FI118507B - Protein solution for use in forming protein film for e.g. food products, contains modified protein, which is modified by cleaving disulfide bond(s) originally present in the protein by sulfitolysis to obtain free sulfhydryl groups - Google Patents

Abstract

A protein solution for use in protein film formation, has a pH of = 7 and contains modified protein, which is modified by cleaving at least one disulfide bond originally present in the protein by sulfitolysis to obtain free sulfhydryl groups. These free sulfhydryl groups are able to cause an interchange reaction to form disulfide bonds between the proteins. Independent claims are also included for: (1) a protein-based film comprising a protein network formed by disulfide bonds between the proteins, where the protein network is formed by treating protein with modified protein in a solution having a pH of = 7; (2) a food product being coated with or containing substances coated with the above film; (3) baby's milk formula containing the above film as emulsion; (4) pharmaceutical product containing at least one therapeutically active agent and being coated with the above film; (5) a container coated with the above film; and (6) method for preparing a protein-based film comprising providing the inventive protein solution, and forming the solution into the protein-based film.

Description

118507

A method for fortifying a proteinaceous product and a proteinaceous product

The present invention relates to a method for enhancing the structure of proteinaceous products using modified protein or fractions made from modified protein. The invention also relates to a proteinaceous product prepared by the process.

Many foods, even those with a high protein content, need a support to support their structure in order to bring their structure to the consumer's requirements and to withstand up to 10 uses as such. Strength of the structure is manifested by a decrease in viscosity or by separation of the liquid phase.

Common protein foods include especially milk-based products, usually low-fat or non-fat, such as yoghurts, filings, puddings, spreads, ice-rolls, and beverages. In these, the desired structure is obtained by raising the protein content to a sufficiently high level, from 6 to 12%, and heating at a sufficiently high temperature for a sufficient time, or by adding a thickening and stabilizing agent, gelatin, modified starch, pectin, carrageenan, locust bean gum.

Gel-forming properties of proteins are generally known and have been extensively studied. However, not all mecca, nism and factors of gel formation are fully known. For example, various milk and whey proteins ·4 have their own, often complex, roles in the formation of a gel-forming protein network.

• · · ···: 25] * According to the state of the art, for example, in the manufacture of yogurt, it is essential to increase the protein content of milk and, according to current methods, i ft strengthens the structure by heating. The preparation begins with raising the protein content of the milk by concentrating the milk by evaporation, heating at a high temperature for 30 minutes or by adding milk powder, usually skimmed milk powder, to increase the milk solids content by 8.5%. From 9.0% M to 10.5-13.0%. The appropriate dry matter content is determined by the fat content.

• · * ···] At this stage, other necessary ingredients are added, such as fat, sugar, and stabilizing and thickening agents.

In the next process step, the temperature of the pretreated milk is raised to 50-65 ° C and homogenized at 150-200 bar. As a result, more fat globules with particles of casein micelles and whey 118507 2 proteins are obtained. Likewise, the free casein micelles are increasing. Casein moieties and whey proteins, both on the surface of fat globules and free, participate in protein network formation 5 An important step in the manufacturing process is the denaturation of milk proteins by heating. At the same time, the number of contaminating bacteria is reduced to maintain hygiene. Denaturation of proteins generally requires a temperature of at least 85 ° C and a duration of 15-30 minutes. The same effect is obtained at higher temperatures and shorter processing times.

10

During heating, as a second step after denaturation of the proteins, the denaturation releases sulfhydryl (SH) groups, particularly in whey proteins and in β-lactoglobulin, which cause an exchange reaction between the SH and disulfide (SS) groups. This reaction results in the formation of a space network of he-15 rapeseed proteins, kappa-casein and all proteins in the mixture which have disulfide bonds or groups in their structure, which manifests itself as gelling when the pH drops below 5. The strength of the network structure depends mainly on the protein content and the effect of heating. The mesh structure strengthens the structure of the yogurt and prevents the whey from separating during storage.

20

Conventional gel formation with whey proteins thus relies on β-. . ·, Succinhydryl groups of lactoglobulins, one in each β- · · · V /. ' laktoglobuliinimonomeerissä. This is generally a limiting factor in gel formation due to the limited amount of β-lactoglobulin present. For example, whey-containing α-lactalbumin, which is also one of the largest whey protein * * components, has no free sulfhydryl groups. The amount of free sulfhydryl groups may be increased by other means, but are generally not usable, for example, in the food industry. An example of this is

Stevenson et al. (J. Agric. Food Chem. 1995, 44: 2825-2828), which discloses chemical thiolation of tap-whey β-casein to yield a synthetic protein containing free sulfhydryl groups and numerous disulfide bonds.

··· * · • · * ···] Cysteine is also capable of opening disulfide bonds as a small '* compound containing the SH group, but does not have the structure-reinforcing properties itself since::; 35 it lacks the reinforcing protein networking property that requires • I * at least two SH groups. In addition, after a certain concentration, cysteine is subject to the Finnish Medicines Act, which limits its use. In Britain, for example, the maximum level is 70 ppm for the production of dough.

118507 3

The structure of the yoghurt is usually affected in the later stages of the preparation mainly by the yeast and its possible structural support, as well as the mixing and its intensity.

5 Especially in the manufacture of non-fat yogurt and filings, it is necessary to increase the amount of protein more than in fat-containing products due to the lack of casein and whey proteins on the surface of fat globules.

However, heating results in the formation of many byproducts that reduce the nutritional value of the proteins and may appear to be unfavorable when ingested, for example as allergens (Walstra, P. et al., Dairy Technology, Principles of Milk Properties and Processes, Marcel Dekker 1999).

The thickeners and stabilizers used to strengthen the structure do not form part of the milk's own constituents but are derived from different animal carcasses, such as gelatin, or plant parts such as pectin, carrageenan, guar gum, etc. and have no significant nutritional value. In addition, the use of certain additives of animal origin, such as gelatin, may be subject to health or ethical constraints that limit their use.

. . ·. Therefore, there is a need for a novel method of reinforcing the structure of foodstuffs, which avoids unnecessary heating of the product or the addition of additional thickeners or stabilizers * · * ··· * · · Methods of converting and fractionating a protein such as whey protein are generally known, one of which is to modify the structure of a protein so that the sulfur bridges or disulfide bonds between the amino acid chains of the proteins are opened.

This is generally accomplished by a sulfonation reaction, whereby the proteins are contacted with a sulfite ion-forming reagent to sulfonate the proteins. Further, an oxidation-reduction reaction is initiated in which one sulfur bridge of the protein *: **: is oxidized to the sulfonate and the other is reduced to the sulfhydryl group. By adding a further oxidizing factor, the oxidizing free sulfhyryl groups are again disulfide bonded, which in turn continue in the reaction until all the sulfhydryl groups are sulfonated or some other factor in the reaction becomes limiting.

4, 1 8507

For example, FI101514 and FI107116 disclose methods for sulfonating and converting proteins for protein isolation. FI101514 discloses a process for modifying the structure of whey proteins by sulfonation without a catalyst. The publications do not disclose specific uses or application methods for isolated proteins.

FI107116 discloses a method for altering the structure of proteins by contacting a protein with a sulfite ion-forming reagent to sulfonate the protein without oxidant. By lowering the pH 10 of the sulfonated protein to the acidic side, the sulfonate groups are released from the protein as sulfur dioxide, which can be removed from the solution by blowing. Part of the modified protein precipitates at low pH and some remains soluble. The protein can be recovered either as a precipitate and as a soluble mixture, as a total whey protein, or as a precipitated and soluble fraction, and subjected to any subsequent treatment. This method is based on the fact that, in the conversion of proteins, sulfitolysis alone already causes sufficient disulfide bonds to be opened and that oxidation is not necessary to alter the conformation of the protein molecule and to precipitate the proteins under acidic conditions. Non-oxidation simplifies and speeds up the process and improves its profitability.

: 20 ···. * ··. The precipitate fraction contains α-lactalbumin, bovine serum albumin (BSA) and some β-lactoglobulin. The soluble fraction essentially contains only β-lactoglobulin. All proteins are modified and have improved function. nal properties. The modified whey protein and both fractions have certain preferred applications, such as soluble fraction film formation and emulsification and structure enhancement of the modified · and precipitate fraction. Out of these three products, you can select, depending on your situation, the application or product • φ! * ·· best suited. In addition, the amounts of the various proteins contained in the fractions can be affected by changing the reaction conditions, for example by raising the pH to be used in the precipitation.

• · · • · ··· · · ·

In the method of FI107116, the disulfide bond opening and conformational elaboration of proteins such as whey or soy proteins is accomplished by sulfite • · V ··olysis in which the sulfite ion specifically reacts with another sulfur bond to form the S-sulfonate derivative. The second sulfur is reduced to a sylphhydryl group. It is preferred to use as the sulphite an alkali metal or alkaline earth metal sulphite, hydrogen sulphite or metabisulphite, or combinations thereof. The most useful sulphites and in this process are soluble and food grade sodium sulphite, sodium hydrogen sulphite and sodium metabisulphite, but other suitable sulphite compounds may also be used. Under the reaction conditions, all of the above sulphites are predominantly sodium sulphite and sodium hydrogen sulphite.

5 The most important factor affecting the conversion rate in sulphitolysis is the amount of sulphite per amount of protein. Surprisingly, it has been found that a sufficient amount of sulfite is lower than, for example, FI107116. The sulphite supplement used in accordance with the invention is about 0.01-0.06% (w / v) of sodium metabisulphite, while the amount of protein in the solution is 10-11% (w / v).

It is an object of the present invention to provide a novel method of enhancing the structure of proteinaceous products, generally foodstuffs, using a modified protein, preferably whey protein, wherein the modification results in free sulfhydryl (SH) groups (Figure 1) originally derived from the protein. disulfide bonds. As a result of a brief heat treatment, such as pasteurization, the free SH groups induce an exchange reaction to form disulfide (SS) bonds and the proteins to form a space network that supports the structure of the proteinaceous product. This avoids lengthy heat treatment of conventional methods, such as the commonly used 85 ° C for 15-30 min or the like (Figure 2).

. ... The method and product of the invention are characterized by what is stated in the independent claims. Some preferred embodiments of the invention are / ·; ' disclosed in the dependent claims.

It is an object of the invention to provide a simple method of strengthening the structure of a foodstuff, preferably a milky foodstuff, such as yoghurt, by means of the milk's own nutritionally valuable proteins without using high temperatures for protein structure modification / denaturation, wherein : * · ": 30 are known to form compounds that reduce the nutritional value of a protein.

• · • «· • · _

Food means in this context any edible product or its precursor to humans or animals. Thus, in addition to conventional foods, food may also mean, for example, animal feed: 35 or pet food. Thus, a foodstuff may also be a semi-finished product or its precursor, such as a dough.

• · 7 118507 6

The invention has realized that, for example, the modified and fractionated whey protein prepared according to FI107116 can be used to strengthen the structure of the proteins by the SH and SS group exchange reaction. Modified whey protein and whey protein fractions, which are the constituents of milk, contain free SH groups, which cause initiation of the conversion and increase of the rate, especially at the pasteurization temperature. Other types of protein, such as soy protein, may also be used. It is a condition that the protein used in the process of the invention initially has at least one disulfide bond which can be opened in the transformation reaction to obtain the free SH groups. Proteins in which disulfide bonds or additional SH groups are artificially created in the native protein are not within the scope of the invention.

By the method of the invention, the amplification of proteins of any foodstuff, such as milk or milk product proteins, or other edible products having disulfide bonds, may be accomplished by adding a suitable amount of modified protein, for example modified whey protein, and heating for a suitable time, for example pasteurization.

An advantage of the invention is that the intense heat treatment of the foodstuff can be reduced, which can degrade the taste or appearance of the product.

. A further advantage of the invention is that products with a stronger structure are obtained.

• · ♦ II ·

• M

• * • ·

A further advantage of the invention is that products with a higher protein content are obtained.

It is a further advantage of the invention that the use of additional thickening and stabilizing agents, for example gelatin and carrageenan, can be avoided. The further advantage of the invention is that the resulting food products have functional properties: * '* ♦ Features.

A further advantage of the invention is that a protein of natural origin is used.

In the following, the invention will be described in detail with reference to food preparation examples which illustrate some embodiments of the invention but are not intended to limit the scope of the invention. Reference is made to the accompanying drawings and descriptions in which:

Figure 1 illustrates the conversion reaction 5

Figure 2 illustrates an exchange reaction and an exchange involving the conversion of protein Pt and the modified protein P to form the first step in the protein network with the original protein Pi. * Figure 3 shows the oxidation of sulfhydryl groups to disulfide groups, reducing the number of SH groups and SS the structure of the network will be strengthened

Figure 4 shows the formation of Amador compound 15 Figure 5 shows the formation of lysinoalanine from lysine and dehydroalanine either free or as part of a peptide

Figure 6 shows the neutralization of acrylamide to form an acryloamide derivative of cysteine without the double acrylic linkage.

A preferred protein for use in the method and product of the invention is a whey protein, such as bovine whey protein, because of its high biological value. high. The biological value of the protein is the ratio of the amount of nitrogen used in tissue formation relative to the amount of nitrogen absorbed from the food, which describes the quality of the protein.

* ♦ I · *: V Egg protein is usually used to determine the biological value of the comparator «·· Luna and its value is denoted by 100. The whey protein is then 104, the cow's milk 91, the casein 77 and the soy protein 74.

: * · *: 30 ♦ · · ** · Modified whey protein has the same nutritional value as the original whey protein, but its nutritional value is enhanced by its better digestibility in the stomach.

• · ** · *] The main proteins in whey are β-lactoglobulin, α-lactalbumin, serum α-burnine and immunoglobulins. The original, i.e. unmodified, whey protein: 35 β-lactoglobulin, which is about half of the whey protein, is virtually non-digested / hydrolysed in the stomach and passes unchanged into the small intestine. This is an important factor in the development of milk allergy in children.

118507 8

The modified protein or protein fractions contain sulfhydryl groups which cause an exchange reaction and consequently an exchange conversion. The exchange conversion results in the strengthening of the structure of the proteinaceous product as a result of a space network formed by disulfide bonded proteins, as can be seen in Figure 5 2. It illustrates the conversion of protein P1 in the exchange reaction and the first step of network formation. The modified protein P forms the first step in the protein network with the original protein Pi. The reaction continues until the network is formed. The number of SH groups remains the same unless oxidation of the SH groups to disulfide bonds is desired. By controlling oxidation, the formation of the desired protein network can be regulated.

The free sulfhydryl groups of the modified protein and protein fractions provide a variety of protective effects in food and also, for example, in pet food. Said proteins have antioxidant activity and, as a result of the exchange conversion, plant and microbial protein toxins having disulfide bonds lose their toxicity. In addition, the modified proteins inhibit the formation of Maillard's upstream compounds, such as Amador's compound, and lysine alanine, and neutralize e.g. acrylamide and other acrylic derivatives (Figures 2, 3, 4, 5 and 6).

20

The Maillard reaction is an event where reducing sugars such as glucose, fructose,. ... maltose or lactose, react with the amino groups in the protein to reduce the biological value of the protein. The Maillard reaction results in a variety of products that can impair the taste and appearance of the food and act as allergens.

• m ·· Φ: V In the exchange reaction, the free SH-φφφ groups of the protein, preferably the modified whey protein, upon heating, cause the SS group to open, and at the same time to form a new SS group with the free SH -: * · *: 30 group. As the reaction proceeds with a suitable number of SH groups, · m; ···. the result is a suitably strong protein structure over a period of time. Formation of the structural network involves • whey proteins and caseins in free solution and on the surface of the fat droplets, where the proteins act as emulsifiers as a protein network.

• «: 35 The number of SH groups is thus not reduced in the exchange reaction. The number of SH groups can be reduced by oxidizing them, for example, with oxygen to disulfide groups 2 SH + Vi 02 --►S-S + H 2 O, which further strengthens the product structure (Figure 3).

118507 9

The functional and other properties of the end products can be affected by the degree of conversion, i.e. the amount of disulfide bonds opened relative to the amount of disulfide bonds in the protein. Depending on the use, the appropriate number of SH groups may be left, depending on the purpose, since the SH groups act as antioxidants, neutralize, by exchange, plant or microbial toxic protein compounds and, for example, acrylamide by reaction with its double bond (Friedman, M., J. Agric. 42 (1994) 3-20) (Figure 6). In addition, free SH groups prevent chemical and enzymatic darkening, and detoxify and neutralize e.g. mold produced by aflatoxin. They also bind nit-10 rites, chelate oxidative Cu2 + and Fe2 + ions and toxic As3 +, Cd2 +, Co3 +, Hg2 +, Pb2 + and Se2 + ions. Thus, a protein containing free SH groups can be used to make a food product a functional, or health-promoting product, which was surprisingly found in the invention. Free SH groups also have therapeutic properties, such as alcohol-enhancing mucosal damage of the food-15 digestive tract (Loguercio C. et al., Gut 34 (1993) 161-165).

Free sulfhydryl groups are added to the product to be prepared, based on the amount of total protein in the product, for example 0.5-60 μιηοΐ / g protein, preferably about 20-5-20 μιηοΐ / g protein, before conversion. Experiments have shown that when there is at least a certain amount of free SH groups, it is found in the product as a metallic aftertaste. The cysteine SH groups had a palatability threshold of 30 ppm or “* 25 μπιοΐ / ΐ”. None of the tasters tasted this amount as a aftertaste in skimmed milk, unflavoured yogurt or low-fat filet. * * ·: SH groups derived from 25 yoghurt-modified whey proteins did not yet taste at 30 μηιοΐ / g protein. However, for example, subsequent treatment of ·· ·: V product, such as during sterilization, may result in the formation of by-products which react with the free SH groups to reduce them, while also lowering the final SH taste threshold. This can already be taken into account when designing the number of free SH groups in 30 basic products.

• m · * · • m m ·

Yoghurt production in the traditional way from skimmed milk (about 0.05% fat) • · ***** usually requires concentration of the milk by evaporation, increasing the dry matter by 2-3 percentage points, the same amount as about 2% (about 20%). : 35 g / 1) Addition of skimmed milk powder gives.

m • *

According to a preferred embodiment of the invention, the same effect is achieved by adding 0.8-1.6 percentage points (8-16 g / l) of a mixture of whey protein modified with skimmed milk powder and whey protein powder 118507 in protein ratio of 10-20% of modified whey protein and 80-90%. percent whey protein concentrate or equivalent or a portion of, for example, soy protein powder or other protein preparation. If it is desired to make low-fat yogurt with a vegetable oil supplement, 5 oils (for example 0.5-1.0%) are added at this point. Smaller amounts, about 0.6 to 1.0 percentage points of protein, are needed to strengthen the structure of low fat milk.

The modified whey protein is of the same composition as the unmodified whey protein. In the modification, some of the disulfide bonds it contains have been opened and formed free SH groups. In the modified whey protein, the amount of free SH groups is generally about 65-85 μιηοΐ / g protein, preferably about 75 μηιοΐ / g protein, as used in the examples below.

The mixture may be slightly homogenized, for example at 50 ° C and 100 bar, to ensure uniform distribution of the ingredients. When adding 0.5 to 1.5% fat or oil for nutritional reasons, for example, rapeseed oil, olive oil, sunflower oil, linseed oil or similar health-promoting product, homogenization is carried out at, for example, 55-60 ° C and 150-200 bar to emulsify the oil less than 1 μηι: η into uniform spheres or droplets.

After homogenization, the milk is pasteurized, for example, at 75-80 ° C for 5 to 10 minutes to 4 · 4 noodles to effect an exchange reaction.

• · • · ·· «• 4 • 44 ··· | After the heat treatment, the milk is cooled by inoculation and acidification * to 42-45 ° C. The acidification lasts for 3 to 6 hours and depends on the temperature and the bacteria used as the acid. The acidification of the yoghurt is stopped at a pH of about · ♦ · 4.3-4.6, from which it decreases slightly during cooling and storage. The structure / gel of the yoghurt is strongest at pH 4.65 and has a viscosity of up to ····: 30.

• * 444 4 4 • ·

«I •

After fermentation, the yoghurt is cooled to below 20 ° C and mixed gently.

• · * ···] The yogurt is packaged in goblets or cans and cooled to a storage temperature of 6-8 ° C.

:! *: 35 ···

According to one embodiment of the invention, the preparation of the fat-free file is possible from milk treated as described above. In a fat-free file, the structure usually remains weak and easily wakes up. Strengthening the structure by using the 118507 11 exchange with the whey protein supplement yields a structurally durable file that does not germinate. Healthy unsaturated oils, such as rapeseed, linseed, or Camelina oil, which are emulsified by modified and exchanged whey protein as a result of homogenization, can be added to the file. Viil's own acid works at 20 ° C 5 and needs time to acidify overnight or 12-14 hours. The surface of the filament develops the usual white and velvety Geoth-richum white homogeneous plant derived from the ferment of the filament.

Usually the basis for making puddings is milk, to which sugar and flavoring ingredients are added, as well as protein for thickening, gelatin or whey protein, plus pectin, starch / modified starch or carrageenan. According to another preferred embodiment of the invention, the use of modified whey protein in puddings provides a nutritionally valuable protein supplement which serves to strengthen the structure under moderate heating and can dispense with commonly used thickening and stabilizing agents such as gelatin or carrageenan.

Protein spreads can be prepared on a milk base such as acidified milk to which fat such as margarine, whey protein, spices and thickeners are added. According to another preferred embodiment of the invention, the addition of the modified whey protein can strengthen the structure of the spread and provide a nourishing protein. ·. line dose in addition to reinforcing the structure. At the same time, other thickeners, such as carrageenan and derivatives, commonly used for this purpose. * "'Sandwich powder, may be reduced or eliminated.

* «»: 25 * * In the prior art, cysteine is commonly used in the manufacture of dough to accelerate kneading: V and to reduce the amount of energy required to mix • * ·. Kneading the dough, that is, mixing the dough effectively, mechanically opens the gluten disulfide bonds of the wheat flour. The addition of cysteine facilitates: * · '* 30 exchange reactions to open disulfide bonds and soften and loosen dough; which is necessary for the final structure of the bread.

Dough refers to any known dough used in baking or in the preparation of food * *, for example, for making bread, pastries, and the like. Wheat flour is preferably used to make the dough structure, since wheat has enough gluten to maintain the structure. Other flours such as rye, barley or oat flour may be used in addition for nutritional or taste reasons.

118507 12

The use of cysteine is generally limited to 70 ppm. The application rate of cysteine is 35-70 ppm depending on the hardness of the wheat / flour. Overdose produces sticky and difficult to handle dough. The nutritional value of the cysteine used is low.

5

Strengthening the dough structure with modified whey protein is very successful. There is no risk of overdose in SH groups and the protein added has nutritional significance. Whey protein powder, protein content 75%, has been used in the dough as an addition of 2-4% of the amount of flour. The results obtained there have been gear-10 algae depending on the treatment. Modified whey protein has been used at an additional 1.5% of the amount of flour as protein and has had a positive effect on the dough properties.

The following examples and related tests illustrate the above invention as applied to various food preparation processes using modified whey protein having a free SH group of about 75 µιηοΐ / g protein. However, it should be noted that any other suitable non-synthetic protein may also be used.

Modified whey protein is used in food preparation processes to provide a protein structure reinforcement by forming a space network. It also acts as an emulsifier in the same way, digesting the unaltered whey protein in the digestive tract as a result of conversion t i »IV.t, and it 1 * *; ' is one of the best proteins of nutritional value.

• · ·! · Ί * 25 '* Example 1 ·· · • · »» ft · «· ·

Non-fat yogurt was prepared in three test samples of different composition and one control sample. Everything was treated in the same way.

: * · *: 30 • ·; ***. The control sample contained 980 ml of skimmed milk and 20.0 g of skimmed milk powder.

• · t

The test samples contained 920 ml and 80 ml protein mixture of skimmed milk. Experimental samples differed in their amount of modified whey protein.

• ·: 35 Test sample 1 contained 27 ml of modified whey protein concentrate with 12% protein content and 53 ml of unmodified whey protein concentrate with 12% protein content. 80 ml of the protein mixture contained 9.6 g of protein.

118507 13

Test sample 2 contained 20% modified whey protein concentrate or 16 ml and 64 ml unmodified whey protein concentrate.

Test sample 3 also contained 16 ml of modified whey protein concentrate and 64 ml of unmodified whey protein concentrate. This protein mixture was heated / pasteurized at 78 ° C for 5 min.

Control and test samples were pasteurized at 78 ° C for 1-2 min and cooled to 45 ° C. The acid was added at this temperature and yogurt acid 0.30 g / l 10 (Yo-Mix VM 1-34; Danisco Cultor) was used as the acid. The acidification lasted at 45 ° C for about 7 hours, at which time the samples reached pH 4.4-4.5 . Samples were cooled to 5-7 ° C, processed, packed in goblets and refrigerated for 1 day prior to assays. The samples were measured for viscosity and subjected to sensory evaluation, which included appearance, smell, texture, taste and mouthfeel.

The viscosity of the samples was measured with a Haage Visco-Tester 7R (spindle R4, 50 rpm) 1-2 days after preparation.

The viscosities of the samples were: 20 ____

Reference sample Test sample 1 Test sample 2 Test sample. . · .___ 1_ .1.1 :: 1 3300 mPa 3210 mPa 3130 mPa 2280 mPa • ·

* · «...... 1 1 1 J

• i · • »· ·« · · · ·

Organoleptic evaluation; a scale of 1-5; 5 Reviewers • · 404 4 4 14 1 1 8507

Test sample 3: a smooth, thinner, slightly acidic aftertaste.

Example 2 5

Fat-free yoghurt was prepared with two test samples and one control sample. All samples were treated in the same way.

The control sample contained 980 ml of skimmed milk and 20.0 g of skimmed milk powder. The test samples contained 920 ml of skimmed milk and 80 ml of protein mixture. 80 ml of protein mixture contained 9.6 g of protein. The same amount of modified whey protein was added to both test samples. Dehydroascorbic acid was added to test sample 1 to oxidize free SH groups to disulfide groups.

The 80 ml of the protein mixture added to the test samples contained 15% or 12 ml of modified whey protein concentrate and 85% or 68 ml of unmodified whey protein concentrate. Both concentrates had a protein content of 12%.

Control and test samples were pasteurized at 80 ° C for 3 min and cooled to 42 ° C. 20 Test sample 1 was supplemented with 50 mg / L of DHAH (dehydroascorbic acid), either purchased ready (Sigma) or prepared as directed (Tolbert, BM & Ward, JB 1982 Adv. Chem. Ser. No. 200, p. 101- 123) and held for 30 min with stirring at 42 ° C and allowed to react before addition of the acid.

»·· • · • m m | Yo-Mix VM 1-34 (Danisco Cultor) was used as the acid. The acid was activated by adding 20 g of melted acid to 200 ml of pasteurized milk at 78 ° C for 3 min and chilled to 42 ° C and incubation for two hours. The activated acid was added to 3.0 µl per liter of yogurt milk.

· * · *: 30 The milk was acidified at 42 ° C until the pH dropped to 4.3. The time required for acidification • · was 4.5 hours. All samples reached the required acidity almost simultaneously, i.e. acidification occurred at the same rate in all samples.

• * • * * ·· Samples were cooled to 4 ° C, homogenized by mixing, packed in · · · · 35 goblets and refrigerated. The samples were measured for viscosity with Haage Visco- ··· ....: Tester 7R (spindle R 4 50 rpm) 1 day after preparation.

118507 15 The viscosities of the samples were

Reference sample__Sample 1__Sample 2_ 3300 mPa 2300 mPa 2300 mPa

The taste of the test samples was sour and there was no pungent metallic aftertaste.

5

Example 3

Non-fat yogurt was prepared in three test samples and one control sample. The control-10 sample contained 980 ml of skimmed milk and 20.0 g of skimmed milk powder. The test samples all had the same composition, 920 ml of skimmed milk and 80 ml of the protein mixture, the proportion of whey protein converted from inine was 15%, that is, 12 ml of the protein mixture of 12%. All samples had a different heat treatment.

The control sample was pasteurized at 90 ° C for 15 min, test sample at 80 ° C for 5 min, test sample 2 at 80 ° C for 10 min and test sample 3 at 80 ° C for 15 min. All samples were cooled to 42-45 ° C.

The acid was added to the yoghurt milk at 42-45 ° C. The acid used was Valio Oy's finished product. 20 of this unflavoured yogurt. It was added at 4% or 40 g / L. The yoghurt was acidified to an acidic pH of 4.6. The acidification time was 4-5 hours.

«M

• 9 • 9 9 :, i | The samples were cooled to 4 ° C, homogenized by mixing, and packed in 2 dl goblets. The cups were stored in a refrigerator at 4 ° C.

IV 25 999 Time of acidification of samples until pH 4,6: ·) Comparative sample__Sample 1__Sample 2__Sample 3_: ***: 5 h 4 h 20 min 4 h 15 min 4 h 30 min

999 * - 1 * iJ

9,999 9,9 ***. The samples were measured for viscosity with Haage Visco-Tester 7R (spindle R 4 at 50 rpm) and * 30 was subjected to organoleptic evaluation 1 day after preparation.

9 9 9 9 9 9 9 999 9 99999 9 9 16 118507 The viscosities of the samples were

Reference sample__Sample 1__Sample 2__Sample 3_ 3900 mPa__3600 mPa__3000 mPa__3800 mPa_

Organoleptic evaluation; a scale of 1-5; 5 Reviewers: 5 __i_ii_i__ Sample__Outdoor Odor Structure Taste Fouling Mean

Reference sample 4.9__4.1 4.1__3.6 4.0__4 ^ 2_

Test Sample 1 4.8__4.2 4.1__4.0 3.9__4j2_

Test Sample 2 4.8__4.1 4.2__4.3 5.0__4.5

Test Sample 3 4.6__4.1 3.8__4.2 4.2__4 ^ 2_

Verbal description

Reference sample: smooth, sour, milky-like, not fresh, stretchy

Test Sample 1: Smooth, smooth, strongly sour, milky, fresh, soft, 10 pungent aftertaste

Test Sample 2: Smooth, smooth, strongly acidic, milky, fresh, soft

Test sample 3: smooth, smooth, sour, milky-like, fresh, sour.

• 15 Example 4.

»» · <«« Two non-fat yoghurt samples and a control sample were prepared. The control sample contained 980 ml of skimmed milk and 20.0 g of skimmed milk powder. Test samples [. the composition differed only slightly. Test sample 1 contained 11 g / l of freeze-dried 20 protein blends with 15% of the total protein converted to 9.6 · · * ··· * g. Test sample 2 contained the same amount of protein mixture per liter but was first dissolved in 80 ml water and added to the milk per liter.

The control sample was pasteurized at 90 ° C for 15 min. Test sample 1 and 2 were pasteurized at 80 ° C. 25 15 mins All samples were cooled to 42-45 ° C.

• * * · ». Unflavoured yoghurt made by Valio Oy was used as the acid. It was added to 4% or 40 g / l of yoghurt milk cooled to 42-45 ° C and acidified to pH 4.5 *: **: acidity. The acidification time was approximately 4 hours.

The samples were cooled to 4 ° C, made homogeneous by mixing and packed in 2 dl goblets. The cups were stored in a refrigerator at 4 ° C.

Of test samples 1 and 2, a portion was passed through the homogenizer without pressure. The viscosities from the original 5 and through the homogenizer (test sample 1 / w and 2 / w) were measured with Haage Visco-Tester 7R (spindle R4 50rpm) and subjected to sensory evaluation 1 day after preparation.

The viscosities of the samples were: 10 ______

Comparative sample Test sample 1 Test sample 2 Test sample 1 / p Test sample 2 / p 4400 mPa 3900 mPa 3700 mPa 1150 mPa 1900 mPa

Organoleptic evaluation; a scale of 1-5; 5 Reviewers: Sample__Outdoor Odor Structure Taste Nudge Mean

Comparative Sample 4.4__4.4 3.9__4.2 3.9__4 ^ 2_

Test Sample 1 4.8__4.4 4.3__4.1 4.2__4 ^ 4_

Test Sample 2 4.8__4.2 4.4 4.2 4.2_4.4. Test sample 1 / p 5.0__4.4 3.6__3.6 2.8 __ ^ 9_. · 1 ·. Test specimen 2 / p 5.0__4.2 4.4__4.4 4.1__4 ^ 4_ '"I1 15 • t« ··· ·. There was no significant difference in the thickness of the structures of the test specimens. The test specimens were found to be even thicker than the reference yoghurt. The structure was similar in each sample • 1 * ··· 1, slightly elongated and thick.

20 H · ί 1. · 'The test sample 1 / p was slightly watery in structure and a slight taste was observed in the taste.

The test sample of 2 / p was considered to be of appropriate thickness, the mouth was soft yogurt. ···. timid with a fresh taste. According to the evaluation summary, the sample was one of the best in the series.

t · ··· § · «• · t • · i * ·« · 118507 18

Example 5

The fat-free file was prepared with three test samples and one control sample. The control sample contained 980 ml of skimmed milk and 20.0 g of skimmed milk powder. The test samples 5 contained 920 ml of skimmed milk and 80 ml of the protein mixture, of which 15% or 12 ml of the protein mixture having a protein content of 15% was modified. The remaining 85% was unmodified whey protein concentrate.

All samples were pasteurized at 78 ° C for 1-2 min. After pasteurization, DHAH (dehydroascorbic acid) was added to test samples 1 10 and 2. Test sample 1 was cooled to 42 ° C, 25 mg / liter DHAH was added and the temperature was maintained for 30 min. Test sample 2 was cooled to 35 ° C. DHAH was added at 50 mg / liter and held for 30 min. All samples were finally cooled to 20 ° C.

The acid was added to 20 ° C filed milk. 5% or 50 g / liter of an unflavoured file made by Valio Oy, containing 1% fat, was used as the acid. The mixture was stirred well and dispensed into 2 dl goblets and acidified overnight at 20 ° C. After acidification, the finished file was transferred to a refrigerator at 4 ° C.

20 Organoleptic evaluation; a scale of 1-5; 5 reviewers:. , ·, Sample__ Appearance Odor Structure Taste Nausea Mean

Comparative sample 4.0__5.0 3.0__4.2 4.3__4l_

Test Sample 1 4.0__5.0 3.0__4 ^ __ 4 ^ 3__4J_ "** i Test Sample 2 4.0 5.0 4.0 4.4 4.5 4.4 ··« ·· ...... ' .

. * Test Sample 3 4.0 5.0 4.5 4.3 4.5 4.5 * · · --Λ. -LLIT. ..... ”^ - • · • ·

• «·. [------- I I 1 .......— 1 I

• · • · »· ·

There was a slightly uneven distribution of Geothrichum- * »• V mold growth on each goblet. The structure was solid in all samples and did not detach upside down:]]]: 25 inverted cups.

• · · • * • ♦

Comparative sample: matt surface, granular, loose electricity, good taste; ♦ * Test sample 1: surface matt, granular, loose, good taste; ί.:. Σ Test Sample 2: Surface glossy, spoon well remains relatively good, *: **: 30 taste good; 118507 19

Test sample 3: surface glossy, best of samples, puncture, well remains, good taste.

Example 6

Two test samples were made of non-fat yogurt. The amount of protein added to the test samples was 10 g protein / liter in sample A and 13 g protein / liter in sample B. The protein mixture was prepared so that 80 ml contained about 10 g of protein. Of that, 15% (12 ml) containing 1.5 g protein was modified whey protein (Lot P75) and 85% (68 ml) containing 8.5 g protein was whey protein concentrate (Juustokaira Oy, Kuusamo). The solution prepared at these weight ratios was freeze-dried to a powder. 12 g of this powder was needed for a 10 g protein supplement.

Sample A weighed 12 g / liter of protein blend powder and Sample B weighed 15.6 g / liter. The protein blend powder was thoroughly mixed with the skimmed milk to a liter. The powder mixes well and dissolves well in milk at room temperature. The test samples were then pasteurized at 80 ° C for 15 min and cooled to 43 ° C.

20 Unflavoured yoghurt produced by Valio Oy was used as a fermentation 4% (0.5 liter can, Tampere). The acidification occurred at 43 ° C. After 4.5 hours the samples had reached pH 4.6 and the acidification was stopped. Samples were cooled to 20 ° C, modified · · · !!! stirring and transferring to the refrigerator.

• «··· 118507 20

According to sensory evaluation, the samples were almost similar; the texture was smooth and thick and the taste velvety soft.

5 Example 7

Non-fat yogurt was prepared in three test samples. The amount of protein mixture added to the test samples was Sample A 10 g protein / liter, Sample B 12.5 g protein / liter and Sample C 15.0 g protein / liter. The protein mixture was prepared by mixing whey protein 10 powder, 75% protein (Juustokaira Oy, Kuusamo) and modified whey protein (Batch P75) freeze dried to a ratio of 85% whey protein powder to 15% modified whey protein powder. To 10 g of protein, 13.0 g of the mixture was needed.

The amount of protein mixture weighed into sample A was 13.0 g / liter, sample B to 16.2 g / liter and sample C to 19.4 g / liter. The protein mixtures were mixed thoroughly with skimmed milk per liter. They were mixed and well soluble in milk. The samples were then pasteurized at 80 ° C for 15 min and cooled to 43 ° C.

20 Unflavoured yoghurt produced by Valio Oy was used as a fermentation 4% (0.5 liter can, Tampere). The acidification took place at 43 ° C for 4 hours at which the samples had pH. 4.6. The samples were cooled to 20 ° C, modified by stirring and transferred to ice · * φ. ···. a cabinet.

a m m • »* · · Φ · ·" [I 25 SH-groups were determined / µmol / g protein with Ellman's reagent:

Koenaytc / SH ftmol / g Protein A__B__C_ • · ........... ^ * ··· * Skimmed milk + protein mix__12,7_ 13,4__14,0

After pasteurisation 14.8 15.5 18.6 ·· * L ~ "1, I I I --- · - • ♦ · • • • Φ · ·

Test samples were tested for viscosity after one day of preparation. At 20 ° C on a Brookfield DV 1 Viscometer (Spindle 3, RPM 12).

!!!!: 30 • ·. The viscosities of the samples were · · · «· · · ·

Test specimen / viscosity__A__B__C__ mPas 3700 3800 4000 118507 21

All samples were uniform and solid in structure; the taste was pleasantly fresh and velvety soft.

5

Example 8

Non-fat mixed yoghurt was prepared in three test samples. Sample A protein supplement contained 12.5 g / liter of whey protein, Sample B protein supplement contained whey protein and soy-10 and total protein 12.5 g / liter, of which soy protein was 10% and Sample C protein contained whey protein and soy protein 12.5 g / liter, of which soy protein was 20%. 12.5 g of protein supplement was weighed 14.7 g / liter of whey protein powder. This was the same freeze-dried whey protein powder as in Example 6.

The 10% protein supplement contains 1.25 g protein. This amount of protein required 1.8 g of soy protein (DANPRO S-900 TS; Central Soya).

Sample A weighed 14.7 g / liter of freeze-dried whey protein powder, Sample B weighed the same whey protein sample 13.2 g / liter and soy protein powder 1.8 to 20 g / liter and Sample C weighed 11.8 g / liter and 3 milligrams of soy protein , 6 g / liter. The protein powders were mixed and added to the non-fat. to one liter of milk, mixing well. The protein mixture was mixed and well solubilized · · · ·. toon. The samples were then pasteurized at 80 ° C for 15 min and cooled to 44 ° C.

• ♦ »25 ... Unflavoured yoghurt made by Valio Oy was used as a sour cream 4% (0.5 • ♦ ♦: Mf liter can, Tampere). The acidification was carried out at 43 ° C for 4.5 hours and the pH of the samples * ··· * ranged from 4.55 to 4.60. The samples were cooled to 20 ° C, modified with stirring and transferred to a refrigerator.

fv 30

Experimental samples were assayed for SH groups, μ mol / g protein with Ellman's reagent: * ··· • · _____ • · I 1 _.

Test Sample / SH jimol / g Protein A__B__C_. Skimmed milk + protein mix 15.2 15.0 14.8

After pasteurization 17.3 17.2 15.3 • *

The test samples were tested for viscosity after one day of manufacture at 35-10 ° C on a Brookfield DV 1 Viscometer (Kara 3, 12 rpm).

118507 22 The viscosities of the samples were

Test sample / viscosity__A__B__C_ mPas 9400 9000 8500 5 Sample A: solid and heavy structure; the taste is pleasant, fresh and soft.

Sample B and C: solid and flat structure; the taste is mild soy in both, but not disturbingly intense.

Example 9

A non-fat yogurt was prepared as a control sample and three test samples. The protein supplement in the control sample was gelatin. The amount of test sample 1 and protein supplement was 10 g / liter and sample 2 and 3 12.5 g / liter. In samples 1 and 2, the freeze-dried protein blend used in Example 15 was used as a protein supplement, and in sample 3, the modified whey protein powder and whey protein concentrate powder mixture used in Example 7 was used.

Sample 1 was weighed in a lyophilized protein mixture at 12.0 g / liter and in 2 at 14.7: · 20 g / liter. Sample 3 weighed 16.2 g / liter of whey protein powder mixture. Comparative sample- ···. * ··. gelatin was weighed at 4 g / liter (Extrago). The protein blend powders were mixed thoroughly: *! ·. per liter of skimmed milk. The protein mixture added to Sample 1 and Sample 2 was quite soluble in cold milk. The protein mixture added to Sample 3 was dissolved in a warm • ♦ ... milk (20-30 ° C) well. The control sample gelatin was mixed with milk at a temperature above 50 ° C. The samples were then pasteurized at 80 ° C for 15 min and cooled to 42 ° C.

·· ·: V Jo-Mix VM 1-30 (Danisco Cultor) yoghurt acid 5 g / liter was used as the yeast.

The acidification occurred at 42 ° C and lasted 3 hours 30 minutes with pH in the control

. ··. The samples were cooled to below 20 ° C, processed by stirring and transferred to a refrigerator.

The samples were measured for pH and viscosity two days after preparation and subjected to organoleptic evaluation. Viscosity was measured on a Bohlin Visco (V) 88 o 30; system 3, speed 1.

118507 23 Sample__pH__Viscosity mPas_

Vertailunäyte__4,32__1413

Test Sample 1__4,33__1467_

Test Sample 2__4,36__1635_

Test Sample 3__4,36__1547_

Organoleptic evaluation: 5 Comparative sample: no waking surface, smooth, thick texture

Test Sample 1: no waking on the surface, thicker control sample, smooth, no bitter taste

Test sample 2: no waking on the surface, thick texture, flaky, no side taste

Test sample 3: does not wake on the surface, thick texture, flaky, no off-taste 10

Example 10

The wheat flour was baked into a dough containing modified whey protein and the like as a control dough without modified protein. The stretchability of the doughs was compared to that of the dough. Elasticity was measured by extensograph.

: The control dough contained 300 g of wheat flour and 2 g of salt dissolved in some water and 212 ml of water. The test dough contained 300 g of wheat flour and 2 g of salt dissolved in ··· water, 211 ml of water and 4.5 g of modified whey protein. Converted whey protein. ". · 20 linins were 1.5% by weight of flour.

Ta · · «· • I I I dough was prepared according to the test dough recipe. The problem with making the comparison dough ***** was that the dough was sticking to the collar. As a result, a small amount of flour had to be used on the surface of the dough ball. An equivalent amount of flour was also added to the surface of the test • · ·:. During the assays, differences in the dough balls could be observed; the control dough was softer and more tacky and harder to handle than the experimental. ***. a dough that was more elastic and less sticky.

• The following table shows the extensogram key figures with reference and test dough.

«1 1 • · t • 1 · 118507 24

Trial and control dough extensogram key figures

The test dough contained 1.5% modified whey protein and 0.7% salt in the flour press and the control dough contained 0.7% salt in the flour.

5

KEY INDICATOR ALSO AS EXPERIMENTAL__ COMPARISON

Setting time (min) __ 45__90__Π5__45__90__135

Elongation A (mm) __ 205__171.5 178__241__205 178

Stretch Resistance B (BU) 380__570__600__305__472.5 515

Area (cm2) __ 98.3 118.9 107.5 82.7 107.5 103.8 B / A_ 1.85 3.32 3.37 1.27 2.30 2.89

Sensory evaluation Dough more elastic and Dough softer and __ shorter_ stretchy_

On the basis of the extensogram results, there are clear differences between the doughs. Ve-10 had a lower depth of test dough than the control dough and had a greater elongation resistance and area. The B / A ratio was higher for the test dough than for the control dough. The test dough was also organically more elastic, shorter and stiffer than the control dough.

From these results, it can be said that the modified whey protein had a clear effect on the dough strengthening dough with 1.5% modified whey protein ··· · stronger and stiffer. The extensiograms took the following forms:. · Such that they anticipate good bread volume potential.

Some embodiments of the invention have been described above. The invention is not limited to the solutions just described. For example, the method may be applied to other proteinaceous products and foods other than those mentioned above. The inventive idea can be applied in numerous ways within the scope of the claims.

* «* • · * • · · •« »« ·· «* ·« · · * *

Claims (18)

118507 A method for strengthening the structure of proteinaceous products during the heat treatment of the product by forming disulfide bonds between the proteins, wherein the proteins form a space network, characterized in that the method comprises the step of: adding to the product a modified protein which is modified by opening at least one a disulfide bond to obtain free sulfhydryl groups, and 10. heating said product at a pasteurization temperature of up to 80 ° C for up to 15 minutes to effect an exchange reaction with said free sulfhydryl groups, wherein said structure-reinforcing disulfide bonds are formed between proteins. Process according to claim 1, characterized in that the protein is modified by contacting it with a sulfite ion-forming reagent to sulfonate the protein. Process according to Claim 2, characterized in that said sulfite-fitting-forming reagent comprises an alkali metal or an alkaline earth metal sulfite, hydrogen sulfite or metabisulfite or combinations thereof. • • * · I " The method according to any one of the preceding claims, characterized in that the free sulfhydryl groups are present in the total protein of the product prior to conversion: from 0.5 to 60 pmol / g of protein. • ··. A method according to any one of the preceding claims, characterized in that: *** the modified protein comprises any edible protein. A method according to any one of the preceding claims, characterized in that: ···. that the modified protein comprises whey protein. M » The method according to any one of the preceding claims, characterized in that the modified protein comprises a soy protein. ·! 35 The method according to any one of the preceding claims, characterized in that the proteinaceous product is food, feed or pet food. 118507 A method according to claim 8, characterized in that said food product is yogurt, filet, pudding, spread, other dairy product or dough. 10. A proteinaceous product comprising a space network of proteins formed by interprotein disulfide bonds that reinforce the structure of the product of heat treatment, characterized in that said protein space network is created by adding at least one disulfide-modified protein to the product prior to heat treatment. to obtain sulfhydryl groups, wherein said structure-reinforcing disulfide-10 bonds are formed by an exchange reaction provided by said free sulfhydryl groups as a result of heat treatment at a temperature of up to 80 ° C for a maximum of 15 minutes. A proteinaceous product according to claim 10, characterized in that said protein is modified by contacting it with a sulfite ion-forming reagent to sulfonate the protein. Protein-containing product according to claim 11, characterized in that said sulfite ion-forming reagent comprises sulphite, hydrogen sulphite or metabisulphite of alkali metal or alkaline earth metal or combinations thereof. A proteinaceous product according to any one of claims 10 to 12, characterized in that the free sulfhydryl groups are present in the total protein of the product prior to substitution / conversion to 0.5-60 pmol / g protein. ί.Γ: 25 A proteinaceous product according to any one of claims 10 to 13, characterized in that said modified protein comprises any edible protein. · * · .. 30 A proteinaceous product according to any one of claims 10 to 14, characterized in that said modified protein comprises whey protein. A proteinaceous product according to any one of claims 10 to 15, characterized in that said modified protein comprises soy protein. A proteinaceous product according to any one of claims 10 to 16, characterized in that said product is a food, feed or pet food. 1 8507 Protein-containing product according to claim 17, characterized in that said food product is a yoghurt, filet, pudding, spread, other dairy product or dough. 5