CN105063130B - With the agaropectin oligose and its preparation method and application for promoting sea cucumber growth and degeneration-resistant effect - Google Patents

With the agaropectin oligose and its preparation method and application for promoting sea cucumber growth and degeneration-resistant effect Download PDF

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CN105063130B
CN105063130B CN201510481550.4A CN201510481550A CN105063130B CN 105063130 B CN105063130 B CN 105063130B CN 201510481550 A CN201510481550 A CN 201510481550A CN 105063130 B CN105063130 B CN 105063130B
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sea cucumber
agaropectin oligose
agar
agaropectin
oligose
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CN105063130A (en
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吴海歌
姚子昂
李倩
崔红利
仇哲
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Dalian University
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Dalian University
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Abstract

There is the agaropectin oligose and its preparation method that promote sea cucumber growth and degeneration-resistant effect and application the present invention provides a kind of, be related to microorganism field.The ectoenzyme degradation agar-agar that the present invention is secreted using the marine bacteria strain QR screened, obtain agaropectin oligose, with high efficiency, product is easy to control, facility is simple, the low advantage of energy consumption, which can improve sea cucumber anti-adversity ability, remarkably promote sea cucumber growth, the sea cucumber growth conditions for taking the oligosaccharides are preferable, pierce strong, body is mellow and full, is ideal sea cucumber bait additive.

Description

With the agaropectin oligose and its preparation method and application for promoting sea cucumber growth and degeneration-resistant effect
Technical field
The present invention relates to microorganism field more particularly to a kind of agaropectin oligoses with promotion sea cucumber growth and degeneration-resistant effect And its preparation method and application.
Background technology
Agar-agar is a kind of polysaccharide extracted from red algae cell wall, is mainly made of the two kinds of polysaccharide in agarose and agaropectin, Although agar-agar has many purposes as natural polysaccharide, due to its viscosity height, dissolubility is low, limits it in food, medicine Etc. application.The agaropectin oligose of certain molecular weight and the degree of polymerization is generated after agar-agar degradation, degradation mode generally divides three kinds:Object Logos, chemical method and enzymatic isolation method.Physical and chemical method degradation effect are preferable, but its degradation condition is difficult to control, and product is inhomogenous, Limit its application.In contrast, sea cucumber is a kind of marine food of high protein and low fat, and with calcium necessary to human body, Iron, zinc, violent and magnesium etc. are micro-.Since the health care enzyme process of sea cucumber has high efficiency, product is easy to control, and facility is simple, consumes energy Low advantage.
Sea cucumber is a kind of marine food of high protein and low fat, and with calcium, iron, zinc, violent and magnesium etc. necessary to human body Trace element.Health care and medicinal function due to sea cucumber is notable, as sea cucumber demand is continuously increased, large-scale sea cucumber occurs Cultivation.Large-scale farming process causes various diseases, and developing and using has pollution-free, noresidue immunopotentiator, to improve Animal body resistivity prevents the somatotrophic novel fodder additive of Animal diseases and has become necessarily becoming for culture fishery Gesture.
Agar-agar can be degraded to agaropectin oligose by agarase.Oligosaccharides is such as anti-because degree of polymerization difference, bioactivity are also not quite similar Viral, antitumor, anti-inflammatory, anti-oxidant etc..These characteristics make agaropectin oligose in the side such as marine drug, health products and cosmetics Face has potential application.Agaropectin oligose has not been reported sea cucumber resistance with growth promoting function, and activity mechanism is still It imperfectly understands, up for further studying.
Invention content
The present invention to solve the above problems, first purpose be to provide it is a kind of have promote sea cucumber growth and degeneration-resistant effect The preparation method of agaropectin oligose and itself.It is a further object to provide added in feed using above-mentioned agaropectin oligose Add the application in agent.
Technical scheme is as follows:A kind of preparation side with the agaropectin oligose for promoting sea cucumber growth and degeneration-resistant effect Method is realized by following steps:
(1) prepared by crude enzyme liquid:By bacterial strain (Pedobacter sp:QR it) is inoculated into fluid nutrient medium, in 30 DEG C of cultures 20h is made seed liquor, is passed on 1% inoculum concentration, in 25-30 DEG C of shake culture 20h, rotating speed 150r/min;Wait for bacterial strain After stabilization, by zymotic fluid, 4000r/min centrifuges 30min under the conditions of 4 DEG C, and it is crude enzyme liquid to take supernatant;
(2) prepared by agaropectin oligose:It is 1 by volume by a concentration of 0.4% agar-agar and crude enzyme liquid:1 mixing, in 35-40 DEG C reaction 20h, 4000r/min centrifuge 10min, and fully degraded agaropectin oligose carries out after rotary evaporation, concentration supernatant cold It is lyophilized dry, obtains thick oligosaccharides, by the purifying of thick oligosaccharides, dry final obtain agaropectin oligose powder.
Preferably, the fluid nutrient medium is:Peptone 3g/L, sodium chloride 20g/L, potassium dihydrogen phosphate 0.5g/L, Magnesium sulfate 0.2g/L, ferrous sulfate 0.01g/L, sodium nitrate 0.25g/L, agar 2g/L, pH 6.8.
The agaropectin oligose degradation bacteria strains QR is isolated from the sea in Daliang City of LiaoNing, China province development zone sea cowry square marine site Mud identifies, which belongs to soil Bacillus (Pedobacter), be named as Pedobacter through Physiology and biochemistry and 16SrDNA sp:QR.
The agaropectin oligose degradation bacteria submits preservation, specific preservation information as follows on June 16th, 2015:
Depositary institution's title:China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC);
Depositary institution address:Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica;
Preservation date:On June 16th, 2015;
Deposit number:CGMCC No.10828.
The agaropectin oligose degradation bacteria strains QR physiology characteristics are:Milky bacterium colony, bacterium colony surface be opaque, gram Feminine gender, bacillus, oxidase positive, 20 DEG C -40 DEG C of optimum growth temperature, most suitable sodium chloride concentration 3%-7%.
It is 5 according to the agaropectin oligose average degree of polymerization that preparation method of the present invention obtains, average relative molecular mass is 861。
The application in feed addictive using the agaropectin oligose is also claimed in the present invention:
(1) agaropectin oligose obtained cooperation basal feed is fed into sea cucumber, the degeneration-resistant effect of sea cucumber can be improved.
(2) agaropectin oligose obtained cooperation basal feed is fed into sea cucumber, the growth rate of sea cucumber can be improved, promote sea cucumber Growth.
The beneficial effects of the invention are as follows:The present invention utilizes the marine bacteria strain Pedobacter sp screened:The born of the same parents of QR secretions Exoenzyme degradation agar-agar, obtains agaropectin oligose, has high efficiency, and product is easy to control, facility is simple, the low advantage of energy consumption, the agar-agar Oligosaccharides can promote sea cucumber to grow and improve sea cucumber anti-adversity ability, increase its resistance and immunity function, remarkably promote sea cucumber life It is long, take add the oligosaccharides bait sea cucumber growth conditions it is preferable, thorn is strong, and body is mellow and full, and present invention gained agaropectin oligose is to open Hair novel fodder additive and immunopotentiator lay the foundation, and are ideal sea cucumber bait additives.
Description of the drawings
Fig. 1 is agaropectin oligose thin layer chromatography analysis result;
Fig. 2 is agaropectin oligose to the degeneration-resistant growing state of sea cucumber;
Fig. 3 is that agaropectin oligose grows promotion situation to sea cucumber.
Specific implementation mode
Technical scheme of the present invention is further described with specific embodiment below, but the present invention is not in any form It is limited to embodiment content.Experimental method described in embodiment is conventional method unless otherwise specified;Unless otherwise specified, The reagent and biomaterial, commercially obtain.
1 agaropectin oligose degradation bacteria (Pedobacter sp of embodiment:QR selection and breeding)
1. the screening of bacterial strain
Take appropriate ooze, fully rinsed with sterile saline, drawing wash liquid in conical flask, shake and by its press than Example is diluted to various concentration gradient and is diluted coating, by coated plate method by 10-2~10-6Dilution be spread evenly across with fine jade Glue is that the tablet coated is placed in 30 DEG C of constant incubators and is cultivated on the solid medium of sole carbon source, and after 48h, observation is solid Whether it is recessed around bacterial strain on body culture medium, transparent circle, picking solid medium whether occurs with Lugol's iodine solution dyeing On occur being recessed and there is the bacterial strain of transparent circle to be isolated and purified, as shown in Figure 1, through 16SrDNA sequencings and bio-chemical characteristics Belong to soil Bacillus after identification, is now named as Pedobacter sp:QR.
Solid screening and culturing medium:Peptone 0.3g/L, yeast extract 0.3g/L, agar 2g/L, NaCl1.5g/L, FeSO4· 7H2O 0.001g/L, pH 6.8.
2. the preparation of agaropectin oligose
(1) preparation of crude enzyme liquid
Bacterial strain QR is inoculated into fluid nutrient medium, 20h is cultivated in 30 DEG C, seed liquor is made, carried out with 1% inoculum concentration Passage, in 30 DEG C of shake culture 20h, rotating speed 150r/min;After bacterial strain stabilization after, by zymotic fluid under the conditions of 4 DEG C 4000r/min 30min is centrifuged, it is crude enzyme liquid to take supernatant;
Fluid nutrient medium:Peptone 3g/L, sodium chloride 20g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.2g/L, sulfuric acid Ferrous 0.01g/L, sodium nitrate 0.25g/L, agar 2g/L, pH 6.8.
Crude enzyme liquid is reacted with agar-agar substrate, carry out enzyme activity determination using DNS methods and is suitably modified.Reaction system For 1ml crude enzyme liquid+1ml substrates (0.4% agar-agar)+2mlDNS, compareed with the bacterium solution of inactivation.Enzyme activity is defined as generation per minute Enzyme amount needed for 1umol reduced sugars.It is 0.53U to measure the bacterium institute producing enzyme
(2) prepared by agaropectin oligose:Volume ratio 1 is added into a concentration of 0.4% agar-agar substrate:1 crude enzyme liquid, in 35- 40 DEG C of reactions 20h, 4000r/min centrifuge 10min, and to remove undegradable polysaccharide, supernatant is carried out through 70 DEG C of rotary evaporations It is concentrated under reduced pressure, the liquid after concentration is spray-dried to obtain thick agaropectin oligose, the purifying of thick agaropectin oligose, drying are finally obtained Obtain agaropectin oligose powder;
Total reducing sugar is carried out to agaropectin oligose and content of reducing sugar measures, total reducing sugar assay method uses sulfuric acid-phynol method, reduced sugar Assay uses DNS methods, using galactolipin as standard items, is computed and show that the average degree of polymerization of agaropectin oligose is 5, average phase It is 861 to molecular mass.
Through TLC identification, the results are shown in Figure 1:Left-hand point 1 is dextrose standard sample, and right-hand point 2 is agaropectin oligose, Agaropectin oligose molecular weight distribution situation can be obtained from Fig. 1, and two clearly oligosaccharides congruent points, molecular weight are shown in thin layer chromatography board It differs in size, is separated clearly, no trailing phenomenon is without trailing phenomenon.
Influence of 2 agaropectin oligose of embodiment to sea cucumber growing state
Agaropectin oligose powder obtained is coordinated into sea cucumber basal feed to feed sea cucumber, is coordinated in basal feed, to sea Ginseng carries out raising in 8 weeks by a definite date.Early feeding, evening change water, monitor water temperature and room temperature in real time, beat oxygen for 24 hours.Five groups of experiment point, by fine jade Glue oligosaccharides additive amount is respectively 0,0.5%, 2.5%, 5%, and 10% ratio is added in basal feed.Sea cucumber weight 4.53 ± It between 0.55g, feeds daily once, feeding amount is the 3-5% of sea cucumber weight.It is primary to change water to sea cucumber daily, clear up residual bait and Excrement.Observation water temperature and room temperature in real time observes sea cucumber state, ingests and defecation situation.
1. influence of the agaropectin oligose to sea cucumber resistance
Experimental results, since sea cucumber has adapted to large-scale farming, are not suitable with real as shown in Fig. 2, when experiment just starts Test the small-scale breeding environment in room and feed mode, when experiment the 10th day, sea cucumber weight that control group, that is, agaropectin oligose additive amount is 0 Drastically decline, growth rate is -13.05%, but the sea cucumber growing state of agaropectin oligose addition group is good, various dose agaropectin oligose The degeneration-resistant effect of sea cucumber can be improved, fall is low compared with control group, growth rate be respectively -11.46%, -4.82%, - 3.19% and 0.51%, when oligosaccharides additive amount is 10%, growth rate 0.51%, and oligosaccharides addition group sea cucumber growing state Preferably, thorn is strong, and freely, power of ingesting is strong for activity, illustrates that oligosaccharides agaropectin oligose improves sea cucumber immunity, enhances its and fights inverse ring border Ability;
2. agaropectin oligose promotes sea cucumber growth
After experimental results are as shown in figure 3, feed sea cucumber 56 days, weigh to sea cucumber, sea cucumber weight significantly increases Adding, agaropectin oligose, which plays the later stage that sea cucumber grows, to be obviously promoted, when testing 30d, 40d and 50d, agaropectin oligose addition Group sea cucumber weight dramatically increases, and reaches 64% and 70%, and agaropectin oligose in 5% and 10% oligosaccharides addition group sea cucumber growth rate Addition group sea cucumber growth conditions are good, and thorn is strong mellow and full, and freely, growth rate is significantly higher than control group, and growth rate is control group for activity 9.63 times.
It is remarkably promoted in conclusion agaropectin oligose plays the growth of sea cucumber;Matched with different agaropectin oligose additive amounts Basal feed is closed to feed sea cucumber, agaropectin oligose addition group sea cucumber average weight dramatically increases, and growth rate is added with agaropectin oligose Increasing for amount and to increase resistance preferable, activities of antioxidant enzymes improves, and correlation factor activity is immunized and increases.
Attachment:
Sequence table
SEQ ID No.1 (the 16S rRNA nucleotide sequences of agaropectin oligose degradation bacteria strains QR)
CTATACATGCAAGTCGAGGGGCAGCGGGGACTTTCGGGTTCGCCGGCGACCGGCGCACGGGTGCGTAACGCGTATGC AACCTACCTTTATCAGGGGGATAGCCCGGAGAAATCCGGATTAACACCGCATAAAATCACAGTACCGCATGGTATAA TGATCAAATATTTATAGGATAAAGATGGGCATGCGTGTCATTAGCTAGTTGGAGAGGTAACGGCTCACCAAGGCGAC GATGACTAGGGGATCTGAGAGGATGACCCCCCACACTGGTACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAG TAAGGAATATTGGTCAATGGAGGGAACTCTGAACCAGCCATGCCGCGTGCAGGAAGACTGCCCTATGGGTTGTAAAC TGCTTTTGTATGGGAACGGAGGATCCAAGCGTTATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGCGGCCTGTTAA GTCAGGGGTGAAAGACATAAACCCTGGTATGTATACCAGGCTGAATGTACCATAAGAATAAGGATCGGCTAACTCCG TGCCAGCAGCCGCGGTAATGGTGGCTCAACCATCGCAGTGCCTTTGATACTGATAGGCTTGAATATAGTTGAGGTAG GCGGAATGTGACAAGTAGCGGTGAAATGCATAGATATGTCACAGAACACCGATTGCGAAGGCAGCTTACTAAACTAA TATTGACGCTGAGGCTCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGATA ACTCGATGTTAGCGATATACAGTTAGCGTCCAAGCGAAAGCGTTAAGTTATCCACCTGGGGAGTACGCCCGCAAGGG TGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGAGGAGCATGTGGTTTAATTCGATGATACGCGAGGAACCT TACCCGGGCTTGAAAGTTACTGCATTACTCAGAGATGAGTAAGACCTTCGGGACAGGAAACTAGGTGCTGCATGGCT GTCGTCAGCTCGTGCCGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATGTTTAGTT GCCAGCATTTAAGGTGGGGACTCTAAACAGACTGCCTGCGCAAGCAGAGAGGAAGGGGCATGGGACGACGTCAAGTC ATCATGGCCCTTACGTCCGGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGCCGCTACCCAGCGATGGGATGC CAATCTCAAAAAGCCGATCACAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTTGGATTCGCTAGTAATCGC GTATCAGCAATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGAAGTTGGGGGTGCC TAAAGTCCGTAACCGCAAGGATCGGCCTAGGGTAAAACCGATAACTGGGGCTAAGTCGTAACAAGGTAGCCGTACCG GAAGGTGC。

Claims (5)

1. a kind of preparation method with the agaropectin oligose for promoting sea cucumber growth and degeneration-resistant effect, which is characterized in that by following Step is realized:
(1) prepared by crude enzyme liquid:By deposit number:CGMCC No.10828 bacterial strainsPedobacter sp:QRIt is inoculated into Liquid Culture In base, 20h is cultivated in 30 DEG C, seed liquor is made, is passed on 1% inoculum concentration, in 25-30 DEG C of shaken cultivation 20h, rotating speed 150r/min;After bacterial strain stabilization, by zymotic fluid, 4000r/min centrifuges 30min under the conditions of 4 DEG C, and it is thick enzyme to take supernatant Liquid;
(2) prepared by agaropectin oligose:It is 1 by volume by a concentration of 0.4% agar-agar and crude enzyme liquid:1 mixing, it is anti-in 35-40 DEG C 20h, 4000r/min is answered to centrifuge 10min, supernatant is freeze-dried by fully degraded agar-agar after rotary evaporation, concentration, Thick agaropectin oligose is obtained, by the purifying of thick agaropectin oligose, dry final acquisition agaropectin oligose powder.
2. the method according to claim 1 for preparing agaropectin oligose, which is characterized in that the fluid nutrient medium is:Egg White peptone 3g/L, sodium chloride 20g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.2g/L, ferrous sulfate 0.01g/L, sodium nitrate 0.25g/L, agar 2g/L, pH 6.8.
3. a kind of agaropectin oligose that preparation method described in accordance with the claim 1 obtains, which is characterized in that the agaropectin oligose Average degree of polymerization is 5, average relative molecular mass 861.
4. a kind of agaropectin oligose answering in preparing feed addictive obtained using preparation method as described in claim 1 With, which is characterized in that agaropectin oligose obtained cooperation basal feed is fed into sea cucumber, the degeneration-resistant effect of sea cucumber can be improved.
5. a kind of agaropectin oligose answering in preparing feed addictive obtained using preparation method as described in claim 1 With, which is characterized in that agaropectin oligose obtained cooperation basal feed is fed into sea cucumber, the growth rate of sea cucumber can be improved, promoted Sea cucumber grows.
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