阅读说明：本技术 一种原生药粉的微生物检测方法及其灭菌方法 (Microorganism detection method and sterilization method for crude drug powder ) 是由 周映佑 滕钰 黄婕 薛咏兰 徐洪 罗曼 王俊 严利娟 孙宜春 李慧馨 于 2020-05-06 设计创作，主要内容包括：本发明提供了一种原生药粉的微生物检测方法,属于中医药领域,具体方法为将供试品直接接种至培养基中培养；取上述培养液至RV沙门增菌液体培养基中再次培养；将该培养物接种于木糖赖氨酸脱氧胆酸盐琼脂培养基培养得疑似菌落。用接种针挑选疑似菌落于三糖铁琼脂培养基高层斜面上进行斜面和高层穿刺接种培养,从而进行根据结果判断微生物的情况,本发明方法简单易行,解决了原生药粉微生物检测的问题。(The invention provides a method for detecting microorganisms of primary medicinal powder, which belongs to the field of traditional Chinese medicine, and specifically comprises the steps of directly inoculating a test sample into a culture medium for culture; taking the culture solution to an RV salmonella enrichment liquid culture medium for secondary culture; the culture is inoculated to a xylose lysine deoxycholate agar culture medium to be cultured to obtain a suspected colony. The suspected bacterial colony is selected by the inoculating needle to be carried out slant and high-level puncture inoculation culture on the high-level slant of the trisaccharide iron agar culture medium, so as to judge the condition of the microorganism according to the result.)

1. A method for detecting microorganisms in crude drug powder is characterized by comprising the following steps

1) Directly inoculating 10g of the test sample into 200ml of trypticase soy peptone liquid culture medium, mixing uniformly, and culturing at 30-35 ℃ for 18-24 hours;

2) inoculating 0.1ml of the culture solution obtained in the step 1) into 10ml of RV salmonella enrichment liquid culture medium, and culturing at 30-35 ℃ for 18-24 hours;

3) taking a small amount of RV salmonella enrichment liquid culture by using an inoculating loop, streaking and inoculating the RV salmonella enrichment liquid culture on a xylose lysine deoxycholate agar culture medium plate, culturing for 18-48 hours at the temperature of 30-35 ℃, wherein salmonella grows well on the xylose lysine deoxycholate agar culture medium plate, and the colony form is light red or colorless, transparent or semitransparent, and black or no black exists in the center;

4) selecting the suspected bacterial colony in the step 3) by using an inoculating needle, performing slant and high-layer puncture inoculation on a high-layer slant of a trisaccharide iron agar culture medium, culturing for 18-24 hours, and judging the result: if bacteria grow on the xylose lysine deoxycholate culture medium and suspected bacterial colonies grow, and the inclined plane on the trisaccharide iron agar culture medium is red, the lower layer is yellow, or the inclined plane is yellow, and the bottom layer is yellow or black, a proper identification test is further carried out to confirm whether the bacteria are salmonella; if the plate grows aseptically, or the identification result is negative although colonies grow, or the inclined plane of the trisaccharide iron agar culture medium does not see red and the bottom layer does not see yellow; or the slant is yellow, and the bottom layer is not yellow or black, so that the test sample is judged to have no salmonella.

2. A method for sterilizing bacterial colonies detected by the method for detecting microorganisms in a crude drug powder according to claim 1, comprising the steps of: the sterilization temperature is 70 ℃, the sterilization time is 18 hours or the sterilization temperature is 75 ℃, and the sterilization time is 12 hours; or the sterilization temperature is 80 ℃ and the sterilization time is 8 hours.

Technical Field

The invention belongs to the field of traditional Chinese medicine, and particularly relates to a crude drug powder microorganism detection method, a sterilization method and a sterilization method.

Background

The causative agent of salmonellosis, belongs to the family Enterobacteriaceae, gram-negative Enterobacteriaceae. Nearly one thousand species (or strains) have been discovered. According to the antigen components, it can be divided into basic bacterial groups of A, B, C, D and E, etc. The main diseases related to human body include A. paratyphi of group A, B. paratyphi and Salmonella typhimurium of group B, C. paratyphi and hog cholera bacillus of group C, and T.typhi and enteritis bacillus of group D. Except for human diseases caused by typhoid bacillus, paratyphoid bacillus and paratyphoid bacillus, most of the typhoid bacillus, paratyphoid bacillus and paratyphoid bacillus only can cause diseases of animals such as livestock, mice, poultry and the like, but sometimes can also pollute food of human to cause food poisoning.

The microbiological examination refers to the examination of the types and amounts of microorganisms contained in the raw materials of medicinal materials, and indicates the degree of microbial contamination of the medicinal materials. The primary medicinal powder is not processed and carries pathogenic microorganisms because of the direct administration form, so the microbial detection of the primary medicinal powder is particularly important.

Disclosure of Invention

The invention provides a microorganism detection method and a sterilization method of crude drug powder, which solve the problem of microorganism pollution in the crude drug powder.

The technical scheme provided by the invention comprises the following steps:

1) directly inoculating 10g of the test sample into 200ml of trypticase soy peptone liquid culture medium, mixing uniformly, and culturing at 30-35 ℃ for 18-24 hours;

2) inoculating 0.1ml of the culture solution obtained in the step 1) into 10ml of an RV salmonella enrichment liquid culture medium, and culturing for 18-24 hours at the temperature of 30-35 ℃;

4) taking a small amount of RV salmonella enrichment liquid culture by using an inoculating loop, streaking and inoculating the RV salmonella enrichment liquid culture on a xylose lysine deoxycholate agar culture medium plate, culturing for 18-48 hours, wherein the salmonella grows well on the xylose lysine deoxycholate agar culture medium plate, and the colony form is light red or colorless, transparent or semitransparent, and black or no black at the center;

5) selecting suspected bacterial colonies by using an inoculating needle, carrying out slant and high-level puncture inoculation on a trisaccharide iron agar culture medium high-level slant, culturing for 18-24 hours, and judging the result: if bacteria grow on the xylose lysine deoxycholate culture medium and suspected bacterial colonies grow, and the inclined plane on the trisaccharide iron agar culture medium is red, the lower layer is yellow, or the inclined plane is yellow, and the bottom layer is yellow or black, a proper identification test is further carried out to confirm whether the bacteria are salmonella; if the plate grows aseptically, or the identification result is negative although colonies grow, or the inclined plane of the trisaccharide iron agar culture medium does not see red and the bottom layer does not see yellow; or the slant is yellow, and the bottom layer is not yellow or black, and the test sample is judged to have no salmonella;

the bacterial colony sterilization method detected by the microbial detection method of the primary medicinal powder comprises the following steps: the sterilization temperature is 70 ℃, the sterilization time is 18 hours or the sterilization temperature is 75 ℃, and the sterilization time is 12 hours; or the sterilization temperature is 80 ℃ and the sterilization time is 8 hours.

Example 1, 10g of the sample was directly inoculated into 200ml of trypticase soy peptone broth, mixed and incubated at 30-35 ℃ for 18-24 hours; inoculating 0.1ml of the culture solution into 10ml of RV salmonella enrichment liquid culture medium, and culturing at 30-35 ℃ for 18-24 hours; taking a small amount of RV salmonella enrichment liquid culture by using an inoculating loop, streaking and inoculating on a xylose lysine deoxycholate agar culture medium plate, and culturing for 18-48 hours. The xylose lysine deoxycholate agar culture medium plate grows bacteria, the culture medium is changed from red to yellow, the colony morphology is round and yellow, the environmental test result is gram positive bacillus, no spore and no flagellum, suspected colonies are selected by an inoculating needle to be inoculated on a trisaccharide iron agar culture medium high-level inclined plane for inclined plane and high-level puncture, the culture is carried out for 18-24 hours, and the result judgment is carried out: the slope of the test sample on the trisaccharide iron agar culture medium grows in a sterile manner, and the salmonella of the test sample is judged not to be detected.

Example 2: in order to verify the colony detection method of the crude drug powder, the tested long bacteria sample is subjected to a high-temperature sterilization test, and the results are as follows:

temperature and time for sterilization and whether or not there is any bacterial growth remark after sterilization

If the content is qualified in appearance after 18 hours at 70 DEG C

The content is not qualified in appearance after 12 hours at 75 DEG C

The content is not qualified after 8 hours at 80 DEG C

And (4) verification result: the bacterial colony detected by the primary medicine powder can be effectively eliminated by using the high temperature of 70 ℃ for 18 hours, 75 ℃ for 12 hours and 80 ℃ for 8 hours, namely: the xylose lysine deoxycholate agar culture medium plate has bacteria growing thereon, the culture medium is changed from red to yellow, the colony morphology is round and yellow, the environmental test result is gram-positive bacillus, no spores and flagella, and the content and appearance of the medicine are not influenced.