Field of the invention

The present invention relates to a method for co-evaporating a eukaryotic cell wall fragment preparation and whey and/or whey derived products. It is also concerned with an intermediate concentrated mixture as well as a substantially dry combination product of these two components, as well as their use in feed or food compositions, for example milk replacers for feeding juvenile animals. Background

Whey is the liquid remaining after milk has been curdled and strained. It is a byproduct of the manufacture of cheese or casein and has several commercial uses. Liquid whey contains lactose, vitamins, protein and minerals along with traces of fat. It can be processed into several streams to be used by other industries. It can be dried to obtain whey powder, it can be crystallized to separate the whey into lactose and delactosed whey, and it may be ultrafiltrated into whey protein concentate and permeate. The permeate may be subjected to crystallization to obtain lactose and delactosed permeate. Cheese whey contains more than 93% of water. In addition, it is comprised of about 5% lactose and about 0.8% protein.

Eukaryotic cell wall fragments, particularly those derived from yeast of the genus

Saccharomyces, are used as a prebiotic additive to animal feed. The yeast cell wall comprises 35% to 40% of the yeast cell. It is largely composed of mannans and β-glucans. Mannan is a non-digestible protein carbohydrate, and is considered a prebiotic. It has been shown that β-glucans interact with the immune system to increase its reaction capabilities. Yeast cell walls are a component part of animal feeds, and have also been recognized for their ability to protect and foster juvenile animals, encouraging their inclusion in formulated commercial milk replacers. The commercially available yeast cell wall products, e.g., Saf- Mannan or Bio-Mos, are available as powders. They are obtained by rupturing of the yeast cell wall, harvesting by centrifugal separation from the yeast extract, washing and drying and pasteurizing on a steam drum dryer. The preparation process of these commercially available yeast cell wall products is cost-intensive, and as such the product is relatively expensive. Low-cost yeast cell wall sources are available, but currently they cannot be used as a nutritional component in spray-drying facilities due to their low percentage of dry matter (about 17%), and prohibitive costs of drying. Both uncrystallized whey and suspensions comprising eukaryotic cell wall fragments are difficult to dry. Yeast cell wall fragments can be dried by means of evaporation to about 17% dry matter. At higher percentage dry matter, the liquid comprising the yeast cell wall fragments becomes too viscous to pass through an industrial scale evaporator. Similarly, whey and whey-derived products are difficult to dry. Using a conventional evaporator, whey or whey derived products can be concentrated to about 40% dry matter. Higher percentages may be obtained using a thermal vapor recompression system.

EP 0 035 062 describes the preparation of a concentrate of yeast cells and byproducts of, among others, the milk industry. A yeast fluid is evaporated, with or without a solution or suspension of by-products to 16 to 50% dry matter. Subsequently, raw materials are mixed in to increase the concentration to 35-60% dry matter. Then, the mixture is dried to over 80% dry matter content.

GB 1 51 1 975 is concerned with a method of utilizing whey for the production of foodstuffs and animal feeds. Whey is mixed with a cereal product and the cereal product is hydrolyzed to sugars in the whey itself. The sugar-containing whey is then subjected to fermentation with yeast to produce single cell proteins. After fermentation, the single cell proteins may be evaporated and dried.

DE 26 43 093 deals with animal feedstuffs in pressed form based on solid, fibrous, agricultural by-products and non-fibrous additives.

GB 1 562 567 relates to a process for the production of a solid water dispersible lipid enriched single cell microbial protein composition by emulsifying the single cell microbial protein. Whey solids and yeast solids in the form of whole cells may be used in the composition.

The documents above refer to whole yeast cells, and not to yeast cell wall fragments. Compared to a suspension of whole yeast cells of the same percentage dry matter, a suspension of yeast cell wall fragments is much more viscous. Also, yeast cell wall fragments are much smaller compared to whole yeast cells. Preparations comprising yeast cell wall fragments retain considerably more water than do preparations comprising whole yeast cells. Additionally, in whole yeast cells the surface consists of the external side of the cell wall which consists mainly of mannoproteins. In yeast cell wall fragments, several surfaces are exposed externally: the inner layer of the cell wall consisting of β-1,3- and β- 1 ,6-glucans complexed with chitine and/or the cell membrane composed of a phospholipid bilayer, sterols, and optionally proteins, as well as the external side of the cell wall which consists mainly of mannoproteins. As such, a yeast cell fragment preparation has properties that are very different from whole yeast cells.

The present inventor has now surprisingly found that co-evaporation of whey or whey-derived products and eukaryotic cell wall fragment preparations, such as yeast cell wall fragment preparations, improves water removal of both products. Evaporation efficiency was strongly enhanced. Since concentrating liquid streams by means of evaporation is about ten times cheaper than drying by means of spray-drying, any water that can be removed using evaporation provides a considerable economic benefit.

Thus, the present invention provides an improved process for drying two components of, for example, animal feed or milk replacers for juvenile mammals. By co-drying whey or whey-derived products and a eukaryotic (preferably yeast) cell wall fragment preparation in a first step, considerably more water can be evaporated than is the case for each of the separate components.

Summary of the Invention

In one aspect, the invention provides a method for preparing a concentrated mixture comprising a eukaryotic cell wall fragment preparation and whey and/or whey-derived products, said method comprising the steps of: i) mixing a first liquid comprising said a eukaryotic cell wall fragment preparation and whey and/or a whey derived product to obtain a liquid mixture, and ii) subjecting said liquid mixture to evaporation to obtain a concentrated mixture.

The method may further comprise the step of spray-drying said concentrated mixture to obtain a combination product comprising a eukaryotic cell wall fragment preparation and solids derived from whey and/or whey-derived products.

The concentrated mixture may comprise about 33 to about 60% dry matter.

In an embodiment, the combination product comprises less than about 10% (w/w) water. The whey and/or whey-derived products may be selected from the group consisting of sweet or sour whey, sweet or sour whey permeate, delactosed whey, and delactosed whey permeate.

In an embodiment, the eukaryotic cell wall fragment preparation comprise yeast cell wall fragments, such as a yeast cell wall fragment preparation, preferably of yeasts of the genus Saccharomyces, preferably belonging to the species Saccharomyces cerevisiae.

In a further aspect, the present invention is concerned with a concentrated mixture obtainable by the method of the invention.

In yet another aspect, the present invention is directed to a combination product obtainable by the method of the invention.

In a further aspect, the present invention is directed to a feed or food composition comprising a concentrated mixture of the present invention or a combination product of the present invention. Such feed or food composition may be a milk replacer for juvenile animals such as calves, lambs, piglets, and the like. Such composition may also be used as a compound in young animal feed, either as a meal, crumble or in pelleted form. Detailed description of the Invention

The present invention provides a method for preparing a concentrated mixture comprising a eukaryotic cell wall fragment preparation and whey and/or a whey-derived product, said method comprising the steps of: i) mixing a first liquid comprising said eukaryotic cell wall fragment preparation and whey and/or a whey-derived product to obtain a liquid mixture, and ii) subjecting said liquid mixture to evaporation to obtain a concentrated mixture.

The term "a concentrated mixture comprising a eukaryotic cell wall fragment preparation and whey and/or a whey-derived product" as used herein refers to a mixture that is more concentrated than a mixture that could be obtained by simply mixing the first liquid comprising a eukaryotic cell wall fragment preparation and whey and/or whey-derived products. As explained above, a eukaryotic cell wall fragment preparation cannot be dried by evaporation beyond about 17% dry matter, and whey or whey derived products cannot be dried by evaporation beyond about 60% dry matter. It was found that when a suspension comprising about 16.1 % (on dry matter basis) yeast cell wall fragments and 60% whey permeate were mixed to yield a suspension comprising 33% dry matter could be concentrated to a concentrated mixture comprising 37.1 % dry matter, whereas each separate component could not be evaporated any further. The concentrated mixture preferably comprises about 34 to about 60% dry matter, more preferably about 35 to about 60% dry matter, yet more preferably about 40 to about 60% dry matter.

The term "a eukaryotic cell wall fragment preparation" refers to a preparation comprising at least 60%, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of eukaryotic cell wall fragments. The term "eukaryotic cell wall fragments" in the context of the present invention refers to the insoluble fraction of eukaryotic cells which is obtained after autolysis or enzymatic hydrolysis, mainly by proteases, of whole eukaryotic cells, leading to the solubilization of at least 50%, and preferably of at least 60% by mass of the dry matter content of the whole eukaryotic cells and preserving the structural polysaccharides of the cell wall. Preferably, a eukaryotic cell wall fragment preparation of the present invention comprises less than about 40%, such as less than about 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1 % intact (or whole) eukaryotic cells. The eukaryotic cell wall fragments may be selected from animal cell wall fragments, plant cell wall fragments, fungal cell wall fragments, and yeast cell wall fragments. It is preferred that the eukaryotic cell wall fragments comprise or consist of yeast cell wall fragments. Preferably, the yeast cell wall fragments of the invention comprise or consist of cell wall fragments of yeasts of the genus Saccharomyces, preferably belonging to the species Saccharomyces cerevisiae. Eukaryotic cell wall fragments may be produced by any method known in the art, e.g., as described in US 6,387,420. As an example, the eukaryotic organism may be grown following common techniques used in food/feed-related fermentations. In case of microorganisms such as fungus or yeast, any of a number of common sugar-containing media, such as diluted molasses, may be used to provide a source for growth of the yeasts. The biomass may then be separated and washed by centrifugation to yield a yeast cream. Following separation, the microorganism is lysed. Any of a number of methods common in the art may be utilized to lyse the microorganisms, including autolysis and hydrolysis. A protease such as papain or any of a number of alkaline or neutral proteases may be added during the lysis phase to accelerate solubilisation of proteins and prevent agglutination of intracellular components. Following autolysis, the resultant cell wall extract may be washed several times to remove intracellular components and concentrate the extract.

In the method of the present invention, a eukaryotic cell wall fragment preparation may comprise cell wall fragments of one eukaryotic organism, or cell wall fragments of two or more different eukaryotic organisms may be employed. Advantageously, at least one of the two or more eukaryotic organisms is yeast, preferably of the genus Saccharomyces, preferably belonging to the species Saccharomyces cerevisiae. In an embodiment, cell wall fragments of plants comprising mannose, mannobiose, oligosaccharides and/or mannans are also included.

The whey and/or whey-derived products may be selected from the group consisting of sweet or sour whey, sweet or sour whey permeate, delactosed whey, and delactosed whey permeate. One of whey or whey-derived products may be used, or they may be mixed prior to combining them with the first liquid comprising the eukaryotic cell wall fragments.

The first liquid comprising the eukaryotic cell wall fragment preparation and the whey and/or whey-derived product(s) may be mixed in any way known in the art, for example, simply by stirring. They may be mixed in various ratios, for example, about 80 to 40% on dry matter basis solids derived from whey and/or whey-derived products and about 20 to 60% on dry matter basis eukaryotic cell wall fragment preparation. Thus far, best results have been obtained using about 70% on dry matter basis solids derived from whey and/or whey- derived products, and about 30% on dry matter basis eukaryotic cell wall fragment preparation. They may also be homogenized.

The skilled person is capable of determining the optimal evaporation conditions. Suitable evaporators include, without limitation, rotary evaporators, falling film evaporators, thin film evaporators, scrapped surface evaporators, forced circulation evaporators, combination evaporators, plate evaporators, and thermal and/or mechanical vapor recompression evaporator. The skilled person will be able to determine suitable operating conditions including temperatures and pressures. Suitable operating conditions depend on the type of evaporator used. In a suitable embodiment, a mechanical vapor recompression evaporator is used. Such evaporator is highly cost- and energy-effective. For example, temperatures employed for a 3-stage mechanical vapor recompression evaporator may be temperatures of 64, 59, and 46°C at stages 1 , 2, and 3, respectively.

The method may further comprise the step of spray-drying said concentrated mixture to obtain a combination product comprising said eukaryotic cell wall fragment preparation and solids derived from whey and/or whey-derived products. The skilled person is capable of establishing suitable spray-dry conditions. Suitable operating conditions depend on the type of evaporator used. For example, a suitable spray-drier is a single-stage spray-drier. For such single-stage spray-drier, suitable settings include, without limitation, an inlet temperature in the range of about 170 to about 200°C, and an outlet temperature in the range of about 70 to about 100°C. The combination product preferably comprises less than about 10% (w/w), preferably less than 8% (w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4% (w/w), 3% (w/w) water.

In a further aspect, the present invention is concerned with a concentrated mixture obtainable by the method of the invention. Said concentrated mixture comprises eukaryotic cell wall fragments as described above, and solids derived from whey or whey-derived products as described above, both in suspension. The concentrated mixture preferably comprises about 30 to about 60% dry matter, more preferably about 35 to about 60% dry matter, yet more preferably about 40 to about 60% dry matter. Preferably, said concentrated mixture consists essentially of eukaryotic cells wall fragments, such as yeast cell wall fragments, as described above, and solids derived from whey or whey-derived products as described above, and water.

The concentrated mixture may further be spray-dried to yield a combination product comprising eukaryotic cell wall fragments as described above, and solids derived from (delactosed) whey, or (delactosed) whey permeate, which combination product is substantially free of water. The combination product preferably comprises less than about 10% (w/w), preferably less than 8% (w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4% (w/w), 3% (w/w) water. The present invention is further directed to such combination product obtainable by the method of the invention. In an embodiment, said combination product consists essentially of eukaryotic cells wall fragments, such as yeast cell wall fragments, as described above, and solids derived from whey or whey-derived products as described above.

The concentrated mixture or combination product of the present invention can be used as a food additive or as a feed additive as such. The feed additive prepared by the method of the invention can be added to a feed composition or food composition as such, moisturised or dried, and it can be generally treated as desired. It can be used in feed compositions for juvenile animals, e.g., pigs, poultry, calves, as well as for pets such as dogs and cats, horses, especially foals, fish and so on. Preferably, it is used for newly weaned or hatched animals, such as, for example, weaning pigs.

Alternatively, the concentrated mixture or combination product of the present invention can be added to any commercially available feed compositions for livestock or companion animals or food compositions for human consumption. Such feed composition may be in any form, e.g., powders, meals, crumbles or pelleted feed.

Thus, in a further aspect, the present invention is directed to a feed or food composition comprising a concentrated mixture of the present invention or a combination product of the present invention. The feed or food composition preferably comprises an amount of about 1 to about 50% (w/w), such as about 2 to 45% (w/w), or about 4 to about 40% (w/w), or about 5 to about 40% (w/w), or about 10, 15, 20, 25 to about 40% (w/w), of the combination product. It is noted that the concentrated mixture of the present invention may have been used in the preparation of the feed or food composition to achieve the desired amounts of combination product of the present invention. For pelleted feed, a content of eukaryotic cell wall fragments (yeast cell wall fragments) of about 5% based on dry matter is very suitable.

In an embodiment, such feed or food composition may be a milk replacer for juvenile animals, preferably juvenile mammals, more preferably juvenile mammals, most preferably juvenile mammals excluding humans, such as calves, lambs, piglets, and the like. Such milk replacer generally comprises about 15 to about 30% (w/w), preferably about 17 to about 25% (w/w), yet more preferably about 19 to about 23% (w/w) protein, preferably including milk protein, preferably derived from whey, and sometimes also egg proteins, animal plasma proteins, wheat proteins, soy proteins, and the like; about 10 to about 25% (w/w), preferably about 15 to about 20% (w/w), fats and oils such as milk fat, lard, choice white grease, and/or vegetable oils such as soy, palm and coconut oil; and about 25 to about 50% (w/w), preferably about 35 to about 45% (w/w) carbohydrates such as lactose, dextrose, and/or galactose. Generally, milk replacers further comprise minerals, and vitamins. Advantageously, the milk replacer comprises about 1 to about 15% (w/w), about 2 to 13% (w/w), or about 4 to about 1 1 % (w/w), or about 5 to about 10% (w/w), eukaryotic cell wall fragments, preferably yeast cell wall fragments, as above, derived from 1 to about 50% (w/w), such as about 2 to 45% (w/w), or about 4 to about 40% (w/w), or about 5 to about 40% (w/w), or about 10, 15, 20, 25 to about 40% (w/w), of the concentrated mixture comprising the combination product of the present invention. It is to be noted that the concentrated mixture of the present invention may have been used in the preparation of the feed or food composition to achieve the desired amounts of combination product of the present invention. For milk replacers, a content of about 10% based on dry matter eukaryotic cell wall fragments is very suitable.

The feed or food composition of the present invention may further comprise mannose, mannobiose, oligosaccharides, mannans, and/or plant cell wall fragments, said plant cell wall fragments preferably comprising mannose, mannobiose, oligosaccharides, and/or mannans.

In a suitable embodiment, the concentrated mixture is mixed with further liquid components of a food or feed composition, optionally homogenized, for example at a pressure of about 60 to about 160 bar, and subsequently spray-dried together with said further liquid components of a feed or food composition. Such method is particularly advantageous when preparing a milk replacer for juvenile animals as indicated above.

"Percentage (%) dm" or "% based on dry matter" as used herein refers to the percentage of dry matter. The dry matter (also known as dry weight) is a measurement of the mass of something when completely dried. The dry matter of a feed/milk replacer composition contains all its constituents excluding water. The percentage of dry matter of a single constituent of such feed/milk replacer composition is the mass of such constituent contained in the feed/milk replacer composition divided by the total mass of all constituents excluding water of said feed/milk replacer composition.

"(w/w)", as herein used, is an abbreviation for "by weight," used to describe the concentration of a substance in a mixture or solution. Herein, a weight percentage of 2, 2% (w/w), means that the mass of the substance is 2% of the total mass.

The term "substantially" herein will be understood by the person skilled in the art. In embodiments the adjective substantially may be removed. Where applicable, the term "substantially" may also include embodiments with "entirely", "completely", "all", etc. Where applicable, the term "substantially" may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. In contrast, the verb "to consist of" and its conjugates do exclude the presence of elements or steps other than those stated. The term "comprise" includes also embodiments wherein the term "comprises" means "consists essentially of" or "consists of".

Likewise, the term "about" may, where applicable, indicate a deviation of 10% or less, or 5% or less, or 1 % or less, or 0.5% or less, or even 01.% or less, and also in an embodiment no (measureable) deviation. As will be clear to the person skilled in the art, small deviations from numerical values may, where applicable, in general be allowed. Hence, except for the values in the definition of about above, numerical values may, where applicable deviate a 10% or less, or 5% or less, or 1 % or less, or 0.5% or less, or even 0.1 % or less from the given value. To stress this, herein sometimes the word "about" is used before numerical values.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

Examples

Example 1: Preparation of co-dried whey permeate and yeast cell wall fragments Whey permeate having a dry matter content on weight basis of about 38% and a liquid suspension comprising yeast cell wall fragments having a dry matter content on weight basis of about 16.1 %, both of which could not be dried any further by means of evaporation, were mixed and co-evaporated in a 3-stage mechanical vapor recompression evaporator (temperature at stages 1-3: 64, 59, and 46°C, respectively). A dry matter content of 37.1 % on weight basis could be yielded.

The total amount of water that has evaporated by means of the evaporator is 7494 kg water. This could only be achieved by means of co-evaporation. The suspension comprising 37.1 % dry matter on weight basis yeast cell wall fragments and permeate solids was spray-dried on a single-stage spray-drier under the following conditions: inlet temperature: 181°C; outlet temperature: 89°C. The resultant after spray- drying comprised the yeast cell wall fragments, whey permeate solids, and about 3.6% (w/w) water.

Example 2: A composition of calf milk replacer for rearing calves

Component Percentage (w/w) on dry matter basis Fat concentrate 50/50 25% (50% sweet whey and 50% fat)

Delactosed whey 15%

Whey Protein Concentrate 35 15%

Soy Protein Concentrate 4%

Hydrolysed wheat protein 6%

Concentrated mixture comprising 37.5%

dry matter (dm) YCW/whey permeate

Pre mix ³

Premix for calf milk replacer may constitute, for example, the following ingredients:

Per kg final

Per kg feed

Nutrient Unit premix composition

Vitamin A I E 1500000 30000

Vitamin D3 I E 250000 5000

Vitamin E I E 10000 200

Vitamin K3 mg 250 5

Vitamin Bl mg 200 4

Vitamin B2 mg 300 6

d-Pantotheic acid mg 1250 25

Nicotinic acid mg 2000 40

Biotin meg 5000 100

Vitamin B12 meg 1500 30

Folic acid mg 40 0.8

Vitamin B6 mg 150 3

Choline Chloride mg 7500 150

Vitamin C mg 5000 100

Fe: Fe(ll)Sulph. mg 5000 100

Cu:CuS04.5H20 mg 350 7

Zn:ZnSulphate mg 4000 80

Mn:Mn(l l)Sulphate mg 1500 30

Co:Co(ll)Carbon. mg 50 1

l :K-Jodide mg 120 2.4

Se:Na-Selenite mg 20 0.4

Calcium formiate mg 150 3.0

Sorbitol mg 41 0.8

Calcium g 50 1.0 Phosphor g 0 0.0 Magnesium g 15 0.3 Natrium g 0 0.0 Chlorine total g 55 1.1 Sulphur total g 5 0.1