acidophilus reached (1.61±0.04) at a Dian-produced Dendrobium officinale ultra-fine powder concentration of 1.0 mg / mL, which was significantly higher than that of the control group and each dose group of Dendrobium officinale polysaccharide, and was superior to other concentration groups (0.5 / 1.5 / 2.0 mg / mL) in the same group, thus determining the optimal concentration.

[0034] (4) Effect of Dian-produced Dendrobium officinale on the growth curve of Lactobacillus acidophilus: Dendrobium officinale ultra-fine powder (concentration 1.0 mg / mL) was added to the culture medium, and 1% activated seed liquid was inoculated, and cultured anaerobically at 37°C; the OD 600 value of the fermentation broth was measured every 4 hours, and each group had 3 replicates. The time was taken as the abscissa, and the OD 600 value was taken as the ordinate to draw the growth curve of Lactobacillus acidophilus.

[0035] The results, as shown in Figure 2 , showed that Dian-produced Dendrobium officinale ultra-fine powder had a promoting effect on the growth of Lactobacillus acidophilus.

[0036] Example 3 Effect of Dendrobium officinale on the gastrointestinal tolerance of Lactobacillus acidophilus (1) Tolerance of Lactobacillus acidophilus to bile salts: 0.3 g of cholic acid salt was dissolved in 100 mL of distilled water, and 1.0 mg / mL of Dian-produced Dendrobium officinale ultra-fine powder (experimental group) and 2.0 mg / mL of inulin (positive control) were added; Inoculate 1% Lactobacillus acidophilus seed liquid, anaerobic culture at 37°C for 6 hours, measure OD value every hour 600 ; (2) Lactobacillus acidophilus tolerance in artificial gastric juice: add 1.0 mg / mL Dian Dendrobium officinale super fine powder to the simulated gastric juice (sterile, pH 3.0), inoculate 1% Lactobacillus acidophilus seed liquid, and then culture at 37°C for 6 hours, measure OD value every hour 600 ; (3) Lactobacillus acidophilus tolerance in artificial small intestinal juice: add 1.0 mg / mL Dian Dendrobium officinale super fine powder to the simulated small intestinal juice (containing trypsin and phosphate), inoculate 1% Lactobacillus acidophilus seed liquid, and then culture at 37°C for 6 hours, measure OD value every hour 600 .

[0037] The results are shown in Figures 3-5 , the viable cell count of Lactobacillus acidophilus in bile salt, gastric juice (pH 3.0) and intestinal juice environment is significantly higher than that of blank group and inulin group, which indicates that Dian Dendrobium officinale super fine powder can improve the colonization ability of Lactobacillus acidophilus in gastrointestinal tract, and make it better to play the role of probiotics. Example 4: A bacterial body preparation The present embodiment provides a bacterial body preparation, which comprises Lactobacillus acidophilus live bacteria and Dendrobium officinale super fine powder, and can improve the survival rate of Lactobacillus acidophilus in gastrointestinal tract.

[0038] The Lactobacillus acidophilus live bacteria can be selected from Lactobacillus acidophilus, which has strong acid and bile salt tolerance and can better survive in gastrointestinal environment. The content of Lactobacillus acidophilus live bacteria is 1×10 8 -1×10 11 CFU / g, preferably 5×10 9 -5×10 10 CFU / g, more preferably 1×10 10 CFU / g.

[0039] The preparation method of Dendrobium officinale super fine powder is the same as that of Example 1.

[0040] The preparation method of the bacterial body preparation comprises the following steps: S1: Lactobacillus acidophilus culture: inoculate Lactobacillus acidophilus into MRS culture medium, and anaerobic culture at 37°C for 24-48 hours to obtain Lactobacillus acidophilus culture solution; S2: Bacterial body collection: centrifuge the Lactobacillus acidophilus culture solution at 4°C and 6000 rpm for 10 minutes to collect bacterial body precipitate; S3: Bacterial body washing: wash the bacterial body precipitate with sterile physiological saline for 2-3 times; S4: Preparation of mixture: mix the washed bacterial body with Dendrobium officinale super fine powder; S5: Freeze-drying: the mixture was pre-frozen at -40℃ for 2 hours, and then freeze-dried at -50℃ for 24-48 hours under vacuum; S6: Powdering and sieving: the freeze-dried product was powdered and sieved through an 80-mesh screen to obtain the final bacterial preparation.

[0041] Example 5: Application of Lactobacillus acidophilus proliferation agent in preparing food for improving intestinal microecology The Dendrobium officinale super-fine powder used as the Lactobacillus acidophilus proliferation agent can significantly increase the growth rate and the number of Lactobacillus acidophilus, and enhance its resistance to cholate, gastric juice and small intestinal juice, thereby improving the survival rate of Lactobacillus acidophilus in the gastrointestinal tract. The Dendrobium officinale used in this example is Dendrobium officinale produced in Yunnan, which is processed by the super-fine grinding process to control the particle size below 10 μm.

[0042] The above Lactobacillus acidophilus proliferation agent is applied to prepare yogurt products for improving intestinal microecology. The specific preparation method is as follows: the Dendrobium officinale produced in Yunnan is processed by the super-fine grinding process to control the particle size below 10 μm, 5 g of the Dendrobium officinale super-fine powder is mixed with 1000 ml of pasteurized milk, Lactobacillus acidophilus is inoculated, and the mixture is fermented at 37℃ for 12 hours to obtain a yogurt product containing high-activity Lactobacillus acidophilus. It is found through detection that the number of Lactobacillus acidophilus in the yogurt added with the Dendrobium officinale super-fine powder is significantly higher than that of the control group, and the bacterial activity is significantly enhanced.

[0043] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A Lactobacillus acidophilus proliferation agent, characterized in that The proliferation agent is ultrafine powder of Dendrobium officinale.

2. The Lactobacillus acidophilus proliferation agent according to claim 1, characterized in that The ultrafine Dendrobium officinale powder is produced in Yunnan and has a particle size of ≤10 μm.

3. The Lactobacillus acidophilus proliferation agent according to claim 1, characterized in that The ultrafine Dendrobium officinale powder increases the growth rate and bacterial count during the culture process of Lactobacillus acidophilus.

4. The Lactobacillus acidophilus proliferation agent according to claim 1, characterized in that The ultrafine Dendrobium officinale powder can enhance the resistance of Lactobacillus acidophilus to bile salt, gastric juice and small intestinal juice, and improve its survival rate in the gastrointestinal tract.

5. The Lactobacillus acidophilus proliferation agent according to claim 1, characterized in that The mass concentration of the Dendrobium officinale ultrafine powder for proliferating Lactobacillus acidophilus is 0.5-2.0 mg / mL.

6. A bacterial preparation, characterized in that: Contains live Lactobacillus acidophilus and ultrafine Dendrobium officinale powder.

7. The bacterial preparation according to claim 6, characterized in that The bacterial preparation can improve the gastrointestinal survival rate of Lactobacillus acidophilus.

8. Use of the Lactobacillus acidophilus proliferator according to any one of claims 1 to 5 or the bacterial preparation according to claim 6 in the preparation of food or medicine for improving intestinal microecology.

9. A method for promoting the growth of Lactobacillus acidophilus and improving its gastrointestinal tolerance, characterized by: The ultrafine powder of Dendrobium officinale is added as a prebiotic to a Lactobacillus acidophilus culture system or bacterial preparation.

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