A method for detoxification and flavor enhancement of distiller's grains by solid state fermentation with probiotics

Abstract

This invention relates to a method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation in the field of distiller's grains reprocessing technology. The method includes the following steps: S1, taking 100 parts by weight of distiller's grains, removing impurities, spreading and drying, adjusting the moisture content to 52-56%, pH to 3.4-3.8, adding 0.8-1.2 parts corn syrup and 0.4-0.6 parts wheat bran, and then mixing evenly; S2, inoculating the pretreated distiller's grains with a 3-4 vol% compound probiotic agent, the total viable bacteria concentration of the agent being ≥1.0 × 10⁻⁶. 9 CFU / mL; S3, In a sealed environment, the inoculated distiller's grains are compacted to a compaction degree of 62-68%, and anaerobic fermented at 30-32℃ for 45-50 hours; S4, The oxygen concentration of the fermentation environment is adjusted to 0.8-1.2 vol%, and solid-state fermentation is carried out at 28-30℃ for 34-38 hours; S5, The product after fermentation in step S4 is dried under negative pressure at 38-42℃ until the moisture content is ≤12%, and then pulverized to obtain the detoxified and aroma-enhanced distiller's grains product. This method solves the problem of mutual inhibition between the detoxification and aroma-enhancing reactions of distiller's grains in current probiotic fermentation.

Description

Technical Field

[0001] This invention relates to the field of distiller's grains reprocessing technology, specifically to a method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation. Background Technology

[0002] Baijiu (Chinese liquor) lees are a byproduct of the grain-based brewing industry, with an annual output exceeding 30 million tons. Rich in crude protein, dietary fiber, and free amino acids, they possess extremely high resource value. However, fresh lees have three major drawbacks: First, mold growth in brewing raw materials and during storage can produce aflatoxin B1 and ochratoxin. Simultaneously, the lees accumulate fusel oils, free tannins, and phenolic anti-nutritional factors, which can easily cause liver and kidney damage and rumen metabolic disorders when directly fed to livestock and poultry, limiting their feed value. Second, lees contain excessive residual ethanol and organic acids, resulting in a pungent, sour odor and extremely poor palatability. Third, natural lees have a short shelf life and are highly susceptible to secondary mold growth and toxicity.

[0003] Current distillers' grains refining technologies are divided into physical detoxification and chemical detoxification. Physical detoxification mainly relies on physical sun-drying and high-temperature cooking, but this method can only remove a small amount of volatile fusel oils, the degradation rate of mycotoxins is less than 35%, and it is extremely energy-intensive. Chemical detoxification methods, which rely on quicklime and oxidants, will destroy the amino acids and protein nutrients in the distillers' grains, produce secondary toxic salts, and the residual chemicals cannot be used in the food and high-end feed industries.

[0004] Patent application number 202211400764.0 discloses a method for preparing fermented baijiu lees and its application. The specific steps of the method for preparing fermented baijiu lees are as follows: Weigh 80% of the wet lees according to the following mass percentages. 90%, bran 5% 15%, glucose and ammonium chloride additives 2% 5% and 2% of the inoculum mixture 5%, of which the microbial mixture includes Saccharomyces cerevisiae, Candida utilis, Bacillus subtilis, and lactic acid bacteria. The weighed raw materials are then spread evenly in the fermentation bed and fermented at 32°C. Fermentation at 37℃ for 36 days After 48 hours of fermentation, the fermented lees are placed in a breathing bag equipped with a breathing valve to obtain fermented distiller's grains. This invention also discloses the application of this fermented distiller's grains in the preparation of feed additives with pathogenic bacteria inhibitory effects. The fermented distiller's grains prepared by this invention have a very strong inhibitory effect on pathogenic bacteria, and when added to feed as a feed additive for growing-finishing pigs, they can effectively improve the growth performance of the animals.

[0005] However, this patent does not target the degradation of aflatoxin and persistent phenolic toxins, resulting in a lack of detoxification function and the product's toxin levels failing to meet national feed standards. Furthermore, the mold-Bacillus co-fermentation process relies on enzymes produced by Aspergillus oryzae and Aspergillus niger for detoxification, but mold metabolism easily generates secondary fungal toxins, simultaneously destroying the natural aroma of the distiller's grains and causing the fermentation products to become sour and bitter, failing to achieve aroma enhancement. Meanwhile, current probiotic fermentation methods all employ constant-aerobic, constant-temperature single-stage solid-state fermentation. Probiotics have weak tolerance to the high ethanol and high organic acid stress of distiller's grains, resulting in a high apoptosis rate. The activity of detoxification enzymes and flavor-synthesizing enzymes decreases simultaneously, leading to mutual inhibition of detoxification and aroma enhancement reactions, preventing synergistic processes. Therefore, a processing method capable of detoxifying and enhancing the aroma of distiller's grains is urgently needed. Summary of the Invention

[0006] The present invention aims to provide a method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation, in order to solve the problem of mutual inhibition between the detoxification and aroma enhancement reactions of distiller's grains in current probiotic fermentation.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation, comprising the following steps: S1, Pretreatment of distiller's grains: Take 100 parts by weight of fresh white wine distiller's grains, remove impurities and spread them out to dry at 40~45℃, adjust the moisture content to 52~56% and pH to 3.4~3.8, add 0.8~1.2 parts of corn syrup and 0.4~0.6 parts of wheat bran, and then mix evenly; S2, Inoculation: Inoculate the pretreated distiller's grains with 3-4 vol% of a compound probiotic agent. The compound probiotic agent is composed of *Lactobacillus plantarum*, *Lactobacillus casei*, *Bacillus coagulans*, and probiotic brewer's yeast in a live bacteria volume ratio of 5:3:2:1, with a total live bacteria concentration ≥1.0 × 10⁻⁶. 9 CFU / mL; S3, a first-stage anaerobic detoxification fermentation: In a closed environment, the inoculated mash is compacted to a compaction degree of 62-68%, and then anaerobic fermented at 30-32℃ for 45-50 hours. S4, two-stage aroma-enhancing fermentation: the oxygen concentration of the fermentation environment is controlled at 0.8~1.2 vol%, and solid-state fermentation is carried out at 28~30℃ for 34~38 hours; S5, Low-temperature post-treatment: The product after fermentation in step S4 is dried under negative pressure at 38~42℃ until the moisture content is ≤12%, and then crushed to obtain the detoxified and aroma-enhanced distiller's grains product.

[0008] Preferably, as an improvement, the compound probiotics in step S2 are all subjected to two generations of directional acclimatization in distiller's grains extract culture medium, and the strains have an ethanol tolerance of ≥8% and a pH tolerance limit of 3.0.

[0009] Preferably, as an improvement, in step S3, the fermentation temperature is 31°C and the fermentation time is 48 hours.

[0010] Preferably, as an improvement, in step S4, the oxygen concentration is 1 vol%, the fermentation temperature is 29°C, and the fermentation time is 36 h.

[0011] A method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation has been developed for use in poultry and livestock feed.

[0012] The principle and advantages of this scheme are as follows: This invention selects four compliant probiotic strains—Lactobacillus plantarum, Lactobacillus casei, Bacillus coagulans, and Saccharomyces cerevisiae—that have been directionally domesticated with distillers' grains extract to construct a symbiotic microbial community. It relies on a two-stage anaerobic-microaerobic coupled solid-state fermentation process to induce differentiated enzyme production within the microbial community, completing detoxification and aroma enhancement metabolism step by step. In the first stage of absolutely anaerobic fermentation, the high-ethanol, low-pH stress environment of the distillers' grains substrate directionally induces Lactobacillus casei, Bacillus coagulans, and Saccharomyces cerevisiae to actively synthesize and secrete extracellular detoxification enzymes, tannin enzymes, and fusel oil oxidants. Enzymes and primary esterifying enzymes, freely distributed in the solid matrix of distiller's grains, directly degrade free aflatoxin B1, ochratoxin, fusel oils, and free phenols and tannins—anti-nutritional factors—within the system. Simultaneously, at the end of anaerobic fermentation, some probiotics undergo physiological programmed autolysis, releasing intrinsic intracellular toxin lysins, glycoside hydrolases, and bound toxin hydrolases accumulated in the cytoplasm and cell membrane binding sites into the fermentation system. These specifically lyse hidden toxins bound to dietary fiber and protein in the distiller's grains, achieving a dual deep degradation of both free and bound toxins. Simultaneously, the peptidoglycan in the cell wall of lactic acid bacteria physically adsorbs trace amounts of residual toxins, thoroughly completing the detoxification of the lees and the degradation of acidic, pungent volatile fatty acids. In the second stage of micro-oxygen fermentation, the oxygen partial pressure regulates the metabolic pathways of the microbial community, inhibiting the reverse synthesis pathway of toxins and activating the transaminase and esterification enzyme systems within the probiotic brewing yeast and lactic acid bacteria. This allows the enzymatic hydrolysis products of the previous stage toxins, endogenous small-molecule monosaccharides in the lees, and free amino acids to serve as flavor synthesis precursor substrates, triggering aldol condensation and bioesterification reactions, and directionally enriching ethyl lactate, ethyl acetate, and ethyl hexanoate. It contains natural aroma compounds such as β-phenylethanol, which optimize the flavor profile of the distiller's grains and remove rancid odors. At the same time, a mutually beneficial symbiotic metabolic system is formed within the microbial community. Lactic acid bacteria produce acid to maintain the antibacterial microenvironment of the system, inhibiting the secondary growth and toxin production of storage bacteria and molds. Bacillus coagulans decomposes the macromolecular polysaccharides of the distiller's grains to supply the substrate for yeast metabolism. Yeast consumes trace amounts of oxygen to stabilize the micro-oxygen environment of the system, avoiding the problem of microbial antagonism. No secondary fungal toxins or chemical toxic byproducts are generated throughout the process. It simultaneously achieves three core effects: detoxification of distiller's grains substrate, targeted enrichment of flavor, and retention of highly active probiotics.

[0013] 1. Compared with existing technologies, this solution overcomes three major technical biases in the industry: conflict between detoxification and flavor enhancement conditions, strain stress and apoptosis, and secondary toxins. It requires no chemical additives, no step-by-step processing, and no exogenous flavorings, and relies on the endogenous metabolism of probiotics to improve quality simultaneously.

[0014] 2. This solution involves low-temperature solid-state fermentation throughout the entire process, with low energy consumption and zero solid-liquid emissions, making it suitable for large-scale industrial production. The finished product can be used as a raw material for high-end probiotic feed for livestock and poultry, as well as as a base for blending liquor and flavoring braised dishes, achieving dual-use resource utilization. Detailed Implementation

[0015] The following detailed description illustrates the specific implementation method: Example 1 A method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation includes the following steps: S1, Pretreatment of distiller's grains: Take 100KG of fresh white wine distiller's grains by weight, sieve to remove coarse rice husks, spread them out to dry at 42℃, adjust the moisture content to 54% and pH to 3.6, add 1KG of corn syrup and 0.5KG of wheat bran, and then mix evenly.

[0016] S2, Inoculation: Inoculate the pretreated distiller's grains with a 3.5 vol% compound probiotic agent at a ratio of 3.5 L / KG of distiller's grains, stirring constantly. The compound probiotic agent is composed of *Lactobacillus plantarum*, *Lactobacillus casei*, *Bacillus coagulans*, and probiotic *Saccharomyces cerevisiae* in a viable cell volume ratio of 5:3:2:1, with a total viable cell concentration ≥1.0 × 10⁻⁶. 9 CFU / mL; all compound probiotics have undergone two generations of directional acclimatization in distiller's grains extract culture medium, and the strains have an ethanol tolerance of ≥8% and a pH tolerance limit of 3.0.

[0017] S3, a stage of anaerobic detoxification fermentation: In a closed environment, the inoculated mash is compacted to a compaction degree of 62-68%, and then anaerobic fermented at 31°C for 48 hours. S4, two-stage aroma-enhancing fermentation: the oxygen concentration of the fermentation environment is controlled to 1 vol%, and solid-state fermentation is carried out at 29℃ for 36 hours; S5, Low-temperature post-treatment: The product after fermentation in step S4 is dried at 40℃ under negative pressure until the moisture content is ≤12%, and then crushed to obtain the detoxified and aroma-enhanced distiller's grains product.

[0018] Example 2 A method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation includes the following steps: S1, Pretreatment of distiller's grains: Take 100KG of fresh white wine distiller's grains by weight, remove impurities, spread them out to dry at 40~45℃, adjust the moisture content to 52% and pH to 3.4, add 0.8KG of corn syrup and 0.4KG of wheat bran, and then mix evenly. S2, Inoculation: Inoculate the pretreated distiller's grains with a 3 vol% compound probiotic agent at a ratio of 3.5 L / KG of distiller's grains, stirring constantly. The compound probiotic agent is composed of *Lactobacillus plantarum*, *Lactobacillus casei*, *Bacillus coagulans*, and probiotic *Saccharomyces cerevisiae* in a viable volume ratio of 5:3:2:1, with a total viable bacteria concentration ≥1.0 × 10⁻⁶. 9 CFU / mL; all compound probiotics have undergone two generations of directional acclimatization in distiller's grains extract culture medium, and the strains have an ethanol tolerance of ≥8% and a pH tolerance limit of 3.0.

[0019] S3, a first-stage anaerobic detoxification fermentation: In a closed environment, the inoculated lees are compacted to a compaction degree of 62%, and then anaerobic fermented at 30°C for 50 hours. S4, two-stage aroma-enhancing fermentation: the oxygen concentration of the fermentation environment is controlled to be 0.8 vol%, and solid-state fermentation is carried out at 28℃ for 34 hours; S5, Low-temperature post-treatment: The product after fermentation in step S4 is dried at 38℃ under negative pressure until the moisture content is ≤12%, and then crushed to obtain the detoxified and aroma-enhanced distiller's grains product.

[0020] Example 3 A method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation includes the following steps: S1, Pretreatment of distiller's grains: Take 100KG of fresh white wine distiller's grains by weight, remove impurities, spread them out to dry at 45℃, adjust the moisture content to 56% and pH to 3.8, add 1.2KG of corn syrup and 0.6KG of wheat bran, and then mix evenly; S2, Inoculation: Inoculate the pretreated distiller's grains with 4 vol% compound probiotic agent at a ratio of 3.5 L / KG distiller's grains, stirring constantly. The compound probiotic agent is composed of *Lactobacillus plantarum*, *Lactobacillus casei*, *Bacillus coagulans*, and probiotic *Saccharomyces cerevisiae* in a viable volume ratio of 5:3:2:1, with a total viable bacteria concentration ≥1.0 × 10⁻⁶. 9 CFU / mL; all compound probiotics have undergone two generations of directional acclimatization in distiller's grains extract culture medium, and the strains have an ethanol tolerance of ≥8% and a pH tolerance limit of 3.0.

[0021] S3, a stage of anaerobic detoxification fermentation: In a closed environment, the inoculated mash is compacted to a compaction degree of 68%, and then anaerobic fermented at 32°C for 45 hours. S4, two-stage aroma-enhancing fermentation: the oxygen concentration of the fermentation environment is controlled to 1.2 vol%, and solid-state fermentation is carried out at 30℃ for 38 hours; S5, Low-temperature post-treatment: The product after fermentation in step S4 is dried under negative pressure at 38~42℃ until the moisture content is ≤12%, and then crushed to obtain the detoxified and aroma-enhanced distiller's grains product.

[0022] The lees prepared in Example 1 were tested, and the aflatoxin B1 content decreased from an initial 68 μg / kg to 4.9 μg / kg, with a degradation rate of 92.7%; the fusel oil content decreased from 1126 mg / kg to 122 mg / kg, with a removal rate of 89.1%; the total flavor ester content was 1.92 mg / g, an increase of 2.6 times compared to the raw material; and the viable probiotic count was 1.3 × 10⁻⁶. 9 CFU / g; no ochratoxin or secondary fungal toxins were detected.

[0023] Comparative Example 1 Single-stage fermentation process using Lactobacillus plantarum: With the same substrate and fermentation time, single-stage anaerobic fermentation using Lactobacillus plantarum at constant temperature resulted in a 31.2% degradation rate of aflatoxin B1, a 27.5% removal rate of fusel oils, and a 28% enhancement rate of flavor esters. The detoxification and aroma enhancement effects were significantly inferior to those of the present invention.

[0024] Comparative Example 2 The existing mold compound fermentation detoxification process, which involves the compound fermentation of Aspergillus oryzae and Bacillus subtilis, achieves a toxin degradation rate of 85%, but trace amounts of secondary ochratoxin are detected, resulting in a sour flavor and no improvement in ester content. Therefore, the product cannot be used as a raw material for catering.

[0025] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation, characterized in that, Includes the following steps: S1, Pretreatment of distiller's grains: Take 100 parts by weight of fresh white wine distiller's grains, remove impurities and spread them out to dry at 40~45℃, adjust the moisture content to 52~56% and pH to 3.4~3.8, add 0.8~1.2 parts of corn syrup and 0.4~0.6 parts of wheat bran, and then mix evenly; S2, Inoculation: Inoculate the pretreated distiller's grains with 3-4 vol% of a compound probiotic agent. The compound probiotic agent is composed of *Lactobacillus plantarum*, *Lactobacillus casei*, *Bacillus coagulans*, and probiotic brewer's yeast in a live bacteria volume ratio of 5:3:2:1, with a total live bacteria concentration ≥1.0 × 10⁻⁶. 9 CFU / mL; S3, a first-stage anaerobic detoxification fermentation: In a closed environment, the inoculated mash is compacted to a compaction degree of 62-68%, and then anaerobic fermented at 30-32℃ for 45-50 hours. S4, two-stage aroma-enhancing fermentation: the oxygen concentration of the fermentation environment is controlled at 0.8~1.2 vol%, and solid-state fermentation is carried out at 28~30℃ for 34~38 hours; S5, Low-temperature post-treatment: The product after fermentation in step S4 is dried under negative pressure at 38~42℃ until the moisture content is ≤12%, and then crushed to obtain the detoxified and aroma-enhanced distiller's grains product.

2. The method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation according to claim 1, characterized in that: The compound probiotics in step S2 were all acclimatized for two generations in a distiller's grains extract culture medium, and the strains had an ethanol tolerance of ≥8% and a pH tolerance limit of 3.

0.

3. The method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation according to claim 2, characterized in that: In step S3, the fermentation temperature is 31℃ and the fermentation time is 48h.

4. The method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation according to claim 3, characterized in that: In step S4, the oxygen concentration is 1 vol%, the fermentation temperature is 29°C, and the fermentation time is 36 hours.

5. The method for detoxifying and enhancing the aroma of distiller's grains using probiotic solid-state fermentation as described in any one of claims 1 to 4, is used in poultry and livestock feed.