Food product containing proline-specific protease, its production method and its application for splitting toxic and allergenic gluten peptides
FIELD: food industry.
SUBSTANCE: pasteurised food product containing a proline-specific protease has water activity equal to at least 0.85. Used as the enzyme is protease extracted from Aspergillus or belonging to the S28 serine proteases family. The optimal activity of the said protease is at a pH value from 1 to 7, preferably - at a pH value from 2 to 6. Additionally proposed is a food product containing less than 1 wt % of protein or peptides. The said food products are produced by way of addition of a proline-specific protease to them.
EFFECT: such products consumption ensures gluten peptides splitting and is recommended to patients suffering from gluten intolerance.
17 cl, 5 dwg, 2 tbl, 6 ex
The technical field to which the invention relates.
The invention relates to a food product containing specific Proline protease, the method of its production and to its use for the breakdown of toxic or allergenic peptides of gluten).
It is known that the consumption of gluten, resulting in a total food protein present in wheat, barley, rye, wheat and the spelt and triticale, causes disease in some individuals. Gluten is a complex mixture rich in glutamine and Proline gliadins and glutenins presumed to be responsible for the development of some diseases. Because the amino acid composition of gluten specific parts are quite resistant to proteolytic cleavage in the gastrointestinal tract of man. The result can accumulate specific Proline-rich peptides, which can lead to undesirable symptoms such as intolerance of various peptides derived from gluten. For example, the literature describes the amino acid sequences of the peptides responsible for the toxicity of gluten observed in patients suffering from coeliac disease coeliac disease (Arentz-Hansen et al., J. Exp.Med. 2000; 6: 337-342; Vader et al., Gastroenterology 2002; 122: 1729-1737).
Celiac disease is widespread autoimmu the Noah disease of the small intestine. In patients with celiac disease there is a significant prevalence of various autoimmune disorders, mainly of type I diabetes, dermatitis herpetiformis, autoimmune thyroiditis, collagenoses, autoimmune alopecia (baldness), and autoimmune hepatitis. Celiac disease is often accompanied by mental and neurological symptoms, which can lead to far-reaching consequences metabolism of peptides rich in Proline.
In such cases, only gluten-free diet throughout life may effectively prevent clinical symptoms in patients with celiac disease. But, unfortunately for these patients, gluten is a cheap protein with concern the potential of its use, so it is widely used in various food products, such as implemented in the commercial network soups, soy sauces, sauces, ice cream, potato chips and hot dogs. That is why patients with gluten intolerance need more detailed list of food products that would allow them to avoid consumption of molecules problematic gluten because gluten intake even in such small quantity as 50 mg per day, can cause a recurrence of clinical symptoms.
Today it is clear that the problematic Proline-rich peptides present in glute is e, are highly resistant to splitting gastric peptidases and peptidase pancreas, such as pepsin, trypsin, chymotrypsin, and others Only specific enzymes that can hydrolyze peptide bonds involving Proline, capable of extensive hydrolysis of Proline-rich sequences with the destruction of epitopes relevant to celiac disease. It was reported on various enzymes, which finds useful application for inaktivirovanie toxic Proline-rich peptides, such as prolyl-oligopeptides (EC 22.214.171.124; Shan et al. Science 297, p.2275-2279), dipeptidyl-peptidase IV (EC 126.96.36.199; US-A-2002/0041871). Published also a number of patent applications mentioning the possible role of specific Proline protease in reducing the antigenicity of foods containing gluten (for example, WO-A-2002/45523), as well as the use of such enzymes to prevent clinical symptoms of celiac disease and related diseases (for example, WO 03068170 and WO 2005/027953). Was recently demonstrated the suitability of permettere for the treatment of patients with celiac disease using duodenal extracts (Cornell et al., Scandinavian Journal of Gastroenterology, 2005; 40: 1304-1312).
WO-A-2002/45523 reveals specific application specific Proline of endoprotease for proteolysis of polypeptides, including peptides rich in Proline. The application also describes the introduction of endo is noteasy in protein foods to suppress the bitterness in them or reduce their allergenicity. In WO-A-2005/027953 emphasizes that this particular endoprotease ideal as a dietary Supplement that supports the digestion of dietary gluten, because it shows a broad pH optimum, which allows the enzyme to be active in the oral cavity, esophagus, stomach and remain active in the duodenum.
WO 2005/027953 aimed at removing toxic Proline-rich peptides from food before consumption, resulting prevented or minimized the impact on the body of toxic Proline-rich peptides. The application discloses the use of a stabilized enzyme compositions as a means of promoting digestion. In the described approach, the enzyme is consumed along with the food product is so rich in Proline and/or rich in glutamine protein sequence of the food product was cut with the passage through the gastro-intestinal tract. However, according to WO 2005/027953 enzyme composition may be injected only rich in Proline and/or rich in glutamine foods with a water activity below 0,85 so that the enzyme remained active enough. In that case, if a food product must be stored for extended periods of time, you should avoid contact with moisture or moist air, i.e. is such cases, it is proposed to use dry food. This approach greatly limits the choice of foods that can be combined with endoproteases.
The use of enzyme compositions in moisture-containing food products such as margarine or spreads like him, it is well known, mainly from the prior art. However, in most cases, enzymes are added as processing AIDS, facilitates the processing, and prolonged enzymatic activity, i.e. the activity, which remains after packaging of the product in these cases is not required. For example, DE-A-101 04 945 discloses low fat spreads containing phospholipids and enzymes, for example, transglutaminase, without the use of emulsifiers or stabilizers. After a short incubation period on the production stage of the product transglutaminase is inactivated by heat treatment at 95°C for 2-3 minutes.
WO-A-95/28092 relates to the use of stabilizers, such as polyols, for stabilizing emulsions, water-in-oil suitable for food products in which the emulsions contain thermolabile compound, such as an enzyme. In contrast to the two aforementioned applications WO-A-95/28092 aimed at long-term stabilization of enzyme activity. With this purpose, the aqueous phase is introduced from 40% to 50% glycerol, as described in the example. However, the introduction of such high amounts for which ilow in food or not Pets, either is unacceptable from an organoleptic point of view.
Description of the invention
The inventors have found that specific Proline protease can be used as an ingredient in food products with high water activity, which do not have high amounts of stabilizers enzymes. Such products can even be subjected to pasteurization in order to ensure adequate stability in storage without significant loss characteristic of enzyme activity. In this application demonstrated that in food products, such as fillings for sandwiches, top toppings, condiments, sauces, drinks, and emulsions, such as low fat spreads specific to Proline protease remain active enough to achieve adequate hydrolysis sequences rich in Proline gluten in the stomach. In most cases, the taste of the food product does not suffer or not changed by the presence of the enzyme.
The fact that the enzyme is resistant to treatment pasteurization, in itself surprising, since in the prior art it has been suggested that under conditions of high water activity of most enzymes are not capable of withstanding the modes of pasteurization. Similarly, it was assumed that a large part of the enzyme loses its activity in products with high and the efficiency of water after one week of storage. So amazing is that according to the invention the enzyme maintains its activity for periods of time up to one year, if the food product with high activity in the water stored in the cooling modes. The phrase "when cooling" refers to the temperature below 10°C., preferably from 0 to 10°C., more preferably from 2°C to 8°C.
Thus, the invention relates to pasteurized and stable in storage of food products having a water activity of at least 0,80, preferably at least 0,85, and contains specific Proline proteolytic activity, which is high enough to ensure detoxification Proline-rich protein sequences. Toxic amounts of Proline-rich protein sequences are present in the gluten quantity of order higher than 1 mg
According to another aspect of the invention disclosed are stable in storage of a food product having a water activity of at least 0,80, preferably at least 0,85, and contains specific Proline proteolytic activity capable of detoxifying a Proline-rich protein sequences, in which the food product contains less than 1% wt./wt., protein or peptides, and preferably a food product contains no gluten.
The present invention relates to sterile specific to the Proline protease. Under "sterile" means that does not contain micro-organisms, preferably not containing bacterial spores. Specific Proline protease is preferably subjected to filtration for removal of microorganisms, preferably and bacterial spores.
Proteins of grain can be divided into albumins, globulins, prolamins and glutelin. Gluten is an insoluble protein fraction of grain, such as wheat, rye, spelt, oats, barley, corn and rice, which remains after washing to remove starch and water-soluble components. It can be divided into gliadins and glutenin. Glutenin can be divided into high - and low-molecular-weight subunit. A detailed discussion of protein gluten, see Wheat Gluten (P.R.Shewry and A.S.Tatham eds., Cambridge: Royal Society of Chemistry, 2000) or review article Wieser (1996) Acta Paediatr. Suppl. 412: 3-9.
In accordance with internationally accepted classification schemes and items of all enzymes from IUMB oligopeptides, dipeptidylpeptidase and endoprotease is enzymes, gidrolizuemye internal peptide bonds, which in turn are subdivided into additional subclasses on the basis of their catalytic mechanism. Preferred specific Proline protease, p is egodnoe for the purpose of the present invention, is acid-resistant and stable to pepsin endoprotease from A.niger disclosed in WO-A-02/45524 and WO-A-2005/027953, which is able to cleave peptides and native proteins with carboxyl side prolinnova residues and which is also able to cleave peptides and native proteins under conditions of very low pH and in the presence of pepsin. This endoprotease persists in the presence of the enzyme pepsin in acidic conditions and, in all likelihood, continue to remain active in the duodenum. The most preferred endoprotease is specific to the Proline endoprotease isolated from fungus food quality Aspergillus or specific to the Proline endoprotease related to the S28 family of serine proteases.
Water activity is the relative availability of water in the substance. It is defined in the prior art as the water vapor pressure divided by the pressure of pure water at the same temperature. Thus, pure distilled water has a water activity equal to exactly one. Water activity is different from the moisture content (% water) in the food product. Moisture content is the total amount of moisture, i.e., the amount of bound plus the amount of free water present in the sample, while the water activity is determined by measuring only free moisture and is usually expressed as awor the percentage of the th content of the equilibrium relative humidity (Equilibrium Relative Humidity) (% ERH). Water activity of the food product is a constant relative humidity in the immediate vicinity of the food product when the food product and the surrounding air is installed balance. This constant relative humidity is indicated as "% ERH tea", if it is expressed in percent (from 0%to 100%), or as "water activity"if it is expressed as a number from 0 to 1.0. Methods of determining water activity detailed in the official methods of analysis of AOAC International (1995), method 978.18.
Under "heat treatment" in the present description refers to heat treatment at a temperature of at least 65°C, preferably at a temperature of at least 70°C for at least 2 seconds, preferably for at least 20 seconds. An example of such a heat treatment may serve as pasteurization, used for milk, i.e. a heat treatment at 72°C for 15 seconds. Pasteurization is a concept known to the skilled technician. The resulting pasteurized food product is microbiologically safe product with a long shelf life.
Under "food product" means a product or food ingredient that is intended for consumption without prior heat treatment such as baking, frying or boiling. Under the food product, having a long shelf life, you should understand the product that has a shelf life of at least one week to one year or more, during which guaranteed the preservation of the organoleptic properties, as well as the microbiological safety of the product. Obviously, the allowable storage period largely depends on the actual modes of storage of the food product. Many perishable foods must be kept chilled to maximize their shelf life.
If the food product of the invention are stabilizers, in particular polyols, such as glycerol, sorbitol, sucrose, polypropylenglycol, trehalose, maltodextrins, lactose and glucose, their number in most cases is less than 10 wt.%, preferably less than 5 wt.% from the food product.
Preferably the food product according to the invention contains less than 1 wt.%/wt., casein, more preferably the food product according to the invention does not contain casein.
Receiving specific Proline protease in the form of pills or tablets would allow patients suffering from gluten intolerance, it's safe to eat food containing gluten products. However, the inventors have now found that the protease can also be convenient to enter in the food products, which themselves can UPO the school not contain gluten or contain it in small quantities, but traditionally combined with containing gluten food. More preferably the food products according to the invention containing endoprotease, are food ingredients that are in the prior art are considered to be "gluten-free". In accordance with the Codex Standard for Gluten-Free Foods (Codex standards for gluten-free foods) (Codex Stan 118-198) Codex Alimentarius Commission (Standards of FAO/who food) nitrogen content in food ingredients derived from cereals containing gluten may not exceed 0.05 g (50 mg) per 100 g of the product in terms of dry substance in the case, if these ingredients are used in gluten-free food product.
According to the present invention is disclosed a food product that contains specific Proline proteolytic activity in the amount of more than 0.5 PPU is based on a portion, i.e. enzymatic activity present in one portion, is able to hydrolyze 25 mg of gluten. One portion is the amount of food consumed at one time within usually one hour, preferably within 40 minutes.
Foods preferred as a carrier of specific Proline protease, are food products that you want to keep chilled. Particularly preferably, the hen containing protease food is seasoning, i.e. food product, which is used to enhance the taste and flavor of other foods, mainly containing gluten food. Spices have the advantage that they are in abundance in households, restaurants, canteens and supermarkets and in typical cases have a long shelf life. Preferred examples of seasonings are tomato sauce or tomato ketchup. Such products typically have a pH below 4.2, more preferably below 4.0, which means that they require only limited processing pasteurization. Examples of other products increased acidity, requires limited processing pasteurization and are particularly suitable as carriers for active specific Proline protease, are fruit juices and fruit concentrates. In fact, even acidic or carbonated bottled water can serve as an excellent carrier of the specified enzyme. "Pop"like vegetable or fruit concentrates, also fall under this category. Similarly, products of high acidity, containing the preservative of food quality, such as benzoate or sorbate, are excellent substrates for the enzyme. For example, icing or filling for sandwiches, usually used in combination with containing gluten food is akimi as bread, having indicators of water activity higher than 0,85. Excellent media can also serve products with very high acidity, which generally do not require pasteurization, such as colas, because specific Proline protease shows significant stability under such conditions. In addition, the enzyme is fully compatible with products containing high concentrations of viable probiotics. Typically, these probiotic foods have a water activity above 0.95 and stabilize the pH below 4.0.
Additionally, the invention relates to a method of manufacturing a food product containing the enzyme composition of the invention, in which specific Proline protease is added to the food product after the food product has been subjected to processing pasteurization. In this approach, the enzyme may be added to the already sterile pasteurized product. An example of a suitable technology for this approach is the technology of aseptic dispensing, such as technology, sold by Tetra Cancer (Terra Aldose TMS; see, for example, http://www.tetrapak-processing.de/produkte/pdf/aldose.pdf).
Another alternative embodiment of the invention is an emulsion of water-in-oil or oil-in-water, spread, preferably margarine or low fat spread. Widely used low-fat spreads, intended for use in the natural the gluten free food product, especially suitable as a carrier for enzyme funds, promoting digestion. The high water content in these products allows you to enter large amounts of enzyme, and the products are usually stored during cooling, traditionally at temperatures of about 7°C or below. Because the enzyme is confined to the aqueous phase, the emulsion also fall under the category of products having a water activity of at least 0,85.
Other important advantages of this variant embodiment of the invention is that the bread or spread thoroughly mixed in the mouth, than is initiated by cleavage of the molecules of gluten specific Proline protease. In addition, it is known that the presence of compounds of the fatty series inhibits gastric emptying after oral-touch mechanism. Both mechanisms lead to more intense and prolonged interaction between the enzyme and gluten, so the enzyme and gluten can best communicate with each other before the chyme (food chyme reaches the duodenum. This is very important because it is known that the duodenum is the initial part of the gastrointestinal tract, which can cause pathogenic reactions of molecules Proline-rich gluten. It was found that specific Proline is retease, in particular acid-specific Proline endoprotease from A.niger, according to WO-A-2002/45523 has a broad pH optimum, which allows it to be active in the oral cavity, esophagus, stomach and duodenum. The fact that specific to the Proline endoprotease has a high residual activity when introducing it into the emulsion, is completely unexpected. The available literature is rather unanimous in their conclusions about what contact with the emulsifiers and the subsequent introduction of the emulsion have a significant stress on enzymes and easily lead to their iactiveaware (see, for example, Gatorae et al. in "Stability and Stabilization of Enzymes, Elsevier Sci. Publish. 1993, p.329, or de Roos and Waistra, "Colloids and Interfaces B"; Biointerfaces 6 (1996) 201-208). It is therefore particularly suitable for the present invention is an enzyme:
with the Proline-specific endoprotease activity, and
- having an amino acid sequence identical to SEQ ID No. 2 (see WO 2002/45523), or having an amino acid sequence that has at least 80%, preferably at least 90%, identity with the amino acid sequence with amino acids 1 to 526 of SEQ ID No. 2 (see WO 2002/45523). The level of identity between amino acid sequences is determined by the method mentioned in WO 2002/45523 on page 15.
The enzyme composition according to the SNO invention must be entered into the food in quantities, which corresponds to the total number of digestible protein. For example, low fat spread is usually applied on a slice of bread. On a slice of bread 18 grams is usually applied five grams of spread. Bread typically contains 8% protein, 7% are gluten, i.e. one slice of bread contains 1.5 grams of protein. To achieve adequate hydrolysis during digestion in the gastrointestinal tract of all proteins, i.e. including gluten fraction, when using the enzyme as described in WO 2002/45523 takes about 20 PPU enzyme per 1 gram of protein present (for definition see Materials and methods). Thus, for the digestion of 1.5 grams of protein required 1.5 times 20 PPU, which corresponds to 30 PPU. These 30 PPU should be provided with five grams of low-fat spread, which implies that the activity of the enzyme 6000 PPU/kg low-fat spread. Introduction the enzyme composition according to the invention is unlikely to affect the production and properties of low-fat spread. However, to avoid bitterness, which is often reported in dairy products after processing enzymes with proteolytic activity, the spread according to the invention preferably avoids the use of hydrophobic proteins, such as casein. So, the taste of spread can be improved through the introduction is of him in fermented whey protein with the to achieve the typical taste and aroma of butter. The inventors found that the residual proteins present in fermented whey, hydrolyzed specific to the enzyme Proline, but this does not have negative effects on organoleptic characteristics. Specific Proline protease is preferably added to the aqueous phase before formation of the emulsion, most preferably specific for Proline protease is added sterile after pasteurization of the aqueous phase, but before formation of the emulsion. There are a number of ways efficient production of low-fat spreads. According to one oil phase containing the emulsifiers, flavoring additives, vitamins and dyes, is supported in a moderately heated and gentle mixed mode in order to avoid negative impacts on the quality of the oil. Then the aqueous phase and oil phase are mixed together and served in votator (heat exchanger-mixer). A more detailed description of the preparation of emulsions can be found in the literature, including Moustafa in "Practical Handbook of Soybean Processing and Utilization"; David R. Erickson editor; co-published by AOCS Press and United Soybean Board. Currently spreads with the introduction of the healthy compounds such as phytosterols/stanely or arachidonic acid, are becoming increasing popular is using. Because these compounds are oil soluble, they do not affect the stability of the enzyme in the aqueous phase.
Thus, the invention relates to a method of manufacturing a food product containing the enzyme composition according to the invention, in which specific Proline protease is added to a food product either before or after exposure of the food product pasteurization.
In principle, it is possible to use also other enzymes that have the ability to hydrolysis of peptide bonds involving prolinnova residues, such as prolyl-oligopeptides (EC 188.8.131.52) or dipeptidyl-peptidase IV (DPP IV, EC 184.108.40.206), or dipeptidyl-peptidase II (DPP II, EC 220.127.116.11). These proteases are mentioned in the list, for example, Augustyns et al. (Current Medicinal Chemistry, 2005, 12, 971-998). However, for specific Proline protease was suitable as facilitating the digestion of funds in the food product, i.e. either in the food product containing gluten or food product intended for use in combination with containing gluten food products specific to Proline protease must: i) show the pH-optimum, which provides adequate activity in the acidic region of pH prevailing in the stomach, preferably at a pH below 5; (ii) to survive in the presence of gastro-intestinal proteolytic enzyme pepsin is ri these acidic conditions; iii) to remain active in the moisture-containing environment for at least the shelf life of the food product. In addition, Pets introduction of the coenzyme to achieve synergies in the hydrolysis of proteins of gluten. Obviously, the coenzyme must meet the same requirements stability that is specific to the enzyme Proline, in the food product. The invention relates also to the use of specific Proline protease as a digestive remedies in the production of pasteurized food product having a water activity of at least 0,85, as mentioned above, in order to prevent clinical symptoms of celiac disease or related disorders. Preferably the food product is intended for use in combination with food containing gluten product.
The cause of celiac disease is an intolerance to some peptides rich in Proline and glutamine. Incomplete cleavage of these peptides led to the development and severity of celiac disease. Celiac disease is often accompanied by mental and neurological symptoms. Already in 1979, Panksepp (Trends in Neuroscience 1979; 2: 174-177) formulated theory of excess opioids, in which he suggested that the disturbance of the metabolism of opioids is part of the pathogenesis in autism. Thus, a food product containing andapr is tease according to the invention, can also be used by patients with mental disorders, including autism, schizophrenia, ADHD, bipolar mood disorders and depression, all of which are associated with the consumption of food proteins rich in Proline. Other disorders associated with celiac disease include autoimmune disorders, mainly type I diabetes, dermatitis herpetiformis, colon cancer, intestinal non-Hodgkin lymphoma, autoimmune thyroiditis, collagen, autoimmune alopecia and autoimmune hepatitis. In addition, the irritable bowel syndrome (IBS) is associated with indigestible Proline rich protein sequences. The benefits of the present invention can be obtained patients suffering from any of the above disorders. These patients can be of any age and to enable adults and children. Children, in particular, may benefit from preventive measures, when the warning at an earlier stage of exposure to toxic gluten peptides can prevent the initial development of the disease. Introduction composition specific to the Proline of endoprotease has particular advantages for the patients, among which are very popular condiments, in particular, mayonnaise and ketchup. Children in need of prevention, can be identified by genetic test Predrag progenote to disease, for example, by classification of HLA-system (antigenic system cells), the study of family history, a breakdown in T-cells or other medical means.
Description of figures
Figure 1 - levels of stimulating T-cell epitopes derived from the "stomach", without specific Proline of endoprotease. Experimental conditions are described in Example 3. "Alpha" refers to the level of reactive molecules alpha-gliadin; "gamma" - to the level of reactive molecules gamma-gliadin; "HMW" - to the level of reactive HMW (high molecular weight)-glutenins and "LMW" - to the level of reactive LMB(low molecular weight)-glutenins.
Figure 2 - levels of stimulating T-cell epitopes derived from the "stomach"containing specific Proline endoprotease. Experimental conditions are described in Example 3. Cm. notes to figure 1, explaining the value of "alpha", "gamma", "HMW and LMW".
Figure 3 - levels of stimulating T-cell epitopes in the sediments extracted from the "stomach", with addition and without adding specific Proline of endoprotease and subjected to Western-blotting with treatment with antibodies specific for alpha-gliadin. Experimental conditions are described in Example 3.
Figure 4 - % of residual enzyme activity in the aqueous phase after melting low-fat spread at 53°C, adding specific to the Strait is well endoprotease in the aqueous phase and shaking a mixture of water/fat in 10, 70 and 100 minutes at 53°C. the Conditions of the experiment such as described in Example 4.
5 is a stability in storage of specific Proline of endoprotease in the aqueous phase of low-fat spread at different temperatures of storage. Experimental conditions are described in Example 5.
Materials and methods
Tests on enzyme activity
Activity polyspecific endoprotease isolated from A.niger was determined test using CBZ-Gly-Pro-pNA (Bachem, Bubendorf, Switzerland) as a substrate at 37°C in citrate/disodium phosphate buffer with a pH of 4.6. The reaction products were monitored spectrophotometrically at 405 nm. The increase in absorbance at 405 nm over time is a measure of enzyme activity. Unit Proline protease (PPU) is defined as the amount of enzyme that releases 1 mmol p-nitroanilide per minute under specified conditions and at a substrate concentration of 0.37 mm Z-Gly-Pro-pNA.
Quantitative detection of stimulating T-cell epitopes
The concentration of stimulating T-cell epitopes as gliadin and glutenin in soluble fractions dynamic gastro-intestinal in vitro model was determined using samples on the competition with the use of monoclonal antibodies (mAB). To this end, the sample was diluted with buffer containing 50 mm Na2HPO4/NaH2PO4(pH 7.0), 50 mm NaCl, 0,1% Tween-20 and a mixture of protease inhibitors (Complete, Roche Diagnostics GmbH, Penzberg, Germany). Samples were run twice, as described previously (Spaenij-Dekking et al., (2004) Gut 53, 1267-1273).
Analysis of proteins by 1D SDS-PAGE and Western blotting
To determine the level of stimulating T-cell epitopes present in the solid (precipitated) fractions of different samples dynamic gastro-intestinal in vitro model was performed experiments on the conduction of 1D SDS-PAGE (one-dimensional polyacrylamide gel electrophoresis using sodium dodecyl sulfate). The solid fraction was solubilities by dissolving samples of protein in a 6-fold volume of buffer (60% glycerol, 300 mm Tris (pH 6,8), 12 mm EDTU (pH 8.0), 12% SDS, 864 mm 2-mercaptoethanol, 0.05% of bromophenol blue) and were applied to 12.5% SDS-PAGE gel. Visualization of proteins was carried out either through their direct staining dye for protein Imperial Protein Stain (Pierce, Rockford I.L., USA), or after Western blotting on PVDF membrane using a mAB (monoclonal antibodies)specific for stimulating T-cell epitopes of α - and γ-gliadin (Spaenij-Dekking et al., (2004) Gut 53, 1267-1273) and HMW(high molecular weight)and LMW(low molecular weight)-glutenins (Spaenij-Dekking et al., Gastroenterology 2005; 797-806).
Competitive analysis using antibodies
To conduct competitive analysis using antibodies in mice With Balb initiated the production of monoclonal ant the phone against the above stimulating T-cell alpha, gamma-gliadin peptide LMW-glutenin. After the unification of the spleens of mice with a line of myeloma cells mice were obtained producing antibodies of hybridoma. Last cloned by the limited dilution, and mAB (monoclonal antibodies), secreted by these cells were tested for suitability of their use in competitive analysis. For each specificdate selected one or two suitable mAB and defined epitopes recognized by different mAB (table 1).
|Specificity||Stimulating T-cell epitope||mAB epitope|
|LMW-glutenin||QPPFSQQQQSPFSQ||GSPFS or PPFSQQ|
Using mAB conducted competitive analyses, which allowed us to quantitatively detect stimulating T-cell epitopes present in the native (intact) proteins and small peptides, approximately 11 amino acids (size epitope T-cells) at low levels of their content.
In the competitive analysis of various sample dilution was mixed with a fixed concentration of biotinylated (i.e. treated with Biotin) peptide-indicator (which encodes the epitope T-cells). For a quantitative assessment of gliadin in this sample was constructed standard curve using the European standard gliadin the IRMM-480 in the concentration range from 10 micrograms (μg)/ml to 10 nanograms (ng)/ml Quantitative assessment of LMW-glutenin was conducted using a synthetic peptide encoding a stimulating T-cell epitope in the range from 1 μg/ml to 1 ng/ml while the quantitative assessment of HMW-glutenin was carried out using the product of hydrolysis by pepsin/trypsin recombinant proteins HMW-glutenins in the range from 1 μg/ml to 1 ng/Junior Visual assessment of the presence associated with antibody biotinylated peptide was performed using streptavidin as a label.
Sterilization by filtration specific Proline endop is ateasy
Filtration is the preferred option sterilization enzymes. Be sterilized solution of the enzyme can be obtained by purification by chromatography, and the solution may contain one or more solvents or other additives to regulate the activity of the enzyme and its subsequent stabilization. Suitable stabilizers are, for example, sorbitol and glycerin. Glycerol solvents can be added in a concentration of from 10 to 70 wt.%/wt. solution of the enzyme, more preferably from 30 to 60 wt.%/wt.
Sterilization by filtration may be carried out by pumping the pump enzyme solution through a sterile filter. Preferably, sterilization by filtration is carried out by pre-filtering with subsequent secondary filtered through a cartridge filter to 0.22 nm. Sterilized in this way, the enzyme solution can be added through a sterile dispensing device into the container for holding, containing previously subjected to pasteurization or sterilization of aquatic food product or food ingredient. Then containing enzyme product or food ingredient can be Packed in the original packaging. If an enzyme solution is required to enter in fat or low-fat spread, then sterilized enzyme solution can be mixed with pasteurized the same phase and the resulting mixture was then subjected to emulsification with the fat or oil at an appropriate elevated temperature.
To get sterilized enzyme solution intended for use in laboratory scale, the solution is specific to the Proline of endoprotease isolated from A.niger, sterilized by filtration by the following method. The syringe was filled with 1 ml of enzyme concentrate, and the top of the syringe was placed sterile filter Millex GV 0.22 μm from the company Millipore with a surface 4,91 see Under the action of pressure by the hand of the enzyme solution was passed through a Millipore filter 0.22 μm, resulting ensured sterilized solution with enzyme activity, the activity of the enzyme solution prior to sterilization by filtration.
The digestion of bread in dynamic gastro-intestinal in vitro model in the absence and in the presence of specific Proline of endoprotease
The passage of food through the gastrointestinal tract is a very dynamic process that cannot be simulated in a static in vitro models. Dynamic gastro-intestinal in vitro model, such as developed by TNO (Zeist, Netherlands)is a generally accepted model of the digestive process, which largely mimics the progressive dynamic processes in the stomach and thin Kish is cnice (Minekus et al., ATLA 1995, 23, 197-209; Larsson et al., J. Sci. Food Agic. 1997, 74, 99-106). The results from these models showed very high similarity with the results obtained in studies on humans and animals.
To study the digestion of white bread in the absence and in the presence of specific Proline of endoprotease isolated from A.niger, experiments were undertaken on the specified dynamic gastrointestinal model. These experiments were performed at average physiological conditions of the gastrointestinal tract, such as described in literature for young adults after administration of semi-solid food. The bread first "projavilsja", i.e. thoroughly mixed with saliva enzyme for 5 minutes in the absence (control) or in the presence of specific Proline of endoprotease from A.niger. In the actual experiment, 70 grams of white bread (containing 5 grams of gluten) gomogenizirovannykh together with 11 ml of gastric juice in the absence or in the presence of 100 PPU specific Proline of endoprotease. After homogenization 25% of the mixture was added to the digestion system in vitro. In gastric Department pH gradually decreased as a result of secretion of gastric juice. "Swallowed the saliva enzyme (amylase) was present initially, while digestive enzymes (pepsin and gastric lipase) secretarials gradually. Pepsin was is active at a pH lower than 5.0. In continuation of 2.5 hours the contents of the stomach gradually came into the small intestine through the pyloric valve of the stomach". In the Department of duodenal pH was regulated at the level of pH of 6.5 due to the secretion of bicarbonate. In the same division gradually secretarials the pancreatic juice containing amylase, lipase and proteolytic enzymes (e.g. trypsin and chymotrypsin), and bile. Secretions mixed with food coming from the stomach by peristaltic mixing and gradually moved to the departments of jejunum and ileum.
After 4-5 hours about 80% of the content of the small intestine gradually moved in the colon" (flask samples) through the ilio-separately valve". Connection - products of digestion was continuously subjected to dialysis at the exit of the departments of the jejunum and ileum model through the membrane system of semi-permeable hollow fibers. Flask for dialysis was replaced every 2 hours.
During the passage of proteins gluten (food) through the departments of the dynamic system from the stomach, duodenum and jejunum were collected in a small volume samples (about 2 ml) at the following time points: t=0, 15, 30, 45, 60, 90, 120, 150, 180 and 240 minutes Immediately after collection, the samples were frozen in dry ice to inhibit the enzyme activity. In the end it is each experiment remains from different departments were collected on dry ice and were stored in two test tubes of 10 ml each at minus 20°C.
Testing digested in vitro bread on the presence of toxic epitopes of gluten
There are two types of reagents that can be used to measure the presence of gluten peptides in samples of food: the clones of T cells isolated from the small intestine of patients with coeliac disease, and monoclonal antibodies specific to different gluten peptides. Clones of T-cells react to gluten peptides, when the last contact with molecules HLA-DQ2 or HLA-DQ8, causing a predisposition to the disease. These responses T-cell inflammation zone are assumed to be the main cause of celiac disease. Clones of T cells specific for peptides of alpha-, gamma-gliadin, LMW-glutenin and HMW-glutenin, readily available (see van de Wal, Y. et al., Eur. J. Immunol. 29, 3133-3139 (1999) & Vader et al., Gastroenterology, 122: 1729-1737 (2002)). Monoclonal antibodies specific for stimulating T-cell alpha-gliadin, γ-gliadin, peptides of low molecular weight (LMW) and high molecular weight (HMW) glutenins are also available and entered into a competitive analysis for the detection of these peptides in samples of food (Spaenij-Dekking et al., Gut 53: 1267-1273 (2004)).
Faction of the stomach and duodenum, collected at time t=0, 15, 30, 45, 60, 90 and 120 minutes, prepared with addition and without adding specific Proline of endoprotease were subjected to preliminary treatment is according to the following method, designed for inaktivirovanie specific Proline of endoprotease. First, the pH of the samples was increased to pH 11-12 with 1 M NaOH, and then immediately have kind of balanced out by using 1 M HCl. After centrifugation in the course of 10 minutes at 14000 rpm supernatant and different precipitation samples were collected and subjected to heat treatment at 85°C for 10 minutes to suppress any residual enzyme activity. From supernatants were prepared cultivation 1:40, 1:200, 1:1000 and 1:5000 in 20 mm phosphate buffer with pH 7, 150 mm NaCl, 0.1% Tween-20, a mixture of 2 protease inhibitors without EDTA. These cultivation, as well as the fraction of precipitation was stored at -20°C prior to the measurement the next day.
After thawing the samples of the water-soluble fraction samples were analyzed in a competitive assay with monoclonal antibodies specific for alpha - and gamma-gliadin and LMW - and HMW-glutenins. To this end, the samples were processed as described under Materials and methods, and measurements were taken of various dilutions. The results obtained for these soluble in water samples from the stomach, is presented in figure 1 (obtained in the absence of specific Proline of endoprotease) and figure 2 (obtained in the presence of specific Proline of endoprotease). Managed to find all the components of gluten except HMW-glutenins. The floor is i.i.d. data clearly show that the addition of specific Proline of endoprotease from A.niger has a pronounced effect on the presence of these gluten proteins in the soluble fraction: even at t=0 (i.e. in less than 1 minute after adding the enzyme to the drug for oral use), it was possible to observe a sharp decline in the availability of proteins/peptides of gluten.
In a separate experiment conducted water-insoluble fraction (i.e. sediment) samples from the stomach were subjected to SDS-PAGE followed by transfer of the separated proteins on a PVDF-membrane. Then the membrane was reacted specific to alpha-gliadin antibody (figure 3). While antibody found gliadin all factions without specific Proline of endoprotease, adding specific Proline of endoprotease led to a significant weakening of the signal after t=45 minutes. At t=90 and t=120 minutes, it was almost impossible to detect gliadin in samples from the stomach, obtained from the material digested in the presence of specific Proline of endoprotease.
Based on all these data we can conclude that specific to the Proline endoprotease is highly effective in breaking down molecules of gluten present in the soluble fraction. As used in this test antibodies are specific for amino acid sequences, shorter, what you eat required to stimulate T cells, this indicates that the processing of specific Proline-endoproteases leads to a strong reduction of potentially harmful molecules of gluten in water-soluble fractions. Due to the fact that these predominantly water-soluble peptides effectively interact, as expected, with receptor sites that are relevant to celiac disease, the data obtained for the water-soluble fraction of gluten, highly relevant to in vivo conditions.
Absolutely amazing way specific to the Proline endoprotease also able to hydrolysis of the gluten molecules present in the water-insoluble phase. After 90 minutes of gliadin molecules could be detected in the fractions treated with the enzyme, whereas in the control samples, these molecules were still present.
Joint results described in this example indicate that specific to the Proline endoprotease according to the invention is able to break down gluten in conditions that simulate conditions in the human stomach. Moreover, the enzyme is able to do this so effectively that the toxic epitopes of gluten in fact remains.
The compatibility of specific Proline of endoprotease with the production of low-fat spread
To verify that saves or not specific to the Proline endoprotease its activity is ü under the influence of emulsifiers at elevated temperatures and the introduction of the emulsion water-in-oil was conducted the following test.
The local supermarket was bought low fat spread "Halvarine voor op brood" (40% fat) produced by Winner Food BV, Lopik, the Netherlands. Specified on its label the ingredients included mono - and diglycerides of fatty acids (E 471) as an emulsifier, sorbic acid (E200) as preservative, citric acid, vegetable oils and fats, water, flavour, vitamins a and D, and salt. The melting behaviour of low-fat spread was evaluated in a cooled incubator equipped with a shaking device. At 53°C low fat spread completely melted, showing the slow separation of the water layer and an oil layer.
To support the introduction of an adequate number of specific Proline of endoprotease in low fat spread 15 ml liquid enzyme concentrate containing about 10 PPU/ml (definition of enzyme, see Materials and methods)were subjected to freeze-drying in vitro of Greiner 50 ml of the Obtained powder (about 2.25 grams) was mixed with 25 grams of low-fat spread, after which the mixture was melted at 53°C in a cooled incubator equipped with a shaking device, which contributed to the dissolution of the obtained freeze-dried powder of the enzyme. Immediately after melting and spontaneous separation of water and fat sample (300 µl) who was sulkala from the aqueous layer in the test tube and cooled to room temperature. After removing the first sample tube containing the diluted emulsion with the enzyme, was intensively shaken for 10 seconds and was maintained at 53°C. After 10, 70 and 100 minutes again was extracted from the aqueous layer samples of 300 μl, after which the extract was shaken intensively. In conclusion, all samples obtained from the aqueous layer was diluted 1000 times with 100 mm acetate buffer with a pH of 4.2, and the residual enzyme activity was measured breakdown with microtiter tablet (ICTR). With this purpose, after hydrolysis of the synthetic peptide substrate Ala-Ala-Pro-pNA (Pepscan, Lelystad, the Netherlands) getting Tripeptide Ala-Ala-Pro and painted pNa molecule was carried out for 10 minutes kinetic measurements at 405 nm and 40°C using a tablet reader (TECAN Genios MTP Reader (Salzburg, Austria). The substrate Ala-Ala-Pro-pNA (rather than Z-Gly-Pro-pNa) was used for the reason that Ala-Ala-Pro-pNA allows the measurement of much smaller quantities of the enzyme. Each hole of the tablet contained 250 μl of substrate solution (3 mm AAP-pNa in 100 mm acetate buffer with a pH of 4.2) and pre-heated in the tablet reader Tecan Genios MTP Reader for 10 minutes at 40°C. the Reaction was initiated by adding 50 µl of the appropriate dilution of enzyme (in this case 1000 times). The release of pNa molecules occurred within 15 minutes. Data collection was carried out by the software Magellan (ecan). There was an increase in optical density at 405 nm; post-treatment were carried out in Excel with getting images are presented in figure 4. Activity specific for Proline endoprotease immediately after the melting of the spread was the result of the determination 100%. These results showed that the activity of the enzyme environment skim spread hardly affected at 53°C. Even shaking melted spread, simulating the process of emulsification, had a negligible impact or no impact, despite the excessive foaming.
The stability in storage of specific Proline of endoprotease in the aqueous phase of low-fat spread
To check the stability in storage of specific Proline of endoprotease in the aqueous phase of low-fat spread was prepared containing lactic acid aqueous phase with a pH of 4.5 and a water activity of 0.98. In order to avoid bacterial contamination of the aqueous phase is added to bring the concentration to 600 ppm (parts per million parts) concentrated solution of sodium benzoate was sterile. Then was added sterile (sterilized by filtration) is specific to the Proline endoprotease to achieve the activity of the enzyme 15 PPU/g. The total solution was distributed over a large number of small sterile flasks. Some of the quiet of the flasks were placed at minus 20°C and served as control, the remaining flasks were kept at 8°C and at 30°C. Every few weeks was measured residual activity specific to the enzyme Proline in different flasks. The enzyme activity was measured according to the method described in section Materials and methods.
The results presented in figure 5 show that during storage of the enzyme at a temperature of 8°C it remains quite active during the period of time of at least 50 weeks at these high values of aw.
The in vitro digestion of toast with raspberry filling on top, containing the enzyme
To demonstrate the concept of application containing the enzyme toppings to facilitate splitting of molecules toxic gluten in the food product was carried out the following experiment. First were concocted 250 ml stable in storage raspberry filling. To the liquid enzyme concentrate in the amount of 204 g (12 PPU/ml) was added 20.7 grams of sucrose, and 1.0 grams of citric acid, 0.02% of raspberry flavour (Givaudan, 76525-36) and 20.7 grams of a 0.5%aqueous solution of saccharin in water. After dissolution of all these ingredients were added 3.11 grams of xanthan gum (Keltrol RD, CP Kelco, Il.), which was dissolved in thorough mixing. The final pH of the viscous mass was 3.7; the final concentration of the enzyme is about 9,8 PPU/g fillings; final water activity of about 0,99. The concentration of benzoate was maintained at about 600 ppm. In the same way it was cooked product placebo containing all the above ingredients, but without active specific to the enzyme Proline. As with the earlier experiments (see Examples 2 and 3) in the test was also introduced non-viscous solution of pure enzyme.
To test the effectiveness of all these different drugs in the breakdown of toxic epitopes of gluten a test was conducted on the digestion in vitro, in which the reduction in the number of epitopes occurred in time. To this end, slice (about 10 grams, containing 1.4 g of protein) were purchased in the trading network of white wheat bread, fried in the form of toast (Bolletje, "Engelse toast"), was applied to the enzyme in a different form. So, one slice was covered containing enzyme raspberry filling; one slice was covered with the filling-placebo (i.e., no enzyme activity), and another piece was added to the pure liquid enzyme. In cases fillings containing the enzyme, and the addition of pure liquid enzyme activity of the enzyme was desirables so that she was 20 PPU/g of protein present. Five minutes after applying different products on the last toast was ground and mixed with 60 ml (control and liquid variants of the enzyme) and from 62.3 ml (gel formation) solution with pH 5.0, they are youseo gastric juice (NaCl (4.8 g/l), KCl (2.2 g/l, CaCl2(0,22 g/l) and NaHCO3(1.5 g/l)). 2,3 ml of the same solution containing porcine pepsin (Sigma P-7012) at a concentration of 500 KU/l, was added to each of toast.
The first sample was obtained at t=0 minutes (i.e. after 5 minutes after adding the solution of pepsin). Then the pH of each mixture was gradually decreased to simulate the slow acidification of the stomach contents. the pH was decreased from pH 5 to pH 4.5, and 15 minutes after sampling the first sample was selected a second sample. In the next 15 minutes the pH was again decreased to pH 4, and at t=30 minutes from the start of the experiment were selected for the third sample. Subsequent samples were selected as follows: at t=45 minutes (after lowering the pH to 3.5); at t=60 minutes (after lowering the pH to 3.0); at t=90 minutes (after lowering the pH to 2.5); then the pH was lowered to 2.0 and additional samples were taken at t=120 minutes, and finally at t=150 minutes. All samples immediately frozen in liquid nitrogen and stored at -80°C.
To measure residual levels of gluten epitopes in different preparations frozen samples were first subjected to procedure a thorough inaktivirovanie enzyme. While still frozen, the samples were subjected to heat treatment at 95°C for 30 minutes, then the pH of the samples was increased to pH 11-12, then decreased to pH 2 and the conclusion was kind of balanced out up to pH 6. Then the samples were again subjected to warm the th-treated at 95°C for 15 minutes, then selected aliquot amount (1 ml) and centrifugals in the continuation of 30 minutes in the Eppendorf centrifuge. The resulting supernatant contained the soluble fraction, and the residue is water-insoluble fraction. Quantitative assessment of the residual level of stimulating T-cell epitopes in the water soluble fraction was carried out experiments on the competition with the use of monoclonal antibodies in accordance with Spaenij-Dekking et al., (2004) Gut 53, 1267-1273 (see also Materials and methods). The results of these tests on competitiveness (average of two independent measurements) are presented in table 2. Residual levels of stimulating T-cell epitopes in the fraction of sediment was reproduced visually Western blotting (see Example 3). The results of the last experiment (pictures not shown), as well as the data obtained for the water-soluble fraction, confirming the effective splitting of stimulating T-cell epitopes by both drugs, containing an enzyme.
As a result, the data clearly indicate that both preparation containing the enzyme, no matter in what form it is present in the form of pure free enzyme or generowania product type jam with high activity in the water, effectively destroyed most stimulating T-cell epitopes of gluten in incubate is for about 30 minutes under the conditions simulate conditions in the human stomach. Only LMW (low molecular weight) fraction was apparently more resistant to cleavage by the enzyme used in in vitro experimental setup.
|Residue levels of stimulating T-cell epitopes (micrograms/ml) in water-soluble fractions|
|Alpha 9||Alpha 20||Gamma 1||LMW||HMW|
|Sample later (minutes)|
|Pure enzyme liquid||0|
1. Pasteurized food product that provides a breakdown of the gluten peptides that have a water activity of at least 0,85, and contains specific Proline protease, which is isolated from Aspergillus or refers to the S28 family of serine proteases and which has an optimum activity at pH values from 1 to 7, preferably at pH values from 2 to 6.
2. The product according to claim 1, which is a gluten-free food product.
3. The product according to claim 1, which is the seasoning and the top of the filling, the filling for a sandwich, sauce, drink or emulsion, such as the spread.
4. The product according to claim 1, which is a low-fat spread.
5. The product according to claim 1, which is recommended to patients suffering from celiac disease.
6. Food product that provides a breakdown of the gluten peptides that have a water activity of at least 0,85 and containing specific Proline protease, which is isolated from Aspergillus or refers to the S28 family of serine proteases and which has an optimum activity at pH values from 1 to 7, preferably at pH values from 2 to 6, and the product contains less than 1 wt.% protein or peptide to the weight of the product.
7. The product according to claim 6, which is a gluten-free food product.
8. the product according to claim 6, which is the seasoning and the top of the filling, the filling for a sandwich, sauce, drink or emulsion, such as the spread.
9. The product according to claim 6, which is a low-fat spread.
10. The product according to claim 6, which is recommended to patients suffering from celiac disease.
11. Method of manufacturing a food product according to any one of p-10, which is specific for Proline protease added to specified food product after the food product is subjected to pasteurization.
12. Method of manufacturing a food product according to any one of claims 1 to 5, which is specific for Proline protease added to pasteurized food product.
13. The use of specific Proline protease, which is isolated from Aspergillus or refers to the S28 family of serine proteases and which has an optimum activity at pH values from 1 to 7, preferably at pH values from 2 to 6, for the breakdown of gluten peptides in the manufacture of a food product or pasteurized food product according to any one of claims 1 to 10.
14. Use item 13 to prevent any harmful effects of the food product, rich in Proline.
15. Use item 13 to prevent clinical symptoms of celiac disease or related disorders.
16. The use according to any one of p-15, wherein said food product is intended for use in the combinations of the emission with food containing gluten product.
17. Use 14 or 15, in which the specified adverse health effects or disorder associated with celiac disease include autoimmune disorders, mainly type I diabetes, dermatitis herpetiformis, colon cancer, intestinal non-Hodgkin lymphoma, irritable bowel syndrome, autoimmune thyroiditis, collagen, the autoimmune alopecia and autoimmune hepatitis, and mental disorders, including autism, schizophrenia, ADHD, bipolar mood disorders and depression.
SUBSTANCE: method of obtaining a yeast extract involves treating Saccharomyces cerevisiae yeast cells with purified phospholipase A and separating the yeast extract from treated yeast cells. Yeast cells undergo proteolysis during treatment with phospholipase. Treatment with phospholipase is carried out at pH 2-10, and the amount of phospholipase ranges from 0.001 to 1 mg enzymatic protein/g dry substance. Yeast extract output is equal to 68.8%, protein output is equal to 67.4% and degree of hydrolysis is equal to 71.8%.
EFFECT: method enables to obtain an end product with high output, protein content and degree of hydrolysis.
6 cl, 3 tbl, 3 ex
SUBSTANCE: method for preparing isotope-marked secondary metabolic products of fungi provides immobilised fungi growing on an inert carrier with an artificial fluid culture medium added. All carbon, nitrogen and/or sulphur atoms in the specified medium are substituted by stable isotopes chosen from the group including 13C, 15N, 33S and 34S. Also, an isotope-marked secondary metabolic product made of fungi is presented.
EFFECT: high-purity end product preparation.
13 cl, 2 dwg, 9 ex
SUBSTANCE: invention relates to biotechnology. Method of obtaining lipids from microorganisms includes processing cells of microorganisms, grown in fermentative medium, for release of intra cellular lipids, dissolving part of present proteins. Fermentative medium is divided into layers: heavy aqueous and light, containing lipids, separation being carried out in medium, which contains less than 5% of non-polar organic solvent, without allowing non-polar organic solvent to extract lipids. Then heavy layer is separated from light layer and lipids are obtained from light layer.
EFFECT: creation of efficient and economical method of obtaining lipids from microorganisms.
45 cl, 6 tbl, 1 ex
FIELD: wood chemical industry.
SUBSTANCE: method involves treatment of balsamic poplar vegetative part with water to isolate essential oil and a solid residue. Treatment with water is carried out by hydrodistillation followed by extraction of solid residue with 94-96% ethyl alcohol to obtain alcoholic extract and oil cake. Wax is isolated from alcoholic extract and evaporated to obtain lipid concentrate, and oil cake is wetted with vat liquid prepared in hydrodistillation and subjected for biodestruction with fungi of genus Trichoderma. Method provides preparing the complex of biologically active substances from balsamic poplar vegetative part.
EFFECT: improved preparing and processing methods.
2 cl, 2 ex
FIELD: biotechnology, microbiology, pharmacology.
SUBSTANCE: invention relates to preparing a medicinal preparation using microorganism-producers. Lecanicillium sp. G16 strain is isolated from a soil sample from Teberdinsky reservation and deposited in Collection of microorganism cultures GNTSA at number 347A. Invention provides preparing a complex preparation possessing high hypolipidemic and antitumor activity and prolonged effect in low concentrations.
EFFECT: valuable medicinal properties of strain and preparation.
3 tbl, 1 ex
FIELD: preparing of fresh citrus fruits for storage and further usage in space feeding.
SUBSTANCE: method involves inspecting citrus fruits and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.005%-citric acid solution and in suspension of preparation produced from biomass of Mortierella polycephala micromycet by predetermined process; covering stems and adjoining surface with medicinal glue; exposing both sides of fruits to ultraviolet radiation; packaging each fruit into smoking paper and laying in polymer trays. Method allows citrus fruits to be transported to space object and stored there during 45 days and used in space feeding.
EFFECT: prolonged shelf life and improved quality of citrus fruits.
FIELD: preparing of fresh citrus fruits for storage and further usage in space feeding.
SUBSTANCE: method involves inspecting citrus fruits and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.005%-citric acid solution and in suspension of preparation produced from biomass of Mortierella parvispora micromycet by predetermined process; covering stems and adjoining surface with medicinal glue; exposing both sides of fruits to ultraviolet radiation; packaging each fruit into smoking paper and laying in polymer trays. Method allows citrus fruits to be transported to space object, stored there during 45 days and used in space feeding.
EFFECT: prolonged shelf life and improved quality of citrus fruits.
FIELD: preparing of fresh citrus fruits for storage and further usage in space feeding.
SUBSTANCE: method involves inspecting citrus fruits and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.005%-citric acid solution and in suspension of preparation produced from biomass of Mortierella pulchella micromycet by predetermined process; covering stems and adjoining surface with medicinal glue; exposing both sides of fruits to ultraviolet radiation; packaging each fruit into smoking paper and laying in polymer trays. Method allows citrus fruits to be transported to space object, stored there during 45 days and used in space feeding.
EFFECT: prolonged shelf life and improved quality of citrus fruits.
FIELD: preparing of fresh citrus fruits for storage and further usage in space feeding.
SUBSTANCE: method involves inspecting citrus fruits and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.005%-citric acid solution and in suspension of preparation produced from biomass of Mortierella gamsii micromycet by predetermined process; covering stems and adjoining surface with medicinal glue; exposing both sides of fruits to ultraviolet radiation; packaging each fruit into smoking paper and laying in polymer trays. Method allows citrus fruits to be transported to space object and stored there during 45 days and used in space feeding.
EFFECT: prolonged shelf life and improved quality of citrus fruits.
FIELD: process for preparing of fresh apples for storage and further usage in space feeding.
SUBSTANCE: method involves inspecting apples and washing at predetermined mode parameters in sweet water; sequentially holding in 0.02%-potassium permanganate solution, 0.5%-citric acid solution and 0.005%-suspension of preparation produced from biomass of Mortierella gamsii micromycet by predetermined process; exposing to ultraviolet radiation; packaging each apple fruit in smoking paper and placing in polymer trays. Method allows apples to be transported to space object, stored there during 45 days and used in space feeding.
EFFECT: prolonged shelf life and improved quality of fresh apples.
SUBSTANCE: disclosed is isolated polypeptide being acid-proof metalloprotease isolated from Thermoascus aurantiacus. Described are strain Thermoascus aurantiacus CGMCC No 0670 for polypeptide production and method for polypeptide production using said strain. Disclosed is method for plant protein treatment to increase digestion value thereof by using said polypeptide.
EFFECT: protease of good acid resistance useful in feed production.
6 cl, 7 ex, 4 tbl
FIELD: biotechnology, microbiology, biochemistry.
SUBSTANCE: invention relates to development of a novel strain used for preparing enzyme representing a complex of acid subacid proteases. Strain is prepared by selection from the known strain Aspergillus oryzae (VKPM F-683) by multistep selection using effective methods of mutagenesis. Strain is stored as lyophilic dried culture and on slants with wort-agar in the biotechnology section of enzyme preparations in the food processing department of the State Scientific Institute VNII of food processing technology in Moscow. Invention provides preparing the strain possessing the high level of synthesis of acid and subacid proteases and high total activity of enzyme in cultural fluid exceeding activity of analogue by 2.0-2.6-fold and in reducing culturing process time.
EFFECT: improved and valuable properties of strain.
2 tbl, 5 ex
FIELD: biotechnology, microbiology, biochemistry.
SUBSTANCE: invention relates to the strain Aspergillus oryzae-12 providing the high level of synthesis of acid proteases and xylanase. The strain is obtained by multistep selection of the strain Aspergillus oryzae-387 (VKPM F-683) using effective methods of mutagenesis. The strain is deposited in collection VKPM at № F-932. Invention provides preparing the enzyme complex showing the high proteolytic and xylanolytic activity in cultural fluid wherein their level exceeds activity in analogue by 2.5-2.8-fold.
EFFECT: valuable properties of strain.
2 tbl, 3 ex
FIELD: biotechnological methods.
SUBSTANCE: invention concerns new polynucleotides, which encode polypeptide having tripeptidylpentidase activity. To produce this polypeptide, host cell is transformed with polynucleotide or polynucleotide-containing vector and then cultured under suitable polynucleotide expression conditions.
EFFECT: enabled production of new protease from thread fungi.
14 cl, 3 tbl, 11 ex
FIELD: biotechnology, microbiology.
SUBSTANCE: invention relates to producing ethyl alcohol or fodder for monogastral animals. The polyenzyme product with glucoamylase, proteolytic and xylanase activities is prepared by fermentation of wheat bran using microorganism Aspergillus niger. Indicated glucoamylase, proteolytic and xylanase activities have the following minimal values: glucoamylase activity - at least 100 U/g of dry matter; proteolytic activity - at least 100 U/g of dry matter; xylanase activity - at least 100 U/g of dry matter under condition that glucoamylase activity has to be at least 750 U/g of dry matter and/or xylanase activity has to be at least 300 U/g of dry matter. Invention provides enhancing the soluble nitrogen content in wort after saccharification, reducing viscosity and effectiveness in using.
EFFECT: improved preparing method, valuable properties of hydrolyzed bran.
14 cl, 10 tbl, 7 ex
FIELD: food industry.
SUBSTANCE: invention relates to a composition for alimentation of babies with low, very low or extremely low body weight at birth as well we for alimentation of premature babies. The composition includes 100-200 mg of threonine per 100 kcal, 5-15 wt % of palmitic acid of the total fatty acids content and prebiotic oligosaccharides capable to reduce pH in the digestive tract (DT). Additionally the composition contains 2 - 25 wt % of medium-chain fatty acids of the total fatty acids content. The composition may contain an undigestible oligosaccharide represented by galactose.
EFFECT: invention allows to enhance oral tolerance, reduce duration of food transit through DT, increase bowels movement periodicity, stimulate the immune system, accelerate initiation of complete enteroalimentation of babies born before 37 pregnancy weeks and/or babies with weight at birth less than 2500 g.
10 cl, 2 tbl, 3 ex
FIELD: food industry.
SUBSTANCE: invention relates to prevention of baby adiposity at a later stage of one's life. One proposes application of a specified composition in alimentation of a baby not suffering from adiposity at an age of no less than 36 months. The composition contains a lipid component, a protein component and a component consisting of digestible carbohydrates. The lipid component includes: linoleic acid (LA) and alfa-linolenic acid (ALA), the weight ratio of LA/ALA being 2 - 7; less than 14.5 wt % of LA in conversion to total weight of fatty acids; 2 wt % - 10 wt % of ALA in conversion to total weight of fatty acids, 0.02 - 0.8 wt % of n-6 long chain polyunsaturated fatty acids (LC-PUFA) in conversion to total weight of fatty acids and 0.2 wt % - 15 wt % of n-3 LC-PUFA in conversion to total weight of fatty acids. The weight ratio of n-6 LC-PUFA to n-3 LC-PUFA is 1.5.
EFFECT: invention allows to prevent occurrence of adiposity at a later stage of one's life.
14 cl, 5 tbl, 4 ex
FIELD: food industry.
SUBSTANCE: this invention covers food for 0-36 month aged infants. A composition comprising free amino acids as a single source of protein, source of fat acids comprising long-chain polyunsaturated fatty acids (LCPUFA), source of carbonhydrates comprising assimilable and non-assimilable carbonhydrates and bifidus bacteria was proposed for treatment of the patients suffering from tormina, astriction, snivel, sniffle, vomit, diarrhea, feces cruentae, mucus in defecation, spots, eczema, gastroesophageal reflux, eosinophilic esophagitis or asthma, allergy for cow milk and/or intolerance to dietary protein, and/or infections. Herewith, non-assimilable carbonhydrates are chosen from milk protein-free source, and bifidus bacteria are dietary protein-free, and the whole composition is essentially intact protein-free. Besides, assimilable carbonhydrate component contains less than 2 mass percent of lactose from overall assimilable carbonhydrates. Fat component includes 0.1-5 mass percent of LCPUFA from overall fat acids content. As an alternative, this composition is applied for stimulation of immune system maturation of the infants suffering from atopic diseases.
EFFECT: improvement of infant health without risks of adverse allergic reaction.
25 cl, 2 dwg, 3 tbl, 1 ex
FIELD: food industry.
SUBSTANCE: invention relates to a non-allergenic food product. The food product includes an amino acid fraction containing at least one component chosen from the group consisting of amino acids and peptides with a degree of polymerisation 7 or less, and a lipid fraction containing at least one fatty acid chosen from the group consisting of arachidonic acid and docosahexanoic acid. The composition content of protein and other peptides with molecular weight 1000 daltons or more (in relation to dry weight) is less than 0.01 wt %, preferably - no less than 0.001 wt %, more preferably - no less than 0.0001 wt %. The food product is used for allergy diagnostics.
EFFECT: invention allows to produce a product which does not impart allergic reactions and is able to attenuate intensity of the patients' allergic reactions.
7 cl, 4 ex
FIELD: food industry.
SUBSTANCE: invention relates to a nutritive composition, to application of the composition in a food product as a protein substitute, to a food supplement and a food product containing the said nutritive composition. The composition consists of particles containing a protein material and micronutrients; 90-100 wt % of the said protein material and micronutrients are coated with a fat-containing layer containing 90-100 wt % of a food fat of the total layer weight and having solid fat content in an amount of 70-100% at a temperature of 37°C.
EFFECT: nutritive composition has a neutral taste, scent, texture and long storage life; the composition is intended for patients suffering from food allergy or phenyl ketonuria.
7 cl, 4 ex
FIELD: food industry.
SUBSTANCE: invention relates to infant alimentation. One proposed application of fat, digestible carbohydrates and protein to produce a composition for therapy and/or prevention of adiposity as well as coexistent diseases. The protein includes at least 25 wt % of peptides with chain length from 25 to 30 amino acids (of total dry protein weight), the composition contains at least 12 mg of indigestible fermentable carbohydrates per 1 g of dry nutritious mixture weight. The composition is applied for baby feeding.
EFFECT: invention allows to prevent overgrowth and proliferation of adipcytes in the period of infancy (and, consequently, children's adiposity and secondary disturbances connected to it) due to combination of hydrolysed proteins and indigestible fermentable carbohydrates.
14 cl, 2 dwg, 1 tbl, 2 ex