CN109007233A - A kind of extracting method of stichopus japonicus intestines protein isolate - Google Patents
A kind of extracting method of stichopus japonicus intestines protein isolate Download PDFInfo
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- CN109007233A CN109007233A CN201811034370.1A CN201811034370A CN109007233A CN 109007233 A CN109007233 A CN 109007233A CN 201811034370 A CN201811034370 A CN 201811034370A CN 109007233 A CN109007233 A CN 109007233A
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- stichopus japonicus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
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Abstract
The invention discloses a kind of extracting methods of stichopus japonicus intestines protein isolate, comprising steps of stichopus japonicus intestines are homogenized, prepare heavy stichopus japonicus intestines skimmed milk, alkali soluble, acid, dialysis and drying.The present invention is using the leftover bits and pieces in stichopus japonicus process --- and stichopus japonicus intestines carry out the recycling of protein as raw material, provide a kind of important channel for the exploitation of stichopus japonicus intestines, that is, reduce resource waste, and improve economic benefit；Compared with other Separation of Proteins extracting methods, extraction process of the present invention is simple, easily operated, and laboratory condition is conducive to control, and economic and practical value is strong, is suitable for large-scale industrial production；Its dissolubility of the protein isolate of preparation is high, and water holding, holding oiliness, emulsibility improve significantly compared with skimmed milk, and the holding oiliness of protein isolate, emulsibility are significantly better than soybean protein isolate.
Description
Technical field
The invention belongs to food processing technology fields, are related to a kind of extracting method of protein isolate, more specifically, being to relate to
And a kind of extracting method of stichopus japonicus intestines protein isolate.
Background technique
Sea cucumber is a kind of important aquatic products, is rich in a variety of active ingredients, and nutritive value is high, is considered as helping meals good merchantable brand and reason
Think invigorant.In recent years, being continuously increased with demand, China's holothruian cultures and secondary industry rapidly develop, and 2015 complete
State's sea cucumber sea-farming yield is more than 200,000 tons, but the leftover bits and pieces during Holothurian machining --- sea cucumber intestine then seldom obtains benefit
With.In sea cucumber production, generally as waste abandoned, this not only causes the wasting of resources and also brings environment dirt sea cucumber intestine
Dye.Protein content is abundant in sea cucumber intestine, if can protein therein be recycled and be utilized, it will bring significant economy
Benefit and social benefit.
At present in terms of the research of sea cucumber intestine is concentrated mainly on some endogenous enzymes, such as isolate and purify out from stichopus japonicus intestines
Beta-1,3-glucanase, cathepsin B, acetylcholinesterase and alkaline phosphatase etc..And to sea cucumber intestine protein isolate
There is not been reported for extraction and isolated research.
Summary of the invention
Stichopus japonicus intestines are rich in protein, are the sources of high-quality polypeptide, but the separation of protein and preparation method be still in stichopus japonicus intestines
It has not been reported.For the recycling for solving the problems, such as protein in stichopus japonicus intestines, the present invention provides a kind of preparations of stichopus japonicus intestines protein isolate
Method, the method for the invention have the characteristics that easy to operate, production cost is low.
A kind of extracting method of stichopus japonicus intestines protein isolate, includes the following steps:
S1, the homogenate of stichopus japonicus intestines: stichopus japonicus intestines are removed into silt, are cleaned, stichopus japonicus intestines homogenate is prepared；
Under preferred embodiment, the parameter of stichopus japonicus intestines homogenate is prepared described in step S1 are as follows: be homogenized under the conditions of 2000~3000rpm
3~5min.
S2, preparation stichopus japonicus intestines skimmed milk: n-hexane/dehydrated alcohol mixing is added into stichopus japonicus intestines homogenate described in step S1
Solution obtains stichopus japonicus intestines skimmed milk by degreasing after filtering, be air-dried at room temperature, crushing, sieving with 100 mesh sieve；
Under preferred embodiment, degreasing described in step S2 specifically: into stichopus japonicus intestines homogenate described in step S1 be added n-hexane/
Dehydrated alcohol mixed solution stirs 5~7h under the conditions of 600~800rpm, 4 DEG C and carries out degreasing, and the skimming processes repeat 1~
3 times；The volume ratio of n-hexane and dehydrated alcohol is 2~3:1 in the n-hexane/dehydrated alcohol mixed solution；N-hexane/anhydrous
The volume ratio of alcohol mixed solution and stichopus japonicus intestines homogenate is 9~11:1.
S3, alkali soluble: being added deionized water for stichopus japonicus intestines skimmed milk obtained by step S2 and mix, be adjusted to alkaline ph values, stir, from
The heart collects supernatant, obtains stichopus japonicus intestines protein isolate extracting solution；
Under preferred embodiment, alkali soluble described in step S3 specifically: stichopus japonicus intestines skimmed milk obtained by step S2 is pressed into solid-liquid ratio (w/v)
Deionized water is added in 1:10~35, mixes, is adjusted to pH8.0~11.5,4~60 DEG C, 300~500rpm item with 1mol/L NaOH
2~3h is stirred under part, 3000~5000r/min is centrifuged 20~30min, collects supernatant, i.e. stichopus japonicus intestines protein isolate extracting solution.
S4, acid are heavy: by stichopus japonicus intestines protein isolate extracting solution obtained by step S3, being adjusted to acid ph value, stand, be centrifuged, collect
Precipitating；
Under preferred embodiment, acid described in step S4 is heavy specifically: stichopus japonicus intestines obtained by step S3 are separated egg with 1mol/L HCl
White extracting solution is adjusted to pH2~4.6, stands 1~5h, and 8000~12000r/min is centrifuged 10~30min, collects precipitating.
S5, dialysis: precipitating obtained by step S4 is dissolved in deionized water, carries out dialysis desalination using phosphate buffer；
It under preferred embodiment, dialyses described in step S5 specifically: be dissolved in precipitating obtained by step S4 with 5~10mL/g ratio
In ionized water, pH6.8~7.0 are adjusted to 0.1mol/L NaOH, dissolved sample is fitted into 3~4KDa bag filter,
24~the 48h that dialyses in the phosphate buffer of 20mmol/L pH7.4 carries out desalination, during which replaces 4~6 dialyzates, every time thoroughly
The amount for analysing liquid is 10~20 times of sample volume, and dialysis procedure is carried out at 4 DEG C.
S6, drying: step S5 products therefrom is freeze-dried, and obtains stichopus japonicus intestines protein isolate freeze-dried powder.
Under preferred embodiment, in step S2, by stichopus japonicus intestines homogenate obtained by step S1 and n-hexane/dehydrated alcohol mixed solution
It is mixed by the volume ratio of 10:1, in 4 DEG C of degreasing 6h, skimming processes are repeated twice, and are filtered, are air-dried at room temperature, and are crushed, and 100 mesh are crossed
After sieve, stichopus japonicus intestines skimmed milk is obtained.
Under preferred embodiment, the alkali soluble extracting condition in step S3 is that gained stichopus japonicus intestines skimmed milk is expected by 1:20 in step S2
Liquor ratio is mixed with deionized water, is adjusted to pH11.5 with 1mol/L NaOH, and 25 DEG C of extraction 2h are centrifuged 20min through 4000r/min, is received
Collect supernatant, obtains the leaching liquor of the Erepsin containing stichopus japonicus.
Under preferred embodiment, the condition of the heavy recycling albumen of acid in step S4 is, by gained leaching liquor 1mol/ in step S3
L HCl is adjusted to pH3.6, stands 3h, 30min is centrifuged under 10500r/min, collect precipitating.
Under preferred embodiment, the extracting method of the stichopus japonicus intestines protein isolate, comprising the following steps:
S1, the homogenate of stichopus japonicus intestines: sea cucumber intestine removes silt, cleans, 5min is homogenized under the conditions of 2000rpm, obtains sea cucumber intestine homogenate
Liquid；
S2, preparation stichopus japonicus intestines skimmed milk: n-hexane/nothing of 9 times of volumes is added into sea cucumber intestine homogenate described in step S1
Water-ethanol (VN-hexane:VDehydrated alcohol=3:1) mixed solution, degreasing is carried out, is filtered, is air-dried at room temperature, crushes, after sieving with 100 mesh sieve, obtains
To stichopus japonicus intestines skimmed milk；The degreasing is that 7h is stirred under the conditions of 600rpm, 4 DEG C, and the degreasing is repeated 1 times；
S3, alkali soluble: using sea cucumber intestine skimmed milk obtained by step S2 as raw material, deionization is added by the solid-liquid ratio of (w/v) 1:20
Water, mixes, and stirring and leaching 2h under the conditions of being adjusted to 11.5,25 DEG C of 300rpm of alkaline pH with 1mol/L NaOH, mixed liquor is put into centrifugation
Machine is centrifuged 20min with 4000r/min, collects supernatant, the leaching liquor of the Erepsin containing stichopus japonicus can be obtained；
S4, acid are heavy: by leaching liquor obtained by step S3, being adjusted to pH3.6 with 1mol/L HCl, stand 3h, under 10500r/min
It is centrifuged 30min, collects precipitating；
S5, dialysis: precipitating obtained by step S4 is dissolved in deionized water with the ratio of 5mL/g, with 0.1mol/L NaOH tune
To pH6.8；Dissolved sample is fitted into 3.5KDa bag filter, is dialysed in the phosphate buffer of 20mmol/L pH7.4
For 24 hours, 4 dialyzates are during which replaced, the amount of each dialyzate is 10 times of sample volume, and entire dialysis procedure is carried out at 4 DEG C；
Thorn can be obtained in S6, drying: product prepared by the step S5 vacuum freeze drying 72h under the conditions of 1Pa, -50 DEG C
Join intestines protein isolate freeze-dried powder.
The beneficial effects of the present invention are:
1, using the leftover bits and pieces in stichopus japonicus process --- stichopus japonicus intestines carry out the recycling of protein as raw material, for thorn
The exploitation of ginseng intestines provides a kind of important channel, that is, reduces resource waste, and improve economic benefit；
2, the present invention recycles protein from stichopus japonicus intestines using alkali extraction-acid precipitation, with other Separation of Proteins extracting methods
It compares, extraction process of the present invention is simple, and easily operated, laboratory condition is conducive to control, and economic and practical value is strong, is suitable for big rule
The industrialized production of mould.
3, n-hexane/dehydrated alcohol mixed liquor degreasing is used after the present invention is homogenized by stichopus japonicus intestines, carries out alkali soluble acid later
It is heavy, by testing repeatedly, just because of the combination for carrying out processing step by said sequence, just make the stichopus japonicus intestines separation egg of preparation
White dissolubility is high, and water holding, holding oiliness, emulsibility improve significantly compared with stichopus japonicus intestines skimmed milk, and the holding oiliness of protein isolate, cream
The property changed is significantly better than food function base-material soybean protein isolate common on the market.
4, stichopus japonicus intestines protein isolate prepared by the present invention is detected using Kjeldahl's method, wherein protein content is up to for discovery
80%.Operation of the present invention is simple, and production cost is low, can protein quick, in high efficiente callback stichopus japonicus intestines, to stichopus japonicus intestines resource
Development and utilization have great importance.
Detailed description of the invention
Fig. 1 stichopus japonicus intestines skimmed milk, the dissolubility of stichopus japonicus intestines protein isolate and soybean protein isolate under the conditions of pH10；
Fig. 2 stichopus japonicus intestines skimmed milk, the emulsibility of stichopus japonicus intestines protein isolate and soybean protein isolate under the conditions of pH8；
Fig. 3 stichopus japonicus intestines skimmed milk, stichopus japonicus intestines protein isolate and soybean protein isolate retentiveness；
The holding oiliness of Fig. 4 stichopus japonicus intestines skimmed milk, stichopus japonicus intestines protein isolate and soybean protein isolate；
Fig. 5 stichopus japonicus intestines skimmed milk prepared by the present invention, stichopus japonicus intestines protein isolate and soybean protein isolate are in condition of different pH
Under dissolubility；
Fig. 6 stichopus japonicus intestines skimmed milk prepared by the present invention, stichopus japonicus intestines protein isolate and soybean protein isolate are in condition of different pH
Under emulsibility；
In FIG. 1 to FIG. 4, skimmed milk is indicated: stichopus japonicus intestines skimmed milk, protein isolate indicate: stichopus japonicus intestines protein isolate.
Specific embodiment
Embodiment 1
S1, the homogenate of stichopus japonicus intestines: sea cucumber intestine removes silt, cleans, 5min is homogenized under the conditions of 2000rpm, obtains sea cucumber intestine homogenate
Liquid；
S2, preparation stichopus japonicus intestines skimmed milk: n-hexane/nothing of 9 times of volumes is added into sea cucumber intestine homogenate described in step S1
Water-ethanol (V:V=3:1) mixed solution, stirring and leaching 7h carries out degreasing under the conditions of 600rpm, 4 DEG C, extracts skimming processes weight
It is 1 time multiple, it is filtered, is air-dried at room temperature, crushed, after sieving with 100 mesh sieve, obtain stichopus japonicus intestines skimmed milk；
S3, alkali soluble: using sea cucumber intestine skimmed milk obtained by step S2 as raw material, deionization is added by the solid-liquid ratio of (w/v) 1:20
Water, mixes, and stirring and leaching 2h under the conditions of being adjusted to 11.5,25 DEG C of 300rpm of alkaline pH with 1mol/L NaOH, mixed liquor is put into centrifugation
Machine is centrifuged 20min with 4000r/min, collects supernatant, the leaching liquor of the Erepsin containing stichopus japonicus can be obtained, recovery rate is
32.23%；
S4, acid are heavy: by leaching liquor obtained by step S3, being adjusted to pH3.6 with 1mol/L HCl, stand 3h, under 10500r/min
It is centrifuged 30min, collects precipitating, protein recovery is 98.04% in extracting solution；
S5, dialysis: precipitating obtained by step S4 is dissolved in deionized water with the ratio of 5mL/g, with 0.1mol/L NaOH tune
To pH6.8.Dissolved sample is fitted into 3.5KDa bag filter, is dialysed in the phosphate buffer of 20mmol/L pH7.4
Desalination is carried out for 24 hours, during which replaces 4 dialyzates, and the amount of each dialyzate is 10 times of sample volume, and entire dialysis procedure is 4
DEG C carry out；
Thorn can be obtained in S6, drying: product prepared by the step S5 vacuum freeze drying 72h under the conditions of 1Pa, -50 DEG C
Join intestines protein isolate freeze-dried powder.
Emulsibility when dissolubility when gained stichopus japonicus intestines protein isolate pH10 is 49.40%, pH8 is 228.39g/m2,
Retentiveness is 5.95g/g, holding oiliness 6.98g/g.
The calculation method of protein extracting ratio described in step S3 is as follows:
Gross protein quality × 100% in protein quality/skimmed milk in extraction rate of protein/%=extracting solution
Wherein, protein concentration uses Takara Bradfrod kit measurement in extracting solution, carries out to specifications；Thorn
The measurement for joining the gross protein quality in intestines skimmed milk uses Kjeldahl nitrogen determination (GB 5009.5-2010)；
The calculation method of protein recovery described in step S4 is as follows:
Before protein recovery/%=(before precipitating in extracting solution after albumen quality-precipitating in extracting solution albumen quality)/precipitating
Albumen quality × 100% in extracting solution
Wherein, protein concentration uses Takara Bradfrod kit measurement in extracting solution, carries out to specifications.
Embodiment 2
S1, the homogenate of stichopus japonicus intestines: sea cucumber intestine removes silt, cleans, 4min is homogenized under the conditions of 2500rpm, obtains sea cucumber intestine homogenate
Liquid；
S2, preparation stichopus japonicus intestines skimmed milk: n-hexane/nothing of 10 times of volumes is added into sea cucumber intestine homogenate described in step 1
Water-ethanol (V:V=3:1) mixed solution, stirring and leaching 6h carries out degreasing and carries out degreasing under the conditions of 700rpm, 4 DEG C, and extraction is de-
Rouge process is repeated 2 times, and is filtered, is air-dried at room temperature, and is crushed, after sieving with 100 mesh sieve, is obtained stichopus japonicus intestines skimmed milk；
S3, alkali soluble: using step 2 skimmed milk as raw material, by 1:30 solid-liquid ratio be added deionized water, mix, use
1mol/L NaOH is adjusted to stirring and leaching 2.5h under the conditions of 11.5,40 DEG C of 400rpm of alkaline pH.Mixed liquor be put into centrifuge with
5000r/min is centrifuged 30min, collects supernatant, the leaching liquor of the Erepsin containing stichopus japonicus, recovery rate 26.60% can be obtained；
S4, acid are heavy: by step 3 gained leaching liquor, be adjusted to pH3.8 with 1mol/L HCl, stand 3h, under 11000r/min from
Heart 30min collects precipitating, and protein recovery is 97% in extracting solution；
S5, dialysis: precipitating obtained by step 4 is dissolved in deionized water with the ratio of 6mL/g, with 0.1mol/L NaOH tune
To pH 6.9, dissolved sample is fitted into 3KDa bag filter, and dialyse 36h in the phosphate buffer of 20mmol/L pH7.4
Desalination is carried out, 5 dialyzates are during which replaced, the amount of each dialyzate is 15 times of sample volume.Entire dialysis procedure 4 DEG C into
Row；
S6, drying: by step 5 dialyse after sample under the conditions of 1Pa, -50 DEG C vacuum freeze drying 72h, can obtain
To stichopus japonicus intestines protein isolate freeze-dried powder.
Emulsibility when dissolubility when gained stichopus japonicus intestines protein isolate pH10 is 48.77%, pH8 is 225.49g/m2,
Retentiveness is 6.46g/g, holding oiliness 7.62g/g.
Embodiment 3
S1, the homogenate of stichopus japonicus intestines: sea cucumber intestine removes silt, cleans, 3min is homogenized under the conditions of 3000rpm, obtains sea cucumber intestine homogenate
Liquid；
S2, preparation stichopus japonicus intestines skimmed milk: n-hexane/nothing of 11 times of volumes is added into sea cucumber intestine homogenate described in step 1
Water-ethanol (V:V=3:1) mixed solution, stirring and leaching 5h carries out degreasing under the conditions of 800rpm, 4 DEG C, which is repeated 2 times,
It is filtered, is air-dried at room temperature, crushed, after sieving with 100 mesh sieve, obtain stichopus japonicus intestines skimmed milk；
S3, alkali soluble: using step 2 skimmed milk as raw material, by 1:20 solid-liquid ratio be added deionized water, mix, use
1mol/L NaOH is adjusted to stirring and leaching extraction 3h under the conditions of 11.5,4 DEG C of 500rpm of alkaline pH.Mixed liquor be put into centrifuge with
5000r/min is centrifuged 20min, collects supernatant, the leaching liquor of the Erepsin containing stichopus japonicus, recovery rate 30.0% can be obtained；
S4, acid are heavy: by step 3 gained leaching liquor, being adjusted to pH2 with 1mol/L HCl, stand 3h, be centrifuged under 12000r/min
20min collects precipitating, and protein recovery is 93.7% in extracting solution；
S5, dialysis: precipitating obtained by step 4 is dissolved in deionized water with the ratio of 7mL/g, with 0.1mol/L NaOH tune
To pH7.0.Dissolved sample is fitted into 4KDa bag filter, and dialyse 48h in the phosphate buffer of 20mmol/L pH7.4
Carry out desalination, during which replace 6 dialyzates, the amount of each dialyzate is 20 times of sample volume, entire dialysis procedure 4 DEG C into
Row；
S6, drying: the sample after dialysing in step 5 is freeze-dried, and the freeze-drying of stichopus japonicus intestines protein isolate can be obtained
Powder.
Emulsibility when dissolubility when gained stichopus japonicus intestines protein isolate pH10 is 49.49%, pH8 is 220.65g/m2,
Retentiveness is 5.44g/g, holding oiliness 6.34g/g.
Embodiment 4
S1, the homogenate of stichopus japonicus intestines: sea cucumber intestine removes silt, cleans, 3min is homogenized under the conditions of 3000rpm, obtains sea cucumber intestine homogenate
Liquid；
S2, preparation stichopus japonicus intestines skimmed milk: n-hexane/nothing of 10 times of volumes is added into sea cucumber intestine homogenate described in step 1
Water-ethanol (V:V=3:1) mixed solution, at 4 DEG C, stirring and leaching 6h carries out degreasing under the conditions of 600rpm, which is repeated 2 times,
It is filtered, is air-dried at room temperature, crushed, after sieving with 100 mesh sieve, obtain stichopus japonicus intestines skimmed milk；
S3, alkali soluble: using step 2 gained skimmed milk as raw material, deionized water is added by the solid-liquid ratio of 1:10, mixes, uses
1mol/L NaOH is adjusted to 9.5,25 DEG C of alkaline pH, stirring and leaching 2.5h under the conditions of 400rpm.Mixed liquor be put into centrifuge with
3000r/min is centrifuged 30min, collects supernatant, the leaching liquor of the Erepsin containing stichopus japonicus, recovery rate 26.97% can be obtained；
S4, acid are heavy: by step 3 gained leaching liquor, be adjusted to pH4.6 with 1mol/L HCl, stand 5h, under 8000r/min from
Heart 30min collects precipitating, and protein recovery is 91.3% in extracting solution；
S5, dialysis: precipitating obtained by step 4 is dissolved in deionized water with the ratio of 8mL/g, with 0.1mol/L NaOH tune
To pH7.0, dissolved sample is fitted into 3.5KDa bag filter, is dialysed in the phosphate buffer of 20mmol/L pH7.4
Desalination is carried out for 24 hours, during which replaces 4 dialyzates, and the amount of each dialyzate is 20 times of sample volume, and entire dialysis procedure is 4
DEG C carry out；
S6, drying: by step 5 dialyse after sample under the conditions of 1Pa, -50 DEG C vacuum freeze drying 72h, pierced
Join intestines protein isolate freeze-dried powder.
Emulsibility when dissolubility when gained stichopus japonicus intestines protein isolate pH10 is 49.22%, pH8 is 224.85g/m2,
Retentiveness is 6.15g/g, holding oiliness 7.18g/g.
Embodiment 5
S1, the homogenate of stichopus japonicus intestines: sea cucumber intestine removes silt, cleans, 3min is homogenized under the conditions of 3000rpm, obtains sea cucumber intestine homogenate
Liquid；
S2, preparation stichopus japonicus intestines skimmed milk: n-hexane/nothing of 10 times of volumes is added into sea cucumber intestine homogenate described in step 1
Water-ethanol (V:V=2:1) mixed solution, at 4 DEG C, stirring and leaching 6h carries out degreasing under the conditions of 600rpm, which is repeated 2 times,
It is filtered, is air-dried at room temperature, crushed, after sieving with 100 mesh sieve, obtain stichopus japonicus intestines skimmed milk；
S3, alkali soluble: using step 2 skimmed milk as raw material, by 1:35 solid-liquid ratio be added deionized water, mix, use
1mol/L NaOH is adjusted to 8,60 DEG C of alkaline pH, stirring and leaching 2h under the conditions of 400rpm.Mixed liquor is put into centrifuge with 5000r/
Min is centrifuged 25min, collects supernatant, the leaching liquor of the Erepsin containing stichopus japonicus, recovery rate 12.09% can be obtained；
S4, acid are heavy: by step 3 gained leaching liquor, being adjusted to pH3 with 1mol/L HCl, stand 5h, be centrifuged under 10000r/min
10min collects precipitating, and protein recovery is 96.14% in extracting solution；
S5, dialysis: precipitating obtained by step 4 is dissolved in deionized water with the ratio of 10mL/g, with 0.1mol/L NaOH tune
To pH7.0, dissolved sample is fitted into 3.5KDa bag filter, is dialysed for 24 hours in 20mmol/L pH7.4 phosphate buffer
Desalination is carried out, 4 dialyzates are during which replaced, the amount of each dialyzate is 20 times of sample volume.Entire dialysis procedure 4 DEG C into
Row；
S6, drying: the sample after dialysing in step 5 is subjected to the vacuum freeze drying 72h under the conditions of 1Pa, -50 DEG C, i.e.,
Stichopus japonicus intestines protein isolate freeze-dried powder can be obtained.
Emulsibility when dissolubility when gained stichopus japonicus intestines protein isolate pH10 is 49.32%, pH8 is 224.85g/m2,
Retentiveness is 5.75g/g, holding oiliness 6.78g/g.
Stichopus japonicus skimmed milk prepared by the present invention, chemical composition are as shown in table 1:
Table 1
Protein isolate dissolubility analyzes result as shown in Figure 1, stichopus japonicus intestines protein isolate prepared by the present invention and soybean separation
Albumen all has preferable dissolubility, and the dissolubility of stichopus japonicus intestines protein isolate is significantly better than stichopus japonicus intestines skimmed milk；Emulsibility
Analysis result is as shown in Fig. 2, the emulsibility of stichopus japonicus intestines skimmed milk is better than soybean protein isolate, and the cream of stichopus japonicus intestines protein isolate
The property changed is substantially better than stichopus japonicus intestines skimmed milk and soybean protein isolate；Water holding/oiliness analysis result is as shown in Figure 3, Figure 4, with thorn
Ginseng intestines skimmed milk compare, the retentiveness and holding oiliness of stichopus japonicus intestines protein isolate are significantly improved, moisture holding capacity be 5.44~
6.46g/g, ability of retaining oil is 6.34~7.62g/g, and the holding oiliness of stichopus japonicus intestines protein isolate will be much higher than soybean protein isolate.
Good holding oiliness can promote food to the absorption and retention of fat, reduces loss fatty in processing, improves food
Taste and flavor.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art within the technical scope of the present disclosure, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (9)
1. a kind of extracting method of stichopus japonicus intestines protein isolate, which comprises the steps of:
S1, the homogenate of stichopus japonicus intestines: stichopus japonicus intestines are removed into silt, cleans, is prepared into stichopus japonicus intestines homogenate；
S2, preparation stichopus japonicus intestines skimmed milk: it is molten that n-hexane/dehydrated alcohol mixing is added into stichopus japonicus intestines homogenate described in step S1
Liquid obtains stichopus japonicus intestines skimmed milk by degreasing after filtering, be air-dried at room temperature, crushing, sieving with 100 mesh sieve；
S3, alkali soluble: deionized water is added in stichopus japonicus intestines skimmed milk obtained by step S2 and is mixed, alkaline ph values are adjusted to, is stirred, centrifugation is received
Collect supernatant, obtains stichopus japonicus intestines protein isolate extracting solution；
S4, acid are heavy: by stichopus japonicus intestines protein isolate extracting solution obtained by step S3, being adjusted to acid ph value, stand, precipitating is collected in centrifugation；
S5, dialysis: precipitating obtained by step S4 is dissolved in deionized water, carries out dialysis desalination using phosphate buffer；
S6, drying: step S5 products therefrom is freeze-dried, and obtains stichopus japonicus intestines protein isolate freeze-dried powder.
2. the extracting method of stichopus japonicus intestines protein isolate according to claim 1, which is characterized in that prepare stichopus japonicus described in step S1
The parameter of intestines homogenate are as follows: be homogenized 3~5min under the conditions of 2000~3000rpm, obtain stichopus japonicus intestines homogenate.
3. the extracting method of stichopus japonicus intestines protein isolate according to claim 1, which is characterized in that degreasing described in step S2 is specific
Are as follows: n-hexane/dehydrated alcohol mixed solution is added into stichopus japonicus intestines homogenate described in step S1, in 600~800rpm, 4 DEG C of items
5~7h is stirred under part and carries out degreasing, and the skimming processes repeat 1~3 time.
4. according to claim 1 or the extracting method of the 3 stichopus japonicus intestines protein isolates, which is characterized in that described in step S1 just oneself
The volume ratio of n-hexane and dehydrated alcohol is 2~3:1 in alkane/dehydrated alcohol mixed solution.
5. according to claim 1 or the extracting method of the 3 stichopus japonicus intestines protein isolates, which is characterized in that described in step S1 just oneself
The volume ratio of alkane/dehydrated alcohol mixed solution and stichopus japonicus intestines homogenate is 9~11:1.
6. the extracting method of stichopus japonicus intestines protein isolate according to claim 1, which is characterized in that alkali soluble described in step S3 is specific
Are as follows: deionized water is added by solid-liquid ratio 1:10~35 in stichopus japonicus intestines skimmed milk obtained by step S2, is mixed, with 1mol/L NaOH tune
To pH8.0~11.5,4~60 DEG C, stir 2~3h under the conditions of 300~500rpm, 3000~5000r/min centrifugation 20~
30min collects supernatant, i.e. stichopus japonicus intestines protein isolate extracting solution.
7. the extracting method of stichopus japonicus intestines protein isolate according to claim 1, which is characterized in that acid described in step S4 is heavy specific
Are as follows: stichopus japonicus intestines protein isolate extracting solution obtained by step S3 is adjusted to pH2~4.6 with 1mol/L HCl, stands 1~5h, 8000~
12000r/min is centrifuged 10~30min, collects precipitating.
8. the extracting method of stichopus japonicus intestines protein isolate according to claim 1, which is characterized in that dialysis described in step S5 is specific
Are as follows: precipitating obtained by step S4 is dissolved in deionized water with the ratio of 5~10mL/g, with 0.1mol/L NaOH be adjusted to pH6.8~
7.0, dissolved sample is fitted into 3~4KDa bag filter, in the phosphate buffer of 20mmol/L pH7.4 dialyse 24~
48h carries out desalination, during which replaces 4~6 dialyzates, and the amount of each dialyzate is 10~20 times of sample volume, dialysis procedure
It is carried out at 4 DEG C.
9. the extracting method of stichopus japonicus intestines protein isolate according to claim 1, which comprises the following steps:
S1, the homogenate of stichopus japonicus intestines: sea cucumber intestine removes silt, cleans, 5min is homogenized under the conditions of 2000rpm, obtains sea cucumber intestine homogenate；
S2, preparation stichopus japonicus intestines skimmed milk: n-hexane/anhydrous second of 9 times of volumes is added into sea cucumber intestine homogenate described in step S1
Mixed alkoxide solution carries out degreasing, is filtered, be air-dried at room temperature, and crushes, after sieving with 100 mesh sieve, obtains stichopus japonicus intestines skimmed milk；Wherein, institute
Stating degreasing is that 7h is stirred under the conditions of 600rpm, 4 DEG C, and the degreasing is repeated 1 times；The n-hexane/dehydrated alcohol mixed solution
In, the volume ratio of n-hexane and dehydrated alcohol is 3:1；
S3, alkali soluble: using sea cucumber intestine skimmed milk obtained by step S2 as raw material, deionization is added by the solid-liquid ratio of mass volume ratio 1:20
Water, mixes, and stirring and leaching 2h under the conditions of being adjusted to 11.5,25 DEG C of 300rpm of alkaline pH with 1mol/L NaOH, mixed liquor is put into centrifugation
Machine is centrifuged 20min with 4000r/min, collects supernatant, the leaching liquor of the Erepsin containing stichopus japonicus can be obtained；
S4, acid are heavy: by leaching liquor obtained by step S3, being adjusted to pH3.6 with 1mol/L HCl, stand 3h, be centrifuged under 10500r/min
30min collects precipitating；
S5, dialysis: precipitating obtained by step S4 is dissolved in deionized water with the ratio of 5mL/g, is adjusted to 0.1mol/L NaOH
pH6.8；Dissolved sample is fitted into 3.5KDa bag filter, is dialysed in the phosphate buffer of 20mmol/L pH7.4 for 24 hours,
Period replaces 4 dialyzates, and the amount of each dialyzate is 10 times of sample volume, and entire dialysis procedure is carried out at 4 DEG C；
Stichopus japonicus intestines can be obtained in S6, drying: product prepared by the step S5 vacuum freeze drying 72h under the conditions of 1Pa, -50 DEG C
Protein isolate freeze-dried powder.
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