CN109221811A - A kind of preparation method of feeding additive aquatic animal brown alga oligose - Google Patents
A kind of preparation method of feeding additive aquatic animal brown alga oligose Download PDFInfo
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- CN109221811A CN109221811A CN201811137102.2A CN201811137102A CN109221811A CN 109221811 A CN109221811 A CN 109221811A CN 201811137102 A CN201811137102 A CN 201811137102A CN 109221811 A CN109221811 A CN 109221811A
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- A—HUMAN NECESSITIES
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- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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Abstract
The present invention provides a kind of preparation method of feeding additive aquatic animal brown alga oligose, belongs to aquatic feeds field.The present invention extracts algin using alkali carries alcohol deposition method from kelp, prepares brown alga oligose using algin catenase hydrolysis of brown algae glue, makes an addition in aquatic feeds, and is applied to the cultivation of Larimichthys crocea and grouper.Addition brown alga oligose can improve the weight, overall length, survival rate of Larimichthys crocea and grouper extremely significantly, the ratio of Larimichthys crocea and protein content, EAA/TAA and EAA/NEAA in lithosporic fish tissue, catalase and lyase bacterium vigor in rheum officinale fish serum, the vigor of acid phosphatase, alkaline phosphatase, catalase and lysozyme in lithosporic fish serum, the proteinase activity (P < 0.01) in Larimichthys crocea and grouper digestive system.Meanwhile the brown alga oligose for digesting preparation has the effect of inhibiting aquatic pathogenic bacterium.
Description
Technical field
Disclosed by the invention is a kind of preparation method of feeding additive aquatic animal brown alga oligose, belongs to aquatic feeds field.
Technical background
The property of functional oligosaccharide is similar with feed fibre, the digestive ferment that cannot be secreted by animal alimentary canal front end or animal
Digestion, but reach the alimentary canal back segment of organism directly for flora utilization, there is benefit in selective stimulating archenteric flora
The breeding of raw bacterium.In order to improve the growth performance of animal, antibiotic is widely used in animal feed.With food
Security knowledge is popularized, and the Consciousness of food security of people is gradually increased, it is understood that antibiotic is largely made using generated pair
With, therefore find novel additive agent for feeding and have been to be concerned by more and more people.Functional oligosaccharide, which has, promotes growth of animal, stabilization
Property strong, noresidue the advantages that, the substitute that can be used as antibiotic is added into feed.
Brown alga oligose as a kind of functional oligose, it is anti-oxidant, antitumor, in terms of it is active, gradually
It is applied in food additives.Have many advantages, such as stable structure, safety, noresidue simultaneously, has to the immune system of organism
Adjustment effect, and body will not generate adverse reaction.With the development of culture fishery, the residue problem of antibiotic in feed
It has been to be concerned by more and more people.The present invention extracts algin using alkali carries alcohol deposition method from kelp, has yield height, without disappearing
Consume excessive chemical reagent, it is at low cost the advantages that；Further traditional chemical method is replaced to degrade using algin catenase brown
Phycocolloid prepares brown alga functional oligosaccharide, analyzes oligosaccharide ingredient, and be applied to aquatic feeds, studies the feed to Larimichthys crocea and lithosporic
The effect of fish growth performance, fish body quality, nospecific immunity etc., and to the fungistatic effect of aquatic pathogenic bacterium, exploitation
A kind of novel brown alga oligose feeding additive aquatic animal out.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of feeding additive aquatic animal brown alga oligose.
To achieve the above object, using following technical scheme:
Using kelp as raw material, algin, process conditions are as follows: liquid-to-solid ratio 70:1(v/w are extracted using alkali carries alcohol deposition method), alkali concentration
1%, 80 DEG C of alkali carries temperature, 4 h of alkali carries time, brown alga glue yield is 30.30%.
Brown alga oligose, process conditions are prepared using algin catenase degradation algin are as follows: concentration of substrate 0.7%, enzyme concentration
0.33 U/mL, pH7.0,50 DEG C of temperature, 3 h of enzymolysis time, degree of hydrolysis is up to 64.60%.
Through thin-layer chromatography, mass spectral analysis, enzymolysis product is brown alga oligose DP1, DP2, DP3, and wherein DP1, DP2 are main produce
Object, DP3 content are relatively fewer.
Brown alga oligose has good fungistatic effect to aquatic pathogenic bacterium Vibrio vulnificus, Vibrio furnissii, vibrio parahaemolytious,
MIC is respectively 1.37 mg/mL, 3.71 mg/mL, 1.11 mg/mL.
The application of feeding additive aquatic animal brown alga oligose:
Compound fish bait used specially for pseudosciaena crocea (extruding buoyancy) main formula: super fish meal 19.90%, domestic fish meal 22.39%, cuttlefish cream
1.99%, proteinic powder of porcine 2.99%, corn protein powder 4.98%, dregs of beans 19.90% of not peeling, Strong flour 19.90%, soya-bean oil
1.00%, fish oil 2.49%, calcium dihydrogen phosphate 1.99%, choline chloride 0.30%, sodium chloride 0.20%, ocean fish premix 1.97%.
Grouper compound feed (heavy property) main formula: super fish meal 19.70%, domestic fish meal 24.63%, cuttlefish cream
1.97%, proteinic powder of porcine 4.93%, Gluten 2.96%, dregs of beans 14.78% of peeling, Strong flour 15.76%, cassava fecula
3.94%, soya-bean oil 1.97%, fish oil 5.91%, calcium dihydrogen phosphate 0.99%, choline chloride 0.30%, sodium chloride 0.20%, ocean fish premix
Material 1.96%.
It is to allow feed to fully absorb in the aqueous solution of 5% oligosaccharides, dry containing mass fraction that above-mentioned feed, which is dissolved in,.
The present invention has the advantages that
Aquaculture experiments have shown that, addition brown alga oligose improves the weight, overall length, survival of Larimichthys crocea and grouper extremely significantly
Rate, the ratio of Larimichthys crocea and protein content, EAA/TAA and EAA/NEAA in lithosporic fish tissue, peroxide in rheum officinale fish serum
Change hydrogen enzyme and lyase bacterium vigor, the work of acid phosphatase, alkaline phosphatase, catalase and lysozyme in lithosporic fish serum
Power, the proteinase activity (P < 0.01) in Larimichthys crocea and grouper digestive system.
Detailed description of the invention
Fig. 1 is algin enzymolysis product thin layer chromatography analysis figure；
Fig. 2 is algin enzymolysis product mass spectral analysis figure；
Fig. 3 is the influence for adding brown alga oligose to fish body weight；
Fig. 4 is the influence for adding brown alga oligose to fish overall length；
Fig. 5 is the influence for adding brown alga oligose to fish survival rate；
Fig. 6 is the influence for adding brown alga oligose to fish meal coefficient；
Fig. 7 is the influence for adding brown alga oligose to fish meat protein content；
Fig. 8 is the influence for adding brown alga oligose to fish serum Acid Phosphatase Activity；
Fig. 9 is the influence for adding brown alga oligose to fish serum alkaline phosphatase activity；
Figure 10 is the influence for adding brown alga oligose to fish serum activity of catalase；
Figure 11 is the influence for adding brown alga oligose to fish serum nitric oxide synthase；
Figure 12 is the influence for adding brown alga oligose to fish serum glutathione peroxidase activity；
Figure 13 is the influence for adding brown alga oligose to fish serum antalzyme activity；
Figure 14 is the influence for adding brown alga oligose to fish digestive system amylase activity；
Figure 15 is the influence for adding brown alga oligose to fish digestive system prolease activity.
Specific embodiment
In 1 kelp of embodiment prepared by the enzymatic hydrolysis of the extraction of algin and brown alga oligose
Smashed kelp and water are with 1:100(w/v) ratio mixing, after 2 h of boiling water bath, it is centrifuged (11000 rpm, 10 min),
Supernatant is abandoned, into filter residue in 1:70(w/v) ratio 1% sodium carbonate liquor of addition, extracts 4 h, centrifugation (11000 at 80 DEG C
Rpm, 10 min) precipitating is abandoned, into supernatant, 95% dehydrated alcohol is added in 1:1 by volume, it is centrifuged (11000 rpm, 10 min),
Precipitating drying is obtained into algin.Brown alga glue yield is 30.30%.
By the disodium hydrogen phosphate of algin pH7.0 --- citrate buffer solution prepares the algin that mass concentration is 0.7%
Solution is cooled to room temperature after heating for dissolving；It is placed in 50 DEG C of shaking baths and preheats, algin catenase (enzyme concentration 0.33 is added
U/mL), 3 h are digested at 50 DEG C, after reaction 5 min of boiling water bath, centrifugation (11000 rpm, 10 min) takes supernatant, past
Ethanol solution is added in supernatant to final concentration 70%, precipitates undegradable polysaccharide and albumen, then be centrifuged (11000 rpm, 10
Min supernatant) is taken, is concentrated to give brown alga oligose, degree of hydrolysis is up to 64.60%.
2 brown alga oligose constituent analysis of embodiment
1. thin layer chromatography analysis
Enzymolysis product is subjected to thin layer chromatography analysis, as shown in Figure 1, enzymatic hydrolysis final product mainly has the brown alga oligose of 3 kinds of degree of polymerization,
The degree of polymerization is respectively DP1, DP2, DP3, and wherein DP1, DP2 are primary product, and DP3 content is relatively fewer.
Mass spectral analysis
ESI-MS result (Fig. 2) display, there are 3 kinds of degree of polymerization oligosaccharides, monosaccharide 175.0267(M-1Na for enzymolysis product+), disaccharides
For 351.0599(M+H+-2Na+), 373.0419(M-1Na+), trisaccharide 527.0929(M+H+-3Na+), 549.0741(M+H+-
2Na+), 571.0567(M-1Na+).
Inhibitory effect of 3 brown alga oligose of embodiment to aquaculture encountered pathogenic bacteria
Using turbidimetry for Determination fungistatic effect, the culture medium of 100 μ L double strengths is added into 96 orifice plates, 50 μ L are through disposable
The brown alga oligose of filter filtering, 50 μ L dilute 1000 times of bacterium solutions being incubated overnight, and 30 DEG C of 24 h of constant temperature incubation in shaking table are used
Microplate reader measures the bacterium solution light absorption value under 600 nm.
Brown alga oligose inhibit Vibrio vulnificus, Vibrio furnissii, vibrio parahaemolytious MIC be respectively 1.37 mg/mL, 3.71
mg/mL、1.11 mg/mL。
Application of the 4 brown alga oligose feeding additive aquatic animal of embodiment in Larimichthys crocea and Grouper cultivating
Compound fish bait used specially for pseudosciaena crocea (extruding buoyancy) main formula: super fish meal 19.90%, domestic fish meal 22.39%, cuttlefish cream
1.99%, proteinic powder of porcine 2.99%, corn protein powder 4.98%, dregs of beans 19.90% of not peeling, Strong flour 19.90%, soya-bean oil
1.00%, fish oil 2.49%, calcium dihydrogen phosphate 1.99%, choline chloride 0.30%, sodium chloride 0.20%, ocean fish premix 1.97%.
Grouper compound feed (heavy property) main formula: super fish meal 19.70%, domestic fish meal 24.63%, cuttlefish cream
1.97%, proteinic powder of porcine 4.93%, Gluten 2.96%, dregs of beans 14.78% of peeling, Strong flour 15.76%, cassava fecula
3.94%, soya-bean oil 1.97%, fish oil 5.91%, calcium dihydrogen phosphate 0.99%, choline chloride 0.30%, sodium chloride 0.20%, ocean fish premix
Material 1.96%.
It is to allow in the aqueous solution of 5% brown alga oligose containing mass fraction that above-mentioned feed (control group) is dissolved in by experimental group respectively
Feed fully absorbs, drying.
Experiment fish farm is located at Ningde City richness shampoo and produces Co., Ltd (national Larimichthys crocea seed farm), will be from big marine sampling
Larimichthys crocea back is grouped, and two barrels of control group, every barrel of 30 tails；Three barrels of experimental group, every barrel of 30 tails；With terramycin pair after dispensing
Larimichthys crocea carries out disinfection processing.And grouper is grouped, one barrel of control group, every barrel of 50 tails, two barrels of experimental group, every barrel 30
Tail carries out disinfection processing to grouper with terramycin after dispensing.
Experiment initial stage measures body length, overall length and the weight of Larimichthys crocea and grouper.It is long by 14.16 to measure the Larimichthys crocea body that is averaged
Cm, 16.57 cm of average length, 40.62 g of average weight；Grouper is averaged body long 11.76 cm, 14.38 cm of average length,
48.59 g of average weight.64 d of formal experiment weigh in each bucket random sampling at the end of experiment, and measure body length, overall length
And weight.
Each group is responsible for by professional, feeds each experimental group respectively, manual bait throwing in, day bait throwing in 2 times (daily 8:40,17:
00), day bait throwing in is the 2% of weight, and the situation of ingesting depending on fish carries out appropriate adjustment.The dead fish of experiment periods is pulled out and is recorded in time, often
Its record food ration and water temperature.Periodically disinfection bucket.
Growth performance measurement
(1) rate of body weight gain
It can be seen from figure 3 that the rate of body weight gain of the Larimichthys crocea of addition brown alga oligose, grouper experimental group is extremely significant few higher than brown alga is not added with
The control group (P < 0.01) of sugar, rate of body weight gain are respectively increased 1.56 times, 6.25 times.
(2) overall length growth rate
As seen from Figure 4, the overall length growth rate of the Larimichthys crocea, grouper experimental group of adding brown alga oligose is extremely significant brown higher than being not added with
The control group (P < 0.01) of algae oligosaccharides, increases separately 3.96 times, 1.50 times.
(3) survival rate
From figure 5 it can be seen that the survival rate of the Larimichthys crocea of addition brown alga oligose, grouper experimental group is extremely significant few higher than brown alga is not added with
The control group (P < 0.01) of sugar, is respectively increased 1.04 times, 1.76 times.
(4) feed coefficient
As seen from Figure 6, the feed coefficient of the Larimichthys crocea, grouper experimental group of adding brown alga oligose is extremely significant lower than being not added with brown alga
The control group (P < 0.01) of oligosaccharides reduces by 2.03 times, 5.31 times respectively.
Fish body quality
(1) in muscle protein content measurement
From fig.7, it can be seen that the addition Larimichthys crocea of brown alga oligose, grouper experimental group muscle in protein content is extremely significant is higher than not
The control group (P < 0.01) for adding brown alga oligose, is respectively increased 1.06 times, 2.08 times.
(2) in muscle amino acid content measurement
Amino acid content is as shown in table 1, table 2 in Larimichthys crocea, the grouper flesh of fish.Amino of the brown alga oligose to Larimichthys crocea and grouper
Acid composition does not make a significant impact, but on NEAA(nonessential amino acid), EAA(essential amino acid) content generates certain influence.
The A1 group NEAA content for adding brown alga oligose is extremely significant lower than being not added with the A group of brown alga oligose, but EAA it is extremely significant higher than A group (P <
0.01), the EAA/TAA(total amino acid content of A1 group and A group) it is respectively 45.43% and 44.91%, EAA/NEAA is respectively
83.26% and 81.52%.The standard of the essential amino acid according to as defined in the World Health Organization, the EAA/TAA and EAA/ of A1 group
NEAA is extremely significant to be higher than A group (P < 0.01), illustrates that the nutritive value of the A1 group flesh of fish is higher than A group；Add the B1 group of brown alga oligose
NEAA content is extremely significant lower than being not added with the B group of brown alga oligose, but EAA is extremely significant is higher than B group (P < 0.01), B1 group and B group
EAA/TAA is respectively 45.26% and 45.03%, and EAA/NEAA is respectively 82.68% and 81.93%, illustrates the nutriture value of the B1 group flesh of fish
It is worth also relatively high.
Amino acid composition and content in 1 Pseudosciaena crocea meat of table
Note: A is the Larimichthys crocea control group for being not added with brown alga oligose, and A1 is the Larimichthys crocea experimental group for adding brown alga oligose.
Amino acid composition and content in the 2 grouper flesh of fish of table
Note: B is the grouper control group for being not added with brown alga oligose, and B1 is the grouper experimental group for adding brown alga oligose.
Nospecific immunity activity
(1) acid phosphatase (ACP) vigor
As seen from Figure 8, it is extremely significant few higher than brown alga is not added with to add ACP activity in the grouper experimental group fish serum of brown alga oligose
The control group (P < 0.01) of sugar illustrates that brown alga oligose can be improved the nospecific immunity activity of grouper.
(2) alkaline phosphatase (AKP) vigor
As seen from Figure 9, brown alga oligose can be improved the activity of AKP in lithosporic fish serum, and effect is extremely significant (P < 0.01)；And it is brown
Algae oligosaccharides is to the activity of AKP in rheum officinale fish serum without significantly acting on (P > 0.05).
(3) catalase (CAT) vigor
As seen from Figure 10, compared to the control group of no addition brown alga oligose, brown alga oligose has the CAT of Larimichthys crocea and grouper
There is extremely significant raising effect (P < 0.01).Illustrate that brown alga oligose can induce the raising of enzyme activity, is adjustable to a certain extent big
The immune defense ability of yellow croaker and grouper.
(4) nitricoxide synthase (NOS) vigor
As can be seen from figure 11 that brown alga oligose does not improve effect to the NOS of Larimichthys crocea and grouper, reason may be brown alga oligose
Do not have inducing action to NOS.
(5) glutathione peroxidase (GSH-Px) vigor
From Figure 12 as it can be seen that brown alga oligose is to the GSH-Px activity of Larimichthys crocea and grouper and the no significant difference of control group
(P > 0.05).
(6) lysozyme (LZM) vigor
From Figure 13 as it can be seen that compared to no control group for adding brown alga oligose, brown alga oligose is living to the LZM of Larimichthys crocea and grouper
Property tool increasing significantly effect (P < 0.01), illustrate brown alga oligose as nonspecific immunity strengthening agent can induce LZM work
Property raising, to a certain extent improve fish nospecific immunity activity.
Digestive enzyme activity
(1) amylase (AMS) vigor
From Figure 14 as it can be seen that compared to the Larimichthys crocea and grouper control group for being not added with brown alga oligose, brown alga oligose to Larimichthys crocea and
Amylase in grouper experimental group digestive system, which does not have, significantly improves effect (P > 0.05).
(2) prolease activity
From Figure 15 as it can be seen that compared to the Larimichthys crocea and grouper control group for being not added with brown alga oligose, brown alga oligose to Larimichthys crocea and
Protease in grouper digestive system has extremely significant raising effect (P < 0.01).
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of preparation method of feeding additive aquatic animal brown alga oligose, it is characterised in that: the following steps are included:
(1) using kelp as raw material, algin, extraction conditions are as follows: liquid-to-solid ratio 70:1(v/w are extracted using alkali carries alcohol deposition method), alkali dense
Degree 1%, 80 DEG C of alkali carries temperature, 4 h of alkali carries time；
(2) brown alga oligose, enzymatic hydrolysis condition are prepared using algin catenase degradation algin are as follows: concentration of substrate 0.7%, enzyme concentration
0.33 U/mL, pH7.0, temperature 50 C, 3 h of enzymolysis time finally obtain brown alga oligose.
2. the brown alga oligose that the method as described in claim 1 obtains is preparing the application in aquatic feeds.
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| CN113045683A (en) * | 2021-04-16 | 2021-06-29 | 中国科学院海洋研究所 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
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| 秦松主编: "《海岸带生物活性物质》", 28 February 2018, 济南：山东科学技术出版社 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113045683A (en) * | 2021-04-16 | 2021-06-29 | 中国科学院海洋研究所 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
| CN113045683B (en) * | 2021-04-16 | 2022-10-11 | 中国科学院海洋研究所 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
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