CN115316497B - Mulberry leaf extract, mandarin fish feed and application thereof - Google Patents
Mulberry leaf extract, mandarin fish feed and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/13—Prevention or treatment of fish diseases
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0207—Control systems
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0261—Solvent extraction of solids comprising vibrating mechanisms, e.g. mechanical, acoustical
- B01D11/0265—Applying ultrasound
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0288—Applications, solvents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0292—Treatment of the solvent
Abstract
The invention provides a mulberry leaf extract, a mandarin fish feed and application thereof, belonging to the technical field of natural products in aquatic feeds. The invention provides a mulberry leaf extract, which takes mulberry leaf polysaccharide and mulberry leaf flavone as main active ingredients. The weight and the body length of the mandarin fish can be obviously improved by adding 5g/kg of mulberry leaf extract into the mandarin fish compound feed, and the survival rate of the mandarin fish is continuously improved along with the improvement of the concentration of the mulberry leaf extract; the addition of a proper amount of mulberry leaf extract can obviously improve the antioxidant capacity of mandarin fish serum, improve liver and intestinal health and improve the intestinal absorption capacity of mandarin fish. Therefore, the invention provides the application of the mulberry leaf extract or the mandarin fish feed containing the mulberry leaf extract in mandarin fish cultivation.
Description
Technical Field
The invention belongs to the technical field of natural products in aquatic feeds, and particularly relates to a mulberry leaf extract, a mandarin fish feed and application thereof.
Background
The mandarin fish (Siniperca chuatsi) belongs to the genus Siniperca (Siniperca) of the family Perciformes (Percichthys), commonly called Osmanthus fragrans, and is a fresh water economic fish with a long-term flourishing name in China. The mandarin fish has delicious meat, no intramuscular penetration, high nutritive value and other features, and is popular with domestic and foreign consumers [1] . After breakthrough is made from the artificial propagation technology of mandarin fish in 70 th century, artificial mandarin fish culture is rapidly developed, and data of the '2021 Chinese fishery statistics annual survey' show that the total yield of mandarin fish culture in 2020 China reaches 37.7 ten thousand tons, and the yield exceeds 200 hundred million yuan [2] . Mandarin fish is carnivorous fish, mainly fed live bait fish, and artificial compound feed cultivation of mandarin fish is preliminarily realized along with the feeding mechanism of mandarin fish and the progress of compound feed domestication technology at present [3 , 4] . In the artificial compound feed cultivation process, fatty liver and intestinal tract injury are the main recessive threats affecting the survival rate of mandarin fish [5] . In addition, the mandarin fish is frequently damaged and has low survival rate, so that a large amount of fishing drugs and antibiotics are used, and the quality safety of the mandarin fish is seriously affected. Therefore, screening a proper feed additive capable of promoting the growth of mandarin fish, enhancing immunity, improving oxidation resistance and protecting liver and intestine functions is a key for promoting the healthy development of mandarin fish breeding industry.
Mulberry (Morus alba L.) has a long history of planting in China, and mulberry leaves are widely used as food and Chinese herbal medicine materials [6 , 7] . The mulberry leaf is rich in polysaccharide, flavonoid, alkaloid, polyphenol and other natural active matters [6 , 8] . Research shows that mulberry leaf has the function of reducing blood sugar [9] Reducing blood fat [10] Antibacterial agent [11] Antioxidant [12] Antiviral agent [13] And anti-inflammatory [14] And the like. The folium Mori extract (mulberry leafextract, MLE) is prepared by sun-drying pulverized folium Mori powder, extracting with solvent such as n-butanol, ethanol and water under heatingThe mixture obtained [15] Contains a large amount of bioactive substances such as polysaccharides, flavonoids, alkaloids, etc [13] . Therefore, extensive researches on mulberry leaves and extracts thereof have been conducted, mainly focusing on the extraction process [6] Pharmacological Activity [16] Clinical application [17] Nutritional and functional ingredients [6] Protein source substitution for feed [18] In aspects of the method, the application of the feed additive as the feed additive in aquaculture is less, and the research conclusion on the growth performance is different. Wangmei, etc [19] It is found that the addition of 10-300 mg/kg of mulberry leaf flavone in the feed has no significant influence on the growth performance and the body composition of the litopenaeus vannamei. Similarly, in the research of tilapia, it is found that the addition of 50-1000 mg/kg of mulberry leaf flavone in the feed has no obvious influence on the growth performance of the tilapia [20] . Early stage in the grass carp research shows that the addition of mulberry leaf powder has no obvious influence on the growth performance of grass carp [21] 。
Reference to the literature
[1] Li Songlin, han Zhihao, wang Xiaoyuan, et al, mandarin culture profile, and research progress on feeding control mechanisms [ J ]. Programming of aquatic products, report 2021:1-8.
LI S L,HAN Z H,WANG X Y,et al.Research progress on aquaculture and feeding regulation mechanism ofMandarin fish[J].Journal ofFisheries ofChina,2021,45(10):1787-1795.(in Chinese)
[2] Chinese mandarin fish industrial development report [ J ]. Chinese aquatic products 2021 (04): 23-32.
[3]LIU L,LIANG X,FANG J,et al.The differentia of nitrogen utilization between fast growth individuals and slow growth individuals in hybrid ofSiniperca chuatsi(♀)×Siniperca scherzeri(♂)mandarin fish fed minced prey fish[J].Aquaculture Research,2017,48(8):4590-4595.
[4]LIANG X F,OKU H,OGATA H Y,et al.Weaning Chinese perch Siniperca chuatsi(Basilewsky)onto artificial diets based upon its specific sensorymodality in feeding[J].Aquaculture Research,2001,32:76-82.
[5]ZHANG Y,FENG H,LIANG X,et al.Dietary bile acids reduce liver lipid deposition via activating farnesoid X receptor,and improve gut health by regulating gut microbiota in Chinese perch(Siniperca chuatsi)[J].Fish&Shellfish Immunology,2022,121:265-275.
[6]WEN P,HU T,LINHARDT R J,et al.Mulberry:A review of bioactive compounds and advanced processing technology[J].Trends in Food Science&Technology,2019,83:138-158.
[7]CHAN E W,LYE P Y,WONG S K.Phytochemistry,pharmacology,and clinical trials ofMorus alba[J].Chin J NatMed,2016,14(1):17-30.
[8]MA G,CHAI X,HOU G,et al.Phytochemistry,bioactivities and future prospects of mulberry leaves:A review[J].Food Chemistry,2022,372:131335.
[9]NAOWABOOT J,PANNANGPETCH P,KUKONGVIRIYAPAN V,et al.Antihyperglycemic,antioxidant and antiglycation activities ofmulberry leaf extract in streptozotocin-induced chronic diabetic rats[J].PlantFoods Hum Nutr.,2009,64(2):116-121.
[10]ZHAO X,FU Z,YAO M,et al.Mulberry(Morus alba L.)leaf polysaccharide ameliorates insulin resistance-and adipose deposition-associated gut microbiota and lipid metabolites in high-fat diet-induced obese mice[J].Food Science&Nutrition,2022,10(2):617-630.
[11]AELENEI P,LUCA S V,HORHOGEA C E,et al.Morus alba leaf extract:Metabolite profiling and interactions with antibiotics against Staphylococcus spp.including MRSA[J].Phytochemistry Letters,2019,31:217-224.
[12]JESZKA SKOWRON M,FLACZYK E,
T.In vitro and in vivo analyses of Morus alba Polish var.wielkolistna zolwinska leaf ethanol-water extract-antioxidant and hypocholesterolemic activities in hyperlipideamic rats[J].European Journal of Lipid Science and Technology,2017,119(10):1600514.
[13]THABTI I,ALBERT Q,PHILIPPOT S,et al.Advances on antiviral activity of Morus spp.plant extracts:human coronavirus and virus-related respiratorytract infections inthe spotlight[J].Molecules,2020,25(8):1876.
[14]OH N S,LEE J Y,LEE J M,et al.Mulberry leaf extract fermented with Lactobacillus acidophilus A4 ameliorates 5-fluorouracil-induced intestinal mucositis inrats[J].LettApplMicrobiol,2017,64(6):459-468.
[15]KIM G,JANG H.Flavonol content in the water extract of the mulberry(Morus alba L.)leafand their antioxidant capacities[J].Journal of Food Science,2011,76(6):C869-C873.
[16]KWON H J,CHUNG J Y,KIM J Y,et al.Comparison of 1-deoxynojirimycin and aqueous mulberry leaf extract with emphasis on postprandial hypoglycemic effects:in vivo and in vitro studies[J].Journal of Agricultural and Food Chemistry,2011,59(7):3014-3019.
[17]SHIN S,SEO H,PARK H,et al.Effects of mulberry leaf extract on blood glucose and serum lipidprofiles in patients with type 2diabetes mellitus:Asystematic review[J].European Journal of Integrative Medicine,2016,8(5):602-608.
[18]ZHU W,XIAO S,CHEN S,et al.Effects of fermented mulberry leaves on growth,serum antioxidant capacity,digestive enzyme activities and microbial compositions of the intestine in crucian(Carassius carassius)[J].Aquaculture Research,2021,52(12):6356-6366.
[19] Wangmei, ice, wang Guoxia, etc. folium Mori flavone has effect on growth performance, oxidation resistance index and stress resistance of Litopenaeus vannamei [ J ]. Chinese aquatic science 2020,27 (10): 1184-1195.
[20] Yang Jihua, ice, huang Yanhua, etc. the feed is added with mulberry leaf flavone for the growth performance, body composition, antioxidant index and nitrite stress resistance of Gift tilapia, animal nutrition report, 2017,29 (09) 3403-3412.
[21] Zhou Donglai, liao Sentai, huang Yong, etc. the effect of mulberry leaf powder on grass carp growth performance and meat quality and flavor [ J ] Guangdong agricultural science 2021,48 (04): 119-130.
Disclosure of Invention
Accordingly, the invention aims to provide the mulberry leaf extract and the mandarin fish feed prepared from the mulberry leaf extract, which are added into daily ration as feed additives for aquaculture by promoting the growth of mandarin fish, improving the oxidation resistance of aquatic animals and improving the liver and intestinal health of the aquatic animals.
The invention provides a mulberry leaf extract, which contains mulberry leaf polysaccharide and mulberry leaf flavone;
the concentration of the mulberry leaf polysaccharide is 350 mg/g-550 mg/g, and the concentration of the mulberry leaf flavone is 30 mg/g-100 mg/g.
The invention provides a preparation method of the mulberry leaf extract, which comprises the following steps:
treating folium Mori with petroleum ether to obtain pretreated folium Mori;
extracting the pretreated mulberry leaves with ethanol water solution, and separating liquid phase to obtain mulberry leaf extract.
Preferably, the feed liquid ratio of the mulberry leaves to the petroleum ether is 1g: 18-22 mL.
Preferably, the petroleum ether treatment comprises petroleum ether extraction and reflux treatment;
the petroleum ether is extracted for 10-14 h;
the time of the reflow treatment is 1.5-2.5 h, and the temperature of the reflow treatment is 55-65 ℃.
Preferably, the feed liquid ratio of the pretreated mulberry leaf to the ethanol water solution is 1g: 28-32 mL;
the volume percentage of the ethanol aqueous solution is 70-80%.
Preferably, the times of the ethanol aqueous solution extraction are 2 to 3 times;
each time of ethanol aqueous solution extraction, accompanied by ultrasonic extraction;
the time of each ultrasonic extraction is 25-35 min; the temperature of each ultrasonic extraction is 38-42 ℃; the power of each ultrasonic extraction is 30-50 KHz.
The invention provides a mandarin fish feed, which comprises mulberry leaf extract or mulberry leaf extract and ration prepared by the preparation method;
the addition amount of the mulberry leaf extract is 1.0g/kg ration, 5.0g/kg ration or 10g/kg ration.
The invention provides an application of the mulberry leaf extract, the mulberry leaf extract prepared by the preparation method or the mandarin fish feed in mandarin fish culture.
The invention provides application of the mulberry leaf extract or the mulberry leaf extract prepared by the preparation method in at least one of improving mandarin fish shape, improving antioxidant activity, protecting liver and intestinal health and improving mandarin fish intestinal nutrient absorption capacity, wherein the addition amount of the mulberry leaf extract is 1.0g/kg ration, 5.0g/kg ration or 10g/kg ration.
The mulberry leaf extract provided by the invention contains mulberry leaf polysaccharide and mulberry leaf flavone; the concentration of the mulberry leaf polysaccharide is 350 mg/g-550 mg/g, and the concentration of the mulberry leaf flavone is 30 mg/g-100 mg/g. In the invention, the mandarin fish is fed by adding the mulberry leaf extract into the daily ration, and the survival rate of the mandarin fish is continuously improved along with the increase of the concentration of the mulberry leaf extract. Meanwhile, the mulberry leaf extract is added in proper amount, so that the mandarin fish serum antioxidant capacity can be improved, the liver and intestinal health effects can be protected, and the mandarin fish intestinal nutrient absorption capacity can be improved. The mulberry leaf extract provided by the invention can be used for culturing mandarin fish, and has a relatively high culturing effect. Meanwhile, the mulberry leaf extract is extracted from plants, and has higher biological safety.
Drawings
FIG. 1 shows the effect of mulberry leaf extract on mandarin fish intestinal tissue morphology (HE staining);
FIG. 2 shows the effect of mulberry leaf extract on mandarin fish liver tissue morphology (HE staining).
Detailed Description
The invention provides a mulberry leaf extract, which contains mulberry leaf polysaccharide and mulberry leaf flavone; the concentration of the mulberry leaf polysaccharide is 350 mg/g-550 mg/g, and the concentration of the mulberry leaf flavone is 30 mg/g-100 mg/g.
The invention provides a preparation method of the mulberry leaf extract, which comprises the following steps:
treating folium Mori with petroleum ether to obtain pretreated folium Mori;
extracting the pretreated mulberry leaves with ethanol water solution, and separating liquid phase to obtain mulberry leaf extract.
In the invention, the feed liquid ratio of the mulberry leaf to the petroleum ether is preferably 1g:18 to 22mL, more preferably 1g:20mL. The petroleum ether treatment preferably includes petroleum ether extraction and reflux treatment. The petroleum ether extraction time is preferably 10 to 14 hours, more preferably 12 hours. The time of the reflux treatment is preferably 1.5 to 2.5 hours, more preferably 2 hours. The temperature of the reflow treatment is preferably 55 to 65 ℃, more preferably 60 ℃. The petroleum ether has the function of removing chlorophyll and lipid components in mulberry leaves.
In the present invention, the ratio of the feed liquid of the pretreated mulberry leaf to the aqueous ethanol solution is preferably 1g:28 to 32mL, more preferably 1g:30mL. The volume percentage of the ethanol aqueous solution is preferably 70-80%, more preferably 75%. The number of times of extraction of the aqueous ethanol solution is preferably 2 to 3 times. At each extraction with aqueous ethanol, it is preferable to accompany the ultrasonic extraction. The time for each ultrasonic extraction is preferably 25 to 35 minutes, more preferably 30 minutes. The temperature of each ultrasonic extraction is preferably 38 to 42 ℃, more preferably 40 ℃. The power of each ultrasonic extraction is preferably 30-50 KHz, more preferably 40Hz.
In the present invention, after extraction, the liquid phases are separated, and after combining the liquid phases, the water is removed. The method for removing water is preferably vacuum concentration and freeze drying. The condition of the reduced pressure concentration is preferably that the vacuum degree is 0.1MPa and the temperature is room temperature. The conditions for freeze-drying are preferably a vacuum degree of 0.1MPa and a temperature of-40 ℃ or lower.
The invention provides a mandarin fish feed, which comprises mulberry leaf extract or mulberry leaf extract and ration prepared by the preparation method; the addition amount of the mulberry leaf extract is 1.0g/kg ration, 5.0g/kg ration or 10g/kg ration.
The composition and the nutrition level of the daily ration are not particularly limited, and the mandarin fish feed well known in the art can be adopted. The preparation method of the mandarin fish feed is not particularly limited, and the preparation scheme of the feed well known in the art is adopted, for example, mulberry leaf extract and ration are uniformly mixed. The daily feeding rate of the mandarin fish feed is preferably 2% -3% of the mass of the mandarin fish, and more preferably 2.5%.
The invention provides an application of the mulberry leaf extract, the mulberry leaf extract prepared by the preparation method or the mandarin fish feed in mandarin fish culture, wherein the addition amount of the mulberry leaf extract is 1.0g/kg ration, 5.0g/kg ration or 10g/kg ration.
In the invention, the mandarin fish culturing condition is evaluated from the aspects of mandarin fish growth character, physical characteristics, liver health, antioxidant capacity and intestinal tissue structure. In the aspect of mandarin fish growth characteristics, compared with a control group, 1g/kg, 5g/kg and 10g/kg of mulberry leaf extract are added into daily ration, so that the average weight, specific growth rate, weight gain rate, feed coefficient and survival rate of the mandarin fish are not obviously influenced, but the survival rate of the mandarin fish is gradually increased along with the increase of the adding proportion of the mulberry leaf extract in the daily ration. In terms of physical characteristics, the mandarin fish is fed with the feed containing 5g/kg and 10g/kg of mulberry leaf extract, so that the body length of the mandarin fish can be effectively improved, but the liver body index, the intestinal body index and the fullness are not obviously influenced; the feed containing 10g/kg of mulberry leaf extract can significantly reduce the visceral body index, but has no significant effect on liver body index, intestinal body index and fullness.
In the invention, the influence of mulberry leaf extract on liver health is measured, and the influence on the enzyme activities of serum glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase (serum transaminase activity is a main index reflecting the damage degree of liver cells) and alkaline phosphatase (plays a role in the absorption and utilization processes of aquatic animals on nutrients) and the total protein, lysozyme and albumin contents and liver tissue morphology are measured respectively at different addition concentrations of the mulberry leaf extract. Compared with a control group, the feed containing 1g/kg of mulberry leaf extract can obviously reduce the activity of siniperca chuatsi serum glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT), obviously improve the content of total protein, but has no obvious influence on the activity of Lysozyme (LYZ); the feed containing 5g/kg mulberry leaf extract obviously reduces the activity of ALT and LYZ of mandarin fish serum and obviously reduces the TP content; a feed containing 10g/kg of mulberry leaf extract significantly reduces AST and LYZ activities and significantly reduces TP content. Meanwhile, liver tissue morphology is detected in the aspect of liver health, and the phenomena of cavitation of liver cells, normal cell morphology, clear structures of liver blood sinuses, cell nuclei and the like, and no phenomena of cavitation of the cell nuclei, offset aggregation of the cell nuclei and the like can be improved after the mulberry leaf extract is added. This shows that 1g/kg of mulberry leaf extract feed can improve liver health of mandarin fish and reduce production cost.
In terms of antioxidant capacity, the present invention evaluates the effect of mulberry leaf extract on total antioxidant capacity (T-AOC), catalase (CAT), reduced Glutathione (GSH), malondialdehyde (MDA) and total superoxide dismutase (T-SOD), respectively. Compared with a control group, the activity of T-SOD in mandarin fish serum can be obviously improved by adding 1g/kg of MLE in daily ration; the feed containing 5g/kg of mulberry leaf extract can remarkably improve GSH activity in mandarin fish serum and remarkably reduce MDA content; the feed containing 10g/kg of mulberry leaf extract can obviously improve GSH activity and MDA content in mandarin fish serum, and has no obvious influence on other indexes. This shows that the mulberry leaf extract can improve the oxidation resistance of mandarin fish to different degrees.
In the aspect of intestinal tissue structure, the invention evaluates the intestinal mucosa tissue morphology of mandarin fish. The feed with 10g/kg mulberry leaf extract increases the intestinal folds of mandarin fish, obviously improves the height and quantity of fluff, and reduces the intestinal cavity rate. This shows that the addition amount of 10g/kg of mulberry leaf extract can improve the intestinal health of mandarin fish and promote nutrient absorption.
Based on the above results, the invention provides the application of the mulberry leaf extract or the mulberry leaf extract prepared by the preparation method in improving the shape of mandarin fish, improving the antioxidant activity, keeping the liver health of mandarin fish and improving the intestinal nutrient absorption of mandarin fish.
The following examples are provided to illustrate a mulberry leaf extract, mandarin fish feed and its application in detail, but they should not be construed as limiting the scope of the invention.
Example 1
Preparation method of mulberry leaf extract
Firstly, pretreatment of mulberry leaf samples: cleaning and sun-drying picked fresh folium Mori, pulverizing, and sieving with 60 mesh sieve. Putting mulberry leaf sample powder into a flask, adding petroleum ether with the volume of 20 times, standing for 12 hours at room temperature, then adopting water bath reflux treatment at 60 ℃ for 2 hours, filtering, and air-drying filter residues for later use. The mulberry leaf extract is extracted by the following steps: weighing a certain amount of pretreated mulberry leaf powder, placing into a flask, adding 30 times of 75% ethanol water solution, performing ultrasonic-assisted extraction at 40 ℃ for 30min each time, and repeating for 3 times. Filtering, discarding residues, mixing filtrates, concentrating under reduced pressure, lyophilizing to obtain folium Mori extract powder, and storing at 4deg.C.
And detecting the polysaccharide content in the mulberry leaf extract powder by adopting an anthrone-sulfuric acid method, and detecting the flavone content in the mulberry leaf extract powder by adopting a rutin standard curve method.
The result shows that the mulberry leaf polysaccharide content is 513.44mg/g; the flavone content of the mulberry leaves is 88.23mg/g.
Example 2
Preparation method of mulberry leaf extract
Firstly, pretreatment of mulberry leaf samples: cleaning and sun-drying picked fresh folium Mori, pulverizing, and sieving with 60 mesh sieve. Putting mulberry leaf sample powder into a flask, adding petroleum ether with the volume of 18 times, standing for 10 hours at room temperature, then adopting water bath reflux treatment at 55 ℃ for 2 hours, filtering, and air-drying filter residues for later use. The mulberry leaf extract is extracted by the following steps: weighing a certain amount of pretreated mulberry leaf powder, placing into a flask, adding 28 times of 70% ethanol water solution, performing ultrasonic-assisted extraction at 38deg.C for 30min each time, and repeating for 3 times. Filtering, discarding residues, mixing filtrates, concentrating under reduced pressure, lyophilizing to obtain folium Mori extract powder, and storing at 4deg.C.
And detecting the polysaccharide content in the mulberry leaf extract powder by adopting an anthrone-sulfuric acid method, and detecting the flavone content in the mulberry leaf extract powder by adopting a rutin standard curve method.
The result shows that the mulberry leaf polysaccharide content is 428.32mg/g; the flavone content of the mulberry leaves is 56.75mg/g.
Example 3
Preparation method of mulberry leaf extract
Firstly, pretreatment of mulberry leaf samples: cleaning and sun-drying picked fresh folium Mori, pulverizing, and sieving with 60 mesh sieve. Putting mulberry leaf sample powder into a flask, adding petroleum ether with volume of 22 times, standing for 14 hours at room temperature, then adopting water bath reflux treatment at 65 ℃ for 2 hours, filtering, and air-drying filter residues for later use. The mulberry leaf extract is extracted by the following steps: weighing a certain amount of pretreated mulberry leaf powder, placing into a flask, adding 32 times of 80% ethanol water solution, performing ultrasonic-assisted extraction at 42 ℃ for 30min each time, and repeating for 3 times. Filtering, discarding residues, mixing filtrates, concentrating under reduced pressure, lyophilizing to obtain folium Mori extract powder, and storing at 4deg.C.
And detecting the polysaccharide content in the mulberry leaf extract powder by adopting an anthrone-sulfuric acid method, and detecting the flavone content in the mulberry leaf extract powder by adopting a rutin standard curve method.
The result shows that the mulberry leaf polysaccharide content is 464.8mg/g; the flavone content of the mulberry leaves is 71.5mg/g.
Example 4
Mandarin fish feed containing mulberry leaf extract and preparation method thereof
According to the nutrition requirement of mandarin fish, preparing basic puffed feed. Since the mulberry leaf extract prepared in example 1 has the highest content of active ingredients, the mulberry leaf extract prepared in example 1 is selected for feed preparation and cultivation experiments. A lipid feed such as 4 groups of nitrogen is prepared by adding 0 (MLE 0), 1g/kg (MLE 1), 5g/kg (MLE 5) and 10g/kg (MLE 10) of mulberry leaf extract to the basic puffed feed. The test feed formulations and nutrient levels are shown in table 1. The preparation method of the puffed feed comprises the following steps: firstly, weighing raw materials according to the proportion of the table 1, then uniformly mixing the weighed raw materials, and crushing the raw materials by a superfine crusher to ensure that all components can pass through a 250 mu m screen; transferring the uniformly mixed raw materials into a modem, introducing water vapor at 102 ℃ for modulation for 8 minutes, and then extruding, puffing and granulating at 95 ℃; and drying the feed particles at 80 ℃, cooling the particle feed, sub-packaging and storing in a refrigerator at-20 ℃ for later use (the feed is processed by the Dairy mountain by the Dairy feed limited company in south sea area of the Florida).
Table 1 test feed raw material composition and nutrient level (dry matter basis)
1) Vitamin premix composition (g/kg): VA20, VD 10,VE 1,VK2,VB15,VB25,VB65, nicotinic acid 20, pantothenic acid 20, biotin 1, folic acid 10, VB121 and defatted rice bran 900.
2) Mineral premix composition (g/kg): cuCO 3 4，FeC 6 H 5 O 7 15，MgO25，MnSO 4 6，KCl240，ZnSO 4 60, naCl 50, zeolite powder Zeolite powder600.
3) The nutrient level is a theoretical calculation.
Example 5
Method for culturing mandarin fish by using feed prepared in example 4
The mandarin fish used in the test is provided by the ocean aquatic products limited company in the three-water area of the bergamot, and the mandarin fish fries can normally eat artificial compound feed after artificial domestication.
The cultivation test was carried out in an indoor circulating water cultivation system of a cultivation base of Jie large feed Limited company in south sea area of Buddha, which consists of 12 cultivation barrels (with an effective volume of 400L) and 1 filtration system.
540 mandarin fish (42.35+ -0.07 g) with strong constitution and uniform size after current year cultivation and domestication is selected, and randomly divided into 4 groups, wherein 3 replicates are arranged in each group, and 45 replicates are arranged in each group. The basic compound feed is firstly used for trial culture for 14d before the formal test, the daily feeding rate is 2-3% of the mass of the mandarin fish for 72d, 1 time is respectively fed for 8:30-9:00 and 16:00-16:30 each day, the feed which is not eaten in half an hour after feeding is fished out, dried and weighed, and the weight of the mandarin fish fed after each feeding is accurately recorded. The water source for cultivation is tap water after filtration, disinfection and aeration treatment, and the water temperature for cultivation is 25-30 ℃ during the test. The dissolved oxygen in the culture water body is ensured to be 6.0-8.5 mg/L, the pH value is 7.2-8.0, the ammonia nitrogen concentration is less than or equal to 0.10mg/L, and the nitrite nitrogen concentration is less than or equal to 0.05mg/L.
Sample collection and preparation
The culture experiment lasts for 72d, and samples are collected after 72d of culture. Before collecting the sample, the test mandarin fish is fasted for 24 hours, then 15 fish (5 fish in each barrel) are randomly taken from each group, after anesthesia is carried out by MS-222, the body length and the body weight are measured, then the mandarin fish is dissected, and the weights of viscera, liver pancreas and intestinal tracts are respectively measured. Randomly taking 9 fish (3 fish per barrel) from each group, separating intestinal tracts, removing surface fat, respectively taking midgut with the length of about 1cm, fixing with 3% paraformaldehyde fixing solution, and using for paraffin tissue sections; separating liver, and cutting at the same location about 0.5cm 3 Is fixed with 3% paraformaldehyde fixing solution for paraffin tissue sections. Randomly taking 9 fish (3 fish in each barrel) from each group, taking blood from tail vein by using a syringe, standing the blood at 4 ℃ for 2 hours, centrifuging for 10 minutes at 3000r/min, and storing the upper serum in a refrigerator at-20 ℃ for serum immunity index, antioxidant index and enzyme activity measurement.
Index measurement
And (3) measuring growth and shape indexes:
measurement of growth index and physical index reference literature experiment method (Zhou Donglai, liao Sentai, huang Yong, etc. the effect of mulberry leaf powder on grass carp growth performance and meat quality flavor is added in feed [ J ]. Guangdong agricultural science 2021,48 (04): 119-130.), specific calculation formulas I to VIII are as follows:
weight gain rate (WGR,%) = (W t -W 0 )/W 0 X 100% formula I;
fullness (condition factor, CF, g/cm) 3 )＝W t /L 3 X 100 formula II;
specific growth rate (specific growthrate, SGR,%/d) = (lnW t -lnW 0 ) T×100% formula III;
visceral volume index (viscera somatic index, VSI,%) =w v /W t X 100% formula IV;
liver index (hepatopancreas somatic index, HSI,%) =w h /W t X 100% formula V;
intestinal body index (viserosomatic index, VI,%) =w i /W t X 100% formula VI;
feed coefficient (feed conversionratio, FCR) =f/(W) t -W 0 ) Formula VII;
survival rate (survivin rate, SR,%) =n t /N 0 X 100% equation VIII.
In which W is t And W is 0 The weight of final mandarin Fish (FBW) and the weight of initial mandarin fish (initial body weight, IBW) (g) of experimental fish, t is the number of days of culture experiment (d), F is the feed intake (g), and N 0 And N t The mantissa (tail) of the experimental fish at the beginning and the end of the experiment, L is the fish body length (cm), W v Visceral weight (g), W h Is the weight of liver and pancreas (g), W i The intestinal tract is heavy (g). MLE effect on mandarin fish growth performance.
Influence result of mulberry leaf extract on growth performance and physical index of mandarin fish
Compared with the control group, the addition of 1g/kg, 5g/kg and 10g/kg of MLE in the daily ration has no obvious influence on the average weight, specific growth rate, weight gain rate, feed coefficient and survival rate of the mandarin fish, but the survival rate of the mandarin fish is gradually increased along with the increase of the addition proportion of the MLE in the daily ration (table 2).
TABLE 2 Effect of mulberry leaf extract on mandarin fish growth performance
Note that: in the same row of data, different lowercase letters indicate that the difference is significant (p < 0.05), while the same lowercase letters indicate that the difference is not significant (p > 0.05). The table below is the same.
Influence of MLE on Mandarin fish
As shown in Table 3, compared with the control group, the addition of 1g/kg of MLE in daily ration has no significant effect on the body length, the dirty body index, the liver body index, the intestinal body index and the fullness of the mandarin fish (p > 0.05); the addition of 5g/kg MLE can obviously improve the body length of mandarin fish (p < 0.05), but has no obvious effect on the visceral body index, the hepatic body index, the intestinal body index and the fullness (p > 0.05); the addition of 10g/kg MLE can significantly improve the body length of mandarin fish, significantly reduce the dirty body index (p < 0.05), but has no significant effect on liver body index, intestinal body index and fullness (p > 0.05). As the MLE addition ratio in the diet increased, the dirty body index of mandarin fish gradually decreased, with the addition of 10g/kg, the dirty body index was lowest and significantly lower than the other groups (p < 0.05) (table 3).
TABLE 3 influence of Mulberry leaf extract on the shape index of Mandarin fish
And (3) measuring an oxidation resistance index:
the oxidation resistance indexes such as total oxidation resistance (T-AOC), catalase (CAT), total superoxide dismutase (T-SOD), malondialdehyde (MDA), and reduced Glutathione (GSH) of serum are all measured by using kits of Nanjing institute of biological engineering, and the specific measuring method is carried out by referring to the specification of each kit.
Influence of MLE on serum immune index of mandarin fish
Compared with the control, the addition of 1g/kg of MLE in daily ration can obviously reduce the activity of siniperca chuatsi serum glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) (p < 0.05), obviously improve the content of Total Protein (TP) (p < 0.05), but has no obvious influence on the activity of Lysozyme (LYZ) (p > 0.05); the addition of 5g/kg MLE significantly reduced the activity of siniperca chuatsi serum ALT and LYZ (p < 0.05), significantly reduced TP content (p < 0.05), but had no significant effect on AST activity (p > 0.05); the addition of 10g/kg of MLE significantly reduced AST and LYZ activity (p < 0.05), significantly reduced TP content (p < 0.05), but had no significant effect on ALT activity (p > 0.05); furthermore, the addition of 1g/kg, 5g/kg and 10g/kg of MLE had no significant effect on both AKP activity and ALB content (p > 0.05) (Table 4).
TABLE 4 Effect of mulberry leaf extract on Mandarin fish serum immune enzyme Activity
Immune index determination:
the immune indexes such as serum Albumin (ALB), total Protein (TP), alkaline phosphatase (AKP), glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and Lysozyme (LZM) are measured by using a kit (Nanjing institute of biological engineering, nanjing), and specific measuring methods refer to the kit specification.
Influence of MLE on the antioxidant capacity of mandarin fish serum
Compared with a control group, the addition of 1g/kg of MLE in daily ration can obviously improve the content of Malondialdehyde (MDA) and the activity of total superoxide dismutase (T-SOD) in mandarin fish serum (p < 0.05), and obviously reduce the activity of total antioxidant capacity (T-AOC) and reducing Glutathione (GSH) in mandarin fish serum (p < 0.05); the addition of 5g/kg MLE can significantly improve GSH activity in mandarin fish serum (p < 0.05), significantly reduce MDA content (p < 0.05), but has no significant effect on T-AOC and T-SOD activity (p > 0.05); the addition of 10g/kg MLE can significantly improve GSH activity and MDA content (p < 0.05) in mandarin fish serum, but has no significant effect on the activity of T-AOC and T-SOD (p > 0.05); whereas the addition of 1g/kg, 5g/kg and 10g/kg of MLE, respectively, to the ration did not significantly affect the activity of Catalase (CAT) (p > 0.05) (Table 5).
TABLE 5 influence of mulberry leaf extract on Mandarin fish serum antioxidant index
Preparation and observation of intestinal tract and liver HE stained tissue sections:
the intestinal and liver tissues fixed with paraformaldehyde solution were taken as described above, and conventional paraffin sections were prepared with a section thickness of 6 μm and stained with hematoxylin-eosin (HE). The slices were scanned using a panoramic slice digital scanner (PANNORAMIC-1000,3DHISTECH) and tissue photographs of the slices were taken using caseviewer2.2 (3 DHISTECH) software. After the screenshot was completed, the intestinal villus height, villus width and myolayer thickness were measured using Image-Pro Plus 6.0 (Media Cybemetics) analysis software, respectively.
Data processing and statistical analysis
The test results are all shown in mean ± standard deviation (mean ± SD), the difference significance analysis is performed using one-way anova and Duncan's multiple comparison test in SPSS 26.0 software, P <0.05 indicates significant difference.
Influence of MLE on liver and intestinal tissue structure of mandarin fish
As shown in fig. 1, the intestinal mucosa tissue of the mandarin fish of the control group is clear, the structure is complete, the arrangement is ordered, and the phenomena of confusion and falling off are avoided. With the increase of the MLE concentration, the intestinal folds of the mandarin fish are increased, the height of the fluff is increased, the quantity of the fluff is increased, and the intestinal cavity rate is reduced. Further statistical analysis found that when the MLE addition concentration was 10g/kg, the fluff height was significantly higher than in the control group (p < 0.05); while there was no significant difference in villus width and myolayer thickness between groups (p > 0.05) (table 6).
TABLE 6 influence of mulberry leaf extract on siniperca chuatsi intestinal villus height, villus width and myometrium thickness
As shown in figure 2, the liver tissue sections of the mandarin fish in each group have regular arrangement of liver cells in the control group and clear cell nucleus and cell membrane limit, but the liver cells have cavitation phenomenon, and a small amount of fat is accumulated in the cells. The method can improve the cavitation phenomenon of liver cells after adding MLE with different concentrations, has normal cell morphology, clear structures of liver blood sinuses, cell nuclei and the like, has no cavitation phenomenon of the cell nuclei, offset aggregation of the cell nuclei and the like, and particularly adds 1g/kg of MLE.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (3)
1. The application of the mulberry leaf extract in preparing the feed for improving the body length of mandarin fish is that the addition amount of the mulberry leaf extract is 5g/kg of daily ration or 10g/kg of daily ration;
the mulberry leaf extract contains mulberry leaf polysaccharide and mulberry leaf flavone; the concentration of the mulberry leaf polysaccharide is 350 mg/g-550 mg/g, and the concentration of the mulberry leaf flavone is 30 mg/g-100 mg/g;
the preparation method of the mulberry leaf extract comprises the following steps:
adding petroleum ether into mulberry leaves, standing for 10-14 h, and then carrying out reflux treatment at 55-65 ℃ for 1.5-2.5 h to obtain pretreated mulberry leaves;
extracting the pretreated mulberry leaves with an ethanol aqueous solution, and separating a liquid phase to obtain a mulberry leaf extract;
the feed liquid ratio of the mulberry leaves to the petroleum ether is 1g: 18-22 mL;
the feed liquid ratio of the pretreated mulberry leaves to the ethanol water solution is 1g: 28-32 mL;
the volume percentage of the ethanol water solution is 70-80%;
the times of the ethanol aqueous solution extraction are 2 to 3 times;
each time ethanol aqueous solution is extracted, ultrasonic treatment is carried out;
the time of each extraction is 25-35 min; the temperature of each extraction is 38-42 ℃; the power of the ultrasonic wave extracted each time is 30-50 KHz.
2. The application of the mulberry leaf extract in preparing the feed for improving the liver health of mandarin fish is that the addition amount of the mulberry leaf extract is 1g/kg ration;
the mulberry leaf extract is the mulberry leaf extract in the application of claim 1;
the improvement of mandarin fish liver health includes improving hepatocyte vacuolation.
3. The application of the mulberry leaf extract in preparing the feed for improving the intestinal health of mandarin fish is that the addition amount of the mulberry leaf extract is 10g/kg ration;
the mulberry leaf extract is the mulberry leaf extract in the application of claim 1;
the improvement of the intestinal health of the mandarin fish comprises increasing the intestinal folds of the mandarin fish, increasing the height of fluff and the quantity of fluff, and reducing the intestinal cavity rate.
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