CN102382200A - Production process for separating and extracting sargasso polysaccharide by enzymatic hydrolysis method - Google Patents
Production process for separating and extracting sargasso polysaccharide by enzymatic hydrolysis method Download PDFInfo
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Abstract
The invention discloses a production process for separating and extracting sargasso polysaccharide by an enzymatic hydrolysis method. According to the invention, papain is taken as deproteinization catalyzing enzyme, sargasso polysaccharideis is obtained by separating and extracting through the enzymatic hydrolysis method. The established better extraction condition are that the ratio of material to liquid is 1:15-1:35, the extraction times is 2-4 times, and the alcohol precipitation concentration is 65-85%. The method enables substantial increase of yield and the total sugar content of the crude polysaccharide, provides good technology route for large scale production of the sargasso polysaccharide, and effectively endure the market supply of vaccine immunopotentiator for fowl animals.
Description
Technical field
The present invention relates to the separation and Extraction of sargassan, especially a kind of production technique of enzymolysis process separation and Extraction sargassan.
Background technology
Sargassan (Sargassum Polysaccharide) can significantly improve the protection ratio and the disease resistance of vaccine as the vaccine immunopotentiator of chicken, has a extensive future.According to statistics, the amount of livestock on hand of present domestic annual chicken is 4,000,000,000, if 1.0% rate of utilization is arranged, and 4,000 ten thousand of promptly annual application chickens, in average every consumption 5 grams, then annual requirement reaches 200 tons.By 200 yuan of market value per kilograms, then annual sales amount reaches 4,000 ten thousand yuan, and it is 1,200 ten thousand yuan (rate of profit is by 30%) that pure profit can reach.Yet there are the problem that polysaccharide yield is low and total sugar content is low in water extraction and alcohol precipitation method, the alkaline extraction of separation and Extraction sargassan at present, so that production efficiency is low, under-supply, are difficult to satisfy great demand.
Summary of the invention
The technical problem that the present invention will solve provides the production technique of the high and enzymolysis process separation and Extraction sargassan that total sugar content is high of a kind of polysaccharide yield.
Adopt following technical scheme for solving the problems of the technologies described above: the production technique of enzymolysis process separation and Extraction sargassan; This technology comprises the steps: that < 1>accurately takes by weighing the sargassun powder,, press solid-liquid ratio and added zero(ppm) water in 1: 15～1: 35; It is 0.5%～1% that the adding papoid makes its mass and size concentration; First 40 ℃ of water-baths dissolving 1h, go out enzyme 0.5h, 80 ℃ of water-bath lixiviates then of 100 ℃ of water-baths again; < 2>in the step < 1>after the lixiviate, the centrifuging and taking supernatant is subsequent use, and deposition press solid-liquid ratio and added zero(ppm) water in 1: 20～1: 35, the continuation lixiviate, and lixiviate is 1～3 time in accordance with the law; < 3>combining step < 2>each time supernatant is concentrated into 1/4 of original volume with it, and the ethanol of liquid concentrator adding 95% is 65～85% to last alcohol precipitation concentration, and alcohol precipitation spends the night, and abandons supernatant, centrifugal must the deposition; < 4>get step < 3>gained deposition, add the zero(ppm) water of 10 times of sargassun grain weight amounts, 100 ℃ of water-bath dissolvings; Solution centrifugal is got supernatant, and adding 95% ethanol in the supernatant is 65～85% to last alcohol precipitation concentration, and alcohol precipitation spends the night; Abandon supernatant, centrifugal must the deposition; < 5>with the lyophilize of step < 4>gained deposition, promptly get the polysaccharide sample.
The polysaccharide sample through grind powdery polysaccharide sample.
Centrifugal 3000r/min carries out 10min.
The polysaccharide yield of the production technique of enzymolysis process separation and Extraction sargassan of the present invention and total sugar content are applied to produce sargassan and can raise the efficiency, meet the need of market apparently higher than present water extraction and alcohol precipitation method and alkaline extraction.
Embodiment
The Research on Process of enzymolysis process separation and Extraction sargassan
The design of first and second factorial
1. secondary factorial plan
Selecting out 6 principal elements that experimental result is had the greatest impact is solid-liquid ratio, enzyme amount, extraction temperature, extraction time, lixiviate number of times, alcohol precipitation concentration, to these 6 principal elements, reserves blank and carries out the orthogonal test factor analysis.Use SAS software, select PB (Plackett-Burman) design of N=12 for use, the path is the new design → 2 grade level of SAS → solution → analysis → experimental design → file → establishment.Each influence factor is all chosen 2 levels and is analyzed, and low-level is-1, and high level is 1, experimental design such as table 1.
Table 1 PB design and result (N=12)
In the table 1: OBS is a scheme; X1 is a solid-liquid ratio; X2 is the enzyme amount; X3 is an extraction temperature; X4 is an extraction time; X5 is the lixiviate number of times; X6 is an alcohol precipitation concentration; SO42-% is a sulfate radical content.
The PB design of the N=12 that designs according to SAS makes an experiment by each factor different levels.Experimentation: accurately take by weighing sargassun powder 10g, the solid-liquid ratio of pressing different levels adds zero(ppm) water, takes by weighing the adding papoid by the enzyme amount of different levels, 40 ℃ of water-baths dissolving 1h, and 100 ℃ of water-baths enzyme 0.5h that goes out is positioned over the different levels temperature and carries out the water-bath lixiviate.After the lixiviate, 3000r/min is centrifugal, and 10min gets supernatant, and deposition adds zero(ppm) water by the solid-liquid ratio of this experimental group, continues lixiviate.According to the extraction time and the lixiviate number of times of each experimental group, the supernatant with each time after centrifugal merges, gained supernatant concentration to 1/4 volume; Liquid concentrator is by the ethanol of the alcohol precipitation concentration adding 95% of each experimental group, and alcohol precipitation spends the night, and abandons supernatant; The centrifugal 10min of 3000r/min must precipitate, and adds 100ml zero(ppm) water, 100 ℃ of water-bath dissolvings; Solution 3000r/min is centrifugal, and 10min gets supernatant, the supernatant of getting continue alcohol precipitation concentration by each experimental group and add 95% ethanol, alcohol precipitation spends the night; Abandon supernatant, the centrifugal 10min of 3000r/min must precipitate, and each experimental group is provided with a horizontal control experiment.The gained deposition is through the freeze drier lyophilize, and weighing deposition gained example weight will precipitate grinding and get powdery polysaccharide sample afterwards.
2. secondary factorial design result and analysis
The result that samples contg is measured is as shown in table 1, analyzes sulfate radical (SO4
2-) content, polysaccharide content and total value content (total value=(sulfate radical content+polysaccharide content) x yield); Utilize SAS software that the PB result of design of N=12 is analyzed; The path is that test design → Report → GenerateReport of SAS → solution → analysis → experimental design → N=12 draws ultimate analysis result report, analytical results such as table 2, table 3, table 4.
Each level of factor of table 2 is selected sulfate radical (SO4
2-) impact effect of content
In the table 2: DF is a degree of freedom; SS is a sum of sguares of deviation from mean; MS is all square; Pr＞F is the probability greater than the F value.
Can know by table 2; In solid-liquid ratio, enzyme amount, extraction temperature, extraction time, lixiviate number of times, these six principal elements of alcohol precipitation concentration; The Pr of alcohol precipitation concentration＞F value less than 0.05, has explained that alcohol precipitation concentration is to sulfate radical (SO4 in the sample of the extraction of whole polysaccharide greater than 0.01
2-) the having the greatest impact of content, promptly the difference of alcohol precipitation concentration is big more, SO4 in the sample that then extracts
2-Content also gap is big more.Other factors also affect SO4 in the sample
2-The size of content, but impact effect is not clearly.
Each level of factor of table 3 is selected the impact effect to polysaccharide content
In the table 3: DF is a degree of freedom; SS is a sum of sguares of deviation from mean; MS is all square; Pr＞F is the probability greater than the F value.
Combine the analysis of SAS software by table 3; Can find out; The value 0.0115＞0.05 of selected solid-liquid ratio, enzyme amount, extraction temperature, extraction time, lixiviate number of times, these six principal element Pr＞F of alcohol precipitation concentration explains that these 6 factor change amounts are not remarkable to polysaccharide content influence in the polysaccharide sample that extracts.
Each level of factor of table 4 is selected the impact effect to total value
In the table 4: DF is a degree of freedom; SS is a sum of sguares of deviation from mean; MS is all square; Pr＞F is the probability greater than the F value.
Combine the analysis of SAS software by table 4; Can find out; The value of solid-liquid ratio (factor 1), lixiviate number of times (factor 5) Pr＞F is respectively 0.0039,0.0007 all less than 0.01; The value 0.0151 of alcohol precipitation concentration (factor 6) Pr＞F greater than 0.01 less than 0.05; The change that solid-liquid ratio, lixiviate number of times are described is remarkable for extremely to total value content influence in the polysaccharide sample that extracts, and the change of alcohol precipitation concentration is significantly to total value content influence in the polysaccharide sample that extracts, so solid-liquid ratio, lixiviate number of times and alcohol precipitation concentration are the main factors that influences extraction efficiency.
Comprehensive above-mentioned analysis; Can see with the sulfate radical content being that index draws the principal element that alcohol precipitation concentration is influence; With the polysaccharide content is that index does not find topmost influence factor; Only with total value be with reference to the time, to testing apparent in view that influential factor just shows, so follow-up test all compares analysis with total value as a reference.
Two, steepest hill climbing test
1. principle of design and step
The gradient direction that the steepest hill climbing test changes with trial value is the climbing direction, confirms the variation size of composition in the sargassan sample according to the size of each factorial effect value, and then can find out near the maximum Polysaccharide from Bee of total value.Result according to above-mentioned analysis; Select three main influence factors; Be variable with solid-liquid ratio, lixiviate number of times, alcohol precipitation concentration respectively, the condition of each variable progressively improves carries out the design of steepest hill climbing test, and each factor condition is as shown in table 5; Experimentation and operation are according to the polysaccharide leaching process, and the result sees table 5.
Climbing experimental design of table 5 steepest and result
2. interpretation of result
From table 5, can be clearly seen that: sargassan is when solid-liquid ratio 1: 15～1: 35, is preferable extraction process in the time of lixiviate number of times 2～4 times, alcohol precipitation concentration 65～85%.
Embodiment 1
< 1>take by weighing sargassun powder 10g, press solid-liquid ratio 1: 30 and add zero(ppm) water, adding papoid, to make its mass and size concentration be 0.5%, 40 ℃ of water-baths dissolving 1h earlier, go out enzyme 0.5h, 80 ℃ of water-bath lixiviates then of 100 ℃ of water-baths again;
< 2>after the middle lixiviate of step < 1 >, it is subsequent use that the centrifugal 10min of 3000r/min gets supernatant, and deposition is pressed solid-liquid ratio and added zero(ppm) water at 1: 30, continues lixiviate, and lixiviate is 3 times in accordance with the law;
< 3>combining step < 2>totally 4 lixiviate centrifugal supernatants are concentrated into 1/4 of original volume with it, and it is 80% to last alcohol precipitation concentration that liquid concentrator adds 95% ethanol, and alcohol precipitation spends the night, and abandons supernatant, and the centrifugal 10min of 3000r/min must precipitate;
< 4>get step < 3>gained deposition, add 100ml zero(ppm) water, 100 ℃ of water-bath dissolvings; Solution 3000r/min is centrifugal, and 10min gets supernatant, and adding 95% ethanol in the supernatant is 80% to last alcohol precipitation concentration, and alcohol precipitation spends the night; Abandon supernatant, the centrifugal 10min of 3000r/min must precipitate;
< 5>step < 4>gained deposition is used the freeze drier lyophilize, weighing deposition gained example weight will precipitate grinding and get powdery polysaccharide sample afterwards.
Embodiment 2
Other conditions are with embodiment 1, and solid-liquid ratio is 1: 15, papoid concentration 1%, and lixiviate totally 2 times, alcohol precipitation concentration is 65%
Embodiment 3
Other conditions are with embodiment 1, and solid-liquid ratio is 1: 25, papoid concentration 0.75%, and lixiviate totally 3 times, alcohol precipitation concentration is 75%
Embodiment 4
Other conditions are with embodiment 1, and solid-liquid ratio is 1: 35, papoid concentration 1%, and lixiviate totally 4 times, alcohol precipitation concentration is 85%
Reference examples 1 water extraction and alcohol precipitation method extracts sargassan
Get sargassun powder 10g, add 150ml zero(ppm) water, boil 1.5h, filter, residue adding distil water 150ml boils 1.5h again, and the centrifugal 10min of 3000r/min merges filtrating twice.
Filtrating is concentrated into 100ml, slowly adds 95% alcohol of 4 times of amounts, and the limit edged stirs, and leaves standstill more than the 8h, and the centrifugal 10min of 3000r/min must precipitate.To precipitate adding 100ml zero(ppm) water, heating for dissolving, 3000r/min is centrifugal, and 10min gets supernatant, adds 95% alcohol of 4 times of amounts, leaves standstill more than the 8h, and the centrifugal 10min of 3000r/min must precipitate.After washing with absolute ethyl alcohol, lyophilize gets sargassan.
Reference examples 2 alkaline extractions extract sargassan
Get sargassun powder 10g, add 150ml10% alcohol (using NAOH to transfer to pH is 9), 80 ℃ of water-bath 1.5h filter, and residue adds 150ml10% alcohol, 80 ℃ of water-bath 1.5h, and the centrifugal 10min of 3000r/min merges filtrating twice.
Filtrating is concentrated into 100ml, slowly adds 95% alcohol of 4 times of amounts, and the limit edged stirs, and leaves standstill more than the 8h, and the centrifugal 10min of 3000r/min must precipitate.To precipitate adding 100ml zero(ppm) water, heating for dissolving, 3000r/min is centrifugal, and 10min gets supernatant, adds 95% alcohol of 4 times of amounts, leaves standstill more than the 8h, and the centrifugal 10min of 3000r/min must precipitate.After washing with absolute ethyl alcohol, lyophilize gets sargassan.
The sargassan of above-mentioned each embodiment and reference examples is measured result such as table 6:
Table 6 sargassan is measured the result
It is thus clear that; Select for use papoid to adopt enzymolysis process separation and Extraction sargassan for the Deproteinization katalaze(enzyme); Crude polysaccharides yield and total sugar content have been improved significantly; For large-scale production sargassan from now on provides good operational path, can effectively guarantee the market supply of the vaccine immunopotentiator of bird.
Claims (3)
1. the production technique of an enzymolysis process separation and Extraction sargassan is characterized in that this technology comprises the steps:
< 1>accurately take by weighing the sargassun powder, press solid-liquid ratio and added zero(ppm) water in 1: 15～1: 35, adding papoid, to make its mass and size concentration be 0.5%～1%, 40 ℃ of water-baths dissolving 1h earlier, go out enzyme 0.5h, 80 ℃ of water-bath lixiviates then of 100 ℃ of water-baths again;
< 2>in the step < 1>after the lixiviate, the centrifuging and taking supernatant is subsequent use, and deposition press solid-liquid ratio and added zero(ppm) water in 1: 20～1: 35, the continuation lixiviate, and lixiviate is 1～3 time in accordance with the law;
< 3>combining step < 2>each time supernatant is concentrated into 1/4 of original volume with it, and the ethanol of liquid concentrator adding 95% is 65～85% to last alcohol precipitation concentration, and alcohol precipitation spends the night, and abandons supernatant, centrifugal must the deposition;
< 4>get step < 3>gained deposition, add the zero(ppm) water of 10 times of sargassun grain weight amounts, 100 ℃ of water-bath dissolvings; Solution centrifugal is got supernatant, and adding 95% ethanol in the supernatant is 65～85% to last alcohol precipitation concentration, and alcohol precipitation spends the night; Abandon supernatant, centrifugal must the deposition;
< 5>with the lyophilize of step < 4>gained deposition, promptly get the polysaccharide sample.
2. the production technique of enzymolysis process separation and Extraction sargassan according to claim 1 is characterized in that: said polysaccharide sample through grind powdery polysaccharide sample.
3. the production technique of enzymolysis process separation and Extraction sargassan according to claim 1 is characterized in that: said centrifugal 3000r/min carries out 10min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105483183A (en) * | 2016-01-07 | 2016-04-13 | 福建农林大学 | Preparation method of sargassum oligosaccharide and application of sargassum oligosaccharide in hypoglycemic drugs |
| CN108727510A (en) * | 2018-06-06 | 2018-11-02 | 广西大学 | The application of spirulina polysaccharide extract and preparation method thereof and enhancing immune function |
| CN115746155A (en) * | 2022-11-22 | 2023-03-07 | 广西大学 | Preparation method and application of sargassum polysaccharide extract SP1 |
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| CN1752110A (en) * | 2004-09-21 | 2006-03-29 | 珠江医院 | Sargassum polysaccharide, its preparation method and use |
| CN101475651A (en) * | 2009-01-16 | 2009-07-08 | 伍曾利 | Preparation of Gracilaria gigas Harvey polysaccharides |
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| JP2001288202A (en) * | 2000-04-05 | 2001-10-16 | Nikken Sohonsha Corp | Preparation method of acidic polysaccharide |
| CN1752110A (en) * | 2004-09-21 | 2006-03-29 | 珠江医院 | Sargassum polysaccharide, its preparation method and use |
| CN101475651A (en) * | 2009-01-16 | 2009-07-08 | 伍曾利 | Preparation of Gracilaria gigas Harvey polysaccharides |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105483183A (en) * | 2016-01-07 | 2016-04-13 | 福建农林大学 | Preparation method of sargassum oligosaccharide and application of sargassum oligosaccharide in hypoglycemic drugs |
| CN105483183B (en) * | 2016-01-07 | 2019-05-10 | 福建农林大学 | A kind of preparation method of sargassum oligosaccharides and its application in hypoglycemic drug |
| CN108727510A (en) * | 2018-06-06 | 2018-11-02 | 广西大学 | The application of spirulina polysaccharide extract and preparation method thereof and enhancing immune function |
| CN115746155A (en) * | 2022-11-22 | 2023-03-07 | 广西大学 | Preparation method and application of sargassum polysaccharide extract SP1 |
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Application publication date: 20120321 |