Application of product for detecting microbial abundance in judging freshness of tiger skin chicken feet

Abstract

The invention relates to application of a product for detecting abundance of microorganisms in judging the freshness of tiger-skin chicken feet. The microorganisms are a combination of lactococcus lactis, lactobacillus coryniformis and lactobacillus farciminis, and the product is a combination of lactobacillus lactis, lactobacillus coryniformis and lactobacillus farciminis. The invention further relates to an application of the product for detecting the abundance of the microorganisms in judging the freshness of the tiger-skin chicken feet and a preparation method of the product for detecting the abundance of the microorganisms in judging the freshness of the tiger-skin chicken feet. It is found for the first time that the abundance of the microorganisms in the tiger-skin chicken feet is closely related to the freshness of the tiger-skin chicken feet, and the abundance of the microorganisms shows significant difference between the deteriorated and rotten tiger-skin chicken feet and normal and non-deteriorated tiger-skin chicken feet and is significantly related to the freshness of samples; the method can be used as a detection target for judging the freshness of the tiger skin chicken feet.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and particularly relates to an application of a product for detecting the abundance of microorganisms in determining the freshness of tiger skin chicken feet. Background Art

[0002] As people's quality of life continues to improve, food safety issues have attracted a lot of attention, especially some foods that are difficult to preserve and easily deteriorate. Therefore, for the food industry, it is particularly important to accurately identify the freshness of food, which can not only protect the health of consumers, but also reduce economic losses.

[0003] At present, the commonly used methods for identifying the freshness of food mainly include appearance identification, odor identification, chemical index identification and microbial identification. Among them, the appearance identification method and the odor identification method are usually used by staff to select spoiled food based on historical experience. When food begins to rot or spoil, its appearance and smell will change, such as spots and mold on the surface, softening of the food surface and obvious odor. These changes can be observed intuitively, but these two food detection methods have a lot of human subjectivity, resulting in low accuracy in the detection results of spoiled food and freshness judgment.

[0004] The chemical index identification method is based on the chemical composition of food. It determines the corruption and deterioration of food by detecting the content or reaction of certain ingredients in food. Commonly used chemical methods include pH value, peroxide value, acid value and hydrogen peroxide value, etc. For foods that are easily oxidized, such as meat or egg products, the peroxide value and acid value methods can be used for identification. These two methods can reflect the degree of oxidation and rancidity of food, but for corrupt or deteriorated foods, the accuracy of these two methods may be reduced.

[0005] Microbial identification is currently one of the most accurate methods for identifying the freshness of food. Since microorganisms are the main cause of food spoilage, the type and quantity of microorganisms in food can be used to clearly determine the extent of food spoilage. However, the detection often requires testing of multiple indicators such as the total colony count, mold and yeast count in the food. Tiger skin chicken feet have a higher fat content than other cooked meat products due to their fried nature, so the changes in their microbial communities during spoilage are different from those of other cooked meat products. Therefore, how to provide a product for detecting microbial abundance that can be specifically used to accurately determine the freshness of tiger skin chicken feet has become an urgent problem to be solved. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a microbial marker for determining the freshness of tiger skin chicken feet and its application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an application of a product for detecting the abundance of microorganisms in determining the freshness of tiger skin chicken feet, wherein the microorganisms are a combination of Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis.

[0009] The judgment criteria are: the tiger skin chicken feet are deteriorated when the concentration of Lactococcus lactis is 8.14 times or more of the initial sample, the concentration of Lactobacillus coryniformis is 6.58 times or more of the initial sample, and the concentration of Lactobacillus farciminis is 3.92 times or more of the initial sample.

[0010] The present invention discovered for the first time that the abundance of three microorganisms, Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis, in tiger skin chicken claws is closely related to the freshness of the tiger skin chicken claws, and their abundance shows significant difference between spoiled tiger skin chicken claws and normal non-spoiled tiger skin chicken claws, and is significantly correlated with the degree of spoilage of the samples. They can be used as detection targets for judging the freshness of tiger skin chicken claws.

[0011] Preferably, the product comprises a reagent, a kit, a device or a prediction system.

[0012] Preferably, the judgment standard is that when the concentration of Lactococcus lactis is 8.14 times or more of the initial sample, the concentration of Lactobacillus coryniformis is 6.58 times or more of the initial sample, and the concentration of Lactobacillus farciminis is 3.92 times or more of the initial sample, the tiger skin chicken feet are deteriorated.

[0013] In a second aspect, the present invention provides a kit for determining the freshness of tiger skin chicken feet, the kit comprising specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting the abundance of microorganisms;

[0014] The microorganisms are a combination of Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis.

[0015] Preferably, the specific primers include primers for amplifying the microbial marker 16SrRNA.

[0016] Preferably, the kit detects the abundance of microorganisms by any one of metagenomic sequencing, 16S sequencing or qPCR quantitative detection, or a combination of at least two of them.

[0017] Preferably, the kit further comprises a reagent for extracting sample DNA.

[0018] In a third aspect, the present invention provides a prediction system for determining the freshness of tiger skin chicken feet, the prediction system comprising a detection unit and a judgment unit;

[0019] The detection unit is used to detect the abundance of microorganisms; the judgment unit is used to determine the freshness of tiger skin chicken feet;

[0020] The microorganism is a combination of Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis;

[0021] The judgment standard is: when the concentration of Lactococcus lactis is 8.14 times or more of the initial sample, the concentration of Lactobacillus coryniformis is 6.58 times or more of the initial sample, and the concentration of Lactobacillus farciminis is 3.92 times or more of the initial sample, the tiger skin chicken feet are deteriorated;

[0022] The initial sample is freshly prepared tiger skin chicken feet.

[0023] The present invention discovers for the first time that the abundance of three microorganisms, Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis, in tiger claws is closely related to the freshness of the tiger claws. The microorganisms of the present invention are used as a biomarker composition for judging the freshness of tiger claws with high accuracy. The microorganisms can be used as detection targets for judging the freshness of tiger claws, which is of great significance to the preservation and quality monitoring of tiger claws. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0025] The term "biomarker" should be understood in a broad sense, including any detectable biological indicator that can reflect an abnormal state, and may include gene markers, species markers (species markers / genus markers) and functional markers. Among them, the meaning of gene markers is not limited to existing genes that can be expressed as proteins with biological activity, but also includes any nucleic acid fragments, which can be DNA or RNA, which can be modified DNA or RNA, or unmodified DNA or RNA. In particular, the biomarker of the present invention is a microbial marker.

[0026] As used herein, the term "volatile organic compounds" will be understood to refer to organic compounds having a high vapor pressure (volatility) at ambient temperature and pressure. Volatile organic compounds generally have a boiling point less than or equal to 250°C when measured at a standard atmospheric pressure of 101.3 kPa (1 atmosphere).

[0027] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0028] Preparation Example

[0029] The prepared tiger skin chicken feet samples were wrapped in 80 μm thick high-density polyethylene / polyamide films and stored at 25 °C, where the O2 permeability was 2.0 cm 3 / (m 2 ·day·atm) to obtain vacuum-packed (VP) tiger skin chicken feet samples.

[0030] Example

[0031] In this example, the total bacterial colony count, odor, volatile organic compound content and microbial concentration / content of the tiger skin chicken feet sample prepared in the preparation example were detected and scored.

[0032] Grouping and preprocessing:

[0033] The samples were just prepared (V0 group), stored for 5 days (V5 group), 5.5 days (V5.5 group), 6 days (V6 group), 10 days (V10 group) and 20 days (V20 group), with three replicates in each group.

[0034] Sample pretreatment: Grind into powder after freezing with liquid nitrogen for relevant testing.

[0035] Test method:

[0036] Total bacterial colony count

[0037] According to the Chinese national standard GB 4789.2-2022, 25g of each sample was homogenized in 225mL of sterile water for 2 minutes. After serial dilution, 1mL was inoculated on an agar plate and incubated at 37°C for 48h before testing.

[0038] Odor Rating:

[0039] Ten trained panelists (4 males and 6 females, aged between 22 and 28 years) were recruited. TSCF samples were placed on opaque disposable plates and evaluated in a sensory laboratory maintained at 25°C. Panelists evaluated samples randomly ordered and coded with three-digit labels using a ten-point scale (8.5-10 for pleasant odor, 7-8.4 for no odor, 4-6.9 for odor but acceptable, and 1-3.9 for overwhelming odor or extremely unpleasant).

[0040] Volatile organic content:

[0041] Headspace solid phase microextraction (HS-SPME) was used to extract volatile organic compounds from TSCF.

[0042] Specifically:

[0043] 1.5 g of each sample was placed in a headspace vial, to which 5 mL of saline and 100 μL of 2-octanol solution (20 mg / L) were added. The sample was equilibrated in a 60 °C water bath for 10 min. The extraction was performed for 30 min using a 50 / 30 μm DVB / CAR / PDMS optical fiber that had been conditioned for 30 min. After extraction, the fiber was desorbed in the GC inlet at 250 °C for 1 min. The chromatographic column used was a TG-WAXA column (60 m*0.25 mm*0.25 μm). The carrier gas was helium with a constant flow rate of 1.2 mL / min, and the inlet temperature was set to 250 °C. The GC operating temperature program was set as follows: the initial temperature was 45 °C for 2 min, then increased to 180 °C at a rate of 6 °C / min, then increased to 230 °C at a rate of 10 °C / min, and finally maintained at 230 °C for 4 min. The mass spectrometer was operated in electron impact ionization mode at 70 eV, with an ion source temperature of 240 °C and a mass scan range of 33–440 m / z. The recorded mass spectra were compared with the NIST 14.0 database and the concentrations of volatile organic compounds were quantified.

[0044] Microbiological content test:

[0045] Bacterial community analysis was performed by Ouyi Biomedical Technology Co., Ltd. (Shanghai, China).

[0046] (1) First, a DNA extraction kit was used to extract genomic DNA from frozen and ground tiger skin chicken feet samples;

[0047] (2) DNA concentration was evaluated by agarose gel electrophoresis and NanoDrop2000 spectrophotometer;

[0048] (3) Genomic DNA was used as a template for amplification using barcode-specific primers customized for the selected sequencing region and Tks Gflex DNA polymerase (Takara). Bacterial diversity was identified in the 16S V3-V4 region (using primers 343F and 798R);

[0049] (4) Check the PCR product by electrophoresis, purify it by magnetic beads, and then use it as a template for the second round of PCR amplification;

[0050] (5) Check the second round of PCR amplification products by electrophoresis, purify them by magnetic beads, and quantify them by Qubit fluorometer;

[0051] (6) Mixing the PCR product with Qubit working solution in equimolar amounts according to the sequencing concentration and sequencing;

[0052] (7) Cutadapt software was used to cut the primer sequence of the raw data sequence. Subsequently, DADA2 was used to perform quality filtering, denoising, merging, and chimera removal on the double-end raw data according to the default parameters in QIIME 2 (2020.11) to obtain representative sequences and ASV abundance;

[0053] (8) The QIIME 2 software package was used to select representative sequences of each ASV and to perform classification annotations with the Silva database (version 138). The default parameters of the q2-feature-classifier software were used for species annotation to obtain the respective proportions of each microorganism in the entire microbial community of the sample. The proportion of microbial content in the V0 group was defined as 1, and the change in the proportion of microbial content in the samples after subsequent storage days was tested and calculated. The above-mentioned related evaluation results are shown in Table 1, and the values ​​are the averages of three replicates. The following ND values ​​indicate that no related substances were detected in the samples.

[0054] Table 1

[0055]

[0056]

[0057] From the data in the table, we can see that with the increase of storage time, the freshness of the tiger skin chicken feet samples changes, and even different degrees of deterioration and corruption occur, and the degree of deterioration and corruption is positively correlated with the storage time. By testing the total bacterial colony count of each group, it can be seen that the total bacterial colony count in the sample after 5 days of storage is 4.53lg CFU / g; the total bacterial colony count in the sample after 5.5 days of storage is 4.96lg CFU / g, which is close to the limit of the total bacterial colony count in the sample in GB 2726-2016; and the total bacterial colony count in the sample after 6 days of storage reaches 6.38lg CFU / g, which exceeds the limit in GB2726-2016 (the limit is 5lg CFU / g), and the tiger skin chicken feet products have deteriorated. The total bacterial colony count in the tiger skin chicken feet samples in the subsequent groups gradually increases with the extension of storage time, and the degree of deterioration and corruption worsens.

[0058] Moreover, the contents of Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis in each sample group were significantly positively correlated with the concentrations of four volatile organic compounds, D-limonene, 2,4-dimethyl-1-decene, 2-butyl-1-octanol and (1S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane, which were related to spoilage during vacuum storage. This indicates that Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis can be used to judge the freshness of tiger skin chicken feet with high accuracy.

[0059] Furthermore, calculations show that when the concentration of Lactococcus lactis in the V5.5 group samples is 8.14 times or more of that in the freshly prepared tiger skin chicken feet, the concentration of Lactobacillus coryniformis is 6.58 times or more of that in the initial sample, and the concentration of Lactobacillus farciminis is 3.92 times or more of that in the initial sample, the tiger skin chicken feet are deteriorated.

[0060] In summary, the present invention discovered for the first time that the abundance of three microorganisms, Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis, in tiger claws is closely related to the freshness of tiger claws; the microorganisms of the present invention are used as a biomarker composition for judging the freshness of tiger claws with high accuracy, and can be used as a detection target for judging the freshness of tiger claws, which is of great significance to the preservation and quality monitoring of tiger claws.

[0061] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0062] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0063] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. Application of a product for detecting microbial abundance in determining the freshness of tiger skin chicken feet, characterized in that: The microorganisms are a combination of Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis.

2. The use according to claim 1, characterized in that The product includes a reagent, a kit, a device or a prediction system.

3. The use according to claim 1 or 2, characterized in that: The judgment standard is that when the concentration of Lactococcus lactis is 8.14 times or more of the initial sample, the concentration of Lactobacillus coryniformis is 6.58 times or more of the initial sample, and the concentration of Lactobacillus farciminis is 3.92 times or more of the initial sample, the tiger skin chicken feet are deteriorated.

4. A kit for determining the freshness of tiger skin chicken feet, characterized in that: The kit includes specific primers, probes, antisense oligonucleotides, aptamers or antibodies for detecting the abundance of microorganisms; The microorganisms are a combination of Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis.

5. The kit according to claim 4, characterized in that The specific primers include primers for amplifying the microbial marker 16SrRNA.

6. The kit according to claim 4 or 5, characterized in that The kit detects the abundance of microorganisms by any one of metagenomic sequencing, 16S sequencing or qPCR quantitative detection or a combination of at least two of them.

7. The kit according to any one of claims 4 to 6, characterized in that The kit also includes reagents for extracting sample DNA.

8. A prediction system for determining the freshness of tiger skin chicken feet, characterized in that: The prediction system includes a detection unit and a judgment unit; The detection unit is used to detect the abundance of microorganisms; the judgment unit is used to determine the freshness of tiger skin chicken feet; The microorganism is a combination of Lactococcus lactis, Lactobacillus coryniformis and Lactobacillus farciminis; The judgment criteria are: when the concentration of Lactococcus lactis is 8.14 times or more of that in the initial sample, the concentration of Lactobacillus coryniformis is 6.58 times or more of that in the initial sample, and the concentration of Lactobacillus farciminis is 3.92 times or more of that in the initial sample, the tiger skin chicken feet are deteriorated.

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