CN117623884A - Method for extracting D-pinitol, isolated soy protein and soy isoflavone by comprehensively utilizing soybean meal - Google Patents

Abstract

The invention discloses a method for comprehensively utilizing soybean meal to extract D-pinitol, isolated soybean protein and isoflavone. The method can efficiently extract the D-pinitol from the soybean meal, the extraction rate of the D-pinitol in the soybean meal is more than or equal to 95%, the purity can reach 80%, the loss rate in the process of purifying the D-pinitol is less than 3%, the impurity sugar removal rate is more than or equal to 98%, the impurity protein removal rate is more than or equal to 87%, the extraction rate of the soybean protein isolate is more than or equal to 75%, the extraction rate of the soybean isoflavone is more than or equal to 93%, and the operation process is green and efficient, so that the comprehensive utilization of active ingredients of the soybean meal raw materials is realized.

Description

Method for extracting D-pinitol, isolated soy protein and soy isoflavone by comprehensively utilizing soybean meal

Technical Field

The invention belongs to the field of extraction and separation of natural products, and particularly relates to a method for comprehensively utilizing soybean meal to extract D-pinitol, soybean protein isolate and soybean isoflavone.

Background

The soybean meal is prepared from a solid part after soybean oil extraction, is mainly applied to the feed industry, and is rich in various nutritional and active ingredients such as soybean protein, soybean phospholipid, soybean fiber, soybean oligosaccharide, soybean isoflavone, soybean saponin, D-pinitol and the like. Wherein the amino acid pattern of the soybean protein is similar to that of human body, and has high nutritive value. Soy isoflavones, also known as "phytoestrogens," have estrogenic activity. In addition, D-pinitol is also an important functional factor in soybean meal, has the effects of regulating blood sugar balance of organisms, relieving insulin resistance, resisting inflammation and edema, relieving cough, eliminating phlegm and the like, and has been widely applied to the fields of medical raw materials, health-care products, additives and the like in recent years.

The preparation method of D-pinitol mainly comprises a chemical synthesis method and a natural extraction method. The chemical synthesis of D-pinitol has the residual problems of various chemical reaction raw materials and has potential safety hazards. The food grade D-pinitol is mainly prepared by separating and purifying from plants. The KR20010016111 patent uses activated carbon to adsorb pinitol and inositol from soybean by-products and separates the pinitol by eluting with ethanol. The D-pinitol product prepared by the process has low yield and purity, and a large amount of protein and sugar impurities exist in the product.

ZL201911256846.0 is prepared from carob by hydrolysis of sucrose, fructose precipitation, glucose oxidation, column chromatography, etc. The method has complex operation and high cost, and is difficult to realize industrial production. ZL200910047048.7 uses locust as raw material, adopts Sevag method to remove protein, uses silica gel column chromatography to prepare high purity D-pinitol, but the method uses various toxic reagents, and has safety risk.

Researches show that the soybean meal contains about 0.2 percent of D-pinitol, is one of raw materials with higher D-pinitol content, and has low cost. The soybean meal also contains about 0.4 percent of soybean isoflavone and 45 percent of crude protein, and if the active ingredients in the soybean meal can be comprehensively utilized, the soybean meal can generate high economic value. In the aspect of comprehensive utilization of soybean meal, ZL200810154689.8 utilizes enzymolysis and membrane separation technology to simultaneously extract four components of soybean peptide, soybean dietary fiber, soybean oligosaccharide and soybean protein isolate from the soybean meal, and the method requires adding a large amount of enzyme preparations, and has high cost and long time consumption. In addition, ZL00122917.6 employs chromatography and recrystallization to simultaneously extract isoflavone, saponin, oligosaccharide and protein from defatted soybean meal, and the method uses various organic reagents, which is not beneficial to safety of food production. At present, the extraction process of the active ingredients of the soybean meal has a plurality of defects, and lacks of a related process for extracting the D-pinitol from the soybean meal and a method for comprehensively extracting the D-pinitol, the protein isolate and the isoflavone in the soybean meal, so that the active ingredients of the D-pinitol in the soybean meal are not fully utilized, and great waste of soybean biological resources is caused.

Disclosure of Invention

The invention provides a method for comprehensively utilizing soybean meal to extract D-pinitol, isolated soybean protein and soybean isoflavone aiming at the defects of the prior art. The invention solves the defects and limitations existing in the preparation process of the D-pinitol, realizes the efficient extraction of the D-pinitol from the soybean meal, and simultaneously can fully utilize two active components of soybean protein isolate and soybean isoflavone in the soybean meal. The invention can save resources, improve the added value of the bean pulp, and has important significance for reducing the production cost of enterprises and changing waste into valuables.

The invention uses microorganism fermentation to remove the impurity sugar, the polyamide method decolors and deproteins, the column chromatography technology prepares D-pinitol with 80 percent of content, and simultaneously extracts and separates the isolated soy protein and the soy isoflavone. The extraction process is green and environment-friendly, efficient and pollution-free, and has simple operation and low cost.

The invention relates to a method for extracting D-pinitol, soy protein isolate and soy isoflavone by comprehensively utilizing soybean meal, which comprises the following steps:

step 1: pulverizing soybean meal, sieving, adding 80% ethanol according to the ratio of 1g to 8-10mL, leaching in water bath at 60deg.C for 30min-1h, repeatedly leaching for 2-3 times, filtering with 8 layers of gauze, and mixing ethanol extracts; draining soybean meal filter residues, adding alkaline water with pH value of 8.5 according to the ratio of 1g to 8-10mL, leaching in a water bath kettle at 60 ℃ for 30min-1h, repeatedly leaching for 2-3 times, filtering through 8 layers of gauze, and combining to obtain a protein solution.

Step 2: concentrating the ethanol extract obtained in the step 1 to a sugar degree of 15-20 DEG Brix, adding 4g/L yeast extract, adjusting pH to 4.5-5.5, sterilizing, inoculating 1.2-1.4g/L activated fruit wine yeast, fermenting and culturing at 120rpm for 48-72h at 28 ℃, concentrating the fermentation broth to 1/20 of the original volume, adding 95% ethanol to a final concentration of 80%, fully stirring, standing at 4 ℃ for 12h, centrifuging at 4000rpm for 10min, and collecting supernatant.

Step 3: concentrating the supernatant obtained in the step 2 to 1/10 of the original volume, uniformly mixing 100-120 mesh polyamide with concentrated solution in a shaking flask according to the feed liquid ratio of 1g to 4mL, shaking for 30min at a shaking table of 200rpm at 37 ℃, washing with deionized water, collecting eluent, concentrating, and freeze-drying to obtain a D-pinitol crude product;

step 4: preparing the D-pinitol crude product obtained in the step 3 into 100mg/mL solution, performing column chromatography by using polystyrene type weak-polarity adsorption resin as a filler, eluting with deionized water at a flow rate of 1mL/min, eluting with 1.5-2BV, and collecting water eluent; then eluting with 40% ethanol at a flow rate of 1.5mL/min and an eluting amount of 1.5-2BV, collecting ethanol eluate, concentrating, spray drying to obtain soybean isoflavone, and ultraviolet spectrophotometry to obtain soybean isoflavone with a content of 40-50% and a yield of 0.31-0.35% (calculated by soybean meal) and an extraction rate of more than or equal to 93%. Adsorbing the protein solution prepared in the step 1 by an activated carbon column, regulating the pH of the eluent to be 4.5, centrifuging at 3000rpm for 5-10min, redissolving the precipitate in deionized water with 4 times of volume, and spray-drying to obtain the soybean protein isolate with low peculiar smell, wherein the yield is 30-35%, and the extraction rate is more than or equal to 75%.

Step 5: and (3) performing chromatographic purification on the water eluent obtained in the step (4) by adopting Amberlyste lRA-21 and Amberlite IR-120 ion exchange resins in sequence, eluting with deionized water with the flow rate of 2mL/min, collecting the eluent, concentrating and freeze-drying. The detection was performed using an Agilent 1260 liquid chromatograph, and D-pinitol analysis standard was purchased from Supelco. Chromatographic conditions: YMC-Pack ployamine II, acetonitrile (A): water (B) =78:22, column temperature 25 ℃, sample injection amount 10 μl, flow rate 1.0mL/min. Detector conditions: the temperature of the drift tube is 110 ℃, the gain coefficient is 1, the gas flow rate is 2.0L/min, the purity of the D-pinitol sample reaches 80%, the yield is 0.20% -0.22%, and the extraction rate is more than or equal to 95%.

The beneficial effects of the invention are as follows:

1. the invention designs a comprehensive utilization process of soybean meal, which combines column chromatography to prepare soybean isoflavone in the process of extracting D-pinitol, wherein the extraction rate of the soybean isoflavone is more than 95 percent, and meanwhile, in the soybean meal extracted by the D-pinitol, the protein loss rate is less than 5 percent, the impurity content in the protein extraction process is obviously reduced, the peculiar smell of soybean protein is also obviously reduced, and the workload of subsequent protein purification is simplified. The invention realizes the comprehensive utilization of the soybean meal, increases the added value of the soybean meal and further widens the application prospect of the soybean meal;

2. the invention uses a microbial fermentation method to remove saccharide impurities with similar properties to D-pinitol in the extract, adopts a polyamide method to rapidly and effectively remove protein and pigment, the impurity sugar removal rate is more than or equal to 98 percent, the protein removal rate is more than or equal to 87 percent, the D-pinitol loss rate is less than 3 percent, the D-pinitol with 80 percent is extracted from the soybean meal raw material with the content of about 0.2 percent, and the extraction rate is more than or equal to 95 percent;

3. by adopting the method, the D-pinitol, the soy protein isolate and the soy isoflavone are comprehensively extracted from the soybean meal, more than 2.0kg (80 percent content) of the D-pinitol can be extracted from each ton of the soybean meal, the value is 3-5 ten thousand yuan, the value of the soy protein isolate is 350kg, the value of the soy isoflavone is more than 3.4kg, the value is nearly ten thousand yuan, the income of more than ten thousand yuan can be realized per ton of the soybean meal after the cost is deducted, and the economic benefit is remarkable;

4. the method is simple, green and efficient, only adopts green solvents such as ethanol, water and the like, does not adopt other toxic and harmful organic reagents, and the extracted D-pinitol, soybean isoflavone and soybean protein isolate are nontoxic and harmless, thereby being beneficial to human health.

Drawings

FIG. 1 is a flow chart of a comprehensive utilization technology of soybean meal provided by the invention;

FIG. 2 is a graph showing the effect of the factors of example 1 on the utilization of the impurity sugars (A is the initial sugar degree, B is the yeast extract, C is the initial pH, and D is the fermentation time);

FIG. 3 is a high performance liquid chromatogram of soybean molasses fermentation of example 2 for 0h (1-D-pinitol, 2-fructose, 3-sucrose, 4-raffinose, 5-stachyose);

FIG. 4 is a high performance liquid chromatogram of soybean molasses fermentation of example 2 for 48h (1-D-pinitol, 2-fructose, 3-sucrose, 5-stachyose);

FIG. 5 is a high performance liquid chromatogram of example 3 (1-D-pinitol, 2-fructose) after decolorization and deproteinization;

FIG. 6 is a high performance liquid chromatogram of example 5D-pinitol (1-D-pinitol) for a sample of 80.5% purity.

Detailed Description

The present invention will be described in detail by way of the following specific examples, which are given for illustration only and are not intended to limit the scope of the present invention.

Example 1: fermentation condition optimization

Step 1: pulverizing soybean meal, sieving (100 mesh), extracting with 80% ethanol at 60deg.C for 30min at a ratio of 1g to 9mL for two times, mixing the extractive solutions, concentrating to sugar degree of 15 ° Brix, adding 3% yeast extract, adjusting pH to 4, and autoclaving at 121deg.C for 30min. Inoculating yeast, culturing at 28deg.C for 50h at 120r/min, and determining the utilization rate of each impurity sugar by HPLC-ELSD method.

Step 2: and screening and optimizing fermentation conditions by adopting a single factor method, and examining the influence of factors such as initial sugar degree, yeast extract addition amount, initial pH, fermentation time and the like on the utilization rate of the mixed sugar. The fixed condition is that the initial sugar degree is 15 DEG Brix,3% yeast extract, the initial pH is 4, and the fermentation is carried out for 50 hours. Sequentially examining the influence of initial sugar degree (5 DEG Brix, 10 DEG Brix, 15 DEG Brix, 20 DEG Brix, 25 DEG Brix), yeast extract addition amount (0%, 1%, 2%, 3%, 4%, 5%), initial pH (3, 4, 5, 6, 7) and fermentation time (0-120 h) on the utilization rate of the impurity sugar;

as a result, as shown in FIG. 2, it was confirmed by analysis of the single factor condition that the optimum conditions for the removal of sugar by fermentation were 15℃Brix,4% yeast extract, initial pH5, 48 hours of fermentation. Experiments prove that under the condition, the utilization rate of the mixed sugar can reach 98.7%, the time consumption is shortest, and the economic benefit is highest.

Example 2: preparation of fermentation broths

Step 1: pulverizing defatted soybean meal, sieving (100 mesh), collecting 100g soybean meal powder, adding 80% ethanol 900mL, extracting at 60deg.C for 30min, extracting twice, mixing the extractive solutions, and retaining residue. Concentrating the extractive solution to about 150mL, measuring sugar degree to 15 ° Brix, adding 0.6g yeast extract (Aladin), adjusting pH to 5, and autoclaving at 121deg.C for 30min;

step 2: inoculating 0.2g of activated fruit wine yeast (Angel SY), culturing at 28deg.C for 120r/min, sampling every 24h, detecting impurity sugar component and content, and fermenting for 72h to obtain soybean molasses fermentation liquid;

step 3: and (5) detecting the impurity sugar. Stachyose, raffinose, sucrose and fructose standard are purchased respectively, quantitatively weighed and dissolved in 70% acetonitrile, and the peak time of each mixed sugar component and the content of each mixed sugar are measured by adopting an HPLC-ELSD method.

As a result, as shown in Table 1, only a small amount of stachyose and raffinose remained in the fermentation broth after 48 hours of fermentation, and the impurity sugar component which was difficult to separate from D-pinitol was consumed and converted by microorganisms, and the impurity sugar utilization rate reached 99.1%.

TABLE 1 variation of the content of miscellaneous sugars in fermentation broths

Example 3: decolorization and deproteinization of fermentation broth

Step 1: concentrating the fermentation liquor to 1/20 of the original volume, adding 5 times of 95% ethanol, standing the mixture at 4deg.C overnight, centrifuging to obtain supernatant, recovering ethanol, and concentrating the supernatant to 1/10 of the original volume;

step 2: and (3) freeze-drying the supernatant concentrate obtained in the step (1), namely CDP (CDP), and deproteinizing and purifying D-pinitol by a polyamide method, an acid precipitation method and a Sevag method respectively.

Polyamide process: 25g of 100-120 mesh polyamide and 100mL of supernatant concentrate are uniformly mixed in a 250mL shaking flask; at room temperature, the polyamide is shaken at 200rpm for 30min to allow the polyamide to fully adsorb proteins, and then filtered, and the filtrate is collected and lyophilized, abbreviated as PDP.

Acid precipitation method: the pH of the supernatant was adjusted to 4.5-4.8 with dilute HCI, centrifuged to remove the precipitate, the supernatant was collected, and lyophilized, abbreviated as ADP.

Sevag method: mixing n-butanol, chloroform and supernatant at a volume ratio of 1:4:5, shaking vigorously for 10min, centrifuging to obtain supernatant, repeating for 3 times, rotary evaporating to remove organic reagent, lyophilizing, and abbreviated as SDP.

Step 3: CDP, PDP, ADP and SDP protein content was determined by Coomassie Brilliant blue G-250 method using bovine serum albumin as a standard. D-pinitol content was determined by HPLC-ELSD.

The results are shown in Table 2. The protein content of PDP and SDP is significantly lower than CDP and ADP compared to the three deproteinization methods. The D-pinitol content SDP is significantly lower than PDP and ADP, indicating that the polyamide process has better deproteinization effect, lower D-pinitol loss, and no introduction of new impurity components compared with the acid precipitation process and Sevag process.

TABLE 2 protein and D-pinitol content

Example 4: separation and detection of soybean isoflavone

Step 1: and (3) dissolving PDP in water, loading the solution to an AB-8 macroporous resin column, washing 1.5BV with water after the adsorption is completed, and collecting water eluent. Eluting with 50% ethanol at a flow rate of 1.5mL/min and an eluting amount of 1.5BV, collecting ethanol eluate, concentrating, and spray drying to obtain soybean isoflavone powder.

Step 2: the soybean isoflavone is measured by ultraviolet spectrophotometry, the soybean isoflavone standard substance (desired) and the sample solution are subjected to spectral scanning on ultraviolet spectrophotometry, the maximum absorption peak of the standard substance and the sample is 259nm, the standard curve is drawn, the soybean isoflavone content in the sample is 40.5%, the yield is 0.32%, and the extraction rate is 93%.

Example 5: separation and detection of D-pinitol

Step 1: sequentially subjecting the AB-8 water eluate to Amberlyste lRA-21 and Amberlite IR-120 ion exchange resins for chromatography, eluting with water to obtain eluate containing D-pinitol, concentrating, and lyophilizing;

step 2: the analysis was performed using an Agilent 1260 liquid chromatograph, D-pinitol analysis standard was purchased from Supelco (Purity. Gtoreq.98%) and the column was YMC-Pack Ployamineii (4.6 mm X250 mm,5 μm), mobile phase acetonitrile (A) water (B) =78:22, column temperature 25 ℃, sample injection volume 10. Mu.L, flow rate 1.0mL/min. Detector conditions: the temperature of the drift tube is 110 ℃, the gain coefficient is 1, the gas flow rate is 2.0L/min, the peak time is 7.8min, the purity is 80.5%, the yield is 0.20%, and the extraction rate is 96%.

Example 6: separation and detection of isolated soy proteins

Adding 900mL of water into the soybean meal filter residue reserved in the step 1 in the example 2, regulating the pH to 8.5 by using food-grade NaOH, heating and extracting for 30min at 60 ℃, repeatedly extracting for 3 times, combining to obtain a protein solution, cooling, carrying out activated carbon column adsorption, regulating the pH of an eluent to 4.5 by using citric acid, centrifuging for 10min at 3000rpm/min, collecting precipitate, redissolving the precipitate in deionized water, drying to obtain soybean protein isolate without obvious beany flavor, and measuring the protein content in a soybean protein isolate sample by using a cow serum albumin (BSA) standard substance by using a Coomassie Brilliant blue G-250 method to obtain a protein content of 91.3%, wherein the yield is 32%, and the extraction rate is 76%.

Claims (6)

1. A method for extracting D-pinitol, soy protein isolate and soy isoflavone by comprehensively utilizing soybean meal is characterized in that:

taking defatted soybean meal as a raw material, removing impurity sugar by microbial fermentation, decoloring and deproteinizing by a polyamide method, obtaining D-pinitol by a column chromatography technology, and simultaneously extracting and separating soybean protein isolate and soybean isoflavone;

the method specifically comprises the following steps:

step 1: pulverizing soybean meal, sieving, adding 80% ethanol, leaching in water bath at 60deg.C for 30-60min, repeatedly leaching for 2-3 times, filtering with 8 layers of gauze, and mixing filtrates; draining bean pulp filter residues, adding alkaline water, leaching in a water bath at 60 ℃ for 30-60min, repeatedly leaching for 2-3 times, filtering with 8 layers of gauze, and mixing to obtain a protein solution;

step 2: concentrating the ethanol extract filtrate obtained in the step 1 to a sugar degree of 15-20 DEG Brix, adding 4g/L yeast extract, adjusting pH to 4.5-5.5, sterilizing, inoculating 1.2-1.4g/L fruit wine yeast, fermenting and culturing, concentrating the fermentation liquor to 1/20 of the original volume, adding 95% ethanol to a final ethanol concentration of 80%, fully stirring, standing at 4 ℃, centrifuging and collecting supernatant;

step 3: concentrating the supernatant obtained in the step 2 to 1/10 of the original volume, uniformly mixing polyamide and concentrated solution in a shaking flask, shaking at 200rpm for 30min at 37 ℃, washing with deionized water, collecting eluent, concentrating, and freeze-drying to obtain a D-pinitol crude product;

step 4: preparing the D-pinitol crude product obtained in the step 3 into a 100mg/mL solution, eluting with deionized water by taking polystyrene type weak-polarity adsorption resin as a filler, and collecting water eluent; eluting with 40% ethanol, collecting ethanol eluate, concentrating, and spray drying to obtain soybean isoflavone; adsorbing the protein solution prepared in the step 1 by an activated carbon column, regulating the pH of the eluent to 4.5, centrifugally collecting the precipitate, redissolving the precipitate in deionized water, and spray-drying to obtain soybean protein isolate with low peculiar smell;

step 5: and (3) performing chromatographic purification on the water eluent obtained in the step (4) by adopting ion exchange resin, eluting by using deionized water with the flow rate of 2mL/min, collecting the eluent, concentrating and freeze-drying to obtain D-pinitol.

2. The method according to claim 1, characterized in that:

in the step 1, the addition amount of 80% ethanol is 1g:8-10mL according to the feed-liquid ratio; the pH value of the alkaline water is 8.5, and the addition amount of the alkaline water is 1g:8-10mL according to the feed-liquid ratio.

3. The method according to claim 1, characterized in that:

in the step 2, the wine yeast is inoculated, and then the wine yeast is fermented and cultured for 48 to 72 hours at the temperature of 28 ℃ by a shaking table at 120 rpm.

4. The method according to claim 1, characterized in that:

in the step 3, the polyamide is 100-120 meshes and is mixed with the concentrated solution according to the ratio of 1g to 4mL of feed liquid.

5. The method according to claim 1, characterized in that:

in the step 4, when deionized water is used for eluting, the eluting flow rate of the deionized water is 1mL/min, and the eluting dosage is 1.5-2BV; when 40% ethanol is used for eluting, the eluting flow rate of the 40% ethanol is 1.5mL/min, and the eluting dosage is 1.5-2BV.

6. The method according to claim 1, characterized in that:

in step 5, the water eluent obtained in step 4 is subjected to chromatographic purification by sequentially adopting Amberlyste lRA-21 and Amberlite IR-120 ion exchange resins.