BG1325U1 - Feed additive containing soluble mannanoligosaccharides - Google Patents

Abstract

The present invention relates to feed additive containing soluble mannanoligosaccharides and can be used in zootechnics. The additive is obtained by yeast cultivation synthesising extracellular mannanoligosaccharides. The yeasts are of stem Candida, Klebsiela, Torula, Rhodotorula or Saccharomyces. The feed additive containing soluble mannanoligosaccharides can be used in feeding certain animal species including pigs, poultry, ruminants, fish and aquaculture, while achieving improved growth of livestock and feed digestibility.

Description

Technical field

A useful model relates to a feed additive that contains soluble mannoligosaccharides and is used in zootechnics. The additive is obtained by culturing yeast that synthesizes outside cellular monoligosaccharides. The application of the feed additive to animals results in improved growth of farmed animals and improved uptake of feed.

BACKGROUND OF THE INVENTION

The use of antibiotics as a nutritional supplement in the diets of industrially-grown animal species is known, which is widely practiced and leads to improved zootechnical parameters, reduced mortality and morbidity in treated animals. The inclusion of antibiotics in the animal's diet can lead to cross-resistance of some pathogens in humans. There is a growing trend to limit the use of antibiotics as growth factors in animal feed and to replace them with probiotic and prebiotic preparations.

Prebiotics are defined as non-absorbable constituents of foods whose positive effect on the health of their host organisms is due to stimulating the growth and / or activity of beneficial microflora or inhibiting the development of unwanted microflora in the digestive tract of animals and / or humans. Most often, prebiotics are non-digestible oligo- or polysaccharides, such as beta-glucans, mannans, mannoligosaccharides and fructooligosaccharides.

There are scientific reports of the positive effect of monoligosaccharides on the feeding of known animal species, including pigs, birds, ruminants, fish and aquaculture. Mananoligosaccharides inhibit the development of pathogens in the gastrointestinal tract by limiting their ability to colonize and exert an immunomodulatory and bifidogenic effect [1].

Yeast cell walls known as the main source of mana noligosaccharides are the yeast cell walls. There is evidence in the literature of the use of feed additives containing mannoligosaccharides obtained by isolation from yeast cell walls. For this purpose, yeast is often used, which is waste from brewing. The disadvantage of these products is the need for pretreatment of the yeast cell walls in order to isolate the monoligosaccharides. The mannan that forms part of the cell walls of yeast cells is in the form of a phosphomannan protein complex. Isolation of mannan from cell walls is difficult to achieve due to the formation of a stable mannan-protein complex.

In the prior art, in the process of isolating the carbohydrate fraction from the yeast cell walls, alkaline extraction is mainly applied, resulting in a significant reduction in the degree of polymerization of mannan as a result of the ongoing depolymerization by the beta-elimination mechanism. In view of the application of these products to animal nutrition, this effect of reducing the degree of polymerization is undesirable. The lower molecular weight products can be more easily hydrolyzed in the acidic environment of the stomach to the constituent monosaccharides, which facilitates their absorption and absorption. Thus, this pretreatment would reduce the expected positive effect on the intestinal microflora and immunomodulatory and bifidogenic function.

WO 1995/004467 discloses the use of beta-glucan isolated from yeast cell walls as a factor improving zootechnical parameters in pig breeding. The main feature of the patent is the pre-treatment of the yeast cell walls in order to isolate the beta-glucan from the other components of the cell wall to enhance its effect. The treatment of insoluble yeast walls used to isolate β-glucans involves treatment with enzymes, highly alkaline and highly acidic solutions.

US 6444448 B1 lists the main methods for isolating the carbohydrate component from the cell walls of yeast:

(i) High temperature extraction with

1325 Ul of concentrated sodium hydroxide, followed by high temperature extraction with acid solution and precipitation with ethanol.

(j) High temperature extraction with concentrated sodium hydroxide, followed by high temperature extraction with acid solution and enzymatic treatment to alter or purify the carbohydrate fraction.

(iii) Extraction from pre-autolysed or enzymatically lysed yeast.

(iv) Extraction with alkaline solutions of organic solvents such as isopropanol, ethanol, acetone or methanol.

The need for these technological processes leads to the difficulty in producing effective feed additives containing mannoligosaccharides. The presence of mannans in yeast cell walls in the form of insoluble phospho-protein complexes and their difficult release from cell walls renders the use of feed additives obtained from such sources either costly or inefficient.

The technical nature of the utility model

The essence of the present utility model is a feed additive containing soluble mannoligosaccharide and obtained by culturing yeast that synthesizes extracellular mannoligosaccharides. The products thus obtained can be used as an adjunct to animal feed and lead to improved growth of farmed animals and improved uptake of feed.

Some microorganisms, including yeast, produce in large quantities extracellular poly- and oligosaccharides. Yeast of the genus Candida, Klebsiela, Rhodotorula and Saccharomyces synthesize in large quantities extracellular mannoligosaccharides, which are in soluble form and are not in complex with other compounds. The amount and composition of the synthesized polysaccharide depends on the biosynthetic ability of the strain used, the culture conditions and the composition of the culture medium. The use of feed additives containing soluble mannoligosaccharides thus obtained is attractive because of the process of preparation and the possibility of higher efficiency of their application, which is due to the fact that they are in free form and soluble in aqueous media.

In particular, the purpose of the utility model is to describe a feed additive containing mannoligosaccharides obtained by fermentation of yeast of the genus Rhodotorula and the application of the resulting preparation to improve zootechnical performance in the cultivation of animal species. As a result of deep cultivation of yeast of the genus Rhodotorula sp. a significant amount of extracellular monoligosaccharide accumulates in the culture fluid. The obtained mannan is in soluble form, which does not require additional procedures to isolate mannan from cell walls. The product thus obtained is easy to obtain and safe to use. It is possible to obtain highly purified mannoligosaccharide by known methods of extraction and purification, which is not possible with the use of yeast cell walls as a source of mannan. Alternatively, the fermentation liquid may be dried to produce a technical product containing soluble mannoligosaccharide.

Yeast fermentation to obtain soluble mannoligosaccharides is carried out by the deep culture method. The optimal development medium, which necessarily contains carbon, nitrogen and trace elements sources, is sterilized and seeded with a pure culture of the producer strain. During the process, the carbon content is monitored and an additional amount may be supplied. During the process, the strain accumulates extracellular monoligosaccharide, which increases the viscosity of the fermentation medium. An exemplary method for fermenting yeasts that produce soluble manganoligosaccharides is given in Examples 1 and 2.

The fermentable soluble monoligosaccharide obtained can be isolated by known methods. Where the purpose is to obtain a purified product, it is best to remove the yeast biomass by filtration or centrifugation and the resulting filtrate (or centrifuge) containing the dissolved mannoligosaccharide to be precipitated by the addition of alcohol. For example, by adding three parts of ethyl alcohol to one part of the supernatant, precipitation of pure monoligosaccharide is achieved which can be separated

1325 W by filtration and dried. The characteristics of the purified mannoligosaccharide are given in more detail in Example 3. It has been found that by precipitation it is possible to obtain a product containing more than 60% carbohydrates with a high content of mannan. Subsequent dissolution in water and alcohol precipitation may result in a more purified product.

When the resulting monoligosaccharide is intended for use as a feed additive, isolation and purification need not be carried out. This is due to the fact that the yeast biomass and fermentation medium contain nutrients that are not harmful and have high nutritional value, as a source of protein, carbohydrates and trace elements. In this case, after the fermentation is complete, the optimal option is to subject the whole culture fluid to drying, by spray drying or other method. This produces a powdered product that can be added to the feed or mixed with other trace elements, vitamins or nutrients before being used as a feed additive. One of the technical difficulties to be overcome in the spray drying of a fermentation fluid containing soluble manganoligosaccharides is to prevent adhesion to the dryer and to reduce the hygroscopicity of the product obtained. This is achieved by the application of minerals or other substances that improve the flowability of the product and reduce its hygroscopicity. Calcium carbonate, silica, sodium sulphate, calcium sulphate are used as fluids. As substances that reduce the hygroscopicity of the product are used silica, calcium silicates and others. The preparation of a flowable powder containing mannoligosaccharides is described in Example 4.

The resulting manganoligosaccharides are included in feed additives. Feed additives are substances that are added to feed in order to improve the nutritional value of feed. Feed additives may consist of one or more substances or components. In this sense, it is clear that the soluble mannoligosaccharides obtained, either in purified form or as a technical product by spray drying, can be used alone as a feed additive or mixed with other such and represent only one part of the feed additive composition.

Improvement of zootechnical characteristics has been observed when using the obtained high-mannan feed additives in the diet of industrially-grown animal species. Zootechnical characteristics are determined by two main indicators: growth and feed absorption efficiency. The increase represents the weight gain of the species grown. It can be improved if a predetermined (regulated) weight is reached faster, or if more weight is reached at a predetermined period.

Feed absorption efficiency is the ratio between the feed consumed and the growth achieved over a given period. The lower this ratio, the greater the efficiency of farm work, since less feed is required to achieve the same weight.

Examples of implementation of the utility model

The following examples illustrate the effect of a utility model, but should not be construed as limiting it in any way.

Example 1.

Preparation of extracellular soluble monoligosaccharide.

This example describes the preparation of extracellular mannan from Rhodotorula mucilaginosa MUCL 45531. The cultivation process is performed in 500 cm ³ Erlenmeyer flasks with a filling volume of 100 cm ³ . The composition of the culture medium is presented in Table-Table 1.

1325 Ul

Table 1. Component composition of the culture medium

Component Concentration, Molasses 10-100 (NHO2SO4 0.1 -10 Brewer's yeast 0.1-100 KN2PO4 0.01-10 MgSO4.TH ₂ O 0.01-10 NaCl 0.01-10 SASJgO 0.01-10

The culture medium was sterilized at 118 ° C for 30 min. After sterilization, the culture medium was cooled to 36 ° C and then inoculated with 10% (v / v) vegetative seed. Cultivation was carried out with continuous shaking of the shaker at 200 rpm for 90-100 h. At the end of the fermentation process, the culture medium is a viscous liquid and contains solanoligosaccharides.

Example 2. Preparation of soluble monoligosaccharide in fermenters.

The purpose of the present example is to describe the production of extracellular mannan from Rhodotorula mucilaginosa MUCL 45531 in a 100 dm ³ bioreactor. The cultivation process is carried out in a bioreactor with a total volume of 100 ³ θ dm ³ and a fill volume of 60 dm ³ . The composition of the culture medium is presented in Table 1. The preparation of the culture medium and the fermentation process were carried out according to the procedure described in Example 1. Cultivation ³⁵ was carried out with continuous stirring, overpressure 0.3-0.5 bar, aeration 0.5 dm ³ / dm ³ .min. and duration 90-100 h. The viscosity of the culture medium is 80.0-90.0 cPa.

Example 3. Isolation of the extracellular mannan produced and characterization of the soluble mannoligosaccharide obtained

This example describes the process of isolating the extracellular mannan produced and its component composition. The extracellular mannan biosynthesis was performed according to Example 1 and Example 2. After termination of the cultivation process, the culture fluid was centrifuged at 1000 rpm for 15 min in order to remove the biomass. The resulting supernatant was decanted and used to isolate extracellular mannan. The isolation of mannan is accomplished by precipitation with chilled ethanol. For complete precipitation of mannan, 4 volumes of cooled ethanol were added to the supernatant. As a result of the addition of ethanol, a white ragged precipitate of precipitated mannan is formed. The precipitate is dried. The quantitative determination of the monosaccharide composition of the obtained products was performed by gas chromatography. Measure about 6.0 mg of test product, then hydrolyze with 2.0 M CF ₃ COOH (2.0 ml) for 3 h at 120 ° C. The excess of CF ₃ COOH was evaporated under nitrogen at 40 ° C to dryness. After drying, the sample was dissolved in 1 ml of a solution of the internal standard ronitrophenyl-beta-O-glucopyranoside in pyridine. The sample was heated in a boiling water bath for 5 min. After cooling, the resulting glycosides were derivatized with hexamethylenedisilazane (0.5 ml) in the presence of CF ₃ COOH (0.15 ml) as a catalyst. The resulting solution was analyzed on a GC / FID gas chromatograph (HewlettPackard 6890) with HP-5 column 5% phenyl-methylsiloxane (30 m; 0.32 m; 0.25 µm). The conditions of analysis are: an initial temperature of 190 ° C, which rises to 280 ° C at a rate of 20 ° C / min. Derivative methylglycosides (standards) have been used to identify and quantify monosaccharides.

The component composition of the precipitate obtained is presented in Table 2.

Table 2. Component composition of isolated extracellular mannan

Component Concentration,% Protein 5.25 Ash 3.00 Moisture 11.6 Total carbohydrates 65.3 Monosaccharide component composition of the carbohydrate fraction (%): Mannose 89.1 Glucose 4.00 Galactose 0.75 Arabinosis 1.00 Fucose 1.15 Fructose 0.90

Example 4. Preparation of a prebiotic feed additive containing soluble β θ mannoligosaccharide.

The purpose of the present experiment is to obtain a high crude mannan preparation for use as a prebiotic feed additive. The extracellular mannan biosynthesis was performed according to Example 1 and Example 2.

After termination of the fermentation, the resulting culture medium was dried by spray drying. 2 g / Ι CaCO ₃ was added to the culture medium before drying to improve the flowability of the product obtained. The resulting product has a composition according to Table 3.

Table 3. Component composition of the resulting product with a high crude manganese content

Component Concentration,% Protein 8.66 Ash 27.6 Moisture 7.50 Total carbohydrates 49.8 Monosaccharide component composition of the carbohydrate fraction (%): Mannose 88.5 Glucose 5.30 Galactose 0.70 Arabia 1.00 Fucose 1.2 Fructose 1.8

The product thus obtained can be used directly as a prebiotic feed additive source of soluble mannoligosaccharides or mixed with fillers or other nutritional components before being incorporated into the feed.

Example 5. Use of prebiotic feed additives containing soluble mannoligosaccharide in bird feeding.

The purpose of the study is to determine the effect of application of the product obtained according to Example 4 as an additive to the feed of broiler chickens. Doses of 1 kg / t, 2 kg / t and 4 kg / t of feed were administered over the entire growing period. The study included 120 1-day-old, sexed broiler chickens from Hybro G hybrid. The chickens were of equal sex, divided into five groups (2 control and 3 experimental), 24 chickens in each (12 males and 12 female). The study lasted 42 days. The results of the study are presented in Table 4.

Table 4.

Parameter (Period: 0-42 days) Groups I P. W IV Negative control Additive lg / kg feed 2g / kg feed Additive 4g / kg feed Number of birds 48 24 24 24

Average weight, g /% 2156.8 ± 277.7 2147.1 ± 282.3 2231.9 ± 357.0 2306.1 ± 3142 100.0 99.6 103.5 106.9 Average daily prrraet, i 50.0 ± 6.60 49.8 ± 6.74 51.8 ± 8.46 53.6 ± 7.48 Conversion to feed, kg /% 2.044 1,987 1.855 1,983 100.0 97.2 90.8 97.0 Mortality, η 1 0 0 2

The use of the additive was found to result in higher productive performance (ultimate average live weight and feed consumption per 1 kg gain) compared to βθ with control (untreated) broiler chickens. A significant improvement in feed conversion is achieved at the dose of 1 kg / t. At doses of 2 kg / t and 4 kg / t, statistically significant differences in weight and conversion of 35 feed were achieved.

Example 6. Comparative use of prebiotic feed additives containing soluble manganoligosaccharide and manganosaccharide from yeast cell walls in feeding birds to

The purpose of the study was to determine the effect of administration of the product obtained according to Example 4 as an adjunct to the feed of chicken broilers and to compare its effect with the effect of administration of yeast wall monoligosaccharides. The Biomos product (Alltech Inc.), which contains monoligosaccharides derived from yeast cell walls, was used as a positive control. Both products were administered at doses of 2 kg / t, 1 kg / t and 0.5 kg / t of feed respectively during the starter (1-15 days, grower (15-28 days) and finishing (28-42 days) growing period The experiment set-up is a complete randomized block design with 13 replicates of each of the 3 groups, respectively. 5.

1325 Ul

Table 5

Parameter (Period: 0-42 days) Groups I P. W Negative control Biomos 2-1-0.5 kg / ton Additive * 2-1-0.5 kg / ton Number of birds 48 24 24 Average weight, g /% _{2779 2777} s 2834 * 100.0 99.9 102.0 Average daily rq »$ aCT, Mr 65.2® 65.2 ⁶ 66.5 * Conversion to feed, kg /% 1.657 * 1.651 * 1.638 * 100.0 99.6 98.9 Mortality, η 1 0 0

The characteristics of the additive are according to the product of Example 4.

There is a statistically significant difference of p <0.05% between results with different indices.

The use of the additive was found to result in higher productive performance (ultimate average live weight and feed consumption per 1 kg gain) compared to control (untreated) broiler chickens and Biomos treated broilers. A third experimental group that was treated with an additive containing soluble mannoligosaccharides ended up with a statistically significant higher average weight, a statistically significant higher average daily gain and a numerically lower feed conversion. At the same time, it appears that, at least in this experiment, the use of Biomos does not lead to a statistically significant or even numerical difference with the control group.

Example 7. Use of prebiotic fu25 rye additives containing soluble manganoligosaccharide for feeding pigs.

The purpose of the study is to determine the effect of administering the product obtained according to Example 4 as an additive to pig feed. Test dose of 1 kg / t feed. The experiment used 620 piglets after weaning at 4 weeks of age with an average weight of about 8.0 kg. The control group, without the addition of monoligosaccharide, included 360 piglets distributed in 36 batteries of 10 piglets per battery. The test group included 260 piglets, distributed in 26 batteries of 10 piglets each. The zootechnical results of the study are shown in Table 6.

1325 Sh

Parameter Units The group Control Additive 1 kg / ton Number of piglets P. 360 260 Average weight after weaning kg 8,025 th most common 8,000 Extended period Give 75 64 S 520.0 * ± 0.047 543.0 ± 0.033 irdno-dnsvn prirst % 100,0 104,4 Feed per 1 kg gain Kg 2,400 th most common 2,040 th most common % 100,0 85.0 Mortality % 3.0 2.92

The use of the additive has been found to result in higher performance. The average daily increase was increased with statistical significance. It also accelerated the growth phase and reduced the amount of feed to achieve 1 kg gain.

Claims (3)

Claims A feed additive comprising soluble manganoligosaccharides obtained by yeast fermentation. Feed additive according to claim 1, characterized in that the yeast is of the genus Candida, Klebsiela, Torula, Rhodotorula or Saccharomyces. Feed additive according to claim 2, characterized in that the yeast is Rhodotorula mucilaginosa MUCL 45531. Literature 1. R. Spring, Competitive Exclusion Products, January 1998, Feed Compounder. 2. WO 1995/004467. 3. US 6444448 Bl.