CN112410113A - Method for preparing pumpkin seed oil by industrial enzyme method - Google Patents
Method for preparing pumpkin seed oil by industrial enzyme method Download PDFInfo
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- CN112410113A CN112410113A CN201910781071.2A CN201910781071A CN112410113A CN 112410113 A CN112410113 A CN 112410113A CN 201910781071 A CN201910781071 A CN 201910781071A CN 112410113 A CN112410113 A CN 112410113A
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/02—Pretreatment
- C11B1/04—Pretreatment of vegetable raw material
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/02—Pretreatment
- C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/16—Refining fats or fatty oils by mechanical means
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Mechanical Engineering (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for preparing pumpkin seed oil by an industrialized enzyme method, which selects pumpkin seed kernels as raw materials, and is subjected to (1) raw material pretreatment, crushing and grinding; (2) carrying out enzymolysis by using compound enzyme; (3) alkali treatment; (4) carrying out enzymolysis by protease; (5) carrying out primary centrifugal separation; (6) performing enzymolysis and demulsification; (7) performing secondary centrifugal separation; (8) the products are combined and these processes yield pumpkin seed oil. The invention optimizes the traditional hydrolase oil extraction process, adopts polysaccharide hydrolase and protease for stepwise enzymolysis, can ensure that the protease and the cell wall polysaccharide hydrolase respectively play the best role under the respective optimum pH, reduces the addition amount of the enzyme, and can improve the oil extraction rate and reduce the production cost without other physical auxiliary modes except wet crushing. In addition, the emulsion breaking process is further optimized by combining an enzyme method with an isoelectric point method, the emulsion breaking effect is obvious, the oil extraction rate is high, the quality of oil is good, and the industrial production can be realized.
Description
Technical Field
The invention belongs to the technical field of food industry, and particularly relates to a method for preparing pumpkin seed oil by an industrial enzyme method.
Background
Because the pumpkin seeds are rich in fat, the pumpkin seeds can be used as oil crops, the fat content is higher, and the crude fat content can reach 35.0-59.4%. The pumpkin seed oil contains various beneficial substances, is particularly rich in linoleic acid, phytosterol, tocopherol, squalene, oleic acid and other bioactive components, is highly unsaturated oil, and has high oxidation stability. Can relieve symptoms related to arthritis; reducing the development of hypertension, and relieving hypercholesterolemia and diabetes; is relevant to the prevention of prostate cancer; is beneficial to improving the immunity of human body and resisting ultraviolet radiation. Is considered as a supplement to the human healthy diet and has potential food and industrial application adaptability.
At present, the extraction technology of the pumpkin seed oil is not perfect, most of the pumpkin seed oil is extracted by traditional squeezing and organic solvent extraction methods, which not only wastes time and labor, but also has low extraction rate, difficult separation, low purity and the like. For example, the traditional squeezing method is used for extracting the oil, the high temperature in the squeezing process causes the color of the finished oil to be darker, functional components in the oil are damaged, the biological function is lost, and the refining is difficult; the organic solvent hexane extraction method has the advantages that a large amount of organic solvent remained in the finished oil needs equipment for removing the organic reagent, so the cost is high and the method is unsafe; the supercritical fluid extraction technology has large equipment capital investment, low production capacity and high production cost; the oil extraction process by aqueous enzymatic method uses water as disperse phase, uses various enzymes to degrade oil cells in the water phase to leach oil, and separates oil and non-oil components (protein and carbohydrate) by affinity difference between oil and water and specific gravity difference between oil and water. The oil obtained by treating the oil material by the aqueous enzymatic method has high quality and mild conditions, and can reduce energy consumption. At present, the research on the oil extraction process by the aqueous enzymatic method is not complete, and the problems of the type of enzyme, the optimal enzymolysis condition, emulsion breaking of an emulsion system, separation of oil and fat and the like need to be further researched and solved.
At present, few reports are reported in the research of pumpkin seed oil in China, and the invention patent CN1766075A in China refers to a method for extracting pumpkin seed oil and pumpkin seed protein by grinding and enzymolysis of germinated pumpkin seeds as raw materials. The adopted extraction method is a conventional enzymolysis method and does not have the advantage of aqueous enzymatic extraction as described above. The dry grinding is not adopted, the wet grinding is directly adopted, and the grease is not fully released. In addition, in the chinese invention patent CN101455240A "method for extracting pumpkin seed oil by aqueous enzymatic method", mention is made of using pumpkin seeds as raw material, drying in the sun, pulverizing, and performing enzymatic hydrolysis to extract pumpkin seed oil. The pumpkin seeds are used for extraction, the pumpkin seed peels have certain influence on the enzymolysis effect, and the recycling of the pumpkin seed protein is difficult, so that the method is not suitable for industrial production. The universal cell crusher is adopted for crushing, no colloid mill is used for wet grinding, the grease is not fully released, and the method is not suitable for industrial production.
There are few foreign patents on the extraction of pumpkin seed oil by the aqueous enzymatic method, and much attention is focused on the pumpkin seed oil as an additive for food or medicine for assisting in the treatment of benign prostate diseases and breast cancer, and U.S. patent application 09/259, 857diet supplement relating to prostate cancer and nettle root suggests that a Dietary supplement containing saw palmetto, pumpkin seed oil and nettle root can help to maintain the health of prostate gland. Anti-aromatic compounds containing extracts of nettle root, pumpkin seed oil, African cherry bark, etc. as taught in U.S. patent No. 09/921, 018 Anti-aromatic pharmaceutical composition for controlling testosterone/estrone rates, are useful in the treatment of benign prostate disease and breast cancer.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for preparing pumpkin seed oil by an industrial enzyme method. The invention solves the problems of improving the extraction rate and safety of the pumpkin seed oil prepared by the aqueous enzymatic method: namely, the physical wall-breaking auxiliary method improves the oil yield, and simultaneously has large energy consumption and high cost; and the organic solvent auxiliary method is adopted to improve the oil yield, and the organic solvent is remained in the finished oil, so that the cost is high, the method is unsafe, and the method is not suitable for the industrialized production of the pumpkin seed oil.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for preparing pumpkin seed oil by an industrial enzyme method comprises the following steps:
(1) raw material pretreatment, crushing and grinding: cleaning pumpkin seed kernels, baking the pumpkin seed kernels in a drying box for 1h at 105 ℃, then crushing the pumpkin seed kernels and sieving the pumpkin seed kernels with a 40-mesh sieve, wherein the grain size of pumpkin seed powder is less than 0.45mm, adding 3-5 times of purified water, and grinding the mixture into pulp by using a colloid mill;
(2) carrying out compound enzyme enzymolysis: adding hydrochloric acid or a dilute sodium hydroxide solution into the feed liquid obtained by grinding, adjusting the pH value to 4.5-6.0, heating to 40-60 ℃, adding 1-2% of cellulase and pectinase respectively, and performing heat preservation and enzymolysis for 2-5 hours;
(3) alkali treatment: adding a proper amount of dilute sodium hydroxide solution into the feed liquid obtained by enzymolysis, adjusting the pH value to 8-9, and slowly stirring for 20-60 min;
(4) and (3) protease enzymolysis: adjusting the pH value of the feed liquid after alkali treatment to 6.8-7.5, adding 1000-5000 (U/g protein) neutral protease, performing enzymolysis at 40-60 ℃ for 3-5 h, heating to 80-90 ℃, performing heat preservation for 5-10 min, inactivating enzyme, and cooling;
(5) primary centrifugal separation: carrying out centrifugal separation by adopting a 5000-10000 r/min automatic slag discharge disc type separator to obtain emulsion liquid, free oil I and residues;
(6) and (3) enzymolysis demulsification: adding 0.5-2 times of purified water into the emulsion, uniformly stirring, adjusting the pH value of the emulsion to 9-11, heating to 40-60 ℃, adding 2000-4000U/g of bacillus licheniformis alkaline protease into the emulsion, carrying out heat preservation and enzymolysis for 2-4 h, heating to 80-90 ℃, preserving the heat for 10-15 min, inactivating the enzyme, and cooling;
(7) and (3) secondary centrifugal separation: adjusting the pH value of the mixed solution after enzymolysis and demulsification to 4.0-5.0, and performing centrifugal separation by using an automatic disc type separator with the speed of 5000-10000 r/min to obtain protein hydrolysate and free oil II;
(8) combining products: and combining the free oil I and the free oil II to obtain the pumpkin seed oil prepared by the enzyme method.
Further, hydrochloric acid solution is added in the step (4) and the step (7) to adjust the pH value.
Further, dilute sodium hydroxide solution is added in the step (6) to adjust the pH value.
Has the advantages that: the invention optimizes the traditional hydrolase oil extraction process, adopts polysaccharide hydrolase and protease for stepwise enzymolysis, can ensure that the protease and the cell wall polysaccharide hydrolase respectively play the best role under the respective optimal pH, reduces the addition amount of the enzyme, can improve the oil extraction rate without wet grinding by a colloid mill, and reduces the production cost. In addition, the emulsion breaking process is further optimized by combining an enzyme method with an isoelectric point method, the emulsion breaking effect is obvious, the oil extraction rate is high, the quality of oil is good, and the industrial production can be realized.
Drawings
FIG. 1 is a flow chart of the manufacturing process of the present invention;
FIG. 2 is a schematic diagram illustrating the influence of the type of enzyme on the determination index in the protease enzymolysis process according to the embodiment of the present invention;
FIG. 3 is a cross-sectional view of the enzyme hydrolysis process of the embodiment of the present invention showing the interaction response among the factors of the enzyme hydrolysis time and temperature, the enzyme hydrolysis temperature and enzyme addition amount, the enzyme hydrolysis time and enzyme, the enzyme hydrolysis temperature and the enzyme addition amount;
FIG. 4 is a schematic diagram illustrating the influence of the type of enzyme on the demulsification rate in the enzymatic demulsification process according to the embodiment of the invention;
FIG. 5 is a cross-sectional view of the liquid-to-material ratio and the enzymolysis pH, the liquid-to-material ratio and the enzyme addition amount, the enzymolysis temperature and the enzymolysis pH in the enzymolysis demulsification process according to the embodiment of the present invention;
FIG. 6 is a schematic diagram showing the effect of different pH values on the Zeta potential of an emulsion in an example of the present invention;
FIG. 7 is a GC chromatogram of fatty acids in pumpkin seed oil in the physicochemical indices in the example of the present invention;
FIG. 8 is a squalene standard HPLC chromatogram;
FIG. 9 is a squalene standard curve;
FIG. 10 is a squalene HPLC chromatogram of pumpkin seed oil in an example of the present invention.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
Example 1
A method for preparing pumpkin seed oil by industrial enzyme method is shown in figure 1, and comprises the following steps:
(1) raw material pretreatment, crushing and grinding: cleaning pumpkin seed kernels, baking the pumpkin seed kernels in a drying oven at 105 ℃ for 1h, then crushing the pumpkin seed kernels, sieving the pumpkin seed kernels with a 40-mesh sieve, wherein the particle size of pumpkin seed powder is less than 0.45mm, adding 3 times of purified water, and grinding the mixture by using a colloid mill to obtain a feed liquid with the liquid-material ratio of 3:1 mL/g;
(2) carrying out compound enzyme enzymolysis: adding hydrochloric acid or sodium hydroxide dilute solution into the feed liquid obtained by grinding, adjusting pH to 5.10, heating to 50 deg.C, adding cellulase and pectase in an amount of 2.5%, wherein cellulase is 1.5% and pectase is 1%, and performing enzymolysis for 3 hr under heat preservation;
(3) alkali treatment: adding appropriate amount of dilute sodium hydroxide solution into the feed liquid obtained by enzymolysis, adjusting pH to 8.5, and slowly stirring for 35 min;
(4) and (3) protease enzymolysis: adding hydrochloric acid solution into the feed liquid after alkali treatment to adjust pH value to 7.2, adding 2500 (U/g protein) neutral protease, performing enzymolysis at 55 deg.C for 4h, heating to 80 deg.C, maintaining the temperature for 10min to inactivate enzyme, and cooling;
(5) primary centrifugal separation: centrifuging with 8212r/min automatic residue discharge disc separator to obtain emulsion, free oil I and residue;
(6) and (3) enzymolysis demulsification: adding 0.9 times of purified water into the emulsion, stirring uniformly, adjusting the liquid-material ratio to 0.9: 1 (mL/g), adding dilute sodium hydroxide solution to adjust the pH value to 10, heating to 51 ℃, adding 3078U/g of Bacillus licheniformis alkaline protease protein, performing enzymolysis for 2.5h while keeping the temperature, heating to 80 ℃, keeping the temperature for 10min, inactivating the enzyme, and cooling;
(7) and (3) secondary centrifugal separation: adding a hydrochloric acid solution into the mixed solution after the enzymolysis and the demulsification to adjust the pH value to 4.0-5.0, and performing centrifugal separation by adopting an 8212r/min automatic disc type separator to obtain protein hydrolysate and free oil II;
(8) combining products: and combining the free oil I and the free oil II to obtain the pumpkin seed oil prepared by the enzyme method.
The pumpkin seed oil prepared by the method has the acid value of 1.43mg/g and the peroxide value of 3.62mmol/kg, meets the hygienic standard of edible vegetable oil, and does not need to be further refined; the relative contents of linoleic acid and linolenic acid are respectively 9.85 percent and 6.08 percent, the content of oleic acid is 50.22 percent, the content of palmitic acid is 8.49 percent, the content of stearic acid is 25.39 percent, and the content of squalene is 1.51 mg/g.
According to the method for preparing pumpkin seed oil by the industrial enzyme method, which is obtained by the embodiment of the invention, the process condition determining process is as follows:
1. determination of process conditions of complex enzyme enzymolysis wall breaking
The single-factor results of enzymolysis temperature, enzyme addition amount, liquid-material ratio and enzymolysis pH are analyzed in detail, and reasonable experimental determination is carried out according to the oil extraction rate and the oil content of residue as reference indexes, wherein the orthogonal test results are shown in Table 1.
TABLE 1 orthogonal experimental results of cell wall disruption process
The influence on the oil content of the residue and the oil extraction rate is in the order of: the temperature is more than the enzyme adding amount and more than the liquid-material ratio and more than the enzymolysis pH, and the oil extraction rate is as follows: the enzyme addition amount is less than the temperature, less than the liquid-material ratio and less than the enzymolysis pH. The optimal condition of the oil content of the residue is A3B2C2D1The optimal condition of oil extraction rate is A3B2C3D1. The optimal oil extraction condition is obtained by considering the production cost and integrating the experimental data and is A3B2C2D1Namely, the liquid-material ratio is 3:1mL/g, the enzymolysis pH value is 5.10, the dosage of the complex enzyme is 2.5 percent, and the enzymolysis temperature is 50 ℃.
2. Determination of protease enzymolysis process conditions
2.1 selection of enzyme species
As shown in FIG. 2, each test index was varied to some extent when different kinds of proteases were used for the enzymatic hydrolysis. For the enzyme types related in the table above, the neutral protease has a significant influence on the oil extraction rate (P < 0.05), and the oil extraction rate is 71.10%; the lowest oil extraction rate is acid protease which is only 51.69%. However, the oil content of the residue is analyzed by the oil content of the residue, which is the opposite of the oil extraction rate, wherein the oil content of the residue of the neutral protease is the minimum, and is 10.80 percent; the oil content of the residue of acidic protease was 19.40% at the maximum. The protease is used for hydrolyzing protein in lipoprotein complex in cells, and breaking the wrapping state of oil drops, so that small molecular oil drops are aggregated and dissociated, and the oil extraction rate is increased.
2.2 Experimental design results of response surface of protease enzymolysis technology
Table 2 response surface experimental protocol and results
| Serial number | A | B | C | D | R1Oil extraction ratio/% | R2Oil content of the |
| 1 | 0 | 0 | 0 | 0 | 90.24 | 4.5 |
| 2 | -1 | -1 | 0 | 0 | 83.72 | 6.4 |
| 3 | 0 | 0 | 1 | 1 | 81.46 | 7.59 |
| 4 | 1 | 0 | 0 | -1 | 79.76 | 9.59 |
| 5 | -1 | 1 | 0 | 0 | 86.56 | 6.39 |
| 6 | -1 | 0 | -1 | 0 | 78.06 | 10.39 |
| 7 | 0 | 0 | -1 | -1 | 73.24 | 15.49 |
| 8 | -1 | 0 | 0 | 1 | 84.29 | 5.79 |
| 9 | 0 | 1 | 1 | 0. | 83.87 | 6.44 |
| 10 | 0 | 0 | -1 | 1 | 73.38 | 10.34 |
| 11 | 0 | 1 | 0 | 1 | 82.45 | 7.04 |
| 12 | 0 | -1 | 1 | 0 | 80.04 | 9.29 |
| 13 | 0 | 0 | 0 | 0 | 88.97 | 5.34 |
| 14 | 0 | -1 | 0 | 1 | 81.32 | 7.94 |
| 15 | 0 | -1 | -1 | 0 | 77.49 | 11.01 |
| 16 | 1 | 0 | 0 | 1 | 81.32 | 7.84 |
| 17 | 1 | 1 | 0 | 0 | 81.87 | 7.35 |
| 18 | -1 | 0 | 0 | -1 | 83.16 | 6.99 |
| 19 | 0 | 0 | 0 | 0 | 89.25 | 5.04 |
| 20 | 0 | 0 | 1 | -1 | 78.34 | 10.09 |
| 21 | 1 | 0 | -1 | 0 | 75.51 | 11.09 |
| 22 | 0 | 1 | -1 | 0 | 73.67 | 12.04 |
| 23 | 1 | -1 | 0 | 0 | 82.73 | 7.44 |
| 24 | 0 | 0 | 0 | 0 | 89.53 | 4.95 |
| 25 | 1 | 0 | 1 | 0 | 82.02 | 7.87 |
| 26 | 0 | 1 | 0 | -1 | 78.91 | 9.89 |
| 27 | 0 | 0 | 0 | 0 | 89.11 | 5.01 |
| 28 | -1 | 0 | 1 | 0 | 85.42 | 5.39 |
As shown in FIG. 3 (a), the pH of the enzymatic hydrolysis was 7.2, the amount of the enzyme added was 2500U/g, and the temperature of the enzymatic hydrolysis was changed with time. When the temperature is lower or higher, the change of the oil extraction rate is smaller; when the enzymolysis time is short, the oil extraction rate is increased and then decreased, and when the enzymolysis time is four to five hours, the oil extraction rate is increased and then slowly decreased. As can be seen from FIG. 3 (b), the pH value of enzymolysis is 7.2, the enzymolysis time is 4 hours, and when the enzyme addition amount is 2000U/g-2600U/g, the oil extraction rate is firstly rapidly increased and then slowly reduced along with the increase of the temperature; when the enzymolysis temperature is between 55 and 60 ℃, the extraction rate obviously changes along with the increase of the addition amount of the enzyme. The interaction between the enzyme addition amount and the enzymolysis temperature has a remarkable response value.
As shown in fig. 3 (c), when the enzyme addition amount is 2500U/g, the pH value of the immobilized enzymolysis is 7.2, and the time is 3-4.5 hours, the oil content of the residue is slowly increased after being reduced along with the temperature increase; when the enzymolysis temperature is between 50 and 55 ℃, the oil content of the residue is obviously reduced along with the increase of the enzymolysis time, and the value between 55 and 60 ℃ is stable. The enzymolysis temperature and the enzymolysis time can be significantly changed. According to the graph in fig. 3 (d), when the enzymolysis temperature is low, the enzymolysis pH value is 7.2 under the experimental condition, and the enzymolysis time is 4 hours, the oil content of the generated residue is high, and particularly, the value of the oil content of the residue is rapidly reduced to the minimum value when the enzymolysis temperature is between 50 ℃ and 55 ℃; when the enzyme is added in a small amount, the value of the oil content of the residue is firstly slowly reduced and then rapidly increased along with the increase of the enzymolysis time.
The results of optimizing the protease enzymolysis process conditions according to Design-expert10.0.4 software by taking the oil extraction rate of pumpkin seed oil and the oil content of residues as investigation indexes are as follows: the pH value of enzymolysis is 7.12, the enzyme adding amount is 2498.79U/g, the enzymolysis temperature is 54.62 ℃, the enzymolysis time is 3.96h, the model prediction oil extraction rate value is 88.03%, and the oil content of residue is 6.03%.
3. Determination of pumpkin seed oil extraction enzyme method demulsification process conditions
3.1 determination of the type of enzyme used for demulsification
As shown in FIG. 4, according to the analysis of the chart, we can obtain that the reaction conditions under the optimal reaction conditions are different with the same addition amount of each enzyme, the highest demulsification rate is Bacillus licheniformis alkaline protease (P < 0.05), the most free oil is obtained by demulsification, the protease is more suitable for demulsification, and finally, the Bacillus licheniformis alkaline protease is selected as the reaction enzyme.
3.2 Experimental results on condition response surface of enzymolysis demulsification process
The RSM experiment designed on the basis of the single factor test of enzymatic demulsification, table 3 is the experimental result performed with the demulsification rate as the response value.
TABLE 3 response surface protocol and results
| Serial number | A | B | C | D | |
| 1 | 0 | 0 | 0 | 0 | 88.34 |
| 2 | -1 | 1 | 0 | 0 | 84.53 |
| 3 | 0 | 0 | 1 | 1 | 83.45 |
| 4 | 1 | -1 | 0 | 0 | 84.90 |
| 5 | 1 | 1 | 0 | 0 | 83.68 |
| 6 | 0 | 1 | 0 | 1 | 83.76 |
| 7 | 0 | -1 | 0 | -1 | 85.87 |
| 8 | 1 | 0 | 1 | 0 | 86.14 |
| 9 | -1 | 0 | 0 | -1 | 84.90 |
| 10 | 0 | 1 | 1 | 0 | 82.45 |
| 11 | 1 | 0 | 0 | -1 | 86.12 |
| 12 | 0 | 0 | 0 | 0 | 88.58 |
| 13 | 0 | 0 | -1 | -1 | 83.58 |
| 14 | 0 | 0 | 0 | 0 | 88.59 |
| 15 | 0 | 0 | 0 | 0 | 87.97 |
| 16 | -1 | 0 | -1 | 0 | 85.05 |
| 17 | 0 | 0 | -1 | 1 | 82.36 |
| 18 | 0 | -1 | 1 | 0 | 84.95 |
| 19 | 0 | 1 | -1 | 0 | 82.80 |
| 20 | -1 | 0 | 0 | 1 | 84.95 |
| 21 | 0 | -1 | -1 | 0 | 83.72 |
| 22 | 0 | 0 | 1 | -1 | 84.18 |
| 23 | 1 | 0 | -1 | 0 | 84.95 |
| 24 | 0 | 0 | 0 | 0 | 88.46 |
| 25 | -1 | -1 | 0 | 0 | 84.29 |
| 26 | 1 | 0 | 0 | 1 | 84.32 |
| 27 | 0 | 1 | 0 | -1 | 81.31 |
| 28 | 0 | -1 | 0 | 1 | 81.46 |
| 29 | -1 | 0 | 1 | 0 | 84.98 |
As shown in table 3 and fig. 5, p value of B, D is less than 0.0001, interaction between B (liquid-to-liquid ratio) and D (enzymatic pH) has a significant effect on emulsion breaking rate, and the value of emulsion breaking rate increases with increasing levels of B (liquid-to-liquid ratio) and D (enzymatic pH); B. the p value of C is less than or equal to 0.05, the demulsification rate is obviously influenced by the interaction of B (feed-liquid ratio) and C (enzyme addition amount), and the demulsification rate is increased and then decreased along with the increase of the interaction water of the feed-liquid ratio and the enzyme addition amount; as the interaction level of A (enzymolysis temperature) and D (enzymolysis pH) is increased, the demulsification rate value changes more smoothly, and compared with the former two interactions, the interaction has no obvious influence on the demulsification rate.
The demulsification process of the enzyme method is optimized through an RSM (remote Red-Green M) experiment: the liquid-material ratio is 0.9: 1 (mL/g), the enzyme addition amount is 3078U/g, the enzymolysis pH is 10.4, and the temperature is 51 ℃. The theoretical demulsification rate of the enzymatic demulsification under the condition is 88.50% by a response surface method.
3.3 Zeta potential of the emulsion at different pH
As shown in fig. 6, the Zeta potential value decreases with increasing pH. According to the change of the pH value, the pH value is close to the isoelectric point of the pumpkin seed protein, the Zeta potential value is close to 0, the worst emulsification force of the protein solubility is minimum, the protein adsorbed between oil drops and water is minimum, the oil drops are easy to gather together due to the absence of electrostatic repulsion, the emulsion is in an extremely unstable state, the pH value is adjusted to be 4.25-4.5 and is close to the isoelectric point of the pumpkin seed protein, an unstable state is formed, free oil is easy to release, and the emulsion breaking rate can be improved to 95.47%.
4. Quality of pumpkin seed oil prepared by enzyme method
4.1 analysis of results of physical and chemical indicators
TABLE 4 physical and chemical index results of pumpkin seed oil
| Index (I) | Pumpkin seed oil |
| Water and volatile matter/%) | 0.05 |
| Acid value (in terms of KOH, mg/g) | 1.43 |
| Iodine value (g/100g) | 168.2 |
| Peroxide number (g/100g) | 0.13 |
| Saponification number (in KOH, mg/g) | 179.6 |
| Insoluble impurities/%) | 0.05 |
| Transparency | Clarifying, and,Is transparent |
| Smell(s) | Has special odor of semen Cucurbitae, but no other foreign odor |
| Color | Brownish green color |
| Refractive index (25 ℃ C.) | 1.475 |
As can be seen from Table 4, the pumpkin seed oil produced by the aqueous enzymatic method has physical and chemical indexes reaching the national standard GB2716-2018 for vegetable oil food safety, wherein the acid value is 1.43mg/g, and the national standard is less than or equal to 3 mg/g; the peroxide value is 0.13g/100g, and the national standard is less than or equal to 0.25g/100 g. The content of insoluble impurities is less than 0.05%, and the product is brownish green and has special flavor of semen Cucurbitae. And all physicochemical indexes of the pumpkin seed oil meet the standard LS/T3250-2017 pumpkin seed oil, so that the pumpkin seed oil extracted by the aqueous enzymatic method is high in quality and can be safely eaten.
4.2 analysis of fatty acid content of pumpkin seed oil
The fatty acid composition and the specific gravity of the pumpkin seed oil are detected by gas chromatography, and the retention time of different fatty acids of the pumpkin seed oil is different according to different standard samples, wherein the retention time of 19.297min is methyl stearate, the retention time of methyl palmitate is slightly shorter than that of methyl stearate, the retention time of methyl linoleate is 13.636min, the retention time of methyl linoleate is 23.589min, and the retention time of linolenic acid is 29.817 min.
The composition and content proportion of fatty acid methyl ester extracted from pumpkin seed oil by aqueous enzymatic method are shown in table 5, and specific substances and content thereof can be directly obtained according to a chart.
TABLE 5 pumpkin seed oil fatty acid methyl ester composition and content
| Fatty acid methyl ester name | Fatty acid methyl ester abbreviation | Relative content |
| Palmitic acid methyl ester | C16:0 | 8.49% |
| Stearic acid methyl ester | C18:0 | 25.39% |
| Oleic acid methyl ester | C18:1n9c | 50.22% |
| Linoleic acid methyl ester | C18:2n6c | 9.85% |
| Linolenic acid methyl ester | C18:3n3 | 6.08% |
As shown in fig. 7, the oil contained five kinds of fatty acids in total, which were palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, respectively, as determined by the chromatogram analysis.
4.3 Squalene content results analysis
Accurately preparing squalene standard solutions with concentrations of 5mg/L, 10mg/L, 20mg/L, 30 mg/L and 40 mg/LAnd sequentially measuring the peak areas of the solutions, wherein a linear regression equation between the mass concentration and the peak area of the squalene standard solution is shown in FIG. 9 as follows: y =99.099x + 2020.3, correlation coefficient R2 And = 0.9961, the linear range of the equation is between 5mg/mL and 40 mg/mL. The standard curve of squalene standard is shown in FIG. 8, and the specific retention time of squalene is 33.084 min. The content of squalene in pumpkin seed oil is 1.51mg/g can be calculated by the peak area at 32.712min in FIG. 10. Meanwhile, the squalene content of the cold-pressed pumpkin seed oil sold in the market is measured, and the value is 1.44 mg/g. And the data show that the average amount of more than ten kinds of pumpkin seed oil shark is higher than that measured by Shijine and the like, and the process conditions optimized by the method are proved to have high oil extraction rate and high quality of finished oil.
Claims (3)
1. A method for preparing pumpkin seed oil by an industrial enzyme method is characterized by comprising the following steps:
(1) raw material pretreatment, crushing and grinding: cleaning pumpkin seed kernels, baking the pumpkin seed kernels in a drying box for 1h at 105 ℃, then crushing the pumpkin seed kernels and sieving the pumpkin seed kernels with a 40-mesh sieve, wherein the grain size of pumpkin seed powder is less than 0.45mm, adding 3-5 times of purified water, and grinding the mixture into pulp by using a colloid mill;
(2) carrying out compound enzyme enzymolysis: adding hydrochloric acid or a dilute sodium hydroxide solution into the feed liquid obtained by grinding, adjusting the pH value to 4.5-6.0, heating to 40-60 ℃, adding 1-2% of cellulase and pectinase respectively, and performing heat preservation and enzymolysis for 2-5 hours;
(3) alkali treatment: adding a proper amount of dilute sodium hydroxide dilute solution into the feed liquid obtained by enzymolysis, adjusting the pH value to 8-9, and slowly stirring for 20-60 min;
(4) and (3) protease enzymolysis: adjusting the pH value of the feed liquid after alkali treatment to 6.8-7.5, adding 1000-5000 (U/g protein) neutral protease, performing enzymolysis at 40-60 ℃ for 3-5 h, heating to 80-90 ℃, performing heat preservation for 5-10 min, inactivating enzyme, and cooling;
(5) primary centrifugal separation: carrying out centrifugal separation by adopting a 5000-10000 r/min automatic slag discharge disc type separator to obtain emulsion liquid, free oil I and residues;
(6) and (3) enzymolysis demulsification: adding 0.5-2 times of purified water into the emulsion, uniformly stirring, adjusting the pH value of the emulsion to 9-11, heating to 40-60 ℃, adding 2000-4000U/g of bacillus licheniformis alkaline protease into the emulsion, carrying out heat preservation and enzymolysis for 2-4 h, heating to 80-90 ℃, preserving the heat for 10-15 min, inactivating the enzyme, and cooling;
(7) and (3) secondary centrifugal separation: adjusting the pH value of the mixed solution after enzymolysis and demulsification to 4.0-5.0, and performing centrifugal separation by using an automatic disc type separator with the speed of 5000-10000 r/min to obtain protein hydrolysate and free oil II;
(8) combining products: and combining the free oil I and the free oil II to obtain the pumpkin seed oil prepared by the enzyme method.
2. The method for preparing pumpkin seed oil by the industrial enzyme method according to claim 1, which comprises the following steps: and (5) adding a hydrochloric acid solution to adjust the pH value in the step (4) and the step (7).
3. The method for preparing pumpkin seed oil by the industrial enzyme method according to claim 1, which comprises the following steps: and (6) adding a dilute sodium hydroxide solution to adjust the pH value.
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