EP0518956A1 - Additif alimentaire pour animaux et procede d'inactivation de mycotoxines presentes dans des aliments pour animaux - Google Patents

Additif alimentaire pour animaux et procede d'inactivation de mycotoxines presentes dans des aliments pour animaux

Info

Publication number
EP0518956A1
EP0518956A1 EP91905765A EP91905765A EP0518956A1 EP 0518956 A1 EP0518956 A1 EP 0518956A1 EP 91905765 A EP91905765 A EP 91905765A EP 91905765 A EP91905765 A EP 91905765A EP 0518956 A1 EP0518956 A1 EP 0518956A1
Authority
EP
European Patent Office
Prior art keywords
sodium
composition
calcium
feed
clay
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP91905765A
Other languages
German (de)
English (en)
Other versions
EP0518956A4 (en
Inventor
Dennis R. Taylor
Keith A. Delaney
Timothy D. Phillips
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Texas A&M University System
BASF Catalysts LLC
Original Assignee
Texas A&M University System
Engelhard Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US07/552,715 external-priority patent/US5165946A/en
Application filed by Texas A&M University System, Engelhard Corp filed Critical Texas A&M University System
Publication of EP0518956A1 publication Critical patent/EP0518956A1/fr
Publication of EP0518956A4 publication Critical patent/EP0518956A4/en
Ceased legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K20/00Accessory food factors for animal feeding-stuffs
    • A23K20/20Inorganic substances, e.g. oligoelements
    • A23K20/28Silicates, e.g. perlites, zeolites or bentonites
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K20/00Accessory food factors for animal feeding-stuffs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K40/00Shaping or working-up of animal feeding-stuffs
    • A23K40/30Shaping or working-up of animal feeding-stuffs by encapsulating; by coating

Definitions

  • This invention relates to a method for inactivating mycotoxins which may be present as contaminants in animal feeds by adding a phyllosiiicate/sequesuant formulation to animal feed which will function as a mycotoxin inactivating agent
  • Mycotoxins chemical substances produced by ubiquitous fungi, can make the difference between profit and loss to the poultry and livestock industnes. Animals are extremely vulnerable to mycotoxins due to the common practice of diversion of mycotoxin contaminated agricultural commodities to animal feed. Thus, mycotoxicoses. or mycotoxin-induced diseases, frequently occur in animals.
  • aflatoxins a group of dosety related polysubstituted coumarin derivatives, which are biosynthesized by flavis and parasiticus species of Asperaill ⁇ s fungi.
  • the aflatoxins have invoked much concern as toxic food and feedbome agents following the discovery that they: 1) are potent carcinogens and mutagens. 2) are stable in foods and feeds and are relatively unaffected by a variety of processing procedures. 3) can be found as residues in the tissues of animals and humans, and 4) are associated with animal and human disease.
  • a preponderance of poultry and livestock exposure to aflatoxins is chronic in nature and occur through the ingestion of low levels of these chemicals such as "marginally contaminated” rations which do not increase the mortality rate nor result in obvious signs of disease. Instead, chronic exposure to aflatoxins results in economically important effects in animals such as depression of growth rates. feed conversion, and alteration of immunocompetency which can result in increase ⁇ susceptibility to infection and decreased ability to resist stress. Numerous approaches to reduction of aflatoxin levels in agricultural commodities have been experimentally assessed. These include mixing and dilution with aflatoxin-free grains in order to obtain a level within regulatory guidelines, i.e.
  • the present applicant has recognized the widespread detrimental effects of aflatoxins in animal feed and has developed an additive which effectively binds aflatoxins or otherwise inactivates the aflatoxins during ing ⁇ stlon by animals.
  • the bound or inactivated aflatoxins are subsequently excreted in the animal faces resulting in little or no detrimental effects on the animals.
  • Clays such as montmorllonite have previously been incorporated into poultry feed at levels as low as one percent of the animal ration as in U.S. Pat .No.3,6.. 87,680. Effects accompanying the addition of montmortionit ⁇ induded increased growth rate and body weight of the chickens and reduced mortailty rate. Dietary additions of zeolites (Smith, J. Animal Science, 1980 Vol. 50(2). pp. 278-285), bento nite (Carson. M.S. Thesis University of Guelph, Canada 1982) and spent bleaching day from canoia oil refi ning (Smith, Can. J. Animal Science, 1984, Vol. 64, pp.
  • an animal feed additive which eliminates the adverse effects of mycotoxins. especially aflatoxins, which are present in the feed without promoting undesirable side effects in the animals such as weight loss. It is a further object of the instant invention to provide a method to prevent the effects of mycotoxin (aflatoxin) intake in animals, especially poultry and swine, through the cojoint administration of minimal amounts of these additives with normal animal feeds.
  • a second Ingredient chosen from a group of sequestrants commonly used in food processing, along with a suitable phyllosilicate capable of inactivating a mycotoxin, preferably a montmorillonite clay, produces a material exhibiting heightened capacity for adsorbing aflatoxin in vitro and further that such materials also exhibit substantially enhanced capabilitieslty for reducing the effect of exposure to aflatoxin in vivo.
  • the additives that is, the phyilosilicate/sequestrant complexes, which are utilized in the present Invention as feed additives and supplements, act as biosequestrants which promote the maintenance of normal body weight gains in animals such as poultry.
  • These additives reduce the levels of parent mycotoxins. especially aflatoxins, which are available for assimilation in their digestive tracts during feeding.
  • These additives effectively bind the mycotoxins and eliminate them in the feces.
  • These additives are effective when used in minimal amounts as feed additives for providing protection against mycotoxicoses during ingestion and digestion of the animal feed which is contaminated with mycotoxins, particularly aflatoxins.
  • the additives of the present invention are combined with a substantially complete animal ration in minor amount, tor example, an amount ranging from 0.05 to 1.5 weight percent of the ration, preferably 0.1 to 0.5 weight percent, most preferably 0.2 to 0.6 weight percent of the feed ration.
  • One aspect of the invention comprises a dry particulate animal feed additive comprising partides
  • Another aspect of the invention comprises a dry solid animal feed composition in which
  • biodegradable feed is contaminated with a mycotoxin and Is admixed with a minor amount of a mycotoxin
  • inactivating agent comprising partides of a phytosMkate mineral capable of inactivating mycotoxins
  • phyllosilicate mineral partides being coated with a sequestering agent in amount sufficient to enhance the
  • the phyllosilicate is a smectite clay. most preferably
  • the preferred phyilosilicates used In practice of the invention are montmorillonite clays which are
  • FIG.B Aflatoxin in blood serum of chickens fed sequestrant/clay versus clay alone: 20 ppb and 80 ppb aflatoxin challenges, respectively.
  • FIG. 8-11 Deoxynivaienol. zearalenone, ochratoxin A, citrinin binding capacity versus
  • Additives of the invention are utilized as small granules or powders and should be thoroughly mixed with the animal feed by any suitable manner prior to feeding.
  • animal feed or "f eed ration” as used in the present invention refer to any natural and processed or otherwise modified organic materials which are susceptible to biodeterioration and which can be consumed by animals or birds for nourishment. Examples of such organic materials would range from freshly harvested grains to pelletized feeds.
  • the preferred animal feeds for use in the instant invention are poultry and livestock feeds.
  • the additives which can be utilized in the instant invention as mycotoxin (such as aflatoxin) inactivating agents indude vanous sequestrant/phyilosiiicate formulations wherein the phyllosilicate portion of the formulation is preferably a smectite type clay.
  • phyilosiiicates are essentially made up of layers formed by condensation of sheets of linked Si(O.OH) 4 tetrahedra with those of linked M 2 .3 (OH) 5 octahedra. where M is either a divalent or trivalent cation or combination thereof.
  • smectite clays also possess a lesser amount of mobile (exchan geable) cations that can be easily solub ⁇ lzed when these clays are added to water.
  • minerals indude pyrophyilite. talc, vermiculite, micas, kadinites. and serpentines as well as smectites.
  • Examples of the preferred smectite clays are: montmorilonite. Na-montmorilionite, Ca-montmorillonite,
  • Na-bentonite Ca-bentonite, beidellite, nontronite. saponite, and hectorite. Most preferred are those
  • montmorionites possessing relatively high (+2,+3/+1) exchangeable cation ratios i.e.- greater than 7).
  • the sequestrant portion of the sequestrant/clay formulation would indude food-grade sequestrant salts used In food processing.
  • a partial list of such substances would indude: sodium, calcium and potassium acetates: sodium, calcium and potassium citrates as well as the free acid and monoisopropyl, monogtyceride stearyt and triethyl derivatives thereof; disodium dihydrogen and disodium calcium salts of ethyienediaminetetraacetic acid (EDTA); calcium and sodium gluconates: oxystearin; ortfio phosphates
  • citrate salts and condensed phosphate salts are clay dispersams.
  • condensed phosphate salts and citrate salts can act as either dispersams (substances for bringing fine partides in water into suspension which usually decreases viscosity) or flocculants (substances for bringing fine partides in water together to form agglomerates which usually increases viscosity)
  • the levels used In these preferred embodiments causes them to act primanly as floccuiants.
  • TSPP tetrasodium pyrophosphate
  • the supernatant was extracted 3 times with 2 ml portions of dichloromethane.
  • the dichloromethane solutions were combined and evaporated to dryness under a nitrogen stream prior to analysis.
  • the dried residue from the aflatoxin binding study was redissolved in a known volume of
  • the tube was gently agitated for 15 sec and then placed in a 37 °C water bath and allowed to equilibrate for 1 hr. After 1 hr, 40 ⁇ g of aflatoxin B1 was introduced (in a 1 ⁇ g/ ⁇ l solution). The tube was vortexed for 15 sec at 15 min intervals (15,30, and 45 min). After 1 hr, the tube was centrifuged for 5 min at 1200 rpm to yield a pellet at the bottom of the tube, and a dear supernatant liquor above. The supernatant was then carefully decanted into a dean test tube. The supernatant was then extracted to recover the remaining mycotoxin to determine (capacity of) binding. The residual clay was extracted to determine strength (stability) of binding.
  • the supernatant was extracted 3 times with 2 ml portions of dichloromethane.
  • the dichloromethane sdutions were combined and evaporated to dryness under a nitrogen stream prior to analysis.
  • the dried residue from the aflatoxin binding study was redissolved in a known volume of chloroform. 2 ⁇ l of this solution was spotted on HPTLC date (Anaitech) and developed using a 9/1 chloroform/acetone (v/v) solv ent system. Quantitation was made by visual comparison to known quantities of aflatoxin B1 spotted on the same plate. Additionally, an aliquot of the chloroform solution was injected onto a Water HPLC system (normal phase radial compression column. Pon's solution was used as a running phase). HPLC detection was by UV absorbance at 365 nm. The quantitations were made by direct comparisons to a standard curve generated with known quantities of pure aflatoxin B1.
  • the residual clay from the 60 min binding studies was extracted by first suspending the clay in 3 ml of methand. This was ailowed to stand at room temperature for 5 mm at which time the suspension was centrifuged for 5 mm at 1200 rpm. The methanol was decanted into a dean test tube. The pellet was then resuspended in 5 ml of dichloromethane and allowed to stand at room temperature for an additional 5 min . At this time the suspension was carefully decanted into the first (methanol) extract The organic phases were evaporated to dryness under a nitrogen stream and analyzed using the same procedure as that used in the capacity study. The amount of tightly bound (stade) toxin was assumed to be the difference between the amount of toxin initially added and the amount recovered from both the aqueous phase and the extract from the residual clay.
  • Table 2 contains the data on the percentage of aflatoxin firmly bound to these formulations when subjected to the stability test procedure descnbed above. As is dear. these formulations form extremely stade Comple xes with the aflatoxin and most retain greater than 95% of the aflatoxin once it is adsorbed.
  • Table 5 lists the chemical and physical properties for the montmonilonite clays used in this senes. As shown by the data in Table 6 (see also Figure 5). those clays possessing relatively high (+2.+3/+1) exchange cation ratios (Clays A, B, D) are also the ones exhibiting enhanced aflatoxin binding capacity after treatment with relatively low levels of various sequestrant salts, regardless of whether that clay possesses a slurry pH on the acidic or basic side.
  • DOSING Between days 13 to 14 (24 hour period) feed consumption was determined per pen of chicks. Based on this feed consumption value, the total amount of 14 C aflatoxin B1 ( 14 C AFB1) and aflatoxin B1 (AFB1) to be given to the birds was determined. The calculated amount of 14 C AFB1 and AFB1 and approximately 0.15 gms of feed (either treated with base clay, base clay /4% sodium acid pyrophospnate[4%], or nontreated. depending on the group) was placed in a small gelatin capsule, capable of dissolving in the crop, and passed to the level of the esophagus in each chicken. After dosing, the birds were placed back into their pens and offered their appropriate diets.
  • ASSAY The samples were assayed for levels of 14 C AFB1 by taking subsamples of individual livers (1.0 gm) which were homogenized in 3X volumes of distilled water and 5 mis of chloroform-methanol (2:1) using a nigh speed blender. The chloroform-methanol layer was removed and placed in a clear glass scintillation vial containing 15 mis of Aqualyte Plus (J.T. Baker) scintillation cocktail. One ml of plasma was added to 19 mls of the scintillation cocktail. Counting of the samples was done on a Beckman LS 7000 Scintillation counter. Each vial was counted over a period of 5 minutes with an external standard quench correction. Counting efficiency was determined by using the 14 C AFB1 standard. Background counts were subtracted from the total counts before dividing by the sample size and correcting for counting efficiency.
  • FEED MIXING A standard com-soy starter ration was used. The feed was mixed in a 100 kg capacity horizontal paddle mixer. Additives were combined with feed at levels sufficient to produce mixed feeds containing either 0.1 % or 0.5% (dry wt basis) additives in the feed. Feeds were mixed for 10 minutes. The nontreated feed consisted of the basal ration.
  • 14 C AFB1 PREPARATION 14 C AFB1 was obtained from Moravek Biochemical. Brea. CA. The specific activity of the 14 C AFB1 was 100-200 ⁇ Cl/mmole. AFB1 was obtained from Sigma Chemicals.
  • the amount of 14 C AFB1 In the liver and plasma was compared between the birds treated at the 0.1% additive level (20 and 80 ppb 14 C AFB1) and 0.5% additive level (20 and 80 ppb 1 4 C AFB1) versus the nontreated birds (20 and 80 ppb 14 C AFB1).
  • the pharmacokinetic parameters of rate of adsorption, rate of elimination and area-under-the-curve were computed for liver and plasma using the curve fitting program ESTRIP (Brown and Manno. J. of Pharmaceutical Sci., 1978. Vol. 67, 1687-1691). Analysis of variance was performed on the 14 C AFB1 content of the liver and plasma at each time point and the pharmacokinetic parameters determined with ESTRIP. Differences among the treatments was determined using Tukey's lsd test The probability of a type 1 error was set at the nominal 5% level.
  • Figures 6A, 6B and 7A, 7B which were derived from the data in Table 7 illustrate graphically what happens to the amount of 14 C AFB1 detected in blood serum and liver tissue versus time, respectively when chicks were fed two levels of radiolabeied aflatoxin in diets containing two different levels of base clay or base clay dus sequestrant. For both bloo d serum and liver tissue. aflatoxin levels peak during the first hour after exposure and then gradually drop off with time. Clearly, diets containing either base clay, or those containing base clay dus sequestrant provide protection against exposure to aflatoxin as evidenced by the fact that significantly lower levels of aflatoxin are detected in blood serum and liver tissues as compared to the levels detected in control groups (i.e. those where feed plus aflatoxin are present, but no additive; see Table 7).
  • Binding experiments were run at two different sorbent levels: 100 mg and 10 mg sorbent/40 ⁇ g toxin.
  • Figures 8 - 11 are bar graphs showing the results obtained when 100 mg of the various clay/sequestrant formulations were used to bind 40 ⁇ g of deoxynivalend. zearalenone. ochratoxin A and citrinin. respectively. With the exception of zearalenone. binding of other toxins was low (i.e. ⁇ 10%) when using only 10 mg of sorbent: therefore only the results obtained at the 100 mg level were graphed. T-2 toxin binding results were not graphed because it was determined that transformation into did and triol derivatives (which were subsequently desorbed) was at least partially responsible for its (apparent) reduction by binding.
  • each of the other toxins exhibit enhancement of binding in the presence of some combination(s) of clay /sequestrant. That not all or the same clay/sequestrant combinations are effective in this regard is believed to be a consequence of not having determined the optimum clay/sequestrant ratio for that particular toxin and sequestrant (eg. - see Example III). Also, it must be kept in mind that the chemical structures (and consequently, reactivities) for these toxins are quite varied (see Figure 12). On this basis, it is not surprising that the optimum clay/sequestrant ratio for promoting enhanced binding of a particular mycotoxin would not necessarily be the optimum for another.
  • a solution of sodium acid pyrophosphate was prepared by adding 50 mg of the salt to water and adjusting to 250 ml in a volumetric flask.
  • An aflatoxin solution was prepared by adding 1 mg of aflatoxin B1 to 1 ml of methanol (reagent grade). 1 ml of the phosphate solution was then added to 100 mg of the sorbent materials described above in a test tube and incubated for 1 hr at 37 °C in a water bath. Then 20 ⁇ I of the B1 solution was added to the material in the test tube and incubated for 2 hrs at 37 °C. in the case of the controls (without phosphate), the same procedures were used, but pure water was used in place of the phosphate solution.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Inorganic Chemistry (AREA)
  • Fodder In General (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Composition alimentaire solide sèche pour animaux dans laquelle un aliment biodégradable est contaminé par une mycotoxine et est mélangé à un agent inactivant la mycotoxine comprenant des particules d'un minéral de phyllosilicate capable d'inactiver les mycotoxines, lesdites particules étant enrobées d'un agent séquestrant en une quantité suffisante pour améliorer la capacité d'inactivation de mycotoxines dudit phyllosilicate.
EP19910905765 1990-03-07 1991-03-05 Animal feed additive and method for inactivating mycotoxins present in animal feeds Ceased EP0518956A4 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US49001390A 1990-03-07 1990-03-07
US490013 1990-03-07
US552715 1990-07-16
US07/552,715 US5165946A (en) 1990-03-07 1990-07-16 Animal feed additive and method for inactivating mycotoxins present in animal feeds

Publications (2)

Publication Number Publication Date
EP0518956A1 true EP0518956A1 (fr) 1992-12-23
EP0518956A4 EP0518956A4 (en) 1993-01-27

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EP19910905765 Ceased EP0518956A4 (en) 1990-03-07 1991-03-05 Animal feed additive and method for inactivating mycotoxins present in animal feeds

Country Status (6)

Country Link
EP (1) EP0518956A4 (fr)
JP (1) JPH05503432A (fr)
AU (1) AU659796B2 (fr)
BR (1) BR9105978A (fr)
MX (1) MX173191B (fr)
WO (1) WO1991013555A1 (fr)

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US5192547A (en) * 1990-10-01 1993-03-09 Engelhard Corporation Animal feed containing selected montmorillonite clay as additive and method for selecting the clay
AT504U1 (de) * 1994-10-19 1995-12-27 Erber Erich Kg Futtermittelzusatz und verwendung desselben zur inaktivierung von mykotoxinen in futtermitteln bzw. im verdauungstrakt von tieren sowie verfahren zur herstellung eines futtermittels
AU4617896A (en) * 1995-02-16 1996-09-04 Maple Leaf Foods Inc Particulate feed supplement
AT406166B (de) 1997-12-30 2000-03-27 Erber Erich Kg Mikroorganismus, verfahren zur gewinnung desselben sowie futtermittelzusatz
DE19821509A1 (de) * 1998-05-13 1999-11-18 Manfred Brunner Verfahren zum Adsorbieren von toxischen Substanzen, insbesondere Mycotoxinen, bei der Herstellung von Human-Nahrungs- oder Tierfuttermitteln und die dabei erhaltenen Produkte
US7347997B1 (en) 1998-12-21 2008-03-25 Erber Aktiengesellschaft Method of using a feedstuff additive
DE19900813A1 (de) * 1999-01-12 2000-07-13 Sued Chemie Ag Mykotoxin-Adsorbens
DE19909770A1 (de) 1999-03-05 2000-09-07 Werner Lubitz Bakterienghosts als Träger- und Targetingvehikel
MXPA00007077A (es) * 2000-07-19 2003-09-08 Nutek S A De C V Proceso para preparar organoaluminosilicatos y su uso en alimentos balanceados para animales.
DE10056634A1 (de) * 2000-11-15 2002-05-29 Sued Chemie Ag Verwendung Aktivierter Schichtsilicate zur Mykotoxinadsorption
AT413540B (de) 2001-12-20 2006-03-15 Erber Ag Mikroorganismus, welcher ochratoxine sowie ochratoxine und zearalenone entgiftet, sowie verfahren und verwendung hiefür
ES2353672T3 (es) 2005-05-10 2011-03-04 Süd-Chemie AG Uso de estevensita para la adsorción de micotoxinas.
EP2170301B1 (fr) 2007-07-27 2014-09-03 Werner Lubitz Fantômes bactériens modifiés par virus
CN102224237B (zh) 2008-01-18 2014-03-12 沃纳·卢比茨 使用β-丙内酯(BLP)进行最终灭活的细菌菌影(BG)生产方法
PL214583B1 (pl) 2010-06-16 2013-08-30 Inst Biotechnologii Przemyslu Rolno Spozywczego Im Prof Waclawa Dabrowskiego Szczep bakterii Lactobacillus plantarum S, zastosowanie szczepu bakterii Lactobacillus plantarum S oraz preparat do kiszenia pasz objetosciowych
US9056894B2 (en) 2010-10-27 2015-06-16 Philip Morris Products S.A. Methods for capturing virus like particles from plants using expanded bed chromatogrpahy
JP5723254B2 (ja) * 2011-10-28 2015-05-27 水澤化学工業株式会社 カビ毒吸着剤
JP5918104B2 (ja) * 2012-10-24 2016-05-18 水澤化学工業株式会社 カビ毒吸着剤
MX338482B (es) 2013-11-25 2016-04-19 Nutek S A De C V Absorbente de micotoxinas y su uso en alimentos balanceados para animales.
US10729127B2 (en) 2015-05-21 2020-08-04 Special Nutrients, Llc Rodenticide binding system
US10058568B2 (en) * 2015-05-21 2018-08-28 Special Nutrients, Inc. Toxin binding system
RU2655739C1 (ru) * 2017-06-05 2018-05-29 Федеральное государственное бюджетное образовательное учреждение высшего образования "Горский государственный аграрный университет" Способ использования аморфного гидротермального нанокремнезема в птицеводстве
EP3840590A4 (fr) * 2018-08-20 2022-05-18 Texas A&M University Entérosorbants comestibles utilisés pour atténuer des expositions aiguës à des toxines environnementales ingérables suite à des épidémies, des catastrophes naturelles et des situations d'urgence
CN115784296B (zh) * 2022-11-25 2024-02-27 广西埃索凯循环科技有限公司 适用于高品质微量元素预混合饲料的高氯硫酸锌的制备方法

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DE3810004A1 (de) * 1988-03-24 1989-10-05 Siegfried Dr Moser Verwendung von bentoniten zur mykotoxinbindung

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See also references of WO9113555A1 *

Also Published As

Publication number Publication date
EP0518956A4 (en) 1993-01-27
MX173191B (es) 1994-02-07
JPH05503432A (ja) 1993-06-10
WO1991013555A1 (fr) 1991-09-19
AU7463291A (en) 1991-10-10
BR9105978A (pt) 1992-11-17
AU659796B2 (en) 1995-06-01

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