NO860075L - PROCESSING OF RAAP PROTEINS. - Google Patents

Description

Industrial fermentation processes for the production of biomass for feedstocks have a long tradition (yeast production of sulphite leachate) and in recent years have been the subject of new process developments (bacterial production based on methanol).

Biomasses from the fermentation process for the application area of animal nutrition such as calf and poultry rearing, fish farming, rye hen feed, pig rearing and livestock feed are also referred to as bioproteins, and contain, depending on the type of microorganism (yeast, bacteria), the carbon source used (sulphite leachate, ethanol , methanol, paraffin fractions) as well as

the type of fermentation and processing technology different amounts of proteins, lipids, carbohydrates and nucleic acids /ribonucleic acids (RNS) and deoxyribonucleic acids, (DNS2?.

While the nutritional value of the biomass is primarily a function of the protein content and amino acid composition and availability, and the lipid and carbohydrate fraction contributes to the energy balance of the overall premix, the nucleic acid part is a secondary component, the nitrogen part of which cannot or only to a small extent be used for building up body material.

Depending on the animal type-specific metabolism, the nitrogenous building blocks of RNS and DNS (purines, pyrimidines) are built up into metabolites, which are excreted in the form of highly water-soluble (allantoin) or poorly soluble products (uric acid).

Therefore, the nucleic content in premixes is limited in order to avoid increased metabolic or organ loads due to nucleic acid degradation products. However, a connection between nucleic acid content and nutritional availability (digestibility and tolerability) of bioproteins depending on the molecular weight of the nucleic acids (RNS and DNS) has not been discussed so far.

It was now found that nucleic acids, in addition to the above-mentioned effects in the metabolism, which arise from the chemical composition of RNS resp. DNS (ribose, deoxyribose, phosphoric acid, purines, pyrimidines) has a hitherto unknown negative effect on the digestibility of bioproteins, which is caused by the high molecular weight of these compounds.

The polynucleotides of DNS in particular achieve molecular weight ranges from 1.10 5 to 1.10 7 , while the shorter polynucleotide strands of RNS have molecular weights of 5,000-50,000.

The impact of the molecular weight of nucleic acid on the digestibility and tolerability of bioproteins was observed for the first time in feeding experiments on chickens, and from this a research model was developed for determining the molecular weight effect on the digestive organs.

The negative effect of high-molecular nucleic acids in premixes causes a concentration-dependent increase in the intestinal weight in relation to the body weight and thereby a general decrease in metabolism due to a negative change in the resorption processes. Poorer growth results and thus a distinctly lower protein value of nucleic acid-holding bioproteins are the primarily observed consequences.

It is known that the molecular weight of RNS and DNS can be reduced by means of pH value, temperature and salt content in an aqueous environment. While RNS hydrolyzes in an alkaline range, DNS can be degraded under acidic conditions. The degree of hydrolysis (reduction in molecular weight) is thereby dependent on the chosen conditions, as even at higher temperatures (80-90°C) and pH values of 8-11 for RNS resp. pH 1.5-5.4 for DNS and treatment times of 1-4 hours only achieve partial hydrolysis to oligonucleotides with molecular weights of 500-10,000 for RNS and 1,000-40,999 for DNS. In addition, the other components of the bioprotein, especially the proteins, are thereby negatively affected (colour, taste, denaturation, salt content, racemisation) and thereby the nutritional value is reduced. Moreover, the partial hydrolysis of RNS and DNS is not satisfactory in order to eliminate their drawbacks on digestibility.

According to the invention, the disadvantages of high-molecular-weight polynucleotides on the tolerability of raw bioproteins are eliminated by RNS and DNS being broken down as best as possible with the help of enzymes under mild conditions, preferably quantitatively, into oligomeric, preferably monomeric, nucleotides.

The invention therefore relates to the use of crude proteins rich in nucleic acids, in which the high molecular weight nucleic acids were cleaved by treatment with nucleases for animal nutrition.

As crude bioproteins, microbial proteins are primarily considered, above all single-cell proteins such as yeast and especially bacterial proteins, but also other nucleic acid-rich materials such as fishmeal or internal eggs.

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The enzymes used are nucleases, above all phosphodiesterases, preferably 5<1->phosphodiesterases, which hydrolyze RNS o and DNS to the corresponding monomers 3'- or 5' nucleotides.

The phosphodiesterases are widespread in nature and can be isolated from microorganisms (streptomycetes, penicillin, staphylococci), animal cells or products (bovine heart, bovine and calf spleen, snake venom) or plant cells (wheat germ).

As the phosphodiesterases only hydrolyze the molecular structure of RNS and DNS, no unwanted side reactions occur on

the other biocomponents (lipids, carbohydrates, egg white).

The phosphodiesterases can penetrate the cell wall of microorganisms, which is why there is no need for a special pre-treatment of the raw bioprotein for the enzymatic hydrolysis of RNS and DNS. Therefore, this method step can be integrated into any processing sequence of fermentatively produced biomass.

The required enzyme concentration (g) corresponds to the substrate concentration (g) in the ratio 1:1,000 to 1:500,000, preferably 1:10,000 to 1:100,000.

Next to crude enzyme preparations from microorganisms and plants, commercially available enzymes such as

Depending on the method, the enzymes can therefore be used in their original form as well as in adsorptive, ionic or covalent form fixed on a carrier.

The reaction environment for the enzymatic hydrolysis is water or mixtures with water-miscible organic solvents, with the nucleic acid concentration depending on the biomass concentration and type being 0.05-10, preferably 0.3-7, particularly preferably 1-5% by weight. The preferred pH range is 4.5-6.5, especially at 5-6, which during the hydrolysis reaction is kept in the preferred range by buffering the formed acid groups of oligo- or monomeric nucleotides with alkali such as NH^OH, NaOH, KOH, Ca(OH)2 or basic salts.

The reaction takes place at a temperature of 30-60°C, preferably 45-55°C. The time required for the hydrolysis depends on the type of biomass used, the temperature and the enzyme and usually amounts to 0.5-8 hours.

To determine the course of hydrolysis of RNS and DNS, the concentration of the nucleotides can be determined using HPLC:

Stationary phase: "LiChrosorb" RP 18 7 |i (Merck)

Column 1 = 20 cm, ø 0.4 cm

Mobile phase: double distilled 1^0 with conc. H^PO^

(pH 2.1)

Flow rate: 2.0 ml/min.

Pressure: ~100 bar

Wavelength: 254 nm

Reference substances

a) for RNS: 5'-nucleotides (5<1->CMP, 5'-UMP, 5'-AMP, 5'-GMP) b) for DNS: Deoxy-5'-nucleotides (d-5'-CMP , d-5'-AMP, d-5'-GMP, d-5'-TMP)

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To determine the relationship between intestinal weight and body weight, broilers are fed after a pre-period of 4 days with a vitamin and trace element poor for with nutrition manager f qualities control and experimental rations of approx. 9 days. Thereby, it does not matter whether the experimental period immediately joins the pre-period/or the experimental period is preceded by a more or less long intermediate period with control (e.g. 1-21 days).

The rations of the actual experimental series contain approx. 31% pure protein (amino acid protein), 1.88% Ca, 1.5% P, 0.18% Na and 0.6% K, they are calculated isoenergetic (on the basis of the exchangeable energy , about 14 MJ/kg). Soya protein serves in the control ration as the only protein source. It is supplemented as needed with DL-methionine and glycine. In the experimental ration, bacterial protein serves as the only protein source. It is adequately supplemented with DL-methionine and L-arginine-HCl.

Table I provides information on the ration composition, for example.

The cuckoo chicks are kept in wire mesh cages with 24 hours of permanent light. They can take food and water (from a Beckendrikker) ad libitum. The feed is generally administered as pellets (ø 3 mm).

The weight development, the absolute and relative weight of the small intestine (without intestinal contents) and its length serve as test parameters.

Results

In a series of different experiments, it could be shown that bacterial protein, also peroxide-treated, leads to a reduction in the live mass increase compared to the soy control group and to an increase in the small intestine weight. Table 2 provides information on the results.

From the comparison of the experimental data, it appears that bacterial protein (raw or peroxide-treated) reduced the live mass increase in the experimental period to 49% (+_ 13%) of the soy control value. The relative intestinal weights were at 152 +_ 12%

of the control group value.

Table 3 shows the analogous values when using nuclease-treated bacterial protein portions.

From comparisons with -nuclease-treated bacterial protein, it appears that the relative intestinal weight with 128 _+ 16% of the control value was significantly less increased than without nuclease treatment. If, in addition, account is taken of the fact that the relative intestinal weight falls with increasing body weight, then these differences are further relativized as the animals fed with bacterial protein were always clearly lighter than the control animals.

To that extent, it could be proven bioexperimentally that the nuclease treatment of bioprotein is a suitable method to reduce side effects that occur when unusually high amounts of bacterial protein are administered to chickens. In particular, the weight of the small intestine is favorably affected. The measured small intestine lengths, which after administration of bacterial protein without nuclease treatment were clearly greater than in the control animals, were adapted to the control values with nuclease-treated bacterial protein.

The following examples show the crude protein processing.

Exem2el_lj_

Methylomonas clara (ATCC 31 226) was grown in a nutrient solution containing methanol as the only carbon source, ammonia as the only nitrogen source, phosphate, iron and magnesium salts as well as other common trace elements under aerobic conditions (US patent 4 166 004). The concentration of the resulting bacterial cell mass was increased by separation from 1.5 to 15% by weight.

Of this paste, 1000 g were heated with slow stirring to 55°C, the pH adjusted from the original 6.9 with sulfuric acid to 5.1, and 1 mg of nuclease (Amano RP8), dissolved in 10 ml of water, was added.

Over a period of 4 hours, while keeping the pH value and the temperature constant, the nucleic acid hydrolysis was carried out and the content of formed monomeric 5<1->nucleotides determined via HPLC measurement. After 3.5 hours the reaction was practically complete, i.e. the content of 5<1->nucleotides (in wt%)

of the biomass used (150 g) roughly corresponded quantitatively (96%) to the content of macromolecular nucleic acids (12.6% by weight) of the biomass.

The pH was then adjusted to pH 6.5 with caustic soda, stirred for 10 minutes at 80°C and the bioprotein mass thus formed dried.

Eczema<pe>l_2:

The procedure was the same as in example 1, however applied

0.05 mg phosphodiesterase (from Crotalus durissus, company Boehringer Mannheim, EC 3.1.4.1). The hydrolysis yield of free 5<1->nucleotides was 89%.

Example_el_3 :

The procedure was the same as in example 1, however applied

0.15 mg nuclease P 1 (the company Sigman, from Penicillium citrinum). The hydrolysis yield was 97% after 3.5 hours.

Eczemagel_4 j_

The procedure was as in example 1, however, 10 g of wheat germ was used as crude nuclease preparation. The yield of 5' nucleotides was 78% after 3.5 hours.

Example_el_5:

The procedure was the same as in example 4, however with a crude enzyme incubation time of 5 hours. The yield of 5<1->nucleotides was 94%.

Exermoel_6 :

The production of the biomass proceeded as in example 1, however, after separation, the crude bioprotein was heated for 10 minutes at 85°C, cooled to 55°C and then mixed with 0.05 mg of nuclease RP8 (Amano). After 2 hours, the hydrolysis yield of 5<1>mucleotides was 97%.

Example_7:

From a continuous fermentation of Methylomonas clara

(ATCC 31 226) according to US patent 4 166 004 with subsequent concentration of the biomass to 30% by weight and spray drying, 15 kg of the thus formed dry product (residual moisture 4.5%, nucleic acid content 11.8%, referred to dry matter) suspended in 85 l of water with stirring, the pH value adjusted with sulfuric acid from 6.8 to 5.4, the temperature raised to 55°C and added 150 mg of nuclease (Sigma,

EC 3.1.30.1), dissolved in 10 ml of water.

Under pH (5.4) and temperature control (55°C) it was stirred for 3 hours, the pH value adjusted with caustic soda to 6.5

and the suspension spray-dried at 95°C. The hydrolysis yield of free 5' nucleotides was determined via HPLC

with 97%.

Example gel_8:

The procedure was carried out as in example 7, however, the lipids were extracted from 15 kg of dry crude bioprotein according to the method..: according to US patent 4 206 243, example 1, and the remaining amount of 13 kg of defatted bioprotein treated under the conditions of example 7 with 75 mg nuclease (Sigma). The hydrolysis yield of free 5<1->nucleotides was 99%.

Example_el_9:

A hydrocarbon-utilizing strain of the yeast Candida lipolytica (ATCC 20,383) was cultured on n-paraffins in the presence of an aqueous nutrient medium and an oxygen-containing gas (US Patent 4,206,243, Example 8). The yeast cell mass was separated from the nutrient solution and dried, the nucleic acid content referred to dry matter was 7.9%.

150 g of this dry mass was suspended at 55°C in 850 ml of water, the pH value adjusted with phosphoric acid to 5.5, and the mixture incubated while stirring and keeping the pH value and temperature constant for 4 hours with 3 mg of nuclease (Amano RP 8). The temperature was then increased for 10 minutes

to 90°C, pH adjusted with caustic soda to 6.5 and dried. The hydrolysis yield of 5<1>nucleotides was 91%.

Claims (10)

1. Use of nucleic acid-rich raw proteins, in which the high-molecular nucleic acids were cleaved by treatment with nucleases for animal nutrition. 2. Application according to claim 1, characterized in that the crude protein is a microbial protein. 3. Use according to claim 1 or 2, characterized in that the crude protein is a single protein. 4. Use according to one or more of the preceding claims, characterized in that the crude protein is a yeast or bacterial protein. 5. Use according to one or more of the preceding claims, characterized in that the crude protein is an isolate from M. clara ATCC 31226. 6. Application according to claim 5, characterized in that the isolate is defatted by extraction with a solution of ammonia in a lower alkanol, lower glycol or an ether from a lower alkanol and a lower glycol, which contains a maximum of 30% water, referred to the weight of the organic solvent. 7. Use according to one or more of the preceding claims, characterized in that the nuclease is 3'- or 5 <1-> phosphodiesterase. 8. Use according to one or more of the preceding claims, characterized in that the cleavage takes place at 30-60°C, a pH range from 4.5-6.5, an enzyme concentration from 0.1-0.005%, referred to the substrate weight , and a nucleic acid concentration from 0.05-10% by weight. 9. Application according to claim 8, characterized in that the cleavage takes place at 45-55°C, a pH range from 5-6, an enzyme concentration from 0.01-0.001%, referred to the substrate weight and a nucleic acid concentration from 0.3-7% by weight. 10. Use according to one or more of the preceding claims, characterized in that the nucleic acids are cleaved up to the stage of mononucleotides.