CN115836709B - Composite seaweed zymolyte and application thereof in large yellow croaker feed additive - Google Patents
Composite seaweed zymolyte and application thereof in large yellow croaker feed additive Download PDFInfo
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Edible Seaweed (AREA)
Abstract
The invention discloses a composite seaweed zymolyte and application thereof in large yellow croaker feed additive, the preparation method comprises the steps of preprocessing laver, ulva and kelp, mixing according to the preset mass portion ratio, adding water to prepare seaweed suspension, adding citric acid and hydrochloric acid solution with preset final concentration, stirring and mixing, adjusting pH to a preset value, adding cellulase and pectase, enabling the mass ratio E/S of the enzyme and the substrate to reach a preset range value, and then adding NaOH and Na 2 HPO 4 ·12H 2 Stirring O to make the final concentration and pH of the mixed solution reach preset values, then adding neutral protease, aminopeptidase and algin lyase to make the mass ratio E/S of enzyme and substrate reach preset range values, finally inactivating enzyme treatment and spray drying to obtain composite seaweed zymolyte; the method has simple process, and the prepared product can increase the diversity of intestinal microflora of the large yellow croaker and the synthesis of butyric acid when being used as a feed additive for feeding the large yellow croaker.
Description
Technical Field
The invention relates to the technical fields of agricultural technology and feed additives, in particular to a composite seaweed zymolyte and application thereof in a large yellow croaker feed additive.
Background
The large yellow croaker (Larimichthys crocea) is a marine fish of the large yellow croaker family native to the North Pacific ocean, generally living in the isothermal zone water area of Taiwan strait, is one of important economic fish species in China, and has annual yield of more than 20 ten thousand tons. At present, large yellow croaker is mainly cultured in a net cage, and is mainly concentrated in eastern coastal areas such as Fujian, guangdong, zhejiang, shandong and the like, wherein the Fujian is a core culture area, and the fish yield is more than 85%. With the continuous growth of the cultivation scale, various problems such as cultivation density, cultivation water and feed quality are continuously developed, so that diseases such as vibriosis, bacterial enteritis, cryptocaryon irritans and the like and diseases such as parasitic diseases and the like are frequently caused in the cultivation of large yellow croakers, wherein the bacterial diseases are main diseases in the cultivation process. The intestinal tract is considered to be the main pathway of bacterial invasion, and intestinal health is beneficial to the overall health of the host organism. Intestinal flora is the basis of intestinal health, and has a close relationship with host health, and complex interactions with hosts exist.
Short Chain Fatty Acids (SCFAs) are saturated fatty acids with 6 or less carbon atoms, mainly including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, caproic acid and isocaproic acid, wherein the content of acetic acid, propionic acid and butyric acid is the highest, and the content of acetic acid, propionic acid and butyric acid is more than 80% of all SCFAs. Short chain fatty acids are metabolites produced by anaerobic fermentation of dietary fibers and proteins by the microflora in the large intestine, are important bands for the intestinal flora to regulate the health of the intestines and hosts, and especially butyric acid, can induce CD4+ T cells and congenital lymphocytes (ILCs) to produce IL-22 in mammals to maintain intestinal homeostasis, inhibit central regulators of inflammatory NF- κB signaling pathways, reduce oxidative stress, and thereby prevent colon damage associated with pathological and inflammatory diseases of the intestines.
The current research proves that butyric acid produced by zebra fish intestinal microflora has anti-inflammatory effects such as reduction of recruitment of neutrophils and M1 type pro-inflammatory macrophages to wounds, and that butyric acid anti-inflammatory effects are conserved in vertebrates and conserved molecular receptors exist in fish. In recent years, researchers in China have conducted careful researches on the aspects of the relationship between the flora and health of the gastrointestinal digestive tract of large yellow croakers, the difference of the flora of the digestive tract in different culture modes and the like, and have researched that the alpha diversity of the intestinal flora is improved by adding different feed raw materials, feeding modes and the like so as to promote the health of the intestinal tract and improve the economic benefit.
The seaweed mainly comprises brown algae (kelp), green algae, red algae (laver) and the like, and the main components which have been determined to have biological significance at present comprise sulfated polysaccharides such as alginic acid (gelatin), laminarin (Fucoidan), fucoidan (Fucoidan), porphyran (Porphyran), green algae polysaccharide (Ulvans), carrageenan and the like, polyphenols (Polyphenols), oligopeptides generated by degradation of seaweed proteins and the like.
Therefore, how to extract effective and beneficial substances in seaweed, and to improve the intestinal tracts of large yellow croakers in feed, so as to promote healthy growth of large yellow croakers, has positive and realistic significance.
Disclosure of Invention
In view of the above, the invention aims to provide a composite seaweed zymolyte which has simple process and can increase intestinal microflora diversity and butyric acid synthesis of large yellow croaker when the prepared product is used as a feed additive for large yellow croaker feeding, and application of the composite seaweed zymolyte in large yellow croaker feed additive.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a process for preparing the enzymolysis product of composite seaweed includes such steps as pretreating thallus Porphyrae, ulva and kelp, mixing, adding water, stirring, adding citric acid and hydrochloric acid, adding cellulase and pectase, stirring, adding NaOH and Na, and stirring 2 HPO 4 ·12H 2 And (3) stirring the O to ensure that the final concentration and the pH value of the mixed solution reach preset values, then adding neutral protease, aminopeptidase and algin lyase to ensure that the mass ratio E/S of the enzyme to the substrate reaches a preset range value, continuously stirring for a preset period of time under a preset condition, and finally carrying out enzyme inactivation treatment and spray drying on the mixed system to obtain the composite seaweed zymolyte.
As a possible implementation mode, the method for pretreating the laver, the ulva and the kelp in the scheme comprises the steps of drying the laver, the ulva and the kelp to ensure that the moisture content is less than or equal to 11%, and smashing the laver, the ulva and the kelp to ensure that the granularity is more than or equal to 80 meshes.
As a preferred implementation choice, preferably, the seaweed suspension comprises 10-30 parts of pretreated laver, 20-40 parts of ulva and 30-70 parts of kelp, after the laver, the ulva and the kelp are uniformly mixed, seaweed powder is prepared, the seaweed powder is added into drinking water according to the proportion of 40-120g/L, and stirring is carried out at the speed of 30-1000rpm for more than or equal to 30min, so that the seaweed suspension is prepared.
As a preferred implementation choice, preferably, citric acid and hydrochloric acid solutions with final concentrations of 0.01M and 0.10M are added into the seaweed suspension to prepare a mixed seaweed suspension, the mixed seaweed suspension is heated to 40-50 ℃, stirring is carried out at a speed of more than or equal to 150rpm for more than or equal to 30min, and hydrochloric acid is also used for adjusting the acidity of the mixed seaweed suspension during stirring, so that the pH value is 4.5-5.0.
As a preferred implementation choice, preferably, the proposal ensures that the mass ratio E/S of enzyme to substrate is more than or equal to 0.50X10 when cellulase and pectase are added into the mixed seaweed suspension 2 U/g, pH is 4.5-5.0, the temperature of the mixed seaweed suspension is 40-50 ℃, then stirring treatment is carried out at a speed of more than or equal to 150rpm for more than or equal to 2 hours, and the mixed seaweed zymolyte is prepared; adding NaOH and Na into the mixed seaweed zymolyte 2 HPO 4 ·12H 2 And (3) when O is added, the final concentration is 0.05M, then stirring is carried out at a speed of more than or equal to 30rpm for more than or equal to 30min, and HCl and/or NaOH are used for adjusting acidity to enable the pH value to be 7.0-8.0.
As a preferred implementation choice, preferably, when neutral protease, aminopeptidase and algin lyase are added in the scheme, the mass ratio E/S of enzyme to substrate is respectively equal to or more than 3000U/g, equal to or more than 300U/g and equal to or more than 1000U/g, the pH is 7.0-8.0, the temperature of the mixed seaweed zymolyte is 40-50 ℃, and then stirring treatment is carried out at the speed of equal to or more than 150rpm, wherein the stirring time period is equal to or more than 2 hours.
As a better implementation choice, the scheme is preferable, the temperature of the mixed system is raised to 85-90 ℃ within 5-30min, then the mixed system is subjected to enzyme inactivation treatment for 15-30min, and then the mixed system is subjected to spray drying treatment and crushing treatment, so that the water content of the product is less than or equal to 10%, and the granularity is more than or equal to 80 meshes, so as to prepare the composite seaweed zymolyte.
As a preferred implementation choice, the scheme comprises the following steps:
(1) Drying thallus Porphyrae, ulva and herba Zosterae Marinae to water content of 11% or less, and pulverizing to obtain granule of 80 mesh or more;
(2) Uniformly mixing 10-30 parts of laver, 20-40 parts of ulva and 30-70 parts of kelp according to a preset mass part ratio to prepare seaweed powder;
(3) Adding the mixed seaweed powder into drinking water according to the proportion of 40-120g/L, and stirring at the speed of 30-1000rpm for more than or equal to 30min to obtain seaweed suspension;
(4) Adding citric acid and hydrochloric acid into the mixed seaweed suspension to make the final concentration of the mixed seaweed suspension be 0.01M and 0.10M respectively, heating the mixed seaweed suspension to 40-50 ℃, and stirring at a speed of more than or equal to 150rpm for more than or equal to 30min;
(5) Adjusting acidity to pH 4.5-5.0 during stirring in the step (4) by using hydrochloric acid;
(6) Adding cellulase and pectase into the mixed seaweed suspension to make the mass ratio E/S of enzyme to substrate not less than 0.50X10 2 U/g, pH 4.5-5.0, mixing seaweed suspension at 40-50deg.C, stirring at speed not less than 150rpm for not less than 2 hr to obtain mixed seaweed enzymolysis product;
(7) Adding NaOH and Na into the mixed seaweed zymolyte 2 HPO 4 ·12H 2 O, wherein the final concentration of O is 0.05M, stirring at a speed of more than or equal to 30rpm for more than or equal to 30min, and adjusting acidity by using HCl and/or NaOH to enable pH to be 7.0-8.0;
(8) Adding neutral protease, aminopeptidase and algin lyase to make the mass ratio E/S of enzyme and substrate be greater than or equal to 3000U/g, greater than or equal to 300U/g and greater than or equal to 1000U/g respectively, pH value is 7.0-8.0, mixing seaweed zymolyte at 40-50deg.C, stirring at speed greater than or equal to 150rpm for longer than or equal to 2h;
(9) Heating the mixed system to 85-90 ℃ within 5-30min, and maintaining for 15-30min for inactivating enzyme;
(10) The composite seaweed zymolyte is prepared by spray drying treatment and crushing treatment, so that the water content of the product is less than or equal to 10 percent and the granularity is more than or equal to 80 meshes.
As a preferred implementation parameter choice, the scheme preferably comprises the following steps:
(1) Drying thallus Porphyrae, ulva and herba Zosterae Marinae to water content of 11% or less, and pulverizing to obtain granule of 80 mesh or more;
(2) Uniformly mixing 29 parts of laver, 24 parts of ulva and 47 parts of kelp according to a preset mass part ratio to prepare seaweed powder;
(3) Adding the mixed seaweed powder into drinking water according to the proportion of 100g/L, and stirring at the speed of 400rpm for 3 hours to prepare seaweed suspension;
(4) Adding citric acid and hydrochloric acid into the mixed seaweed suspension to make the final concentration of the mixed seaweed suspension be 0.01M and 0.10M respectively, heating the mixed seaweed suspension to 48+/-0.5 ℃, and stirring at 300rpm for 30min;
(5) Adjusting acidity to pH 4.8+ -0.2 with hydrochloric acid during stirring in step (4);
(6) Adding cellulase and pectase into the mixed seaweed suspension to make the mass ratio E/S of enzyme to substrate be 0.30X10 respectively 3 U/g、0.50×10 3 U/g, pH 4.8+ -0.2, mixing seaweed suspension at 48+ -0.5deg.C, stirring at 300rpm for 3 hr to obtain mixed seaweed zymolyte;
(7) Adding NaOH and Na into the mixed seaweed zymolyte 2 HPO 4 ·12H 2 O, wherein the final concentration of O is 0.05M, stirring at 300rpm for 30min, regulating acidity with HCl and/or NaOH to pH 7.8+ -0.2, and cooling mixed seaweed zymolyte to 43+ -0.5deg.C;
(8) Adding neutral protease, aminopeptidase and algin lyase to make the mass ratio E/S of enzyme to substrate be 6000U/g, 600U/g and 3000U/g respectively, pH 7.8+ -0.2, mixing seaweed zymolyte at 43+ -0.5deg.C, stirring at 300rpm for 4h;
(9) Heating the mixed system to 85+/-0.1 ℃ within 20min, and maintaining for 15+/-2 min for inactivating enzyme treatment;
(10) The composite seaweed zymolyte is prepared by spray drying treatment and crushing treatment, so that the water content of the product is less than or equal to 10 percent and the granularity is more than or equal to 80 meshes.
Based on the above, the soluble solids of the composite seaweed zymolyte prepared by the scheme is more than or equal to 400mg/g, the total polysaccharide is more than or equal to 30mg/g, the reducing sugar is more than or equal to 20mg/g, the protein content is more than or equal to 1000ug/g, the polypeptide content is more than or equal to 50ug/g, the alginic acid oligosaccharide is more than or equal to 30mg/g, and the total antioxidation is more than or equal to 30mM FeSO 4 And/g, wherein sulfate radical combined in the zymolyte is more than or equal to 3mg/g.
Based on the above, the invention also provides a large yellow croaker feed additive for increasing the diversity of intestinal microflora of large yellow croaker and the synthesis of butyric acid, which comprises the composite seaweed zymolyte prepared by the preparation method.
By adopting the technical scheme, compared with the prior art, the invention has the beneficial effects that: the scheme is ingenious in that the kelp, the ulva and the Porphyra yezoensis which are easy to obtain are mixed and balanced according to a certain proportion, the cell wall is fully destroyed by cellulase and pectase to release alginic acid, sulfated polysaccharide, protein and the like in the cell periplasm space and the cell, then the algin lyase is used for enzymolysis of long-chain sugar which is difficult to be absorbed and utilized by the organism into reducing sugar and short-chain sugar, the protease is used for degrading seaweed protein to generate oligopeptide with biological activity, and a composite seaweed zymolyte is prepared, and is added into an anaerobic culture medium in an amount of 0.5%, so that alpha diversity and butyric acid synthesis of a large yellow croaker intestinal microorganism community after in vitro culture can be remarkably improved, and the composite seaweed zymolyte can be used as a feed additive for feeding large yellow croaker, and the large yellow croaker intestinal microorganism community diversity and butyric acid synthesis can be improved; in addition, the scheme has the following advantages:
(1) The seaweed has multiple varieties and larger content difference of functional components in different seaweed, the kelp, the ulva and the laver which are easy to obtain are selected and mixed according to a certain proportion, so that the functional components such as alginic acid, sulfated polysaccharide, seaweed protein and the like are balanced, and experiments prove that the effect is superior to that of single component;
(2) Cellulases and pectinases destroy cell walls sufficiently to release alginic acid, sulfated polysaccharides and proteins in the periplasmic space and cells; the algin lyase is used for hydrolyzing long-chain sugar which is difficult to be absorbed and utilized by an organism into reducing sugar and short-chain sugar, and the protease is used for degrading seaweed protein to generate oligopeptide with biological activity;
(3) The composite seaweed zymolyte is added to obviously increase the intestinal microflora diversity and the butyric acid synthesis of the large yellow croaker.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the comparison of differences in the synthesis of short chain fatty acids under different in vitro culture conditions of intestinal microflora of large yellow croakers;
FIG. 2 is a comparative chart of PCoA analysis after cultivation of intestinal microflora of large yellow croaker under different in vitro cultivation conditions;
FIG. 3 is a comparison chart of the significance test of the differences of the group colony structures after the large yellow croaker intestinal microbial colony is cultured under different in vitro culture conditions;
FIGS. 4a to 4f are graphs showing comparison of Alpha diversity after cultivation of a large yellow croaker intestinal microflora under different in vitro cultivation conditions, wherein FIG. 4a is a graph showing differences between the set of observed_patterns, FIG. 4b is a graph showing differences between the set of shannon-fingers, FIG. 4c is a graph showing differences between the set of simpson-fingers, FIG. 4d is a graph showing differences between the set of chao 1-fingers, FIG. 4e is a graph showing differences between the set of ace-fingers, and FIG. 4f is a graph showing differences between the set of PD whole tree-fingers;
FIG. 5 is a Venn diagram of groups based on OTUs after in vitro culture of a large yellow croaker intestinal microbiota;
FIG. 6 is a graph showing the comparison of Alpha diversity changes of the same intestinal flora of large yellow croaker after culture under different conditions in vitro;
FIG. 7 is a graph showing comparison of structural features at portal level after in vitro culture of intestinal microflora of large yellow croakers;
FIG. 8 is a graph showing the transformation comparison of the main dominant phylum after in vitro culture of the intestinal microflora of large yellow croakers;
FIG. 9 is a heat map comparison of abundance of species at the genus level after in vitro culture of intestinal microflora of large yellow croaker;
FIG. 10 is a bar graph showing LDA distribution of differential flora between groups after in vitro culture of intestinal microflora of large yellow croaker.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples. It is specifically noted that the following examples are only for illustrating the present invention, but do not limit the scope of the present invention. Likewise, the following examples are only some, but not all, of the examples of the present invention, and all other examples, which a person of ordinary skill in the art would obtain without making any inventive effort, are within the scope of the present invention.
The preparation method of the composite seaweed zymolyte comprises the following steps:
(1) Drying thallus Porphyrae, ulva and herba Zosterae Marinae to water content of 11% or less, and pulverizing to obtain granule of 80 mesh or more;
(2) Uniformly mixing 10-30 parts of laver, 20-40 parts of ulva and 30-70 parts of kelp according to a preset mass part ratio to prepare seaweed powder;
(3) Adding the mixed seaweed powder into drinking water according to the proportion of 40-120g/L, and stirring at the speed of 30-1000rpm for more than or equal to 30min to obtain seaweed suspension;
(4) Adding citric acid and hydrochloric acid into the mixed seaweed suspension to make the final concentration of the mixed seaweed suspension be 0.01M and 0.10M respectively, heating the mixed seaweed suspension to 40-50 ℃, and stirring at a speed of more than or equal to 150rpm for more than or equal to 30min;
(5) Adjusting acidity to pH 4.5-5.0 during stirring in the step (4) by using hydrochloric acid;
(6) Adding cellulase and pectase into the mixed seaweed suspension to make the mass ratio E/S of enzyme to substrate not less than 0.50X10 2 U/g, pH 4.5-5.0, mixing seaweed suspension at 40-50deg.C, stirring at speed not less than 150rpm for not less than 2 hr to obtain mixed seaweed enzymolysis product;
(7) Adding NaOH and Na into the mixed seaweed zymolyte 2 HPO 4 ·12H 2 O, wherein the final concentration of O is 0.05M, stirring at a speed of more than or equal to 30rpm for more than or equal to 30min, and adjusting acidity by using HCl and/or NaOH to enable pH to be 7.0-8.0;
(8) Adding neutral protease, aminopeptidase and algin lyase to make the mass ratio E/S of enzyme and substrate be greater than or equal to 3000U/g, greater than or equal to 300U/g and greater than or equal to 1000U/g respectively, pH value is 7.0-8.0, mixing seaweed zymolyte at 40-50deg.C, stirring at speed greater than or equal to 150rpm for longer than or equal to 2h;
(9) Heating the mixed system to 85-90 ℃ within 5-30min, and maintaining for 15-30min for inactivating enzyme;
(10) The composite seaweed zymolyte is prepared by spray drying treatment and crushing treatment, so that the water content of the product is less than or equal to 10 percent and the granularity is more than or equal to 80 meshes.
Quality index
The indexes of the composite seaweed zymolyte prepared in the above example follow the indexes shown in table 1,
TABLE 1 composite seaweed enzymolysis quality Standard index
The detection method corresponding to the quality standard index comprises the following steps:
1) Determination of the content of soluble solids
Measured according to GBT 12729.11-2008 determination of cold water soluble extracts of spices and condiments.
2) Soluble solids protein content
The soluble solid protein content was determined according to the determination of protein in national food Standard for food safety, GB 5009.5-2016.
3) Determination of the Total polysaccharide content in solubles
Measured according to appendix A of SNT4260-2015 crude polysaccharide-phenol sulfate method
4) Determination of the content of reducing sugars in solubles
Measured according to GB5009.7-2016 determination of reducing sugar in food.
5) Vulcanized polysaccharide assay (sulfate radical assay)
Total sulfate and free sulfate were determined according to SCT3404-2012 fucoidan (total sugar, sulfate).
6) Content of alginate enzymolysis oligosaccharide
Measured according to the detection of the oligosaccharide content of DB21T 2598-2016 alginic acid.
7) Polypeptide content determination
Pretreatment:
1. accurately weighing 1.000g of composite seaweed enzymolysis product, adding water to a constant volume to 50mL of water, stirring for 4-6h, refrigerating for 18h, recovering to room temperature, filtering with medium-speed filter paper, collecting filtrate, and centrifuging at room temperature for 5min at 8,000Xg;
2. taking 10mL of supernatant, performing alcohol precipitation (4 ℃ C., 12 h) with the final volume of ethanol concentration of 80%, centrifuging for 15min at 4,000Xg, and removing polysaccharide and impurities;
3. taking supernatant, drying, and redissolving by using 5mL PBS;
4. the solution was concentrated by ultrafiltration (membrane 3 kDa), and the filtrate with a molecular weight of <3kDa was collected.
The polypeptide content was determined according to the determination of protein in national food safety Standard of food GB 5009.5-2016.
8) Total antioxidant Activity
The antioxidant property of the GBT39100-2020 polypeptide is measured according to the specification of GBT39100-2020 polypeptide antioxidant property.
Examples of the embodiments
Based on the foregoing, this embodiment illustrates the present scheme by way of the following examples and comparative examples:
a preparation method of a composite seaweed zymolyte comprises the following steps:
(1) Drying thallus Porphyrae, ulva and herba Zosterae Marinae to water content of 11% or less, and pulverizing to obtain granule of 80 mesh or more;
(2) Uniformly mixing 29 parts of laver, 24 parts of ulva and 47 parts of kelp according to a preset mass part ratio to prepare seaweed powder;
(3) Adding the mixed seaweed powder into drinking water according to the proportion of 100g/L, and stirring at the speed of 400rpm for 3 hours to prepare seaweed suspension;
(4) Adding citric acid and hydrochloric acid into the mixed seaweed suspension to make the final concentration of the mixed seaweed suspension be 0.01M and 0.10M respectively, heating the mixed seaweed suspension to 48+/-0.5 ℃, and stirring at 300rpm for 30min;
(5) Adjusting acidity to pH 4.8+ -0.2 with hydrochloric acid during stirring in step (4);
(6) Adding cellulase and pectase into the mixed seaweed suspension to make the mass ratio E/S of enzyme to substrate be 0.30X10 respectively 3 U/g、0.50×10 3 U/g, pH 4.8+ -0.2, mixing seaweed suspension at 48+ -0.5deg.C, stirring at 300rpm for 3 hr to obtain mixed seaweed zymolyte;
(7) Adding NaOH and Na into the mixed seaweed zymolyte 2 HPO 4 ·12H 2 O, wherein the final concentration of O is 0.05M, stirring at 300rpm for 30min, regulating acidity with HCl and/or NaOH to pH 7.8+ -0.2, and cooling mixed seaweed zymolyte to 43+ -0.5deg.C;
(8) Adding neutral protease, aminopeptidase and algin lyase to make the mass ratio E/S of enzyme to substrate be 6000U/g, 600U/g and 3000U/g respectively, pH 7.8+ -0.2, mixing seaweed zymolyte at 43+ -0.5deg.C, stirring at 300rpm for 4h;
(9) Heating the mixed system to 85+/-0.1 ℃ within 20min, and maintaining for 15+/-2 min for inactivating enzyme treatment;
(10) The composite seaweed zymolyte is prepared by spray drying treatment and crushing treatment, so that the water content of the product is less than or equal to 10 percent and the granularity is more than or equal to 80 meshes.
The composite seaweed zymolyte prepared in this example was tested by the method mentioned above, the test results are shown in table 2,
table 2 quality and composition detection of composite seaweed zymolyte prepared in example
Comparative example
The comparative example provides a preparation method of kelp zymolyte, which comprises the following steps:
(1) Drying kelp to make the moisture content less than or equal to 11%, and pulverizing to 80 mesh or more;
(2) Preparing kelp zymolyte by using kelp powder as a raw material;
(3) Adding single kelp powder into drinking water according to the proportion of 100g/L, and stirring for 3 hours at the speed of 400 rpm;
(4) Adding citric acid and hydrochloric acid into the mixed seaweed suspension to obtain final concentrations of 0.01M and 0.10M respectively, heating the mixed seaweed suspension to 48+ -0.5deg.C, stirring at 300rpm for 30min, and adjusting pH to 4.8+ -0.2 with hydrochloric acid;
(5) Adding cellulase and pectase to make the enzyme/substrate mass (E/S) 0.30X10 respectively 3 U/g and 0.50X10 3 U/g, stirring and hydrolyzing at the pH of 4.8+/-0.2, 48+/-0.5 ℃ and the speed of 300rpm for 3 hours;
(6) Adding NaOH and Na into the mixed seaweed zymolyte 2 HPO 4 ·12H 2 O, the final concentration is 0.05M, stirring is carried out for 30min at the speed of 300rpm, hydrochloric acid is used for regulating the pH value to be 7.8+/-0.2 during the period, and meanwhile, the temperature is reduced to 43+/-0.5 ℃;
(5) Adding neutral protease, aminopeptidase and algin lyase to make the enzyme/substrate mass (E/S) be 6000U/g, 600U/g and 3000U/g respectively, stirring at pH 7.8+ -0.2, 43+ -0.5 deg.C and 300rpm for 4 hr;
(6) Heating to 85+ -1.0deg.C within 20min, maintaining for 15+ -2.0 min, and inactivating;
(7) Spraying powder and drying to make the water content less than or equal to 10%, and further pulverizing to 80 mesh or more to obtain kelp zymolyte.
The kelp zymolyte prepared in this comparative example was examined by the method mentioned above, and the results obtained are shown in table 3,
quality and composition detection for composite seaweed zymolyte prepared in Table 3
Application contrast
Collecting and processing intestinal flora of large yellow croaker
Collecting 4 tails of 1 year old healthy large yellow croaker (with weight of 150-200g and body length of 20-24 cm) cultured in a net cage without antibiotics within 3 months. Under a sterile environment, wiping the body surface of a living large yellow croaker with 75% alcohol, dissecting, extracting digestive tracts, collecting the small intestine and rectum contents of each fish, uniformly mixing, preparing intestinal bacterial solutions YC1, YC2, YC3 and YC4 by using a sterilized GAM culture solution according to the ratio of 1g to 9m L, and refrigerating for later use.
In vitro culture of intestinal flora
The specific culture method using GAM anaerobic culture medium is as follows:
(1) First the tests were divided into three groups, which are:
BC: GAM culture;
TA: adding 0.5% (W/V) mixed seaweed zymolyte into GAM;
TD: adding 0.5% (W/V) kelp zymolyte into GAM;
wherein, 4 large glass test tubes are prepared in each group, 9 mL/branch of culture solution is split, 3-5 g/branch of hard wax is put in each group, 3 branches (GAM culture medium) of pollution control group are simultaneously prepared, and the sterilization is carried out at 121 ℃ for 15min under high pressure.
(2) After cooling to room temperature, wax was sealed off with an alcohol lamp and added into 1mL (10% added) of prepared intestinal bacteria liquid YC1, YC2, YC3, YC4 to test tubes of each group, after heat-sealing and cooling the hard wax, the mixture was thoroughly mixed upside down, and the pollution control group was operated in parallel with sterilized GAM culture liquid.
(3) Culturing at 25deg.C and 100rpm for 24 hr, sampling test group under the precondition that the pollution control group is clear and free of turbidity, mixing each test tube upside down, taking 3mL therefrom, and preserving at-80deg.C.
Short chain fatty acid and 16s sequencing
Qualitative and quantitative analysis of Short Chain Fatty Acids (SCFAs), sequencing of 16S rDNA amplicon of intestinal flora cultures, analysis, and association of SCFAs with intestinal flora were all carried out by the company wunmei metabolic limited.
The 16S rDNA amplicon is sequenced by using NovaSeq PE250 scheme, through Reads splicing and filtering, OTUs (Operational Taxonomic Units) clustering, species annotation and abundance analysis, alpha Diversity analysis (Alpha Diversity) and Beta Diversity analysis (Beta Diversity) and other analysis.
The SCFAs sample preparation process is as follows:
(1) After the sample is thawed, vortex for 1min, mix well, centrifuge for 10min at 12000r/min and 4 ℃;
(2) Taking 50 mu L of a sample supernatant, adding the sample supernatant into a corresponding 1.5mL centrifuge tube, adding 100 mu L of 0.5% phosphoric acid solution, and swirling for 3min;
(3) 150. Mu.L of MTBE solvent containing an internal standard is added, vortex is carried out for 3min, ultrasonic treatment is carried out for 5min under ice bath, and then centrifugation is carried out for 10min under 12000r/min and 4 ℃;
(4) After centrifugation, 90. Mu.L of supernatant was aspirated into a glass liner tube-containing sample bottle and stored in a refrigerator at-20℃until GC-MS/MS analysis.
The chromatographic conditions were as follows: the chromatographic column was Agilent DB-FFAP (30 m. Times.0.25 mm,0.25 μm); setting the temperature of a sample inlet at 200 ℃, the temperature of a detector at 230 ℃ and the sample injection amount at 2 mu L; heating to 90deg.C within 1min, then 25 deg.C/min (90-100deg.C), 20 deg.C/min (100-150deg.C) 0.6min,25 deg.C/min (150-200deg.C), 200deg.C 0.5min, and operating for 3min; setting the split ratio to be 1:1, a step of; the fluence was 1.2.
Short chain fatty acid detection
SCFAs mainly containing acetic acid can be produced under the in-vitro anaerobic culture of the intestinal microflora of the large yellow croaker, the ratio of the acetic acid to the propionic acid to the butyric acid is about 94:5:1, the ratio of the butyric acid to the butyric acid can be improved by adding 0.5% of mixed seaweed zymolyte to 92:4:4, the synthesis amount of the butyric acid is increased from 0.04mM to 0.27mM, the production of the valeric acid is increased from 0.0001mM to 0.0004mM, and the synthesis amount of the propionic acid is reduced to 0.23mM; the addition of 0.5% kelp zymolyte (TD group) only significantly increased the yield of valeric acid, corresponding comparison is shown in figure 1.
The results shown in FIG. 1 are obtained by collecting the microbial flora of small intestine and rectum of large yellow croaker under aseptic condition, diluting 10 times, inoculating to GAM culture medium containing different seaweed zymolytes at 10% ratio, anaerobic culturing at 25deg.C and 100rpm for 24 hr, and detecting.
The numbers in fig. 1 correspond to: AA: acetic acid; BA: butyric acid; CA: caproic acid; IBA: isobutyric acid; IVA: isovaleric acid; PA: propionic acid; VA: valeric acid;
error bars are shown as mean ± SD; * P <0.01;
BC, blank: anaerobic culture medium (GAM);
TD, kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte;
TA, mixed seaweed enzymolysis product addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate.
Packet diversity analysis
The butyric acid and the valeric acid produced by the intestinal microflora of the large yellow croaker under different in vitro culture conditions have extremely remarkable differences, and the change of the falling body of the intestinal microflora is the reason for the differences, so that the 16S rDNA amplicon sequencing technology is used for further research. After sequencing the in vitro culture of intestinal microbial community by 16S rDNA amplicon, PCoA (principle Co-ordinates Analysis) analysis is firstly carried out based on Unweighted Unifrac distance, and the closer the sample distance is, the more similar the species composition structure is, so that samples with high similarity of community structure tend to be gathered together, and samples with large community differences are far apart. PCoA analysis shows that the samples of the TA group are basically gathered in relatively independent communities, and have obvious differences from the samples of the TD group and the BC group; the sample communities of the TD group and the BC group are separated, but the distance is not obvious, and the comparison is shown in figure 2.
As can be seen from fig. 2, based on Unweighted Unifrac distance PCoA analysis, ANOVA: p=0.004. Wherein the abscissa represents one principal component, the ordinate represents the other principal component, and the percentage represents the contribution value of the principal component to the sample difference; each point in the graph represents a sample, the samples of the same group are represented by the same color, and the color region represents the confidence interval. In addition, the present test 3 group corresponds to the following:
BC (BC.1, BC.2, BC.3, BC.4), blank group: anaerobic culture medium (GAM);
TD (TD.1, TD.2, TD.3, TD.4), kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte;
TA (TA.1, TA.2, TA.3, TA.4), mixed seaweed hydrolysate addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate.
An Anosim analysis was used to verify if the difference between the groups was significantly greater than the difference in the groups and to perform a group difference significance test based on the rank order of the Bray-Curtis distance values. The results showed that the differences between BC and TD groups were not significant (r= -0.146), the differences between TA and BC groups (r=0.240) and TD groups were significant (r=0.208), but showed that the confidence of the statistical analysis was not significant, as compared to fig. 3.
As can be seen from fig. 3, the Anosim analysis was used to examine whether the difference between groups was significantly greater than the difference within the group. R is between (-1, 1), R > 0, which indicates that the difference between groups is significant; r <0, indicating that the intra-group variance is greater than the inter-group variance; p represents the confidence level of the statistical analysis, and p <0.05 represents that the statistics are significant.
BC_vs_TD，R＝-0.146，p＝0.741；BC_vs_TA，R＝0.240，p＝0.242；TD_vs_TA，R＝0.208，p＝0.128。
Wherein, BC, blank group: anaerobic culture medium (GAM);
TD, kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte;
TA, mixed seaweed enzymolysis product addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate.
Differential analysis of samples between groups
The Alpha diversity index is used for carrying out the differential analysis of samples among groups, and box-shaped graphs capable of intuitively reflecting the median, the discrete degree, the maximum value, the minimum value and the abnormal value of the diversity of the species in the groups are used for displaying. The observed_features index, shannon index, simpson index, chao1 index, ace index and PD white tree index all show that there are differences in the three groupings, except for the BC group index > TD group index in the median value of simpson index, the median value of each of the other indexes all represents TA group > TD group > BC group, as compared to fig. 4.
The subgraphs of fig. 4 are expressed as: a: box plot of differences between the set of observed_patterns, b: box plot of differences between shannon sets of indices, c: box plot of differences between simpson index sets, d: difference box plot between chao1 index sets, e: difference box plot between ace index sets, f: the box diagram of the differences between the PD white tree index sets.
Wherein, BC, blank group: anaerobic culture medium (GAM);
TD, kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte;
TA, mixed seaweed enzymolysis product addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate.
Characterization and variation of microbial communities under different culture conditions
The intestinal microflora of the same large yellow croaker synchronously cultures the BC group, the TD group and the TA group, and statistics and comparison of classification operation units (OTUs, operational Taxonomic Units) show that 653 BC groups, 626 TD groups and 945 TA groups are counted; the TD group and TA group are respectively unique to 192 and 371 as compared to BC group; the TA group was unique to 347 as compared to the TD group, see figure 5 for comparison.
In fig. 5, BC, blank: anaerobic culture medium (GAM);
TD, kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte;
TA, mixed seaweed enzymolysis product addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate.
Comparing the Alpha diversity of the intestinal microbiota of the same large yellow croaker under each culture condition, the observed_features index, simpson index, shannon index, chao1 index, ace index and PD white tree index show that each index of Fish 1 is obviously different from the other three fishes, and each index of other fishes shows that the TA group is larger than the BC group and the TD group, and the comparison is shown in fig. 6.
The subgraphs of fig. 6 are shown as: a: the observed_patterns index, b: simpson index, c: shannon index, d: char 1 index, e: ace index, f: PD white tree index.
Wherein, BC, blank group: anaerobic culture medium (GAM);
TD, kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte;
TA, mixed seaweed enzymolysis product addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate.
After in vitro culture of intestinal microflora of large yellow croaker, the dominant mycota of BC group is Thick wall mycota (Firmic), proteus (Proteus) and Fusobacterium (Fusobacteria) as main, accounting for more than 98% of all sequences; after kelp zymolyte is added (TD group), dominant mycota is converted into Firmicutes and Proteobacteria (Proteus) accounting for more than 90% of all sequences; the dominant mycota after adding the mixed seaweed zymolyte (TA group) is Thick-walled mycota (Firmic), proteus (Proteus) and Bacteroides (Bacteroideta), and accounts for more than 95% of all sequences, and the comparison is shown in figure 7. In addition, there are individual differences in intestinal microbiota dominant phylum conversion among different additives, and the comparison is shown in fig. 8.
In fig. 7, BC, blank: anaerobic culture medium (GAM); TD, kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte; TA, mixed seaweed enzymolysis product addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate. Other represents the sum of the relative abundances of all but the 10 gates in the graph.
In fig. 8, BC (BC.1, BC.2, BC.3, BC.4), blank group: anaerobic culture medium (GAM); TD (TD.1, TD.2, TD.3, TD.4), kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte; TA (TA.1, TA.2, TA.3, TA.4), mixed seaweed hydrolysate addition group: anaerobic culture medium (GAM) +0.5% mixed seaweed zymolyte; the same numerical numbers indicate the intestinal microbiota of the same fish. Other represents the sum of the relative abundances of all but the 10 gates in the graph.
In addition, according to quantitative information of microbial communities of all samples, the microorganisms with the top 35 rank are clustered from the species and sample two layers, a heat map is drawn, and the microorganisms in the TA group are obviously different from the microorganisms in the BC and TD groups on the genus level, meanwhile, the microorganisms in the same group have larger differences, and the comparison is shown in fig. 9.
In fig. 9, the top 35 microorganism classification is selected at the genus level based on its quantitative information in each sample, clustered from both species and sample levels, and mapped to a heat map.
Wherein BC (BC.1, BC.2, BC.3, BC.4), blank group: anaerobic culture medium (GAM);
TD (TD.1, TD.2, TD.3, TD.4), kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte;
TA (TA.1, TA.2, TA.3, TA.4), mixed seaweed hydrolysate addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate.
Biomarker analysis
To determine species with significant differences between groups, species with significant differences between BC and TA groups were analyzed and screened using the Metastats method from species abundance tables of different levels, and microorganisms with significant differences between BC and TA groups were bacteroides (bacterioidota), categorised underdetermined bacteria (Unidentified Bacteria), deironickened bacilli (Deferrobacteria), wart micro-bacilli (Verrucomicrobiota) and Cyanobacteria (Cyanobacteria), and microorganisms with significant differences between TD and TA groups were bacteroides (bacterioidota), categorised underdetermined bacteria (Unidentified Bacteria) and other bacteria (other).
The intestinal microflora cultures of the large yellow croakers in different groups are subjected to intensive research through colony structure difference statistical analysis. Species with obvious differences in abundance change among groups are to be found out in a targeted manner, and the enrichment condition of different species among different groups is clarified. Biomarkers (biomarkers) with statistical differences were found from group to group using LEfSe (LDA Effect Size) tool, which showed no significant microbial communities between TD and BC groups, but more microbial communities between TA and TD and BC groups, see fig. 10.
In fig. 10, the species with LDA Score greater than the set point (default set 4), i.e. Biomarker with statistical differences between groups, are shown in the LDA value distribution bar graph. Species with significant differences in abundance in different groups are shown, and the length of the histogram represents the magnitude of the effect of the different species (i.e., LDA Score).
Wherein, TD group is deleted: there were no species of significant difference in the TD group, so this group was absent.
BC, blank: anaerobic culture medium (GAM);
TD, kelp zymolyte addition group: anaerobic culture medium (GAM) +0.5% kelp zymolyte;
TA, mixed seaweed enzymolysis product addition group: anaerobic Medium (GAM) +0.5% of mixed seaweed substrate. Phylum (P_), class (C_), order (O_), family (F_), genus (G_), species (S_).
The foregoing description is only a partial embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent devices or equivalent processes using the descriptions and the drawings of the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (3)
1. A large yellow croaker feed additive for increasing intestinal microflora diversity and butyric acid synthesis of large yellow croaker, comprising a complex seaweed zymolyte, characterized in that the preparation method of the complex seaweed zymolyte comprises the following steps:
(1) Drying thallus Porphyrae, ulva and herba Zosterae Marinae to water content of 11% or less, and pulverizing to obtain granule of 80 mesh or more;
(2) Uniformly mixing 10-30 parts of laver, 20-40 parts of ulva and 30-70 parts of kelp according to a preset mass part ratio to prepare seaweed powder;
(3) Adding seaweed powder into drinking water according to the proportion of 40-120g/L, and stirring at the speed of 30-1000rpm for more than or equal to 30min to obtain seaweed suspension;
(4) Adding citric acid and hydrochloric acid into the seaweed suspension to obtain final concentrations of 0.01M and 0.10M respectively, heating to 40-50deg.C, and stirring at speed of at least 150rpm for at least 30min;
(5) Adjusting acidity to pH 4.5-5.0 during stirring in the step (4) by using hydrochloric acid;
(6) Adding cellulase and pectase to make the mass ratio E/S of enzyme to substrate not less than 0.50X10 2 U/g, pH 4.5-5.0, temperature 40-50deg.C, stirring at speed not less than 150rpm for not less than 2h to obtain mixed seaweed zymolyte;
(7) Adding NaOH and Na into the mixed seaweed zymolyte 2 HPO 4 ·12H 2 O, the final concentration of which is 0.05M, and then stirring at a speed of more than or equal to 30rpm for more than or equal to 30min, wherein HCl and/or NaOH are used for adjustingAcidity, pH 7.0-8.0;
(8) Adding neutral protease, aminopeptidase and algin lyase to make the mass ratio E/S of enzyme and substrate be greater than or equal to 3000U/g, greater than or equal to 300U/g and greater than or equal to 1000U/g respectively, pH being 7.0-8.0, temperature being 40-50 ℃, stirring at speed being greater than or equal to 150rpm for longer than or equal to 2h;
(9) Heating the mixed system to 85-90 ℃ within 5-30min, and maintaining for 15-30min for inactivating enzyme;
(10) The composite seaweed zymolyte is prepared by spray drying treatment and crushing treatment, so that the water content of the product is less than or equal to 10 percent and the granularity is more than or equal to 80 meshes.
2. A feed additive for large yellow croaker for increasing the diversity of intestinal microflora and the synthesis of butyric acid according to claim 1, wherein: the method comprises the following steps:
(1) Drying thallus Porphyrae, ulva and herba Zosterae Marinae to water content of 11% or less, and pulverizing to obtain granule of 80 mesh or more;
(2) Uniformly mixing 29 parts of laver, 24 parts of ulva and 47 parts of kelp according to a preset mass part ratio to prepare seaweed powder;
(3) Adding the mixed seaweed powder into drinking water according to the proportion of 100g/L, and stirring at the speed of 400rpm for 3h to obtain a seaweed suspension;
(4) Adding citric acid and hydrochloric acid into the seaweed suspension to obtain final concentrations of 0.01M and 0.10M respectively, heating to 48+ -0.5deg.C, and stirring at 300rpm for 30min;
(5) Adjusting acidity to pH 4.8+ -0.2 with hydrochloric acid during stirring in step (4);
(6) Adding cellulase and pectase to make the mass ratio E/S of enzyme to substrate 0.30X10 respectively 3 U/g、0.50×10 3 U/g, pH 4.8+ -0.2, temperature 48+ -0.5deg.C, stirring at 300rpm for 3h to obtain mixed seaweed zymolyte;
(7) Adding NaOH and Na into the mixed seaweed zymolyte 2 HPO 4 ·12H 2 O, the final concentration of which is 0.05M, stirring at 300rpm for 30min, adjusting acidity by HCl and/or NaOH to pH 7.8+ -0.2, and cooling to 43+ -0.5 ℃;
(8) Adding neutral protease, aminopeptidase and algin lyase to make the mass ratio E/S of enzyme to substrate be 6000U/g, 600U/g and 3000U/g respectively, pH 7.8+ -0.2, temperature 43+ -0.5 deg.C, stirring at 300rpm for 4h;
(9) Heating the mixed system to 85 ℃ within 20min, and maintaining for 15min for inactivating enzyme treatment;
(10) The composite seaweed zymolyte is prepared by spray drying treatment and crushing treatment, so that the water content of the product is less than or equal to 10 percent and the granularity is more than or equal to 80 meshes.
3. A feed additive for large yellow croaker for increasing the diversity of intestinal microflora and the synthesis of butyric acid according to claim 1 or 2, wherein: the soluble solids of the composite seaweed zymolyte are more than or equal to 400mg/g, the total polysaccharide is more than or equal to 30mg/g, the reducing sugar is more than or equal to 20mg/g, the protein content is more than or equal to 1000ug/g, the polypeptide content is more than or equal to 50ug/g, the alginic acid oligosaccharide is more than or equal to 30mg/g, and the total antioxidation is more than or equal to 30mM FeSO 4 Per gram, the sulfate radical combined in the zymolyte is more than or equal to 3mg/g.
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