Method for detecting viable bacterium types and dominant bacteria in dairy products and application

Abstract

The invention belongs to the technical field of microbial flora detection, and particularly relates to a method for detecting viable bacteria types and dominant bacteria in dairy products and application. A to-be-detected milk sample is optimized before high-throughput sequencing, firstly, milk is cultured in nutrient broth, peptone and beef powder contained in the nutrient broth can provide carbon and nitrogen sources, vitamins and growth factors for growth of microorganisms, and most microorganisms can breed in the nutrient broth. The method comprises the following steps: inoculating a milk sample to be analyzed into nutrient broth for culture by utilizing the characteristics that the nutrient broth is rich in nutrient substances, live microorganisms have a large amount of DNA information in the broth through reproduction, decay bacteria cannot reproduce and the nutrient broth is finally diluted through transfer culture, so that the live microorganisms in the milk sample are reproduced in quantity; the DNA of the microorganisms which decline originally in the milk is diluted, and finally, the information of the types of the living microorganisms in the milk sample is obtained by combining a high-throughput sequencing analysis means.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial flora detection, and particularly relates to a detection method and application of live bacteria species and dominant bacteria in dairy products. Background Art

[0002] In the quality risk analysis of dairy products, it is often necessary to study the types of residual microorganisms in samples after heat treatment or other sterilization treatments in order to evaluate the effectiveness of quality control measures and further assess the level of food safety risk.

[0003] At present, the analysis and identification methods for microbial species mainly include identification techniques that rely on traditional microbial separation and culture (referring to methods that require separation to pure strains for identification, such as biochemical experiments, MALDI-TOF mass spectrometry, 16S / ITS sequencing) and non-culture techniques, such as high-throughput sequencing technology. Both methods have their own advantages and disadvantages. For example, although the experimental process of traditional separation and culture identification technology is relatively simple, there are many steps in the process of separation, purification, and identification, and all isolated bacteria need to be purified and analyzed, which is a huge workload. High-throughput sequencing can simultaneously sequence more DNA or RNA samples, which is very convenient and fast to obtain a large amount of gene sequence data. It has been widely used in a large number of microbial related studies. However, the object of high-throughput sequencing analysis is all DNA in the sample, which makes it impossible to distinguish whether the detected DNA comes from decaying microorganisms or surviving microorganisms. The sequencing results contain a large number of dead bacteria after sterilization, which makes it impossible to accurately obtain live bacteria and dominant bacteria in the sample. Therefore, the current high-throughput sequencing technology cannot meet the analysis of live bacteria and dominant bacteria in dairy samples. Summary of the invention

[0004] The purpose of the present invention is to overcome the technical defect in the prior art that high-throughput sequencing analysis cannot analyze the types of live bacteria remaining in dairy samples after sterilization, and to provide a method and application for detecting the types of live bacteria and dominant bacteria in dairy products.

[0005] In a first aspect, the present invention provides a method for detecting the types of live bacteria and dominant bacteria in dairy products, comprising the following steps:

[0006] Step 1, taking a milk sample to be tested and adding it into sterilized nutrient broth, culturing for 18-24 hours to obtain a culture for standby use; the nutrient components of the nutrient broth include peptone, beef powder, and glucose;

[0007] Step 2: Perform high-throughput sequencing analysis on the cultures to obtain experimental data, wherein the bacteria with a relative abundance greater than 1% are the dominant bacteria.

[0008] In the technical scheme of the present invention, the milk sample to be tested is optimized before high-throughput sequencing, and the nutrient-rich nature of the nutrient broth is utilized. The living microorganisms will have a large amount of DNA information in the broth through reproduction, while the dead bacteria will not reproduce and will eventually be diluted through transfer culture. The milk sample to be analyzed is inoculated into the nutrient broth for culture, so that the living microorganisms in the milk sample are reproduced in large quantities. Among them, the peptone and beef powder contained in the nutrient broth can provide carbon and nitrogen sources, vitamins and growth factors for the growth of microorganisms, and most microorganisms can reproduce in it. The DNA of the originally dead microorganisms in the milk is diluted, and the milk sample is cultured and then combined with high-throughput sequencing analysis. Finally, the types of living microorganisms and dominant bacteria information in the milk sample can be obtained through data analysis.

[0009] Transferring milk to nutrient broth for cultivation can proliferate the viable bacteria remaining in milk, but the composition of the above-mentioned nutrient broth cannot satisfy that all types of bacteria in milk proliferate well. Common genus in raw milk generally includes bacillus, lactobacillus, enterobacter, and cocci such as enterococcus, staphylococcus, streptococcus, and some pseudomonads. In these genus, common bacteria in dairy products such as thermophilic streptococcus, bacillus subtilis, lactobacillus grow extremely weak in nutrient broth. Taking the above-mentioned nutrient broth to carry out milk sample cultivation has the technical defect that the accuracy of some genus data is low, therefore, as the preferred technical scheme of the present invention, the nutrients of the nutrient broth also include mannitol and sodium pyruvate.

[0010] By optimizing the milk samples before high-throughput sequencing, the nutrient broth formula was further optimized according to the types of live bacteria in dairy products. In the preferred nutrient broth, peptone, beef powder and glucose can provide carbon and nitrogen sources, vitamins and growth factors for microbial growth, mannitol and sodium pyruvate can compensate for certain cell damage, so that most microorganisms in milk can reproduce in it.

[0011] Preferably, the nutrient broth comprises the following raw materials in parts by weight: 10-15 parts of peptone, 3-5 parts of beef powder, 4-6 parts of sodium chloride, 10-20 parts of glucose, 4-6 parts of mannitol, 9-11 parts of sodium pyruvate, and 1000 parts of water.

[0012] Further preferably, the nutrient broth comprises the following raw materials in parts by weight: 10-12 parts of peptone, 3-4 parts of beef powder, 4-5 parts of sodium chloride, 10-15 parts of glucose, 4-5 parts of mannitol, 9-10 parts of sodium pyruvate, and 1000 parts of water.

[0013] Preferably, the nutrient broth is prepared as follows: corresponding ingredients are weighed according to weight ratio, mixed evenly in an aqueous solution to form a mixed solution, and the mixed solution is sterilized at 120° C.-125° C. for 10-20 minutes to obtain the nutrient broth.

[0014] Preferably, the volume ratio of the milk sample to be tested to the nutrient broth is 1:(8-12).

[0015] Preferably, the volume ratio of the milk sample to be tested to the nutrient broth is 1:(9-10).

[0016] Preferably, the parameters of the high-throughput sequencing analysis are: the sequencing project type is 16S / 18S / ITS microbial classification sequencing, the bacterial amplification type is "bacteria", and the amplification region is "bacteria V3-V4"; the fungal amplification type is "fungus", and the amplification region is "fungus ITS1-ITS2".

[0017] In step 2, the sample processing method for high-throughput sequencing analysis is kept uniform, and the sampling amount is consistent. If necessary, a quality control strain can be added to determine the error of the high-throughput detection process.

[0018] In a second aspect, the present application provides an application of the method for detecting dominant bacteria in determining dominant bacteria in milk.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] In the technical scheme of the present invention, the milk sample to be tested is optimized before high-throughput sequencing, and the nutrient-rich nature of the nutrient broth is utilized. The living microorganisms will have a large amount of DNA information in the broth through reproduction, while the dead bacteria will not reproduce and will eventually be diluted through transfer culture. The milk sample to be analyzed is inoculated into the nutrient broth for culture, so that the living microorganisms in the milk sample are reproduced in large quantities. Among them, the peptone and beef powder contained in the nutrient broth can provide carbon and nitrogen sources, vitamins and growth factors for the growth of microorganisms, and most microorganisms can reproduce in it. The DNA of the originally dead microorganisms in the milk is diluted, and the milk sample is cultured and then combined with high-throughput sequencing analysis. Finally, the types of living microorganisms and dominant bacteria information in the milk sample can be obtained through data analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a bar graph showing the significant difference in absorbance values ​​of different bacterial strains before and after the improvement of nutrient broth ingredients; DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0023] Example 1

[0024] This embodiment provides a method for detecting dominant bacteria in milk, which specifically comprises the following steps:

[0025] Step 1: two portions of the milk sample to be tested are set, and are marked as sample #1 and reference sample respectively; the processing of sample #1 includes the following steps: adding sample #1 to sterilized nutrient broth, culturing for 18-24 hours to obtain a culture, and setting aside;

[0026] Specifically, the composition of the nutrient broth is: 10 parts of peptone, 3 parts of beef powder, 5 parts of sodium chloride, 1 part of glucose, and 1000 parts of water; the peptone and beef powder contained in the nutrient broth can provide carbon and nitrogen sources, vitamins and growth factors for the growth of microorganisms, and most microorganisms can reproduce in it. Mix the above ingredients evenly and sterilize at 121°C for 15 minutes. After sterilization, divide into 10mL for later use;

[0027] 1 mL of sample #1 was added to 10 mL of the nutrient broth to obtain a culture.

[0028] Step 2: Perform high-throughput sequencing analysis on the milk sample to be tested (reference sample) that has not been cultured in the nutrient broth and the culture to obtain experimental data, and compare and analyze the experimental data to obtain the dominant bacteria species in the milk. The relative abundance data is used as the final data result.

[0029] As shown in Table 1, Table 1 shows the types of bacteria with a relative abundance greater than 1% in this sample sequencing.

[0030] Table 1: Summary of relative abundance changes of bacteria in sample #1 and reference samples

[0031] microorganism Reference samples Sample #1 Pseudomonas 66.54％ 0.01％ Acinetobacter 13.52％ 3.84％ Lelliottia_sp. 2.67％ 0％ Anoxybacillus_flavithermus 1.76％ 0.12％ Pseudomonas_mosselii 1.24％ 0 Ralstonia_pickettii 0.27％ 23.41％ Caulobacteraceae_sp. 0.21％ 24.05％ Streptococcus 0.01％ 9.07％ Chitinophaga_cf. 0.03％ 3.43％ Blastomonas_sp. 0.02％ 2.36％

[0032] From the data in Table 1, it can be seen that the relative abundance of Ralstonia_pickettii, Caulobacteraceae_sp., Streptococcus and other bacteria in the untreated reference sample is significantly increased compared with the sample #1 cultured in nutrient broth, and it can be judged that these bacteria are the dominant bacteria in the milk sample #1. The growth rate determines that these bacteria have been reproduced after being cultured in broth, that is, they are live bacteria in the sample. Most of the bacteria with a relative abundance greater than 1% in the reference sample have a relative abundance less than 1% after being cultured in broth, indicating that these bacteria are already dead microorganisms in milk.

[0033] In the technical solution of this embodiment, sample #1 is optimized before high-throughput sequencing, and the nutrient-rich nature of the nutrient broth is utilized. The living microorganisms will have a large amount of DNA information in the broth through reproduction, while the dead bacteria will not reproduce, so the relative abundance in the sample will be reduced. The milk sample to be analyzed is inoculated into the nutrient broth for cultivation, so that the living microorganisms in the milk sample are reproduced in large quantities, and the DNA of the originally dead microorganisms in the milk is diluted. Finally, high-throughput sequencing analysis is performed to obtain information on the types of living microorganisms in the milk sample. This is something that cannot be accurately determined by directly performing high-throughput sequencing on the sample.

[0034] Example 2

[0035] This embodiment uses a modified broth to conduct a verification test; a determined strain is selected and placed in sterilized milk to form a "milk sample to be tested", and the "milk sample to be tested" is subjected to direct high-throughput sequencing analysis and high-throughput sequencing after modified broth culture.

[0036] The specific method is as follows:

[0037] Step 1: Select different types of bacteria commonly found in dairy products, such as Bacillus, Negative Bacillus, Positive Bacillus, Positive Cocci, Streptococcus, Staphylococcus, and Pseudomonas.

[0038] Bacillus selection: Bacillus subtilis and Bacillus thermoamylophilus;

[0039] Negative bacilli selection: Citrobacter freundii and Escherichia coli;

[0040] Positive bacilli selection: Lactobacillus helveticus;

[0041] Streptococcus selection: Streptococcus thermophilus;

[0042] Staphylococcus selection: Staphylococcus epidermidis;

[0043] Pseudomonas selection: Pseudomonas aeruginosa;

[0044] Positive cocci selection: Enterococcus faecalis;

[0045] Table 2: Standard strain name and number information

[0046] Strain name (Chinese) Strain name (Latin) Strain number Bacillus subtilis Bacillus subtilis FSCC 115037 Citrobacter freundii Citrobacter freundii FSCC 135002 Enterococcus faecalis Enterococcus faecalis FSCC 146002 Escherichia coli Escherichia coli FSCC 149002 Streptococcus thermophilus Streptococcus thermophilus FSCC 225005 Staphylococcus epidermidis Staphylococcus epidermidis FSCC 223011 Lactobacillus helveticus Lactobacillus helveticus CICC 6032

[0047] Table 3: Name and number information of self-isolated strains

[0048] Strain name (Chinese) Strain name (Latin) Gene accession number Similarity Pseudomonas aeruginosa Pseudomonas aeruginosa JQ669958.1 99％ Bacillus thermomylovora Bacillus Thermoamylovorans / /

[0049] The formula of the nutrient broth in this embodiment is a further preferred formula. In order to distinguish it from the nutrient broth in the embodiment, the nutrient broth in Example 1 is marked as a basic nutrient broth, and the nutrient broth in this embodiment is an improved nutrient broth.

[0050] The above different strains were inoculated into the basic nutrient broth of Example 1 and the improved nutrient broth of this example, respectively, and cultured at 36°C for 24 hours. The absorbance at 600 nm was detected by SpectraMax ABS enzyme label analyzer. Three tubes of each strain were measured in parallel in the two broths. After the absorbance value of the strain was subtracted from the blank value of the corresponding broth, it was calculated whether there was a significant difference, so as to compare the growth of each type of bacteria in the two broths. For details, the significant difference analysis is shown in Figure 1 .

[0051] Note 1: Ingredients and preparation method of improved nutrient broth: peptone 10g, beef powder 3g, sodium chloride 5g, glucose 10g, mannitol 5g, sodium pyruvate 10g, water 1000mL, sterilized at 121℃ for 15 minutes. After sterilization, it can be divided into 10mL for later use.

[0052] Step 2: inoculate Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii, Enterococcus faecalis, Bacillus subtilis, and Staphylococcus epidermidis into nutrient broth and culture for 24 hours, and inoculate Streptococcus thermophilus into MRS broth and culture for 24 hours.

[0053] Step 3: Heat and inactivate Escherichia coli, Pseudomonas aeruginosa, and Streptococcus thermophilus (heating at 100°C for 10 minutes) to obtain the dead bacterial DNA of these three bacteria. Take 1 ml of the heated sample and pour it into PCA (Escherichia coli, Pseudomonas aeruginosa) or MC (Streptococcus thermophilus) culture medium to detect the number of live bacteria to determine whether all of them are inactivated. The remaining 4 bacteria are kept as live bacteria for standby use.

[0054] Step 4: Add all the "dead bacteria" DNA to the sterile pure milk sample, mix well and set aside.

[0055] Step 5: Add the remaining four types of "live bacteria" to step 4 as the sample "stock solution" for high-throughput sequencing. This sample stock solution includes both dead bacteria DNA and live bacteria DNA.

[0056] Step 6: Take 1 mL of the sample "stock solution" in step 5 and add it to 10 mL of sterilized modified nutrient broth and culture for 18-24 hours.

[0057] Note 2: The reason for taking only 1 ml here is that the standard strain has a high concentration, and 1 ml of sample is sufficient for analysis. However, for daily samples, 25 mL is still needed for more representativeness.

[0058] Step 7: Perform high-throughput sequencing analysis on the samples processed in steps 5 and 6. The sequencing project type is 16S / 18S / ITS microbial classification sequencing, the amplification type is "bacteria", and the amplification region is "bacteria V3-V4".

[0059] The growth of various bacteria in the two broths is shown in Table 4, which shows the absorbance data of different bacteria after being cultured in the basic nutrient broth of Example 1 and the improved nutrient broth of this example.

[0060] Table 4: Absorbance data of different strains after culture in basic nutrient broth and improved nutrient broth

[0061]

[0062]

[0063] By culturing different bacterial strains in basic nutrient broth and modified nutrient broth and obtaining the absorbance values ​​respectively, and then deducting the corresponding blank broth absorbance values, the average absorbance value of each bacterial strain was calculated. It can be seen that 7 out of 9 bacterial strains had higher absorbance values ​​in modified broth than in basic broth, especially Bacillus subtilis, Streptococcus thermophilus, and Lactobacillus helveticus. These three strains had no absorbance values ​​in basic nutrient broth, while the absorbance values ​​in modified nutrient broth increased to 0.05-0.49.

[0064] Combination Figure 1 From the absorbance value significant difference analysis chart, it can be seen that 7 out of 9 strains of bacteria have significant differences in absorbance values ​​between basic nutrient broth and improved nutrient broth, and the absorbance values ​​of the improved broth are all higher than those of the basic broth, indicating that the improved nutrient broth is more suitable for the growth of these types of bacteria. In particular, the improvement effect is obvious for Bacillus subtilis, Streptococcus thermophilus, and Lactobacillus helveticus, which are common bacteria in dairy products. Escherichia coli grows well in both broths, with no significant difference. The absorbance value of Citrobacter freundii in the improved nutrient broth is slightly lower than that of the broth before improvement, but it can still grow well in the improved broth. This shows that the improved nutrient broth is more advantageous than the basic broth in culturing live bacteria remaining in milk.

[0065] Analysis of high-throughput sequencing experiment results:

[0066] Table 5: Comparison of the results of the method provided in this experiment with the direct sequencing method

[0067]

[0068] Note 3: When the relative abundance of a sample is greater than 1%, it is considered as the dominant bacteria detected.

[0069] Note 4: Relative abundance is a term used in high-throughput sequencing to reflect the proportion of a species or gene in a sample to all species or genes. It can be considered that the greater the relative abundance, the greater the amount of this species or gene.

[0070] From Table 5 we can see that:

[0071] 1) When using direct high-throughput sequencing, it is impossible to distinguish whether the dominant bacteria are dead or live. For example, Escherichia coli, Streptococcus thermophilus, and Pseudomonas aeruginosa added as dead bacteria are all detected as dominant bacteria.

[0072] 2) After the method of this experiment was optimized, the relative abundance trend of dead and live bacteria could be intuitively seen, and the relative abundance of the three added dead bacteria dropped below 1%, so that the detected bacteria could be quickly distinguished as dead or live.

[0073] In the technical solution of the present invention, the milk sample to be tested is optimized before high-throughput sequencing, and the milk is cultured in the basic nutrient broth or improved nutrient broth provided by the present invention. The broth is rich in nutrients, and the living microorganisms reproduce and have a large amount of DNA information in the broth, while the dead bacteria do not reproduce and are diluted by transfer culture. The living microorganisms in the milk sample are reproduced in large quantities, while the DNA of the originally dead microorganisms in the milk is diluted. Finally, through high-throughput sequencing analysis, the information of the types of living microorganisms in the milk sample and the highly credible information of the dominant bacteria are obtained.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the types of live bacteria and dominant bacteria in dairy products, characterized in that: The steps include: Step 1, taking a milk sample to be tested and adding it into sterilized nutrient broth, culturing for 18-24 hours to obtain a culture for standby use; the nutrient components of the nutrient broth include peptone, beef powder, and glucose; Step 2: Perform high-throughput sequencing analysis on the cultures to obtain experimental data, wherein the bacteria with a relative abundance greater than 1% are the dominant bacteria.

2. The method for detecting the types of live bacteria and dominant bacteria in dairy products according to claim 1, characterized in that: The nutrients in the nutrient broth also include mannitol and sodium pyruvate.

3. The method for detecting the types of live bacteria and dominant bacteria in dairy products according to claim 2, characterized in that: The nutrient broth comprises the following raw materials in parts by weight: 10-15 parts of peptone, 3-5 parts of beef powder, 4-6 parts of sodium chloride, 10-20 parts of glucose, 4-6 parts of mannitol, 9-11 parts of sodium pyruvate and 1000 parts of water.

4. The method for detecting the types of live bacteria and dominant bacteria in dairy products according to claim 3, characterized in that: The nutrient broth comprises the following raw materials in parts by weight: 10-12 parts of peptone, 3-4 parts of beef powder, 4-5 parts of sodium chloride, 10-15 parts of glucose, 4-5 parts of mannitol, 9-10 parts of sodium pyruvate and 1000 parts of water.

5. The method for detecting the types of live bacteria and dominant bacteria in dairy products according to claim 3, characterized in that: The nutrient broth is prepared in the following manner: corresponding components are weighed according to weight proportions, mixed evenly in an aqueous solution to form a mixed solution, and the mixed solution is sterilized at 120° C.-125° C. for 10-20 minutes to obtain the nutrient broth.

6. The method for detecting the types of live bacteria and dominant bacteria in dairy products according to claim 1, characterized in that: The volume ratio of the milk sample to be tested to the nutrient broth is 1:8-12.

7. The method for detecting the types of live bacteria and dominant bacteria in dairy products according to claim 6, characterized in that: The volume ratio of the milk sample to be tested to the nutrient broth is 1:9-10.

8. The method for detecting the types of live bacteria and dominant bacteria in dairy products according to claim 1, characterized in that: The parameters of the high-throughput sequencing analysis are: the sequencing project type is 16S / 18S / ITS microbial classification sequencing, the bacterial amplification type is "bacteria", and the amplification region is "bacteria V3-V4"; the fungal amplification type is "fungus", and the amplification region is "fungus ITS1-ITS2".

9. The method for detecting live bacteria species and dominant bacteria in dairy products according to claim 1, characterized in that: In step 2, the sample processing method for high-throughput sequencing analysis remains unified and the sampling amount is consistent.

10. Use of the method for detecting the types of live bacteria and dominant bacteria in dairy products according to any one of claims 1 to 9 in determining the dominant bacteria in milk.

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