FI129256B

FI129256B - Method for modification of whey and plant proteins and use of modified proteins in microencapsulation and films

Info

Publication number: FI129256B
Application number: FI20185303A
Authority: FI
Inventors: Jouko Savolainen; Anne Kallioinen
Original assignee: Uniq Bioresearch Oy
Priority date: 2018-03-29
Filing date: 2018-03-29
Publication date: 2021-10-15
Also published as: WO2019185991A1; FI20185303A1

Abstract

The disclosure relates to a method for the modification of whey and plant proteins. Furthermore, the invention relates to the use of modified proteins in the production of microencapsulated compositions and films.

Description

METHOD FOR MODIFICATION OF WHEY AND PLANT PROTEINS AND USE OF MODIFIED PROTEINS IN MICROENCAPSULATION AND FILMS

TECHNICAL FIELD The invention relates to a method for the modification of proteins and, especially to the modification of whey and plant proteins. Furthermore, the invention relates to the use of modified proteins in the production of microcapsules and film coatings and for nutritional purposes.

BACKGROUND Proteins are biopolymers that consist of amino acids connected to each other. They are necessary components in healthy nutrition and used in various food products. Whey protein is a nutritionally superior protein, especially due to its’ high methionine and lysine contents. It is a side product obtained from cheese manufacture. Plant proteins, such as proteins from soy beans, pea or other legumes and rice protein are interesting alternatives for consumers, and especially suitable for vegetarians. Most of proteins contain disulphide bridges between amino acid cysteine residues which affect the properties and secondary structure of proteins. Functional properties of proteins can be modified by various chemical reactions such as reduction, acylation or hydrolysis (Nesterenko 2012; Ruan et al., 2014), but many of these reactions are not suitable for food applications. The properties of whey protein and soy protein isolate can also be modified by food grade chemicals in sulphitolysis as described in US 6797810. The modified whey proteins contain typically two to four free SH-groups, whereas the unmodified whey proteins contain on average less than one sulfhydryl group. The modification results in several advantageous properties such as improved formation of protein network, emulsion and gel, as well as improved digestibility and antioxidant = 25 capacity. For soy, modification also results in decreased trypsin inhibitor activity. N In microencapsulation the active ingredient or the core is shielded by the shell material. = Microencapsulation can be applied for different purposes: to protect the sensitive core N substances from surroundings, to develop controlled release of substances, to mask E unpleasant tastes and odors, or to transform liguid substances into solid particles. Whey 2 30 and plant proteins have been applied as a shell material in microencapsulation of various 3 substances such as oils, vitamins and probiotic bacteria (Nesterenko et al., 2012, 2013; > Ying et al., 2010). These proteins can also be used in combination with other shell materials such as starch or glucose syrup. The limitations of existing microencapsulation methods are the weak, permeable shells that do not provide adeguate stability to the microencapsulated product. If carbohydrates are combined with proteins in shell, formation of harmful Maillard reaction products, such as carsinogenic substances, is likely during heat treatments in microencapsulation process. Use of vegetable protein for making high added value products has been limited by the extraction cost to obtain high-quality proteins, low solubility of some proteins and large polydispersity in the size of naturally occurring protein chains.In patent application WO2009/050333 modified whey protein was applied in the microencapsulation of liposomes from bovine or pig and ubiguinone. The whey protein was modified according to US 6797810 and the modified whey protein was mixed with whey protein concentrate. Patent publication WO 2015/019307 describes a method to microencapsulate probiotics. The microcapsules consisted of chick pea, pea, soy, faba bean, or lentil protein which was mixed with k-carrageenan, genipin or alginate as a croslinking agent. The protein- crosslinking agent mixture was mixed with bacterial suspension and then mixed with canola oil to obtain emulsion. In FI20031508 modified whey protein was applied in formation of emulsion, films and microcapsules. Although in the disclosures described above the modification of proteins is carried out, the modification process as a whole still has stages that prolong processing time and complicate the method. Therefore, despite the ongoing research and development of processes and methods for the modification of proteins, there is still need to provide improved method where particularly whey and plant proteins are modified.

SUMMARY OF THE INVENTION An object of the present disclosure is to provide a method and uses to alleviate the above disadvantages. Disclosed herein are improved processes to modify whey and plant protein by sulphitolysis. In addition, their application in microencapsulation and in films are © 25 presented. The object of the disclosure is achieved by a method and uses which are N characterized by what is stated in the independent claims. The preferred embodiments of g the disclosure are disclosed in the dependent claims. N The disclosure is based on the idea of modifying whey or plant protein by bringing the E proteins in contact with a reagent that forms sulphite ions to sulphonate a part of the © 30 proteins thiol groups. After removing sulphonate groups, free sulfhydryl groups are D obtained in protein. Modified protein is used for preparing microcapsules and protein- = based films. Disclosed herein is a method for the modification of whey or plant protein, wherein a) a whey or plant protein concentrate containing 9-12% by weight of protein is brought into contact with a reagent containing 0.02-0.20 M of sulphite for 20 to 40 minutes at a temperature of 40-70°C and a pH of 5.5-7.0 to sulphonate the protein without using oxidizing agent to form a protein suspension; and b) the pH of the protein suspension is adjusted to pH 4.5-5.0; and c) the pH of the protein suspension is adjusted to and kept at pH 2.0 for 5-20 minutes with mixing; and d) the pH of the protein suspension is adjusted to pH 4.5-5.5 and the suspension is washed and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and d) the protein suspension is dissolved by adjusting the pH to pH 5.0-6.0; and e) the obtained modified protein is spray dried at an inlet and outlet temperature of 170- 180°C and 70-80°C, respectively. In an embodiment, the protein is whey protein and the whey protein is recovered by ultrafiltration. In another embodiment, the protein is plant protein and the plant protein is recovered by filtration or centrifugation. A plant protein may be pea protein, rice protein or soy protein. The plant protein may comprise a mixture of pea, rice and soy proteins. In an embodiment, in the above-mentioned step a) the contact time is 20-40 minutes, preferably 30 minutes. In an embodiment, the protein suspension is after the above-mentioned step b) washed and recovered by ultrafiltration, filtration or centrifugation to remove extra sulphite and salts. In an embodiment, in the above-mentioned step c), wherein the pH of the protein suspension is adjusted to pH 2.0, the contact time is 5-20 minutes, preferably 15 minutes. 00 > Disclosed herein is also a use of the modified protein, obtainable by the above disclosed & 25 modification method of whey or plant protein, for preparing a microencapsulated o composition, comprising

N I a) preparing an aqueous protein solution comprising protein concentrate or isolate, and * said modified protein at a ratio from 4:1 to 3:1, and 0.1-1.0 % ascorbic acid, and adjusting

O 3 pH to pH 6.5 and temperature to 45-65°C; and 0 5 30 b) preparing a mixture comprising oil and a phospholipid, preferably lecithin, at ratio from N 110:10 to 110:20, and 0.05-0.2% tocopherol, and adjusting temperature to 45-65°C; and c) combining the aqueous protein solution obtained at step a) and the mixture obtained at step b) to form a protein-oil composition, and adjusting pH to pH 6.5 and temperature to 50-65°C; and d) homogenizing the protein-oil composition to obtain an emulsion; and e) pasteurizing the emulsion at a temperature of 70-85°C for 5 minutes or less; and f) spray drying the emulsion at an inlet and outlet temperature of 170-180°C and 70-80°C, respectively. In an embodiment, the heating time at step e) is 15 seconds to 5 minutes, preferably 15 seconds. Disclosed herein is also a use of the modified protein obtainable by the above disclosed modification method of whey or plant protein, for preparing a protein-based film comprising a) providing an aqueous protein solution comprising 9-12% protein, wherein protein concentrate or isolate, and said modified protein are at a ratio of 3:1, 5-7% glycerol, and 1-2% sorbitol; and b) heating said aqueous protein solution to a temperature of 70-80°C and cooling said solution to room temperature; and c) forming said solution into said protein-based film, and evaporating water. Disclosed herein is also a microencapsulated composition comprising oil, phospholipid preferably lecithin, protein concentrate or isolate, modified protein, 0.1-1.0% ascorbic acid, and 0.5-0.5% tocopherol, wherein the modified protein is obtainable by the above disclosed modification method of whey or plant protein, wherein the ratio of protein concentrate, or isolate to modified protein is from 4:1 to 3:1, and the ratio of oil to phospholipid is from 110:10 to 110:20. 00 S In an embodiment, the oil is selected from the group of fish oil, sunflower oil, corn gromwell

N 0 25 oil, rape oil, rapeseed oil, olive oil, linseed oil, coconut oil, and seed oil from a berry such = as blueberry, cloudberry or sea buckthorn. In a preferred embodiment, the oil is corn N gromwell oil. = = Disclosed herein is also a protein-based film comprising 9-12% protein, wherein protein = concentrate, or isolate, and modified protein are at a ratio of 3:1, and 5-7% glycerol and 1- ® 30 2% sorbitol, wherein the modified protein is obtainable by the above disclosed modification Q method of whey or plant protein. In an embodiment, said protein-based film comprises 5% glycerol and 1% sorbitol.

The present invention improves the existing processes by many ways. An advantage of the method of the present disclosure is that the present process is simpler and processing time is decreased by 10% compared to the state of the art methods. In the state of the art methods, the sulphur dioxide gases that are blowed out of whey protein suspension are 5 toxic and bad smelling. In the present modified process modification of proteins is carried out without blowing sulphur dioxide gases by air flow, Thus, in the present novel improved process, process safety is increased, and harmful health effect decreased. The blowing step is very complicated step as modified whey protein is prone to foam formation even more than the normal whey protein. Without blowing step in the herein disclosed process, the use of antifoam agents can be decreased or even stopped. The present modification process increases the amount of free SH groups in protein. High concentration of free SH groups has positive effects on the safety of the food products providing healthy and functional properties to the product. The benefits of free SH groups are: toxins are made harmless and removed from the human body, microbial and plant based toxins, even aflatoxins, are neutralized. Furthermore, acetaldehyde and acrylamide are neutralized. Furthermore, free SH groups act as antioxidants, as free radical traps of highly reactive hydroxyl, nitroxyl and other radicals and as nitrite traps. In addition, free SH groups prevent Maillard reaction, prevent the formation of Amador compounds, and prevent enzymatic and non-enzymatic browning. In the present process formation of harmful Maillard, reaction products, such as carsinogenic substances can be avoided. Covalent bonds, which are based on free SH groups, are stronger than the bonds formed by other microencapsulation and film making materials thus resulting in more stable cores and improved shelf-life of the encapsulated products.

DEFINITIONS o 25 The term “microencapsulation” refers here to a process in which small particles or droplets > are surrounded by a coating to give small capsules, of useful properties. se Microencapsulation is used to incorporate food ingredients, enzymes, cells or other o materials on a micro metric scale. Microencapsulation can also be used to enclose solids, - liguids, or gases inside a micrometric wall made of hard or soft soluble film. a N 30 The term “microcapsule” refers here to a small sphere with a uniform wall around it. The 3 material inside the microcapsule is referred to as the core, internal phase, or fill, whereas 2 the wall is sometimes called a shell, coating, or membrane. Most microcapsules have N pores with diameters between a few micrometers and a few millimeters.

The term “spray drying” refers here to a continuous process to convert an initial liquid into a solid powder of microparticles. It is a dehydration process used to form a continuous matrix surrounding the active substances. The initial liquid (solution, emulsion or suspension) containing wall and core materials is sprayed into a stream of heated air. The solvent, typically water, is evaporated to give instantaneous powder production. Spray drying may be used in microencapsulation. The term “protein concentrate” refers here to a protein suspension or dry powder where protein content is below 80%. The term “protein isolate” refers here to a dry protein powder where protein content is 80% or above.

BRIEF DESCRIPTION OF THE DRAWINGS In the following the disclosure will be described in greater detail by means of preferred embodiments with reference to the accompanying drawings, in which Figure 1 presents a flow chart describing one preferable embodiment. A process for modification of whey and plant protein comprises an additional washing and recovering step to minimize the release of sulphur dioxide gas. Figure 2 presents a flow chart describing another preferred embodiment. A process for modification of whey and plant protein, without additional washing and recovering step, suitable for closed processes and processes with efficient ventilation to remove sulphur dioxide.

DETAILED DESCRIPTION It should be understood that although an illustrative implementation of one or more embodiments are provided below, the disclosed methods and processes can be implemented using any number of techniques. The disclosure should in no way be limited 0 to the illustrative implementation, drawings, or technigues illustrated below, including the > 25 exemplary designs described therein, but can be modified within the scope of appended 0 claims along with their full scope of eguivalents.

O 2 Disclosed herein is a method for modification of the isolated proteins by bringing the T proteins, especially whey or plant proteins or a concentrate or isolate thereof in contact N with a reagent that forms sulphite ions to sulphonate the protein. The sulphonate groups 3 30 are then removed by pH adjustment and free sulfhydryl groups are obtained. 00 5 A method for modification of proteins

N The disclosure relates to a method for the modification of whey or plant protein, wherein a) a whey or plant protein concentrate containing 9-12% by weight of protein is brought into contact with a reagent containing 0.02-0.20 M of sulphite for 20 to 40 minutes at a temperature of 40-70°C and a pH of 5.5-7.0 to sulphonate the protein without using oxidizing agent to form a protein suspension; and b) the pH of the protein suspension is adjusted to pH 4.5-5.0; and c) the pH of the protein suspension is adjusted to and kept at pH 2.0 for 5 to 20 minutes with mixing; and d) the pH of the protein suspension is adjusted to pH 4.5-5.5 and the suspension is washed and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and e) the protein suspension is dissolved by adjusting the pH to pH 5.0-6.0; and f) the obtained modified protein is spray dried at an inlet and outlet temperature of 170- 180°C and 70-80°C, respectively.

Whey or plant protein concentrate may contain 9%, 10%, 11%, or 12% by weight of protein. In sulphitolysis of whey or plant proteins a suitable amount of sulphite is 0.02-0.20 M, more preferred amount is 0.05-0.10 M, such as 0.06, 0.07, 0.08, or 0.09 M.

In an embodiment, the protein is whey protein. In another embodiment, the protein is plant protein. In a preferable embodiment protein is pea protein, rice protein or soy protein, or a mixture thereof. An optional amino acid content and the amount of cysteine are preferred characteristics in a suitable plant.

Above mentioned step a), wherein a whey or plant protein concentrate containing 9-12% by weight of protein is brought into contact with a reagent containing 0.02-0.20 M of sulphite for 20 to 40 minutes at 40-70°C and a pH of 5.5-7.0 in order to sulphonate the protein without using oxidizing agent to form a protein suspension, is carried out for 20, co 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 minutes, & 25 preferably said step is carried out for 30 minutes, i.e. the contact time is 30 minutes. The 2 temperature is 40-70°C, such as 40, 45, 50, 55, 60, 65, or 70°C.

O N In above mentioned step a) pH is adjusted to 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, E 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

S After above mentioned step b) the protein suspension is optionally washed and recovered DS 30 by ultrafiltration, filtration or centrifugation. Said washing step is carried out to remove extra > salts and free sulphite. The amount of sulphite in the sulphitolysis step needs to be sufficient and therefore there might be guite a lot of free sulphite left in subseguent steps. This extra sulphite is washed off.

Above mentioned step c), wherein the pH of the protein suspension is adjusted to pH 2.0 for 5 to 20 minutes, is carried out for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, preferably said step is carried out for 15 minutes, i.e. the contact time is 15 minutes. In an embodiment, whey protein is recovered by ultrafiltration. In another embodiment, plant protein is recovered by filtration or centrifugation. Prior to obtaining whey or plant protein concentrate, protein material is optionally ground to a fine powder. There are differences in grinding efficiency between different plants. If particles are too large, disulphide bonds are not properly available in reactions. The ground protein may not, however, be fluffy or dusty. Grinding can be carried out by a grinder, e.g. a hammer mill, pulverizer, impactor, roller mill, or ball mill. Protein concentrate, or isolate is suspended in water, heated to 40-70°C and mixed efficiently until homogenous. The temperature may be raised directly or gradually to a suitable temperature. For example, the protein concentrate, or isolate may be suspended in water, heated to 40°C, mixed efficiently until homogenous, and thereafter heated to 55°C. A reagent containing sulphite, such as sodium metabisulphite (Na»>S20s), is dissolved in water to obtain a reagent containing 0.02-0.20 M of sulphite. Sulphite may also be in the form of sodium bisulphite (NaHSO;) or sodium sulphite (Na>SOs). The sulphite reagent is added to protein solution. The temperature may be kept at 55°C, for 30 minutes and pH is adjusted to pH 6.0. After reaction protein suspension is cooled, for example to 30°C. For example, protein suspension is cooled by adding cold water. pH of the protein suspension is decreased by adding HCI, The pH of the protein suspension is adjusted to pH 4.5-5.0, such as pH 4.5, 4.6, 4.7, 4.8,

4.9, or 5.0. The suspension is optionally washed using ultrafiltration, filtration or o 25 centrifugation in to remove extra salts. When pH is adjusted to 4.5-5.0, the protein is in > suspension. In this washing step extra salts and free sulphite present in the modified se protein are washed away. When the protein to be modified is whey protein, ultrafiltration > is performed. When the protein to be modified is plant protein, filtration is performed. - Filtration may be carried out through filter paper. Alternatively, orin addition, centrifugation & 30 may be used for plant protein suspensions. The conductivity of the protein suspension is S determined, and the suspension is washed with water, as many times and as much as & necessary to remove free sulphite and other salts. The term 'washing' means that water is 2 added once or several times. In previous steps HCI is used to adjust pH and there might be extra salts left in the protein suspension.

An advantage of removing free sulphite is that sulphur (S) is not released as a harmful sulphur dioxide gas during subsequent reaction stages. Thus, work safety is improved, and the process is made smoother. In prior art methods, blowing was used to remove extra sulphite. Blowing, however, requires additional equipment. Thus, the present process is simpler. Foaming or froth formation has been a problem in protein modification in blowing step. Foaming is harmful in processing and to the quality of product. In the present method foaming and the use of anti-foaming agents is avoided The pH of the protein suspension is adjusted to and kept at pH 2.0. Protein becomes more dissolved, when pH is lowered to pH 2.0. When pH is 2.0, SO; is released. In an embodiment, the contact time is 5-20 minutes, preferably 15 minutes. After that, pH is increased by adding NaOH solution. pH may be increased gradually to pH 3.0 and then to pH 4.5. The pH of the protein suspension is adjusted to pH 4.5-5.5, such as pH 4.5, 4.6, 4.7, 4.8,

4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5 and the suspension is washed to remove sulphite, and extra salts, which have mainly appeared to the reaction during the addition of HCI, and recovered by ultrafiltration, filtration or centrifugation. When the protein to be modified is whey protein, ultrafiltration is performed. When the protein to be modified is plant protein, filtration or centrifugation is performed. The added amount of water is approximately twice the original volume. The protein suspension is dissolved by adjusting the pH to pH 5.0-6.0, such as pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. Optionally, the suspension may be homogenized prior to or after sulphitolysis by homogenizer using 10 000 — 20 000 r/min, such as 10 000, 11 000, 12 000, 13 000, 14 000, 15000, 16000, 17000, 18000, 19000, or 20000 r/min. Optionally, homogenization may be carried out with a pressure homogenizer at 50 — 400 bar, such as 70, 100, 150, 200, 300 or 350 bar.

[20] > The obtained modified protein is spray dried at an inlet and outlet temperature of 170- se 180°C and 70-80°C, respectively. > A method for preparing microcapsules Ek The disclosure also relates to a use of the modified protein obtainable by a method N 30 described above, for preparing a microencapsulated composition, comprising 2 a) preparing an agueous protein solution comprising protein concentrate or isolate, and = said modified protein at a ratio of from 4:1 to 3:1, and 0.1-1.0% ascorbic acid, and adjusting N pH to pH 6.5 and a temperature to 45-65°C; and b) preparing a mixture composition comprising oil and a phospholipid, preferably lecithin, at ratio from 110:10 to 110:20, and 0.05-0.5% tocopherol, and adjusting temperature to 45-65°C; and c) combining the aqueous protein solution and the mixture obtained at step b) to form a protein-oil composition, and adjusting pH to 6.5 and temperature to 50-65°C; and d) homogenizing protein-oil solution to obtain an emulsion; and e) pasteurizing the emulsion at a temperature of 70-85°C for 5 minutes or less; and f) spray drying the emulsion at an inlet and outlet temperature of 170-180°C and 70-80°C, respectively. Accordingly, the disclosure relates to a use of the modified protein obtainable by a method wherein a) a whey or plant protein concentrate containing 9-12% by weight of protein is brought into contact with a reagent containing 0.02-0.20 M of sulphite for 20 to 40 minutes at a temperature of 40-70°C and a pH of 5.5-7.0 to sulphonate the protein without using oxidizing agent to form a protein suspension; and b) the pH of the protein suspension is adjusted to pH 4.5-5.0 and the suspension is optionally washed and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and c) the pH of the protein suspension is adjusted to and kept at pH 2.0 for 5 to 20 minutes with mixing; and d) the pH of the protein suspension is adjusted to pH 4.5-5.5 and the suspension is washed and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and © e) the protein suspension is dissolved by adjusting the pH to pH 5.0-6.0; and a f) the obtained modified protein is spray dried at an inlet and outlet temperature of 170- g 25 180°C and 70-80°C, respectively, Q for preparing a microencapsulated composition, comprising

I a a) preparing an agueous protein solution comprising protein concentrate or isolate and 2 said modified protein at a ratio of 4:1 to 3:1, and adjusting the pH to pH 6.5 and a

O O temperature to 45-65°C and adding 0.1-1.0% ascorbic acid of protein; N 30 b) preparing a mixture comprising oil and a phospholipid, preferably lecithin, at ratio from 110:10 to 110:20, and 0.05-0.5% tocopherol of oil and adjusting temperature to 45-65°C;

c) combining the aqueous protein solution obtained at step a) and the mixture obtained at step b) to form a protein-oil composition, and adjusting pH to 6.5 and temperature to 50- 65°C; and d) homogenizing protein-oil solution to obtain an emulsion; and e) pasteurizing the emulsion at a temperature of 70-85°C for 5 minutes or less; and f) spray drying the emulsion at an inlet and outlet temperature of 170-180°C and 70-80°C, respectively. Preparation of an aqueous protein solution comprising protein concentrate or isolate, and said modified protein at a ratio of 4:1 to 3:1, and 0.1-1.0% ascorbic acid at pH 6.5 is carried out at a temperature of 45-65°C. Preferably, the temperature is 45, 50, 55, 60, or 65°C. A mixture comprising oil and a phospholipid, preferably lecithin, is prepared at ratio from 110:10 to 110:20, and 0.5-0.5% tocopherol and the temperature is adjusted to 45-65°C. Preferably, the ratio of oil and phospholipid is 110:10, 110:13, 110:16, 110:18 or 110:20. Preferably, the temperature is 45, 50, 55, 60, or 65°C. The amount of ascorbic acid is 0.1-

1.0% of protein. The amount of ascorbic acid is calculated from the total amount of protein. The amount of ascorbic acid is such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% of protein. The amount of tocopherol is 0.05-0.5% of oil. The amount of tocopherol is calculated from the total amount of oil. The amount of tocopherol is such as 0.05, 0.1, 0.15,

0.2, 0.25, 0.3, 0.35. 0.4, 0.45, or 0.5% of oil. The oil is selected from the group of fish oil, sunflower oil, corn gromwell oil, rape oil, rapeseed oil, olive oil, linseed oil, coconut oil, seed oil from berries such as blueberry, cloudberry or sea buckthorn. In a preferred embodiment, the oil is corn gromwell oil. A commercial name of corn gromwell is Ahiflower®. Corn gromwell (Buglossoides arvensis or Lithospermum arvense) is also known as field gromwell.

[20] > 25 Oil may be any oil from plant or animal origin, which oil is meant for food or animal feed, se or for medical or cosmetic purposes. Animal oil may be fish oil. The phospholipid may be o selected from lecithin, phosphatidylcholine, glycerophospholipids, phosphosphingolipids

N I and purified or a mixture of different phospholipids. a JN The agueous protein solution and the mixture of oil and a phospholipid obtained at step b) 3 30 is combined to form a protein-oil composition, pH is adjusted to pH 6.5 and temperature to 2 50-65°C. Preferably, the temperature is 50, 55, 60, or 65°C.

O

N Protein-oil solution is homogenized to obtain an emulsion using any known suitable homogenizing eguipment or method known in the art, such as a homogenizer.

Homogenization is carried out for example 10 000-20 000 r/min. such as 10 000, 11 000, 12 000, 13 000, 14 000, 15 000, 16 000, 17 000, 18 000, 19 000, or 20 000 r/min. Optionally, homogenization may be carried out with a pressure homogenizer at 50- 400 bar, such as 70, 100, 150, 200, 300 or 350 bar. Pasteurizing of the protein-oil solution is carried out at a temperature of 70-85°C for 5 minutes or less, preferably for 15 seconds to 5 minutes, more preferably 15 seconds. The pasteurizing or heating time is preferably 15, 30, 45 or 60 seconds, or 1, 2, 3, 4, or 5 minutes. Pasteurizing or heating is carried out at temperature of 70-85°C, preferably at 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85°C. Suitably the pasteurizing temperature is 73°C. The emulsion is spray dried at inlet and outlet temperature of 170-180°C and 70-80°C, respectively. The inlet temperature may be 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 or 180°C. The outlet temperature may be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80°C. A method for preparing protein-based film The disclosure also relates to a use of the modified protein concentrate or isolate obtainable by a method described above for preparing a protein-based film comprising a) providing an aqueous protein solution, comprising 9-12% protein, wherein protein concentrate or isolate, and modified protein are at a ratio of 3:1, and 5-7% glycerol, and 1- 2% sorbitol; and b) heating said aqueous protein solution to a temperature of 70-80°C and cooling said solution to room temperature; and c) forming said solution into said protein-based film, and evaporating water. 0 Accordingly, the disclosure relates to a use of the modified protein obtainable by a method o 25 wherein

N 3 a) a whey or plant protein concentrate containing 9-12% by weight of protein is brought 2 into contact with a reagent containing 0.02-0.20 M of sulphite for 20 to 40 minutes at a T temperature of 40-70°C and a pH of 5.5-7.0 to sulphonate the protein without using N oxidizing agent to form a protein suspension; and

O 3 30 b) the pH of the protein suspension is adjusted to pH 4.5-5.0 and the suspension is 5 optionally washed and recovered by ultrafiltration, filtration or centrifugation to remove

N extra salts; and c) the pH of the protein suspension is adjusted to pH 2.0 for 10 to 20 minutes with mixing; and d) the pH of the protein suspension is adjusted to pH 4.5-5.5 and the suspension is washed and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and e) the protein suspension is dissolved by adjusting the pH to pH 5.0-6.0; and f) the obtained modified protein is spray dried at an inlet and outlet temperature of 170- 180°C and 70-80°C, respectively, for preparing a protein-based film comprising a) providing an aqueous protein solution, comprising 9-12% protein, wherein protein concentrate or isolate and modified protein are at a ratio of 3:1, and 5-7% glycerol and 1- 2% sorbitol; and b) heating said aqueous protein solution to a temperature of 70-80°C and cooling said solution to room temperature; and c) forming said solution into said protein-based film and evaporating water.

An aqueous protein solution, comprising protein concentrate or isolate and modified protein at a ratio of 3:1, and 5-7% glycerol, and 1-2% sorbitol is prepared. The aqueous protein solution may comprise 5, 6, or 7% glycerol. Preferably, the amount of glycerol is 5%. The aqueous protein solution may comprise 1, or 2 % sorbitol. Preferably, the amount of sorbitol is 1%. The amount of protein is 9-12% comprising protein concentrate or isolate and modified protein at a ratio of 3:1. The amount of protein may be 9, 10, 11, or 12%. The aqueous protein solution is heated to a temperature of 70-80°C and cooled to room temperature. The heating or pasteurizing temperature may be 70, 71, 72, 73, 74, 75, 76, 77,78, 79, or 80°C. Room temperature means typically a temperature ranging from 15 to = 25%. In other words, cooling temperature may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, N 25 or 25°C. Cooling temperature may be such as 20-22°C, or 20-24°C, or 21-25°C.

O

O o The aqueous protein solution is formed into protein-based film. Forming the film may be

N - carried out for example by pouring the protein solution into a shallow container, such as a n. petri dish. The formed film may be dried at room temperature, or in a heating chamber or 2 an incubator, at a temperature of 30-40°C, for example 30, 31, 32, 33, 34, 35, 36, 37, 38,

O DS 30 39, or 40°C. In an embodiment, the film is formed on a substance to coat the substance. > In another embodiment, the film is formed around lipid, oil, or lipophilic compound to form an emulsion or microcapsule.

Microencapsulated composition The disclosure also relates to a microencapsulated composition comprising oil, and phospholipid preferably lecithin, and protein concentrate or isolate, and modified protein, wherein the modified protein is obtainable by a disclosed method for the modification of whey of plant protein.

Accordingly, the disclosure relates to a microencapsulated composition comprising oil, phospholipid preferably lecithin, protein concentrate or isolate, modified protein, ascorbic acid, and tocopherol, wherein the modified protein is obtainable by a method wherein a) a whey or plant protein concentrate containing 9-12% by weight of protein is brought into contact with a reagent containing 0.02-0.20 M of sulphite for 20 to 40 minutes at a temperature of 40-70°C and a pH of 5.5-7.0 to sulphonate the protein without using oxidizing agent to form a protein suspension; and b) the pH of the protein suspension is adjusted to pH 4.5-5.0 and the suspension is optionally washed and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and c) the pH of the protein suspension is adjusted to and kept at pH 2.0 for 10-20 minutes with mixing; and d) the pH of the protein suspension is adjusted to pH 4.5-5.5 and the suspension is washed and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and e) the protein suspension is dissolved by adjusting the pH to pH 5.0-6.0; and f) the obtained modified protein is spray dried at an inlet and outlet temperature of 170- 180°C and 70-80°C, respectively.

The disclosure relates to a microencapsulated composition comprising oil, phospholipid © preferably lecithin, protein concentrate or isolate, modified protein, 0.1-1.0% ascorbic acid, & 25 and 0.05-0.5% tocopherol, wherein the modified protein is obtainable by a method of the 3 present disclosure, wherein the ratio of protein concentrate or isolate to modified protein 2 is from 4:1 to 3:1, and the ratio of oil to phospholipid is from 110:10 to 110:20.

E In an embodiment the oil is selected from the group of fish oil, sunflower oil, corn gromwell 0 oil, rape oil, rapeseed oil, olive oil, linseed oil, coconut oil, and seed oil from a berry such 2 30 as blueberry, cloudberry or sea buckthorn.

0 > Preferably, the ratio of oil and phospholipid is 110:10, 110:13, 110:16, 110:18 or 110:20. The amount of ascorbic acid is 0.1-1.0% of protein. The amount of ascorbic acid is calculated from the total amount of protein. The amount of ascorbic acid is such as 0.1,

0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0% of protein. The amount of tocopherol is 0.05-

0.5% of oil. The amount of tocopherol is calculated from the total amount of oil. The amount of tocopherol is such as 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35. 0.4, 0.45, or 0.5% of oil. In an embodiment the ratio of protein concentrate, or isolate to modified protein is from 4:1 to 3:1. In an embodiment the ratio of oil to phospholipid is from 110:10 to 110:20. Microencapsulated compositions may also comprise additives, such as antimicrobial agents and excipients and antioxidants. Antioxidants are used in the present compositions in an amount of 0.001-1.0 weight-% to prevent oxidation of the product. Antioxidants protect both shell and core materials from oxidation during storage. Suitable antioxidants are for example ascorbic acid, tocopherols, ascorbyl palmitate, BHA, BHT, plant extracts having antioxidative properties such as rosemary extract, and other known suitable antioxidants. Protein-based film The disclosure also relates to a protein-based film comprising 9-12% protein, wherein protein concentrate, or isolate, and modified protein are at a ratio of 3:1, 5-7% glycerol and 1-2% sorbitol, wherein the modified protein concentrate, or isolate is obtainable by a method described above. Accordingly, the disclosure relates to a protein-based film comprising 9-12% protein, wherein protein concentrate, or isolate, and modified protein are at a ratio of 3:1, and 5- 7% glycerol, and 1-2% sorbitol, wherein the modified protein concentrate, or isolate is obtainable by a method wherein a) a whey or plant protein concentrate containing 9-12% by weight of protein is brought into contact with a reagent containing 0.02-0.20 M of sulphite for 20 to 40 minutes at a temperature of 40-70°C and a pH of 5.5-7.0 to sulphonate the protein without using © 25 oxidizing agent to form a protein suspension; and

O N b) the pH of the protein suspension is adjusted to pH 4.5-5.0 and the suspension is O optionally washed and recovered by ultrafiltration, filtration or centrifugation to remove

O N extra salts; and

I E c) the pH of the protein suspension is adjusted to and kept at pH 2.0 for 10-20 minutes S 30 with mixing; and

O

LO 00 d) the pH of the protein suspension is adjusted to pH 4.5-5.5 and the suspension is washed

O N and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and e) the protein suspension is dissolved by adjusting the pH to pH 5.0-6.0; and f) the obtained modified protein is spray dried at inlet and outlet temperature of 170-180°C and 70-80°C, respectively. In an embodiment, said protein based film comprises 5% glycerol, and 1% sorbitol. The aqueous protein solution may comprise 5, 6, or 7% glycerol. Preferably, the amount of glycerol is 5%. The aqueous protein solution may comprise 1, or 2 % sorbitol. Preferably, the amount of sorbitol is 1%. The protein-based film may comprise 9-12% protein, such as 9, 10, 11, or 12% protein. In a preferred embodiment, said protein-based film comprises 10% protein, 5% glycerol and 1% sorbitol.

EXAMPLES The following examples are illustrative embodiments of the present invention, as described above, and they are not meant to limit the invention in any way. Example 1 22 kg of whey protein concentrate (WPC77) was suspended in 120 | of water and heated to temperature 40°C and mixed efficiently until homogeneous. Then, whey protein solution was heated to 55°C. 510 g of sodium metabisulphite was dissolved in 8 | of water and added to whey protein solution. Temperature was kept at 55°C and pH was adjusted to

6.0. Reaction time at 55°C, pH 6.0 was 30 min. After reaction, protein suspension was cooled to 30°C. pH of the protein suspension was decreased by adding HCI gradually first to pH 4.5 and then to pH 2.0 in efficient stirring. Suspension was mixed for 15 min at pH

2.0. After that, pH was increased gradually to pH 3.0 and then 4.5 by adding NaOH solution. The obtained solution was ultrafiltrated using MWCO 10000 Da membranes. To remove sulphite and salts, suspension was diafiltrated. The added amount of water was approximately 2 x the original volume. After ultrafiltration and concentration to 15% d.m. content, pH was adjusted to pH 5.0. Concentrated whey protein was spray dried using inlet o 25 air temperature 160-180°C and outlet air temperature 75-80°C. N Example 2 g 270 g of whey protein concentrate (WPC77) was suspended in 1600 ml of water and 2 heated to temperature 40°C and mixed efficiently until homogeneous. Then, whey protein =E solution was heated to 55°C. 6.1 g of sodium metabisulphite was dissolved in 125 ml of N 30 water and added to whey protein solution. Temperature was kept at 55*C and pH was 3 adjusted to 6.0. Reaction time at 55°C, pH 6.0 was 30 min. After reaction, suspension was 2 cooled to 30*C. pH of the protein suspension was decreased by adding HCI gradually to N pH 4.5. The obtained solution was diafiltered using MWCO 10000 Da ultrafiltration membranes to remove salts and excess sulphite from the whey protein solution. After ultrafiltration, the pH was adjusted to pH 2.0 in efficient stirring by adding HCI gradually. Suspension was mixed for 15 min at pH 2.0. After that, pH was increased gradually to pH

3.0 and then pH 4.5 by adding NaOH solution. The obtained solution was ultrafiltrated using MWCO 10000 Da membranes. To remove the rest of the sulphite and salts, suspension was diafiltrated. The added amount of water was approximately 2 x the original volume. After ultrafiltration and concentration to 15% d.m. content, pH was adjusted to pH

5.0. Concentrated whey protein was spray dried using inlet air temperature 160-180 °C and outlet air temperature 75-80°C. Example 3 22 g of modified whey protein as prepared in Example 1, 89 g of whey protein concentrate, and 230 mg ascorbic acid were mixed with 800 ml of water. pH was adjusted to 6.5 and mixture was heated to 60°C. 120 g Ahiflower oil was mixed with 17 g of lecithin (Epikuron 135 F IP, from soy) and 120 mg of tocopherol (Tocomix 170-1P) and was heated to 55°C. After complete dissolution and heating, oil phase was added to whey protein suspension in continuous mixing. pH was adjusted to 6.5. Mixture was heated to 60°C, homogenized at 15000 rpm and then pasteurized by heating to 73°C in water bath and then cooling immediately. The suspension was spray dried to produce whey protein microencapsulated Ahiflower oil. The yield was 204 g. Example 4 111 g of soy protein isolate (protein content 90%) was suspended in 800 ml of water and heated to 50°C. 3.4 g sodium metabisulphite was dissolved in 50 ml of water and added into soy protein suspension. 100 ml of water was added. pH was adjusted to pH 6.0 and mixture was heated to 60 °C. Mixture was kept at 60°C for 30 min in continuous stirring. After reaction, mixture was cooled. pH was decreased to pH 4.5 to precipitate the = suspension and was filtered and washed with water through Whatman 40 filter paper to N remove excess sulphite reagent. The filter cake was suspended in 1000 ml water and pH P was decreased to pH 2.0 by HCI. After 15 min mixing, pH was increased to pH 4.5 by & adding NaOH. The precipitated protein was separated by filtration through whatman 40 = 30 filter paper and was washed with water to remove salts from protein and suspended in 800 2 ml of water. The conductivity was decreased from 9 mS/cm to 5 uS/cm by washing. The 3 pH was increased to pH 6.0 and suspension was mixed for 1 h. Mixture was homogenized D by a Ika mixing homogenizer using 18000 rpm. The homogenized protein was spray dried N using 180 inlet air temperature and 80°C outlet air temperature.

Example 5

27.5 g of modified soy protein as prepared in Example 4, 82.5 g of soy protein isolate and 230 mg ascorbic acid were mixed with 800 ml of water. pH was adjusted to 6.5 and mixture was heated to 55°C. Mixture was homogenized at 18000 rpm. Ahiflower oil was mixed with 16 g of lecithin (Epikuron 135 F IP, from soy) and 110 mg of tocopherol (Tocomix 170-IP) and was heated to 55°C. After complete dissolution and heating, oil phase was added to soy protein suspension in continuous mixing. Mixture was homogenized at 18000 rpm and then pasteurized by heating to 73°C in water bath and then cooling immediately. The suspension was spray dried to produce soy protein microencapsulated Ahiflower oil. The yield was 200 g. Example 6 112 g of pea protein isolate was suspended in 800 ml of water and was heated to 50°C.

3.4 g of sodium metabisulphite was dissolved in 50 ml of water and was added to protein solution. pH was adjusted to pH 6.0 by adding 3 M HCI. The suspension was heated to 60°C. Mixture was kept at 60°C for 30 min in continuous stirring. After reaction, temperature is cooled to 40 °C. pH was adjusted to 4.5 by HCI to precipitate the protein. The precipitated protein was separated and washed on Whatman 40 filter paper to remove excess sulphite reagent. The filter cake was suspended in 1000 ml of water and the pH was decreased to pH 2.0 and kept for 15 min in efficient mixing. After that the pH was increased to 4.5 by adding NaOH to precipitate the protein again. Precipitate was separated by filtration through Whatman 40 filter paper and was washed with water to remove salts from protein and suspended in 800 ml of water. The pH was increased to pH

6.0 and suspension was mixed for 1 h. Mixture was homogenized by a IKA mixing homogenizer using 18000 rpm. The homogenized protein was spray dried using 180°C inlet air temperature and 80°C outlet air temperature. The modification of pea protein was co evaluated by analyzing the amount of SH-groups by Ellmans method. The amount of SH > groups in modified pea protein was 34 umol/g compared to the 9 umol/g of normal pea g protein isolate. 2 Example 7 = 30 27.5 g of modified pea protein as prepared in Example 6, 82.5 g of pea protein isolate and N 230 mg ascorbic acid were mixed with 800 ml of water. pH was adjusted to 6.5 and mixture 3 was heated to 55°C. 110 g of Ahiflower oil was mixed with 16 g of lecithin (Epikuron 135 = F IP, from soy) and 110 mg of tocopherol (Tocomix 170-1P) and was heated to 55 °C. After N complete dissolution and heating, oil phase was added to pea protein suspension in continuous mixing. Volume was adjusted to 1000 ml by adding water. Mixture was homogenized at 15000 rpm and then pasteurized by heating to 73°C for 15 seconds in water bath and then cooling immediately. The suspension was spray dried to produce pea protein microencapsulated Ahiflower oil. The yield was 200 g. Example 8 112 g of rice protein isolate was suspended in 800 ml of water and was heated to 50°C.

3.4 g of sodium metabisulphite was dissolved in 50 ml of water and was added to protein solution. pH was adjusted to pH 6.0 by adding 3 M HCI. The suspension was heated to 60°C. Mixture was kept at 60°C for 30 min in continuous stirring. After reaction, temperature is cooled to 40°C. pH was adjusted to 4.5 by HCI to precipitate the protein. The precipitated protein was separated and washed on Whatman 40 filter paper to remove excess sulphite reagent. The filter cake was suspended in 1000 ml of water and the pH was decreased to pH 2.0 and kept for 15 min in efficient mixing. After that the pH was increased to 4.5 by adding NaOH to precipitate the protein again. Precipitate was separated by filtration through Whatman 40 filter paper and was washed with water to remove salts from protein and suspended in 800 ml of water. The pH was increased to pH

6.0 and suspension was mixed for 1 h. Mixture was homogenized by a IKA mixing homogenizer using 18000 rpm. The homogenized protein was spray dried using 180°C inlet air temperature and 80°C outlet air temperature. Example 9 125 g of rice protein isolate was suspended in 800 ml of water and was heated to 50°C.

4.4 g of sodium metabisulphite was dissolved in 50 ml of water and was added to protein solution. pH was adjusted to pH 6.0 by adding HCI. The suspension was heated to 65°C. Mixture was kept at 65°C for 30 min in continuous stirring. After reaction, temperature was cooled to 40°C. pH was adjusted to 4.5 and then pH 2.0 by HCI and kept for 15 min in efficient mixing. After that the pH was increased to pH 4.5 by adding NaOH to precipitate 00 the protein. Precipitate was separated by filtration through Whatman 40 filter paper and N was washed with water to remove salts from protein and suspended in 800 ml of water. 3 The pH was increased to pH 6.0 and suspension was mixed for 1 h. Mixture was z homogenized by a IKA mixing homogenizer using 18000 rpm. The homogenized protein = 30 was spray dried using 180°C inlet air temperature and 80°C outlet air temperature. The N modification of rice protein was evaluated by analyzing the amount of SH-groups by 3 Ellmans method. The amount of SH groups in modified rice protein was 65-101 umol/g = compared to the 20 umol/g of normal rice protein isolate.

N

Example 10

27.5 g of modified rice protein as prepared in Example 9, 82.5 g of rice protein isolate and 230 mg ascorbic acid were mixed with 800 ml of water. pH was adjusted to 6.5 and mixture was heated to 55°C. 110 g of Ahiflower oil was mixed with 16 g of lecithin (Epikuron 135 F IP, from soy) and 110 mg of tocopherol (Tocomix I70-IP) and was heated to 55°C. After complete dissolution and heating, oil phase was added to rice protein suspension in continuous mixing. Volume was adjusted to 1000 ml by adding water. Mixture was homogenized at 15000 rpm and then pasteurized by heating to 73°C in water bath and then cooling immediately. The suspension was spray dried to produce rice protein microencapsulated Ahiflower oil. Example 11

13.1 g of soy protein isolate, 4.4 g of modified soy isolate from example 4 were dissolved in 70 ml of water. 6.25 g of glycerol and 1.25 g of sorbitol were added. pH was adjusted to

6.5 and solution was diluted to 125 ml by adding water. Mixture was heated to 75°C and kept there for 3-5 min with continuous mixing. Mixture was cooled down to room temperature. 7.5 ml of suspension was pipetted into petri dishes and kept at 22°C, relative humidity 51% until films were dried.

REFERENCES Ruan, O, Chen, Y, Kong, X., Hua, Y., 2014. Heat induced aggregation and sulphydryl /disulphide reaction products of soy protein with different sulphydryl contents. Food Chem. 156, 14-22. Nesterenko, A., Alric, |., Silvestre, F., Durrieu, V., 2013. Vegetable proteins in microencapsulation: a review of recent interventions and their effectiveness. Ind. Crops Prod. 42, 469-479. © 25 Nesterenko, A., Alric, |., Silvestre, F., Durrieu, V., 2012. Influence of soy protein's structural N modifications on their microencapsulation properties: a-tocopherol microparticles g preparation. Food Res. Int. 48, 387-396. Q Ying, D.Y., Phoon, M.C., Sanguansri, L., Weerakkody, R., Burgar, I., Augustin, M. A, E 2010. Microencapsulated Lactobacillus rhamnosus GG powders: relationship of powder © 30 physical properties to probiotic survival during storage. J. Food Sci. 75 (9) E588-E595. O WO2009/050333, US 6797810, WO 2015/019307, FI20031508

O N

Claims

1. A method for the modification of whey or plant protein, wherein a) a whey or plant protein concentrate containing 9-12% by weight of protein is brought into contact with a reagent containing 0.02-0.20 M of sulphite for 20-40 minutes at a temperature of 40-70°C and a pH of 5.5-7.0 to sulphonate the protein without using oxidizing agent to form a protein suspension; and b) the pH of the protein suspension is adjusted to pH 4.5-5.0; and c) the pH of the protein suspension is adjusted to and kept at pH 2.0 for 5-20 minutes with mixing; and d) the pH of the protein suspension is adjusted to pH 4.5-5.5 and the suspension is washed and recovered by ultrafiltration, filtration or centrifugation to remove extra salts; and e) the protein suspension is dissolved by adjusting the pH to pH 5.0-6.0; and f) the obtained modified protein is spray dried at an inlet and outlet temperature of 170-180°C and 70-80°C, respectively.

2. The method according to claim 1, characterized in that the protein is whey protein and the whey protein is recovered by ultrafiltration.

3. The method according to claim 1, characterized in that the protein is plant protein and the plant protein is recovered by filtration or centrifugation.

4. The method according to claim 3, characterized in that the protein is pea protein.

5. The method according to claim 3, characterized in that the protein is rice protein.

N S 6.

The method according to claim 3, characterized in that the protein is soy protein.

S N 7. The method according to any one of claims 1 to 6, characterized in that in step a) the > contact time is 30 minutes. E25 8. The method according to any one of claims 1 to 7, characterized in that after step b) S the protein suspension is washed and recovered by ultrafiltration, filtration or 3 centrifugation to remove extra salts. &

9. The method according to any one of claims 1 to 8, characterized in that in step c) the contact time is 15 minutes.