CN103951761A - Method for degrading enteromorpha prolifera polysaccharides by enzymic method - Google Patents
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Abstract
The invention discloses a method for degrading enteromorpha prolifera polysaccharides by an enzymic method. The method disclosed by the invention is characterized by comprising the following steps: preparing a polysaccharide solution with concentration of 1-20mg/mL from crude enteromorpha prolifera polysaccharides or enteromorpha prolifera polysaccharides; then with glucoamylase as hydrolase, reacting for 1-5 hours under the conditions that the pH value is 3.5-5.5 and the temperature is 40-60 DEG C, thus obtaining a solution of polysaccharides degraded through enzymolysis for the first time; boiling the solution of polysaccharides degraded through enzymolysis for the first time for 4-6 minutes and then centrifuging the solution of polysaccharides degraded through enzymolysis for the first time to remove glucoamylase, thus obtaining supernatant containing enteromorpha prolifera polysaccharides degraded through enzymolysis for the first time; and dialyzing and freeze-drying the supernatant, thus obtaining powdery enteromorpha prolifera polysaccharides (with molecular weight range of 7.2-122kDa) degraded through enzymolysis. The method has the characteristics of high hydrolysis efficiency and high in vitro antioxidative activities of obtained polysaccharides.
Description
Technical field
The invention belongs to technical field of food biotechnology, relate to a kind of method of enzymic degradation sea grass polysaccharide.
Background technology
Enteromorpha is a kind of large-scale economy green alga of China's southeastern coast, aboundresources, and itself is edible, and contains various active material, as sea grass polysaccharide, lipid pigment, phenols etc.According to the literature, sea grass polysaccharide has and improves mammalian immune power, reducing blood-fat, antiphlogistic antibacterial and the physiological function such as anti-oxidant.Therefore Enteromorpha is developed to function food good prospect.Enteromorpha water extraction polysaccharide is mainly water miscible sulfated polysaccharide, and its composition is mainly glucuronic acid-wood sugar-rhamnosyl polymkeric substance.Sea grass polysaccharide molecular weight is large, if be degraded into the product that molecular weight is relatively little, owing to dissociating more active group, can make its biological activity be improved.
It is reported, sea grass polysaccharide sugar key type is mainly β-Isosorbide-5-Nitrae, α-1,2, α-Isosorbide-5-Nitrae, α-1,2,4 and β-1,6, also contain β-1 of small amount, and 3 and α-1,3 glycosidic links.
Acid degradation is the traditional method of polysaccharide degraded, can be by regulating acidity, temperature and obtaining the hydrolysate of different palliating degradation degrees action time.But sulfated polysaccharide its sulfate in degradation process is easily hydrolyzed and comes off, the reduction of sulfate content can cause the physiologically active of polysaccharide to reduce; The degradation rate of the method is 40.05%; Molecular weight ranges is generally 5.32～95.6kDa.
Though oxidative degradation can reduce the sulfate of polysaccharide in degradation process, come off, oxidation products is relatively complicated, has increased the difficulty of separation and purification.The degradation rate of the method is 61.32%; Molecular weight ranges is generally 2.28～55.9kDa.
Enzymic degradation action condition is gentle, also can avoid coming off of sulfate, but be difficult to obtain the enzyme that hydrolysis efficiency is high.But at present the enzymic degradation of sea grass polysaccharide is seldom had been reported.
Summary of the invention
The technical problem to be solved in the present invention is to provide the method for the enzymic degradation sea grass polysaccharide that a kind of hydrolysis efficiency is high, antioxidation activity in vitro is good.
In order to solve the problems of the technologies described above, the invention provides a kind of method of enzymic degradation sea grass polysaccharide, Enteromorpha Crude polysaccharides or sea grass polysaccharide are configured to the polysaccharide soln that concentration is 1～20mg/mL (being preferably 4～6mg/mL); Then using saccharifying enzyme (glucoamylase) as lytic enzyme, in polysaccharide soln, add saccharifying enzyme until the final concentration of saccharifying enzyme is 2～100U/mL (being preferably 10～20U/mL); In pH be 3.5～5.5, temperature is 40～60 ℃ of reactions 1～5 hour (being preferably in pH is 4.5,49 ℃ of reactions 2 hours), enzymolysis degradation of polysaccharide solution first; Enzymolysis degradation of polysaccharide solution boiled after 4～6 minutes first, centrifugal to remove saccharifying enzyme, must contain the supernatant liquor of the rear sea grass polysaccharide of enzymolysis degraded first.
Remarks explanation: Enteromorpha Crude polysaccharides or sea grass polysaccharide are configured to the polysaccharide soln that concentration is 1～20mg/mL, are: when selecting Enteromorpha Crude polysaccharides, the concentration of Enteromorpha Crude polysaccharides is 1～20mg/mL; When selecting sea grass polysaccharide, the concentration of sea grass polysaccharide is 1～20mg/mL.
Centrifugal 4～the 6min of centrifugal referring to: 7000～9000r/s.
Improvement as the method for enzymic degradation sea grass polysaccharide of the present invention: dialyse containing the dialysis tubing that the supernatant liquor of sea grass polysaccharide is 500 with molecular weight cut-off after enzymolysis degraded first, to remove inorganic salt; Obtain dialyzate; By dialyzate lyophilize, obtain pulverous enzymolysis degraded sea grass polysaccharide (molecular weight is lower, and molecular weight ranges is between 7.2～122kDa).
Remarks explanation: above-mentioned dialysis time is 20～28 hours; Lyophilize refers at-45～-55 ℃ and is dried 20～28 hours.
Another kind as the method for enzymic degradation sea grass polysaccharide of the present invention improves: by containing the supernatant liquor of sea grass polysaccharide after enzymolysis degraded first, be again hydrolyzed, described hydrolysis again comprises the steps:
After containing enzymolysis degraded first, in the supernatant liquor of sea grass polysaccharide, add polygalacturonase, the consumption of described polygalacturonase is 1～10% (being preferably 6～9%) of Enteromorpha Crude polysaccharides or sea grass polysaccharide weight; In pH be 3.5～5.5, temperature is 45～60 ℃ of reactions 1～5 hour (being preferably pH is 4.5, and temperature is 56 ℃ of reactions 3 hours), enzymolysis degradation of polysaccharide solution again; Enzymolysis degradation of polysaccharide solution boiled after 4～6 minutes again, centrifugal to remove polygalacturonase, must contain again the supernatant liquor of the rear sea grass polysaccharide of enzymolysis degraded.
Remarks explanation: the enzyme of polygalacturonase is lived as 30U/mg.Centrifugal 4～the 6min of centrifugal referring to: 7000～9000r/s.
Further improvements in methods as enzymic degradation sea grass polysaccharide of the present invention:
The dialysis tubing dialysis that is 500 with molecular weight cut-off by the supernatant liquor that contains again the rear sea grass polysaccharide of enzymolysis degraded, to remove inorganic salt; Obtain dialyzate; By dialyzate lyophilize, obtain pulverous enzymolysis degraded sea grass polysaccharide (molecular weight is lower, and molecular weight ranges is between 7.2～122kDa).
Remarks explanation: above-mentioned dialysis time is 20～28 hours; Lyophilize refers at-45～-55 ℃ and is dried 20～28 hours.
In the present invention, the acetic acid-sodium acetate buffer of available pH3.5～5.5 carries out the adjusting control of pH value.
The purity of Enteromorpha Crude polysaccharides used in the present invention (mass content) is 75～80%.
In the present invention, Enteromorpha Crude polysaccharides or sea grass polysaccharide all can obtain by known technology, and for example, Enteromorpha Crude polysaccharides also can be prepared by the following method and obtain:
1), the Enteromorpha that will be dried after (water ratio≤8.21%, quality %) pulverizes with stamp mill, crosses 120 mesh sieves, obtains Enteromorpha powder.
2), ethanol removal of impurities:
By dehydrated alcohol and Enteromorpha powder according to the solid-liquid ratio of 20ml/1g, thereby under 90 ℃ of water bath condition, stir and remove the fat-soluble component in marine alga, described removal of impurities process repeats (each stir about 30 minutes) 2～4 times, filters, and gets filter cake.
3), water extraction sea grass polysaccharide:
By step 2) " Enteromorpha water-soluble polysaccharide extraction process " in the technical study > > that extracts according to < < Enteromorpha water-soluble polysaccharide of the filter cake of gained, prepare the Enteromorpha Crude polysaccharides aqueous solution.
4), the removal of albumen:
By step 3) the Enteromorpha Crude polysaccharides aqueous solution of gained carries out enzyme process deproteinated according to " sea grass polysaccharide enzyme process deproteinated technique " in the research > > of < < sea grass polysaccharide method for removing protein: the consumption of papoid is 3% (W:V), pH value is 6.5, temperature 50 C, enzymolysis time is 2.5h.
Again the enzymolysis solution of gained is utilized Sevag method to remove albumen repeatedly, centrifugal until upper strata is almost without albumen, vacuum rotary steam is removed chloroform and propyl carbinol, add dehydrated alcohol to make its purity be not less than 80%, after 4 ℃ of refrigerator alcohol precipitation 12h, more centrifugal, precipitation is repeatedly washed by the order of dehydrated alcohol, ether, acetone, finally carry out lyophilize, obtain Enteromorpha Crude polysaccharides.Wherein, the mass content of polysaccharide is 75.45%, and purity is 75.45%.
The enzymolysis degraded sea grass polysaccharide powder that adopts the inventive method preparation and obtain, molecular weight ranges is between 7.2～122kDa.
The enzymolysis degraded sea grass polysaccharide powder that adopts the inventive method preparation and obtain, measures its anti-oxidant activity, with efficient gel permeation chromatography method determining molecular weight.
The present invention has following technique effect:
1. compare with the traditional method such as acid degradation, adopt method degraded sea grass polysaccharide action condition of the present invention gentle, the reaction times is short.
2. the degradation of polysaccharide molecular weight obtaining is lower; Molecular weight ranges is between 7.2～122kDa.
3. compare with undegradable sea grass polysaccharide, there is better antioxidation activity in vitro.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the enzymolysis sea grass polysaccharide that obtained by embodiment of the present invention 1-1 and the test result of the DPPH radical scavenging activity of degradation of polysaccharide not.
Fig. 2 be the enzymolysis sea grass polysaccharide that obtained by embodiment of the present invention 1-1 and not the ultra-oxygen anion free radical of degradation of polysaccharide remove the test result of ability.
Fig. 3 be the enzymolysis sea grass polysaccharide that obtained by embodiment of the present invention 1-1 and not the hydroxyl radical free radical of degradation of polysaccharide remove the test result of ability.
Fig. 4 is the efficient gel permeation chromatography figure of the enzymolysis sea grass polysaccharide that obtains of embodiment of the present invention 1-1.
Embodiment
The method of embodiment 1-1, enzymic degradation sea grass polysaccharide:
Get purity and be 75.45% Enteromorpha Crude polysaccharides and be dissolved into acetic acid-sodium acetate buffer (total concn of acetic acid and sodium acetate is 0.1mol/L) of pH4.5 the Crude polysaccharides solution that Enteromorpha Crude polysaccharides concentration is 5mg/mL.
First by 10 * 10
4the saccharifying enzyme of U/mL (glucoamylase) is diluted to 100U/mL with acetic acid-sodium acetate buffer (total concn of acetic acid and sodium acetate is 0.1mol/L) of pH4.5, obtains glucoamylase enzyme solution.Get 710 μ L glucoamylase enzyme solution and complement to 1mL with distilled water, glucoamylase enzyme solution after must diluting.
Then in 4mL Crude polysaccharides solution, adding the rear glucoamylase enzyme solution of 1ml dilution, is 14.2U/mL thereby make the concentration of saccharifying enzyme, and the concentration of Enteromorpha Crude polysaccharides is 4mg/mL; Now pH is 4.5, in temperature, is 2 hours 49 ℃ of reaction times.
Polysaccharide soln after enzymolysis is boiled to 5min, the centrifugal enzyme (8000r/s, 5min) of removing, the dialysis tubing that is then 500 by gained supernatant liquor with molecular weight cut-off dialysis 24h, to remove inorganic salt; Obtain dialyzate; Finally dialyzate is carried out to lyophilize (be dried 24h in vacuum freeze drying, temperature is-50 ℃).Obtain degradation rate and be 7.6% enzymolysis sea grass polysaccharide (that is, obtaining enzymolysis degraded sea grass polysaccharide).
Remarks explanation:
1, " enzymolysis polysaccharide ", refers to " the enzymolysis sea grass polysaccharide " of final gained.
2, " sea grass polysaccharide " refers to not " the Enteromorpha Crude polysaccharides " of enzymolysis.
Embodiment 1-2, by sea grass polysaccharide saccharifying enzyme enzymolysis:
Reaction times in embodiment 1-1 was made into 4 hours by 2 hours, and all the other are with embodiment 1-1.
Obtain degradation rate and be 11.6% enzymolysis sea grass polysaccharide.
Experiment 1,
Adopt document (Espin J C, Soler-Rivas C, Wichers H J, etal.Anthocyanin-based natural colorants:a new source of antiradical activity for foodstuff[J] .Journal Agricultural Food Chemistry, 2000,48 (5): 1588-1592.) report method, the enzymolysis sea grass polysaccharide that embodiment 1-1 is obtained carries out the test of DPPH radical scavenging activity, and compares with degradation of polysaccharide not.As seen from Figure 1, sea grass polysaccharide is after enzymolysis, and DPPH radical scavenging activity is significantly improved.When concentration is 5mg/mL, the sea grass polysaccharide of degrading with saccharifying enzyme and the not clearance rate of the DPPH free radical of degradation of polysaccharide are respectively 21.03 ± 1.67% and 12.2 ± 1.9%.But the activity of the removing DPPH free radical of enzymolysis sea grass polysaccharide is still less than vitamins C.
Experiment 2,
Adopt document (Xu Yan, Qu Tingting. Radix Glycyrrhizae is eliminated the in vitro study [J] of oxyradical. food research and development, 2006,27 (8): 63-65.) report method, the enzymolysis sea grass polysaccharide that embodiment 1-1 is obtained carries out the test that ultra-oxygen anion free radical is removed ability, and compares with degradation of polysaccharide not.As seen from Figure 2, enzymolysis sea grass polysaccharide to the removing ability of ultra-oxygen anion free radical apparently higher than degradation of polysaccharide not.When concentration is 5mg/mL, the clearance rate of the ultra-oxygen anion free radical of saccharifying enzyme degradation of polysaccharide and not degradation of polysaccharide is respectively 56.96 ± 1.67% and 42.09 ± 1.95%.
Experiment 3,
Adopt document (Chen H W, Chen A H, Shao Y, etal.Studies on the antioxidant capacity of zinc rich exopolysaccharide of cordyceps militaris[J] .Food and Fermentation Industries, 2009,35 (6): 54-57.) report method, the enzymolysis sea grass polysaccharide that embodiment 1-1 is obtained carries out the test that hydroxyl radical free radical is removed ability, and compares with degradation of polysaccharide not.As seen from Figure 3, enzymolysis sea grass polysaccharide to the removing ability of hydroxyl radical free radical higher than degradation of polysaccharide not.When concentration is 5mg/mL, the clearance rate of the hydroxyl radical free radical of saccharifying enzyme degradation of polysaccharide and not degradation of polysaccharide is respectively 44.68 ± 1.72% and 32.37 ± 2.02%.
Experiment 4,
Adopt High Performance Gel Permeation Chromatography (HPGPC) to measure the molecular weight of the enzymolysis sea grass polysaccharide being obtained by embodiment 1-1.From the atlas analysis of Fig. 4, this Enteromorpha enzymolysis polysaccharide is heterogencity polysaccharide, and its retention time is from 19.312min to 24.567min, show that its molecular weight ranges is between 7.2～122kDa.
Comparative example 1-1, the enzymolysis time in embodiment 1-1 was made into respectively 0.5 hour, 7 hours by 2 hours, all the other contents are with embodiment 1.
The enzymolysis degraded sea grass polysaccharide of gained is detected according to the method for above-mentioned experiment 1～experiment 3, and acquired results is as shown in table 1 below.
Remarks explanation: result when following data are polysaccharide concentration and are 5mg/ml.
Table 1
The method of embodiment 2, enzymic degradation sea grass polysaccharide
Get purity and be 75.45% Enteromorpha Crude polysaccharides and be dissolved into acetic acid-sodium acetate buffer (0.1mol/L) of pH4.5 the Crude polysaccharides solution that concentration is 5mg/mL.
First by 10 * 10
4the saccharifying enzyme of U/mL (glucoamylase) is diluted to 100U/mL with acetic acid-sodium acetate buffer (0.1mol/L) of pH4.5, obtains glucoamylase enzyme solution.
The polygalacturonase of 156mg (enzyme is lived as 30U/mg) is settled to 100mL with acetic acid-sodium acetate buffer (0.1mol/L) of pH4.5; Obtain polygalacturonase enzyme liquid.
Then in 4mL Crude polysaccharides solution, adding the rear glucoamylase enzyme solution of 1ml dilution, is 14.2U/mL thereby make enzyme concn, and the concentration of Enteromorpha Crude polysaccharides is 4mg/mL; Now pH is 4.5, in temperature, is 2 hours 49 ℃ of reaction times.
Polysaccharide soln after enzymolysis is boiled to 5min, the centrifugal enzyme (8000r/s, 5min) of removing, then carries out following hydrolysis again:
Adding polygalacturonase enzyme liquid 1mL, that is, is 7.8% (% by weight) of Enteromorpha Crude polysaccharides thereby make the enzyme concentration of polygalacturonase again; At pH, be 4.5, temperature is 56 ℃ of reaction 180min, the polysaccharide soln after enzymolysis is boiled to 5min, the centrifugal polygalacturonase (8000r/s, 5min) of removing.Then the dialysis tubing that is 500 by gained supernatant liquor with molecular weight cut-off dialysis 24h, to remove inorganic salt; Obtain dialyzate; Finally dialyzate is carried out to lyophilize (be dried 24h in vacuum freeze drying, temperature is-50 ℃).Obtain degradation rate and be 15.28% enzymolysis sea grass polysaccharide (that is, enzymolysis degraded sea grass polysaccharide).
The enzymolysis degraded sea grass polysaccharide of the composite enzyme gained of embodiment 2 is detected according to the method for above-mentioned experiment 1～experiment 3, and acquired results is as shown in table 2 below.
Remarks explanation: result when following data are polysaccharide concentration and are 5mg/ml.
Table 2
| ? | Embodiment 2 |
| DPPH clearance rate (%) | 66.36±1.39 |
| Ultra-oxygen anion free radical clearance rate (%) | 85.86±1.86 |
| Hydroxyl radical free radical is removed (%) | 56.64±1.52 |
Comparative example 2-1, saccharifying enzyme and polygalacturonase in embodiment 2 are used simultaneously, and the consumption of saccharifying enzyme and polygalacturonase is constant.Specific as follows:
Getting 4mL Crude polysaccharides solution, add 0.5ml glucoamylase enzyme solution (142U/ml) and 0.5ml polygalacturonase solution (3.12mg/ml), is 14.2U/mL thereby make final saccharifying enzyme concentration, and the enzyme concentration of polygalacturonase is 7.8% of Crude polysaccharides total amount.Now pH is 4.5, in temperature, is 3 hours 50 ℃ of reaction times.Polysaccharide soln after enzymolysis is boiled to 5min, the centrifugal enzyme (8000r/s, 5min) of removing.Then the dialysis tubing dialysis 24h that gained supernatant liquor is 500 with molecular weight cut-off, to remove inorganic salt; Obtain dialyzate; Finally dialyzate is carried out to lyophilize (i.e. dry 24h in vacuum freeze drying).Obtain degradation rate and be 12.96% enzymolysis sea grass polysaccharide.
The use of comparative example 2-2, cancellation saccharifying enzyme, only adopts polygalacturonase to be hydrolyzed; That is, in 4mL Crude polysaccharides solution, add polygalacturonase enzyme liquid 1mL, according to " hydrolysis again " described in embodiment 2, carry out.
The enzymolysis degraded sea grass polysaccharide of above-mentioned comparative example 2-1 and 2-2 gained is detected according to the method for above-mentioned experiment 1～experiment 3, and acquired results is as shown in table 3 below.
Remarks explanation: result when following data are polysaccharide concentration and are 5mg/ml.
Table 3
| ? | Comparative example 2-1 | Comparative example 2-2 |
| DPPH clearance rate (%) | 59.76±2.31 | 53.32±2.05 |
| Ultra-oxygen anion free radical clearance rate (%) | 78.27±2.02 | 74.31±1.72 |
| Hydroxyl radical free radical is removed (%) | 51.16±2.72 | 43.86±1.56 |
Comparative example 2-3, make the saccharifying enzyme in embodiment 2 into cellulase, enzyme concn still remains 14.2U/mL; All the other contents are equal to embodiment 2.
Comparative example 2-4, make the saccharifying enzyme in embodiment 2 into α-amylase, enzyme concn still remains 14.2U/mL; All the other contents are equal to embodiment 2.
Comparative example 2-5, make the saccharifying enzyme in embodiment 2 into lipase, enzyme concn still remains 14.2U/mL; All the other contents are equal to embodiment 2.
The enzymolysis degraded sea grass polysaccharide of above-mentioned comparative example 2-2～comparative example 2-5 gained is detected according to the method for above-mentioned experiment 1～experiment 3, and acquired results is as shown in table 4 below.
Remarks explanation: result when following data are polysaccharide concentration and are 5mg/ml.
Table 4
| ? | Comparative example 2-3 | Comparative example 2-4 | Comparative example 2-5 |
| DPPH clearance rate (%) | 14.54±1.63 | 13.12±1.02 | 14.56±1.24 |
| Ultra-oxygen anion free radical clearance rate (%) | 41.86±1.25 | 38.43±1.86 | 39.43±1.67 |
| Hydroxyl radical free radical is removed (%) | 34.76±1.66 | 33.67±1.72 | 34.08±1.35 |
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (4)
1. the method for enzymic degradation sea grass polysaccharide, is characterized in that: Enteromorpha Crude polysaccharides or sea grass polysaccharide are configured to the polysaccharide soln that concentration is 1～20mg/mL; Then using saccharifying enzyme as lytic enzyme, in polysaccharide soln, add saccharifying enzyme until the final concentration of saccharifying enzyme is 2～100U/mL; In pH be 3.5～5.5, temperature is 40～60 ℃ of reactions 1～5 hour, enzymolysis degradation of polysaccharide solution first; Enzymolysis degradation of polysaccharide solution boiled after 4～6 minutes first, centrifugal to remove saccharifying enzyme, must contain the supernatant liquor of the rear sea grass polysaccharide of enzymolysis degraded first.
2. the method for enzymic degradation sea grass polysaccharide according to claim 1, is characterized in that: the dialysis tubing that is 500 with molecular weight cut-off by the supernatant liquor that contains the rear sea grass polysaccharide of enzymolysis degraded is first dialysed, to remove inorganic salt; Obtain dialyzate; By dialyzate lyophilize, obtain pulverous enzymolysis degraded sea grass polysaccharide.
3. the method for enzymic degradation sea grass polysaccharide according to claim 1, is characterized in that: the supernatant liquor that contains the rear sea grass polysaccharide of enzymolysis degraded is first hydrolyzed again, and described hydrolysis again comprises the steps:
After containing enzymolysis degraded first, in the supernatant liquor of sea grass polysaccharide, add polygalacturonase, the consumption of described polygalacturonase is 1～10% of Enteromorpha Crude polysaccharides or sea grass polysaccharide weight; In pH be 3.5～5.5, temperature is 45～60 ℃ of reactions 1～5 hour, enzymolysis degradation of polysaccharide solution again; Enzymolysis degradation of polysaccharide solution boiled after 4～6 minutes again, centrifugal to remove polygalacturonase, must contain again the supernatant liquor of the rear sea grass polysaccharide of enzymolysis degraded.
4. the method for enzymic degradation sea grass polysaccharide according to claim 3, is characterized in that: the dialysis tubing that is 500 with molecular weight cut-off by the supernatant liquor that contains again the rear sea grass polysaccharide of enzymolysis degraded is dialysed, to remove inorganic salt; Obtain dialyzate; By dialyzate lyophilize, obtain pulverous enzymolysis degraded sea grass polysaccharide.
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