CN115074271B - Enrichment medium for salmonella in tea polyphenol food, preparation method of enrichment medium and enrichment method - Google Patents
Enrichment medium for salmonella in tea polyphenol food, preparation method of enrichment medium and enrichment method Download PDFInfo
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Abstract
The invention discloses a salmonella enrichment medium in tea polyphenol food, a preparation method thereof and an enrichment method, wherein the enrichment medium is calculated by 1L of total volume of the enrichment medium and 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 grease without antibacterial components, 10-50 mmol of amino acid substances and the balance of water; the preparation method comprises the following steps: adding peptone, sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, liquid oil and amino acid substances which do not contain antibacterial components into water, stirring, heating and dissolving, and sterilizing to obtain a bacteria increasing culture medium; the method for increasing bacteria comprises the following steps: comprises the preparation of a enrichment medium and enrichment culture. The invention can effectively proliferate and grow salmonella in tea polyphenol food by improving the enrichment medium of salmonella, thereby being successfully detected.
Description
Technical Field
The invention relates to the technical field of pathogenic bacteria detection, in particular to a salmonella enrichment medium in tea polyphenol food, a preparation method thereof and an enrichment method.
Background
The polyhydroxyphenols in tea leaves are collectively called tea polyphenols, which contain more than 30 phenols, the main component being catechins and derivatives thereof. The tea polyphenol has biological activities of antioxidation, antibiosis, bacteriostasis, anti-tumor and the like, so the tea polyphenol is used as a natural and efficient food additive and is widely used for production, fresh-keeping and storage of aquatic products, meat and meat products, fruits and vegetables and cakes. Salmonella is an important food-borne pathogenic bacterium, the incidence rate of food poisoning is 2, and the food types of food poisoning mainly comprise meat, meat products, aquatic products, cakes and the like.
In the prior art, salmonella in food is detected by adding a enrichment medium (25 g of food is weighed and 225mL of enrichment medium) in an amount of 1:10, so as to promote recovery of salmonella and enable the salmonella to grow to a sufficient amount. However, in foods added with tea polyphenols, the examination of salmonella has the following problems: firstly, due to the antibacterial and bacteriostatic effects of tea polyphenol, salmonella in food is in a damaged state, and the current enrichment medium used for detecting salmonella is insufficient for repairing and resuscitating the damaged salmonella; secondly, tea polyphenol used in food continuously plays an antibacterial and bacteriostatic role, and inhibits the growth of salmonella in a bacteria increasing culture medium. The detection of salmonella in tea polyphenol foods is greatly influenced by the two conditions.
Disclosure of Invention
Aiming at the problem that tea polyphenol affects salmonella detection in the prior art, the invention provides a salmonella enrichment medium for salmonella in tea polyphenol foods, a preparation method thereof and a enrichment method.
The technical scheme adopted for solving the technical problems is as follows: an enrichment medium for salmonella in tea polyphenol food, which comprises the following components in terms of 1L of total volume of enrichment medium: 8-12 g peptone, 3-8 g sodium chloride, 7-11 g disodium hydrogen phosphate, 1-2 g monopotassium phosphate, 10-50 mL liquid grease without antibacterial components, 10-50 mmol amino acid substances and the balance of water.
Preferably, the peptone is tryptone, fish meal peptone or lunar peptone.
Preferably, the liquid oil and fat free of bacteriostatic components 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 salmonella enrichment medium in the 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 grease without antibacterial components and amino acid substances with the content of 10-50 mmol into water to 1L of total volume, stirring, heating and dissolving, and sterilizing to obtain the enrichment medium.
Preferably, the sterilizing pressure of the culture medium is 103-137 kPa, the sterilizing temperature is 110-130 ℃, and the sterilizing 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 salmonella enrichment method in the tea polyphenol food, which comprises the following steps:
s1, preparing a bacteria increasing culture medium: adding 8-12 g peptone, 3-8 g sodium chloride, 7-11 g disodium hydrogen phosphate, 1-2 g monopotassium phosphate, 10-50 mL liquid grease without antibacterial components and amino acid substances with the content of 10-50 mmol into water to 1L total volume, stirring, heating and dissolving, sterilizing for 10-20 min at the temperature of 103-137 kPa and 110-130 ℃ to obtain the bacteria increasing culture medium with the pH value of 7.0-7.4;
s2, enrichment culture: adding the tea polyphenol food into a sterile bag containing the enrichment medium in the step S1, homogenizing, and performing enrichment culture; the volume ratio of tea polyphenol food to 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, liquid grease without bacteriostatic components is added on the basis, and because of the characteristic of strong lipid solubility of tea polyphenol, the tea polyphenol of food is extracted into the liquid grease without bacteriostatic components, and the salmonella in the water-soluble liquid of the enrichment culture medium can be eliminated due to the fact that the salmonella is not contacted with the tea polyphenol, the antibacterial and bacteriostatic effects for inhibiting the growth of the salmonella are eliminated, and the salmonella can grow rapidly.
(2) According to the invention, amino acid substances are added into the culture medium, so that the repairing and reviving effects of damaged bacteria are promoted, and the repairing and reviving effects of damaged salmonella in tea polyphenol food can be promoted.
In conclusion, the enrichment medium disclosed by the invention can promote the repair and recovery of 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 can be effectively proliferated and grown, and can be smoothly 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 result of separation on a Salmonella chromogenic medium in a validation test after pre-enrichment using the enrichment medium of example 1;
FIG. 2 is the result of separation on a salmonella chromogenic medium in a validation test after pre-enrichment using the enrichment medium of comparative example 1.
Detailed Description
The present invention will be further described in detail with reference to the following examples, which are only for explaining the present invention and are not to be construed as limiting the scope of the present invention, for the purpose of making a clearer understanding of the technical features, objects and effects of the present invention.
An enrichment medium for salmonella in tea polyphenol food, which comprises the following components in terms of 1L of total volume of enrichment medium: 8-12 g peptone, 3-8 g sodium chloride, 7-11 g disodium hydrogen phosphate, 1-2 g monopotassium phosphate, 10-50 mL liquid grease without antibacterial components, 10-50 mmol amino acid substances and the balance of water.
The total volume of the enrichment medium is 1L, different components are limited, and the proportion relation of the components is conveniently represented.
Wherein the peptone is tryptone, fish meal peptone or moon peptone. Peptone is used as a main raw material of the enrichment medium to provide a nitrogen source for the growth of salmonella.
The liquid oil and fat without antibacterial component is soybean oil, corn oil or salad oil. Because of the characteristic of strong fat solubility of tea polyphenol, the tea polyphenol in food is extracted into liquid grease without antibacterial components, and salmonella in water-soluble liquid of an enrichment medium can not contact the tea polyphenol, so that the antibacterial and bacteriostatic effects of inhibiting the growth of the salmonella are eliminated, and the salmonella can grow rapidly.
The amino acid substance is L-cysteine, DL-serine or methionine, and 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 by adding the amino acid substance into the enrichment medium.
The invention also provides a preparation method of the salmonella enrichment medium 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 grease without antibacterial components and amino acid substances with the content of 10-50 mmol into water to 1L of the total volume of the enrichment medium, stirring, heating and dissolving, and sterilizing for 10-20 min at the temperature of 103-137 kPa and the temperature of 110-130 ℃ to obtain the enrichment medium with the pH value of 7.0-7.4.
The invention also provides a salmonella enrichment method in the tea polyphenol food, which comprises the following steps:
s1, preparing a bacteria increasing culture medium: adding 8-12 g peptone, 3-8 g sodium chloride, 7-11 g disodium hydrogen phosphate, 1-2 g monopotassium phosphate, 10-50 mL liquid grease without antibacterial components and amino acid substances with the content of 10-50 mmol into water to 1L total volume, stirring, heating and dissolving, sterilizing for 10-20 min at the temperature of 103-137 kPa and 110-130 ℃ to obtain the bacteria increasing culture medium with the pH value of 7.0-7.4;
s2, enrichment culture: adding tea polyphenol food into a sterile bag containing the enrichment culture medium in the step S1, beating for 1-2 min by a beating type homogenizer, and performing enrichment culture at 34-38 ℃ for 16-20 h; the volume ratio of tea polyphenol food to enrichment medium is 1 (7-12).
According to the invention, the salmonella in the tea polyphenol food is revived and grows to a sufficient amount by improving the salmonella enrichment medium, so that salmonella can be successfully detected, and the damage to the health of consumers is prevented.
The following is described by way of specific examples:
example 1
An enrichment medium of salmonella in tea polyphenol food, based on 1L of total volume of the medium, comprises the following components: 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate, 1.5g of potassium dihydrogen phosphate, 30mL of soybean oil, 30mmol of L-cysteine and the balance of water.
A preparation method of a salmonella enrichment medium 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 1L of total volume, stirring, heating and dissolving, sterilizing at 120kPa and 121 ℃ for 15min to obtain the enrichment medium with the pH value of 7.2.
A method for increasing bacteria of salmonella in tea polyphenol food comprises the following steps:
s1, preparing a bacteria increasing culture medium: 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 1L of total volume of the enrichment medium, stirring, heating and dissolving, and sterilizing at 120kPa and 121 ℃ for 10min to obtain the enrichment medium with the pH value of 7.2;
s2, enrichment culture: 25g of tea polyphenol food is added into a sterile bag containing 225mL of the enrichment medium of the step S1, and the bacteria are enriched and cultured for 18 hours at 36 ℃ after being beaten for 1.5min by a beating type homogenizer.
Example 2
An enrichment medium of salmonella in tea polyphenol food, based on 1L of total volume of the medium, comprises the following components: 8g of fish meal peptone, 3g of sodium chloride, 7g of disodium hydrogen phosphate, 1g of potassium dihydrogen phosphate, 10mL of corn oil, DL-serine with the content of 10mmol and the balance of water.
According to the method for preparing the enrichment medium, based on the total volume of the enrichment medium being 1L, 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 are added into water until the total volume is 1L, stirred, heated and dissolved, and sterilized at 103kPa and 110 ℃ for 20min to obtain the enrichment medium with the pH value of 7.0.
A method for increasing bacteria of salmonella in tea polyphenol food comprises the following steps:
s1, preparing a bacteria increasing culture medium: adding 8g of fish meal peptone, 3g of sodium chloride, 7g of disodium hydrogen phosphate, 1g of potassium dihydrogen phosphate, 10mL of corn oil and DL-serine with the content of 10mmol into water to 1L of total volume of the enrichment medium, stirring, heating and dissolving, and sterilizing at 103kPa and 110 ℃ for 20min to obtain the enrichment medium with the pH value of 7.0;
s2, enrichment culture: 25g of tea polyphenol food is added into a sterile bag containing 175mL of the enrichment medium of the step S1, and after being beaten for 1min by a beating type homogenizer, the tea polyphenol food is subjected to enrichment culture for 20h at 34 ℃.
Example 3
An enrichment medium of salmonella in tea polyphenol food, based on 1L of total volume of the medium, comprises the following components: 12g peptone, 8g sodium chloride, 11g disodium hydrogen phosphate, 2g potassium dihydrogen phosphate, 50mL salad oil, methionine with a content of 50mmol, and the balance being water.
According to the method for preparing the enrichment medium, based on the total volume of the medium being 1L, 12g of lunar peptone, 8g of sodium chloride, 11g of disodium hydrogen phosphate, 2g of potassium dihydrogen phosphate, 50mL of salad oil and methionine with the content of 50mmol are added into water until the total volume is 1L, stirred, heated and dissolved, and sterilized at the temperature of 137kPa and 130 ℃ for 10min to obtain the enrichment medium with the pH value of 7.4.
A method for increasing bacteria of salmonella in tea polyphenol food comprises the following steps:
s1, preparing a bacteria increasing culture medium: adding 12g of 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 1L of total volume of the enrichment medium, stirring, heating and dissolving, and sterilizing at 137kPa and 130 ℃ for 10min to obtain the enrichment medium with the pH value of 7.4;
s2, enrichment culture: 25g of tea polyphenol food is added into a sterile bag containing 300mL of enrichment medium, and after being beaten for 2min by a beating type homogenizer, the tea polyphenol food is subjected to enrichment culture for 16h at 38 ℃.
It will be appreciated that in the above embodiment and alternatives thereof, 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
In comparison with example 1, the enrichment medium of this comparative example was Buffered Peptone Water (BPW), and the components thereof contained no amino acids and no liquid oils and fats containing antibacterial ingredients.
An enrichment medium of salmonella in tea polyphenol food, based on 1L of total volume of the medium, comprises the following components: 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate, 1.5g of monopotassium phosphate and the balance of water.
10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate and 1.5g of potassium dihydrogen phosphate are added into water to 1L of the total volume of the culture medium, and the mixture is stirred, heated and dissolved, and sterilized at 120kPa and 121 ℃ for 15min to obtain the enrichment culture medium with the pH value of 7.2.
A method for increasing bacteria of salmonella in tea polyphenol food comprises the following steps:
s1, preparing a bacteria increasing culture medium: adding 10g of tryptone, 5g of sodium chloride, 9g of disodium hydrogen phosphate and 1.5g of potassium dihydrogen phosphate into water to obtain a total volume of 1L, stirring, heating and dissolving, and sterilizing at 120kPa and 121 ℃ for 10min to obtain the enrichment medium with a pH value of 7.2;
s2, enrichment culture: 25g of tea polyphenol food is added into a sterile bag containing 225mL of enrichment medium, and the mixture is subjected to enrichment culture for 18h at 36 ℃ after being beaten for 1.5min by a beating type homogenizer.
Comparison test:
test materials: salmonella Abutila standard strain (Salmonella enterica subsp. Enterica serovar Abaetetuba ATCC 35640), matcha cake, RVS broth medium, sodium sulfosuccinate brilliant green (TTB) medium, bismuth Sulfite (BS) agar medium plates, salmonella chromogenic medium plates.
The test steps are as follows:
the test was conducted by referring to the salmonella test method specified in GB 4789.4-2016, and the salmonella in tea polyphenol food was pre-enriched using the enriched medium of the modified salmonella of example 1 of the present invention and the buffered peptone water of comparative example 1, and the positive rate was compared, and the test results are shown in tables 1 and 2.
(1) Preparing a sample: preparing a bacterial suspension of a salmonella abamectin standard strain (ATCC 35640), adding the bacterial suspension into a green tea cake, fully and uniformly mixing the green tea cake until the final concentration of salmonella abamectin is 1-5 CFU/25g, and placing the green tea cake added with the bacterial strain in a refrigerator at 4 ℃ for overnight storage.
(2) Pre-enrichment: 10 parts of a total of 25g of green tea cake are weighed, each part is placed in a sterile homogenizing bag containing 225mL of an enrichment medium of the improved salmonella, and the mixture is subjected to pre-enrichment for 18h at 36 ℃ after being beaten for 1.5min by a slapping homogenizer. Simultaneously weighing 10 parts of 25g of green tea cake, placing each part of green tea cake into a sterile homogenizing bag containing 225mL of buffer peptone water, beating for 1.5min by a beating type homogenizer, and pre-sterilizing at 36 ℃ for 18h.
(3) And (3) increasing bacteria: the pre-enriched culture was gently shaken, 0.1mL of the culture was transferred to 10mL of RVS broth, and incubated at 42℃for 20h. 1mL of the pre-enriched culture is transferred into 10mL of TTB culture medium, and is placed at 36 ℃ for 20h for selective enrichment.
(4) Separating: after shaking and mixing the selectively enriched cultures, 1 loop of each selectively enriched culture was streaked onto a BS agar plate and a Salmonella chromogenic medium plate, respectively, and cultured at 36℃for 44h and 21h, respectively, and colonies grown on each plate were observed.
(5) Biochemical test: and after the typical colony on the BS agar plate and the salmonella chromogenic medium plate is selected and separated for purification and culture, a biochemical test is carried out by using a full-automatic microorganism biochemical identification system.
(6) Serological identification: representative colonies on the BS agar plates and salmonella chromogenic medium plates were picked and serological identification experiments were performed with salmonella O multivalent and H multivalent diagnostic serum.
TABLE 1 Matcha cake Standard Strain added indoor verification test results (example 1)
Note that: "-" is a negative reaction; "+" is a positive reaction.
TABLE 2 Matcha cake Standard Strain added indoor verification test results (comparative example 1)
Note that: "-" is a negative reaction; "+" is a positive reaction.
The salmonella in tea polyphenol food is subjected to enrichment culture by using the enrichment culture medium of the comparative example 1 and the enrichment culture medium of the example 1, typical bacterial colonies on a BS agar plate and a salmonella chromogenic plate are subjected to biochemical identification test, the identification result of the full-automatic microorganism biochemical identification system shows that the bacterial colonies all accord with the biochemical characteristics of salmonella, and salmonella O and H multivalent diagnosis serum agglutinators are positive to salmonella.
FIGS. 1 and 2 show the results of the culture of the enrichment culture on a plate of a chromogenic medium for Salmonella, and the results of the experiments in Table 1 and Table 2 show that the positive rate of Salmonella after pre-enrichment using the enrichment medium for improved Salmonella of the present invention is 50% significantly higher than the positive rate after pre-enrichment using buffered peptone water by 10%. Therefore, compared with the common salmonella pre-enrichment medium buffer peptone, the salmonella-modified enrichment medium can eliminate the antibacterial and bacteriostatic effects of tea polyphenol and promote the repair and recovery of salmonella damaged by tea polyphenol food, so that salmonella can be detected.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Claims (3)
1. The method for increasing the bacteria of salmonella in tea polyphenol food is characterized by comprising the following steps of:
s1, preparing a bacteria increasing culture 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 grease without antibacterial components and amino acid substances with the content of 10-50 mmol into water to 1L of total volume of the enrichment medium, stirring, heating and dissolving, and sterilizing for 10-20 min at the temperature of 103-137 kPa and 110-130 ℃ to obtain the enrichment medium with the pH value of 7.0-7.4;
the peptone is tryptone, fish meal peptone orpeptone; the liquid grease without antibacterial components is soybean oil, corn oil or salad oil; the amino acid substance is L-cysteine, DL-serine or methionine;
s2, enrichment culture: adding the tea polyphenol food into a sterile bag containing the enrichment medium in the step S1, homogenizing, and performing enrichment culture; the volume ratio of tea polyphenol food to enrichment medium is 1 (7-12).
2. The method for increasing salmonella in tea polyphenol foods according to claim 1, wherein the homogenization time in step S2 is 1-2 min.
3. The method for increasing bacteria of salmonella in tea polyphenol food according to claim 1, wherein the temperature of the bacteria increasing culture in the step S2 is 34-38 ℃ and the time is 16-20 h.
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| US5871944A (en) * | 1996-07-09 | 1999-02-16 | The University Of Maryland | Salmonellae preferential media |
| 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 |
| 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 |
| KR101701064B1 (en) * | 2015-12-04 | 2017-01-31 | 건국대학교 산학협력단 | A Salmonella Single Enrichment Medium composition, a method thereof and use of the same |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5871944A (en) * | 1996-07-09 | 1999-02-16 | The University Of Maryland | Salmonellae preferential media |
| 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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