Method for killing spores and use thereof

Abstract

The application discloses a spore killing method and application thereof, and the method is to add glucosamine to mix with spores and then to perform heat treatment to efficiently inactivate the spores. According to the technical scheme of the application, the spore cortex is first damaged by glucosamine to release DPA in the nucleus, the DPA destroys the disulfide bond on the surface of the spores, and then the integrity of the spore coat is destroyed to reduce the resistance of the spores, and the killing of the spores is realized by cooperating with a mild heat treatment step; compared with the prior art, the spores can be inactivated by 100% by using a lower heat treatment temperature, and better sterilization effect is achieved by lower energy consumption and cost. The method is applied to the field of food processing, and can provide a novel, effective, highly applicable and more practically applicable spore killing method for the storage of food and the keeping of quality, flavor and nutrient components of the food.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for killing spores and its application. Background Technology

[0002] Bacillus species are widely distributed in soil, water, air, and animal intestines, easily invading the food system at every stage of food production, processing, storage, and transportation. Statistics show that cases of food spoilage and foodborne illnesses caused by spore contamination are common worldwide. For example, spore contamination is particularly prominent in the production of canned foods, dairy products, meat products, and beverages. When spores in food are not effectively killed, under suitable conditions, they can rapidly revive and germinate into vegetative cells, multiplying rapidly and causing food spoilage, severely damaging the food's flavor, texture, color, and nutritional value.

[0003] Traditional food sterilization techniques, such as heat sterilization, chemical sterilization, and irradiation sterilization, have revealed numerous limitations when dealing with Bacillus. Heat sterilization is a widely used method in the food industry, including pasteurization (65-90℃), high-temperature sterilization (110-121℃), and autoclaving. However, Bacillus spores have a unique heat-resistant structure, making it difficult for conventional heat sterilization to completely kill them. While increasing the temperature and extending the time can enhance the sterilization effect, it inevitably causes serious damage to the nutritional components, flavor, and color of food, reducing food quality and commercial value, and increasing energy consumption and production costs. For example, high-temperature treatment may lead to the loss of nutrients such as vitamins and proteins in food, resulting in unpleasant flavor and color changes. Chemical sterilization uses disinfectants, such as chlorine-containing disinfectants and peroxide disinfectants, which have good bactericidal effects on general microorganisms, but Bacillus spores have strong resistance to chemical disinfectants. The multi-layered, dense structure of Bacillus spores effectively blocks the penetration of disinfectants, making it difficult for them to reach the key life-sustaining substances inside the spores, thus making effective killing difficult. Furthermore, the residue of chemical disinfectants poses potential risks to food safety and the environment, and long-term use may lead to spore resistance. While irradiation sterilization can kill spores to some extent, the high cost and complex operation of irradiation equipment, coupled with consumer concerns about the safety of irradiated food, limit its widespread application. In addition, spores produced by strains possessing the Tn1546 transposon often exhibit higher resistance; these heat-resistant spores are more difficult to kill than ordinary spores, posing a serious threat to food safety and quality. Chinese invention patent CN119908386A discloses a method and application for spore killing, which achieves efficient killing of ordinary spores and effective killing of heat-resistant spores through the synergistic effect of ethylenediaminetetraacetic acid (EDTA) and / or EDTA salts with heat sterilization. The killing rate of ordinary spores can reach 99.9997%, and the killing rate of heat-resistant spores can reach 99.98%. This invention provides a new, effective, highly applicable, and practically significant spore control method for food safety and quality maintenance. However, this method has limited effectiveness in killing heat-resistant spores, which are widely present in heat-processed foods. While the spores that are not killed remain dormant for a short period, they germinate into vegetative cells within the food and, through metabolic activity, decompose food components or produce secondary metabolites, leading to spoilage. Therefore, ordinary heat treatment methods are ineffective in killing spores, especially heat-resistant ones, which are a major cause of the short shelf life of heat-processed foods. Adding food additives to inhibit the germination and subsequent reproduction of heat-resistant spores to extend shelf life inevitably poses a potential threat to food safety.

[0004] Given the serious threat that Bacillus poses to food safety and the limitations of traditional sterilization methods, it is urgent to develop efficient, safe, environmentally friendly spore-killing technologies that have minimal impact on food quality. Summary of the Invention

[0005] The main objective of this invention is to provide a method for killing spores and its application, so as to overcome the shortcomings of the prior art.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution.

[0007] One aspect of the present invention proposes a method for killing spores, the core of which is to achieve highly efficient inactivation of spores by synergistic action of glucosamine and heat treatment.

[0008] Preferably, the above-mentioned spores include Bacillus (Bacillus) Bacillus The spores produced by the present invention include one or more of the common spores and the heat-resistant spores produced by Bacillus transposonis containing Tn1546; it should be noted that the common spores referred to in the present invention are spores produced by Bacillus transposonis not containing Tn1546.

[0009] Furthermore, the scope of the Bacillus includes, but is not limited to, Bacillus stearothermiae (…). Bacillus spizizenii Bacillus subtilis ( Bacillus subtilis ), Bacillus cereus ( Bacillus cereus ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus belesi ( Bacillus velezensis ) and Bacillus pumilus ( Bacillus pumilus ).

[0010] Specifically, the method for killing spores in food provided by the present invention includes the following steps:

[0011] S1. Resuspend the spore mud in 20-100 mM potassium phosphate buffer solution with 0.1-10 g / L glucosamine added to obtain a pretreated heat-resistant spore suspension;

[0012] S2. The pretreated spore suspension obtained in S1 is subjected to heat treatment to obtain an inactivated spore suspension.

[0013] Preferably, the pH of the potassium phosphate buffer in S1 is between 4.2 and 6.5; more preferably, the pH is between 5 and 6; and most preferably, the pH is 6.2.

[0014] Preferably, the number of spores in the pretreated spore suspension in S1 is 1~9×10⁻⁶. 7 CFU / mL.

[0015] Preferably, the heat treatment in S2 simulates the food heat processing and sterilization process. The specific process is as follows: first, it is treated in a water bath at 90~100℃ for 10~60 min, then cooled to 0~30℃ within 10~60 min, and finally sterilized at 80~100℃ for 10~60 min.

[0016] Preferably, the method for preparing heat-resistant spores in S1 includes the following steps:

[0017] (1) Inoculate Bacillus subtilis into a medium containing Mn 2+ The spores were cultured in a nutrient agar medium, and then washed and collected to obtain crude spore extract.

[0018] (2) The crude spore liquid obtained in step (1) is centrifuged and washed to obtain spore mud.

[0019] More preferably, in step (1), the activated bacteria are inoculated into 8-12 mL of solution containing 0.3-1.5 μmol / L Mn. 2+ The spores were cultured in nutrient agar medium at 30-40℃ for 6-8 days, then shaken, washed and collected with 2-8 mL of sterile deionized water to obtain crude spore solution.

[0020] More preferably, in step (2), the crude spore liquid is centrifuged and washed with 10-30 mL of sterile deionized water at a speed of 8000-10000 r / min for 10-15 min, and this operation is repeated 3-6 times to obtain spore mud.

[0021] In another aspect, the present invention also provides the application of the above-mentioned method for killing spores in heat-processed foods.

[0022] Preferably, the heat-processed food includes, but is not limited to, braised and marinated products and emulsified meat products.

[0023] Preferably, braised or marinated products include braised meat products, bean products, or vegetable products.

[0024] Preferably, the preparation method of the braised product includes: adding the raw materials of the braised product into a sachet containing 0.01~1% glucosamine, braising and cooling to room temperature, and then sterilizing at 80~100℃ for 15~60 min to obtain the sterilized braised product.

[0025] In a preferred embodiment, taking braised beef as an example, the specific steps of the spore-killing method for sterilization include:

[0026] X1. Soak the beef in cold water at a weight ratio of 1:2 to 3, add ginger slices equivalent to 2 to 3% of the meat weight, boil for 3 to 5 minutes, remove and rinse, then put it in 2 to 3 volumes of cold water, add 0.01 to 1% glucosamine, 2 to 4% white sugar, 2 to 3% ginger, 2 to 3% salt, 0.2 to 0.6% MSG, 0.5 to 0.7% chicken essence, 3 to 4% light soy sauce, 1 to 2% dark soy sauce and 1 to 3% mixed spice packet, braise for 30 to 60 minutes, vacuum pack, and cool to room temperature with ice water to obtain braised beef;

[0027] X2. Sterilize the above-mentioned braised beef at 80~100℃ for 15~60 min to obtain sterilized braised beef.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] (1) This invention provides a highly efficient method for killing heat-resistant spores in heat-processed foods. This method uses glucosamine to contact the spores, causing damage to the spore coat and releasing DPA from the nucleus. DPA induces the breakage of disulfide bonds in the spore coat protein, destroying the structural integrity of the spore coat and significantly reducing the heat resistance of the spores. Based on this, combined with heat treatment, highly efficient inactivation of spores is achieved, with a sterilization rate of up to 100% for both ordinary and heat-resistant spores.

[0030] (2) Compared with traditional sterilization technology, the technical solution adopted in this invention achieves efficient inactivation while reducing energy consumption and cost, especially the efficient killing of heat-resistant spores, which has significant technical and economic benefits.

[0031] (3) The technical solution of the present invention can be applied to the sterilization treatment of heat-processed food. It adopts a milder heat sterilization temperature, avoiding the adverse effects of high-temperature sterilization on product quality and nutrition. The heat-processed food after treatment does not need to add other additives. It is a new control solution with green, safe, efficient and broad-spectrum applicability, and has great industrialization potential.

[0032] (4) This technology is easy to operate, low in cost, green and environmentally friendly, highly safe and suitable for large-scale promotion. Attached Figure Description

[0033] Figure 1 The diagram shows a comparison of the logarithm and inactivation rate of spores treated with the methods provided in Examples 3-4 and Comparative Examples 1-2, respectively.

[0034] Figure 2 The diagram shows a comparison of the logarithm and inactivation rate of spores treated with the methods provided in Examples 6-7 and Comparative Examples 3-4, respectively.

[0035] Figure 3 The diagram shows a comparison of the amount of spore nucleic acid leakage treated using the methods of Example 3 and Comparative Example 1, respectively.

[0036] Figure 4 SEM images of spore morphological changes treated with the methods of Example 3 and Comparative Example 1 are shown.

[0037] Figure 5 The diagram shows a logarithmic comparison of damage to the spore coat and inner membrane of spores pretreated using the methods of Example 3 and Comparative Example 1, respectively.

[0038] Figure 6 The diagram shows a comparison of the disulfide bond content on the spore surface treated by the methods of Example 3 and Comparative Example 1, respectively.

[0039] Figure 7 The SDS-PAGE comparison images of spores treated with the methods of Example 3 and Comparative Example 1 are shown.

[0040] Figure 8 The diagram shows a comparison of the changes in the protein secondary structure of spores treated in Example 3 and Comparative Example 1 by peak fitting using infrared spectroscopy.

[0041] Figure 9 The infrared spectra of the changes in spore protein structure and membrane lipid phase state treated in Example 3 and Comparative Example 1 are shown.

[0042] Figure 10 A comparison graph showing the DPA release levels of spores treated in Example 3 and Comparative Example 1 is presented.

[0043] Figure 11 The diagram shows a comparison of spore cortex damage in Example 3 and Comparative Example 1 after pretreatment (without heat treatment). Detailed Implementation

[0044] The technical solution of the present invention will be explained in more detail below with reference to several embodiments.

[0045] The sources and preparation methods of some of the raw materials used in the following examples and comparative examples are as follows:

[0046] (1) PCA medium: PCA medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd. and used for counting Bacillus subtilis.

[0047] (2) LB medium: LB medium was purchased from Qingdao Haibo Biotechnology Co., Ltd. and used for the activation and subculture of Bacillus subtilis.

[0048] (3) Contains 0.91 μmol / L Mn 2+Nutrient agar medium: 0.91 μmol Mn 2+ Add to 1 L of nutrient agar medium, sterilize at 121℃ for 15 min, cool, and prepare as a slant for use. Nutrient agar medium was purchased from Guangdong Huankai Microbial Technology Co., Ltd.; Mn 2+ Derived from MnCl2·4H2O, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; this culture medium is used for the preparation of spores.

[0049] (4) Activation of Bacillus subtilis and Bacillus spp.: Under aseptic conditions, Bacillus subtilis was picked up with an inoculation loop and inoculated into 10-15 mL of LB medium. After vortexing and culturing at 37°C for 16-18 h, activated Bacillus subtilis was obtained.

[0050] (5) The seasonings used in this invention include salt (China Salt Industry Group Co., Ltd.), white sugar (Henan Lianhua Sugar Industry Co., Ltd.), monosodium glutamate (Henan Lianhua Sugar Industry Co., Ltd.), chicken essence (Shanghai Taitole Food Co., Ltd.), light soy sauce (Foshan Haitian Seasoning Food Co., Ltd.), dark soy sauce (Foshan Haitian Seasoning Food Co., Ltd.), and mixed spice packet (Foshan Haitian Seasoning Food Co., Ltd.), all of which are commercially available.

[0051] Example 1

[0052] This embodiment provides a method for preparing heat-resistant spores. The method for preparing heat-resistant spores includes the following steps:

[0053] (1) Inoculate Bacillus stearothermia containing the Tn1546 transposon into 10 mL of solution containing 0.5 µmol / L Mn 2+ The spores were cultured in nutrient agar medium at 37°C for 7 days, and then shaken, washed and collected with 5 mL of sterile deionized water to obtain the crude spore solution.

[0054] (2) The crude spore liquid obtained in step (1) is centrifuged and washed, and then centrifuged and washed with 10 mL of sterile deionized water at 8000 r / min for 15 min. This process is repeated 5 times to obtain the heat-resistant spore mud.

[0055] Example 2

[0056] This embodiment provides a method for preparing common spores, which is basically the same as that in Example 1, except that the strain used in step (1) is Bacillus subtilis 168 which does not contain the Tn1546 transposon.

[0057] Example 3

[0058] This embodiment provides a method for killing heat-resistant spores, and the specific steps are as follows:

[0059] S1. The heat-resistant spore mud obtained in Example 1 was resuspended in a 100 mM potassium phosphate buffer solution containing 5 g / L glucosamine at pH 6.2 to achieve a spore count of 10. 7 CFU / mL was used to obtain a pretreated heat-resistant spore suspension;

[0060] S2. The pretreated heat-resistant spore suspension obtained in S1 is subjected to heat treatment to simulate the food heat processing and sterilization process, namely, in a water bath at 100°C for 30 min, cooled at 4°C for 15 min, and sterilized at 95°C for 30 min to obtain an inactivated spore suspension.

[0061] This embodiment can kill 99.99996% of heat-resistant spores.

[0062] Example 4

[0063] This embodiment provides a method for killing common spores, and the specific steps are as follows:

[0064] S1. The ordinary spore mud obtained in Example 2 was resuspended in a 100 mM potassium phosphate buffer solution containing 5 g / L glucosamine at pH 6.2 to achieve a spore count of 10. 7 CFU / mL was used to obtain a pretreated ordinary spore suspension;

[0065] S2. The pretreated ordinary spore suspension obtained in S1 is subjected to heat treatment to simulate the food heat processing and sterilization process, that is, in a water bath at 100°C for 30 min, cooled at 4°C for 15 min, and sterilized at 95°C for 30 min to obtain an inactivated spore suspension.

[0066] This embodiment can kill 100% of ordinary spores.

[0067] Example 5

[0068] This embodiment provides an application of a spore-killing method in braised beef, specifically including the following steps:

[0069] X1. Soak the beef in cold water at a weight ratio of 1:2, add ginger slices equivalent to 3% of the meat weight, boil for 5 minutes, remove and rinse, then put it in 2 volumes of cold water, add 0.5% glucosamine, 2-4% white sugar, 2-3% ginger, 2% salt, 0.4% MSG, 0.6% chicken essence, 3.2% light soy sauce, 1.5% dark soy sauce and 2% mixed spice packet, braise for 60 minutes, vacuum pack, and cool to room temperature with ice water to obtain braised beef;

[0070] X2. The above-mentioned braised beef is sterilized at 95℃ for 30 min to obtain sterilized braised beef.

[0071] No microorganisms (including spore-forming bacteria) were detected in the sterilized braised beef in this embodiment.

[0072] Example 6

[0073] This embodiment provides a method for killing spores and its application in braised beef with high levels of spore contamination, specifically including the following steps:

[0074] X1. Soak the beef in cold water at a weight ratio of 1:2, add ginger slices equivalent to 3% of the meat weight, boil for 5 minutes, remove and rinse, then put it in 2 volumes of cold water, add 2-4% white sugar, 2-3% ginger, 2% salt, 0.4% MSG, 0.6% chicken essence, 3.2% light soy sauce, 1.5% dark soy sauce and 2% mixed spice packet, braise for 30 minutes, vacuum pack, cool to room temperature with ice water, and sterilize at 121℃ for 15 minutes to obtain sterile braised beef;

[0075] X2. The heat-resistant spores prepared in Example 1 were resuspended in an aqueous solution containing 5 g / L glucosamine and inoculated into the sterile braised beef obtained in step X1, so that the spore count was 10. 7 The concentration of CFU / g was then increased, and the mixture was treated at 100℃ for 30 min, cooled to room temperature in an ice bath, and then sterilized at 95℃ for 30 min to obtain sterilized braised beef.

[0076] This embodiment can kill 99.9995% of heat-resistant spores in braised beef with high spore contamination levels.

[0077] Example 7

[0078] This embodiment provides a method for killing spores and its application in braised beef with low levels of spore contamination, specifically including the following steps:

[0079] X1. Soak the beef in cold water at a weight ratio of 1:2, add ginger slices equivalent to 3% of the meat weight, boil for 5 minutes, remove and rinse, then put it in 2 volumes of cold water, add 2-4% white sugar, 2-3% ginger, 2% salt, 0.4% MSG, 0.6% chicken essence, 3.2% light soy sauce, 1.5% dark soy sauce and 2% mixed spice packet, braise for 30 minutes, vacuum pack, cool to room temperature with ice water, and sterilize at 121℃ for 15 minutes to obtain sterile braised beef;

[0080] X2. The heat-resistant spores prepared in Example 1 were resuspended in an aqueous solution containing 5 g / L glucosamine and inoculated into the sterile braised beef obtained in step X1, so that the spore count was 10. 3 The product was then processed at 100℃ for 30 min, vacuum packaged, cooled to room temperature in an ice bath, and then sterilized at 95℃ for 30 min to obtain sterilized braised beef.

[0081] This embodiment can kill 100% of heat-resistant spores (microorganisms not detected) in braised beef with low spore contamination levels.

[0082] Comparative Example 1

[0083] This comparative example provides a method for killing spores, which is basically the same as that in Example 3, except that in S1, the spore suspension is prepared by resuspending the heat-resistant spore mud prepared in Example 1 in a 100 mM potassium phosphate buffer solution with a pH of 6.2, so that the spore count is 10. 7 CFU / mL.

[0084] This comparative sample can kill 17.14% of heat-resistant spores.

[0085] Comparative Example 2

[0086] This comparative example provides a method for killing spores, which is basically the same as that in Example 4, except that in S1, the spore suspension is prepared by resuspending the ordinary spore mud prepared in Example 2 in a 100 mM potassium phosphate buffer solution with a pH of 6.2, so that the spore count is 10. 7 CFU / mL.

[0087] This control ratio can kill 99.95% of common spores.

[0088] Comparative Example 3

[0089] This comparative example provides a method for killing spores in braised beef with high spore contamination levels. It is basically the same as Example 6, except that 0.5% glucosamine was not added in step X1.

[0090] This comparative sample can kill 95.48% of heat-resistant spores in braised beef with high spore contamination levels.

[0091] Comparative Example 4

[0092] This comparative example provides a method for killing spores and its application in braised beef with low spore contamination levels. It is basically the same as Example 7, except that 0.5% glucosamine is not added in step X1.

[0093] This comparative sample can kill 90.20% of heat-resistant spores in braised beef with low spore contamination levels.

[0094] Comparative Example 5

[0095] This comparative example provides a method for killing heat-resistant spores, which is basically the same as that in Example 3. The difference is that in S1, glucosamine is not added, and in S2, the pretreated heat-resistant spore suspension obtained in S1 is subjected to heat treatment simulating the food heat processing and sterilization process, that is, in a water bath at 100°C for 60 min to obtain an inactivated spore suspension.

[0096] This control can kill 37.18% of heat-resistant spores.

[0097] Comparative Example 6

[0098] This comparative example provides a method for killing heat-resistant spores, which is basically the same as that in Example 3. The difference is that in S2, the pretreated heat-resistant spore suspension obtained in S1 is subjected to heat treatment to simulate the food heat processing and sterilization process, that is, in a water bath at 100°C for 60 min, to obtain an inactivated spore suspension.

[0099] This comparative sample can kill 96.17% of heat-resistant spores.

[0100] Comparative Example 7

[0101] This comparative example provides a method for killing spores in braised beef, specifically including the following steps:

[0102] X1. Soak the beef in cold water at a weight ratio of 1:2, add ginger slices equivalent to 3% of the meat weight, boil for 5 minutes, remove and rinse, then put it in 2 volumes of cold water, add 2-4% white sugar, 2-3% ginger, 2% salt, 0.4% MSG, 0.6% chicken essence, 3.2% light soy sauce, 1.5% dark soy sauce and 2% mixed spice packet, braise for 30 minutes, vacuum pack, and cool to room temperature with ice water to obtain braised beef;

[0103] X2. Sterilize the braised beef at 95℃ for 30 minutes to obtain sterilized braised beef.

[0104] This comparative sample can kill 86.71% of spores in braised beef with low spore contamination levels.

[0105] Test case

[0106] Spore counting: 1 mL of samples prepared in Examples 3-4, 6-7, and Comparative Examples 1-4 were serially diluted in 9 mL of 0.9% NaCl solution, and then spread onto PCA medium using appropriate gradients. The spread media were incubated at 37°C for 12 h, and spore counts were performed to determine the spore count. The spore counting results for Examples 3-4, 6-7, and Comparative Examples 1-4 are shown below. Figure 1 and 2 As shown.

[0107] Nucleic acid leakage determination: Spore suspensions from Example 3 and Comparative Example 1, after heat treatment for 0, 30, and 60 min, were centrifuged at 1000g for 10 min. The absorbance of the supernatant at A260 and A280 was measured, and the nucleic acid leakage content after spore inactivation was calculated by A260 / A280. The results are shown in [Figure number missing]. Figure 3 .

[0108] Characterization of spore morphology changes: The microstructure of spores was observed using scanning electron microscopy (SEM). 5 mL of spore suspensions (10 mL each) subjected to heat treatment for 0, 30 min, and 60 min, respectively, as described in Example 3 and Comparative Example 1, were used. 6 After centrifugation at 4°C for 5 min (7000 × g) using CFU / mL spores, the supernatant was removed, and the spores were resuspended in 2.5% glutaraldehyde and fixed overnight at 4°C. The spores were washed three times with PBS buffer (0.1 M, pH 7.0) by centrifugation, followed by dehydration with ethanol solutions of 30%, 50%, 70%, 80%, 90%, and 100% concentrations, respectively. Finally, the spores were fixed on a glass slide, sputtered with metallic gold under vacuum, and the ultrastructure of the spores was observed using SEM (S-4800-I, Hitachi, Japan). The results are shown in the figure below. Figure 4 .

[0109] Spore coat and inner membrane damage assay: Spore suspensions pretreated in Example 3 and Comparative Example 1 were centrifuged at 4600g for 10 min to harvest spores, and washed with 0.9% NaCl. Then, the spores were resuspended in buffer solutions (50 mmol / L) containing 0.15% hypochlorous acid (pH 7.0) and 3% NaCl, respectively, and treated at 25°C for 20 min. The number of surviving spores was then counted using the spread assay. The loss of spores after treatment with available chlorine and NaCl was calculated, corresponding to the degree of damage to the spore coat and inner membrane, respectively. The results are shown in [Figure number missing]. Figure 5 .

[0110] Determination of disulfide bond content on spore surface: 0.6 mol / L NaCl + 8 mol / L urea (SB) and 0.6 mol / L NaCl + 8 mol / L urea + 0.5 mol / L β-mercaptoethanol (SC) solutions were prepared. The protein solubility of spores treated with heat in Example 3 and Comparative Example 1 for 0 min, 30 min, and 60 min in different buffer solutions was measured. The supernatant was collected by centrifugation at 15000×g for 10 min. The content of disulfide bonds (SC-SB) on the spore surface was then calculated based on the difference in protein solubility in the supernatant. The results are shown in [Figure number missing]. Figure 6 .

[0111] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of spore coat proteins: 100 μL of spore suspension (prepared by heat treatment for 0, 30, and 60 min as in Example 3 and Comparative Example 1) was centrifuged at 4°C for 5 min (7000 × g) to remove the supernatant. The suspension was then resuspended in 40 μL of SDS-PAGE loading buffer, boiled for 5 min, vigorously mixed for 1 min, and boiled again for 5 min. After brief centrifugation, 15 μL of the sample was separated on an acrylamide gel containing 12% SDS using a large-size 1-D electrophoresis system (Protean II Biorad). The gel was stained with Coomassie Brilliant Blue R-250 for 30 min, then destained. The results are shown in [Figure number missing]. Figure 7 .

[0112] Determination of secondary structure of spore proteins: Changes in spore protein structure and membrane lipid phase were determined using Fourier transform infrared spectrometry (FT-IR) to analyze the changes in protein structure and membrane lipid phase of Bacillus subtilis spores before and after treatment. Spore suspensions from Examples 3 and Comparative Example 1, after heat treatment for 0, 30, and 60 min, were centrifuged at 4°C for 5 min (7000 × g), the supernatant was removed, and the samples were then freeze-dried under vacuum. The samples were then ground and mixed with dry potassium bromide powder at a mass ratio of 1:150, and compressed into tablets using a tablet press. Using pure potassium bromide tablets as a background, the spectra were analyzed at 4000–500 cm⁻¹. -1 The area was scanned 32 times, with a resolution of 4 cm. -1 .

[0113] See Figure 9 Located at 1700–1600 cm -1 The infrared absorption peak at this point mainly characterizes the changes in the protein amide I band, involving the CO stretching vibration and NH in-plane bending vibration of the peptide chain, with the α-helix concentrated at 1660–1650 cm⁻¹. -1 At this location, β-folds are concentrated between 1640 and 1600 cm. -1 At this point, the β-turn is concentrated between 1690 and 1660 cm. -1 At this point, the irregular curls are concentrated between 1650 and 1640 cm. -1 Location. For different treatment groups, 1700–1600 cm. -1 Peak fitting was performed on the infrared spectrum at the location to quantitatively analyze the changes in the secondary structure of spore proteins. The results are shown in [Figure number missing]. Figure 8 .

[0114] Determination of DPA release rate from spores: Spore suspensions pretreated according to Example 3 and Comparative Example 1 were centrifuged at 12000×g for 2 min, and the supernatant was collected. Subsequently, this supernatant was mixed with an equal volume of 50 μmol / L terbium chloride solution (pH 5.6), and the fluorescence intensity of the mixture was measured at an excitation wavelength of 545 nm and an emission wavelength of 270 nm. The positive control was spores inactivated at 121 °C for 15 min. The DPA release rate was calculated as follows: DPA% = (F2-F0) / (F1-F0), where F0, F1, and F2 represent the fluorescence intensity of the spore supernatant from the untreated, treated, and positive control spores, respectively. Results are as follows: Figure 10 As shown.

[0115] Characterization of spore cortex damage: SYTO9 dye is a membrane-permeable dye, but it cannot penetrate the intact spore cortex. Only when the cortex is hydrolyzed or destroyed can it penetrate the inner membrane of the spore and bind to nucleic acids, emitting green fluorescence upon excitation. Therefore, SYTO9 staining can be used to observe the damage to the spore cortex. Spore suspensions pretreated in Example 3 and Comparative Example 1 were centrifuged at 4600×g for 10 min to harvest spores, and washed with 0.9% NaCl. 1 μL of SYTO9 dye was added to each 1 mL of spore suspension, mixed well, and incubated at room temperature in the dark for 15 min. 5 μL of the stained spore suspension was transferred to a glass slide, covered with an 18 mm square coverslip, and observed through a suitable channel on a Leica SP8 laser confocal microscope. The results are as follows: Figure 11 As shown.

[0116] Statistical analysis: All experiments were repeated three times, and results are expressed as mean ± standard deviation. Experimental data were statistically analyzed using the Linear Models program in the Statistix 8.1 software package. Significance of differences was determined by... P Analysis with values ​​<0.05) was performed using the TukeyHSD program. It should be noted that... Figures 1-2 Lowercase letters (a, b, c, d) indicate significant differences between different treatment groups. P <0.05); Figure 3 and Figure 5 Uppercase letters (A, B, C, D) and lowercase letters (a, b, c, d) indicate significant differences between different treatment times and different treatment groups, respectively. P <0.05).

[0117] Results analysis:

[0118] like Figure 1 and Figure 2As shown, compared with Comparative Examples 1-4, in both the simulated system Examples 3-4 and the meat system Examples 6-7, the synergistic treatment of glucosamine and heat can kill almost all common spores and heat-resistant spores. In particular, the kill rate of common spores can reach 100%, and the kill rate of heat-resistant spores can reach 99.99996%, indicating that the synergistic treatment of glucosamine and heat can efficiently inactivate spores.

[0119] like Figure 3 As shown, compared with Comparative Example 1 which was heat-treated alone, the leakage of nucleic acid material from the heat-resistant spores treated in Example 3 increased significantly with heat treatment time, while Comparative Example 1, which only used heat treatment, had almost no nucleic acid material in the heat-resistant spores. This indicates that the synergistic treatment of glucosamine and heat can effectively inactivate the heat-resistant spores, causing them to rupture and release nucleic acid materials such as DNA from the nucleus.

[0120] like Figure 4 As shown, the morphology of the spores in Comparative Example 1, which underwent heat treatment alone, did not change significantly with the extension of heat treatment time; however, in Example 3, the spores treated with added glucosamine ruptured, and the contents were completely released. After 60 minutes, almost all the spores were deformed, compared to... Figures 1-3 The results were consistent, and the number of spores ruptured increased with increasing heat treatment time.

[0121] like Figure 5 As shown, by measuring the effects of glucosamine on the inner membrane and outer coat of spores without heat treatment, it was found that glucosamine can damage the outer coat, thereby reducing resistance to hypochlorous acid. However, glucosamine has no significant effect on the inner membrane of spores; therefore, the addition of glucosamine does not affect spore germination and growth.

[0122] The resistance of spores to various environmental stresses such as chemicals and heat is partly attributed to their outermost spore coat. For example... Figure 7 As shown, by extracting spore coat proteins from spores of Example 3 and Comparative Example 1 after heat treatment for 0, 30 min, and 60 min, and performing SDS-PAGE, it was found that at a heat treatment time of 0 min, compared with Comparative Example 1, the high molecular weight spore coat protein bands in Example 3 were lighter and more blurred, and gradually disappeared with increasing heat treatment time. This indicates that the addition of glucosamine does indeed lead to the degradation of certain high molecular weight proteins (especially those above 180 kDa) on the spore coat, and that heat treatment further exacerbates the degradation of these proteins, resulting in weakened spore resistance and inactivation.

[0123] The protein components of the spore coat exhibit extensive cross-linking properties; the higher the level of protein cross-linking, the higher the thermal stability. The most important cross-linking interaction is the disulfide bond. Therefore, this invention further determined the effect of glucosamine on the disulfide bonds in the spore coat, and the results are as follows: Figure 6 As shown in the figure. The results indicate that the addition of glucosamine in Example 3 significantly reduced the disulfide bonds on the spore surface, while the heat treatment alone in Comparative Example 1 did not affect the disulfide bond cross-linking of the spore coat. This demonstrates that glucosamine can reduce the cross-linking level of proteins by disrupting the disulfide bonds of the spore coat proteins on the spore surface, thereby damaging the spore coat and reducing the resistance of the spores, making them more susceptible to inactivation upon further heat treatment.

[0124] Because glucosamine-assisted heat treatment damages and kills spores, it leads to changes in protein structure, which are associated with alterations in the spore coat. For example... Figure 8 As shown in Example 3, the addition of glucosamine reduced the β-turn and α-helix content of the protein while increasing the β-sheet content. This indicates that glucosamine can unfold the secondary structure of the spore coat protein, and heat treatment exacerbates this phenomenon. Furthermore, the conversion of α-helices to β-sheets, peptide chain unfolding, and decreased protein stability; the reduction in β-turn also signifies decreased protein stability. This suggests that glucosamine's influence on the secondary structure of the spore coat protein may lead to its entry into the spore and its interaction with the cortex. The changes in the secondary structure of the spore coat protein demonstrate that glucosamine can affect the stability of the spore coat, and further heat treatment exacerbates the damage to the spore coat structure, causing the spores to lose resistance and become inactive.

[0125] like Figure 10 As shown, the addition of glucosamine causes spores to release DPA during germination. The released DPA disrupts the cross-linking of the CotE protein in the spore coat (mainly disulfide bonds). Therefore, the addition of glucosamine can promote the release of DPA, thereby disrupting the disulfide bonds on the spore coat and causing damage to the spore coat structure.

[0126] like Figure 11 As shown, Example 3 exhibited obvious green fluorescence, indicating that the STYO9 dye had entered the spores treated with glucosamine and could penetrate the cortex. However, the spores treated with Comparative Example 1 did not exhibit green fluorescence, indicating that glucosamine caused damage to the spore cortex. This may be because the peptidoglycan in the spore cortex is polymerized from acetylglucosamine and acetylmuramic acid. The molecular structure of glucosamine is highly similar to that of acetylglucosamine, differing by only one acetyl group. The two destroy the polymer structure of peptidoglycan through "competitive binding" and release DPA, thereby causing damage to the spore coat structure.

[0127] In summary, the present invention employs a combination of glucosamine and heat treatment, which can destroy the spore coat structure by adding glucosamine, thereby causing the spores to lose their resistance and become inactive, and then achieve spore inactivation by combining it with heat treatment.

[0128] Furthermore, the present invention has been described using *Bacillus stearothermiae* as an example in the preferred embodiments described above. It can be inferred that the present invention may employ other *Bacillus* species (…). BacillusBacteria can also achieve the same or similar results.

[0129] Therefore, glucosamine can work synergistically with heat treatment to effectively kill spores.

[0130] Furthermore, analysis of Example 3 and Comparative Examples 5-6 shows that the staged heat treatment method of the present invention can more effectively kill spores.

[0131] Analysis of Examples 5-7, and Comparative Examples 3-4 and 7 shows that the addition of glucosamine in the synergistic heat treatment process can effectively kill heat-resistant spores. Further comparison of Examples 5 and 7 indicates that the addition of glucosamine also promotes the killing of heat-resistant spores in braised products. Heat treatment alone cannot completely kill heat-resistant spores. However, by using the technical solution of this invention, vacuum packaging the heat-processed food treated by this invention can greatly extend the shelf life of braised products. Moreover, a gentler heat sterilization temperature is used, avoiding the adverse effects of high temperature on product quality and nutrition.

[0132] It should be noted that the above description is only a preferred embodiment of this application and the foregoing embodiments have been used to describe this application in detail. It is not intended to limit this application. Although those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for killing spores, characterized in that, The addition of glucosamine and heat treatment work synergistically to stimulate spores; The spores include one or more of the spores produced by Bacillus stearothermiae and Bacillus subtilis. The method for killing the spores includes the following steps: S1. Resuspend the spore mud in 20-100 mM potassium phosphate buffer solution with 0.1-10 g / L glucosamine to obtain a pretreated spore suspension; S2. The pretreated spore suspension is subjected to heat treatment to obtain an inactivated spore suspension; The heat treatment includes: first treating in a water bath at 90~100℃ for 10~60 min, then cooling to 0~30℃ within 10~60 min, and finally sterilizing at 80~100℃ for 10~60 min.

2. The method for killing spores according to claim 1, characterized in that, The preparation method of the spore-forming mud described in S1 includes the following steps: (1) Inoculate Bacillus subtilis into a medium containing Mn 2+ The spores were cultured in a nutrient agar medium, and then washed and collected to obtain crude spore extract. (2) The crude spore liquid obtained in step (1) is centrifuged and washed to obtain the spore mud.

3. The application of the spore-killing method as described in any one of claims 1-2 in heat-processed foods, characterized in that, The heat-processed foods include braised and marinated products and / or emulsified meat products.

4. The application of the spore-killing method according to any one of claims 1-2 in the processing of braised and marinated products, characterized in that, The braised and marinated products include braised meat products, bean products, or vegetable products.

5. The application of a method for killing spores in braised and marinated products, characterized in that, The sterilization method includes the following steps: adding the raw materials of the braised products into a 0.01-1% glucosamine spice packet, braising and cooling to room temperature, and then sterilizing at 80-100℃ for 15-60 minutes to obtain sterilized braised products.

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