EP3930476A1

EP3930476A1 - Method for reducing deoxynivalenol levels in a composition, feed additive and feed composition

Info

Publication number: EP3930476A1
Application number: EP20709522.5A
Authority: EP
Inventors: Mathieu CASTEX; Paul Gerard Bruinenberg
Original assignee: Danstar Ferment AG; Nutreco IP Assets BV
Current assignee: Danstar Ferment AG; Nutreco IP Assets BV
Priority date: 2019-03-01
Filing date: 2020-02-28
Publication date: 2022-01-05
Also published as: WO2020178184A1

Abstract

The present disclosure is directed to a method for biologically inactivating or detoxifying mycotoxins, e.g., doxynivalenol, zearalenone, and/or ochratoxin A, such as trichothecenes, in particular deoxynivalenols, in food products and animal feeds by the aid of a yeast hydrolysate.

Description

METHOD FOR REDUCING DEOXYNIVALENOL LEVELS IN A COMPOSITION,

FEED ADDITIVE AND FEED COMPOSITION

FIELD OF THE INVENTION

The present invention is in the field of animal feed and food products, and more particular of prevention and/or reduction of mycotoxicosis, and even more particular in the reduction of trichothecene, in particular deoxynivalenol, levels in animal feed.

BACKGROUND OF THE INVENTION

Mycotoxicosis is a growing concern in the field of animal and human nutrition. Mycotoxins are secondary metabolites produced by fungi which can contaminate plants and therefore plant based feed or food materials to be used for human and animal consumption. Mycotoxin formation may occur when the causative fungi grow on crops in the field, at harvest, in storage, or during feed processing; essentially whenever favourable conditions for their formation prevail. There are hundreds of mycotoxins known, but few have been extensively researched and even fewer have good commercially available methods for analysing them. The primary classes of mycotoxins are Aflatoxins (B1 , B2, G1 , G2) of which aflatoxin B1 (AFB1) is the most prevalent, zearalenone (ZEA), trichothecenes such as deoxynivalenol (DON) and T-2 toxin (T-2), fumonisins (FUM: FB1 , FB2, FB3) and ochratoxin A (OTA). The major mycotoxin-producing fungal genera are Aspergillus, Fusarium and Penicillium. Many species of these fungi produce mycotoxins in commodities, feeds and feed ingredients. Mycotoxin contamination in animal feed and human food is a worldwide problem. Rodriguez and Naehrer (Phytopathol. Mediterr. 2012; 51 : 175-192) reviewed mycotoxin contamination of diverse feedstuffs samples from throughout the world for five toxins (AFB1 , DON, ZEA, FUM and OTA).

Mycotoxins are toxic when contaminated feeds or feed ingredients are consumed by animals. Mycotoxicoses are diseases caused by exposure to feeds contaminated with mycotoxins. Mycotoxins exhibit a variety of biological effects in animals, which include liver and kidney toxicity, neurological, estrogenic and teratogenic effects, to name a few. Some mycotoxins are carcinogenic. Additionally, the mycotoxin-contaminated feed consumption in animals can cause loss of appetite, decreased feed efficiency, feed refusal, poor weight gain, immunosuppression, and mortality. Each mycotoxin has its own particular effect, and all can be devastating. Co-contamination by multiple types of mycotoxin occurs naturally, and exerts a greater negative impact on health and productivity of livestock than contamination by individual mycotoxins. The mycotoxin contamination of feed results in considerable economic losses to animal husbandry world-wide and in some cases in health damage to human consumers due to transfer of contamination via dairy products, eggs and meats.

Several strategies exist to cope with mycotoxins in feed through the use of feed additive with specific properties: (1) mycotoxins binders: product able to bind mycotoxins in the gut of the animal and therefore limit their absorption, (2) mycotoxin detoxifying agent: enzymes and/or microorganisms able to metabolize mycotoxins in less toxic compounds, (3) products strengthening the natural defences of the animal through immune modulation, antioxidant response and barrier integrity, or products supporting liver functions.

Mycotoxin binders reduce the exposure to mycotoxins by decreasing their bioavailability, including various mycotoxin adsorbing agents in the feed, which leads to a reduction of mycotoxin uptake as well as distribution to the blood and target organs. Various mycotoxin binders exist on the market for the last 20 years. Those products are usually based on mineral binders (various types of clays), yeast components (mainly yeast cell walls), algae and active carbon. Products available on the market range from generic products (basic clay and inactive yeast) to specialty products (specific mixes of yeast cell walls and minerals, processed clays, processed clay with algae, process yeast derivatives, and the like). These products supposedly cover a broad range of toxins: aflatoxins, zearalenone and fumonisins. However, for tricothecenes (DON, T2-toxins), no efficient binders are available. In particular, yeast components, and more specifically yeast cell walls are fairly good binders for ZEA and to some extends FB1 and OTA, but compounds that bind tricothecenes substantially are yet to be reported. For example, WO 99/53772 discloses a composition for binding and inactivating a mycotoxin in an animal feed comprising a yeast cell wall extract and a mineral clay. Also, it is described in Kogan et al. (Livostock Science, vol. 109, no. 1-3, April 2007) that commercial yeast polysaccharides (MTB100®, Alltech Inc.) have been shown to absorb a wide range of mycotoxins at low inclusion levels.

It is an object of the present invention to provide a method for reducing tricothecene levels, in particular deoxynivalenol levels, and/or zealarone and/or ochratoxin A, preferably at least deoxynivalenol levels, in or on a product, and/or to provide an animal feed product, complementary feed product, premix or feed additive comprising a compound effective in reducing tricothecene levels, preferably deoxynivalenol levels.

SUMMARY OF THE INVENTION

The present disclosure relates to a method for reducing deoxynivalenol, zearalenone, and/or ochratoxin A, preferably at least deoxynivalenol, and optionally further one, or both, of zearalenone and ochratoxin A, levels in or on a product, said method comprising the step of contacting said product with a yeast hydrolysate.

In an embodiment, the yeast hydrolysate is obtained by an alkaline hydrolysis method comprising the steps of:

i. providing yeast cell material; and

ii. subjecting said yeast cell material to a chemical treatment with an alkali solution at a pH of above 8 and a temperature of above 50°C to obtain a yeast alkaline hydrolysate.

In an embodiment, the alkali solution has a pH in the range of 8.5-13, or in the range of about 8.5-11.5.

In an embodiment, the temperature is in the range of 50-120°C, or is in the range of 80-110 °C.

In an embodiment, the alkaline hydrolysis method is carried out for sufficient time to allow the yeast alkaline hydrolysate to form, such as at least about 30 minutes, or at least about one hour, or for 1-10 hours.

In another embodiment, the yeast hydrolysate is obtained by an enzymatic hydrolysis method comprising the steps of:

i. providing yeast cell material

ii. subjecting said yeast cell material to an enzymatic treatment with a protease at a pH, time and temperature sufficient to obtain enzymatic hydrolysis of said yeast cell material (e.g., at a pH in the range of 4-6, preferably in the range of 4.5-5.5, and a temperature in the range of 45-70°C, preferably 50-65°C); and

iii. neutralizing said yeast cell material to obtain a yeast enzymatic hydrolysate.

In an embodiment, the protease is an endoprotease, an exoprotease or a mixture thereof.

In an embodiment, the protease is papain, alkaline protease, subtilisin-type protease, neutrase or a mixture thereof.

In an embodiment, the subtilisin-type protease is an alcalase.

In an embodiment, the protease is papain. In an embodiment, the enzymatic treatment of step ii) is carried out at a pH in the range of 4- 6, preferably in the range of 4.5-5.5, and a temperature in the range of 45-70°C, preferably 50-65°C.

In an embodiment, the enzymatic treatment of step ii) is carried out for sufficient time to allow the yeast enzymatic hydrolysate to form, such as at least about 10 to 25 hours, preferably 15 to 20 hours.

In an embodiment, the yeast of the yeast hydrolysate is a species from the genera Saccharomyces, Kluyveromyces, Hanseniaspora, Metschnikowia, Pichia, Starmerella, Torulaspora or Candida ; preferably, the yeast is a species from the genus Saccharomyces, even more preferably the yeast is S. cerevisiae.

In an embodiment, the yeast of the yeast hydrolysate is a strain of S. cerevisiae deposited on February 27, 2019 at the CNCM under number I-5405.

In an embodiment, the yeast hydrolysate comprises soluble and insoluble components derived from the yeast cell material.

In an embodiment, the product is selected from the group consisting of a food product, feed product, beverage, grain, plant, and bioethanol.

The present disclosure further provides an animal feed product, complementary feed product, premix or feed additive comprising a yeast hydrolysate as defined herein.

The present disclosure also provides a yeast strain deposited on February 27, 2019 at the CNCM under number I-5405.

The present disclosure further relates to use of the yeast hydrolysate as taught herein, the yeast alkaline hydrolysate as taught herein or the yeast enzymatic hydrolysate as taught herein for reducing deoxynivalenol, zearalenone, and/or ochratoxin A levels in or on a product.

The present disclosure also pertains to an animal feed product, complementary feed product, premix or feed additive comprising a yeast enzymatic hydrolysate, wherein the yeast enzymatic hydrolysate is prepared from a strain of S. cerevisiae deposited on February 27, 2019 at the CNCM under number I-5405. The present invention also pertains to a method for decreasing oral bioavailability of deoxynivalenol, zearalenone, and/or ochratoxin A to an animal, said method comprising administering a yeast enzymatic hydrolysate as taught herein.

The present invention further provides a method for decreasing systemic exposure of an animal to deoxynivalenol, zearalenone, and/or ochratoxin A, said method comprising administering a yeast enzymatic hydrolysate as taught herein.

In an embodiment, yeast enzymatic hydrolysate as taught herein is administered orally.

GENERAL DEFINITIONS

In the following description and examples, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided. Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The disclosures of all publications, patent applications, patents and other references are incorporated herein in their entirety by reference.

The terms“comprising” or“to comprise” and their conjugations, as used herein, refer to a situation wherein said terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb“to consist essentially of” and“to consist of”.

Reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The term“mycotoxin” means a secondary metabolite produced by fungi (mold).

“Mycotoxicosis” refers to all diseases caused by the effects of toxins produced by moulds. Disease is often subclinical and may be difficult to diagnose. Problems occur worldwide, but especially in climates with high temperature and humidity and where grain is harvested with high water content. Economic impact is considerable in some countries. Mortality is variable but all are detrimental to health and are resistant to heat inactivation. The route of infection is by ingestion of fungal spores, which are readily carried in the air. High grain humidity, and damage due to insects, as well as poor storage conditions are major predisposing causes. Once toxins have been formed it is difficult to avoid their biological effects; they also increase susceptibility to bacterial diseases.

The term “mycotoxin binder” means a binding agent, which adsorbs or absorbs and/or deactivates mycotoxins present in foods and feeds, and thus reverses the adverse effects of mycotoxins.

The term "hydrolysis" as used in the context of the present disclosure is defined as the enzymatic and non-enzymatic breakdown of yeast cells using, for example, endogenous and/or exogenous enzymes. The endogenous yeast enzymes may or may not be inactivated, for instance by a heat shock. Alternatively, the yeast cells may be treated chemically or mechanically.

"Yeast hydrolysate" is defined herein as the digest of yeast obtained by hydrolysis of yeast, such as by chemical treatment and/or enzymatic hydrolysis using endogenous and/or exogenous enzymes. In the context of the present disclosure, the term“autolysis” of a yeast is defined as a process wherein degradation of the yeast cells and of the polymeric yeast material is at least partially effected by active native yeast enzymes (i.e., endogenous enzymes) released in the medium after (partially) damaging and/or disrupting the yeast cell wall. A“yeast hydrolysate” according to the present disclosure can be a“yeast alkaline hydrolysate” or a “yeast enzymatic hydrolysate”. A “yeast alkaline hydrolysate” may be obtained by chemical treatment as taught herein and a“yeast enzymatic hydrolysate” may be obtained by enzymatic hydrolysis using exogenous enzymes as taught herein.

A "yeast hydrolysate" preferably contains both soluble and insoluble components derived from the whole yeast cell. A“yeast hydrolysate” differs from a“yeast extract” because the yeast hydrolysate, in addition to all the interesting components present in yeast extracts, also contains interesting cell wall components (mainly composed of b-glucans, mannoproteins, chitin and proteins) which are not separated from the soluble fraction.

The term “animal” includes all animals, including human beings. In an embodiment, an animal is non-human animal. Examples of such animals are livestock animals, such as cattle, (including but not limited to cows and calves); mono-gastric animals, e.g., pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys and chicken (including but not limited to broiler chicks, layers); and fish. Also companion animals are included in the term. The term“feed” or“feedstuff” or“feed ingredient” or“feed product” means any compound, grain, nut, forage, silage, preparation, mixture, or composition suitable for, or intended for intake by an animal, preferably a livestock animal or a companion animal.

BRIEF DESCRIPTION OF FIGURES AND DRAWINGS

Figure 1 illustrates the adsorption level (%) of DON and ZEA in a buffer at pH 3.0, 5.0 and 8.5 by the yeast product AHSC1 (alkaline hydrolysate of Saccharomyces cerevisiae).

Figure 2 illustrates the adsorption level (%) of DON and ZEA in a buffer at pH 3.0, 5.0 and 8.5 by the yeast product EHSC2 (enzymatic hydrolysate of Saccharomyces cerevisiae strain CNCM I-5405 in combination with a consortium of inactivated S. cerevisiae yeast strains).

Figure 3 illustrates the adsorption level (%) of DON and ZEA in a buffer at pH 3.0, 5.0 and 8.5 by the yeast product EHSC3 (enzymatic hydrolysate of a single strain of Saccharomyces cerevisiae - strain CNCM I-5405).

Figure 4 illustrates the adsorption level (%) of DON and ZEA in a buffer at pH 3.0, 5.0 and 8.5 by the yeast product ASC4 (autolysate of Saccharomyces cerevisiae - single strain).

Figure 5 illustrates the adsorption level (%) of DON and ZEA in a buffer at pH 3.0, 5.0 and 8.5 by the yeast product IWYSC5 (inactive whole yeast of Saccharomyces cerevisiae - single strain).

DETAILED DESCRIPTION

The present inventors have surprisingly found that chemical and/or enzymatic hydrolysis of yeast material improves its capacity to bind tricothecenes, in particular deoxynivalenol and ochratoxin A, as well as zearalenone.

Thus, in a first aspect, the present invention provides for a method for reducing deoxynivalenol (DON) levels, zearalenone (ZEA) levels, and/or ochratoxin A (OTA) levels, preferably at least DON levels, and optionally further one or both of ZEA levels and OTA levels, in or on a product, and/or a method for detoxifying a mycotoxin contaminated feed or food product, and/or a method for reducing the bioavailability of mycotoxin in a mycotoxin contaminated feed or food product, said method comprising the step of contacting said product with a yeast hydrolysate. In an embodiment, the present invention provides for a method for reducing deoxynivalenol (DON) levels in or on a product, and/or a method for detoxifying a DON contaminated feed or food product, and/or a method for reducing the bioavailability of DON in a DON contaminated feed or food product, said method comprising the step of contacting said product with a yeast hydrolysate as taught herein.

In an embodiment, the present invention provides for a method for reducing zearalenone (ZEA) levels in or on a product, and/or a method for detoxifying a ZEA contaminated feed or food product, and/or a method for reducing the bioavailability of ZEA in a ZEA contaminated feed or food product, said method comprising the step of contacting said product with a yeast hydrolysate as taught herein.

In an embodiment, the present invention provides for a method for reducing ochratoxin A (OTA) levels in or on a product, and/or a method for detoxifying a OTA contaminated feed or food product, and/or a method for reducing the bioavailability of OTA in a OTA contaminated feed or food product, said method comprising the step of contacting said product with a yeast hydrolysate as taught herein.

In an embodiment, the yeast hydrolysate taught herein is capable of reducing both DON and ZEA levels, both ZEA and OTA levels, both DON and OTA levels, or all of DON, ZEA, and OTA levels.

The method is applicable to different kinds of products in which contamination with DON, ZEA and/or OTA, in particular DON, may be an issue. Such products include, without limitation, a food product, an animal feed product, beverage such as beer or wine, grains, plants, and bioethanol. The method taught herein is especially of interest to the feed industry, and is especially well suited for feeds comprising or 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 such as wheat, barley and triticale.

The first sign that animal 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. Furthermore, DON causes loss of intestinal barrier function via impairment of the intestinal tight junction network preceding an inflammatory response. The method according to the present invention can accordingly be used to mitigate the adverse effects of DON in an animal.

The yeast hydrolysate can be made using many yeast strains, including yeast strains of the genus Saccharomyces like wine and beer yeast strains, baker’s yeast strains ( Saccharomyces cerevisiae) and probiotic yeast strains ( Saccharomyces cerevisiae var. boulardii). Other suitable yeast strains include Kluyveromyces, Hanseniaspora, Metschnikowia, Pichia, Starmerella, Torulaspora or Candida. Both liquid yeast hydrolysate and dry yeast hydrolysate (e.g., a powder) can be used. In a highly suitable embodiment, the yeast hydrolysate is made using a strain of Saccharomyces cerevisiae deposited February 27, 2019 at the CNCM (Collection Nationale de Cultures de Microorganismes, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France) under the number I-5405.

The yeast hydrolysate may be any yeast product obtained from yeast cells using any type of hydrolysis. In a suitable embodiment, the yeast hydrolysate is obtained by alkaline treatment of yeast cells and is referred to as being a yeast alkaline hydrolysate. In another suitable embodiment, the yeast hydrolysate is obtained by enzymatic treatment of yeast cells using at least one exogenous enzyme, preferably a protease, and is referred to as being a yeast enzymatic hydrolysate. The yeast hydrolysate may comprise both soluble and insoluble components derived from the yeast cell material.

The yeast hydrolysate may be in any form, e.g., a liquid form or in the form of a dry powder.

i. providing yeast cell material; and

ii. subjecting said yeast cell material to a chemical treatment with an alkali solution at a pH of above 8 and a temperature of above 50°C. Such yeast hydrolysate is also called“yeast alkaline hydrolysate”.

i. providing yeast cell material, ii. subjecting said yeast cell material to an enzymatic treatment with a protease at a pH, time and temperature sufficient to obtain enzymatic hydrolysis of said yeast cell material (e.g., at a pH in the range of 4-6, preferably in the range of 4.5-5.5, and a temperature in the range of 45-70°C, preferably 50-65°C); and

The yeast cell material may be any yeast cell material. For example, the method for the production of yeast hydrolysate from yeast cells may start with an aqueous suspension of yeast cells such as a fermentation broth of yeast cells, in which case such aqueous suspension may qualify as yeast cell material. Fermentation processes suitable to produce suspensions of yeast cells are well-known in the art. In some cases the fermentation broth may be concentrated prior to its use in the hydrolysis method of the present disclosure, for example, by centrifugation or filtration to yield a yeast cream.

Suitably, the hydrolysis methods taught herein may be initiated by breaking and/or rupturing the yeast cell walls of the yeast cell material. The content of the cells, in part or entirely, may then be released via the partial openings created by the disruption of the yeast cell walls. In order to break or rupture or disrupt the yeast cell walls, the yeast cells can be treated mechanically, chemically or enzymatically according to methods well known in the art.

Mechanical treatments include homogenization techniques. At this purpose, use of high- pressure homogenizers is possible. Other homogenization techniques may include mixing with particles, e.g. sand and/or glass beads, or the use of a milling apparatus (e.g. a bead mill).

Preparation of the yeast alkaline hydrolysate

The yeast alkaline hydrolysate taught herein may be produced by chemical treatments which include the use of salts, alkali and/or one or more surfactants or detergents. In an embodiment, the yeast cells are heated and alkali treated. For example, the chemical treatment may be performed at a temperature of 50 to 120°C, under alkaline conditions (pH 7.0-13.0) for sufficient time to allow the yeast alkaline hydrolysate to form.

The temperature in the method taught herein may, for example, be between 50 and 130 °C, such as between 60 and 120 °C, between 70 and 110 °C, between 80 and 100 °C, or between 90 and 100 °C. The pH is preferably alkaline, i.e. , above pH 7, more preferably above pH 8. In an embodiment, the pH is in the range of 7.0-13.0. The chemical treatment may be performed for sufficient time to allow the yeast alkaline hydrolysate to form, e.g., any time between 0.25-40 hours, such as between 0.5-30 hours, or between 1-20 hours.

In an embodiment, the chemical treatment is performed at a temperature of 80 to 110°C, under alkaline condition at a pH 8.5 to 11.5, for 1-10 hours. In a further embodiment, the chemical treatment is performed at a temperature of 90 to 100°C, under alkaline condition at a pH above 8.5 for 3-8 hours.

Any alkaline solution known in the art can be used. For example, the alkaline agent can be calcium hydroxide (Ca(OH)₂), calcium oxide (CaO), ammonia (NH₃), sodium hydroxide (NaOH), sodium carbonate (NaCCh), potassium hydroxide (KOH), urea, and/or combinations thereof. In an embodiment, sodium hydroxide can be used as an alkaline solution.

The yeast hydrolysate produced by the chemical treatment taught herein can be formulated according to methods known to those skilled in the art. For example, the yeast alkaline hydrolysate obtained by the methods taught herein, being an aqueous suspension, may be centrifuged and/or ultra-filtered. It is also possible to concentrate the aqueous suspension by evaporation. The resultant suspension may be dried into powder according to any suitable manners known in the art such as spray drying, roller drying, freeze drying, fluidized bed treatment or a combination of these methods. In an embodiment, the resultant suspension is dried into powder by roller or spray drying.

In an embodiment, the yeast alkaline hydrolysate of the present disclosure is made from a yeast of the genus Saccharomyces. In an embodiment, the yeast is Saccharomyces cerevisiae.

Preparation of the yeast enzymatic hydrolysate

The yeast hydrolysate of the present disclosure may also be produced by enzymatic treatments. Several enzymes or enzyme mixtures can be used for the hydrolysis of the yeast cells such as cellulases, glucanases, hemicellulases, chitinases, proteases or peptidases and/or pectinases. In an embodiment, a protease or a peptidase is used. These enzymes can be subdivided into two major categories, exopeptidases and endopeptidases, depending upon their site of action. Commercially-useful proteases have been isolated from plants (e.g. papain, bromelain, and keratinases), animals (e.g., trypsin, pepsin, and rennin), and microbes such as fungi (e.g., Aspergillus oryzae proteases) and bacteria (especially well- known proteases isolated from the genus Bacillus). In the context of the present disclosure, the yeast enzymatic hydrolysate is preferably obtained by enzymatic hydrolysis with at least one protease or peptidase as, for example, papain, subtilisin-type protease (e.g. an alkaline protease such as alcalase), neutrase and/or mixture thereof.

One specific example of subtilisin-type protease is alcalase. Alcalase is a serine protease commercially produced from Bacillus licheniformis. Alcalase is a protease preparation of subtilisin, which is a non-glycosylated single polypeptide chain without disulfide bonds and a MW of 15-30 KDa (Gupta, R., Beg, Q. K., & Lorenz, P. (2002). Bacterial alkaline proteases: molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59, 15-32). As used herein, the term“alcalase” is not limited to the specific enzyme sold by Novozymes under the name Alcalase®.

Another form of endopeptidase used in the context of the present disclosure is neutrase. Neutrase® (Novozyme Corp., Copenhagen, Denmark) is a bacterial protease produced by a selected strain of Bacillus amyloliquefaciens.

As used herein, the term“papain family” refers to a family of related proteins with a wide variety of activities, including endopeptidases, aminopeptidases, dipeptidyl peptidases and enzymes with both exo- and endopeptidase activity (Rawlings N D, Barrett A J (1994). “Families of cysteine peptidases”. Meth. Enzymol. 244: 461-486). Members of the papain family are widespread, found in baculovirus, eubacteria, yeast, and practically all protozoa, plants and mammals. As used herein the term“papain” is a complex mixture of various enzymes including proteases obtained from papaya fruits.

An example of papain enzymes that could be used in the context of the present disclosure is Promod™ 144GL (Biocatalysts). Furthermore, Promod™ 144P, 144MDP, 144SP, 144GL, Promod™ 439L, P439L or Promod™ 950L can also be used in the context of the present disclosure.

The conditions to initiate the hydrolytic and autolytic processes are dependent on the type of enzyme used and can be easily determined by the person skilled in the art. The enzymatic treatment is performed at a particular pH range combined with a particular temperature. In the first step of the process, the hydrolysis and/or autolysis is performed at a temperature between 50 and 65°C, preferentially between 55 and 60 °C, at a pH of 4.5 to 5.5 for a period of, for example, 10 to 25 hours, preferentially 15 to 20 hours. The yeast enzymatic hydrolysate can be deactivated by heat treatment, with methods known to the skilled practitioner, e.g., at a temperature between 75 and 85 °C for 15 to 60 minutes.

The yeast hydrolysate produced by enzymatic treatments can be formulated according to methods known to those skilled in the art. For example the aqueous suspension comprising the yeast hydrolysate may be centrifuged and/or ultra-filtered. It is also possible to concentrate the aqueous suspension by evaporation. The resultant suspension may be dried into powder according to any suitable manners known in the art such as spray drying, roller drying, freeze drying, fluidised bed treatment or a combination of these methods. In an embodiment, the resultant suspension is dried into powder by roller or spray drying.

In an embodiment, the yeast enzymatic hydrolysate of the present disclosure is made from a yeast of the genus Saccharomyces. In an embodiment, the yeast is Saccharomyces cerevisiae. In a further embodiment, the yeast is a strain of Saccharomyces cerevisiae deposited February 27, 2019 at the CNCM (Collection Nationale de Cultures de Microorganismes, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France) under the number I-5405.

Yeast hydrolysate

The alkaline yeast hydrolysate and enzymatic yeast hydrolysate taught herein can comprise a suitable carrier or may be used as is. Non limiting examples of carrier include organic or inorganic carrier such as, for example, malto-dextrin, starches, calcium carbonate, cellulose, whey, ground corn cobs, silicon dioxide or minerals and inactive yeast filler.

Products and uses

The yeast hydrolysates taught herein may be used as a detoxification aid to reduce the DON, ZEA and/or OTA content in fermented beverages such as beer and wine, and in the spent grain product after the bioethanol fermentation process. Alternatively, the yeast hydrolysate taught herein may be used to protect crops against fungal infections, the most important cause of spoilage of crops like wheat, barley, and of fruits, causing large economic losses around the globe. Without wishing to be bound by theory, it is hypothesized that the mode of action of the yeast hydrolysate taught herein is based on two traits: 1) binding of DON, ZEA, and/or OTA will reduce the concentration of fungal virulence factor, resulting in inhibition of fungal colonization in plants and furthermore in reduction of the phytotoxic effects of DON, ZEA and/or OTA, such as growth retardation, inhibition of seedling and green plant regeneration; and 2) B-glucans and chitin, both present in the yeast product, are known to induce the plant defence mechanisms against fungi. In an aspect, the present disclosure provides an animal feed product, complementary feed product, premix or feed additive comprising a yeast hydrolysate as taught herein.

The animal feed product, complementary feed product, premix or feed additive taught herein can be fed to any animal, for example, a farming animal, a zoo animal, a laboratory animal and/or a companion animal. In some embodiments, the animal can be, but is not limited to, a bovine (e.g., domestic cattle (cows (e.g., dairy and/or beef)), bison, buffalo), an equine (e.g., horse, donkey, zebra, and the like), an avian (e.g., a chicken, a quail, a turkey, a duck, and the like; e.g., poultry), a sheep, a goat, an antelope, a pig (e.g., swine), a canine, a feline, a rodent (e.g., mouse, rat, guinea pig); a rabbit, a fish, and the like. In some embodiments, the animal can be a cow. In some embodiments the animal can be poultry. In other embodiments, the animal can be a chicken. In further embodiments, the animal can be swine.

The yeast hydrolysate taught herein may be included in the animal feed product, and accordingly complementary feed product, premix or feed additive, in an amount suitable to reduce DON, ZEA, and/or OTA levels, preferably at least DON levels, and optionally further one or both of ZEA and OTA levels, in animal feed. Such amounts may be in the range of about 0.0001% to 30%, such as about 0.001 % to 20%, about 0.01% to 10%, about 0.02% to 5%, about 0.05% to 2%, or about 0.1 % to 0.5% by weight of feed.

The yeast hydrolysate taught herein may be added to an animal feed product, complementary feed product, premix or feed additive as taught herein to reduce DON levels, ZEA levels, and/or OTA levels, preferably at least DON levels, and optionally further one or both of ZEA and OTA levels, in said animal feed product, complementary feed product, premix or feed additive.

The present invention is further illustrated, but not limited, by the following examples. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the teaching and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

EXAMPLES Example 1

Preparation of the yeast alkaline hydrolysate (sample AHSC1)

Industrial cream yeast from Saccharomyces cerevisiae (Lallemand) was treated with sodium hydroxide to adjust the pH above 9 and the mixture was incubated for 3 to 8 hours at a temperature of at least 90°C. The hydrolysate was then dried by roller or spray drying into powder.

Preparation of the yeast enzymatic hydrolysates (samples EHSC2 and EHSC3)

Cream yeast from strain CNCM I-5405 of Saccharomyces cerevisiae (EHSC2 and EHSC3) was heated to at least 50°C. Subsequently, protease (papain) was added and the mixture was incubated for 15 to 20 hours at a pH of above 5 for autolysis. Next, the mixture or hydrolysate was heated for 1 hour at a temperature above 70°C to inactivate all enzyme activity. The pH of the mixture or hydrolysate is then adjusted with NaOH to about 6.0, heated to 75°C for 60 seconds and then dried by roller or spray drying into powder. Then, EHSC2 was combined with a consortium of industrial inactivated S. cerevisiae yeast strains (Lallemand). Sample EHSC3 corresponds to the enzymatic hydrolysate prepared from the single strain CNCM I-5405 of Saccharomyces cerevisiae.

Preparation of the control sample ASC4 ( autolysate of Saccharomyces cerevisiae)

Industrial cream yeast from Saccharomyces cerevisiae (Lallemand) was treated with sodium hydroxide or sulfuric acid to adjust the pH at 5.5. The mixture is heated to 55°C for at least 20 hours. The mixture was then heated 30 minutes at 85°C. Yeasts cells were harvested by centrifugation (15 minutes at 4000 rpm), washed 1 : 1 with sterile water, centrifuged again and then lyophilized.

Preparation of the control sample IWYSC5 (inactive whole yeast of Saccharomyces cerevisiae)

Industrial cream yeast of a strain of Saccharomyces cerevisiae (Lallemand) is pasteurized at 70°C for 15 minutes. The yeast cells are harvested and lyophilized.

Mycotoxin binding assay

The binding assay was performed essentially as described by Sabater-Vilarand and co workers (Department of Veterinary, Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Utrecht University, The Netherlands & Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Urmia University, Iran) in Mycopathologia, 2007, vol. 163, p. 81-90. Experimental procedure

The yeast products were suspended in PBS solution (CaCI₂-2H₂0, 1.2 mM; KCI, 2.7 mM; KH2PO4, 1.5 mM; MgCI₂-6H₂0, 1.1 M; NaCI, 138 mM; Na₂HP0₄-2H₂0, 8.1 mM; pH = 5.0) to reach a final concentration 2 mg of product/ml: i.e., the generally applied dosage of mycotoxin binder product in feed. DON and ZEA (Sigma Aldrich) were added to this suspension at a final concentration of 1 ppm and 0.5 ppm, respectively. The pH of the mixtures was adjusted to 3.0 with 1 M HCI and incubated at 37 °C for 2 hours under constant agitation to simulate the pH condition during gastric passage in a monogastric animal. After this first incubation step, a sample was taken for further analysis. The incubations were continued in the same flask by raising the pH to pH 5.0 with 1 M NaOH and leaving the incubation mixture for 2 hours under constant agitation at 37 °C. After sampling, incubations were continued at a final pH of 8.5. These latter two incubation steps simulate the pH conditions during intestinal passage of a monogastric animal.

Samples were immediately centrifuged to separate the binder from the aqueous phase and the supernatants were stored at -20 °C until further analysis by LC-MS. All binding assays were performed in triple.

Results

The results of the binding study are displayed in Figures 1 to 5. The data are presented as average values of 3 replicates per sample. The standard deviation (SD) is displayed in the graphs. As shown in Figure 1 , the yeast alkaline hydrolysate (sample AHSC1) exhibited a greater activity against DON than towards ZEA. Surprisingly, as shown in Figures 2 and 3, the results demonstrated that EHSC2 and EHSC3, which comprise yeast enzymatic hydrolysates of S. cerevisiae strain CNCM I-5405, presented a greater capacity to bind DON in comparison with their capacity to bind ZEA.

In contrast, the control yeast products ASC4 and IWYSH5, a yeast product prepared by inactivation and drying, appeared to bind only ZEA and not DON (Figures 4 and 5). The binding efficiency of DON was shown not to be affected by the pH of incubation.

Example 2

The effects of the yeast products AHSC1 and EHSC3 on the oral absorption of the mycotoxins deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEA) in female pigs was determined as follows. A toxicokinetic study was carried out focusing on the plasma concentration-time profiles of DON, OTA, ZEA and ZEA-GIcA in female pigs, after an oral bolus administration of the mycotoxins with or without AHSC1 or EHSC3. ZEA was administered at relative high concentration to enable measurement in blood plasma of main phase II metabolite ZEA-glucuronide (ZEA-GIcA).

The study was conducted with 18 healthy female piglets of the same breed and about the same body weight (BW) of 15-20 kg at arrival. Following an acclimatization period of at least 5 days, the pigs were fasted 12 hours before the treatment, but water was still available. The treatment exists of an intragastrical bolus with either the mycotoxins or the mycotoxins in combination with AHSC1 or EHSC3 (0.05 mg DON/kg BW, 0.05 mg OTA/kg BW, 0.5 mg ZEA/kg BW and 100 mg mycotoxin binder/kg BW) administered in a parallel study design. The dosage of AHSC1 and EHSC3 resembled 2 g/kg feed based on daily feed intake, whereas DON and OTA resembled a dosage 1 ppm and ZEA 10 ppm in feed. The animals were fed again 4 hours post administration.

Blood samples of the 18 piglets were taken from the vena jugularis externa at indicated time intervals post administration. The analysis of DON, OTA, ZEA and the main phase II metabolite of ZEA, namely ZEA-glucuronide (ZEA-GIcA), in plasma was performed using a validated UHPLC-MS/MS method. For ZEA-GIcA peak areas were corrected for the internal standard (IS) and results are presented as peak area ratio (= peak area ZEA-GIcA/peak area IS).

Toxicokinetic modeling of the plasma concentration-time profiles of DON, OTA and ZEA- GIcA was done by non-compartmental analysis (Phoenix 8.1 , Pharsight Corporation, USA). No ZEA was quantifiable in plasma hence the use of ZEA-GIcA as biomarker for exposure. Following parameters were calculated: area under the curve from time zero to 2, 4 and 0.5 hour (AUCo_®x_h) representing the absorption phase of the mycotoxins DON, OTA and ZEA, respectively; maximal plasma concentration (DON and OTA) or maximal plasma chromatographic peak area (ZEA-GIcA) (Cmax) , time at Cmax (T_max) .

The relative oral bioavailability expressed as a percentage, F = ((AUCo_®x_h mycotoxin + binder / AUCo_®x_h mycotoxin)* 100), was evaluated for DON, OTA and ZEA-GIcA as marker for efficacy of the mycotoxin binder. The effect of the mycotoxin binder on the oral absorption of the mycotoxin was evaluated by comparing toxicokinetic parameters between the mycotoxin and mycotoxin+binder treated piglets.

Toxicokinetics of DON and the effect of the mycotoxin binders AHSC1 and EHSC3

Table 1 shows the results of the most important toxicokinetic parameters of DON after oral bolus administration of DON, whether or not combined with one of the binders. Table 1. Major toxicokinetic characteristics of DON after single oral bolus administration of DON to 6 pigs, whether or not combined with one of the mycotoxin binders. Values are presented as mean.

AUCo->2 _h: area under the plasma concentration-time curve from time 0 to 2 h post administration, Cmax: maximum plasma concentration, Tmax: time at maximum plasma concentration, Relative oral bioavailability (Relative F).

The mean AUCo 2 _h was 30.03 h.ng/mL for DON and 25.12 h.ng/mL and 25.76 h.ng/mL for DON combined with yeast product AHSC1 and EHSC3, respectively. The relative oral bioavailability in the absorption phase was 83.66% and 85.79% for yeast product AHSC1 and EHSC3, respectively. The max concentration of DON in blood plasma was reduced with 20 % and 23 % for yeast product AHSC1 and EHSC3, respectively.

Toxicokinetics of OTA and the effect of the mycotoxin binders AHSC1 and EHSC3

Table 2. Major toxicokinetic characteristics of OTA after single oral bolus administration of OTA to 6 pigs, whether or not combined with one of the mycotoxin binders. Values are presented as mean.

AUCO- _h: area under the plasma concentration-time curve from time 0 to 4 h post administration, Cmax: maximum plasma concentration, Tmax: time at maximum plasma concentration, Relative oral bioavailability (Relative F).

Table 2 shows the results of the most important toxicokinetic parameters of OTA after oral bolus administration of OTA, whether or not combined with one of the mycotoxin binders to 6 pigs per group. The mean AUCo 4 _h of OTA was 1.42 h.pg/mL for OTA and 1.16 h.pg/mL and 1.14 h.pg/mL for OTA combined with yeast product AHSC1 and EHSC3, respectively. The relative oral bioavailability was 81.75% and 79.86% for yeast product AHSC1 and EHSC3, respectively. The maximum concentration of OTA in blood plasma was reduced with 15 and 17 % for yeast product AHSC1 and EHSC3, respectively.

Toxicokinetics of ZEA-GIcA and the effect of the mycotoxin binders AHSC1 and EHSC3

Table 3. Major toxicokinetic characteristics of ZEA-GIcA after single oral bolus administration of ZEA to 6 pigs, whether or not combined with one of the mycotoxin binders.

AUCO_{®O 5 h}: area under the plasma concentration-time curve from time 0 to 0.5 h post administration, Cmax: maximum plasma concentration, Tmax: time at maximum plasma concentration, Relative oral bioavailability (Relative F).

Table 3 shows the results of the most important toxicokinetic parameters of ZEA-GIcA after oral bolus administration of ZEA, whether or not combined with one of the mycotoxin binders to 6 pigs per group. The mean AUCo o.5 _h of ZEA-GIcA was 505.92 h.peak area/mL for ZEN and 362.54 h.peak area/mL and 407.48 h.peak area/mL for ZEA combined with yeast product AHSC1 and EHSC3, respectively. The relative oral bioavailability was 71.66% and 80.54% for yeast product AHSC1 and EHSC3, respectively. The maximum concentration of ZEA-GIcA in blood plasma was reduced with 25 and 20 % for yeast product AHSC1 and EHSC3, respectively.

In conclusion, the results showed that the mycotoxin binders AHSC1 and EHSC3 decreased the oral bioavailability and, consequently, the systemic exposure to DON, OTA and ZEA after single oral administration in piglets.

Claims

1. Method for reducing deoxynivalenol, zearalenone, and/or ochratoxin A levels in or on a product, said method comprising the step of contacting said product with a yeast hydrolysate.

2. The method according to claim 1 , in which the yeast hydrolysate is obtained by an alkaline hydrolysis method comprising the steps of:

i. providing yeast cell material; and

3. The method according to claim 2, wherein the alkali solution has a pH in the range of 8.5-13, or in the range of about 8.5-11.5.

4. The method according to claim 2 or 3, wherein the temperature is in the range of 50- 120°C, or is in the range of 80-110 °C.

5. The method according to any one of claims 2-4, in which the alkaline hydrolysis method is carried out for sufficient time to allow the yeast alkaline hydrolysate to form, such as at least about 30 minutes, or at least about one hour, or for 1-10 hours.

6. The method according to claim 1 , in which the yeast hydrolysate is obtained by an enzymatic hydrolysis method comprising the steps of:

i. providing yeast cell material;

ii. subjecting said yeast cell material to an enzymatic treatment with a protease at a pH, time and temperature sufficient to obtain enzymatic hydrolysis of said yeast cell material; and

7. The method according to claim 6, wherein the protease is an endoprotease, an exoprotease or a mixture thereof.

8. The method according to claim 7, wherein the protease is papain, alkaline protease, subtilisin-type protease, neutrase or a mixture thereof.

9. The method according to claim 8, wherein the subtilisin-type protease is an alcalase.

10. The method according to claim 8, wherein the protease is papain.

11. The method according to any one of claims 6 to 10, wherein the enzymatic treatment of step ii) is carried out at a pH in the range of 4-6, preferably in the range of 4.5-5.5, and a temperature in the range of 45-70°C, preferably 50-65°C.

12. The method according to any one of claims 6 to 11 , wherein the enzymatic treatment of step ii) is carried out for sufficient time to allow the yeast enzymatic hydrolysate to form, such as at least about 10 to 25 hours, preferably 15 to 20 hours.

13. Method according to any one of claims 1 to 12, in which the yeast of the yeast hydrolysate is a species from the genera Saccharomyces, Kluyveromyces, Hanseniaspora, Metschnikowia, Pichia, Starmerella, Torulaspora or Candida.

14. The method according to any one of claims 1 to 13, wherein the yeast is a species from the genus Saccharomyces.

15. The method according to claim 14, wherein the yeast is S. cerevisiae.

16. The method according to any one of claims 1 to 15, wherein the yeast of the yeast hydrolysate is a strain of S. cerevisiae deposited on February 27, 2019 at the CNCM under number I-5405.

17. The method according to any one of claims 1 to 16, in which the yeast hydrolysate comprises soluble and insoluble components derived from the yeast cell material.

18. The method according to any one of claims 1 to 17, wherein the product is selected from the group consisting of a food product, feed product, beverage, grain, plant, and bioethanol.

19. Animal feed product, complementary feed product, premix or feed additive comprising a yeast hydrolysate as defined in any one of claims 1 to 18.

20. Yeast strain deposited on February 27, 2019 at the CNCM under number I-5405.

21. Use of the yeast hydrolysate of any one of claims 1 to 16, the yeast alkaline hydrolysate of any one of claims 2 to 5 or the yeast enzymatic hydrolysate of any one of claims 6 to 12 for reducing deoxynivalenol, zearalenone, and/or ochratoxin A levels in or on a product.

22. Animal feed product, complementary feed product, premix or feed additive comprising a yeast hydrolysate, wherein the yeast hydrolysate is prepared from a strain of S. cerevisiae deposited on February 27, 2019 at the CNCM under number I-5405.