CN113584122B - Liquid medium for recovering dry injury from Cronobacter and Salmonella - Google Patents

Liquid medium for recovering dry injury from Cronobacter and Salmonella Download PDF

Info

Publication number
CN113584122B
CN113584122B CN202110924195.9A CN202110924195A CN113584122B CN 113584122 B CN113584122 B CN 113584122B CN 202110924195 A CN202110924195 A CN 202110924195A CN 113584122 B CN113584122 B CN 113584122B
Authority
CN
China
Prior art keywords
cronobacter
salmonella
culture medium
food
resuscitating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110924195.9A
Other languages
Chinese (zh)
Other versions
CN113584122A (en
Inventor
叶应旺
凌娜
偶德馨
董晶晶
蒋秀婷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hefei University of Technology
Original Assignee
Hefei University of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hefei University of Technology filed Critical Hefei University of Technology
Priority to CN202110924195.9A priority Critical patent/CN113584122B/en
Publication of CN113584122A publication Critical patent/CN113584122A/en
Application granted granted Critical
Publication of CN113584122B publication Critical patent/CN113584122B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/255—Salmonella (G)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to a liquid culture medium for recovering dry injury from Cronobacter and salmonella, belonging to the technical field of biological product detection. The resuscitating culture medium consists of peptone, mannitol, trehalose, betaine, proline, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate and water, and the results of verification by using a large amount of food-borne pathogenic bacteria show that the effect of the resuscitating culture medium is obviously improved compared with that of the existing pre-enrichment culture medium such as BPW used in international standards and national food safety standards, and the resuscitating culture medium has good resuscitating universality, and experimental tests show similar experimental conclusion including 7 Cronobacter strains, salmonella standard strains and food isolates. The method is suitable for rapid recovery and enrichment culture of the Cronobacter and salmonella in dry food or low water activity food, is beneficial to improving the detection rate of the Cronobacter and salmonella damaged by drying, and avoids food poisoning caused by missed detection in the existing method.

Description

Liquid medium for recovering dry injury from Cronobacter and Salmonella
Technical Field
The invention belongs to the technical field of detection of biological products, and relates to a liquid culture medium for recovering dry injury from Cronobacter and salmonella.
Background
Drying is a common process unit for food processing that controls the growth and reproduction of harmful microorganisms in food by causing dehydration inactivation of bacteria. Cronobacter and salmonella are important food-borne pathogenic bacteria, have strong dry tolerance, and are important subjects for safety risk monitoring in infant formula in the food safety standard of China.
Safety of infant formulas is of social concern. The infant formula milk powder needs to be dried, cooled and stored for a long time with low water activity in the processing process, and bacteria in the milk powder are easy to damage in a dry environment and are in a sublethal state. Therefore, in the food microorganism inspection process, bacterial damage can be caused by a drying process and a long-term drying storage environment, so that false negative results are easily caused in the inspection of Cronobacter and salmonella in food, and infant formula milk powder polluted by the Cronobacter and salmonella is brought into the market; these damaged sublethal bacteria, once they enter the infant's intestines through food, can revive under appropriate conditions, causing food-borne diseases of cronobacter and salmonella infections. Therefore, the pre-enrichment culture medium for efficiently recovering the dry and damaged bacteria is very important for improving the detection rate of the dry and damaged bacteria and guaranteeing the safety of related foods.
In the existing national food safety standard inspection process, the detection methods of salmonella and Cronobacter both use buffer peptone water as a pre-enrichment resuscitating medium, and the buffer peptone water lacking a protective agent easily causes that the dry and damaged bacteria cannot be resuscitated normally or effectively.
Disclosure of Invention
In order to prevent the occurrence of the condition of no detection of the Cronobacter and salmonella in the process of testing the infant formula milk powder, improve the detection rate of the Cronobacter and salmonella in the milk powder, and avoid the outbreak of food-borne diseases caused by the fact that the Cronobacter and salmonella which are dry and damaged enter the infant body through food, the invention provides a liquid culture medium for recovering and drying the Cronobacter and salmonella.
A liquid medium for resuscitating dry lesions of cronobacter and salmonella consisting of the following mass: 10g of peptone, 1.5g of potassium dihydrogen phosphate, 9.0g of sodium dihydrogen phosphate, 5.0 g sodium chloride, 2-5g mannitol, 2-6g of trehalose, 2-5g of betaine, 2-6g of proline and 1000 mL water;
the sodium dihydrogen phosphate is sodium dihydrogen phosphate with 12 crystal waters;
and uniformly mixing the substances at normal temperature to obtain the liquid recovery culture medium.
The technical scheme is as follows:
a liquid medium for resuscitating dry lesions of cronobacter and salmonella consisting of the following mass: 10g peptone, 1.5g potassium dihydrogen phosphate, 9.0g sodium dihydrogen phosphate, 5.0 g sodium chloride, 3.0g mannitol, 2.5g trehalose, 3.0g betaine, 2.5g proline and 1000 mL water.
The beneficial technical effects of the invention are as follows:
1. the invention is based on buffer peptone water culture medium, and repair factors mannitol, trehalose, betaine and proline are added, so that cell rupture death caused by rehydration or osmotic pressure difference of dry damaged bacteria is reduced, and metabolic enzyme activity of the damaged bacteria is improved, and fine normal physiological metabolism activity is maintained.
2. Good recovery effect on dry injury
Bacterial solutions of Cronobacter sakazakii ATCC29544 and Salmonella CMCC50071, which had been cultured to mid-log phase respectively, were centrifuged at 5000 rpm for 15 minutes, the supernatant was discarded, and the remaining medium components were washed out with physiological saline in the same manner. After that, 50. Mu.L of milk (250 g milk powder/L) was used to resuspend the cells, 50. Mu.L of the milk cell suspension was aspirated and added to a 12-well plate, and the plate was dried in a desiccator for 20 days, 30 days and 40 days. Adding the optimized liquid culture medium 2 mL into 12-hole plates for drying damaged bacteria in different time periods, uniformly mixing, and placing into a constant-temperature incubator 37 o C culture 18 h. The blank group was BPW peptone water and LB broth without addition of substances. Then, 200. Mu.L of the culture medium was aspirated, and OD was measured 600 As a result, it was found that the colony growth amount (OD 600 ) The method is obviously higher than other culture mediums, and shows that the resuscitating liquid culture medium has good bacterium increasing effect and high resuscitating efficiency.
3. The resuscitation effect has universality
Cronobacter sakazakii standard strain ATCC29544 @Cronobacter sakazakiiATCC 29544), mo Jinsi An Keluo Norbacter ATCC51329Cronobacter muytjenii) Seven species of Cronobacter equi-and Salmonella strains(comprising Salmonella typhimurium, salmonella paratyphi and Salmonella isolates) were cultured to medium logarithmic phase, and the bacterial solutions were centrifuged at 5000 rpm for 15 min, the supernatant was discarded, and the residual medium components were washed off with physiological saline, and then the bacterial cells were resuspended with 50. Mu.L of milk (250 g milk powder/L), and 50. Mu.L of the bacterial suspension was sucked up and added to the center of a 12-well plate, and dried in a desiccator for 20 days. Adding the optimized liquid culture medium 2 mL into 12-well plate of dry damaged bacteria, mixing, and placing into a constant temperature incubator 37 o C18, h, blank group was BPW peptone water and LB broth without addition of substance. Then, 200. Mu.L of the culture medium was aspirated, and OD was measured 600 As a result, it was found that the growth amounts (OD of the strains of Cronobacter and Salmonella in the medium of the present invention 600 ) The method is obviously higher than liquid culture media such as BPW and LB, and the like, and shows that the resuscitating liquid culture medium has good bacteria increasing effect and good universality.
Detailed Description
The invention is further described below with reference to examples.
Example 1
The preparation of liquid medium for recovery of dry lesions from Cronobacter and Salmonella was performed as follows:
accurately weighing each component of the culture medium: 10g of peptone, 1.5g of monopotassium phosphate, 9 g of sodium dihydrogen phosphate with 12 crystal water, 5g of sodium chloride, 3.0g of mannitol, 2.5g of trehalose, 2.5g of betaine and 2.5g of proline are dissolved in 1000 mL of water and uniformly mixed at normal temperature; sterilizing at 121deg.C for 15 min to obtain liquid culture medium for recovering and drying Cronobacter and Salmonella, and sealing and storing.
Cronobacter sakazakii ATCC29544 and Salmonella typhi CMCC50071 were cultured to logarithmic phase, the bacterial solutions were centrifuged, the medium components were washed with physiological saline, then the bacterial cells were resuspended with 50. Mu.L of milk (250 g milk powder/L), 50. Mu.L of the milk bacterial suspension was sucked up into the center of the 12-well plate, and placed in a desiccator for 20 days, 40 days, 60 days.
2.0. 2.0 mL of the resuscitation Medium of the present invention was addedAdding into 12-well plate of Cronobacter dry for different time periods, mixing with Cronobacter dry, culturing at 37deg.C for 18 h, and then sucking 200 μl of 96-well plate to determine OD of resuscitated bacterial solution 600 Repeating the values for three times, and taking an average value;
as can be seen from Table 1, the Cronobacter and Salmonella bacteria were dried for various periods of time (20 days, 40 days and 60 days), and were subjected to resuscitative culture by adding BPW, LB and the culture medium of the present invention, and then subjected to constant temperature culture at 37℃for 18 h, and the OD of the resuscitative bacteria-increasing liquid of the present invention was obtained 600 Compared with BPW and LB, the recovery medium has obviously improved performance, and has good dry injury repair capability.
Example 2
The preparation of liquid medium for recovery of dry lesions from Cronobacter and Salmonella was performed as follows:
accurately weighing each component of the culture medium: 10g of peptone, 1.5g of monopotassium phosphate, 9 g of sodium dihydrogen phosphate with 12 crystal water, 5g of sodium chloride, 3.0g of mannitol, 2.5g of trehalose, 2.5g of betaine and 2.5g of proline are dissolved in 1000 mL of water, uniformly mixed at normal temperature, sterilized at high temperature of 121 ℃ for 15 minutes, and a liquid culture medium for recovering, drying and damaging the Cronobacter and salmonella is obtained, and the liquid culture medium is stored in a sealed manner for standby.
Cronobacter (strains including Cronobacter sakazakii ATCC29544, norobacter Mo Jinsi An Keluo ATCC51329, cronobacter dublinii Cro1018, cronobacter malonate 362, cronobacter zurilensis CICC 21543, cronobacter You Niwo, LMG 26149, cronobacter Kang Dimeng, LMG 26250) and salmonella (strains including Salmonella typhimurium, salmonella typhimurium CMCC50115, salmonella paratyphi CMCC50093 and Salmonella isolates) suspensions were prepared as in example 1 and placed in a desiccator for 20 days.
2.0 mL of the resuscitation Medium of the invention was added to the desiccated Cronobacter and CronobacterUniformly mixing the salmonella with the dried Cronobacter in a 12-well plate, culturing at a constant temperature of 37 ℃ for 18 h, and then sucking 200 mu L of the recovered bacterial liquid OD in a 96-well plate 600 Values were repeated three times and averaged, see table 2;
as can be seen from Table 2, cronobacter (including 7 species of the genus) and Salmonella (strains including Salmonella typhimurium, salmonella typhimurium CMCC50115, salmonella paratyphi A CMCC50093 and Salmonella isolates) were dried for 20 days, and were subjected to resuscitative culture by adding BPW, LB and the medium of the present invention, and culturing at 37℃for 18 h, the OD of the resuscitative enrichment solution of the present invention 600 The recovery medium is remarkably higher than BPW and LB, and has good dry injury repair capability and good universality on Cronobacter and salmonella.

Claims (2)

1. A liquid medium for resuscitating dry lesions in cronobacter and salmonella consisting of: 10g of peptone, 1.5g of potassium dihydrogen phosphate, 9.0g of sodium dihydrogen phosphate, 5.0 g sodium chloride, 2-5g mannitol, 2-6g of trehalose, 2-5g of betaine, 2-6g of proline and 1000 mL water;
the sodium dihydrogen phosphate is sodium dihydrogen phosphate with 12 crystal waters;
and uniformly mixing the substances at normal temperature to obtain the liquid recovery culture medium.
2. A liquid medium for resuscitating dry injury from cronobacter and salmonella according to claim 1, consisting of: 10g peptone, 1.5g potassium dihydrogen phosphate, 9.0g sodium dihydrogen phosphate of 12 crystal waters, 5.0 g sodium chloride, 3.0g mannitol, 2.5g trehalose, 3.0g betaine, 2.5g proline and 1000 mL water.
CN202110924195.9A 2021-08-12 2021-08-12 Liquid medium for recovering dry injury from Cronobacter and Salmonella Active CN113584122B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110924195.9A CN113584122B (en) 2021-08-12 2021-08-12 Liquid medium for recovering dry injury from Cronobacter and Salmonella

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110924195.9A CN113584122B (en) 2021-08-12 2021-08-12 Liquid medium for recovering dry injury from Cronobacter and Salmonella

Publications (2)

Publication Number Publication Date
CN113584122A CN113584122A (en) 2021-11-02
CN113584122B true CN113584122B (en) 2023-09-15

Family

ID=78257426

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110924195.9A Active CN113584122B (en) 2021-08-12 2021-08-12 Liquid medium for recovering dry injury from Cronobacter and Salmonella

Country Status (1)

Country Link
CN (1) CN113584122B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113416765B (en) * 2021-08-12 2022-08-16 合肥工业大学 Detection method of Cronobacter sakazakii in infant formula milk powder

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103865849A (en) * 2014-03-07 2014-06-18 合肥工业大学 Pre-enrichment liquid culture medium for repairing cold and thermal injuries of Cronobacter
WO2015044472A1 (en) * 2013-09-27 2015-04-02 Universidad De Oviedo Method for the simultaneous detection of pathogenic micro-organisms
CN104651247A (en) * 2013-11-25 2015-05-27 刘汉斌 Enterobacter sakazakii chromogenic medium
KR20160052828A (en) * 2014-10-10 2016-05-13 건국대학교 산학협력단 A COMPOSITION FOR DETECTING Cronobacter spp. AND Salmonella spp.AND A METHOD THEREOF

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015044472A1 (en) * 2013-09-27 2015-04-02 Universidad De Oviedo Method for the simultaneous detection of pathogenic micro-organisms
CN104651247A (en) * 2013-11-25 2015-05-27 刘汉斌 Enterobacter sakazakii chromogenic medium
CN103865849A (en) * 2014-03-07 2014-06-18 合肥工业大学 Pre-enrichment liquid culture medium for repairing cold and thermal injuries of Cronobacter
KR20160052828A (en) * 2014-10-10 2016-05-13 건국대학교 산학협력단 A COMPOSITION FOR DETECTING Cronobacter spp. AND Salmonella spp.AND A METHOD THEREOF

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Margot,Heike et al..Evaluation of different buffered peptone water (BPW) based enrichment broths for detection of Gram-negative foodborne pathogens from various food matrices.International Journal of Food Microbiology.2015,第214卷109-115. *
Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA;Dezhao Kong et al.;Food and Agricultural Immunology;第 26卷(第4期);566-576 *
海藻糖对阪崎肠杆菌温度耐受性的影响研究;逯玉东等;现代农业科技(第9期);296-297 *

Also Published As

Publication number Publication date
CN113584122A (en) 2021-11-02

Similar Documents

Publication Publication Date Title
Rouse et al. Dependence of adenovirus replication on arginine and inhibition of plaque formation by pleuropneumonia-like organisms
Stern et al. A differential-selective medium and dry ice-generated atmosphere for recovery of Campylobacter jejuni
Donnelly Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury
CN108103031B (en) Wide-spectrum phage preparation for aquaculture and preparation method thereof
CN111944766B (en) Salmonella typhimurium bacteriophage T156 and application thereof
Bi et al. Sublethal injury and recovery of Escherichia coli O157: H7 by high pressure carbon dioxide
CN108102982B (en) Vacuum freeze-drying protective agent for vibrio metschnikovii and preservation method thereof
CN113584122B (en) Liquid medium for recovering dry injury from Cronobacter and Salmonella
Cheng et al. Influence of acid adaptation on the tolerance of Escherichia coli O157: H7 to some subsequent stresses
Gale Determination of amino acids by use of bacterial amino acid decarboxylases
JP2020518284A (en) Methods and compositions for storing bacteria
CN112457990A (en) Freeze-drying protective agent and application thereof
Marques et al. Acid tolerance and survival of Escherichia coli O157: H7 inoculated in fruit pulps stored under refrigeration
CN103865849B (en) For enrichment liquid body substratum before the cold and hot injury repairing of Cronobacter bacterium
CN113444653B (en) Method for improving control efficiency of yeast on postharvest diseases of fruits and vegetables through induction
Byelashov et al. Fate of post-processing inoculated Listeria monocytogenes on vacuum-packaged pepperoni stored at 4, 12 or 25 C
CN115029247A (en) Lactobacillus plantarum composite freeze-drying protective agent
CN111621461B (en) Method for improving activity and tolerance of bacillus coagulans BC99 and application thereof
CN113201459A (en) Method for quick freeze-drying preservation of microorganisms
Stachura et al. Incomplete bacteriophage-like particles in ultraviolet-irradiated haemophilus
KR101847130B1 (en) A composition, and method for separation of microorganism in food
Zeng et al. Developing a de novo designed broth to rapidly recover lactic acid-injured Escherichia coli to ensure almost no multiplication during repair for precise enumeration
Ferraro et al. Microbiology of Frozen Orange Concentrate: IV. Further Studies of Enterococci in Frozen Concentrate
CN115537365B (en) Resuscitation medium and method for detecting VBNC (viable but non-viable) state acetic acid bacteria in food
CN115786204B (en) Vacuum freeze-drying protective agent and freeze-drying method suitable for vibrio

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant