A method for determining the optimal parameters of a pigeon meat preservation method

Abstract

The present invention discloses a method for determining the optimal parameters of pigeon meat preservation methods, belonging to the field of preservation technology. By establishing the correlation relationship between the core microbial genera of pigeon meat and the main differential volatile compounds, the source strains of the main off-flavor substances are identified, and then the pigeon meat preservation method with the least odor generation is analyzed based on the flavor of the volatile compounds. GC-IMS is used for the detection of flavor substances, which has high sensitivity, does not require enrichment and concentration of samples, can maintain the true flavor, and has simple data processing. This analysis method can simply and quickly analyze the changes of flavor substances during the preservation process of pigeon meat. Through this analysis method, a pigeon meat preservation method with the least odor generation is found, solving the problem that the existing preservation technology is prone to produce off-flavors.

Description

Technical Field

[0001] The present invention relates to a method for determining the optimal parameters of pigeon meat preservation method, belonging to the technical field of preservation. Background Art

[0002] Pigeon meat is the fourth largest high-yield poultry meat variety in the world. The rapid development of the industry has exposed the problem of insufficient pigeon meat preservation technology. Inhibiting food spoilage and extending the product shelf life are the research directions for the development of preservation technology. The spoilage of pigeon meat caused by microorganisms is the biggest obstacle to the development of the industry. The entire circulation process of poultry meat from slaughter to consumers is likely to be contaminated by microorganisms, which will lead to food safety problems. Therefore, microbial contamination is a major problem in poultry meat preservation. The main body of microbial contamination of poultry meat is spoilage bacteria, which will ultimately cause the loss of safety and edibility of poultry meat and result in economic losses. Pseudomonas, Enterobacter, and Lactobacillus are common spoilage flora, which are detected in many studies on the spoilage of poultry meat. The spoilage bacteria in poultry meat consume organic substances such as glucose to grow and reproduce, and are accompanied by the deterioration of meat quality such as color, flavor, and texture. To solve this problem, many preservation technologies have been developed and applied, including modified atmosphere packaging (MAP), electron beam irradiation (EBI), and low voltage electrostatic field (LVEF).

[0003] Modified atmosphere packaging (MAP) is a preservation technology that fills a certain proportion of mixed gas into the packaging material containing food to inhibit the growth of microorganisms and maintain the quality of food. The gases commonly used for fresh poultry meat are O2, CO2, and N2, which are added to the headspace of the package in different proportions and combinations. O2 usually promotes the growth of microorganisms, but has a good inhibitory effect on anaerobic microorganisms. CO2 is non-polar and easily dissolves in the lipid bilayer of the microbial cell membrane, which will increase the fluidity of the membrane, thereby exposing the cytoplasm to a toxic environment. In addition, the cytoplasm of microorganisms contains water, and carbon dioxide will react with water to form carbonic acid, resulting in a decrease in the pH value of the cytoplasm. This change will pressurize the cell. N2, as a gas without antibacterial activity, is mainly used to displace oxygen and prevent package collapse. It has been widely used in the preservation and storage of livestock and poultry meat products.

[0004] Electron beam irradiation preservation (EBI) technology mainly refers to a preservation technology that uses an electron accelerator to generate a large number of electron beams to treat food. The electron beam destroys the higher-order structures of proteins and DNA to achieve the effect of killing pests and microorganisms in food, greatly reducing the degree of microbial contamination in food, and is conducive to reducing food spoilage and safety problems. It has been widely used in the preservation and storage of livestock, poultry meat, and aquatic products, and is welcomed by consumers because of its characteristics of cold sterilization and no residue.

[0005] Low-voltage electrostatic field (LVEF) is currently widely used in the preservation of fruits and vegetables and has achieved good preservation effects. The positive and negative charges in a unit in the low-voltage electrostatic field move directionally, changing the surface charge of enzymes, affecting normal physiological metabolism. In addition, it affects the stability of cell membranes, changes the permeability of biological membranes, causing the death of microorganisms and the loss of product juice.

[0006] The unique flavor of pigeon meat makes it highly favored by consumers. However, the flavor of pigeon meat will change due to the physiological metabolism of the carcass itself during the preservation process. In addition, the flavor of pigeon meat is also affected by the microbial metabolic reactions during the preservation process. The contribution of each microbial metabolic reaction to the flavor depends on the microbial abundance and composition of pigeon meat. This microbial diversity will produce some different flavors and affect the sensory color, nutritional value, and quality characteristics of the meat. Many studies have tried to link bacterial diversity with the formation of volatile compounds in the research object to better understand and maintain the flavor of meat. Currently, the preservation methods of pigeon meat, such as refrigeration preservation, modified atmosphere preservation, etc., still have problems such as the large reproduction of microorganisms, the intensification of endogenous enzyme reactions, the loss of nutrients, and the generation of off-flavors. Therefore, it is of great significance to study a preservation technology that can inhibit the reproduction of microorganisms and reduce the increase in off-flavor content during the preservation process. Summary of the Invention

[0007] To solve one or more of the above existing problems, the present invention provides a method for determining the optimal parameters of pigeon meat preservation methods. The metabolism of microorganisms has a huge impact on the composition of volatile flavor substances, and its metabolites can bring off-flavors to pigeon meat. Identifying the source strains of the main off-flavor substances and exploring the feasibility of composite preservation technologies based on this preservation method in subsequent preservation research, and studying the method of directional inhibition of spoilage bacteria can more effectively inhibit the generation of off-flavors and improve the flavor of pigeon meat after preservation.

[0008] A method for determining the optimal parameters of pigeon meat preservation methods, the technical solution is as follows:

[0009] Step 1: Pretreat the pigeon meat, then use various preservation methods. Divide the experimental groups according to different parameters of the preservation methods, place the experimental groups under refrigeration at 3±0.5°C, and take samples on the 0th, 5th, 10th, 15th, and 20th days of the preservation experiment.

[0010] Step 2: Measure the total number of colonies of the pigeon meat after preservation to determine the inhibitory effect of various preservation methods on the total number of colonies; and measure the microbial genera of the pigeon meat after preservation, analyze the changes in the relative abundances of the flora at the phylum and genus levels after different preservation treatments, and screen out the core microbial genera; conduct principal component analysis to compare the differences in the effects of different preservation methods on microbial genera; draw a clustering heatmap to analyze the correlation between microbial genera and preservation methods.

[0011] Step 3: Detect the volatile flavor substances of the pigeon meat, analyze the differences in the composition and content of volatile substances after different preservation treatments, and screen out the main different volatile compounds; conduct clustering heatmap analysis on the detected volatile substances to identify the correlation between preservation methods and flavor substances.

[0012] Step 4: Draw a heatmap of the correlation between the core microbial genera and the main different volatile compounds, and analyze the influence of the core bacterial genera on volatile compounds; find out the off-odor substances among the main different volatile compounds, find the corresponding core microbial genera according to the correlation relationship, sum up the relative abundances of the core microbial genera corresponding to the off-odor substances in each experimental group, and screen out the experimental groups with the sum of the relative abundances of microbial genera less than 70%.

[0013] Step 5: Analyze the volatile compounds of the experimental groups screened in Step 4. The volatile flavor substances are detected in Step 3. Classify the volatile compounds according to flavor types, sum up the contents of the volatile compounds belonging to off-odors, compare the sums of the contents of off-odor volatile compounds in each group, and screen out the minimum value of the sum of off-odor volatile compounds. The corresponding treatment method is the preservation method that can maintain the flavor of pigeon meat.

[0014] In one embodiment, the pretreatment in Step 1 is to slaughter the pigeon, remove the feathers, remove the head and neck, cut it in half along the spine, remove the internal organs, wash the blood, and then dry it.

[0015] In one embodiment, the preservation methods in Step 1 include modified atmosphere packaging, irradiation, and electrostatic field.

[0016] In one embodiment, the specific steps for determining the microbial genera of pigeon meat after preservation in step two are as follows: Total DNA is extracted using a DNA extraction kit. Meanwhile, the DNA is quantified using Nanodrop, and the quality of DNA extraction is detected by 1.2% agarose gel electrophoresis. The DNA concentration is adjusted to 1 μg / μL with sterile water. The V3-V4 region of the bacterial 16S rDNA gene is amplified with barcodes using specific primers SEQ ID NO.1: 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and SEQ ID NO.2: 806R (5’-GGACTACH VGGGTWTCTAAT-3’). The PCR thermal cycle is as follows: 98°C for 1 min; 30 cycles of 98°C (10 seconds), 50°C (30 seconds), and 72°C (30 seconds); 72°C for 5 min. The PCR products are detected by 2% agarose gel electrophoresis and purified using a Qiagen gel extraction kit. A sequencing library is prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit. The library is quantified using the Quant-iT PicoGreen dsDNA Assay Kit on a Promega QuantiFluor fluorescence quantification system. The concentration of the qualified library should be above 2 nM. After gradient dilution of each qualified library for sequencing, they are mixed according to the required sequencing amount in the corresponding proportion, and denatured with NaOH into single strands for sequencing on the machine. Paired-end sequencing is performed using a MiSeq sequencer. The optimal sequencing length of the target fragment is 250 bp. The Alpha diversity level of each sample and the beta diversity difference between different samples (groups) are evaluated according to the QIIME2 dada2 analysis process to obtain the microbial genus composition result.

[0017] In one embodiment, in step two, the correlation between the preservation method and the microbial genera is determined by analyzing PCA, PLS-DA, and clustering heat maps and judging according to the distance of the Euclidean distance. The smaller the Euclidean distance, the stronger the correlation between the preservation method and the microbial genera.

[0018] In one embodiment, the number of core microbial genera screened in step two is 10.

[0019] In one embodiment, the number of mainly different volatile compounds screened in step two is 20.

[0020] In one embodiment, the clustering heat map analysis of volatile substances in step three is to analyze the similarity of the flavor substance composition of pigeon meat after different preservation treatments.

[0021] In one embodiment, the volatile flavor substances of pigeon meat described in step three are detected by GC-IMS.

[0022] In one embodiment, the clustering heat map analysis of the volatile substances in step three is to analyze the similarity of the flavor substance compositions of pigeon meat after different preservation treatments.

[0023] In one embodiment, 6 kinds of volatile compounds with peculiar smells are screened out in step four, and the corresponding core microbial genera are 5 kinds.

[0024] In one embodiment, in step four, the Pearson algorithm is used to draw the heat map of the correlation between the core microbial genera and the main differential volatile compounds.

[0025] A preservation method for pigeon meat, which is obtained by screening using the above method.

[0026] A preservation method for pigeon meat, which is to use electron beam irradiation of 3-5 kGy for preservation and is obtained by screening using the above method.

[0027] The beneficial effects of the present invention are:

[0028] By establishing the correlation relationship between the core microbial genera of pigeon meat and the main differential volatile compounds, identifying the source strains of the main bad flavor substances, and then analyzing the pigeon meat preservation method with the least peculiar smell according to the flavor of the volatile compounds, through this analysis method, a pigeon meat preservation method with the least peculiar smell is found, solving the problem that the existing preservation technology is prone to produce bad flavors; using GC-IMS for the detection of flavor substances, with high sensitivity, no need for sample enrichment and concentration, maintaining the true flavor, and simple data processing. This analysis method can simply and quickly analyze the changes of flavor substances during the preservation process of pigeon meat. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to these drawings.

[0030] Figure 1A It is a change diagram of the total viable count during the pigeon meat preservation process;

[0031] Figure 1B It is an influence diagram of different treatment groups on the relative abundance at the phylum level;

[0032] Figure 1C It is an influence diagram of different treatment groups on the relative abundance at the genus level;

[0033] Figure 2A Figure showing the effects of different preservation treatments on the differences in the bacterial communities of pigeon meat;

[0034] Figure 2B Figure showing the variable importance in projection (VIP) scores of microbial genera during the preservation of pigeon meat;

[0035] Figure 2C Figure showing the heatmap analysis between preservation treatment methods and the main bacterial genera;

[0036] Figure 3A Figure showing the 2D ion mobility spectrometry of flavor compounds;

[0037] Figure 3B Figure showing the 3D ion mobility spectrometry of flavor compounds;

[0038] Figure 3C Figure showing the comparison of 2D ion mobility spectrometry of flavor compounds;

[0039] Figure 4A Figure showing the PCoA analysis of the flavor of pigeon meat under different preservation methods;

[0040] Figure 4B Figure showing the variable importance in projection (VIP) scores of the flavor of pigeon meat under different preservation methods;

[0041] Figure 4C Figure showing the heatmap analysis of the flavor of pigeon meat under different preservation methods;

[0042] Figure 5 Figure showing the correlation heatmap between the core bacterial community and 20 main volatile compounds;

[0043] Figure 6 Figure showing the total content of off-flavor compounds in different treatment groups. Detailed implementation manners

[0044] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will further describe the embodiments of the present invention in detail with reference to the accompanying drawings.

[0045] Example 1:

[0046] 1. Pretreatment of pigeon meat

[0047] After slaughtering the pigeons, remove the feathers and heads and necks, cut them in half along the spine, remove the internal organs, wash the blood, and wipe them dry.

[0048] (1) Grouping

[0049] CR group: Fresh samples, which can be sampled immediately.

[0050] Group C4: The dried half pigeons were placed in black fresh-keeping boxes and packaged using a compound modified atmosphere packaging machine, filling with air. They were stored under refrigerated conditions.

[0051] MAP group: The dried half pigeons were placed in black fresh-keeping boxes and packaged using a compound modified atmosphere packaging machine. They were divided into three groups, MA, MB, and MC. The filling gas for the samples in group MA was 50% CO2 + 50% N2; the filling gas for the samples in group MB was 10% O2 + 40% CO2 + 50% N2; the filling gas for the samples in group MC was 20% O2 + 30% CO2 + 50% N2.

[0052] EBI group: The dried half pigeons were placed in black fresh-keeping boxes and packaged using a compound modified atmosphere packaging machine, filling with air. Then, irradiation treatment was carried out. According to the irradiation dose, they were divided into three groups, EA, EB, and EC, where EA: 2 kGy, EB: 4 kGy, EC: 8 kGy. The irradiation treatment time was 5 min for all.

[0053] LV group: The dried half pigeons were placed in black fresh-keeping boxes and packaged using a compound modified atmosphere packaging machine, filling with air. They were under an electric field of 1.2 kV / m throughout the fresh-keeping process.

[0054] (2) Storage and preservation

[0055] The pigeon meat samples of all treatment groups were refrigerated under the condition of 3 ± 0.5 °C. Samples were taken on the 0th, 5th, 10th, 15th, and 20th days of the fresh-keeping experiment, and the number of sample parallels was 5.

[0056] 2. Microbiological analysis

[0057] The total number of colonies of the pigeon meat after preservation was determined according to the national standard method. Total DNA was extracted using a DNA extraction kit. At the same time, DNA was quantified using Nanodrop, and the quality of DNA extraction was detected by 1.2% agarose gel electrophoresis. The DNA concentration was adjusted to 1 μg / μL with sterile water. The V3-V4 region of the bacterial 16S rDNA gene was amplified with the specific primers SEQ ID NO.1: 515F (5’-GTGCCAGCMG CCGCGGTAA-3’) and SEQ ID NO.2: 806R (5’-GGACTACHVGGGTWTCTAAT-3’).

[0058] The PCR thermal cycling was as follows: 98°C for 1 min; 30 cycles of 98°C (10 s), 50°C (30 s), and 72°C (30 s); 72°C for 5 min. The PCR products were detected by 2% agarose gel electrophoresis and purified using the Qiagen Gel Extraction Kit. The sequencing library was prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit. The library was quantified using the Quant-iT PicoGreen dsDNA Assay Kit on the Promega QuantiFluor fluorescence quantification system, and the concentration of the qualified library should be above 2 nM. Paired-end sequencing was performed using the MiSeq sequencer, and the optimal sequencing length of the target fragment was 250 bp.

[0059] (1) Analysis of the relative abundance and dynamic changes of microbial colonies

[0060] Microorganisms are the main factors causing the spoilage of meat and meat products. By analyzing the dynamic changes of microorganisms in pigeon meat, the edible value of pigeon meat can be better determined. The changes in the total number of colonies in each treatment group during the preservation process are as Figure 1A shown, and the total viable count in each treatment group continuously increases over time. The acceptable upper limit of the total viable count of microorganisms is 5 log CFU / g. The C4, MA, MB, MC, EA, EB, EC, and LV samples exceeded this limit after 5, 5, 5, 5, 10, 15, 20, and 5 days respectively, which confirmed the different degrees of inhibition of microbial growth and reproduction by different preservation technologies. Among them, electron beam irradiation can effectively inhibit and kill microorganisms. Based on 16S rDNA sequencing, pigeon meat was analyzed at the phylum level ( Figure 1B ) and genus level ( Figure 1C) Classification analysis was carried out. The ten most abundant phyla and genera were selected for dynamic analysis. The ten most abundant phyla were Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Elusimicrobia, Acidobacteria, Cyanobacteria, and Verrucomicrobia. Among them, Proteobacteria, Firmicutes, and Actinobacteria had the relatively largest proportions. The combined relative abundances of these three phyla in each sample were all above 90%, except for the samples of EB10, EC10, and LV10 (EB10 represents the sample of the EB group at the 10th day of preservation, and the same applies to EC10 and LV10, and the following is the same). At the same time, it was also found that Actinobacteria had the highest relative abundance in these three groups of samples. This indicates that irradiation and low-voltage electrostatic fields can effectively kill or inhibit Proteobacteria and Firmicutes, but have a weaker effect on Actinobacteria. However, the phenomenon in the samples of EB10 and EC10 groups did not occur in EA10, which may be caused by insufficient irradiation dose. The relatively high relative abundance of Firmicutes in the CR group samples may come from the slaughter environment. Compared with the CR group samples, the relative abundances of Proteobacteria in the samples of each treatment group increased and showed an upward trend over time. Relatively, the relative abundance of Firmicutes decreased, indicating that Proteobacteria had a stronger competitive advantage. The relative abundances of Proteobacteria in the samples of the MA group were lower than those of the CR group both at 10d and 20d, indicating that nitrogen and carbon dioxide can effectively stabilize the growth and reproduction of Proteobacteria. However, a similar phenomenon did not occur in the samples of the MB and MC groups, indicating that the presence of oxygen can promote the growth of Proteobacteria.

[0061] At the genus level ( Figure 1C) Ten genera with the highest relative abundances were still selected, namely Lactococcus, Psychrobacter, Pseudomonas, Lactobacillus, Aeromonas, Hafnia-Obesumbacterium, Streptococcus, Shewanella, Megasphaera, and Enhydrobacter. These genera were mainly concentrated in Proteobacteria and Firmicutes, and this result was consistent with the phylum abundances. The three genera with the highest relative abundances in the samples of the CR group were Lactobacillus, Streptococcus, and Megasphaera, and this result was consistent with the phylum composition of the CR group samples, with the highest relative abundance of Firmicutes. After different treatments, the bacterial community composition in pigeon meat changed greatly. In the C410 group, Psychrobacter, Pseudomonas, and Aeromonas in Proteobacteria had the highest relative abundances. However, from day 0 to day 20, the relative abundance of Lactococcus continuously increased (0.89%, 29.39%), which was related to Lactococcus reducing the pH of the meat and inhibiting the growth of other microorganisms. At the same time, it also indicated that low temperature seemed to inhibit the growth of Lactobacillus, Streptococcus, and Megasphaera. The relative abundance of Lactococcus in the samples treated with MAP (MA, MB, MC) was much higher than that in other treatment groups, which was attributed to the change in gas composition. Lactobacilli are facultative anaerobes and can reproduce better in a low-oxygen environment. On day 10, Aeromonas was the second most abundant genus in the MB and MC group samples, which was related to the oxygen content and showed a trend of decreasing abundance with increasing oxygen concentration. By day 20, the abundance of Aeromonas decreased significantly, which was related to the exhaustion of oxygen during long-term storage. The same theory could also explain that Hafnia-Obesumbacterium, a facultative anaerobe, had the second highest relative abundance in MB20 and MC20. Compared with CR, the relative abundance of Lactobacillus (Lactobacillus, Firmicutes) in the EBI group decreased, which was related to the irradiation killing most of the microorganisms, and this could also explain the continuous decrease in the concentration of Lactobacillus in the EBI group until day 20. On day 10, with the continuous increase in the electron beam irradiation dose, the relative abundance of Psychrobacter (Proteobacteria) continuously decreased. This showed that irradiation could effectively kill Psychrobacter bacteria. However, on day 20, the relative abundance of Psychrobacter in the irradiated group was the highest. This was because almost all Lactococcus was killed after irradiation and could not compete with Psychrobacter to become the dominant bacterium during the preservation process. On day 10, the genera with the highest abundances in the LV group were Lactococcus, Psychrobacter, and Pseudomonas. By day 20, the relative abundances of Lactococcus and Pseudomonas continuously increased, while the relative abundance of Psychrobacter decreased. According to the analysis of colony composition, the combined use of EBI with MAP or LVEF might effectively inhibit the reproduction of Lactococcus, Psychrobacter, and Pseudomonas.

[0062] (2) Similarity analysis of colony composition

[0063] To better characterize the changes and similarities in community composition, principal component analysis (PCA) was performed based on the relative abundance levels of the microbiota. As can be seen from Figure 2A , PC1: 49.96%, PC2: 29.71%. It can be found that it can be roughly divided into four regions. The first region is mainly concentrated in the upper left corner, including six treatment groups: C410, EA10, EA20, EB20, EC20, and LV10. This indicates that the colony compositions of the samples in these six treatment groups are similar. The second region is concentrated in the upper middle, and the groups are C420 and LV20. The third region is located in the upper right corner and consists of the samples on the 10th and 20th days of the MAP group. This shows that the relative abundance of the microbiota in the pigeon meat samples after modified atmosphere packaging tends to be stable in the middle and late stages of preservation. The fourth region is concentrated in the lower right corner, including CR, EB10, and EC10. This seems to indicate that irradiation doses higher than 4 kGy have a relatively weak impact on the relative abundance of microorganisms. The above results show that during storage, different preservation methods have a significant impact on the composition of pigeon meat colonies, which is due to different antibacterial principles. Compared with the C4 group, the impact of low-voltage electrostatic fields on colony growth is less than that of other treatment methods. After low-dose irradiation kills some microorganisms, it quickly loses its effect during the subsequent preservation process, and the colonies grow rapidly and tend to be stable. There is little difference from the C410 group in the middle stage of preservation, but the colony succession in the C4 group and the LV group continues, and there is a large gap from the EBI group on the 20th day. Medium and high doses (≥4 kGy) of irradiation may cause the microbiota to re-succeed, but the succession path is similar to that of the C410 group. The PCA distribution of the MAP group is relatively concentrated, which seems to indicate that more than 30% carbon dioxide or less than 20% oxygen has little impact on the overall relative abundance of the pigeon meat microbiota. To better understand the colony composition among different treatment methods, we performed PLS-DA analysis. As shown in Figure 2B , VIP>1 is the standard for co-differential genera. Microorganisms of 7 genera showed significant differences under different preservation methods of pigeon meat. They include Lactococcus, Psychrobacter, Pseudomonas, Lactobacillus, Aeromonas, Hafnia-Obesumbacterium, and Shewanella.

[0064] (3) Heat map

[0065] To quickly compare the correlations between each treatment group and the top 30 genera with the highest abundances, a heat map was drawn, as shown in Figure 2CThe results are shown as follows. The results showed that the CR group was positively correlated with the abundances of Megasphaera, Flavobacteriaceae, Streptococcus, Enterococcus, Lactobacillus, Staphylococcus, and Luteimonas, which was the same as the results of relative abundances. The EB10 group was positively correlated with Faecalibacterium, Deinococcus, Bacillus, and Escherichia. The abundance of Corynebacterium was relatively high in the LV10 group. However, the samples in the LV20 group were positively correlated with Shewanella, Pseudomonas, and Jensenella, indicating that the succession of the microbial community in pigeon meat was ongoing and not yet stable, which was similar to the PCA results. It should be noted that the samples treated with MAP were all positively correlated with Lactococcus on the 10th day. In addition, MB10 and MC10 were also positively correlated with Aeromonas and Weissella. However, on the 20th day, the positive correlation of the samples in the MB and MC groups changed to Hafnia-Obesumbacterium, and the MA group was still Lactococcus. Generally speaking, after different preservation treatments, the dominant microbial communities in pigeon meat changed greatly. For example, there were no common dominant genera between the EBI group and the MAP group.

[0066] Finally, 10 core microbial genera were identified as Lactococcus, Psychrobacter, Pseudomonas, Lactobacillus, Aeromonas, Hafnia-Obesumbacterium, Streptococcus, Shewanella, Megasphaera, and Enhydrobacter.

[0067] 3. Analysis of Volatile Flavor Compounds

[0068] (1) Ion Mobility Spectrometry Plots

[0069] The GC-IMS test method was used to obtain global IMS information to help identify the composition and variation patterns of flavor compounds in pigeon meat under different preservation methods, as Figure 3A shown. The ordinate represents the retention time of gas chromatography, and the abscissa represents the migration time of ions. The background of the whole graph is blue, and the red vertical line at 1.0 on the abscissa is the RIP peak (reaction ion peak, after normalization). Each point on both sides of the RIP peak represents a flavor compound. The color represents the concentration of the substance; white indicates a lower concentration, red indicates a higher concentration, and the darker the color, the higher the concentration. The three-dimensional spectra of volatile compounds in pigeon meat under different preservation treatments are also as Figure 3B shown. It can be clearly seen that the volatile compounds vary due to different pigeon meat preservation methods. To more clearly compare the differences, the spectral diagram of one sample (CR) was selected as a reference, and the spectral diagrams of other samples were subtracted as references. If two flavor compounds are the same, the background after subtraction is white, red indicates that the substance concentration is higher than the reference, and blue indicates that the substance concentration is lower than the reference. The results are as Figure 3CAs shown, the volatile substances of MA20 had the smallest difference from the samples in the CR group, indicating that 50% nitrogen + 50% carbon dioxide might help stabilize the flavor of pigeon meat. At the same time, it was also observed that with the increase in oxygen concentration or irradiation dose, the content of volatile substances continuously increased, which might be related to the oxidative hydrolysis of macromolecular substances such as lipids in the meat.

[0070] (2) Effects of different preservation methods on the content of volatile flavor substances in pigeon meat

[0071] The types and contents of volatile compounds in pigeon meat under different preservation methods were determined by GC-IMS method. The volatile compounds identified by gas chromatography-IMS are shown in Table 1. A total of 91 peaks and 76 volatile substances were detected during the pigeon meat preservation stage, including 16 alcohols, 16 aldehydes, 14 ketones, 14 esters, 2 acids, 2 ethers, 4 alkenes, 2 furans, 1 alkane, and 5 other substances. Since 15 volatile substances showed double peaks due to the presence of monomers and dimers, including isopropanol, n-butanol, n-hexanol, 1-octen-3-ol, hexanal, heptanal, trans-2-octenal, n-octanal, n-nonanal, 2-butanone, 3-hydroxy-2-butanone, 2-octanone, 2-nonanone, ethyl acetate, and α-pinene.

[0072] Table 1 Types and contents of volatile compounds in pigeon meat under different preservation methods

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Alcohols are closely related to lipid oxidation, amino acid degradation, methyl ketone reduction, and acid degradation. During the preservation process, propanol, n-hexanol-M, and n-hexanol-D all showed an upward trend under the action of MAP and EBI, and the maximum value appeared in the MC group, but in the C420 group and the LV20 group, they showed a downward trend. Propanol and n-hexanol can provide special fragrance and fatty odor, which can provide good flavor for pigeon meat. 1-Octen-3-ol has the aroma of mushrooms, lavender, roses, and hay, but it showed a downward trend during the preservation process, especially in the samples of the C420 group.

[0081] Aldehydes are the main degradation products of lipid oxidation. Due to their low flavor thresholds, aldehyde compounds become one of the main volatile components contributing to the flavor of meat. Butyraldehyde has an asphyxiating odor, and its content significantly decreases in the C420 and LV20 groups, which is beneficial to improving the flavor of pigeon meat. However, MAP and EBI can effectively inhibit the decomposition and conversion of butyraldehyde, and there is no statistical significance compared with the CR group. The opposite situation occurs in the content change of propionaldehyde. The contents of hexanal-D and n-valeraldehyde significantly increase under the treatments of MAP and EBI, and continuously increase with the increase of oxygen concentration or irradiation dose. The special odors of oil and grass possessed by hexanal-D and n-valeraldehyde contribute greatly to the composition of pigeon meat flavor as their contents increase. The reason for the decrease in the contents of hexanal and n-valeraldehyde in the EC group samples can be explained by the over-oxidation that destroys the cell structure, resulting in their loss with the juice. Similar situations occur in alcohols, aldehydes, and ketones.

[0082] Ketones are produced by amino acid degradation and microbial metabolism, with special flavors. In Table 1, 2-butanone-D shows an obvious downward trend in groups C420, MC20, EC20, and LV20. The special fragrance of 2-butanone may contribute to enhancing the richness of pigeon meat flavor. The content of 2-heptanone in pigeon meat shows an inhibitory decrease with the increase of oxygen and irradiation dose, and C420 and LV20 also show inhibitory effects.

[0083] Esters come from the esterification reaction of short-chain acids and alcohols, which is also related to the esterase activity of Staphylococcus. The detected isopentyl isovalerate, ethyl acetate-D, γ-valerolactone, ethyl isovalerate, benzyl acetate, and ethyl heptanoate play important roles in constituting the flavor of pigeon meat. The resulting fruity and sweet flavors may mask the unpleasant odors generated during the preservation process, such as spoilage.

[0084] Acids mainly come from lipid oxidation, hydrolysis of phospholipids and triglycerides; short-chain acids (C<6) produced by lipid oxidation have a relatively low odor threshold and have a greater impact on the aroma of pigeon meat. Only two acids were identified in this study, and their contents were low. It is speculated that they and other acid volatile substances participated in the esterification reaction and their contents decreased.

[0085] The two furan substances detected in the present invention, 2-methyl-3-mercaptofuran and 2-butylfuran, show an upward trend in content during refrigeration. Their coffee-like aroma improves the flavor level of pigeon meat and is beneficial to the formation of good flavor of pigeon meat. However, acrylonitrile in pigeon meat has a slightly stinky smell, and its content only significantly decreases in C420, MB20, MC20, EB20, and LV20 in the treatment groups.

[0086] (3) PCoA, PLS-DA analysis

[0087] To better compare the differences in flavor substances after different preservation treatments, PCoA and PLS-DA were used to analyze the original data. The PCoA analysis of the volatile components in the samples is as Figure 4A shown. The differences between PCoA1 and PCoA2 were 57.53% and 16.68% respectively. On the 20th day of preservation storage, there were significant differences in the contents of volatile substances in each treatment group (P<0.05), which were caused by the differences in the action mechanisms and inhibitory effects of each preservation technology on microorganisms and endogenous enzymes. The flavor showed different development directions with the action of microorganisms and endogenous enzymes. It should be noted that on the 20th day of preservation storage, the contents of volatile substances in the EA, EB, EC, MB, and MC groups were relatively similar, and this result was similar to the analysis result of Table 1. The main influencing factor was the oxidation of pigeon meat. Electron beam irradiation could promote the generation of reactive oxygen species, which would lead to the oxidative hydrolysis of proteins and lipids, but the influence of microorganisms in this process still needed to be further explored. At the same time, it was observed that the difference in volatile substances between the C420 group samples and the LV20 group samples was small, indicating that the effect of low-voltage electrostatic field on stabilizing volatile substances was not ideal. Relatively speaking, among all treatment groups, the composition of volatile substances in the MA20 group samples was most similar to that of the CR group samples, and it could well stabilize the flavor changes during the preservation process. To better understand the differential volatiles between different stages, we conducted PLS-DA analysis, as Figure 4BAs shown, VIP>1 is the standard for common differential metabolites. Twenty volatile compounds (VIP>1, P<0.05) showed significant differences (P<0.05) during the preservation process due to different preservation technologies. These include Butanal, (Z)-3-Hexen-1-yl acetate, Hexanal-D, Acetic acid ethyl ester-D, Valeraldehyde, 1-Hexanol-D, ethyl 3-methylbutanoate, 1-Hexanol-M, 2-Butanone-D, Propanal, 2-methyl-3-furanthiol, 2-Cyclohexen-1-one, ethyl heptanoate, 2-butylfuran, 2-Methylpropyl acetate, Anisole, Gamma-Valerolactone, 2-Heptanone, acrylonitrile, Dihydromyrcenol.

[0088] (4) Volatile substance clustering heatmap analysis

[0089] As Figure 4CAs shown, there is a two-way aggregation between volatile flavor compounds and groups. The peak volume of each volatile flavor is marked with different colors in the heat map. The darker the red, the higher the peak volume, and the darker the blue, the lower the peak volume. Samples clustered in the same category show a high degree of correlation. The shorter the Euclidean distance, the higher the similarity of the samples. From the vertical clustering, due to the complexity of flavor substances, each treatment group can be roughly divided into five categories before finally clustering into one group. The first category consists of the EB20 group, which has relatively high contents of five volatile substances: n-valeraldehyde, 2-methyl-3-mercaptofuran, ethyl heptanoate, hexanal-D, and dihydromyrcenol. The second category is composed of the groups MC20 and MB20. Compared with EB20, the contents of propanol, anisole, and (Z)-3-hexen-1-yl acetate are increased. The third category consists of the groups C420 and LV20. The high-content volatile substances in this category are opposite to those in the first and second categories, and the contents of three volatile substances, propionaldehyde, 2-heptanone, and benzyl acetate, are the highest in the third category. The MA20 group is the preservation treatment group with the flavor substance composition closest to the CR group, and the same phenomenon also appears in the PCoA analysis, indicating that 50% CO2 + 50% N2 can stabilize the flavor of pigeon meat. Therefore, the fourth category consists of the groups CR and MA20, with the highest contents of two flavor substances, ethyl isovalerate and acrylonitrile. The fifth category includes the groups EA20 and EC20. Compared with the CR group, the contents of ethyl acetate, ethyl isovalerate, and acrylonitrile in the groups EA20 and EC20 are equally rich, but the contents of dihydromyrcenol, 2-cyclohexen-1-one, and 2-butylfuran are significantly higher than those of the samples in the CR group.

[0090] Finally, 20 major differential volatile compounds were identified as Butanal, (Z)-3-Hexen-1-yl acetate, Hexanal-D, Acetic acid ethyl ester-D, Valeraldehyde, 1-Hexanol-D, ethyl 3-methylbutanoate, 1-Hexanol-M, 2-Butanone-D, Propanal, 2-methyl-3-furanthiol, 2-Cyclohexen-1-one, ethyl heptanoate, 2-butylfuran, 2-Methylpropylacetate, Anisole, Gamma-Valerolactone, 2-Heptanone, acrylonitrile, Dihydromyrcenol.

[0091] 4. Correlation analysis of core microbial genera and major differential volatile compounds

[0092] Volatile compounds produced during the entire fermentation process are related to microbial metabolism. A heatmap of the correlation between 10 core microbial genera and 20 major differential volatile compounds was plotted using the Pearson algorithm, as Figure 5As shown in the figure. Among the 20 volatile flavor substances, only butyraldehyde, hexanal-D, 2-butanone-D, propionaldehyde, 2-cyclohexen-1-one, and acrylonitrile showed unpleasant odors such as pungency, aldehyde smell, or ketone smell. At the same time, it was also observed that Pseudomonas was significantly negatively correlated with 5 volatile substances except propionaldehyde (P<0.05). In addition, it was also observed that Shewanella, Hafnia-Obesumbacterium, and Aeromonas were significantly negatively correlated with acrylonitrile (P<0.05), which means that the metabolism of related unpleasant odors may be inhibited by Pseudomonas, Shewanella, Hafnia-Obesumbacterium, and Aeromonas. Lactococcus was strongly significantly positively correlated with cis-3-Hexenyl acetate (strong green grass smell), isobutyl acetate (fruity smell), and anisole (aromatic smell) (P<0.01), which may be related to the esterification reaction participated by lactic acid bacteria. At the same time, it was observed that Lactococcus became the dominant genus in the MAP group in the later stage of preservation, promoting the process of esterification reaction, increasing the content of ester substances in the MAP group, and being beneficial to the formation of good flavor. Pseudomonas was strongly significantly positively correlated with propionaldehyde (suffocating and irritating smell) (P<0.01). Pseudomonas became the dominant strain in group LV20, which also led to a significantly higher content of propionaldehyde in the LV20 group than in other groups, contributing to the generation of off-flavors in pigeon meat. Both Aeromonas and Hafnia-Obesumbacterium were significantly positively correlated with 4 volatile substances, namely anisole, isobutyl acetate, cis-3-Hexenyl acetate, and n-valeraldehyde (P<0.05), and could provide a fruity-like taste for pigeon meat.

[0093] In summary, Lactobacillus, Psychrobacter, Streptococcus, and Enhydrobacter had no significant relationship with the generation of off-flavors. According to the results in Table 2, the sum of the relative abundances of these four genera was more than 70% only in groups EA10, EA20, EB20, and EC20.

[0094] Table 2 Relative abundances of core microbial genera in each treatment group

[0095]

[0096]

[0097] Due to the relatively low relative abundances of the genera related to the generation of off-flavors in the CR group, it could still maintain good flavor during the preservation process. Figure 4A The results showed that the flavor compositions of EA10, EA20, EB20, and EC20 were quite different from those of the CR group, which was directly related to their genus compositions. It should be noted that the dominant strains in these groups had no significant correlation with the generation of off-flavor substances (P>0.05). Therefore, the reason for the large gap between these groups and the CR group was presumably the generation of a large number of volatile substances with good flavor. At the same time Figure 4AAmong the results, the CR group was the most similar to the MA20 group. However, the main genus of bacteria in the MA20 group was Lactococcus, which had a significant positive correlation with the increase in acrylonitrile content (P < 0.05). This was the direct reason for the difference between the MA20 group and the CR group. Pigeon meat after electron beam irradiation could produce fewer off-flavor substances. Although its flavor composition was quite different from that of fresh samples after preservation, among them, the EB group produced the least off-flavor. At the same time, its dominant genus - Psychrobacter had a significant positive correlation with isobutyl acetate, cis-3-hexenyl acetate, anisole, and ethyl isovalerate. It was worth noting that lipid oxidation caused by irradiation seemed to play a role.

[0098] 5. Screening of preservation methods

[0099] The present invention found that different preservation methods had differences in the microorganisms and volatile components of pigeon meat. The present invention identified a total of 10 main spoilage microorganism genera during the preservation of pigeon meat. Lactococcus, Psychrobacter, Pseudomonas, and Lactobacillus had relatively high abundances in each preservation method. In addition, the present invention also identified 76 volatile flavor substances in pigeon meat after 20 days of preservation, including 16 alcohols, 16 aldehydes, 14 ketones, 14 esters, 2 acids, 2 ethers, 4 alkenes, 2 furans, 1 alkane, and 5 other substances. A total of 20 flavor substances were analyzed as the sources of differences between different preservation methods. Pearson correlation analysis found that 5 genera of bacteria, namely Lactococcus, Pseudomonas, Aeromonas, Hafnia-Obesumbacterium, and Shewanella, were positively correlated with 6 off-flavors among the 20 differential flavor substances, making the preservation methods with these microorganisms as the dominant genera able to produce a large amount of off-flavor substances. The preservation methods represented by the remaining 5 dominant genera of bacteria could produce fewer off-flavor substances, although the flavor gap from fresh pigeon meat was relatively large. Among the 76 volatile substances analyzed and detected, 11 substances, namely acrolein, propionaldehyde, butyraldehyde, hexanal, heptanal, 2-butanone, 2-cyclohexen-1-one, ethyl acrylate, ethylene glycol monobutyl ether, dimethyl disulfide, and acrylonitrile, were determined as off-flavor substances. As Figure 6 shown, by analyzing the total content of off-flavor substances, it was found that the content of off-flavor substances in the EB group was significantly lower than that of other treatment groups, but there was still a significant difference from the CR group (fresh sample). This indicated that it was beneficial to reduce the generation of off-flavor substances in pigeon meat and inhibit the deterioration of quality.

[0100] Therefore, it was finally determined that the preservation parameters of the EB group in the experimental group set in this example were the optimal preservation parameters. In practical applications, relevant technical personnel could set more experimental groups, and then determine the best preservation parameters through the method provided in this application.

[0101] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for determining the optimal parameters of pigeon meat preservation method, characterized in that, It includes the following steps: Step 1: Pretreat the pigeon meat, then use various preservation methods to process the pigeon meat. Divide the experimental groups according to different parameters of the preservation methods, place the experimental groups under the condition of 3±0.5°C for refrigeration, and take samples on the 0th day, 5th day, 10th day, 15th day, and 20th day of the preservation experiment; Step 2: Measure the total number of colonies of the pigeon meat after preservation to determine the inhibitory effect of various preservation methods on the total number of colonies; and measure the microbial genera of the pigeon meat after preservation, analyze the changes in the relative abundances of the flora at the phylum and genus levels after different preservation treatments, and screen out the core microbial genera; conduct principal component analysis to compare the differences in the effects of different preservation methods on microbial genera; draw a clustering heat map to analyze the correlation between microbial genera and preservation methods; Step 3: Detect the volatile flavor substances of the pigeon meat, analyze the differences in the composition and content of volatile substances after different preservation treatments, and screen out the main different volatile compounds; conduct a clustering heat map analysis on the detected volatile substances to identify the correlation between the preservation method and the flavor substances; Step 4: Draw a heat map of the correlation between the core microbial genera and the main different volatile compounds, and analyze the influence of the core microbial genera on the volatile compounds; find out the off-flavor substances among the main different volatile compounds, find the corresponding core microbial genera according to the correlation relationship, sum up the relative abundances of the core microbial genera corresponding to the off-flavor substances in each experimental group, and screen out the experimental groups with the sum of the relative abundances of microbial genera less than 70%; Step 5: Analyze the volatile compounds of the experimental groups screened out in Step 4. The volatile flavor substances are detected in Step 3. Classify the volatile compounds according to the flavor type, sum up the contents of the volatile compounds belonging to off-flavors, compare the sums of the contents of off-flavor volatile compounds in each group, and screen out the minimum value of the sum of off-flavor volatile compounds. The corresponding treatment method is the preservation method that can maintain the flavor of the pigeon meat; In Step 2, the comparison of the correlation between the preservation method and the microbial genera is to judge according to the distance of Euclidean distance through PCA, PLS-DA analysis and clustering heat map. The smaller the Euclidean distance, the stronger the correlation between the preservation method and the microbial genera; The core microbial genera screened out in Step 2 are 10 species; In Step 3, the clustering heat map analysis of the volatile substances is to analyze the similarity of the composition of the flavor substances of the pigeon meat after different preservation treatments; The main different volatile compounds screened out in Step 3 are 20 species; The preservation methods described in Step 1 include modified atmosphere packaging, irradiation, and electrostatic field.

2. The method according to claim 1, wherein In Step 2, the screening out of the core microbial genera is to analyze the changes in the relative abundances of the flora at the phylum and genus levels after different preservation treatments by using 16S rDNA sequencing technology.

3. The method according to claim 1, wherein The specific steps for determining the microbial genera of pigeon meat after preservation in Step 2 are as follows: Extract total DNA using a DNA extraction kit, quantify the DNA simultaneously, and detect the quality of DNA extraction by gel electrophoresis; Amplify the V3-V4 region of the bacterial 16S rDNA gene using the specific primers shown in SEQ ID NO.1 and SEQ ID NO.2; Purify and recover the PCR amplification products with magnetic beads; Perform fluorescence quantification on the PCR amplification and recovery products. According to the fluorescence quantification results, mix each sample in the corresponding proportion according to the sequencing amount requirement of each sample; Prepare a sequencing library; Perform fragment selection and purification on the library; After gradient dilution of each qualified sequencing library for sequencing, mix them in the corresponding proportion according to the required sequencing amount, and denature them into single strands for sequencing on the machine; Perform paired-end sequencing with a sequencing instrument, and the sequencing length of the target fragment is 200-300 bp; Evaluate the Alpha diversity level of each sample and the beta diversity difference between different samples according to the QIIME2 dada2 analysis process to obtain the results of the microbial genera composition.

4. The method according to claim 1, characterized in that, For detecting the volatile flavor substances of pigeon meat described in Step 3, GC-IMS is used for detection.

Images