Method for rapidly judging fermentation degree of fermented sour meat based on Raman spectrum

Abstract

The invention discloses a method for rapidly discriminating the fermentation degree of fermented sour meat based on Raman spectroscopy, which is characterized in that a discrimination standard of the fermentation degree of a fermented meat product is formed by starting with physicochemical properties such as protein degradation degree and amino acid content change in different stages of the fermentation process of the meat product; the Raman spectrum is applied to the rapid and standardized detection of the fermented sour meat product for the first time, especially the discrimination of the fermentation degree of the fermented chicken, and the condition of the characteristic peak of the collected Raman spectrum of the fermented sour meat is directly compared with the characteristic of the spectrum peak of the obtained fermented sour meat with different fermentation degrees, so that the detection accuracy of the fermented sour meat product is improved. The method is simple, lossless, rapid and efficient, and provides more choices for judging the fermentation degree of the fermented meat product.

Description

Technical Field

[0001] The invention relates to the technical field of fermented products, and in particular to a method for quickly judging the fermentation degree of fermented sour meat based on Raman spectroscopy. Background Art

[0002] Fermented meat products have attracted much attention due to their good nutritional value and preservation performance. During the fermentation process, the protein in the meat is broken down into amino acids, which are more conducive to human absorption, and a large amount of flavor substances are produced, forming a unique flavor.

[0003] A large number of studies have shown that the longer the fermentation time of fermented meat products, the better. Excessive fermentation can lead to problems such as low pH, accumulation of bitter amino acids and rearrangement of protein secondary structure, which not only affect the quality and taste of fermented meat products, but also cause loss of nutrients. How to accurately monitor the fermentation degree of fermented meat products is an urgent problem to ensure product quality and nutritional content.

[0004] At present, there are two main methods to judge the fermentation degree of fermented meat products: one is to rely on the operator's experience and sensory evaluation, which is basically used in the family workshop production process. However, relying on manual labor means that it is highly subjective, difficult to standardize, and cannot guarantee safety, and the operator needs to accumulate considerable experience. The second is to monitor the degree of fermentation by measuring pH value, organic acid content, and microbial composition. Although this method is more objective and standardized than manual labor, it often has problems such as complex operation, lengthy steps, long time consumption, the need for specialized technicians and large volumes of samples. It is often difficult to accurately determine the fermentation degree of fermented meat products, and it cannot meet the actual detection needs of relevant production companies and testing agencies to supervise and test the fermentation degree of large quantities / different batches of fermented meat products.

[0005] Protein is the most important substance and quality component of meat products. Protein undergoes a complex degradation and transformation process during the fermentation of meat products. Moderate protein degradation can effectively improve the flavor and nutritional value of sour meat, but excessive fermentation and oxidation of some proteins will have an adverse effect on the quality of meat, texture, color and flavor, and even produce unhealthy factors such as biogenic amines. In addition, the oxidation induction of protein may affect its digestion and utilization, reduce the content of essential amino acids, reduce the nutritional value of sour meat, and even cause deterioration, which seriously affects the edible safety of the product.

[0006] At present, the main methods for determining the degree of protein degradation in fermented meat products are non-protein nitrogen and amino nitrogen content determination, gel electrophoresis, amino nitrogen content determination, gel permeation chromatography and free amino acid content. These methods are complicated to operate, time-consuming, and inefficient, and it is difficult to meet the needs of rapid on-site detection of large sample quantities. Raman spectroscopy can reflect the most authentic information about the composition and molecular structure of the sample through the molecular vibration and rotation information of the scattering spectrum of the target detection object. It is a rapid detection technology with simple sample pretreatment, no interference from water molecules, and convenient and non-destructive. However, there are no reports on the rapid determination of the fermentation degree of fermented meat products by non-destructive testing through Raman spectroscopy. Summary of the invention

[0007] Therefore, based on the above background, the present invention provides a method for quickly determining the fermentation degree of fermented sour meat based on Raman spectroscopy. The present invention applies Raman spectroscopy to the detection of the fermentation degree of fermented meat products for the first time. The method is simple, non-destructive, fast and efficient, and provides more options for determining the fermentation degree of fermented meat products, especially fermented chicken.

[0008] The technical solution of the present invention is:

[0009] A method for quickly determining the fermentation degree of fermented sour meat based on Raman spectroscopy comprises the following steps:

[0010] S1: taking the fermented sour meat sample to be tested and processing it;

[0011] S2: Detecting the fermented sour meat sample to be tested in step S1 using a Raman spectrometer to obtain a SERS spectrum of the fermented sour meat sample to be tested;

[0012] S3: Analyze the SERS spectrum of the fermented sour meat sample to be tested, compare the result with the SERS spectrum peak characteristics of sour meat with different fermentation degrees, and judge the fermentation degree of the fermented sour meat sample to be tested.

[0013] Furthermore, the grading of the fermentation degree in step S3 includes the following steps:

[0014] S3.1: Collect fermented sour meat samples at different fermentation stages;

[0015] S3.2: testing the physical and chemical properties of the fermented sour meat samples collected in step S3.1 respectively; the physical and chemical properties include pH, microbial diversity, TVB-N value, protein molecular weight distribution, free amino acids, and tissue microstructure;

[0016] S3.3: Based on the results of the physical and chemical properties measured in step S3.2, the fermentation degree is graded.

[0017] Furthermore, the fermentation degree of fermented sour meat is graded and divided into under-fermentation, moderate fermentation and over-fermentation.

[0018] Furthermore, the SERS peak characteristics of the sour meat with different fermentation degrees in step S2 are obtained by comprehensive analysis of the SERS spectra of the fermented sour meat samples with different fermentation degrees collected by Raman spectrometer in step S3.3; the peak characteristics include Raman characteristic peaks and characteristic peak area ratios;

[0019] Furthermore, the fermented sour meat is fermented chicken.

[0020] Furthermore, in step S3, the criteria for judging the fermentation degree according to the SERS spectrum analysis results of the fermented chicken are:

[0021] ① When the SERS spectrum of the fermented chicken sample to be tested also has 1007cm -1 、1156cm -1 、1438cm -1 、1521cm -1 and 1656cm -1 There are five significant Raman characteristic peaks with a characteristic peak area of ​​S1521 cm -1 / S1007 cm -1 >2, it is determined that the fermented chicken sample to be tested is insufficiently fermented;

[0022] ② When the SERS spectrum of the fermented chicken sample to be tested also contains 1007cm -1 、1301cm -1 、1438cm -1 and 1521cm -1 If there are 4 Raman characteristic peaks, and the characteristic peak area 1<S1521 / S1007<2, it is judged that the fermented chicken sample to be tested is moderately fermented;

[0023] ③ When the SERS spectrum of the fermented chicken sample to be tested has 1007cm -1 and 1301cm -1 、1438cm -1 There are three Raman characteristic peaks, and the characteristic peak area is 0<S1521 cm -1 / S1007 cm -1 <1, it is judged that the fermented chicken sample is over-fermented.

[0024] Furthermore, in step S2, aluminum foil is used as the base material when the fermented sour meat sample to be tested is detected by Raman spectrometer.

[0025] Furthermore, when using a Raman spectrometer to collect SERS spectra of fermented sour meat with different fermentation degrees, aluminum foil is used as the substrate material.

[0026] The beneficial effects achieved by the present invention are as follows:

[0027] The present invention starts with the physical and chemical properties of meat products at different stages of the fermentation process, including the degree of protein degradation and the change in amino acid content, to form a standard for distinguishing the fermentation degree of fermented meat products. For the first time, Raman spectroscopy is applied to rapid and standardized detection of fermented sour meat products, especially for distinguishing the fermentation degree of fermented chicken. The fermentation degree of the fermented sour meat can be judged by directly comparing the characteristic peaks of the collected Raman spectrum of the fermented sour meat with the spectral peak characteristics of the fermented sour meat with different fermentation degrees. The method is simple, non-destructive, rapid and efficient, and provides more options for distinguishing the fermentation degree of fermented meat products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Attached Figure 1 This is a flow chart of the fermentation degree classification and characteristic spectrum peak determination of fermented chicken according to an embodiment of the present invention.

[0029] Attached Figure 2 It is the detection of the physical and chemical indicators of the fermentation process of the low-fat fermented sour meat according to the embodiment of the present invention;

[0030] Among them: (A) pH changes of low-fat fermented sour meat at different fermentation stages; (B) microbial diversity analysis of low-fat fermented sour meat at different fermentation stages, TOP10 species abundance diagram at the genus level; (C) changes in TVB-N value of low-fat fermented sour meat at different fermentation stages; (D) molecular weight distribution of crude protein extract of low-fat fermented sour meat at different fermentation stages.

[0031] Attached Figure 3 The cross-sectional microstructure diagram of fermented chicken at different fermentation stages according to an embodiment of the present invention is as follows:

[0032] Among them: (A)-(F) are cross-sectional microstructure images of low-fat fermented sour meat at 0d, 15d, 30d, 45d, 60d and 75d of fermentation respectively.

[0033] Attached Figure 4 Aluminum foil paper according to an embodiment of the present invention is used as a SERS substrate to enhance the chicken breast sample:

[0034] Among them: a, Raman spectrum of aluminum foil; b, Raman spectrum of 0d chicken breast; c, Raman spectrum of the "aluminum foil + 0d chicken breast" sample.

[0035] Attached Figure 5 The results of the Raman spectrum reproducibility experiment of the embodiment of the present invention are as follows:

[0036] Among them: (A)~(F) are three-dimensional maps of low-fat fermented sour meat with fermentation times of 0d, 15d, 30d, 45d, 60d and 75d respectively; (A-1)~(F-1) are 2D mapping barcodes of low-fat fermented sour meat with fermentation times of 0d, 15d, 30d, 45d, 60 and 75d respectively.

[0037] Attached Figure 6 This is the SERS spectrum analysis of low-fat fermented sour meat at different fermentation stages according to the embodiment of the present invention:

[0038] Among them: (A) 400-1800cm -1 (A) Average SERS spectra of low-fat fermented sour meat at different fermentation stages within the spectral band; (B) spectral intensity plot of the average SERS spectrum; (C) comparison of Raman intensities of basically the same peak positions in the SERS spectrum; (D) secondary structure of proteins in low-fat fermented sour meat at different fermentation stages; (E) PCA three-dimensional scatter plot of SERS spectra of low-fat fermented sour meat at different fermentation stages; (F) SERS spectrum classification scatter plot of low-fat fermented sour meat at different fermentation stages analyzed by LDA.

[0039] Attached Figure 7 Application and verification of actual samples of embodiments of the present invention:

[0040] Among them: (A)-(C) SERS spectra of three batches of low-fat fermented sour meat were verified by LDA model. (D) S1521 cm in SERS spectra of three batches of low-fat fermented sour meat -1 / S1007 cm -1 Ratio statistics chart. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0043] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0044] The technical solution of the present invention:

[0045] A method for quickly determining the fermentation degree of fermented sour meat based on Raman spectroscopy comprises the following steps:

[0046] S1: taking the fermented sour meat sample to be tested and processing it;

[0047] S2: Detecting the fermented sour meat sample to be tested in step S1 using a Raman spectrometer to obtain a SERS spectrum of the fermented sour meat sample to be tested;

[0048] S3: Analyze the SERS spectrum of the fermented sour meat sample to be tested, compare the result with the SERS spectrum peak characteristics of sour meat with different fermentation degrees, and judge the fermentation degree of the fermented sour meat sample to be tested.

[0049] The grading of the fermentation degree in step S3 comprises the following steps:

[0050] S3.1: Collect fermented sour meat samples at different fermentation stages;

[0051] S3.2: testing the physical and chemical properties of the fermented sour meat samples collected in step S3.1 respectively; the physical and chemical properties include pH, microbial diversity, TVB-N value, protein molecular weight distribution, free amino acids, and tissue microstructure;

[0052] S3.3: Based on the results of the physical and chemical properties measured in step S3.2, the fermentation degree is graded.

[0053] The fermentation degree of fermented sour meat is graded into under-fermentation, moderate fermentation, and over-fermentation.

[0054] The SERS peak characteristics of the sour meat with different fermentation degrees in step S2 are obtained by collecting the SERS spectra of the fermented sour meat samples with different fermentation degrees in step S3.3 using a Raman spectrometer and then performing a comprehensive analysis; the peak characteristics include Raman characteristic peaks and characteristic peak area ratios;

[0055] Example 1: Example 1 is an application of the present invention for determining the fermentation degree of fermented chicken.

[0056] (1) Classification of fermentation degree of fermented chicken

[0057] 1) Preparation of fermented chicken samples;

[0058] Fresh chicken breast, Northeastern rice, table salt and white wine were all purchased from a supermarket in Jinshui District, Zhengzhou, Henan Province.

[0059] Cut the chicken breast into strips (8cm×4cm×4cm), fry the rice until golden and crush it into powder (sieve 20 mesh). The ingredients include 25wt% rice flour, 4.5wt% salt, 70.5wt% chicken, and an appropriate amount of white wine. Then put the mixture of chicken breast and ingredients into a fermentation tank (the net mass of each tank of mixture is 1000g), store the tank at a constant temperature of 20℃, and ferment in triplicate. At the fermentation time points of 0d, 15d, 30d, 45d, 60d and 75d, take out samples from the center of the tank under a sterile environment, and store them in a low-temperature refrigerator at -80℃ for subsequent experiments.

[0060] 2) Determination of the physical and chemical properties of chicken at different fermentation stages

[0061] ① Determination of pH value

[0062] The sour meat samples were cut into small pieces, ground with a meat grinder for 2 min, and 10 g was taken, added with 90 mL of deionized water, homogenized in a homogenizer (T25, IKA company, Staufen, Germany), and homogenized for 3 min. The pH value of the filtrate was measured with a pH meter (Mettler-Toledo Instrument Co., Ltd., Shanghai, China), and each meat sample was repeated three times. Commercial standard pH 4.00, pH 6.86, and pH 9.18 calibration solutions were used for calibration.

[0063] ②Microbial diversity analysis

[0064] The total DNA of the microbial community was extracted according to the kit operation instructions. The kit used was Soil DNA Kit (OmegaBio-Tèk, Norcross, GA, USA). The quality of DNA extraction was tested by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined by NanoDrop 2000 UV-visible spectrophotometer. The samples were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for sequencing. The V3V4 region of bacterial 16S rDNA was used as the target DNA sequence for PCR amplification. The front primer 343F (5'-TACGGRAGGCAGCAG-3') and the back primer 798R

[0065] (5'-AGGGTATCTAATCCT-3'), the V3V4 region of 16SrDNA of bacteria in low-fat fermented chicken at six fermentation periods was amplified and then subjected to high-throughput sequencing on the Illumina-HiSeq platform.

[0066] ③Determination of TVB-N value

[0067] TVB-N was measured according to the method of Chinese standard (GB 5009.228-2016). The ground low-fat fermented chicken (10 g) and trichloroacetic acid solution (100 ml, 20 g / L) were mixed in a conical flask, left to stand for 30 minutes, and then the filtrate was filtered out. Then, the filtrate (10 mL) and MgO (5 mL, 10 g / L) were added to the reaction chamber, and the reaction chamber was quickly sealed for distillation. Finally, the received liquid was titrated to the end point with a standard HCl solution.

[0068] ④Molecular weight distribution

[0069] Weigh 10g of sour meat and add 60mL of water to beat and homogenize, adjust pH=11.0 with 1mol / L NaOH, let stand for 1h, centrifuge at 6000r / min for 10min, adjust the supernatant to pH=7.0 with 0.1mol / L HCl, add 90% ethanol at 4℃ for 12h, centrifuge at 6000r / min to take the supernatant, and evaporate the supernatant ethanol at 50℃ to obtain a crude protein extract. Resuspend 2mg / mL solutions in ultrapure water in 6 samples of different fermentation degrees, centrifuge at 10000 speed for 15min at 20℃ to remove insoluble matter, and use HPLC SCL-10AVP (Shimadzu Corporation, Tokyo, Japan) to measure the molecular weight distribution of 6 low-fat sour meat crude protein solutions of different fermentation times.

[0070] ⑤ Determination of free amino acids

[0071] Accurately weigh 2 g of sample, mix with 10 mL of 10% sulfosalicylic acid, homogenize at 7000 r / min for 40 s, centrifuge the homogenized mixture at 4°C and 10000 r / min for 15 min, take 4 mL of supernatant, and mix and shake with 4 mL of petroleum ether, then remove the organic layer, dilute 10 times, pass through a 0.22 μm aqueous filter membrane, take 1.5 mL of the filtrate into a sampling bottle, and measure with a fully automatic amino acid analyzer.

[0072] ⑥Determination of tissue microstructure

[0073] The samples were cut into slices of 5×5×2 mm (perpendicular to the muscle fibers) and fixed with 2.5% glutaraldehyde solution at 4°C for 24 hours. Then, they were washed three times with 0.1 mol / L phosphate buffer solution at pH 7.3, each time for 10 minutes. Then, they were dehydrated twice in different concentrations (50%, 70%, 80%, 90% and anhydrous ethanol), each for 10 minutes. Finally, the dehydrated samples were dried with a vacuum freeze dryer. The dried samples were sprayed with gold, and then the microstructure was observed and photographed on a scanning electron microscope.

[0074] Measurement results:

[0075] Changes in pH

[0076] Depend on Figure 2 (A) It can be seen that the pH value dropped rapidly in the early stage of fermentation (0-30 days), from 5.93 on day 0 to 4.37, which is conducive to inhibiting the growth of some harmful bacteria and ensuring the quality of low-fat fermented chicken. As the fermentation time increases, the pH value gradually decreases, reaching a minimum value of 4.07 at 60 days of fermentation, and slowly recovers to 4.43 at 60 days to 75 days of fermentation. This phenomenon is because the decrease in pH in the early stage of sour meat fermentation is related to the large-scale reproduction of lactic acid bacteria to become the dominant flora and acid production, and with the continuous consumption of carbon sources and the accumulation of metabolites, the growth of lactic acid bacteria is inhibited, and sour meat protein may produce alkaline amino acids, TVB-N, ammonia and other alkaline substances under the action of endogenous proteases or spoilage microorganisms, causing the pH to slowly rise.

[0077] Microbial diversity analysis

[0078] In order to analyze the changes in microbial diversity during the fermentation of low-fat fermented chicken in detail, the relative abundance of the microbial community at the genus level was Figure 2 (B). At the genus level ( Figure 2 B), Lactobacillus was observed to be the most abundant genus in the six samples, and the three main bacterial communities at the lactic acid bacteria species level were Lactobacillus sakei, Lactobacillus ginsenosidimutans and Lactobacillus oligofermentans. Lactic acid bacteria will produce a large amount of lactic acid to lower the pH value and change the quality of the fermented product. The next are Unclassified Leuconostoc and Unclassified Lactobacillales. Leuconostoc can decompose citric acid and produce diacetyl and aromatic substances, which have a significant effect on the aroma and flavor of meat products. In the production process of fermented meat, Leuconostoc often works together with other microorganisms such as yeast and lactic acid bacteria to promote the fermentation and maturation of meat products through complex microbial metabolic activities, thereby improving its texture and taste. Figure 2B shows that during the fermentation time of 0d-60d, as the fermentation time increases, the ratio of the community abundance percentage of Lactobacillus at the species level gradually increases. At 60d of fermentation time, the sum of the community abundance percentages of Lactobacillus, Unclassified Lactobacillales and Unclassified Leuconostoc at the species level reached a maximum value of 96.28%, specifically, Lactobacillus sakei (51.82%), Lactobacillus ginsenosidimutans (8.35%), Lactobacillus oligofermentans (21.12%), Unclassified Lactobacillales (1.06%) and Unclassified Leuconostoc (13.93%). Brochothrix thermosphacta is a cold-resistant spoilage bacterium that can produce various enzymes and metabolites in low-temperature environments, resulting in excessive spoilage bacteria detection and decreased sensory quality of food. The genus Brochothrix thermosphacta accounted for the lowest percentage of community abundance at the species level in low-fat fermented chicken at 60 days of fermentation (1.77%), but increased to 41.66% at the species level in low-fat fermented chicken at 75 days of fermentation, which may lead to bad odor and spoilage of low-fat fermented chicken.

[0079] Changes in TVB-N Values

[0080] The changes of TVB-N value of low-fat fermented chicken at 6 different fermentation time points are as follows Figure 2(C) As shown. The TVB-N values ​​of sour meat with different fermentation degrees showed a trend of continuous increase. The TVB-N content of low-fat fermented chicken from 0 to 60 days was 5.12 to 13.79 mg / 100g. Because during the fermentation process of sour meat, microorganisms began to use protein to grow and reproduce, and the alkaline nitrogen-containing substances produced by protein decomposition continued to increase, causing the TVB-N value to continue to rise. Compared with many fresh meat or aquatic meat products in storage, the TVB-N content of low-fat fermented chicken increased slowly, which may be due to the lactic acid and antibiotics produced by lactic acid bacteria fermentation neutralizing part of the TVB-N. When the fermentation time reached 75 days, TVB-N reached 18.24 mg / 100g, exceeding the Chinese limit of TVB-N in meat products of 15 mg / 100g. This shows that after 60 days of fermentation, the content of low-level ammonia compounds such as volatile ammonia and trimethylamine in low-fat sour meat increased, causing it to produce a certain ammonia smell, affecting its safety and flavor.

[0081] Molecular weight distribution

[0082] The molecular weight distribution of crude protein solution of low-fat fermented chicken at 6 different fermentation time points is as follows Figure 2 (D) As shown. Unfermented sour meat does not contain soluble proteins less than 500Da, and is mainly concentrated in 500-2000Da. As the fermentation progresses, the proportion gradually decreases, while the proportion of soluble proteins of 180-500Da gradually increases. When the fermentation time is 60d, the content of soluble proteins with a molecular weight of less than 3000Da reaches 78.98%, and soluble proteins with a molecular weight of less than 180Da appear, indicating that as the fermentation progresses, the protein is degraded to form small molecular peptides, amino acids or other substances. There is no significant difference between fermentation to 75d and 60d.

[0083] Changes in free amino acids

[0084] Amino acids are one of the main sources of umami substances in meat and are also an important indicator for evaluating the nutritional value of protein. With the extension of fermentation time, the content of free amino acids (FAA) in low-fat fermented chicken increased. As shown in Table 1, the total free amino acid content of low-fat fermented chicken reached a maximum value of 1515.74 mg / 100 g after 60 days of fermentation, which is 6.22 times that of chicken breast in the unfermented group. The rich amino acid content improves the nutritional value of low-fat fermented chicken and enriches the unique flavor of the product. Although the total amount of amino acids increased at 75 days of fermentation, the bitter amino acids increased exponentially (2.43 times compared to 60 days), and the bitter amino acid content accounted for nearly 60% of the total amount of amino acids, which would lead to an unharmonious flavor of the product and affect the taste quality of the product.

[0085] Table 1 Changes of free amino acids in low-fat fermented chicken at different fermentation stages

[0086]

[0087] Microstructural changes

[0088] The microstructural changes of low-fat fermented chicken during the fermentation process (SEM images) are shown in Figure 2. Figure 3 As shown in the figure, at 0d of fermentation, the muscle fiber bundle structure was complete, tightly arranged, with clear boundaries between muscle bundles and small gaps, clear texture, and regular and uniform structure. At 15d of fermentation, the muscle tissue was slightly damaged, the gaps between muscle fiber bundles slightly increased, and the structure began to become loose. During the 15d to 45d of fermentation, the muscle fibers were further damaged, the endomysium began to rupture, the gaps between muscle fiber bundles became more obvious, the muscle fiber arrangement became more chaotic, the muscle fiber boundaries became more blurred, and there was obvious rupture. At 60d of fermentation, the muscle fiber boundaries disappeared significantly, the gaps between muscle fiber bundles were obvious, and the structure was loose. During the 60d to 75d of fermentation, the perimysium was severely damaged, possibly due to further degradation of protein and intensified gelation, which led to the collapse of some muscle fiber bundles and caused them to stick together, and the texture of the muscle cross section became very irregular. Among them, the complete rupture of the endomysium caused the dense structure to be damaged and the nutrients to be lost.

[0089] Based on the results of the physical and chemical properties determination in step S3.2, the fermentation degree is graded.

[0090] When the fermentation time of low-fat fermented chicken is less than 60 days (0-55 days), the degree of muscle fiber degradation and protein degradation are not sufficient, and the overall fermentation degree is evaluated as insufficient fermentation. The optimal fermentation stage of low-fat fermented chicken is about 60 days (55-65 days). At this stage, the content of probiotics in low-fat fermented chicken is the highest, the pH value is the lowest, and the TVB-N value is less than 15 mg / 100g. The protein is fully degraded and the free amino acid content is high. The overall fermentation degree is evaluated as moderate fermentation. When the fermentation time of low-fat fermented chicken exceeds 60 days (greater than 65 days), the protein in the sour meat is severely gelled and the nutritional value is lost, the proportion of spoilage bacteria increases and the TVB-N value exceeds 15 mg / 100g, the bitter amino acids increase, affecting the product quality and taste, and the overall fermentation degree is evaluated as excessive fermentation.

[0091] (2) Obtaining Raman peak characteristics of fermented chicken samples with different fermentation degrees

[0092] ①Enhancement effect and spectral repeatability verification

[0093] In order to understand the enhancement effect of aluminum foil as a SERS substrate material on the chicken samples in this study, a laser Raman instrument was used to collect spectra of 0d chicken breast and "aluminum foil + 0d chicken breast" samples, and the Raman spectrum of aluminum foil was used as a blank control. The results are shown in Figure 2. Figure 4As shown in Table 2. When aluminum foil is not used as the enhanced substrate material, the intensity of the Raman spectrum of the chicken breast sample is very low. After using aluminum foil as the enhanced substrate material, the Raman signal of the chicken breast sample is significantly enhanced, and the characteristic peaks in the SERS spectrum are significantly different. Figure 4 As shown in the figure, the SERS spectrum of 0d chicken breast collected on aluminum foil as SERS substrate has four obvious characteristic peaks, which can be observed at 1007cm-1, 1156cm-1, 1438cm-1 and 1656cm-1, and the SERS signal intensity at 1656cm-1 is the largest. In this study, the enhancement factor (EF) of aluminum foil as an enhanced substrate was calculated by the change of the Raman characteristic peak of 0d chicken breast at 1656cm-1, and was calculated by equation (1). Where ISERS and INR are the SERS intensity and unenhanced Raman intensity of chicken breast at 1656cm-1, respectively. It was calculated that the EF value of aluminum foil as a SERS substrate material for chicken breast samples is 2.13×10 1 (Calculation details are provided in the Supporting Information ). Figure 5 The results show the reproducibility of aluminum foil as SERS substrate material for chicken breast samples at different fermentation stages. The barcodes in the three-dimensional mapping and two-dimensional mapping generated by 20 SERS spectra of chicken breast samples at 6 different fermentation stages are very uniform, which reflects the high reproducibility of aluminum foil as SERS substrate detection. The dominant characteristic peaks in the barcodes of the two-dimensional mapping of chicken breast samples at different fermentation stages were selected to calculate the corresponding relative standard deviations, and the maximum RSD was 8.28%. The Raman signal intensity of aluminum foil basically does not affect the SERS spectrum of chicken breast sample detection and has good signal enhancement ability and detection reproducibility, indicating that aluminum foil is a suitable choice as a substrate material for Raman detection of fermented chicken breast samples.

[0094] Table 2 Enhancement factor of aluminum foil as SERS substrate

[0095]

[0096] ②Spectrum collection and analysis

[0097] The SERS spectra of chicken at different fermentation stages were collected using a Raman spectrometer. The collected SERS spectra were eliminated by cosmic rays using LabSpec 6 software (Horiba, France), and the multi-point baseline correction and baseline background were removed to obtain the average SERS spectra of low-fat fermented chicken at 6 different fermentation stages. Figure 6 (A) and Table 3 show that the SERS spectra of low-fat fermented chicken at 6 different fermentation stages are at 1007, 1156, 1438, 1521, and 1656 cm -1 There are 5 main Raman characteristic peaks, 1261cm -1and 1296cm -1 It only exists in low-fat fermented chicken with a fermentation time of 30 to 45 days. Figure 6 (B) 2D mapping of SERS spectra of low-fat fermented chicken at 6 different fermentation stages and Figure 6 (C) The comparison results of the peak intensities of the SERS spectra of low-fat fermented chicken at 6 different fermentation stages at the above 5 same Raman shifts show that the peak intensities of the SERS spectra of low-fat fermented chicken at 6 different fermentation stages at the 5 same Raman shifts are significantly different, and the peak at 1007 cm -1 The peak intensity of 1438 cm -1 The peak intensity of 1521 cm has the largest change. It is worth noting that in the SERS spectra of low-fat fermented chicken from 0 to 60 days, -1 The peak intensities of 1521 cm -1 The peak intensity is the smallest.

[0098] PeakFit 4.12 software (SeaSolve software Inc., USA) was used to determine the secondary structures of proteins in low-fat fermented chicken at different fermentation stages, including the proportion of α-helix, β-sheet, β-turn and random coil. Figure 6 D). The amide I region in the Raman spectrum (1600-1700 cm －1 The change of the amide I region is closely related to the change of the secondary structure of the protein. The amide I region contains the information of C=O, C-N and N-H stretching vibrations. The main form of the secondary structure of the protein is α-helix (1650-1660cm -1 ), β-folding (1600~1640cm -1 ), β-angle (1661-1700cm -1 ) and irregular curls (1640~1650cm -1). On the 0th day, the relative content of α-helix was the highest at 42.89%, followed by β-fold (21.86%) and β-turn (19.15%), and the relative content of random coil was the lowest at 16.10%. As the fermentation time increased, the content of α-helix gradually decreased, and its relative content was 29.35% on the 60th day of fermentation; the relative content of β-fold and β-turn gradually increased, and the increase was large, from 21.86% and 19.15% to 29.23% and 22.17% respectively; the relative content of random coil increased slowly from 16.10% to 19.25%. The changes in the secondary structure of the protein indicate that with the extension of the fermentation time, the protein has undergone a certain degree of denaturation, which causes the α-helix structure to rotate into β-fold, β-turn and random coil, which means that the protein molecular structure changes from regular to loose during the fermentation process, which is consistent with the SEM results. Since protein is the main component of chicken breast tissue, changes in the spatial structure of protein will cause changes in the binding force between proteins, which will affect the water retention and texture characteristics of low-fat fermented chicken to a certain extent. It is worth noting that the α-helix content increased slightly at 75 days of fermentation, and structural reorganization may have occurred at this time. The cause of the reorganization may be related to protein degradation or a certain degree of enzymatic hydrolysis of protein, which causes changes in protein structure, protein isoelectric point, and hydrophobicity, and excessive denaturation and mutual squeezing of proteins to rearrange.

[0099] Furthermore, this embodiment also constructs a rapid recognition model for the SERS spectra of low-fat sour meat at 6 different fermentation stages collected based on aluminum foil as the SERS substrate material. -1 In the spectral band range, classification and recognition algorithms such as PCA and LDA were used to classify and recognize 120 SERS spectra of 6 low-fat sour meat samples with different fermentation degrees. The appropriate visual qualitative recognition model was screened by comparing the classification capabilities of different classification algorithms. The results are shown in the figure. Figure 6 (E)-(F) and Table 2. Figure 6 (E) It can be seen that PCA analysis can clearly cluster the SERS spectra of low-fat sour meat at 6 different fermentation stages into 6 independent clusters. Each cluster is independent and non-repetitive, and the SERS spectra of low-fat sour meat at each fermentation degree can be distinguished from each other. The variance contribution rate of PC1 is 37.4%, the variance contribution rate of PC2 is 17.2%, and the variance contribution rate of PC3 is 29.0%. The cumulative contribution rate is 83.6%, representing most of the spectral information. Figure 6(F) and Table 4 show that LDA completely distinguishes the SERS spectra of low-fat fermented chicken samples at 6 different fermentation stages. All SERS spectra of low-fat fermented chicken samples at each fermentation stage are concentrated in a relatively small area near the centroid. There is no overlap between the areas and the recognition rate reaches 100%. The 120 SERS spectra of low-fat fermented chicken samples at 6 different fermentation stages collected were cross-validated for the discrimination model by the "leave one out" method, and the sensitivity and specificity were both 100%. LDA takes into account the category information of different samples, and can minimize the difference in SERS spectra of low-fat fermented chicken samples at the same fermentation stage, and maximize the difference in SERS spectra of low-fat fermented chicken samples at the same fermentation stage. The recognition rate, sensitivity and specificity of the classification model are all 100%.

[0100] Table 3: Attribution of SERS characteristic peaks of low-fat fermented sour meat at six fermentation stages

[0101]

[0102]

[0103] Table 4: Classification of LDA models based on SERS spectra of low-fat fermented sour meat at 6 different fermentation stages

[0104]

[0105] ③ Comprehensively determine the spectral peak characteristics of fermented chicken with different fermentation degrees based on the spectra of fermented chicken at different fermentation stages

[0106] In terms of the intensity and quantity of the characteristic Raman spectra, the 1007 cm -1 、1156cm -1 、1438cm -1 、1521cm -1 and 1656cm -1 A total of five significant Raman characteristic peaks exist simultaneously; in the low-fat fermented chicken sample with a moderate degree of fermentation (55-65 days), the peak at 1007 cm -1 、1301cm -1 、1438cm -1 and 1521cm -1 A total of four significant Raman characteristic peaks exist simultaneously; in the low-fat fermented chicken sample with excessive fermentation (55-65 days), only the peak at 1007 cm -1 and 1301cm -1 、1438cm -1 There are three significant Raman characteristic peaks at the same time. 1007cm -1It is a relatively stable Raman characteristic peak in the SERS spectra of low-fat fermented chicken samples at six fermentation stages, 1521 cm -1 It is the most significant Raman characteristic peak in the SERS spectra of low-fat fermented chicken samples at the six fermentation stages. Further, the SERS spectra of low-fat fermented chicken samples at the six fermentation stages were calculated at 1007 cm -1 and 1521cm -1 The peak area of ​​the Raman spectrum at , and the ratio between the two is calculated (S 1521 / S 1007 ), and the results are shown in Table 5. As shown in Table 5, in the SERS spectra of low-fat fermented chicken with insufficient fermentation degree (0-55d), S1521 cm -1 / S1007 cm -1 >2; in the SERS spectrum of low-fat fermented chicken with moderate fermentation degree (55-65 days), 1 < S1521 cm -1 / S1007 cm -1 <2, and S1521cm -1 / S1007 cm -1 ≈1; in the SERS spectrum of low-fat fermented chicken with excessive fermentation (greater than 65 days), 0<S1521 cm -1 / S1007 cm -1 <1, and S1521 cm -1 / S1007 cm -1 ≈0.

[0107] In summary, the fermentation progress judgment criteria of low-fat fermented sour meat based on aluminum foil as SERS substrate material are: -1 、1156cm -1 、1438cm -1 、1521cm -1 and 1656cm -1 There are 5 significant Raman characteristic peaks at the same time, and S1521 cm -1 / S1007 cm -1 >2, it can be judged that the low-fat fermented sour meat is insufficiently fermented; when 1007cm -1 、1301cm -1 、1438cm -1 and 1521cm -1 When there are 4 significant Raman characteristic peaks at the same time, and 1<S1521 / S1007<2(S1521 cm -1 / S1007 cm -1 ≈1), it can be judged that the fermented sour meat with low fat content is moderately fermented; -1and 1301cm -1 、1438cm -1 When there are three significant Raman characteristic peaks at the same time, and 0<S1521 cm -1 / S1007 cm -1 <1(S1521 cm -1 / S1007 cm -1 ≈0), it can be judged that the fermented sour meat is over-fermented.

[0108] Table 5: SERS spectra of low-fat sour meat samples at 1007 cm in 6 fermentation stages -1 and 1521cm -1 Peak area and ratio

[0109]

[0110] (3) Validation of the method

[0111] Three batches of low-fat fermented sour meat samples with different fermentation times (0d, 15d, 30d, 45d, 60d and 75d) were collected for SERS spectra collection and verification. The sample processing method and SESR collection method were the same as above, and 20 SERS spectra were randomly collected for each sample. The average spectrum of the SERS spectra of the existing six fermentation stages of low-fat fermented sour meat was retrieved, and each 21 spectra of the three batches of low-fat fermented sour meat samples at different fermentation stages were used for qualitative identification verification of the LDA model. The results are as follows: Figure 5 The results show that in the LDA model, all 21 SERS spectra of low-fat fermented sour meat samples from three batches at different fermentation stages were correctly identified, with an identification accuracy of 100%. -1 and S1521cm -1 The peak area ratio at S1521 cm -1 / S1007cm -1 The statistical results are as follows Figure 5 As shown in (D), the S1521 / S1007 ratios of the samples with insufficient fermentation degree (0-55 days) of the three batches of low-fat fermented sour meat were all greater than 2, while the S1521 / S1007 ratios of the samples with moderate fermentation degree (55-65 days) were greater than 2. -1 / S1007cm -1 The ratios of the samples with excessive fermentation (greater than 65 days) were all within the range of 1 to 2. The S1521cm -1 / S1007cm -1 The ratios were all in the range of 0 to 1, which verified the above-mentioned method for rapid identification of the fermentation process of low-fat fermented sour meat based on Raman spectroscopy technology.

[0112] Furthermore, the chicken was fermented at 15°C and 35°C in the laboratory. Except for the different fermentation temperatures, other operations were the same as above. Each temperature had three batches. Each batch was randomly sampled at different fermentation time points 75 days before fermentation, with 6 samples respectively. The samples fermented at 15°C were sample 1, sample 2, and sample 3, and the samples fermented at 35°C were sample 4, sample 5, and sample 6. The physical and chemical properties of the fermented chicken were tested as above, confirming that sample 1 was under-fermented, sample 2 was under-fermented, and sample 3 was moderately fermented; sample 4 was over-fermented, sample 5 was over-fermented, and sample 6 was under-fermented; and the fermented sour meat samples were collected for SERS spectra to identify the characteristic peaks of the spectra. The spectra of sample 1, sample 2, and sample 6 simultaneously contained 1007cm -1 、1156cm -1 、1438cm -1 、1521cm -1 and 1656cm -1 There are five significant Raman characteristic peaks with a characteristic peak area of ​​S1521 cm -1 / S1007 cm -1 >2; The spectrum of sample 3 also has 1007cm -1 、1301cm -1 、1438cm -1 and 1521cm -1 There are 4 Raman characteristic peaks, and the characteristic peak area 1<S1521 / S1007<2; there is 1007cm in the spectra of sample 4 and sample 5 -1 and 1301cm -1 、1438cm -1 There are three Raman characteristic peaks, and the characteristic peak area is 0<S1521 cm -1 / S1007 cm -1 <1.

[0113] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for quickly determining the fermentation degree of fermented sour meat based on Raman spectroscopy, characterized in that: The steps include: S1: taking the fermented sour meat sample to be tested and processing it; S2: Detecting the fermented sour meat sample to be tested in step S1 using a Raman spectrometer to obtain a SERS spectrum of the fermented sour meat sample to be tested; S3: Analyze the SERS spectrum of the fermented sour meat sample to be tested obtained in step S2, compare the result with the SERS spectrum peak characteristics of fermented sour meat with different fermentation degrees, and judge the fermentation degree of the fermented sour meat sample to be tested.

2. The method for rapidly determining the fermentation degree of fermented sour meat based on Raman spectroscopy according to claim 1, characterized in that: The grading of the fermentation degree in step S3 comprises the following steps: S3.1: Collect fermented sour meat samples at different fermentation stages; S3.2: testing the physical and chemical properties of the fermented sour meat samples collected in step S3.1 respectively; the physical and chemical properties include pH, microbial diversity, TVB-N value, protein molecular weight distribution, free amino acids, and tissue microstructure; S3.3: Based on the results of the physical and chemical properties determination in step S3.2, the fermentation degree is graded.

3. The method for rapidly determining the fermentation degree of fermented sour meat based on Raman spectroscopy according to claim 2, characterized in that: The fermentation degree of fermented sour meat is divided into under-fermentation, moderate fermentation and over-fermentation.

4. The method for rapidly determining the fermentation degree of fermented sour meat based on Raman spectroscopy according to claim 3, characterized in that: The SERS peak characteristics of the fermented sour meat with different fermentation degrees in step S2 are obtained by comprehensive analysis of the SERS spectra of the fermented sour meat samples with different fermentation degrees after being graded and divided in step S3.3 using a Raman spectrometer; the peak characteristics include Raman characteristic peaks and characteristic peak area ratios.

5. The method for rapidly determining the fermentation degree of fermented sour meat based on Raman spectroscopy according to claim 3, characterized in that: The fermented sour meat is fermented chicken.

6. The method for rapidly determining the fermentation degree of fermented sour meat based on Raman spectroscopy according to claim 5, characterized in that: In step S3, the criteria for judging the fermentation degree according to the SERS spectrum analysis results of the fermented chicken are: ① When the SERS spectrum of the fermented chicken sample to be tested also has 1007cm -1 、1156cm -1 、1438cm -1 、1521cm -1 and 1656cm -1 There are five significant Raman characteristic peaks with a characteristic peak area of ​​S1521 cm -1 / S1007 cm -1 >2, it is determined that the fermented chicken sample to be tested is insufficiently fermented; ② When the SERS spectrum of the fermented chicken sample to be tested also contains 1007cm -1 、1301cm -1 、1438cm -1 and 1521cm -1 If there are 4 Raman characteristic peaks, and the characteristic peak area 1<S1521 / S1007<2, it is judged that the fermented chicken sample to be tested is moderately fermented; ③ When the SERS spectrum of the fermented chicken sample to be tested has 1007cm -1 and 1301cm -1 、1438cm -1 There are three Raman characteristic peaks, and the characteristic peak area is 0<S1521 cm -1 / S1007 cm -1 <1, it is judged that the fermented chicken sample is over-fermented.

7. A method for rapidly determining the fermentation degree of fermented sour meat based on Raman spectroscopy according to any one of claims 1 to 6, characterized in that: In step S2, aluminum foil is used as the base material when the fermented sour meat sample to be tested is tested by Raman spectrometer.

8. The method for rapidly determining the fermentation degree of fermented sour meat based on Raman spectroscopy according to claim 4, characterized in that: Aluminum foil was used as the substrate material when using Raman spectrometer to collect SERS spectra of fermented sour meat with different fermentation degrees.

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