Patent Application
20160235082
The present invention relates to whey protein products and methods for making such products. More particularly, the present invention relates to heat stable whey protein products and methods for making such heat stable whey proteins, even more particularly whey proteins subjected to specific heat treatments in the presence of certain concentrations of hydrogen peroxide.
Whey protein is considered as high quality protein because of its branch chain amino acid. Growing awareness about the nutritional benefits of whey proteins and technological advancements in improving functional properties has led to ever increasing demand of whey proteins in form of commercial whey protein ingredients such as whey protein concentrates (WPC) and whey protein isolates (WPI). These ingredients have Generally Regarded As Safe (GRAS) status and are widely used in a variety of products such as whey protein fortified sports beverages, nutritional and meal replacement beverages. In most case, these beverage products are subjected to severe heat treatments such as temperatures of about 120° C. for about 15-20 minutes (retort sterilization) or temperatures of about 140° C. for about 3-4 seconds (UHT treatment) for food safety reasons and to extend their shelf-life. However, the heat treatment of whey protein solutions above 85° C. leads to denaturation and aggregation of whey proteins; and at sufficiently high protein concentration (typically beyond 8-10% proteins content), whey proteins tend to form heat-induced gels. Because of their poor heat stability, whey proteins show precipitation, aggregation, sedimentation or gelation of whey protein in the beverages, particularly if the beverages are subjected to severe heat treatment such as retort or UHT treatment. While aggregation and gelation is desirable in the application of whey proteins in many products such as bakery, meat, pudding or sauces, such aggregation, gelation, precipitation or thickening is considered highly undesirable for incorporation of whey proteins in high protein beverages and for processing of liquid food products.
In recent years, consumer demand for whey protein fortified nutritional, meal replacement beverages containing high protein (preferably more than 10% protein) is increasing. To be able to formulate heat-stable, high protein beverages having a long shelf-life, it is important to ensure that the whey protein used in such high protein beverage formulations are heat stable. Various approaches have been used to improve the heat stability of whey proteins, and extensive literature is available on this topic. Some examples of approaches to improve heat stability of whey proteins include, tailoring protein-carbohydrate interactions (Rich and Foegeding, 2000, Foegeding et al., 2002, Baier and McClements, 2005), the treatment with glycerol (Kulmyrzaev et al., 2000, Rich and Foegeding, 2000), the application of transglutaminase (Tanimoto and Kinsella, 1988, Zhang and Zhong, 2009), partial enzymatic hydrolysis of whey proteins (Doucet and Foegeding, 2005), using high pressure processing (Patel et al., 2005) or combination of high pressure and binding ligands to whey proteins (Considine et al., 2005a, Considine et al., 2005b, Considine et al., 2007a, Considine et al., 2007b), the use of milk protein concentrate as a chaperone agent (Zhang et al., 2005, Yong and Foegeding, 2008) and treatment of WPI solutions with combination of enzymatic hydrolysis and succinic anhydride induced electrostatic repulsion (Yilmaz-Gemili, 2012), application of stabilizing and calcium cheating agent such as sodium phosphate and EDTA (Kuhn and Foegeding, 1991, Xiong, 1992), ultrasonication of whey proteins (Zisu et al., 2010), tailored polysaccharide-whey protein interactions (Zhang et al., 2004), formation of protein-soluble-aggregates (Ryan et al., 2012) and blocking free —SH groups by use of various chemicals such as N-ethylmaleimide, mercaptoethanol, iodoacetamide, lipoic acid and dihydrolipoic acid interfere in sulfhydryl-disulfide interchange reaction and improved heat stability of whey proteins (Sawyer, 1968, Iametti et al., 1996, Wijayanti et al., 2013). However, limited success has been achieved to date.
Hydrogen peroxide (H2O2) has a GRAS status. It is permitted for use by the Code for Federal Regulation (CFR, 2014b) under 21 CFR 184.1366 to use at a maximum allowed concentration for the various purposes such as: antimicrobial agent in the starch and in the milk intended for use during the cheese making process (500 ppm), for bleaching of whey (up to 400 ppm) as well as a bleaching agent in beef, tripe, instant tea and whey (500 ppm), an oxidizing and reducing agent in corn syrup, dried eggs and wine (21 CFR 184.1366). It is also allowed for use in washing or to assist in the peeling of fruits and vegetables (21 CFR 173.315) as well as a sterilizing agent for food packaging materials.
H2O2 is a strong oxidizing agent. Many reports in the literature suggested use of H2O2 in the dairy industry for various purposes. For example, H2O2 has been used as a preservative in milk (Williams, 1966; Mistry & Kosikowski, 1985), bleaching agent for whey (Croissant et al., 2009; Jervis & Drake, 2013), and in milk for cheese making (Roundy, 1958; Kosikowski & Fox, 1968). Some reports are available on the effect of H2O2 on sensory and functional properties of WPC (Jervis et al., 2012). However, no application of H2O2 has been reported for improving heat stability of whey protein, as well as manufacturing WPC and WPI with improved heat stability.
Accordingly, there is a need in the industry for improved whey proteins with improved functionality and nutrition, including heat stable whey proteins, either in liquid or dried form. There is also a need in the industry for improved manufacturing processes of such heat stable whey proteins and whey protein ingredients.
The present invention is directed to a process for the preparation of heat stable whey proteins, either in liquid or dried form, by optimizing specific combinations of hydrogen peroxide and heat treatment at specific stages during the manufacturing process of whey protein ingredients. In this process, hydrogen peroxide can be added as a processing aid, which plays a specific role in altering the intermolecular sulfhydryl-disulfide interchange reaction responsible for heat-induced aggregation and gelation of whey proteins. The process of the present invention by which heat stable intermediate compounds can be formed at sufficiently higher protein concentration does not form precipitate or gel when whey protein solutions are subjected to severe heat treatments, such as the case with retort or UHT. Instead, these high temperature conditions can be tolerated by the whey proteins to make them heat stable according to certain aspects of the present invention.
The present invention is also directed to at least two different versions of heat stable whey protein ingredients (1) heat stable liquid retentate of WPI, WPC or any other form of whey protein ingredients and (2) heat stable powder WPI, WPC or other whey protein powders. According to certain aspects of the present invention, the heat stable liquid retentate of WPI, WPC or any other form of whey protein ingredients are formed according to the process of the present invention. According to certain aspects of the present invention, the heat stable powder WPI, WPC or any other whey protein powders are formed according to the process of the present invention.
In certain aspects of the present invention, whey is provided, which may be stored in a storage tank, to a series of ultra filtration and/or micro filtration steps to separate and/or concentrate the whey proteins. According to certain aspects of the present invention, hydrogen peroxide is added in a suitable amount of water to make a hydrogen peroxide solution having a desired concentration. The whey solution and hydrogen peroxide solution are combined in a mixing or reaction tank, such that the hydrogen peroxide reacts with the whey protein. In certain aspects of the present invention, the whey solution and hydrogen peroxide solution are mixed in a reaction tank, such that the hydrogen peroxide reacts with the whey protein. The mixture of the whey protein solution treated with the hydrogen peroxide solution undergoes a pasteurization process at a desired temperature for a desired period of time to form a heat stable solution or liquid retentate of WPI, WPC or other whey protein ingredients. In certain aspects, an excess amount of hydrogen peroxide may be removed. The resultant whey protein solution may undergo ultra-filtration to remove excess lactose. The whey protein solution may also undergo an evaporation step to concentrate the whey protein to a desired whey protein concentration. In certain aspects, the whey protein solution may be dried to form a powder.
In certain aspects of the present invention, the heat stable solutions or liquid retentate of WPI, WPC or other whey protein ingredients can be prepared by subjecting the reaction mixture containing whey protein to specific heat treatments in the presence of a specific amount of hydrogen peroxide during specific stages of the manufacturing process.
In certain other aspects of the present invention, the liquid retentate of WPI, WPC or other whey protein ingredients can be prepared by subjecting the reaction mixture containing whey protein and hydrogen peroxide to a non-thermal method that normal denatures proteins, including high pressure processing, pulsed electric field, or the like, with the presence of a specific amount of hydrogen peroxide being in the range of about 300 ppm to about 500 ppm.
In certain aspects, the heat stable solutions or liquid retentate of WPI, WPC or other whey protein ingredients can be prepared by subjecting the reaction mixture of whey protein and hydrogen peroxide to specific heat treatments at temperatures in the range of about 60° C. to about 200° C. for about 5 seconds or longer in the presence of a specific amount of hydrogen peroxide in the range of about 300 ppm to about 500 ppm.
In certain other aspects, the specific heat treatment of the whey protein solution and hydrogen peroxide solution reaction mixture is at a temperature of about 60° C. to about 200° C., in other aspects about 70° C. to about 180° C., in other aspects about 80° C. to about 160° C., and in other aspects about 85° C. to about 145° C.
In certain other aspects, the specific heat treatment of the whey protein solution and hydrogen peroxide solution reaction mixture for about 1 second to about 25 minutes, in some other aspects about 5 seconds to about 20 minutes, in some other aspects about 15 seconds to about 18 minutes, and in some other aspects about 1 minute to about 10 minutes.
In certain other aspects, the hydrogen peroxide concentration is about 300 ppm to about 500 ppm, in some other aspects about 325 ppm to about 480 ppm, in some other aspects about 340 ppm to about 470 ppm, in some other aspects about 350 ppm to about 450 ppm, and in some other aspects about 375 ppm to about 425 ppm.
In certain aspects, after the specific heat treatment in the presence of hydrogen peroxide, the excess residual hydrogen peroxide can then be removed by methods such as catalase treatment, ultraviolet (UV) light; electrochemical reduction and oxidation, activated carbon catalysts, a pyrolusite catalyst or any other method or chemicals which can break the hydrogen peroxide into water and oxygen molecules or any other molecules.
In certain aspect of the present invention, after the removal of excess hydrogen peroxide the treated solutions can then be concentrated by ultra-filtration to obtain a heat-stable concentrated liquid or retentate of WPI, WPC or any other whey protein ingredients containing about 6% protein or higher. In certain aspect of the present invention, after the removal of excess hydrogen peroxide the treated solutions can then be concentrated by ultra-filtration to obtain a heat-stable concentrated liquid or retentate of WPI, WPC or any other whey protein ingredients containing about 6% protein to about 20% protein, in some aspects about 8% protein to about 18% protein, and in some other aspects about 10% protein to about 16% protein.
In certain aspects of the present invention, the concentrated forms of liquid or retentate of WPI or WPC can be dried to obtain a heat-stable powder of WPI, WPC or any other whey proteins ingredients. In certain aspects of the present invention, the liquid or retentate of WPI or WPC can be freeze dried to obtain a heat stable WPI powder, WPC powder or whey protein powder.
In certain aspects of the present invention, during the heat treatment of a solution containing whey proteins in the presence of a sufficient concentration of hydrogen peroxide, one or more of the free sulfhydral groups of major whey protein (β-lactoglobulin) are converted into non-reactive compounds (e.g. cystine sulfonic acid), which is a precursor for taurine group of compounds. Taurine is generally used in the energy drinks and is regarded as non-harmful for human consumption. In certain aspects, the cystine of the starting whey protein product is converted to essentially a non-reactive compound, such as cystine sulfonic acid in the resulting heat stable whey proteins.
The above summary is not intended to describe each illustrated embodiment or every implementation of the subject matter hereof. The figures and the detailed description that follow more particularly exemplify various embodiments.
Subject matter hereof may be more completely understood in consideration of the following detailed description of various embodiments in connection with the accompanying figures, in which:
While various embodiments are amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the claimed inventions to the particular embodiments described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the subject matter as defined by the claims.
The method of the present invention can produce at least two different versions of heat stable whey protein ingredients. For example, the method according to certain aspects of the present invention can make (1) heat stable liquid retentate of WPI, WPC or any other form of whey protein ingredients, and (2) heat stable WPI, WPC or other whey protein powders. The inventive method (
The present invention involves preparation of heat stable whey proteins by subjecting the dilute whey protein solutions to heat treatment in the presence of hydrogen peroxide. Referring generally to
In certain aspects of the present invention, the heat stable solutions or liquid retentate of WPI, WPC or other whey protein ingredients can be prepared by subjecting the reaction mixture containing whey protein to specific heat treatments in the presence of a specific amount of hydrogen peroxide during specific stages of the manufacturing process.
In certain aspects, the heat stable solutions or liquid retentate of WPI, WPC or other whey protein ingredients can be prepared by subjecting the reaction mixture of whey protein and hydrogen peroxide to specific heat treatments at temperatures in the range of about 60° C. to about 200° C. for about 60 seconds or longer in the presence of a specific amount of hydrogen peroxide in the range of about 300 ppm to about 500 ppm.
In certain other aspects, the specific heat treatment of the whey protein solution and hydrogen peroxide solution reaction mixture is at a temperature of about 60° C. to about 200° C., in other aspects about 70° C. to about 180° C., in other aspects about 80° C. to about 160° C., and in other aspects about 85° C. to about 145° C.
In certain other aspects, the specific heat treatment of the whey protein solution and hydrogen peroxide solution reaction mixture for about 60 seconds to about 25 minutes, in some other aspects about 60 seconds to about 20 minutes, in some other aspects about 60 seconds to about 10 minutes.
In certain other aspects, the hydrogen peroxide concentration is about 300 ppm to about 500 ppm, in some other aspects about 325 ppm to about 480 ppm, in some other aspects about 340 ppm to about 470 ppm, in some other aspects about 350 ppm to about 450 ppm, and in some other aspects about 375 ppm to about 425 ppm.
In certain aspects, after the specific heat treatment in the presence of hydrogen peroxide, the excess residual hydrogen peroxide can then be removed by methods such as catalase treatment, ultraviolet (UV) light; electrochemical reduction and oxidation, activated carbon catalysts, a pyrolusite catalyst or any other method or chemicals which can break the hydrogen peroxide into water and oxygen molecules or any other molecules.
In certain aspect of the present invention, after the removal of excess hydrogen peroxide the treated solutions can then be concentrated by ultra-filtration to obtain a heat-stable concentrated liquid or retentate of WPI, WPC or any other whey protein ingredients containing about 6% protein or higher. In certain aspect of the present invention, after the removal of excess hydrogen peroxide the treated solutions can then be concentrated by ultra-filtration to obtain a heat-stable concentrated liquid or retentate of WPI, WPC or any other whey protein ingredients containing about 6% protein to about 20% protein, in some aspects about 8% protein to about 18% protein, and in some other aspects about 10% protein to about 16% protein.
In certain aspects of the present invention, the concentrated forms of liquid or retentate of WPI or WPC can be dried to obtain a heat-stable powder of WPI, WPC or any other whey proteins ingredients. In certain aspects of the present invention, the liquid or retentate of WPI or WPC can be freeze dried to obtain a heat stable WPI powder, WPC powder or whey protein powder.
In certain aspects of the present invention, during the heat treatment of a solution containing whey proteins in the presence of a sufficient concentration of hydrogen peroxide, one or more of the free sulfhydral groups of major whey protein (β-lactoglobulin) are converted into non-reactive compounds (e.g. cystine sulfonic acid), which is a precursor for taurine group of compounds. Taurine is generally used in the energy drinks and is regarded as non-harmful for human consumption. As such, in certain aspects of the present invention, the cystine of the starting whey protein product is converted to essentially a non-reactive compound, such as cystine sulfonic acid in the heat stable whey protein.
It has been known in the art that β-lactoglobulin contains two free-SH (Cys) in its structure. These free —SH groups are highly reactive. Upon heat treatment, these free —SH reacts with other —SH groups in the structure of the protein and forms disulfide-linked aggregation, and at sufficiently higher concentration of proteins (e.g. >7-8% protein), it forms gel. Therefore, this free, reactive —SH is believed to be mainly responsible for inducing the chain reaction leading to denaturation and inter and intra-molecular disulphide-linked aggregation.
In the present inventive process, it is believed that hydrogen peroxide plays a specific role in altering the intra- and intermolecular sulfhydryl-disulfide interchange reaction responsible for heat-induced aggregation and gelation of whey proteins by binding with free —SH group and converting it to non-reactive intermediate compounds such as Cysteic Acid and Cysteine Sulphonic acid. The process of the present invention, which forms heat stable intermediate compounds, does not form precipitate or gel when whey protein solutions are subjected to severe heat treatments such as retort or UHT at sufficiently higher protein concentrations; but instead, these conditions can be tolerated by the whey proteins to make them heat stable.
During the heat treatment of solution containing whey proteins in the presence of a sufficient concentration of hydrogen peroxide according to certain aspects of the present invention, the free sulfhydral group of major whey protein (β-lactoglobulin) are converted into non-reactive compounds cysteic acid and/or cysteine sulphonic acid, which is a precursor for taurine group of compounds. Taurine is generally used in the energy drinks and is regarded as non-harmful for human consumption. In this process, significant quantity of Free-SH group at least 60%, in some aspects at least 70%, and in some other aspects at least 80% is converted to nonreactive compound such as Cysteic acid/Cysteine Sulphonic Acid form. This process mainly alters the free-SH group; therefore the inventors without wishing to be bound to theory believe that only free —SH groups take part in this reaction. Typical whey protein isolate powder contains about 2400 to 2600 mg of cysteine/100 gm protein. Whereas WPI used in the present study have a calculated concentration of Taurine like compound or taurine precursor being around 350 mg/100 gm protein (2500*0.14=350 mg).
The present invention involves preparation of heat stable whey proteins by subjecting the dilute whey protein solutions to heat treatment in the presence of hydrogen peroxide. The whey protein solutions in the range of about 0.05% to about 20.0% protein content, in certain other aspects in the range of about 1% to about 16% protein content, while a heat treatment can be in the range of about 72° C. to about 145° C. for about 1 second to about 1200 seconds or higher in the presence of hydrogen peroxide in the concentration range of about 100 ppm to about 1000 ppm, in certain other aspects in the range of about 300 to about 400 ppm.
The excess residual hydrogen peroxide can then be removed by any conventional method such as the treatment with a catalase enzyme. The resulting heat stable whey protein solution can be concentrated by any known means such as ultra-filtration or evaporation to obtain a heat stable liquid WPI concentrate with up to at least 6% protein, in some aspects up to about 20% protein, in certain other aspects up to about 20% protein, and in some further aspects between about 18% and about 20% protein. Additionally, the stable liquid WPI, WPC or any other liquid, solutions or beverages containing whey proteins can be converted into a dried form, such as a powder, by any drying techniques, such as freeze drying, spray drying or any other drying methods known in the art to obtain a heat stable WPI, WPC or any other form of whey protein. In certain aspects, the WPI, WPC or any other form of heat stable whey proteins are in the form of a dried powder ingredients.
Heat stability tests have been performed on the whey protein solutions at 121° C. for 20 minute for the samples treated with varying concentration of hydrogen peroxide, including a WPI sample treated with a hydrogen peroxide concentration according to certain aspects of the present invention. Referring now to
The heat coagulation time test was performed at 120° C. and 140° C. using an oil bath. 5 ml of WPI (12.8% protein) samples were filled in 8 mL wheaton glass tube (D-17 mm×H-61 mm) and submerged in an oil bath (on rotating platform operating at medium speed) maintained at 120° C. and 140° C. Heat coagulation time (HCT) was defined as the time required for forming visible set precipitation or coagulation or gelling at 120° C. and 140° C.
A dynamic oscillatory shear test was performed in a controlled stress rheometer (STRESSTECH ATS, Bordentown, N.J.) fitted with a bob and cup geometry (25 mm diameter, gap 1 mm). A peltier system in the bottom plate enabled fast and accurate temperature control. 15 ml of sample used for analysis and sample surface was covered with a thin layer of paraffin oil at the start of the test, in order to prevent evaporation.
Each sample was pre-sheared and equilibrated for 5 min followed by a temperature ramp from 30° C. to 90° C., at a rate of 2° C./min, after which the temperature was maintained at 90° C. for 10 min. At the end of this time sweep, the sample was cooled down to 30° C. at a rate of 2° C./min, and the temperature was maintained at 30° C. for 10 min. A maximum shear strain of 0.5% and a frequency of 1 Hz were used for the temperature-time sweep. A strain sweep from 0.1 to 1% was then made to verify that the product temperature-time sweep was conducted within the linear viscoelastic region. From this method G′ (elastic modulus) values were obtained to compare the elasticity difference (gel strength) between control and test samples.
WPI solutions containing 4% protein and different levels of H2O2 were preheated at 120° C. for 2 min. WPI samples were analyzed using SDS-PAGE under non reducing condition as described by Tovar Jimenez and Rosario (2012) with slight modifications. The electrode buffer (pH 8.3) contained 0.025 M Tris-Base, 0.192 M glycine and 0.1% SDS. The WPI samples were diluted in the SDS sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS and 0.01% (W/v) bromophenol blue. An aliquot of each sample containing 0.015 mg protein and 10 μl of MW standards were loaded into each well. The samples were electrophoresed at voltage 200 V for 55 min. After electrophoresis gels were removed from slabs and stained for 1 hour using 0.1% (w/v) amido black 10B in 10% (v/v) acetic acid and 25% (v/v) 2-propanol. After staining the gels, they were de-stained using 10% acetic acid (v/v) solution for a total of 18 hours with 2 destaining. The stained gels were then photographed using a Bio-5000 Microtek scanner (Hsinchu, Taiwan) and the images were analyzed using the Bio image Intelligent Quantifier v 3.3.7 system (Jackson, Mich.) to quantify the intensity of whey protein bands.
These terms and specifications, including the examples, serve to describe the invention by example and not to limit the invention. It is expected that others will perceive differences, which, while differing from the forgoing, do not depart from the scope of the invention herein described and claimed. In particular, any of the function elements described herein may be replaced by any other known element having an equivalent function.
Various embodiments of systems, devices, and methods have been described herein. These embodiments are given only by way of example and are not intended to limit the scope of the claimed inventions. It should be appreciated, moreover, that the various features of the embodiments that have been described may be combined in various ways to produce numerous additional embodiments. Moreover, while various materials, dimensions, shapes, configurations and locations, etc. have been described for use with disclosed embodiments, others besides those disclosed may be utilized without exceeding the scope of the claimed inventions.
Persons of ordinary skill in the relevant arts will recognize that the subject matter hereof may comprise fewer features than illustrated in any individual embodiment described above. The embodiments described herein are not meant to be an exhaustive presentation of the ways in which the various features of the subject matter hereof may be combined. Accordingly, the embodiments are not mutually exclusive combinations of features; rather, the various embodiments can comprise a combination of different individual features selected from different individual embodiments, as understood by persons of ordinary skill in the art. Moreover, elements described with respect to one embodiment can be implemented in other embodiments even when not described in such embodiments unless otherwise noted.
Although a dependent claim may refer in the claims to a specific combination with one or more other claims, other embodiments can also include a combination of the dependent claim with the subject matter of each other dependent claim or a combination of one or more features with other dependent or independent claims. Such combinations are proposed herein unless it is stated that a specific combination is not intended.
Any incorporation by reference of documents above is limited such that no subject matter is incorporated that is contrary to the explicit disclosure herein. Any incorporation by reference of documents above is further limited such that no claims included in the documents are incorporated by reference herein. Any incorporation by reference of documents above is yet further limited such that any definitions provided in the documents are not incorporated by reference herein unless expressly included herein.
For purposes of interpreting the claims, it is expressly intended that the provisions of 35 U.S.C. §112(f) are not to be invoked unless the specific terms “means for” or “step for” are recited in a claim.
The present application claims the benefit of U.S. Provisional Application No. 62/116,951, filed Feb. 17, 2015, which is hereby incorporated herein in its entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62116951 | Feb 2015 | US |
