Method for improving quality of rapeseed meal by medium-high temperature staged solid state fermentation

Abstract

This invention discloses a method for improving the quality of rapeseed meal through segmented solid-state fermentation at medium and high temperatures, belonging to the field of bio-fermentation and animal feed technology. The method uses unsterilized rapeseed meal directly as raw material. In the first stage, *Bacillus subtilis* is inoculated under medium-temperature (40℃) conditions for aerobic solid-state fermentation, utilizing its high protease production characteristics to deeply hydrolyze large protein molecules, generating easily digestible and absorbable peptides. In the second stage, *Bacillus stearothermophilus* is inoculated under high-temperature (55℃) conditions to continue aerobic fermentation. The high-temperature environment inhibits the growth of secondary metabolic flora, making the fermentation process more controllable, increasing the content of total phenols and flavonoids, and simultaneously degrading anti-nutritional factors. The technology disclosed in this invention is simple to operate and easy to scale up, which can broaden the application of rapeseed meal in the field of high-quality protein feed.

Description

Technical Field

[0001] This invention belongs to the fields of biological fermentation and animal feed, specifically relating to a method for improving the quality of rapeseed meal through medium- and high-temperature segmented solid-state fermentation. Background Technology

[0002] Rapeseed meal is the solid residue obtained after oil pressing and extraction from rapeseed. Its nutrient content varies depending on the rapeseed variety, oil extraction process, and storage conditions. As an important source of plant protein, rapeseed meal contains 35%–45% crude protein and has a relatively complete amino acid composition, making it an ideal substitute for soybean feed. However, due to the oil extraction process, rapeseed meal often suffers from poor protein solubility, resulting in inefficient nutrient utilization. Furthermore, rapeseed meal contains anti-nutritional factors such as glucosinolates, which not only reduce its nutritional value and affect palatability but can also cause damage to organs if consumed excessively, raising concerns about its safety. Therefore, it is necessary to pre-treat rapeseed meal to reduce anti-nutritional factors and improve its feed value.

[0003] To improve the feed value of rapeseed meal, it is necessary to detoxify it and enhance its nutritional value. Various technologies exist for processing rapeseed meal, including high-temperature puffing, acid-base leaching, microbial fermentation, and enzymatic hydrolysis. However, most methods struggle to balance economic efficiency and processing efficiency, failing to simultaneously reduce anti-nutritional factors and improve nutritional value. Numerous studies have shown that solid-state fermentation, as a mainstream technology in actual production, can utilize different microorganisms to produce enzymes during rapeseed meal fermentation, achieving varying treatment effects. Patent CN119144508A discloses a Bacillus subtilis strain and its application in rapeseed meal fermentation. This patent achieves the goal of effectively degrading anti-nutritional factors in rapeseed meal and improving its nutritional properties through strain screening. Patent CN119931873A discloses a method for preparing rapeseed meal peptides through mixed-culture fermentation of rapeseed meal. This patent increases the content of soluble peptides and reduces the content of anti-nutritional factors in the fermentation products by using a mixed-culture combination fermentation of rapeseed meal. However, most fermentation studies are based on fermentation after sterilizing the raw materials. While this effectively inhibits the growth of unwanted microorganisms, the high temperature and pressure can cause secondary denaturation of proteins, resulting in the loss of some nutrients. Furthermore, large-scale material processing in production requires significant energy consumption. Therefore, direct fermentation of unsterilized rapeseed meal can better preserve heat-sensitive nutrients in the protein while reducing energy costs. Multi-strain mixed-culture fermentation and high-temperature fermentation can effectively inhibit the growth of unwanted microorganisms and increase peptide yield without sterilizing the raw materials. However, current mixed-culture fermentation is mostly mesophilic, which easily leads to the formation of secondary metabolic flora in the later stages, and different strains may exhibit antagonistic effects during co-fermentation. While high-temperature fermentation can effectively inhibit unwanted microorganisms and make the process more controllable, the peptide conversion rate is relatively low. Based on this, this invention fully utilizes the characteristics of different fermentation methods, selecting high-protease-producing strains and thermophilic strains for sequential, staged fermentation of rapeseed meal at medium and high temperatures to increase peptide content and make the fermentation process more controllable. The first stage utilizes mesophilic fermentation to efficiently hydrolyze proteins, producing peptides and reducing glucosinolate content. Then, the second stage employs high-temperature fermentation to inhibit the growth of secondary metabolic flora, making the fermentation process more controllable and further increasing peptide yield while reducing glucosinolate content. A search revealed no reports on methods or applications of mesophilic-high-temperature segmented solid-state fermentation for improving rapeseed meal quality. Summary of the Invention

[0004] This invention addresses the problems associated with the solid-state fermentation of unsterilized rapeseed meal mentioned above, providing a method for improving rapeseed meal quality through medium- and high-temperature segmented solid-state fermentation. By combining the different process characteristics of medium- and high-temperature solid-state fermentation, it significantly increases the peptide content of rapeseed meal while reducing glucosinolate content, making the fermentation process more controllable and generating fewer secondary metabolic microorganisms. This technology can serve as a method for obtaining high-quality fermented rapeseed meal feed, with the aim of expanding the application of rapeseed meal in the field of microbial fermented feed.

[0005] This invention discloses a method for improving the quality of rapeseed meal through medium- and high-temperature segmented solid-state fermentation, which is carried out according to the following steps:

[0006] (1) Pretreatment of raw materials: rapeseed meal is crushed, passed through a 60-mesh sieve, and degreased with low-temperature subcritical propane to obtain rapeseed meal raw material;

[0007] (2) Preparation of seed culture: Bacillus subtilis and Geobacillus stearothermophilus were inoculated from cryovials onto LB solid medium and activated at 37 ℃ and 55 ℃, respectively. Single colonies were picked and inoculated onto LB liquid medium and amplified by shaking at 37 ℃ and 55 ℃ for 16 h. After the culture was completed, the number of bacteria was adjusted to 4×10⁻⁶. 8 CFU / mL was used to prepare a seed culture.

[0008] (3) Solid-state fermentation of rapeseed meal: medium-temperature fermentation, the Bacillus subtilis seed liquid prepared in step (2) is inoculated into the defatted rapeseed meal in step (1) at a mass ratio of 15%, and water at a mass ratio of 50% of the total rapeseed meal is added. After thorough mixing, the mixture is transported to a rotary fermenter and fermented at 40 ℃ for 24-72 h. When entering the high-temperature stage, the fermentation temperature is raised to 55 ℃, and Bacillus limonene seed liquid at a mass ratio of 25% of the total rapeseed meal is inoculated. The mixture is fermented at high temperature for 24 h, and the oxygen volume concentration in the fermentation chamber is maintained at no less than 15% during the fermentation process.

[0009] (4) Drying treatment: The rapeseed meal that has undergone two stages of solid-state fermentation in (3) is directly dried in the fermentation chamber, and the water content of the material is controlled to be less than 10%. The dried material is crushed and sealed.

[0010] Compared with the prior art, the beneficial effects of the present invention are:

[0011] (1) This invention is the first to utilize medium-high temperature sequential segmented solid-state fermentation of rapeseed meal, providing a new process for fermenting unsterilized rapeseed meal, making the fermentation process more controllable and the fermentation more thorough, and can broaden the application scope of solid-state fermentation of rapeseed meal.

[0012] (2) This invention achieves deep fermentation of rapeseed meal through medium- and high-temperature staged fermentation, thereby improving its nutritional characteristics. This is mainly reflected in increased nitrogen solubility index, polypeptide and crude protein content, total phenol and total flavonoid content, and enhanced antioxidant capacity. Simultaneously, it leads to more thorough degradation of glucosinolates and phytic acid. Experiments have shown that the best results in improving the nutritional characteristics of rapeseed meal were achieved in the first stage with a 15% inoculum of Bacillus subtilis at 40°C for 72 h, and in the second stage with a 25% inoculum of Bacillus steatophilus at 55°C for 24 h. Compared with uninoculated rapeseed meal, the nitrogen solubility index increased by 419.92%, the polypeptide and crude protein contents increased by 377.75% and 8.17% respectively, the phytic acid and glucosinolate contents decreased by 20.13% and 84.14% respectively; the total phenol and total flavonoid contents increased by 108.04% and 19.28% respectively; and the antioxidant capacity increased by 30.84%, which is significantly better than the results of mesophilic fermentation and uninoculated meal. Detailed Implementation

[0013] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below.

[0014] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0015] The nitrogen solubility index is measured by including crude protein content and water-soluble protein content. The crude protein content is determined according to GB / T5009.5-2016. The water-soluble protein content is measured by the Kjeldahl method after extraction. The nitrogen solubility index is calculated according to the following formula.

[0016]

[0017] The peptide content was determined according to the method of Rawdkuen et al. (Rawdkuen, et al., Biochemical and gelling properties of tilapia surimi and protein recovered using an acid-alkaline process. Food Chemistry, 2008, 112(1), 112–119), using the Folin-Ciocalteu method, with absorbance measured at 760 nm. The total phenol and total flavonoid content were determined according to the method of Yin et al. (Yin et al., Varietal differences in nutritional profiles and functionality of radish sprouts: Food application potential. Food Research International, 2026, 224: 117970–117970). Total polyphenols were measured using the Folin-Ciocalteu method, with gallic acid as the standard, and absorbance measured at 760 nm. Total flavonoids were measured using the aluminum nitrate colorimetric method, with rutin as the standard, and absorbance measured at 500 nm.

[0018] The glucosinolate content was measured according to the method of Shi et al. (Shi et al. Physicochemical Properties Analysis and Secretome of Aspergillus niger in Fermented Rapeseed Meal. PLoSOne, 2016, 11(4): 16). The phytic acid content was determined according to the method of Dai et al. (Dai et al. Dual-frequency ultrasound, an innovative technique for enhancing oats germination to improve nutritional and functional properties [J]. Journal of Cereal Science, 2025, 126: 104275–104275). The antioxidant activity was determined according to the method of Shao et al. (Shao F, Zhang Y, Wan X. et al., Improving the properties of whey protein isolate-zeinnanogels with novel acidifiers: Re-dispersity, stability and quercetinbioavailability. International Journal of Biological Macromolecules, 2024, 266: 137567). The DPPH working solution was mixed with the sample solution and incubated in the dark for 30 min. The absorbance was measured at 517 nm, and the DPPH scavenging capacity was calculated according to the following formula.

[0019] Where A1 is the absorbance after the sample-working solution reaction, A2 is the absorbance of the sample control, and A0 is the absorbance of the blank control.

[0020] Bacterial community diversity was determined using the method of Hou et al. (Hou et al. Thermophilic solid-state fermentation of rapeseed meal and analysis of microbial community diversity. LWT, 2019, 116: 108520–108520), with species abundance analysis at the genus level.

[0021] The Bacillus subtilis and Geobacillus stearothermophilus used in this invention were purchased from the China Industrial Microbial Culture Collection Center, with culture numbers CICC 21927 and CICC 10782, respectively.

[0022] Example 1

[0023] Bacillus subtilis and Geobacillus stearothermophilus were inoculated from cryovials onto LB solid medium and activated at 37 ℃ and 55 ℃, respectively. Single colonies were then picked and inoculated onto LB liquid medium and cultured with shaking at 37 ℃ and 55 ℃ for 16 h. After the culture, the bacterial count was adjusted to 4 × 10⁻⁶. 8 CFU / mL was used to prepare a seed culture. 50% water (by weight) was added to the defatted rapeseed meal after pretreatment, and the mixture was stirred thoroughly. Then, 15% (by weight) of Bacillus subtilis seed culture was added, stirred thoroughly, and transferred to a rotary fermenter. The fermentation temperature was set at 40 ℃ and the relative humidity at 50%, and solid-state fermentation was carried out at a medium temperature for 24 hours. The temperature of the fermentation chamber was then increased to 55 ℃, and 25% (by weight) of Bacillus steatophilus seed culture was inoculated with the rapeseed meal, stirred thoroughly, and the relative humidity was controlled at 50%. High-temperature solid-state fermentation was carried out for 24 hours, maintaining an oxygen volume concentration of no less than 15% throughout the fermentation process. After fermentation, the material was dried directly in the fermentation chamber, controlling the moisture content to be below 10%. The dried material was then pulverized and sealed. Rapeseed meal fermented at medium to high temperatures for 48 hours was obtained.

[0024] Example 2

[0025] The steps are the same as in Example 1, except that the solid-state fermentation time at medium temperature is 48 hours. Rapeseed meal fermented at medium to high temperature for 72 hours is obtained.

[0026] Example 3

[0027] The steps are the same as in Example 1, except that the solid-state fermentation time at medium temperature is 72 hours. Rapeseed meal fermented at medium to high temperature for 96 hours is obtained.

[0028] Comparative Example 1

[0029] The steps are the same as in Example 1, except that the rapeseed meal was not inoculated with Bacillus subtilis and Bacillus thermophilus seed liquid.

[0030] Comparative Example 2

[0031] The steps are the same as in Example 1, except that only mesophilic solid-state fermentation is performed for 72 hours. Rapeseed meal fermented at a mesophilic temperature for 72 hours is obtained.

[0032] Table 1 compares the nutrient content of fermented rapeseed meal in each example and comparative example. Nitrogen solubility index (NSOI) and polypeptide content are important indicators of whether protein quality has been fundamentally improved; high NSOIs indicate deep protein hydrolysis and easy digestion and absorption. Therefore, increasing NSOI and polypeptide content can effectively improve the feed value of rapeseed meal. As shown in Table 1, this invention achieves efficient conversion of protein into functional polypeptides. From Comparative Example 1 to Example 3, the polypeptide content in the fermentation product increased from 3.73 g / 100 g in Comparative Example 1 to 17.82 g / 100 g in Example 3, an increase of 377.75%. The NSOI increased from 15.46% in Comparative Example 1 to 80.38% in Example 3, with the NSOI from Example 1 to Example 3 increasing by 97.15% to 419.92%. This indicates that through a segmented fermentation process, utilizing the combined action of inoculated high-protease-producing bacteria and thermophilic bacteria, the nutritional value of rapeseed meal can be significantly improved, protein digestibility and utilization can be enhanced, and a large amount of small molecules such as polypeptides can be generated. The purpose of Comparative Example 2 was to compare the effects of mesophilic fermentation and mesophilic-high-temperature fermentation of rapeseed meal with Example 3. Compared with Comparative Example 2, the nitrogen solubility index of Example 3 increased by 6.58%, and the peptide content increased by 15.41%, indicating that high-temperature fermentation can further enhance nutritional value, make protein hydrolysis more thorough, and achieve deep fermentation. In addition, fermentation can also significantly increase the crude protein content of rapeseed meal. The crude protein content of Examples 1 to 3 increased by 3.88% to 8.17%, indicating that a large amount of microbial protein was produced during the fermentation process.

[0033] Table 1 Comparison of nutrient content in fermented rapeseed meal of the examples and comparative examples.

[0034]

[0035] Note: Different letters in the same column represent significant differences (p < 0.05).

[0036] Table 2 compares the content of anti-nutritional factors in fermented rapeseed meal from each example and comparative example. Glucosinolates and phytic acid are known to be the main anti-nutritional factors in rapeseed meal, especially glucosinolates, which are the primary detoxification target. Table 2 shows that the glucosinolate content was significantly degraded after fermentation, decreasing from 17.50 µmol / g in Example 1 to 3.33 µmol / g in Example 3, a decrease of 16.67%–84.14% compared to 21.00 µmol / g in Comparative Example 1. Furthermore, compared to Comparative Example 2 (a reduction of 57.33%), Example 3 (84.14%) further degraded glucosinolates by 62.83%, indicating that the staged fermentation, through the combined effects of microbial enzymes and high temperature, resulted in more thorough glucosinolate degradation. In addition, the phytic acid content was also reduced to a certain extent, decreasing by 7.26%–20.13% from Example 1 to Example 3.

[0037] Table 2 Comparison of antinutritional factor content in fermented rapeseed meal of the examples and comparative examples.

[0038]

[0039] Note: Different letters in the same column represent significant differences (p < 0.05).

[0040] Table 3 compares the active substances and free radical scavenging abilities in the fermented rapeseed meal of the Examples and Comparative Examples. Total polyphenols are known to be a class of phenolic compounds with antioxidant and anti-inflammatory activities, while total flavonoids are unique polyphenolic substances in rapeseed meal. Both exhibit antioxidant properties through metal chelation and free radical scavenging. As shown in Table 3, after the staged fermentation of this invention, the total polyphenol content shows a trend of increasing with the depth of fermentation. The total polyphenol content increased from 13.26 μg GAE / mg in Comparative Example 1 to 27.59 μg GAE / mg in Example 3, an increase of 108.1%. This indicates that the cellulase secreted by the microorganisms effectively destroyed the cell wall structure of the rapeseed meal, causing a large amount of bound polyphenols to be released into a free state. The total flavonoid content steadily increased from 12.19 mg / g in Comparative Example 1 to 14.68 mg / g in Example 3, an increase of 20.43%. The stability of flavonoids from Comparative Example 2 to Example 3 was good, indicating that high-temperature fermentation did not cause the loss of flavonoid active substances. The enhancement of antioxidant activity is closely related to active substances such as peptides and polyphenols, and the antioxidant activity of fermentation products can be evaluated by DPPH· scavenging rate. The antioxidant activity of Comparative Example 1 was 48.28%, which was significantly increased to 63.17% after the staged fermentation in Example 3. The free radical scavenging ability of Examples 1 to 3 increased by 11.35% to 30.84%.

[0041] Table 3 Comparison of active substances and free radical scavenging ability in fermented rapeseed meal of the examples and comparative examples.

[0042] Note: Different letters in the same column represent significant differences (p < 0.05).

[0043] Table 4 compares the changes in colony structure at the genus level in fermented rapeseed meal of the examples and comparative examples. The succession of colony structure reveals which bacterial groups dominate and play a role during fermentation. Table 4 shows that at the genus level, Comparative Example 1 exhibits greater diversity of bacterial species. Due to the absence of Bacillus subtilis and thermophilic bacteria inoculation, the bacterial types are mainly those naturally present in the raw materials, resulting in a relatively dispersed distribution. In Comparative Example 2, while Bacillus subtilis dominates and significantly inhibits the growth of other miscellaneous bacteria, the relative abundance of secondary metabolic bacteria such as Enterococcus, Marine Bacillus, and Weissella increases. In Example 3, however, their content significantly decreases. Enterococcus content drops from 23.23% to 13.73%, Marine Bacillus is virtually undetectable, while the relative abundance of Bacillus subtilis increases to 30%. This indicates that with the introduction of high-temperature environment and thermophilic bacteria, secondary metabolic bacteria are significantly suppressed, and the overall microbial community is dominated by Bacillus subtilis and thermophilic bacteria, resulting in a more stable microbial structure.

[0044] Table 4. Comparison of the relative abundance of bacterial colony structures at the genus level in fermented rapeseed meal of the examples and comparative examples.

[0045] In summary, the medium- and high-temperature segmented solid-state fermentation method provided by this invention drives the deep hydrolysis of large molecular proteins in rapeseed meal, improving the nitrogen solubility index, peptide content, and overall nutritional composition. It also makes the microbial community during fermentation more controllable and significantly reduces the content of anti-nutritional factors, thereby increasing the feed value of rapeseed meal. Furthermore, solid-state fermentation enhances bioactive substances and antioxidant function, showing promising application prospects in high-value-added fields such as functional feed ingredients.

[0046] The invention has been described through embodiments that are currently considered to be the most preferred and practical. It should be understood that the invention is not limited to the disclosed embodiments. Rather, its purpose is to cover various modifications and equivalent arrangements within the spirit and scope of the appended claims, the scope of which is to be interpreted in the broadest possible sense to include all such modifications and equivalent structures permitted under the law.

Claims

1. A method for improving the quality of rapeseed meal through medium- and high-temperature segmented solid-state fermentation, comprising the following steps: (1) Pretreatment of raw materials: rapeseed meal is crushed, passed through a 60-mesh sieve, and degreased with low-temperature subcritical propane to obtain rapeseed meal raw material; (2) Preparation of seed culture: Bacillus subtilis and Geobacillus stearothermophilus were inoculated from cryovials onto LB solid medium and activated at 37 ℃ and 55 ℃, respectively. Single colonies were picked and inoculated onto LB liquid medium and amplified by shaking at 37 ℃ and 55 ℃ for 16 h. After the culture was completed, the number of bacteria was adjusted to 4×10⁻⁶. 8 CFU / mL was used to prepare a seed solution; (3) Solid-state fermentation of rapeseed meal: medium-temperature fermentation, the Bacillus subtilis seed liquid prepared in step (2) is inoculated into the defatted rapeseed meal in step (1) at a mass ratio of 15%, and water of 50% of the total mass of rapeseed meal is added. After thorough mixing, the mixture is transported to a rotary fermenter and fermented at 40 ℃ for 24-72 h. In the high-temperature stage, the fermentation temperature is raised to 55 ℃, and Bacillus limonene seed liquid of 25% of the total mass of rapeseed meal is inoculated. The mixture is fermented at high temperature for 24 h. During the entire fermentation process, the oxygen volume concentration in the chamber is maintained at no less than 15%. (4) Drying treatment: The rapeseed meal that has undergone two stages of solid-state fermentation in (3) is directly dried in the fermentation chamber to control the water content of the material to be less than 10%.

2. The method for improving rapeseed meal quality through medium- and high-temperature segmented solid-state fermentation according to claim 1, characterized in that, The seed liquid for the first stage of mesophilic fermentation was Bacillus subtilis, and the seed liquid for the second stage of hyperthermic fermentation was Bacillus stearothermophilus.

3. The method for improving rapeseed meal quality through medium- and high-temperature segmented solid-state fermentation according to claim 1, characterized in that, Fermentation improves the nutritional quality of rapeseed meal, with the nitrogen solubility index increasing by 97.15%–419.92%, and the content of polypeptides and crude protein increasing by 93.03%–377.75% and 3.88%–8.17%, respectively. The content of phytic acid and glucosinolates decreases by 7.26%–20.13% and 16.67%–84.14%, respectively. The content of total phenols and total flavonoids increases by 108.04% and 19.28%, respectively, and the antioxidant capacity increases by 30.84%.

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