A method for extracting D-pinitol, soy protein isolate and soy isoflavones by comprehensive utilization of soybean meal

Abstract

The invention discloses a method for extracting D-pinitol, soy protein isolate and soy isoflavones from soybean meal by comprehensive utilization of soybean meal. The method uses defatted soybean meal as raw material, and the method is carried out through steps such as crushing and extraction, protein extraction, microbial sugar removal, polyamide treatment, column chromatography adsorption, etc. to achieve the simultaneous extraction and preparation of three active ingredients, D-pinitol, soy protein isolate and soy isoflavones, from soybean meal. The method can efficiently extract D-pinitol from soybean meal, with an extraction rate of ≥95% and a purity of up to 80%. During the purification of D-pinitol, the loss rate is less than 3%, the removal rate of miscellaneous sugars is ≥98%, the removal rate of miscellaneous proteins is ≥87%, the extraction rate of soy protein isolate is ≥75%, and the extraction rate of soy isoflavones is ≥93%. The operation process is green and efficient, and the comprehensive utilization of the active ingredients of the soybean meal raw material is achieved.

Description

Technical Field

[0001] The invention belongs to the field of extraction and separation of natural products, and particularly relates to a method for extracting D-pinitol, soy protein isolate and soy isoflavones by comprehensively utilizing soybean meal. Background Art

[0002] Soybean meal, made from the solid portion of soybeans after oil extraction, is primarily used in the feed industry. It is rich in various nutrients and active ingredients, including soy protein, soy lecithin, soy fiber, soy oligosaccharides, soy isoflavones, soy saponins, and D-pinitol. The amino acid pattern of soy protein is similar to that of the human body, making it highly nutritious. Soy isoflavones, also known as "phytoestrogens," possess estrogenic activity. D-pinitol is also a key functional factor in soybean meal, known for its effects on regulating blood sugar levels, alleviating insulin resistance, combating inflammation, reducing edema, and relieving cough and expectoration. In recent years, D-pinitol has been widely used in pharmaceutical raw materials, health supplements, and additives.

[0003] The preparation methods for D-pinitol are primarily chemical synthesis and natural extraction. Chemically synthesized D-pinitol is subject to residual residues from various chemical reaction raw materials, posing a safety hazard. Edible-grade D-pinitol is primarily produced through isolation and purification from plants. Patent KR20010016111 utilizes activated carbon to adsorb pinitol and inositol from soybean byproducts, followed by ethanol elution to separate the pinitol. This process produces D-pinitol with low yield and purity, and contains significant amounts of protein and sugar impurities.

[0004] Patent ZL201911256846.0 uses carob as a raw material, producing D-pinitol through a complex process involving sucrose hydrolysis, fructose precipitation, glucose oxidation, and column chromatography. This method is complex and expensive, making it difficult to commercialize. Patent ZL200910047048.7 uses Robinia pseudoacacia as a raw material, employing the Sevag method to remove proteins and silica gel column chromatography to produce high-purity D-pinitol. However, this method uses multiple toxic reagents, posing safety risks.

[0005] Research has shown that soybean meal contains approximately 0.2% D-pinitol, making it one of the most cost-effective raw materials. It also contains approximately 0.4% soy isoflavones and 45% crude protein. Utilizing these active ingredients in soybean meal could yield significant economic value. Regarding the comprehensive utilization of soybean meal, ZL200810154689.8 utilizes enzymatic hydrolysis and membrane separation techniques to simultaneously extract soy peptides, soy dietary fiber, soy oligosaccharides, and soy protein isolate from soybean meal. This method requires the addition of large amounts of enzymes, resulting in high costs and a long time-consuming process. Furthermore, ZL00122917.6 utilizes chromatography and recrystallization to simultaneously extract isoflavones, saponins, oligosaccharides, and protein from defatted soybean meal. This method utilizes multiple organic reagents, which poses a safety risk to food production. At present, there are not only many deficiencies in the extraction process of active ingredients in soybean meal, but also a lack of related processes for extracting D-pinitol from soybean meal, as well as a method for comprehensively extracting D-pinitol, isolated protein and isoflavones from soybean meal. This has led to the underutilization of D-pinitol, the active ingredient in soybean meal, and caused a great waste of soybean biological resources. Summary of the Invention

[0006] The present invention addresses the deficiencies of the above-mentioned prior art and provides a method for extracting D-pinitol, soy protein isolate, and soy isoflavones from soybean meal. This method addresses the defects and limitations of the D-pinitol preparation process, achieving efficient extraction of D-pinitol from soybean meal while fully utilizing the two active ingredients in the soybean meal: soy protein isolate and soy isoflavones. This method conserves resources and increases the added value of soybean meal, significantly reducing production costs for enterprises and transforming waste into valuable resources.

[0007] The invention utilizes microbial fermentation to remove sugars, polyamide decolorization and protein removal, and column chromatography to produce 80% D-pinitol, while simultaneously extracting and separating soy protein isolate and soy isoflavones. The extraction process is environmentally friendly, highly efficient, pollution-free, simple to operate, and low in cost.

[0008] The method for extracting D-pinitol, soy protein isolate and soy isoflavones by comprehensively utilizing soybean meal comprises the following steps:

[0009] Step 1: Grind and sieve the soybean meal, add 80% ethanol at a solid-liquid ratio of 1g:8-10mL, extract in a 60°C water bath for 30min-1h, repeat the extraction 2-3 times, filter through 8 layers of gauze, and combine the alcohol extracts; drain the soybean meal residue, add alkaline water with a pH value of 8.5 at a solid-liquid ratio of 1g:8-10mL, extract in a 60°C water bath for 30min-1h, repeat the extraction 2-3 times, filter through 8 layers of gauze, and combine to obtain a protein solution.

[0010] Step 2: The alcohol extract obtained in step 1 is concentrated to a sugar content of 15-20° Brix, 4 g / L yeast extract is added, the pH is adjusted to 4.5-5.5, and after sterilization, 1.2-1.4 g / L activated fruit wine yeast is inoculated. The mixture is fermented at 28°C and 120 rpm on a shaker for 48-72 hours. The fermentation liquid is concentrated to 1 / 20 of the original volume, 95% ethanol is added to a final ethanol concentration of 80%, the mixture is stirred thoroughly, and the mixture is allowed to stand at 4°C for 12 hours. The mixture is centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected.

[0011] Step 3: The supernatant obtained in step 2 was concentrated to 1 / 10 of its original volume, and 100-120 mesh polyamide was mixed with the concentrate in a shaker at a material-liquid ratio of 1 g:4 mL. The mixture was shaken at 200 rpm at 37°C for 30 minutes, and then rinsed with deionized water. The eluate was collected, concentrated, and lyophilized to obtain crude D-pinitol.

[0012] Step 4: The crude D-pinitol obtained in step 3 is configured into a 100 mg / mL solution, and a polystyrene-type weak polar adsorption resin is used as a filler for column chromatography. The solution is eluted with deionized water at a flow rate of 1 mL / min and an elution amount of 1.5-2 BV, and the water eluate is collected. The solution is then eluted with 40% ethanol at a flow rate of 1.5 mL / min and an elution amount of 1.5-2 BV. The alcohol eluate is collected, concentrated, and spray-dried to obtain soybean isoflavones. The content of the soy isoflavones is 40-50% as measured by ultraviolet spectrophotometry, with a yield of 0.31-0.35% (based on soybean meal) and an extraction rate of ≥93%. The protein solution obtained in step 1 is adsorbed on an activated carbon column, the eluate is adjusted to pH 4.5, and centrifuged at 3000 rpm for 5-10 min. The precipitate is redissolved in 4 volumes of deionized water and spray-dried to obtain a low-odor soy protein isolate with a yield of 30%-35% and an extraction rate of ≥75%.

[0013] Step 5: The aqueous eluate obtained in Step 4 was purified by chromatography using Amberlyste IRA-21 and then Amberlite IR-120 ion exchange resins, eluting with deionized water at a flow rate of 2 mL / min. The eluate was collected, concentrated, and lyophilized. Detection was performed using an Agilent 1260 liquid chromatograph. D-pinitol analytical standards were purchased from Supelco. Chromatographic conditions included: YMC-Pack Polyamine II, mobile phase: acetonitrile (A): water (B) = 78:22, column temperature: 25°C, injection volume: 10 μL, and flow rate: 1.0 mL / min. Detector conditions included: drift tube temperature: 110°C, gain factor: 1, and gas flow rate: 2.0 L / min. The D-pinitol sample had a purity of 80%, a yield of 0.20%-0.22%, and an extraction efficiency of ≥95%.

[0014] The beneficial effects of the present invention are embodied in:

[0015] 1. The present invention designs a process for the comprehensive utilization of soybean meal. During the extraction of D-pinitol, column chromatography is combined with the production of soy isoflavones. The extraction rate of soy isoflavones is greater than 95%. Simultaneously, the protein loss rate in the soybean meal after D-pinitol extraction is less than 5%. The impurity content during the protein extraction process is significantly reduced, and the off-flavor of the soy protein is also significantly reduced, simplifying the workload of subsequent protein purification. This invention achieves the comprehensive utilization of soybean meal, increasing its added value and further broadening its application prospects.

[0016] 2. The present invention utilizes microbial fermentation to remove sugar impurities with similar properties to D-pinitol in the extract, and adopts a polyamide method to quickly and effectively remove proteins and pigments. The removal rate of miscellaneous sugars is ≥98%, the protein removal rate is ≥87%, and the D-pinitol loss rate is <3%. 80% D-pinitol can be extracted from a soybean meal raw material with a content of about 0.2%, and the extraction rate is ≥95%;

[0017] 3. The present invention is used to comprehensively extract D-pinitol, soy protein isolate and soy isoflavones from soybean meal. Per ton of soybean meal, more than 2.0 kg of D-pinitol (80% content) can be extracted, which is worth 30,000 to 50,000 yuan, 350 kg of soy protein isolate and more than 3.4 kg of soy isoflavones, which are worth nearly 10,000 yuan. After deducting the cost, each ton of soybean meal can still achieve a profit of more than 10,000 yuan, which has significant economic benefits.

[0018] 4. The operation method of the present invention is simple, green and efficient. It only uses green solvents such as ethanol and water, and does not use other toxic or harmful organic reagents. The extracted D-pinitol, soy isoflavones and soy protein isolate are non-toxic and harmless, and are beneficial to human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a flow chart of the soybean meal comprehensive utilization technology provided by the present invention;

[0020] Figure 2 The effects of various factors on the utilization rate of miscellaneous sugars in Example 1 (A is the initial sugar content, B is the yeast extract, C is the initial pH, and D is the fermentation time);

[0021] Figure 3 This is the HPLC chromatogram of soybean molasses fermentation at 0 h in Example 2 (1-D-pinitol, 2-fructose, 3-sucrose, 4-raffinose, 5-stachyose);

[0022] Figure 4 This is the HPLC chromatogram of soybean molasses fermented for 48 hours in Example 2 (1-D-pinitol, 2-fructose, 3-sucrose, 5-stachyose);

[0023] Figure 5 This is the HPLC chromatogram of Example 3 after decolorization and deproteinization (1-D-pinitol, 2-fructose);

[0024] Figure 6 This is the HPLC chromatogram of the sample of Example 5 (1-D-pinitol) with a purity of 80.5%. DETAILED DESCRIPTION

[0025] The present invention is described in detail below through specific examples. The examples are only used to explain the present invention and are not used to limit the scope of protection of the present invention.

[0026] Example 1: Optimization of fermentation conditions

[0027] Step 1: Soybean meal was pulverized and sieved (100 mesh). 1 g of soybean meal was extracted with 80% ethanol at 60°C for 30 min at a ratio of 9 mL. The extracts were combined and concentrated to a Brix of 15°. 3% yeast extract was added, the pH was adjusted to 4, and the mixture was autoclaved at 121°C for 30 min. Yeast was inoculated and cultured at 28°C at 120 rpm for 50 h. The utilization rate of various sugars was determined by HPLC-ELSD.

[0028] Step 2: Use the single factor method to screen and optimize the fermentation conditions, and investigate the effects of factors such as initial sugar content, yeast extract addition, initial pH, and fermentation time on the utilization rate of miscellaneous sugars. The fixed conditions are an initial sugar content of 15°Brix, 3% yeast extract, an initial pH of 4, and a fermentation time of 50h. The effects of initial sugar content (5°Brix, 10°Brix, 15°Brix, 20°Brix, 25°Brix), yeast extract addition (0%, 1%, 2%, 3%, 4%, 5%), initial pH (3, 4, 5, 6, 7), and fermentation time (0-120h) on the utilization rate of miscellaneous sugars were investigated in turn;

[0029] The results are as follows Figure 2 As shown in the figure, through single-factor analysis, the optimal conditions for fermentation sugar removal were determined to be an initial sugar content of 15° Brix, 4% yeast extract, an initial pH of 5, and a fermentation time of 48 hours. Experimental verification confirmed that under these conditions, the utilization rate of sugars reached 98.7%, with the shortest fermentation time and the highest economic benefits.

[0030] Example 2: Preparation of fermentation broth

[0031] Step 1: Grind defatted soybean meal and sieve (100 mesh). Take 100g of soybean meal powder and add 900mL of 80% ethanol. Extract twice at 60°C for 30 minutes. Combine the extracts and retain the filter residue. Concentrate the extract to approximately 150mL and determine the sugar content to be 15°Brix. Add 0.6g of yeast extract (Aladdin), adjust the pH to 5, and sterilize by autoclaving at 121°C for 30 minutes.

[0032] Step 2: Inoculate 0.2 g of activated fruit wine yeast (Angel SY), culture at 28°C and 120 rpm, sample every 24 hours during fermentation to detect the composition and content of miscellaneous sugars, and finally ferment for 72 hours to obtain soybean molasses fermentation liquid;

[0033] Step 3: Miscellaneous sugar detection method: Purchase standard products of stachyose, raffinose, sucrose, and fructose, weigh them quantitatively, and dissolve them in 70% acetonitrile. Use HPLC-ELSD to determine the peak time and content of each miscellaneous sugar component.

[0034] The results are shown in Table 1. After 48 hours of fermentation, only a small amount of stachyose and raffinose remained in the fermentation broth. The miscellaneous sugar components that were difficult to separate from D-pinitol had been consumed and converted by the microorganisms, and the miscellaneous sugar utilization rate reached 99.1%.

[0035] Table 1 Changes in sugar content in fermentation broth

[0036]

[0037] Example 3: Decolorization and deproteinization of fermentation broth

[0038] Step 1: Concentrate the fermentation broth to 1 / 20 of its original volume, add 5 times the volume of 95% ethanol, and let the mixture stand overnight at 4°C. After centrifugation, retain the supernatant and recover the ethanol, and concentrate the supernatant to 1 / 10 of its original volume;

[0039] Step 2: The supernatant concentrate obtained in step 1 was freeze-dried and referred to as CDP. The D-pinitol was purified by deproteinization using the polyamide method, acid precipitation method and Sevag method, respectively.

[0040] Polyamide method: 25 g of 100-120 mesh polyamide and 100 mL of supernatant concentrate were mixed in a 250 mL shake flask; at room temperature, shaken at 200 rpm for 30 minutes to allow the polyamide to fully adsorb the protein, then filtered, the filtrate was collected, and freeze-dried, abbreviated as PDP.

[0041] Acid precipitation method: Use dilute HCl to adjust the pH of the supernatant to 4.5-4.8, centrifuge to remove the precipitate, collect the supernatant, freeze-dry, and abbreviate it as ADP.

[0042] Sevag method: n-butanol, chloroform and supernatant were mixed in a volume ratio of 1:4:5, shaken vigorously for 10 minutes, centrifuged and supernatant was collected, repeated 3 times, organic reagents were removed by rotary evaporation, and freeze-dried, which was abbreviated as SDP.

[0043] Step 3: Determination of the protein content of CDP, PDP, ADP, and SDP was performed by the Coomassie Brilliant Blue G-250 method using bovine serum albumin as the standard. The D-pinitol content was determined by the HPLC-ELSD method.

[0044] The results are shown in Table 2. Compared with the three deproteinization methods, the protein content of PDP and SDP was significantly lower than that of CDP and ADP. The D-pinitol content of SDP was significantly lower than that of PDP and ADP, indicating that the polyamide method has a better deproteinization effect than the acid precipitation method and the Sevag method, with lower D-pinitol loss and no introduction of new impurities.

[0045] Table 2 Protein and D-pinitol content

[0046]

[0047] Example 4: Separation and Detection of Soy Isoflavones

[0048] Step 1: Dissolve PDP in water and load onto an AB-8 macroporous resin column. After adsorption is complete, rinse with water for 1.5 BV and collect the water eluate. Elute with 50% ethanol at a flow rate of 1.5 mL / min and a volume of 1.5 BV. Collect the alcohol eluate, concentrate it, and spray-dry it to obtain soy isoflavone powder.

[0049] Step 2: Determination of soy isoflavones by ultraviolet spectrophotometry. The soy isoflavone standard (Desite) and sample solution were spectrally scanned on an ultraviolet spectrophotometer to obtain the maximum absorption peak of the standard and sample at 259 nm. The calibration curve was drawn and the soy isoflavone content in the sample was measured to be 40.5%, the yield was 0.32%, and the extraction rate was 93%.

[0050] Example 5: Separation and Detection of D-Pinitol

[0051] Step 1: The water eluate of AB-8 is chromatographed on Amberlyste IRA-21 and Amberlite IR-120 ion exchange resins in sequence, and the eluate containing D-pinitol is obtained by water elution, which is then concentrated and lyophilized;

[0052] Step 2: An Agilent 1260 liquid chromatograph was used for detection. D-pinitol analytical standards were purchased from Supelco (purity ≥ 98%). The chromatographic column was a YMC-Pack Polyamine II (4.6 mm x 250 mm, 5 μm). The mobile phase consisted of acetonitrile (A) and water (B) in a ratio of 78:22. The column temperature was 25°C, the injection volume was 10 μL, and the flow rate was 1.0 mL / min. Detector conditions included a drift tube temperature of 110°C, a gain factor of 1, and a gas flow rate of 2.0 L / min. The sample was measured with a peak elution time of 7.8 min, a purity of 80.5%, a yield of 0.20%, and an extraction efficiency of 96%.

[0053] Example 6: Separation and Detection of Soy Protein Isolate

[0054] The soybean meal residue retained in step 1 of Example 2 was added with 900 mL of water, the pH was adjusted to 8.5 with food-grade NaOH, and the mixture was heated and extracted at 60° C. for 30 min. The extraction was repeated 3 times, and the resulting protein solution was combined to obtain a protein solution. After cooling, the solution was adsorbed on an activated carbon column. The pH of the eluate was adjusted to 4.5 with citric acid, and the solution was centrifuged at 3000 rpm / min for 10 min. The precipitate was collected, redissolved in deionized water, and dried to obtain a soy protein isolate with no obvious beany odor. The protein content of the soy protein isolate sample was determined by Coomassie Brilliant Blue G-250 method using bovine serum albumin (BSA) as a standard, and the yield was 32%, with an extraction rate of 76%.

Claims

1. A method for extracting D-pinitol, soy protein isolate and soy isoflavones by comprehensive utilization of soybean meal, characterized in that: Using defatted soybean meal as raw material, microbial fermentation is used to remove miscellaneous sugars, polyamide method is used for decolorization and deproteinization, column chromatography technology is used to obtain D-pinitol, and soy protein isolate and soy isoflavones are extracted and separated at the same time; The specific steps include: Step 1: Grind and sieve the soybean meal, add 80% ethanol, and extract in a 60°C water bath for 30-60 minutes. Repeat the extraction 2-3 times, filter through 8 layers of gauze, and combine the filtrates; drain the soybean meal residue, add alkaline water, and extract in a 60°C water bath for 30-60 minutes. Repeat the extraction 2-3 times, filter through 8 layers of gauze, and combine to obtain the protein solution; Step 2: The alcohol-extracted filtrate obtained in step 1 was concentrated to a sugar content of 15-20° Brix, 4 g / L yeast extract was added, the pH was adjusted to 4.5-5.5, and after sterilization, 1.2-1.4 g / L fruit wine yeast was inoculated for fermentation. The fermentation broth was concentrated to 1 / 20 of the original volume, 95% ethanol was added to a final ethanol concentration of 80%, and the mixture was stirred thoroughly and allowed to stand at 4°C. The supernatant was collected by centrifugation. Step 3: The supernatant obtained in step 2 was concentrated to 1 / 10 of its original volume, the polyamide and the concentrate were mixed in a shaker, shaken at 200 rpm at 37°C for 30 minutes, rinsed with deionized water, and the eluate was collected, concentrated, and lyophilized to obtain crude D-pinitol; Step 4: The crude D-pinitol obtained in step 3 is prepared into a 100 mg / mL solution, filled with a polystyrene-type weakly polar adsorption resin, eluted with deionized water, and the water eluate is collected; then eluted with 40% ethanol, the alcohol eluate is collected, concentrated, and spray-dried to obtain soy isoflavones; the protein solution obtained in step 1 is adsorbed on an activated carbon column, the pH of the eluate is adjusted to 4.5, the precipitate is collected by centrifugation, the precipitate is redissolved in deionized water, and spray-dried to obtain a low-odor soy protein isolate; Step 5: The water eluate obtained in step 4 is subjected to chromatography purification using an ion exchange resin, eluted with deionized water at a flow rate of 2 mL / min, the eluate is collected, concentrated and lyophilized to obtain D-pinitol; In step 3, the polyamide is 100-120 mesh and is mixed with the concentrate at a material-liquid ratio of 1 g:4 mL; In step 4, when eluting with deionized water, the elution flow rate of deionized water is 1 mL / min, and the elution volume is 1.5-2 BV; when eluting with 40% ethanol, the elution flow rate of 40% ethanol is 1.5 mL / min, and the elution volume is 1.5-2 BV; In step 5, the water eluate obtained in step 4 is purified by chromatography using Amberlyste IRA-21 and Amberlite IR-120 ion exchange resins in sequence.

2. The method according to claim 1, wherein: In step 1, the amount of 80% ethanol added is 1g:8-10mL according to the solid-liquid ratio; the pH value of the alkaline water is 8.5, and the amount of alkaline water added is 1g:8-10mL according to the solid-liquid ratio.

3. The method according to claim 1, wherein: In step 2, the fruit wine yeast was inoculated and fermented at 28° C. and 120 rpm for 48-72 h.

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