Preparation method and application of lactobacillus fermentum metagen

Abstract

The invention discloses a method for preparing and purifying lactobacillus fermentum metaplasm and application of the lactobacillus fermentum metaplasm to improvement of texture and quality stability of meat products. The method comprises the following steps: carrying out fermentation, inactivation, centrifugation, filtration and freeze-drying on lactobacillus fermentum to obtain crude biomass; after dissolving, separating and enriching antioxidant components through macroporous resin, so as to obtain the purified biomass. Experiments prove that the prepared purified biogen can significantly improve the antioxidant activity (the antioxidant activity is improved by about 3 times compared with that of an unpurified group), can effectively inhibit myofibrillar protein oxidation, improves the hardness, elasticity, chewiness and water binding capacity of meat paste, and delays quality deterioration during storage. The method is simple and convenient in process and high in efficiency, and has relatively high industrialization and popularization and application values.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food processing and biotechnology, in particular to a method for preparing and purifying Lactobacillus fermentum postbiotics and application thereof. BACKGROUND

[0002] Lactobacillus fermentum is a common probiotic widely used in food fermentation, with good probiotic properties and safety. Postbiotics refers to non-living probiotics and their metabolites, which are usually inactivated by heat treatment, high pressure or ultrasonic, etc. to lyse the cells and release the contents, such as short-chain fatty acids, exopolysaccharides, teichoic acids, polypeptides and other active ingredients. Compared with probiotics, postbiotics is more stable and is not affected by storage and processing conditions, while avoiding the risk of colonization of live bacteria in the body. The various active ingredients in postbiotics have significant antioxidant and protein structure regulating effects, which reduce protein oxidative damage through mechanisms such as inhibiting free radical generation and chelating metal ions, and play an important role in meat products.

[0003] Macroporous adsorption resin, as a kind of organic polymer adsorption material, has excellent performance in the field of separation and purification. It has excellent chemical stability, can withstand acid and alkali environment and various organic solvents, and has good selectivity and is less affected by inorganic substances. From its own characteristics, it has larger specific surface area, faster ion exchange speed, stronger mechanical strength, lower degree of pollution to the extracted substance, and good thermal stability. Compared with other separation technologies, macroporous adsorption resin has obvious advantages, not only can improve the relative content of effective components, but also the obtained product has the characteristics of not absorbing moisture, the production cycle is shortened, and the resin can be regenerated and reused. Due to these advantages, in recent years, macroporous adsorption resin has been widely used in the field of separation and purification of natural products. Some studies, such as patent CN120114502A, use macroporous resin to enrich and reuse ethanol elution to realize the simultaneous collection of functional components such as saponins, flavonoids, and polyphenols, and the obtained mixed extract has multiple functional activities, which significantly improves the biological activity.

[0004] Traditional postbiotic preparation methods mostly use simple centrifugation, coarse filtration and other processes, which often have the defects of low purity, many impurities, unstable components, etc., limiting the exertion of antioxidant effect. Therefore, it is of great scientific significance and industrial application prospect to develop a method for enriching and purifying Lactobacillus fermentum postbiotics based on macroporous resin, efficiently extract postbiotic antioxidant components, and apply them to the quality improvement of meat products. SUMMARY

[0005] The application aims to provide a preparation method of Lactobacillus fermentum postbiotic based on macroporous resin and application thereof. The postbiotic is enriched and purified by macroporous resin, which can significantly improve the antioxidant activity, effectively remove impurities, and improve the application value in the field of food processing such as meat products.

[0006] The application first provides a preparation method of Lactobacillus fermentum postbiotic for improving meat quality, which comprises the following steps: centrifuging the inactivated Lactobacillus fermentum fermentation broth to obtain supernatant, and freeze-drying the supernatant after filtration to obtain crude postbiotic; and using macroporous resin to purify and enrich antioxidant components of the crude postbiotic to obtain macroporous resin enriched and purified postbiotic.

[0007] The Lactobacillus fermentum is obtained from the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO.1.1694. Lactobacillus fermentum The Lactobacillus fermentum is obtained from the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO.1.1694.

[0008] The application takes the antioxidant capacity as the quality control index, and selects the macroporous resin which is better for purifying and enriching antioxidant components of the Lactobacillus fermentum postbiotic. Further, the macroporous resin is selected from one of DA201, S-8, D101, NKA-9, X-5 or AB-8 resin; preferably, the macroporous resin is selected from one of DA201, D101, NKA-9, X-5 macroporous resin; more preferably, the macroporous resin is selected from one of DA201, NKA-9, X-5 macroporous resin.

[0009] Further, the preparation step of the Lactobacillus fermentum crude postbiotic comprises: inoculating the Lactobacillus fermentum (obtained from GDMCC, NO.1.1694) into the sterilized MRS solid culture medium in a sterile environment, and culturing in a 37℃ constant temperature incubator for 24h; picking a single colony and inoculating into the sterilized MRS liquid culture medium, and standing in a 37℃ constant temperature incubator for 24h to obtain a seed liquid; inoculating the cultured seed liquid into the sterilized MRS culture medium, and culturing at 35~37℃ and 50-180 rpm for 18~24h with an inoculation ratio of 3~5%, and harvesting the fermentation culture liquid when the pH is 4.0~4.25 and the OD600 is 6~7; and heating the fermentation culture liquid in a water bath at 70~75℃ for 15~35min for inactivation treatment. Then centrifuging the fermentation broth (6500~8000 rpm for 15min). Taking the supernatant and filtering it by using a water-based MCE mixed cellulose filter membrane (0.22mm) to remove intact cells, freeze-drying the filtered supernatant to obtain the crude postbiotic.

[0010] Further, the enrichment and purification of the Lactobacillus fermentum antioxidant components by using the macroporous resin comprises the following steps: (1) Pre-treating the macroporous resin to obtain treated resin.

[0011] (2) Dissolve the crude postbiotic in water to obtain a crude postbiotic solution, take the pretreated resin, and pass the crude postbiotic solution with a mass concentration of 20-40 mg / mL at a loading flow rate of 2-6 BV / h through the macroporous resin for adsorption and purification, after sufficient adsorption, wash with deionized water at 1 BV to remove the crude postbiotic solution that is not adsorbed, and then elute with 40-70% ethanol at an elution flow rate of 2-6 BV / h to obtain an eluate.

[0012] (3) Separation and purification: collect the eluate obtained in the above step, recover the solvent by distillation under reduced pressure, and freeze-dry to obtain the purified postbiotic.

[0013] Further, the concentration of ethanol used in the elution of step (2) is preferably 70%.

[0014] Further, the concentration of the loading solution in step (2) is preferably 20 mg / mL.

[0015] Further, the loading flow rate in step (2) is preferably 4 BV / h.

[0016] Further, the elution flow rate in step (2) is preferably 4 BV / h.

[0017] Further, the maximum loading amount in step (2) is 2 BV, and the amount of eluate used is 5 BV.

[0018] Further, the pretreatment of the macroporous resin includes the following steps: soaking the macroporous resin in 95% ethanol for 24 h for sufficient swelling; after column installation, washing the resin with ethanol multiple times until no turbidity appears in the column; then washing the resin with deionized water until no ethanol odor is detected, soaking in 5% hydrochloric acid for 2 h and then washing with pure water until neutral, then soaking in 5% sodium hydroxide solution for 2 h and then washing with pure water until neutral, and the pretreatment is completed.

[0019] The present application further provides a fermented lactobacillus postbiotic with antioxidant capacity, which is prepared by the above method.

[0020] The present application further provides a use of the macroporous resin-enriched fermented lactobacillus postbiotic in improving meat quality.

[0021] Further, the use of the enriched postbiotic in meat patties includes the following steps: (1) Take fresh pig back long straight ridge, remove visible fascia and connective tissue of raw meat, cut into small pieces and put into a meat grinder for mincing; (2) Mix the chopped meat with edible salt (0.5-1 wt%) and continue chopping for 30-90 s to obtain low-salt meat paste, and control the temperature during the whole process to be no more than 20℃. (3) Adding the enriched postbiotic to the meat paste, and rolling and kneading in a vacuum rolling machine for 10-30 min to prepare a pure postbiotic pork paste; (4) Pressing the chopped and mixed meat paste into a meat cake with a mold, and storing in a 2-4℃ refrigerator.

[0022] In the technical scheme of the present application: the pure postbiotic added in step (3) is 0.5-2wt% of the mass of the meat paste.

[0023] The present application has the following beneficial effects: 1. The present application constructs a method for preparing Lactobacillus fermentum postbiotic with antioxidant capacity as a quality control index. Compared with traditional technology, the present application has simple process, low cost, easy operation, high efficiency, and the antioxidant capacity of the enriched postbiotic is enhanced, the free radical activity half clearance rate (IC 50 ) of the purified postbiotic is improved by about 3 times, and a best process for purifying Lactobacillus fermentum is provided, which has good development and utilization value and provides a reference for further postbiotic purification and component analysis.

[0024] 2. The purified postbiotic as a natural functional additive applied in meat product processing can effectively improve the texture characteristics, improve the water retention performance, and delay the quality deterioration problem during storage.

[0025] 3. The method of the present application can be applied to the production practice of other different meat products, and the postbiotic does not need to be in a live state, is heat stable, and can be widely applied in processed meat products; the components are natural, suitable for food regulations, and have broad market application prospects. DETAILED DESCRIPTION

[0026] Figure 1 Influence trend chart of different types of macroporous resin static adsorption and desorption; Figure 2 Effect of sample concentration on macroporous resin adsorption; Figure 3 Effect of eluent ethanol concentration on macroporous resin desorption; Figure 4 Effect of sample flow rate on macroporous resin adsorption; Figure 5 Effect of elution flow rate on macroporous resin desorption; Figure 6 DPPH, ABTS + radical scavenging rate and IC 50 of Lactobacillus fermentum before and after purification; Figure 7 Change rule chart of cooking loss rate of Lactobacillus fermentum during storage before and after purification; Figure 8A graph showing the changes in water-holding capacity of *Lactobacillus fermentum* before and after purification during storage; Figure 9 Texture analysis diagrams of *Lactobacillus fermentationus* before and after purification during storage; Figure 10 To investigate the effect of Lactobacillus fermentation before and after purification on the carbonyl content of myofibrillar proteins. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention, and any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

[0028] Key experimental material sources and physicochemical parameters: The fermenting lactobacillus is ( Lactobacillus fermentum The sample was purchased from Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO.1.1694; the total antioxidant capacity (T-AOC) test kit (reduced iron method) was purchased from Nanjing Jiancheng Biotechnology Institute.

[0029] Example 1: Determination of adsorption and desorption rates of crude postbiotic antioxidant components by six macroporous resins

[0030] (1) Under aseptic conditions, *Lactobacillus fermentum* (purchased from GDMCC, NO.1.1694) was inoculated onto sterilized MRS solid medium and cultured at 37°C for 24 h. Single colonies were picked and inoculated onto sterilized MRS liquid medium and cultured at 37°C for 24 h to obtain seed culture. The cultured seed culture was inoculated into sterilized MRS medium at an inoculation ratio of 3% and cultured at 35°C and 180 rpm for 24 h until the pH reached 4.0 and the OD600 reached. The fermentation culture was then inactivated by heating in a water bath at 70°C for 30 min. The fermentation broth was then centrifuged (6500 rpm for 15 min). The supernatant was filtered through an aqueous MCE mixed cellulose membrane (0.22 mm) to remove intact cells. The filtered supernatant was then freeze-dried to obtain crude metagenic bacteria.

[0031] (2) Weigh 1.0 g of each of the six pretreated macroporous resins (DA201, S-8, D101, NKA-9, X-5 and AB-8), add 10 ml of crude post-generic solution with a concentration of 20 mg / mL, and shake and adsorb for 12 h at 25 ℃ and 150 r / min. The total antioxidant capacity of the adsorbed solution is determined by the total antioxidant capacity (T-AOC) test kit, and the adsorption rate of different macroporous resins is determined according to the following formula.

[0032]

[0033] A0 (U / mL) is the total antioxidant capacity before adsorption; A1 is the total antioxidant capacity (U / mL) after equilibrium.

[0034] (3) The macroporous resin after adsorption is taken out, and the impurities remaining on the surface are removed by elution with distilled water. Then the water in the resin is absorbed by suction filtration, and finally the desorption is carried out by oscillating with an equal volume of 70% ethanol solution at 25°C and 150 r / min for 12 h. The total antioxidant capacity of the desorbed solution is measured by using the total antioxidant capacity (T-AOC) test box, and the desorption rate of different macroporous resins is determined according to the following formula.

[0035]

[0036] A0 (U / mL) is the total antioxidant capacity before adsorption; A1 (U / mL) is the total antioxidant capacity after equilibrium; and A2 (U / mL) is the total antioxidant capacity of the desorption solution.

[0037] The experimental results are shown in Table 1. Figure 1 As shown in Table 1, the adsorption rate of DA201 is 81.3%, and the desorption rate is 74.1%, which is higher than the purification effect of other macroporous resins. Therefore, DA201 is selected as the best resin for purifying and enriching the antioxidant components of the postbiotic.

[0038] Example 2: Determination of sample mass concentration

[0039] 1.0 g of pretreated macroporous resin was accurately weighed and placed in a 50 mL conical flask. 20 mL of crude postbiotic solution with different concentrations (10, 20, 30, 40, 50 mg / mL) was added respectively. The conical flask was placed in a constant temperature oscillator, and the adsorption was continuously oscillated at 25°C and 150 rpm for 24 h to ensure that the macroporous resin and postbiotic components were fully contacted and reached adsorption equilibrium. After adsorption, the filtrate was collected by filtration, and the total antioxidant capacity of the filtrate was measured to calculate the adsorption rate of the macroporous resin (the calculation formula is the same as that of Example 1) and evaluate the adsorption effect under different sample concentrations.

[0040] The experimental results are shown in Table 2. Figure 2As shown in Figure 2, with the increase of sample solution concentration, the adsorption rate of macroporous resin to the antioxidant components of the metagenomic crude solution showed a trend of first increasing and then decreasing. When the sample solution concentration was 20 mg / mL, the adsorption rate reached the maximum value. The possible reason for this is that the interaction between the various antioxidant components in the sample solution and the macroporous resin is weak at a lower concentration, resulting in a lower adsorption rate. When the concentration is too high, the macromolecules or high-concentration components in the solution may cause the pore of the macroporous resin to be blocked, reducing its effective adsorption area, and thus inhibiting the entry and binding of subsequent components, resulting in a decrease in adsorption efficiency. Therefore, when purifying the metagenomic solution, the concentration should be around 20 mg / mL.

[0041] Example 3: Determination of the ethanol concentration of the eluent

[0042] The saturated macroporous resin was washed with pure water to ensure that the surface was free of metagenomic crude solution residue. The surface water was absorbed with suction filtration. 1.0 g of macroporous resin was accurately weighed and placed in a 50 mL conical flask. 20 mL of ethanol solution was added to each conical flask for desorption. The ethanol concentration was set at 20%, 30%, 40%, 50%, 60%, 70%, and 80%, respectively. The conical flasks were placed in a constant temperature shaker at 25°C and 150 rpm for continuous shaking for 24 h. After the macroporous resin reached desorption equilibrium, it was filtered, and a certain amount of filtrate was taken to determine the total antioxidant capacity. The desorption rate of the macroporous resin was calculated (the calculation formula is the same as that in Example 1).

[0043] As can be seen from Figure 3, with the increase of the ethanol concentration of the eluent, the desorption rate of the macroporous resin to the total antioxidant capacity of the metagenomic crude solution showed a trend of first increasing and then tending to be stable. When the ethanol concentration reached 70%, the desorption rate reached the maximum value. Further increasing the ethanol concentration did not significantly change the desorption rate (P>0.05). Considering that using too high a concentration of ethanol would increase the production cost, when purifying the metagenomic solution, the ethanol concentration of the eluent should be selected to be around 70%.

[0044] Example 4: Dynamic adsorption and desorption test of macroporous resin

[0045] (1) Determination of sample flow rate and sample volume

[0046] 10.0 g of macroporous resin was accurately weighed and packed into a glass chromatographic column (Φ1.5 cm x 30 cm). The sample concentration of the metagenomic crude solution was set at 20 mg / mL. The flow rate was controlled at 2, 4, and 6 BV / h, respectively, for dynamic adsorption. 10 mL of effluent was collected from each test tube and the total antioxidant capacity was measured. When the total antioxidant capacity of the effluent was 1 / 10 of that of the sample solution, it was the leakage point, which was observed and recorded. The dynamic adsorption curve was plotted with the effluent volume as the abscissa and the total antioxidant capacity of the effluent as the ordinate.

[0047] Experimental results: According to Figure 4, it can be found that as the loading flow rate increases, the time of the leakage point appears correspondingly. When the total antioxidant capacity of the effluent reaches 1 / 10 of the loading solution, the appearance of the leakage point can be observed. When the loading flow rate is 2, 4, and 6 BV / h, the leakage point appears at about 50 mL, 40 mL, and 30 mL, respectively. When the loading flow rate is 2 BV / h, the slow loading flow rate leads to an increase in adsorption time and a decrease in adsorption efficiency. When the loading flow rate is 6 BV / h, the high flow rate and the unsaturated macroporous resin result in a short sample residence time, which reduces the adsorption efficiency and increases the cost. Therefore, the loading flow rate is selected to be 4 BV / h, and under this condition, the loading volume should be 2 BV.

[0048] (2) Determination of elution flow rate and elution volume

[0049] Precisely weigh 10.0 g of macroporous resin and fill it into a glass chromatographic column (Φ1.5 cm x 30 cm). Set the loading concentration of the metagenomic crude solution to be 20 mg / mL and the loading flow rate to be 4 BV / h. Under this condition, perform dynamic adsorption. After the macroporous resin is saturated, wash it with a large amount of pure water until there is no metagenomic crude solution on the surface of the macroporous resin. Then, perform dynamic elution with a 70% ethanol solution. Control the elution flow rate to be 2, 4, and 6 BV / h, respectively. Collect 5 mL of effluent in each test tube and measure its total antioxidant capacity. Plot the dynamic desorption curve with the effluent volume as the horizontal coordinate and the total antioxidant capacity of the eluent as the vertical coordinate.

[0050] Experimental results: According to Figure 5, when the elution flow rate is 6 BV / h, the peak value of the elution peak is low, the peak shape is wide, and the tailing is serious. Not only is the elution effect poor, but also the amount of eluent used later will be large. When the elution flow rate is 2 BV / h and 4 BV / h, the elution peak is concentrated, and there is no obvious tailing phenomenon, indicating that the elution effect is good. However, a slow elution flow rate will prolong the production cycle. Therefore, considering comprehensively, when purifying the metagenomic crude solution, the elution flow rate should be controlled at 4 BV / h, and under this condition, the elution volume should be 5 BV.

[0051] Example 5

[0052] (1) Prepare the crude metagenomic according to the procedure in Example 1 (1).

[0053] (2) Take the pretreated DA201 resin wet column, the ratio of column diameter to height is 1:10, 2 BV volume of 20 mg / ml crude probiotic solution, 4 BV / h flow rate for column adsorption; After adsorption, 1 BV of deionized water is used for elution to remove unabsorbed samples and impurities, and finally the macroporous resin is eluted with elution solvent, the elution solvent is 70% ethanol solution, the elution volume and flow rate are 5 BV and 4 BV / h respectively, the eluate is collected, the obtained eluate is subjected to vacuum distillation at 40°C and 11 kPa pressure to remove ethanol, and the product after concentration is freeze-dried for 48-60 hours, and finally the purified probiotic freeze-dried powder is obtained.

[0054] (3) Antioxidant test of Lactobacillus fermentum probiotic before and after purification

[0055] DPPH determination method: The crude probiotic freeze-dried powder and the purified probiotic freeze-dried powder are prepared into sample solutions with a mass concentration of 0, 0.1, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0 mg / ml, and glutathione (GSH) is used as a reference substance. 3 mL of 0.04 mg / ml DPPH free radical solution (dissolved in anhydrous ethanol) is added to 1 ml of sample solution, and the reaction is carried out in the dark for 30 min, and the absorbance is measured at 517 nm. 3 ml of DPPH + 1 ml of probiotic is used as a sample, 1 mL of sample + 3 ml of 95% ethanol is used as a blank, and 3 ml of DPPH + 1 mL of distilled water is used as a control. The measurement wavelength is 517 nm. The test is repeated 3 times, and the DPPH clearance rate is calculated.

[0056]

[0057] In the formula: As sample absorbance; Ac control group absorbance; Ab blank group absorbance

[0058] ABTS + Determination method: The probiotic freeze-dried powder and the purified probiotic freeze-dried powder are prepared into sample solutions with a mass concentration of 0, 0.1, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0 mg / ml, and GSH is used as a reference substance. 20 mg of ABTS and 3.44 mg of potassium persulfate solution are dissolved in 5 ml of distilled water, and left overnight at room temperature, and diluted with 95% ethanol (v / v) to an absorbance of 0.7 ±0.02 at 734 nm wavelength. 1.8 mL of ABTS + free radical solution + 0.2 mL of probiotic is used as a sample. 1.8 mL of ABTS +A free radical solution + 0.2 ml distilled water was used as a control. After thorough mixing, the solutions were reacted at room temperature in the dark for 5 min, and the absorbance at 734 nm was measured. ABTS was calculated. + Free radical scavenging rate.

[0059]

[0060] In the formula: Ab and As represent the absorbance of the sample and control, respectively.

[0061] Experimental results: such as Figure 6 As shown, crude post-biotics, purified post-biotics, and glutathione (GSH) have effects on DPPH and ABTS. + The scavenging rate of free radicals showed a positive correlation with increasing concentration, and the scavenging ability of purified biotics was significantly better than that of unpurified biotics. P＜0.05 The half-maximal scavenging capacity (IC50) of the purified DPPH free radical activity was obtained. 50 The concentration of crude metabolite was 1.63 ± 0.26 mg / ml. 50 The concentration was 5.56 ± 0.39 mg / ml, and the antioxidant activity of the biogenic ABTS increased by 3.4 times after purification. + Free radical activity half-maximal scavenging rate (IC50) 50 The concentration of crude metabolite was 2.68 ± 0.71 mg / ml, and the IC50 of crude metabolite was... 50 The concentration was 6.81 ± 0.52 mg / ml, and the antioxidant activity of the post-biotic increased by 2.5 times after purification. The results indicate that purification effectively enriched the functional components with antioxidant activity while removing some inactive impurities, thus significantly enhancing the free radical scavenging ability of the post-biotic.

[0062] Example 6: Analysis of components before and after purification of post-fermented Lactobacillus spp.

[0063] (1) Chromatographic conditions

[0064] Column: ACQUITY UPLC ®HSS T3 (2.1 x 100 mm, 1.8 µm) (Waters, Milford, MA, USA); 0.3 mL / min flow rate, 40 ℃ column temperature, 2 μL injection volume. Positive ion mode, mobile phase was 0.1% formic acid in acetonitrile (B2) and 0.1% formic acid in water (A2), gradient elution program was: 0~1 min, 10% B2; 1~5 min, 10%~98% B2; 5~6.5 min, 98% B2; 6.5~6.6 min, 98%~10% B2; 6.6~8 min, 10% B2. Negative ion mode, mobile phase was acetonitrile (B3) and 5 mM ammonium formate in water (A3), gradient elution program was: 0~1 min, 10% B3; 1~5 min, 10%~98% B3; 5~6.5 min, 98% B3; 6.5~6.6 min, 98%~10% B3; 6.6~8 min, 10% B3.

[0065] (2) Mass spectrometry conditions

[0066] Electrospray ion source (ESI), positive and negative ion modes were collected respectively. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 arb, the auxiliary gas was 10 arb. The capillary temperature was 325 ℃, the first order full scan was performed at a resolution of 60000, the first order ion scan range was m / z 100~1000, and the HCD was used for secondary fragmentation, the collision energy was 30%, the secondary resolution was 15000, and the signals of the first 4 ions were collected for fragmentation, and the unnecessary MS / MS information was removed by dynamic exclusion.

[0067] The obtained spectral data was identified by public databases such as HMDB, massbank, LipidMaps, mzcloud, KEGG, etc., to obtain the qualitative results of the components of the crude and purified postbiotics. As shown in Table 1, among them, the relative content of carboxylic acids and derivatives changed the least, indoles and derivatives were significantly enriched, which increased by 102.8% compared with the crude postbiotics; the content of steroids and derivatives increased by 42%; the contents of flavones and isoflavones doubled; the content of prenyl lipids increased slightly. The purification process effectively enriched the antioxidant substances such as indoles, flavones and isoflavones, so that the antioxidant activity of the purified postbiotics was significantly enhanced.

[0068] Table 1 Components of Lactobacillus fermentum postbiotics before and after purification

[0069] Application Example 1

[0070] The purified Lactobacillus fermentum postbiotic was prepared according to the method of Example 2. Fresh pig back long straight ridge was taken, and the visible fascia and connective tissue of the raw meat was removed, and then cut and put into a meat grinder for mincing. The minced meat was mixed with edible salt (1 wt%) and continuously chopped for 60 s to obtain a low-salt meat paste, and the temperature during the whole process was controlled not to exceed 20 ℃. 0.5 wt% of the purified Lactobacillus fermentum postbiotic was added to the above-mentioned meat paste, and the mixture was rolled in a vacuum tumbling machine for 20 min to prepare a Lactobacillus fermentum postbiotic-containing pork paste. The pork paste was pressed into a meat patty with a diameter of 4.5 cm and a thickness of 1.6 cm, and stored in a 4 ℃ refrigerator.

[0071] Application Example 2

[0072] The pork patty was prepared according to the method of Application Example 1, except that 1 wt% of the purified Lactobacillus fermentum postbiotic was added.

[0073] Application Example 3

[0074] The pork patty was prepared according to the method of Application Example 1, except that 2 wt% of the purified Lactobacillus fermentum postbiotic was added.

[0075] Application Example 4

[0076] The pork patty was prepared according to the method of Example 1, except that 2 wt% of the crude Lactobacillus fermentum postbiotic was added.

[0077] Application Example 5

[0078] The pork patty was prepared according to the method of Example 1, except that no purified Lactobacillus fermentum postbiotic was added.

[0079] Comparison of application example results

[0080] In order to prove the beneficial effects of the present application, the inventors stored the pork patties obtained in Application Example 1, Application Example 2, Application Example 3 and Application Example 4 in a 4 ℃ refrigerator, and respectively at 0 h, 24 h, 48 h, 96 h, the products were detected as follows, and the specific determination method and determination results are as follows: The determination method is as follows: (1) Cooking loss rate An appropriate amount of sample was taken from the pork patty, the surface moisture of the sample was absorbed with kitchen paper, and the net weight was weighed and recorded as m1. It was put into a boiling water bath for 8 min, taken out and weighed as m2. Then, the percentage of cooking loss was calculated according to the following formula:

[0081] (2) Determination of water holding capacity

[0082] Weigh the sample and record the mass as m1. Wrap it in filter paper and place it in a centrifuge tube. Centrifuge (3000 g, 10 min, 4 ℃). Remove the filter paper and weigh the sample again, recording the mass as m2. Calculate the mass using the following formula:

[0083] (3) Determination of texture

[0084] The samples were cut into 1cm×1cm×1cm pieces and cross-sectional analysis was performed using a TMS-TOUCH texture analyzer. Four textural properties—hardness, elasticity, chewiness, and cohesion—were selected to characterize the textural changes of the meat patties. A TMS-100mm probe was used, and the measurement parameters are shown in Table 2.

[0085] Table 2. Parameter settings for texture profile analysis

[0086] (4) Extraction of myofibril proteins

[0087] Add the minced meat to a ratio of 1:5 ( m / v Add 5 times the volume of buffer solution (0.1 mol / L NaCl, 2 mmol / L MgCl2, 50 mmol / L PBS buffer, 1 mmol / L EGTA, pH 7.0) at a ratio of 1: ... g Centrifuge for 20 min, wash the precipitate three times, then wash again with 5 volumes of 0.1 mol / L NaCl solution, filter through 4 layers of gauze, and centrifuge the filtrate. The resulting precipitate is myofibrillar protein. Resuspend the myofibrillar protein in 50 mmol / L PBS buffer containing 0.6 mol / L NaCl, determine the protein concentration using the biuret method, store at 4 ℃, and use within 48 h.

[0088] (5) Determination of carbonyl content

[0089] Take 0.5 mL of 5 mg / mL myofibrillar protein suspension and add 2 mL of 20 mmol / L 2,4-dinitrophenylhydrazine. Incubate in the dark for 30 min, then add 2 mL of 20% TCA solution and centrifuge (4℃, 4000 rpm). g (20 min) Take the precipitate and add an ethanol-ethyl acetate mixture (1:1). V:V Wash the precipitate, repeating 3 times. Add 5 mL of 6 mol / L guanidine hydrochloride to the precipitate, react at 37℃ for 15 min, centrifuge, and measure the absorbance of the supernatant at 370 nm. Calculate the absorbance using the following formula.

[0090]

[0091] In the formula: b is the carbonyl content (nmol / mg); A is the sample absorbance; C is the sample mass concentration (mg / mL); ε is the molar extinction coefficient, 22000 L / (mol·cm); n is the dilution factor.

[0092] Analysis of cooking loss rate and water retention: From Figure 7 and Figure 8 The experimental data show that the addition of purified biogenics has a significant impact on the water-holding capacity and cooking loss rate of minced pork. With increasing addition amount, the water-holding capacity of the minced pork increases, while the cooking loss rate decreases, showing a negative correlation. After 96 hours of storage, compared with the control group without purified biogenics (Application Example 5), the water-holding capacity of samples with 0.5wt%, 1wt%, and 2wt% purified biogenics increased by 1.4%, 2.9%, and 5.6%, respectively; simultaneously, the cooking loss rate decreased by 7.4%, 17.8%, and 29.3%, respectively. This indicates that adding purified biogenics helps improve the water-holding capacity of minced pork, reduces the loss of moisture and soluble substances during heating, thereby improving product yield and sensory quality.

[0093] Furthermore, compared to the sample with 2 wt% crude Lactobacillus post-fermentation prebiotic (Application Example 4), the sample with 2 wt% purified post-fermentation prebiotic showed significant improvements in water retention and cooking loss. This indicates that the purification process effectively enriched antioxidants, reduced the interference of impurities on the stability of the meat product system, improved water retention capacity, and reduced moisture loss.

[0094] Texture analysis: Hardness, elasticity, chewiness, and cohesiveness are key indicators for evaluating the quality of minced meat. Figure 9 As shown, the elasticity and chewiness of the control group and the example group gradually increased, while the hardness showed a trend of first increasing and then decreasing with the extension of storage time. Cohesion significantly improved with increasing concentration of purified biogener. The control group (Application Example 3) with 2wt% purified biogener showed the best improvement in the hardness, elasticity, chewiness, and cohesion of the minced meat; at 96 hours of storage, the textural indices of Application Example 3 were far superior to those of Applications 4 and 5. In conclusion, purified biogener can effectively improve the texture of minced meat and mitigate product quality deterioration.

[0095] Carbonyl content analysis: Protein oxidation is an important cause of meat aging and nutrient loss, and carbonyl content is a key indicator for measuring protein oxidative damage. Figure 10 The results showed that at 96 hours of storage, the carbonyl content was highest in the group without added biogenic elements (Application Example 5). After adding 0.5 wt%, 1 wt%, and 2 wt% purified biogenic elements, the carbonyl content significantly decreased by 11.86%, 24.92%, and 37.27%, respectively. PThe carbonyl content of the meat batter with 2wt% purified postbiotic was reduced by 23.61%, and the antioxidant effect was almost halved. In summary, the purified postbiotic showed stronger antioxidant activity, which helped to maintain the structural integrity of myofibrillar protein, thereby improving the texture properties and overall quality of the meat batter.

[0096] The above only describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a Lactobacillus fermentum postbiotic for improving meat quality, characterized by, The method comprises the following steps: centrifuging a fermentation broth of Lactobacillus fermentum after inactivation to obtain supernatant, and then filtering and freeze-drying the supernatant to obtain the crude metagenome.

2. The method of claim 1, wherein the preparation of the Lactobacillus fermentum postbiotic for improving meat quality is characterized by, The macroporous resin is selected from one of DA201, S-8, D101, NKA-9, X-5 or AB-8.

3. The preparation method of Lactobacillus fermentum postbiotic for improving meat quality according to claim 1, characterized in that, The preparation steps of the crude metagenome comprise the following steps: under a sterile environment, inoculating seed liquid of Lactobacillus fermentum into sterilized MRS medium at an inoculation ratio of 3-5%, and culturing at a temperature of 35-37 DEG C and a speed of 50-180 rpm for 18-24 hours until the pH is 4.0-4.25 and the OD600 is 6-7; heating the fermentation broth in a water bath at 70-75 DEG C for 15-35 min for inactivation treatment; centrifuging and filtering the fermentation broth with a microporous filter, freeze-drying the filtered liquid to obtain the crude metagenome.

4. The preparation method of Lactobacillus fermentum postbiotic for improving meat quality according to claim 1, characterized in that, The preservation number of the Lactobacillus fermentum is GDMCC NO.1.1694.

5. The preparation method of Lactobacillus fermentum postbiotic for improving meat quality according to claim 1, characterized in that, The macroporous resin is used to enrich the antioxidant components, and the method comprises the following steps: dissolving the crude metagenome in water to obtain a crude metagenome solution with a mass concentration of 20-40 mg / mL; fully adsorbing the crude metagenome solution on the macroporous resin at a feeding speed of 2-6 BV / h, eluting the unabsorbed crude metagenome with deionized water, and then eluting with 40-70% ethanol at a speed of 2-6 BV / h; collecting the eluate, reducing pressure to concentrate, and freeze-drying to obtain the macroporous resin-enriched metagenome.

6. The method of claim 5, wherein the preparation of the Lactobacillus fermentum postbiotic for improving meat quality is characterized by, The maximum feeding amount is 2 BV, and the eluate is used in an amount of 5 BV.

7. The Lactobacillus fermentum metagenome prepared by the method of any one of claims 1-6.

8. The application of the Lactobacillus fermentum metagenome of claim 7 in improving meat quality.

9. Use according to claim 8, characterized in that, The addition amount of the Lactobacillus fermentum metagenome is 0.5-2 wt% of the mass of the meat.

10. Use according to claim 9, characterized in that, The specific method of the application comprises the following steps: adding the Lactobacillus fermentum metagenome into low-salt pork paste, and rolling and kneading in a vacuum rolling and kneading machine for 10-30 min; uniformly chopping and mixing the paste, and pressing the paste into a meat patty with a mold, and storing the meat patty in a refrigerator at 2-4 DEG C.

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