CN106260502A - A kind of method prepared containing surface element feed additive - Google Patents
A kind of method prepared containing surface element feed additive Download PDFInfo
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- CN106260502A CN106260502A CN201510240490.7A CN201510240490A CN106260502A CN 106260502 A CN106260502 A CN 106260502A CN 201510240490 A CN201510240490 A CN 201510240490A CN 106260502 A CN106260502 A CN 106260502A
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Abstract
The invention discloses a kind of method prepared containing surface element biology feed additive, the method is the bacillus subtilis mutant strain (B.S.surfactin 1) utilizing and having high surface element (surfactin) yield, semi-solid ferment is carried out as substrate using Semen Glycines, again by the Semen Glycines after fermentation after being dried and being crushed into an analysis for soybean powder, obtain a feed additive containing surface element；Wherein containing surface element about 6-7g in per kilogram fermentation analysis for soybean powder.Present invention discover that feeding contains the cabrilla of the feed additive of surface element, its growth rate is very fast, the expression of immunologic pattern gene higher (such as: AMP, Mx and IFN-induced protein) and the Fish of bacteria infection, its mortality rate declines to a great extent, and shows that this feed additive containing surface element has the effect promoting growth, increasing the infection of immunity and opposing source of disease bacterium.
Description
Technical field
The invention belongs to the technical field of aquaculture in agriculture field and biological technical field, relate to one and utilize withered
The production method on microorganism lipopeptid prepared by grass bacillus cereus-surface element (surfactin) and answering in aquatic feeds thereof
With.
Background technology
The secondary metabolic product that bacillus subtilis (Bacillus subtilis) produces has beta-lactam (β-lactams), amine
Base glucosides (aminoglycoside), policapram derivant (polyketides) and small-sized win peptide (small more
Polypeptides), wherein relatively attracted attention with lipoprotein (lipopeptides and lipoproteins) with lipopeptid, because of
Having antibacterial effect for lipopeptid and lipoprotein, be otherwise known as antibacterial victory peptide (Antimicrobial Peptide, AMP).
The antibacterial victory peptide having now been found that can be divided into three families, and one is Feng Yuansu (Fengycin), by ten in molecule
Amino acid molecular composition ring bark peptide chain, in victory peptide chain N termination, 14 to 18 fatty acid side chains not waited, have
Good fungal resistance, but obvious effect (Schneider et al., 1999) is not had for yeast and antibacterial；Separately
One is her subtilicin (Iturin), connects containing seven α-amino acid ring and coupled to beta-amido acid and alkyl chain,
Being connected to 14 to 17 fatty acid side chains not waited on victory peptide chain, molecular size range about 1044～1081kD, function is
To antifungal and antibacterial, have the advantage such as biological degradability, high surface and hypotoxicity (Bonmatin,
Laprevote,&Peypoux,2003)；3rd surface element (Surfactin) then inquired into by the present invention, it contains
7 cyclic aminocarbonyl acid and 13 to 16 fatty acid side chains not waited, bacillus subtilis is when producing surface element, logical
Often meeting occurs along with a subtilicin, in the presence of two kinds of antibacterial victory peptides are common, and can be because mixing micella
Cell is had high-affinity so that erythrocyte hemolysis rate increase, the most also can increase antifungal activity (Feignier,
Besson,&Michel,1995；R Maget-Dana,Thimon,Peypoux,&Ptak,1992；Sandrin,
Peypoux,&Michel,1990)。
Arima et al. finds a white, needle-shaped crystals in nineteen sixty-eight in bacillus subtilis bacteria culture fluid, because it has
There is interfacial activity therefore by its named surface element (Kakinuma, Hori, Isono, Tamura, 1969).?
The research in past is pointed out, when the surface containing 20 μMs in water is plain, and they can be by the surface tension of aqueous solution by 72
MN/m is down to 27mN/m, be have now been found that the optimal bioelectric interface activating agent of interfacial activity (Arima, Kakinuma,
&Tamura,1968).Surface element is a ring-like fat victory peptide, and it contains 7 cyclic aminocarbonyl acid with 13 to 16 not
Deng fatty acid side chain (Kakinuma, Sugino, Isono, Tamura, &Arima, 1969).In the solution, fat
Acid carbochain is hydrophobic property, and ring-type amino acid is then water-wet side, the acid, aspartic (aspartic in amino acid
Acid) and glutamic acid (glutamic acid) is the most electronegative, two electronegative amino acids can form tong-like, makes surface
Element molecule form electronegative saddle structure, belong to anion bioelectric interface activating agent a member (Tsan, Volpon,
Besson ， &Lancelin, 2007), according to the difference of Amino acid sequence, surface element can be divided four types: A, B,
C、D.Difference (the Shaligram& of the fatty acid carbon chain number that it is connect after main difference is that cyclic aminocarbonyl acid
Singhal,2010；Singh&Cameotra,2004).
Due to surface element non-specific biological activity, make it can by reduce surface tension to destroy antibacterial
Cell membrane, and do not result in Drug resistance, therefore it is considered to have antibacterial potentiality, and is classified as antibacterial lipopeptid
A member (Cho, Lee, Cha, Kim, &Shin, 2003).Research in the past point out surface element can suppress fungus,
The growth of mycoplasm hyopneumoniae, gram-negative bacteria and gram positive bacteria (P.Das, Mukherjee, &Sen, 2008；
Singh&Cameotra,2004)；Heiko et al. finds i.e. have sterilization energy when surface element concentration at 12～50 μ g/ml
Power (Heerklotz&Seelig, 2001)；Lipopeptid biological interfacial activity produced by Bacillus circulans in ocean
Agent (lipopeptide biosurfactant), for minority anti-multi-medicament bacterial strain (multidrug-resistant strain,
MDR) and resistance to penicillin staphylococcus aureus (methicillin-resistant Staphylococcus aureus,
MRSA) there is the effect (Pan et al., 2007) of suppression；Surface element also has the characteristic of anti-mycoplasm hyopneumoniae, can recover
The kenel feature of the cell polluted by mycoplasm hyopneumoniae, and injury will not be produced for cell metabolism and propagation
(Vollenbroich,Pauli,Ozel,&Vater,1997)；Find that surface element can make mycoplasm hyopneumoniae via ultramicroscope
Film forms perforated phenomenon, osmotic pressure inside and outside mycoplasm hyopneumoniae now can be caused uneven and dead (R é gine Maget-Dana
&Ptak,1995)。
Current research thinks that the Antiviral Mechanism of surface element is the lipid-coat film by virus and the thing of surface element
After Physicochemical effect, cause viral coat film and protein coat structural collapse, and suppress viral nucleic acid material to enter
Row duplication (Vollenbroich,,Vater,Kamp,&Pauli,1997).Past research points out that surface element can be right
Anti-good several viruses, including mankind's acquired immunity not totivirus (Human Immunodeficiency Virus,
HIV), herpesvirus suis the first type (Suid Herpes virus type 1, SHV-1), herpes simplex virus the first type
(Herpes Simplex Virus 1, HSV-2), stomatitis follicularis virus (Vesicular Stomatitis Virus,
VSV), simian immunodeficiency virus (Simian Immunodeficiency Virus, SIV), cat flu virus
(Feline Calicivirus, FCV), murine encephalomyocarditis virus (Murine Encephalomyocarditis Virus,
MECV), Pseudorabies virus (Pseudorabies Virus, PRV), pig parvoviral (Porcine Parvovirus,
PPV), new castle disease virus (Newcastle Disease Virus, NDV), infectiousness French capsule syndrome virus
(Infectious Bursal Disease Virus, IBDV) (Huang et al., 2006；Itokawa et al.,1994；
Vollenbroich,,et al.,1997)。
Antibiotic antibacterial mechanisms is that antibiotic is combined with the receptor of pathogen specific part, makes the normal framework of pathogen
Destroyed or hindered some biosynthesis, to reach the antibacterial or effect of sterilization.There is research at present towards antibacterial victory
The angle of peptide inquires into surface element, and owing to the plain and antibacterial victory peptide in surface is all amphiphatic molecule, but the structure of surface element is
Ring-like, and antibacterial victory peptide is according to the difference of structure, it is divided into four classes: α-helix, β-sheet, β-turn loop,
Boat-shaped.Antibacterial victory peptide and cell membrane mechanism of action are to utilize victory peptide lipid reciprocal action (Peptide-lipid
Interaction), four patterns it are divided into: accumulation mode (Aggregate model), annular hole pattern (Toroidal
Pore model), tubular gives a farfetched interpretation formula (Barrel-Stave model) and carpet cover type (Carpet model), wherein
These binding modes are the reciprocal action via victory peptide lipid mostly, cause osmotic pressure inside and outside film uneven, make thalline
Dead.Mechanism of action between surface element and cell membrane there is no final conclusion at present, but mainly has several hypothesis: 1. class is clear
Clean dose (detergent-like): this kind of mechanism is to win a part of intermediate layer inserting film of peptide chain and then cause the state of film
Extremely unstable (stronger destabilization) in turn results in film and produces crack (leak) and separate
(partitioning)(Lohner and Epand,1997；Heeklotz et al.,2004)；2. the formation of hole (vesicle):
This mechanism can be divided into three parts, (1) surface element by and film between hydrophobic forces (hydrophobic
Interaction) film surface is inserted；(2), in surface element structure, electronegative amino acid can be with electronegative lipid head
Portion (lipid headgroups) produces electrical charge rejection (charge repulsions), in turn results in film bending；(3) film body pole is not
Stable and produce the structure of similar micella (micelle) cause film disintegrate (Sebastien Buchoux et al., 2008；
Huang et al.,2009).Current research thinks that the non-polar end of surface element can be with the virus having mantle (envelope)
Effect, and on film, form hole (pore), and then generation ion channel (ion channels) destroys the film of whole virus
Body (Vollenbroich et al., 1997)；The outer sheath protein (capsid) destroying the virus not having mantle about surface element grinds
Study carefully less, but Vollenbroich et al. thinks that surface element is mainly non-fat by victory peptide moiety (peptide moiety)
Fat acid carbochain with virus epitheca effect and cause virus injury (Vollenbroich et al., 1997).These two kinds of theories
Difference be one advocate crack generation, and another one advocate hole formation.Comprehensive above saying is permissible
That learns surface element can cause the impaired of film itself really, and then has influence on the normal physiological function of cell.At present
Research finds, gram positive bacteria, gram negative bacteria, fungus and protozoacide can be had broken by antibacterial victory peptide
Bad ability (Powers and Hancock, 2003)；Document is additionally had to point out, antibacterial victory peptide and procaryotic core
After acid combines, the generation (Powers and Hancock, 2003) of protein can be suppressed.Compared with traditional antibiotic,
The activity of antibacterial victory peptide has broad-spectrum, high efficiency, stability, quick sterilization ability and immune phase interaction
With etc. feature.
Report is pointed out, the microbial balance using heavy damage animal intestinal of Antibiotic Additive, and easily animal
Internal residual, has a strong impact on the quality of livestock products and the health of the mankind, and uses antibacterial victory peptide as feed additive
Can solve the problem that the problems referred to above, this is also that antibacterial victory peptide is the most directly applied in animal husbandry.In higher mammal intestinal
Antibacterial victory Toplink suppresses exogenous pathogen, and the normal flora that there is animal intestinal and cell are without lethal effect.
Antibacterial victory peptide components is the amino acid of absorption easy to digest, can replace mesh as additive for farm animal feed and replacement or part
Antibiotic used by front animal, reduces the antibiotic harm to animal body.Antibacterial victory peptide can replace conventional antibiotic,
The various thorny diseases such as treatment piglet diarrhea, swine fever, newcastle disease and mammitis of cow, will not develop immunity to drugs
Antibacterial, nontoxic residue-free, livestock and poultry treatment with prevent in have broad application prospects.Antibacterial victory peptide is made
For the many merits of feed additive, just causing the extensive concern of people, be expected to become substitute antibiotics product it
One.At present, both at home and abroad as the mainly giant silkworm antibacterial victory peptide AD-yeast preparation of feed additive production application,
The test of most poultry is also around what this antibacterial victory peptide was carried out.
Feedstuff and feedstuff are global problems by mould contamination.Occur to reduce mould contamination, right
The most contaminated feedstuff takes suitable process and to reduce the mycotoxin harm to animal health to greatest extent, is
International herding for many years, veterinary, the important topic of feed technology circle research.Initially people are through controlling to contain in feedstuff
The amount that means such as the water yield, ambient temperature, humidity and reducing are gone mouldy.Along with chemical and microbiological development, people
Developing chemical preservative, currently used more mainly have organic acid, acylate (ester) and composite mildewproof
Agent three major types, but they all have toxicity to edible.And antibacterial victory peptide preservatives is a kind of protein, in digestion
System is easily degraded into amino acid, edible safety little to mankind's side effect, such as nisin (Nisin),
In the whole world more than 50 country to be widely used in food antiseptic fresh-keeping.Originate antibacterial victory natural, that have no side effect
The trend of peptide preservatives the most substituted traditional chemical preservative, it is believed that following antibacterial victory peptide is in feedstuff industry and phase
The anticorrosion of some industry products closed will take on important role.
Due to the antibiotic a large amount of uses in poultry and aquatic animal, medicine is serious in poultry and aquatic animal body
Residual and the generation of antibiotic-resistant bacteria make the safety problem of animal food increasingly severe, threaten the body of the mankind
The healthy development with aquaculture of body, and huge loss is brought for the livestock products export trade.Antibacterial victory peptide because
There is broad-spectrum antibacterial activity, quick sterilization, be not likely to produce drug-fast strain and can have collaborative with conventional antibiotic
The features such as effect, and be considered to have broad application prospects.The more important thing is, antibacterial victory peptide is at sequence and framework
On there is multiformity, for new drug design provide the biggest imagination and play space, the product of stable performance can be produced
Product.
When antibacterial victory peptide is as feed additive, feed additive production can be met with high temperature during resistance to feed granulating
The requirement of flow process.When antibacterial victory peptide is as antibiotic, its bactericidal mechanism is unique, has broad-spectrum antibacterial action, right
Aquatic animal and poultry have acceleration of growth, keep healthy and treat the function of disease, and have no side effect, nothing
Residual, without problems such as Drug resistance generations, and production cost is low the most not by the shadow of the external environment such as season and climate change
Ring.Industrialization produces the antibacterial victory peptide of the aquatic microorganisms containing natural activity, can meet Aquatic product and animal husbandry development
Demand, the development of expansion green feed additive industry.
Antibacterial victory peptide is just to grow up as the research of feed additive recent years, also in the exploratory stage, away from
A lot of problems are also had to need to solve 1. antibacterial victory peptides in forming the technology of maturation, being applied on a large scale produce
Natural resources are limited, and chemosynthesis and genetic engineering become the Main Means into obtaining antibacterial victory peptide；2. the one-tenth of synthesis
This costliness, the most also cannot produce on a large scale；3., through genetic engineering, microorganism is directly expressed antibacterial peptide
Gene, is likely to result in microorganism dead, or the problem that Product Expression amount is very few.The amount of current general antibacterial victory peptide
Product pattern is to utilize synthesis bacterial strain to carry out liquid fermentation to be produced mostly, and its shortcoming is that the liquid fermentation of 1. initial stages produces
Equipment is the highest with investment cost, and investment repayment is slow；If 2. without purifying procedure, directly will synthesis bacterial strain as
Feed additive, has GMO base and changes the doubt of additive.Therefore research and develop natural, (resisting) bacterium, valency can be killed by broad-spectrum
The antibacterial victory peptide of the substitute antibiotics of the cheap and non-GMO of lattice is medicine, the important topic of feed additive research and development.
The present invention is to utilize the bacillus subtilis mutant strain of tool surface element yield, with Semen Glycines as fermentation substrate, carries out
Semi-solid ferment, then by after the Semen Glycines drying after fermentation and pulverizing, by the analysis for soybean powder of gained as feed additive.
Summary of the invention
A kind of method that it is an object of the invention to provide feed additive prepared containing surface element.
The method comprises the following steps,
A, the bacterium solution of bacillus subtilis (Bacillus subtilis) mutant of high yield surface element is seeded to inorganic salt
Fluid medium, with the mixture of Semen Glycines, carries out semi-solid ferment, obtains a Semen Glycines fermented, wherein, institute
The bacillus subtilis mutant strain of the high yield surface element stated, named B.S.surfactin 1, Classification And Nomenclature is hay bud
Spore bacillus (Bacillus subtilis), is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, and its culture presevation is numbered
For CGMCC No.10270, preservation date is December in 2014 31.
B, by this Semen Glycines fermented dry, grind and sieve, obtain a Semen Glycines powder；This Semen Glycines powder is
Feed additive containing surface element (surfactin).
In the present invention, it is preferred to, connecing of the bacterium solution of the bacillus subtilis mutant strain of the high yield surface element of step A
Planting volume with the ratio of Semen Glycines volume in this mixture is 5:100-10:100.
In the present invention, it is preferred to, the volume of inorganic salt liquid culture medium and Semen Glycines body in this mixture in step A
Long-pending ratio is 25:100-35:100.
In the present invention, it is preferred to, the semi-solid ferment of step A is at temperature 30-40 DEG C, humidity 80-90%,
Ferment 2-3 days.
In the present invention, it is preferred to, the drying of step B is at temperature 50-60 DEG C, 2-3 hour time, carries out
Dry.
In the present invention, it is preferred to, the Semen Glycines powder of step B, its surface cellulose content is 6-7 g/kg.
It is an object of the invention to provide a kind of method prepared containing surface element feed additive, this feed additive is should
For the feedstuff of aquatic biological (such as: cabrilla or South America white shrimp), there is the promotion rate of growth of aquatic biological, increase
Immunity and the activity of antibacterium, antifungal and antiviral.
Accompanying drawing explanation
Fig. 1 is the neutralization Potency Analysis utilizing the surface element of variable concentrations to process virus infected cell strain.
Fig. 2 A of Fig. 2 is the kenel impact that nervous necrosis virus granule is caused by surface element, (A) 1%L-15 process group；
(B) 1%L-15+100 μ g/ml surface element mixed processing group；(C) 1%L-15 nervous necrosis virus mixed processing group, arrow
Head is pointed out to represent normal virion；(D) 40 μ g/ml surface elements+nervous necrosis virus mixed processing group, arrow refers to
Go out and represent abnormal virion；(E) 100 μ g/ml surface element and nervous necrosis virus mixed processing group, arrow refers to
Go out and represent abnormal virion.Fig. 2 B be surface element irido virus granule is caused kenel impact, (A) just
Normal irido virus granule group；(B) 10 μ g/ml surface elements+irido virus mixed processing group, this virion outward appearance
Deformation；(C) 40 μ g/ml surface elements+irido virus mixed processing group, has between surface element and virion and is stained with glutinous showing
As.
Fig. 3 is to promote that South America white shrimp is grown up containing surface element feed additive to test.
Fig. 4 is to promote that cabrilla is grown up containing surface element feed additive to test.
Fig. 5 is the expression increasing immunologic pattern gene containing surface element feed additive.
Fig. 6 A of Fig. 6 is containing the mortality rate of cabrilla after surface element feed additive reduction infection nervous necrosis virus.
Fig. 6 B is containing the mortality rate of cabrilla after surface element feed additive reduction infection irido virus.
Detailed description of the invention
Further describing the present invention, advantages of the present invention and feature below in conjunction with specific embodiments and the drawings will be with
Description and apparent.But embodiment is only exemplary, the scope of the present invention is not constituted any restriction.
It will be understood by those skilled in the art that can be to the technology of the present invention under without departing from the spirit and scope of the present invention
The details of scheme and form are modified or replace, but these amendments and replacement each fall within protection scope of the present invention
In.
Embodiment one. prepare the feed additive containing surface element in semi-solid ferment mode.
The present invention is to use the bacillus subtilis mutant strain (depositing numbering CGMCC No.10270) of high yield surface element
With Semen Glycines as substrate, carrying out semi-solid ferment, the Semen Glycines rich surface that this fermented contains secondary metabolic product-surface element；
Fermentation substrate may be used without the cheapest fermentation substrate of other prices such as the dish son dregs of rice, Petiolus Trachycarpi dregs, coconut palm son's dregs of rice, agricultural
Side-product or leftover bits and pieces (peel of Rhizoma Solani tuber osi, Maninot esculenta crantz. skin, Pericarpium Mali pumilae).
Being soaked 16 hours by organic Semen Glycines (U.S.'s product), the ratio with 10% water yield of Semen Glycines amount as addition is in 121 DEG C
Steaming and decocting 30 minutes, after cooling to room temperature the bacillus subtilis sudden change of the high yield surface element of inoculation Semen Glycines volume 10%
The bacterium solution of strain (CGMCC No.10270), and add the inorganic salt liquid culture medium of Semen Glycines volume 30% respectively,
Carrying out semi-solid ferment, its condition is temperature 30 DEG C, humidity 80%, and fermentation time is 48 hours, then by after fermentation
Semen Glycines through 55 DEG C dry 2-3 hour, pulverize after through 60 eye mesh screens, be placed in 4 DEG C of preservations, wherein per kilogram Semen Glycines
Powder contains the surface element of 6-7 gram.
This inorganic salt liquid culture medium includes 2-6% (v/v) glucose, 35-45mM dibastic sodium phosphate (Na2HPO4)、
25-35mM potassium dihydrogen phosphate (KH2PO4), 45-55mM ammonium nitrate (NH4NO3), 5-10mM calcium chloride
(CaCl2), 2-6mM sodium ethylene diamine tetracetate (Sodium EDTA), the aqueous magnesium sulfide of 750-850mM
(MgSO4.7H2O), the aqueous iron sulfide of 1-3mM (FeSO4.7H2O)。
Embodiment two. the bacteriostatic test of surface element
In order to inquire into whether surface element has antibacterial activity, therefore carry out extracorporeal maleate sensibility test.
Antibacterial bacterium solution is prepared respectively by escherichia coli (Escherichia coli DH5 α), Vibro harveyi (Vibrio
Harveyi), molten bath vibrio (Vibrio alginolyticus), Vibrio anguillarum (Vibrio anguillarum), salmon vibrio (Vibrio
Salmonicida), Aerononas punctata (Aeromonas hydrophila), staphylococcus epidermidis (Staphylococcus
Epidermidis) it is incubated on LBA (E.coli DH5 α) or TSA (+1.5%NaCl), cultivates 16 hours in 37 DEG C
After, scrape bacterium colony and be dissolved in appointment culture fluid；Make OD540When being 1, (concentration is about 1 × 109/ c.c. bacterium number), take 100 μ l
Bacterium solution adds LB or TSB (+1.5%NaCl) of 900 μ l, makes bacterial concentration reach 5 × 105CFU/ml。
Prepared by mycete bacterium solution
Aspergillus niger (Aspergillus niger) is incubated on MEAIII flat board, after 25 DEG C of trainings 48 hours, scrapes bacterium
Fall to being dissolved in appointment culture fluid.Via serial dilution and be applied on MEAIII flat board, calculate clump count and calculate original liquid concentration
It is 1 × 106CFU/ml, takes the MEB that 500 μ l bacterium solution add 500 μ l afterwards, bacterial concentration is diluted to 5 × 105
CFU/ml。
Sensitivity tests (Susceptibility test)
Adding the bacterium solution of 130 μ l at 96 hole culture dishs at each groove, bacterial concentration is 5 × 105CFU/ml, adds
20 μ l are diluted to the surface element of variable concentrations by titration mode, after cultivating 16 hours in 37 DEG C, use light splitting luminance meter
Calculating thalli growth concentration, the data obtained is in comparison with original initial value (OD540When being 1), with equal to initial value (thalline
There is no hypertrophy) experimental group be minimal inhibitory concentration (Minimun inhibiting concentration, MIC), Mei Geshi
Test group and be respectively arranged with three repetitions and matched group.
Result of the test is as shown in Table 1: the surface element produced with bacillus subtilis ATCC21332 compares, by height
Producing the bacillus subtilis mutant strain of surface element, the surface produced its minimal inhibitory concentration of element is 96.5 μ Μ.
The bacillus subtilis mutant strain of table one, bacillus subtilis ATCC21332 and high yield surface element is produced
The minimal inhibitory concentration (μ Μ) of surface element compares.
Embodiment three. surface element with nervous necrosis virus in and reaction test
In order to inquire into whether surface element has antiviral activity, therefore it is neutralized reaction test.In testing the previous day,
GF-1 cell is cultivated at 96 hole microflat bottom culture plates, each hole inoculation 5 × 103Individual cell, the next day for the treatment of (16 hours)
Cell grow into eight points full time, and power valency mensuration can be carried out.The most first by virus liquid to be determined, with 1%
FBS-L15 does continuous ten times of dilutions, and extension rate is by 101～1010, then the virus liquid diluted is seeded to respectively
In 96 porose discs, 100 μ l are inoculated in each hole, uniformly rock culture plate dispersed by virus liquid, and totally 8 repeat；Will training
Foster dish is placed in 28 DEG C of incubators, allows viruses adsorption after 90 minutes, changes old culture fluid, fills into 200 μ l's
1%FBS-L15 culture fluid, puts back in 28 DEG C of incubators by culture plate, and after cultivating 9 days, observation of cell becomes situation.Sick
Toxic effect valency (50%tissue culture infection dose, TCID50) is based on the side of Reed-Muench Method
Method calculates, and represents with TCID50, and calculating neutralization index Neutralization index (the NI=former power valency of virus/through table
Virus titer NI after the element neutralization of face), NI value is the highest, represents that the plain neutralization titer obtained in surface is the highest.Neutralize reaction
Operation is 10 times of dilutions of series, neutralizes the decision procedure of titer, and when neutralization index, less than 10, (i.e. Log10NI value is less than
1), for without neutralizing effect；Neutralization index between 10～50 (Log10NI value between 1～1.6) for neutralizing effect not
Significantly, the scope that leaves a question open is belonged to；And when neutralization index is more than 50 (Log10NI value is more than 1.7), then it is judged to have aobvious
The neutralization effect (Grace, 1979) write.Result of the test is as shown in Figure 1.
Embodiment four. surface element and virus function mechanism
Being dissolved in aquesterilisa by surface element, after filtering with 0.2 μm filter membrane, recycling 1%FBS-L15 is diluted to
40 μ g/ml and 100 μ g/ml.Surface element process group: surface element is mixed with nervous necrosis venom with 1:1 volume ratio
Close；Matched group: 1%FBS-L15 culture fluid mixes with nervous necrosis venom, two groups mix respectively after at 28 DEG C
Cultivate one day, then with transmission electron microscope (Transmission electron microscope, Hitachi H-600)
Observe.Take 6 μ l surface element process groups drip on copper mesh and make virus sedimentation about 10 minutes, then with the 1% of pH 7.4
Phosphotungstic acid (PTA, Sigma) dyes 10 minutes, after having dyeed, sucks unnecessary mixing with filter paper
Close liquid and place room temperature 30 minutes, making copper mesh observe immediately after drying in the shade.Transmission electron microscope use voltage is
75KV, egative film time of exposure is 4 seconds.Egative film rinses Yu Haiyang university electricity Xian center dark place, the egative film that will take out
Be positioned on punching frame with developing agent D19 develop 4 minutes, after develop again with 0.33% glacial acetic acid stopping development 2
Minute, then soak fixative 10 minutes, in the way of flowing water, wash away unnecessary medicament the most again after 60 minutes, cloudy
Dry bottom sheet.Result of the test is as shown in Figure 2.
Embodiment five. the feed additive containing surface element promotes South America white shrimp growth test
Fermentation Semen Glycines powder is pulverized with commercially available shrimp feed, after adding sterilized water mix homogeneously, utilizes feedstuff squeezer to make
Graininess, places baking oven and is dried 3 hours in 70 DEG C, the moisture in feedstuff is down to 10%, and by dried feedstuff
It is placed in 4 DEG C of Refrigerator stores.Test of growing up divides four feedstuff process groups, and respectively 1. add fermentation its surface elements of Semen Glycines powder contains
Amount is 10ppm；2. adding fermentation its surface cellulose content of Semen Glycines powder is 20ppm；3. add water washed after fermentation Semen Glycines powder 2%；
4. the water washed after fermentation Semen Glycines powder 5% of interpolation and matched group: commercial diet.Often group is 25 white shrimp shrimp Seedlings, and weight in wet base is about
0.6g, experimental trough is the clear glass cylinder of 30 centimeters × 24 centimeters × 60 centimeters, and interior water content is about 45 liters, real
Testing period not temperature control, persistently inflate, each quantity of exchanged water is about 1/3.Every day, feeding volume was about the 5% of shrimp weight, threw
The time of feeding is morning 8 every day and point in afternoon 5, its weight in wet base of scale per week, records and calculate its rate of growth.Test knot
Fruit is as shown in Figure 3.
Embodiment six. the feed additive containing surface element promotes cabrilla growth test
Feed protein is tested formulated with lipid content with reference to Shiau and Lan (1996), and test feed formula is
General feeds, its crude protein is 47%, and lipid is 10%.HONGYU FEN, Semen Maydis muscle albumen, Semen Tritici aestivi muscle albumen and crow
Thief's powder, grinds via pulverizer and sieves (35mesh screen cloth), thereby increases feedstuff cohesiveness, all feedstuffs
After squeezing grain, pelletizing via crowded grain machine and dry, adding 2-5% surface element, ice is stored in-20 DEG C of refrigerators to guarantee feedstuff matter
Amount.Result of the test is as shown in Figure 4.
Embodiment seven. the feed additive containing surface element can induced animal innate immunity gene expression amount
The continuous feedstuff thrown something and fed cabrilla containing 0%, 2% and 5% surface element, can induce cabrilla after the 7th day
Immunologic pattern Gene A MP (Antimicrobial peptide), MX (MX albumen be that interferon (interferon) induces one
Kind of GTPase), the expression of interferon inducible protein (INF-inducted protein).Result of the test is as shown in Figure 5.
Embodiment eight. the feed additive containing surface element increases animal disease resistant ability
Malaber reefcod Seedling, through 7 days feedstuffs containing surface element of throwing something and feeding continuously, infects nervous necrosis for cabrilla
After poison or irido virus, the change of mortality rate.Experiment is divided into 4 groups, 1. negative control group: feeding general feeds+
Injection PBS；2. positive controls: feeding general feeds+injecting virus；3. experimental group: feeding is containing 2% surface element
Feedstuff+injecting virus；4. experimental group: feeding is containing the feedstuff+injecting virus of 5% surface element.Result of the test such as Fig. 6 institute
Show.
Above example discloses the plain aquaculture that must be applied in surface produced by the inventive method and throws something and feeds feedstuff, and
It is adapted to environmental change and multiple cause of disease in water, there is acid and alkali-resistance, high temperature resistant, broad spectrum activity bactericidal effect (gram sun
Property with feminine gender, fungus, parasite, virus), do not result in Drug resistance, and can be degraded by microorganisms, be one
Environment is friendly, green feed additive, does not have drug residue problem, is highly suitable as new antibiotic with anti-
Sick type feed additive.
The inventive method use solid state fermentation, the convection drying that fermented pulverize, feed additive, obtain replacement
General antibiotic, the method production technology is simple, low production cost.
Above-mentioned multinomial effect, the real Statutory Invention patent requirement fully complying with novelty and progressive that belongs to, whence carries in accordance with the law
Go out application, earnestly ask your office and check and approve this part application for a patent for invention case to encourage invention.
Claims (12)
1. the method preparing the feed additive plain containing surface, the method comprises the following steps:
A, the bacterium solution of bacillus subtilis (Bacillus subtilis) mutant of high yield surface element is seeded to inorganic salt
In the mixture of fluid medium and Semen Glycines, carry out semi-solid ferment, obtain a Semen Glycines fermented, wherein,
The bacillus subtilis mutant strain of described high yield surface element, named B.S.surfactin 1, it is deposited in China
Microbiological Culture Collection administration committee common micro-organisms center, the numbered CGMCC of its culture presevation
No.10270；
B, by this Semen Glycines fermented dry, grind and sieve, obtain a Semen Glycines powder；This Semen Glycines powder is
Feed additive containing surface element (surfactin).
2. preparation as claimed in claim 1 is containing the method for the feed additive of surface element, the wherein high yield surface of step A
The inoculation volume of the bacterium solution of the bacillus subtilis mutant strain of element with the ratio of Semen Glycines volume in this mixture is
5:100-10:100。
3. preparation as claimed in claim 1 is containing the method for the feed additive of surface element, wherein inorganic salt liquid training in step A
The volume supporting base is 25:100-35:100 with the ratio of Semen Glycines volume in this mixture.
4. preparation as claimed in claim 1 is containing the method for the feed additive of surface element, and wherein the semisolid of step A is sent out
Ferment is at temperature 30-40 DEG C, humidity 80-90%, ferments 2-3 days.
5. preparation as claimed in claim 1 is containing the method for the feed additive of surface element, wherein the drying of step B be in
At temperature 50-60 DEG C, 2-3 hour time, dry.
6. preparation as claimed in claim 1 is containing the method for the feed additive of surface element, the wherein analysis for soybean powder of step B
End, its surface cellulose content is 6-7 g/kg.
7. preparation as claimed in claim 1 is containing the method for the feed additive of surface element, and this feed additive is to be applied to
The feedstuff of aquatic biological.
8. preparation as claimed in claim 6 is containing the method for the feed additive of surface element, and wherein this aquatic biological is Fish
Or shrimps.
9. preparation as claimed in claim 7 is containing the method for the feed additive of surface element, and its Mesichthyes is cabrilla；Should
Shrimps are South America white shrimp.
10. preparation as claimed in claim 1 is containing the method for the feed additive of surface element, and this feed additive is to promote
Enter the rate of growth of aquatic biological.
11. methods that preparation contains the plain feed additive in surface as claimed in claim 1, this feed additive is to increase
Add the immunity of aquatic biological.
12. methods that preparation contains the plain feed additive in surface as claimed in claim 1, this feed additive has anti-thin
The activity of bacterium, antifungal and antiviral.
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