CN113116939B

CN113116939B - Microbial preparation and preparation method thereof

Info

Publication number: CN113116939B
Application number: CN201911419274.3A
Authority: CN
Inventors: 胡义成; 闫天文; 沈玉保; 束妍; 胡程程; 张辉
Original assignee: Hangzhou Grand Biologic Pharmaceutical Inc
Current assignee: Hangzhou Grand Biologic Pharmaceutical Inc
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2023-07-25
Anticipated expiration: 2039-12-31
Also published as: CN113116939A

Abstract

The invention provides a microbial preparation. The microbial preparation comprises: lactobacillus acidophilus, bifidobacterium infantis, enterococcus faecalis and bacillus cereus. The microbial preparation provided by the embodiment of the invention is tetrad bacteria, can effectively improve the immunity of organisms, and can keep good stability of the number of living bacteria under the normal temperature condition.

Description

Microbial preparation and preparation method thereof

Technical Field

The invention relates to the field of biological products, in particular to a microbial preparation and a preparation method thereof.

Background

Probiotics are a class of active microorganisms beneficial to a host by colonizing the human body and altering the flora composition of a part of the host. There is a continuing need for improvements in probiotics.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, an object of the present invention is to provide a microbial preparation which can effectively maintain the viable count of probiotics and improve the storage stability of the microbial preparation.

In view of this, a first aspect of the present invention proposes a microbial preparation. According to an embodiment of the invention, the microbial preparation comprises lactobacillus, bifidobacterium, enterococcus faecalis and bacillus cereus. The microbial preparation provided by the embodiment of the invention is tetrad bacteria, can effectively improve the immunity of organisms, and can keep good stability of the number of living bacteria under the normal temperature condition.

According to an embodiment of the present invention, the above-mentioned microbial preparation may further include at least one of the following additional technical features:

according to an embodiment of the invention, the lactobacillus is lactobacillus acidophilus and the bifidobacterium is bifidobacterium infantis.

According to an embodiment of the present invention, the microbial preparation comprises: lactobacillus acidophilus, bifidobacterium infantis, enterococcus faecalis and bacillus cereus. According to an embodiment of the present invention, there is provided: 35-150 parts by weight of the bifidobacterium infantis; 35-150 parts by weight of lactobacillus acidophilus; 5-35 parts by weight of enterococcus faecalis; and 5 to 35 parts by weight of the Bacillus cereus. Preferably, according to an embodiment of the present invention, there is provided: 50 parts by weight of said bifidobacterium infantis; 55 parts by weight of the lactobacillus acidophilus; 7 parts by weight of the enterococcus faecalis; and 8 parts by weight of the Bacillus cereus. The microbial preparation provided by the embodiment of the invention can be used for regulating the flora of the gastrointestinal tract by compounding the bacterial strain, so that various problems caused by dysbacteriosis, such as diarrhea, constipation, functional dyspepsia and the like, can be solved.

According to an embodiment of the invention, the microbial preparation further comprises pharmaceutically acceptable excipients.

According to an embodiment of the present invention, the pharmaceutically acceptable excipients include fillers and lubricants.

According to an embodiment of the invention, the filler comprises at least one selected from the group consisting of starch, sucrose, dextrin, calcium sulfate, dibasic calcium phosphate, calcium carbonate, microcrystalline cellulose, mannitol, lactose, pregelatinized starch, glucose, maltodextrin.

According to an embodiment of the invention, the sweetener comprises at least one selected from lactose, mannitol, sucrose, glucose.

According to an embodiment of the invention, the lubricant comprises at least one selected from stearic acid, magnesium stearate, aerosil, sodium/magnesium lauryl sulfate, talc, sodium stearyl fumarate, glyceryl behenate.

According to an embodiment of the invention, the filler comprises microcrystalline cellulose, sucrose, corn starch and lactose.

According to an embodiment of the invention, the lubricant is magnesium stearate.

Through intensive analysis, the inventor discovers that the water content in auxiliary materials is a key factor for maintaining the viable count of microorganisms at normal temperature through a large number of experiments, the water content of various commonly used auxiliary materials such as aluminum hydroxide and magnesium trisilicate is relatively high, and the water content of aluminum hydroxide and magnesium trisilicate is not required to be controlled in Chinese pharmacopoeia, so that the water content difference of the auxiliary materials such as the aluminum hydroxide and the magnesium trisilicate which are currently marketed is relatively large, and the quality of each batch of solid preparation is easy to be uneven. To this end, the inventors of the present invention propose an adjuvant formulation that does not use aluminum hydroxide and magnesium trisilicate. The water content in the solid preparation such as tablets can be effectively reduced, and experiments prove that the microbial preparation using microcrystalline cellulose, sucrose, corn starch, lactose and magnesium stearate as auxiliary materials has better viable bacteria stability.

According to an embodiment of the present invention, the microbial preparation contains: 15-25% by weight of said sucrose; and 10 to 20 wt% of the corn starch. According to an embodiment of the present invention, the microbial preparation contains: 18 to 21 wt% of the sucrose; and 14 to 16 wt.% of said cornstarch. According to an embodiment of the present invention, the microbial preparation contains: 19-20% by weight of said sucrose; and 15 to 16 wt% of said corn starch. According to the examples of the present invention, the inventors found that after excluding both the aluminum hydroxide and magnesium trisilicate, it was found that further adjustment of the proportions of corn starch and sucrose was necessary, which would otherwise deteriorate the compressibility of the formulation, for example, the compressed tablets failed to meet the hardness requirement under the condition of a large pressure, the tablets were liable to fracture, and in some cases the disintegration time period was also lengthened, which failed to meet the formulation requirement. For this reason, the inventors have conducted a large number of screening experiments, and unexpectedly obtained sucrose and corn starch in the above-mentioned proportions can provide advantageous compressibility, so that tablets capable of satisfying hardness requirements can be obtained.

According to an embodiment of the present invention, the microbial preparation contains: 12 to 18% by weight of said lactose. According to an embodiment of the present invention, the microbial preparation contains: 12 to 13% by weight of said lactose. According to an embodiment of the present invention, the microbial preparation contains: 45 to 55 wt% of the microcrystalline cellulose or 48 to 51 wt% of the microcrystalline cellulose. According to an embodiment of the present invention, the microbial preparation contains: 49 to 50.5% by weight of said microcrystalline cellulose. According to the embodiment of the invention, the inventor further optimizes other auxiliary materials, and the content ratio of lactose and microcrystalline cellulose is proposed by considering comprehensive consideration of compressibility, fluidity, physical and chemical indexes of tablets and the like. Specifically, according to an embodiment of the present invention, the microbial preparation contains: 50 parts by weight of said bifidobacterium infantis; 55 parts by weight of the lactobacillus acidophilus; 7 parts by weight of the enterococcus faecalis; 8 parts by weight of the bacillus cereus; 2500 parts by weight of the microcrystalline cellulose; 976 parts by weight of said sucrose; 780 parts by weight of the corn starch; 645 parts by weight of the lactose; and 25 parts by weight of said magnesium stearate. The inventor finds that the fluidity and the compressibility of the finally obtained formula are further improved, all physicochemical indexes meet the quality requirements, and the stability of the preparation is inspected through the optimized formula of the preparation, so that the stability of bifidobacterium infantis and lactobacillus acidophilus in the preparation is greatly improved.

According to an embodiment of the present invention, the microbial preparation is at least one of a tablet, a drop, a powder, and a capsule.

According to an embodiment of the invention, the tablet has a hardness of 40 to 100N. The inventors found that the tablet has a hardness of 40-100N, the number of viable bacteria in the tablet is high and stable, the tablet with a hardness lower than 40N is easy to break, and the number of viable bacteria in the tablet with a hardness higher than 100N is lower.

In a second aspect of the invention, the invention proposes a method that can be used for preparing the aforementioned microbial preparation, according to an embodiment of the invention, the method comprising: mixing predetermined amounts of said lactobacillus acidophilus, said bifidobacterium infantis, said enterococcus faecalis and said bacillus cereus, and preferably said pharmaceutically acceptable excipients; and tabletting the obtained mixture to obtain the microbial preparation, wherein the hardness of the microbial preparation is 40-100N. According to the embodiments of the present invention, the aforementioned microbial preparation can be efficiently prepared by using the present invention, and the features and advantages regarding the preparation are equally applicable to the method, and are not described herein. As will be appreciated by those skilled in the art, the term "predetermined amount" as used herein refers to an appropriate amount based on the final formulation.

Drawings

FIG. 1 is a graph showing the results of a Bifidobacterium infantis stability study in formulas 6, 7, 8 after modification of the adjuvant according to an embodiment of the present invention;

fig. 2 shows the results of stability investigation of lactobacillus acidophilus in formulations 6, 7 and 8 after modification of the auxiliary materials according to an embodiment of the present invention;

FIG. 3 is a graph showing the results of stability studies of enterococcus faecalis in formulations 6, 7, 8 after modification of the adjuvant according to the examples of the present invention;

fig. 4 shows the results of examining the stability of bacillus cereus in formulations 6, 7 and 8 after changing the auxiliary materials according to the embodiment of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.

Example optimization of tablet formulation

(1) Auxiliary material investigation

The water content and flowability (angle of repose) of the existing 7 excipients (microcrystalline cellulose, corn starch, sucrose, lactose, aluminum hydroxide, magnesium trisilicate, talc) and microcrystalline cellulose were tested and the results are shown in table 1.

Table 1:

as can be seen from the above test results, the water content of the aluminum hydroxide and the magnesium trisilicate is high and exceeds 10%, so that the 2 auxiliary materials are selected to be removed. Because magnesium trisilicate has good fluidity, the fluidity of the formula is poor after being removed as a formula glidant. The inventors found through studies that when the angle of repose is 45 ° or more, it is difficult for the powder to freely flow out of the container.

According to the result of measuring the repose angle of the auxiliary materials, the fluidity of the sucrose is poor and is higher than 45 degrees, and the integral fluidity of the formula can be reduced by increasing the proportion of the sucrose. By combining the characteristics of the product, from the angles of moisture, process angle and the like, the applicable solid preparation auxiliary materials are as follows:

filler: starch, sucrose, dextrin, calcium sulfate, dibasic calcium phosphate, calcium carbonate, microcrystalline cellulose, mannitol, lactose, pregelatinized starch.

Sweetener: lactose, mannitol, sucrose, glucose.

And (3) a lubricant: selected from stearic acid, magnesium stearate, aerosil, sodium/magnesium lauryl sulfate, talc, sodium stearyl fumarate, and glyceryl behenate.

The auxiliary materials can meet the production requirement of the product preparation, microcrystalline cellulose, corn starch, sucrose and lactose are common auxiliary materials of the solid preparation, and the magnesium stearate is the most common lubricant of the solid preparation at present, and the magnesium stearate has low dosage and wide source. Microcrystalline cellulose, corn starch, sucrose, lactose, and magnesium stearate are used for formulation screening.

(2) Single factor investigation

The scheme is as follows: the total weight of aluminum hydroxide, magnesium trisilicate and talcum powder in the commercial product SiLikang is 17 percent. According to the water content results examined above, the inventors decided to delete the aluminum hydroxide and magnesium trisilicate and replace the talc powder with magnesium stearate (the inventors found that the tabletting compressibility is deteriorated after the water content of the formulation is reduced, the use amount of the talc powder in the existing formulation is large (the proportion is up to 10%), the compressibility of the formulation is greatly affected, the tabletting process requirements cannot be met, the compressibility of the formulation can be improved by reducing the content of the talc powder, but serious sticking phenomenon occurs in tabletting after the talc powder is reduced, the talc powder is a lubricant of an oral solid preparation widely used at one time, but the use amount of the magnesium stearate is low, the lubricating effect is good, the present optimization uses magnesium stearate instead of talcum powder as lubricant, the inventor finds that magnesium stearate is hydrophobic, the addition proportion is too high, which leads to low hardness of tabletting tablets, so that the lowest concentration of 0.5 weight percent is used in the prescription for single factor examination.

Prescription composition: prescription components (g/10000 tablets): the addition amount of the bacterial powder is related to the viable count of the raw material bacterial powder, and is adjusted according to the different viable count, but the addition amount of all bacterial powder is not a fixed value, but is limited to a certain range, specifically the addition range of bifidobacterium infantis and lactobacillus acidophilus is 35-150, and the addition range of enterococcus faecalis and bacillus cereus is 5-35, and when formula screening is carried out, research is carried out according to the common proportion of bifidobacterium infantis bacterial powder 50, lactobacillus acidophilus bacterial powder 55, enterococcus faecalis bacterial powder 7 and bacillus cereus bacterial powder 8, and the addition amount of upper and lower limit bacterial powder is used for verification after the formula is determined; the proportion of the auxiliary materials is unchanged.

Acidophilic adjuvants are formulated as shown in table 2.

Table 2:

material name	Formulation 1	Formulation 2	Formulation 3	Formulation 4
					Bifidobacterium infantis	50	50	50	50
Lactobacillus acidophilus	55	55	55	55
					Enterococcus faecalis	7	7	7	7
Bacillus cereus	8	8	8	8
					Microcrystalline cellulose	2845	2020	2020	2020
Sucrose	976	1801	976	976
					Corn starch	780	780	1605	780
Lactose and lactose	300	300	300	1125
					Magnesium stearate	25	25	25	25
Total amount of	5046	5046	5046	5046

Note that: the actual adding proportion of the bacterial powder is adjusted according to the number of the viable bacteria

The preparation process comprises the following steps: adopting a powder direct tabletting process, and collecting four bacterial powders for standby after passing through a 60-mesh screen; the sucrose is crushed and filtered by a 60-mesh screen for standby. Then sequentially adding microcrystalline cellulose, corn starch, sucrose, lactose and mixed bacterial powder into a mixer, mixing for 25min, then adding magnesium stearate, and mixing for 5min. Tabletting the final mixture on a tablet press, and adjusting the tablet weight to be within 500+/-25 mg by adopting a shallow concave round punch, controlling the tablet hardness to be 40-100N, and tabletting.

Screening results show that the formula compressibility is poor when the corn starch dosage is increased, and the compressed tablet cannot meet the hardness requirement under the condition of larger pressure, so that the tablet is easy to fracture. Increasing the proportion of sucrose has poor flowability and poor compressibility, requires a greater pressure to compress tablets meeting the hardness requirement, and increases the proportion of sucrose results in a longer disintegration time. So the formulation ratio of the corn starch and the sucrose is kept unchanged and is not increased.

(3) Screening of compound formulas

The microcrystalline cellulose content is increased, the formula compressibility is higher (compared with the formula 3 and the formula 4), and each physical and chemical index of the tablet meets the requirements. However, the tablets are thicker, difficult to swallow, and have poor mouthfeel, and there is a feeling of dryness in swallowing and chewing. Increasing lactose content, the formulation was more compressible but slightly inferior to formulation 1. And (3) comprehensively comparing, further optimizing and adjusting the proportion of microcrystalline cellulose and lactose, and comprehensively comparing the compressibility, the fluidity and the disintegration time limit to obtain the optimal formula. The design recipe is shown in Table 3.

Table 3:

the results are shown in Table 4.

Table 4:

from the above results, it can be seen that the formulations 5 and 6 have a smaller repose angle and good fluidity compared with the existing formulations; under the same pressure condition, the tablet has high hardness, good compressibility and shortened disintegration time. So formulations 5 and 6 are superior to existing formulations. Because fluidity and compressibility are key indexes affecting the production process, the disintegration time limit meets the pharmacopoeia requirements, so that the fluidity and compressibility of the formula 6 are better compared comprehensively, and the formula 6 is selected as the investigation formula.

(4) And (3) process investigation: influence of tabletting process on viable count

Experiments show that different tablet press models are used for pressing tablets with the same hardness, the tablet press displays larger pressure value difference, the tablet press with the same model of the same manufacturer is used for pressing tablets with the same hardness, the tablet press displays different pressure values, and finally, the tablet hardness is used as the final reference for evaluating the quality of the preparation.

The results of viable count under different hardness conditions are shown in Table 5.

Table 5:

the formula 6 is adopted to press tablets with different hardness by setting different tabletting pressures, the friability, disintegration time limit and viable count of the tablets with different hardness are inspected, and the hardness control range is confirmed. From the above results, it can be seen that when the hardness is less than 40N, the tablet is directly broken, and the friability is not acceptable; the friability decreases with increasing hardness, but the disintegration time increases and the viable count survival rate decreases. When the hardness exceeds 100N, the disintegration time exceeds the existing prescription, and the survival rate of viable bacteria is lower than 50%. Therefore, the hardness is comprehensively considered to be controlled within the range of 40-100N, the viable count is high, the preparation is not easy to break, and the pressure control range of a tablet press is determined by the hardness index in future practical application.

(5) Workshop pilot experiment investigation

And carrying out 1 batch of 10 ten thousand pieces of pilot-scale experiments on the formula 6 in a workshop, verifying the feasibility of the production of the formula 6 under the industrialized production condition, and simultaneously verifying the influence of the upper and lower limit fungus powder feeding amounts on the formula.

The formulation is shown in Table 6.

Table 6:

material name	Formulation 6	Formulation 7	Formulation 8
				Bifidobacterium infantis	50	35	150
Lactobacillus acidophilus	55	35	150
				Enterococcus faecalis	7	5	35
Bacillus cereus	8	5	35
				Microcrystalline cellulose	2500	2500	2425
Sucrose	976	976	946
				Corn starch	780	780	756
Lactose and lactose	645	645	626
				Magnesium stearate	25	25	25
Total amount of	5046	5006	5148

The results are shown in Table 7.

Table 7:

formulation of	Thickness mm	Hardness N	Disintegration time	Moisture content
					Existing formulations	4.6-4.8	52.0	5min	3.8％
Formulation 6	4.7-4.9	64.2	1min	2.5％
					Formulation 7	4.7-4.9	70.2	1min	2.4％
Formulation 8	4.7-4.9	65.8	2min	2.4％

From the experimental results, the compressibility of the formulation after changing the auxiliary materials is similar to that of the existing formulation. And the moisture of the tablet is reduced after the auxiliary materials are changed, so that the low-moisture preservation of the product can be realized, meanwhile, the indexes of the pressed tablets with different bacterial powder addition amounts are similar, no obvious difference exists, and the fact that bacterial powder feeding fluctuation in the existing bacterial powder feeding range has no influence on the production process of the preparation is shown.

Living bacteria investigation: the number of viable bacteria in the existing formula and the formula after changing auxiliary materials by using the same batch of fungus powder in the pilot scale test pre-experiment is shown in table 8.

Table 8:

	bifidobacterium strain	Lactobacillus acidophilus	Enterococcus faecalis	Bacillus cereus
					Existing formulations	2.6E+08	5.1E+08	3.2E+07	3.4E+06
Formulation 6	2.6E+08	5.4E+08	3.9E+07	3.8E+06
					Formulation 7	1.8E+08	3.3E+08	2.6E+07	2.1E+06
Formulation 8	8.1E+08	1.4E+09	1.6E+08	1.5E+07

The results show that the existing formula with the same bacterial powder feeding amount and the formula 6 are not different in the whole tablet after tabletting, and the change of auxiliary materials has no influence on the contents of four living bacteria. Meanwhile, the formula 7 and the formula 8 with the formula changed in the feeding amount of the bacterial powder are calculated according to the feeding proportion of the bacterial powder, and the bifidobacterium infantis, the lactobacillus acidophilus, the enterococcus faecalis, the bacillus cereus and the existing formula are not different. Therefore, the process for finally obtaining the probiotic tablet by compounding microcrystalline cellulose, sucrose, corn starch, lactose and magnesium stearate can produce the rapidly disintegrated probiotic tablet, the prepared probiotic tablet has the advantages of rapid disintegration speed, convenient administration, obvious treatment effect, low cost, wide auxiliary material sources, stable process conditions, good reproducibility and the like.

(6) Pilot test stability test results

Stability of viable bacteria: under the pilot-scale conditions, the inventor prepares 1 batch of samples according to the formula 6, the formula 7 and the formula 8, prepares 1 batch of samples according to the existing formula, simultaneously puts 4 batches of samples into the conditions of 25 ℃ and 65% humidity, and samples and detects the viable count in 0, 1, 2 and 3 months respectively. The results are shown in Table 9.

Table 9:

the results of plotting the 4 bacteria on the abscissa and the number of viable bacteria of LOG10 on the ordinate are shown in FIGS. 1 to 4.

As can be seen from table 9 and fig. 1 to 4, under the condition of normal production of pilot-scale test, the stability of bifidobacterium infantis and lactobacillus acidophilus in the formulation after the auxiliary materials are changed is obviously improved compared with the stability of the existing formulation during storage, the stability of enterococcus faecalis in the formulation after the auxiliary materials are changed is improved to a certain extent compared with the stability of the existing formulation during storage, and the stability of three viable bacteria of bifidobacterium infantis, lactobacillus acidophilus and enterococcus faecalis is better than the stability of 3 bacteria in the existing formulation. There was no significant difference in the stability of the live bacillus cereus in the 2 formulations. Meanwhile, the formula 7 and the formula 8 after the formula is changed in the feeding amount of the bacterial powder have similar stability trends of live bacteria of bifidobacterium infantis, lactobacillus acidophilus, enterococcus faecalis and bacillus cereus, and have no obvious difference, so that the bacterial powder feeding fluctuation in the existing bacterial powder feeding range has no influence on the stability of the preparation.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A microbial formulation, wherein the microbial formulation is in the form of a tablet comprising: 35-150 parts by weight of lactobacillus, 35-150 parts by weight of bifidobacterium, 5-35 parts by weight of enterococcus faecalis and 5-35 parts by weight of bacillus cereus, wherein the lactobacillus is lactobacillus acidophilus, and the bifidobacterium is bifidobacterium infantis;

the microbial preparation further comprises pharmaceutically acceptable auxiliary materials, wherein the pharmaceutically acceptable auxiliary materials comprise a filling agent, a lubricating agent and a sweetener;

the filler comprises microcrystalline cellulose, sucrose, corn starch and lactose;

the lubricant is magnesium stearate;

the corn starch comprises 15-25% by weight of sucrose, 10-20% by weight of corn starch, 12-13% by weight of lactose, 49-50.5% by weight of microcrystalline cellulose and 0.5% by weight of magnesium stearate;

the tablet has a hardness of 40-71.2N.

2. A microbial preparation according to claim 1, comprising:

50 parts by weight of said bifidobacterium infantis;

55 parts by weight of the lactobacillus acidophilus;

7 parts by weight of the enterococcus faecalis;

8 parts by weight of the Bacillus cereus.

3. The microbial preparation according to claim 1, wherein the sweetener comprises at least one selected from lactose and sucrose.

4. A microbial preparation according to any one of claims 1 to 3, wherein the microbial preparation comprises:

50 parts by weight of said bifidobacterium infantis;

55 parts by weight of the lactobacillus acidophilus;

7 parts by weight of the enterococcus faecalis;

8 parts by weight of the bacillus cereus;

2500 parts by weight of the microcrystalline cellulose;

976 parts by weight of said sucrose;

780 parts by weight of the corn starch;

645 parts by weight of the lactose;

25 parts by weight of magnesium stearate.

5. A method for preparing the microbial preparation according to any one of claims 1 to 4, comprising:

mixing predetermined amounts of said lactobacillus acidophilus, said bifidobacterium infantis, said enterococcus faecalis and said bacillus cereus and said pharmaceutically acceptable excipients;

and tabletting the obtained mixture to obtain the microbial preparation, wherein the hardness of the microbial preparation is 40-71.2N.