CN103284158B - Goose oil diacylglycerol microcapsules - Google Patents
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Abstract
A purpose of the present invention is to provide goose oil diacylglycerol microcapsules, which comprise a core material and a wall material, and is characterized in that the core material is goose oil diacylglycerol, and the wall material is a beta-cyclodextrin and sodium caseinate mixture. According to the present invention, the core material and the wall material are respectively subjected to an ultrasonic treatment, such that the wall material is easily dissolved in water, and the core material is easily dissolved in an emulsified liquid so as to substantially increase an emulsified liquid concentration, reduce a freeze-drying time, and improve a microcapsule encapsulation rate and production efficiency; and a freeze drying method is adopted so as to effectively prevent goose oil diacylglycerol loss during the preparation process, avoid the disadvantage of high temperature of the existing method, filling the goose oil diacylglycerol encapsulation technology research gap, provide a new method for new goose oil product research and development, and provide important social values and economic values.
Description
Technical field
The invention belongs to grease microcapsule preparing technical field, be specifically related to a kind of goose oil diglyceride microcapsules.
Background technology
Goose oil has that fusing point is low, fragrance is unique, cholesterol level is low, unsaturated fatty acid content high.But at present less for the research of goose oil, most of goose oil is all used as feed and causes waste.The fat of goose with fat liver belly and the position such as subcutaneous deposition of usining is prepared diglyceride as aliphatic acid donor, can turn waste into wealth and greatly improve the economic use value of goose oil, improves value-added content of product.
Diglyceride is the product obtaining by after glycerine (glycerine) and two fatty acid esterifications.It is divided into 1,3-DAG and two kinds of isomers of 1,2-diglyceride.Research shows, diglyceride (DG) in reducing blood lipid, reduce interior fat, suppress the aspects such as body weight increase and have critical function.This function is mainly accumulated realization in vivo by suppressing triglycerides (TG).TG is in enteron aisle, and two ends aliphatic acid, due to lipase effect, is 2-monoglyceride (MG) and free fatty (FA) by enzymolysis, and is absorbed at intestinal epithelial cell.In intestinal epithelial cell, FA and 2-MG are synthesized rapidly TG(neutral fat again), as neutral fat in blood, in all-around exercises, those neutral fats that are not used as energy utilization are just accumulated as body fat.And DG is broken down into 1-MG and the aliphatic acid of synthctic fat mostly again, because the position that 1-MG is combined with glycerine from aliphatic acid in 2-MG is different, therefore as neutral fat synthesis material, there is very big difference, again synthetic extremely slow to neutral fat in small intestine.Endocellular liberation fatty acid concentration uprises, and is finally broken down into water and release of carbonate dioxide by beta oxidation approach, so DG decomposes and capacity usage ratio raising at small intestine lipid.Make the neutral fat in blood after edible DG be difficult to rise simultaneously, like this, if continue edible DG, just can reduce body fat accumulation.Infer thus, diglyceride has retained the trophic function that triglycerides has on the one hand; On the other hand, due to diglyceride and the triglycerides difference human body metabolism, and the pancreatic lipase in human body is 1,3 selectivity enzyme, and therefore, diglyceride has again physicochemical property and the health-care efficacy that some triglycerides do not have.In addition, because diglyceride has unique some physics and biochemical characteristic, make it at aspects such as food, cosmetics, medicine, have wide exploitation prospect.
At present, consume with liquid form in the market of goose oil both at home and abroad, is used for the industries such as feed processing, still there is no goose diglyceride product.In order to expand the consumption market of goose oil, improve its added value of product, present inventor successfully explores the preparation method of goose oil diglyceride.Yet the goose oil diglyceride of preparation is solid-liquid admixture, poor fluidity, gives transportation, preserves and eats and all bring very big inconvenience, is unfavorable for the market sale of goose oil diglyceride.Therefore, be necessary to provide a kind of use or storage configuration of more scientific goose oil diglyceride.
Summary of the invention
The object of this invention is to provide a kind of goose oil diglyceride microcapsules, adopt cryodesiccated method to prepare goose oil diglyceride microcapsules, thereby make up the deficiencies in the prior art.
Applicant is devoted to study the best approach of goose oil diglyceride microencapsulation always, by long research, through the screening of various factors, obtain each compositional selecting and the proportioning of wall material, determine vacuum freeze-drying method simultaneously and prepare the optimum process condition of goose oil diglyceride microcapsules, and the product of preparing under this condition has been carried out to quality determination.
Goose oil diglyceride microcapsules of the present invention, include core and wall material, it is characterized in that, described core is goose oil diglyceride, and wall material is the mixture of beta-schardinger dextrin-and casein sodium.
Above-mentioned wall material and the mass ratio of core are 1～5:1, are preferably 2.44:1;
In above-mentioned wall material, the quality ratio of beta-schardinger dextrin-and casein sodium is 0.5～2.5:1, is preferably 1.22:1.
For core goose oil diglyceride, take following steps preparation:
1) preparation of fatty acid mixed: by the goose oil of refining treatment by volume the ratio of 1:4 add in 1mol/L NaOH-ethanolic solution, after magnetic agitation is dissolved it completely, with water-bath backflow saponification under nitrogen protection, obtain saponification liquor; Decompression steams ethanol, adds water and makes saponified dissolving completely, under the condition stirring, with 10%HCl, is acidified to pH2～3 in constant temperature constant speed, adds n-hexane extraction, and Warm Wash is to neutral, and decompression steams solvent, adds anhydrous sodium sulfate dehydration, and suction filtration, obtains fatty acid mixed;
So not only make reaction more abundant, also shortened the reaction time to a certain extent, improved diglyceride production rate.Wherein, fatty acid mixed preparation condition: 82 ℃ of saponification temperatures, reaction time are 60min; The ethanol reclaiming in reaction and n-hexane all can reuse.
2) preparation of diglyceride: take glycerine and fatty acid mixed mixes, add candida antarctica lipase B, by centrifugal that enzyme is separated, can obtain high diglyceride mixt after isothermal reaction; Wherein, the temperature of isothermal reaction is 30 ℃～70 ℃, and the mass ratio of glycerine and fatty acid mixed is 1:0.5～3, and enzyme concentration is 1%～5% of glycerine and fatty acid mixed total amount, and the reaction time is 8～12h.
The preparation method of goose oil diglyceride microcapsules of the present invention, three steps are processed in the preparation and the freeze drying that include the processing of core wall material, emulsion:
1) core wall material is processed: core and wall material adopt respectively ultrasonic wave to process;
2) preparation of emulsion: the core wall material solution of processing in step 1) is fully mixed, add emulsifier for mixing and make emulsion;
3) freeze drying is processed: after putting into freeze drier vacuum freeze drying after emulsion precooling, take out.
Above-mentioned 1) ultrasonic wave processing power is 500W, and the processing time is 3min.
Above-mentioned 2) in the preparation of emulsion, emulsifying agent is sodium carboxymethylcellulose, and addition is 0.7%.
Above-mentioned 3) condition of vacuum freeze drying is :-35 ℃～-45 ℃, 20Pa～100Pa.
Core of the present invention and wall material adopt respectively ultrasonic wave to process, and make wall material more soluble in water, and core is more soluble in emulsion, has greatly increased concentration of emulsion used, thereby has reduced sublimation drying, has improved microcapsule embedded rate and production efficiency.In addition, adopt freeze-drying, effectively prevented the loss of goose oil diglyceride in preparation process, avoided the shortcoming of existing method excess Temperature, filled up the blank of goose oil diglyceride embedding techniques research, for China's goose oil new-product development provides new method, there is important society and economic worth.And goose oil diglyceride, after freeze-drying microencapsulation is processed, can overcome the defect of grease itself, has effectively prevented the loss of diglyceride in process, its stability and convenience are all increased, and the shelf life of product extends greatly.Foregoing invention, for full-scale development goose oil eries product, is widened goose oil and is utilized market to have important research meaning.
Accompanying drawing explanation:
Fig. 1: interaction response face figure between mesospore material of the present invention and core ratio and the ratio of wall material ratio;
Fig. 2: interaction response face figure between mesospore material of the present invention and core ratio and solid content;
Fig. 3: interaction response face figure between mesospore material of the present invention and core ratio and emulsifier;
Fig. 4: interaction response face figure between solid content and wall material ratio in the present invention;
Fig. 5: interaction response face figure between emulsifier and wall material ratio in the present invention;
Fig. 6: interaction response face figure between solid content and emulsifier in the present invention;
Fig. 7: the microcapsules of preparing under optimum condition in the present invention are photo (10 μ m) under Electronic Speculum;
Fig. 8: photo (10 μ m) under the microcapsules Electronic Speculum that the present invention is not prepared under optimal conditions;
Fig. 9: goose foie gras oil microcapsule heat stability test figure of the present invention;
Figure 10: goose foie gras oil microcapsule storage-stable of the present invention detects figure.
The specific embodiment
The present invention adopts the method for vacuum freeze drying to prepare goose oil diglyceride microcapsules, has optimized the optimum condition of preparing microcapsules.Adopt freeze-drying method simultaneously, beta-schardinger dextrin-in varing proportions and casein sodium are wall material, sodium carboxymethylcellulose is emulsifying agent, take microcapsule embedded rate as index, emulsifier, solid content, core wall material ratio and wall material ratio are factor, the test of design response surface, obtains preparing the optimum process condition of goose oil diglyceride microcapsules, and preparing product quality and storage-stable is measured.
The coating material of selecting is beta-schardinger dextrin-and casein sodium, and wherein casein sodium itself is the most perfect protein, as food additives, safe, selects it as wall material, has enriched the nutrition of goose oil diglyceride microcapsules; The design feature of beta-schardinger dextrin-itself, makes it have dissolubility good, easily crystallization, purification, and the feature of good stability, can improve the concentration of system solid content, is conducive to reduce energy consumption and reduces cost.
Core in the present invention is goose oil diglyceride, selects the method for being prepared as follows:
1) preparation of fatty acid mixed: the goose oil of refining treatment is added to 1mol/L NaOH-ethanolic solution in the ratio of 1:4, magnetic agitation a period of time is transferred to the there-necked flask of 1000ml after it is dissolved completely, water-bath backflow saponification a period of time, obtain saponification liquor, decompression steams ethanol, add water and make saponified dissolving completely, under the condition stirring in constant temperature constant speed, with 10%HCl, be acidified to pH2～3, the n-hexane extraction that adds certain volume, Warm Wash is to neutral, and decompression steams solvent, adds anhydrous sodium sulfate dehydration, suction filtration, obtains fatty acid mixed.Wherein, fatty acid mixed preparation condition: 82 ℃ of saponification temperatures, reaction time are 60min.
2) preparation of diglyceride: 1:1.05 takes glycerine and fatty acid mixed in little flask in proportion, be placed in permanent shaking table, reactant is mixed, wait rising to the immobilized candida antarctica lipase B that adds 2.25% after 50 ℃, start timing, after isothermal reaction 9.2h, by centrifugal, can, by enzyme separation, can obtain high diglyceride mixt.
Microcapsule preparation method of the present invention comprises the steps:
1) wall material is selected: beta-schardinger dextrin-dissolubility is good, is difficult for the moisture absorption, good stability, and there is good filling effect; Casein sodium itself is the most perfect protein, as food additives, safe; Select this bi-material as wall material, not only enriched the nutrition of goose oil diglyceride microcapsules; And the concentration of raising system solid content, reduced the loss of core, be conducive to reduce energy consumption, reduce cost.
2) core wall material is processed: core and wall material adopt respectively 500W ultrasonic wave to process 3min.The microcapsule embedded rate making after processing is apparently higher than untreated samples, and ultrasonic wave processing rear wall material is more soluble in water, has greatly increased concentration of emulsion used, thereby has reduced sublimation drying, has improved microcapsule embedded rate and production efficiency.
3) emulsion preparation: sodium carboxymethylcellulose (CMC) is not only good emulsion stabilizer, thickener, and there is the excellent stability of freezing, melt, and can improve the local flavor of product, prolongation storage time.FAO and WHO approved are by pure CMC for food, and it is just to get the Green Light after strict biology, toxicologic study and test, and the safe intake (ADI) of international standard is 25mg/ (kgd), i.e. about everyone 1.5g/d.If emulsifying agent use amount is too low in system, can make the easy layering and precipitating of emulsion of preparation, stability of solution also can correspondingly reduce, and easily causes microcapsule embedded rate to decline.And emulsifying agent use amount is too high, can make emulsion viscosity increase, this is unfavorable for the abundant dispersion of grease and the carrying out of follow-up homogeneous.In preparation process, the addition of emulsifying agent sodium carboxymethylcellulose is 0.7%, makes solution homogeneous good when meeting the safe intake of international standard, reduces layering.
4) during freeze drying is processed, sample is in-40 ℃ of precoolings, this is because cryodesiccated principle for directly distilling the steam in material out under vacuum condition, thereby and material itself is stayed in the ice shelf freezing, reach and remove the wherein object of moisture, before freeze drying, material must be carried out to thorough freezing could realization; As freezing in material incomplete, in dry processing, there will be a large amount of foams, meeting is the distillation of obstruction moisture seriously, and reduces the rate of recovery of material.
Below in conjunction with embodiment, method of the present invention is described in detail.
Embodiment 1: microencapsulation optimum process condition screening test
(1) preparation of emulsion
By beta-schardinger dextrin-and casein sodium in mass ratio for 1.5:1 is dissolved in the water, ultrasonic wave is processed 3min, after it fully dissolves, adds 1% sodium carboxymethylcellulose (CMC), goose oil diglyceride after while stirring ultrasonic wave being processed slowly adds, and makes it abundant homogeneous and mixes.
(2) emulsion freeze drying is processed
Emulsion is poured in plastic culture dish, taken out after putting into freeze drier freeze drying 15h after subzero 40 ℃ of precoolings.
(3) mensuration of microcapsule embedded rate
1. the assay method of surface of microcapsule oil content: get 2.0g sample, add 50ml benzinum, after sharp pounding 2min, suction filtration, collects filtrate and steam solvent in Rotary Evaporators, and weight reduction is measured surface oil quality.
2. the assay method of the total oil quality of microcapsules: soxhlet extraction
3. the mensuration of microcapsule embedded rate: the ratio of the oily total amount that embedding rate adds while referring in microcapsule product by the oil content of embedding with embedding.The higher core of embedding rate is larger by the amount of embedding, and effect is better.
Embedding rate=(the total oil quality of 1-surface of microcapsule oil quality/microcapsules) * 100%
(4) selection of wall material
Choose different wall materials and emulsifying agent goose oil diglyceride is carried out to embedding test, get respectively xanthans, gelatin, starch, maltodextrin, carragheen, soybean protein isolate, sucrose, Arabic gum, casein sodium, beta-schardinger dextrin-, sodium carboxymethylcellulose etc. in carrying out embedding test under 1.5:1 ratio uniform temperature, by embedding rate, choose beta-schardinger dextrin-and casein sodium, sodium carboxymethylcellulose are embedded material.
(5) goose oil diglyceride microencapsulation optimised process determines
1. the screening of core and wall material ratio: be 25% in solid content (concentration of wall material solution mesospore material), beta-schardinger dextrin-and casein sodium ratio be 1.5:1, emulsifying agent use amount is under 1% condition, set that core and wall material ratio are respectively 1:1,1:2,1:3,1:4,1:5 tests, measure microcapsule embedded rate.So that the ratio of its core and wall material is carried out to Preliminary screening.
2. wall material ratio (beta-schardinger dextrin-: screening casein sodium): test adopts beta-schardinger dextrin-and casein sodium compound wall materials to prepare microcapsules, in solid content, be 25%, core wall material ratio is that 1:3, emulsifying agent use amount are under 1% condition, the ratio of setting beta-schardinger dextrin-and casein sodium ammonium is respectively 0.5:1,1:1,1.5:1,2:1,2.5:1 tests, measure microcapsule embedded rate, so that the ratio of its wall material is carried out to Preliminary screening.
3. the screening of solid content (wall material use quantity): be 1:3 in core wall material ratio, beta-schardinger dextrin-and casein sodium ratio are 1.5:1, emulsifying agent use amount is under 1% condition, regulating solid content (being the concentration of wall material solution mesospore material) content is 15%, 20%, 25%, 30%, 35% to test respectively, measure microcapsule embedded rate, so that its solid content is carried out to Preliminary screening.
4. the screening of emulsifier: be 1:3 in core wall material ratio, beta-schardinger dextrin-and casein sodium ratio are 1.5:1, solid content is under 25% condition, regulating emulsifying agent use amount is 0.33%, 0.67%, 1%, 1.33%, test respectively, measure microcapsule embedded rate, so that its emulsifier is carried out to Preliminary screening.
5. response surface testing program and result
Table 1: response surface scheme and result
6. the Establishment and analysis of response-surface model
After result of the test being analyzed by response surface software design expert8.06, show that its equation of linear regression is as follows:
Y=93.0-1.93A-1.33B+0.73C+2.12D+3.35AB+0.49AC+2.05AD+3.64BC-0.63BD+0.97CD-4.09A
2-4.34B
2-1.97C
2-2.16D
2
In formula: A represents wall material and core ratio; B represents wall material ratio; C represents solid content; D represents emulsifier
Above-mentioned response surface test is carried out to variance analysis, and result is as table 2.
Table 2: response surface variance analysis
As can be seen from Table 2, regression model is (model F<0.0001) extremely significantly, lose and intend a p=0.1010>0.05, without conspicuousness impact, illustrate that residual error causes by random error, model regulation is suitably, available regression equation replaces the true point of test to analyze result of the test, it proofreaies and correct coefficient of determination is 0.9574, shows that this models fitting degree is fine, has the variability of 95.74% test data to explain with this regression model.Therefore, regression equation can be described the relation between each factor and response preferably, and each concrete test factor is not simple linear relationship on the impact of response face amount.A, D, AB, BC, A in each factor
2, B
2result of the test is had to impact (P<0.01) extremely significantly, B, AD, C
2, D
2result of the test is had to remarkable impact (P<0.05), and the magnitude relationship of 4 factor impacts is: wall material and core ratio=emulsifier > wall material ratio > solid content.
Use design expert8.06 software data processing and analysis, goose oil diglyceride microencapsulation optimum condition is wall material: core=2.44:1, beta-schardinger dextrin-: casein sodium=1.22, and solid content is 22.95%, emulsifier is 0.7%.Take this method goose oil diglyceride embedding rate to reach 93.71%.Refer to accompanying drawing 1,2,3,4,5,6.
Embodiment 2: microencapsulation optimum process condition Orthogonal Rotational Regressive Tests
134g beta-schardinger dextrin-is added in the distilled water that is preheating to 50 ℃, after it is dissolved completely, then according to beta-schardinger dextrin-and casein sodium ratio, be that 1.22:1 slowly adds 110g casein sodium, after it fully dissolves, add sodium carboxymethylcellulose (CMC) 7.56g that is equivalent to 0.7% quality, under high-speed stirred, 100g goose oil diglyceride is slowly added to rear homogeneous.Emulsion is poured in plastic culture dish, after subzero 40 ℃ of precoolings, taken out after putting into freeze drier freeze drying 15h, the condition of vacuum freeze drying is :-40 ℃ and 80Pa.Through 5 repeated tests, the microcapsule product embedding rate obtaining is respectively 93.27%, 93.45%, 93.37%, 93.26%, 93.05%, and average embedding rate is 93.28%, basically identical with the result of case study on implementation 1; Show that technique of the present invention has fine stability.
The prepared microcapsules of method of the present invention have good physicochemical property, and goods are white powder, free from extraneous odour, and slightly with frankincense taste, water content is 3.63%.Prepared microcapsules sample is observed under ESEM, compares shape matching rule with the goose oil diglyceride microcapsules under optimal conditions not, and quality is comparatively tight; Illustrate that these microcapsules have good embedding effect to goose oil diglyceride.Refer to accompanying drawing 7,8.
Embodiment 3: the test of micro-encapsulation technology conditional filtering
According to the step of embodiment 1, carry out the preparation of emulsion, wherein beta-schardinger dextrin-and casein sodium ratio are that 2:1, solid content are 35%, wall material and core ratio be 3:1, and add 1% sodium carboxymethylcellulose (CMC), while stirring goose oil diglyceride is slowly added, homogeneous carries out freeze drying processing after mixing, and carries out according to the method described above the mensuration of microcapsule embedded rate, and result shows, under this condition, microcapsule embedded rate is 50.28%, lower than 93.71% under optimum condition.
Embodiment 4: the test of micro-encapsulation technology conditional filtering
According to embodiment 1 processing step, again test completely, just core and wall material do not adopt ultrasonic wave to process.Through 5 repeated tests, the microcapsule product embedding rate obtaining is respectively 90.27%, 90.45%, 90.37%, 90.26%, 90.05%, and average embedding rate is 90.28%, all low than the result of case study on implementation 1 and case study on implementation 2; Show that core and wall material adopt ultrasonic wave to process and can make embedding rate improve.
Embodiment 5: microencapsulation goose oil diglyceride stability contrast test
(1) heat endurance contrast test
Accurately take microcapsule product and the goose oil diglyceride of 2g embodiment 1 preparation, be positioned over and in 100 ℃ of baking ovens, heat respectively 1,2,3,4,5,6,7, after 8h, weigh each sample quality reduction, and compare.Result shows, in whole process, microcapsule product and goose oil diglyceride reduction are all rising, but the reduction of goose oil diglyceride is all the time higher than microcapsule product, and the reduction of microcapsule product after 4 hours is quite small, and the reduction of goose oil diglyceride is still rising.This explanation microcapsule product heat endurance is higher than goose oil diglyceride, and product has good heat endurance.Refer to accompanying drawing 9.
Through microcapsule product is carried out to thermal stability determination, show that the stability of the goose oil diglyceride of microencapsulation is better than the not goose oil diglyceride of microencapsulation.Therefore the microcapsule product of, preparing according to method of the present invention has heat endurance well.
(2) storage-stable test
The goose oil diglyceride microcapsules that take 2g embodiment 1 preparation, are put in measuring cup, in temperature is 65 ℃ and the baking oven that ventilates, place 14 days, within every 2 days, measure a microcapsules quality reduction, and measure the percentage that surplus accounts for gross mass.Result demonstration, within 2 years, microcapsules storage rate can reach 89.28%, illustrates that this product has good storage-stable.Refer to accompanying drawing 10.
Method of the present invention has adopted ultrasonic wave processing, makes wall material more soluble in water, and core is more soluble in emulsion, has greatly increased concentration of emulsion used, thereby has reduced sublimation drying, has improved microcapsule embedded rate and production efficiency.In addition, adopt freeze-drying, effectively prevented the loss of goose oil diglyceride in preparation process, avoided the shortcoming of existing method excess Temperature, reduce the loss of goose oil diglyceride, improve microcapsule embedded rate, production efficiency, product stability and shelf life, make it to apply convenient.Technique can be applied to the further exploitation of goose oil product, and to aquatic bird secondary industry byproduct, comprehensive utilization provides new way, will produce tremendous economic social benefit.
Claims (4)
1. goose oil diglyceride microcapsules, include core and wall material, it is characterized in that, described core is goose oil diglyceride, and wall material is the mixture of beta-schardinger dextrin-and casein sodium; Described wall material and the mass ratio of core are 1～5:1, and in its mesospore material, the mass ratio of beta-schardinger dextrin-and casein sodium is 0.5～2.5:1; And in preparation process, be added with emulsifying agent sodium carboxymethylcellulose.
2. goose oil diglyceride microcapsules as claimed in claim 1, is characterized in that, described wall material and the mass ratio of core are 2.44:1.
3. goose oil diglyceride microcapsules as claimed in claim 1, is characterized in that, in described wall material, the mass ratio of beta-schardinger dextrin-and casein sodium is 1.22:1.
4. goose oil diglyceride microcapsules as claimed in claim 1, is characterized in that, the preparation method of described core goose oil diglyceride is as follows:
1) preparation of fatty acid mixed: by the goose oil of refining treatment by volume the ratio of 1:4 add in 1mol/LNaOH-ethanolic solution, after magnetic agitation is dissolved it completely, with water-bath backflow saponification under nitrogen protection, obtain saponification liquor; Decompression steams ethanol, adds water and makes saponified dissolving completely, under the condition stirring, with 10%HCl, is acidified to pH2～3 in constant temperature constant speed, adds n-hexane extraction, and Warm Wash is to neutral, and decompression steams solvent, adds anhydrous sodium sulfate dehydration, and suction filtration, obtains fatty acid mixed;
Wherein, fatty acid mixed preparation condition: 82 ℃ of saponification temperatures, reaction time are 60min;
2) preparation of diglyceride: take glycerine and fatty acid mixed mixes, add candida antarctica lipase B, by centrifugal that enzyme is separated, can obtain the diglyceride mixt of high concentration after isothermal reaction; Wherein the temperature of isothermal reaction is 30 ℃～70 ℃, and the mass ratio of glycerine and fatty acid mixed is 1:0.5～3, and enzyme concentration is 1%～5% of glycerine and fatty acid mixed total amount, and the reaction time is 8～12h.
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