WO2018203812A1

WO2018203812A1 - Method for reduction of deoxynivalenol levels in animal feed

Info

Publication number: WO2018203812A1
Application number: PCT/SE2018/050453
Authority: WO
Inventors: Hugo HJELM
Original assignee: Perstorp Ab
Priority date: 2017-05-04
Filing date: 2018-05-03
Publication date: 2018-11-08
Also published as: SE541250C2; SE1730126A1

Abstract

The present invention refers to a method for reduction of deoxynivalenol concentration in animal 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, salts of propionic acid, glyceryl propionates, formic acid and salts of formic acid to an animal feed, wherein said composition comprises at least 40% by weight of acid content and wherein said composition is added to said feed in an amount of 0.5-10 g/kg b) anaerobically fermenting said feed. The present invention also refers to the use of a mehod for reduction of deoxynivalenol concentration in animal feed.

Description

Method for reduction of deoxynivalenol levels in animal feed

FIELD OF THE INVENTION

The present invention refers to a method for reduction of deoxynivalenol concentration in animal 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, salts of propionic acid, glyceryl propionates, formic acid and salts of formic acid to an animal feed, wherein said composition comprises at least 40% by weight of acid content and wherein said composition is added to said feed in an amount of 0.5-10 g/kg b) anaerobically fermenting said feed.

The present invention also refers to the use of a mehod for reduction of deoxynivalenol concentration in animal feed,

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 of 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.

Danicke 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 Danicke 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 has now quite surprisingly been found that a two-step method involving a fermentation step, wherein no sulfites are used, can considerably reduce DON concentration in feeds. In order for the method according to the present invention to work, the feed needs to have a water content of at least 25% by weight; so that a fermentation process can take place. The method is cheap and easy to perform directly on the farm. Crops or grains can be taken directly from harvest, without drying, and be treated with the method according to the present invention. The result will be a feed that is ensiled and that has DON levels which are reduced by approximately 50-60%. The method according to the present invention does not lead to complete detoxification of the feed,

nevertheless, this method will considerably improve the quality of the feed, which is of great importance to the individual farmer.

One of the studies performed by Danicke et al (Archives of Animal Nutrition . 2010, Vol 64, No. 3, pages 190-203) relates to investigations on the kinetics of the concentration of DON and on spoilage by moulds and yeasts of wheat grains preserved with sodium metabisulfite and propionic acid at various moisture contents. Danicke et al concluded that the addition of propionic acid alone to cereal grains stored at higher moisture contents did not decrease the DON concentration consistently, as the reduction rate was obviously higher when the moisture content was lower (~90% reduction at 15% moisture vs. 32 and 39% reduction at moisture contents of 17.5% and 20% respectively). In contrary, the studies leading to the method according to the present invention show that a higher moisture content of the feed is favorable for the method, hence the DON reduction is greater when the water content of the feed is higher. In fact, the method according to the present invention only works for feeds with a water content of at least 25% by weight. At lower water concentrations in the feed, as is the case in the study performed by Danicke et al, there will be no fermentation process taking place in the feed.

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 and reduction of deoxynivalenol concentration in animal 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, salts of propionic acid, glyceryl propionates, formic acid and salts of formic acid to an animal feed, wherein said composition comprises at least 40% by weight of acid content and wherein said composition is added to said feed in an amount of 0.5-10 g kg

b) anaerobically fermenting said feed.

The composition added to the feed in step a) should comprise at least 40% by weight of acid content. This means that the weight of the acid molecules and the weight of the acid residues present in the glyceryl propionates or the salts 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 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 or formic 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 in a) comprises propionic acid and glyceryl propionates and said composition comprises at least 40% by weight of propionic acid content.

According to a preferred embodiment of the present invention, the composition added to the feed in step a) comprises 25-80% by weight, such as 50-70% by weight, of propionic acid; 15-65% by weight, such as 20-40% by weight, of glyceryl propionates; and less than 4% by weight of glycerol; balance to 100% being water. The glyceryl propionates are then 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.

According to one embodiment of the present invention the composition added in a) additionally comprises at least one substance selected from the group consisting of; hexanoic acid, sorbic acid, salts of hexanoic acid, salts of sorbic acid, glycerol esters of hexanoic acid and glycerol esters of sorbic acid.

It is believed that by treating an animal feed with the method according to the present invention, certain bacteria responsible for breaking down the DON molecule are favored. An alternative route to providing conditions preferred by these bacteria is to provide the bacteria itself by inoculation of bacterial cultures into the feed. According to one embodiment of the present invention, the composition added in a) additionally comprises at least one inoculated bacterial culture.

The composition added in step a) is added to the feed in an amount of between 0.5-10 g/kg, preferably in an amount of 2.0-6.0 g/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 25% by weight. A higher water content will favor the fermentation process. Preferably, the water content should be at least 30% by weight and still more preferably at least 35% 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.

The present invention also refers to the use of a mehod for reduction of deoxynivalenol concentration in animal feed.

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. By using the method according to the present invention, a decreased feed intake or feed refusal in an animal can be prevented.

Besides a decreased feed intake or feed refusal, DON also causes immunosuppression in mammals, and this already at moderate concentrations. The method according to the present invention can accordingly also be used to prevent immunosuppression in an animal.

The present invention is illustrated in the below Embodiment Examples, which are to be construed as merely illustrative and not limiting in any way.

EMBODIMENT EXAMPLES

Example 1: Deoxynivalenol (DON) levels in crimped maize treated with the method according to the invention

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 Formic acid/propionic acid mix. The contents of the three additives tested are given in Table 1 :

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 g kg (fresh matter). After thorough blending of grain and additive in the bags, equal quantities of treated maize were filled into 1.7 1 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. 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 μΐ). Concentrations of deoxynivalenol expressed as μg/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.

Table 2. DON levels decreased in all the samples that were ensiled compared with the sample taken before ensiling. The greatest reduction of DON is seen in the sample that was ensiled with propionic acid. In this case, the method according to the invention gives a 50% reduction of initial DON concentrations in the tested feed. It can be noted that the average DON level for the "untreated ensiled" sample also decreased with 24%, which shows that the anaerobic fermentation step is important for the technical effect of the present invention.

Example 2: Deoxynivalenol (DON) levels in whole crop maize treated with the method according to the invention

30 kg of whole crop maize was brought from a farm in Skane, Sweden the day before the trial started and stored in a cool location. The water content of the maize was approximately 66%. The maize was homogenized by thorough mixing on a plastic sheet. Three replicate samples, of 600 g each, were taken as a reference of the feed before ensiling and put in the freezer. 7 plastic bags with portions of 2 kg maize in each were prepared. The bags with whole crop maize were then allocated to the following treatments: Propionic acid, ProSid™ MI 700, ProSid™ MI 700 with 20% hexanoic acid, Promyr™ XR 680, Na formate/K sorbate and Na formate (one bag was left untreated). The contents of the additives tested are given in Table 3:

Table 3. The additives were applied by spraying with a paintbrush in each bag. Dry additives were poured into the bags. The application rate was 4 g/kg. The bags were shaken 2*50 times each. The contents of each treated bag was re-packed in 3 vacuum bags (3 replicates), with 600 g maize in each bag. All bags were stored at 20°C for 87 days. To resemble real on-farm conditions, artificial air stress was produced by weekly replacing 35 ml air in each bag with 35 ml fresh air, using a syringe and needle.

The quantitative analysis of deoxynivalenol concentration in the samples was conducted at the Analytical Laboratory at Perstorp AB, Perstorp, Sweden.

Quantitative analysis of DON with an LC/MS-QTOF based method was performed. External calibration was performed using serial dilutions of a DON stock solution. 2 g of homogenized sample were extracted using 8 ml of acetonitrile/water/acetic acid 79/20/1, v/v/v, for 90 min, on a shaker. The raw extracts were diluted 1+1 using water and subsequently injected (5 μΐ).

Concentrations of deoxynivalenol expressed as μg kg sample in the different samples are shown below in Table 4 and in Figure 2. Each treatment was performed in triplicate (three different bags per treatment) and the DON concentrations below are mean values of these three samples.

Table 4. The DON levels decreased in all samples during the fermentation step. For all the samples that had been treated with the method according to the invention, the DON level decreased with 55-58%, which means that the quality of the feed was significantly improved.

The method according to the present invention is easy to perform directly on the farm. It is an excellent way for the individual farmer to ensile the feed for further storage and at the same time greatly reduce the DON concentration in the feed, thereby improving the feed quality.

Claims

1. A method for reduction of deoxynivalenol concentration in animal 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, salts of propionic acid, glyceryl propionates, formic acid and salts of formic acid to an animal feed, wherein said composition comprises at least 40% by weight of acid content and wherein said composition is added to said feed in an amount of 0.5-10 g/kg

b) anaerobically fermenting said feed.

2. A method according to claim 1 characterized in, that the water content of said feed is at least 30% by weight.

3. A method according to claim 1 characterized in, that the water content of said feed is at least 35% by weight.

4. A method according to claim 1 characterized in, that the composition added in a) comprises at least 50% by weight of acid content.

5. A method according to claim 1 characterized in, that the composition in a) comprises

propionic acid and glyceryl propionates and that said composition comprises at least 40% by weight of propionic acid content.

6. A method according to claim 5 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.

7. A method according to claim 6 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.

8. A method according to any of the claims 1-7 characterized in, that the composition added in a) additionally comprises at least one substance selected from the group consisting of; hexanoic acid, sorbic acid, salts of hexanoic acid, salts of sorbic acid, glycerol esters of hexanoic acid and glycerol esters of sorbic acid.

9. A method according to any of the claims 1-8 characterized in, that the composition added in a) additionally comprises at least one inoculated bacterial culture.

10. A method according to any of the claims 1-9 characterized in, that said composition is added to said animal feed in an amount of between 2.0-6.0 g/kg.

11. A method according to any of the claims 1-10 characterized in, that said feed is selected from the group consisting of grains, moist-rolled grains, grain crops and/or whole crop grains.

12. A method according to any of the claims 1-11 characterized in, that said feed is selected from the group consisting of maize, oats, wheat, barley and/or triticale. Use of a method for reduction of deoxynivalenol concentration in animal feed, wherein said feed has a water content of at least 25% by weight and wherein said method consists of the following consecutive steps:

anaerobically fermenting said feed.