A solid-state fermentation process for soy sauce lees and its application

Abstract

This invention relates to the field of feed resource development technology, specifically to a solid-state fermentation process for soy sauce residue. Through the synergistic effect of desalination pretreatment and mixed-culture solid-state fermentation, the nutritional quality of the soy sauce residue is significantly improved. This invention also provides the application of solid-state fermented soy sauce residue in broiler diets. The beneficial effects of this invention are: fermented soy sauce residue can be directly used as a protein feed ingredient to partially replace soybean meal, with a stable source and low cost. This not only reduces breeding costs and dependence on imported soybean meal but also realizes the resource utilization of soy sauce residue, conforming to the concepts of circular economy and sustainable development in the feed industry.

Description

Technical Field

[0001] This invention relates to the field of feed resource development technology, specifically to a solid-state fermentation process for soy sauce lees and its application. Background Technology

[0002] With the rapid development of the feed industry and the expansion of poultry farming, the scarcity and price fluctuations of protein feed resources have become key factors restricting the sustainable development of the poultry industry. Soybean meal, as the most important source of plant protein in broiler diets, is highly dependent on imports, and its price is significantly affected by the international market, resulting in persistently high farming costs. Therefore, developing unconventional protein feed resources that are stable in source, inexpensive, and have suitable nutritional value to partially replace soybean meal is of significant practical importance.

[0003] Soy sauce residue is a major byproduct of soy sauce production. Global annual production has increased in scale along with the growth of the soy sauce industry, with approximately 0.67 kg of fresh soy sauce residue generated for every 1 kg of soy sauce produced. Soy sauce residue contains 15%-30% crude protein, 8%-25% crude fat, as well as amino acids, residual polysaccharides, minerals, and soy isoflavones, possessing potential value as a feed ingredient. However, untreated soy sauce residue suffers from high crude fiber content, poor digestibility, high salt content (5%-17%), and residual anti-nutritional factors, directly limiting its application in broiler diets. Direct feeding can lead to electrolyte imbalances due to high salt content, while high crude fiber reduces feed digestibility and utilization. Indiscriminate disposal not only wastes resources but also creates environmental pressure.

[0004] Solid-state fermentation technology is considered an effective means of improving the feed quality of by-products due to its simplicity, low energy consumption, and strong adaptability. Microbial solid-state fermentation can degrade crude fiber and macromolecules in soy sauce residues, reduce salt content and anti-nutritional factors, and simultaneously enrich small peptides, free amino acids, and functional metabolites, thereby improving its nutritional value and feed safety. Currently, there are some studies on the fermentation of soy sauce residues to prepare feed, but existing technologies mostly use single-strain fermentation, which has problems such as limited nutritional improvement effects and unclear process parameters. Therefore, developing a multi-strain synergistic fermentation technology for soy sauce residues with optimized processes and clearly defined nutritional value has become an urgent technical problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a solid-state fermentation process for soy sauce lees, which significantly improves the nutritional quality of soy sauce lees through the synergistic effect of desalination pretreatment and mixed-culture solid-state fermentation.

[0006] This invention also provides the application of solid-state fermented soy sauce residue in broiler diets.

[0007] An optimized solid-state fermentation process for soy sauce lees includes the following steps: S1: Desalination pretreatment: After crushing the soy sauce lees raw material, sieve it, add distilled water at a material-to-water ratio of 1:5-1:7 (m / v), soak at room temperature for 18-22 h, filter with gauze and squeeze to dehydrate, and obtain desalted wet lees; then place the wet lees in a constant temperature forced-air drying oven to dry to constant weight, crush and sieve again to obtain desalted soy sauce lees powder.

[0008] S2: Mixed culture: using Bacillus subtilis, Aspergillus niger and Lactobacillus plantarum as the compound fermentation strains; Bacillus subtilis was inoculated into LB medium and cultured at 35°C and 180 rpm for 10 h until the initial plateau phase, at which the viable cell concentration reached 1.83 × 10⁻⁶. 7 CFU / mL; *Lactobacillus plantarum* was inoculated into MRS medium and cultured at 37℃ and 200 rpm for 20 h until the initial plateau phase, at which the viable cell concentration reached 2.29 × 10⁻⁶. 7 CFU / mL; *Aspergillus niger* was inoculated into PDA medium and incubated statically at 30°C until spores covered the plate, preparing a concentration ≥ 1 × 10⁻⁶. 7 CFU / mL spore suspension.

[0009] S3: Solid-state fermentation: The material with adjusted moisture content is placed into a well-ventilated fermentation bag or shallow fermentation container, ready for inoculation with microorganisms for fermentation; first, Bacillus subtilis bacterial solution is inoculated at 1% and Aspergillus niger spore suspension is inoculated at 1%; aerobic fermentation is carried out at 30℃ for 75 h; then, Lactobacillus plantarum bacterial solution is inoculated at 2%; anaerobic fermentation is carried out at 37℃ for 72 h. After fermentation, the mixture is dried, crushed and sieved to obtain solid-state fermented soy sauce lees.

[0010] This invention also provides the application of solid-state fermented soy sauce residue in broiler diets.

[0011] Preferably, the broiler diet includes soybean meal and fermented soy sauce residue; the proportion of soybean meal added to the broiler diet is 15%-26%, and the proportion of fermented soy sauce residue added to the broiler diet does not exceed 15%.

[0012] Preferably, the proportion of the fermented soy sauce lees to the total amount of fermented soy sauce lees and soybean meal is no more than 50%.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention adopts a "desalination pretreatment-aerobic-anaerobic two-stage mixed fermentation" process, which specifically solves the core problems of high salt, high fiber and low digestibility of soy sauce lees. After fermentation, the crude protein content of soy sauce lees is significantly increased, while the content of crude fiber and anti-nutritional factors is greatly reduced, and the nutritional quality and feed safety are improved simultaneously.

[0014] 2. The selected composite microbial strains exhibit complementary functions: Bacillus subtilis has a strong enzyme-producing capacity, which can degrade anti-nutritional factors and improve protein utilization; Aspergillus niger can secrete cellulase and hemicellulase, disrupting plant cell wall structure; Lactobacillus plantarum rapidly produces acid in the anaerobic stage, inhibiting the growth of other microorganisms and promoting stable protein accumulation. These three strains synergistically achieve the dual effect of "reducing fiber and increasing protein." Fermentation process parameters were systematically optimized using response surface methodology, with high model prediction accuracy (deviation rate of only 0.89%) and strong process stability, making it suitable for large-scale production applications. The fermentation products have a crude protein content ≥ 38.44%, crude fiber content ≤ 15.05%, neutral detergent fiber content ≤ 36.16%, and acid detergent fiber content ≤ 24.89%.

[0015] 3. Fermented soy sauce residue is used to replace part of the soybean meal, and the replacement ratio does not exceed 50% of the total soybean meal usage. Through systematic nutritional value evaluation, the nutrient utilization rate and amino acid digestibility of fermented soy sauce residue in broilers have been clarified. The standard ileal digestibility of most essential amino acids is ≥85%, which provides accurate data support for its addition ratio in broiler diets and avoids the breeding risks caused by blind application.

[0016] 4. Fermented soy sauce residue can be used directly as a protein feed ingredient to partially replace soybean meal. It has a stable source and low cost, which not only reduces breeding costs and dependence on imported soybean meal, but also realizes the resource utilization of soy sauce residue, which is in line with the concept of circular economy and sustainable development of feed industry. Attached Figure Description

[0017] Figure 1 The effect of anaerobic fermentation time on the crude fiber content of soy sauce lees; Figure 2 The effect of aerobic fermentation time on the crude fiber content of soy sauce lees; Figure 3 The effect of moisture content on the crude fiber content of soy sauce lees; Figure 4 The effect of inoculum size during the aerobic stage on the crude fiber content of soy sauce lees; Figure 5 The effect of inoculum size during the anaerobic stage on the crude fiber content of soy sauce lees; Figure 6 Response surface plot of the effect of the interaction between aerobic and anaerobic fermentation time on the degradation rate of crude fiber in soy sauce lees; Figure 7 Response surface plot of the effect of the interaction between moisture content and aerobic fermentation time on the degradation rate of crude fiber in soy sauce lees; Figure 8 Response surface plot of the effect of the interaction between moisture content and anaerobic fermentation time on the degradation rate of crude fiber in soy sauce lees; Figure 9Contour plot of the effect of the interaction between aerobic and anaerobic fermentation time on the degradation rate of crude fiber in soy sauce lees; Figure 10 Contour plot showing the effect of the interaction between moisture content and aerobic fermentation time on the degradation rate of crude fiber in soy sauce lees; Figure 11 Contour plot showing the effect of the interaction between moisture content and anaerobic fermentation time on the degradation rate of crude fiber in soy sauce lees; Detailed Implementation

[0018] The technical solution of the present invention will be further explained and illustrated below through specific embodiments. Example 1

[0019] Raw materials and strains: Soy sauce lees were provided by Qianhe Flavoring Food Co., Ltd., and were crushed and passed through a 40-mesh sieve for later use; Bacillus subtilis, Lactobacillus plantarum, and Aspergillus niger powder were purchased from China National Research Institute of Food Fermentation Industries Co., Ltd.; LB medium, MRS medium, and PDA medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0020] The present invention provides a solid-state fermentation process for soy sauce lees, comprising the following steps: S1: Desalination Pretreatment: Soy sauce lees were mixed with distilled water at a material-to-water ratio of 1:5 (m / v) and soaked at room temperature for 18 hours, with intermittent stirring to promote salt dissolution. After soaking, the mixture was filtered through gauze and squeezed to remove water, yielding desalted wet lees. The wet lees were then dried at a constant temperature of 65℃ until constant weight, and pulverized through a 40-mesh sieve to obtain desalted soy sauce lees powder. The salt content of the soy sauce lees was measured to decrease from 9.08% to 3.64%.

[0021] The desalination pretreatment process reduces the salt content of soy sauce residue by 47%-69% compared to the original residue, ensuring desalination while reducing nutrient loss and water consumption.

[0022] S2: Mixed culture: Bacillus subtilis, Aspergillus niger and Lactobacillus plantarum were selected as the mixed fermentation strains; Bacillus subtilis was inoculated onto LB agar plates and streaked to isolate the bacteria. Single colonies were picked and transferred to LB liquid medium, and incubated at 35°C and 180 rpm for 10 h to prepare a solution with a concentration of 1.83 × 10⁻⁶. 7 CFU / mL bacterial suspension; Lactobacillus plantarum was streaked onto MRS agar plates for isolation. Single colonies were picked and transferred to MRS liquid medium, and cultured at 37°C and 200 rpm for 20 h to prepare a solution with a concentration of 2.29 × 10⁻⁶. 7 CFU / mL bacterial suspension; Aspergillus niger was inoculated onto PDA agar plates and incubated statically at 30°C for 72 h. Spores were collected by rinsing with sterile distilled water and prepared to a concentration of 1.2 × 10⁻⁶. 7CFU / mL spore suspension; S3: Weigh 10 kg of the desalted soy sauce residue powder obtained in S1, and spray it evenly with sterile water while stirring to ensure uniform moisture distribution. Then seal the material and let it stand for 30-60 minutes to allow for full water absorption. Take a small amount of material and check the moisture content using a rapid moisture meter or drying method, ensuring it is within the range of 55%-60%. If the moisture content is too low, add sterile water as needed and mix again; if it is too high, extend the ventilation and drying time appropriately. Box-Behnken experiments were conducted to determine the interaction of various factors on the degradation rate of crude fiber in the soy sauce residue, ultimately determining 75 h of aerobic fermentation and 72 h of anaerobic fermentation as the optimal fermentation conditions.

[0023] S4: The 10 kg of desalted soy sauce residue powder described in S3 is placed into a fermentation bag, and 1% Bacillus subtilis suspension and 1% Aspergillus niger spore suspension are added. Aerobic fermentation is carried out at 30°C for 75 h. Then, 2% Lactobacillus plantarum suspension is added. Anaerobic fermentation is carried out at 37°C for 72 h. After fermentation, the mixture is dried at 65°C, pulverized and passed through a 40-mesh sieve to obtain solid-state fermented soy sauce residue.

[0024] Product testing was conducted on this embodiment: the test results showed that the fermented soy sauce lees contained 95.54% dry matter, 38.34% crude protein, 14.52% crude fiber, 4.50% crude ash, 35.95% neutral detergent fiber, 24.09% acid detergent fiber, and 3.64% salt.

[0025] In this embodiment, the crude fiber degradation rate of soy sauce residue reached 23.24%, and the crude protein content increased from 30.01% in the original soy sauce residue to 38.34%. Compared with the pretreated soy sauce residue, the content of neutral detergent fiber, acid detergent fiber and crude ash was significantly reduced, and the nutritional structure was significantly optimized.

[0026] Table 1. Comparison of chemical composition before and after soy sauce lees treatment (%) Example 2

[0027] Experimental materials: Fermented soy sauce lees were the product prepared in Example 1; the experimental animals were 1-day-old healthy AA broilers (which were fed routinely from day 1 in preparation for the experiment), provided by Sichuan Suining Zhengda Food Co., Ltd.

[0028] This invention also provides the application of solid-state fermented soy sauce residue in broiler diets.

[0029] Nutritional value assessment: Through broiler metabolism test and standard ileal amino acid digestibility test, the apparent digestibility of conventional nutrients such as dry matter, crude protein, and crude fiber of fermented soy sauce lees, as well as the apparent ileal digestibility (AID) and standard ileal digestibility (SID) of 17 amino acids, were systematically evaluated to clarify its nutrient utilization efficiency in broilers aged 19-21 days and 40-42 days.

[0030] The broiler diet includes soybean meal and fermented soy sauce residue; the soybean meal is added at a rate of 15%-26% in the broiler diet, and the fermented soy sauce residue is added at a rate not exceeding 15%.

[0031] 1. Metabolic test: 48 broilers were selected at 16 days and 37 days of age and randomly divided into 2 groups, with 6 replicates in each group and 4 chickens in each replicate. The test period was 6 days, with the first 3 days being the adaptation period and the last 3 days being the formalization period. Apparent nutrient digestibility and metabolizable energy were measured at 19-21 days of age and 40-42 days of age, respectively. Fecal samples were collected daily during the period, dried at 65℃, pulverized, and then tested.

[0032] 2. Standard ileal amino acid digestibility test: 24 broilers were selected at 18 days and 39 days of age, with 6 replicates in each group. 4 chickens in each replicate were fed a nitrogen-free diet for 3 days to adapt. At the same time, a basal diet group and a soy sauce residue group were set up. At 21 days and 42 days of age, the broilers were fasted for 4 hours and then fed for 1 hour. After 3 hours, the ileal digest was dissected and collected. The amino acid content was then measured after freeze-drying.

[0033] The nitrogen-free diets in the experimental groups used corn starch and sucrose as the main energy sources. The nutritional levels of the diets in each group met the growth requirements of broilers and complied with the requirements of NRC (1994) and the Chicken Feeding Standard (NY / T33—2004).

[0034] Table 2. Composition and Nutritional Levels of the Basal Diet

[0035] 1 Each kg of diet provides: Copper 16 mg; Iron 20 mg; Iodine 1.25 mg; Manganese 120 mg; Zinc 110 mg; Selenium 0.3 mg; Vitamin A 11,000 IU; Vitamin D3 4,750 IU; Vitamin E 72.5 IU; Vitamin K3 3.1 mg; Vitamin B2 7.55 mg; Vitamin B1 2.85 mg; Vitamin B6 3.75 mg; Vitamin B... 12 0.017 mg; pantothenic acid 19 mg; niacin 62.5 mg; biotin 0.2 mg; folic acid 2.05 mg; choline chloride 250 mg; antifungal agent 50 mg; antioxidant 200 mg.

[0036] 2 Each kg of diet provides: Copper 16 mg; Iron 20 mg; Iodine 1.25 mg; Manganese 120 mg; Zinc 110 mg; Selenium 0.3 mg; Vitamin A 9000 IU; Vitamin D3 4000 IU; Vitamin E 55 IU; Vitamin K3 2.2 mg; Vitamin B2 5.4 mg; Vitamin B1 2.2 mg; Vitamin B6 2.2 mg; Vitamin B... 12 0.011 mg; Pantothenic acid 15 mg; Niacin 45 mg; Biotin 0.15 mg; Folic acid 1.6 mg; Choline chloride 250 mg; Antifungal agent 50 mg; Antioxidant 200 mg.

[0037] Table 3. Dietary composition and nutrient levels in the nutrient metabolism trial.

[0038] Note: Premixed feed is the same as basal ration.

[0039] Table 4. Composition and nutrient levels of nitrogen-free diets

[0040] Note: Premixed feed is the same as basal ration.

[0041] Measurement indicators and methods: The dry matter, crude protein, crude fiber, neutral detergent fiber, acid detergent fiber and other conventional nutrients in feed, feces and digest were determined according to the national standard method. The amino acid content was determined by high performance liquid chromatography. The apparent digestibility, apparent ileal amino acid digestibility (AID) and standard ileal amino acid digestibility (SID) were calculated.

[0042] Experimental results: 1. Nutrient utilization rate: In 19-21 day old broilers, the apparent digestibility of dry matter from fermented soy sauce residue was 89.15%, crude protein was 60.12%, crude fiber was 78.84%, and apparent metabolizable energy was 14.50 MJ / kg; in 40-42 day old broilers, the apparent digestibility of dry matter was 87.46%, crude protein was 50.45%, crude fiber was 73.01%, and apparent metabolizable energy was 15.66 MJ / kg.

[0043] 2. Amino acid digestibility: In 19-21 day old broilers, the SID of essential amino acids was 95.51% for methionine, 95.47% for arginine, and 92.03% for lysine; in 40-42 day old broilers, the SID of methionine was 96.53%, arginine was 96.22%, and lysine was 93.96%, with most essential amino acids having an SID ≥ 85%.

[0044] Table 5. Apparent digestibility (%) of conventional nutrients in broilers from fermented soy sauce lees

[0045] Table 6. Apparent and standard ileal amino acid digestibility (%) of fermented soy sauce lees in the early and late stages.

[0046] In this embodiment, through broiler metabolism and amino acid digestibility tests, the nutritional value of fermented soy sauce residue was fully revealed, indicating that it can be directly applied in broiler diets.

Claims

1. A solid-state fermentation process for soy sauce lees, characterized in that, Includes the following steps: S1: Desalination pretreatment: After crushing the soy sauce lees raw material, sieve it, add distilled water at a material-to-water ratio of 1:5-1:7 (m / v), soak at room temperature for 18-22 h, filter with gauze and squeeze to dehydrate, and obtain desalted wet lees; then place the wet lees in a constant temperature forced-air drying oven to dry to constant weight, crush and sieve again to obtain desalted soy sauce lees powder. S2: Mixed culture: using Bacillus subtilis, Aspergillus niger and Lactobacillus plantarum as the compound fermentation strains; Bacillus subtilis was inoculated onto LB medium and cultured at 35°C and 180 rpm for 10 h until the initial plateau phase, at which the viable cell concentration reached 1.83 × 10⁻⁶. 7 CFU / mL; *Lactobacillus plantarum* was inoculated into MRS medium and cultured at 37℃ and 200 rpm for 20 h until the initial plateau phase, at which the viable cell concentration reached 2.29 × 10⁻⁶. 7 CFU / mL; *Aspergillus niger* was inoculated into PDA medium and incubated statically at 30°C until spores covered the plate, preparing a concentration ≥ 1 × 10⁻⁶. 7 CFU / mL spore suspension; S3: Solid-state fermentation: The material with adjusted moisture content is placed into a well-ventilated fermentation bag or shallow fermentation container, ready for inoculation with microorganisms for fermentation; first, Bacillus subtilis bacterial solution is inoculated at 1% and Aspergillus niger spore suspension is inoculated at 1%; aerobic fermentation is carried out at 30℃ for 75 h; then Lactobacillus plantarum bacterial solution is inoculated at 2%; anaerobic fermentation is carried out at 37℃ for 72 h. After fermentation, the mixture is dried at 65℃, pulverized and sieved to obtain solid-state fermented soy sauce lees.

2. The application of the solid-state fermented soy sauce residue according to claim 1 in broiler chicken diets.

3. The application of solid-state fermented soy sauce residue according to claim 2 in broiler chicken diets, characterized in that, The broiler diet includes soybean meal and fermented soy sauce residue; the soybean meal is added at a rate of 15%-26% in the broiler diet, and the fermented soy sauce residue is added at a rate not exceeding 15%.

4. The application of the solid-state fermented soy sauce residue according to claim 2 in broiler chicken diets, characterized in that, The proportion of the fermented soy sauce lees to the total amount of fermented soy sauce lees and soybean meal used shall not exceed 50%.

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