CN1341664A - Preparation method of polysaccharide polymannuronate and its application - Google Patents
Preparation method of polysaccharide polymannuronate and its application Download PDFInfo
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Abstract
一种聚甘露糖醛酸多糖的制备方法,包括使褐藻酸钠溶于水,加入褐藻胶裂合酶进行反应,用沸水浴加热使酶失活,离心后除去沉淀杂质,将所得溶液调pH,离心除去所产生的沉淀物,用碱液溶解并调pH,加入乙醇产生沉淀,干燥得到聚甘露糖醛酸多糖,其特征是所述的将所得溶液调pH的范围为2.5～3.2,所述的酶是通过弧菌产生的专一性褐藻胶裂合酶。该产品具有提高对虾免疫力活性、抗肿瘤活性、广谱抑菌活性、可分别用于制备对虾饲料添加剂、抗肿瘤药物、抗菌药物。A preparation method of polymannuronic acid polysaccharide, comprising dissolving sodium alginate in water, adding alginate lyase to react, heating in a boiling water bath to inactivate the enzyme, removing precipitated impurities after centrifugation, and adjusting the pH of the obtained solution , remove the produced precipitate by centrifugation, dissolve it with lye and adjust the pH, add ethanol to produce a precipitate, and dry to obtain polymannuronic acid polysaccharide, which is characterized in that the range of adjusting the pH of the obtained solution is 2.5 to 3.2. The enzyme described is a specific alginate lyase produced by Vibrio. The product has the activity of improving prawn immunity, antitumor activity and broad-spectrum antibacterial activity, and can be used to prepare prawn feed additives, antitumor drugs and antibacterial drugs respectively.
Description
The present invention relates to a kind of organic polymer, particularly relate to a kind of the have prawn of raising immunizing power activity, anti-tumor activity and the active polysaccharide polymannuronate of broad-spectrum antibacterial.
Nisizawa etc. 1971 (Further studies on the fine structure of alginic acid.Proc.Int.seaweed symp.7:485-490) use the polysaccharide product that obtains different hydrolysis degrees from isolated mannuronic acid lyase of mollusk and the isolated guluronic acid lyase of bacterium hydrolysis of brown algae glue, do not report any biological activity of product.
The purpose of this invention is to provide a kind of method for preparing polysaccharide polymannuronate, and this product has and improve prawn immunizing power activity, can be used for manufacturing the prawn feed additive; This product has anti-tumor activity, can be used for preparing antitumor drug; This product also has the broad-spectrum antibacterial activity, can be used for preparing antibacterials.
A kind of preparation method of polysaccharide polymannuronate, comprise that to make sodium alginate water-soluble, adding alginate lyase reacts, make enzyme deactivation with the boiling water bath heating, remove precipitated impurities after centrifugal, gained solution is transferred pH, the centrifugal throw out that is produced of removing, with alkali lye dissolving and accent pH, add ethanol and produce precipitation, drying obtains polysaccharide polymannuronate, it is characterized in that described scope with gained solution accent pH is 2.5～3.2, described enzyme is the specificity alginate lyase that produces by vibrios, and the amount that every gram sodium alginate adds alginate lyase is 0.5～1.5 unit.
A kind of polysaccharide polymannuronate is characterized in that having and improves prawn immunizing power activity, is used to prepare the prawn feed additive.
A kind of polysaccharide polymannuronate is characterized in that having anti-tumor activity, is used to prepare antitumor drug.
A kind of polysaccharide polymannuronate is characterized in that having the broad-spectrum antibacterial activity, is used to prepare antibacterials.
Polysaccharide polymannuronate product of the present invention has the prawn of raising immunizing power, anti-tumor activity and broad-spectrum antibacterial activity, can be used for manufacturing prawn feed additive, antibacterials and antitumor drug.
Below by embodiment the present invention is described.
Accompanying drawing 1 is that polysaccharide polymannuronate is to culturing the influence of Chinese prawn serum bacteriolyze vigor
Accompanying drawing 2 is that polysaccharide polymannuronate is to culturing the influence of Chinese prawn serum haemolysis vigor
Get the 50g sodium alginate and be dissolved in the 1000ml water, (enzyme activity unit of alginate lyase is that every min makes 0.2% alginate solution absorbancy increase by 1 required enzyme amount to add 50 units.) alginate lyase liquid, react 2h down at 28 ℃, the boiling water bath heating makes enzyme deactivation then, the centrifugal decon in cooling back, gained solution is transferred pH to 2.85, and the centrifugal precipitation of removing is with the NaOH accent pH to 7 of 4mol/L, add ethanol and make the product precipitation, obtain polysaccharide polymannuronate (M3) after the drying.The molecular weight of gained polysaccharide polymannuronate is about 5000, and wherein the mannuronic acid polysaccharide accounts for about 75%, and guluronic acid accounts for about 25%.
The reaction times of alginate lyase degraded sodium alginate is 2～6 hours.
Test product of the present invention is to the influence of Chinese prawn immunizing power below
At first choose the healthy close breed Chinese prawn of individual size, adopt the method for prawn belly body cavity injection, the experiment employing is 1.0% polysaccharide polymannuronate solution with physiological saline preparation weight percent concentration, sterilization back injection 0.1ml/ tail, the stroke-physiological saline solution of control group injection equivalent, every group 20 tail prawn.At 24h, 48h, 72h get prawn 20 tails respectively, and from the aseptic extraction blood of heart, every tail prawn is got 0.1ml, places centrifuge tube, at 4 ℃, behind the centrifugal 10min of 3500r/min, get supernatant liquor and are prawn serum.At 24h, 48h, 72h gets hepatopancreas and the muscle of 20 tail prawns respectively, homogenate in ice bath after weighing, add stroke-physiological saline solution, make the concentration of liver pancreas line and muscle reach 0.1g/ml, at 4 ℃, behind the centrifugal 10min of 3500r/min, remove hepatopancreas and muscle tissue extracting solution that post precipitation obtains prawn.Mensuration with prawn immunity related enzyme activity
1. the mensuration of lysozyme activity: will be through the micrococcus lysodeikticus (Micrococcuslysodekticus) of re-activation, inoculate and carry out shaking table in the sub-liquid nutrient medium and cultivate 48h, it is centrifugal to take out the back, collects thalline.Phosphate buffered saline buffer (pH=6.4) with 0.1mol/L is diluted to initial absorbance value A
0=0.303, be made into the substrate suspension and use for test.
2 haemolysis vitality tests: will be stored in chicken red blood cell in Alsever ' the s liquid and be mixed with 3% red cell suspension (pressing the hematocrit volume calculation) after with stroke-physiological saline solution washing for several times.Get the 2.5ml red cell suspension and add 0.1ml serum, control group is with the physiologic saline for substitute serum of 0.1ml, 37 ℃ of insulation 30min, and ice bath immediately, the centrifugal 5min of 3000r/min, supernatant liquor is in 540nm place photometry absorption value.With the hemolytic activity of surveying the hepatopancreas tissue extract with quadrat method.Hemolysin content (homolysin concentration) is expressed as: the OD value x30 that surveys (sample extension rate).
Measuring inject the Chinese prawn serum of above-mentioned 1% polysaccharide polymannuronate solution and the haemolysis vigor of hepatopancreas tissue extract, the results are shown in Figure 2.Experimental result shows that polysaccharide polymannuronate has tangible raising effect to the hemolytic activity of cultured prawn serum.Behind the body cavity injection polysaccharide polymannuronate solution 24h, the serum haematolysis ability brings up to 3.45 by 3.03 of control group, still keeps higher level to 72h.The hemolysin of prawn contains in serum, in hepatopancreas, also contain a large amount of hemolysins, its haematolysis ability is far above the serum of equivalent, so also measure the influence of polysaccharide polymannuronate to the hepatopancreas hemolytic action in this experiment simultaneously, the result shows the haemolysis vigor almost not influence of polysaccharide polymannuronate to hepatopancreas.
The mensuration of 3 acid phosphatases:
The mensuration of acid phosphatase (ACP) adopts the disodium phenyl phosphate method., produce 1mg phenol and be defined as an enzyme activity unit at 37 ℃ and substrate-function 60min with every 100ml serum.Active influence the results are shown in Table 1 to polysaccharide polymannuronate to Chinese prawn ACP.
Table 1 polysaccharide polymannuronate is tested group ALP activity (U/100ml) to the sampling point that influences of ACP activity (U) in the Chinese prawn body
24h 48h
The poly-seminose 0.37 0.48 0.56 aldehydic acid polysaccharide of 72h serum contrasts the poly-seminose 6.82 6.40 9.69 aldehydic acid polysaccharide of 0.37 0.25 0.49 hepatopancreas and contrasts the poly-seminose 0.33 0.28 0.31 aldehydic acid polysaccharide of 3.52 3.53 3.21 muscle and contrast 0.27 0.26 0.25
Experimental result shows that Chinese prawn is after the polysaccharide polymannuronate injection stimulates, and the ACP activity in the hepatopancreas strengthens greatly, promptly reaches higher level behind the 24h, and it is the highest that 72h reaches.The ACP of serum and muscle is active close with control group.
The mensuration of 4 alkaline phosphatases (ALP): adopt the disodium phenyl phosphate method., produce 1mg phenol person and be defined as an enzyme activity unit at 37 ℃ and substrate-function 15min with every 100ml serum.
Polysaccharide polymannuronate the results are shown in Table 2 to Chinese prawn to the influence of ALP.
Table 2 polysaccharide polymannuronate is to the influence of ALP activity (U) in the Chinese prawn body
Sampling point experiment group ALP activity (U/100ml)
24h 48h 72h
Serum polymannuronic acid 0.50 0.60 0.63
Polysaccharide 0.36 0.42 0.45
Contrast
Hepatopancreas polymannuronic acid 12.3 13.6 13.2
Polysaccharide 4.12 4.25 4.09
Contrast
Muscle polymannuronic acid 0.45 0.41 0.44
Polysaccharide 0.36 0.42 0.45
Contrast
Experiment shows, all has alkaline phosphatase in the serum of Chinese prawn, muscle and the liver extracting solution, and the distribution of its activity in these three kinds of tissues is comparatively similar to acid phosphatase, promptly activity is the highest in liver, activity in muscle and the serum is lower, and this and the intravital ALP distribution situation of higher animal also have similarity.Prawn is after the polysaccharide polymannuronate injection, and the ALP in the hepatopancreas is active obviously to be strengthened, and the enzymic activity behind the 24h promptly reaches 12.3U, is 3 times of control group, and the 48h activity reaches the highest, is 13.6U; 72h still keeps higher level and ALP activity change in serum and the muscle is little.The mensuration of embodiment 2 bacteriostatic activities: the polysaccharide polymannuronate solution that present embodiment is used is identical with embodiment 1.The mensuration of antimicrobial spectrum adopts the method for measuring the antibacterial circle diameter size, has selected multiple Lu Sheng bacterium and marine bacteria, wherein except common Gram-negative bacteria (G
-) and gram-positive microorganism (G+) outside, some important pathogenic bacterium are also arranged.The inhibition zone of intestinal bacteria (Escherichia coli) is 8mm, enteroaerogen (Enterobacter aerogenes) is 13mm, Fu Shi shiga bacillus (Shigella flexneri) 15mm, micrococcus lysodeikticus (Micrococcus lysodekticus) 9.5mm, Salmonellas (Salmonella paratyphiB) 16mm, streptococcus aureus (Staphalococcus aureus) 12mm, pseudomonas aeruginosa (Pseudomonas aeruginosa) 14mm, Bacillus subtilus (Bacillus svbtilis) 17mm, Vibrio harveyi (Vibrio harveyi) 9mm, Vibrio vulnificus (Vibrio vulnificus) 14mm, rivers and creeks vibrios (Vibriofluvialis) 9mm, ocean vibrios (Vibrio pelagius) 8.5mm, vibrio alginolyticus (Vibrioalginolyticus) 12mm.Experimental result shows that the polysaccharide polymannuronate polysaccharide polymannuronate has the bacteriostatic action of wide spectrum, and the restraining effect of marine bacteria is higher than the land endophytic bacteria.Prove from bacteriostatic experiment, polysaccharide polymannuronate has best fungistatic effect, adopt micro-dilution method to measure the minimal inhibitory concentration of polysaccharide polymannuronate to bacterial growth, intestinal bacteria 0.312 μ g/ml, Salmonellas 0.225 μ g/ml, streptococcus aureus 0.016 μ g/ml, Bacillus subtilus 0.325 μ g/ml, pneumobacillus 0.016 μ g/ml tetrads 0.225 μ g/ml result show, polysaccharide polymannuronate has broad spectrum antibacterial, to intestinal bacteria, Salmonellas, streptococcus aureus, Pseudomonas aeruginosa, Bacillus subtilus, acinetobacter calcoaceticus, tens kinds of Gram-positives such as pneumobacillus and negative bacteria all have significant inhibitory effect.The used polysaccharide polymannuronate solution of the mensuration present embodiment of embodiment 3 anti-tumor activities is identical with embodiment 1.With tetrazolium bromide (TTM) determination of experimental method the anti-tumor activity of polymannuronic acid of method of the present invention preparation.During experiment the Ovcar-3 Proliferation of Human Ovarian Cell is made into 10 with the RPMI1640 substratum of 10% calf serum (FCS)
4Individual/the ml suspension, in 37 ℃, 5%CO
2, 24h is fostered in 100 μ l/ holes (96 orifice plate), and each is organized correspondence and adds above-mentioned polysaccharide polymannuronate solution, make its concentration be respectively 1,10,100ug/ml, blank group correspondence adds substratum, every group of 5 parallel holes, add the TTM 20ul of 5mg/ml behind the cultivation 24h, cultivate 4h, go supernatant liquor to add 150ul dimethyl sulfoxide (DMSO) (DMSO), measure 570nm with microplate reader, (630nm is for setting wavelength) light absorption value.The tumour inhibiting rate that records polysaccharide polymannuronate with this method is 24.7%, 20.16%, 31.22%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003099870A3 (en) * | 2002-05-29 | 2004-04-01 | Ct D Etude Et De Valorisation | Methods for obtaining oligomannuronates et guluronates, products obtained and use thereof |
| WO2005089776A1 (en) * | 2004-03-24 | 2005-09-29 | Ocean University Of China | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| US7090856B2 (en) | 2003-06-19 | 2006-08-15 | Hong Kong University Of Science And Technology | Anti-fouling exopolysaccharides isolated from cultures of Vibrio alginolyticus and Vibrio proteolyticus |
| CN100508985C (en) * | 2004-03-24 | 2009-07-08 | 中国海洋大学 | Algin oligose and derivative thereof and producing method and use thereof |
| CN104489356A (en) * | 2014-12-15 | 2015-04-08 | 青岛海洋生物医药研究院股份有限公司 | Application of oligomerization mannuronic acid in preparation of feed additive |
-
2000
- 2000-09-07 CN CN 00111367 patent/CN1341664A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003099870A3 (en) * | 2002-05-29 | 2004-04-01 | Ct D Etude Et De Valorisation | Methods for obtaining oligomannuronates et guluronates, products obtained and use thereof |
| US7090856B2 (en) | 2003-06-19 | 2006-08-15 | Hong Kong University Of Science And Technology | Anti-fouling exopolysaccharides isolated from cultures of Vibrio alginolyticus and Vibrio proteolyticus |
| WO2005089776A1 (en) * | 2004-03-24 | 2005-09-29 | Ocean University Of China | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| EA011417B1 (en) * | 2004-03-24 | 2009-02-27 | Оушн Юниверсити Оф Чайна | Algin oligosaccharides and the derivatives thereof, the manufacture and the use of the same |
| CN100508985C (en) * | 2004-03-24 | 2009-07-08 | 中国海洋大学 | Algin oligose and derivative thereof and producing method and use thereof |
| US8835403B2 (en) | 2004-03-24 | 2014-09-16 | Meiyu Geng | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| US9493496B2 (en) | 2004-03-24 | 2016-11-15 | Ocean University Of China | Algin oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| US10213456B2 (en) | 2004-03-24 | 2019-02-26 | Ocean University Of China | Alginate oligosaccharides and the derivatives thereof as well as the manufacture and the use of the same |
| CN104489356A (en) * | 2014-12-15 | 2015-04-08 | 青岛海洋生物医药研究院股份有限公司 | Application of oligomerization mannuronic acid in preparation of feed additive |
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