CN101528050A - Feed additive and feed - Google Patents
Feed additive and feed Download PDFInfo
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- CN101528050A CN101528050A CNA2007800387084A CN200780038708A CN101528050A CN 101528050 A CN101528050 A CN 101528050A CN A2007800387084 A CNA2007800387084 A CN A2007800387084A CN 200780038708 A CN200780038708 A CN 200780038708A CN 101528050 A CN101528050 A CN 101528050A
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- feed
- feed addictive
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- mel
- rhamnolipid
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Images
Abstract
The present invention provides a safe and convenient means for preventing or treating diseases of birds or mammals, particularly livestock. In particular, means for preventing or treating an infectious disease of livestock caused by gram-positive bacteria are provided. In addition, rumen fermentation of ruminants is improved, contribution is made to inhibiting greenhouse gas generation, and feed efficiency is further improved. Make birds or mammals take Mannose Erythritol Lipid (MEL) and/or Rhamnolipid (RL).
Description
Technical field
The birds and the mammiferous method for breeding that the present invention relates to contain feed addictive, the feed of glycolipid and use these.
Background technology
The infection disease of domestic animal makes losing weight of domestic animal, or causes various symptoms etc., and its commodity value is significantly reduced.As, staphylococcus aureus (Staphylococcus aureus) is dermexanthesis, the pig of mastitis, hypodermic tumour, pyaemia, the horse of ox, sheep, goat, the arthritis of chicken, dermatitis, the reason bacterium of septicemia.Streptococcus suis (Streptococcussuis) is meningitis, septicemia, endocarditis, the arthritic reason bacterium of pig in addition, and bargen's streptococcus (Streptococcus bovis) is the reason bacterium of the bulging disease of ox.
The 1940's found by adding antibiotic to can promote domestic animal in the feed stripped growth on a small quantity, henceforth add antibiotic and extensively carried out in the feed of domestic animal as the growth that promotes domestic animal or prophylactic means.Think that antibiotic demonstrates the pathogenic bacterial infection that prevents domestic animal, the effect that improves the propagation of metabolism, inhibition intestinal toxic bacterium, prevent disease, promotion as a result grown up, but details is still unclear.On the other hand, mix antibiotic in the feed, the result makes antibiotic widely be dispersed in external environment, even if the appearance of antibiotic resistant bacterium also becomes problem in animal industry.As, there is report to be referred to as representational antibiotic resistant bacterium MRSA (methicillin BRL-1241 patience staphylococcus aureus (Staphylococcusaureus)) and in the domestic animal of horse etc., also is found.
Under such background, in recent years, add antibiotic to feed and got up by strict regulations.As, antibiotic feed was completely forbidden in Europe till 2006, reduce in that the operable antibiotic quantity of Japan is also positive interimly.In addition, adapt, just becoming from the producer's expectation and becoming heavy antibiotic substitute with such trend.
Be subjected to so alternative antibiotic tendency, in the part, use the polypeptide class of the streptococcus lactis peptide of lactic acid bacteria production, the iturin that bacillus produces etc. also to occur to replace antibiotic trend.In addition, to add glycolipid in the canned coffee etc. to be sucrose ester that the antibacterial action of bacillus etc. is added as emulsifying agent in expectation.
In addition, the ruminant domestic animal of ox or sheep etc. makes feed link cross microorganism digestion fermentation in cud, utilizes its fermentation to produce the thing survival.Therefore, producing methane by cud is the loss of the energy efficiency of feed.Even methane is the greenhouse oxidizing gases that global greenhouseization is brought influence, and the interior methane generation of cud that therefore reduces ruminant is important.
Methane in the cud generates bacterium and utilizes hydrogen that carbon dioxide reduction is generated methane.Methane is inferior high after carbon dioxide to the contribution rate of greenhouseization, and the methane from the ruminant domestic animal discharging in total discharge of methane amount accounts for 15～20%.
Ionophore class as antibiotic coban etc. is widely used in ruminant feed.Coban shows that to rumen microorganism selectivity suppresses effect, and the result makes methane generate minimizing, has the effect that promotes that propionic acid generates.Propionic acid is compared ATP production rate height with other volatile fatty acid, therefore the generation by propionic acid promotes that feed efficiency improves.
Because such background, people expect to develop the substitute of the coban that makes an addition to ruminant feed etc.Thing as an alternative, research have that plant extract oil (non-patent literature 1), anti-lactic acid generate bacteria vaccine (non-patent literature 2), anti-lactic acid generates bacterium egg antibody (non-patent literature 3) etc.Yet these technology are residual to produce effect not necessarily, do not reached practicability by the problem of approval etc. as the registration of feed.
On the other hand, have the various character that act as representative with surface-active, be intended to carry out various uses as described below with the glycolipid headed by mannose erythrose alcohol ester (MEL) or the rhamnolipid (RL).As knowing: use the liposome contain MEL improve the importing efficient of gene technology (patent documentation 1), hinder the formation of the liposome of the gene that contains drug resistance etc. with MEL, the technology (patent documentation 3) of reduce the method (patent documentation 2) of the generation of drug resistance bacterium etc., MEL being used as the active ingredient of antiinflammatory and anti-allergic agent etc.Know in addition: with rhamnolipid improve natural fiber absorptive technology (patent documentation 4), with rhamnolipid from the non-handled thing that contains harmful usability organic compound separate the technology (patent documentation 5) of described organic compound, by carrying the ice slurry of usefulness, the aggegation of anti-stagnant ice and the technology (patent documentation 6) of unification etc. with rhamnolipid modulation high density cold heat regenerator.Also have, though the antibiotic property of MEL or rhamnolipid has been reported a part (non-patent literature 4, non-patent literature 5), but just do not study as yet, in the livestock products field, do not use the example of MEL or rhamnolipid for the antibiotic property of the bacterium of the infection disease that causes domestic animal.
Patent documentation 1: the Japan Patent spy opens the 2006-174727 communique
Patent documentation 2: the Japan Patent spy opens the 2006-158387 communique
Patent documentation 3: the Japan Patent spy opens the 2005-68015 communique
Patent documentation 4: the Japan Patent spy opens the 2002-105854 communique
Patent documentation 5: the Japan Patent spy opens the 2001-327803 communique
Patent documentation 6: the Japan Patent spy opens the 2001-131538 communique
Non-patent literature 1:Benchaar et al., Can.J.Anim.Sci.86,91-96 (2006)
Non-patent literature 2:Shu et al., FEMS Immunology﹠amp; Medical Microbiology, 26 (2), 153-158 (1999)
Non-patent literature 3:DiLorenzo et al., J.Anim.Sci., 84,2178-2185 (2006)
Non-patent literature 4:Fat.Sci.Technol., 91,363-366,1989
Non-patent literature 5:Biotechnol., 29,91-96,1993
Summary of the invention
Problem of the present invention provides and is used to prevent or treat birds and the mammality especially safety of the disease of domestic animal and easy means.The present invention especially will be used to prevent or the means of infection disease for the treatment of the domestic animal that causes by gram-positive bacterium as problem.
In addition, the present invention will improve ruminant lumen fermentation, the generation that suppresses the greenhouse oxidizing gases is contributed even improves feed efficiency as problem.
The inventor finds that for solving the result that above-mentioned problem is attentively studied the glycolipid of mannose erythrose alcohol ester (MEL) and rhamnolipid (RL) etc. has antibacterial activity to the gram-positive bacterium that causes animal infection disease, so that finished invention.Further, the inventor finds that the glycolipid of mannose erythrose alcohol ester (MEL) and rhamnolipid (RL) etc. suppresses methane and generates in cud, and promotes propionic acid to generate, so that finished invention.
That is, the present invention is as described below.
(1) feed addictive used of a kind of birds or mammality contains mannose erythrose alcohol ester and/or rhamnolipid.
(2) as the feed addictive of (1) record, it is characterized in that, be used for domestic animal.
(3) as the feed addictive of (2) record, it is characterized in that domestic animal is chicken, pig or ox.
(4) as the feed addictive of (1) record, it is characterized in that, be used for ruminant.
As the feed addictive of each record of (1)～(4), it is characterized in that (5) mannose erythrose alcohol ester is subordinated to the yeast that false Aspergillus (Pseudozyma) belongs to and obtains.
As the feed addictive of each record of (1)～(5), it is characterized in that (6) rhamnolipid is subordinated to the bacterium that pseudomonad (Pseudomonas) belongs to and obtains.
(7) as the feed addictive of each record of (1)～(6), it is characterized in that, be used for the prevention or the treatment of disease.
(8) as the feed addictive of (7) record, it is characterized in that described disease is the infection disease that is caused by gram-positive bacterium.
As the feed addictive of (8) record, it is characterized in that (9) gram-positive bacterium is the bacterium that belongs to staphylococcus (Staphylococcus) or streptococcus (Streptococcus) genus.
(10) feed addictive of putting down in writing as (9), it is characterized in that gram-positive bacterium is staphylococcus aureus (Staphylococcus aureus), MRSE (Staphylococcus epidermidis), Streptococcus suis (Streptococcus suis) or bargen's streptococcus (Streptococcus bovis).
(11) contain (1)～feed of the feed addictive of each record of (10).
(12) a kind of birds or mammiferous method for breeding is characterized in that, make the feed of record in birds or the mammality picked-up (11).
Description of drawings
Fig. 1 shows RL and the MEL influence to gas generating amount in cud and composition.
Fig. 2 shows RL and the MEL influence to the concentration and the ratio of the volatile fatty acid in cud.
The specific embodiment
The feature of feed addictive of the present invention is to contain mannose erythrose alcohol ester (MEL) and/or rhamnolipid (RL).
MEL is biosurfactant a kind of of glycolipid type, has the structure of mannose, erythrite and aliphatic acid combination, represents with following general formula (1).
[changing 1]
In general formula (1), R
1And R
2It is aliphatic acyl radical independent separately, carbon number 3～25.Especially it is desirable to R
1And R
2It is aliphatic acyl radical independent separately, carbon number 5～14.In addition, R
1And R
2It also can be aliphatic acyl radical independent separately, carbon number 5～13.These aliphatic acyl radicals can be that the straight chain shape also can be to contain chain, can be saturated also can be undersaturated.In addition, R
3And R
4One is acetyl group, another is hydrogen, and perhaps two all is acetyl group.
Also has R
3And R
4The MEL that is acetyl group is called MEL-A, R
3Be hydrogen, R
4For the MEL of acetyl group is called MEL-B, R
3Be acetyl group, R
4For the MEL of hydrogen is called MEL-C.
In addition, the MEL in the feed addictive of the present invention can only be a kind of, also can be multiple mixture.
The MEL that uses among the present invention can cultivate mushroom, especially culture yeasts class etc. microorganism and obtain.For example, can use and belong to false Eurotium, candida albicans (Candia) belong to, gram formula load spore yeast (Kurtzmanomyces) belongs to yeast etc.In addition, also can use shizonella melanogramma.Wherein, it is desirable to use the yeast that belongs to false Eurotium.As the yeast that belongs to false Eurotium Pseudozyma aphidis, Pseudozyma antarctica etc. are arranged for example.Particularly, can use Pseudozyma aphidis NBRC 10182 bacterial strains, Pseudozyma antarctica NBRC10260 bacterial strain, Peudozyma antarctica NBRC 10736 bacterial strains.
NBRC 10182 bacterial strains, NBRC 10260 bacterial strains, NBRC 10736 bacterial strains are the strains that are registered in the Biological Genetic Resources department (NBRC) of independent administrative corporation's product evaluation technology basal disc mechanism.
In addition, MEL can use synthetic or commercially available product.
Rhamnolipid is biosurfactant a kind of of glycolipid type, has the structure of rhamnose and aliphatic acid combination.The rhamnolipid that uses among the present invention is not particularly limited, as using the rhamnolipid of the structure with following general formula (2) or general formula (3) expression.
[changing 2]
In general formula (2), R
5The expression hydrogen atom ,-CH
2-[CH (OH)]
m-CH
2(OH) ,-(XO)
nThe alkyl of H or carbon number 1～36, alkenyl or aliphatic acyl radical.Here, alkyl, alkenyl can be that the straight chain shape also can be to contain chain, and aliphatic acyl radical can be that the straight chain shape also can be to contain a chain, can be saturated also can be undersaturated.In addition, m is 0～8 integer, and X represents at least a of ethylidene (エ チ レ Application), propylidene (プ ロ ピ レ Application) and butylidene (Block チ レ Application), and n is 1～1000 integer.R
6Be hydrogen atom or 2-decenoyl.R
5And R
6Be independently.
[changing 3]
In general formula (3), R
7The expression hydrogen atom ,-CH
2-[CH (OH)]
m-CH
2(OH) ,-(XO)
nThe alkyl of H or carbon number 1～36, alkenyl or aliphatic acyl radical.Here, alkyl, alkenyl can be that the straight chain shape also can be to contain chain, and aliphatic acyl radical can be that the straight chain shape also can be to contain a chain, can be saturated also can be undersaturated.In addition, m is 0～8 integer, and X represents at least a of ethylidene (エ チ レ Application), propylidene (プ ロ ピ レ Application) and butylidene (Block チ レ Application), and n is 1～1000 integer.R
8Be hydrogen atom or 2-decenoyl.R
7And R
8Be independently.
In addition, the rhamnolipid in the feed addictive of the present invention can only be a kind of, also can be multiple mixture.
The rhamnolipid that uses among the present invention can culture of bacteria and is obtained.For example, can use and belong to pseudomonad (Pseudomonas) belongs to, Bai Kehuoerdeshi (Burkholderia) belongs to bacterium etc.Wherein, it is desirable to use the bacterium that belongs to pseudomonas.As the bacterium that belongs to pseudomonas Pseudomonas aeruginosa, Pseudomonas chlororaphis etc. are arranged for example.Also can use Pseudomonas Sp.As the bacterium that belongs to the Bai Kehuoerdeshi genus glander-like disease Burkholderia etc. is arranged for example.This wherein uses Pseudomonas aeruginosa desirable especially.Particularly, can use Pseudomonas aeruginosa NBRC 3924 bacterial strains, PseudomonasSp.DSM 2874 bacterial strains etc.
NBRC 3924 bacterial strains are the bacterial strains that are registered in the Biological Genetic Resources department (NBRC) of independent administrative corporation's product evaluation technology basal disc mechanism.
DSM 2874 bacterial strains are the bacterial strains that are registered in Deutsche Sammulung von Mikroorganismen und ZellkulturenGmbH (DSMZ).
In addition, rhamnolipid can use synthetic or commercially available product.
In order to use above-mentioned microorganism to make it to produce MEL and rhamnolipid, can use method as described below.
Be production MEL,, adopt the cultivation temperature that is generally used for its microorganism cultivation to cultivate and get final product as long as in the raw material of natural oil lipid, aliphatic acid, alcohol, ketone, hydro carbons, positive chain alkane etc., select to be suitable for the raw material of employed microorganism.It is desirable to natural oil lipid as raw material, can use soybean oil, sunflower oil, coconut oil, cottonseed oil, corn oil, palm wet goods, wherein soybean oil especially uses ideal.
In addition, in order to produce rhamnolipid, as long as in the raw material of natural oil lipid, aliphatic acid, alcohol, ketone, hydro carbons, positive chain alkane etc., select to be suitable for the raw material of employed microorganism, adopt the cultivation temperature that is generally used for its microorganism cultivation to cultivate and get final product.As such method, as using the method for putting down in writing in the Japanese patent laid-open 10-75796 communique.
No matter be any situation, cultural method is not restriction especially, can use the liquid culture method or the plate method that leave standstill cultivation, past double-lift shaken cultivation, rotation dynamic formula shaken cultivation, fermentation tank culture etc.
When the yeast that use belongs to false Eurotium is produced MEL, cultivate down at 20～35 ℃ as long as in the culture medium of the cultivation of the yeast that is generally used for belonging to false Eurotium, add the natural oil lipid of soybean wet goods.
Again, when the bacterium that use belongs to pseudomonas produces rhamnolipid, cultivate down at 20～40 ℃ as long as in the culture medium of the cultivation of the bacterium that is generally used for belonging to pseudomonas, add the alcohols of the carbohydrate, ethanol etc. of the natural oil lipid, glucose etc. of soybean wet goods.
In addition, use micro-organisms MEL with and/or during rhamnolipid, can be to culture make with extra care, use MEL with and/or the highly finished product of rhamnolipid, also can the centrifugation culture, use contain MEL with and/or the separator (draw and divide) of rhamnolipid.In addition, also can intactly use culture, for example can use nutrient solution or solid culture are carried out material of drying and crushing etc.
Feed addictive of the present invention can contain any of MEL and rhamnolipid, also can contain these two kinds.In addition, MEL with and/or the content of rhamnolipid do not have special restriction, from obtaining the viewpoint of effect of sufficient, it is desirable to more than the 10 quality ppm, better is more than the 1 quality %.
In addition, feed addictive of the present invention except MEL with and/or rhamnolipid, can also further contain to prevention or treatment birds or the effective composition of mammiferous disease, to promoting ruminant the effective composition of growth, nutrition auxiliary element, improve any composition that the one-tenth of storage stability grades.As so any composition, the bacteria-promoting agent of enterococcus class, bacillus class, Bifidobacterium class etc. is arranged for example; The enzyme of amylase, lipase etc.; The vitamin of L-ascorbic acid, Choline Chloride, inositol, folic acid etc.; Amino acid such as the mineral of potassium chloride, ironic citrate, magnesia, phosphoric acid salt etc., DL-alanine, DL-methionine, L-Lysine mono Hydrochloride; The organic acid of fumaric acid, butyric acid, lactic acid, acetate and their salt etc.; The antioxidant of ethoxyquin, dibutyl hydroxy toluene etc.; The mould inhibitor of calcium propionate etc.; The binding agent of CMC, casein-sodium, Sodium Polyacrylate etc.; The emulsifying agent of fatty glyceride, sorbitan fatty esters etc.; The pigment of astaxanthin, canthaxanthin etc.; Various esters, ether, ketone etc. add spices.
The formulation of feed addictive of the present invention does not have special restriction, for example can use form arbitrarily such as powder, liquid, tablet.Feed addictive of the present invention can be made by mixing MEL and/or rhamnolipid and any component preparationization as required.
In addition, MEL and rhamnolipid demonstrate antibacterial activity to the bacterium that causes birds or mammiferous disease, and therefore, feed addictive of the present invention can be used for prevention or treat by these bacterial birds or mammiferous disease.
Feed addictive of the present invention especially is suitable for the prevention or the treatment of the infection disease that is caused by gram-positive bacterium.
As such gram-positive bacterium, the Micrococcus of belonging to is for example arranged for example, staphylococcus, streptococcus, Planococcus, Stomatococcus belongs to, enterococcus spp, Peptococcus, Peptostreptococcus, Ruminococcus, Leuconostoc, Pediococcus, Aerococcus, twin Pseudomonas, Coprecoccus, Sarcina, Bacillus, fusobacterium, lactobacillus, listeria, erysipelothrix, corynebacterium, Rhod, Propionibacterium, Eubacterium, actinomyces, Bifidobacterium, Mycobacterium, Nocardia, the bacterium that Dermatophilus etc. belong to.
Feed addictive of the present invention especially is suitable for by the prevention of the bacterial disease that belongs to staphylococcus or streptococcus or treatment, specifically the prevention or the treatment of the disease that is caused by staphylococcus aureus, MRSE, Streptococcus suis, bargen's streptococcus etc.
Feed addictive of the present invention can be used for birds or mammiferous feed, pet food, pet and mix the feed of using as birds or mammality with other feed ingredients of nutritious supplementary pharmaceutical (below, be called feed).The kind of feed or composition do not have special restriction.In addition, also can add above-mentioned any one-tenth that can add in the feed of the present invention in feed addictive assigns to modulate.In addition, feed of the present invention also can be used as the feed of prevention or treatment birds or mammiferous disease.
MEL in the feed of the present invention with and/or the content of rhamnolipid suitably regulate according to the kind of the kind of the animal that gives, health status, feed, feed ingredient, age, sex, body weight etc., there is not special restriction, every dry matter amount, it is desirable to 1～10000 quality ppm, further it is desirable to 10～10000 quality ppm, further it is desirable to 10～1000 quality ppm.
Feed of the present invention can intactly add feed addictive in feed ingredient mixing manufacture.At this moment, when using the feed addictive of Powdered, solid shape, become easily, also can make feed addictive become aqueous or gelatinous form in order to make to mix.At this moment, can make the water-soluble high-molecular compound of vegetable oil, liquid animal oil, polyvinyl alcohol or polyvinylpyrrolidone, polyacrylic acid etc. of water, soybean oil, rapeseed oil, corn wet goods as liquid-carrier.In addition, for keep in the feed MEL with and/or the homogeneity of rhamnolipid, the water-soluble polysaccharides that mixes alginic acid, mosanom, xanthane glue, casein-sodium, Arabic gum, guar gum, tamarind seed polysaccharide class etc. is also more satisfactory.
The animal species that absorbs feed of the present invention is birds or mammality.As, can be used for the pets of domestic animal or dog, cat etc.Feed of the present invention is particularly suited for domestic animal, is particularly suitable for the raising of chicken, pig, ox.In addition, also suitable to the raising of ruminant.For example, feed of the present invention is fit to the raising of ox, goat, sheep etc.The amount of the feed of picked-up can suitably be regulated according to the kind of animal, body weight, age, sex, health status, feed ingredient etc.
The method of picked-up feed and the method for raising can be taked the method for employing usually according to the kind of animal.
Embodiment
The evaluation of [I] antibiotic property
＜1〉production of MEL
(1) cultivation of false Eurotium yeast
(preceding cultivation)
10ml Solanum tuberosum dextrosum culture medium is put into test tube, block with the silicon plug.After the autoclave sterilization, 10182,30 ℃ of following shaken cultivation of inoculation Pseudozyma aphidis NBRC 24 hours.
(the main cultivation)
To contain ion exchange water, soybean oil 8%, NaNO
30.2%, KH
2PO
40.02%, MgSO
47H
2The culture medium 50ml of O 0.02%, yeast extract 0.1% puts into the 500ml conical flask, blocks with the silicon plug, sterilizes with autoclave.Toward the preceding nutrient solution that wherein adds aforesaid NBRC10182,30 ℃/220rpm shaken cultivation 7 days.
(2) extraction of MEL is refining
(extraction)
Get the nutrient solution 50ml that obtains in main the cultivation with separatory funnel, carry out 2 extractions, add that the ethyl acetate distillation is except that desolvating with the ethyl acetate of equivalent.Afterwards, in 25ml methyl alcohol, dissolve, clean 2 times with the hexane of 50ml after distillation for removing methanol, obtain the thick refining thing (is 69% by anthrone reaction purity described later) of MEL.
(making with extra care)
Above-mentioned thick refining thing 1g is dissolved in a spot of chloroform, separates with the silica dioxide gel post.Flow through separation successively with chloroform 500ml, chloroform/ethyl acetate=4/1500ml, acetone 500ml, methyl alcohol 500ml.
Each separator is launched (to launch solvent C HCl with thin-layered chromatography
3/ MeOH/ water=65/15/2), screen following document 1) in the separator of Rf value (Rf=0.52,0.58,0.63,0.77) of the various MEL of demonstration of record, comprehensively these separators are as standard sample.
1)Agric.Biol.Chem.，54(1)31-36，1990
(purity testing: anthrone reaction)
To be put into test tube by the ethyl acetate dilution for the thick refining thing of debita spissitudo, distillation removes and desolvates.Toward wherein adding anthrone reagent (0.2% anthrone, 75% sulfuric acid liquid) 5ml, reaction is 10 minutes in the boiling water, measures the absorption of 620nm.Rely on standard sample to compare, carry out the slightly purity calculating of refining thing.
＜2〉production of rhamnolipid
(1) cultivation of pseudomonas bacterium
(preceding cultivation)
10ml Solanum tuberosum dextrosum culture medium is put into test tube, block with the silicon plug.After the autoclave sterilization, 3924,30 ℃ of following shaken cultivation of inoculation Pseudomonas aeruginosa NBRC 24 hours.
(the main cultivation)
To contain ion exchange water, CaCO
30.2%, K
2HPO
40.05%, MgSO
47H
2The culture medium 50ml of O 0.05%, yeast extract 0.5%, soy meal 0.5% puts into the 500ml conical flask, blocks with the silicon plug, sterilizes with autoclave.Toward wherein add filtration sterilization ethanol 1ml and the preceding nutrient solution of aforesaid NBRC 3924, added filtration sterilization in per 2 days ethanol 0.75ml, 28 ℃/220rpm shaken cultivation 8 days.
(2) extracting and refining of rhamnolipid
(extraction)
Separatory funnel is got the nutrient solution 50ml that obtains in main the cultivation, carries out 2 extractions with methyl alcohol/chloroform=1/1, adds the organic layer distillation except that desolvating, and obtains the thick refining thing (is 55% by anthrone reaction purity described later) of rhamnolipid.
(making with extra care)
The nutrient solution 450ml that obtains in above-mentioned main the cultivation is modulated to pH3, removes thalline by centrifugation.Make the post of supernatant by the TSKgel DEAE-ト ヨ パ one Le 650M that packs into, this post has used 0.5M Tris-HCl buffer solution (pH9.0) to carry out pre-treatment, uses 0.5MTris-HCl buffer solution (pH9.0) to clean post afterwards.Divide by post with 2.3ml/ in the gradient mode in the scope of NaCl concentration 0～0.4M (0.5MTris-HCl buffer solution (pH9.0)), make the rhamnolipid wash-out of being caught and separate by gel.
Each separator is launched (to launch solvent C HCl with thin-layered chromatography
3/ MeOH/ water=65/25/4), screen following document 2) in the separator of Rf value (Rf=0.32,0.52) of the various rhamnolipids of demonstration of record, with methyl alcohol/chloroform-1/1 extraction, these separators are lumped together distillation except that desolvating, as standard sample.
2)Biotechnology Letters 54(12)1213-1215，1997
(purity testing: anthrone reaction)
To be put into test tube by the methyl alcohol dilution for the thick refining thing of debita spissitudo, distillation removes and desolvates.Wherein add anthrone reagent (0.2% anthrone, 75% sulfuric acid liquid) 5ml, reaction is 10 minutes in the boiling water, measures the absorption of 620nm.Rely on standard sample to compare, carry out the slightly purity calculating of refining thing.
＜3〉evaluation of antibiotic property
About MEL and rhamnolipid, measure the minimum of the various bacteriums shown in the table 1 with following main points and grow obstruction concentration (MIC).
The preceding cultivation use experience mensuration of various bacteriums is carried out with broth bouillon (day water).The bacteria concentration that is modulated to nutrient solution with physiological saline becomes about 1.0 * 10
5～10
6After the CFU/ml, with various microbionations to measuring in the culture medium.As measuring culture medium, at staphylococcus aureus, MRSE, bacillus subtilis use experience mensuration culture medium (day water), use blood agar culture-medium (heart infusion medium: day water, the aseptic defiber blood of sheep: kohjin bio) at Streptococcus suis, bargen's streptococcus.About cultivating, staphylococcus aureus, MRSE, bacillus subtilis are carried out aerobic cultivation, Streptococcus suis, bargen's streptococcus are cultivated with 5% carbon dioxide, whichsoever all under 37 ℃, carried out about 20 hours.After cultivating end, measure MIC.
MEL uses＜1〉in the thick refining thing (purity 69%) of the MEL that obtains, rhamnolipid uses＜2〉in the thick refining thing (purity 55%) of the rhamnolipid that obtains.The SIGMA-ALDRICHJapan product), mannose (with the pure medicine industrial goods of light), rhamnose (SIGMA-ALDRICH Japan product) also measured MIC in addition, be relatively, sucrose ester (CT-1695:.
The result represents at table 1.
[table 1]
Table 1
| Bacterial classification | MEL | Rhamnolipid | Sucrose ester | Mannose | Rhamnose |
| Staphylococcus aureus | 50 | 12.5 | ＜1600 | ＜1600 | ＜1600 |
| MRSE | 25 | 12.5 | ＜1600 | ＜1600 | ＜1600 |
| Streptococcus suis | 50 | 50 | 800 | ＜1600 | ＜1600 |
| Bargen's streptococcus | 50 | 12.5 | 800 | ＜1600 | ＜1600 |
| Bacillus subtilis | 12.5 | 6.25 | 400 | ＜1600 | ＜1600 |
MEL and rhamnolipid are compared with the CT-1695 of identical glycolipid, the bacterium of staphylococcus, streptococcus, Bacillus are demonstrated the antibacterial activity of several times～tens of times high.On the other hand, mannose and the rhamnose as the constituting body of glycolipid do not show antibacterial activity.
Thus, raise, can expect to prevent or treat effect by above-mentioned various bacterial diseases by making the picked-up of birds or mammality MEL and rhamnolipid.
The evaluation of the growing amount of [II] gas and volatile fatty acid
＜1〉production of mannose erythrose alcohol ester (MEL)
(1) cultivation of false Eurotium yeast
(preceding cultivation)
10ml Solanum tuberosum dextrosum culture medium is put into test tube, block with the silicon plug.After the autoclave sterilization, 10182,30 ℃ of following shaken cultivation of inoculation Pseudozyma aphidis NBRC 24 hours.
(the main cultivation)
To contain ion exchange water, soybean oil 8%, NaNO
30.2%, KH
2PO
40.02%, MgSO
47H
2The culture medium 50ml of O 0.02%, yeast extract 0.1% puts into the 500ml conical flask, blocks with the silicon plug, sterilizes with autoclave.Toward the preceding nutrient solution that wherein adds aforesaid Pseudozyma aphidis NBRC 10182,30 ℃/220rpm shaken cultivation 10 days.
(2) MEL's is refining
(making with extra care)
After above-mentioned nutrient solution 50ml adding 1N HCl is adjusted to pH3, remove supernatant by centrifugation.Add in the precipitation part after the pure water 50ml stirring, carrying out centrifugation operation, reclaim the precipitation part.Precipitation is partially dissolved in after the MeOH of 10ml, the hexane that adds 10ml again cleans (3 times).Add 10ml water in the MeOH solution after cleaning, carry out the extracting operation (3 times) of MEL with chloroform 10ml.Lump together distillation except that desolvating with chloroform layer, obtain slightly refining thing.By anthrone reaction purity 90%.
(standard sample)
Above-mentioned thick refining thing 1g is dissolved in a spot of chloroform, separates with the silica dioxide gel post.Flow through separation successively with chloroform 500ml, chloroform/ethyl acetate=4/1500ml, acetone 500ml, methyl alcohol 500ml.
Each separator is launched (to launch solvent C HCl with thin-layered chromatography
3/ MeOH/ water=65/15/2), screening Agric.Biol.Chem., 54 (1) 31-36, the separator (the Rf value that shows various MEL, Rf=0.52,0.58,0.63,0.77) of the Rf value of record in 1990, comprehensively these are as standard sample.
(purity testing: anthrone reaction)
To be put into test tube by the ethyl acetate dilution for the thick refining thing of debita spissitudo, distillation removes and desolvates.Toward wherein adding anthrone reagent (0.2% anthrone, 75% sulfuric acid liquid) 5ml, reaction is 10 minutes in the boiling water, measures the absorption of 620nm.Rely on standard sample to compare, carry out the slightly purity calculating of refining thing.
＜2〉rhamnolipid (RL)
BFL biosurfactant (rhamnolipid) drying that Bio Future Co., Ltd makes is also used.
＜3〉rhamnolipid (RL) and mannose erythrose alcohol ester (MEL) the influence that gas generates and volatile fatty acid generates to producing in the cud
(1) sample
Above-mentioned rhamnolipid (RL) and mannose erythrose alcohol ester (MEL) are used for test.Cultivate inoculum and used all rumen fluids of taking from Holstein kind cow (the cud sleeve pipe is housed) (4 layers of gauze filtrate) in biological production research farm, grass science center, biosphere, the Hokkaido University north.Inoculum uses after being 2 times with the dilution of the artificial saliva (pH6.8) of McDougal.
(2) (cultivation)
Test nutrient solution concentration is set at 500 μ g/ml, is set at 500 μ g/ml with MEL with RL, implements to cultivate.Each 0.05g of RL, MEL is dissolved in the ethanol of 1ml respectively, and each 100 μ l adds in the Heng Gaite pipe.Place a few hours, make the ethanol volatilization.Add cornstarch 0.15g, mixed fodder powder 0.025g and orchard grass hay powder 0.025g as culture matrix therein.Add above-mentioned dilution rumen fluid 10ml, the limit is blown into nitrogen in head space (headspace), and the limit covers butyl rubber lid and plastic screw cap, carries out anaerobism with water-bath and cultivates (37 ℃, 18 hours).
The check plot), add RL (RL district) and add MEL (MEL district) be treated to and do not have add and (have only ethanol:, carry out 5 times (5 Even) cultivation respectively.
(2) analyze
With TCD gc analysis methane, hydrogen, carbon dioxide.General volatile aliphatic acid (VFA) concentration and composition FID gas chromatography determination.
(4) result
(i) gas generates
In RL district and MEL district, total gas flow of cultivating after 18 hours all reduces (reducing 51% and 48% respectively).Wherein, the minimizing of methane is especially remarkable, reduces 96% and 99% respectively in RL district and MEL district, and methane does not almost generate.Carbon dioxide reduces 37% and 35% respectively in RL district and MEL district.Observation is shared ratio in total gas, with regard to methane, compares with 23.7% of check plot, is reduced to 2.1% in the RL district, is reduced to 0.3% in the MEL district.
The result shows at table 2 and Fig. 1.
[table 2]
Table 2
A, b, c: there were significant differences between the distinct symbols
Have between b and the c and a and b between identical significant difference
Italic: have significant difference with respect to contrast
The (ii) generation of volatile fatty acid (VFA)
Though the influence that total VFA concentration is not caused by processing, the methane that each VFA produces alters a great deal.That is, acetate, butyric acid, isobutyric acid, valeric acid and isovaleric acid concentration are owing to adding RL and adding MEL and significantly reduce.On the other hand, propionic acid concentration significantly increases (increase by 85% in the RL district, increase by 53% in the MEL district).The molar ratio that each is sour, propionic acid increases (25.8% is increased to 46.7% and 41.1%), acetate and butyric acid and reduces (60.9% is reduced to 49.7% and 53.4%, 10.6% respectively is reduced to 2.0% and 5.0%), and is all remarkable.Especially, the propionic acid ratio rises to the degree of can not see at common cud.
The result shows at table 3 and Fig. 2.
[table 3]
Table 3
A, b, c: there were significant differences between the distinct symbols
Have between b and the c and a and b between identical significant difference
Italic: have significant difference with respect to contrast
Utilize possibility on the industry
Can make birds or mammality picked-up, prevention or treatment disease in the feed by feed addictive of the present invention is mixed into. Particularly, can prevent or treat the infection disease that is caused by gram-positive bacterium. The feed that contains feed addictive of the present invention goes in the raising of domestic animal of chicken, pig, ox etc. In addition, feed addictive Biodegradable of the present invention is very high, and is very high to the security of organism and environment.
In addition, can make the ruminant picked-up in the feed by feed addictive of the present invention is mixed into, suppress the methane generation and promote propionic acid to generate, the result can promote the growth of ruminant, improves feed efficiency. The feed that contains feed addictive of the present invention goes in the raising of ruminant of ox, goat, sheep etc. In addition, feed addictive Biodegradable of the present invention is very high, and is very high to the security of organism and environment.
Claims (12)
1. the feed addictive used of birds or mammality contains mannose erythrose alcohol ester and/or rhamnolipid.
2. as the feed addictive of claim 1 record, it is characterized in that, be used for domestic animal.
3. as the feed addictive of claim 2 record, it is characterized in that domestic animal is chicken, pig or ox.
4. as the feed addictive of claim 1 record, it is characterized in that, be used for ruminant.
5. as the feed addictive of each record of claim 1～4, it is characterized in that the yeast that mannose erythrose alcohol ester is subordinated to false Eurotium obtains.
6. as the feed addictive of each record of claim 1～5, it is characterized in that rhamnolipid is subordinated to the bacterium that pseudomonad (Pseudomonas) belongs to and obtains.
7. as the feed addictive of each record of claim 1～6, it is characterized in that, be used for the prevention or the treatment of disease.
8. as the feed addictive of claim 7 record, it is characterized in that described disease is the infection disease that is caused by gram-positive bacterium.
9. as the feed addictive of claim 8 record, it is characterized in that gram-positive bacterium is to belong to the bacterium that staphylococcus (Staphylococcus) belongs to or streptococcus (Streptococcus) belongs to.
10. as the feed addictive of claim 9 record, it is characterized in that gram-positive bacterium is staphylococcus aureus (Staphylococcus aureus), MRSE (Staphylococcus epidermidis), Streptococcus suis (Streptococcus suis) or bargen's streptococcus (Streptococcus bovis).
11. the feed of the feed addictive of each record that contains claim 1～10.
12. birds or mammiferous method for breeding is characterized in that, make the feed of record in birds or the mammality picked-up claim 11.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP282697/2006 | 2006-10-17 | ||
| JP2006282697 | 2006-10-17 | ||
| JP144393/2007 | 2007-05-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101528050A true CN101528050A (en) | 2009-09-09 |
Family
ID=41095650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007800387084A Pending CN101528050A (en) | 2006-10-17 | 2007-10-11 | Feed additive and feed |
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| Country | Link |
|---|---|
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| CN103070167A (en) * | 2010-03-30 | 2013-05-01 | 湖州紫金生物科技有限公司 | Application of rhamnolipid as additive |
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