阅读说明：本技术 重组人源化胶原蛋白的微生物限度检查方法 (Microbial limit inspection method for recombinant humanized collagen ) 是由 张莉莉 赵霜 邵秀萍 钱文娟 于 2021-07-20 设计创作，主要内容包括：本发明公开了一种重组人源化胶原蛋白的微生物限度检查方法。所述检查方法采用pH7.0无菌氯化钠-蛋白胨缓冲液或pH7.2磷酸盐缓冲溶液作为稀释剂,能够真实反映由保藏编号为CGMCC No.5021的巴斯德毕赤酵母Pichia pastoris发酵产生的重组人源化胶原蛋白中的微生物生长情况,提高重组人源化胶原蛋白的微生物限度检查准确度。(The invention discloses a microbial limit inspection method for recombinant humanized collagen. The inspection method adopts a pH7.0 sterile sodium chloride-peptone buffer solution or a pH7.2 phosphate buffer solution as a diluent, can truly reflect the microbial growth condition of the recombinant humanized collagen generated by fermentation of Pichia pastoris with the preservation number of CGMCC No.5021, and improves the microbial limit inspection accuracy of the recombinant humanized collagen.)

1. The microbial limit detection method of the recombinant humanized collagen is characterized by comprising the following steps:

(1) diluting a recombinant humanized collagen to be detected by taking a pH7.0 sterile sodium chloride-peptone buffer solution or a pH7.2 phosphate buffer solution as a diluent, fully oscillating and uniformly mixing, standing, taking a supernatant as a bacteria detection solution, and fermenting the recombinant humanized collagen by Pichia pastoris with the preservation number of CGMCC No. 5021;

(2) injecting the bacteria detection solution into a sterilization plate, pouring a trypticase soy peptone agar culture medium which is melted and cooled to 45-50 ℃ into the sterilization plate, rotating the plate immediately to fully and uniformly mix the bacteria detection solution and the culture medium, turning the plate after the agar is solidified, and culturing for 3-5 days in an incubator at 36 +/-1 ℃; and taking another empty sterilization plate without the bacteria detection liquid, adding a diluent which is equal to the bacteria detection liquid in volume, adding a trypticase soy peptone agar culture medium, turning the plate after the agar is solidified, culturing for 3-5 days in an incubator at 36 +/-1 ℃ to serve as a negative control test, wherein the negative control test needs aseptic growth, and if bacterial growth exists, deviation investigation needs to be carried out.

2. The method for examining a limitation of a microorganism according to claim 1, wherein in the step (1), the dilution ratio is 1:10 to 1: 10000.

3. The microbial limit test method according to claim 1, wherein in the step (2), the tryptone soy agar medium is a mixture of tryptone soy agar and water at a ratio of 4 g: 100mL of the solution was prepared.

4. The method for checking a limitation of a microorganism according to claim 1, wherein in the step (2), the volume ratio of the bacterial sample solution to the trypticase Soy agar medium is 1: 15.

Technical Field

The invention belongs to the technical field of microbial limit inspection of non-sterile products, and relates to a microbial limit inspection method of recombinant humanized collagen.

Background

The diluent used in the microbial limit test has an influence on the detection rate of the microorganisms, the growth and the applicability of the microorganisms, and mainly comprises 0.9% of sterile sodium chloride solution, pH7.0 sterile sodium chloride-peptone buffer solution, pH7.2 phosphate buffer solution and purified water.

The 0.9% sterile sodium chloride solution as diluent has the characteristics that the osmotic pressure is equivalent to that of human blood, and microorganisms waiting in the diluent are equivalent to that waiting in the blood and can not absorb water and crack due to the osmotic pressure.

The pH7.0 sterile sodium chloride-peptone buffer solution is mainly characterized by 'buffering', namely, the pH change is not very large under the buffering action, the osmotic pressure regulating effect is realized, the buffer solution is suitable for microorganisms sensitive to the pH value, and in addition, the peptone can provide nutrient substances such as a nitrogen source, amino acid, a carbon source and the like for the microorganisms.

The pH7.2 phosphate buffer solution is composed of a mixed solution of monohydrogen phosphate and dihydrogen phosphate, wherein the monohydrogen phosphate is basic, the dihydrogen phosphate is acidic, and reacts with the monohydrogen phosphate to form dihydrogen phosphate when the microorganism secretes acidic substances, and reacts with the dihydrogen phosphate to form monohydrogen phosphate when the microorganism secretes basic substances, so that the whole system is maintained in a relatively stable pH range. As the phosphate buffer solution is close to neutral, is exactly in the physiological pH range and has lower cost, the phosphate buffer solution becomes the most widely applied and popular inorganic salt buffer solution in the field of life science and is also one of the common buffer solutions necessary for laboratories of immunology, cell biology and molecular biology.

The recombinant humanized collagen is a water-soluble product, can promote the growth of fibroblasts, can permeate into dermis after being dissolved in water, has 100 percent of gene sequence the same as that of the humanized collagen, has anti-sensitivity, can promote the growth of cells to form a protective barrier, belongs to the cosmetic grade, and needs to be checked according to the technical Specification for cosmetic safety on the related limit of microorganisms. Document 1 establishes a recombinant human collagen microorganism limit test method (li dao, hou zeng 28156, cradle of a gem, etc.. construction and verification of the recombinant human collagen microorganism limit test method [ J ] scientific and technological innovation and application, 2017(35):98-99.) using a 0.9% sterile sodium chloride solution as a diluent. However, the recombinant human collagen described in the above documents is produced by the same company, and different recombinant humanized collagens have different proteins due to completely different fermentation conditions of fermentation tubes, and thus the diluent has no universality.

Disclosure of Invention

The invention aims to provide a microbial limit detection method of recombinant humanized collagen.

The technical scheme for realizing the purpose of the invention is as follows:

the microbial limit detection method of the recombinant humanized collagen comprises the following steps:

(1) diluting a recombinant humanized collagen to be detected by taking a pH7.0 sterile sodium chloride-peptone buffer solution or a pH7.2 phosphate buffer solution as a diluent, fully oscillating and uniformly mixing, standing, taking a supernatant as a bacteria detection solution, and fermenting the recombinant humanized collagen by Pichia pastoris with the preservation number of CGMCC No. 5021;

(2) injecting the bacteria detection solution into a sterilization plate, pouring a trypticase soy peptone agar culture medium which is melted and cooled to 45-50 ℃ into the sterilization plate, rotating the plate immediately to fully and uniformly mix the bacteria detection solution and the culture medium, turning the plate after the agar is solidified, and culturing for 3-5 days in an incubator at 36 +/-1 ℃; and taking another empty sterilization plate without the bacteria detection liquid, adding a diluent which is equal to the bacteria detection liquid in volume, adding a trypticase soy peptone agar culture medium, turning the plate after the agar is solidified, culturing for 3-5 days in an incubator at 36 +/-1 ℃ to serve as a negative control test, wherein the negative control test needs aseptic growth, and if bacterial growth exists, deviation investigation needs to be carried out.

In the step (1), the dilution factor is adjusted according to the bacterial content of the recombinant humanized collagen to be detected, and the higher the bacterial content is, the larger the dilution factor is. The dilution factor can be 1: 10-1: 10000.

In step (2) of the present invention, the tryptic soy agar medium is a tryptic soy agar medium conventionally used in the art, and the ratio of tryptic soy agar to water is 4 g: 100mL of the solution was prepared.

In the step (2), the volume ratio of the bacteria detection solution to the trypticase soy peptone agar medium is 1: 15.

Compared with the prior art, the invention has the following advantages:

according to the invention, different diluents are used for carrying out microbial limit inspection on the recombinant humanized collagen, and the detection rate of the microorganisms in the recombinant humanized collagen, the growth and the applicability of the microorganisms are compared, so that the recombinant humanized collagen of the application is determined to be suitable for being used as the diluent for a sterile sodium chloride-peptone buffer solution with pH7.0 or a phosphate buffer solution with pH7.2, and the microbial limit inspection accuracy of the recombinant humanized collagen is improved.

Detailed Description

The present invention will be described in more detail with reference to specific examples.

Example 1

1. Preparation of diluent and culture medium:

0.9% sodium chloride solution: adding 0.9g of sodium chloride into 1000mL of distilled water, dissolving, subpackaging, and autoclaving at 121 ℃ for 20 min.

ph7.0 sterile sodium chloride-peptone buffer: dissolving 3.56g of dipotassium phosphate, 7.23g of disodium phosphate, 4.30g of sodium chloride and 1.0g of peptone in 1000mL of purified water, and sterilizing.

ph7.2 phosphate buffer solution: 41g of distilled water of pH7.2 phosphate buffer solution powder was added to 1000mL, and autoclaved at 121 ℃ for 15 min.

Sterilizing distilled water: subpackaging with distilled water, and autoclaving at 121 deg.C for 20 min.

Trypticase soy peptone agar medium: dissolving trypticase soy agar 12g in 300mL water, and autoclaving at 121 deg.C for 20 min.

2. Examination of suitability of culture Medium used for controlled bacteria examination of test sample

(1) The strains, strains used and culture media are shown in Table 1, and the number of passages of the strains for the test should not exceed 5.

TABLE 1

Test strains	Culture medium for bacterial strain	Characteristics of	Control Medium
				Staphylococcus aureus	Trypticase soy peptone agar medium	Growth promoting ability	Mannitol sodium chloride agar culture medium

(2) And (3) adaptability checking: respectively using 0.9% sodium chloride aqueous solution, pH7.0 sterile sodium chloride-peptone buffer solution, pH7.2 phosphate buffer solution and purified water as bacterial suspensions to prepare 1mL of golden yellow staphylococcus liquid with not less than 100cfu, respectively inoculating 1mL of the golden yellow staphylococcus liquid on two plates, pouring into tryptic soy peptone agar medium, and simultaneously using the negative control. And another two plates are respectively used for taking 1mL of golden yellow staphylococcus liquid which is not less than 100cfu on the two plates, and the golden yellow staphylococcus liquid is poured into a mannitol sodium chloride agar culture medium and is used as a negative control. Culturing for 18-24 hours at the same temperature of 30-35 ℃, and carrying out aseptic growth in a negative control test, and carrying out deviation investigation if bacterial growth exists.

(3) And (5) judging a result: after 24 hours, the number of colonies on the detected culture medium is checked, the number is not less than 70% of the average number of colonies on the control culture medium, the shape and the size of the colonies are consistent with those on the control culture medium, and therefore the applicability of the culture medium is judged to be in accordance with the specification.

3. Aerobic bacteria detection of test articles

(1) Weighing 1.0g of recombinant humanized collagen (generated by fermentation of Pichia pastoris with the preservation number of CGMCC No. 5021), respectively adding into a triangular flask filled with glass beads, 0.9% sodium chloride aqueous solution, pH7.0 sterile sodium chloride-peptone buffer solution, pH7.2 phosphate buffer solution and purified water, fully oscillating, mixing uniformly, and standing for 15 min. Taking the supernatant as a 1:10 bacteria detection solution, and sucking 1: the 10-diluted 2mL of the bacterial sample solution was poured into two sterile plates, each containing 1mL of the bacterial sample solution. Another 1mL of the suspension was added to 9mL of a 0.9% aqueous sodium chloride solution, pH7.0 sterile sodium chloride-peptone buffer, pH7.2 phosphate buffer solution, and purified water in a test tube (note that the pipette was not brought into contact with the liquid surface), and the mixture was thoroughly mixed with one pipette to prepare a 1:100 cell test solution. 2mL of the solution was aspirated and injected into two sterile plates, 1mL each. If the bacteria content of the sample is high, the sample can be further diluted into 1:1000, 1:10000 and the like, and 1 straw should be replaced for each dilution. Pouring the melted and cooled tryptone soy peptone agar medium (45-50 ℃) into a plate, wherein each plate is about 15mL, rotating the plate immediately to fully and uniformly mix the sample and the medium, turning the plate after the agar is solidified, and culturing in an incubator at 36 +/-1 ℃ for 3-5 days. And taking 4 sterilized empty plates without samples, respectively adding 1mL of 0.9% sodium chloride aqueous solution, pH7.0 sterile sodium chloride-peptone buffer solution, pH7.2 phosphate buffer solution and purified water, adding about 15mL of tryptone soybean peptone agar medium, turning the plates after agar is solidified, culturing in an incubator at 36 +/-1 ℃ for 3-5 days, and performing negative control test, wherein the negative control test needs aseptic growth, and if bacterial growth needs deviation investigation.

(2) Results of determination of aerobic bacteria:

TABLE 20.9% sodium chloride solution as diluent set

TABLE 3 pH7.0 sterile NaCl-peptone buffer as diluent group

TABLE 4 pH7.2 phosphate buffer solution as diluent set

TABLE 5 sterile distilled water as diluent group

From the above results, it can be seen that the recombinant humanized collagen produced by fermentation of Pichia pastoris with accession number CGMCC No.5021 uses 0.9% sodium chloride aqueous solution as diluent, and the size and morphological characteristics of aerobic bacterial colony show that the growth of bacteria is rare and slow, which does not meet the actual situation of microbial growth in the recombinant humanized collagen produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021; the bacteria grow well by using the pH7.0 sterile sodium chloride-peptone buffer solution as a diluent; phosphate buffer solution with the pH value of 7.2 is used as diluent, corresponding nutrition is absorbed by microorganisms, and the bacteria grow well; the effect is not ideal when the purified water is used as the diluent, the microorganism grows weakly and slowly, and the actual situation of the microorganism growth in the recombinant humanized collagen generated by the fermentation of the Pichia pastoris with the preservation number of CGMCC No.5021 is not met.

In summary, when the microbial limit of the recombinant humanized collagen produced by fermentation of Pichia pastoris with the preservation number of CGMCC No.5021 is checked, the pH7.0 sterile sodium chloride-peptone buffer solution and the pH7.2 phosphate buffer solution are both suitable as diluent, so that the microbial growth condition in the recombinant humanized collagen can be reflected really.