EP0518956A4

EP0518956A4 - Animal feed additive and method for inactivating mycotoxins present in animal feeds

Info

Publication number: EP0518956A4
Application number: EP19910905765
Authority: EP
Inventors: Dennis R. Taylor; Keith A. Delaney; Timothy D. Phillips
Original assignee: Texas A&M University System; Engelhard Corp
Current assignee: Texas A&M University System; BASF Catalysts LLC
Priority date: 1990-03-07
Filing date: 1991-03-05
Publication date: 1993-01-27
Also published as: MX173191B; BR9105978A; EP0518956A1; AU7463291A; AU659796B2; JPH05503432A; WO1991013555A1

Abstract

A dry solid animal feed composition in which biodegradable feed is contaminated with a mycotoxin and is admixed with a mycotoxin inactivating agent comprising particles of a phyllosilicate mineral capable of inactivating mycotoxins, said particles being coated with a sequestering agent in an amount sufficient to enhance the mycotoxin inactivating capacity of said phyllosilicate.

Description

ANIMAL FEED ADDITIVE AND METHOD FOR INACTIVATING MYCOTOXINS PRESENT IN ANIMAL FEEDS

BACKGROUND OF THE INVENTION

This invention relates to a method for inactivating mycotoxiπs which may be present as contaminants in animal feeds by adding a phyllosiiicate/sequesuaπt 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 industπes. 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.

It is readily apparent from a review of scientific literature that the most studied and prevalent of these agents are the aflatoxins, a group of osety related polysubstituted coumariπ derivatives, which are biosyπthesized by ffaws and parasiticus species of Asperσillυs 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 iπgestion of low levels of these chemicals such as "marginally contaminated" rations which d not increase the mortality rate nor result in obvious signs of disease. Instead, chronic exposure t aflatoxins results in economically important effects in animals such as depression of growth rates^, fee conversion, and alteration of immunocompetency which can result in increaseα susceptibility to infectio and decreased ability to resist stress. Numerous approaches to reduction of aflatoxin levels in agricultural commodities have bee experimentally assessed. These iπdude mixing and dilution with aflatoxin-free grains in order to obtain level within regulatory guidelines, i.e. 20 ppb or less; physical methods of separation such as deanin density segregation and preferential fragmentation; solvent extraction; biologiral iπactivation; therm iπactivation; and chemical iπactivation with a variety of acids, aldehydes, oxidizing agents and alkalie These approaches have been relatively unsuccessful on a commercial scale due to lack of efficac economic constraints of the protocol, unacceptable alteration of feed quality, or the introduction potentially deleterious substances. Consequently, simple, cost effective, practical and safe processes which animal feeds can be decontaminated or detoxified are in great demand.

The present applicant has recognized the widespread detrimental effects of aflatoxins in ani feed and has developed an additive which effectively binds aflatoxins or otherwise inactivates the aflatoxi during ingβstlon by animals. The bound or inactivated aflatoxins are subsequently excreted in the ani faces resulting in little or no detrimental effects on the animals.

Clays such as moπtmorllonite have previously been incorporated into poultry feed at levels as l as one percent of the animal ration as in U.S. PaL No. 3,687,680. Effects accompanying the additio moπtmortionitβ induded increased growth rate and body weight of the chickens and reduced mort rate. Dietary additions of zeolites (Smith, J. Animal Science. 1980 Vol. 50(2). pp. 278-285), bβπto (Carson. M.S. Thesis University of Guelph. Canada 1982) and spent bleaching day from caπoia oil refi (Smith, Can. J. Animal Science. 1984, Vol. 64, pp. 725-732), have been shown to diminish the adv effects of T-2 toxin and zearalenone in rats and immature swine. The adsorption of aflatoxin B1 f various liquid media by various day minerals, iπduding montmorilionites, has been reported (Masim et al.. Ann, de Nutrition t Alimentation. 1973 Vol. 23, pp. 137-147). SUMMARY OF THE INVENTION

Accordingly, it is the object of the instant invention to provide 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 cojoiπt administration of minimal amounts of these additives with normal animal feeds.

The present inventors surprisingly have discovered that the incorporation of a second Ingredient, chosen from a group of sequestraπts commonly used in food processing, along with a suitable phyllosilicate capable of inactivating a mycotoxin, preferably a moπtmoriilonite clay, produces a material exhibiting heightened capacity for adsorbing aflatoxin in vitro and further that such materials also exhibit substantially enhanced capabBtty for reducing the effect of exposure to aflatoxin in vivo.

Further, it has been discovered that such formulations can be utilized as feed additives to effectively bind mycotoxins, such as aflatoxins, which are ingested in conjunction with animal feed. The bound mycotoxin-addltlve complex is not significantly adsorbed during digestion and it is then excreted In the feces of the animal.

It appears that the additives, that is, the phyilosϋicate/sequβstraπt complexes, which are utilized In the present Invention as feed additives and supplements, act as biosequestrants which promote th maintenance of normal body weight gains in animals such as poultry. These additives reduce the levels o parent mycotoxins. especially aflatoxins, which are available for assimilation in their digestive tracts durin feeding. These addith es effectively bind the mycotoxins and eliminate them in the feces. These additive are effective when used in minimal amounts as feed additives for providing protection again mycotoxicoses during iπgestioπ and digestion of the animal feed which is contaminated with mycotoxin particularly aflatoxins. The additives of the present invention are combined with a substantially comol^β animal ration in minor amount, tor example, an amount ranging from 0.05 to 1.5 weight percent of t 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 of a suitable phyllosilicate mineral coated with a minor amount erf a water-soluble sequestering agent in an amount suffkaeπt to enhance the mycotoxin inactivating capacity of the phyllosyicate mineral.

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, the phyllosiilcate mineral partides being coated with a sequestering agent in amount sufficient to enhance the mycotoxin inactivating capacity of the phytlosilcate mineral.

In the preferred embodiments of the invention, the phyllosilicate is a smectite day. most preferably a montmorilionit_β day in which the ratio of divalent plus trivalβ /monovaieπt exchangeable cations is greater than 7.

The preferred phyilosϋicates used In practice of the invention are mont orillonite days which are known to possesses two kinds of binding sites: 1) those located on the basal planes of the clay partides, and 2) those located at the edges of the day partides. Although the identity of the sites involved in binding aflatoxin is unknown, three possibilities exisr. 1) binding is occurring only on basal sites: 2) binding is occurring only on edge sites: or 3) binding is occurring on both basal and edge sites. One surprising aspect of the present invention is that the incorporation of various sequestrants actually enhances binding of aflatoxin even though some of the sequestrants used in the present invention are phosphate and pdyphosphate salts which are known (Theng, The Chemistry of Clay-Organic Reaction", John Wley & Sons, NY, 1974, pp. 264-268) to bind selectively to edge sites (thereby rendering them unfit for binding other molecules). Under these drcumstaπces it was expected that if case i were operatⁱve, there should be no effect of the added phosphate; rf case 2 were operative, there should be complete inhibition of aflatoxin binding, and if case 3 were operative, there should be some degree of inhibition. But for none of these cases was it expeαed that there would be an increase in aflatoxin binding even though that is what was actually observed. -J-

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Aflatoxin binding capacity versus sequestraπt "AP" loading on day A.

Figure 2. Aflatoxin binding capacity versus sequestraπt type and loading on day B.

Figure 3. Aflatoxin binding capacity versus sequestraπt "AP^* loading ^n days B.C.D, and E.

Figure 4. Aflatoxin binding capacity versus sequestraπt type and loading on day A.

Figure 5. Aflatoxin binding capacity versus selected sequestraπt/day combinations, low sequestraπt loadings.

Figure 6A.B. Aflatoxin in blood serum of chickens fed sequestrant/day versus clay alone: 20 ppb and 80 ppb aflatoxin challenges, respectively.

Figure 7A.B. Same as above expect liver tissue.

Figure 8-11. Deoxyπivaienol. zearalenone. ochratoxin A, citrinin binding capacity versus sequestraπt type and loading on clay B.

Figure 12. Structural formulas for some common mycotoxins.

DESCRIPTION OF PREFERRED EMBODIMENTS

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. The terms 'animal feed^* or feed 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 sequestraπt/phyilosiiicate formulations wherein the phyllosilicate portion of the formulation is preferably a smectite type day. Structurally, phyilosiiicates are essentially made up of layers formed by condensation of sheets of linked Si(0.0H)₄ tetrahedra with those of linked M.₃(OH)_s octahedra. where M is either a divalent or trivalent cation or combination thereof. In addition to possessing the aforementioned properties, smectite clays also possess a lesser amount of mobile (excftaπgeab/e) cations that can be easily soiubϊlzed wnen these days are added to water. Phyllosilicate minerals indude pyrophyilite. talc, vermicul^'ite. micas, kadinrtes. and serpentines as weli as smectites. Closely related to the phyilosiiicates are the fibrous day minerals induding attapulgite and sepiotite. Examples of the preferred smectite days are: montmorilonite. Na-montmorilionite, Ca-moπtmorillonite, Na-bentonite. Ca-beπtonite, beidellite, noπtronite. saponite, and hectorite. Most preferred are those moπtmorionites possessing relatively high (+2.+3/+1) exchangeable cation ratios (i.e.- greater than 7).

The sequestraπt portion of the sequestraπt/ciay formulation would indude food-grade sequestraπt 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 moπoisopropyi, monogtyceride stβaryt and triethyt derivatives thereof; disodium dihydrogen and disodium calcium salts of ethyienediaminetβtraacetic acid (EDTA); calcium and sodium giuconates: oxystβarin; ortfio phosphates (monocaldum add. dibasic potassium, sodium aluminum, dibasic sodium, monobasic sodium, tribasic sodium); meta phosphates (caldum hexameta. sodium hexameta); pyro phosphates (tetrasodium, sodium acid); sodium tripdyphosphate; caldum phytatβ; sodium and sodium potassium tartrates as well as the free acid; and sodium thiosutfate and mixtures thereof.

Many of these sequestrants. for example citrate salts and condensed phosphate salts (e.g., pdy and pyrophosphates) are day dispersams. It Is worth noting that although condensed phosphate salts and citrate salts can act as either dispersa s (substances for bringing fine partides in water into suspension which usually decreases viscosity) or flocculaπts (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 pπmaπly as floccuiants. Accordingly, almost ail of the sequestrant/day siumes prepared in the fdlowing examples exhibited much higher viscosities than the corresponding pure day siumes. This would seem to rule out simple dispersion (and increased available surface area⁾ of the day partides as the mechanism by which enhanced adsorption of aflatoxin is achieved. A typical preparation of a preferred seαuestraπt /phyllosilicate formulation is as follows:

1) Dissolve sodium tripdyphosphate (STP) In water (10:90 paπs/wt. ratio).

2) Add STP solution to dried (-25 wt% loss-on-ignition) and ground (60-100 mesh) montmoriilonite day in pugging machine such that the STP and day are present in a 4:96 parts /wt. ratio (dry basis).

3) Pug day/STP /water combination for 15-30 minutes to effect intimate mixture of ingredients.

4) Extrude the day/STP /water mixture (5/16^* or 5/8^* die).

5) Dry resultant pellets (15-25 wt% ioss-on-ignltlon)in tray or rotary drier.

6) Grind pellets to form granules (16-60 mesh) or powder (100-200 mesh).

Another example of a procedure to prepare a preferred formulation would be as follows:

1) Dissolve tetrasodium pyrophosphate (TSPP) In water.

2) Add the TSPP sdutlon to montmoriilonite day such that the TSPP:day ratio is 4:96 and the resultant day/TSPP/water slurry contains 15 wt% soiids (day + TSPP).

3) Mix components for 30 minutes using a Talboy mixer.

4) Spray dry the mixture using any suitable type of spray drier such that resultant microspheres have most of their diameters in the 60-80 micron range and possess 3-5 wt% free moisture.

The fdlowing examples illustrate the invention and are not considered restrictive of the invention as otherwise descnbed herein.

EXAMPLE l

The fdlowing in vitro tests were performed to demonstrate the enhancement in aflatoxin B1 binding capacity that results when a Ca-montmoriilonite day obtained in Mississippi (Clay A) was coated with various levels of sodium acid pyrophosphate. In this example, a clay slurry was prepared by mⁱxin the απed (8-12% free moisture) and ground (93-97% T-100 mesh) clay in water (20 wt% soiids), and the adding different amounts of sodium acid pyrophosphate to the stirred slurry such that the desire sequestrant level (dry wt. basis) was obtained. The components were mixed for 30 minutes and the poureα into evaporating dishes and placed in an oven at 90-110 °C overnight After drying, the samples were putveπzed in a hammer mill and suosequeπtly screened to obtain a -325 mesh fraction for further testing. Moisture content, surface area, and pore vdumes for these preparations are listed in Table 1.

In vitro binding studies were conducted using these materials as follows: A weighed sample of additive was placed into a dean 16X125 mm disposable glass test tube. To this was added 5.00 ml of distilled water. The tube was vortexed for 15 sec and then placed in a 37 °C water bath ana allowed to equilibrate for 1 hr. After 1 hr, 40 μg of aflatoxin B1 was Introduced (in a l μg/μi solution). The tube was vortexed for 15 sec and then returned to the water bath and allowed to stand at 37 °C for 5 mm. The supernatant was carefully decanted Into a dean test tube. The supernatant was then extracted to recover the remaining mycotoxin.

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 redissdved in a known vdume of

TABLE 1 AFLATOXIN B1 BINDING VS SALT LOADING

CLAY A/SODIUM ACID PYROPHOSPHATE

0 I 15 i 0.044 I 20 I 46.7 ' 99.4

(1) LOD * Wt% loss on drying at 110 degrees C for 4 hrs. :2) Measured by BET method. (3) Measured by BHJ metnod.

Level 1 - 1 mg soroent 40 ug toxin.

Level 2 - 100 mg soroent/40 ug toxin. chioroform. 2 μl of this sdution was spotted on HPTLC plate (Anaitech) and developed using a 9/1 chloroform/acetone (v/v) solvent system. Quaπtitattoπ was made by visual comparison to known quantities of aflatoxin B1 spotted on the same plate. Analysis of the aflatoxin controls was by GC/MS as well as HPTLC. All samples were run in tridicate. Tetrahydrofuran (THF) which had been freshly πistiled from sodium metal was used as the delivery solvent for aflatoxin B1. The percent recovery of aflatoxin B1 from the supernatant was determined from control samples run for each experiment. The percent recovery of aflatoxin B1 was consistently found to be 100% for each experiment - as confirmed by GC/MS, TLC and HPLC quaπtitatton.

As shown by the data in Table 1 and the associated graph (Figure 1), there is a substantial enhancement in the amount of aflatoxin bound when the day contains sodium acid pyrophosphate sequestraπt According to the graph shown in Figure 1 , a maximum in enhancement is achieved when the clay contains about 6 wt% of the phosphate salt Clearly, higher levels of phosphate salt lead to reduced effteiβncy of binding; In the extreme case when the day contains 20 wt% of the salt the binding capacity Is actually somewhat below that of the untreated day. While the reasons for this reduced binding efficiency at higher salt loadings is unknown. It Is worth noting (Table 1) that both surface area and porosity decrease as salt levels increase. This suggests there may be a trade off between the enhancement in binding caused by adding the sequestraπt and the reductions It causes to surface area and porosity. As expected^, higher levels of additive bind more of the toxin (compare Level 1 vs. Level 2) - when 100 mg of soroent are used with the standard aflatoxin sdution. essentially all of the mycotoxin is bound.

EXAMPLE II

The fdlowing m vitro tests were performed to demonstrate the enhancement in aflatoxⁱn B binding capacity and stability that results when a Ca-montmorillonite day obtained in Mississⁱppi (Clay was treated with vanous levels of different (salt) sequestrants. Oven dried samples were prepared descnbed in Examde i; spray dried samdes were prepared as previously descπbed (vide suora⁾. In vitro binding studies were conducted using these materials as fdlows: A weighed saπφie additive was placed into a dean 16X125 mm disposable glass test tube. To this was added 5.00 ml of Type 111 water (equivalent to double-distilled deioπized water). 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 μg of aflatoxin B1 was introduced (in a I μg/μl sdution). The tube was vortexed for 15 sec at 15 min intervals (15,30, and 45 min). After 1 hr, the tube was ceπtrifuged 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 day was extracted to determine strength (stability) of binding.

/n vrtro CanacitvTpςts-

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 redissdved in a known vdum^β of chloroform. 2 μl of this sdution was spotted on HPTLC date (Anaitech) and developed using a 9/1 chloroform/acetone (v/v) sdveπt system. Quaπtitation was made by visual comparison to known quantities of aflatoxin B1 spotted on the same plate. Additionally, an aliquot of the chloroform sdution was injected onto a Water HPLC system (normal phase radial compression cdumn. Pon's sdution was used as a running phase). HPLC detection was by UV absorbance at 365 nm. The quantitations were made b direct comparisons to a standard curve generated with known quantities of pure aflatoxin B1.

In vitro Stability Tests:

The residual day from the 60 min binding studies was extracted by first suspending the day in 3 of methand. This was ailowed to stand at room temperature for 5 mm at which time the suspension wa centπfuged for 5 mm at 1200 rpm. The methand was decanted into a dean test tube. The pellet was the resuspendeα in 5 mi of dichloromethane and allowed to stand at room temperature for an additional 5 m . At this time the suspension was carefully decanted into the first (methanol) extract The organic phases were evaporated to dryness unαer 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.

With regard to stability of binding, 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 Comdexes with the aflatoxin and most retain greater than 95% of the aflatoxin once it is adsoroeo.

TABLE 2

BINDING CONDITIONS: 1 mg sorbent 40 ug toxin 50 mg sorbent 40 ug i oxin Dichloromethane MeOH/Acetone 60 mιn/37 oC 60 mιn 37 oC TABLE 3 PHYSICAL PROPERTIES VS. SALT LOADING

As snown in Figure 2. practically all of the sequestrant salts utilized in clay/seαuestrant formulations enhance the binding capacity for aflatoxin over the base day (0% loading). The only exception is calcium phytate which seems not to improve binding capacity. Although the percentage salt loading required to achieve maximum enhancement In aflatoxin binding capacity vanes from one salt to the next; most seem to reach a maximum before 10 wt% of salt Is added. As mentioned above, surface area and pore vdume for these formulations generally decrease with increasing salt loadings (see Tade 3).

EXAMPLE 111

The fdlowing in vitro tests were performed to demonstrate that the enhancement in aflatoxin B1 binding rapacity that results when a variety of montmoriilonite clays are treated with vanous levels of different (salt) sequestrants depends on complex interactions between the particular day/seouestrant combination being utilized, as well as the level of the sequestrant being used. Samples were prepared a αescπbed in Examde I and in vitro capacity tests were performed as descπbec in Examde II

The data in Tade 4 (see also Figure 3) snows dearty that only one of the four source ciays (Clay B exniDits eπnanceo capacity for aflatoxin B1 wnen treated with sodium acid pyropnospnate ⁽AP) at levels i the range 7-21 wt%. This snows that with some days, the point of maximum eπnancement in bindin capacity is reached even before 7 wt% of the sequestraπt salt has been added. This result is in essential agreement with the data presented in Examde I. Ftgure 4 shows what happens when another source day (Clay A) Is similarly treated with vanous other sequestrant salts.

TABLE 4. EFFECT OF CLAY AND SALT TYPE AFLATOXIN B1 BINDING ON CLAY/SALT COMPLEXES

% AFB1 BOUND (1 MG SORBENT/40 uG TOXIN)

CLAY I SALT LOADING (WT%) SALT §ALI CODE 0% 7% 14% I 21% CODE

A 69.5 | 73.7 ; 66.4 I 65.0 I M Na EOTA ED I

A 69.5 | 74.6 I 62.4 60.1 NaCltraia SC | A 69.5 j 60.1 j 69.3 61.7 Na Hacaπwtaøtnaonata HP j A 69.5 I 57.0 I 53.0 41.4 01 Na Phαsonate DP I B 89.3 I 95.8 I 92.2 I 93.8 I Na Acid Pyroonospnata AP ι

C 82.9 I 82.9 i 81.1 j 82.2 I Ditto AP l D 77.8 I 62.8 63.9 46.2 Ditto AP I

E 92.6 ϊ 91.2 I 77.1 l 75.9 I Ditto APJ

I % AFB1 BOUND (10 MG SORBENT/40 uG TOXIN) I

I CLAY I SALT LOADING (WT%) SALT §ALΣ !

I CODE i 0% I 7% I 14% I 21% CODE !

A 94.9 95.0 92.5 91.7 DINa EOTA ED I A 94.9 93.4 87.8 88.2 Na Citrate SC A 94.9 93.4 93.7 90.4 NaHβα βtaotwβpftaiβ HP A 94.9 81.8 80.4 85.0 Di Na Phosphate DP

C 95.2 97.2 96.6 95.1 Na Aod Pyropnosonata AP D 97.7 95.2 94.7 92.1 Ditto AP ε 96.3 93.9 95.9 94.6 Ditto AP

This examde. therefore, illustrates the fact that the optimum salt level for achieving maximu aflatoxin binding is not the same for all combinations of sorbeπts and salts. Thus, for instance, t disodium salt of EDTA and sodium citrate exhibit enhanced binding when 7 wt% salt levels are utilized, b two other salts, hexametaohosphate. and disodium phosphate apparentiy have already exceeded th optimum oy me time 7 wt% levels are utilized. This suggests that optimum av/seαuestrant com^pilatⁱo must be determined individually. EXAMPLE IV

The fdlowing in vitro tests demonstrate that the desired ennancement in aflatoxin B1 binding capacity is optimum at around 4% salt loadings when montmoπiionite αays are coated with different sequestrants. in addition, the data presented in this examde indicate that certain types of montmonllonite. particularly those characterized by high dl- and trtvaiβπt/monαvaleπt exchangeable cation ratios, are most suitade for preparing enhanced capacity toxin sorbeπts by the methods ot the instant invention descnbed herein. Samdβs were prepared as descnbed In Examde I except that generally higher sdids contents were emdoyed (24-38 wt%) with the exception of Clay C where only 9% sdids were employed. In vitro capacity tests were performed as descnbed in Examde 11.

Tade 5 lists the chemical and physical properties for the montmonllonite days used in this seπes. As snown by the data in Tade 6 (see also Figure 5). those days possessing relatively high (f2.+3/+i) exchange cation ratios (Clays A, B, D) are also the ones exhibiting enhanced aflatoxin binding capacity after treatment with relatively low levels of various- sequestraπt salts, regardless of whether that day possesses a slurry pH on the acidic or basic side.

TABLE S CHEMICAL AND PHYSICAL PROPERTIES OF SOURCE CLAYS

SSS5 SA' y^l SEC² aϋ viscosity³

(m²/g) (cc/g) (meq/100 gj (slurry) (cps)

A 67 0.11 104-112 7.95 357 @ 36% Sdids

B 62 0.089 84-100 8.75 375 @ 38% sdids C 30 0.064 111 9.68 1420 @ 9% solids D 52 0.10 80 5.51 354 @ 38% soiids E 39 0.078 . 5.10 323 @ 24% solids

^* Surface area (BET method) and p_dre volumes (BJH metnoα) were measured simultaneously wⁱth Micromeπtics ASAP 2400. 2. Cation excnange capacitv 3. Measured with Brooκfield viscomet i #3 soincle except sample C. #6 spindle). TABLE 5 (Contd.)

CHEMICAL ANALYSIS ( TSi

HQ, A' £S flfi ≤lfi ϋlafi * CLAY TYPE

^A 67.6 20.7 2.82 5.44 3.32 0.37 0.19 Ca-mcntmonllonite

B 67.1 19.4 5.87 3.85 3.10 0.30 0.44 Ca-montmonilonite

C 63.5 21.1 5.08 3.41 4.10 2.12 0.70 Na-montmonllonite

0 67.2 22.1 3.80 4.16 1.27 0.32 1.16 Ca/AI-montmonllonite

^E 70.7 20.0 1.19 4.67 2.29 0.66 0.51 Ca/Na-montmonllonite

EXCHANGEABLE CATIONS (meg/100 ol EXCHANGE CATION RATIO

C *^z lag*² UT JS* Al±Ef*³ (Dlviiaπt +tτtvalβπt/monovιl«ιt)

^A 137.9 15.8 4.25 .38 33.2

B 115.4 19.2 5.18 .94 • 22.0

C 58.0 10.0 41.7 2.01 - 1.56

0 25.7 10.2 5.74 32 45.4 13.4

E 57.1 20.2 11.2 41 - 6.67

in contrast those days exhibiting relatively low (+2. +3/ + 1) exchange cation ratios (Gays C. E) show littl or no improvement In toxin binding rapacity when treated with either of two different sequestrant salts. T latter two days also generally possess lower surface areas and pore vdumes than do the other days whi show ennaπcement in toxin binding capacity upon treatment with seαuestraπt salts. TABLE 6 PHYSICAL PROPERTIES/AFLATOXIN B1 BINDING CLAY/SALT COMPLEXES: EFFECT OF CLAY/SALT TYPE

Salt csαa: TP-soαium triooiypnosonatβ: TS-tβtrasooium oyroonosonata: AP-soαium acid oyroonosoπai HP-soαium naxamataonosonaiβ: SC-soαiu citrata: ST-soαium tniosuttata: OP-disooium onosonata. TABLE 6 (C0NTD.) PHYSICAL PROPERTIES/AFLATOXIN B1 BINDING CLAY/SALT COMPLEXES: EFFECT OF CLAY/SALT TYPE

Salt coca: TP-sooium tnpdypnospnata: TS-iβtrasodlum pyropiwsoπate; AP-sodlum acid oyroonospnat HP-soαium naxa ataonospnata: SC-soαiu citrata: ST-aodium tniosuilatβ: DP-dlsooium pnospnata.

EXAMPLE V

The fdlowing in vrvo tests were performed to illustrate the improvement in binding of aflatoxin that occurs when a formulation of Clay B and 4% sodium acid pyrophosphate is employed in a living system as compared to using untreated base clay (CLAY B). In these experiments, 1 week old Arbor Acres X Peterson broiler chickens were wing banded and randomly placed in Pβtersime battery cages, 25/pβn, 2 pens/group, except for group 1 which contained only 10 birds. They were provided brooding heaters at 95 °F± 5 °F, water and feed ad libitum. On day 7 the brooder heaters were turned down to 90 °F± 5 °F. On day 10 the birds were transferred to a Petersime growing battery according to a randomization schedule. Ten birds were placed in each pen and maintained on their appropriate diets. Each group of 10 birds represented a saπφiing period. Ambient temperatures were then maintained at 85 °F ± 5 °F. Trough type feeders and waterers were used.

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 ¹ C aflatoxin B1 (¹ C AFB1) and aflatoxin B1 (AFB1) to be given to the birds was determined. The calculated amount of C AFB1 and AFB1 and approximately 0.15 gms of feed (either treated with base day, base day/4% sodium acid pyrophospnate(4%], or nontreated. depending on the group) was placed in a small gelatin capsule, capade 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.

SAMPLING: Sample times for each group of birds was 1/2 hour. 1 hour. 2 hours, 4 hours and 6 hours. Liver and Wood samples were obtained at these samding times. The liver saπφles were immediately frozen at -20 °C. Blood was drawn into 10 ml hepaπnized vacutainer tubes and immediately refrigerated (maximum of 6 hours) until ceπtrifuged and the piasma removed and frozen at -20 °C.

ASSAY: The samdes were assayed for levels of C AFB1 by taking subsarndes of individual livers (1.0 gm) which were homogenized in 3X vdumes of distilled water and 5 mis of chlorof orm-methan (2:1) using a nigh speed^' dender. The chloroform-methand layer was removed and placed in a dear glas scmtillation vial containing 15 mis of Aqualyte Plus (J.T. Baker) scintillation cocktail. One ml of dasma wa added to 19 mis 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 ¹ C AFB1 standard. Background counts were subtracted from the total counts before dividing by the samde 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.

¹ C AFB1 PREPARATION: C AFB1 was obtained from Moravek Biochemical. Brea. CA. The specific activity of the ¹⁴C AFB1 was 100-200 μCt/mmdβ. AFB1 was obtained from Sigma Chemicals.

DATA ANALYSIS: The amount of ¹⁴C AFB1 In the liver and plasma was compared between the birds treated at the 0.1% additive level (20 and 80 ppb ¹ C AFB1) and 0.5% additive level (20 and 80 ppb ¹ C AFB1) versus the nontreated birds (20 and 80 ppb ¹ C AFB1). The pharmacokinetic parameters o* rate of adsorption, rate of elimination and aree-under-the-curvβ were computed for liver and plasma using the curve fitting program ESTRIP (Brown and Manno. J. of Pharmaceutical ScL 1978. Vd. 67, 1687-1691). Analysis of variance was performed on the ¹ 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 isd test The probability of a type 1 error was set at the nominal 5% level.

RESULTS: Figures 6A, 6B and 7A, 7B which were derived from the data in Table 7 illustrate graphically what happens to the amount of ¹ C AFB1 detected in blood serum and liver tissue versus time, respectively when chicks were fed two levels of radidabeied aflatoxin in diets containing two different level of base clay or base day dus sequestrant. For both dood serum and liver tissue, aflatoxin levels pea duπng the first hour after exposure and then gradually drop off with time. Clearly, diets containing eithe base day, or those containing base day dus sequestrant provide protection against exposure to aflatoxi -20-

"Sodium Acid Pyropnosohate as evidenced by the fact that significantly lower ieveis 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 Tade 7).

However, as is also apparent the diets treated with base day dus sequestrant are significantly better at reducing aflatoxin detected in blood serum (by a factor of 2 to 4X) and liver tissues (by a factor rf 2 to 8X) at the 1 hr peak time as compared to diets containing only base day (without sequestraπt). This expenment. then, provides in vivo verification of the enhancement in aflatoxin binding capacity afforded by treating a susceptide montmoriilonite day with a typical sequestraπt salt used in food processing.

EXAMPLE VI

The fdlowing in vitro tests were performed to demonstrate that other mycotoxins besides aflatoxin exhibit enhanced binding when exposed to appropriate day/sequestraπt formulations. In this sat ot experiments, five other commercially significant mycotoxins were examined: deoxynivalend, zearalenone, ochratoxin A, citrinin and T-2 toxin. The ay/sequestraπt samples used in these experiments were the same ones as used in Examde II.

The fdlowing modifications to the in vitro capacity test described in Examde II were utilized in the extraction and analysis of these toxins:

Extraction Procedures

1 ) For zearalenone. the extraction procedure was the same as for aflatoxin B1.

2) For ochratoxin A and citrinin. the aqueous phase was acidified with 5 drops of 10% aqueous HO and then extracted twice with 3 mi portions of dichloromethane. The organic extracts were combined and evaporated to dryness under nitrogen pπor to analysis.

3) For T-2 and deoxynivalend, the aqueous phase was saturated with sodium chloride and then extracted three times with 3 ml portions of ethyl acetate. The organic extracts were combined and evaporated to dryness under nitrogen pπor to analysis. Analvsis Procedures

1 ) For ochratoxin A and citrinin, the analysis procedure was the same as for aflatoxin B1.

2) For T-2. deoxynivalenol and zearalenone, the residue from the extraction of the aqueous phase was dissolved in 40 μi of ethyl acetate. 0ne (1) μl of this sdution was added by on-cdumn injection onto a 12 m cross-linked methyl silicon capillary cdu n (0.2 mm I.D. with 33 micron film thickness). Initial temperature was held at 40 °C for 1 min and then ramped at 40 °/min to a final temperature of 270 °C. Peaks were quaπtitated by computer comparison of integration values for the total Ion chromatograms of Individual samde runs with known standards which were subiected to the same chromatographic conditions. Standards were routinely analyzed to ensure that the sensitivity of the GC/MS did not significantly change during the experiment Due to the extensive time requirements for GC/MS analysis. TLC analysis for zearalenone was found to be advantageous.

Binding experiments were run at two different sorbeπt levels: 100 mg and 10 mg sorber*/40 μg toxin. Figures 8 - 11 are bar graphs showing the results obtained when 100 mg of the various day/seαuestraπt formulations were used to bind 40 μg of deoxynivalend. zearalenone. ochratoxin A and citrinin. respectively. With the exception of zearalenone. binding of other toxins was low (i.e. < 10%) when using orty 10 mg of sorbeπt: 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 trid derivatives (which were subsequently desorbed) was at least partially responsible for its (apparent) reduction by binding.

Nevertheless, as is dear from the figures, each of the other toxins exhibit enhancement of binding in the presence of some combination^) of day /sequestrant. That not all or the same day/seαuestrant combinations are effective in this regard is beiieved to be a consequence of not having determⁱned the optimum day/sequestrant ratio for that particular toxin and seαuestrant (eg. - see Examde M⁾. Also. 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 day/sequestraπt ratio for promoting enhanced binding of a particular mycotoxin would not necessarily be the optimum for another.

EXAMPLE VII

The fdlowing in vitro test was performed to demonstrate that sequestrants enhanced the effectiveness of sorbeπt materials other than moπtmorillonites in binding a mycotoxin. in this experiment aflatoxin bonding to phosphate treated (and untreated) pseudoboehmite alumina (a high surface area. partially crystalline oxyhγdroxxie of alumina) and pyrophyllite (a 2:1 phytlosiiicate possessing a structure identical to montmoriilonite but devoid of intβrtayer cations) were compared to a Ca-montmorϋlonite obtained in Arizona. The phosphate salt used In these experiments was sodium acid pyrophosphate.

Procedure A sdution of sodium add pyrophosphate was prepared by adding 50 mg of the salt to water and adjusting to 250 ml in a vdumetric flask. An aflatoxin sdution was prepared by adding 1 mg of aflatoxin B1 to 1 mi of methand (reagent grade). 1 mi of the phosphate sdution was then added to 100 mg of the sorbent materials descnbed above in a test tube and incubated for 1 hr at 37 °C in a water bath. Then 20 μ I of the B1 sdution 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 dace erf the phosphate sdution.

After extraction as per Examde II, the residue was dissolved in 100 μi of chloroform. 2 μi of thi sdution was spotted on a 10x10 cm Analtech HPTLC-HLF silica gel date (lot # 20888). The plate wa developed with a chloroform /acetone sdution (9/1 , v/v) and quantitation was made by visual companso of fluorescence (365 nmi with known standards of aflatoxins B1. B2. and G1 spotted on the same date. Resuts. reported in Tade 8. show that the addition of phosphate salt enhances aαsorptton of aflatoxin B1. regardless of the soroent type emdoyed. Significantly, when the pnosphate salt is present, it also inhibits the formation of related species d aflatoxin (l.e. - B2. G1) from B1 as. for examde. wnen alumina and pyrophylllte are the soroeπts .

These results show that the use of sequestrants such as sodium acid pyrophospnate with vanous soroent matenals to enhance the binding of aflatoxin is a general phenomenon and not restncted to a narrow class d day minerals (Le. • montmortlonitβs).

TABLE B EFFECT OF ADDING Na ACID PYROPHOSPHATE: AFLATOXIN BINDING ON OTHER SORBENTS

SORBENT I TREATED , WT. AFLATOXINS EXTRACTED FROM SORBE T (ng)

I . - YES ' 81 | B2 ι G1

Alumina I + 37 j 0 0 I

Alumina - 380 10 I 0

Pyropnyilitβ + 410 0 0

Pyroonyllitβ - 650 37 12

IMontmonitonM 0 0 0

I Montmonllonite ' - I 230 0 0 I

Claims

CLAIMS:

1. A dry sdid paniculate animal feed additive comprising partides of a phyllosilicate mineral capade of inactivating mycotoxins. said particles being coated with a water-soiude sequestering agent in an amount sufficient to enhance the mycotoxin inactivating capacity of said partides of mineral.

2. The feed additive of daim 1 wherein said sequestering agent is further charaαerized by being caoade of dispersing said partides of phyllosilicate and is present in amount in excess of that required to disperse said partides of phyllosilicate in water.

3. The feed additive of daim 1 wherein said phyliosiiicate is a high calcium/low sodium montmoriilonite day and said sequestering agent is present in amount in the range of about 2 to 10% based on the weight d said day.

4. The feed additive of daim 1 wherein said sequestenng agent is selected from the group consisting of sodium, calcium and potassium acetates: sodium, calcium and potassium citrates and free acid and monoisopropyl, monogiyceride stearyi and triethyi derivatives thereof; disodium dihydrogen and disodium caldum salts of ethyienediaminetetraacetic acid: caldum and sodium gluconates; oxystearin; monocaicium acid, dibasic potassium, sodium aluminum, dibasic sodium, monobasic sodium, tribasic sodium orthophosphates: calcium and sodium hexametaphosphate: tetrasodium and sodium pyrophosphate: sodium tripdyphosonate: calcium phytate: sodium and sodium potassium taπrates and the free acid thereof: sooium thiosulfate: and mixtures of the aforementioned.

5. The feed additive of claim 2 wherein said sequestenng agent is a phosphate or citrate salt. 6. The feed additive of claim 3 wherein said montmoriilonite has exchangeade cations whose divalent dus trfvaieπt/monovaieπt cation ratio is greater than 7.

7. The feed additive of daim 3 wherein said montmoriilonite has a surface area in the range d about 40 to 80 rr^/gm and a total pore vdume in the range of about 0.1 to 0.3 cc/gm.

8. The feed additive of daim 1 wherein said phyilosϋicatβ is coated by spray drying an aqueous slurry of phyilosϊicate and sequestraπt

9. The feed additive of daim 1 wherein said phyliosiiicate is coated by mixing an aqueous slurry d phyllosilicate with sequestrant and then drying and pulverizing the mixture.

10. A dry sdid animal feed composition in which biodegradable feed is contaminated with a mycotoxin and is admixed with a mycotoxin inactivating agent comprising partides of a phyllosilicate mineral capade of inactivating mycotoxins. said partides being coated with a sequestering agent in an amount sufficient to enhance the mycotoxin inactivating capacity rf said phyllosilicate.

11. The feed composition of claim 10 wherein said sequestering agent is further characterized by being capade of inactivating mycotoxins when coated onto partides of phyliosiiicate in an amount sufficient to enhance the mycotoxin inactivating capacity of said phyllosilicate.

12. The composition of daim 10 wherein said phyllosilicate is a calcium montmoriilonite.

13. The composition of daim 10 wherein said calcium montmoriilonite has exchangeade cations whose divalent dus trivalent/monovalent cation ratio is greater than 7. 14. The composition of daim 13 wherein said montmoriilonite has a surface area in the range of about 40 to 80 m²/gm and a total pore vdu e in the range of about 0.1 to 0.3 cc/gm.

15. The composition of daim 10 wherein said sequestering agent is selected from the group consisting of sodium, caldum and potassium acetates; sodium, calcium and potassium citrates and free acid and monoisopropyi, monogiyceride stearyi and triethyl derivatives thereof; disodium dihydrogen and disodium calcium salts of ethyteπediaminetetraacetic acid; calcium and sodium gluconates: oxystearin; monocalcium acid, dibasic potassium, sodium aluminum, dibasic sodium, monobasic sodium, tribasic sodium orthophosphates: calcium and sodium hβxametaphosphate: tetrasoαium and sodium pyrophosphate; sodium tnpdyphosphate; caldum phytate; sodium and sodium potassium tartrates and the free acid thereof; sodium thiosulfate; and mixtures of the aforementioned.

16. The composition of claim 10 wherein said coated phyliosiiicate is present in an amount in the range of about 0.025 to 1.5% by weight of the animal feed.

17. The feed composition of daim 10 wherein said animal feed is poultry, swine or dairy feed.

18. The composition of daim 10 wherein said mycotoxin is aflatoxin.

19. The composition of daim 10 wherein said mycotoxin is aflatoxin. said phyllosilicate is a high caicium/low sodium montmoriilonite day and said sequestrant is a phosphate or citrate salt present in an amount of about 2 to 10% based on the weight of said calcium montmoriilonite.

20. The composition of claim 10 wherein said phosphate salt is a condensed phosphate. 21. The composition of claim 20 wherein said condensed phosphate salt is sodium acid pyrophosphate.

22. The composition of daim 20 wherein said condensed phosphate salt is sodium acid hβxametaphosphate.

23. The composition of daim 20 wherein said condensed phosphate salt is tetrasodium pyrophosphate.

24. The composition of daim 10 wherein said sequestraπt is sodium citrate.

25. A dry sdid animal feed composition in which the feed is contaminated with a mycotoxin selected from a group consisting of aflatoxin B1, deoxynivalend, zearalenone. ochratoxin A, citrinin or T-2 toxin, admixed with from about 0.025 to 1.5% by weight of a high calcium/low sodium beπtonite that is coated with from about 2 to 10% by weight of a sequestraπt

26. The composition of daim 25 wherein said feed Is a dry poultry, swine or dairy feed.