CN112250739A

CN112250739A - Myceliophthora additive and application thereof

Info

Publication number: CN112250739A
Application number: CN202011019873.9A
Authority: CN
Inventors: 张志清; 黎卓莹; 邓远钊; 代珍青; 曾文年
Original assignee: Guangdong Hinabiotech Co ltd
Current assignee: Guangdong Hinabiotech Co ltd
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2021-01-22
Anticipated expiration: 2040-09-25
Also published as: CN112250739B

Abstract

The invention provides a mycelial mycin additive and application thereof, wherein the additive is prepared into embedded particles, the embedded particles comprise contents and semipermeable membranes wrapping the contents, and the content of the mycelial mycin is 1% -30% by the total weight of the embedded particles. 1% -30% of plectasin is used as the embedded content, and a semi-permeable membrane is formed on the surface of the content, so that the storage and use stability of the plectasin can be effectively improved. In addition, after the plectasin is prepared into the embedded particles, the plectasin has a slow-release effect in the feeding process, so that the continuous and stable drug effect in the animal body is ensured, pathogenic bacteria are effectively inhibited, the morbidity of the animal body is reduced, and the cure rate of inflammatory infection is further improved.

Description

Myceliophthora additive and application thereof

Technical Field

The invention relates to feed preparation, and particularly relates to a myceliophthora additive and application thereof.

Background

Plectasin belongs to defensive antibacterial peptide, has extremely low expression level or no expression under natural conditions, can be expressed only by being induced by microorganisms and proinflammatory cytokines, contains 3-4 disulfide bonds in a molecule, and is difficult to ensure correct pairing of polypeptides so as to exert the antimicrobial activity of the plectasin. The biological activity of the compound mainly shows that the compound has stronger bactericidal action on gram-positive bacteria, and especially has the antibacterial action on streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus and the like which are equivalent to penicillin and vancomycin. In addition, plectasin has the advantages of no cytotoxicity, no hemolysis, good cerebrospinal fluid permeability and the like in vitro antibacterial tests, animal tests and some clinical tests, and is not easy to cause drug resistance of pathogenic bacteria (including resistance to traditional antibiotics), so that plectasin has great treatment potential as a new generation antibiotic substitute. However, the natural plectasin has high separation and purification cost and low yield, is difficult to obtain in large quantities by direct separation and purification, and the purified plectasin has poor stability on the whole, so that the plectasin has large loss and low utilization rate when applied to the preparation of the feed, and the application effect of the plectasin is greatly reduced.

In conclusion, the above problems still remain to be solved in the field of mycelial mycin preparation and application.

Disclosure of Invention

Based on the above, in order to solve the problems that mycelial mycin obtained by purification has high separation and purification cost and low yield, and the purified mycelial mycin has poor stability, so that the mycelial mycin is consumed greatly in the process of preparing the feed, the utilization rate is low, and the application effect is greatly reduced, the invention provides a mycelial mycin additive, and the specific technical scheme is as follows:

the myceliophthora additive is characterized in that myceliophthora is prepared into embedded particles, the embedded particles comprise contents and semipermeable membranes wrapping the contents, and the content of the mycelial is 1% -30% of the total weight of the embedded particles.

Further, the preparation method of the plectasin additive comprises the following steps:

purifying plectasin;

preparing contents;

and spraying a semipermeable membrane coating on the surface of the content to form the embedded granular plectasin additive with a semipermeable membrane on the surface.

Further, the purification treatment comprises the following steps:

placing myceliomycin obtained by induction culture of recombinant pichia pastoris in a centrifugal tube at the temperature of 4-8 ℃ and the rotating speed of 3500-;

putting the supernatant into a rotary evaporator at 45-65 ℃ for rotary evaporation, and concentrating through an ultrafiltration tube to obtain a concentrated solution;

and purifying the concentrated solution by His affinity chromatography to obtain purified plectasin.

Further, the semipermeable membrane coating material is obtained by mixing cellulose acetate, sodium chloride, hydroxypropyl cellulose and triacetin.

Further, the method for preparing the content comprises the following steps: adding sodium alginate and shell powder into a proper amount of water, heating to 75-95 ℃ under the condition of 500-1000r/min, stirring until the sodium alginate and the shell powder are completely dissolved, cooling to 35-50 ℃, adding purified plectasin, stirring uniformly, adding calcium chloride, and continuously stirring for 20-40 min to form a gelatinous content.

Further, the spraying method comprises the following steps: heating the semipermeable membrane coating to 30-65 ℃, and spraying the content for 15-30 min by adopting an intermittent atomization spraying mode.

Further, according to the mass percentage, the addition amount of the cellulose acetate is 45-75%; the addition amount of the sodium chloride is 8% -12%; the addition amount of the hydroxypropyl cellulose is 4-18%; the addition amount of the triacetin is 1% -8%.

Furthermore, the mesh number of the embedded particles is 80-250 meshes.

In addition, the application of the mycelial additive is provided, and the embedded mycelial has better stability and can be applied to products for inhibiting the growth of streptococcus agalactiae.

Further, the plectasin is applied to preparation of feeds.

In the scheme, 1% -30% of mycelial mycin is used as the embedded content, and a semi-permeable membrane is formed on the surface of the content, so that the storage stability and the use stability of the mycelial mycin can be effectively improved. In addition, after the plectasin is prepared into embedded particles and is applied to antibacterial products as an additive, the plectasin has a slow-release effect in the feeding process, so that the continuous and stable drug effect in the animal body is ensured, pathogenic bacteria are effectively inhibited, the morbidity of the animal body is reduced, and the cure rate of inflammatory infection is further improved.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to embodiments thereof. It should be understood that the detailed description and specific examples, while indicating the scope of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

1% -30% of plectasin is used as the embedded content, and a semi-permeable membrane is formed on the surface of the content, so that the storage and use stability of the plectasin can be effectively improved. In addition, after the plectasin is prepared into the embedded particles, the plectasin is applied to antibacterial products, plays a role in slow-release in the feeding process, ensures the continuous and stable drug effect in the animal body, effectively inhibits pathogenic bacteria, reduces the morbidity of the animal body, and further improves the cure rate of inflammatory infection.

In one embodiment, the used producing strain for producing plectasin is a pichia pastoris recombinant strain GS115/pPicZaMF57-PLE118, and the construction process of the pichia pastoris recombinant strain is as follows: constructing, screening and transforming recombinant plasmids: and (3) optimizing and artificially synthesizing the plectasin nucleotide sequence according to the bias of the yeast exogenous protein expression codon. The artificially synthesized plectasin nucleotide sequence is connected between Aval and BamH I enzyme cutting sites of pUC57 to construct a recombinant plasmid pUC57-plectasin containing plectasin target gene, wherein the plectasin nucleotide sequence is GGTTTTGGTTGTAACGGTCCATGGGATGAAGATGATATGCAATGTCATAACCATTGTAAGTCTATTAAGGGTTACAAGGGTGGTTACTGTGCTAAGGGTGGTTTTGTTTGTAAGTGTTAC (SEQ ID NO. 1).

The recombinant plasmid pUC57-Plectasin is digested with Aval and BamH I to obtain Plectasin gene fragment, which is connected to the multiple cloning sites (Aval and BamH I) of Pichia expression vector pPicZaMF57 (an alpha-factor signal peptide mutant) and located at the downstream of alpha-factor signal peptide sequence to construct the recombinant expression vector pPicZaMF57-Plectasin containing Plectasin target gene.

Constructing, screening and identifying recombinant pichia pastoris: after the recombinant expression vector pPicZaMF57-Plectasin is subjected to enzyme digestion linearity by Pme1 enzyme, the recombinant expression vector pPicZaMF57-Plectasin is converted into Pichia pastoris GS115 by electric shock, screened by YPD culture medium containing 0.5-1mg/ml Zeocin (bleomycin), cultured at 30 ℃ for 2-3 days, screened out positive multicopy transformants, and preliminarily determined that the gene sequence of the bleomycin is recombined on a Pichia pastoris chromosome group.

The positive transformants obtained by the above screening were inoculated into 20ml centrifuge tubes containing 2ml of BMGY medium, and shake-cultured at 30 ℃ for 24 hours at 220 r/mim. Transferring 1ml of thallus to 10ml of BMMY induction culture medium, continuously culturing at 30 ℃ by a 200r/mim shaking table, and supplementing 100u l 00% methanol every 24 h. Sampling every 24h, centrifuging the culture medium for 5min at a speed of 5000r/mim, collecting supernatant, and performing antibacterial activity detection on the supernatant; and simultaneously, performing PCR amplification molecular identification on the myceliophthora target gene of the positive transformant obtained by primary screening. According to the antibacterial activity detection and PCR amplification molecule identification results, the plectasin recombinant pichia pastoris strain with optimal antibacterial activity and containing plectasin target genes is named as GS115/pPicZaMF57-PLE 118. And the amino acid sequence of the plectasin produced by the strain is GGCNGWDDDMCHNHCKSKGYKGGYCAKGGVCKCY (SEQ ID NO. 2).

In one embodiment, the mycelial mycin additive is prepared by the following steps:

purifying plectasin;

preparing contents;

The method for preparing the plectasin additive is simple and low in cost, a semi-permeable membrane is formed on the surface of the content in a spraying mode, and the stability of the plectasin additive is improved while the bleeding-out delaying effect is achieved under the condition that no additional expanding agent is added.

In one embodiment, the purification treatment is:

In one embodiment, the method of preparing the contents is: adding sodium alginate and shell powder into a proper amount of water, heating to 75-95 ℃ under the condition of 500-1000r/min, stirring until the sodium alginate and the shell powder are completely dissolved, cooling to 35-50 ℃, adding purified plectasin, stirring uniformly, adding calcium chloride, and continuously stirring for 20-40 min to form a gelatinous content. By mixing with sodium alginate, shell powder, calcium chloride and water, gel-like content is formed, variation probability of myceliophthorin during storage period is reduced, and gel-like content is easier to transport and store than liquid-like content, and has high stability. In the application process, the gel-like content is easily absorbed by organism, and has high safety.

In one embodiment, the addition amount of the sodium alginate is 15-30 parts by weight; the addition amount of the shell powder is 8-16 parts; the addition amount of the calcium chloride is 2-7 parts.

In one embodiment, the semipermeable membrane coating is formed by mixing cellulose acetate, sodium chloride, hydroxypropyl cellulose, and triacetin. The semipermeable membrane coating prepared by mixing a plurality of components has good semipermeable effect and good embedding effect.

In one embodiment, the cellulose acetate is added in an amount of 45-75% by mass; the addition amount of the sodium chloride is 8% -12%; the addition amount of the hydroxypropyl cellulose is 4-18%; the addition amount of the triacetin is 1% -8%.

In one embodiment, the spraying method comprises the following steps: heating the semipermeable membrane coating to 30-65 ℃, and spraying the content for 15-30 min by adopting an intermittent atomization spraying mode.

In one embodiment, the intermittent atomization spraying refers to spraying once every 25-45 min, and 35-60 s every time.

In one embodiment, the spraying pressure of the atomized spraying is 0.25-0.36Mp, and the spraying flow rate is 8-12L/h.

In one embodiment, the embedded particles have a mesh size of 80-250 mesh.

In one embodiment, the mycelial mycin additive, which has improved stability after encapsulation, can be used in products that inhibit the growth of Streptococcus agalactiae.

In one embodiment, the mycelial supplement is used in feed preparation.

In one embodiment, the plectasin additive is added in 1-5 parts by weight in the feed preparation.

In one embodiment, the feed comprises the following components in parts by weight: 20-40 parts of corn flour, 12-30 parts of soybean meal, 3-8 parts of fermented soybean meal, 1-2 parts of glucose, 5-16 parts of bran, 0.3-0.8 part of calcium hydrophosphate, 0.11-0.3 part of lysine, 0.1-0.5 part of sodium chloride, 1.2-1.5 parts of stone powder, 0.15-0.2 part of composite trace element premix, 0.01-0.05 part of methionine, 0.01-0.05 part of threonine, 0.03-0.10 part of choline and 1-5 parts of plectasin additive.

In one embodiment, the feed is prepared as follows:

adding corn flour, fermented soybean meal and bran into a stirring kettle according to the weight parts of the feed, and heating and curing; adding calcium hydrogen phosphate, glucose, lysine, sodium chloride, stone powder and choline under the condition of stirring; cooling to 45-65 ℃, adding the composite trace element premix, methionine, threonine and choline, and uniformly stirring; cooling to below 45 deg.C, adding plectasin additive, stirring, and freeze drying until water content is less than 18% to obtain compound feed.

Embodiments of the present invention will be described in detail below with reference to specific examples.

Example 1:

a preparation method of a myceliophthora additive comprises the following steps:

placing myceliophthorns obtained by induction culture of recombinant pichia pastoris in a centrifugal tube at 4 ℃ and the rotating speed of 3500r/min for centrifugation for 20min, and collecting supernatant; putting the supernatant into a rotary evaporator at 65 ℃ for rotary evaporation, and concentrating through an ultrafiltration tube to obtain a concentrated solution; purifying the concentrated solution by His affinity chromatography to obtain purified plectasin;

adding 30 parts by weight of sodium alginate and 8 parts by weight of shell powder into a proper amount of water, heating to 75 ℃ under the condition of 500r/min, stirring until the sodium alginate and the shell powder are completely dissolved, cooling to 35 ℃, adding 1% of purified plectasin, stirring uniformly, adding 2 parts by weight of calcium chloride, and continuously stirring for 20min to form a gelatinous content;

according to the mass percentage, the adding amount of the cellulose acetate is 72 percent; the addition amount of the sodium chloride is 8 percent; the addition amount of the hydroxypropyl cellulose is 12 percent; the additive amount of the triacetin is 8 percent, and the triacetin is mixed to obtain a semipermeable membrane coating;

and heating the semipermeable membrane coating to 30 ℃, spraying the content for 15min in a mode of spraying once every 25min and 35 min every time, and forming the plectasin additive with the semipermeable membrane on the surface.

Example 2:

placing myceliophthorns obtained by induction culture of recombinant pichia pastoris in a centrifugal tube at 4 ℃ and the rotating speed of 3500r/min for centrifugation for 30min, and collecting supernatant; putting the supernatant into a rotary evaporator at 65 ℃ for rotary evaporation, and concentrating through an ultrafiltration tube to obtain a concentrated solution; purifying the concentrated solution by His affinity chromatography to obtain purified plectasin;

adding 30 parts by weight of sodium alginate and 16 parts by weight of shell powder into a proper amount of water, heating to 95 ℃ under the condition of 1000r/min, stirring until the sodium alginate and the shell powder are completely dissolved, cooling to 50 ℃, adding 15% of purified plectasin, stirring uniformly, adding 7 parts by weight of calcium chloride, and continuously stirring for 40min to form a gelatinous content;

according to the mass percent, the adding amount of the cellulose acetate is 69 percent; the addition amount of the sodium chloride is 12 percent; the addition amount of the hydroxypropyl cellulose is 18 percent; the additive amount of the triacetin is 1 percent, and the triacetin is mixed to obtain a semipermeable membrane coating;

and heating the semipermeable membrane coating to 65 ℃, spraying the content once every 45min for 30min in a mode of 60s every time, and forming the plectasin additive with the semipermeable membrane on the surface.

Example 3:

placing myceliomycin obtained by induction culture of recombinant pichia pastoris in a centrifugal tube at the temperature of 5 ℃ and the rotating speed of 4500r/min for centrifugation for 25min, and collecting supernatant; putting the supernatant into a rotary evaporator at 55 ℃ for rotary evaporation, and concentrating through an ultrafiltration tube to obtain a concentrated solution; purifying the concentrated solution by His affinity chromatography to obtain purified plectasin;

adding 20 parts by weight of sodium alginate and 12 parts by weight of shell powder into a proper amount of water, heating to 80 ℃ under the condition of 800r/min, stirring until the sodium alginate and the shell powder are completely dissolved, cooling to 45 ℃, adding 30% of purified plectasin, stirring uniformly, adding 6 parts by weight of calcium chloride, and continuously stirring for 30min to form a gelatinous content;

according to the mass percentage, the adding amount of the cellulose acetate is 66 percent; the addition amount of the sodium chloride is 8 percent; the addition amount of the hydroxypropyl cellulose is 18 percent; the additive amount of the triacetin is 8 percent, and the triacetin is mixed to obtain a semipermeable membrane coating;

and heating the semipermeable membrane coating to 45 ℃, spraying the content once every 40min for 40s every time for 20min to form the plectasin additive with the semipermeable membrane on the surface.

Application test examples 1 to 3:

the mycelial mycin additive prepared in the application test examples 1-3 is applied to the preparation of feed, the raw materials and the weight parts of the raw materials in the feed are shown in table 1, and the mycelial mycin additive added in the application test example 1 in the table 1 is the mycelial mycin additive prepared in the example 1; the mycelial mycin additive added in application test example 2 was the mycelial mycin additive prepared in example 2; the mycelial mycin additive added in application test example 3 was the mycelial mycin additive prepared in example 3.

Table 1:

the preparation method of the feed described in application test examples 1 to 3 was as follows:

adding corn flour, fermented soybean meal and bran into a stirring kettle according to the weight parts of the components of the compound feed, and heating and curing; adding calcium hydrogen phosphate, glucose, lysine, sodium chloride, stone powder and choline under the condition of stirring; cooling to 45-65 ℃, adding the composite trace element premix, methionine, threonine and choline, and uniformly stirring; cooling to below 45 ℃, adding the embedded particles, uniformly stirring, and freeze-drying until the water content is lower than 18% to obtain the compound feed.

Comparative example 1:

and heating the cellulose acetate to 65 ℃, spraying the content once every 45min for 30min in a mode of 60s every time, and forming the mycelial mycin additive with a semi-permeable membrane on the surface.

The mycelial mycin additive prepared in comparative example 1 was applied in the preparation of feed:

adding 30 parts of corn flour, 5.1 parts of fermented soybean meal and 12 parts of bran into a stirring kettle according to the weight parts of the feed, and heating and curing; under the condition of stirring, adding 0.5 part of calcium hydrophosphate, 1.2 parts of glucose, 0.10 part of lysine, 0.3 part of sodium chloride, and 0.3 part of stone powder and choline; cooling to 65 ℃, adding 0.16 part of composite trace element premix, 0.03 part of methionine, 0.02 part of threonine and 0.07 part of choline, and uniformly stirring; and cooling to below 45 ℃, adding 3.2 parts of plectasin additive, uniformly stirring, and freeze-drying until the water content is lower than 18% to obtain the feed containing the plectasin additive.

Comparative example 2:

a preparation method of the feed comprises the following steps:

adding 30 parts of corn flour, 5.1 parts of fermented soybean meal and 12 parts of bran into a stirring kettle according to the weight parts of the components of the compound feed, and heating and curing; under the condition of stirring, adding 0.5 part of calcium hydrophosphate, 1.2 parts of glucose, 0.10 part of lysine, 0.3 part of sodium chloride, and 0.3 part of stone powder and choline; cooling to 65 ℃, adding 0.16 part of composite trace element premix, 0.03 part of methionine, 0.02 part of threonine and 0.07 part of choline, and uniformly stirring; cooling to below 45 deg.C, and freeze drying until the water content is less than 18% to obtain compound feed.

Comparative example 3:

a preparation method of the feed comprises the following steps:

adding 30 parts of corn flour, 5.1 parts of fermented soybean meal and 12 parts of bran into a stirring kettle according to the weight parts of the components of the compound feed, and heating and curing; under the condition of stirring, adding 0.5 part of calcium hydrophosphate, 1.2 parts of glucose, 0.10 part of lysine, 0.3 part of sodium chloride, and 0.3 part of stone powder and choline; cooling to 65 ℃, adding 0.16 part of composite trace element premix, 0.03 part of methionine, 0.02 part of threonine and 0.07 part of choline, and uniformly stirring; cooling to below 45 ℃, adding 3.2 parts of plectasin, uniformly stirring, and freeze-drying until the water content is lower than 18% to obtain the compound feed.

And (3) stability determination:

the mycelial mycin additive is subjected to an application test, namely a stability test is carried out on the feed of the test examples 1-3, and the stability test is carried out on the feed prepared in the comparative examples 1-3, and the specific operation is as follows:

the test method comprises the following steps:

1) the feed containing the myceliophthora additive prepared in application test examples 1-3 and the feed prepared in comparative examples 1-3 are respectively stored at the temperature of minus 5 ℃ for 30 days, then unfrozen, and the ratio of the feed to the liquid is 1:8, forming mixed liquid, and taking 1mL of the mixed liquid to carry out enzyme activity test.

2) The feed containing the myceliophthora additive prepared in application test examples 1-3 and the feed prepared in comparative examples 1-3 are stored at 85 ℃ for 30 days, and then the ratio of the feed to the liquid is 1:8, forming mixed liquid, and taking 1mL of the mixed liquid to carry out enzyme activity test.

3) The feed containing the myceliophthora additive prepared in application test examples 1-3 and the feed prepared in comparative examples 1-3 are stored at normal temperature for 100 days, and then the ratio of the feed to the liquid is 1:8, forming mixed liquid, and taking 1mL of the mixed liquid to carry out enzyme activity test.

The results are shown in table 2 below:

as can be seen from the data analysis in Table 2, the feeds prepared in examples 1-3 still have relatively stable activity under the conditions of 30 days at-5 ℃, 30 days at 85 ℃ and 100 days at normal temperature, which indicates that the mycelial mycin additive has high storage stability after embedding treatment, is less influenced by the environment and still has significant activity. In comparative example 1, the single cellulose acetate is used as the component of the semipermeable membrane, the embedding effect is not as good as that in the application test example, which shows that the selection of the semipermeable membrane material also plays an obvious role in the embedding effect in the application. In comparative example 2, no embedded particles are added, and mycelial mycin is directly added and used in comparative example 3, but the amylase activity and the neutral protease activity in comparative example 3 are obviously poorer than those in examples 1-3, which shows that the mycelial mycin is embedded and then used as an additive in the invention, so that the storage and use stability of the mycelial mycin can be ensured, and the activity is high.

And (3) determining the antibacterial activity:

and (3) determination of a test solution: the mycelial mycin additives prepared in examples 1-3, the mycelial mycin additive prepared in comparative example 1 and mycelial mycin were allowed to stand at room temperature for 100 days, and then distilled water was added to the mycelial mycin additives prepared in examples 1-3, distilled water was added to the mycelial mycin additive prepared in comparative example 1 and distilled water was added to mycelial mycin, and 1mL of each of the solutions was used as a sample solution, at a ratio of 1: 8.

The determination method comprises the following steps: antibacterial activity was measured by a filter paper assay method, in which 0.5ml of a suspension of Streptococcus agalactiae was inoculated onto an NA plate by a coating method (preparation method: boiling 1000ml of distilled water, adding 10g of glucose, 20g of agar, 3g of beef extract, 10g of peptone, pH 7), filter paper pieces of the measurement test solutions soaked respectively were taken with sterilized forceps and placed flat in a petri dish containing the test bacteria, each set was repeated 3 times, and sterile water was used as a blank control. The prepared culture dish is placed in a constant temperature incubator at 30 ℃, observed after 48 hours, and the diameter of the inhibition zone is measured by a cross method. The results of the test and the measurement of the diameter of the zone of inhibition for 5 repetitions are shown in table 3 below.

Table 3:

as can be seen from the data analysis in Table 3, the embedded mycelial mycin additive still has significant bacteriostatic activity after being placed for 100 days, while the semi-permeable membrane in comparative example 1 has a single component, which results in the embedding effect not similar to that of examples 1-3, and the mycelial mycin has a bacteriostatic activity which is reduced to a certain extent without being embedded, and is significantly poorer than that of examples 1-3.

Determination of buffer analysis:

the test method comprises the following steps: taking the plectasin additive prepared in example 3 and plectasin as test materials, randomly selecting 20 mice, averagely dividing into 2 groups, marking as a first group and a second group, feeding the plectasin additive prepared in example 3 to the first group and feeding the plectasin to the second group in an amount of 0.5g in the same test environment, randomly selecting 3 mice for dissection 0.5 hours after feeding, randomly selecting 3 mice for dissection 1.5 hours after feeding, dissecting the rest mice 2 hours after feeding, and detecting whether the plectasin is contained in the mice. The results are shown in Table 4.

Table 4:

from the preliminary analysis in table 4, it can be known that the embedded mycelial mycin exists in the body for a longer time, can keep the concentration relatively stable within a certain time, shows a slow-release effect, and can act in the body for a long time; while the non-embedded mycelial mycin has higher concentration in the first 0.5 hours, the concentration of the non-embedded mycelial mycin is reduced rapidly along with the increase of time, and the action time of the non-embedded mycelial mycin in the body is short.

Application test example 4:

the test method comprises the following steps: 250 cattle are randomly selected for testing and are respectively marked as a test group 1, a comparison test group 2, a comparison test group 3 and a comparison group for 5 groups, and the basic ration and feeding management modes of the 5 groups are the same. The pre-test period is 10 days, and the formal test period is 2 months. The test group 1 corresponds to feeding the feed containing the myceliophthora additive prepared in the application test example 3, the comparative test group 1 corresponds to feeding the feed prepared in the comparative example 1, the comparative test group 2 corresponds to feeding the feed prepared in the comparative example 2, the comparative test group 3 corresponds to feeding the feed prepared in the comparative example 3, and the comparative group is feeding the conventional daily ration. Before the test, fresh cow dung was collected and measured, and after the test time was over, a fresh cow dung sample was collected again, and the change of microbial flora was measured, and the results are shown in table 5.

Table 5:

as can be seen from the analysis of the data in Table 5, when comparing the Salmonella and Streptococcus agalactiae detected in cow dung before the test with those detected after the test, it can be seen that the feed containing the myceliophthora additive in test group 1 has significant inhibitory effect on Salmonella and Streptococcus agalactiae. The plectasin in the comparative test group 1 is embedded by a single semipermeable membrane material, and the expressed bacteriostatic effect is not as good as that in the test group 1; compared with the comparative test group 2, no plectasin is added, and streptococcus agalactiae tends to increase; the comparative test group 3 had a certain bacteriostatic effect, but was significantly inferior to the test group 1.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Guang Donghai Nachuan Biotech Co., Ltd

<120> a myceliophthora additive and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 129

<212> DNA

<213> Manual example

<400> 1

ggttttggtt gtaacggtcc atgggatgaa gatatgcaat gtcataacca ttgtaagtct 60

attaagggtt acaagggtgg ttacaagggt ggttactgtg ctaagggtgg ttttgtttgt 120

aagtgttac 129

<210> 2

<211> 34

<212> PRT

<213> myceliophthorin

<400> 2

Gly Gly Cys Asn Gly Trp Asp Asp Asp Met Cys His Asn His Cys Lys

1 5 10 15

Ser Lys Gly Tyr Lys Gly Gly Tyr Cys Ala Lys Gly Gly Val Cys Lys

20 25 30

Cys Tyr

Claims

1. The myceliophthora additive is characterized in that myceliophthora is prepared into embedded particles, the embedded particles comprise contents and semipermeable membranes wrapping the contents, and the content of the myceliophthora is 1% -30% of the total weight of the embedded particles.

2. The mycelial supplement of claim 1, wherein the mycelial supplement is prepared by a method comprising:

purifying plectasin;

preparing contents;

3. The plectasin additive according to claim 2, wherein the purification treatment comprises:

4. The plectasin additive as claimed in claim 2, wherein the semipermeable membrane coating is prepared by mixing cellulose acetate, sodium chloride, hydroxypropyl cellulose and triacetin.

5. The plectasin additive of claim 2, wherein the method for preparing the contents comprises: adding sodium alginate and shell powder into a proper amount of water, heating to 75-95 ℃ under the condition of 500-1000r/min, stirring until the sodium alginate and the shell powder are completely dissolved, cooling to 35-50 ℃, adding purified plectasin, stirring uniformly, adding calcium chloride, and continuously stirring for 20-40 min to form a gelatinous content.

6. The plectasin additive of claim 2, wherein the spraying method comprises: heating the semipermeable membrane coating to 30-65 ℃, and spraying the content for 15-30 min by adopting an intermittent atomization spraying mode.

7. The plectasin additive according to claim 4, wherein the cellulose acetate is added in an amount of 45-75% by mass; the addition amount of the sodium chloride is 8% -12%; the addition amount of the hydroxypropyl cellulose is 4-18%; the addition amount of the triacetin is 1% -8%.

8. The plectasin additive according to claim 1, wherein the mesh number of the embedded particles is 80-250 mesh.

9. Use of a mycelial additive according to any one of claims 1-8, wherein the mycelial additive is embedded for better stability and for use in a product for inhibiting the growth of Streptococcus agalactiae.

10. The use according to claim 9, wherein the mycelial supplement is used in the preparation of feed.