Peanut cake meal fermented detoxified marine aquatic feed and preparation method thereof

Abstract

The invention belongs to the technical field of feeds, and discloses a peanut cake meal fermented detoxified marine aquatic feed and a preparation method thereof. The feed is mainly prepared from peanut meal which is subjected to synergistic fermentation treatment by compound probiotics (bacillus subtilis and saccharomyces cerevisiae) and compound enzymes (phytase and alkaline protease), and is supplemented with an antioxidant and a mildew preventive; according to the preparation method, two-stage temperature-control solid-state fermentation is adopted, pH is dynamically regulated and controlled through flora metabolism in a micro-aerobic environment, and synchronous efficient degradation of phytic acid and lectin and directional hydrolysis of protein are achieved. A biotransformation system which is complete in structure, synergetic in function and feasible in engineering is constructed, efficient removal of phytic acid and agglutinin in the peanut cakes and directional hydrolysis activation of protein are synchronously achieved under a single process path for the first time, and the technical bottleneck of application of the peanut cakes in high-end marine aquatic feed is fundamentally broken through; and a new technical normal form is provided for high-value utilization of plant protein resources.

Description

Technical Field

[0001] This invention belongs to the field of feed technology and relates to fermented and detoxified peanut meal marine aquatic feed and its preparation method. Background Technology

[0002] Peanut meal, as a byproduct of oil processing, has long been considered a potential high-quality plant-based protein feed ingredient due to its high protein content, relatively balanced amino acid composition, and wide availability. However, its practical application is significantly limited, mainly due to the presence of various endogenous anti-nutritional factors, particularly phytic acid and lectins.

[0003] Phytic acid not only chelates essential mineral ions such as calcium, iron, and zinc, reducing their bioavailability, but it can also form complexes with proteins, hindering the effective hydrolysis of nutrients by digestive enzymes. Meanwhile, lectins can interfere with nutrient absorption and induce inflammatory responses by specifically binding to glycosyl groups on the surface of intestinal epithelial cells, potentially leading to growth retardation or even poisoning and death in aquatic animals. Therefore, effectively removing or neutralizing these anti-nutritional factors is a core prerequisite for the resource utilization of peanut meal.

[0004] Existing technologies commonly employ microbial fermentation for detoxification, the basic principle of which lies in utilizing the enzyme systems or acidic environments produced during the metabolism of specific bacterial strains to degrade or transform anti-nutritional components. For example, fermentation systems using only Bacillus subtilis or yeast have been shown to reduce phytic acid content or weaken lectin activity to some extent.

[0005] However, phytic acid is a highly phosphorylated inositol derivative, requiring stepwise dephosphorylation by specific phytases; while lectins are thermostable glycoproteins, and their inactivation often depends on the disruption of their spatial conformation by proteases or the intervention of a strong reducing environment. A single microbial strain can usually only secrete a limited number of extracellular enzymes, making it difficult to simultaneously and efficiently act on two very different types of anti-nutritional factors. Summary of the Invention

[0006] To achieve the above-mentioned objectives, this invention provides a fermented and detoxified peanut meal marine aquatic feed and its preparation method. The feed achieves efficient removal of phytic acid and lectins and targeted hydrolysis of proteins simultaneously in a single fermentation process through the synergistic effect of compound probiotics and compound enzymes, thereby significantly improving the safety, palatability, and nutritional bioavailability of peanut meal as a marine aquatic feed ingredient.

[0007] The peanut meal fermentation and detoxification marine aquatic feed of this invention comprises, by weight percentage: 90%-98% peanut meal treated with synergistic fermentation of compound probiotics and compound enzymes, 0.1%-0.5% antioxidant, 0.1%-0.3% mold inhibitor, and the remainder is moisture, with the moisture content controlled at 8%-12%. In the synergistically fermented peanut meal, the phytic acid residue is less than 0.3%, the lectin activity is less than 5 HAU / g, the water-soluble protein content is greater than 15%, and small peptides (molecular weight less than 5000 Da) account for more than 40% of the total protein.

[0008] The compound probiotics consist of Bacillus subtilis and Saccharomyces cerevisiae, with a total live bacteria count greater than 1×10⁻⁶. 8 The enzyme contains CFU / g of Bacillus subtilis and Saccharomyces cerevisiae, with a live cell ratio of 3:1 to 5:1. The Bacillus subtilis is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2023123; the Saccharomyces cerevisiae is deposited at the same center, accession number CCTCC NO: M2023124. The complex enzyme consists of phytase and alkaline protease, with a total enzyme activity greater than 5000 U / g, of which phytase activity is greater than 3000 U / g and alkaline protease activity is greater than 2000 U / g. The phytase is 3-phytase derived from Aspergillus niger, with an optimal pH of 5.5, an optimal reaction temperature of 55℃, and a specific activity of 8000 U / mg; the alkaline protease is a serine protease derived from Bacillus licheniformis, with an optimal pH of 9.0, an optimal reaction temperature of 60℃, and a specific activity of 12000 U / mg.

[0009] The method for preparing fermented and detoxified peanut meal marine aquatic feed according to the present invention includes the following steps: Step 1, Raw material pretreatment: Peanut meal is crushed to a particle size of 0.5-1.5 mm and passed through a 40-mesh sieve to obtain pretreated peanut meal powder. The initial phytic acid content of the peanut meal is 1.8%-2.5%, the lectin activity is 80-120 HAU / g, and the crude protein content is 45%-50%.

[0010] Step 2, Preparation of the bacterial-enzyme mixture: After separately culturing Bacillus subtilis and Saccharomyces cerevisiae in liquid seed culture, they are mixed at a live bacteria ratio of 4:1 to obtain a compound probiotic suspension with a live bacteria concentration of 2×10⁻⁶. 9 CFU / mL; Phytase and alkaline protease were dissolved in phosphate buffer at pH 6.0 at a mass ratio of 3:2 to obtain a complex enzyme solution with a total enzyme activity of 10000 U / mL. The complex probiotic suspension and the complex enzyme solution were mixed at a volume ratio of 1:1 to form a bacterial-enzyme mixture.

[0011] Step 3, Solid-state fermentation: Place the pretreated peanut meal powder in a stainless steel fermentation tank, add the bacterial enzyme mixture at a material-to-liquid ratio of 1:0.4, and mix thoroughly to achieve a material moisture content of 55%-60%. Seal the fermentation tank and circulate sterile air to maintain a micro-aerobic environment inside the tank, controlling the oxygen concentration at 2%-5%. The fermentation process is divided into two stages: Stage 1 is constant temperature fermentation at 30℃ for 12 hours. In this stage, Bacillus subtilis rapidly proliferates and secretes alkaline protease, simultaneously initiating the initial hydrolysis of proteins; Stage 2 involves raising the temperature to 37℃ and continuing fermentation for 36 hours. In this stage, the Saccharomyces cerevisiae metabolizes and produces acid, lowering the system pH to 4.8-5.2, activating phytase activity. At the same time, Bacillus subtilis continuously secretes protease, achieving deep degradation of phytic acid and destruction of lectin conformation.

[0012] Step 4, Termination of Fermentation and Drying: After fermentation, immediately introduce saturated steam at 121℃ into the material for 15 minutes to completely inactivate the enzymes and kill any unwanted bacteria, thus terminating the fermentation reaction. The material is then transferred to a belt dryer and dried at 60℃ until the moisture content is 10%, yielding fermented peanut meal powder.

[0013] Step 5, finished product preparation: Add butylated hydroxyanisole as an antioxidant to the fermented peanut meal dry powder at a dosage of 0.3%; add calcium propionate as a mold inhibitor at a dosage of 0.2%; after mixing evenly, crush and pass through a 60-mesh sieve to obtain the finished product of fermented and detoxified marine aquatic feed.

[0014] In a preferred embodiment of the present invention, the fermenter in step 3 is equipped with an online pH and temperature monitoring system. A pH probe and temperature sensor are directly inserted into the center of the material, and data is fed back to the central controller in real time. When the pH deviates from the set range of ±0.3 or the temperature fluctuates by more than ±1℃, the system automatically adjusts the aeration rate or the jacket cooling water flow rate to maintain process stability. The inner wall of the fermenter is equipped with a spiral agitator rotating at 10 rpm, running in both forward and reverse directions for 5 minutes every 2 hours to ensure uniform mass transfer of the material and avoid localized anaerobic conditions or overheating.

[0015] In another preferred embodiment of the present invention, before the compound probiotic suspension is prepared, Bacillus subtilis is cultured at 37°C and 200 rpm for 18 hours with shaking, and Saccharomyces cerevisiae is cultured at 30°C and 180 rpm for 24 hours with shaking, both reaching the late logarithmic growth stage; the compound enzyme solution is refrigerated at 4°C before use and restored to room temperature before use to avoid enzyme activity loss.

[0016] The technical solution of this invention is based on a systematic reconstruction of the essential differences in chemical properties and degradation mechanisms between two major anti-nutritional factors in peanut meal—phytic acid and lectins. Phytic acid is inositol hexaphosphate, and its degradation depends on phytase gradually hydrolyzing phosphate ester bonds from specific positions, a process that is most efficient under weakly acidic conditions. Lectins, on the other hand, are glycoproteins with a β-sheet structure, and their biological activity depends on their intact tertiary conformation. They can be inactivated by protease hydrolysis of their peptide chains or by the disruption of disulfide bonds in a reducing environment. A single microbial system cannot simultaneously provide a suitable pH environment and a matching enzyme combination. This invention constructs a Bacillus subtilis-Saccharomyces cerevisiae dual-strain system, utilizing the ability of Bacillus subtilis to efficiently secrete alkaline proteases under neutral to weakly alkaline conditions, combined with the characteristic of Saccharomyces cerevisiae to produce organic acids (mainly acetic acid and succinic acid) during its growth process, which lowers the environmental pH, thus forming a dynamically evolving microenvironment. In the early stages of fermentation, the system pH is maintained at around 6.5, which is conducive to the proliferation of Bacillus subtilis and the release of proteases, initiating the hydrolysis of lectins and large protein molecules. As yeast metabolizes and produces acid, the pH gradually decreases to around 5.0, which is exactly within the optimal range for phytase activity, thus efficiently catalyzing the dephosphorylation of phytic acid. This dynamic pH regulation mechanism does not require the addition of exogenous acid-base regulators, achieving a green and self-consistent process.

[0017] The exogenous addition of compound enzymes compensates for the insufficient enzyme production capacity of microorganisms themselves. Although Bacillus subtilis can secrete a certain amount of phytase, its activity is far lower than that of dedicated phytase preparations; Saccharomyces cerevisiae produces almost no phytase. By precisely proportioning high-activity phytase and alkaline protease, sufficient enzyme concentration is ensured during the critical reaction window, enabling the detoxification reaction to be completed within 48 hours, shortening the time by more than 30% compared to single-strain fermentation. In addition, the targeted hydrolysis of proteases not only destroys the lectin structure but also degrades large molecular weight antigens such as peanut globulin into small peptides with a molecular weight of less than 5000 Da. These small peptides can be directly absorbed by the intestines of aquatic animals, significantly improving protein digestibility, stimulating intestinal development, and enhancing the immune response.

[0018] In the preparation method described in this invention, the two-stage temperature control strategy is key to achieving synergistic detoxification and quality improvement. The 30℃ stage ensures rapid colonization of Bacillus subtilis and early protease accumulation; the 37℃ stage balances yeast metabolic activity and phytase catalytic efficiency. A microaerobic environment (2%-5% O2) inhibits the excessive growth of anaerobic bacteria such as lactic acid bacteria, prevents a sudden drop in pH that could lead to protease inactivation, and maintains the aerobic metabolic advantage of Bacillus subtilis. The steam sterilization step terminates the reaction while simultaneously performing pasteurization, ensuring that the product's microbiological indicators meet the feed hygiene standards (GB13078-2017), with a total bacterial count below 1×10⁻⁶. 4 CFU / g, Escherichia coli and Salmonella were not detected.

[0019] The peanut meal fermentation and detoxification marine aquatic feed of this invention is suitable for marine economic aquatic animals such as flounder, grouper, large yellow croaker, and shrimp. It can replace 15%-25% of fishmeal usage, reducing feed costs by 10%-15% while ensuring growth performance. Its preparation method has clear process parameters, conventional equipment requirements, and a controllable process, making it suitable for industrial-scale scaling up of feed production lines with a capacity of tens of thousands of tons. It solves the core defects of existing technologies, such as non-synergistic detoxification and nutrient conversion, uncontrollable processes, and unstable product quality.

[0020] Compared with the prior art, the beneficial effects of the present invention are: This invention constructs a structurally complete, functionally synergistic, and engineering-feasible biotransformation system through the rational design of compound probiotics and compound enzymes, the self-regulation mechanism of dynamic microenvironment, and the integration of a two-stage precision fermentation process. For the first time, it simultaneously achieves the efficient removal of phytic acid and lectins from peanut meal and the targeted hydrolysis and activation of proteins under a single process pathway. This fundamentally breaks through the technical bottleneck of peanut meal application in high-end marine aquaculture feed and provides a new technical paradigm for the high-value utilization of plant protein resources. Detailed Implementation

[0021] This invention provides a fermented and detoxified peanut meal feed for marine aquaculture and its preparation method. This technical solution utilizes the synergistic effect of compound probiotics and compound enzymes to simultaneously achieve efficient removal of phytic acid and lectins, as well as targeted hydrolysis of proteins, in a single solid-state fermentation process. This significantly improves the safety, palatability, and nutritional bioavailability of peanut meal as a marine aquaculture feed ingredient.

[0022] The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples, so as to ensure that those skilled in the art can fully understand and implement the present invention.

[0023] Example 1: Bacillus subtilis: Saccharomyces cerevisiae = 4:1; phytase: alkaline protease = 3:2; two-stage fermentation: 30℃ for 12 hours + 37℃ for 36 hours; oxygen concentration: 3%; material-to-liquid ratio: 1:0.4; moisture content: 58%; Preparation process: Peanut cake meal crushing → preparation of bacterial enzyme mixture → solid-state fermentation → steam-terminated fermentation → drying → addition of antioxidants and antifungal agents → crushing and sieving → finished product.

[0024] Example 2: Bacillus subtilis: Saccharomyces cerevisiae = 3:1, the rest of the formula and process are the same as in Example 1; Preparation process: Same as in Example 1.

[0025] Example 3: Bacillus subtilis: Saccharomyces cerevisiae = 5:1, the rest of the formula and process are the same as in Example 1; Preparation process: Same as in Example 1.

[0026] Example 4: Phytase activity 2500 U / g, alkaline protease activity 2000 U / g (total enzyme activity 4500 U / g), the rest of the formula and process are the same as in Example 1; Preparation process: Same as in Example 1.

[0027] Example 5: Phytase activity 3500 U / g, alkaline protease activity 2500 U / g (total enzyme activity 6000 U / g), the rest of the formula and process are the same as in Example 1; Preparation process: Same as in Example 1.

[0028] Example 6: Fermentation oxygen concentration 2%, other formulas and processes are the same as in Example 1; Preparation process: Same as in Example 1.

[0029] Example 7: Fermentation oxygen concentration 5%, other formulas and processes are the same as in Example 1; Preparation process: Same as in Example 1.

[0030] Example 8: Fermentation moisture content 55%, the rest of the formula and process are the same as in Example 1; Preparation process: Same as in Example 1.

[0031] Comparative Example 1: Fermentation with Bacillus subtilis only; no compound enzymes added; the rest of the formulation and process are the same as in Example 1; Preparation process: Peanut cake powder crushing → probiotic suspension preparation → solid-state fermentation → steam termination → drying → addition of auxiliary agents → finished product.

[0032] Comparative Example 2: No compound probiotics or compound enzymes; natural fermentation; the rest of the formula and process are the same as in Example 1; Preparation process: Peanut cake meal is crushed → water is added and mixed well → natural fermentation → drying → additives are added → finished product.

[0033] Test method: Detoxification efficiency test: phytic acid residue was determined by high performance liquid chromatography; lectin activity was determined by hemagglutination assay; and the removal rate of anti-nutritional factors was detected.

[0034] Nutritional performance testing: The Kjeldahl method was used to determine the content of water-soluble protein; gel filtration chromatography was used to determine the proportion of small peptides; and protein conversion efficiency was tested.

[0035] Safety and aquaculture testing: Total colony count determination using colony counting method; detection of Escherichia coli and Salmonella; evaluation of weight gain and health indicators in marine aquaculture trials.

[0036] The test data comparisons are shown in Table 1 and Table 2.

[0037] Table 1. Comparison of Phytic Acid Residue, Lectin Activity, and Water-Soluble Protein Table 2 Comparison of Small Peptide Proportion, Total Colony Count, and Shrimp Weight Gain Rate Examples 1-8 showed phytic acid residue ≤0.30% and small peptide ratio ≥38%, which were far superior to the comparative examples. Comparative example 1 lacked enzyme synergy due to the single probiotic, and comparative example 2 had extremely low natural fermentation detoxification and conversion efficiency, confirming that the core process is the key to efficient detoxification and quality improvement.

[0038] The increased proportion of probiotics (Examples 2→1→3) resulted in simultaneous improvement in detoxification efficiency and protein conversion; enhanced enzyme activity (Examples 4→1→5) further reduced phytic acid residue; and the synergistic effect of bacteria and enzymes was ensured within the oxygen concentration range of 2%-5%.

[0039] The product tested in this example showed no harmful bacteria and met feed safety standards; it significantly improved the weight gain rate of aquatic animals and was suitable for various marine aquatic products such as flounder and shrimp; it could replace 15%-25% of fishmeal, reducing feed costs.

[0040] Compared to single probiotic fermentation (Comparative Example 1), the phytic acid removal rate of the example was increased by 71%, and the proportion of small peptides was increased by 91%; compared to traditional natural fermentation (Comparative Example 2), the detoxification efficiency was increased by 83%, and the weight gain rate of aquaculture was increased by 484%, solving the industry problem of difficult removal of anti-nutritional factors and low protein utilization in peanut meal.

[0041] The process described in this invention achieves efficient detoxification and nutrient activation through synergistic bacterial and enzyme fermentation and two-stage fermentation, with different parameter combinations, making it suitable for large-scale production of marine aquatic feed.

[0042] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fermented and detoxified peanut meal marine aquatic feed, characterized in that, It contains the following components by weight percentage: Peanut meal treated with synergistic fermentation of compound probiotics and compound enzymes has a 90%-98% purity. Antioxidant 0.1%-0.5%; Antifungal agent 0.1%-0.3%; The rest is water.

2. The peanut meal fermentation and detoxification marine aquatic feed according to claim 1, characterized in that, The peanut meal contains less than 0.3% phytic acid residue, less than 5 HAU / g lectin activity, more than 15% water-soluble protein, and more than 40% small peptides in total protein.

3. The peanut meal fermentation and detoxification marine aquatic feed according to claim 1, characterized in that, The compound probiotics consist of Bacillus subtilis (CCTCC NO: M2023123) and Saccharomyces cerevisiae (CCTCC NO: M2023124), with a total viable count greater than 1×10⁻⁶. 8 CFU / g, with a live count ratio of Bacillus subtilis to Saccharomyces cerevisiae of 3:1-5:

1.

4. The peanut meal fermentation and detoxification marine aquatic feed according to claim 1, characterized in that, The complex enzyme is composed of 3-phytase derived from Aspergillus niger and alkaline protease derived from Bacillus licheniformis, with a total enzyme activity greater than 5000 U / g, of which phytase activity is greater than 3000 U / g and alkaline protease activity is greater than 2000 U / g.

5. The peanut meal fermentation and detoxification marine aquatic feed according to claim 3, characterized in that, The ratio of live Bacillus subtilis to Saccharomyces cerevisiae is 4:

1.

6. The peanut meal fermentation and detoxification marine aquatic feed according to claim 4, characterized in that, The optimal pH of the 3-phytase is 5.5, the optimal reaction temperature is 55℃, and the specific activity is 8000 U / mg; the optimal pH of the alkaline protease is 9.0, the optimal reaction temperature is 60℃, and the specific activity is 12000 U / mg.

7. A method for preparing fermented and detoxified marine aquatic feed from peanut meal as described in any one of claims 1-6, characterized in that, Includes the following steps: S10, Raw material pretreatment: Peanut cake meal is crushed and sieved to obtain pretreated peanut cake meal powder; S20, Preparation of bacterial-enzyme mixture: Bacillus subtilis and Saccharomyces cerevisiae are mixed at a live bacteria ratio of 4:1 to obtain a compound probiotic suspension with a live bacteria concentration of 2×10⁻⁶. 9 CFU / mL; 3-phytase and alkaline protease were dissolved in phosphate buffer at pH 6.0 at a mass ratio of 3:2 to obtain a complex enzyme solution with a total enzyme activity of 10000 U / mL; the complex probiotic suspension and the complex enzyme solution were mixed at a volume ratio of 1:1 to form a bacterial enzyme mixture; S30, solid-state fermentation: Place the pretreated peanut cake powder in a stainless steel fermentation tank, add the bacterial enzyme mixture at a material-to-liquid ratio of 1:0.4, mix well to make the moisture content 55%-60%; after sealing, introduce sterile air to maintain the oxygen concentration in the tank at 2%-5%; first ferment at a constant temperature of 30℃ for 12 hours, then raise the temperature to 37℃ and continue fermentation for 36 hours. S40, Fermentation Termination and Drying: After fermentation, saturated steam is introduced for treatment, followed by drying until the moisture content is 10%; S50, finished product preparation: Add butylated hydroxyanisole as an antioxidant to the dried material at a dosage of 0.3%; add calcium propionate as a mildew inhibitor at a dosage of 0.2%; mix evenly, then pulverize and sieve to obtain the finished product.

8. The preparation method according to claim 7, characterized in that, The stainless steel fermenter in step S30 is equipped with an online pH and temperature monitoring system. The pH probe and temperature sensor are inserted into the center of the material, and the data is fed back to the central controller in real time. When the pH deviates from the set value of ±0.3 or the temperature fluctuates by more than ±1℃, the ventilation rate or the jacket cooling water flow rate is automatically adjusted.

9. The preparation method according to claim 8, characterized in that, The stainless steel fermentation tank is equipped with a spiral stirring paddle on its inner wall, which rotates at 10 rpm and runs in both directions for 5 minutes every 2 hours.

10. The preparation method according to claim 7, characterized in that, The Bacillus subtilis was cultured at 37°C and 200 rpm for 18 hours before preparation, and the Saccharomyces cerevisiae was cultured at 30°C and 180 rpm for 24 hours, both of which were in the late logarithmic growth stage.

Images