Compound bacterium wet feed breeding method for improving flavor and nutrition of pork

Abstract

The invention discloses a compound bacterium wet feed breeding method for improving flavor and nutrition of pork, which comprises the following steps: 5-30% of wet fermentation feed is added into basic ration during feeding, and the wet fermentation feed is prepared by fermenting mixed strains containing bacillus and lactic acid bacteria by taking the basic ration as a raw material. Through combined fermentation of bacillus, enterococcus faecalis and lactobacillus acidophilus, triple synergy of enzymolysis, acidification and bacteriostasis is achieved, efficient fiber and protein degradation is achieved, and the flavor and nutritional value of pork are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of livestock breeding and feed processing, and particularly relates to a compound bacteria wet feed breeding method for improving the flavor and nutrition of pork. Background Art

[0002] With the improvement of consumers' requirements for pork quality, traditional breeding feeds have problems such as insufficient flavor substances and low nutrient conversion rate. In recent years, wet feeds for pigs have been widely used in large-scale breeding due to their advantages such as good palatability, high digestibility, and reduced feed waste (LYDERIK K K, MADSEN J G, LARSEN C, et al. An increased weaning age and liquid feed enhances weight gain compared to piglets fed dry feed pre-weaning[J]. animal, 2023, 17(5):100801.). The wet feed fermentation technology improves the palatability and nutritional value of the feed through microbial metabolism. However, most of the existing technologies use single-strain fermentation (such as only using lactic acid bacteria), which has problems such as single strain function, difficulty in fully enzymolyzing, effectively acidifying, and long-term antibacterial of the feed, resulting in limited improvement in pork flavor and insufficient feed degradation (ZHANG Qiang, CHEN Rongqiang, KONG Zhiwei, et al. Effects of fermentation with different strains on the true protein content of navel orange waste feed[J]. Swine Industry Science, 2017, 34(09):56-58.). In addition, wet feeds are prone to spoilage, and the existing technology has insufficient preservation ability, which affects large-scale application.

[0003] The co-fermentation of composite strains (Bacillus + Lactobacillus) can comprehensively exert multiple functions such as protein degradation, acid production and antibacterial activity, and flavor substance generation ([3] WANGL, CHEN J, ZHANG J B, et al. Changes in intestinal microbiota, immunity and metabolism caused by mixed Lactiplantibacillus plantarum and Bacillus subtilis-fermented feed in Bamei pigs[J]. Chemical and Biological Technologies in Agriculture, 2024, 11:76.). For example, Bacillus (such as Bacillus subtilis) has strong cellulose and protein degradation abilities and can decompose anti-nutritional factors in feed (HOU Yongshuai, GAO Liang, JIANG Song, et al. Physiological functions of Bacillus subtilis and its application in ruminant production[J]. Shanghai Journal of Animal Husbandry and Veterinary Medicine, 2025, (01):47-51.); while Lactobacillus (such as Enterococcus faecalis, Lactobacillus acidophilus) can rapidly produce acid, reduce the pH value, inhibit the growth of harmful bacteria, and at the same time promote the colonization of probiotics in the intestine ([5] LIU H, WANG S X, CHEN M X, et al. Effects of Lactobacillus-fermented low-protein diets on the growth performance, nitrogen excretion, fecal microbiota and metabolomic profiles of finishing pigs[J]. Scientific Reports, 2024, 14:8612.).The synergistic effect of the two can not only improve the digestion and utilization rate of feed, but also enhance the immunity of pigs and reduce the dependence on antibiotics, which is in line with the development trend of modern green and healthy breeding (CHI Z H, ZHANG M Q, FU B T, et al. Branched short-chain fatty acid-rich fermented protein food improves the growth and intestinal health by regulating gut microbiota and metabolites in young pigs[J]. Journal of Agricultural and Food Chemistry, 2024, 72(39):21594–21609.). Summary of the Invention

[0004] Aiming at the deficiencies of existing problems, the purpose of the present invention is to provide a method for cultivating compound bacteria wet feed to enhance the flavor and nutrition of pork. The present invention realizes the efficient degradation of fiber and protein through the combined fermentation of Bacillus, Enterococcus faecalis, and Lactobacillus acidophilus, and significantly improves the flavor and nutritional value of pork by triple synergy of enzymatic hydrolysis, acidification, and antibacterial action.

[0005] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0006] First invention, the present invention provides a method for cultivating compound bacteria wet feed to enhance the flavor and nutrition of pork, and the method is as follows: When feeding, 5-30% of wet fermented feed is added to the basal diet, and the wet fermented feed is prepared by fermenting a mixed strain containing Bacillus and lactic acid bacteria with the basal diet as the raw material.

[0007] In a specific embodiment, the basal diet is conventionally selected in the art. For example, one or more of soybean meal, corn, and wheat bran can be selected as the basal diet.

[0008] In a more specific embodiment, the basal diet consists of soybean meal, corn, and wheat bran in a mass ratio of 4-10:0.5-3:1-5, preferably 6.5:1.5:2.

[0009] In a specific embodiment, the lactic acid bacteria are Enterococcus faecalis, Lactobacillus acidophilus, etc.

[0010] In a specific embodiment, the preparation method of the wet fermented feed is as follows:

[0011] (1) Pretreatment of strains (activation and expansion culture):

[0012] (a1) Liquid expansion culture of Bacillus subtilis: Take 0.5 - 3 g (preferably 1 g) of Bacillus subtilis, add 100 mL of sterile water, and culture with shaking at 37 °C and 200 rpm for 12 hours (OD 60 0 ≈ 1.0); Transfer to 20 L of sterilized culture medium according to an inoculation amount of 5%, and continue to culture for 24 hours (the viable count ≥ 1×10 8 CFU / mL).

[0013] (a2) Compound lactic acid bacteria (Enterococcus faecalis + Lactobacillus acidophilus): Take 25 - 50 g of Enterococcus faecalis microcapsules and 50 g of freeze-dried Lactobacillus acidophilus powder, mix them, and add 1 L of warm water at 35 °C (containing 5 g of glucose), and let it stand for half an hour for activation.

[0014] (2) Raw material mixing and inoculation

[0015] (b1) Dry material mixing: Pour 1000 kg of basal diet into a blender and mix at low speed for 10 minutes until uniform.

[0016] (b2) Adjust moisture: Add water in portions, and stir while adding until it forms a ball when squeezed by hand, and water seeps out between the fingers but does not drip (the water content is about 48%).

[0017] (b3) Bacterial liquid inoculation: First add 20 L of Bacillus subtilis expansion culture liquid (final concentration 1×10 6 CFU / g), stir for 5 minutes, then add 1 L of compound lactic acid bacteria liquid (Enterococcus faecalis + Lactobacillus acidophilus, final concentration 5×105 CFU / g each), and continue to stir for 5 minutes.

[0018] (3) Anaerobic fermentation management

[0019] (c1) Loading: Load the mixed material into a fermentation bag with a breathing valve (or a sealed plastic bucket), and compact it once every 30 cm (to reduce oxygen residue).

[0020] (c2) Sealing: Cover the top layer with a plastic film, press with heavy objects for sealing (or perform vacuum treatment).

[0021] (c3) Fermentation monitoring: Monitor pH (the end-point pH is controlled at 4.0 - 4.5), temperature (the first stage (0 - 24 h) is 37 °C, the second stage (24 - 72 h) is 30 °C), and fermentation time (72 h).

[0022] In a more specific embodiment, the mixed bacteria are composed of Bacillus, Enterococcus faecalis, and Lactobacillus acidophilus, wherein Bacillus subtilis is purchased from Beijing Dabeinong Technology Group Co., Ltd., Enterococcus faecalis is purchased from Hunan Youtell Biochemical Co., Ltd., and Lactobacillus acidophilus is purchased from Jiangsu Runkang Biotechnology Co., Ltd.

[0023] Beneficial effects

[0024] The compound bacteria wet feed breeding method for improving the flavor and nutrition of pork provided by the present invention has the following beneficial effects compared with the prior art:

[0025] (1) In the experimental group of pigs fed with the wet pig feed prepared by fermenting the compound bacterium agent (Bacillus subtilis + Enterococcus faecalis + Lactobacillus acidophilus) added in the present invention, the contents of essential amino acids, arginine, inosinic acid and taste amino acids in the pork tenderloin were significantly increased compared with the control group (P<0.05).

[0026] (2) The pork of the present invention has a better flavor. The results of gas chromatography-mass spectrometry (GC-IMS) show that the relative content of aldehyde substances in the pork obtained by the present invention is higher than that of the control group, and the relative contents of butyraldehyde and heptanol are significantly higher than those of the control group (P<0.05).

[0027] (3) In addition, the saturated fatty acid content of the pork obtained by the present invention is reduced by 9.23%, while the content of monounsaturated fatty acids is increased by 9.32%, and the relative content of linoleic acid is increased by 24.91%. The results show that adding the compound bacterium agent (Bacillus subtilis + Enterococcus faecalis + Lactobacillus acidophilus) to ferment the prepared wet pig feed can significantly improve the flavor and nutritional value of pork. Description of the Drawings

[0028] Figure 1 Percentages of different flavor nucleotides in the pork tenderloin. Data are expressed as mean ± SE. The differences between the two groups of data were compared by independent sample t-test. Different lowercase letters indicate significant differences between the two groups of data (P<0.05). Abbreviations: GMP: guanylic acid; CMP: cytidylic acid; IMP: inosinic acid; AMP: adenylic acid; Hx: hypoxanthine; In: inosine.

[0029] Figure 2 Flavor map of pig pork tenderloin obtained by electronic nose. (a) Principal component analysis of the flavor of pork tenderloin fed with two kinds of feeds. (b) Radar chart of the flavor of pork tenderloin of pigs fed with two kinds of feeds. Abbreviations: OF: ordinary feed; WFF: wet fermented feed.

[0030] Figure 3 Gallery map of volatile organic compounds in the pork tenderloin. Each row represents a sample. Each column represents a volatile compound. The color scale represents the concentration of the volatile compound ("D" represents dimer). In the group fed with ordinary feed, the content of the substances in the red box is higher, while in the group fed with wet fermented feed, the content of the substances in the yellow box is higher. Abbreviations: OF: ordinary feed; WFF: wet fermented feed.

[0031] Figure 4 Percentages of six volatile organic compounds in the pork tenderloin of the group fed with ordinary feed (a) and the group fed with wet fermented feed (b). Detailed Embodiments

[0032] The following further elaborates on the present invention in conjunction with embodiments. Reagents or equipment not indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0033] Bacillus subtilis was purchased from Beijing Dabeinong Technology Group Co., Ltd., Enterococcus faecalis was purchased from Hunan Youtell Biochemical Co., Ltd., and Lactobacillus acidophilus was purchased from Jiangsu Runkang Biotechnology Co., Ltd.

[0034] Example 1

[0035] Step 1: Selection and grouping of experimental animals

[0036] Select 240 three-way crossbred pigs at 115 days of age, in good health condition and with similar initial body weights (about 45 kg). Using a completely randomized design, divide the 240 pigs into 2 groups (control group and experimental group), with 120 pigs in each group, ensuring that there is no statistically significant difference in the initial body weights between groups (P > 0.05).

[0037] Step 2: Preparation of experimental diets

[0038] Control group: Prepare a basal diet (the mass ratio of soybean meal, corn, and wheat bran is 6.5:1.5:2), without adding fermented feed.

[0039] Experimental group: First, perform liquid expansion culture of Bacillus subtilis. That is, take 1 g of commercial bacterial powder, add 100 mL of sterile water, and culture with shaking at 37°C and 200 rpm for 12 hours (OD 600 ≈1.0), then transfer it to 20 L of sterilized medium at an inoculation amount of 5%, and continue to culture for 24 hours (the viable bacteria count ≥ 1×10 8 CFU / mL). Then, perform the compounding of lactic acid bacteria. That is, take 25 g of Enterococcus faecalis microcapsules and 50 g of Lactobacillus acidophilus freeze-dried powder, mix them and add to 1 L of warm water at 35°C (containing 5 g of glucose), and let it stand for half an hour for activation. Then, perform raw material mixing and inoculation. That is, using 1000 kg of basal diet as the raw material, first add 20 L of Bacillus subtilis expansion culture solution (final concentration 1×10 6 CFU / g), stir for 5 minutes, then add 1 L of lactic acid bacteria compounding solution (Enterococcus faecalis + Lactobacillus acidophilus, final concentration of each 5×10 5 CFU / g), and continue to stir for 5 minutes to prepare wet fermented feed by anaerobic fermentation. Anaerobic fermentation management: Filling: Fill the mixed material into a fermentation bag with a breathing valve, and compact it once every 30 cm. Sealing: Cover the top layer with a plastic film and seal it with a heavy object. Fermentation monitoring: Monitor pH (the end point pH is controlled at 4.0 - 4.5), temperature (the first stage (0 - 24 h) is 37°C, the second stage (24 - 72 h) is 30°C), and fermentation time (72 h).

[0040] The experimental group's diet was mixed with wet fermented feed and the basal diet at a ratio of 10%.

[0041] Step 3: Implementation of feeding management

[0042] The free-feeding and free-drinking mode was adopted to ensure that the feeding conditions of the two groups were the same. The standardized immunization program was strictly implemented (vaccines were inoculated according to the company's regulations), and the environment of the pens was kept clean and disinfected regularly.

[0043] Step 4: Experimental period and data collection

[0044] From 115 days of age until slaughter at 175 days of age (a total of 60 days). Slaughtering and sampling were carried out at Jiangsu Huaian Shifenweidao Food Co., Ltd. The pork tenderloin was taken 60 minutes after slaughter, divided and packed into cryotubes, placed in liquid nitrogen, and then transferred to a -80°C ultra-low temperature freezer for storage, for the determination of flavor and nutritional indicators.

[0045] Example 2

[0046] Step 1: Selection and grouping of experimental animals

[0047] Two hundred and forty three-way crossbred pigs at 115 days of age, in good health and with similar initial weights (about 45 kg) were selected. Using a completely randomized design, the 240 pigs were divided into 2 groups (control group and experimental group), with 120 pigs in each group, ensuring that there was no statistically significant difference in the initial weights between the groups (P>0.05).

[0048] Step 2: Preparation of experimental diets

[0049] Control group: Prepare the basal diet (the mass ratio of soybean meal, corn, and wheat bran is 5:2:3), without adding fermented feed.

[0050] Experimental group: First, perform liquid expansion culture of Bacillus subtilis. That is, take 1 g of commercial bacterial powder, add 100 mL of sterile water, and culture with shaking at 37°C and 200 rpm for 12 hours (OD 600 ≈1.0), then transfer it to 20 L of sterilized medium at an inoculation amount of 5% and continue to culture for 24 hours (the viable bacteria count ≥ 1×10 8 CFU / mL). Then, perform the compounding of lactic acid bacteria. That is, take 25 g of Enterococcus faecalis microcapsules and 50 g of freeze-dried Lactobacillus acidophilus powder, mix them and add them to 1 L of warm water at 35°C (containing 5 g of glucose), and let it stand for half an hour for activation. Then, perform raw material mixing and inoculation. That is, using 1000 kg of basal diet as the raw material, first add 20 L of Bacillus subtilis expansion culture solution (final concentration 1×10 6 CFU / g), stir for 5 minutes, and then add 1 L of lactic acid bacteria compounding solution (Enterococcus faecalis + Lactobacillus acidophilus, final concentration of each 5×10 5(CFU / g), continue stirring for 5 minutes, and prepare wet fermented feed through anaerobic fermentation. Anaerobic fermentation management: Loading: Load the mixed material into a fermentation bag with a breathing valve, and compact it once every 30 cm of loading. Sealing: Cover the top layer with a plastic film and seal it with a heavy object. Fermentation monitoring: Monitor pH (the end-point pH is controlled at 4.0 - 4.5), temperature (the first stage (0 - 24 h) is 37 °C, the second stage (24 - 72 h) is 30 °C), and fermentation time (72 h).

[0051] In the experimental group diet, the wet fermented feed was mixed with the basal diet at a ratio of 10%.

[0052] Step 3: Implementation of feeding management

[0053] Adopt the free-feeding and free-drinking mode to ensure that the feeding conditions of the two groups are the same. Strictly implement the standardized immunization program (vaccinate according to the company's regulations), keep the pen environment clean, and disinfect regularly.

[0054] Step 4: Experimental period and data collection

[0055] From 115 days of age until slaughter at 175 days of age (a total of 60 days). Slaughtering and sampling were carried out at Jiangsu Huaian Shifen Weidao Food Co., Ltd. Take the pork tenderloin of the pig 60 min after slaughter, sub-pack it into cryopreservation tubes, put it into liquid nitrogen, and then transfer it to a -80 °C ultra-low temperature freezer for storage, for the determination of flavor and nutritional indicators.

[0056] Example 3

[0057] Step 1: Selection and grouping of experimental animals

[0058] Select 240 three-way crossbred pigs at 115 days of age, in good health and with similar initial weights (about 45 kg). Using a completely random design, divide the 240 pigs into 2 groups (control group and experimental group), with 120 pigs in each group, ensuring that there is no statistically significant difference in the initial weights between the groups (P > 0.05).

[0059] Step 2: Preparation of experimental diets

[0060] Control group: Prepare a basal diet (the mass ratio of soybean meal, corn, and wheat bran is 6.5:1.5:2), without adding fermented feed.

[0061] Experimental group: First, perform liquid expansion culture of Bacillus subtilis. That is, take 1 g of commercial bacterial powder, add 100 mL of sterile water, and culture it at 37 °C and 200 rpm for 12 hours (OD 600 ≈1.0), then transfer it to 20 L of sterilized culture medium at an inoculation amount of 5%, and continue to culture for 24 hours (the viable bacteria count ≥ 1 × 10 8(CFU / mL), and then perform the compounding of lactic acid bacteria. That is, take 50 g of freeze-dried powder of Enterococcus faecalis and 50 g of freeze-dried powder of Lactobacillus acidophilus, mix them, and add 1 L of warm water at 35 °C (containing 5 g of glucose), and let it stand and activate for half an hour. Then, carry out raw material mixing and inoculation. That is, using 1000 kg of the basic diet as the raw material, first add 20 L of the Bacillus subtilis expanded culture solution (final concentration 1×10 6 (CFU / g), stir for 5 minutes, then add 1 L of the lactic acid bacteria compound solution (Enterococcus faecalis + Lactobacillus acidophilus, final concentration 5×10 5 (CFU / g) each), and continue to stir for 5 minutes, and prepare wet fermented feed by anaerobic fermentation. Anaerobic fermentation management: Loading: Put the mixed material into a fermentation bag with a breathing valve, and compact it once every 30 cm of loading. Sealing: Cover the top layer with a plastic film and seal it with a heavy object. Fermentation monitoring: Monitor pH (the end-point pH is controlled at 4.0 - 4.5), temperature (the first stage (0 - 24 h) is 37 °C, the second stage (24 - 72 h) is 30 °C), and fermentation time (72 h).

[0062] The experimental group diet mixes the wet fermented feed and the basic diet in a ratio of 10%.

[0063] Step 3: Implementation of feeding management

[0064] Adopt the free-feeding and free-drinking mode to ensure that the feeding conditions of the two groups are the same. Strictly implement the standardized immunization program (vaccinate according to the company's regulations), and keep the pen environment clean and disinfect regularly.

[0065] Step 4: Test cycle and data collection

[0066] From 115 days of age until slaughter at 175 days of age (a total of 60 days). Slaughtering and sampling are carried out at Jiangsu Huai'an Shifen Weidao Food Co., Ltd. Take the pork tenderloin after slaughter and sub-pack it into cryopreservation tubes 60 minutes after slaughter, put it into liquid nitrogen, and then transfer it to a -80 °C ultra-low temperature freezer for storage, for the determination of flavor and nutritional indicators.

[0067] The following tests are carried out for Example 1, and the specific test methods are as follows:

[0068] 1. Determination of free amino acids

[0069] The method for determining the content of free amino acids refers to the method of ZHANG et al. Weigh 4 g of minced loin sample, add 20 mL of sulfosalicylic acid solution (mass concentration: 3 g / 100 mL), and homogenize the solution three times in an ice-water bath using a high-speed homogenizer at a speed of 10,000 r / min for 20 s each time. Then centrifuge at 4 °C and 10,000 g for 15 min. After that, take the supernatant, add 2 mL of n-hexane, and shake well. After standing for layer separation, filter the lower aqueous phase through a 0.22-μm aqueous filter membrane. Use an amino acid analyzer equipped with a BioBasic SCX cation exchange column (4.6 mm × 60 mm, 5 μm) and an ultraviolet detector to determine the content of free amino acids.

[0070] 2 Determination of flavor nucleotides

[0071] The content of flavor nucleotides is determined by high-performance liquid chromatography, referring to the method of ZOU et al. [2] and modified according to the actual situation. Weigh 4 g of minced meat sample, accurate to 0.0001 g, place it in an 80-mL centrifuge tube, add 20 mL of 5% perchloric acid solution (pre-cooled), and homogenize in an ice-water bath using a homogenizer (rotation speed: 10,000 r / min, 3 times, 30 s each time, with an interval of 5 s); then centrifuge at 4 °C and 10,000 g for 10 min, and take the supernatant into a 50-mL centrifuge tube. Add 10 mL of 5% perchloric acid solution (pre-cooled) to the precipitate for vortex mixing and washing, and centrifuge at 4 °C and 10,000 r / min for 15 min. Combine the two supernatants, adjust the pH of an appropriate amount of supernatant to 4.5 with 0.5 mol / L KOH solution, and make up the volume of the supernatant to 10 mL with ultrapure water. Finally, take 2 mL of the liquid with a syringe, filter it through a 0.22-μm aqueous filter membrane, place it in a 1-mL brown liquid-phase screw vial for storage at 4 °C, and use a high-performance liquid chromatograph to analyze the content of each nucleotide in the sample.

[0072] Configuration of nucleotide standards: Select six nucleotides, namely CMP, GMP, IMP, AMP, Hx, and In, as the determination indicators. Accurately weigh 10 mg of each standard and make up the volume to 10 mL respectively to obtain single-standard solutions with a concentration of 1 mg / mL; weigh 5 mg of each nucleotide standard again, mix them and make up the volume to 20 mL to obtain a mixed standard solution with a concentration of 250 μg / mL. Other concentration mixed standard solutions are diluted based on this.

[0073] The main parameters of high-performance liquid chromatography are shown in Table 1.

[0074] Table 1 Parameters of high-performance liquid chromatography gradient elution method

[0075]

[0076] 3 Electronic nose analysis

[0077] Refer to the method of Zhou Hui and make slight modifications. Weigh 5 g of the minced meat sample into a headspace vial, seal it, heat it in a water bath at 50 °C for 30 min, equilibrate it at room temperature for 30 min, and then start the measurement. Turn on the instrument, insert the plug of the electronic nose into the headspace vial to aspirate the top gas, and the instrument starts to collect data. Set the data collection time to 120 s and the cleaning time to 60 s.

[0078] Table 2 Performance description of PEN3 portable electronic nose sensors

[0079]

[0080]

[0081] 4 Determination of volatile flavor substances

[0082] After mincing the samples of each treatment group, weigh 3 g of each and place them in 20 mL headspace vial, seal and wait for measurement, and analyze them with gas chromatography-ion mobility spectrometry (GC-IMS). The parameter settings are shown in Table 3.

[0083] Table 3 Main parameters of GC-IMS

[0084]

[0085] 5 Fatty acid determination

[0086] The determination of muscle fatty acids refers to the method of LI et al. [4] and make slight modifications. Mince the muscle and weigh 6 g into a 50 mL round-bottom centrifuge tube. Add 30 mL of chloroform-methanol (2:1, V / V) solution, homogenize it at 8000 r / min for 3 times, 30 s each time. After standing in a fume hood for 24 h, filter it with gauze. Add 8 mL of 0.9% normal saline to the filtrate, vortex and mix well, and centrifuge at 5000 r / min and 4 °C for 20 min. Take the lower organic phase liquid and dry it with a nitrogen blower to obtain the pure oil product.

[0087] The saponification and methylation were carried out with reference to the method of CHEN et al. 0.1 g of oil was pipetted into a 50 mL centrifuge tube, and 4 mL of 0.5 mol / L sodium hydroxide - methanol solution was added. It was heated in a water bath at 70 °C for 10 min. Then 5 mL of 14% boron trifluoride - methanol solution was added to the centrifuge tube, and it was heated in a water bath at 70 °C again for 15 min. Immediately, 7 mL of n - hexane and 10 mL of saturated sodium chloride solution were added, and it was vortexed for 15 s. It was centrifuged at 15000 g and 4 °C for 5 min, and the upper clear liquid was pipetted and passed through a 0.22 μm organic filter membrane into a sample vial. The gas chromatography operating conditions were referred to GB 5009.168 - 2016 National Food Safety Standard - Determination of Fatty Acids in Foods, as shown in Table 4. A mixed standard solution of 37 fatty acids was used as the external standard. The fatty acids in the sample were quantified by standard peak area normalization.

[0088] Table 4 Gas Chromatography Operating Conditions

[0089]

[0090] 6. Data Statistics and Analysis

[0091] A completely randomized design was adopted, with two treatments and three replicates. Hypotheses were used to evaluate the significance of differences between different groups, and P < 0.05 was considered significant. A mixed model was designed to evaluate the differences in pork flavor, including electronic nose and GC - IMS data. Feed type and flavor substances were used as fixed effects, while loin, slaughter batch, and repeated measurements were used as random effects. The design concept of the models for the contents of free amino acids, free fatty acids, and flavor nucleotides was similar to that of the flavor evaluation model, with loin, slaughter batch, and repeated measurements as random effects. In the free amino acid content model, amino acid type, feed type, and their interaction were used as fixed effects. In the free fatty acid content model, fatty acid type, feed type, and their interaction were fixed terms. In the flavor nucleotide content model, nucleotide type, feed type, and their interaction were used as fixed effects. The data were analyzed by SPSS 25.0 (IBM Corporation, USA). The data were expressed as mean ± standard error of the mean (SEM). Duncan's multiple range analysis was used for post - hoc tests. Data visualization was performed using GraphPad Prism 8 (GraphPad Software Inc.). PCA analysis of the electronic nose measurement results was carried out using the matching Win Muster software, and a radar chart was plotted in Excel. When qualitatively and analytically detecting volatile flavor compounds, Library Search software and Laboratory Analytical Viewer (LAV) were used, and a fingerprint map of volatile compounds was plotted using the built - in plug - in of LAV.

[0092] The results are as follows:

[0093] 1 Free Amino Acid Composition

[0094] Some essential amino acids such as glutamic acid, aspartic acid, phenylalanine, alanine, glycine, and tyrosine are regarded as delicious amino acids in meat due to their unique flavors. Their composition and concentration directly affect the flavor of meat. Besides the wonderful flavor, the sweetness of meat is also an important taste that forms the meaty flavor. Glycine, threonine, and lysine are sweet amino acids, which contribute to the production of sweetness. Table 5 lists the relative concentrations of free amino acids in this study. In the experimental group, the relative concentration of sweet amino acids was much higher than that in the control group, indicating that the pork from pigs fed with fermented feed was generally more palatable. The percentage of sweet amino acids (glycine, threonine, and lysine) in the group fed with wet fermented feed was much higher than that in the group fed with common feed (Table 5), indicating that the meat in the group fed with wet fermented feed was sweeter and more palatable.

[0095] It is worth noting that the relative concentrations of essential amino acids and arginine in the group fed with wet fermented feed were much higher than those in the group fed with common feed, which means that the pork from pigs fed with wet fermented feed had higher nutritional value. Amino acids are the basic components of proteins and contribute to physiological and biochemical activities such as protein synthesis, playing an important role in human nutritional metabolism. There are eight amino acids called essential amino acids, and the content of essential amino acids in the group fed with wet fermented feed was significantly higher than that in the group fed with common feed. In particular, the contents of phenylalanine, threonine, and lysine were significantly different between the two groups of samples (Table 5). Phenylalanine is a necessary raw material for the synthesis of adrenaline, thyroxine, and other neurotransmitters and hormones in the human body. Threonine can promote the synthesis of phospholipids and the oxidation of fatty acids, while lysine has important nutritional significance for growth and normal physiological activities. The results of the present invention show that the relative concentration of essential amino acids in the wet fermented feed group was significantly higher than that in the wet fermented feed group, indicating that wet fermented feed can improve the flavor and nutritional value of pork. The reason may be that wet fermented feed is more easily digested and absorbed by pigs.

[0096] Table 5 Composition of Free Amino Acids in the Tenderloin

[0097]

[0098]

[0099] 2 Flavor Nucleotides

[0100] Nucleotides are important active substances in organisms and have various biological functions. Some nucleotides have unique tastes. Inosine-5'-monophosphate (IMP) and guanosine-5'-monophosphate (GMP) have the effect of blocking salty, sour, bitter, fishy, and burnt tastes, so they have a strong ability to improve the flavor of pork. In this study, the percentages of GMP and IMP contents in the tenderloin of pigs in the wet fermentation group were significantly higher than those in the group fed with common feed, while the relative contents of other nucleotides decreased (Figure 1 )。This result indicates that feeding wet fermented feed can change the composition ratio of flavor nucleotides through nutritional management, thereby improving the flavor of pork. In a previous study, this phenomenon was attributed to the upregulation of the expression of genes related to flavor nucleotide synthesis by wet fermented feed.

[0101] 3 fatty acids

[0102] An important way to produce meat flavor is lipid oxidation. The main fatty acids in this study were palmitic acid, stearic acid, oleic acid, and linoleic acid (Table 6), which is consistent with the results of most studies on the fatty acid composition of pigs. Palmitic acid is a fatty acid that has a good correlation with meat flavor, and its content in the group fed wet fermented feed was much higher than that in the group fed ordinary feed. The content of saturated fatty acids in the group fed wet fermented feed decreased by 9.23%, while the content of monounsaturated fatty acids increased by 9.32%. In addition, the relative content of linoleic acid in the group fed wet fermented feed increased by 24.91%, and arachidonic acid was only present in the group fed wet fermented feed. These results support the conclusion of Lu et al. that feeding wet fermented feed can greatly increase the relative proportion of monounsaturated fatty acids in pork. The study also shows that the pork of pigs fed wet fermented feed contains more oxidizable lipids, thus producing a stronger flavor. In addition, some studies have shown that excessive intake of saturated fatty acids is harmful to human health, can lead to fat deposition, and cause hypertension, hyperlipidemia, cardiovascular diseases, and cerebrovascular diseases. Unsaturated fatty acids can improve liver lipoprotein metabolism, remove blood clots, lower blood lipids, prevent cardiovascular diseases, and have anti-cancer effects. Therefore, the research results on the relevant fatty acid components indicate that the pork of pigs fed wet fermented feed has higher nutritional value and is beneficial to human health.

[0103] Table 6 Composition of fatty acids in the loin

[0104]

[0105]

[0106] Note: The differences between the two groups of data were compared by independent samples t-test. "-" indicates that the substance was not detected.

[0107] 4 Analysis of volatile flavor substances

[0108] (1) Electronic nose

[0109] Electronic nose analysis is a bionic method for quickly perceiving food flavor. Figure 2A describes the principal component analysis (PCA) of two loins detected by an electronic nose. The first principal component (PC1) accounts for 78.7%, and the second principal component (PC2) accounts for 9.95%. The sum of the contribution rates of PC1 and PC2 is equal to 88.65%, which may explain most of the sample flavor information. On the PC1 dimension, feeding with ordinary feed and wet fermented feed are strongly distinguished, indicating that there are significant differences in the flavors of the two groups. The source of flavor differences can be further detected using a radar chart ( Figure 2 B). The response value of the W5C sensor (short-chain alkane aromatic components) to the wet fermented feed sample is higher than that of the W1S sensor (methyl). This indicates that the flavor of the ordinary feed sample tends to be methane, while the flavor of the wet fermented feed sample tends to be alkanes. This may mean that the flavor components of the wet fermented feed sample are more complex and the carbon chain is longer.

[0110] (2) Gas chromatography-ion mobility spectrometry analysis

[0111] Gas chromatography-ion mobility spectrometry is an advanced analytical method that is effective in detecting complex samples. This method requires no pretreatment and has good repeatability in determining the flavor of loin. 57 chemical substances were found, including 11 aldehydes, 17 alcohols, 13 ketones, 7 esters, 6 acids, 1 sulfur compound, 1 phenol, and 1 terpene. The main volatile components in the sample include aldehydes, alcohols, and ketones, which is consistent with the previous research results.

[0112] Aldehydes have high volatility and low flavor thresholds and are very important for forming the flavor of the outer loin. This study found that the contents of nonanal and heptanal in both treatment groups are high and there is no significant difference. However, n-butanal has a banana and green aroma, and its relative content in the wet fermented feed group is much higher than that in the ordinary feed group. Generally, aldehydes are produced by lipid oxidation; considering that the content of monounsaturated fatty acids, especially linoleic acid, in the wet fermented feed group is higher, this may be the main reason for the different aldehyde contents in the two groups.

[0113] Although alcohols have less influence on meat flavor than aldehydes, they are also important. It was found that the contents of pentanol and n-hexanol are high and often have a fruity flavor. In addition, the relative content of heptanol in the wet fermented feed group increased by 51.51%, enhancing the flavor of pork. The formation of alcohols is often related to the metabolism of sugars and amino acids, and many amino acids were widely found in the wet fermented feed group, which explains the source of some alcohols.

[0114] Ketones have a synergistic effect on the odor of pork. In the wet fermented feed group, the relative content of 2-butanone has no substantial difference from that in the ordinary feed group, but the relative content of 2,3-pentanedione drops sharply. According to the fingerprint ( Figure 3) Among the treatment groups, there are both common chemicals and specific chemicals.

[0115] Volatile organic chemicals such as nonanal, heptanal, pentanol, n-hexanol, 2-butanone, 2,3-pentanedione, propyl butyrate, and hexyl acetate, which have a greater impact on pork flavor, have relatively high concentrations in both groups of samples. However, the relative concentration of aldehydes in the loin fed with wet fermented feed is higher than that in the loin fed with ordinary feed, while the relative contents of alcohols, esters, and other compounds are lower ( Figure 4 ). The composition and content of volatile organic compounds have a great impact on the flavor of pork. Therefore, based on the content of volatile organic compounds, aldehydes are the basis for the improved flavor of the loin of pigs fed with fermented feed.

[0116] In summary, feeding wet fermented feed during the fattening stage of pigs helps to improve the flavor and nutritional components of pork. As a feasible method for nutritional improvement, the feeding of wet fermented feed in the present invention improves the nutritional status of fattening pigs. Pigs fed with wet fermented feed have higher levels of amino acids, free fatty acids, and flavor nucleotides. This contributes to the development of aldehydes and alcohols in their bodies. This also indicates that wet fermented feed improves the flavor and nutritional value of pork.

[0117] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, variations and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims.

Claims

1. A composite bacteria wet feed breeding method for improving pork flavor and nutrition, characterized in that: The method comprises the following steps: adding 5-30% of wet fermented feed to the basic diet during feeding and fattening, wherein the wet fermented feed is made from the basic diet and is fermented with mixed strains containing bacillus and lactic acid bacteria.

2. The method for cultivating a composite bacterial wet feed for improving pork flavor and nutrition according to claim 1, characterized in that: The basic diet consists of soybean meal, corn and bran in a mass ratio of 4-10:0.5-3:1-5, preferably 6.5:1.5:

2.

3. The composite bacteria wet feed breeding method for improving pork flavor and nutrition according to claim 2, characterized in that: The basic diet consists of soybean meal, corn, and bran in a mass ratio of 6.5:1.5:

2.

4. The composite bacteria wet feed breeding method for improving pork flavor and nutrition according to claim 1, characterized in that: The mixed bacteria consisted of Bacillus, Enterococcus faecalis and Lactobacillus acidophilus.

5. The composite bacteria wet feed breeding method for improving pork flavor and nutrition according to claim 1, characterized in that: The preparation method of the wet fermented feed is as follows: (1) Pretreatment of bacterial strains: Bacillus subtilis is cultured, and then lactic acid bacteria Enterococcus faecalis and Lactobacillus acidophilus are compounded; (2) Raw material mixing and inoculation: dry material mixing, moisture adjustment and bacterial liquid inoculation; (3) Anaerobic fermentation management: filling, sealing and fermentation monitoring.

6. The composite bacteria wet feed breeding method for improving pork flavor and nutrition according to claim 5, characterized in that: In step (1), the Bacillus subtilis expansion step is as follows: 0.5-3 g of Bacillus subtilis is added to 100 mL of sterile water, and cultured at 37° C. and 200 rpm for 12 hours; then transferred to 20 L of sterile culture medium at a 5% inoculum volume, and continued to culture until the number of viable bacteria is ≥ 1×10 8 CFU / mL.

7. The composite bacteria wet feed breeding method for improving pork flavor and nutrition according to claim 5, characterized in that: In the step (1), the compounding step of Enterococcus faecalis and Lactobacillus acidophilus is as follows: 25-50 g of Enterococcus faecalis and 50 g of Lactobacillus acidophilus are taken, 1 L of 35° C. warm water is added after mixing, and the mixture is allowed to stand for activation for half an hour.

8. The composite bacteria wet feed breeding method for improving pork flavor and nutrition according to claim 5, characterized in that: In the step (2), the moisture content is 48±5%.

9. The method for cultivating pork with composite bacteria wet feed for improving pork flavor and nutrition according to claim 5, characterized in that: In step (2), the bacterial solution inoculation step is as follows: first add 20L of Bacillus subtilis expansion solution to a final concentration of 1×10 6 CFU / g, stirred for 5 minutes, and then added 1L of lactic acid bacteria compound solution, with a final concentration of 5×10 5 CFU / g, and continue stirring for 5 minutes.

10. The method for cultivating pork with composite bacteria wet feed for improving pork flavor and nutrition according to claim 5, characterized in that: In step (3), the specific steps of anaerobic fermentation are as follows: (c1) Filling: The mixed material is placed into a fermentation bag with a breathing valve or a sealed plastic barrel, and compacted every 30±5 cm; (c2) Sealing: The top layer is covered with a plastic film and sealed with a weight or vacuumed; (c3) Fermentation monitoring: The endpoint pH was controlled at 4.0-4.5, the first stage of fermentation was 0-24h, 37°C, and the second stage was 24-72h, 30°C.

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