RU2288590C1 - Method for producing of feed product from lupine seeds - Google Patents

Abstract

FIELD: biotechnological industry, in particular, production of feeds for farm animals and poultry by processing of farm crops, more particular, preparing of protein-containing feeds.

SUBSTANCE: method involves providing double soaking of lupine seeds in water, followed by at least one-staged fermentative treatment with enzymic preparations having cellulose, hemi-cellulase, xylanase, pectinase, alpha-galactosidase, phytase, protease activities, and with multi-enzymic preparations having indicated activities; after fermentative treatment, providing additional soaking of lupine seeds.

EFFECT: reduced production costs, and provision for producing of feeds with reduced content of anti-nutrient alpha-galactooligosaccharides.

2 cl, 1 tbl, 6 ex

Description

The invention relates to the field of biotechnology industry, in particular to the field of obtaining feed for farm animals and poultry by processing seeds of agricultural crops, and can be used in the preparation of protein-containing feeds, as well as food products.

Leguminous seeds (peas, fodder beans, lupins, etc.) are an important source of vegetable protein. Legumes have a number of healing properties, are characterized by a high content and high biological value of the protein, as well as a low level of inhibitors of digestive enzymes, phytohemagglutinins and other anti-nutritional substances. The main factors limiting the use of leguminous seeds in the feed of farm animals and poultry are the presence of toxic alkaloids, which cause a bitter taste, as well as the presence of α-galactoside oligosaccharides (raffinose, stachyose and verbascose), which are unable to break down by enzymes of the gastrointestinal tract and therefore cause flatulence legume consumption.

A known method of removing sugar, causing flatulence, from legumes (US Patent 4645677, 1987), which consists in suspending the flour of seeds of legumes in water, followed by ultrafiltration of the suspension through a membrane with a cutoff threshold of 30 to 100 kDa, and ultrafiltration is carried out in two stages.

The disadvantage of this method is the impossibility of using it for the treatment of whole seeds of lupine.

There is also a method of eliminating the bitter taste and characteristic of pea aroma from pea flour in order to use it as an additive to food systems (US patent 4022919, 1977). When implementing the method, the flour is contacted with wet steam for a time sufficient to remove the bitterness from the pea flour and transfer the aromatic components of the pea to the gas phase, followed by the removal of steam and volatile aromatic components, moreover, this operation is carried out before a noticeable development of the aroma of cooked pea in the flour .

The main disadvantage of this method should be recognized as unsuitable for the treatment of whole seeds of lupine. In addition, the use of the method leads to high energy consumption, its implementation leads to the destruction of protein (i.e., the nutritional value of the finished product decreases), and α-galactoside oligosaccharides that cause flatulence are present in the finished product.

The closest analogue of the proposed method can be recognized (RU, patent 2207012,2003) as a way to eliminate the bitter taste and aroma inherent in the flour of leguminous seeds, such as peas, fodder beans and lupins, as well as to reduce the content of leguminous oligosaccharides in the flour. According to a known method, the flour of the seeds of legumes is soaked in water for 10-50 minutes at 20-50 ° C and the ratio of flour: water is 1: 2-1: 5, the suspension is acidified with diluted food acid to a pH of 3.2-5.0, subjected to the action of a protease active in an acidic environment, for example pepsin, pectofoetidine, at 20-50 ° C for 40-90 minutes, increase the pH of the suspension to 6-8 and maintain at this pH for 3-8 hours at room temperature, then subjected to spray drying or directly introduced into the formulation of the food product subjected to heat treatment boot.

The disadvantage of this method should be recognized as unsuitable for the treatment of lupine seeds.

The technical problem solved by implementing the proposed method is to enable the use of lupine seeds as one of the components of the feed of farm animals and birds.

The technical result obtained by the implementation of the proposed method is to reduce the cost of feed of farm animals and poultry by replacing soybeans with de-alkaloidized lupine seeds with a reduced content of anti-nutritional α-galactooligosaccharides.

To achieve the technical result, it is proposed to use a method for producing a feed product from lupine seeds, characterized by double soaking lupine seeds in water followed by at least one-stage enzymatic treatment, and enzymatic preparations using enzyme preparations with cellulase, hemicellulase, xylanase, pectinase, α -galactosidase, phytase, protease activity, as well as multienzyme preparations possessing the above activities, moreover, le enzymatic treatment is carried out additional soaking lupine seeds. In a preferred embodiment, the membrane is removed from the lupine seeds before enzymatic treatment.

Usually, the first soaking process is carried out at a temperature of 40-80 ° C for 3-5 hours. The second soaking is preferably carried out at a temperature of 40-80 ° C for 2-5 hours. The need for each subsequent soaking sets the content of alkaloids to be removed in the water after the previous soaking. In addition to water, a slightly acidic buffer with a pH of 3-7 (for example, 0.1 M Na-acetate buffer with a pH of 5) can be used for soaking to maintain optimal enzymatic activity (depends on the type of enzyme used for processing), as well as to improve the solubility of alkaloids in the aqueous phase. A dilute solution of a weak acid, such as acetic acid, can also be used to acidify the medium. When soaked, the lupine seeds can be mixed in any known manner. The volume of liquid medium is approximately 3-5 times the volume of the treated seeds. The enzymatic treatment process can be repeated two times. During the enzymatic treatment, lupine seeds can also be mixed. The need for repeated enzymatic treatment (or soaking without an enzyme) is determined by the content of alkaloids removed in the liquid medium of the previous treatment. In some cases, lupine seeds are soaked simultaneously with enzymatic treatment. Usually this is the second (and subsequent) soaking. In a preferred embodiment, the membrane is removed from the lupine seeds before enzymatic treatment.

To remove the maximum amount of alkaloids and α-galactooligosaccharides with minimal loss of lupine protein, individual enzyme preparations or mixtures of enzyme preparations with cellulase, hemicellulase, xylanase, pectinase, α-galactosidase, phytase, protease activity are used. The best results were obtained using enzyme preparations with high cellulase, xylanase, pectinase, α-galactosidase activity. It was found that regardless of the type of activity of the used enzymatic preparations, it is necessary to pre-soak the lupine seeds with subsequent enzymatic treatment.

When implementing the proposed method, both during soaking and during enzymatic treatment, alkaloids, protein, α-galactooligosaccharides are transferred from the lupine seeds to a liquid medium and reducing sugars are formed. It was found that with one-step soaking and a one-time enzymatic treatment, the amount of alkaloids on average decreases by 40-60% of the initial amount, protein - by 2-4% of the initial amount. With two-stage soaking and one-time enzymatic treatment, the amount of alkaloids on average decreases by 75% of the initial amount, protein - by 5-6% of the initial amount.

With two-stage soaking and double enzymatic treatment, the amount of alkaloids decreases on average by 90% of the initial amount, protein - by 8-9% of the initial amount, and the content of anti-nutritional α-galactooligosaccharides (the sum of raffinose, stachyose, verbascose) decreases by 90-99% . The decrease in the content of galactooligosaccharides depends on the presence of α-galactosidase activity in the enzyme preparation.

On average, at each additional processing stage after the first, the amount of alkaloids in the grain decreases by 40-60%, the amount of protein by 0.7-2.0% of the initial amount. Consequently, the number of soaking stages, ceteris paribus, is determined by the initial content of alkaloids in the feedstock. The concentration of protein (determined by the Lowry method) in the drained liquid after each processing step after the first (with or without enzyme) is approximately 0.7-1.5 g / l, reducing sugars (determined by the Shomody-Nelson method) 0.8 -4 g / l

The process was carried out in a thermostatically controlled shaker. The processed lupine seeds were placed in an airtight plastic canister mounted on a shaker platform. The rotation speed of the platform ranged from 120 to 180 rpm. The processing time at each stage ranged from 2.5 to 4 hours. The enzymatic preparation was added in an amount of from 0.1 to 0.5% by weight of the treated lupine seeds. At each stage, the lupine seeds in the canister were poured with fresh water and the contents of alkaloids, protein, and reducing sugars in the drain from the canister were analyzed after the stage. To determine the content of these components used standard analytical methods.

Below are examples of the implementation of the proposed method.

1. For the treatment of Decaf-14 lupine seeds with an alkaloids content of 0.056%, an embodiment of the method with two-stage soaking before a single-stage enzymatic treatment was used. The experiment was carried out with stirring. For 1 kg of lupine at the stages of soaking and enzymatic treatment used 4 l of water. Soaking was carried out in water at a temperature of 50 ° C for 3 hours. After two-stage soaking, the content of alkaloids in the seeds, calculated on the dry weight, was 74% of the initial one, the protein loss with the solution was 0.9% of the initial one. After draining the water used for soaking, the swollen lupine seeds were poured with a solution of an enzyme preparation with xylanase, α-galactosidase and phytase activity, for which a laboratory enzyme preparation produced by the microscopic fungus Penicillium sp. with xylanase activity (at pH 5.0, 50 ° С, the conditions for measuring activity are further the same, the activity is expressed in international units) - 1500 units / g, α-galactosidase activity 125 units / g, phytase activity 12680 units / g, content protein 406 mg / g. The drug content at both stages of the enzymatic treatment was 0.5% of the initial dry seed weight. Enzymatic treatment was carried out at a temperature of 50 ° C for 3 hours in the presence of 20 mm acetate buffer with a pH of 4.5. After the enzymatic treatment, the grain was subjected to another additional stage of soaking in a volume of 4 l of water with stirring at a temperature of 50 ° C for 3 hours. After this complex treatment, the content of alkaloids in the seeds was 0.03% of the initial one, the protein loss with the solution (in plums) was 3% of the initial one, and the content of reducing sugars in the solution was 1.6 g / l (in glucose equivalent). Since the permissible alkaloid content in the seeds of lupine is 0.01%, this treatment was sufficient.

2. The experiment was carried out as in example 1, but used the same laboratory enzyme preparation produced by the microscopic fungus Penicillium sp. with xylanase activity - 1550 units / g, α-galactosidase activity 114 units / g, phytase activity 12430 units / g, protein content 433 mg / g. After treatment, the alkaloids content in the lupine grains was 0.026%, protein loss with the solution (in plums) amounted to 5.5% of the original. The treatment used was sufficient to obtain the specified technical result.

3. The experiment was carried out as in example 2, but after the enzymatic treatment of the grain was subjected to another similar stage of the enzymatic treatment under similar conditions. After treatment, the content of alkaloids in the lupine grains amounted to 9% of the original, protein loss with solution (total in plums) amounted to 10% of the original. The content of reducing sugars in the solution is 0.9 g / l (in glucose equivalent). The permissible alkaloids content in the seeds of lupine is 0.01%, therefore, this treatment was sufficient, since the achieved alkaloids content in the lupine grains was 0.005%. 91% of grain alkaloids were removed with draining waters. In addition, the content of α-galactooligosaccharides was significantly reduced. Oligosaccharides were analyzed chromatographically on a column with the inoculated aminophase Diasorb-130-amine (Biokhimmak, Russia), 4 * 250 mm, 6 μm, eluent acetonitrile: water 70: 30.1 ml / min, with refractometric detection.

The content of anti-nutritional oligosaccharides (the sum of raffinosis, stachyosis, verbascosis) in the grain after processing was 0.11%, and in the initial 3.2% by weight. The content of anti-nutritional oligosaccharides was reduced by 97%. Thus, in this parameter, the lupine grains after processing significantly exceed feed soy flour, the total content of anti-nutritional oligosaccharides, in which an average of 3%.

4. The experiment was carried out as in example 3 with different enzyme preparations in the treatment of lupine seeds of the narrow-leaved variety "Crystal" with the content of alkaloids 0,040%. The experiment was carried out with stirring at a ratio of lupine: water = 1:10 at a temperature of 50 ° C for 8 hours. To maintain the pH in the reaction mixture, 100 mM sodium acetate buffer with a pH of 5.0 was used. The dosage of enzyme preparations was 0.5% of the weight of lupine (drugs were dosed according to the protein content per “average” preparation with a protein content of 50 mg / g). Used the following enzyme preparations:

1) phytase preparation, producer of Penicillium sp. (protein 625 mg / g, phytase activity - 23125 units / g, α-galactosidase - 328 units / g);

2) α-galactosidase, phytase, pectin-lyase preparation, producer Penicillium sp. (protein 9.4 mg / ml, phytase 116 units / ml, α-galactosidase - 276 units / ml, pectin lyase - 61 units / ml);

3) pectin-lyase enzyme preparation, producer of Penicillium sp. (protein 554 mg / g, α-galactosidase - 90 units / g, pectin-lyase - 5170 units / g);

4) multienzyme pectinase-hemicellulase enzyme preparation, producer of Aspergillus foetidus (protein 254 mg / g, α-galactosidase - 80 units / g, pectin lyase - 4.0 units / g, polygalacturonase - 20400 units / g, xylanase - 515 units / g);

5) cellulase-hemicellulase enzyme preparation, producer of Trichoderma reesei (protein 280 mg / ml, CMCase - 4700 units / ml, xylanase - 1750 units / ml);

6) α-galactosidase enzyme preparation, producer of Aspergillus sp. (Protein 420 mg / ml, α-galactosidase - 13,700 units / g, pectin lyase - 74 units / g);

7) multienzyme preparation, producer Aspergillus sp. (Protein 56 mg / g, α-galactosidase - 43 units / g, pectin lyase - 16 units / g, polygalacturonase - 4985 units / g, CMCase - 200 units / g, xylanase - 80 u / g);

8) multienzyme preparation, producer Penicillium sp. (protein 406 mg / g, phytase - 12,680 units / g, α-galactosidase 125 units / g, pectin lyase - 22 units / g, CMCase - 350 units / g, xylanase - 1,500 units / g).

After treatment, the content of alkaloids in the seeds in terms of dry weight decreased (table 1). The loss of protein with the solution and the yield of reducing sugars in the solution are given in table 1.

Table 1. No. of drug The content of alkaloids,% Loss of protein,% The output of reducing sugars in solution, g / l one 0.005 3.6 2,5 2 0.008 3.8 2,4 3 0.007 3.3 1,5 four 0.009 3.6 2.6 5 0.009 4.1 2.1 6 0.010 4.0 4,5 7 0.010 3.9 2.7 8 0.010 3,7 3,7 No enzyme (control) 0,021 3.8 1,5 Source 0,040 - -

After two-stage soaking and two-stage enzymatic treatment, the content of alkaloids in the seeds amounted to 23-53% of the initial one, the loss of protein with solution was 3.3-4.1%, depending on the type of enzyme preparation. Since the permissible alkaloid content in the seeds of lupine is 0.01%, this treatment turned out to be sufficient, since the final content of alkaloids in the grains was 0.009-0.01% (see table 1). At the same time, treatment without an enzyme (control) was not enough, since the final content of alkaloids in the grains was 0.021%.

5. The experiment was carried out as in example 4 with the enzyme preparation No. 8 but at two different temperatures of 40 and 50 ° C. After enzymatic treatment, the content of alkaloids in the seeds was 0.017 and 0.010%, the loss of protein with solution was 1.3 and 3.3%, respectively. The content of alkaloids was reduced by 57 and 75%, respectively. Since the permissible alkaloid content in the seeds of lupine is 0.01%, treatment with the variant with 50 ° C was enough, and with the variant with 40 ° C it is not enough.

6. The experiment was carried out as in example 4 with the enzyme preparation No. 8, but using two different varieties of lupine in the low-alkaloid "Crystal" - with the initial content of alkaloids 0.04% and highly alkaloid "Siderat-38" with the initial content of alkaloids 0.63% After enzymatic treatment in seeds, the alkaloid content was reduced by 76 and 77% of the initial one, protein and solution losses were 3.3 and 5.5%, respectively. Since the permissible alkaloid content in lupine seeds is 0.01%, processing of a low-alkaloid grade Crystal has provided it’s enough, because the final alkaloids content was 0.01%, and in the case of the high alkaloid grade Siderat-38, the final alkaloids content was 0.15% and further processing steps are required. It is important to note that this embodiment of the method led to the same decrease alkaloids content by 76-77% regardless of the initial alkaloids content; therefore, this method is applicable to grain with different initial alkaloids content.

The developed method allows replacing soybean processing products with lupine seed processing products in feeds for farm animals and poultry with a reduction in feed costs by 18-22%, and the product obtained in principle can also be used for food purposes.

Claims (2)

1. A method of obtaining a feed product from lupine seeds, characterized by double soaking lupine seeds in water followed by at least one-stage enzymatic treatment, and enzymatic preparations using cellulase, hemicellulase, xylanase, pectinase, α-galactosidase, are used in the enzymatic treatment protease activity, as well as multienzyme preparations possessing the above activities, and after enzymatic treatment, additional soaking of seeds is carried out pin. 2. The method according to claim 1, characterized in that the membrane is removed from the lupine seeds before enzymatic treatment.