Application of an exogenous additive in improving the quality of fermented beef jerky and extending its shelf life

Abstract

The present invention belongs to the field of food processing, and specifically relates to the use of an exogenous additive in improving the quality of fermented beef jerky and extending its shelf life. The exogenous additive includes a Sichuan pepper extract, which has the best effect when added in an amount of 16%. The present invention extracts and purifies cathepsin L in beef, studies the effect of adding Sichuan pepper extract to beef jerky fermented by Xylella fastidiosa on the myofibrillar protein structure, protein degradation and quality of beef jerky. Through experiments, the changes in quality indicators such as color, sensory perception, shear force, water activity and pH, as well as cathepsin L activity and total colony count of beef jerky added with 16% Sichuan pepper extract when stored at different temperatures are explored, and the shelf life of beef jerky is predicted. Finally, an exogenous additive that inhibits enzyme activity is obtained to improve the quality of fermented beef jerky and extend its shelf life.

Description

Technical Field

[0001] The invention belongs to the field of food processing, and particularly relates to the application of an exogenous additive in improving the quality of fermented dried beef and extending its shelf life. Technical Background

[0002] Fermented beef jerky is a traditional fermented meat product unique to the ethnic minority regions of southwestern China. Fermentation imparts its unique, beloved fermented aroma and makes the macromolecules in the beef more digestible. However, due to the complex production process, inconsistent product quality, long production cycles, short shelf life, and low production efficiency, traditional fermented beef jerky cannot meet market demand. Furthermore, it has been found that beef jerky is prone to structural degradation and reduced chewiness during storage. To shorten the fermentation time of beef jerky, many studies have inoculated selected starter cultures during the fermentation process. This not only shortens the fermentation time but also improves the quality of the product. For example, Wang Chan et al. (2022) screened numerous bacterial communities in traditional beef jerky and found that the presence of high-quality microorganisms improved the fermentation process. Liu Hanyu et al. (2019) used high-throughput sequencing technology to analyze the bacterial communities of traditional fermented beef jerky.

[0003] With the progress of research and people's deeper understanding of fermented dried beef, many problems of fermented dried beef have been improved to a certain extent. Through the study of processing technology, people have provided accurate and detailed process parameters for fermented dried beef, and selected excellent strains that can shorten the fermentation time and improve the fermentation quality of fermented dried beef. However, there is a lack of research on the structural deterioration of fermented dried beef during storage, which leads to the reduction of the quality of dried beef and poor toughness and chewiness of dried beef. Therefore, improving the quality of dried beef and extending its shelf life has become an important topic.

[0004] Cathepsin L is a major lysosomal cathepsin that plays a crucial role in the maturation process and exhibits endopeptidase activity. It rapidly degrades troponins T and I, and slowly degrades myosin, actin, α-actinin, tropomyosin, actin, heavy myosin, and light myosin. Numerous studies have shown that the production of cathepsin L in meat products can irreversibly affect myofibrillar proteins, leading to their fragmentation and consequently affecting the structural stability of the meat product. Beef jerky contains approximately 45.6% protein by weight. Myofibrillar proteins are the primary component of meat protein and primarily determine the textural properties of meat products, comprising approximately 55% of total muscle protein. The higher-order conformations of actin, myosin, and collagen within the fibrillar proteins are maintained by numerous hydrogen bonds. Myofibrillar proteins are considered the structural building blocks of muscle. Because myofibrillar proteins are composed of filamentous protein gels, these filaments are arranged in parallel bundles and are directly involved in muscle contraction. When myofibrillar proteins are destroyed, the shape and organization of the muscle fibers are also disrupted, leading to changes in muscle structure. Myofibrillar proteins can be destroyed by many factors, including degradation by endogenous enzymes present in muscle. Therefore, providing an exogenous additive that inhibits enzyme activity to improve the quality and extend the shelf life of fermented dried beef is of great significance. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of an exogenous additive in improving the quality of fermented dried beef and extending its shelf life. By exploring the changes in quality indicators such as color, sensory perception, shear force, water activity and pH, as well as indicators such as cathepsin L activity and total colony count when dried beef with added Sichuan pepper extract is stored at different temperatures, an exogenous additive that can improve the quality of dried beef and extend its shelf life is finally obtained.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] The present invention provides an application of an exogenous additive in improving the quality of fermented dried beef and extending its shelf life. The exogenous additive comprises a Sichuan pepper extract.

[0008] Furthermore, the added amount of the Zanthoxylum bungeanum extract is 16%.

[0009] Furthermore, in the process of improving the quality of fermented dried beef and extending its shelf life, the storage temperature range of the exogenous additive is 5° C.-45° C.

[0010] Furthermore, the exogenous additive utilizes the pepper extract to inhibit the degradation of beef myofibrillar protein by cathepsin L in beef, thereby improving the quality of fermented beef jerky and extending its shelf life.

[0011] Furthermore, the components in the Zanthoxylum bungeanum extract that inhibit the degradation of beef myofibrillar protein by cathepsin L in beef include histone H3 and 50S ribosomal protein L14.

[0012] The beneficial effects of the present invention are as follows: the present invention extracts and purifies cathepsin L in beef, explores the effect of the purified cathepsin L pure enzyme activity on beef myofibrillar protein, and simultaneously screens additives that can inhibit enzyme activity, and studies the effect of adding a pepper extract to dried beef fermented by Xylella fastidiosa on the myofibrillar protein structure, protein degradation and quality of the dried beef. Experiments are conducted to explore changes in quality indicators such as color, sensory perception, shear force, water activity and pH, as well as indicators such as cathepsin L activity and total colony count when dried beef added with 16% pepper extract and stored at different temperatures, and the shelf life of the dried beef is predicted, ultimately obtaining an exogenous additive for improving the quality of fermented dried beef and extending its shelf life. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Diagram of cathepsin L purification, including (a) DEAE cellulose ion exchange fractionation process; (b) G-75 gel filtration fractionation process; (c) SDS-PAGE electrophoresis of cathepsin L fractionation. M: Marker; Lane 1: Crude cathepsin L enzyme; Lane 2: Ion fractionation of cathepsin L; Lane 3: Gel fractionation of cathepsin L.

[0014] Figure 2 : A bar graph showing the effect of the addition of potato and Zanthoxylum bungeanum extracts on cathepsin L;

[0015] Figure 3 : A bar graph showing the effect of ginger and garlic extract addition on cathepsin L activity;

[0016] Figure 4 : The purification diagram of Zanthoxylum bungeanum extract protein, where M: Marker; Lane 1: Zanthoxylum bungeanum crude extract; Lane 2: Zanthoxylum bungeanum dialyzate;

[0017] Figure 5 : Line graph showing the effect of Zanthoxylum bungeanum extract on the degradation of beef myofibrillar protein by cathepsin L;

[0018] Figure 6 :The SDS-PAGE image of the effect of Zanthoxylum bungeanum extract on the degradation of myofibrillar protein by cathepsin L;

[0019] Figure 7 : This is a line graph showing the effect of the amount of pepper extract added on the pH value of dried beef;

[0020] Figure 8: This is a line graph showing the effect of the amount of pepper extract added on the color of dried beef jerky;

[0021] Figure 9 : This is a line graph showing the effect of Zanthoxylum bungeanum extract on the activity of cathepsin L in dried beef;

[0022] Figure 10 : Bar graph showing the effect of Zanthoxylum bungeanum extract on TCA-soluble peptides from dried beef;

[0023] Figure 11 : Microstructural changes of dried beef with 0% pepper added, with the left image magnified 300 times and the right image magnified 600 times;

[0024] Figure 12 : Microstructural changes of dried beef with 4% added Sichuan peppercorns, with the left image magnified 300 times and the right image magnified 600 times;

[0025] Figure 13 : Microstructural changes of dried beef with 8% added Sichuan peppercorns, with the left image magnified 300 times and the right image magnified 600 times;

[0026] Figure 14 : Microstructural changes of dried beef with 12% added Sichuan peppercorns, with the left image magnified 300 times and the right image magnified 600 times;

[0027] Figure 15 : Microstructural changes of dried beef with 16% added Sichuan peppercorns, with the left image magnified 300 times and the right image magnified 600 times;

[0028] Figure 16 : Microstructural changes of dried beef with 20% added Sichuan peppercorns, with the left image magnified 300 times and the right image magnified 600 times;

[0029] Figure 17 : This is a line graph showing the effect of the amount of Zanthoxylum bungeanum extract added on the fragmentation index of the dried beef muscle;

[0030] Figure 18 : Fourier transform infrared spectra of dried beef with different addition amounts of pepper extract;

[0031] Figure 19 : This is the effect of pepper extract on the secondary structure of dried beef;

[0032] Figure 20 : The secondary structure analysis diagram of the myofibrillar protein of dried cattle by Zanthoxylum bungeanum extract;

[0033] Figure 21 :The SDS-PAGE image of the effect of Zanthoxylum bungeanum extract on myofibrillar protein of cattle dried basil;

[0034] Figure 22 : This is a graph showing the effect of Zanthoxylum bungeanum extract on the surface hydrophobicity, free sulfhydryl groups and disulfide bond content of dried beef;

[0035] Figure 23 : Correlation analysis between cathepsin L and various indicators of dried beef;

[0036] Figure 24 : The pH value change of dried beef at different storage temperatures;

[0037] Figure 25 : The change of water activity (Aw) value of dried beef at different storage temperatures;

[0038] Figure 26 : The change of brightness L of dried beef at different storage temperatures;

[0039] Figure 27 : This is a graph showing the changes in sensory quality of dried beef at different storage temperatures;

[0040] Figure 28 : The change of volatile basic nitrogen (TVB-N) in dried beef at different storage temperatures;

[0041] Figure 29 : The change of TBARS of dried beef at different storage temperatures;

[0042] Figure 30 : The diagram of the change of shear force of dried beef at different storage temperatures;

[0043] Figure 31 : Changes of TCA-soluble peptides in dried beef stored at different storage temperatures;

[0044] Figure 32 : The diagram showing the changes in cathepsin L activity of dried beef at different storage temperatures;

[0045] Figure 33 : The graph showing the changes in the number of viable bacteria in dried beef at different temperatures;

[0046] Figure 34 : Arrhenius curve of the change of bovine dry cathepsin L;

[0047] Figure 35 : Arrhenius curve of shear force variation of cattle trunk. DETAILED DESCRIPTION

[0048] The following experimental examples and embodiments are intended to further illustrate the present invention, but are not intended to limit the present invention.

[0049] Experimental Example 1

[0050] 1.1 Extraction and detection of cathepsin L (CATL) in beef

[0051] 1.1.1 Extraction of crude cathepsin L (CATL) enzyme from beef

[0052] A 10g beef sample was taken, fat and connective tissue removed, and four volumes of 20mmol / L phosphate buffer A (pH 6.0) were added. Homogenize at 8000rpm for 30 seconds, then extract at 4°C for 30 minutes. The sample was then centrifuged at 10,000g for 30 minutes at 4°C. The supernatant was collected and 80% ammonium sulfate (solid) was added to the supernatant in an ice bath with stirring. The sample was then allowed to stand at 4°C for 1 hour before centrifugation. The resulting precipitate was collected and dissolved in 20mmol / L phosphate buffer B (pH 6.0) containing 5mmol / L L-Cys. After complete dissolution, the precipitate was centrifuged at 8000g for 10 minutes at 4°C. The supernatant was then dialyzed against phosphate buffer B at 4°C for at least 24 hours. The resulting enzyme solution was crude beef cathepsin L.

[0053] 1.1.2 Purification of crude cathepsin L from beef

[0054] 1.1.2.1 DEAE purification

[0055] The crude enzyme solution after complete dialysis was centrifuged again (4°C, 8000g, 10 min) to obtain the supernatant, which was then filtered through a 0.45 μm filter membrane. The filtered dialysate was added to a DEAE prepacked column pre-equilibrated with 20 mmol / L pH 6.0 phosphate buffer (buffer B) containing 5 mmol / L L-Cys, and gradient eluted with buffer B containing 0-2 mol / L NaCl (the liquid added to the prepacked column needed to be filtered through a 0.45 μm filter membrane to avoid clogging the prepacked column). The flow rate was 0.5 mL / min, and 4 mL was collected in each tube. The absorbance value and enzyme activity of each tube at 280 nm were measured.

[0056] Depend on Figure 1 (a) It can be found that during the entire process of ion exchange chromatography, there are two main protein absorption peaks. Figure 1 Lane 2 in (c) shows that the enzyme solution after ion exchange chromatography contains significantly less protein. However, after DEAE ion exchange chromatography, cathepsin L was purified 2.91-fold, with an enzyme activity recovery rate of 51.30%.

[0057] 1.1.2.1 Sephadex G-75 gel chromatography

[0058] Preparation of gel: Weigh 10g of Sephadex G-75 powder, add 400mL of deionized water to soak, let it stand for 30 minutes, remove the upper suspended particles by pouring after it settles, then add 400mL of deionized water to soak, let it stand, pour, repeat at least 3 times until there are no suspended particles in the upper layer, then vacuum the gel to remove bubbles; Column filling: Take a clean chromatography column with an inner diameter of 0.8cm and a length of 1m, place a layer of wire mesh inside the bottom of the tube, fix the chromatography column vertically, then close the water outlet of the chromatography column, add 1 / 2 column volume of deionized water, and then Slowly add the gel to the chromatography column while opening the column outlet to allow the gel to settle naturally. Equilibrate: Equilibrate the column with buffer solution B for 1-2 column volumes. When the pH at the outlet is the same as the inlet, add the sample. Add sample: When 2-3 mm of eluent remains above the bed, close the column outlet and slowly add the sample solution along the column inner wall. Then, open the column outlet to allow the sample to penetrate the gel bed. Elute: When the upper liquid level is level with the bed, elute with buffer solution C (buffer solution B with 0.2 mol / L NaCl, pH 6.0). Rinse the inner wall with a small amount before eluting. Collect 3 mL of eluate in a centrifuge tube for two column volumes. Stop elution when the absorbance of the eluate at 280 nm is less than or equal to 0.0003. Measure the absorbance and enzyme activity of each tube.

[0059] The concentrated enzyme solution was further eluted and separated through a G-75 gel column. Figure 1 (b) It can be seen that three protein absorption peaks appeared during the elution process. The corresponding SDS-PAGE gel electrophoresis purity was identified by Figure 1 As can be seen in lane 3 of (c), a single band was obtained at approximately 70 kDa. After gel chromatography, cathepsin L was purified 3.27-fold with a recovery rate of 23.08%.

[0060] 1.1.2.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

[0061] 5% stacking gel and 12% polyacrylamide separation gel were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the extracted proteins. The protein samples were mixed with 5× buffer (containing 2 mL distilled water, 300 μL 1 mol / LTris-HCl, pH 6.8), 0.005 g bromophenol blue, 0.1 g SDS, 2.5 mL glycerol, and 250 μL DTT) in a volume ratio of 4:1. The mixture was heated in boiling water for 5 minutes, and then 20 μL of each sample was loaded onto the polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250 solution for more than half an hour, and the gel was destained with a decolorization solution (ethanol: acetic acid: aqueous solution = 5:1:4) at 37°C for at least 2 hours until the protein bands were clear.

[0062] 1.1.3 Determination of protein concentration

[0063] Protein was determined using the BCA kit. First, BCA reagent and copper reagent were mixed in a ratio of 50:1 to form BCA working solution. Then, 10 μL of BSA protein standard (5 mg / mL) was diluted to 100 μL with PBS diluent to a final concentration of 0.5 mg / mL. Then, 0, 2, 4, 6, 8, 12, 16, and 20 μL of the diluted standard were added to a 96-well plate and diluted to 20 μL with PBS. Then, 200 μL of BCA working solution was added to each well, and the plate was reacted at 37°C for 30 minutes. The absorbance value was measured at 562 nm using a microplate reader, and the absorbance value and the standard concentration were used as a calibration curve.

[0064] Take 20 μL of sample and add 200 μL of working solution, react at 37°C for 30 min, and then measure the absorbance at 562 nm using a microplate reader.

[0065] 1.1.4 Determination of cathepsin L activity

[0066] Cathepsin L activity is measured by taking 100 μL of the enzyme solution and adding 100 μL of reaction buffer (150 mmol / L Bis-Tris, 30 mmol / L EDTA, 6 mmol / L DTT, pH 5.5). Preheat the reaction at 30°C for 10 minutes. Then, add 100 μL of 90 μmol / L fluorescent substrate Z-Phe-Arg-AMC. Incubate the reaction in a 30°C water bath for 15 minutes. Add 3 mL of reaction stop solution (1% (w / v) SDS, 50 mmol / L Bis-Tris, pH 7.0), immediately place the reaction in an ice bath, and measure fluorescence intensity within 10 minutes. Fluorescence is measured on a microplate reader at an excitation wavelength of 380 nm and an emission wavelength of 469 nm. One unit (U) of enzyme activity is defined as the amount of enzyme required to hydrolyze the substrate and release 1 nmol of AMC product per minute at 30°C (1 nmol / min).

[0067] Prepare the AMC standard substance into a 22 ng / μL solution and take different volumes to draw the AMC standard curve.

[0068] 1.2 Detection of the effects of different additives on cathepsin L activity

[0069] The effects of aqueous extracts of potato, Zanthoxylum bungeanum, ginger, and garlic on the activity of cathepsin L were investigated. Potato, Zanthoxylum bungeanum, ginger, garlic, and sesame seeds were ultrasonically extracted in distilled water and then added to the reaction system for the enzyme activity assay. The activity of cathepsin L was measured after addition, aiming to identify the additive with the best inhibitory effect.

[0070] Among them, the effects of potato and pepper water extracts on cathepsin L are as follows Figure 2 As shown in the results, the pepper extract has a significant inhibitory effect on the activity of cathepsin L (P<0.05). Ginger and garlic were added to the cathepsin L activity reaction system, and the results were as follows: Figure 3 As shown in the figure, the extract of Zanthoxylum bungeanum has the greatest effect on the activity of cathepsin L. Therefore, Zanthoxylum bungeanum was used as the main research object.

[0071] 1.3 Extraction and identification of Zanthoxylum bungeanum protein

[0072] 1.3.1 Extraction of Zanthoxylum bungeanum protein

[0073] 80% ammonium sulfate was added to the Zanthoxylum bungeanum extract, and then the extract was allowed to stand at 4°C for 1 hour, and then centrifuged at 8000g for 10 minutes. The precipitate obtained by salting out was collected, dissolved in 20 mmol phosphate buffer, and dialyzed thoroughly. The SDS-PAGE image after dialysis is shown in the figure below. Figure 4As shown in the figure, the Zanthoxylum bungeanum protein bands are few. After ammonium sulfate precipitation and dialysis, three bands are visible on the SDS-PAGE gel: 63-75 kDa, 35-48 kDa, and below 17 kDa. After dialysis, centrifuge the supernatant and perform SDS-PAGE according to the method in step 1.1.2.3.

[0074] 1.3.2 Analysis of the inhibitory rate of Zanthoxylum bungeanum protein on cathepsin L activity

[0075] 20 μL of Zanthoxylum bungeanum protein extract from the purification process was added to the cathepsin L activity assay reaction system described in Method 2.2.5, and the inhibitory rate of the extracted protein on cathepsin L activity was calculated. The initial purification of the Zanthoxylum bungeanum protein extract and its effect on cathepsin L activity are shown in Table 1.

[0076] Table 1 Preliminary purification of Zanthoxylum bungeanum extract protein and its effect on cathepsin L activity

[0077]

[0078]

[0079] 1.3.2 Identification of Zanthoxylum bungeanum protein

[0080] 1.3.2.1 Sample processing

[0081] 1.3.2.1.1 Protein extraction

[0082] 1) Take the sample, add 4 times acetone, and place it at -20℃ overnight;

[0083] 2) Remove the solution, centrifuge at 12000g for 10 min, discard the supernatant, and evaporate the acetone in a fume hood;

[0084] 3) Add 200 μL of pre-chilled lysis buffer and pipette to dissolve the precipitate;

[0085] 4) Ultrasonication on ice: sonication for 2 seconds, pause for 2 seconds, and sonication for 2 minutes;

[0086] 5) Centrifuge at 17000 g, 4°C, for 10 min. Pipette the supernatant into a new 1.5 mL centrifuge tube.

[0087] 1.3.2.1.2 Protease cleavage

[0088] 1) Take the protein solution, add DTT reagent to a final concentration of 50 mmol / L, and react at 37°C for 1 hour;

[0089] 2) Add insulin autoantibody (IAA) reagent to a final concentration of 100 mmol / L and react at room temperature in the dark for 40 minutes;

[0090] 3) Add the protein solution after reduction and alkylation to a 10KD ultrafiltration tube, centrifuge at 12000g for 20 minutes, and discard the solution at the bottom of the collection tube;

[0091] 4) Add 100 μL of 8 mol / L urea (pH 8.5), centrifuge at 12,000 g for 20 min, discard the solution at the bottom of the collection tube, and repeat twice;

[0092] 5) Add 100 μL of 25 mmol / L ammonium bicarbonate solution, centrifuge at 12,000 g for 20 min, discard the solution at the bottom of the collection tube, and repeat three times;

[0093] 6) Replace the collection tube with a new one and add 25 mmol / L ammonium bicarbonate solution (containing trypsin) to the ultrafiltration tube to make a final volume of 50 μL. The ratio of trypsin to protein was 1:50. Incubate at 37°C overnight.

[0094] 7) The next day, trypsin was added (trypsin to protein ratio 1:100) and the mixture was reacted at 37°C for 4 hours. After the reaction, the mixture was centrifuged at 12,000 g for 20 minutes. The peptide solution after enzymatic digestion was collected at the bottom of the centrifuge tube.

[0095] 8) Add 50 μL of 25 mmol / L ammonium bicarbonate solution to the ultrafiltration tube and centrifuge again at 12,000 g for 20 min. Combine with the sample from the previous step and collect the bottom of the tube to obtain 100 μL of enzymatically hydrolyzed sample.

[0096] 9) Add 100 μL of 0.2% trifluoroacetic acid (TFA) solution to the peptide sample and mix well;

[0097] 10) C 18 The column was activated with acetonitrile (ACN) and then washed twice with 1 mL of 0.1% TFA solution;

[0098] 11) Transfer the peptide sample to C 18 Small column, discard the dripping liquid;

[0099] 12) Elute with 500 μL 0.1% TFA and discard the remaining liquid;

[0100] 13) Elute with 500 μL of 70% cerium ammonium nitrate solution, collect the liquid, and dry it in vacuum.

[0101] 1.3.2.1.3 Using ZipTipC 18 Desalination

[0102] 1) Rinse triisopropylsilyl acetylene (TIP) 10 times with 50 μL 60% ACN / 0.1% TFA;

[0103] 2) Wash the TIP 10 times with 10 μL 0.1% TFA;

[0104] 3) Aspirate and expel the sample into the TIP 20 times to drain the liquid;

[0105] 4) Wash the TIP with 10 μL 0.1% TFA five times;

[0106] 5) Elute the peptide fragments with 10 μL of 60% ACN, 0.1% TFA into a new centrifuge tube and vacuum dry;

[0107] 6) Test on the computer.

[0108] 1.3.2.1.5 Database Analysis

[0109] This project used PEAKS software to search the database. The specific search parameters are shown in Table 2.

[0110] Table 2 Identification parameters

[0111]

[0112] The proteins in Zanthoxylum bungeanum extract were digested with trypsin and then analyzed by RPLC-MS and database search. A total of 5 proteins were identified, including ubiquitin extension protein 1, ATP synthase subunit α, ATP synthase subunit β, histone H3 and 50S ribosomal protein L14. The molecular weight of the proteins was concentrated between 9 and 55 kDa, which indicated that the protein species in Zanthoxylum bungeanum extract were not large. Figure 4 The SDS-PAGE results of Zanthoxylum bungeanum extract were relatively high in the 5 proteins, including ATP synthase subunit α, ATP synthase subunit β and histone H3.

[0113] HH3 and RPL14 were extracted. The effects of HH3 and RPL14 on the activity of cathepsin L are shown in Table 3.

[0114] Table 3 Effects of HH3 and RPL14 on cathepsin L activity

[0115]

[0116] 1.4 Effect of Zanthoxylum bungeanum extract on myofibrillar protein degradation by cathepsin L

[0117] Based on the degradation of myofibrillar protein by cathepsin L, 0, 10, 15, 20, and 25 μL of Zanthoxylum bungeanum extract were added to 2 mL of a 2 mg / mL myofibrillar protein solution. The mixture was then incubated at 30°C for 2 h with 200 μL of 40 U cathepsin L. The degradation of myofibrillar protein in each group was observed by SDS-PAGE gel electrophoresis, following the procedure described in 1.1.2.3. Surface hydrophobicity was determined as follows:

[0118] The myofibrillar protein concentration was adjusted to 2 mg / mL. 40 μL of bromophenol blue (1 mg / mL) solution was then mixed with 2 mL of the protein solution. Phosphate buffer was used as a blank. The mixture was shaken constantly for 15 minutes at 7000 g for 5 minutes at 4°C. The supernatant was then measured for absorbance at 595 nm. Changes in hydrophobicity and solubility were calculated.

[0119] Protein solubility and surface hydrophobicity change due to Figure 5 It can be seen that Zanthoxylum bungeanum can inhibit the degradation of myofibrillar protein by cathepsin L.

[0120] like Figure 6 As shown, when the Zanthoxylum bungeanum extract was added to myofibrillar protein with cathepsin L, the degradation of myofibrillar protein was gradually reduced.

[0121] 2.1 Study on the effect of the amount of Zanthoxylum bungeanum extract on the activity of cathepsin L and its quality in dried beef

[0122] 2.1.1 Materials and methods

[0123] 2.1.1.1 Experimental Materials

[0124] Fresh beef, salt, sesame oil, tea polyphenols, glucose, pepper, MSG and liquor were purchased from local supermarkets; Staphylococcus xylosus was isolated and purified from a homemade laboratory specimen.

[0125] 2.1.1.2 Instruments and experimental reagents

[0126] The main experimental instruments are shown in Table 4, and the main experimental reagents are shown in Table 5.

[0127] Table 4 Main experimental instruments

[0128]

[0129]

[0130] Table 5 Main experimental reagents

[0131]

[0132] 2.1.2 Test methods and results

[0133] 2.1.2.1 Production of Dried Beef

[0134] The visible connective tissue and adipose tissue of the beef shank were trimmed and stored at 4°C. The rested beef was cut into 5×10×2 cm pieces, soaked in white wine for 10 min, washed, and then added with 2% sesame powder, 1.2% salt, 3% glucose, 3.5% five-spice powder, 0.6% monosodium glutamate, 0.15 g / kg tea polyphenols, and pepper extract (0%, 4%, 8%, 12%, 16%, 20%), mixed evenly, and 1% xylose Staphylococcus aureus culture was added. The meat was fermented at 20°C for 60 h, baked in an oven at 90°C until the moisture content was 30%, cooled, and stored for later use.

[0135] 2.1.2.2 Determination of pH value and color

[0136] pH value: A mixture of 2 g of sample and 14 mL of distilled water was homogenized at 5000 rpm for 30 s, and the final pH of the beef sample was measured using a pH meter.

[0137] Color: The color of the dried beef samples was measured using an HP-2132 colorimeter, and the L* (lightness value), a* (redness value), and b* (yellowness value) were recorded.

[0138] Effect of Sichuan pepper extract on pH of fermented dried beef Figure 7 Indicated by Figure 7 It can be seen that the pepper extract has a significant effect on the pH value of beef jerky.

[0139] like Figure 8 As shown, the pepper extract has a significant effect on the color surface displayed by the dried beef.

[0140] 2.1.2.3 Texture determination

[0141] Three pieces of beef samples were randomly selected and their hardness, elasticity, chewiness, cohesion, adhesiveness and shear force were measured using a CT3 texture analyzer to evaluate their texture characteristics.

[0142] The study found that increasing the amount of Sichuan peppercorn extract added also altered the texture of the beef jerky, demonstrating a significant effect of Sichuan peppercorn extract on its texture (P < 0.05). The firmness of the beef jerky also changed significantly with increasing Sichuan peppercorn extract levels. The firmness of meat during chewing is closely related to its tenderness and is a key indicator of consumer preference, influenced by the amount and distribution of fat. This effect was most pronounced when Sichuan peppercorn extract was added at 16%.

[0143] 2.1.2.4 Sensory evaluation

[0144] Twenty master's students (aged 25-30) from the School of Food Science were selected to evaluate the samples. Samples were evaluated for color, odor, flavor, texture, and overall acceptability in a dedicated room with a constant temperature, no noise or odor, and adequate lighting. All evaluators underwent a brief discussion and training at the beginning of the sensory evaluation. Each team member evaluated three replicate samples in a random order and assigned a value between 1 and 9, with 9 indicating highly acceptable and 1 indicating extremely unacceptable. The following items were evaluated: color (reddish-brown with a glossy cut surface = 9; green with a dull cut surface = 1); odor (with a strong aroma like beef jerky = 9; without a beef jerky aroma, with a very light aroma = 1); flavor (moderately salty with no beefy aftertaste and a good aftertaste = 9; too salty or too light with a distinct beefy aftertaste = 1); texture (smooth cut surface with dense texture = 9; rough cut surface with loose texture = 1); and overall acceptability (acceptable = 9; unacceptable = 1). After each test, the assessor rinsed his mouth with an appropriate amount of warm water.

[0145] It was finally found that the taste was best when the addition amount of pepper extract was 16%.

[0146] 2.1.2.5 Cathepsin L enzyme activity assay

[0147] The method is the same as step 1.1.4.

[0148] in, Figure 9 The activity of cathepsin L in roasted dried beef with or without Zanthoxylum bungeanum extract was shown.

[0149] 2.1.2.6 TCA-solubilized peptides

[0150] After cutting the dried beef sample into pieces, 3 g was mixed with 27 mL of 5% TCA solution and homogenized. After ice bathing for 30 min, the mixture was centrifuged at 4°C (10,000 g × 10 min). The supernatant was collected and the content of TCA-soluble peptides was determined using a BCA protein quantification kit (Beiyang Time Biotechnology, China). The results were expressed as μmol Tyr / g meat, and each group was repeated 3 times.

[0151] Effect of Zanthoxylum bungeanum extract on the content of TCA-soluble peptides in dried beef Figure 10 shown.

[0152] 2.1.2.7 Scanning electron microscopy of dried cattle group

[0153] The dried beef sample was cut into 2mm×5mm strips with a double-sided blade; fixed with 2.5% pH6.8 glutaraldehyde at 4°C for about 1.5 hours; rinsed with 0.1mol / L pH 6.8 phosphate buffer solution for 10 minutes, and repeated 2-3 times; then dehydrated with 50%, 70%, and 90% ethanol for 10 minutes respectively, and then dehydrated with 100% ethanol 3 times, also for 10 minutes each time; the dehydrated sample needed to be placed in the freezer of a refrigerator at -20°C for 30 minutes, and freeze-dried with an ES-2030 freeze dryer; the treated sample was fixed on the scanning electron microscope sample stage with the observation surface facing up with conductive tape, and a layer of metal film was plated on the fixed sample surface by an ion sputtering coater; the coated sample was observed with an EM-30 scanning electron microscope.

[0154] In order to observe the effect of the amount of pepper extract added on the quality of beef jerky, the ultrastructure of the intramuscular connective tissue was observed using a scanning electron microscope (SEM). Figure 11-16 As shown, scanning electron microscopy (SEM) analysis clearly showed that the addition of 4%, 8%, 12%, 16% and 20% of Zanthoxylum bungeanum extract changed the microstructure of beef dried bark.

[0155] 2.1.2.8 Determination of myofibril fragmentation index

[0156] Take 2.00g of crushed dried beef sample and mix it with 20 volumes of pre-cooled MFI buffer solution (100mmol / L KCl, 11.2mmol / L K2HPO4, 8.8mmol / L KH2PO4, 1mmol / L EGTA, 1mmol / L MgCl2). Then homogenize at 8000rpm for 1min, filter through three layers of degreased gauze to remove connective tissue, then centrifuge at 4℃ and 11000g for 10min, and remove the supernatant after centrifugation. Add 20 volumes of pre-cooled MFI buffer to the centrifuge tube again, repeat the centrifugation 2-3 times to make a suspension, centrifuge at 11000g for 10min at 4℃, discard the supernatant, and precipitate with MFI buffer to make a suspension with a protein concentration of 0.50±0.05mg / mL. Take the suspension and use the biuret method.

[0157] 1. Determination of the standard curve: Take six identical test tubes and add 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard protein solution to each tube, respectively. Make up any volume less than 1 mL with deionized water. Then, add 4 mL of biuret reagent. Shake well and place in a dark place at room temperature for 30 minutes. Measure the absorbance at 540 nm. The blank tube is the first test tube without protein solution. Plot a standard curve with protein content as the horizontal axis and absorbance as the vertical axis.

[0158] 2. Sample Determination: Take three identical test tubes and determine the protein concentration of the unknown sample according to the above method. The sample concentration should not exceed 10 mg / mL. Measure the absorbance (A) at 540 nm. MFI = A540 × 200. Prepare three replicates for each sample.

[0159] Figure 17 This study shows the effect of Zanthoxylum bungeanum extract on the myogenic fragmentation index in dried beef.

[0160] 2.1.2.9 Fourier Infrared

[0161] 2 mg of freeze-dried sample powder was mixed and ground thoroughly with 200 mg of sample powder in an agate mortar, then put on the table and placed in the FTIR with a measurement range of 4000 to 400 cm -1 The spectra and relative contents of protein secondary structures were calculated using OMNIC software.

[0162] The Fourier infrared spectra of dried beef with different addition amounts of pepper extract are shown in the figure below. Figure 18 shown.

[0163] like Figure 19 As shown, with the increase in the amount of pepper extract added, the β-sheet content in the dried beef increased from 31.97% to 49.29% (P>0.05), indicating increased rigidity and decreased conformational flexibility. The α-helix content decreased significantly from 17.17% to 12.63% (P<0.05), and the changes in β-turns and random coils showed the same trend as α-helices. This means that the pepper extract promoted the unfolding of the α-helical structure in the dried beef and increased the percentage of β-sheet content.

[0164] At the same time, the effect of pepper extract on the secondary structure of myofibrillar protein in beef jerky was also explored. Figure 20 As shown, this shows that when the addition amount of pepper extract is 16%, the degree of unfolding of myofibrillar protein structure decreases and the hardness of beef jerky increases.

[0165] 2.1.2.10 Effect of different addition amounts of Sichuan pepper extract on myofibrillar protein in beef.

[0166] Myofibrillar protein was extracted from beef. SDS-PAGE analysis was then performed to observe changes in myofibrillar protein. The concentration of the extracted myofibrillar protein was determined using the Coomassie Brilliant Blue method.

[0167] The electrophoresis diagrams of myofibrillar proteins extracted from dried beef that was fermented and baked without adding pepper extract and dried beef that was fermented and baked with different pepper extracts are as follows: Figure 21 express.

[0168] 2.1.2.10 Surface hydrophobicity determination

[0169] Follow the method for measuring surface hydrophobicity in step 1.4.

[0170] like Figure 22 As shown in the figure, the addition of Sichuan pepper extract to dried beef had a significant effect on the surface hydrophobicity of myofibrillar protein (P<0.05), and the surface hydrophobicity of myofibrillar protein decreased significantly with the increase of Sichuan pepper extract.

[0171] 2.1.2.11 Disulfide bonds and free sulfhydryl groups

[0172] To 0.9 mL of buffer A (1 mmol / L EDTA, 0.1 mol / L phosphate buffer, pH 8.0), add 100 μL of a 4 mg / mL myofibrillar protein solution and 100 μL of buffer B (10 mmol / L DTNB, 0.2 mol / L Tris-HCl, pH 8.0). Replace the protein solution in the blank sample with 0.6 mol / L NaCl-20 mmol / L phosphate buffer. After mixing, incubate at 37°C in the dark for 25 min. Measure absorbance at 412 nm using a microplate reader (200 μL per well).

[0173] Dissolve 100 mg of DTNB reagent in 10 mL of 1 mol / L sodium sulfite at 37°C until the bright red solution turns pale yellow, indicating the formation of NTSB. Add 1 mL of buffer C (8 mol / L urea, 3 mmol / L EDTA, 1% SDS, 0.1 mol / L NaSO, 1% NTSB, 0.2 mol / L Tris-HCl (pH 9.5)) to the mixture, followed by the addition of 100 μL of 4 mg / mL myofibrillar protein solution. Mix well and incubate at 37°C, protected from light, for 25 minutes. Measure absorbance at 412 nm using a microplate reader (200 μL per well). Perform this in triplicate.

[0174] Depend on Figure 22 It can be seen that after adding the pepper extract, the free sulfhydryl content in dried beef increased with the increase of the pepper extract addition, increasing from 30.408 nmol / mg to a maximum of 43.739 nmol / mg, which indicates that the degradation degree of free sulfhydryl in dried beef decreased. 2.1.2.12 Correlation analysis between cathepsin L and various indicators of dried beef

[0175] The correlation between cathepsin L activity and various indicators of dried beef is as follows Figure 23As shown in the figure, it can be seen that the activity of cathepsin L is correlated with the amount of Zanthoxylum bungeanum extract added, pH value of dried beef, shear force, myogenic fragmentation MFI, TCA-soluble peptide, surface hydrophobicity, free thiol groups, and disulfide bonds.

[0176] 3.1 Effect of Sichuan pepper extract on the quality of dried beef during storage and prediction of its shelf life

[0177] 3.1.1 Materials and methods

[0178] 3.1.1.1 Experimental materials, instruments and reagents

[0179] Same as steps 2.1.1.1 and 2.1.1.12

[0180] 3.1.2 Test methods

[0181] 3.1.2.1 Sample processing

[0182] The method is the same as step 2.1.2.1. The dried beef sample with 16% Sichuan pepper extract was placed in a constant temperature environment of 5℃, 15℃, 25℃, 35℃ and 45℃. Samples were taken at intervals of 15 days at 5℃, 15℃ and 25℃, and every 10 days at 35℃ and 45℃. The samples were used for subsequent experiments.

[0183] 3.1.2.2 Determination of pH value

[0184] The method is the same as the pH value determination method in step 2.1.2.2.

[0185] like Figure 24 The initial pH value of the dried beef was 5.65. As storage time increased, the pH value generally decreased. The decreasing trend in pH became more pronounced as storage temperature increased, reaching 5.015 and 4.894 at 37 and 45°C, respectively, on day 70.

[0186] 3.1.2.3 Determination of water activity

[0187] Turn on the water activity meter 20 minutes in advance to preheat, take a certain amount of sample, mince it, and spread it evenly in the water activity measuring dish (it is best to cover the bottom of the dish so that it is opaque), put the dish into the sample cell for measurement, and record the water activity of the sample.

[0188] like Figure 25 As shown in the figure, the changes in water activity of dried beef at different storage temperatures are shown. The water activity in dried beef changes with storage time and temperature, which has a significant effect (P<0.05). It shows an overall downward trend, and the higher the storage temperature, the more obvious the downward trend.

[0189] 3.1.2.4 Determination of color brightness

[0190] The method is the same as the color determination method in step 2.1.2.2.

[0191] like Figure 26 As the storage time prolonged, the lightness L* of dried beef gradually increased at different storage temperatures, with significant differences (P<0.05). The higher the temperature and the longer the storage time, the more obvious the L* change.

[0192] 3.1.2.5 Sensory evaluation

[0193] The method is the same as the sensory evaluation step in step 2.1.2.4.

[0194] Depend on Figure 27 The results show that at all five storage temperatures, the sensory evaluation indicators (color, texture, odor, and overall acceptability) of dried beef decreased with increasing storage time. When the sensory index score fell below 5, the dried beef was considered unacceptable and not recommended for consumption. These results indicate that storing dried beef at low temperatures can delay its quality deterioration and extend its shelf life.

[0195] 3.1.2.6 Determination of Volatile Basic Nitrogen (TVB-N)

[0196] The total volatile alkaline nitrogen content can be used to judge the freshness of animal foods.

[0197] Cut the dried beef into small pieces, accurately weigh 3g, and place in a beaker. Add Volatile Basic Nitrogen Extraction Solution No. 1 to the beaker and mix thoroughly until the minced meat turns mostly white. Add 16.0mL of distilled or purified water, mix thoroughly, and extract in an ultrasonic extractor for 10 minutes. Remove the sample and add 0.5mL of Volatile Basic Nitrogen Extraction Solution No. 2 and 10.0mL of distilled or purified water. Mix thoroughly, centrifuge, and collect the supernatant as the sample treatment solution. Transfer 0.25mL of the sample treatment solution to a cuvette, add 1.75mL of distilled or purified water, add 3 drops of Detection Solution A, place in an instrument and zero, add 3 drops of Detection Solution B, and mix thoroughly. Place in a 37°C constant temperature water bath for 10 minutes. Remove, wipe the cuvette clean, and place in the instrument for testing.

[0198] Depend on Figure 28 The changes in volatile basic nitrogen (TVB-N) in dried beef at different storage temperatures showed that the TVB-N content in dried beef increased continuously with the increase of storage time.

[0199] 3.1.2.7 Determination of thiobarbituric acid (TBARS)

[0200] The minced sample (10 g) was mixed with a 20% trichloroacetic acid (TCA) (w / v) solution (25 mL) and homogenized at 10,000 rpm for 30 s. The supernatant was centrifuged at 5500 g for 15 min at 4°C and filtered twice. 2 mL of the filtrate and 2 mL of 0.02 mol / L TBA were mixed in a test tube and heated in an 80°C water bath for 20 min. The solution was then cooled in an ice bath for 10 min. The absorbance of the sample was measured at 532 nm using water as a blank. The TBARS value was expressed as milligrams of malondialdehyde (MDA) equivalent per kilogram of meat sample.

[0201] according to Figure 29 The study found that the initial TBARS content in beef jerky was 0.9 mg MDA / kg. With the increase of storage time at different temperatures, the TBARS content in beef jerky increased significantly (P<0.05).

[0202] 3.1.2.8 Determination of shear force

[0203] The method is the same as the shear force determination method in step 2.1.2.3.

[0204] Changes in shear force during storage of dried beef at different temperatures Figure 30 shown.

[0205] 3.1.2.9 Determination of TCA-soluble peptides

[0206] The method is the same as that for TCA-solubilized peptide assay in step 2.1.2.6.

[0207] Changes of TCA-soluble peptides during storage of dried beef at different temperatures Figure 31 As shown in the figure, it can be seen that storage at different temperatures has a significant effect on the changes of TCA-soluble peptides in beef jerky (P<0.05).

[0208] 3.1.2.10 Cathepsin L enzyme activity assay

[0209] The method is the same as step 1.1.4.

[0210] The changes of cathepsin L in dried beef stored at different temperatures are as follows Figure 32 shown.

[0211] 3.1.2.11 Determination of total colony count

[0212] The total bacterial count method was carried out in accordance with GB4789.2-2022 National Food Safety Standard (National Health Commission, 2022).

[0213] Plate count medium (PCA): 5.0 g tryptone; 2.5 g yeast extract powder; 15.0 g glucose agar; 1000 mL distilled water; pH 7.0 ± 0.2.

[0214] The dried beef was stored in different incubators at temperatures of 5°C, 15°C, 25°C, 35°C, and 45°C, and samples were taken at intervals. The changes in the number of viable bacteria in the samples were as follows: Figure 33 express.

[0215] 3.1.2.12 Shelf Life Prediction

[0216] 3.1.2.12.1 Establishing the Shelf Life of Dried Beef Using the Arrhenius Equation

[0217] Under normal circumstances, the quality of dried beef will gradually deteriorate over time, thus affecting its sales and consumption. Many changes in food quality conform to the zero-order reaction (4-1) or first-order reaction (4-2) model. The reaction equation is as follows:

[0218] A=A0-K a t (4-1)

[0219]

[0220] Where t is the storage time; A is the quality value of the product on storage day t; A0 is the initial value of the product quality; Ka is the chemical reaction rate constant.

[0221] The Arrhenius equation (4-3) is as follows:

[0222]

[0223] Taking the logarithms of both sides of formula (4-3) we get:

[0224]

[0225] Where K a0 is the frequency factor; Ea is the activation energy (kJ / mol); R is the ideal gas constant (8.314 J / mol﹒K); T is the absolute temperature (K).

[0226] According to formula (4-3), there is a good linear relationship between LnKa and 1 / T. Therefore, the activation energy Ka can be calculated based on the slope of the Arrhenius equation of LnKa and 1 / T, thereby establishing a shelf life prediction model for dried beef.

[0227] The shelf life of dried beef was predicted based on the Pearson correlation coefficient between various indicators stored at 5°C. It can be seen that there is a significant correlation between various indicators at 5°C. Among them, the sensory evaluation was positively correlated with pH and Aw, and negatively correlated with other indicators. In the correlation analysis, it was found that indicators such as shear force and cathepsin L activity had the highest correlation, with a correlation coefficient of 0.992. Therefore, cathepsin L activity and shear force were used as key indicators. According to the change rules of cathepsin L activity and shear force, the changes in key indicators measured at 5, 15, 25, 35 and 45°C (converted to thermodynamic temperatures of 278.15K, 288.15K, 298.15K, 308.15K, 318.15K) were brought into zero-order or first-order kinetics for linear fitting. It was found that R in the zero-order reaction of the two groups 2 Greater than R in the first-order reaction 2 This result shows that the zero-order reaction kinetic model can better explain the quality changes of dried beef during storage. The obtained quality change kinetic parameters are shown in Table 6.

[0228] Table 6 Kinetic parameters of quality change

[0229]

[0230] The values ​​of cathepsin L activity and shear force at different storage temperatures of dried beef are now brought into the zero-order reaction kinetic model to predict the shelf life of dried beef. The reaction rate constant Ka obtained after fitting is brought into the Arrhenius equation (4-3), and the relationship between the reaction rate constant Ka of cathepsin L activity and shear force and absolute temperature T is shown in Table 7. Taking the natural logarithm of both sides of the equation, a linear relationship between lnKa and 1 / T is obtained. The results are as follows Figure 34 and Figure 35 shown.

[0231] Table 7 Shelf life model parameters

[0232]

[0233] according to Figure 34 and 35 , combined with the Arrhenius equation (4-3), we can obtain the Arrhenius curve based on the activity of cathepsin L and the shear force, where the activation energy Ea of cathepsin L activity is related to K a0 They are: 34.477 (kJ / mol) and e 14.4361 ; Activation energy Ea and K of shear force a0 They are: 27.6022 (kJ / mol) and e 10.2265 Substitute the result into formula (4-4) to construct the rate of change of cathepsin L activity parameter Ka during the storage of dried beef1 and shear stress parameter change rate Ka 2 The Arrhenius equation:

[0234]

[0235]

[0236] Substituting formulas (4-7) and (4-8) into the zero-order kinetic model (4-1), we obtain

[0237]

[0238]

[0239] Formulas (4-9) and (4-10) represent shelf-life models for dried beef, based on the kinetics of cathepsin L activity and shear stress degradation during storage. These two models indicate that by determining the changes in cathepsin L activity and shear stress at the beginning and end of storage at a specific temperature, the shelf life of dried beef can be calculated.

[0240] 4.3.12.2 Prediction Model Validation

[0241] The accuracy and applicability of the model were verified based on the predicted model and actual values, as shown in Table 8. This table demonstrates the reliability of the shelf life of dried beef calculated using the established kinetic model of cathepsin L activity and shear force. The relative error between the predicted and actual shelf life predictions indicates that the model is more accurate at low temperatures, better preserving the quality of dried beef. The acceptable range of relative error is approximately 20%, and the mean relative error for shear force is less than 20%, making it more accurate than the cathepsin L activity prediction model.

[0242] Table 8 Predicted and actual shelf life of dried beef at different temperatures

[0243]

[0244] 3.1.2.13 Data Processing

[0245] All data presented are means of at least three replicates using one-way analysis of variance (ANOVA). Significant differences between means were determined by Tukey's test and were considered significant at P < 0.05. Statistical analyses were performed using IBM SPSS Statistics 26.0 and Microsoft Excel 2019, and Origin 2021 was used for graphics.

[0246] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. An application of an exogenous additive in inhibiting the degradation of beef myofibrillar protein by cathepsin L in beef to improve the quality of fermented beef jerky and extend its shelf life, characterized in that: The exogenous additive is Zanthoxylum bungeanum extract; The Zanthoxylum bungeanum extract is prepared by extracting Zanthoxylum bungeanum in distilled water through ultrasonic extraction; The added amount of the Zanthoxylum bungeanum extract is 16%; The exogenous additive improves the quality of fermented dried beef and prolongs its shelf life, and its storage temperature range is 5° C.-45° C.

2. The use of the exogenous additive according to claim 1 in inhibiting the degradation of beef myofibrillar protein by cathepsin L in beef to improve the quality of fermented beef jerky and extend its shelf life, characterized in that: The components in the Zanthoxylum bungeanum extract that inhibit the degradation of beef myofibrillar protein by cathepsin L in beef include histone H3 and 50S ribosomal protein L14.

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