SE541250C2 - Method for reduction of deoxynivalenol levels in feed - Google Patents

Method for reduction of deoxynivalenol levels in feed

Info

Publication number
SE541250C2
SE541250C2 SE1730126A SE1730126A SE541250C2 SE 541250 C2 SE541250 C2 SE 541250C2 SE 1730126 A SE1730126 A SE 1730126A SE 1730126 A SE1730126 A SE 1730126A SE 541250 C2 SE541250 C2 SE 541250C2
Authority
SE
Sweden
Prior art keywords
feed
weight
propionic acid
composition
glyceryl
Prior art date
Application number
SE1730126A
Other versions
SE1730126A1 (en
Inventor
Hugo Hjelm
Original Assignee
Perstorp Ab
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
Application filed by Perstorp Ab filed Critical Perstorp Ab
Priority to SE1730126A priority Critical patent/SE541250C2/en
Priority to PCT/SE2018/050453 priority patent/WO2018203812A1/en
Publication of SE1730126A1 publication Critical patent/SE1730126A1/en
Publication of SE541250C2 publication Critical patent/SE541250C2/en

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Classifications

    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/30Removing undesirable substances, e.g. bitter substances
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K20/00Accessory food factors for animal feeding-stuffs
    • A23K20/10Organic substances
    • A23K20/105Aliphatic or alicyclic compounds
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K20/00Accessory food factors for animal feeding-stuffs
    • A23K20/10Organic substances
    • A23K20/158Fatty acids; Fats; Products containing oils or fats
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K30/00Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • A23K30/10Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K50/00Feeding-stuffs specially adapted for particular animals
    • A23K50/10Feeding-stuffs specially adapted for particular animals for ruminants
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K50/00Feeding-stuffs specially adapted for particular animals
    • A23K50/20Feeding-stuffs specially adapted for particular animals for horses
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K50/00Feeding-stuffs specially adapted for particular animals
    • A23K50/30Feeding-stuffs specially adapted for particular animals for swines
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K50/00Feeding-stuffs specially adapted for particular animals
    • A23K50/40Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/30Removing undesirable substances, e.g. bitter substances
    • A23L11/34Removing undesirable substances, e.g. bitter substances using chemical treatment, adsorption or absorption

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Food Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Husbandry (AREA)
  • Zoology (AREA)
  • Birds (AREA)
  • Nutrition Science (AREA)
  • Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Agronomy & Crop Science (AREA)
  • Fodder In General (AREA)
  • Feed For Specific Animals (AREA)

Abstract

The present invention refers to a method for reduction of deoxynivalenol concentration in feed, said method comprising the following consecutive steps:a) adding a composition selected from the group consisting of propionic acid and/or glyceryl propionates to a feed, wherein said composition comprises at least 40 % by weight of propionic acid content and wherein said composition is added to said feed in an amount of between 0.5-10 ml/kg.b) anaerobically fermenting said feed.The present invention additionally refers to an animal feed that has been treated with the method.In a further aspect, the present invention refers to a composition selected from the group consisting of propionic acid and/or glyceryl propionates, for use in preventing a decreased feed intake or feed refusal in mammals.The present invention also refers to a composition selected from the group consisting of propionic acid and/or glyceryl propionates, for use in preventing immunosuppression in mammals.

Description

Method for reduction of deoxynivalenol levels in feed FIELD OF THE INVENTION The present invention refers to a method for reduction of deoxynivalenol concentration in feed, said method comprising the following consecutive steps: a) adding a composition selected from the group consisting of propionic acid and/or glyceryl propionates to a feed, wherein said composition comprises at least 40 % by weight of propionic acid content and wherein said composition is added to said feed in an amount of between 0.5-10 ml/kg. b) anaerobically fermenting said feed.
The present invention additionally refers to an animal feed that has been treated with the method.
In a further aspect, the present invention refers to a composition selected from the group consisting of propionic acid and/or glyceryl propionates, for use in preventing a decreased feed intake or feed refusal in mammals.
The present invention also refers to a composition selected from the group consisting of propionic acid and/or glyceryl propionates, for use in preventing immunosuppression in mammals.
BACKGROUND OF THE INVENTION Mycotoxins are highly toxic secondary metabolic products of various moulds, mainly those belonging to the genera Fusarium, Aspergillus and Penicillium. It has been estimated that at least 300 of these fungal metabolites are potentially toxic to animals and humans. The mycotoxins causing the most problems in agriculture, and also the ones most extensively investigated are aflatoxin Bi, zearalenone, deoxynivalenol (DON), T-2 toxin, ochratoxin A and fumonisin Bi. The individual toxicity of mycotoxins is extremely variable. It depends on the physical and chemical properties of each toxin, but also on the level of intake, the duration of exposure, the animal species, sex, age, breed and physiological status, nutritional status, environmental conditions and the synergy that can occur between mycotoxins simultaneously present in feed.
The side effects of ingesting mycotoxins are called mycotoxicosis, which can be a variety of medical conditions in both humans and animals. Some mycotoxins are reported to be carcinogenic, genotoxic, to cause liver disease, hematological changes and/or digestive disorders. However, in general, decreased performance, reproductive disorders and immunosuppression (resulting in a higher susceptibility to disease) are of major concern, leading to big economic losses at all levels of the food and feed production.
Deoxynivalenol (DON) belongs to the trichothecenes group of mycotoxins and is produced by different Fusarium species. The side effects of ingesting these mycotoxins are called trichotecenes toxicosis. Given in sublethal toxic doses via any route, the trichothecenes are immunosuppressive in mammals. Irritation of the skin and mucous membranes and gastroenteritis are another set of signs typical of trichothecene toxicosis. Hemorrhagic diathesis can occur and damage to dividing cells is expressed as lymphopenia or pancytopenia.
Eventually, hypotension may lead to death. Many of the severe effects described for experimental trichothecene toxicosis are due to dosing by gavage. From a practical perspective, high concentrations of trichothecenes often cause feed refusal and therefore are often self-limiting as a toxic problem. Because of the immunosuppressive action of trichothecenes, secondary bacterial, viral, or parasitic infections may mask the primary injury.
Refusal to consume contaminated feedstuffs is the typical sign of DON contamination of the feed, which limits development of other signs. If no other food is offered, animals may eat reluctantly, but in some instances, excessive salivation and vomiting may occur (because of its ability to cause vomiting, DON is also called vomitoxin).
In many parts of the world, DON is a substantial concern because of its common occurrence in feed grains and its well-known ability to cause feed refusal. Swine appears to be most sensitive to feed refusal, with greater tolerance by horses and dogs and even higher acceptance by ruminants. In swine, reduced feed intake may occur at dietary concentrations as low as 1 ppm, and refusal may be complete at 10 ppm.
Prevention of fungal infections during plant growth, harvest, storage and distribution would seem the most rational and efficient way to avoid mycotoxins in agricultural commodities. Common practical measures include planting of more resistant strains cereals, selection of high quality seeds, avoiding high plant densities and keeping a balanced fertilization. Careful selection of harvest date and harvesting procedure to minimize crop damage may reduce mould infections. Proper storage and the addition of antifungal agents may also diminish fungal growth but cannot detoxify already contaminated feedstuffs.
Despite all prevention efforts, contamination of agricultural products with mycotoxins still occurs and has a major economic impact on crop and animal producers as well as on food and feed processors. This has led to the development of a number of decontamination procedures. There are a number of physical treatments that have been tried, like washing, polishing, mechanical sorting and separation, density segregation, microwave heating and UV irradiation, however, the efficiency of these techniques varies a lot depending on the level of contamination and the distribution of toxins through the grain. Results obtained are thus uncertain and often connected with high product losses. Chemical methods require suitable reaction facilities and also additional treatment, such as drying and cleaning, that can make them time consuming and expensive. Also, many of the chemicals tested for this purpose diminish the feed’s nutritional value or palatability.
The most extensively investigated method for decontamination of mycotoxin contaminated feed is the addition of adsorbents with the ability to tightly bind and immobilize mycotoxins directly in the gastrointestinal tract of animals, resulting in a major reduction of the toxin bioavailability. This method is very successful for counteracting aflatoxins, however adsorption of other mycotoxins is limited (zearalenone, ochratoxin A) and does not work at all for still other toxins, such as DON.
Dänicke et al have disclosed methods for detoxification of DON contaminated feeds, such as maize, wheat and triticale using a wet preservation method with sulfites (sodium sulfite or sodium metabisulfite) and propionic acid. Very good results were obtained with regard to DON reduction in the feed. However, the problem with the methods disclosed by Dänicke et al is that sulfites are not considered to be safe from a health perspective, and hence are not allowed as feed additives in the EU. It was further on concluded by Dänicke et al that propionic acid does not contribute to a destruction of already existing DON which is formed prior to harvest by various Fusarium species. Wet preservation with only propionic acid added to the feed gave only a slight DON reduction by 14 to 16% of the initial concentration after a storage period of 63 days (Mycotoxin Research, 25/4, 215-223, 12/2009).
It has now quite surprisingly been found that a method using propionic acid and/or glyceryl propionates, wherein no sulfites are used, can reduce DON concentration in feed considerably. The method is cheap and easy to perform directly on the farm. The method according to the present invention does not lead to complete detoxification of the feed, rather to a reduction of about 50 % of DON levels in the feed. Nevertheless, this method will considerably improve the quality of the feed, which is of great importance to the individual farmer.
DETAILED DESCRIPTION OF THE INVENTION As described above, different kinds of mycotoxins require different approaches for dealing with them. For deoxynivalenol, there is today no method that meets the requirement of complete detoxification and at the same time is easy to perform and free from chemicals that are not considered safe to ingest for the animal.
The present invention refers to a method for reduction of deoxynivalenol concentration in feed, said method comprising the following consecutive steps: a) adding a composition selected from the group consisting of propionic acid and/or glyceryl propionates to a feed, wherein said composition comprises at least 40 % by weight of propionic acid content and wherein said composition is added to said feed in an amount of between 0.5-10 ml/kg. b) anaerobically fermenting said feed.
The composition added to the feed in step a) should comprise at least 40 % by weight of propionic acid content. This means that the weight of the propionic acid molecules and the weight of the propionic acid residues present in the glyceryl propionates should together constitute at least 40 % of the weight of the composition.
According to a preferred embodiment of the present invention, the composition comprises at least 50 % by weight of propionic acid content.
To use a composition comprising glyceryl propionates in the method according to the present invention is beneficial for practical reasons. Said composition is far less corrosive both to skin and to metals than is pure propionic acid, with the consequence that it is non-classified and much easier to handle for the individual farmer.
According to a preferred embodiment of the present invention, the composition added to the feed in step a) comprises 25-80 % by weight of propionic acid, 15-65 % by weight of glyceryl propionates and less than 4 % by weight of glycerol, balance to 100 % being water, such as reaction water and water emanating from the water content of the used raw materials.
According to a still more preferred embodiment of the present invention, the composition added to the feed in step a) comprises 50-70 % by weight of propionic acid and 20-40 % by weight of glyceryl propionates.
The glyceryl propionates are preferably a mixture comprising 30-50 % by weight of glyceryl monopropionate, 40-60 % by weight of glyceryl dipropionate and 5-20 % by weight of glyceryl tripropionate, balance being 100 % by weight.
Optionally, the composition further comprises 0.5-5 % by weight of an alkali metal salt, preferably a formate, such as potassium or sodium formate.
The composition added in step a) is added to the feed in an amount of between 0.5-10 ml/kg, preferably 2.0-6.0 ml/kg.
In order for the fermentation process in the present invention to start off spontaneously, the feed needs to have a water content of at least about 20 % by weight. Preferably, the water content should be more than 25 % by weight. Grains and grain crops that should be treated with the method according to the present invention is ideally harvested at an early stage, when the water content, and also the nutritional content, is high.
The method is applicable to different kinds of feed, but is especially well suited for feeds selected from the group consisting of grains, moist-rolled grains, grain crops and/or whole crop grains. Particularly maize and oats are sensitive to the Fusarium species that produce DON and it is often with these crops the problems with DON are most troublesome. However, the method according to the present invention can also be applied to other crops like wheat, barley and triticale.
In a further aspect the present invention refers to an animal feed that has been treated with the method described above.
As discussed above, the first sign that a feed is contaminated with DON is often a decreased feed intake shown by the animal being fed the contaminated feed. The higher the DON concentration in the feed, the more pronounced will the decreased feed intake be, leading eventually to feed refusal by the animal.
In yet another aspect, the present invention refers to a composition selected from the group consisting of propionic acid and/or glyceryl propionates, wherein said composition comprises at least 40 % by weight of propionic acid content, for use in preventing a decreased feed intake or feed refusal in mammals, wherein said decreased feed intake or feed refusal is caused by deoxynivalenol contamination of the feed fed to said mammal and wherein said composition is added to said feed, the feed is thereafter anaerobically fermented and thereafter fed to said mammal.
Besides a decreased feed intake or feed refusal, DON also causes immunosuppression in mammals, and this already at moderate concentrations.
The present invention additionally refers to a composition selected from the group consisting of propionic acid and/or glyceryl propionates, wherein said composition comprises at least 40 % by weight of propionic acid content, for use in preventing immunosuppression in mammals, wherein said immunosuppression is caused by ingestion of a feed contaminated with deoxynivalenol by said mammal and wherein said composition is added to said feed, the feed is thereafter anaerobically fermented and thereafter fed to said mammal.
The present invention is illustrated in the below Embodiment Example, which is to be construed as merely illustrative and not limiting in any way.
EMBODIMENT EXAMPLE Deoxynivalenol (DON) levels in crimped, maize treated with the method according to the invention and with comparative methods.
DON levels were measured in crimped maize before ensiling and in crimped maize ensiled with three different additives.
The crimping was done at a farm nearby Uppsala and storage in lab-scale silos was conducted at the Swedish Agricultural University’s facility in Uppsala, Sweden.
Moist maize grain was treated to obtain a target moisture content of 35-40%. The grain was crimped with a Murska crimper (model 350 S2 from Aimo Kortteen Konepaja Oy, Finland). Three replicate samples were taken as a reference of the feed before ensiling and put in the freezer. After thorough mixing of the crimped maize, portions of grains were weighed into large fresh plastic bags (125 L volume). Grain bags were then allocated to the following treatments: ProSid™ MI 700, Propionic acid and Crimpstore 2000S (clone). The contents of the three additives tested are given in Table 1: Image available on "Original document" Table 1.
The additives were applied with the atomizer of a spray bottle to the spread-out bag contents at a rate of 4.0 ml/kg (fresh matter). After thorough blending of grain and additive in the bags, equal quantities of treated maize were filled into 1.71 glass jar mini-silos and slightly compacted. The samples were made in triplicate, so there were three silos per treatment. Silo lids were equipped with water-filled plastic siphones, which allowed fermentation gases to escape without any ingress of air and helped to identify badly sealed silos. To resemble real on farm conditions, artificial air stress was produced by removing silicone stoppers from four holes (diameter 6 mm); one hole situated at the lid and three holes situated at the lower part of the glass jar. These stoppers were removed three times a week for 2 hours at each occasion. Silos were stored for 62 days at 20 ± 1°C and were protected from direct sunlight. After 62 days silos were opened for sampling and samples were taken and freezed for later analysis.
The quantitative analysis of deoxynivalenol concentration in the samples was conducted at the University of Natural Resources and Life Sciences in Vienna, Austria. The samples tested were: fresh grain (before ensiling), ensiled with ProSid™ MI 700, ensiled with Propionic acid and ensiled with Crimpstore 2000S (clone). Quantitative analysis of mycotoxins with an HPLC-MS/MS based multi-mycotoxin method was performed. External calibration was performed using serial dilutions of a multi-analyte stock solution. Results were corrected for apparent recoveries obtained during validation of the method for the matrix maize.
Samples were milled and homogenized using a laboratory mill (RAS-mill, Romer Labs). 5 g of homogenized sample were extracted using 20 ml of acetonitrile/water/acetic acid 79/20/1, v/v/v for 90 min on a rotary shaker. The raw extracts were diluted 1+1 using acetonitrile/water/acetic acid 20/79/1, v/v/v and subsequently injected (5 ?1).
Concentrations of deoxynivalenol expressed as ?g/kg sample in the different samples are shown below in Table 2 and in Figure 1. Each treatment was performed in triplicate (three different silos per treatment) and the DON concentrations below are mean values of these three samples.
Image available on "Original document" Table 2.
J Image available on "Original document" DON levels decreased in all the samples that were ensiled compared with the sample taken before ensiling, however, the greatest reduction was seen in the two samples that were treated according to the method disclosed in the present invention (ensiled with ProSid™ MI 700 and ensiled with Propionic acid). This is quite unexpected, since none of these additives in combination with anaerobic fermentation have been described earlier to lower the level of toxins already present in a feed.
The method according to the present invention gives as much as a 50% reduction of initial DON concentrations in the tested feed, resulting in a feed that is of a higher quality and safer to ingest for the animal.
The method according to the present invention is easy to perform directly on the farm and it involves only chemicals that are already allowed as feed additives all over the world, making it an excellent method for the individual farmer to lower the DON concentration in feed.

Claims (9)

1. A method for reduction of deoxynivalenol concentration in feed, wherein said feed has a water content of at least 25% by weight, said method consisting of the following consecutive steps: a) adding a composition comprising at least one substance selected from the group consisting of propionic acid and glyceryl propionates to a feed, wherein said composition comprises at least 40 % by weight of propionic acid content and wherein said composition is added to said feed in an amount of between 0.5-10 ml/kg. b) anaerobically fermenting said feed.
2. A method according to claim 1 characterized in, that said composition comprises at least 50 % by weight of propionic acid content.
3. A method according to claim 1 or 2 characterized in, that said composition comprises 25-80 % by weight of propionic acid, 15-65 % by weight of glyceryl propionates and less than 4 % by weight of glycerol, balance to 100 % being water.
4. A method according to claim 3 characterized in, that said glyceryl propionates is a mixture comprising 30-50% by weight of glyceryl monopropionate, 40-60% by weight of glyceryl dipropionate and 5-20% by weight of glyceryl tripropionate, balance being 100% by weight.
5. A method according to any of the claims 1-4 characterized in, that said composition further comprises at least one alkali metal salt added in an amount of 0.5-5 % by weight.
6. A method according to claim 5 characterized in, that said alkali metal salt is potassium and/or sodium formate.
7. A method according to any of the claims 1-6 characterized in, that said composition is added to said feed in an amount of between 2.0-6.0 ml/kg.
8. A method according to any of the claims 1-7 characterized in, that said feed is selected from the group consisting of grains, moist-rolled grains, grain crops and/or whole crop grains.
9. A method according to any of the claims 1-7 characterized in, that said feed is selected from the group consisting of maize, oats, wheat, barley and triticale.
SE1730126A 2017-05-04 2017-05-04 Method for reduction of deoxynivalenol levels in feed SE541250C2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
SE1730126A SE541250C2 (en) 2017-05-04 2017-05-04 Method for reduction of deoxynivalenol levels in feed
PCT/SE2018/050453 WO2018203812A1 (en) 2017-05-04 2018-05-03 Method for reduction of deoxynivalenol levels in animal feed

Applications Claiming Priority (1)

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SE1730126A SE541250C2 (en) 2017-05-04 2017-05-04 Method for reduction of deoxynivalenol levels in feed

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SE541250C2 true SE541250C2 (en) 2019-05-14

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WO2022217031A1 (en) * 2021-04-08 2022-10-13 Eastman Chemical Company Methods of using formaldehyde-free antimicrobial compositions in animal by-product compositions

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012161633A1 (en) * 2011-05-24 2012-11-29 Perstorp Ab Animal feed additive effective as preservative
WO2012176151A1 (en) * 2011-06-24 2012-12-27 Bio-Energy Ingredients Limited Glycerol compositions and solutions

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Publication number Priority date Publication date Assignee Title
DE10222358A1 (en) * 2002-05-21 2003-12-11 Nutrinova Gmbh ß-Glucan-containing sorbic acid preparation as a feed additive in livestock rearing

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2012161633A1 (en) * 2011-05-24 2012-11-29 Perstorp Ab Animal feed additive effective as preservative
WO2012176151A1 (en) * 2011-06-24 2012-12-27 Bio-Energy Ingredients Limited Glycerol compositions and solutions

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Commission recommendation of 17 August 2016 on the prevention and reduction of Fusarium toxins in cereals and cereal products. Official Journal of th European Union. 2006-08-29, L 234/35 *
DÄNICKE, S. et al "Investigation on the kinetics of the concentration of deoxynivalenol (DON) and on spoilage by moulds and yeasts of wheat grain preserved with sodium metabisulfite (Na2S2O5, SBS) and propionic acid at various moisture contents". Archives of Animal Nutrition. 2010, Vol. 64, No. 3, pages 190-203 *
UEGAKI, R. et al "Changes in the concentrations of fumonisin, deoxynivaalenol and zearalenone in corn silage during ensilage". Animal Science Journal. 2013, Vol. 84, No. 9, pages 656-662 *
VEVERS, W. F. "Deoxynivalenol: toxicological profile and potential for reducing cereal grain contamination using bacterial additivies in fermented animal feed". A thesis submitted to Plymouth University in partial fulfilment for the degree of doctor of philosophy, University of Plymouth. [online]. 2015 [retrieved on 2018-06-14]. Retrieved from the Internet: <URL: https://pdfs.semanticscholar.org/be4e/57ee1483bed3fedf522c992165ccc54a297a.pdf >. *

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