CN108289478A

CN108289478A - Include the feed addictive for aquatic animal of essential oil and lysozyme

Info

Publication number: CN108289478A
Application number: CN201680058693.7A
Authority: CN
Inventors: 薇薇安·维尔哈克
Original assignee: Novozymes AS; DSM IP Assets BV
Current assignee: Novozymes AS; DSM IP Assets BV
Priority date: 2015-10-13
Filing date: 2016-10-12
Publication date: 2018-07-17
Also published as: CL2018000925A1; EP3361882A1; WO2017064092A1; CA3001389A1

Abstract

The present invention relates to the combinations of natural active matter and the polypeptide with lysozyme activity selected from α firpenes, α terpineols, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene to be used for aquatic animal in manufacture, in particular for the purposes in cold water fish (such as salmon, bream, perch) and the fodder compound of warm water fish (such as carp, Tilapia mossambica, catfish).More particularly it relates to which substance is used to improve the feed conversion rate and/or daily gain, the microorganism species for adjusting enteron aisle and for protecting animal from the purposes that is infected caused by pathogenic microorganisms (especially gram-positive microorganism) of fish as defined above.

Description

Include the feed addictive for aquatic animal of essential oil and lysozyme

The present invention relates to the combinations of natural essential oil active matter and at least one polypeptide with lysozyme activity to use in manufacture In aquatic animal (including fish and shrimp), in particular for cold water fish (such as salmon, bream, perch) and warm water fish (such as Carp, Tilapia mossambica, catfish) feed in purposes.

Sequence table explanation

The application includes the sequence table of computer-reader form, is incorporated herein by reference.

SEQ ID NO:1 is as described in WO2013/076253 from the wild of Acremonium alcalophilum The mature amino acid sequence of type GH25 lysozymes.

SEQ ID NO:2 be the gene order from the Tri-chophaea saccata GH24 lysozymes detached.

SEQ ID NO:3 be from SEQ ID NO:2 amino acid sequences derived.

SEQ ID NO:4 be the mature amino acid sequence of the wild type GH24 lysozymes from Trichophaea saccata Row.

SEQ ID NO:5 be the maturation of the wild type GH22 lysozymes (hen egg-white lysozyme) from Gallus gallus Amino acid sequence.

SEQ ID NO:6 be primers F -80470.

SEQ ID NO:7 be primer R-80470.

SEQ ID NO:8 be primer 8643.

SEQ ID NO:9 be primer 8654.

Introduction

The present invention relates to selected from australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinol The natural essential oil active matter of alkene manufacture be used for aquatic animal (including fish and shrimp), in particular for cold water fish (such as salmon, Bream, perch) and the feed of warm water fish (such as carp, Tilapia mossambica, catfish) in purposes.

More particularly it relates at least one, preferably at least two kinds essential oil active matters as defined above and tool There are feed conversion rate and/or daily gain of the combination of the polypeptide of lysozyme activity for improving fish, for by adjusting enteron aisle Microorganism species and/or by protecting animal to reduce the purposes of the death rate from being infected caused by pathogenic microorganisms.

Lysozyme is a kind of by many biological as the O- glycosyl hydrolases generated to antibacterial defensive mechanism.It should Enzyme causes the hydrolysis of bacteria cell wall by cutting the glycosidic bond of peptide glycan；The peptide glycan is a kind of important knot in bacterium Structure molecule.After the cell wall of bacterium dies down because of bacteriolyze enzyme effect, bacterial cell is cracked due to osmotic pressure.

Lysozyme is present in many biologies, such as virus, plant, insect, birds, reptile and mammal.It is feeding In newborn animal, lysozyme has been isolated from nasal discharge, saliva, tears, enteron aisle, urine and breast.Cleavage N- acetyl Glycosidic bond between No. 1 carbon of base muramic acid and No. 4 carbon of N- acetyl group-d-glucosamine.In vivo, both sugar polymerizations are formed Cell wall polysaccharides.

Lysozyme has been classified as five kinds of different glycoside hydrolase (GH) families (CAZy, www.cazy.org)：Ovum gallinaceum Clear lysozyme (GH22), goose hen egg white lysozyme (GH23), bacteriophage T4 Lysozyme (GH24), Sphingomonas flagellum egg whites (GH73) and Chalaropsis lysozymes (GH25).Lysozyme from GH23 and GH24 families initially knows from bacteriophage, most Closely just identified in fungi.It has been found that bacteriolyze enzyme family GH25 and other bacteriolyze enzyme families are structurally independent.

Hen egg-white lysozyme is obtainable major product on commercial market, but it does not cut such as Staphylococcus N in aureus cell walls, 6-O- diacetyl muramic acid, therefore cannot especially crack this important human pathogen (Masschalck B,Deckers D,Michiels CW(2002),“Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure”,J Food Prot.65(12):1916-23)。

In addition, the present invention relates to a kind of novel fish feed composition, it includes it is at least one, preferably at least two kinds or three Kind selected from australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene reactive compound with The combination of at least one antimicrobial polypeptide with lysozyme activity is as active constituent.

A key factor in aquaculture is turnover rate.Turnover rate by fish how soon can grow into trappable size Lai It determines.For example, by atlantic salmon (Atlantic salmon) from samlet (when atlantic salmon can be turned for the first time from fresh water Physiological stage when moving on to seawater) raising needs 12-18 a month to trappable size.Fast turnover has several positive effects Fruit.First, it contributes to cash flow.Secondly, it improves risk management.Particularly, high mortality is weight for the person of breeding fish Big risk.

It is well known that unbalance microorganism species and/or the infection caused by pathogenic microorganisms can cause the death rate to improve. Fish disease is common, and the possibility broken out within long growth period is higher.There is also fishes due to (such as when draping ) unexpected or due to bad weather causes fish column broken and escapes risk.

For other farm-animals, prevent disease to develop to be well known using antibiotic and vaccine.It is supported in aquatic products In growing, the use of antibiotic less extensively-at least in cold aqueous aquaculture so-this is because in fact transmission Must be very fast, the fish of illness eats seldom, and due also to negative effect of the drug containing feed discarded to environment.Vaccine is available When be widely used, but they are developed for all diseases.

As the substitute of synthetic drug, the purposes of plant extracts and essential oil in animal feed is described in document. For example, patent WO2011/006993 describes purposes of the essential oil in fish meal.It is kept away however, WO2011/006993 is not recorded Exempt to infect caused by gram-positive microorganism.In addition, in example disclosed above, only there is limited work(using essential oil Effect.

Therefore, there is still a need for (include for the anti-of gram-positive microorganism by any preventive means in aquaculture Microbial activity) develop to prevent disease, to reduce the death rate.

Present inventor it was unexpectedly found that：The combination of substance as defined above has in fish meal Great potential, for example, for improving feed conversion rate (FCR) and/or weightening and/or for adjusting intestinal flora.In addition, invention People it was unexpectedly found that：Novel fish feed composition also has the antimicrobial acivity for gram-positive microorganism, It is reduced so as to cause the death rate.Unique selection of the reactive compound of the present invention allows control to be drawn by many different pathogens for the first time The many fish diseases risen.

Therefore, in the first specific implementation mode, the present invention relates at least one, preferably at least two are used in fish meal Kind selected from australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene reactive compound with The combination of microbic muramidase come improve feed conversion rate (FCR) and/or weightening and/or (by adjust intestinal microflora And/or by preventing the disease caused by pathogenic microorganisms, preferably caused by gram-positive microorganism) reduce the death rate Method.

For example, having shown that：The present invention selected compounds (such as：The group of cinnamic acid and australene and bacterium lysozyme Close) it is shown in terms of the growth for inhibiting Lactococcus garvieae (a kind of disease especially found in cold water fish) Go out excellent effect.Combination according to the present invention also exhibits confrontation Streptococcus iniae, and (one kind is especially in warm water The disease found in property fish) excellent effect.

In alternative embodiment, australene and/or α-terpineol and/or cinnamic acid and/or dihydroeugenol and/or Eugenol and/or metacresol and/or terpinolene are applied in combination at least one antimicrobial polypeptide with lysozyme activity, with Improve animal feed digestibility and/or maintains animal health by supporting function of immune system.

In another embodiment, the present invention relates to use at least three kinds to be selected from australene, α-terpin in fish meal Alcohol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene reactive compound there is lysozyme at least one The combination of active antimicrobial polypeptide come improve feed conversion rate (FCR) and/or weightening and/or (by adjust enteric microorganism Flora and/or pass through prevent the disease caused by pathogenic microorganisms) reduce the death rate method.

In one preferred embodiment, australene and cinnamic acid are applied in combination with bacterium lysozyme to improve animal feeding Expect digestibility and/or maintains animal health by supporting function of immune system.

Definition

Term feed or fodder compound refer to any compound for being suitable for or being intended to be taken in by animal, product, mixing Object or composition.

FCR can be measured based on fish growth experiment, and the experiment includes the first processing, wherein by least two bases The mixture of the compound of the present invention is added to every suitable concentration of kg feeds in animal feed；With second processing (control), The compound is not added into animal feed.

As is generally known, improved FCR is less than control FCR.In a particular embodiment, compared with the control, FCR improves (reduce) at least 1.0%, preferably at least 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4% or at least 2.5%.

Term as used herein " enteron aisle " indicates gastrointestinal tract or alimentary canal (also referred to as digest tube), refers to that many cells are dynamic Dietary intake, digestion food are to extract energy and nutrients and the tract of residual waste is discharged in object.

Term as used herein enteron aisle " microorganism species " refer to be present in enteron aisle and by help appropriate digestion come The natural microbial culture kept fit.

Term " adjusting " related with intestinal microflora used herein generally means that normal in health and function Animal in change, manipulate, its function of variations or modifications or state, i.e. non-therapeutic use.

Term as used herein " supporting function of immune system " refers to being made by the immunostimulation that the compound obtains With.

Term as used herein " death rate " refer to growth phase at the end of surviving animals and pond in include initially Animal quantity ratio.It can be measured based on fish challenge trial (fish challenge trail), the fish attack Experiment is comprising by two groups of fishes of specific fish disease pathogen attack, it is therefore an objective to cause the death rate of the animal 40-80% in untreated fish group. However, in the attack group for the mixture for feeding at least two compound according to the present invention per Kg feed suitable concentrations, with Untreated fish group is compared, and the death rate reduces at least 5%, preferably at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or at least 50%.

Animal feed：Term " animal feed " refer to any compound for being suitable for or being intended to be taken in by animal, product or Mixture.Animal feed for nonruminant generally comprises concentrate and the micro- life of vitamin, minerals, enzyme, Direct-fed Object, amino acid and/or other feed ingredients (such as in pre-composition), and the animal feed for being used for ruminant generally comprises grass Expect (including roughage and ensilage), and concentrate and vitamin can be further included, minerals, enzyme, directly raised Feed microorganism, amino acid and/or other feed ingredients (such as in pre-composition).

Antimicrobial acivity：Term " antimicrobial acivity " is defined herein as killing microorganism (such as algae, Gu Bacterium, bacterium, fungi and/or protozoan) or inhibit growth activity.For example, antimicrobial acivity can be bactericidal (mean kill bacterium) or inhibit (the meaning to prevent bacterial growth) of bacterium.Antimicrobial acivity may include that being catalyzed peptide gathers It is residual with N- acetyl group-d-glucosamine in chitosan between N- acetylmuramic acids and N- acetyl group-d-glucosamine residue in sugar The hydrolysis of 1,4- β-key between base.Antimicrobial acivity can also be combined with the surface of microorganism including lysozyme and inhibit it Growth.Anti-microbial effect can also include using the lysozyme of the present invention, by inhibiting or reducing bacteriotoxin and pass through tune Reason plain (opsonin) acts on to activate bacterial autolysin, as immunostimulant.

For the purposes of the present invention, the (" survey of antimicrobial acivity of the anti-microbial test described in embodiment 6 It is fixed ") measure antimicrobial acivity.Antimicrobial acivity is determined by the diameter of clear area (clearing zone), if used Clear area is more than 2mm when 50%Mueller-Hinton culture mediums (pH 6), then antimicrobial acivity is assessed as significantly.

Concentrate：Term " concentrate " means there is high protein and the feed of energy concentration, for example, fish meal, molasses, oligosaccharides, Sorghum, seed and cereal (come from such as corn, oat, rye, barley, wheat, it is complete or by the systems such as crushing, grinding It is standby), oily seed cake of press is (such as from cottonseed, safflower, sunflower, soybean (such as soy meal), rapeseed, peanut or fallen flowers It is raw), palm kernel cake, the substance of yeast sources and vinasse (such as wet vinasse (WDS) and distiller's dried grain (DDGS) containing soluble matter).

The European production efficiency factor (EPEF)：The European production efficiency factor is to compare a kind of mode of animal performance.The list One number helps to compare performance between farm inside farm, and can be used for Evaluation Environment, weather and management variable.EPEF It is calculated as [(survival rate (%) × live-weight (kg))/(service life (day) × FCR when exhausting)] × 100, wherein survival rate is to slaughter The percentage of surviving animals when government official, live-weight are the average weights of animal when butchering, and exhaust the service life that the service life is animal when butchering, FCR is feed conversion rate when butchering.

Feed conversion rate (FCR)：FCR is measurement of the animal by feed material conversion for the increased efficiency of desired output.It is right In the domesticated animal (such as pig, poultry and fish) in order to meat, the output is the increased quality of animal.Specifically, FCR It is calculated as：The weightening in feed divided by set period taken in set period.FCR improvement means the reduction of FCR values.FCR Improving 2% means that FCR reduces 2%.

Segment：Term " segment " refers to amino from mature polypeptide or structural domain and/or carboxyl-terminal deletion one or more The polypeptide or catalyst structure domain of multiple (such as several) amino acid；The wherein described segment has lysozyme or phytase activity. On one side, segment includes SEQ ID NO:1 at least 170 amino acid, such as at least 175 amino acid, at least 177 ammonia Base acid, at least 180 amino acid, at least 185 amino acid, at least 190 amino acid, at least 195 amino acid or at least 200 A amino acid and have lysozyme activity.

On the other hand, segment includes SEQ ID NO:4 at least 210 amino acid, such as at least 215 amino Acid, at least 220 amino acid, at least 225 amino acid, at least 230 amino acid, at least 235 amino acid or at least 240 Amino acid and have lysozyme activity.

It is separated：Term " separated " refers to the substance in the form being not present in nature or environment.Through The non-limiting examples of the substance of separation include (1) any non-naturally occurring substance, and (2) include but not limited to any enzyme, change Any substance of body, nucleic acid, protein, peptide or co-factor, the associated one or more at least partly from nature It is taken out in kind or all naturally occurring ingredients；(3) pass through manually modified any object relative to the substance found in nature Matter；Or any substance (example that (4) are modified by relative to the amount of the substance is increased with its natural relevant other components Such as, multiple copies of the gene of the substance are encoded；Using than with encode the substance gene naturally relevant promoter is stronger Promoter).Separated substance may be present in fermentation broth sample.

Lysozyme activity：Term " lysozyme activity " means N- acetylmuramic acids and the Portugals N- acetyl group-D- in peptide glycan The hydrolysis of Isosorbide-5-Nitrae-β-key between glucosamine residue and chitosan between N- acetyl group-d-glucosamine residue, leads to cell Dissolving.Lysozyme belongs to the other EC 3.2.1.17 of enzyme.Lysozyme activity is usually measured by turbidimetric assay.This method is based on The turbidity of 4698 suspended matters of Micrococcus luteus ATCC caused by splitting action by lysozyme changes.Suitable Under experiment condition, these variations are proportional to the amount of lysozyme in culture medium (to advise referring to FAO's food additives The INS 1105 (www.fao.org) that model Symphyla is wanted).For the purposes of the present invention, the turbidity described in embodiment 5 is surveyed (" measurement of lysozyme activity ") is determined to measure lysozyme activity.In one aspect, polypeptide of the invention has SEQ ID NO:1 Lysozyme activity at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100%.On the other hand, polypeptide of the invention has SEQ ID NO:4 lysozyme activity At least 20%, for example, at least 40%, at least 50%, at least 60%%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100%.

Mature polypeptide：Term " mature polypeptide " refers to translation and any posttranslational modification (such as processing of the ends N-, the ends C- section Short, glycosylation, phosphorylation etc.) after final form polypeptide.

Microbic muramidase：Term " microbic muramidase " refers to the polypeptide for having lysozyme activity, is obtained from or can obtain From microbe-derived.Microbe-derived example has fungi.Specifically, microbic muramidase be obtained from or available from Ascoycota, such as Pezizomycotina subphylums.

Sequence identity：Correlation between two amino acid sequences or between two nucleotide sequences is by parameter " sequence Homogeneity " describes.

For the purposes of the present invention, using EMBOSS packets (EMBOSS:The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), preferred version 5.0.0 or more Implement in the Needle programs of highest version Needleman-Wunsch algorithms (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) determine the sequence identity between two amino acid sequences.Parameter used is that vacancy opens Point penalty 10, gap extension penalties 0.5 and EBLOSUM62 (the EMBOSS versions of BLOSUM62) substitute matrix.Needle is labeled as The output (acquisition of use-nobrief options) of " longest homogeneity " is used as homogeneity percentage, following to calculate：

(identical residue × 100)/(comparing the vacancy sum in length-comparison).

Substantially pure polypeptide：Term " substantially pure polypeptide " refers to such product, contain at most 10 weight %, At most 8 weight %, at most 6 weight %, at most 5 weight %, at most 4 weight %, at most 3 weight %, at most 2 weight %, at most 1 Weight % and at most 0.5 weight %'s natively or recombinates other relevant peptide materials with it.It is preferably based in product The weight of existing total peptide material, the polypeptide are at least 92% pure, and for example, at least 94% is pure, at least 95% pure, at least 96% is pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.5% pure and mild 100% pure.The polypeptide of the present invention is excellent It is selected as substantially pure form.This can rely on for example prepares polypeptide by well known recombination method or by classical purification process To realize.

Variant：Term " variant " means the polypeptide with lysozyme or phytase activity, in one or more (examples Such as, several) change that includes one or more (several) amino acid residues at position, that is, it replaces, be inserted into and missing.Replacement is Refer to the amino acid to plant oneself to be substituted by different amino acid；Missing refers to the amino acid that removal plants oneself；Insertion refers to neighbour Amino acid that is close and and then planting oneself adds 1,2 or 3 amino acid.

In one aspect, bacteriolyze enzyme variants according to the present invention can include 1-5；1-10；1-15；1-20；1-25；1- 30；1-35；1-40；1-45；Or 1-50, i.e., 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, 20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、 45,46,47,48,49 or 50 changes, and there is parent's phytase (such as SEQ ID NO:1) phytase activity is at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100%.On the other hand, bacteriolyze enzyme variants according to the present invention can include 1-5；1-10；1-15；1-20；1-25；1-30； 1-35；1-40；1-45；Or 1-50, i.e., 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、 46,47,48,49 or 50 changes, and there is parent's lysozyme (such as SEQ ID NO:4) lysozyme activity is at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100%.

Detailed description of the invention

Specifically, present inventor it was unexpectedly found that：Compound combination according to the present invention is to cold aqueous It is effective with many pathogenic gram-positive microorganisms of warm water fish.Show that composition according to the present invention is shown pair Lactococcus garvieae (especially a kind of found in cold water fish disease) and to Streptococcus iniae The inhibiting effect of (a kind of disease especially found in warm water fish).

In addition, at least one antimicrobial polypeptide with lysozyme activity is applied in combination as described above further improves day The existing inhibiting effect of right essential oil active matter.

The known inhibiting effect of essential oil has：

Display australene, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene are shown to Aeromonas The inhibiting effect of salmonicida, Aeromonas salmonicida are the pathogen for the disease for causing to be referred to as dothienesis.

Display australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene are shown pair The inhibiting effect of Edwardsel-lia tarda, Edwardsel-lia tarda cause fish general infection.

Display australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol and terpinolene are shown pair The inhibiting effect of Lactococcus garvieae, Lactococcus garvieae are the pathogen of Latococcosis, Latococcosis is a kind of sudden illness, when water temperature increases above 16 DEG C in summer months, influences many fish objects It plants and all causes heavy economic losses in seawater and fresh water aquaculture.

Cinnamic acid, dihydroeugenol, eugenol and metacresol show the excellent suppression to Yersinia ruckeri growths It makes and uses, Yersinia ruckeri are the pathogenic microorganisms that one kind causing intestines red-mouth disease (ERM), and ERM is that a kind of be especially exists The disease found in salmon.

Display cinnamic acid and metacresol are shown for inhibiting effect below：

- Vibrio salmonicida are psychrophilic bacterias, are the causative agents of atlantic salmon cold water vibriosis；

- Aeromonas hydrophila, cause ulcer and bovine pasteurellosis.This pathogen is to conventional simple The resistance of antimicrobial (such as chlorine) is very big；

- Photobacterium damselae are formerly referred to as Pasteurella piscicida, are economically heavy The high pathogen lost, for example Japanese Yellowtail of the economically important fish are caused in the marine fish culture industry wanted (seriola) and epinephelus akaara (red grouper) and the striped perch (striped bass) and sciaenoid in the U.S. (white perch)；

- Streptococcus iniae are high pathogenic in fish and are high lethal：Outburst can be with 30- 50% death rate is associated with.

Other water-borne pathogens can also be inhibited by compound mixture of the present invention, such as

- Piscirickettsia salmonis are piscirickettsiosis or salmons septicaemia (SRS) causative agent,

- Vibrio viscosus are renamed into Moritella viscosa, are known as in teiology recently The reason of disease of " winter ulcer ",

- Ich (parasite) is one of most common fish protozoon parasite,

- Vibrio harveyi are the reason of causing photism vibriosis, and photism vibriosis is a kind of influence business Cultivate the disease of shrimp.

In second aspect, the present invention provides a kind of fish feed compositions, and it includes at least one to have lysozyme activity Microbial polypeptide with selected from australene (CAS 99-86-5), α-terpineol (CAS98-55-5), cinnamic acid (CAS 14371-10- 9/104-55-2), dihydroeugenol (CAS2785-87-7), eugenol (CAS 97-53-0), cresols (CAS 108-39-4) and The combination of at least two reactive compounds of terpinolene (CAS 554-61-0).Compound according to the present invention is commercially available, or Person can easily be prepared by those skilled in the art using technique commonly known in the art and method.

As fish feed composition, the compound of the present invention can be used alone or be used with its mixture, naturally may be used Obtain the form of extract or extract mixtures or in the form of natural materials.

The term as used herein " extract " include the composition (also referred to as " extract oil ") obtained by solvent extraction, It is method known to those skilled in the art by the composition (also referred to as " essential oil ") of steam distillation acquisition or by other The composition of acquisition.Suitable Extraction solvent includes alcohols, such as ethyl alcohol.

Term " natural " is herein understood to be made of following compounds, and the compound is naturally occurring and from day Right product is obtained or is obtained by synthesizing.

A small amount of other ingredients can be added into active essential oil compounds or natural materials or extract.These ingredients Example has：Capsaicine, tannic acid, pipering, trimethylamine, 3,4, dimethlbenzene, furfuryl alcohol and its mixture.

The mixture of at least two essential oil compounds if as described above is preferred, then cinnamic acid and/or metacresol It is used as the key component of mixture.Suitably, mixture contains the metacresol and/or cinnamic acid of 10-90 weight %, 1-50 weights The australene of %, the dihydroeugenol of 1-50 weight % are measured, wherein total amount of the amount based on the component calculates.These activity The total amount of ingredient can change in a wide range, but 10-5000ppm, preferably 100-1000ppm are finally used in fish meal, Dry weight of the amount based on fish meal calculates.

The most preferred mixture of at least two essential oil compounds as described above includes cinnamic acid and australene as mixing The key component of object.Suitably, mixture includes the australene of 30-70 weight %, the cinnamic acid of 30-70 weight %, 1-20 weights The metacresol of %, the dihydroeugenol of 1-20 weight % are measured, wherein total amount of the amount based on the component calculates.These activity The total amount of ingredient can change in a wide range, but 10-5000ppm, preferably 100-1000ppm are finally used in fish meal, Dry weight of the amount based on fish meal calculates.

All essential oil compounds (reactive compound and added ingredient) being defined above can be with emulsifying surfactant group It closes and uses.Emulsifier can be selected advantageously from those quite hydrophilic emulsifiers, for example, aliphatic acid polyglycerol ester, example The ricinoleic acid of such as esterification or propylene glycol ester of aliphatic acid, sugared -ester class or sugar-glyceride type, polyethylene glycol, lecithin.

In a preferred embodiment of the present invention, blend of essential oils can contain the cinnamic acid of 20 weight %, 20 weights Measure the metacresol of %, the dihydroeugenol of 20 weight %, the australene of 20 weight %, the trimethylamine of 3 weight %, 1.8 weight % Pipering and 4 weight % furfuryl alcohol.

In another preferred embodiment of the present invention, the mixture of essential oil reactive compound includes 40-60 weight % Australene, the cinnamic acid of 40-60 weight %, and the trimethylamine of 1-5 weight %, the pepper of 1-5% can be further included The furfuryl alcohol of alkali and 3-8 weight %.

As described above, having unexpectedly discovered that：With microbic muramidase supplement animal feed in aquatic animal (including fish And shrimp) in produce significant performance benefit.This is unexpected because using microbic muramidase improve animal performance with It is preceding to be never proved.

Therefore, the invention further relates to a kind of method improving the European production efficiency factor (EPEF), the method includes：To Animal applies animal feed or animal feed comprising at least two essential oil compounds and one or more of microbic muramidases Additive.

In one preferred embodiment, animal feed or the animal that will improve with microbic muramidase is wherein not present Feed addictive (herein referred as compareing) is compared.

In one embodiment, compared with the control, EPEF improves at least 1%, and such as at least 1.5%, at least 2.0%, at least 2.5%, at least 3%, at least 3.5%, at least 4% or at least 5%.In another embodiment, with compare It comparing, EPEF improves 1%-15%, such as 1%-12%, 1%-10%, 1.5%-8%, 2.0%-7% or these sections Any combinations.

In one embodiment, with every kg animal feeds 8-250ppm zymoproteins, such as per kg animal feeds 9- 200ppm, 10-150ppm, 11-125ppm, 12-100ppm, 13-75ppm, 15-50ppm, 17.5-40ppm, 25-75ppm or 30-60ppm zymoproteins or any combination of horizontal dispensing microbic muramidase in these sections.

In one embodiment, microbic muramidase is originated from fungus.In one embodiment, microorganism bacteriolyze Enzyme is obtained from or available from Ascoycota, such as Pezizomycotina subphylums.

In one embodiment, microbic muramidase includes one or more structural domains selected from GH24 and GH25.

In one preferred embodiment, the present invention relates to a kind of European production efficiency factor (EPEF) of improvement and/or The method of feed conversion rate (FCR), the method includes：The animal for including one or more of microbic muramidases is applied to fish Feed or animal feed additive, wherein：

(a) microbic muramidase is that the microorganism comprising one or more structural domains selected from GH24 and GH25 is molten Bacterium enzyme, with the horizontal dispensing of every kg animal feeds 10-150ppm zymoproteins；

(b) aquatic animal is to be selected from shrimp, cold water fish such as salmon, bream, perch and warm water fish such as carp, Tilapia mossambica, catfish, and；

(c) compared with the control, the European production efficiency factor (EPEF) and/or feed conversion rate (FCR) improve at least 1%.

In one embodiment, microbic muramidase and SEQ ID NO:1 have at least 50%, for example, at least 60%, At least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% sequence identity.

In one embodiment, microbic muramidase includes SEQ ID NO:1 amino acid sequence or its allelic variant Or by SEQ ID NO:1 amino acid sequence or its allelic variant composition；Either SEQ ID NO:1 amino acid sequence Segment with lysozyme activity, wherein the segment includes at least 170 amino acid, for example, at least 175 amino acid, at least 177 amino acid, at least 180 amino acid, at least 185 amino acid, at least 190 amino acid, at least 195 amino acid or At least 200 amino acid.In another embodiment, microbic muramidase includes SEQ ID NO:1 amino acid sequence or Its allelic variant and the ends N- and/or C- terminal His tags and/or HQ labels or by SEQ ID NO:1 amino acid sequence Row or its allelic variant and the ends N- and/or C- terminal His tags and/or HQ labels composition.On the other hand, polypeptide includes SEQ ID NO:1 amino acid 1-208 is made from it.

In another embodiment, microbic muramidase is SEQ ID NO:1 variant, wherein the variant is with molten Bacterium enzymatic activity and 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50 positions include one or more replacements and/or one or more missings and/or one or more insertions or its What is combined.In another embodiment, SEQ ID NO:In 1 comprising one or more amino acid substitutions and/or one or More amino acid deletions and/or one or more amino acid be inserted into or any combination thereof the number of position be 1-45, example Such as 1-40,1-35,1-30,1-25,1-20,1-15,1-10 or 1-5 positions.In one embodiment, SEQ ID NO:1 In include one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino Acid be inserted into or any combination thereof the number of position be no more than 10, such as 1,2,3,4,5,6,7,8,9 or 10.In another implementation In mode, SEQ ID NO:The number of replacement, deletion and/or insertion in 1 is no more than 10, such as 1,2,3,4,5,6,7,8,9 Or 10.In another embodiment, SEQ ID NO:Replacement in 1, the preferably conservative number replaced be no more than 10, such as 1, 2,3,4,5,6,7,8,9 or 10.In another embodiment, SEQ ID NO:The number replaced is guarded in 1 is no more than 10, example Such as 1,2,3,4,5,6,7,8,9 or 10.

Amino acid change can be not too important to property, i.e., does not significantly affect the folding of protein and/or active conservative Amino acid substitution or insertion；The small missing of usual 1-30 amino acid；Small amino-or carboxyl-tenninus extend, such as amino- Terminus methionine residue；The small connection peptide of at most 20-25 residue；Or it is pure to promote by changing net charge or other functions The small extension changed, such as polyhistidine beam, epitope or binding domain.

The conservative example replaced is with the following group：Basic amino acid group (arginine, lysine and histidine), acid ammonia Base acid group (glutamic acid and aspartic acid), polar amino acid group (glutamine and asparagine), hydrophobic amino acid group (bright ammonia Acid, isoleucine and valine), aromatic amino acid group (phenylalanine, tryptophan and tyrosine) and p1 amino acid group (sweet ammonia Acid, alanine, serine, threonine and methionine).The amino of specific activity (specific activity) is not changed usually It is known in the art that acid, which is replaced, and for example by H.Neurath and R.L.Hill, and 1979 in The Proteins, Described in Academic Press, New York.Common replacement has Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/ Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、 Leu/Val, Ala/Glu and Asp/Gly.

Can according to program known in the art such as direct mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989,Science 244:1081-1085) identify the essential amino acid in polypeptide.In latter technique, in the every of molecule Single alanine mutation is introduced at a residue, and detects the lysozyme activity of gained mutating molecule, to identify for molecular activity Crucial amino acid residue.Referring to Hilton etc., 1996, J.Biol.Chem.271:4699-4708.The active site of enzyme or its Its biological interaction can also be determined by the physical analysis to structure, such as pass through these following technologies：Such as nuclear-magnetism is total It shakes, crystallography, electronic diffraction or photoaffinity labeling, is determined together with the mutation of the contact site amino acids of presumption.See, for example, De Vos etc., 1992, Science 255:306-312；Smith et al.,1992,J.Mol.Biol.224:899-904； Wlodaver et al.,1992,FEBS Lett.309:59-64.The homogeneity of essential amino acid can also from related polypeptide Comparison infer.

As disclosed in WO2013/076253, withResolution ratio cracked Acremonium alcalophilum The crystal structure of CBS114.92 lysozymes.These atomic coordinates, which can be used for generating, describes Acremonium alcalophilum The threedimensional model of the structure or homologous structure (such as variant of the present invention) of CBS114.92 lysozymes.It, will using x/ ray structures Amino acid residue D95 and E97 (use SEQ ID NO:1 number) it is accredited as catalytic residue.

In one embodiment, microbic muramidase and SEQ ID NO:4 have at least 50%, for example, at least 60%, At least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% sequence identity.

In one embodiment, microbic muramidase includes SEQ ID NO:4 amino acid sequence or its allelic variant Or by SEQ ID NO:4 amino acid sequence or its allelic variant composition；Either SEQ ID NO:4 amino acid sequence Segment with lysozyme activity, wherein the segment includes at least 210 amino acid, for example, at least 215 amino acid, at least 220 amino acid, at least 225 amino acid, at least 230 amino acid, at least 235 amino acid or at least 240 amino acid. In another embodiment, microbic muramidase includes SEQ ID NO:4 amino acid sequence or its allelic variant and N- End and/or C- terminal His tags and/or HQ labels or by SEQ ID NO:4 amino acid sequence or its allelic variant with And the ends N- and/or C- terminal His tags and/or HQ labels composition.On the other hand, polypeptide includes SEQ ID NO:4 amino Sour 1-245 is made from it.

In another embodiment, microbic muramidase is SEQ ID NO:4 variant, wherein the variant is with molten Bacterium enzymatic activity and 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50 positions include one or more replacements and/or one or more missings and/or one or more insertions or its What is combined.In another embodiment, SEQ ID NO:In 4 comprising one or more amino acid substitutions and/or one or More amino acid deletions and/or one or more amino acid be inserted into or any combination thereof the number of position be 1-45, example Such as 1-40,1-35,1-30,1-25,1-20,1-15,1-10 or 1-5 positions.In one embodiment, SEQ ID NO:4 In include one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino Acid be inserted into or any combination thereof the number of position be no more than 10, such as 1,2,3,4,5,6,7,8,9 or 10.In another implementation In mode, SEQ ID NO:The number of replacement, deletion and/or insertion in 4 is no more than 10, such as 1,2,3,4,5,6,7,8,9 Or 10.In another embodiment, SEQ ID NO:Replacement in 4, the preferably conservative number replaced be no more than 10, such as 1, 2,3,4,5,6,7,8,9 or 10.In another embodiment, SEQ ID NO:The number replaced is guarded in 4 is no more than 10, example Such as 1,2,3,4,5,6,7,8,9 or 10.

Described above is amino acid change, the examples of conservative replacement and N- and/or C- end fittings.

The enzyme of the present invention can be formulated into liquid or solid.For liquid preparation, preparaton may include that polyalcohol is (such as sweet Oil, ethylene glycol or propylene glycol), salt (such as sodium chloride, sodium benzoate, potassium sorbate) or sugar or sugar derivatives (such as dextrin, Portugal Grape sugar, sucrose and D-sorbite).Therefore, in one embodiment, composition is enzyme and one or more comprising the present invention The liquid composition of kind of preparaton, the preparaton be selected from glycerine, ethylene glycol, 1,2-PD, 1,3-PD, sodium chloride, Sodium benzoate, potassium sorbate, dextrin, glucose, sucrose and D-sorbite.Liquid preparation can be sprayed onto after granulation on feed, Or it can be added in the drinking water for giving animal.

For solid pharmaceutical preparation, preparation can be such as particle, spray-dried powders or agglomerate.Preparaton can include salt (zinc salt, sodium salt, sylvite or the calcium salt of organic or inorganic, for example, calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, Calcium sorbate, calcium sulfate, potassium acetate, Potassium Benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, acetic acid Sodium, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulphate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, lemon Sour zinc, sorbic acid zinc, zinc sulfate), starch or sugar or sugar derivatives (such as sucrose, dextrin, glucose, lactose, D-sorbite).

In one embodiment, solid composite is in granular form.Particle can have matrix structure, wherein each component Uniformly mixing.But particle generally comprises core particle and one or more of coatings, the coating is typically salt and/or wax Coating.Core particle can be that the enzyme of the present invention optionally combines and with one or more of other enzymes optionally together with one The homogeneous blend of kind or more salt；Either inert particulate, wherein the present invention enzyme optionally with coating on it one The other enzyme combination of kind or more.

In one embodiment, the material of core particle be selected from inorganic salts (such as calcium acetate, calcium benzoate, calcium carbonate, Calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, Potassium Benzoate, potassium carbonate, potassium chloride, potassium citrate, sorbic acid Potassium, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulphate, zinc acetate, zinc benzoate, carbonic acid Zinc, zinc chloride, zinc citrate, sorbic acid zinc, zinc sulfate), starch or sugar or sugar derivatives (such as sucrose, dextrin, glucose, Lactose, D-sorbite), sugar or sugar derivatives (such as sucrose, dextrin, glucose, lactose, D-sorbite), small organic molecule, Starch, powder, cellulose and minerals.

Salt coating typically at least 1 μ m-thick, and can be the mixture of a kind of specific salt or salt, such as Na₂SO₄、 K₂SO₄、MgSO₄And/or sodium citrate.Other examples are such as WO 2008/017659, WO 2006/034710, WO 1997/ 05245, WO 1998/54980, WO 1998/55599, those of described in WO 2000/70034 or such as WO 2001/ Polymer coating described in 00042.

In another embodiment, composition is solid composite, and it includes the enzyme of the present invention and one or more Preparaton, the preparaton are selected from sodium chloride, sodium benzoate, potassium sorbate, sodium sulphate, potassium sulfate, magnesium sulfate, thiosulfuric acid Sodium, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, D-sorbite, lactose, starch and cellulose.It is preferred real at one It applies in mode, preparaton is selected from one or more of following compounds：Sodium sulphate, dextrin, cellulose, sodium thiosulfate and carbonic acid Calcium.In one preferred embodiment, solid composite is in granular form.In one embodiment, solid composite is in Particle form and include core particle, include the present invention enzyme enzyme layer and salt coating.

In another embodiment, preparaton is selected from one or more of following compounds：Glycerine, ethylene glycol, 1,2- Propylene glycol or 1,3- propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulphate, potassium sulfate, magnesium sulfate, sodium thiosulfate, Calcium carbonate, sodium citrate, dextrin, glucose, sucrose, D-sorbite, lactose, starch and cellulose.In a preferred implementation In mode, preparaton is selected from one or more of following compounds：1,2- propylene glycol, 1,3- propylene glycol, sodium sulphate, dextrin, fibre Dimension element, sodium thiosulfate and calcium carbonate.

The fish feed composition containing reactive compound can be mixed in fish meal as described in example 1 above.Then will The mixture of reactive compound is directly prepared into oil, then mixes the oil with the oil being sprayed onto on feed pellets, such as embodiment 1 Described in.

Or it can be by preparing the pre-composition of active constituent and other suitable additives to which reactive compound will be contained Fish feed composition incorporation fish meal in.This pre-composition can include the active mixture or natural goods of 2-10 weight % Matter or extract, other conventional additives such as flavoring agent of 0-40 weight % and any Conventional absorbent of 50-98 weight % carry Body (support).

Carrier can contain the wood-fibred of such as 40-50 weight %, the tristearin of 8-10 weight %, the turmeric of 4-5 weight % Powder, the rosemary powder of 4-5 weight %, the lime stone of 22-28 weight %, the natural gum such as gum arabic of 1-3% weight, 5-50% The water of the sugar and/or starch and 5-15% weight of weight.

Then by the pre-composition and vitamin (such as vitamin C), mineral salt and other feed addictive ingredients (such as containing Have the yeast extract of nucleotide, glucan and other intestinal microflora conditioning agents) mixing, it is then final so that feed The amount of active constituent according to the present invention containing 10-5000ppm, preferably 100-1000ppm or 100-500ppm is added to In feed.

In addition, the composition of the present invention is preferably used together with containing nucleotide with the yeast extract of glucan.

In addition, optional feed addictive ingredient is colorant, for example, carotenoid such as beta carotene, astaxanthin and Lutein；Aromatic compound；Stabilizer；Antimicrobial peptide；Polyunsaturated fatty acid；And/or it is at least one selected from phytase (EC 3.1.3.8 or 3.1.3.26)；Zytase (EC 3.2.1.8)；Galactanase (EC 3.2.1.89)；Alpha-galactosidase (EC 3.2.1.22)；Protease (EC 3.4.)；Phospholipase A1 (EC 3.1.1.32)；Phospholipase A2 (EC 3.1.1.4)；Haemolysis Phosphatidase (EC 3.1.1.5)；Phospholipase C (EC 3.1.4.3)；Phospholipase D (EC 3.1.4.4)；Amylase, such as alphalise starch Enzyme (EC 3.2.1.1)；And/or the enzyme of 1,4 beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

The example of polyunsaturated fatty acid has C18, C20 and C22 polyunsaturated fatty acid, such as arachidonic acid, 22 Carbon acid, eicosapentaenoic acid and gamma-linoleic acid.

Fish meal as described herein with 20-60 weight % protein and 1-45 weight % moisture and lipid it is close Like composition.

In some specific examples, fish meal includes the source of one or more of following substances：

Protein, carbohydrate and lipid (such as fish meal, fish oil, blood meal, feather meal, poultry powder, chicken powder and/or by The other kinds of powder that other slaughterhouse wastes generate)；

Animal tallow (such as poultry oil)；

Vegetable powder (such as soy meal, feather fan bean powder, peameal, bean powder, ground oil seeds and/or sunflower powder)；

Vegetable oil (such as rapeseed oil, soybean oil)；

Seitan (such as gluten or corn gluten) and

The amino acid (such as lysine) of addition.

The term as used herein " fish meal " includes fish feed composition according to the present invention and component as described above.It is logical Often, fish meal includes fish meal as component.Suitably, fish meal is in the form of sheets or pellet (such as squeezing out pellet) form.

In the third aspect, the present invention relates to for aquatic animal fodder compound and the composition for feeding fish Purposes.This feed is particularly suitable for feeding salmon, including atlantic salmon (Ontario salmon (Salmo salar)), other salmons Fish species and trout and non-salmon such as gadus, perch, madai and sea eel.However, this feed can feed it is all types of Fish, such as turbot, halibut, Yellow Tail (yellow tail) and tuna.

Described and claimed herein is not limited in range by particular implementation disclosed herein, because These embodiments are intended as the explaination of several aspects of the invention.Any equivalent embodiment is intended to the present invention's In range.It is of the invention various to repair other than those modifications shown and described herein in fact, according to the description of front Change and is apparent to practitioners skilled in the art.These modifications are also intended to fall within the scope of the appended claims.

Embodiment 1：Suppress the preparation of fish meal

Main raw material(s) is ground and mixed.Then micro constitutent is added in mixer, and by into preprocessor Material in addition water and steam adjust homogeneous mixture.This has started the cooking in starch fraction (binding component) (cooking) process.Material is fed in granulator.Mold of the material by granulator is forced, and will in the outside of mold Thread (string) is broken into pellet.Moisture is low, therefore dry feed is not required.

Then the other oil comprising fish feed composition according to the present invention is sprayed onto on pellet surface, but due to pellet It is quite fine and close, so total lipid content rarely exceeds 24%.The oil of addition can be fish oil or vegetable oil, such as rapeseed oil or beans The mixture of the mixture or fish oil and vegetable oil of oil or vegetable oil.After oil cladding, pellet is cooled down and filled in cooler Bag.Final compacting fish meal contains the composition of the present invention of 10-5000ppm.

Embodiment 2：The method for preparing extruding fish feed

Main raw material(s) is ground and mixed.Then the micro constitutent comprising fish feed composition according to the present invention is added Enter in mixer.Homogeneous mixture is adjusted by adding water and steam in the material into preprocessor.It is gone back in this stage Additional oil can be added into material.This has started boiling (cooking) process in starch fraction (binding component).By object Material is fed in extruder.Extruder can be single-bolt type or double-screw type.Due to the rotary motion of material in an extruder, Material is further mixed.Additional oil, water and steam can be added into the material in extruder.In the end of extruder, object Expect that there is the temperature higher than 100 DEG C and the pressure higher than environmental pressure.Material is forced to pass through the opening in extruder template.Due to The release of temperature and pressure, some moisture can evaporate (flash distillation) immediately, therefore extruded material becomes porous.By rotating knife by line Shape object is cut into pellet.Water content is quite high (18-28%), therefore immediately contains the water that pellet is dried to about 10% in drier Amount.

It, can be by the way that oil be sprayed onto on feed surface or by the way that feed is dipped in oil, thus to feeding after drier More oil are added in material.Oil is added in feed (vacuum coated) in closed container of the wherein air pressure less than environmental pressure It is advantageous, this makes porous feed pellets absorb more heavy wool.It can be in this way containing the feed for having more than 40% lipid Production.After coating machine, by feed cooling and pack.In method as described above, oil can be added in several positions, and And can be fish oil or vegetable oil, such as the mixture of the mixture or fish oil and vegetable oil of rapeseed oil or soya-bean oil or vegetable oil.

In order to grow and keep good health state, fish to need protein, fat, minerals and vitamins.Eat meat fish Diet it is especially important.Initially, in the cultivation of carnivorous fish, full fish (whole fish) or broken fish (ground fish) are used To meet the nutritional need of cultivation fish.The broken fish mixed with various dry raw materials (such as fish meal and starch) is referred to as soft feed or half Moist forage.With aquaculture industry, soft feed or semi-wet feed are pressed dry feed replacement.Its own is gradually extruded dry feed It substitutes.

Nowadays, feed is squeezed out in the cultivation of many fish species (such as various types of salmons, gadus, perch and madai) It is almost universal.

Main protein source in the dry feed of fish is the fish meal of different qualities.Other animal protein sources are also used for doing Fish meal.Therefore, using blood meal, bone meal, feather meal and the other kinds of powder such as chicken generated by other slaughterhouse wastes Powder is known.They are usually cheaper than fish meal and fish oil.However, in some geographic areas, it is forbidden to use this kind of raw material life Produce the feed of the animal and fish for producing food.

Use vegetable protein such as gluten, maize (corn) seitan, soybean protein, feather fan bean powder, peameal, beans Powder, ground oil seeds, sunflower powder and rice flour are also known.

Embodiment 3：Assess reactive compound according to the present invention to Lactococcus garvieae and The influence of the survival of Streptococcus iniae

The present composition is measured in vitro to resist Lactococcus garvieae and Streptococcus iniae Microbial activity.

Feeding scheme：

1. individual lysozyme continues 48 hours

2. lysozyme continues 24 hours；Essential oil is added above, continues next 24 hours

3. mixing and adding lysozyme and essential oil, continue 48 hours

Lysozyme：007 batch 9PPL 38353T.reesei, 52g EP/kg of LBF

- 1/2 essential oil combination

50% α firpenes

50% cinnamic acid

In test, using following biology, growth medium, condition of culture and appraisal procedure：

Bacterium：The pathogenic strain of all detections belong to University of Bern (Switzerland) Veterinary Pathological Research fish and The bacterial strain preservation in wild animal health center.

The determination of suitable bacterium dilution：It is thin from 24 hour age on sheep blood agar (Biomerieux, Geneva) It is shifted in bacterium subculture in extremely sterile NaCl on a small quantity, until obtaining 0.5 McFarland values.Since the solution, having There is the dilution that in TSB 3%, 1.5% and 0.75% is formed on 96 orifice plates of round bottom.Each hole receives 100 μ l total volumes.22 At DEG C after 24 hours, the growth of bacterium is assessed.The dilution for covering the apparent spot in boundary line of round bottom hole half is caused to be selected For testing.

The measurement of solvent effect：The detection substance in TSB agar is dissolved using ethyl alcohol (ETOH).It can in order to determine ethyl alcohol It can influence, using the ethyl alcohol final concentration (0.1%, 0.05% and 0.025%) calculated in the Bacteria Detection detection hole of various concentration, Any influence to bacterial growth is not shown.

Detect the concentration of substance：In view of dietary concentration likely is at least 1000ppm and daily uptake ratio is 2%, estimate external dosage range.Potential concentration in enteron aisle is determined as 0.1 μ l/100 μ l of highest.Then detection series 2 is dilute Degree of releasing, so as to cause the final concentration of 0.85 μ g/ml of substance；0.42 μ g/ml and 0.21 μ g/ml are (when being adjusted to average essential oil density When).

From each substance, the liquid storage being made of 2 μ l substances, 18 μ l ETOH and 180 μ l PBS is prepared.

The preparation of plate：Detect three concentration (0.21 μ g/ml, 0.42 μ g/ml and 0.85 μ g/ml) each three of each substance Part.Include the positive control and blank control (PBS) being made of the bacterium in TSB on every block of plate.Also include ETOH on every block of plate Three dilution in addition each three parts.

The reading of plate：After being incubated 24 hours at 22 DEG C, using 0 point (no bacterial growth=hole bottom is without point) to 3 minutes (just Be frequently grown=spot size is comparable to the spot size of positive control) read plate.

It obtains following result and summarizes in fig 1 and 2.

Claims

1. at least one selected from australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene Natural active matter is used to improve the feed of aquatic animal with the combination of the polypeptide with lysozyme activity in fodder compound The purposes of conversion ratio and/or daily gain.

2. selected from australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene at least two Natural active matter is used to adjust the enteron aisle of aquatic animal with the combination of the polypeptide with lysozyme activity in fodder compound The purposes of microorganism species.

3. selected from australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene at least two Natural active matter with lysozyme activity polypeptide combination in fodder compound for reducing the aquatic animal death rate Purposes.

4. purposes according to any one of claim 1-3, wherein the aquatic animal is cold water fish, such as salmon, Trout, bream or perch.

5. according to the purposes described in any one of claim 1 or 3, wherein the aquatic animal is warm water fish, such as carp, Tilapia mossambica or catfish.

6. purposes according to any one of claims 1-5, wherein the fodder compound contains australene and cinnamic acid.

7. purposes according to claim 6, wherein the fodder compound contain concentration every Kg feeds 10mg-5g it Between australene, cinnamic acid, dihydroeugenol and metacresol.

8. purposes according to claim 6, wherein the fodder compound contain concentration every Kg feeds 0.1g-1g it Between australene, cinnamic acid, dihydroeugenol and metacresol.

9. the purposes described in any one of claim 1-8, wherein the microbic muramidase is originated from fungus.

10. the purposes described in any one of claim 1-9, wherein the microbic muramidase be obtained from or available from Ascomycota.

11. the purposes described in any one of claim 1-10, wherein the microbic muramidase be obtained from or available from Pezizomycotina subphylums.

12. the purposes described in any one of claim 1-11, wherein the microbic muramidase includes one or more choosings From the structural domain of GH24 and GH25.

13. the purposes described in any one of claim 1-12, wherein the microbic muramidase is selected from：

(a) with SEQ ID NO:1 have at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity it is more Peptide；

(b)SEQ ID NO:1 variant, wherein the variant have lysozyme activity and 1,2,3,4,5,6,7,8,9, 10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50 positions include one or more amino acid It replaces and/or one or more amino acid deletions and/or one or more amino acid is inserted into；

(c) (a) with lysozyme activity or (b) segment of the polypeptide, wherein the segment includes at least 170 amino Acid, for example, at least 175 amino acid, at least 177 amino acid, at least 180 amino acid, at least 185 amino acid, at least 190 amino acid, at least 195 amino acid or at least 200 amino acid；

(d) with SEQ ID NO:4 have at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity it is more Peptide；

(e)SEQ ID NO:4 variant, wherein the variant have lysozyme activity and 1,2,3,4,5,6,7,8,9, 10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50 positions include one or more amino acid It replaces and/or one or more amino acid deletions and/or one or more amino acid is inserted into；With

(f) (d) with lysozyme activity or (e) segment of the polypeptide, wherein the segment includes at least 210 amino Acid, for example, at least 215 amino acid, at least 220 amino acid, at least 225 amino acid, at least 230 amino acid, at least 235 amino acid or at least 240 amino acid.

14. the purposes described in any one of claim 1-13, wherein the microbic muramidase is selected from SEQ ID NO:1 ammonia Base acid 1-208 and SEQ ID NO:4 amino acid 1-245.

15. selected from australene, α-terpineol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene at least two The combination of kind natural active matter and the polypeptide with lysozyme activity according to any one of claim 9-14 is used for Treat and prevent the purposes of the disease caused by pathogenic microorganisms in aquatic animal.

16. for the fodder compound or premix composition or feed addictive of aquatic animal, it includes selected from australene, α-terpene Product alcohol, cinnamic acid, dihydroeugenol, eugenol, metacresol and terpinolene at least two natural active matters with according to power Profit requires the combination of the polypeptide with lysozyme activity described in any one of 9-14 as main component.