CN111676179A

CN111676179A - Parahemolytic vibrio culture medium, culture method and application

Info

Publication number: CN111676179A
Application number: CN202010732442.0A
Authority: CN
Inventors: 胡智恺; 索一平; 宋丽萍; 王丹; 蔡雪凤; 任岩; 刘凯; 张跃川; 巩有博; 毕思丹
Original assignee: Beijing Food Safety Monitoring And Risk Assessment Center (beijing Food Inspection Institute)
Current assignee: Beijing Food Safety Monitoring And Risk Assessment Center (beijing Food Inspection Institute)
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2020-09-18

Abstract

The invention relates to the technical field of food pathogen detection, and particularly discloses a parahemolytic vibrio culture medium, a culture method and application. The nitrogen source of the parahemolytic vibrio culture medium comprises bovine heart soaking powder, yeast soaking powder and soybean peptone in sequence from large to small according to the dosage. The specific conditions for culturing by using the culture medium are as follows: 35-37 ℃, pH 7.5-8.0, 160-. The culture method can promote the repair of microorganisms to the damaged area without adding other nutritional components except a nitrogen source, ensure the permeability of cell membranes, realize the quick and effective proliferation of vibrio parahaemolyticus, and provide powerful technical support for really realizing the quick detection of food-borne pathogenic bacteria.

Description

Parahemolytic vibrio culture medium, culture method and application

Technical Field

The invention relates to the technical field of food pathogen detection, and particularly relates to a parahemolytic vibrio culture medium, a culture method and application.

Background

With the increasing concern on food safety, higher requirements are provided for the speed and accuracy of food-borne pathogenic bacteria detection. The downstream detection speed of the food-borne pathogenic bacteria is greatly improved due to the occurrence of the PCR technology. At present, the pre-enrichment process of upstream detection of food-borne pathogenic bacteria becomes the bottleneck of really realizing rapid detection of the food-borne pathogenic bacteria. Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a gram-negative Vibrio halophilus, widely distributed in estuaries and marine environments, is the main cause of bacterial gastroenteritis caused by seafood, and is often related to edible raw or uncooked seafood. As the vibrio parahaemolyticus shows the trend of increasing infection, the demand for rapid detection of the vibrio parahaemolyticus is increasing.

The existing vibrio parahaemolyticus detection needs 8-18 hours in the bacteria increasing stage before the first step, and the rapid detection speed of food-borne pathogenic bacteria is seriously hindered. Further, Vibrio parahaemolyticus is halophilic basophilic spore-free Vibrio. Among them, salinity is critical to the survival of Vibrio parahaemolyticus. Vibrio parahaemolyticus is not easily viable or weakly growing in a sodium chloride-free environment. And because the food system has complex components, the factors interfering the growth of the vibrio parahaemolyticus are more, and the vibrio parahaemolyticus thalli are damaged and even died in the transportation and storage processes, so that the detection rate of the vibrio parahaemolyticus is low. The pre-enrichment is to recover the damaged thallus, so that the detection accuracy is improved, but the timeliness of the detection is also influenced if the pre-enrichment is unreasonable. Therefore, the development of an effective pre-enrichment scheme is the key for improving the timeliness and the accuracy of the vibrio parahaemolyticus test.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a culture medium capable of effectively improving the proliferation efficiency of vibrio parahaemolyticus.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a parahemolytic vibrio culture medium comprises bovine heart infusion powder, yeast infusion powder and soybean peptone in sequence from large to small according to dosage.

According to a great deal of research, the invention discovers that three specific components, namely the bovine heart infusion powder, the yeast infusion powder and the soybean peptone, are used as nitrogen sources for culturing the vibrio parahaemolyticus in sequence from large to small according to the dosage, and other nutritional components are not required to be added, so that the repair of microorganisms to a damaged area can be promoted, the permeability of cell membranes is ensured, and the rapid and effective proliferation of the vibrio parahaemolyticus is realized. And the effect is not ideal when other nitrogen source combination modes are used.

In the invention, the mass ratio of the bovine heart extract powder, the yeast extract powder and the soybean peptone is (1.8-4.5): (1.1-2.4): 1, preferably (2.6-3): (1.1-1.5): 1, more preferably 2.78: 1.32: 1.

further, when bovine heart meal, yeast meal and soy peptone are used in combination in the specific ratio of the present invention, a better culture effect can be exerted. The method not only can ensure that the target bacteria are rapidly propagated within the time specified by the existing standard method to reach the detection limit, but also can cover different types of samples, thereby avoiding the result of false negative. And the raw materials of the scheme are convenient and easy to obtain, and no toxic or harmful substances are generated. The pre-enrichment liquid prepared according to the proportion can quickly restore the damaged vibrio parahaemolyticus, and is the optimal proportion for restoring the vibrio parahaemolyticus. And the added bovine heart extract powder and yeast extract powder can form better synergistic effect with the nutrient components of the soybean peptone. Especially, the effect is better under the preferable proportion. If the proportion deviates from the proportion of the invention, the repair effect of the vibrio parahaemolyticus is influenced, and the ineffective use of raw material resources is caused. In addition, the scheme of the invention also provides technical support for explaining the requirement of the nutritional characteristics of the vibrio parahaemolyticus and provides theoretical reference for controlling the vibrio parahaemolyticus.

In the invention, the concentration of the bovine heart infusion powder is (18.4-22.8) g/L, preferably 20.6 g/L;

and/or the concentration of the yeast extract powder is (7.6-12.0) g/L, preferably 9.8 g/L;

and/or the concentration of the soybean peptone is (5.1-10.1) g/L, preferably 7.4 g/L.

The concentration of each component in the culture medium is proper, and the ideal osmotic pressure can be maintained, so that the culture effect is improved, the cost is saved, and the waste of raw materials is avoided.

In the present invention, the medium further comprises 28-32g/L NaCl, preferably 30 g/L.

As a preferred embodiment of the present invention, each 1L of the medium comprises: 20.6g of the bovine heart meal, 9.8g of the yeast meal, 7.4g of the soy peptone and 30g of the NaCl, the balance being water.

The invention also provides a method for culturing the vibrio parahaemolyticus, which uses the culture medium to culture the vibrio parahaemolyticus.

In the present invention, the specific conditions of the culture are: 35-37 ℃, pH 7.5-8.0, 160-. Preferably, the culture is carried out aerobically at 180rpm at 36 ℃.

The invention researches the culture condition of vibrio parahaemolyticus in the culture medium of the invention, and obtains the culture condition which can fully exert the function of the culture medium of the invention and can quickly and effectively realize the enrichment effect.

The culture temperature of the invention is beneficial to the metabolic growth of the vibrio parahaemolyticus. In addition, the invention finds that the aerobic environment is more suitable for the propagation of the vibrio parahaemolyticus, the change of the content of dissolved oxygen in the culture solution can be caused by adopting the shaking table culture, the distribution of the nutrient components in the culture medium is uniform, and the propagation speed of the vibrio parahaemolyticus can be obviously improved. The invention also finds that the culture is carried out at the pH value of 7.5-8.0, which can avoid the influence on the extracellular protease of the vibrio parahaemolyticus and is more beneficial to the formation of the biofilm. By combining the synergistic consideration of the factors, the culture method can be used for acting with the culture medium to realize the rapid proliferation of the vibrio parahaemolyticus.

The invention also provides application of the culture medium or the method in a bacterium increasing process before the vibrio parahaemolyticus test.

In the present invention, the culture of Vibrio parahaemolyticus using the medium is carried out for 6 to 6.5 hours, preferably 6 hours.

According to the invention, after the vibrio parahaemolyticus is cultured for 6 hours, downstream detection can be carried out by using a PCR technology, so that the detection time of the vibrio parahaemolyticus can be greatly shortened.

The invention has the beneficial effects that:

according to the invention, vibrio parahaemolyticus is taken as a research object, and culture conditions and culture medium formulas in the pre-enrichment process are optimized, so that other nutrient components except a nitrogen source are not required to be added, the proliferation efficiency of vibrio parahaemolyticus is greatly improved, the culture time is shortened, and powerful technical support is provided for really realizing the rapid detection of food-borne pathogenic bacteria.

Drawings

FIG. 1 shows the PCR results of the shrimp paste labeling test;

FIG. 2 shows the PCR result of the squid silk labeling test;

FIG. 3 shows PCR results of oyster sauce spiking test;

in each figure, a is an amplification curve after the culture in the medium and culture method of example 1, b is an amplification curve after the culture in the APW medium and culture method of example 1, and c is an amplification curve after the culture in the culture method of example 1 except that the shaking culture is replaced by the static culture in the APW medium.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Some materials and instruments used in the examples of the present invention are as follows:

vibrio parahaemolyticus ATCC 17802; peptone, 3% NaCl Tryptone Soy Agar (TSA), and tryptone broth (TSB) containing 3% NaCl were purchased from Beijing Luqiao, Inc.; NaCl (analytically pure) was purchased from chinese medicine; soy peptone, yeast extract powder were purchased from BD ltd, usa; bovine heart infusion powder was purchased from Solarbio ltd.

Analytical balance, precision pH meter purchased from mettler-toledo instruments ltd; UV-visible spectrophotometers, biosafety cabinets from ThermoFisher, Inc.; biochemical incubators were purchased from Memmert, Inc.; the gas bath constant temperature oscillator was purchased from suzhou peying experimental facilities ltd; the McLeod and McLeod turbidimeters were purchased from Bio-Metrei.

Example 1

This example provides a culture medium for Vibrio parahaemolyticus according to the present invention and culturing Vibrio parahaemolyticus.

Specifically, each liter of the parahemolytic vibrio culture medium comprises: 20.6g of bovine heart extract powder, 9.8g of yeast extract powder, 7.4g of soybean peptone and 30g of NaCl, and the balance of water. The pH was 8.0.

The specific culture method comprises the following steps:

1. preparation of seed liquid

Inoculating Vibrio parahaemolyticus ATCC 17802 to a TSA culture medium containing 3% NaCl, culturing at 36 ℃ for 24h, picking out a single colony by using an inoculating loop, inoculating to 75mL of TSB culture medium (250mL triangular flask) containing 3% NaCl, and culturing at 36 ℃ for 18h to obtain a seed solution.

2. Inoculation of

1mL of seed solution was put in a Mach's turbidimetric tube, a TSB medium containing 3% NaCl was added thereto by aseptic technique until the Mach's turbidity became 0.5, 100. mu.L of the bacterial suspension was aspirated respectively and added to a 250mL triangular flask containing 75mL of the above-mentioned medium, and the flask was sealed with a gas-permeable sealing film.

3. The cells were incubated at 36 ℃ for 6 hours on a shaker at 180 rpm.

Example 2

Specifically, each liter of the parahemolytic vibrio culture medium comprises: 18.4g of bovine heart extract powder, 10.5g of yeast extract powder, 7g of soybean peptone and 32g of NaCl, and the balance of water. The pH was 7.5.

The specific cultivation method was the same as in example 1 in steps 1-2 except that in step 3, the cultivation was carried out at 35 ℃ for 6.5 hours on a shaker at 160 rpm.

Example 3

Specifically, each liter of the parahemolytic vibrio culture medium comprises: 22.8g of bovine heart extract powder, 8.4g of yeast extract powder, 7.6g of soybean peptone and 28g of NaCl, the balance being water. The pH was 7.8.

The specific cultivation method was the same as in example 1 in steps 1-2 except that in step 3, the cultivation was carried out at 37 ℃ for 6 hours on a shaker at 200 rpm.

Example 4

Specifically, each liter of the parahemolytic vibrio culture medium comprises: 20.2g of bovine heart extract powder, 12g of yeast extract powder, 10.1g of soybean peptone and 28g of NaCl, the balance being water. The pH was 7.8.

The specific culture method was the same as in example 1.

Example 5

Specifically, each liter of the parahemolytic vibrio culture medium comprises: 20.2g of bovine heart extract powder, 7.6g of yeast extract powder, 5.1g of soybean peptone and 28g of NaCl, the balance being water. The pH was 7.8.

The specific culture method was the same as in example 1.

Comparative examples 1 to 5

This comparative example was carried out by culturing Vibrio parahaemolyticus according to the culture method of example 1, except that in the specific formulation of the medium, peptone, acid hydrolyzed casein, casein peptone, fish peptone, No. 3 month peptone were used instead of soybean peptone, respectively.

Comparative examples 6 to 7

The comparison example carries out the culture of the vibrio parahaemolyticus according to the culture method of the example 1, and the difference is that in the specific formula of the culture medium, beef extract powder and bovine brain extract powder are respectively used for replacing bovine heart extract powder.

Comparative example 8

This comparative example was carried out for the culture of Vibrio parahaemolyticus according to the culture method of example 1, except that in the specific formulation of the medium, 20.6g of bovine heart extract powder, 8g of yeast extract powder and 10g of soybean peptone were used.

Comparative example 9

This comparative example was carried out for the culture of Vibrio parahaemolyticus according to the culture method of example 1, except that in the specific formulation of the medium, the amount of bovine heart extract was 22.2g, the amount of yeast extract was 18.5g, and the amount of soybean peptone was 7.4 g.

Comparative example 10

This comparative example was carried out for the culture of Vibrio parahaemolyticus according to the culture method of example 1, except that in the specific formulation of the medium, the amount of bovine heart extract was 15g, the amount of yeast extract was 18g, and the amount of soybean peptone was 9 g.

Comparative example 11

This comparative example was conducted in accordance with the culture method of example 1 except that the pH of the medium was 8.5.

Comparative example 12

This comparative example was carried out by culturing Vibrio parahaemolyticus according to the culture method of example 1, except that in the 3 rd step of the culture method, the culture conditions were as follows: the cells were cultured at 36 ℃ for 6 hours.

Comparative example 13

This comparative example was conducted in accordance with the culture method of example 1 except that in the 2 nd step of the culture method, the air-impermeable plastic sealing film was used in place of the air-permeable sealing film for sealing.

Experimental example 1

This example examined the cell concentration of Vibrio parahaemolyticus cultured by the methods of examples 1 to 5 and comparative examples 1 to 13.

The method for measuring the thallus concentration comprises the following steps:

taking out the cultured culture solution, shaking thoroughly, adding 200 μ L into the enzyme labeling plate, and measuring OD value at wavelength of 570nm with the uncultured culture medium as blank control. Each flask of culture medium was set in 3 replicates. The test results are shown in Table 1.

TABLE 1

Test object	OD_570nmValue of	Test object	OD_570nmValue of
				Example 1	0.676±0.01	Comparative example 5	0.610±0.03
Example 2	0.673±0.02	Comparative example 6	0.655±0.01
				Example 3	0.671±0.02	Comparative example 7	0.525±0.05
Example 4	0.666±0.01	Comparative example 8	0.659±0.02
				Example 5	0.665±0.02	Comparative example 9	0.650±0.03
Comparative example 1	0.625±0.02	Comparative example 10	0.635±0.03
				Comparative example 2	0.573±0.01	Comparative example 11	0.594±0.01
Comparative example 3	0.607±0.01	Comparative example 12	0.215±0.02
				Comparative example 4	0.599±0.01	Comparative example 13	0.391±0.05

Experimental example 2

In the experimental example, 3 different samples are selected according to the food category of GB 29921-2013 food safety national standard food pathogenic bacteria limit, namely prawn paste, squid shreds and oyster sauce, 1000CFU/mL vibrio parahaemolyticus is artificially added, and the sample is flapped for 2min by a flapped homogenizer to prepare a labeled sample. 25g of each sample was added to 225mL of the Vibrio parahaemolyticus enrichment medium of example 1, and cultured by the culture method of example 1. While APW was used as a control medium (peptone 10g, NaCl30g, water 1000mL), and after Vibrio parahaemolyticus was added, a part of the cells were cultured in the same manner as in example 1, and the other part was cultured in the same manner as in example 1 except that static culture was used instead of shake culture. After the culture was completed, PCR was performed. The test results are shown in fig. 1-3, fig. 1 is the PCR result of the shrimp sauce labeling test, fig. 2 is the PCR result of the squid silk labeling test, and fig. 3 is the PCR result of the oyster sauce labeling test. In each figure, a is an amplification curve after the culture in the medium and culture method of example 1, b is an amplification curve after the culture in the APW medium and culture method of example 1, and c is an amplification curve after the culture in the culture method of example 1 except that the shaking culture is replaced by the static culture in the APW medium.

As can be seen from the figure, the culture medium of the embodiment 1 of the invention can remarkably promote the growth and the propagation of vibrio parahaemolyticus in each sample within 6h, reach the detection limit of a fluorescent quantitative PCR method, reduce the omission factor of positive samples and integrally improve the detection efficiency.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The parahemolytic vibrio culture medium is characterized in that a nitrogen source of the culture medium sequentially comprises bovine heart soaking powder, yeast soaking powder and soybean peptone from large to small according to the dosage.

2. The culture medium according to claim 1, wherein the mass ratio of the bovine heart extract powder, the yeast extract powder and the soybean peptone is (1.8-4.5): (1.1-2.4): 1, preferably (2.6-3): (1.1-1.5): 1.

3. the culture medium according to claim 1 or 2, wherein the concentration of bovine heart meal is (18.4-22.8) g/L;

and/or the concentration of the yeast extract powder is (7.6-12.0) g/L;

and/or the concentration of the soybean peptone is (5.1-10.1) g/L.

4. The culture medium according to any one of claims 1 to 3, further comprising 28 to 32g/L NaCl.

5. The culture medium according to claim 4, comprising per liter of said medium: 20.6g of the bovine heart meal, 9.8g of the yeast meal, 7.4g of the soy peptone and 30g of the NaCl, the balance being water.

6. A method for culturing Vibrio parahaemolyticus, wherein the culture of Vibrio parahaemolyticus is carried out using the medium according to any one of claims 1 to 5.

7. The method according to claim 6, wherein the specific conditions of the culture are: 35-37 ℃, pH 7.5-8.0, 160-.

8. Use of the culture medium according to any one of claims 1 to 5 or the method according to claim 6 or 7 for enrichment prior to the Vibrio parahaemolyticus test.

9. The use according to claim 8, wherein the culture of Vibrio parahaemolyticus is carried out using the medium for 6 to 6.5 hours.