CN108395468B - Method for recovering protein in bean curd yellow slurry by using astringent persimmon tannin - Google Patents
Method for recovering protein in bean curd yellow slurry by using astringent persimmon tannin Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Separation Of Suspended Particles By Flocculating Agents (AREA)
Abstract
The invention discloses a method for recovering protein in bean curd yellow slurry by using astringent persimmon tannin. The method has the advantages of simple process, low production cost, simple operation and higher efficiency on protein recovery, the modified persimmon tannin has less influence on the chemical action of the protein, the environment cannot be further polluted, the recovery time is short, and a new way is provided for processing and utilizing the persimmon. The method can also be used for treating protein-containing wastewater generated in various fields, such as minced fillet rinsing liquid, starch wastewater, soybean protein wastewater, dairy wastewater and the like.
Description
Technical Field
The invention belongs to the technical field of protein recovery, and particularly relates to a method for recovering protein in bean curd yellow slurry by using modified astringent persimmon tannin as a flocculating agent.
Background
During the preparation process of the bean curd, the soybean is firstly added with water and ground into soybean milk, and then coagulants such as calcium sulfate, gluconolactone and the like are added into the boiled soybean milk to denature and coagulate substances such as protein, amino acid and the like in the soybean milk. The coagulated protein is separated from the juice and pressed into bean curd, and the remaining juice is called bean curd yellow serofluid, which is called yellow serofluid for short. The yellow serofluid is rich in nutrient substances, in particular nitrogen-containing substances such as protein, amino acid and the like which are not solidified and soluble. It was analyzed that when bean curd was produced using 1kg of raw soybean, the amount of discharged soybean milk was about 8.95 kg. The bean curd yellow serofluid contains 0.4-0.5% of whey protein, which accounts for about 17% of the raw material soybean; in addition, many domestic enterprises producing soybean protein isolate from soybeans emit 60-80 m of soybean protein isolate for every 1 ton of soybean protein isolate3If 4 ten thousand tons of soybean protein isolate are produced every year, the discharged yellow serofluid is as high as 200 ten thousand tons. At present, most bean curd production enterprises and soybean protein isolate enterprises use yellow serofluid as wastewater for sewage treatment, and the yellow serofluid is not well utilized. Separating protein from yellow serofluidOn one hand, the whey protein in the yellow serofluid is high-quality soluble protein and has important nutritional effect, and can be used as an important food additive through separation and extraction; on the other hand, organic matters in the yellow slurry water can be reduced through protein recovery, and the sewage treatment load of a bean product factory is reduced.
At present, chitosan is mostly used as a flocculant in protein recovery technologies, for example, protein in rice bran wastewater is treated by chitosan in Lei et al (food industry, 2015,36(07): 165-167); the method comprises the following steps of (1) recovering proteins in surimi rinsing liquid by adopting chitosan in the Shirai (surimi rinsing liquid protein recovery research [ D ]. Zhejiang university, 2012); the method comprises the following steps of (1) recovering protein from marine product processing wastewater by using chitosan, aluminum sulfate, ferric sulfate and polyacrylamide as flocculating agents by the people in the province of Ministry of Zhejiang industry, 2008(02): 133-; gaojie et al adopt flocculants such as carboxymethyl cellulose, chitosan, sodium alginate, polyacrylamide and the like to recover proteins in potato wastewater (recovery and property research of proteins in potato starch wastewater [ D ]. Shanxi university of science and technology, 2012). The chitosan, a natural polymeric flocculant, has the advantages of nature and environmental protection, but the required amount is large when the wastewater is treated, the chitosan is expensive, the cost of wastewater treatment is increased, and other flocculants such as aluminum sulfate, polyacrylamide and polymeric ferric sulfate for treating the wastewater in the prior art also have the defects of poor safety, adverse effect on human bodies and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for recovering protein in bean curd yellow slurry by adopting astringent persimmon tannin.
The technical scheme adopted for solving the technical problems comprises the following steps:
1. preparation of astringent persimmon powder
Picking immature astringent persimmon fruit in 6-month middle ten days, cleaning, removing sepals and fruit stalks with stainless steel knife, cutting into pieces, crushing into paste with beater, making into persimmon pulp, heating to 95 deg.C with jacketed pan, keeping the temperature for 5min for enzyme inactivation, cooling to 30 deg.C, inoculating beer yeast activated with malt juice, and inoculating the inoculum size of beer yeastThe content of cerevisiae Fermentum in the wort is 3 wt%8～109Fermenting for 36-60 h at 30 ℃ per mL; freeze drying the fermented persimmon pulp with a vacuum freeze dryer to obtain astringent persimmon powder.
2. Extraction of tannins
Uniformly mixing astringent persimmon powder and 40-60% volume concentration ethanol water solution according to a material-liquid ratio of 10g:100mL, treating the mixture for 20min at a constant temperature of 50 ℃ under the power of 100W by using an ultrasonic processor, centrifuging the mixture by using a centrifugal machine to remove impurities and insoluble substances, concentrating the supernatant by using a vacuum reduced pressure concentrator at the vacuum degree of 0.009MPa and the temperature of 50-55 ℃ until the soluble solid is 20% tannin concentrated solution, and dehydrating and drying the concentrated solution by using a vacuum freeze dryer at the temperature of-25 ℃ until the water content is 5-7% to obtain the astringent persimmon tannin freeze-dried powder.
3. Modification of tannins
Mixing the astringent persimmon tannin freeze-dried powder with a dimethylamine aqueous solution with the mass fraction of 33% and a formaldehyde aqueous solution with the mass fraction of 37% according to the mass ratio of 1:1: 1.5-1: 2:2, adjusting the pH value to 6-6.5 by using 1mol/L hydrochloric acid, reacting for 3 hours at the temperature of 75-90 ℃, and dehydrating and drying at the temperature of-25 ℃ by using a vacuum freeze dryer until the water content is 5-7% to obtain the modified astringent persimmon tannin.
4. Pretreatment of bean curd yellow serofluid
Filtering bean curd yellow serofluid with a 100 mesh stainless steel screen filter, heating yellow serofluid with plate heat exchanger to 100 deg.C, maintaining the temperature for 20min, and cooling to room temperature.
5. Flocculating proteins
And (3) regulating the pH value of the bean curd yellow serofluid pretreated in the step (4) to 4-5 by using 1mol/L hydrochloric acid, adding 10-20% of modified astringent persimmon tannin aqueous solution according to the addition amount of the modified astringent persimmon tannin of 40-56 mg/L, placing in an environment of 10-30 ℃, uniformly stirring, standing for 80-140 min, centrifuging by using a centrifugal machine, removing supernatant, and taking precipitate.
6. Purification of
Mixing the precipitate obtained in the step 5 with n-hexane according to a feed-liquid ratio of 1g: 5-10 mL, stirring for 50min at a constant temperature of 40 ℃, and volatilizing the n-hexane in a ventilated kitchen to obtain degreased protein; fully and uniformly stirring the degreased protein and the purified water according to the feed liquid mass ratio of 1:8, adjusting the pH to 9 by using 1mol/L sodium hydroxide aqueous solution to dissolve the protein, adjusting the pH to 4 by using 1mol/L hydrochloric acid, and settling the protein; and centrifuging by using a centrifugal machine, removing supernatant, taking out precipitate, and drying and dehydrating by using a hot air drier at the temperature of 50-60 ℃ until the water content is less than 10%.
In the above step 1, fermentation is preferably carried out at 30 ℃ for 48 hours.
In the step 2, preferably, the astringent persimmon powder is uniformly mixed with 50% ethanol water solution according to the material-liquid ratio of 10g to 100 mL.
In the step 3, preferably, the astringent persimmon tannin freeze-dried powder is mixed with a dimethylamine aqueous solution with the mass fraction of 33% and a formaldehyde aqueous solution with the mass fraction of 37% according to the mass ratio of 1:1.5:1.5, the pH value of the mixture is adjusted to 6-6.5 by using 1mol/L hydrochloric acid, the mixture reacts for 3 hours at the temperature of 80 ℃, and the mixture is dehydrated and dried by using a vacuum freeze dryer at the temperature of-25 ℃ until the water content is 5-7%, so that the modified astringent persimmon tannin is obtained.
In the step 5, preferably, the pH of the bean curd yellow serofluid pretreated in the step 4 is adjusted to 4 by 1mol/L hydrochloric acid, and then 10-20% by mass of modified astringent persimmon tannin aqueous solution is added according to the addition of 48mg/L modified astringent persimmon tannin, and the mixture is placed in an environment with the temperature of 20 ℃ and is stirred uniformly and then is kept stand for 100 min.
The method takes the modified persimmon tannin with low price as the flocculating agent to recover the protein in the bean curd yellow slurry, and the result shows that the protein recovery has higher efficiency, and the added modified astringent persimmon tannin has less influence on the chemical action of the protein and can not cause further pollution to the environment. The method of the invention can also be used for the treatment of protein-containing wastewater generated in various fields, such as minced fillet rinsing liquid, starch wastewater, soybean protein wastewater, dairy wastewater and the like.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1
1. Preparation of astringent persimmon powder
Picking immature astringent persimmon fruit in 6-month middle ten days, cleaning, removing sepals and fruit stalks with a stainless steel knife, cutting into pieces, crushing into paste with a beater to obtain persimmon pulp, heating the persimmon pulp to 95 deg.C with a jacketed pan, keeping the temperature for 5min for enzyme inactivation, cooling to 30 deg.C, inoculating beer yeast activated with malt extract, wherein the inoculation amount of beer yeast is 3 wt% of the persimmon pulp, and the amount of beer yeast in the malt extract is 10%8～109Keeping the temperature at 30 ℃ for fermentation with alcohol, converting sugar in the persimmon pulp into alcohol, and removing saccharide substances in the persimmon pulp, wherein the fermentation time is 48 h. Freeze drying the fermented persimmon pulp with a vacuum freeze dryer to obtain astringent persimmon powder.
2. Extraction of tannins
Adding 10L ethanol water solution with volume concentration of 50% into 1kg astringent persimmon powder, treating with ultrasonic processor at power of 100W at constant temperature of 50 deg.C for 20min, centrifuging with centrifuge at 4000r/min for 10min to remove impurities and insoluble substances, collecting supernatant, and determining tannin extraction amount by Folin-phenol method to 48.15 mg/g. And concentrating the supernatant by using a vacuum reduced pressure concentrator at the vacuum degree of 0.009MPa and the temperature of 50 ℃ until the soluble solid content is 20% of tannin concentrated solution, dehydrating and drying by using a vacuum freeze dryer at the temperature of-25 ℃ until the water content is 5% -7%, thus obtaining the astringent persimmon tannin freeze-dried powder, and storing the astringent persimmon tannin freeze-dried powder at the temperature of-4-0 ℃ for later use.
3. Modification of tannins
Adding 1.5g of a dimethylamine aqueous solution with the mass fraction of 33% and 1.5g of a formaldehyde aqueous solution with the mass fraction of 37% into 1.0g of the astringent persimmon tannin freeze-dried powder, adjusting the pH value to 6-6.5 by using 1mol/L hydrochloric acid, reacting at a constant temperature of 80 ℃ for 3 hours, dehydrating and drying at the temperature of-25 ℃ by using a vacuum freeze dryer until the water content is 5% -7%, thus obtaining the modified astringent persimmon tannin, and storing at the temperature of 4 ℃ for later use.
4. Pretreatment of bean curd yellow serofluid
Passing the bean curd yellow serofluid through a filter with a 100-mesh stainless steel screen to remove bean curd residue and other insoluble precipitate; then heating yellow serofluid with plate heat exchanger to 100 deg.C, maintaining the temperature for 20min, and cooling to room temperature to sterilize and prevent spoilage and odor.
5. Flocculating proteins
And (3) taking 100L of the bean curd yellow serofluid pretreated in the step (4), adjusting the pH value of the bean curd yellow serofluid to be 4 by using 1mol/L hydrochloric acid, then taking 4.8g of the modified astringent persimmon tannin obtained in the step (3) to prepare an aqueous solution with the mass fraction of 10%, adding the aqueous solution into the bean curd yellow serofluid, placing the aqueous solution in an environment with the temperature of 20 ℃, uniformly stirring, and standing for 100min to ensure that the modified astringent persimmon tannin powder and the protein are fully flocculated and precipitated. Centrifuging the flocculated and precipitated bean curd yellow serofluid for 7min at 4000r/min, measuring the light absorption values of the supernatant at 215nm and 225nm by using an ultraviolet spectrophotometer, and calculating the recovery rate of the protein in the yellow serofluid to be 85.51%. The supernatant was removed and the precipitate was taken.
6. Purification of
Mixing the precipitate obtained in step 5 with n-hexane at a feed-liquid ratio of 1g:10mL, stirring at 40 deg.C for 50min, leaching for defatting, removing lipid component in crude protein, and volatilizing n-hexane in a ventilated kitchen to obtain defatted protein. And fully and uniformly stirring the degreased protein and the purified water according to the feed liquid mass ratio of 1:8, adjusting the pH to 9 by using a 1mol/L sodium hydroxide aqueous solution to dissolve the protein, adjusting the pH to 4 by using 1mol/L hydrochloric acid, and settling the protein, namely purifying the protein by using an alkali-soluble acid precipitation method. And then centrifuging the mixture in a low-speed centrifuge at the rotating speed of 3000r/min for 30min, removing supernatant, taking out precipitates, and drying and dehydrating the precipitates by using a hot air dryer at the temperature of 50-60 ℃ until the water content is less than 10%.
Comparative example 1
In the step 5 of the example 1, 100L of the pretreated soybean curd yellow serofluid is taken, 1mol/L of hydrochloric acid is used for adjusting the pH value to 4, then 4.8g of the astringent persimmon tannin freeze-dried powder obtained in the step 2 is taken to prepare an aqueous solution with the mass fraction of 10%, the aqueous solution is added into the soybean curd yellow serofluid, the soybean curd yellow serofluid is placed in an environment with the temperature of 20 ℃, the mixture is stirred uniformly, the mixture is kept stand for 100min, the centrifugal operation is carried out for 7min under the condition of 4000r/min, the light absorption values of the supernatant at 215nm and 225nm are measured by an ultraviolet spectrophotometer, and the recovery rate of the protein in the soybean serofluid is calculated to be 70.56%.
In order to determine the process conditions of the present invention, the inventors conducted a number of research experiments, and the specific experimental conditions were as follows:
1. index and method of measurement
Measurement of sugar degree: hand-held saccharimeter
And (3) tannin content determination: forlin-phenol process
Protein content determination: ky type nitrogen determination method
Determination of protein recovery: taking 20mL, 40mL, 60mL, 80mL and 100mL of bean curd yellow serofluid respectively, fixing the volume to 100mL by using distilled water, preparing yellow serofluid with concentration gradients of 20%, 40%, 60%, 80% and 100%, and determining the protein content in each sample by adopting a Kjeldahl method; taking 1mL of yellow serofluid with each concentration gradient to reach a constant volume of 100mL, taking distilled water as a blank sample, respectively measuring the absorbance values A of each sample under 215nm and 225nm by using an ultraviolet spectrophotometer, taking the protein content as an abscissa and A215-A225Establishing a standard curve y of 0.0779x +0.0102 and a correlation coefficient R for the ordinate20.9901; the protein content in the raw soya bean curd water is determined to be 4.016 mg/mL. And protein recovery was calculated from the standard curve.
The protein recovery rate calculation method comprises the following steps: the protein recovery (%) was (1-protein content in supernatant/protein content in raw soybean curd pulp) x 100%
2. Test results and analysis
(1) Determination of persimmon pulp fermentation time
Picking immature astringent persimmon fruit in 6-month middle ten days, cleaning, removing sepals and fruit stalks with a stainless steel knife, cutting into pieces, crushing into paste with a beater to obtain persimmon pulp, heating the persimmon pulp to 95 deg.C with a jacketed pan, keeping the temperature for 5min for enzyme inactivation, cooling to 30 deg.C, inoculating beer yeast activated with malt extract, wherein the inoculation amount of beer yeast is 3% of the weight of persimmon pulp, and the amount of beer yeast in malt extract is 10%8～109Fermenting at 30 deg.C for alcohol fermentation to convert sugar in fructus kaki pulp into wineAnd (4) refining and removing sugar substances in the persimmon pulp. The sugar degrees of the fermentation time periods of 0, 12h, 24h, 36h, 48h, 60h and 72h were measured by a handheld sugar meter.
TABLE 1 variation in sugar content of fermented persimmon pulp
| Fermentation time (h) | 0 | 12 | 24 | 36 | 48 | 60 | 72 |
| Sugar degree (%) | 16 | 15 | 11 | 9 | 8 | 8 | 8 |
As can be seen from Table 1, the sugar degree of persimmon pulp during initial fermentation is 16%, after the beer yeast activated by wort is inoculated, yeast can grow and propagate in a large amount in a short time to consume sugar in fermentation liquor for alcohol fermentation, the fermentation time is continuously increased after 48 hours, and the sugar degree of the fermentation liquor is not changed any more, so that the fermentation time is selected to be 36-60 hours, and preferably 48 hours.
(2) Extraction of tannins
Taking 10g of astringent persimmon powder, respectively adding into 100mL of ethanol water solution with volume concentration of 20%, 40%, 50%, 60% and 80%, treating for 20min at constant temperature of 50 ℃ under power of 100W by using an ultrasonic processor, then centrifuging for 10min under the condition of 4000r/min by using a high-speed centrifuge, removing impurities and insoluble substances, measuring the tannin content in the supernatant, and calculating the tannin extraction amount.
TABLE 2 influence of ethanol concentration on tannin extraction
| Concentration of ethanol | 20％ | 40％ | 50％ | 60％ | 80％ |
| Tannin (mg/g) | 42.78 | 44.92 | 48.15 | 44.01 | 40.62 |
As can be seen from Table 2, the ethanol concentration is from 20% to 50%, the tannin content is continuously increased along with the increase of the ethanol concentration, when the ethanol concentration exceeds 50%, the tannin extraction amount is not increased any more but gradually reduced when the ethanol concentration is continuously increased, and the change trend occurs because the ethanol with high concentration can promote the precipitation of some alcohol-soluble substances to influence the extraction rate of the tannin, so when the ethanol concentration is 50%, the tannin has the maximum extraction value of 48.15 mg/g. Therefore, the ethanol volume concentration of the invention is selected to be 40-60%, preferably 50%.
(3) Effect of heating temperature on protein recovery ratio during tannin modification
Adding 1.5g of a dimethylamine aqueous solution with the mass fraction of 33% and 1.5g of a formaldehyde aqueous solution with the mass fraction of 37% into 1.0g of the astringent persimmon tannin freeze-dried powder, adjusting the pH value to be 6-6.5 by using 1mol/L hydrochloric acid, reacting for 3h at the conditions of 70 ℃, 75 ℃, 80 ℃, 85 ℃ and 90 ℃, respectively, and dehydrating and drying at the temperature of-25 ℃ by using a vacuum freeze dryer until the water content is 5-7% to obtain the modified astringent persimmon tannin. Taking 100mL of pretreated bean curd yellow serofluid, adjusting the pH value of the bean curd yellow serofluid to be 6 by using 1mol/L hydrochloric acid, respectively adding 0.32mL of aqueous solution with the mass fraction of 1% prepared by the modified astringent persimmon tannin obtained after modification at different temperatures (because the addition amount of the modified astringent persimmon tannin in each liter of bean curd yellow serofluid is very small, aqueous solution with the mass concentration of 1% is prepared for convenient sampling test so as to reduce test error), wherein the addition amount of the modified astringent persimmon tannin is 32mg/L, placing the modified astringent persimmon tannin in an environment of 40 ℃, uniformly stirring, standing for 80min, then centrifuging for 7min under the condition of 4000r/min, taking supernatant, measuring the absorbance values of the supernatant at 215nm and 225nm by using an ultraviolet spectrophotometer, and calculating the recovery rate of protein in the bean curd yellow serofluid.
TABLE 3 Effect of different reaction temperatures on protein recovery
| Reaction temperature | 70 | 75 | 80 | 85 | 90 |
| Protein recovery (%) | 69.46 | 72.31 | 73.60 | 72.15 | 72.16 |
As can be seen from table 3, when the mass ratio of the astringent persimmon tannin freeze-dried powder, the dimethylamine aqueous solution with the mass fraction of 33% and the formaldehyde aqueous solution with the mass fraction of 37% is 1:1.5:1.5, the flocculation effect is improved along with the improvement of the reaction temperature, and when the addition amount of the modified astringent persimmon tannin is 32mg/L, the flocculation effect of the modified astringent persimmon tannin corresponding to the temperature of 80 ℃ is the best, the protein recovery rate reaches 73.60%, and the temperature is continuously increased, so that formaldehyde and dimethylamine are volatilized, and the probability of participating in the liquid phase reaction is reduced, therefore, the reaction temperature is selected to be 75-90 ℃, and the best is 80 ℃.
(4) Influence of raw material ratio on protein recovery rate during tannin modification
Mixing 1.0g of astringent persimmon tannin freeze-dried powder, a dimethylamine aqueous solution with the mass fraction of 33% and a formaldehyde aqueous solution with the mass fraction of 37% according to the mass ratio of 1:1:1, 1:1:1.5, 1:1.5:1.5 and 1:2:2 respectively, adjusting the pH value to be 6-6.5 by using 1mol/L hydrochloric acid, reacting for 3 hours at the temperature of 80 ℃, and dehydrating and drying by using a vacuum freeze dryer at the temperature of minus 25 ℃ until the water content is 5-7% to obtain the modified astringent persimmon tannin. Taking 100mL of pretreated bean curd yellow serofluid, adjusting the pH value of the bean curd yellow serofluid to be 6 by using 1mol/L hydrochloric acid, respectively adding 0.32mL of aqueous solution with the mass fraction of 1% prepared from the modified astringent persimmon tannin obtained under different raw material proportions (because the addition amount of the modified astringent persimmon tannin in each liter of bean curd yellow serofluid is very small, aqueous solution with the mass concentration of 1% is prepared for convenient sampling test so as to reduce test errors), wherein the addition amount of the modified astringent persimmon tannin is 32mg/L, placing the modified astringent persimmon tannin in an environment of 40 ℃, uniformly stirring, standing for 80min, centrifuging for 7min under the condition of 4000r/min, taking supernatant, measuring the absorbance values of the supernatant at 215nm and 225nm by using an ultraviolet spectrophotometer, and calculating the recovery rate of protein in the bean curd yellow serofluid.
TABLE 4 Effect of different raw material ratios on protein recovery
| Raw material ratio | 1:1:1 | 1:1:1.5 | 1:1.5:1 | 1:1.5:1.5 | 1:2:2 |
| Protein recovery (%) | 70.20 | 73.18 | 72.54 | 73.60 | 73.16 |
As can be seen from Table 4, under the conditions that the addition amount of the modified astringent persimmon tannin is 32mg/L and the temperature is 80 ℃, when the mass ratio of the astringent persimmon tannin freeze-dried powder to the dimethylamine aqueous solution with the mass fraction of 33% to the formaldehyde aqueous solution with the mass fraction of 37% is increased, the protein recovery rate is improved, the raw material ratio is 1:1.5:1.5, and the protein recovery rate reaches 73.60%. The raw materials are selected according to the ratio of 1:1: 1.5-1: 2:2, and the optimal ratio is 1:1.5: 1.5.
(5) Influence of addition amount of modified astringent persimmon tannin on protein recovery rate
Taking 50mL of pretreated bean curd yellow serofluid, adjusting the pH value to 6 by using 1mol/L hydrochloric acid, then respectively adding 0.04, 0.08, 0.12, 0.16, 0.20, 0.24 and 0.28mL of modified astringent persimmon tannin aqueous solution with the mass fraction of 1%, placing in an environment with the temperature of 40 ℃, uniformly stirring, standing for 80min, then centrifuging for 7min under the condition of 4000r/min, taking supernatant, measuring the absorbance values of the supernatant at 215nm and 225nm by using an ultraviolet spectrophotometer, and calculating the recovery rate of protein in the bean curd yellow serofluid. Meanwhile, unmodified freeze-dried powder of astringent persimmon tannin is used for comparison test.
TABLE 5 influence of the amount of added modified astringent persimmon tannin on the recovery of yellow serofluid
| Addition amount (mL) | 0.04 | 0.08 | 0.12 | 0.16 | 0.20 | 0.24 | 0.28 |
| Recovery ratio of modified tannin (%) | 24.70 | 48.49 | 61.80 | 73.60 | 76.60 | 80.40 | 74.45 |
| Tannin recovery (%) | 23.45 | 41.44 | 52.80 | 60.60 | 64.19 | 67.95 | 61.81 |
Table 5 shows that the recovery effect of the modified tannin on protein is obviously higher than that of tannin, and the addition amount of the modified tannin has obvious influence on the recovery of the protein. With the increasing of the addition amount of the modified astringent persimmon tannin from 8-48 mg/L, the recovery rate of the protein is remarkably improved and can reach 80.40% at most, because the modified tannin is combined with protein molecules to form floccule settlement, the higher the concentration of the modified tannin is, the more the combined protein is, and the recovery rate is increased. Therefore, the modified astringent persimmon tannin is adopted to recover the protein in the bean curd yellow slurry, and the addition amount of the modified astringent persimmon tannin is 40-56 mg/L, and the optimal addition amount is 48 mg/L.
(6) Effect of flocculation time on protein recovery
Taking 50mL of pretreated bean curd yellow serofluid, adjusting the pH value of the bean curd yellow serofluid to 6 by using 1mol/L hydrochloric acid, then respectively adding 0.16mL of modified astringent persimmon tannin aqueous solution with the mass fraction of 1%, placing the modified astringent persimmon tannin aqueous solution in an environment of 40 ℃, uniformly stirring, respectively standing for 20, 40, 60, 80, 100, 120 and 140min, centrifuging for 7min under the condition of 4000r/min, taking a supernatant, measuring absorbance values of the supernatant at 215nm and 225nm by using an ultraviolet spectrophotometer, and calculating the recovery rate of protein in the bean curd yellow serofluid. Meanwhile, unmodified freeze-dried powder of astringent persimmon tannin is used for comparison test.
TABLE 6 Effect of flocculation time on protein recovery
| Time (min) | 20 | 40 | 60 | 80 | 100 | 120 | 140 |
| Recovery ratio of modified tannin (%) | 62.71 | 62.90 | 66.90 | 73.60 | 76.70 | 75.90 | 73.64 |
| Tannin recovery (%) | 40.90 | 41.58 | 53.90 | 60.60 | 68.67 | 62.40 | 62.15 |
Table 6 shows that the recovery rate of protein is increased and then decreased along with the increase of flocculation time, the recovery rate of the modified tannin and the tannin to the protein is maximized when the mixture is kept still for 100min, when the standing time exceeds 100min, the desorption phenomenon can occur in overlong time, so that part of the protein is re-dissolved in water, the recovery rate of the protein is reduced, when the standing time is 100min, the maximum recovery rate of the modified tannin is 76.70%, and the maximum recovery rate of the tannin is 68.67%, which indicates that the recovery effect of the modified tannin to the protein is obviously higher than that of the tannin. Therefore, the invention adopts the modified astringent persimmon tannin to recover the protein in the bean curd yellow slurry, and the flocculation time is 80-140 min, preferably 100 min.
(7) Effect of flocculation temperature on protein recovery
Taking 50mL of pretreated bean curd yellow serofluid, adjusting the pH value of the bean curd yellow serofluid to 6 by using 1mol/L hydrochloric acid, then respectively adding 0.16mL of modified astringent persimmon tannin aqueous solution with the mass fraction of 1%, respectively placing the modified astringent persimmon tannin aqueous solution in the environments of 10 ℃,20 ℃, 30, 40, 50, 60 and 70 ℃, uniformly stirring, standing for 80min, centrifuging for 7min under the condition of 4000r/min, taking supernatant fluid, measuring absorbance values of the supernatant fluid under 215nm and 225nm by using an ultraviolet spectrophotometer, and calculating the recovery rate of protein in the bean curd yellow serofluid. Meanwhile, unmodified freeze-dried powder of astringent persimmon tannin is used for comparison test.
TABLE 7 Effect of flocculation temperature on protein recovery
| Temperature (. degree.C.) | 10 | 20 | 30 | 40 | 50 | 60 | 70 |
| Recovery ratio of modified tannin (%) | 76.18 | 80.48 | 75.39 | 73.60 | 70.10 | 69.60 | 68.66 |
| Tannin recovery (%) | 64.65 | 68.18 | 63.40 | 60.60 | 53.90 | 53.90 | 53.90 |
Table 7 shows that the modified tannin has a significantly higher protein recovery than tannin and that the protein recovery decreases gradually when the flocculation temperature is higher than 20 ℃. Therefore, the flocculation temperature of the protein in the bean curd yellow slurry recovered by adopting the modified astringent persimmon tannin is 10-30 ℃, and the optimal flocculation temperature is 20 ℃.
(8) Effect of pH on protein recovery
Taking 50mL of pretreated bean curd yellow serofluid, adjusting the pH value to 3, 4, 5, 6, 7, 8 and 9 by using 1mol/L hydrochloric acid or 1mol/L sodium hydroxide aqueous solution respectively, then adding 0.16mL of modified astringent persimmon tannin aqueous solution with the mass fraction of 1%, placing in an environment at 40 ℃, uniformly stirring, standing for 80min, centrifuging for 7min under the condition of 4000r/min, taking supernatant fluid, measuring the absorbance values of the supernatant fluid at 215nm and 225nm by using an ultraviolet spectrophotometer, and calculating the recovery rate of protein in the bean curd yellow serofluid. Meanwhile, unmodified freeze-dried powder of astringent persimmon tannin is used for comparison test.
TABLE 8 Effect of pH on protein recovery
| pH | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| Recovery ratio of modified tannin (%) | 72.90 | 77.89 | 76.60 | 73.60 | 72.90 | 72.67 | 66.90 |
| Tannin recovery (%) | 54.12 | 64.40 | 63.12 | 60.60 | 60.20 | 60.19 | 60.15 |
Table 8 shows that the recovery of protein reached the maximum at pH 4, the recovery of protein by modified tannin was 77.89% at the maximum, and the recovery of protein by tannin was 64.40% at the maximum, with increasing pH and decreasing recovery of protein gradually. Therefore, the pH value of the protein in the bean curd yellow slurry recovered by adopting the modified astringent persimmon tannin is 4-5, and the best pH value is 4.
Claims (5)
1. A method for recovering protein in bean curd yellow slurry by using astringent persimmon tannin is characterized by comprising the following steps:
(1) preparation of astringent persimmon powder
Picking immature astringent fructus kaki in 6-month middle ten days, cleaning, removing sepals and fruit stalks with stainless steel knife, cutting into pieces, crushing into paste with beater, making into fructus kaki pulp, heating to 95 deg.C with jacketed pan, keeping the temperature for 5min for enzyme inactivation, cooling to 30 deg.C, inoculating beer yeast activated with succus Hordei Germinatus, and making into beerThe inoculation amount of Saccharomyces cerevisiae is 3% of persimmon pulp weight, and the amount of cerevisiae Fermentum in wort is 108～109Fermenting for 36-60 h at 30 ℃ per mL; freeze drying the fermented persimmon pulp by using a vacuum freeze dryer to prepare astringent persimmon powder;
(2) extraction of tannins
Uniformly mixing astringent persimmon powder and 40-60% volume concentration ethanol water solution according to a material-liquid ratio of 10g:100mL, carrying out constant temperature treatment at 50 ℃ for 20min by using an ultrasonic processor under the power of 100W, centrifuging by using a centrifugal machine, removing impurities and insoluble substances, concentrating the supernatant by using a vacuum reduced pressure concentrator at the vacuum degree of 0.009MPa and the temperature of 50-55 ℃ until the soluble solid is 20% tannin concentrate, and dehydrating and drying by using a vacuum freeze dryer at-25 ℃ until the water content is 5-7% to obtain the astringent persimmon tannin freeze-dried powder;
(3) modification of tannins
Mixing the astringent persimmon tannin freeze-dried powder with a dimethylamine aqueous solution with the mass fraction of 33% and a formaldehyde aqueous solution with the mass fraction of 37% according to the mass ratio of 1:1: 1.5-1: 2:2, adjusting the pH value to 6-6.5 by using 1mol/L hydrochloric acid, reacting for 3 hours at the temperature of 75-90 ℃, and dehydrating and drying at the temperature of-25 ℃ by using a vacuum freeze dryer until the water content is 5-7% to obtain modified astringent persimmon tannin;
(4) pretreatment of bean curd yellow serofluid
Filtering bean curd yellow serofluid with a filter of 100 mesh stainless steel screen, heating yellow serofluid with plate heat exchanger to 100 deg.C, maintaining the temperature for 20min, and cooling to room temperature;
(5) flocculating proteins
Regulating the pH value of the bean curd yellow serofluid pretreated in the step (4) to 4-5 by using 1mol/L hydrochloric acid, adding 10-20% of modified astringent persimmon tannin aqueous solution according to the addition amount of the modified astringent persimmon tannin of 40-56 mg/L, placing in an environment of 10-30 ℃, uniformly stirring, standing for 80-140 min, centrifuging by using a centrifuge, removing supernatant, and taking precipitate;
(6) purification of
Mixing the precipitate obtained in the step (5) with n-hexane according to a feed-liquid ratio of 1g: 5-10 mL, stirring for 50min at a constant temperature of 40 ℃, and putting the mixture in a fume hood to volatilize the n-hexane to obtain degreased protein; fully and uniformly stirring the degreased protein and the purified water according to the feed liquid mass ratio of 1:8, adjusting the pH to 9 by using 1mol/L sodium hydroxide aqueous solution to dissolve the protein, adjusting the pH to 4 by using 1mol/L hydrochloric acid, and settling the protein; and centrifuging by using a centrifugal machine, removing supernatant, taking out precipitate, and drying and dehydrating by using a hot air drier at the temperature of 50-60 ℃ until the water content is less than 10%.
2. The method for recovering protein from bean curd yellow slurry with astringent persimmon tannin as claimed in claim 1, wherein: in the step (1), fermentation is carried out for 48h at 30 ℃.
3. The method for recovering protein from bean curd yellow slurry with astringent persimmon tannin as claimed in claim 1, wherein: in the step (2), the astringent persimmon powder and 50% ethanol water solution are uniformly mixed according to the material-liquid ratio of 10g to 100 mL.
4. The method for recovering protein from bean curd yellow slurry with astringent persimmon tannin as claimed in claim 1, wherein: in the step (3), the astringent persimmon tannin freeze-dried powder is mixed with a dimethylamine aqueous solution with the mass fraction of 33% and a formaldehyde aqueous solution with the mass fraction of 37% according to the mass ratio of 1:1.5:1.5, the pH value of the mixture is adjusted to 6-6.5 by using 1mol/L hydrochloric acid, the mixture reacts for 3 hours at the temperature of 80 ℃, and the mixture is dehydrated and dried by using a vacuum freeze dryer at the temperature of-25 ℃ until the water content is 5-7%, so that the modified astringent persimmon tannin is obtained.
5. The method for recovering protein from bean curd yellow slurry with astringent persimmon tannin as claimed in claim 1, wherein: in the step (5), the pH of the bean curd yellow serofluid pretreated in the step (4) is adjusted to 4 by 1mol/L hydrochloric acid, and then 10-20% of modified astringent persimmon tannin aqueous solution is added according to the addition of 48mg/L modified astringent persimmon tannin, and the mixture is placed in an environment with the temperature of 20 ℃, stirred uniformly and then kept stand for 100 min.
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| CN102229628A (en) * | 2011-06-16 | 2011-11-02 | 五峰赤诚生物科技有限公司 | Method for extracting persimmon tannin from persimmons |
| CN104311812A (en) * | 2014-09-30 | 2015-01-28 | 西安华陆环保设备有限公司 | Preparation method for natural vegetable tannin quaternary ammonium salt modified flocculant |
| CN104530145A (en) * | 2014-12-30 | 2015-04-22 | 桂林得坤生物科技股份有限公司 | Method for extracting high-purity persimmon tannin from persimmon fruits |
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| CN102229628A (en) * | 2011-06-16 | 2011-11-02 | 五峰赤诚生物科技有限公司 | Method for extracting persimmon tannin from persimmons |
| CN104311812A (en) * | 2014-09-30 | 2015-01-28 | 西安华陆环保设备有限公司 | Preparation method for natural vegetable tannin quaternary ammonium salt modified flocculant |
| CN104530145A (en) * | 2014-12-30 | 2015-04-22 | 桂林得坤生物科技股份有限公司 | Method for extracting high-purity persimmon tannin from persimmon fruits |
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