A production method of a selenium-rich black peanut sauce system

Abstract

This invention belongs to the field of food processing, specifically disclosing a production method for a selenium-enriched black peanut soy sauce system, comprising: S1, using the selenium-enriched black peanut residue after pressing as raw material, inoculating it with selenium yeast for cultivation; S2, adding neutral protease and cellulase for enzymatic hydrolysis; S3, inoculating with Aspergillus oryzae and Aspergillus niger for fermentation; S4, inoculating with Lactobacillus plantarum and Streptococcus halophilus for fermentation; S5, inoculating with Saccharomyces roximatee and Saccharomyces cerevisiae for fermentation; S6, using supercritical CO₂ extraction to obtain selenium-enriched black peanut soy sauce. This invention, by using selenium-enriched black peanut residue as raw material and employing a process of "bioactivation-enzymatic hydrolysis-multi-stage fermentation-high-efficiency extraction," not only improves the decomposition rate of protein and cellulose in the residue, achieving efficient resource utilization, but also increases the total selenium content of the soy sauce and the proportion of organic selenium through selenium yeast conversion, probiotic integration, and low-temperature extraction, thereby improving bioavailability. At the same time, the synergistic fermentation of multiple strains generates a variety of flavor substances.

Description

Technical Field

[0001] This invention belongs to the field of food processing, and specifically relates to a production method of a selenium-enriched black peanut soy sauce system. Background Technology

[0002] Soy sauce is a condiment made from grains, salt, and water through fermentation, and is widely used in cooking in East Asia and other regions. It has a delicious salty and savory flavor and is rich in umami components, which can enhance flavor, color, and aroma.

[0003] Traditional soy sauce production primarily uses soybeans and wheat as raw materials, resulting in a limited range of flavor compounds and a lack of distinctive characteristics. Furthermore, peanut residue (containing protein, cellulose, polyphenols, etc.) after oil extraction is often treated as waste, leading to resource waste. Statistics show that peanut residue accounts for over 60% of the raw material's mass, with a protein content of 25%-30%, yet traditional processes fail to utilize it efficiently.

[0004] Selenium is an essential trace element for the human body. Traditional selenium-enriched foods are mostly made by adding inorganic selenium (such as sodium selenite). However, inorganic selenium has low bioavailability and excessive intake poses a toxic risk. In addition, it is difficult to effectively combine with food matrix, and selenium is easily lost in the form of H2Se during high-temperature fermentation. Summary of the Invention

[0005] The purpose of this invention is to provide a production method for a selenium-enriched black peanut soy sauce system, in order to solve the problems mentioned in the background art, such as the reliance on soybean fermentation in traditional processes, the limited variety of flavor substances, and the low selenium content resulting from the direct addition of inorganic selenium.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for producing selenium-enriched black peanut soy sauce includes:

[0008] S1. Using the selenium-enriched black peanut residue after pressing as raw material, add 0.5% sodium selenite solution to the raw material until the water content is 45%, and inoculate with selenium yeast and culture at 30℃ for 24h to obtain activated residue.

[0009] S2. Neutral protease and cellulase are added to the activated residue for enzymatic hydrolysis to obtain enzymatic hydrolysate;

[0010] S3. Inoculate the enzymatic hydrolysate with Aspergillus oryzae and Aspergillus niger, and ferment to obtain the initial fermentation material;

[0011] S4. Inoculate Lactobacillus plantarum and halophilic tetrastreptococcus into the early fermentation material, and add selenomethionine and glutathione at the same time to ferment and obtain the middle fermentation material.

[0012] S5. Inoculate the intermediate fermentation material with *Saccharomyces rouxii* and *Saccharomyces globulus*, and ferment to obtain the late fermentation material;

[0013] S6. The post-fermentation material was treated with ultrasound, and then supercritical CO2 extraction was used to obtain selenium-enriched black peanut soy sauce.

[0014] Preferably, the mass percentages of the neutral protease and cellulase are 0.1-0.3% and 0.05-0.15% of the activated residue, respectively, and the enzymatic hydrolysis conditions are pH 6-7, temperature 48-52℃, and time 2.5-3.5h.

[0015] Preferably, the mass ratio of Aspergillus oryzae to Aspergillus niger is 1:1, the fermentation temperature is 36-40℃, and the fermentation time is 5-9 days.

[0016] Preferably, the mass ratio of Lactobacillus plantarum to Halophilic tetrastreptococcus is 2:1, the mass percentages of selenomethionine and glutathione are 0.01-0.03% and 0.05-0.15% of the initial fermentation material, respectively, and the fermentation temperature is 40-45℃ for 8-12 days.

[0017] Preferably, the mass ratio of *Saccharomyces rouxii* to *Saccharomyces globosa* is 3:1, and the fermentation temperature of the late-stage fermentation material is 28-32℃ with a total acid content ≥1.5g / 100mL.

[0018] Preferably, the ultrasonic treatment is performed at a frequency of 18-22 kHz for 28-32 min, the supercritical CO2 extraction is performed at a pressure of 35 MPa, a temperature of 45 °C, a 10% ethanol solution as the entrainer, and a flow rate of 1.2 mL / g·min.

[0019] Preferably, after obtaining the selenium-enriched black peanut soy sauce, the selenium-enriched black peanut soy sauce is treated with pulsed intense light and then bottled.

[0020] Preferably, the wavelength of the pulsed intense light treatment is 200-1100nm, the single pulse energy is 1.2J, and the total dose is 12J / cm².

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention uses selenium-rich black peanut residue as raw material and employs a process of "bioactivation-enzymatic hydrolysis-multi-stage fermentation-high-efficiency extraction" to not only improve the decomposition rate of protein and cellulose in the residue and achieve efficient resource utilization, but also increases the total selenium content of soy sauce and the proportion of organic selenium through selenium yeast conversion, probiotic integration and low-temperature extraction, thereby improving bioavailability. At the same time, the synergistic fermentation of multiple strains generates a variety of flavor substances.

[0023] This invention combines ultrasonic-assisted extraction with pulsed light sterilization to shorten the fermentation cycle while ensuring nutrient retention. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0027] Example 1: This example provides a production method for a selenium-enriched black peanut soy sauce system, including:

[0028] S1. Using the selenium-enriched black peanut residue after pressing as raw material, add 0.5% sodium selenite solution to the raw material until the water content is 45%, and inoculate with selenium yeast and culture at 30℃ for 24h to obtain activated residue.

[0029] Sodium selenite, as an inorganic selenium source, is metabolized by selenium yeast into bioactive selenium (such as selenoamino acids). Selenium yeast proliferates at a suitable temperature, decomposes macromolecules in the residue, and converts bound selenium into water-soluble selenium, thereby converting bound selenium into bioactive selenium and improving the bioavailability of selenium.

[0030] The selenium content was determined by atomic fluorescence spectrometry: 1 g of activated residue was extracted with 10 mL of 0.1 mol / L hydrochloric acid solution by ultrasonic extraction for 30 min. After centrifugation, the supernatant was collected and the selenium content was determined by atomic fluorescence spectrometry. The proportion of water-soluble selenium in the total selenium was calculated.

[0031] Test results: The proportion of water-soluble selenium increased from 35% to 82%.

[0032] S2. Neutral protease and cellulase are added to the activated residue for enzymatic hydrolysis to obtain enzymatic hydrolysate;

[0033] The mass percentages of neutral protease and cellulase were 0.2% and 0.1% of the activation residue, respectively. The enzymatic hydrolysis conditions were pH 6.5, temperature 50℃, and time 3h.

[0034] Neutral proteases break down proteins to generate small peptides and amino acids, while cellulases disrupt plant cell walls, releasing polysaccharides and small molecules. This provides readily available carbon and nitrogen sources for subsequent microbial fermentation, while also increasing the soluble solids content of the system and enhancing the flavor precursors of soy sauce.

[0035] High performance liquid chromatography (HPLC) was used: after centrifugation of the enzymatic hydrolysate, the supernatant was filtered through a 0.22 μm filter membrane and separated by a C18 column (4.6 mm × 250 mm). The mobile phase was 0.1% trifluoroacetic acid aqueous solution-acetonitrile (gradient elution), and the sample was detected by a UV detector (220 nm). The proportion of peptides <1000 Da was calculated.

[0036] Test results: 75% of peptides had a molecular weight <1000 Da.

[0037] S3. Inoculate the enzymatic hydrolysate with Aspergillus oryzae and Aspergillus niger (total 3%) and ferment to obtain the initial fermentation material;

[0038] The mass ratio of Aspergillus oryzae to Aspergillus niger was 1:1, the fermentation temperature was 38℃, and the fermentation time was 7 days.

[0039] Aspergillus oryzae and Aspergillus niger secrete proteases and amylases, which further break down peptides and polysaccharides to produce amino acids, glucose, etc. The protease activity reaches 2200 U / g, which accelerates protein degradation and produces rich umami amino acids (such as glutamic acid) and sweet substances, laying the foundation for the basic flavor of soy sauce, while providing nutrition for probiotics in the middle stage of fermentation.

[0040] The Folin-phenol method was used: 10 g of fermentation material was taken, 100 mL of phosphate buffer (pH 7.5) was added, and the mixture was extracted by shaking at 40 °C for 1 h. The supernatant was collected by centrifugation. Casein was used as the substrate, and after the reaction, Folin reagent was used for color development. The absorbance was measured at 660 nm, and the protease activity (U / g) was calculated.

[0041] Test results: Protease activity reached 2200 U / g.

[0042] S4. Inoculate the early fermentation material with Lactobacillus plantarum and halophilic tetrastreptococcus (total 2%), and add selenomethionine and glutathione at the same time to obtain the mid-stage fermentation material.

[0043] The mass ratio of Lactobacillus plantarum to Halophilic tetrastreptococcus was 2:1, and the mass percentages of selenomethionine and glutathione were 0.02% and 0.1% of the initial fermentation material, respectively. The fermentation temperature was 42℃ and the fermentation time was 10 days.

[0044] Lactobacillus plantarum produces acid (lowers pH), inhibiting the growth of other bacteria, while simultaneously producing organic acids (such as lactic acid) and flavor compounds through metabolism; Halophilic tetrastreptococcus participates in the synthesis of flavor compounds, enhancing ester aromas; selenomethionine, as an organic selenium precursor, is integrated by microorganisms into selenoproteins or small molecule selenium compounds; glutathione, as an antioxidant, protects the stability of selenium compounds, increases the proportion of organic selenium, and at the same time, the acidity of the fermentation system increases, allowing flavor compounds (such as esters and organic acids) to begin to accumulate.

[0045] High performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was used: the fermentation material was precipitated with 5% trichloroacetic acid solution, the supernatant was collected after centrifugation, and the selenomethionine content was separated by HPLC (C18 column) and detected by ICP-MS.

[0046] Test results: The selenomethionine content increased to 18.4 μg / g.

[0047] S5. Inoculate the intermediate fermentation material with Saccharomyces rouxii and Saccharomyces globosa (total 1.5%), and ferment to obtain the late fermentation material;

[0048] The mass ratio of *Saccharomyces rouxii* to *Saccharomyces globosa* was 3:1, the fermentation temperature of the late-stage fermentation material was 30℃, and the total acid content was 1.8 g / 100 mL.

[0049] The metabolism of *Saccharomyces rouxii* produces ethanol, which reacts with organic acids to form esters (such as ethyl acetate), giving soy sauce its ester aroma. *Saccharomyces globosa* produces furans and phenolic compounds (such as 4-ethylguaiacol, with a content ≥12.7 mg / L), which enhance the caramel aroma and complex flavor of soy sauce, thus forming the unique flavor and aroma profile of soy sauce. At the same time, the low temperature and high acid environment of the later fermentation stage ensure the stable existence of selenium compounds.

[0050] Gas chromatography-mass spectrometry (GC-MS) was used: the fermentation material was extracted with diethyl ether, concentrated, and then analyzed by GC-MS (DB-5MS column, temperature programmed), with ion monitoring mode (SIM) selected for quantification.

[0051] Test results: 4-ethylguaiacol content ≥12.7mg / L.

[0052] S6. The post-fermentation material was treated with ultrasound, and then supercritical CO2 extraction was used to obtain selenium-enriched black peanut soy sauce.

[0053] The ultrasonic treatment was performed at a frequency of 20 kHz for 30 min, the supercritical CO2 extraction pressure was 35 MPa, the temperature was 45 ℃, the entrainer was a 10% ethanol solution, and the flow rate was 1.2 mL / g·min.

[0054] Ultrasound disrupts the structure of microbial cells and residues through cavitation, releasing intracellular selenium compounds and flavor substances; supercritical CO2 combines the solubility of liquids with the diffusion ability of gases, efficiently extracting small molecule organics (such as selenoamino acids and flavor esters), while ethanol entrainer enhances the solubility of polar substances, and the low extraction temperature avoids the degradation of selenium and flavor substances.

[0055] After obtaining the selenium-enriched black peanut soy sauce, it is treated with pulsed intense light and then bottled.

[0056] The wavelength of the pulsed intense light treatment is 200-1100nm, the single pulse energy is 1.2J, and the total dose is 12J / cm².

[0057] Pulsed intense light destroys the DNA / RNA structure of microorganisms through the ultraviolet band, while the visible and infrared bands generate a thermal effect, achieving sterilization through a dual action that meets food sterilization standards without affecting the nutrition and flavor of soy sauce.

[0058] The total selenium content was determined by microwave digestion-atomic fluorescence spectrometry. The proportion of organic selenium was separated by protein precipitation. The inorganic selenium content was determined by taking the supernatant. The organic selenium content was obtained by subtracting the inorganic selenium from the total selenium content, and the proportion was calculated.

[0059] Test results: The selenium content of the soy sauce was 23.6 mg / kg, with organic selenium accounting for 95.3%.

[0060] Sterilization effect: Plate count method was used: 1 mL of soy sauce sample was taken, serially diluted and spread on nutrient agar plates, incubated at 37℃ for 48 h, and the total number of colonies was counted.

[0061] Test results: Total bacterial count <100 CFU / mL.

[0062] As can be seen from the above, using selenium-enriched black peanut residue as raw material, through a systematic process of "bioactivation-enzymatic hydrolysis-multi-stage microbial fermentation-physicochemical extraction", selenium can be efficiently converted and retained, while simultaneously generating soy sauce flavor substances.

[0063] Among them, selenium yeast and probiotics convert inorganic selenium into organic selenium, glutathione protects the stability of selenium compounds, and finally, through extraction and enrichment of highly active organic selenium, enzymatic hydrolysis and multi-strain fermentation synergistically decompose macromolecules such as proteins and cellulose to generate flavor substances such as amino acids, peptides, organic acids, and esters, forming the color, aroma and taste of soy sauce. Ultrasonic waves destroy cell structures to promote the release of substances, supercritical extraction accurately separates target components, and pulsed strong light ensures product safety without destroying nutrients.

[0064] By integrating microbial fermentation, enzyme engineering, and supercritical extraction, the bioavailability of selenium is improved, while the problems of residue waste and monotonous flavor in traditional soy sauce production are solved, achieving dual optimization of functionality (selenium enrichment) and taste (flavor).

[0065] Example 2: This example is basically the same as the previous example, except that:

[0066] S2, cellulase addition amount is 0.05, enzymatic hydrolysis temperature is 52℃, and time is 2.5h;

[0067] The residue after enzymatic hydrolysis was dried, weighed, and tested: the residue dissolution rate decreased by 38% compared to Example 1.

[0068] HPLC detection: Peptide yield ≥70% (<1000Da account for 68%).

[0069] S5. The fermentation temperature during the later stage of fermentation is 35℃.

[0070] Acid-base titration method for detection: total acid 1.2 g / 100 mL.

[0071] S3 protease activity: 1496 U / g (32% lower than in Example 1, detection method is the same as in Example 1).

[0072] S4 selenomethionine content: 16.2 μg / g (detection method is the same as in Example 1).

[0073] S6 soy sauce selenium content: 20.1 mg / kg (organic selenium content 92.1%, detection method is the same as in Example 1).

[0074] This invention is achieved through:

[0075] Step one converts inorganic selenium into bioactive selenium. Step four promotes the integration of selenium by microorganisms through the addition of selenomethionine and glutathione. Finally, step six extracts and retains high organic selenium content, forming a complete chain of "selenium activation-selenium integration-selenium retention" to avoid the loss of selenium in traditional processes.

[0076] Step two, enzymatic hydrolysis, provides small molecule substrates; step three, mold fermentation generates basic umami flavor; step four, probiotics produce acid for flavoring; and step five, yeast fermentation generates complex aromas. The microbial metabolites (such as amino acids, organic acids, and esters) of each stage are layered together to ultimately form a flavorful soy sauce system.

[0077] The residue is fully decomposed through activation in step one and enzymatic hydrolysis in step two. Multi-strain fermentation in steps three to five further transforms it into soluble substances. Extraction in step six achieves efficient extraction of effective components. The entire process transforms the pressing residue into high-value-added products and reduces resource waste.

[0078] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0079] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A production method for a selenium-enriched black peanut soy sauce system, characterized in that, include: S1. Using the selenium-enriched black peanut residue after pressing as raw material, add 0.5% sodium selenite solution to the raw material until the water content is 45%, and inoculate with selenium yeast and culture at 30℃ for 24h to obtain activated residue. S2. Neutral protease and cellulase are added to the activated residue for enzymatic hydrolysis to obtain enzymatic hydrolysate; S3. Inoculate the enzymatic hydrolysate with Aspergillus oryzae and Aspergillus niger, and ferment to obtain the initial fermentation material; S4. Inoculate Lactobacillus plantarum and halophilic tetrastreptococcus into the early fermentation material, and add selenomethionine and glutathione at the same time to ferment and obtain the middle fermentation material. S5. Inoculate the intermediate fermentation material with *Saccharomyces rouxii* and *Saccharomyces globulus*, and ferment to obtain the late fermentation material; S6. The post-fermentation material was treated with ultrasound, and then supercritical CO2 extraction was used to obtain selenium-enriched black peanut soy sauce.

2. The production method of a selenium-enriched black peanut soy sauce system according to claim 1, characterized in that, The neutral protease and cellulase are present in mass percentages of 0.1-0.3% and 0.05-0.15% of the activated residue, respectively, and the enzymatic hydrolysis conditions are pH 6-7, temperature 48-52℃, and time 2.5-3.5h.

3. The production method of a selenium-enriched black peanut soy sauce system according to claim 1, characterized in that, The mass ratio of Aspergillus oryzae to Aspergillus niger is 1:1, the fermentation temperature is 36-40℃, and the fermentation time is 5-9 days.

4. The production method of a selenium-enriched black peanut soy sauce system according to claim 1, characterized in that, The mass ratio of *Lactobacillus plantarum* to *Streptococcus halophilus* is 2:1, the mass percentages of selenomethionine and glutathione are 0.01-0.03% and 0.05-0.15% of the initial fermentation material, respectively, and the fermentation temperature is 40-45℃ for 8-12 days.

5. The production method of a selenium-enriched black peanut soy sauce system according to claim 1, characterized in that, The mass ratio of *Saccharomyces rouxii* to *Saccharomyces globosa* is 3:1, and the fermentation temperature of the late-stage fermentation material is 28-32℃ with a total acid content ≥1.5g / 100mL.

6. The production method of a selenium-enriched black peanut soy sauce system according to claim 1, characterized in that, The ultrasonic treatment is performed at a frequency of 18-22 kHz for 28-32 min, and the supercritical CO2 extraction is performed at a pressure of 35 MPa, a temperature of 45 °C, a 10% ethanol solution as the entrainer, and a flow rate of 1.2 mL / g·min.

7. The production method of a selenium-enriched black peanut soy sauce system according to claim 1, characterized in that, After obtaining the selenium-enriched black peanut soy sauce, the selenium-enriched black peanut soy sauce is treated with pulsed intense light and then bottled.

8. The production method of a selenium-enriched black peanut soy sauce system according to claim 7, characterized in that, The wavelength of the pulsed intense light treatment is 200-1100nm, the single pulse energy is 1.2J, and the total dose is 12J / cm².