Culture medium for rapidly culturing psychrophilic bacteria in cow milk and preparation method of culture medium

Abstract

The invention provides a culture medium for rapidly culturing psychrophilic bacteria in cow's milk, the culture medium is prepared from deeply hydrolyzed lactoprotein, glucose, agar and ultra-high temperature sterilized milk, and the material ratio of the deeply hydrolyzed lactoprotein to the glucose to the agar to the ultra-high temperature sterilized milk to deionized water is (1.0-10.0 g): (0.2-3 g): 15.0 g: (50-500 ml): (950-500 ml). The invention also provides a preparation method of the culture medium for rapidly culturing psychrophilic bacteria in cow milk. The culture medium can effectively shorten the culture time and improve the detection and research efficiency in the aspect of rapidly culturing the psychrophilic bacteria in cow milk, the cultured microorganisms are basically the same as MPC and PCA common psychrophilic bacteria culture media in variety, and the culture medium has wide application prospects in the fields of food microorganism detection, dairy product quality control and the like.

Description

Technical Field

[0001] The invention belongs to the field of biology and relates to a microbial culture technology, in particular to a culture medium for rapidly culturing psychrophilic bacteria in milk and a preparation method thereof. Background Art

[0002] Psychrophilic bacteria is a general term for microorganisms that can grow and reproduce at temperatures of 7°C or lower. This type of bacteria can grow and reproduce in a low temperature environment of -20°C to 15°C. Its most suitable growth temperature generally does not exceed 15°C. Because this temperature is much lower than the most suitable growth temperature of other bacteria (the suitable growth temperature of ordinary bacteria is 25°C - 40°C), it is called psychrophilic bacteria.

[0003] Psychrophiles are the most active type of microorganisms when storing milk and food at low temperatures. Compared with other microorganisms, their cell membranes contain a large amount of polyunsaturated fatty acids, which lowers the melting point of the cell membrane and allows it to maintain good fluidity at low temperatures. In addition, psychrophiles also contain antifreeze proteins and cold shock proteins, which help psychrophiles grow at low temperatures.

[0004] The presence of psychrophilic bacteria has a serious impact on the product quality and shelf life of dairy products. The proteases and lipases produced and secreted by psychrophilic bacteria are highly heat-resistant, which will reduce the physical and sensory quality of raw milk and affect its shelf life and shelf life. At the same time, psychrophilic bacteria are also infectious bacteria that can grow under refrigerated conditions, which may cause adverse reactions such as food poisoning, and even death in severe cases. Therefore, rapid cultivation and detection of psychrophilic bacteria is conducive to the control of food safety testing and rapid clinical diagnosis.

[0005] At present, the method of detecting the content of psychrophilic bacteria in milk is time-consuming. For example, the first method of 6.5℃ colony counting method in the national industry standard SN / T 2552.4-2010 "Microbiological Examination Methods for Hygiene of Milk and Dairy Products-Part 4: Counting Colonies of Psychrophilic Microorganisms" requires 10 days of cultivation; the second method of 21℃ colony counting method is time-consuming, but the colony growth is small and counting is inconvenient, which affects the accuracy. In practical applications, the culture media commonly used for psychrophilic bacteria in milk, such as PCA medium and MPC medium, grow slowly when culturing psychrophilic bacteria in milk, and it takes more than 6 days to obtain colonies for selection.

[0006] The method of plate detection of psychrophilic bacteria has the advantages of being simple, easy to observe, and accurate in counting, but its limitation is that it takes too long. At present, there are also some more advanced rapid detection methods, such as indirect detection of protease activity, direct fluorescence filtration, electrical impedance, PCR, flow cell method, enzyme-linked immunosorbent assay, etc. The advantages of these methods are rapid and accurate, but their limitations are complicated operation steps and high cost. In summary, the existing culture medium culture cycle is too long to meet the needs of rapid culture and detection of psychrophilic bacteria, and the more advanced rapid detection methods are cumbersome to operate and too expensive, which is not conducive to food safety detection and control and rapid clinical diagnosis. Therefore, it is of great significance to develop a culture medium that can quickly culture psychrophilic bacteria in milk.

[0007] Deeply hydrolyzed milk protein is a commercially available substance that is produced by deeply decomposing intact milk protein (mainly casein and whey protein) through an enzymatic hydrolysis process. Generally speaking, deep hydrolysis will degrade milk protein molecules into smaller peptides, the molecular weight of which is usually around 300-1000 Daltons, and some are even further decomposed into free amino acids (deeply hydrolyzed milk protein can be prepared using the method provided in CN110679958A).

[0008] Extensively hydrolyzed milk protein belongs to infant formula for special medical purposes. The national standard is: "General Rules for Infant Formula for Special Medical Purposes" (GB 25596-2010).

[0009] Ultra-high temperature sterilized milk (UHT milk) is a commercially available substance that refers to a dairy product that is heated by a heat exchanger in a continuous flow state, heated to 135-150°C in a very short time (usually 2-8 seconds), maintained for a period of time (several seconds-tens of seconds), and then packaged in a sterile environment.

[0010] Ultra-high temperature sterilization is mainly based on the killing effect of high temperature on microorganisms. The cell structure and physiological functions of microorganisms (such as bacteria, spores, etc.) will be destroyed under high temperature. When the temperature reaches the range of 135-150℃, biological macromolecules such as proteins and nucleic acids in microorganisms will denature. For example, bacterial enzymes will lose their activity at high temperatures. These enzymes are necessary for bacterial metabolism. After the enzymes are inactivated, bacteria cannot survive and reproduce normally. Spores are a dormant form of bacteria with strong stress resistance, but in an ultra-high temperature environment, the structure and function of spores will also be destroyed, thereby achieving a thorough sterilization effect. The ultra-high temperature sterilization process kills almost all microorganisms and their spores, and the aseptic packaging prevents the re-contamination of external microorganisms, so that the product can maintain stable quality for a long time.

[0011] Compared with traditional long-term high-temperature sterilization methods, ultra-high temperature sterilized milk (UHT milk) has relatively less nutritional loss: although it has been treated with ultra-high temperature, the loss of nutrients such as protein and calcium in ultra-high temperature sterilized milk is relatively small due to the short heating time. For example, the whey protein in cow's milk has limited denaturation under ultra-high temperature treatment, and most of its nutritional value can still be retained. Summary of the invention

[0012] In view of the above problems existing in the prior art, the present invention provides a culture medium for rapidly culturing psychrophilic bacteria in bovine milk and a preparation method thereof. The culture medium for rapidly culturing psychrophilic bacteria in bovine milk and a preparation method thereof are intended to solve the technical problem that the culture cycle of the culture medium in the prior art is too long and cannot meet the needs of rapidly culturing and detecting psychrophilic bacteria.

[0013] The invention provides a culture medium for rapidly culturing psychrophilic bacteria in milk (deeply hydrolyzed milk protein medium, abbreviated as DHMP medium), which is prepared by mixing deeply hydrolyzed milk protein, glucose, agar, ultra-high temperature sterilized milk and deionized water. The material ratio of the deeply hydrolyzed milk protein, glucose, agar, ultra-high temperature sterilized milk and deionized water is (1.0g-10.0g): (0.2g-3g): 15.0g: (50ml-500ml): (500ml-950ml).

[0014] Furthermore, the material ratio of the deep hydrolyzed milk protein, glucose, agar, ultra-high temperature sterilized milk and deionized water is 5.0g:1g:15.0g:250ml:750ml.

[0015] The present invention also provides a method for preparing the culture medium for rapidly culturing psychrophilic bacteria in milk, comprising the following steps: 1) Weigh each raw material according to the material ratio; 2) Place the extensively hydrolyzed milk protein in a container; 3) Measure the ultra-high temperature sterilized milk, add it to the above container, and stir it thoroughly to ensure that the deep hydrolyzed milk protein is completely dissolved; 4) Then add glucose and continue stirring until dissolved; 5) Add agar and stir to mix all ingredients evenly; 6) Add some deionized water to 80% of the volume of the culture medium, adjust the pH value of the culture medium to 6.8-7.2, add the remaining deionized water and mix well; 7) Sterilize the plate, mix all ingredients, cool to 45-55°C, pour into a plate in a clean bench, wait for the plate to cool, and refrigerate for later use.

[0016] Specifically, low-pressure steam sterilization can be used for sterilization, specifically at 115° C. and keeping warm for 30 minutes.

[0017] The beneficial effects of the present invention are as follows: 1. Rapid growth advantage: The deep hydrolyzed milk protein culture medium of the present invention is used to isolate and culture psychrophilic bacteria in raw milk. When cultured at 6.5°C, it only takes 4 days to grow colonies for selection. Compared with the commonly used PCA culture medium and MPC culture medium (which requires more than 6 days), the culture time is significantly shortened and the detection efficiency is greatly improved.

[0018] 2. Easy operation: During the preparation of the culture medium, the raw materials are widely available and easy to obtain. The preparation steps are relatively simple and do not require complex equipment and technology, making it easy to promote and apply in laboratories or production environments.

[0019] 3. Cost-effectiveness: The culture medium of the present invention has relatively simple ingredients and low cost, and can reduce the cost of testing or research while ensuring the culture effect.

[0020] Compared with the prior art, the technical effect of the present invention is positive and obvious. The culture medium of the present invention has significant advantages in rapidly culturing psychrophilic bacteria in milk, can effectively shorten the culture time, improve the detection and research efficiency, and the types of microorganisms cultured are basically the same as those of MPC and PCA, two commonly used psychrophilic bacteria culture media, and has broad application prospects in the fields of food microbiological detection, dairy product quality control, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The relationship between the total number of colonies on MPC, PCA, and DHMP medium plates and time in a 10-³ dilution gradient is shown.

[0022] Figure 2 Shown are photos of the gradient plates of the three culture media cultured at 6.5°C for 132 hours.

[0023] Figure 3 A heat map (species level) of the relative abundance of microorganisms grown on MPC, PCA, and DHMP medium plates is shown.

[0024] Figure 4 The bar graphs (species level) and similarity of microbial composition of the microorganisms grown on MPC, PCA, and DHMP culture medium plates are shown.

[0025] The raw materials used in the following experimental procedures are all commercially available products unless otherwise specified. DETAILED DESCRIPTION

[0026] Example 1 MPC, PCA, and DHMP culture media were used to dilute and plate psychrophilic bacteria in milk and then cultured and counted 1.1 Formula of each culture medium: 1. Psychrophilic bacteria count agar medium (MPC)

[0027] pH 7.0±0.2 (25℃).

[0028] 2. Plate Count Agar (PCA)

[0029] pH 7.0±0.2 (25℃).

[0030] 3. Deeply hydrolyzed milk protein medium (DHMP)

[0031] pH 7.0±0.2 (25℃).

[0032] 1.2 Preparation and sterilization of DHMP medium oAccording to the above preparation method, accurately weigh 5.0 g of extensively hydrolyzed milk protein and put it into a 1L beaker.

[0033] o Use a measuring cylinder to measure 250 ml of UHT milk and slowly pour it into the beaker while stirring with a glass rod to fully dissolve the extensively hydrolyzed milk protein.

[0034] o Weigh 1g of glucose into the beaker and continue stirring until it is completely dissolved.

[0035] o Add 15.0 g agar and mix well.

[0036] o Add deionized water to 800ml, measure the pH value of the solution with a pH meter, and adjust the pH to 7.0 with an acid-base regulator (such as hydrochloric acid or sodium hydroxide), add deionized water to 1000ml, and mix well; oPut the wrapped culture dish into the autoclave and sterilize at 115℃ for 30 minutes. Take it out after sterilization and wait for the culture medium to cool to 50℃ before pouring it into a plate for later use.

[0037] o Dispense the culture medium into 90mm diameter culture dishes while it is still hot, about 20ml per dish. After cooling, refrigerate at 4℃ for later use.

[0038] 1.3 Psychrophilic bacteria culture experiment o Take fresh raw milk sample and dilute it in 10-fold series with sterile saline, such as 1, 10⁻¹, 10⁻², 10⁻³, 10⁻4 Sample solutions of different dilutions.

[0039] o Take 0.1 ml of sample solution with different dilutions respectively and inoculate it on the prepared MPC, PCA, and DHMP culture medium plates. Make three parallels for each dilution.

[0040] oAt the same time, the samples were inoculated on PCA medium and MPC medium plates in the same way as controls.

[0041] o Invert the inoculated culture medium plate and place it in a constant temperature incubator at 6.5℃.

[0042] oObserve and record the growth of psychrophilic bacterial colonies on the culture medium every day, including the time, size, morphology, and number of colonies.

[0043] 1.4 Culture results 1. After 4 days of culture, obvious psychrophilic bacterial colonies appeared on the extensively hydrolyzed milk protein culture medium. The colonies were moderate in size and clear in morphology, and could be selected for subsequent identification and analysis.

[0044] 2. On the PCA medium and MPC medium, the colony growth was not obvious at 4 days, and it was not until 6 days that colonies of similar size to those on the extensively hydrolyzed milk protein medium grew.

[0045] 3. 10 - ³ Relationship between the total number of colonies on MPC, PCA, and DHMP culture medium plates and time under dilution gradient (see Figure 1 On the DHMP medium plate, after culturing at 6.5℃ for 84 hours, a small number of countable colonies can be seen. A large number of colonies appear after culturing at 6.5℃ for 120 hours and 132 hours. The number of colonies increases and quickly enters the plateau phase after 132 hours. On the MPC culture plate, after culturing at 6.5℃ for 132 hours ( Figure 2 ), a small number of countable colonies can be seen, and a large number of colonies appeared after culturing at 6.5℃ for 192 hours and 204 hours. The number of colonies increased and quickly entered the plateau phase after 204 hours; on the PCA culture plate, a small number of countable colonies can be seen after culturing at 6.5℃ for 144 hours, and a large number of colonies appeared after culturing at 6.5℃ for 204 hours and 216 hours. The number of colonies increased and quickly entered the plateau phase after 216 hours; The above results show that the colony growth rate of psychrophilic bacteria in milk on DHMP medium plates is much faster than that on MPC and PCA medium plates, and the number of psychrophilic bacteria finally cultured by the three culture media is similar, which proves that the number of colonies cultured by DHMP medium can be similar to that on MPC and PCA medium plates.

[0046] Example 2 After the psychrophilic bacteria in milk were diluted and plated using MPC, PCA, and DHMP culture media, all the cultured colonies were washed off and the diversity of microorganisms was detected by high-throughput sequencing.

[0047] According to the experimental results of the culture in Example 1, the colonies on the MPC, PCA, and DHMP culture medium plates were selected at a 10-³ dilution gradient, and the bacteria were eluted with 1 ml of sterilized saline, mixed, and 100 μL of the bacterial solution was taken to extract the genome using the proteinase K lysis method.

[0048] 2.1 Genomic DNA extraction steps: 1. Transfer 100 μL of eluted bacterial solution to a sterilized 2 ml EP tube and add sterile PBS to 1 mL.

[0049] 2. Add 1 / 10 volume of lysozyme (50 mg / mL) and incubate in a 37°C water bath for 30 min.

[0050] 3. Add 15 μL proteinase K (10 mg / mL) and 1 / 20 volume of 20% SDS to a final concentration of 1%, invert to mix, and incubate in a 55°C water bath for 2 h.

[0051] 4. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to 2 mL, mix thoroughly until a suspension forms, and centrifuge at 11,000 rpm for 10 min.

[0052] 5. Transfer the upper liquid phase into a clean 2 mL centrifuge tube and repeat the above steps until the supernatant is clear.

[0053] 6. Transfer the upper liquid phase into a clean 2 mL centrifuge tube, add 1 μL of glycogen (20 mg / mL) and 2 / 3 volume of ice isopropanol, invert several times to mix, and precipitate at -20°C for 2 h.

[0054] 7. Centrifuge at 11000rpm for 10min, discard the supernatant, rinse the DNA twice with 500μL 70% ethanol, centrifuge at 11000rpm for 5min after each wash, and carefully remove the ethanol.

[0055] 8. Centrifuge for 1 minute, aspirate the remaining ethanol with a pipette, air-dry the DNA pellet for 5-10 minutes, add 100μ TE, shake briefly and centrifuge, place in a 37 degree water bath for 30 minutes 9. Take 3 μL of sample for detection.

[0056] 2.2 Primers for 16S amplicon high-throughput sequencing According to the requirements of Illumina high-throughput sequencing, bidirectional sequencing was performed, and the target region and fusion primers with "5' adapter-barcode-sequencing primer-specific primer-3'" were designed. The library was constructed by a two-step PCR amplification method (the construction method was the conventional literature method).

[0057] Primer sequences: F 5'-AATGATACGGCGACCACCGAGATCTACAC-- barcode F2 TCTTTCCCTACACGACGCTCTTCCGATCT- barcodeF1- specific primer-3' (SEQ ID NO.1) R 5'-CAAGCAGAAGACGGCATACGAGAT- barcode R2- GTGACTGGAGTTCCTTGGCACCCGAGAATTCCA- barcode R1- specific primer-3' (SEQ ID NO.2) The specific primer sequences are as follows:

[0058] 2.3 16S amplicon high-throughput sequencing PCR amplification The library construction adopts a two-step PCR amplification method. First, specific primers (described as inner primers below) are used to amplify the target fragment, the target fragment is recovered by gel, and then the recovered product is used as a template for secondary PCR amplification (described as amplification by outer primers below). The purpose is to add the adapters, sequencing primers, and barcodes required for Illumina platform sequencing to both ends of the target fragment.

[0059] According to the experimental requirements, the primers are designed as follows: F inner primer: 5'-TTCCCTACACGACGCTCTTCCGATCT-barcodeF1 specific primer-3' (SEQ ID NO. 5).

[0060] F outer primer: 5′-AATGATACGGCGACCACCGAGATCTACAC- barcode F2-TCTTTCCCTACACGACGCTC -3′ (SEQ ID NO. 6).

[0061] R inner primer: 5'-GAGTTCCTTGGCACCCGAGAATTCCA- barcodeR1 specific primer-3' (SEQ ID NO. 7).

[0062] R outer primer: 5′-CAAGCAGAAGACGGCATACGAGAT- barcodeR2-GTGACTGGAGTTCCTTGGCACCCGAGA-3′ (SEQ ID NO. 8).

[0063] 2.4 PCR amplification PCR amplification system:

[0064] PCR amplification procedure:

[0065] The PCR amplification products were recovered by 2% agarose gel electrophoresis (keep the electrophoresis tank clean and replace the electrophoresis buffer). The AxyPrep DNA gel recovery kit from AXYGEN was used to recover the products. The recovered products were quantified.

[0066] All PCR products were recovered using the AxyPrep DNA gel recovery kit and quantitatively detected using Qubit 3.0. The samples were mixed in equal proportions and then amplified by secondary PCR, and the adapters required for sequencing were added.

[0067] 2.5 Secondary PCR amplification and sequencing PCR amplification system:

[0068] PCR amplification procedure:

[0069] After the secondary PCR amplification, the PCR products were subjected to subsequent gel recovery experiments. The PCR amplification products were recovered by cutting the gel on 2% agarose gel electrophoresis (the electrophoresis tank was kept clean and the electrophoresis buffer was replaced).

[0070] The target product was recovered using the AxyPrep DNA gel recovery kit from AXYGEN. After recovery, the product was sequenced using the Illumina MiSeq sequencer, and high-throughput sequencing was performed according to the sequencer's instructions to obtain sequencing data of the 16S amplicon, providing a basis for subsequent analysis of microbial community structure and diversity.

[0071] 2.6 Microbial diversity analysis results Table 1: Microbial diversity composition (species level) grown on MPC, PCA, and DHMP culture plates

[0072] like Figure 3 and 4 As shown in the figure, the data analysis of the diversity of microorganisms grown on MPC, PCA, and DHMP culture medium plates showed that in terms of the number of species, the microbial species detected by DHMP culture medium were highly similar to those obtained by MPC and PCA culture medium, with little difference.

[0073] Various microorganisms, including Pseudomonas_fragi, Pseudomonas_azotoformans, and Pseudomonas_azotoformans, appeared on all three culture media, indicating that there are certain commonalities in the core microbial species.

[0074] In the three culture media, Pseudomonas_fragi was one of the dominant species. Although its relative abundance (37.826%) was lower than that in MPC (74.265%) and PCA (83.127%), it still occupied an important position. Pseudomonas_gessardii (22.321%) became one of the dominant species on DHMP medium, while its relative abundance was lower on MPC and PCA medium (0.787% and 0.719%, respectively), which showed that DHMP medium had certain advantages in cultivating this species, but it also cultivated dominant species commonly found on the other two culture media, and there was no obvious loss of dominant species.

[0075] The nutrient combination of DHMP medium may be more conducive to the growth of a variety of microorganisms. Although there are differences in relative abundance with MPC and PCA medium, it can support the growth of most of the same types of microorganisms and provide better growth opportunities for some species with relatively low abundance on other media (such as Pseudomonas_gessardii). This shows that its nutritional selectivity is broader and may better reflect the actual situation of the microbial community in some cases.

[0076] Based on the above analysis, DHMP medium has a high similarity with MPC and PCA medium in terms of cultured microbial species. Although there are differences in species relative abundance and individual dominant species, the species information cultured has not been significantly reduced. Under specific microbial culture test requirements, especially when the main microbial species of concern have commonalities on the three culture media, DHMP medium has the potential to replace MPC and PCA medium for microbial culture tests, which provides more options and convenience for microbial culture research.

Claims

1. A culture medium for rapidly culturing psychrophilic bacteria in milk, characterized in that: The product is prepared from deep hydrolyzed milk protein, glucose, agar, ultra-high temperature sterilized milk and deionized water, wherein the material ratio of the deep hydrolyzed milk protein, glucose, agar, ultra-high temperature sterilized milk and deionized water is (1.0g~10.0g):(0.2g~3g):15.0g:(50ml~500ml):(500ml~950ml).

2. A culture medium for rapidly culturing psychrophilic bacteria in milk according to claim 1, characterized in that: The material ratio of the deep hydrolyzed milk protein, glucose, agar, ultra-high temperature sterilized milk and deionized water is 5.0g:1g:15.0g:250ml:750ml.

3. The method for preparing a culture medium for rapidly culturing psychrophilic bacteria in milk according to claim 1, characterized in that The steps include: 1) Weigh each raw material according to the material ratio; 2) Place the extensively hydrolyzed milk protein in a container; 3) Measure the ultra-high temperature sterilized milk, add it to the above container, and stir it thoroughly to ensure that the deep hydrolyzed milk protein is completely dissolved; 4) Then add glucose and continue stirring until dissolved; 5) Add agar and stir to mix all ingredients evenly; 6) Add some deionized water to 80% of the volume of the culture medium, adjust the pH value of the culture medium to 6.8-7.2, add the remaining deionized water and mix well; 7) Sterilize the plate, mix all ingredients, cool to 45-55°C, pour into a plate in a clean bench, wait for the plate to cool, and refrigerate for later use.

4. The method for preparing a culture medium for rapidly culturing psychrophilic bacteria in milk according to claim 3, characterized in that: Sterilization is carried out by low-pressure steam sterilization, the temperature of which is 115°C and the temperature is kept for 30 minutes for sterilization.

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