Preparation method of quality control sample for determining total number of bacterial colonies
Through the measured quality control samples prepared by the total number of colonies prepared through steps of bacterial fermentation, vacuum frozen and drying, the problem of high quality control market prices is solved, and the quality control sample preparations with low cost and reliable stability are significantly reduced, which significantly reduces the economy. burden.
Patent Information
- Application Number
- CN202411403722.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-09
- Publication Date
- 2025-01-24
AI Technical Summary
The market price of existing colony total measurement and quality control samples is relatively high, which brings economic pressure to food manufacturers and regulatory testing institutions.
Quality control samples for total bacterial colony determination are prepared using the steps of mixed bacterial fermentation, vacuum freeze-drying, mixing and packaging, and quality control sample preparation. The concentration is determined first and then packaged, which reduces production costs.
The quality control samples prepared by this method are low in cost, reliable in stability, and have a long shelf life, which significantly reduces the economic burden on food production enterprises and regulatory testing agencies.
Smart Images
Figure CN119351515A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of food inspection and detection, and in particular to a method for preparing a quality control sample for total colony count determination. Background Art
[0002] Food hygiene and safety are related to the health and life safety of the general public, and the total colony count is the most important indicator for evaluating food hygiene and safety. The total colony count determination is a must-check item for all food production companies and food testing agencies. In order to accurately determine the total colony count, it is inseparable from the total colony count determination quality control sample. However, the market price of the quality control samples used for the total colony count determination is relatively expensive, which brings huge economic pressure to food production and processing companies and regulatory testing agencies. In order to solve this common problem for food production companies and regulatory testing agencies, our team has developed a low-cost total colony count determination quality control sample. Summary of the invention
[0003] In order to develop a quality control sample for total colony count determination which has low cost, reliable stability and a long shelf life, the present invention provides a method for preparing a quality control sample for total colony count determination.
[0004] The present invention is implemented by the following technical scheme: A method for preparing a quality control sample for total colony count determination comprises the following steps: (1) mixed bacterial fermentation: the quality control mixed strain is streaked and inoculated on a TSA plate for constant temperature culture to obtain a mixed bacterial moss, and then the mixed bacterial moss is inoculated into a liquid culture medium and filtered through a filter membrane to obtain a mixed bacterial fermentation filtrate; (2) vacuum freeze drying: the mixed bacterial fermentation filtrate is placed in a vacuum freeze dryer and subjected to vacuum freeze drying to obtain a freeze-dried powder; (3) mixing and packaging: repeating steps (1) and (2) multiple times to obtain freeze-dried powder, and the freeze-dried powder obtained multiple times is mixed, and then ground with a freeze grinder to obtain a quality control sample raw powder; (4) quality control sample preparation: according to the required quality control sample concentration, the freeze-dried powder raw powder corresponding to the required quality control sample concentration is weighed into a vial, and aseptically packaged to prepare a quality control sample of a certain concentration of total colony count.
[0005] In one embodiment of the application, the quality control mixed strain includes Bacillus subtilis and Bacillus licheniformis.
[0006] In one embodiment of the application, the step (1) includes: streaking the quality control mixed strain onto a TSA plate, culturing at 36° C. for 24 h, scraping the bacterial moss from the TSA plate with a sterilized inoculation loop and inoculating it into 200 mL of liquid culture medium, then adding 5% trehalose-5% sucrose-5% skim milk powder in a 1:1 ratio, mixing, filtering through a 5 um filter membrane, and filtering the filtrate with a 0.45 um filter membrane to obtain the mixed bacteria fermentation filtrate.
[0007] In one application embodiment, the step (2) includes: placing the mixed bacteria fermentation filtrate on a tray, pre-freezing it in a refrigerator at -18°C, and then placing it in the vacuum freeze dryer, setting the cold trap temperature to below -55°C and the vacuum degree to below 15Pa, and obtaining the freeze-dried powder after vacuum freeze drying for about 24 hours.
[0008] Compared with the existing method for preparing quality control samples for total colony count determination, the present invention has the following beneficial effects:
[0009] The present invention abandons the process of first packaging and then determining the concentration, and adopts the process of first determining the concentration and then packaging, which greatly reduces the cost. The main reason why the cost can be greatly reduced after the process change is that the commercially available quality control samples are first packaged and then the content of each batch is determined. When determining the content, 30-50 testing institutions are required to conduct testing, and the median is taken to obtain the bacterial content; while the quality control sample in the present application is to measure the content of the freeze-dried powder after mixing multiple batches of freeze-dried powder. It is only necessary to test the bacterial count of the original freeze-dried powder, and there is no need to test each batch of quality control samples, thereby greatly reducing the cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 This is a flow chart of a method for preparing a quality control sample for total colony count determination according to an embodiment of an application of the present invention. DETAILED DESCRIPTION
[0011] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0012] See also Figure 1 In the embodiment of the present application, a method for preparing a quality control sample for total colony count determination is proposed, and the preparation method comprises the following steps:
[0013] (1) Mixed bacterial fermentation: The quality control mixed bacterial strains were streaked and inoculated on TSA plates for constant temperature culture to obtain mixed bacterial mosses, and then the mixed bacterial mosses were inoculated into liquid culture medium and filtered through a filter membrane to obtain mixed bacterial fermentation filtrate;
[0014] (2) vacuum freeze drying: placing the mixed bacteria fermentation filtrate in a vacuum freeze dryer and subjecting it to vacuum freeze drying to obtain a freeze-dried powder;
[0015] (3) Mixing and packaging: Repeat steps (1) and (2) multiple times to obtain freeze-dried powder, mix the freeze-dried powder obtained multiple times, and then grind it with a cryo-grinder to obtain the quality control sample powder;
[0016] (4) Preparation of quality control samples: According to the required quality control sample concentration, weigh the lyophilized powder of the corresponding weight into a vial, and aseptically package it into a quality control sample of a certain concentration of total colony count.
[0017] In the embodiment of the present application, the process of first packaging and then determining the concentration is abandoned, and the process of first determining the concentration and then packaging is adopted, which greatly reduces the cost. The main reason for the significant cost reduction after the process change is that the commercially available quality control samples are first packaged and then the content of each batch is determined. When determining the content, 30-50 testing institutions are required to conduct testing, and the median is taken to obtain the bacterial content; while the quality control sample in the present application is to measure the content of the freeze-dried powder after mixing multiple batches of freeze-dried powder. It is only necessary to test the bacterial count of the original freeze-dried powder, and there is no need to test each batch of quality control samples, thereby greatly reducing the cost.
[0018] In one embodiment of the application, the quality control mixed strain includes Bacillus subtilis and Bacillus licheniformis.
[0019] The present invention uses Bacillus subtilis and Bacillus licheniformis for mixed fermentation. On the one hand, the unique spore structure of spore-forming bacteria is utilized, which has strong resistance to high turbulence, ultraviolet rays, drying, radiation and other toxic chemicals, and the prepared quality control sample has good transportation stability and storage stability. On the other hand, Bacillus subtilis and Bacillus licheniformis can work synergistically in the fermentation process, give play to their respective advantages, and improve the overall benefits of the fermentation product.
[0020] In one embodiment of the application, the step (1) comprises: streaking the quality control mixed strain onto a TSA plate, culturing at 36°C for 24 hours, scraping the bacterial moss from the TSA plate with a sterilized inoculation loop and inoculating it into 200 mL of liquid culture medium, adding 5% trehalose-5% sucrose-5% skim milk powder in a ratio of 1:1, mixing, filtering through a 5um filter membrane, and filtering the filtrate with a 0.45um filter membrane to obtain the mixed bacteria fermentation filtrate; the step (2) comprises: placing the mixed bacteria fermentation filtrate on a tray, pre-freezing it in a refrigerator at -18°C, and then placing it in the vacuum freeze dryer, setting the cold trap temperature to be lower than -55°C and the vacuum degree to be lower than 15Pa, and vacuum freeze-drying for about 24 hours to obtain the lyophilized powder.
[0021] In the embodiment of the present application, the concentrated bacterial liquid is obtained by two filtrations, which reduces the cost of vacuum freeze drying. The bacterial cell size range of Bacillus subtilis and Bacillus licheniformis is 1-5um, and they are filtered with a 5um filter membrane and a 0.45um filter membrane respectively. The 5um filter membrane is used to remove large impurities, and then the 0.45um filter membrane is used to enrich the bacterial cells. In this way, the moisture content entering the freeze dryer is greatly reduced, reducing the cost of vacuum freeze drying.
[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing a quality control sample for total colony count determination, characterized in that: The method for preparing a quality control sample for total colony count determination comprises the following steps: (1) Mixed bacterial fermentation: The quality control mixed bacterial strains were streaked and inoculated on TSA plates for constant temperature culture to obtain mixed bacterial mosses, and then the mixed bacterial mosses were inoculated into liquid culture medium and filtered through a filter membrane to obtain mixed bacterial fermentation filtrate; (2) vacuum freeze drying: placing the mixed bacteria fermentation filtrate in a vacuum freeze dryer and subjecting it to vacuum freeze drying to obtain a freeze-dried powder; (3) Mixing and packaging: Repeat steps (1) and (2) multiple times to obtain freeze-dried powder, mix the freeze-dried powder obtained multiple times, and then grind it with a cryo-grinder to obtain the quality control sample powder; (4) Preparation of quality control samples: According to the required quality control sample concentration, weigh the lyophilized powder of the corresponding weight into a vial, and aseptically package it into a quality control sample of a certain concentration of total colony count.
2. The method for preparing a quality control sample for total colony count determination according to claim 1, characterized in that: The quality control mixed strain includes Bacillus subtilis and Bacillus licheniformis.
3. The method for preparing a quality control sample for total colony count determination according to claim 1, characterized in that: The step (1) comprises: streaking the quality control mixed strain onto a TSA plate, culturing at 36° C. for 24 h, scraping the bacterial moss from the TSA plate with a sterilized inoculation loop and inoculating it into 200 mL of liquid culture medium, then adding 5% trehalose-5% sucrose-5% skim milk powder in a ratio of 1:1, mixing, filtering through a 5 μm filter membrane, and filtering the filtrate through a 0.45 μm filter membrane to obtain the mixed bacteria fermentation filtrate.
4. The method for preparing a quality control sample for total colony count determination according to claim 1, characterized in that: The step (2) comprises: placing the mixed bacteria fermentation filtrate on a tray, pre-freezing it in a refrigerator at -18°C, and then placing it in the vacuum freeze dryer, setting the cold trap temperature to be lower than -55°C and the vacuum degree to be lower than 15 Pa, and performing vacuum freeze drying for about 24 hours to obtain the freeze-dried powder.