Transcript Slide 1
Mycotoxins and Use of Mycotoxin Binders to Alleviate Mycotoxicoses
By Dennis R. Taylor, Ph.D. Sponsored by
BROOKSIDE-AGRA
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About Mycotoxins
Over 370 known mycotoxins * Mycotoxins are produced by molds and fungi that grow on grains Mycotoxins are specific chemicals (called “metabolites”) produced by the molds and fungi
Apspergillus flavus
→ produces aflatoxins Aspergillus flavus growing on corn * Handbook of Toxic Fungal Metabolites – Cole / Cox (1981) Aspergillus flavus magnified DRTaylor Consulting
About Mycotoxins
Fusarium toxins - Deoxynivalenol (vomitoxin), T-2, zearalenone
Fusarium graminearum
→ produces vomitoxin, T-2, zearalenone DRTaylor Consulting
About Mycotoxins
Mycotoxins can adversely affect animal health and performance.
Mycotoxins are potent chemicals and can produce toxic effects at very low levels – parts per million (ppm) and parts per billion (ppb) range.
Mycotoxins are very common – it is hard to avoid eventually getting some contaminated grains.
Once mycotoxins are produced, they are hard to get rid of…
• • •
They cannot be destroyed by heating – even to 340
°
C.
They cannot be washed off – low solubility in water.
There are no effective chemical treatments.
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About Mycotoxins
Fatty liver due to aflatoxin (left) compared to normal liver (right) (Ledoux UMC)
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About Mycotoxins
Only six mycotoxins are of real commercial concern… Aflatoxin B1 Deoxynivalenol (“vomitoxin”) Ochratoxin A Zearalenone Fumonisin B T-2 toxin DRTaylor Consulting
Effects of Mycotoxins on Animals
TOXIN Aflatoxins B1, B2, G1, and G2 Ochratoxins Affected Commercial Species Duckling, turkey poult, chicks, mature chickens, piglets, calves, pregnant sows, sheep, human, fish Swine,duckling, chicken, human EFFECTS on ANIMALS Carcinogenic; attacks liver; reduced growth rate; hemmorrhagic enteritis; suppression of natural immunity to infection; decreased production of meat, milk, and eggs.
Toxic to kidneys and liver; abortion; poor feed conversion, reduced growth rate, reduced immunity to infection.
Deoxynivalenol (Vomitoxin) T-2 Toxin Zearalenone Fumonisin B1, B2 Food refusal by swine; vomiting and diarrhea; reduction in weight gain. Swine, cattle, chicken, turkey, horse, human Oral lesions. Severe inflammation of gastrointestinal tract and possible hemorrhage; edema; infertility; degeneration of bone marrow; reduced weight gain; slow growth; sterility. Swine, dairy cattle, turkey, lamb Estrogenic effects (edema of vulva, prolapsed vagina, enlargement of uterus), abortion, infertility, stunting. Atrophy of testicles, ovaries, enlargement of mammary glands.
Horses Leucoencephalomalacia, "blind staggers," in horses.
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Recommended Acceptable Levels
FDA Guidelines on maximum levels of Aflatoxin, Vomitoxin and Fumonisin in feedstuffs for animals Aflatoxin M1 Aflatoxin B1 < 0.5 ppb in milk < 20 ppb in feeds Vomitoxin 5 ppm swine 10 ppm cattle, poultry Fumonisins 5 ppm horses 10 ppm swine 50 ppm beef cattle and poultry THE ROLE OF MYCOTOXINS IN FOOD AND FEED SAFETY Jon Ratcliff — Food and Agriculture Consultancy Services, UK www.facs.org.uk
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Recommended Acceptable Levels
EU Maximum permitted levels of mycotoxins in animal feed and foods for human consumption Aflatoxin B1 5 ppb animal feedstuffs – cattle, sheep 2 ppb animal feeding stuffs – adult poultry and swine 1 ppb animal feeding stuffs – piglets and chicks Ochratoxin A 5 ppb dried fruit and nuts THE ROLE OF MYCOTOXINS IN FOOD AND FEED SAFETY Jon Ratcliff — Food and Agriculture Consultancy Services, UK www.facs.org.uk
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What can be done? Limited options
Only buy uncontaminated grains
•
But difficult to accomplish because even if you analyze for toxins you may miss them.
•
Usually contamination is not uniformly distributed throughout the sample.
• •
Sometimes nothing but contaminated grains are available.
Remember – not possible to remove toxins by heating or washing.
Use mycotoxin binding sorbents to sequester toxins
•
This approach – first reported in 1988 by Phillips & Taylor, et al. – has over 30 years of peer reviewed research and commercial use proving its viability and utility.
•
At last count, there were over 100+ companies world-wide offering mycotoxin binders – and new offerings are made practically every day.
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Early History of HSCAS as Aflatoxin Binder
1988 – Phillips, Taylor , Kubena, Harvey show 0.5 wt% hydrated sodium calcium aluminosilicate (HSCAS) protects CHICKENS against 7.5 ppm AFB 1 (Poultry Sci., 67, 243-247) 1989 – Harvey, Phillips, Kubena, et al. show HSCAS protects SWINE against AFB 1 (Amer. J. Vet. Res., 50, 416-420)
1991 – Kubena, Huff, Harvey, et al. show HSCAS protects TURKEYS against AFB 1 (Poultry Sci., 70, 1823-1830) 1991 – Harvey, Kubena, Phillips, et al. show HSCAS protects LAMBS against AFB1 (Amer. J. Vet. Res., 57, 152-156)
1994 – Phillips, Harvey, Kubena, et al., show HSCAS protects GOATS against AFB 1 (J. Anim. Sci., 72, 677-682) DRTaylor Consulting
Evaluating Mycotoxin Binders
Two possible approaches…
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In-vivo testing
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Uses live animals
•
Uses mycotoxin contaminated feeds
•
Uses mycotoxin binder mixed with contaminated & uncontaminated feeds.
•
In-vitro testing
•
Does not use live animals
• •
Generally uses low level of mycotoxin dissolved in water.
Uses mycotoxin binder to remove the mycotoxin from the water.
•
Usually does not use mycotoxin contaminated feeds. DRTaylor Consulting
Comparison: in-vitro vs. in-vivo testing
IN-VIVO
•
In-vivo tests are very expensive. It usually cost $15K-$20K to conduct an in-vivo evaluation with perhaps 3 or 4 treatment groups (poultry least expensive).
• •
In-vivo tests take time (~40 days / evaluation for poultry) There are too many competitive mycotoxin binders to evaluate in a single in-vivo test.
IN-VITRO
• • •
In-vitro tests are much less expensive. It usually costs about $350 to conduct an in-vitro evaluation of a sorbent (+4 toxins).
In-vitro tests are much quicker – usually about 1 week.
In-vitro tests are much more reproducible because all conditions can be carefully controlled.
•
Any number of competitive sorbents can be evaluated, and at different periods in time.
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Does in-vitro binding correlate with in-vivo binding?
The answer is YES… …at least for aflatoxin B1 in-vitro versus in-vivo testing.
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Correlation: In-vivo Wt. Gain vs. In-vitro Binding Activity 1 Broilers: Aflatoxin Challenge - 3000 PPB 2,4 2,3 2,2 2,1 2 Conclusion:
In-vitro
binding correlates with
in-vivo
response
Pos. Control (0 ppb AFB1/ 0 binder) 1,9 1,8 Binders: Increasing Binding Activity (3000 ppb AFB1/ 0.5% binder) 1,7 1,6 Neg. Control (3000 ppb AFB1/ 0 binder) 1,5 0 0,05 0,1 0,15 0,2 In-vitro Toxin Bound/Binder Ratio (@ 50% binding) 1 2002, Dr. Carlos Mallman, Univ. Federal de Santa Maria, Brazil 0,25 DRTaylor Consulting
So what makes one mycotoxin binder Good – and another not so good – or even Poor?
An in-vitro approach to finding the answer
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Physical properties of montmorillonite Raw clay
• Density (Kg/m 3 ) • Porosity (cc/g) • Pore diameter (µm) • Surface area (m 2 /g)
Scanning Electron Microscopy at high magnification DRTaylor Consulting
Chemical properties of montmorillonite Exchangeable cations → Ca ++
• Surface acidity (pK a ) • Cation Exchange Capacity (meq/100 g) • Exchange cations (Na + , K + , H + , Mg ++ , Ca ++ ) • pH of clay slurry in water
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Protocol of the study
Obtain a representative group of commercially available binders.
Measure aflatoxin B 1 binding under a constant set of conditions … (20 µg toxin / 1 mg binder / 1 mL)
Obtain XRD & measure complete set of physical & chemical properties.
Determine if aflatoxin binding correlates to any particular property … or combination of properties.
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Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Mineralogy Product Aflatoxin-% Binding Mineralogical Composition Code ( 20 µg / mg / mL ) ( basis XRD analysis ) ---------------------------------------------------------------------------------------------------------------------------------------------- A B C D E 99.2
97.1
95.2
95.9
94.6
Excellent Binding Ca montmorillonite…………..……. + Qz, FS Ca/Na montmorillonite………..….. + Qz Ca montmorillonite (low level)….. + Opal CT, Qz Attapulgite + Ca montmorillonite. + Qz Sepiolite F G H 90.7
Ca/Na montmorillonite……………. + Qz ---------------------------------------------------------------------------------------------------------------------------------------------- 87.5
Na montmorillonite………………… + FS I Good 92.6
87.3
Ca montmorillonite Na/Ca montmorillonite……………. + Qz J Binding 86.4
Na montmorillonite K 85.2
Attapulgite (low level) L S Binding 83.6
Attapulgite M N 80 Ca montmorillonite……….…….….. + Qz ---------------------------------------------------------------------------------------------------------------------------------------------- 73.9
Ca montmorillonite…………….…… + Opal CT O Poor 70.5
Ca montmorillonite (low level)….… + Qz, FS P Binding 66.2
Ca montmorillonite Q 55.9
Clinoptilolite, mordenite…….…….. + Qz R Inadequate 47.7
Kaolinite + mica/illite 44 Ca montmorillonite (low level) T U 25.2
16.9
Amorphous silica Amorphous silica DRTaylor Consulting
Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Physical Properties Product Code Slurry Loose Density Hg PV Hg Pore BET Surface pH (Kg/m 3 ) (cc/g) Diameter (µm) Area (m²/g) -------------------------------------------------------------------------------------------------------------------------- A B C D E F G H I J K L M N O P Q R S T U 6.9
9.2
4.0
7.5
8.5
9.5
9.82
9.1
9.12
9.2
9.5
6.8
8.35
6.84
8.4
9.1
9.7
5.1
6.4
10 6.8
806 1050 602 607 559 751 701 1048 632 1067 663 812 777 761 657 830 990 342 693 215 350 0.5119
0.1533
0.4156
0.5081
0.4432
0.1213
0.2713
0.2473
0.5546
0.0848
0.3849
0.1785
0.1292
0.212
0.3088
0.2414
0.1844
0.6061
0.1776
0.9556
1.6362
0.02
0.028
0.09; 0.015
0.05
0.028
0.03
0.03
0.038
0.065
0.07
0.03
0.035
0.03
0.03
0.4
0.04
0.05
0.68
0.025
0.19; 1.0
a 0.11
a a Bimodal distribution of porosity; two maxima in pore volume versus pore diameter plots.
65 25 97 140 195 57 77 21 31 21 32 64 81 82 14 75 18 25 8 66 72 DRTaylor Consulting
Binding of Aflatoxin by Commercially Available Mycotoxin Binders: Chemical Properties Product Surface Acidity ( meq/g ) Cation Exchange Capacity ( meq/100 g ) Code ------------------------------------------------------------------------------------------------------------------------------------------ A pKa <1.5 pKa >1.5 Total Ca 0.079
0.089
0.168
++ 52.0
Na 9.0
+ Total 76 B C D I E F G H J K L M N O P Q R S T U 0.021
0.080
0.080
0.180
0.060
0.021
0.039
0.010
0.042
0.020
0.080
0.030
0.117
0.039
0.050
0.021
0.020
0 0 N/A 0.053
0.080
0.130
0.040
0.200
0.029
0.142
0.032
0.106
0.110
0.020
0.140
0.076
0.100
0.130
0.020
0.001
0.020
0.060
N/A 0.074
0.160
0.210
0.220
0.260
0.049
0.181
0.042
0.148
0.130
0.100
0.170
0.193
0.139
0.180
0.040
0.020
0.020
0.060
N/A 62.0
28.6
20.0
54.1
59.8
110.0
38.0
49.0
42.0
63.6
39.3
68.0
111.0
76.0
38.7
144.0
2.8
41.2
1.3
1.2
53.0
0.4
3.0
1.5
3.0
86.0
75.0
53.0
67.0
26.3
31.8
12.0
2.0
2.0
19.9
57.0
0.6
21.1
183.2
245.9
119 48 39 77 102 207 121 127 125 121 79 102 122 82 63 215 7 75 185 248
In-Vitro Binding vs. Physical / Chemical Properties of Mycotoxin Binders
RESULTS No single physical property correlates with in-vitro binding of aflatoxin
No single chemical property correlates with in-vitro binding of aflatoxin
… however, there is a weak correlation with combination of strong surface acidic sites (of pKa <1.5) and Mg ++ concentration 100 80 60 40 20 0 0 20 40 60 80 ln [ (<1.5 pKa) x (Mg ++ conc.) ] 100 120
Optimum binding – like tumblers in a lock… Optimum set of physical / chemical properties in binder … …like tumblers in a lock… Another toxin Aflatoxin fits perfectly… …so lock and key mechanism work together to bind aflatoxin If toxin does not match physical / chemical properties of binder - lock and key mechanism won’t work… DRTaylor Consulting
So - how does FLO-BOND compare to the competition?
Some more in-vitro and in-vivo studies
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Flo-Bond is the only mycotoxin binder with Organic approval in the USA DRTaylor Consulting
Recent Dioxin Analysis on Flo-Bond
All samples below 1.5 ppt EEC limit for sum (dioxins + PCB’s) Except sample B&N 8511 which was mining sample not used for Flo-Bond
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2010 Competitive in-vitro Mycotoxin Binder Study METHODOLOGY Mycotoxins / Concentration Binder Level: Binding Conditions: Aflatoxin B1, Deoxynivalenol, Fumonisin B1, Ochratoxin A, T-2 Toxin, Zearalenone – 0.5 wt% all @ 2000 ppb Adsorption phase: 3 reps. @ pH 3.0
Desorption phase: 3 reps. @ pH 8.0
% Efficie ncy Binders Studied Flo-Bond, Flo-Bond AZ (experimental), Flo-Bond X (experimental), NovaSil Plus, MycoAd New experimental products (Brookside-Agra currently conducting in-vivo trials) DRTaylor Consulting
2010 Competitive in-vitro Mycotoxin Binder Study Flo-Bond % Adsorption pH3 % Desorption pH 8 % Efficiency New Flo-Bond AZ (AFB1/ZONE) % Adsorption pH 3 % Desorption pH 8 % Efficiency NEW Flo-Bond X (Full Spectrum) % Adsorption pH 3 % Desorption pH 8 % Efficiency NS Plus MycoAd % Adsorption pH 3 % Desorption pH 8 % Efficiency % Adsorption pH 3 % Desorption pH 8 % Efficiency Results by Trilogy Analytical Lab, Missouri, USA 99.9
0.3
99.6
100 0.1
99.9
100 0 100 RESULTS AFB1 100 0 100 100 0 100 FUM 95 87 8 90 49.4
40.6
95.3
7.9
87.4
99.2
86.2
13 98.4
94.7
3.7
DON 20.6
15.9
4.7
22.6
15.6
7 36.7
12.8
23.9
27.3
20.8
6.5
17.6
14.6
3 OCHRA 77.8
77.8
0 94.4
93.3
1.1
98.1
9.2
88.9
95.2
95.1
0.1
91.8
89.3
2.5
40.8
0 40.8
35.4
0 35.4
T-2 23.7
0 23.7
67.2
7.3
59.9
98.3
0 98.3
ZONE 31 30.8
0.2
96.5
4.4
92.1
99.8
0.3
99.5
74.9
35.6
39.3
50.7
42.6
8.1
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2010 Competitive in-vitro Mycotoxin Binder Study General Aflatoxin B1 Experimental Blends… CONCLUSIONS While all mycotoxins studied (except DON) were significantly bound in-vitro under acidic (pH 3) conditions, some were significantly, or completely desorbed under basic (pH 8) conditions.
Aflatoxin strongly bound by all binders in-vitro under acidic (pH 3) & basic (pH 8) conditions. Numerous studies support in-vivo efficacy.
New experimental blends being studied by Brookside-Agra are showing promise for extending binder efficacy for other mycotoxins DRTaylor Consulting
Why does aflatoxin bind so strongly compared to other mycotoxins?
Aflatoxin B 1 between these …but none here Deoxynivalenol This grouping causes strong binding Ochratoxin A Zearalenone DRTaylor Consulting
Binding of the diketone moiety to cationic sites: the reason for the strong binding of aflatoxin The 1,3-diketone structure of aflatoxin possesses high electron density and is therefore strongly attracted to positively charged sites Ca ++ Clay Structure This forms a type of complex called a CHELATE DRTaylor Consulting
Binding of the diketone moiety to cationic sites: the reason for the strong binding of aflatoxin Broken bonds at crystal edges can also generate cationic sites + These sites can also bind aflatoxin as a CHELATED complex Ca ++ Clay Structure DRTaylor Consulting
IN-VIVO DATA USING FLO-BOND
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Chicken Feeding Trial: FLO-BOND vs. NovaSil : 2500 ppb Aflatoxin B1
0 ppb AFB1 / no binder 350 300 250 200 150 100 50 0 2500 ppb AFB1 / .75% FB 2500 ppb AFB1 / .5% NS 2500 ppb AFB1 / .5% FB 2500 ppb AFB1 / .25% FB 2500 ppb AFB1 / no binder Therefore 0.25% FLO-BOND = 0.5% NovaSil 0.5% FLO-BOND better than 0.5% NovaSil 1 2 3 Week 4 DRTaylor Consulting
2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge
Evaluation of FLO-BOND in growing pigs fed 0.9 ppm and 1.8 ppm deoxynivalenol (DON) contaminated diets
Test: A 7-d (64-pen) pig study was conducted comparing live performance (average daily gain, feed conversion, and feed consumption) of pigs fed FLO-BOND at levels of 0%, 0.25% and 0.50% in commercial type diets contaminated with DON mycotoxin at levels of 1.8 and 0.9 ppm
Location: Virginia Diversified Research, Corp., Harrisonburg, VA; investigator: Michael D. Sims DRTaylor Consulting
2010 In-Vivo Swine Trial Using FLO-BOND against Deoxynivalenol Challenge RESULTS FLO-BOND vs. DON @ 0.9 ppm FLO-BOND lbs/ton DON (ppm) Day 0-7ADG (lbs/d) Day 0-7 Feed/Gain (lb/1b) Feed Consumption (lb) 0 0 1.982a 1.324a 18.36a 5 0 1.893a 1.284a 16.99ab 0 0.9 0.693c 2.415c 12.30c 5 0.9 1.430b 1.632b 16.66b 10 0.9 1.307b 1.663b 15.09b FLO-BOND vs. DON @ 1.8 ppm FLO-BOND lbs/ton: DON (ppm): Day 0-10 ADG (lbs/d) Day 0-10 Feed/Gain (lb/lb) Feed Consumption (lb) 0 0 1.982a
1.324a
18.36a
5 0 1.893a
1.284a
16.99a
0 1.8
0.566c
8.544c
11.41d
5 1.8
1.079b
1.834b
13.4b
10 1.8
0.779bc
2.050b
10.61c
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FLO-BOND Does Not Hinder Uptake of Nutrients
Nutrient Effect on Level of HSCAS Nutrient Utilization Riboflavin Riboflavin Vitamin A Vitamin A Manganese Manganese Zinc Zinc Phosphorus, inorganic Phosphorus, inorganic Phosphorus, phytate Phosphorus, phytate 0.5 % 1.0 % 0.5 % 1.0 % 0.5 % 1.0 % 0.5 % 1.0 % 0.5 % 1.0 % 0.5 % 1.0 % None None None None None None None Slight tibia Zn decrease None None None None Chung, T.K.et al., 1990 Poultry Science 69: 1364-1370 Chung, T.K. and Baker, D.H., 1990 J Animal Science 68: 1992-1998 DRTaylor Consulting
Effect of Flo-Bond Plus on Mold Reduction
Summary Conclusions
Inclusion 0.5% Flo-Bond Plus 1.0% Flo-Bond Plus 2.0% Flo-Bond Plus Controls 1% Propionic Acid 5% Propionic Acid No Inclusions 25% Moisture Corn Mold Count(CFU/g) 108,500 2,450 100 949 699 700 900,000 % Reduction 87.94* 99.73* 99.99* 99.89* 99.92* This evaluation demonstrates that Flo Bond Plus can significantly reduce the mold count in a high moisture corn sample. * Compared to 25% Moisture Corn without any Inclusions CFU/g = Colony Forming Units / gram (after 48 hr. incubation @30ºC )
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What about processing?
What about quality control?
Can they affect product quality?
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FLO-BOND Process – No Added Ingredients – Only Drying & Grinding Selective Mining of HSCAS Strata Manufacturing Plant Product Dried & Ground into Powder Quality Control Check Bagging & Sample Retention Containerization & Shipping FLO-BOND DRTaylor Consulting
Effect of Thermal Processing on Surface Area vs. Binding of Aflatoxin B 1 100 Therefore, it is very important not to overheat clay that is to be used as mycotoxin binder Increasing Processing Temperature 90 80 70 BET Surface Area (m²/g) 60 100 90 80 70 60 50 50 40 DRTaylor Consulting
Effect of Particle Size (Grind) vs. Binding of Aflatoxin B 1 95 90 85 80 75 70 65 60 55 50 65 Therefore, it is very important to get good grinding in order to get maximum binding 70 75 80 % AFB1 Bound 85 90 95 DRTaylor Consulting
Important Considerations Before Buying a Mycotoxin Binder
Does the manufacturer have both in-vitro and in-vivo data demonstrating efficacy for his product?
Does the manufacturer have a proven track record for delivering a quality product?
Does the manufacturer have control over his source materials and manufacturing process?
Does the manufacturer maintain good quality control during the manufacturing process?
To all these questions, Brookside Agra can say “ YES ” with regard to its FLO-BOND mycotoxin binding product. DRTaylor Consulting
SUMMARY AND CONCLUSIONS
Montmorillonite is the most common commercially available mycotoxin binder.
Aflatoxin B1 is the most strongly adsorbed mycotoxin.
FLO-BOND is (HSCAS) montmorillonite binder that possess superior mycotoxin binding characteristics for many different mycotoxins.
Manufacturing conditions (temperature, grind) affect binding performance, so good quality control is absolutely essential.
Brookside-Agra is committed to good quality control during the manufacturing of FLO-BOND
USDA approved as organic product
FLO BOND is dioxin free –
(
i.e. - below EEC limits for dioxin content
)
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Thank you for your kind attention The End
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