CN115074271A - Enrichment culture medium for salmonella in tea polyphenol food, preparation method of enrichment culture medium and enrichment method - Google Patents
Enrichment culture medium for salmonella in tea polyphenol food, preparation method of enrichment culture medium and enrichment method Download PDFInfo
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Abstract
The invention discloses a enrichment medium for salmonella in tea polyphenol food, a preparation method thereof and an enrichment method, wherein the enrichment medium comprises the following components in percentage by volume of 1L of the total volume of the enrichment medium: 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of monopotassium phosphate, 10-50 mL of liquid oil containing no antibacterial component, amino acid substances with the content of 10-50 mmol, and the balance of water; the preparation method comprises the following steps: adding peptone, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, liquid oil without antibacterial components and amino acid substances into water, stirring, heating for dissolving, and sterilizing to obtain enrichment medium; the enrichment method comprises the following steps: comprises the preparation of enrichment culture medium and enrichment culture. The invention improves the enrichment culture medium of the salmonella, so that the salmonella in the tea polyphenol food can effectively proliferate and grow, thereby being successfully detected.
Description
Technical Field
The invention relates to the technical field of pathogenic bacteria detection, and particularly relates to a bacterium increasing culture medium for salmonella in tea polyphenol food, a preparation method of the bacterium increasing culture medium and a bacterium increasing method.
Background
The polyhydroxy phenolic compounds in tea are collectively called tea polyphenol, and contain more than 30 phenolic substances, and the main components are catechin and derivatives thereof. The tea polyphenol has biological activities of oxidation resistance, antibiosis, bacteriostasis, tumor resistance and the like, so the tea polyphenol is used as a natural and efficient food additive and widely applied to production, fresh keeping and storage of aquatic products, meat products, fruits, vegetables and cakes. Salmonella is an important food-borne pathogenic bacterium, the incidence of food poisoning is 2 th, and the food poisoning mainly comprises meat, meat products, aquatic products, cakes and the like.
In the prior art, the salmonella in the food is firstly tested by adding a bacterium-increasing culture medium (25 g of food is weighed and 225mL of bacterium-increasing culture medium is added) in an amount of 1:10, so that the recovery of the salmonella is promoted and the salmonella is grown to a sufficient amount, and the salmonella can be detected. However, the test for salmonella in foods containing tea polyphenols has the following problems: firstly, because of the antibacterial and bacteriostatic actions of tea polyphenol, most of salmonella in food is in a damaged state, and the existing bacterium increasing culture medium for detecting salmonella is not enough to repair and recover the damaged salmonella; secondly, the tea polyphenol used in the food continuously plays the role of antibiosis and bacteriostasis, and inhibits the growth of salmonella in the enrichment medium. The two conditions greatly influence the detection of the salmonella in the tea polyphenol food.
Disclosure of Invention
The invention aims to solve the technical problem of providing a bacterium increasing culture medium for salmonella in tea polyphenol food, a preparation method thereof and a bacterium increasing method thereof aiming at the detection of the salmonella influenced by tea polyphenol in the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows: a enrichment culture medium for salmonella in tea polyphenol food, which is calculated by taking the total volume of the enrichment culture medium as 1L, comprises the following components: 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of monopotassium phosphate, 10-50 mL of liquid oil containing no antibacterial component, amino acid substances with the content of 10-50 mmol, and the balance of water.
Preferably, the peptone is tryptone, fish meal peptone or monthly peptone.
Preferably, the liquid oil without bacteriostatic component is soybean oil, corn oil or salad oil.
Preferably, the amino acid is L-cysteine, DL-serine or methionine.
The invention also provides a preparation method of the enrichment medium for salmonella in tea polyphenol food, which comprises the steps of adding 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of potassium dihydrogen phosphate, 10-50 mL of liquid oil containing no antibacterial ingredients and 10-50 mmol of amino acid substances into water until the total volume is 1L, stirring, heating and dissolving, and sterilizing to obtain the enrichment medium.
Preferably, the sterilization pressure of the culture medium is 103-137 kPa, the sterilization temperature is 110-130 ℃, and the sterilization time is 10-20 min.
Preferably, the pH value of the enrichment medium after sterilization is 7.0-7.4.
The invention also provides a bacterium increasing method of salmonella in tea polyphenol food, which comprises the following steps:
s1, preparation of a enrichment medium: adding 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of monopotassium phosphate, 10-50 mL of liquid oil containing no antibacterial ingredients and 10-50 mmol of amino acid substances into water to make the total volume be 1L, stirring, heating and dissolving, and sterilizing at 103-137 kPa and 110-130 ℃ for 10-20 min to obtain a bacteria-increasing culture medium with the pH value of 7.0-7.4, wherein the total volume of the bacteria-increasing culture medium is 1L;
s2, enrichment culture: adding tea polyphenol food into a sterile bag containing the enrichment culture medium in the step S1, homogenizing, and carrying out enrichment culture; the volume ratio of the tea polyphenol food to the enrichment medium is 1 (7-12).
Preferably, the homogenization time in step S2 is 1-2 min.
Preferably, the temperature of the enrichment culture in the step S2 is 34-38 ℃, and the time is 16-20 h.
The invention has the beneficial effects that:
(1) in the invention, peptone, sodium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate are dissolved in water to form a basic culture medium, and liquid oil containing no bacteriostatic components is added on the basis, because the tea polyphenol has the characteristic of strong lipid solubility, tea polyphenol of food is extracted into the liquid oil containing no bacteriostatic components, salmonella in water-soluble liquid of the enrichment culture medium can not contact the tea polyphenol, the antibacterial and bacteriostatic action for inhibiting the growth of the salmonella can be eliminated, and the salmonella can grow rapidly.
(2) According to the invention, the amino acid substances are added into the culture medium, and the culture medium has the effect of promoting the repair and recovery of damaged bacteria, so that the repair and recovery of damaged salmonella in tea polyphenol food can be promoted.
In conclusion, the enrichment culture medium can promote the repair and recovery of the damaged salmonella in the tea polyphenol food and eliminate the antibacterial and bacteriostatic effects of the tea polyphenol, so that the salmonella in the tea polyphenol food is effectively proliferated and grown, and is successfully detected, and the salmonella in the tea polyphenol food is prevented from harming the health of consumers.
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FIG. 1 is the results of the isolation on a chromogenic Salmonella medium in a validation experiment after pre-enrichment using the enrichment medium of example 1;
FIG. 2 is the result of isolation on a Salmonella chromogenic medium in a validation test after pre-enrichment using the enrichment medium of comparative example 1.
Detailed Description
In order to clearly understand the technical features, objects and effects of the present invention, the present invention will be further described in detail with reference to the following embodiments, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
A enrichment culture medium for salmonella in tea polyphenol food, which is calculated by taking the total volume of the enrichment culture medium as 1L, comprises the following components: 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of monopotassium phosphate, 10-50 mL of liquid oil containing no antibacterial component, amino acid substances with the content of 10-50 mmol, and the balance of water.
The total volume of the enrichment culture medium is 1L, different components are limited, and the proportional relation of the components is conveniently expressed.
Wherein the peptone is tryptone, fish meal peptone or monthly peptone. The peptone is used as a main raw material of the enrichment medium and provides a nitrogen source for the growth of the salmonella.
The liquid oil without antibacterial component is soybean oil, corn oil or salad oil. Because the tea polyphenol in the food has strong fat solubility, the tea polyphenol in the food is extracted into liquid oil without bacteriostatic components, and because the salmonella in the water-soluble liquid of the enrichment culture medium can not contact the tea polyphenol, the antibacterial and bacteriostatic action for inhibiting the growth of the salmonella is eliminated, so the salmonella can grow quickly.
The amino acid substance is L-cysteine, DL-serine or methionine, which has the function of promoting the repair and recovery of damaged bacteria, so that the amino acid substance added into the enrichment culture medium can promote the repair and recovery of damaged salmonella in the tea polyphenol food.
The invention also provides a preparation method of the enrichment medium for salmonella in tea polyphenol food, which comprises the steps of adding 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of potassium dihydrogen phosphate, 10-50 mL of liquid oil containing no antibacterial ingredients and 10-50 mmol of amino acid substances into water to reach a total volume of 1L, stirring, heating and dissolving, and sterilizing at 103-137 kPa and 110-130 ℃ for 10-20 min to obtain the enrichment medium with the pH value of 7.0-7.4, wherein the total volume of the enrichment medium is 1L.
The invention also provides a bacterium increasing method of salmonella in tea polyphenol food, which comprises the following steps:
s1, preparation of a enrichment medium: adding 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of monopotassium phosphate, 10-50 mL of liquid oil containing no antibacterial ingredients and 10-50 mmol of amino acid substances into water to make the total volume be 1L, stirring, heating and dissolving, and sterilizing at 103-137 kPa and 110-130 ℃ for 10-20 min to obtain a bacteria-increasing culture medium with the pH value of 7.0-7.4, wherein the total volume of the bacteria-increasing culture medium is 1L;
s2, enrichment culture: adding tea polyphenol food into a sterile bag containing the enrichment culture medium in the step S1, beating the tea polyphenol food for 1-2 min by using a beating type homogenizer, and carrying out enrichment culture at 34-38 ℃ for 16-20 h; the volume ratio of the tea polyphenol food to the enrichment medium is 1 (7-12).
The invention revives the salmonella in the tea polyphenol food and grows to enough quantity by improving the enrichment culture medium of the salmonella, thereby leading the salmonella to be detected successfully and preventing the salmonella from harming the health of consumers.
The following is illustrated by specific examples:
example 1
A bacterium increasing culture medium for salmonella in tea polyphenol food comprises the following components by taking the total volume of the culture medium as 1L: 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate, 1.5g of monopotassium phosphate, 30mL of soybean oil, 30mmol of L-cysteine and the balance of water.
A preparation method of a bacterium enrichment medium for salmonella in tea polyphenol food comprises the steps of adding 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate, 1.5g of potassium dihydrogen phosphate, 30mL of soybean oil and 30mmol of L-cysteine into water to enable the total volume to be 1L, stirring, heating and dissolving, and sterilizing at 120kPa and 121 ℃ for 15min to obtain the bacterium enrichment medium with the pH value of 7.2, wherein the total volume of the bacterium enrichment medium is 1L.
A method for increasing the bacteria of salmonella in tea polyphenol food comprises the following steps:
s1, preparation of a enrichment medium: adding 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate, 1.5g of monopotassium phosphate, 30mL of soybean oil and 30mmol of L-cysteine into water to enable the total volume to be 1L, stirring, heating and dissolving, and sterilizing at 120kPa and 121 ℃ for 10min to obtain a enrichment medium with the pH value of 7.2, wherein the total volume of the enrichment medium is 1L;
s2, enrichment culture: adding 25g tea polyphenols into sterile bag containing 225mL of the enrichment medium of step S1, beating with beating type homogenizer for 1.5min, and enrichment culturing at 36 deg.C for 18 h.
Example 2
A bacterium increasing culture medium for salmonella in tea polyphenol food comprises the following components by taking the total volume of the culture medium as 1L: 8g of fish meal peptone, 3g of sodium chloride, 7g of disodium hydrogen phosphate, 1g of monopotassium phosphate, 10mL of corn oil, 10mmol of DL-serine and the balance of water.
A method for preparing the enrichment medium comprises the steps of adding 8g of fish meal peptone, 3g of sodium chloride, 7g of disodium hydrogen phosphate, 1g of monopotassium phosphate, 10mL of corn oil and 10mmol of DL-serine into water to make the total volume of 1L, stirring, heating and dissolving, and sterilizing at 103kPa and 110 ℃ for 20min to obtain the enrichment medium with the pH value of 7.0, wherein the total volume of the enrichment medium is 1L.
A method for increasing the bacteria of salmonella in tea polyphenol food comprises the following steps:
s1, preparation of a enrichment medium: adding 8g of fish meal peptone, 3g of sodium chloride, 7g of disodium hydrogen phosphate, 1g of monopotassium phosphate, 10mL of corn oil and 10mmol of DL-serine into water to make the total volume be 1L, stirring, heating and dissolving, sterilizing at 103kPa and 110 ℃ for 20min to obtain the enrichment medium with the pH value of 7.0, wherein the total volume of the enrichment medium is 1L;
s2, enrichment culture: adding 25g tea polyphenols into sterile bag containing 175mL of the enrichment medium of step S1, beating with beating type homogenizer for 1min, and enrichment culturing at 34 deg.C for 20 h.
Example 3
A bacterium increasing culture medium for salmonella in tea polyphenol food comprises the following components by taking the total volume of the culture medium as 1L: 12g of peptone, 8g of sodium chloride, 11g of disodium hydrogen phosphate, 2g of potassium dihydrogen phosphate, 50mL of salad oil, methionine with the content of 50mmol, and the balance of water.
A method for preparing the enrichment medium comprises the steps of adding 12g of monthly peptone, 8g of sodium chloride, 11g of disodium hydrogen phosphate, 2g of monopotassium phosphate, 50mL of salad oil and methionine with the content of 50mmol into water to enable the total volume to be 1L, stirring, heating and dissolving, and sterilizing at 137kPa and 130 ℃ for 10min to obtain the enrichment medium with the pH value of 7.4, wherein the total volume of the enrichment medium is 1L.
A method for increasing the bacteria of salmonella in tea polyphenol food comprises the following steps:
s1, preparation of a enrichment medium: adding 12g of peptone, 8g of sodium chloride, 11g of disodium hydrogen phosphate, 2g of monopotassium phosphate, 50mL of salad oil and 50mmol of methionine into water to make the total volume of 1L, stirring, heating and dissolving, sterilizing at 137kPa and 130 ℃ for 10min to obtain the enrichment medium with the pH value of 7.4, wherein the total volume of the enrichment medium is 1L;
s2, enrichment culture: adding 25g tea polyphenols into sterile bag containing 300mL enrichment medium, beating with beating type homogenizer for 2min, and enrichment culturing at 38 deg.C for 16 h.
It is understood that, in the above-mentioned examples and their alternatives, the volume ratio of the tea polyphenol food and the enrichment medium in step S2 of the enrichment method is 1 (7-12).
Comparative example 1
Compared with the example 1, the enrichment medium of the comparative example is Buffer Peptone Water (BPW), and the components of the enrichment medium do not contain amino acid substances and liquid oil and fat without bacteriostatic components.
A bacterium increasing culture medium for salmonella in tea polyphenol food comprises the following components by taking the total volume of the culture medium as 1L: 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate, 1.5g of potassium dihydrogen phosphate and the balance of water.
A method for preparing the enrichment medium comprises the steps of adding 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate and 1.5g of potassium dihydrogen phosphate into water to enable the total volume to be 1L, stirring, heating and dissolving, and sterilizing at 120kPa and 121 ℃ for 15min to obtain the enrichment medium with the pH value of 7.2, wherein the total volume of the enrichment medium is 1L.
A method for increasing the bacteria of salmonella in tea polyphenol food comprises the following steps:
s1, preparation of a enrichment medium: adding 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate and 1.5g of potassium dihydrogen phosphate into water to enable the total volume to be 1L, stirring, heating and dissolving, and sterilizing at 120kPa and 121 ℃ for 10min to obtain the enrichment medium with the pH value of 7.2, wherein the total volume of the enrichment medium is 1L;
s2, enrichment culture: adding 25g tea polyphenols into sterile bag containing 225mL enrichment medium, beating with beating type homogenizer for 1.5min, and enrichment culturing at 36 deg.C for 18 h.
And (3) comparison test:
test materials: bactria abalonella (Salmonella enterica subsp. enterica serovar abeateuba ATCC 35640), Matcha cake, RVS broth, sodium tetrasulfosulfonate Brilliant Green (TTB) medium, Bismuth Sulfite (BS) agar medium plate, Salmonella chromogenic medium plate.
The test steps are as follows:
tests were carried out with reference to the Salmonella test method specified in GB 4789.4-2016, and Salmonella in tea polyphenol food products were subjected to pre-enrichment using the enrichment medium for modified Salmonella of example 1 of the present invention and the buffer peptone water of comparative example 1, respectively, and the positive rates thereof were compared, with the test results shown in tables 1 and 2.
(1) Preparing a sample: preparing bacterial suspension of the abamectin Basalmonellae standard strain (ATCC 35640), adding the bacterial suspension into a matcha cake, fully and uniformly mixing to enable the final concentration of the abamectin Basalmonellae to be 1-5 CFU/25g, and placing the matcha cake added with the bacterial strain in a refrigerator at 4 ℃ for overnight storage.
(2) Pre-enrichment: weighing 10 parts of 25g matcha cake, placing each part into a sterile homogenizing bag containing 225mL of enrichment medium for improving salmonella, beating with a beating type homogenizer for 1.5min, and placing at 36 deg.C for pre-enrichment for 18 h. Meanwhile, weighing 10 parts of 25g matcha cake, placing each part in a sterile homogenizing bag containing 225mL buffer peptone water, beating with a beating type homogenizer for 1.5min, and placing at 36 ℃ for pre-enrichment for 18 h.
(3) And (3) enrichment: the cultures of the pre-enrichment were gently shaken and 0.1mL of the transfer was transferred to 10mL of RVS broth and cultured at 42 ℃ for 20 h. Another 1mL of the pre-enrichment culture was inoculated into 10mL of TTB medium and cultured at 36 ℃ for 20h for selective enrichment.
(4) Separation: after the selectively enriched cultures were shaken and mixed well, 1 ring of each selectively enriched culture was taken with an inoculating loop having a diameter of 3mm, streaked on a BS agar plate and a Salmonella color medium plate, cultured at 36 ℃ for 44 hours and 21 hours, respectively, and the colonies grown on each plate were observed.
(5) And (3) biochemical test: after typical bacterial colonies on an isolated BS agar plate and a salmonella chromogenic medium plate are picked and purified and cultured, a full-automatic microbial biochemical identification system is used for biochemical test.
(6) Serological identification: typical colonies on separate BS agar plates and Salmonella chromogenic medium plates were picked and serological identification tests were performed with Salmonella O-multivalent and H-multivalent diagnostic sera.
TABLE 1 Matcha cake Standard Strain addition laboratory test results (example 1)
Note: "-" is a negative reaction; "+" indicates a positive reaction.
TABLE 2 Matcha cake Standard Strain addition laboratory test results (comparative example 1)
Note: "-" is a negative reaction; "+" indicates a positive reaction.
The enrichment culture of the salmonella in the tea polyphenol food is carried out by using the enrichment culture medium of the comparative example 1 and the example 1, the typical colonies on a BS agar plate and a salmonella chromogenic plate are subjected to biochemical identification tests, the identification result of the full-automatic microorganism biochemical identification system shows that the colonies all accord with the biochemical characteristics of the salmonella, and the salmonella O and H polyvalent diagnosis serum agglutinate is positive.
FIG. 1 and FIG. 2 show the culture results of enrichment culture on a Salmonella color development medium plate, and the test results in Table 1 and Table 2 show that the positive rate of Salmonella after pre-enrichment by using the enrichment culture medium of the improved Salmonella of the invention is 50%, which is obviously higher than the positive rate of Salmonella after pre-enrichment by using buffer peptone water by 10%. Therefore, compared with the common salmonella pre-enrichment culture medium buffer peptone water, the improved salmonella enrichment culture medium can eliminate the antibacterial and bacteriostatic effects of the tea polyphenol and promote the repair and recovery of the damaged salmonella in tea polyphenol food, so that the salmonella can be detected.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. The enrichment medium for salmonella in tea polyphenol food is characterized by comprising the following components by taking the total volume of the enrichment medium as 1L: 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of monopotassium phosphate, 10-50 mL of liquid oil containing no antibacterial component, amino acid substances with the content of 10-50 mmol, and the balance of water.
2. The enrichment medium for salmonella in tea polyphenol food as claimed in claim 1, wherein the peptone is tryptone, fish meal peptone or monthly peptone.
3. The enrichment medium for salmonella in tea polyphenol food as claimed in claim 1, wherein the liquid oil without bacteriostatic component is soybean oil, corn oil or salad oil.
4. The enrichment medium for salmonella in tea polyphenol food as claimed in claim 1, wherein the amino acid is L-cysteine, DL-serine or methionine.
5. The preparation method of the enrichment medium for salmonella in tea polyphenol food is characterized by adding 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of potassium dihydrogen phosphate, 10-50 mL of liquid oil containing no antibacterial ingredients and amino acid substances with the content of 10-50 mmol into water to make the total volume of the enrichment medium be 1L, stirring, heating and dissolving, and sterilizing to obtain the enrichment medium.
6. The preparation method of the enrichment medium for salmonella in tea polyphenol food as claimed in claim 5, wherein the sterilization pressure of the medium is 103-137 kPa, the sterilization temperature is 110-130 ℃, and the sterilization time is 10-20 min.
7. The method for preparing the enrichment medium for the salmonella in the tea polyphenol food as claimed in claim 5, wherein the pH value of the enrichment medium after sterilization is 7.0-7.4.
8. A method for increasing the bacteria of salmonella in tea polyphenol food is characterized by comprising the following steps:
s1, preparation of a enrichment medium: adding 8-12 g of peptone, 3-8 g of sodium chloride, 7-11 g of disodium hydrogen phosphate, 1-2 g of monopotassium phosphate, 10-50 mL of liquid oil containing no antibacterial ingredients and 10-50 mmol of amino acid substances into water to make the total volume be 1L, stirring, heating and dissolving, and sterilizing at 103-137 kPa and 110-130 ℃ for 10-20 min to obtain a bacteria-increasing culture medium with the pH value of 7.0-7.4, wherein the total volume of the bacteria-increasing culture medium is 1L;
s2, enrichment culture: adding tea polyphenol food into a sterile bag containing the enrichment culture medium in the step S1, homogenizing, and carrying out enrichment culture; the volume ratio of the tea polyphenol food to the enrichment medium is 1 (7-12).
9. The method for increasing Salmonella bacteria in tea polyphenol food according to claim 8, wherein the homogenization time in the step S2 is 1-2 min.
10. The method for increasing the number of salmonella in tea polyphenol food as claimed in claim 8, wherein the temperature for the bacteria increasing cultivation in step S2 is 34-38 ℃ for 16-20 h.
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| JP2000116395A (en) * | 1998-10-09 | 2000-04-25 | Eiken Chem Co Ltd | Culture medium for separating salmonella |
| CN102864204A (en) * | 2012-09-07 | 2013-01-09 | 四川大学 | Enrichment culture solution for detection of Salmonella in poultry eggs |
| KR101701064B1 (en) * | 2015-12-04 | 2017-01-31 | 건국대학교 산학협력단 | A Salmonella Single Enrichment Medium composition, a method thereof and use of the same |
| CN106047781A (en) * | 2016-08-22 | 2016-10-26 | 四川华汉三创生物科技有限公司 | Enrichment culture medium and preparation method thereof, and enrichment culture method |
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