CN113045683B - Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed - Google Patents
Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed Download PDFInfo
- Publication number
- CN113045683B CN113045683B CN202110413226.4A CN202110413226A CN113045683B CN 113045683 B CN113045683 B CN 113045683B CN 202110413226 A CN202110413226 A CN 202110413226A CN 113045683 B CN113045683 B CN 113045683B
- Authority
- CN
- China
- Prior art keywords
- oligosaccharide
- laver
- porphyra
- fish
- molecular sieve
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Animal Husbandry (AREA)
- Materials Engineering (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Insects & Arthropods (AREA)
- Marine Sciences & Fisheries (AREA)
- Birds (AREA)
- Fodder In General (AREA)
- Feed For Specific Animals (AREA)
Abstract
The invention provides a preparation method of laver oligosaccharide and application of the laver oligosaccharide in fish feed. The laver oligosaccharide is mainly composed of 3-linked beta-D-galactose and 4-linked alpha-L-galactose-6-sulfate linkage, the polymerization degree is 2-20, the galactose content is 60-80%, and the sulfate content is 15-30%. The porphyra oligosaccharide is added into the fish feed, so that the meat quality of the cultured fish can be improved, the fat content of the fish body is reduced, and the protein content of the fish body is increased, so that the meat quality and flavor of the fish are improved. The porphyra oligosaccharide is a brand-new, green and safe fish feed additive, can effectively solve the problems of loose meat quality, low protein content and poor taste of cultured fish, can improve the immunity of the fish, and can broaden the market of the cultured fish.
Description
Technical Field
The invention relates to the field of aquaculture, in particular to a preparation method of porphyra oligosaccharide and application of the porphyra oligosaccharide in fish feed.
Background
With the development of aquaculture industry, the problem of restricting aquaculture production is increasingly prominent. One of the current problems seriously affecting the marketing quantity of the cultured fish is that the flavor of the cultured fish is poor, and the serious fishy smell, loose meat quality and high fat content all make consumers stand away. The lower quality of farmed fish has severely affected its commercial value and development. Poor meat quality of the cultivated fish is directly related to the bad cultivation environment and low feed quality. The polluted water environment will cause accumulation of fish diseases and fishy smell substances, while the energy of the common formula feed is higher, so that the fish body fat is higher and the lean meat percentage is low. Therefore, the development of safe, nontoxic and green aquaculture disease-resistant preparations has already formed a trend. At present, the meat quality of the cultured fishes is improved by adjusting the feed formula on one hand and paying attention to the enhancement of the immunity of the cultured fishes on the other hand. Researches show that microbial polysaccharide, chitin, animal and plant extracts and the like have immune enhancement effects on aquatic animals to different degrees, but the application of the immune enhancement agents needs to be further improved at present in consideration of preparation cost and technical difficulty.
Porphyra polysaccharide is a sulfated polysaccharide, which is a specific polysaccharide component of plants belonging to the genus Porphyra, and is present in the cell wall of laver, accounting for 20-40% of the dry weight of laver. Its main structural unit is → 3) β -D-galactose- (1 → 4) - α -L-6-sulfate-galactose- (1 →. Wherein part of the alpha-L-6-sulfato-galactose is replaced by L-3, 6-lacto-galactose to form the structural unit: → 3) - β -D-galactose- (1 → 4) - α -L-3, 6-lacto-galactose- (1 →. The porphyra polysaccharide has multiple biological activities of anticoagulation, blood sugar reduction, blood fat regulation, thrombus resistance, tumor inhibition, cell immunity enhancement and the like, and is widely applied to industries of food, medicine and the like. The laver oligosaccharide is prepared from laver directly or by degrading laver polysaccharide, and has polymerization degree of 2-20. However, the current preparation method of the laver oligosaccharide mainly comprises an acid degradation method, an oxidative degradation method, a microwave and ultrasonic auxiliary degradation method and an enzyme degradation method. The hydrogen peroxide degradation prospect in the chemical degradation method is optimal, the method is low in cost and easy to realize industrial production, however, the oxidizing agent does not completely participate in the reaction process, and meanwhile, the temperature is increased, so that the product is more prone to browning; the biological enzyme is utilized to assist the reaction, so that the side effect can be effectively avoided, the external reaction condition is easy to control, and the cost is increased due to the use of the enzyme reaction; the degradation reaction by physical methods is incomplete. The molecular structure determines the biological function, and the heterogeneity of the oligosaccharide chains of the laver in the microstructure such as molecular size, sequence arrangement, configuration conformation and the like can cause great difference of the biological function. Therefore, the establishment of the preparation method of the laver oligosaccharide is a precondition for the application of the laver oligosaccharide.
Compared with the laver polysaccharide, the laver oligosaccharide has better antioxidant and immunostimulating activities and better absorbability, and has wider application prospect. The research results show that the porphyra oligosaccharide serving as the meat quality modifier acts on the fish body, so that the meat quality of the cultured fish can be remarkably improved, and the immunity of the cultured fish can be improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation process of the laver oligosaccharide, which has high yield, maximally reserves the content of sulfate groups in the oligosaccharide and improves the activity of the laver oligosaccharide. On the other hand, from the perspective of improving fish flavor, starting from a fish meat quality improving agent, an application of porphyra oligosaccharide in fish feed is provided.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the preparation method of the laver oligosaccharide comprises the following specific preparation steps:
step 1, pulverizing laver, and adding 20-40 times of water, preferably 30 times of water; adding immobilized cellulase to a final concentration of 0.1-2%, preferably 0.8-1%; adjusting the pH to 4.5-6.5, preferably 5.0, with dilute HCl; hydrolyzing at 30-60 deg.C for 1-5 hr, preferably 50 deg.C for 3-4 hr under stirring;
step 2, adding a Fenton catalyst loaded by a molecular sieve into the solution subjected to enzymolysis in the step 1 to ensure that the mass concentration of the catalyst is 0.1-1%, preferably 0.5-0.7%, and adding hydrogen peroxide with the mass concentration of 28-30% to the final concentration of 10-50mM, preferably 30-40mM; centrifuging to remove precipitate, nano-filtering the obtained supernatant for desalting, concentrating, and drying to obtain thallus Porphyrae oligosaccharide with oligosaccharide yield of 10-22%.
The preparation method of the immobilized cellulase comprises the following steps: soaking the dried molecular sieve in 0.5-5 wt% cellulase solution, preferably 2-3 wt%; to a final concentration of 0.5-5%, preferably 2-3%, of the molecular sieve; stirring at room temperature for 6-24h, preferably 12-18h, centrifugally separating, washing with distilled water, and vacuum drying the precipitate to obtain an immobilized enzyme catalyst; the molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48 and MCM-50.
The preparation method of the molecular sieve loaded Fenton catalyst comprises the following steps: soaking the molecular sieve in 10-20 times volume of 0.5-5M ferric sulfate aqueous solution, preferably 12-15 times volume and 2-3M ferric sulfate aqueous solution; stirring at room temperature for 4-12h, preferably 8h, drying at 110 deg.C, and calcining in muffle furnace at 500-600 deg.C for 2-5 h.
The molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48 and MCM-50.
The preparation method of the molecular sieve-loaded Fenton catalyst and the preparation method of the immobilized cellulase can be the same or different.
The laver oligosaccharide is formed by alternately connecting 3-connected beta-D-galactose-6-sulfate and 4-connected alpha-L-galactose, the polymerization degree of the oligosaccharide is 2-20, the content of galactose is 60-80%, and the content of sulfate is 15-30%.
The porphyra oligosaccharide is directly added into basic fish feed as a feed additive, and the addition amount is 0.05-5%, preferably 0.1-1%.
Compared with the prior art, the invention has the following advantages:
1. improve the high-value utilization level of the laver. China is a large laver culture country, and the laver has poor taste and low additional value in the later period. The laver oligosaccharide is applied to the fish feed in the item, so that the added value of the laver can be greatly improved.
2. The preparation process of the laver oligosaccharide is simple and efficient. The method provides a new technology for efficiently preparing the porphyra oligosaccharide through biochemical synergistic heterogeneous catalysis for the first time, has simple process and low cost, is suitable for industrial production, and lays a foundation for the application of the porphyra oligosaccharide.
3. Compared with the common cellulase and catalyst, the immobilized cellulase and supported catalyst technology improves the use efficiency of the enzyme and the catalyst and saves the cost.
4. Broadens the application field of the laver oligosaccharide. The laver oligosaccharide is added into the fish feed for the first time, can improve the meat quality, is suitable for various cultured fishes, has simple and convenient operation, and does not need to change the original feed formula.
5. The laver oligosaccharide is natural, safe and non-toxic. The laver oligosaccharide has no toxic or side effect on a using object and the environment, does not generate drug resistance, and does not have the risk of causing harm to human health because of residue in the fish body.
6. The laver oligosaccharide has strong bioactivity. The laver oligosaccharide has the functions of improving meat quality and enhancing immunity, and also has pharmacological activities of resisting oxidation, aging and tumor. Because the environmental pollution is increasingly serious, the fish tumor diseases are more and more common, and the laver oligosaccharide has certain effect of preventing the fish tumor.
In addition, the laver oligosaccharide has immunoregulation effect on cultured fishes, thereby accelerating the metabolism of the fishes and reducing the accumulation of fishy smell substances. The porphyra oligosaccharide is a brand-new, green and safe fish feed additive, can effectively solve the problems of loose meat quality, low protein content and poor taste of cultured fish, can improve the immunity of the fish, and can broaden the market of the cultured fish.
Drawings
FIG. 1 influence of PS oligosaccharide feedstuff for Porphyra tenera on crude protein and crude fat of Epinephelus tenera, P <0.05 compared with control group, and P <0.01 compared with control group
FIG. 2 influence of oligosaccharide feed PS for laver on the amount of active oxygen produced by respiratory burst of head kidney of rockfish, P <0.05 in comparison with control group, and P <0.01 in comparison with control group
FIG. 3 influence of oligosaccharide feed PS for laver on the weight gain and survival rate of grouper, P <0.05 compared with control group, and P <0.01 compared with control group
FIG. 4 the effect of PS oligosaccharide feedstuff for laver on crude protein and crude fat of weever, P <0.05 compared with control group, and P <0.01 compared with control group
FIG. 5 Effect of different algal oligosaccharide feeds on the respiratory burst active oxygen production of weever head kidney P <0.05 compared with control group P <0.01 compared with control group
FIG. 6 the effect of different algal oligosaccharide feeds on crude protein and crude fat of weever bodies, P <0.05 compared with control group, P <0.01 compared with control group
FIG. 7 influence of Porphyra tenera oligosaccharide and Porphyra tenera polysaccharide feed on crude protein and crude fat of Epinephelus tenera body, P <0.05 compared with control group, and P <0.01 compared with control group
FIG. 8 the effect of aquatic feeds supplemented with different amounts of Porphyra tenera oligosaccharide OP on crude protein and crude fat in Epinephelus rockfish compared with control group P <0.05 and control group P <0.01
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications can be made by those skilled in the art after reading the contents of the present invention, and these equivalents also fall within the scope of the invention defined by the present invention.
Example 1 preparation of Porphyra oligosaccharide
Method I, biochemical synergistic biocatalysis high-efficiency preparation technology of laver oligosaccharide (method used in the invention)
(1) Preparation of immobilized enzyme catalyst (enzyme-MCM-48)
Weighing 3g of cellulase, adding the cellulase into 50mL of acetic acid-sodium acetate buffer solution (pH = 4.5), stirring for 1h, centrifuging to collect supernatant, continuously stirring the precipitate for 1h, centrifuging until the precipitate is completely dissolved, and fixing the volume of the solution to 100mL to obtain the cellulase liquid. 10g of dried MCM-48 molecular sieve was weighed, immersed in 100mL of 3% cellulase solution, stirred at room temperature for 12 hours, centrifuged, and washed with distilled water. And (4) drying the precipitate in vacuum to obtain the immobilized enzyme catalyst.
(2) Preparation of molecular sieve supported Fenton catalyst (Fe-MCM-48)
100mL of 2M ferric sulfate aqueous solution is prepared, 10g of dried MCM-48 molecular sieve is weighed and dipped in the aqueous solution, the mixture is stirred at room temperature for 8h, dried at 110 ℃, and roasted at 500 ℃ for 3h in a muffle furnace for standby.
(3) Process research for degrading laver oligosaccharide by enzyme-MCM-48 and Fe-MCM-48 in cooperation
Firstly, enzyme-MCM-48 is adopted to carry out enzymolysis on the laver. Adding 30 times of water into the crushed laver, adding enzyme-MCM-48 until the final concentration is 1%, adjusting the pH value to 5.0, stirring at 50 ℃ for hydrolysis for 3 hours, and filtering out the catalyst after the reaction is finished. Fe-MCM-48 was added to the solution after the enzymatic hydrolysis to a final concentration of 0.5%, and 30% hydrogen peroxide was added to a final concentration of 30mM. Hydrolyzing at 60 deg.C for 3h under stirring, centrifuging to remove precipitate, subjecting the obtained supernatant to nanofiltration desalination, concentrating, and drying to obtain thallus Porphyrae oligosaccharide OP with yield of 15.4%. Through measurement, the content of galactose in OP is 66.2%, the content of sulfate radical is 24.3%, and the degree of polymerization is 2-20.
Method II, sulfuric acid hydrolysis preparation technology of laver oligosaccharide (preparation method of laver oligosaccharide in literature)
(1) Preparation method of thallus Porphyrae polysaccharide
Adding 20 times of water into pulverized laver, extracting at 120 deg.C under high pressure for 3h, centrifuging at 4000r/min for 15min, directly dialyzing the supernatant, collecting dialysate after dialysis, concentrating, and drying to obtain porphyra polysaccharide (P), with yield of 18%.
(2) Preparation of laver oligosaccharide
5g of porphyra polysaccharide is added with 150mL of water and stirred overnight at room temperature to be dissolved. Diluting 5.435mL of concentrated sulfuric acid to 50mL, adding the porphyra polysaccharide solution, stirring while adding, uniformly mixing, and reacting for 3 hours under stirring at 80 ℃ (adding reflux). 31.546g of Ba (OH) was weighed out at the end of the reaction 2 ·8H 2 And O, preparing a saturated solution for neutralization (continuously stirring). Centrifuging at 4000r/min for 8min, collecting supernatant, filtering, concentrating, dialyzing with dialysis bag with molecular weight cutoff of 1kDa, and lyophilizing to obtain thallus Porphyrae oligosaccharide OM with yield of 55% and 9.9% in terms of thallus Porphyrae. Through determination, the content of galactose in OM is 55.6%, the content of sulfate radical is 14.3%, and the polymerization degree is 2-20.
Comparing the first method with the second method, the laver oligosaccharide OP yield and the content of galactose and sulfate radical obtained by the first method are higher than the laver oligosaccharide OM obtained by the second method, and the method has simple steps and is more suitable for industrial production. Meanwhile, we also tried enzymolysis method using only cellulase, agarase, etc. and only H 2 O 2 Method for preparing laver oligosaccharide by oxidative degradation, wherein the molecular weight of samples obtained by the two methods is more than 5000Da, no oligosaccharide sample with low degree of polymerization could be obtained. Compared with the prior art, the method for preparing the porphyra oligosaccharide is simple and efficient, the obtained oligosaccharide sample is good in quality, and the method is suitable for industrial scale-up production and has good application potential.
Example 2 preparation of Porphyra oligosaccharide feed (PS, PO), porphyra polysaccharide feed (PP) and Porphyra powder feed (PM)
The laver oligosaccharide extract (OP) obtained in the first step of the example 1 is added into the feed in an amount of 0.05-5% by weight of the total weight of the feed, and any concentration within the range can obviously improve the meat quality and flavor of the cultured fish, enhance the immunity of the organism cells and improve the disease resistance of the cultured fish, and only a plurality of different concentrations are taken as representative samples in the example. The preparation method comprises the following steps: firstly, preparing the laver oligosaccharide extract into 8-12% solution, preferably 10%; then adding anhydrous ethanol under stirring to make the total concentration of ethanol be 10% -20%, preferably 15%; and uniformly spraying the prepared solution on the feed to prepare the feed with the laver oligosaccharide accounting for 0.1% of the total weight of the feed, and storing the feed after drying or airing for later use, wherein the feed is marked as PS. The same procedure was followed to prepare PO feed containing 0.1% of laver oligosaccharide OM obtained by the method II of example 1, PP feed containing 3% of laver polysaccharide obtained by the method II of example 1, and PM feed containing 20% of laver powder.
Example 3 Effect of feeding of Aquaculture feed containing Porphyra tenera oligosaccharide on meat quality improvement and immunoregulation of Epinephelus tenera
The experiment uses the aquatic feed containing the porphyra oligosaccharide to feed the grouper, and the effect of the grouper on the improvement of the grouper meat quality and the immunoregulation is demonstrated through the change of the body composition, the muscle texture and the related indexes of the physiological function of the grouper.
The test method comprises the following steps: the healthy and uniform groupers are randomly selected and divided into 3 groups, and different baits are respectively used according to a normal feeding management program. Group 1 is commercial aquatic feed F control group, group 2 is PM control group, group 3 is PS experimental group, the experimental period is 42 days, 9 fishes are sampled every 14 days, and the head kidney is aseptically taken for measuring the respiratory burst of head kidney macrophage. Taking out fish head kidney from clean bench, placing into 1.5ml centrifuge tube containing 1ml PBS, grinding on ice, and usingSieving with 100 mesh cell sieve, removing tissue mass, centrifuging at 500g to remove supernatant, washing with PBS for 2 times, and resuspending to obtain single cell suspension with cell count not less than 10 6 And (4) cells. And adding a DCFH-DA fluorescent probe into the single-cell suspension for incubation. The subsequent operations were performed according to the instructions of Nanjing's built-up Reactive Oxygen Species (ROS) test kit. After the test is finished, the weight gain rate and the survival rate are measured, the dorsal muscle (2 cm multiplied by 1.5 cm) is taken for carrying out muscle Texture (TPA) characteristic analysis, and the tissue fish body is not taken for measuring crude protein and crude fat. The activities of antioxidant enzyme (SOD, CAT) total complement CH50 and alkaline phosphatase AKP in serum are determined by Nanjing reagent kit.
As a result:
during the test period, the weight gain rate and the survival rate of the groupers are shown in figure 1, the weight gain rate of the 3 rd group is obviously improved compared with that of a basal feed group, and the weight gain rate of the 3 rd group is also higher than that of a laver powder adding group with the weight gain rate of 20%, so that the laver oligosaccharide has an obvious promotion effect on the weight gain of the groupers, and the weight gain effect of the laver oligosaccharide is better than that of the laver powder directly added with high dosage. The survival rate of grouper group 3 was also higher during the test than that of the basal feed group and the 20% laver powder-supplemented group.
As can be seen from Table 1, the SOD activity of the superoxide dismutase in the group 3 added with the laver oligosaccharide is obviously improved compared with that of the control group, and in the feeding process of the previous 28 days, the SOD activity of the superoxide dismutase in the group 3 is always in an increasing trend, but is reduced after 28 days, which is consistent with the trend of the control group. For total complement CH 50 The value of group 3 was also significantly increased compared to the basal diet group, but the overall trend was similar to the control group and decreased with the duration of the feeding experiment. For catalase CAT, the experimental group to which the laver oligosaccharide was added was higher than the control group, and decreased and then increased with the lapse of time. For alkaline phosphatase AKP, all three groups decreased with time, but the third group was more active than the control group and the laver powder-added group. It is worth mentioning that all indexes of the laver oligosaccharide adding group are superior to those of the laver powder adding group. SOD, CH 50 CAT and AKP are closely related to the immunity of the grouper, and the increase of the indexes proves that the feed added with the laver extract has positive immunoregulation function.
TABLE 1 Effect on specific indices in Epinephelus coioides serum
* Comparison with control group P<0.05, ** Comparison with control group P<0.01
As can be seen from FIG. 2, the ROS level of active oxygen generated by respiratory burst of the porphyra oligosaccharide-added group head kidney macrophages is significantly higher than that of the control group and the porphyra powder-added group, which indicates that the nonspecific immunity of the porphyra oligosaccharide-added group is significantly improved.
From fig. 3, it can be seen that the laver oligosaccharide-added group effectively increases the crude protein content of fish bodies and reduces the crude fat content of fish bodies, compared with the control group.
As can be seen from Table 2, the hardness, elasticity, adhesiveness, chewiness and recoverability of the muscle of the PS group are superior to those of the control group and the laver powder-added group.
TABLE 2 grouper Texture (TPA) characterization results
* Comparison with control group P<0.05, ** Comparison with control group P<0.01
Example 4 Effect of feeding of Aquaculture feed containing Porphyra tenera oligosaccharide on meat quality improvement and immunoregulation of Perch
The experiment uses the aquatic feed containing the laver oligosaccharide to feed the weever, and the effect of the weever on the meat quality improvement and the immunoregulation is demonstrated through the change of the body composition, the muscle texture and the related indexes of the physiological function of the weever.
The test method comprises the following steps:
selecting healthy and uniform weever, randomly dividing the weever into 3 groups, respectively using different baits, and carrying out the breeding according to a normal breeding management program. Group 1 is commercial aquatic feed F control group, group 2 is 20% laver powder added feed (PM) control group, group 3 is PS experimental group, the experimental period is 28 days, 10 tails of each group of experimental fishes are randomly taken respectively at 0 day, 14 days and 28 days, tail vein blood is taken, each tail is 0.5ml, whole blood stands for 3h at room temperature, stands for 5h at 4 ℃, serum is centrifugally collected, and samples are reserved at-20 ℃ for serum nonspecific immunity index determination (SOD, AKP, LZM). After the feeding experiment, the dorsal muscle (2 cm × 2cm × 1.5 cm) was taken for the analysis of the texture of muscle meat (TPA), and the fish body without tissue was used for the measurement of crude protein and crude fat.
As a result:
from fig. 4, it can be seen that the crude protein content of the weever body in the laver oligosaccharide addition group is significantly increased and the crude fat content is significantly reduced compared with the weever body in the control group.
As can be seen from Table 3, compared with the control group, the nonspecific immunity indexes of the PS group are increased and are increased more obviously than those of the PM group, which indicates that the nonspecific immunity of aquatic animals can be effectively improved by adding the porphyra oligosaccharide
TABLE 3 influence on nonspecific immune index of weever
* Comparison with control group P<0.05, ** Comparison with control group P<0.01
From table 4, it can be seen that the laver powder addition group and the laver oligosaccharide addition group are superior to the control group in four aspects of hardness, adhesiveness and chewiness, wherein the laver oligosaccharide addition group has more significant improvement in muscle properties, and the three groups do not show significant difference in the adhesiveness of the weever muscle.
TABLE 4 measurement results of Perch flesh Texture (TPA)
| Group of | Hardness (N) | Elasticity (mm) | Adhesion (g) | Degree of adhesion (N) | Degree of mastication (N) |
| Control group | 47.87±0.62 | 0.33±0.02 | 0.21±0.01 | 6.38±0.07 | 1.72±0.23 |
| PM group | 46.23±1.03 | 0.35±0.04 | 0.23±0.02 | 7.72±0.12 * | 2.14±0.03 * |
| PS group | 50.21±0.33 * | 0.38±0.01 * | 0.22±0.03 | 8.92±0.08 ** | 3.32±0.16 ** |
* Comparison with control group P<0.05, ** Comparison with control group P<0.01
Example 5 comparison of the Effect of several aquatic feeds supplemented with different Marine oligosaccharides on meat quality improvement and immunomodulation of weever
In the test, the weever is fed by using aquatic feeds containing 0.1 percent of chitosan oligosaccharide, fucooligosaccharide, algin oligosaccharide and laver oligosaccharide, and the effects of different oligosaccharides on the improvement of weever meat quality and the immunoregulation are demonstrated through the change of weever body composition, the change of muscle texture and related indexes of physiological function.
Selecting healthy and uniform weever, randomly dividing the weever into 5 groups, respectively using different baits, and carrying out the breeding according to a normal breeding management program. The group 1 is a commercial aquatic feed F control group, the group 2 is a PS experimental group, the group 3 is an experimental group for adding 0.1% chitosan oligosaccharide, the group 4 is an experimental group for adding 0.1% fucoidan oligosaccharide, and the group 5 is an experimental group for adding 0.1% algin oligosaccharide. The test period was 28 days. Taking the head kidney to measure the respiratory burst, and taking the fish body without tissue to measure crude protein and crude fat.
As a result:
as can be seen from figure 5, the oligosaccharide addition group has a promoting effect on the ROS level of active oxygen generated by respiratory burst of weever head kidney macrophages compared with a basal feed control group, wherein the ROS level of the laver oligosaccharide addition group is highest, and chitosan oligosaccharide is second.
It can be seen from fig. 6 that the crude protein content of the fish body of the porphyra oligosaccharide added group is significantly increased compared with the control group, the crude fat content of the fish body is significantly reduced compared with the control group, the change of other oligosaccharide body components is not significant, and the crude protein content of the fish body of the algin oligosaccharide added group is slightly reduced compared with the control group.
Example 6 comparison of meat quality improvement effects of Aquaculture feed supplemented with Porphyra yezoensis oligosaccharide and Porphyra yezoensis polysaccharide on Epinephelus
This experiment shows the difference in the meat quality improvement effect of porphyra oligosaccharides and porphyra polysaccharides from grouper by the change in body composition and the change in texture of muscles of grouper by feeding grouper with aquaculture feeds (PS and PO) containing porphyra oligosaccharides prepared in example 1 and aquaculture feed (PP) containing undegraded porphyra polysaccharides, respectively.
The healthy and uniform groupers are randomly divided into 4 groups, different baits are respectively used, and the operation is carried out according to a normal feeding management program. Group 1 is a commercial aquatic feed F control group, group 2 is a PS experimental group, group 3 is a PO experimental group, and group 4 is a PP experimental group. The test period was 28 days. The dorsal muscle (2 cm. Times.2 cm. Times.1.5 cm) was taken for analysis of the texture of muscle meat (TPA) and the tissue fish body was not taken for measurement of crude protein and crude fat.
As can be seen from fig. 7, the PS group had a higher crude protein content and a lower crude fat content than the basal feed group, and the PO group and the porphyra polysaccharide-supplemented group did not show a significant difference in fish body composition. The activity of the laver oligosaccharide prepared by different processes is different, and the process adopted by the invention can furthest reserve the biological activity of the laver oligosaccharide. This is probably because the process has less damage to sulfate groups, which are important groups for the activity of the laver oligosaccharide.
As can be seen from Table 5, the PO and PP groups did not show significant difference in the index of fleshy texture characteristics compared to the control group, which is probably due to the fact that the porphyra polysaccharide has a large molecular weight and is not easily absorbed by aquatic animals, while the oligosaccharide PO group has a low sulfate group and is not significant in activity. The laver oligosaccharide PS group shows remarkable improvement in elasticity, adhesiveness, chewiness and resilience.
TABLE 5 texture of grouper muscle (TPA) determination results
* Comparison with control group P<0.05, ** Comparison with control group P<0.01
Example 7 comparison of meat quality improvement effects of Epinephelus altivelis by aquatic feeds supplemented with different dosages of Porphyra oligosaccharide OP
In this test, the aquatic feeds PS1, PS2, PS3, PS4, PS5 and PS6 containing 0.05%, 0.1%, 1%, 5%, 10% and 20% of laver oligosaccharides were prepared according to the method of example 2 and fed to grouper, and the differences in the effects of laver oligosaccharides and laver polysaccharides on the improvement of meat quality of grouper were demonstrated by the changes in body composition and muscle texture of grouper.
The healthy and uniform groupers are randomly selected and divided into 7 groups, different baits are respectively used, and the normal feeding management procedure is carried out. Group 1 is a commercial aquatic feed F control group, groups 2-7 are PS1-PS6 experimental groups respectively, and the experimental period is 28 days. Dorsal muscle (2 cm. Times.2 cm. Times.1.5 cm) was taken for analysis of muscle Texture (TPA) characteristics, and fish body tissue was taken for measurement of crude protein and crude fat.
As can be seen from fig. 8, the PS groups containing different dosages of oligosaccharides had higher fish body crude protein content and reduced crude fat content compared to the basal feed group, and when the added amount of porphyra oligosaccharide was greater than 5%, each group had little effect on fish body crude protein and crude fat content. When the addition amount of the laver oligosaccharide is less than 5%, the content of crude protein in fish bodies can be gradually increased and the content of crude fat in fish bodies can be gradually reduced in each group along with the increase of the addition amount of the laver oligosaccharide. This shows that when the addition amount of the laver oligosaccharide exceeds a certain amount, the crude protein and crude fat components of the fish body are not influenced with the increase of the addition amount, and the addition amount is more suitable to be 0.05-5%.
As can be seen from table 6, PS groups containing different dosages of oligosaccharides showed significant differences in muscle texture characteristics indexes as compared to the control group. The elasticity, the adhesive degree, the chewiness and the resilience are all obviously improved. When the addition amount of the porphyra oligosaccharide is more than 5%, the change of each index relatively tends to be stable, which indicates that the addition amount of the porphyra oligosaccharide is more appropriate from 0.05 to 5 percent.
TABLE 6 texture of grouper muscle (TPA) determination results
* Comparison with control group P<0.05, ** Comparison with control group P<0.01
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A preparation method of laver oligosaccharide is characterized in that:
the preparation method comprises the following specific steps:
step 1, pulverizing laver and adding 20-40 times of water; adding immobilized cellulase into the solution to a final concentration of 0.1-2%; adjusting pH to 4.5-6.5 with HCl with concentration of 0.1-1M; hydrolyzing at 30-60 deg.C for 1-5h under stirring;
step 2, adding a Fenton catalyst loaded by a molecular sieve into the solution subjected to enzymolysis in the step 1 to ensure that the mass concentration of the catalyst is 0.1-1%, and adding hydrogen peroxide with the mass concentration of 28-30% to the final concentration of 10-50 mM; centrifuging to remove precipitate, nano-filtering the obtained supernatant for desalting, concentrating, and drying to obtain laver oligosaccharide; the preparation method of the immobilized cellulase comprises the following steps: soaking the dried molecular sieve in cellulase liquid with the mass concentration of 0.5-5%; the final concentration of the molecular sieve is 0.5-5%; stirring for 6-24h at room temperature, performing centrifugal separation, washing with distilled water, and performing vacuum drying on the precipitate to obtain an immobilized enzyme catalyst; wherein the molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48 and MCM-50;
the preparation method of the molecular sieve supported Fenton catalyst comprises the following steps: soaking the molecular sieve into 10-20 times volume of 0.5-5M ferric sulfate aqueous solution; stirring at room temperature for 4-12h, drying at 110 deg.C, and roasting in muffle furnace at 500-600 deg.C for 2-5 h.
2. The method of claim 1,
in the step 1, adding immobilized cellulase into the solution until the final concentration is 0.8-1%; hydrolyzing at 50 deg.C for 3-4h under stirring; and 2, adding a Fenton catalyst loaded by a molecular sieve into the solution subjected to enzymolysis in the step 1 to ensure that the mass concentration of the catalyst is 0.5-0.7%, and adding hydrogen peroxide with the mass concentration of 28-30% to ensure that the final concentration is 30-40 mM.
3. The method of claim 1,
the preparation method of the immobilized cellulase comprises the following steps: soaking the dried molecular sieve in cellulase liquid with the mass concentration of 2-3%; the final concentration of the molecular sieve is 2-3%; stirring for 12-18h at room temperature, performing centrifugal separation, washing with distilled water, and performing vacuum drying on the precipitate to obtain the immobilized enzyme catalyst.
4. The method according to claim 1, wherein the molecular sieve is one or more of M41S series mesoporous molecular sieves MCM-41, MCM-48, MCM-50.
5. Use of the porphyra oligosaccharide prepared by the preparation method of any one of claims 1 to 4 in fish feed.
6. The use of the porphyra oligosaccharide in fish feed according to claim 5, wherein the porphyra oligosaccharide is directly added to the fish basal feed as a feed additive in an amount of 0.05-5%.
7. The use of the porphyra oligosaccharide in fish feed according to claim 6, wherein the porphyra oligosaccharide is directly added into the basic feed of fish as a feed additive, and the addition amount is 0.1-1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110413226.4A CN113045683B (en) | 2021-04-16 | 2021-04-16 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110413226.4A CN113045683B (en) | 2021-04-16 | 2021-04-16 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113045683A CN113045683A (en) | 2021-06-29 |
| CN113045683B true CN113045683B (en) | 2022-10-11 |
Family
ID=76519599
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110413226.4A Active CN113045683B (en) | 2021-04-16 | 2021-04-16 | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113045683B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1772915A (en) * | 2004-11-10 | 2006-05-17 | 中国科学院大连化学物理研究所 | Chitosn producing process with immobilized enzyme |
| CN1931882A (en) * | 2006-10-09 | 2007-03-21 | 北京联合大学生物化学工程学院 | Industrial prepn process of chitosan oligomer |
| CN108210504A (en) * | 2017-12-27 | 2018-06-29 | 中国科学院海洋研究所 | The application of sulphation galactooligosacchari(es and pharmaceutical composition |
| CN109221811A (en) * | 2018-09-28 | 2019-01-18 | 福州大学 | A kind of preparation method of feeding additive aquatic animal brown alga oligose |
| CN109395766A (en) * | 2018-11-12 | 2019-03-01 | 济南大学 | A kind of molecular sieve carried Fenton catalyst for chitosan oxidative degradation |
-
2021
- 2021-04-16 CN CN202110413226.4A patent/CN113045683B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1772915A (en) * | 2004-11-10 | 2006-05-17 | 中国科学院大连化学物理研究所 | Chitosn producing process with immobilized enzyme |
| CN1931882A (en) * | 2006-10-09 | 2007-03-21 | 北京联合大学生物化学工程学院 | Industrial prepn process of chitosan oligomer |
| CN108210504A (en) * | 2017-12-27 | 2018-06-29 | 中国科学院海洋研究所 | The application of sulphation galactooligosacchari(es and pharmaceutical composition |
| CN109221811A (en) * | 2018-09-28 | 2019-01-18 | 福州大学 | A kind of preparation method of feeding additive aquatic animal brown alga oligose |
| CN109395766A (en) * | 2018-11-12 | 2019-03-01 | 济南大学 | A kind of molecular sieve carried Fenton catalyst for chitosan oxidative degradation |
Non-Patent Citations (1)
| Title |
|---|
| "坛紫菜多糖的降解及其产物的活性研究和结构解析";党甜甜;《中国优秀博硕士学位论文全文数据库(硕士)医药卫生科技辑》;20190115;正文第1-3、18-19页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113045683A (en) | 2021-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105001352B (en) | Beta-1,3/1,6-glucan, preparation method therefor, and application thereof in preparing immune enhancement and anti-tumor medicine and functional food | |
| CN105231058A (en) | Feed additive for broiler chickens, capable of promoting appetite and prepared by comprehensively utilizing coconut shells | |
| CN112430546A (en) | Isoaerobic fermentation preparation method and application of chlamydomonas reinhardtii and chlamydomonas reinhardtii powder | |
| CN102134285A (en) | Method for preparing polysaccharide of defective porphyra haitanensis | |
| CN110257296B (en) | Culture method and application of chromium-enriched spirulina | |
| CN113045683B (en) | Preparation method of porphyra oligosaccharide and application of porphyra oligosaccharide in fish feed | |
| CN113913362A (en) | Sorbus commixta stem cell capable of increasing anthocyanin content, culture medium and culture method | |
| CN103330083A (en) | Composite feed for improving immunity of herring and preparation method thereof | |
| CN103205352B (en) | Oyster polysaccharide wine | |
| CN116158490A (en) | A feed additive rich in stachyose extract and Bacillus licheniformis | |
| CN114287521B (en) | Compound enzymolysis preparation method of laminarin for replacing fish meal for cottonseed protein compound feed and application thereof | |
| CN108261418B (en) | Preparation and drying method of pachyman powder | |
| CN105146083A (en) | Chicken immunity enhancing feed additive prepared based on comprehensive utilization of coconut shells | |
| CN113439838B (en) | Fermentation method, fermentation product and kit containing fermentation product | |
| CN114009626A (en) | Fish meal substitute containing yeast extract and aquatic animal premix composition | |
| CN108047344B (en) | Preparation method of soybean spirulina compound extract and application of soybean spirulina compound extract in tipping paper | |
| CN102178066B (en) | Compound polysaccharide feed additive capable of replacing antibiotics in poultry daily ration and preparation method thereof | |
| KR101326267B1 (en) | Composition for anti-obesity effect from fermented sea squirt with acid and alkali treatment | |
| CN111449241A (en) | Method for improving antioxidant capacity of sea cucumber saponin extracting solution | |
| CN101816377A (en) | O-acyl carrageenin oligosaccharide feed additive | |
| CN111620965A (en) | Preparation method of flammulina velutipes chitin | |
| CN115553402B (en) | Biological polypeptide preparation capable of improving nonspecific immunity function of Haliotis discus hand-Mazz and application thereof | |
| CN109452466A (en) | A kind of application of composition of plant extracts in feed addictive | |
| CN108323622A (en) | A kind of preparation method of the feed addictive containing composite vegetables extractive | |
| CN106666094A (en) | Nano feed additive for adjusting metabolism of body fat of duck |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230630 Address after: 066303 east of Yu Guan Village, Yu Guan Town, Funing District, Qinhuangdao, Hebei, China Patentee after: Qinhuangdao Yi Er Biological Technology Co.,Ltd. Address before: 266071 Shandong province Qingdao City Nanhai Road No. 7 Patentee before: Institute of Oceanology Chinese Academy of Sciences |