A solid-state fermentation method for improving the nutritional value of moringa oleifera seed meal by using a compound probiotic

Abstract

The present application relates to the technical fields of microbial fermentation and deep processing of agricultural and sideline products, and discloses a compound probiotic solid-state fermentation method for improving the nutritional value of moringa seed meal, which comprises the following steps: uniformly mixing moringa seed meal powder with water and sterilizing and cooling to obtain a solid-state fermentation substrate; based on the dry basis mass of the moringa seed meal powder, inoculating a compound fermentation inoculum into the substrate at an inoculation amount of 3%-5% (v / w), and fermenting at 34-38 DEG C for 16-20 hours. The compound fermentation inoculum is prepared by mixing 1 part of Lactococcus lactis liquid, 1 part of Bacillus subtilis liquid and 1-2 parts of Rhizopus oryzae liquid in a volume ratio. The present application utilizes the metabolic complementation of the compound flora, which helps to loosen the substrate structure of the moringa seed meal and adjust the fermentation microenvironment, promotes the release of polyphenols in the moringa seed meal, increases the extractable polyphenol content in the fermentation product, and realizes the improvement of the nutritional value of the moringa seed meal by-product.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation and deep processing of agricultural and sideline products, specifically to a compound probiotic solid-state fermentation method for enhancing the nutritional value of Moringa seed meal. Background Technology

[0002] After extracting moringa oil, a large amount of residual byproducts, namely moringa seed meal, are generated from moringa seeds. Moringa seed meal is rich in crude protein, crude fiber, and functional components such as polyphenols, and has the potential to be used as a high-quality feed or food industry raw material. However, current conventional treatment methods often involve directly discarding moringa seed meal or simply downgrading it for use as low-value ordinary feed. This extensive treatment approach not only wastes high-quality plant resources but also puts pressure on environmental pollution to some extent.

[0003] Among the active ingredients in Moringa seed meal, polyphenols have high nutritional and application value. However, most polyphenols in natural plant tissues exist in a bound form, often undergoing physical cross-linking or chemical bonding with the dense cell walls, polysaccharides, or protein macromolecules of plants. This bound state results in low bioavailability when directly extracted or consumed by animals, hindering the full realization of its nutritional value.

[0004] To improve the nutritional structure of agricultural by-products, industrial processes often employ microbial fermentation technology. However, existing microbial fermentation methods have certain limitations when processing Moringa seed meal. Current solid-state fermentation often relies on a single microbial strain. Because single strains secrete relatively limited extracellular enzyme systems during metabolism, they struggle to effectively target the dense cell walls and matrix network of Moringa seed meal, resulting in insufficient release of polyphenols and limiting the increase in extractable polyphenol content. Furthermore, single strains are susceptible to substrate nutrient depletion and microenvironmental fluctuations during fermentation, posing risks of decreased metabolic activity or interference from other microorganisms, and making it difficult to establish a long-lasting and stable enzymatic hydrolysis environment in the substrate. These shortcomings of existing technologies hinder the deep breakdown of the dense structure of Moringa seed meal, making it difficult to substantially convert and improve the bioavailability of nutrients, thus restricting the high-value utilization of Moringa seed meal resources. Summary of the Invention

[0005] The technical problem solved by this invention is as follows: Moringa seed meal, a byproduct of moringa oil extraction, is rich in crude protein, crude fiber, and polyphenols. However, polyphenols in moringa seed meal exist mostly in bound form, tightly bound to plant cell walls, polysaccharides, or protein macromolecules, resulting in low bioavailability during direct extraction. Current treatment methods often involve direct disposal or use as ordinary feed, leading to a waste of plant resources. Existing single-strain fermentation methods, due to their limited enzyme systems, are unable to effectively target the relatively dense cell walls and matrix structure of moringa seed meal, resulting in insufficient release of polyphenols and a low content of extractable polyphenols, thus hindering the full enhancement of the nutritional value of moringa seed meal.

[0006] To address the above problems, the present invention provides the following technical solution: A method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal includes the following steps: Moringa seed meal powder is mixed evenly with water to keep it solid, then sterilized and cooled for later use to obtain a solid fermentation substrate. Based on the dry weight of the Moringa seed meal powder, a compound fermentation agent is inoculated into the solid fermentation substrate at an inoculation rate of 3% to 5% (v / w), thoroughly mixed, and then fermented at 34 to 38°C for 16 to 20 hours to obtain fermented Moringa seed meal. Here, v / w represents the ratio of the volume of the compound fermentation agent to the dry weight of the Moringa seed meal powder. The compound fermentation agent is prepared by mixing 1 part of Lactococcus lactis liquid, 1 part of Bacillus subtilis liquid, and 1-2 parts of Rhizopus oryzae liquid in a volume ratio.

[0007] The term "compound probiotic solid-state fermentation" as used in this specification refers to solid-state fermentation of the solid-state fermentation substrate using the compound fermentation agent composed of Lactococcus lactis liquid, Bacillus subtilis liquid, and Rhizopus oryzae liquid.

[0008] By adopting the above technical solution, a compound probiotic fermentation system is constructed by using a specific ratio of Lactococcus lactis liquid, Bacillus subtilis liquid, and Rhizopus oryzae liquid, and the solid-state fermentation is carried out by controlling the corresponding temperature, time, and inoculation amount. Therefore, it helps to loosen the cell walls and matrix structure of Moringa seed meal, promotes the release of polyphenols in Moringa seed meal, increases the content of extractable polyphenols in the fermented Moringa seed meal, and improves the nutritional value of Moringa seed meal.

[0009] In this specification, improving the nutritional value of Moringa seed meal includes at least increasing the content of extractable polyphenols in Moringa seed meal; the present invention uses the polyphenol content measured by the Folin-Ciocalteu method as the main indicator for evaluating the effect of compound fermentation.

[0010] The reaction mechanism of the above-mentioned technical solution mainly involves the synergistic effect of microbial community metabolism and enzymatic reactions. After the compound fermentation agent is inoculated into the solid fermentation substrate, microorganisms such as Bacillus subtilis and Rhizopus oryzae in the system can utilize the nutrients in the solid fermentation substrate to grow and reproduce, and can produce hydrolytic enzymes such as proteases, cellulases, and esterases. These hydrolytic enzymes help to act on the cell walls and substrate components of Moringa seed meal, loosening the relatively dense substrate network. Some polyphenols in Moringa seed meal may be bound to polysaccharides, proteins, or cell wall components. The related hydrolytic enzymes produced during the compound fermentation process help to weaken the binding relationship between polyphenols and the above-mentioned substrate components, thereby promoting the release of polyphenols.

[0011] In terms of regulating the fermentation microenvironment, the lactic acid and other organic acids produced by Lactococcus lactis metabolism lower the pH of the system. This slightly acidic condition helps regulate the microenvironment of the fermentation system and improves the stability of the complex fermentation process.

[0012] Furthermore, controlling the solid-state fermentation time to 16 to 20 hours is beneficial to the growth, metabolism, and hydrolytic enzyme activity of the complex microbial community, and reduces the risk of a decrease in polyphenol content due to excessively long fermentation time; a temperature range of 34 to 38°C helps maintain the growth and metabolic state of the complex microbial community and is conducive to the related enzymatic reactions; while an inoculum size of 3% to 5% is beneficial to the growth of the complex microbial community in the early stage of fermentation and reduces the accelerated nutrient consumption and increased cell competition that may be caused by excessively high inoculum size.

[0013] Preferably, the compound fermentation agent is prepared by mixing the following components in parts by volume: 1 part of Lactococcus lactis liquid, 1 part of Bacillus subtilis liquid and 1 part of Rhizopus oryzae liquid.

[0014] By adopting the above technical solution, when the three fermentation bacteria are mixed in equal volume proportions, the metabolic activities within the system can tend to be in a relatively balanced state. Specifically, Lactococcus lactis moderately produces acid to regulate the environment, while Bacillus subtilis and Rhizopus oryzae grow and secrete enzymes simultaneously. This can reduce the risk of excessively rapid acid production or intensified substrate competition caused by an excessively high proportion of a single bacterial species, and better leverage the synergistic advantages of metabolic complementarity in the complex microbial community.

[0015] Preferably, the *Lactococcus lactis* solution, the *Bacillus subtilis* solution, and the *Rhizopus oryzae* solution are cultured independently, wherein the final culture conditions for the *Lactococcus lactis* solution are 32°C for 9 hours, the final culture conditions for the *Bacillus subtilis* solution are 32°C for 6 hours, and the final culture conditions for the *Rhizopus oryzae* solution are 28°C for 12 hours. After the final culture is completed, they are mixed according to the corresponding volume ratio.

[0016] By adopting the above technical solution, the culture temperature and culture time are controlled according to the growth characteristics of different strains, so that the three bacterial solutions can be compounded after the corresponding culture is completed. This is beneficial for starting the fermentation process after the compound fermentation agent is introduced into the Moringa seed meal substrate, and improves the stability of the fermentation process.

[0017] Preferably, the method for preparing the Lactococcus lactis solution is as follows: a single colony of the activated Lactococcus lactis is picked and inoculated into MRS liquid medium, and cultured at 32°C for 12 hours to obtain Lactococcus lactis seed solution; the Lactococcus lactis seed solution is inoculated into fresh MRS liquid medium at an inoculation amount of 1% by volume, and cultured at 32°C for 9 hours.

[0018] Preferably, the preparation method of the Bacillus subtilis liquid is as follows: a single colony of the activated Bacillus subtilis is picked and inoculated into LB liquid medium, and cultured at 32°C for 12 hours to obtain Bacillus subtilis seed liquid; the Bacillus subtilis seed liquid is inoculated into fresh LB liquid medium at a volume percentage of 1%, and cultured at 32°C for 6 hours.

[0019] Preferably, the method for preparing the *Rhizopus oryzae* suspension is as follows: *Rhizopus oryzae* is inoculated into PDA solid medium and cultured for 72 hours until spores are formed; the spores on the surface of the culture are washed off with sterile physiological saline; the filtrate is collected after filtration as a spore suspension; and the spore concentration of the spore suspension is adjusted to 1 × 10⁻⁶. 6 The spore suspension was inoculated into PDA liquid medium at an inoculation rate of 1% (v / v) and cultured at 28°C for 12 hours.

[0020] By adopting the above technical solution and setting corresponding culture times based on the growth curves of each bacterial species, bacterial solutions can be obtained at their optimal growth stages. Bacillus subtilis cultured for 6 hours is in the late logarithmic growth phase to the early stationary phase, exhibiting a high OD600 value; Lactococcus lactis cultured for 9 hours also shows a high OD600 value; and Rhizopus oryzae reaches a high cell dry weight after approximately 12 hours of culture. Obtaining and combining these three bacterial solutions within the specified culture times facilitates the initiation of the fermentation process after the composite fermentation agent is introduced into the solid-state fermentation substrate.

[0021] Preferably, before preparing the solid fermentation substrate, the moringa seed meal raw material is pre-treated as follows: the remaining by-products after moringa oil extraction are pulverized and passed through an 80-mesh sieve to obtain the moringa seed meal powder.

[0022] By adopting the above technical solution, the specific surface area of ​​Moringa seed meal particles is increased after pretreatment by crushing and passing through an 80-mesh sieve. This physical refinement provides more attachment sites for microorganisms and also increases the contact area between hydrolytic enzymes and substrates, thus promoting fermentation and transformation to a certain extent.

[0023] Preferably, in preparing the solid-state fermentation substrate, 1 mL of water is added per 1 g of Moringa seed meal powder on a dry basis, so that the moisture content of the solid-state fermentation substrate is 50%. By adopting the above technical solution, controlling the substrate moisture content at 50% can provide the water required for microbial growth and enzymatic reactions, and maintain suitable porosity between solid powder particles. This physical structure facilitates air diffusion into the substrate, thereby meeting the dissolved oxygen requirements of aerobic bacteria such as Bacillus subtilis.

[0024] Preferably, when preparing the solid fermentation substrate, the sterilization process parameters are sterilization at 121°C under high-pressure steam for 15 minutes.

[0025] By employing the above-mentioned technical solution, high-temperature and high-pressure steam treatment can effectively kill native contaminants carried by the Moringa seed meal substrate. This provides a relatively pure substrate environment for inoculation, reduces competitive interference from non-target microorganisms in the early stages of fermentation, and thus improves the stability of the fermentation process.

[0026] Preferably, the compound fermentation agent is inoculated into the solid fermentation substrate at an inoculation rate of 5% (v / w) of the dry basis weight of the Moringa seed meal powder, and solid fermentation is carried out at 36°C for 18 hours, wherein v / w represents the ratio of the volume of the compound fermentation agent to the dry basis weight of the Moringa seed meal powder.

[0027] By adopting the above technical solution, under the controlled parameters of 18 hours of fermentation time, 36℃ temperature, and 5% inoculum amount, a good balance can be achieved between the initial microbial community construction rate and the substrate nutrient consumption rate. At this time, the hydrolytic enzymes produced by Bacillus subtilis and Rhizopus oryzae in the compound fermentation agent can act more fully on the Moringa seed meal substrate, and the organic acids produced by Lactococcus lactis help regulate the fermentation microenvironment, thereby facilitating the release of polyphenols and enabling the extractable polyphenol content in the fermented Moringa seed meal to reach a high level.

[0028] This invention provides a compound probiotic solid-state fermentation method for enhancing the nutritional value of Moringa seed meal. It has the following beneficial effects: 1. This invention utilizes a compound fermentation agent constructed from Lactococcus lactis, Bacillus subtilis, and Rhizopus oryzae in a specific ratio, taking advantage of the metabolic complementarity mechanism among different microbial species. During their growth, Bacillus subtilis and Rhizopus oryzae produce hydrolytic enzymes such as proteases, cellulases, and esterases, which help act on the cell walls and matrix components of Moringa seed meal, promoting the release of polyphenols. The organic acids produced by Lactococcus lactis help regulate the pH of the fermentation system, improving the stability of the fermentation process.

[0029] 2. This invention limits the temperature and time parameters in the solid-state fermentation process to a suitable range, promoting the growth and development of microorganisms to adapt to the enzymatic hydrolysis process of the substrate. An environment of 34 to 38°C is conducive to maintaining the growth state of the microbial community and the catalytic activity of related hydrolytic enzymes. Combined with a fermentation cycle of 16 to 20 hours, this facilitates the release of polyphenols in Moringa seed meal and reduces the risk of a decrease in polyphenol content due to excessively long fermentation time.

[0030] 3. This invention, by combining an initial inoculum of 3% to 5% with substrate preparation steps such as raw material crushing and water control, effectively balances the community building rate and nutrient consumption rate in the early stages of fermentation. This process combination allows the complex microbial community to establish a microecological advantage in the solid substrate and fully exert its enzymatic depolymerization effect. Compared with unfermented raw materials, the extractable polyphenol content in the fermented Moringa seed meal is significantly increased, thereby improving the overall nutritional value of Moringa seed by-products. Attached Figure Description

[0031] Figure 1 This is a line graph showing the growth curve of Bacillus subtilis according to the present invention. Figure 2 This is a line graph showing the growth curve of *Lactococcus lactis* according to the present invention. Figure 3 This is a line graph showing the growth curve of Rhizopus oryzae in this invention. Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0034] Lactococcus lactis ( Lactococcus lactis, L. lactis The strain used was preserved using slant culture, with accession number BNCC195305. Bacillus subtilis ( Bacillus subtilis, B. subtilis The strain used was preserved using slant culture, with accession number BNCC109047. *Rhizopus oryzae* ( Rhizopus oryzae, R. oryzae The strain used was preserved using slant culture, with the preservation number BNCC336321. Moringa seed meal is a byproduct of moringa oil extraction, originating from Yunnan Province. Before use, it was pulverized and passed through an 80-mesh sieve to obtain moringa seed meal powder.

[0035] The gallic acid standard used in the determination and extraction process had a purity of ≥99% and CAS number 149-91-7. Folin-Ciocalteu reagent was a commercially available 2 mol / L solution. Sodium carbonate was analytical grade and CAS number 497-19-8. Anhydrous ethanol had CAS number 64-17-5.

[0036] Among the biochemical reagents used to prepare the culture medium, bacteriological peptone, beef extract, yeast extract, and agar powder were all commercially available biochemical reagent-grade products. Tryptone, yeast extract, and potato extract were all conventional commercially available microbial culture reagents. Glucose was analytical grade, CAS number 50-99-7. Dipotassium hydrogen phosphate CAS number 7758-11-4. Triammonium citrate CAS number 3458-72-8. Anhydrous sodium acetate CAS number 127-09-3. Magnesium sulfate CAS number 7487-88-9. Manganese sulfate CAS number 7785-87-7. Tween-80 CAS number 9005-65-6. Sodium chloride CAS number 7647-14-5.

[0037] Preparation Examples 1-3: Preparation Example 1: This preparation example provides a method for preparing a compound fermentation agent, including the following steps: (1) Select a single colony of Lactococcus lactis activated by MRS medium and inoculate it into 10 mL of MRS liquid medium without agar. Culture it in a shaker at 32℃ and 120 rpm for 12 h to prepare seed culture. Transfer the seed culture to 50 mL of fresh MRS liquid medium at an inoculation rate of 1% v / v and culture it in a shaker at 32℃ and 120 rpm for 9 h to obtain Lactococcus lactis liquid. (2) Select a single colony of Bacillus subtilis activated by LB medium and inoculate it into 10 mL of LB liquid medium without agar. Incubate at 32℃ and 120 rpm for 12 h to prepare seed culture. Transfer the seed culture to 50 mL of fresh LB liquid medium at an inoculation rate of 1% v / v and incubate at 32℃ and 120 rpm for 6 h to obtain Bacillus subtilis liquid. (3) Inoculate Rhizopus oryzae into PDA solid medium and culture for 72 h until spores are formed. Wash the spores off the surface of the culture with sterile physiological saline, filter through a filter cloth and collect the filtrate as a spore suspension. Adjust the spore concentration to about 1×10⁻⁶ using a hemocytometer.6 spores / mL; spore suspension was inoculated into 100mL PDA liquid medium at an inoculation rate of 1% (v / v), and cultured with shaking at 28℃ and 120rpm for 12h to obtain Rhizopus oryzae solution; (4) The above-prepared Lactococcus lactis liquid, Bacillus subtilis liquid and Rhizopus oryzae liquid are mixed in a volume ratio of 1:1:1 to obtain a compound fermentation agent.

[0038] Preparation Example 2: This preparation example provides a method for preparing a compound fermentation agent, including the following steps: (1) Select a single colony of Lactococcus lactis activated by MRS medium and inoculate it into 10 mL of MRS liquid medium without agar. Shake culture at 32℃ and 120 rpm for 12 h to prepare Lactococcus lactis seed culture. Transfer the Lactococcus lactis seed culture to 50 mL of fresh MRS liquid medium at an inoculation rate of 1% v / v and shake culture at 32℃ and 120 rpm for 9 h to obtain Lactococcus lactis liquid. (2) Pick a single colony of Bacillus subtilis activated by LB medium and inoculate it into 10 mL of LB liquid medium without agar. Shake culture at 32℃ and 120 rpm for 12 h to prepare Bacillus subtilis seed liquid. Transfer the Bacillus subtilis seed liquid to 50 mL of fresh LB liquid medium at an inoculation rate of 1% v / v. Shake culture at 32℃ and 120 rpm for 6 h to obtain Bacillus subtilis liquid. (3) Inoculate Rhizopus oryzae into PDA solid medium and culture for 72 h until spores are formed. Wash the spores off the surface of the culture with sterile physiological saline, filter through a filter cloth and collect the filtrate as a spore suspension. Adjust the spore concentration to about 1×10⁻⁶ using a hemocytometer. 6 spores / mL; spore suspension was inoculated into 100mL PDA liquid medium at an inoculation rate of 1% (v / v), and cultured with shaking at 28℃ and 120rpm for 12h to obtain Rhizopus oryzae solution; (4) The above-prepared Lactococcus lactis liquid, Bacillus subtilis liquid and Rhizopus oryzae liquid are mixed in a volume ratio of 1:1:2 to obtain a compound fermentation agent.

[0039] Preparation Example 3: This preparation example provides a method for preparing a compound fermentation agent, including the following steps: (1) Select a single colony of Lactococcus lactis activated by MRS medium and inoculate it into 10 mL of MRS liquid medium without agar. Shake culture at 32℃ and 120 rpm for 12 h to prepare Lactococcus lactis seed culture. Transfer the Lactococcus lactis seed culture to 50 mL of fresh MRS liquid medium at an inoculation rate of 1% v / v and shake culture at 32℃ and 120 rpm for 9 h to obtain Lactococcus lactis liquid. (2) Pick a single colony of Bacillus subtilis activated by LB medium and inoculate it into 10 mL of LB liquid medium without agar. Shake culture at 32℃ and 120 rpm for 12 h to prepare Bacillus subtilis seed liquid. Transfer the Bacillus subtilis seed liquid to 50 mL of fresh LB liquid medium at an inoculation rate of 1% v / v. Shake culture at 32℃ and 120 rpm for 6 h to obtain Bacillus subtilis liquid. (3) Inoculate Rhizopus oryzae into PDA solid medium and culture for 72 h until spores are formed. Wash the spores off the surface of the culture with sterile physiological saline, filter through a filter cloth and collect the filtrate as a spore suspension. Adjust the spore concentration to about 1×10⁻⁶ using a hemocytometer. 6 spores / mL; spore suspension was inoculated into 100mL PDA liquid medium at an inoculation rate of 1% (v / v), and cultured with shaking at 28℃ and 120rpm for 12h to obtain Rhizopus oryzae solution; (4) The above-prepared Lactococcus lactis liquid, Bacillus subtilis liquid and Rhizopus oryzae liquid are mixed in a volume ratio of 3:1:2 to obtain a compound fermentation agent.

[0040] Examples 1-5: Example 1: This embodiment provides a method for solid-state fermentation with compound probiotics to enhance the nutritional value of Moringa seed meal. Specifically, the compound fermentation agent from Preparation Example 1 is used for solid-state fermentation, which includes the following steps: (1) Weigh 15.0g of Moringa seed meal powder that has passed through an 80-mesh sieve and place it in a 250mL Erlenmeyer flask. Add 1mL of water and 15mL of distilled water for every 1g of Moringa seed meal powder, based on the dry weight of the Moringa seed meal powder. Mix thoroughly so that the theoretical moisture content of the solid fermentation substrate is about 50%. Place it in a high-pressure steam sterilizer and sterilize at 121℃ for 15min. Cool to room temperature for later use to obtain the solid fermentation substrate. (2) The compound fermentation agent prepared in Preparation Example 1 was inoculated into the sterilized and cooled solid fermentation substrate at an inoculation amount of 5% (v / w) of the dry basis of Moringa seed meal powder, and mixed thoroughly. Solid fermentation was carried out at 36°C for 18 hours to obtain fermented Moringa seed meal. Here, v / w represents the ratio of the volume of the compound fermentation agent to the dry basis of Moringa seed meal powder.

[0041] Example 2: This embodiment provides a method for solid-state fermentation with compound probiotics to enhance the nutritional value of Moringa seed meal. Specifically, the compound fermentation agent from Preparation Example 1 is used for solid-state fermentation, which includes the following steps: (1) Weigh 15.0g of Moringa seed meal powder that has passed through an 80-mesh sieve and place it in a 250mL Erlenmeyer flask. Add 15mL of distilled water and mix thoroughly to make the substrate moisture content about 50%. Place the mixed substrate in a high-pressure steam sterilizer and sterilize at 121℃ for 15min. Cool to room temperature to obtain a solid fermentation substrate. (2) The compound fermentation agent prepared in Preparation Example 1 was inoculated into the sterilized and cooled solid fermentation substrate at an inoculation amount of 3% (v / w) of the dry basis of Moringa seed meal powder, and mixed thoroughly. Solid fermentation was carried out at 34°C for 16 hours to obtain fermented Moringa seed meal.

[0042] Example 3: This embodiment provides a method for solid-state fermentation with compound probiotics to enhance the nutritional value of Moringa seed meal. Specifically, the compound fermentation agent from Preparation Example 1 is used for solid-state fermentation, which includes the following steps: (1) Weigh 15.0g of Moringa seed meal powder that has passed through an 80-mesh sieve and place it in a 250mL Erlenmeyer flask. Add 15mL of distilled water and mix thoroughly to make the substrate moisture content about 50%. Place the mixed substrate in a high-pressure steam sterilizer and sterilize at 121℃ for 15min. Cool to room temperature to obtain a solid fermentation substrate. (2) The compound fermentation agent prepared in Preparation Example 1 was inoculated into the sterilized and cooled solid fermentation substrate at an inoculation amount of 4% (v / w) of the dry basis of Moringa seed meal powder, and mixed thoroughly. Solid fermentation was carried out at 36°C for 18 hours to obtain fermented Moringa seed meal.

[0043] Example 4: This embodiment provides a method for solid-state fermentation with compound probiotics to enhance the nutritional value of Moringa seed meal. Specifically, the compound fermentation agent from Preparation Example 1 is used for solid-state fermentation, which includes the following steps: (1) Weigh 15.0g of Moringa seed meal powder that has passed through an 80-mesh sieve and place it in a 250mL Erlenmeyer flask. Add 15mL of distilled water and mix thoroughly to make the substrate moisture content about 50%. Place the mixed substrate in a high-pressure steam sterilizer and sterilize at 121℃ for 15min. Cool to room temperature to obtain a solid fermentation substrate. (2) The compound fermentation agent prepared in Preparation Example 1 was inoculated into the sterilized and cooled solid fermentation substrate at an inoculation amount of 5% (v / w) of the dry basis of Moringa seed meal powder, and mixed thoroughly. Solid fermentation was carried out at 38°C for 16 hours to obtain fermented Moringa seed meal.

[0044] Example 5: This embodiment provides a method for solid-state fermentation with compound probiotics to enhance the nutritional value of Moringa seed meal. Specifically, the compound fermentation agent from Preparation Example 2 is used for solid-state fermentation, which includes the following steps: (1) Weigh 15.0g of Moringa seed meal powder that has passed through an 80-mesh sieve and place it in a 250mL Erlenmeyer flask. Add 15mL of distilled water and mix thoroughly to make the substrate moisture content about 50%. Place the mixed substrate in a high-pressure steam sterilizer and sterilize at 121℃ for 15min. Cool to room temperature to obtain a solid fermentation substrate. (2) The compound fermentation agent prepared in Preparation Example 2 was inoculated into the sterilized and cooled solid fermentation substrate at an inoculation amount of 5% (v / w) of the dry basis of Moringa seed meal powder, and mixed thoroughly. Solid fermentation was carried out at 36°C for 18 hours to obtain fermented Moringa seed meal.

[0045] Comparative Examples 1-4: Comparative Example 1: Compared with Example 1, the difference is that the compound fermentation agent was not inoculated in step (2), and it was used as a blank control group without inoculation. All other aspects are the same.

[0046] Because the compound fermentation agent was not inoculated, the Moringa seed meal matrix lacked the action of hydrolytic enzymes such as protease, cellulase, and esterase produced by Bacillus subtilis and Rhizopus oryzae. As a result, the cell walls and matrix network of Moringa seed meal were difficult to loosen sufficiently, the release of polyphenols was relatively low, and the content of extractable polyphenols was difficult to increase effectively. At the same time, the lack of microenvironmental regulation by lactococcus lactis production in the system was not conducive to the stable progress of the fermentation process.

[0047] Comparative Example 2: Compared with Example 1, the difference is that the inoculated agent in step (2) is the compound fermentation agent prepared in Example 3, that is, the volume ratio of compound Lactococcus lactis: Bacillus subtilis: Rhizopus oryzae = 3:1:2, and the rest are the same.

[0048] Comparative Example 2 adjusted the volume ratio of *Lactococcus lactis*, *Bacillus subtilis*, and *Rhizopus oryzae* in the compound fermentation agent to 3:1:2, forming a process control compared to the 1:1:1 ratio in Example 1. Test Example 3 screened different compound ratios in a symbiotic culture medium, with the 1:1:1 group showing a total growth grade of 15 and the 3:1:2 group showing a total growth grade of 5. The results of Test Example 3 are used to illustrate the selection criteria for the compound fermentation agent ratio and are not considered as direct results of the polyphenol content determination in the solid-state fermentation product of Comparative Example 2.

[0049] Comparative Example 3: The difference from Example 1 is that the solid-state fermentation time in step (2) is 6 hours, while the rest are the same.

[0050] Comparative Example 3, by shortening the solid-state fermentation time from 18 h in Example 1 to 6 h, forms a process control against Example 1 in terms of fermentation time. Test Example 2 conducted a single-factor experiment on fermentation time under fixed conditions of a fermentation temperature of 32°C and an inoculum amount of 6% (v / w) of compound fermentation agent. The results showed that the polyphenol contents at 6 h and 18 h of fermentation were 3.12 mg GAE / g DW and 3.98 mg GAE / g DW, respectively. This single-factor experiment result indicates that, under its set fixed conditions, extending the fermentation time to 18 h is beneficial to increasing the extractable polyphenol content; however, this value is not considered a direct quantitative comparison between Comparative Example 3 and Example 1.

[0051] Comparative Example 4: The difference from Example 1 is that the solid-state fermentation temperature in step (2) is 44°C, while the rest are the same.

[0052] Comparative Example 4 was designed to provide a process control over fermentation temperature by increasing the solid-state fermentation temperature from 36°C in Example 1 to 44°C. Test Example 2 conducted a single-factor experiment on fermentation temperature under fixed conditions of 18 hours of fermentation and 6% (v / w) of the compound fermentation agent inoculum. The results showed that the polyphenol contents in the 36°C and 44°C treatment groups were 4.15 mg GAE / g DW and 2.89 mg GAE / g DW, respectively. This single-factor experiment indicates that, under the established fixed conditions, the extractable polyphenol content in the 36°C treatment group was higher than that in the 44°C treatment group; however, this value is not considered a direct quantitative comparison between Example 1 and Comparative Example 4.

[0053] Test Examples 1-5: Test Example 1: This test case was used to determine the growth curves of Bacillus subtilis, Lactococcus lactis, and Rhizopus oryzae to identify the optimal culture time for these three bacteria when preparing a compound fermentation agent.

[0054] (1) Select a single colony of activated Bacillus subtilis and inoculate it into 10 mL of LB liquid medium. Incubate at 32℃ and 120 rpm for 12 h with shaking to obtain seed culture. Transfer the seed culture to 50 mL of fresh LB liquid medium at an inoculation rate of 1% v / v and incubate at 32℃ and 120 rpm with shaking. Starting from 0 h of incubation, take samples every 1 h, using uninoculated LB liquid medium as a blank, and measure the OD600 value at a wavelength of 600 nm. Set up 3 parallel samples at each time point.

[0055] (2) Select a single colony of activated Lactococcus lactis and inoculate it into 10 mL of MRS liquid medium. Incubate at 32℃ and 120 rpm for 12 h with shaking to obtain a seed culture. Transfer the seed culture to 50 mL of fresh MRS liquid medium at an inoculation rate of 1% v / v and incubate at 32℃ and 120 rpm with shaking. Starting from 0 h of incubation, take samples every 1 h, using uninoculated MRS liquid medium as a blank, and measure the OD600 value at a wavelength of 600 nm. Set up 3 parallel samples at each time point.

[0056] (3) Inoculate Rhizopus oryzae into PDA solid medium and culture for 72 h until spores are formed. Wash the spores off the surface of the culture with sterile physiological saline, filter through a filter cloth and collect the filtrate as a spore suspension, and adjust the spore concentration to about 1×10⁻⁶. 6 The spore suspension was inoculated at a rate of 1% (v / v) into 100 mL of PDA liquid medium and cultured with shaking at 28 °C and 120 rpm. Starting from 0 h of culture, samples were taken every 4 h. The samples were centrifuged at 4000 rpm for 10 min, the cells were collected, and washed twice with distilled water. Then, the cells were dried at 100 °C to constant weight, and the dry weight of the cells was determined.

[0057] The growth curves of the three bacteria are as follows: Figures 1-3 As shown, the growth stages and optimal culture times obtained from the curves are summarized in Table 1.

[0058] Table 1: Growth stages and optimal culture time for the three bacteria

[0059] Based on the growth curves of the three bacteria, Bacillus subtilis cultured for 6 hours, Lactococcus lactis cultured for 9 hours, and Rhizopus oryzae cultured for 12 hours all exhibited high biomass or growth activity. Therefore, the subsequent preparation of the compound fermentation agent used Bacillus subtilis cultured for 6 hours, Lactococcus lactis cultured for 9 hours, and Rhizopus oryzae cultured for 12 hours, respectively.

[0060] Test Example 2: This test example was used to investigate the effects of fermentation time, fermentation temperature, and inoculum size of the compound fermentation agent on the polyphenol content of Moringa seed meal fermentation products. Unless otherwise stated, the compound fermentation agent used was the one prepared in Preparation Example 1, and three replicates were set up for each experiment.

[0061] (1) Weigh 15.0g of Moringa seed meal powder that has passed through an 80-mesh sieve and place it in a 250mL Erlenmeyer flask. Add 15mL of distilled water, mix thoroughly, sterilize under high pressure steam at 121℃ for 15min, and cool to room temperature to obtain solid fermentation substrate.

[0062] When examining the fermentation time, the compound fermentation agent was inoculated into the sterilized and cooled solid fermentation substrate at an inoculation rate of 6% (v / w) of Moringa seed meal powder dry basis, and fermented at 32℃ for 6h, 9h, 12h, 15h and 18h ​​respectively.

[0063] When investigating the fermentation temperature, the compound fermentation agent was inoculated into the sterilized and cooled solid fermentation substrate at an inoculation rate of 6% (v / w) of Moringa seed meal powder dry basis, and fermented for 18 hours at 28℃, 32℃, 36℃, 40℃ and 44℃ respectively.

[0064] When examining the inoculation amount, the compound fermentation agent was inoculated into the sterilized and cooled solid fermentation substrate at inoculation amounts of 2%, 4%, 6%, 8% and 10% (v / w) of Moringa seed meal powder dry basis, and fermented at 36℃ for 18 hours.

[0065] (2) After fermentation, 0.2 g of each group of fermentation samples were weighed and 10 mL of 70% ethanol was added. The samples were extracted by ultrasonication at 55℃ for 60 min. After extraction, the samples were centrifuged at 4000 rpm for 10 min, and the supernatant was collected and diluted 5 times. 0.5 mL of the diluted sample solution was taken, 2.5 mL of diluted Folin-Ciocalteu reagent was added, the mixture was mixed and allowed to stand for 5 min, and then 7.5 mL of 7.5% w / v sodium carbonate solution was added. The mixture was reacted at room temperature in the dark for 90 min, and the absorbance was measured at a wavelength of 765 nm. The polyphenol content was calculated based on the gallic acid standard curve.

[0066] The complete single-factor experiments investigated various settings for fermentation time, fermentation temperature, and inoculum size of the compound fermentation agent. Representative experimental results are summarized in Table 2. The polyphenol content listed in Table 2 is the average of the results measured in three parallel samples for each treatment group.

[0067] Table 2: Summary of representative results of single-factor experiments

[0068] Based on the results of the single-factor experiment, the polyphenol content was 3.12 mg GAE / g DW after 6 hours of fermentation, and increased to 3.98 mg GAE / g DW after 18 hours of fermentation. Therefore, within the range of 6-18 hours investigated, a fermentation time of 18 hours is more suitable.

[0069] Within the investigated temperature range of 28–44℃, the 36℃ treatment group exhibited the highest polyphenol content, at 4.15 mg GAE / g DW. When the fermentation temperature was increased to 40℃ and 44℃, the polyphenol content decreased to 3.41 mg GAE / g DW and 2.89 mg GAE / g DW, respectively. Therefore, a fermentation temperature of 36℃ is considered most suitable.

[0070] When the inoculum size of the compound fermentation agent increased from 2% (v / w) to 4% (v / w), the polyphenol content increased from 3.55 mg GAE / g DW to 4.25 mg GAE / g DW. When the inoculum size was further increased to 6%, 8%, and 10% (v / w), the polyphenol content decreased to 4.01, 3.68, and 3.42 mg GAE / g DW, respectively. Therefore, a 4% (v / w) inoculum size is more suitable for the compound fermentation agent in single-factor experiments.

[0071] Test Example 3: The experimental subjects were symbiotic culture systems of Lactococcus lactis, Bacillus subtilis and Rhizopus oryzae mixed in different volume ratios.

[0072] (1) Prepare a symbiotic culture medium according to the following ratio: 10.0g potato extract powder, 5.0g glucose, 5.0g peptone, 2.5g yeast extract powder, 1.0g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 1.0mL Tween-80 and 1000mL distilled water. After dispensing, sterilize under high pressure steam at 121℃ for 15min and cool for later use.

[0073] (2) Obtain Lactococcus lactis culture for 9 h, Bacillus subtilis culture for 6 h, and Rhizopus oryzae culture for 12 h, respectively, and mix them at volume ratios of 1:1:1, 1:1:2, 2:1:1, 2:1:3, 3:1:2, and 3:1:3. Inoculate each group of compound bacterial cultures into the symbiotic culture medium at a total inoculum of 5% (v / v) and incubate at 32℃ for 24 h.

[0074] (3) After the culture was completed, Lactococcus lactis was counted on MRS agar medium, Bacillus subtilis was counted on LB agar medium, and Rhizopus oryzae was counted on PDA agar medium. The relative growth grade was indicated by "+" based on the relative number of viable bacteria in each group in the same batch of experiments. The more "+" signs there were, the higher the relative number of viable bacteria of the corresponding species in the same batch of experiments. This grade was only used for relative comparison between each compound ratio group. The total growth grade was the sum of the relative growth grades of the three bacteria.

[0075] The growth of the three bacteria under different compound ratios is shown in Table 3.

[0076] Table 3: Comparison of growth of various bacteria under different compound ratios

[0077] When *Lactococcus lactis*, *Bacillus subtilis*, and *Rhizopus oryzae* were mixed in a volume ratio of 1:1:1, the total growth grade of the three bacteria was 15, the highest among all experimental groups. When mixed in ratios of 1:1:2 and 3:1:3, the total growth grades were 14 and 13 respectively, both lower than the 1:1:1 group. Therefore, the optimal volume ratio of *Lactococcus lactis*, *Bacillus subtilis*, and *Rhizopus oryzae* was determined to be 1:1:1.

[0078] Test Example 4: The experimental subjects were solid-state fermentation products of Moringa seed meal under different process parameters constructed using orthogonal experimental design. Unless otherwise stated, the compound fermentation agents used in this test example were all prepared in Preparation Example 1.

[0079] (1) Using the compound fermentation agent prepared in Preparation Example 1, fermentation time, fermentation temperature, and inoculum size of the compound fermentation agent were investigated as factors. Each factor was set with 3 levels, and L9(3) was used. 4 Nine experiments were designed using an orthogonal array. The fermentation time levels were 16h, 18h, and 20h, the fermentation temperature levels were 34℃, 36℃, and 38℃, and the inoculum amounts of the compound fermentation agent were 3%, 4%, and 5% (v / w). Each group had three replicates.

[0080] (2) Moringa seed meal samples were prepared and fermented according to the orthogonal experimental parameters of each group. After fermentation, 0.2g of sample was weighed and 10mL of 70% ethanol was added. The samples were extracted by ultrasonication at 55℃ for 60min. After extraction, the samples were centrifuged at 4000rpm for 10min, and the supernatant was collected and diluted 5 times. The absorbance was measured at 765nm using the Folin-Ciocalteu method. The polyphenol content per gram of dry basis was calculated based on the gallic acid standard curve.

[0081] The results of the orthogonal experimental design and polyphenol content are shown in Table 4.

[0082] Table 4: Orthogonal Experimental Design and Results

[0083] The results of the orthogonal experiment range analysis are shown in Table 5.

[0084] Table 5: Range Analysis of Orthogonal Experiments

[0085] In Table 5, K1, K2, and K3 represent the average extractable polyphenol content of each investigated factor at levels 1, 2, and 3, respectively; the range R is the difference between the maximum and minimum values ​​of the average values ​​of each level for the same factor.

[0086] According to Table 4, the polyphenol content in the fifth group of experiments was the highest, at 4.7157±0.20 mg GAE / g DW. The fermentation time for this group was 18 h, the fermentation temperature was 36 ℃, and the inoculum amount of the compound fermentation agent was 5% (v / w).

[0087] According to Table 5, the ranges R for fermentation time, fermentation temperature, and inoculum amount of compound fermentation agent were 0.4984, 0.3023, and 0.1308, respectively. The influence of the three factors on polyphenol content was in the order of fermentation time, fermentation temperature, and inoculum amount of compound fermentation agent.

[0088] Based on the results of orthogonal experiments and range analysis, the influence of fermentation time, fermentation temperature, and inoculum size of the compound fermentation agent on polyphenol content was in the following order: fermentation time, fermentation temperature, and inoculum size of the compound fermentation agent. Considering the polyphenol content of each group and the range analysis results, the optimal fermentation conditions were determined to be: fermentation time 18 h, fermentation temperature 36 °C, and inoculum size of the compound fermentation agent 5% (v / w).

[0089] Test Example 5: The experimental subjects were the fermentation product group prepared using the process of Example 1, and the uninoculated blank treatment group under the conditions of Comparative Example 1.

[0090] (1) In Example 1, three groups of parallel fermentation samples were set up, and each group of samples was measured twice in parallel, resulting in a total of six measurement results; in Comparative Example 1, two uninoculated blank treatment parallel samples were set up. 0.2g of each sample was weighed and placed in a centrifuge tube, 10mL of 70% ethanol was added, and the samples were extracted by sonication at 55℃ for 60min; after extraction, the samples were centrifuged at 4000rpm for 10min and the supernatant was collected.

[0091] (2) Dilute the obtained supernatant 5 times, take 0.5 mL of the diluted sample solution, add 2.5 mL of diluted Folin-Ciocalteu reagent, mix well and let stand for 5 min, then add 7.5 mL of 7.5% w / v sodium carbonate solution, and react for 90 min at room temperature in the dark. After the reaction is completed, measure the absorbance at a wavelength of 765 nm, and calculate the polyphenol content of each sample according to the gallic acid standard curve.

[0092] The polyphenol content test results of Example 1 and Comparative Example 1 are shown in Table 6.

[0093] Table 6: Results of polyphenol content tests in experimental and comparative groups

[0094] In Table 6, the total average of the six test results in Example 1 was 4.0957 mg GAE / g DW; the two test results in Comparative Example 1 were 2.0509 mg GAE / g DW and 2.6528 mg GAE / g DW, respectively, with a total average of 2.3519 mg GAE / g DW.

[0095] The average total extractable polyphenol content of Example 1 group was about 74.1% higher than that of Comparative Example 1 group, indicating that the compound fermentation agent prepared by using Lactococcus lactis liquid, Bacillus subtilis liquid and Rhizopus oryzae liquid in a volume ratio of 1:1:1, and carried out under solid-state fermentation conditions of 5% (v / w) inoculum, fermentation temperature of 36℃ and fermentation time of 18h, can significantly increase the extractable polyphenol content in Moringa seed meal.

Claims

1. A method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal, characterized in that, Includes the following steps: Moringa seed meal powder is mixed evenly with water to keep it solid, then sterilized and cooled for later use to obtain a solid fermentation substrate. Based on the dry weight of the Moringa seed meal powder, the compound fermentation agent is inoculated into the solid fermentation substrate at an inoculation rate of 3% to 5% (v / w), thoroughly mixed, and solid fermentation is carried out at 34 to 38°C for 16 to 20 hours to obtain fermented Moringa seed meal. Here, v / w represents the ratio of the volume of the compound fermentation agent to the dry weight of the Moringa seed meal powder. The compound fermentation agent is prepared by mixing 1 part of Lactococcus lactis liquid, 1 part of Bacillus subtilis liquid, and 1-2 parts of Rhizopus oryzae liquid in a volume ratio.

2. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, The compound fermentation agent is prepared by mixing the following components in parts by volume: One part of the Lactococcus lactis solution, one part of the Bacillus subtilis solution, and one part of the Rhizopus oryzae solution.

3. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, The Lactococcus lactis solution, the Bacillus subtilis solution, and the Rhizopus oryzae solution were cultured independently. The lactococcus lactis solution was obtained by transferring lactococcus lactis seed culture and culturing it at 32°C for 9 hours. The Bacillus subtilis liquid was obtained by transferring Bacillus subtilis seed liquid and culturing it at 32°C for 6 hours. The Rhizopus oryzae solution was obtained by inoculating a spore suspension and then culturing it at 28°C for 12 hours. After the culture is complete, mix according to the volume ratio stated.

4. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, The method for preparing the lactococcus lactis solution is as follows: Single colonies of activated Lactococcus lactis were picked and inoculated into MRS liquid medium and cultured at 32℃ for 12 h to obtain Lactococcus lactis seed culture. The Lactococcus lactis seed culture was inoculated into fresh MRS liquid medium at an inoculation rate of 1% (v / v) and cultured at 32°C for 9 hours.

5. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, The preparation method of the Bacillus subtilis liquid is as follows: Single colonies of activated Bacillus subtilis were picked and inoculated into LB liquid medium and cultured at 32°C for 12 h to obtain Bacillus subtilis seed culture. The Bacillus subtilis seed culture was inoculated into fresh LB liquid medium at an inoculation rate of 1% (v / v) and cultured at 32°C for 6 hours.

6. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, The method for preparing the Rhizopus oryzae solution is as follows: Rhizopus oryzae was inoculated into PDA solid medium and cultured for 72 h until spores formed. Spores on the surface of the culture were washed off with sterile physiological saline. The filtrate was collected after filtration through a filter cloth as a spore suspension, and the spore concentration of the suspension was adjusted to 1 × 10⁻⁶. 6 cells / mL; The spore suspension was inoculated into PDA liquid medium at an inoculation rate of 1% (v / v) and cultured at 28°C for 12 h.

7. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, Before preparing the solid fermentation substrate, the Moringa seed meal raw material is pretreated as follows: The remaining byproducts after extracting moringa oil are pulverized and passed through an 80-mesh sieve to obtain the moringa seed meal powder.

8. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, When preparing the solid fermentation substrate, 1 mL of water is added to every 1 g of Moringa seed meal powder on a dry basis, so that the water content of the solid fermentation substrate is 50%.

9. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, When preparing the solid fermentation substrate, the sterilization process parameters are sterilization at 121°C under high-pressure steam for 15 minutes.

10. The method for solid-state fermentation of compound probiotics to enhance the nutritional value of Moringa seed meal according to claim 1, characterized in that, The compound fermentation agent was inoculated into the solid fermentation substrate at an inoculation rate of 5% (v / w), and solid fermentation was carried out at 36°C for 18 hours.

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