NZ510680A - Process for controlling maillard-type glycation of whey proteins and products with enhanced functional properties - Google Patents

Abstract

A method of storing whey protein-containing dry powdered material comprising the steps; i) Predetermining the initial level of glycation of the whey protein-containing dry powdered material; and either ii) increasing the glycation level of the whey protein-containing dry material of step i) by subjecting the material to Maillard-type glycation reaction by storing the whey-protein containing dry powdered material under controlled temperature and moisture conditions for a sufficient time to produce a desired level of glycation without causing denaturation of the whey protein, followed by termination of the Maillard-type reaction after a desired level of glycation has been reached by storing the whey protein-containing dry powdered material under controlled temperature and moisture conditions which inhibit the glycation reaction; or iii) inhibiting further glycation of the whey protein-containing dry powdered material of step i) by storing the material under controlled temperature and moisture conditions to inhibit the glycation reaction.

Description

PCT/NZ99/00163 : PROCESS FOR CONTROLLING MAILLARD-TYPE GLYCATION OF WHEY PROTEINS AND PRODUCTS WITH ENHANCED FUNCTIONAL PROPERTIES FIELD OF THE INVENTION This invention relates to a dairy product and process, particularly although by no means exclusively, to a process of controlling Maillard-type glycation of whey proteins to produce a product having enhanced functional properties.

BACKGROUND OF THE INVENTION Reducing sugars and compounds that contain them have been shown to react with the free amino groups of milk proteins by the Maillard reaction. The Maillard reaction, which occurs even at low temperatures, has been extensively studied since 1912 (Maillard, 1912). The Maillard reaction is a complex group of many reactions occurring "'in vivo" as well as during processing and storage. The reaction is pH and temperature dependent. It is also affected by relative humidity/water activity of the samples, reducing sugar and metal ions.

The first irreversible product originated by the non-enzymatic interaction of a saccharide/carbonyl group and the a or e-amino groups of proteins is known as the Amadori compound. The Amadori compound can be formed by many reducing sugars, oligosaccharides and their derivatives. Examples are e-lactosyl-lysine, fructosyl-lysine or tagatosyl-lysine. All of these compounds under acid hydrolysis generate furosine. Quantitation of furosine is therefore an excellent way of monitoring the extent of modification of proteins due to the Maillard reaction (Erbersdobler, 1986).

The covalent attachment of sugar compounds to proteins gives rise to glycated proteins, with the consequent modification of their structure as well as functionality. These effects have been studied on a laboratory scale by many groups, such as the reaction between purified (3-lactoglobulin and lactose or glucose (Matsuda et al, 1985), the conjugation of p-lactoglobulin with an alginic acid oligosaccharide conjugate (Hattori et al 1997) and the covalent binding of glycosyl residues (gluconic and melibionic acids) to (3-lactoglobulin to improve solubility and heat stability (Kitabake et al 1985). The heat stability and emulsifying properties of (3-lactoglobulin were improved by conjugation with glucose-6- phosphate (Aoki et al, 1997) or with carboxymethyldextran (Nagasawa et al, 1996). The emulsifying properties of casein were improved by conjugating them with dextran and galactomannan (Kato et al 1992). Japanese researchers pioneered studies on the antigenicity of the browning product between (3-lactoglobulin and lactose (Matsuda et al 1985 and 1987; Otani & Tokita, 1980; 1982a, b; Otani & Hosono, 1987; Otani et al, 1984; Otani et al, 1985, a,b,c,). Their findings were not surprising because although (3-lactoglobulin is a valuable protein according to nutrition scientists, it is also known as a potent stimulant of milk allergies. About 82% of milk allergy patients are sensitive to (3-lactoglobulin (Spies, 1976).

Therefore, it was strongly desirable to develop a glycated protein with decreased allergenicity and enhanced functional properties. Hattori et al (1997) reported that the conjugation of (3-lactoglobulin with oligosaccharides from alginic acid by the Maillard reaction had high heat stability, improved emulsifying activity and depressed aggregation. Shida et al (1994) reported enhanced heat stability of glycated (3-lactoglobulin and a-lactalbumin formed during heat treatment of skim milk. The two glycated proteins also have enterotoxin-binding properties. Watanabe et al (1984) showed altered heat stability of whey protein preparations as well as an isolated a-lactalbumin and (3-lactoglobulin whereby glycation with lactose increased the temperature of denaturation and coagulation of these protein preparations.

Other beneficial properties are also developed by the Maillard reaction including the antioxidant properties of heated whey powders (Browdy & Harris 1995, 1997). They found that whey powders, and Maillard reaction products (MRP) produced from heated whey, retard lipid oxidation. The antioxidant properties of MRP are well studied in model systems (Alais et al, 1997; Yoshimura et al 1997). Natural antioxidants are destroyed by processing but at the same time new ones are formed during processing and storage (Nicoli et al, 1997).

The heat stability of other proteins has been improved by this mechanism. Bovine serum albumin conjugated with glucose by Maillard reaction was reported by Morales et al (1976) and the glycation of ovalbumin by glucose was reported by Matsuda et al (1985). Kato et al (1995) improved the heat stability and emulsifying activity of ovalbumin by modification with glucose-6-phosphate. Plasma protein prepared from animal blood was complexed with galactomannan and shown to have improved emulsifying activity (Matsudomi et al, 1995).

The major focus of academic research has been on the interaction between isolated milk proteins and the natural milk sugar lactose that may occur during processing of dairy streams. Morgan et al (1997) and Leonil et al (1997), characterised the adducts formed by lactose and p-lactoglobulin during mild heat treatment. Leonil et al (1997) evaluated the extent of the early Maillard reaction by mass spectrometry and provided the first direct evidence of specific lactosylation of P-lactoglobulin. Morgan et al (1998) monitored Amadori product formulation between lactose and P-lactoglobulin directly using electrospray ionisation mass spectrometry and found the glycation reaction was faster at lower water activity, and further observed different heterogenous P-lactoglobulin glycoforms. Jones et al- (1998) analysed skimmed milk powders using capillary electrophoresis and showed that Malliard reactions occur during ambient storage of skimmed milk powder as well as during spray drying, but do not occur in fresh pastuerised milk. Nacka et al (1998) found that the substitution of sugars on amino groups of a-lactalbumin and P-lactoglobulin was of a purely random nature and further that proteins substituted with hexoses and lactoses exhibited higher solubility and improved emulsifying properties compared with non-glycated proteins or proteins substituted with ribose and glyceraldehyde.

However, to date, no commercial process has been developed to produce a glycated whole milk or whey protein product having improved functional properties.

It is an object of this invention to go some way to obtaining these desiderata or at least provide the public with a useful choice.

SUMMARY OF THE INVENTION According to the present invention there is provided a process for controlling Maillard-type glycation of a whey protein-containing material comprising the steps: i) predetermining the initial level of glycation of the whey protein-containing material; ii) subjecting the whey protein-containing material of step i) to Maillard-type glycation reaction by incubation of the whey protein-containing material with a reducing sugar under controlled temperature and moisture conditions for a sufficient time to produce a desired level of glycation without causing denaturation of the whey protein; WO 00/18249 PCT/NZ99/00163 iii) terminating the Maillard-type reaction after a desired level of glycation has been reached and removing any excess reducing sugars; and iv) recovering the glycated whey protein product.

The preferred reaction conditions enable the reaction to proceed efficiently to produce a whey protein product having enhanced functional properties on a commercially viable scale.

The reaction is preferably -carried out at a ratio of lg whey protein: 0.02-0.6g reducing sugar, water activity of 0.3-0.8aw and temperature of 30-75°C for 18-240 hours.

The water activity is preferably 0.6 aw .

The temperature is preferably held at between 40°C and 50°C.

Preferably the reaction is terminated at the required degree of glycation in step iii) by further drying the powder to a water activity of less than 0.20 and reducing the temperature to 20°C or below.

Preferably the process of step ii) is carried out at a pH of between 6.0-8.0.

The process may be precisely controlled by monitoring the level of glycation by mass spectrometry or any other method known in the art, such as, for example, by monitoring ^^>5 the production of furosine, in order to produce a product having the desired level of glycation and therefore, the desired enhanced functional properties.

Additionally or alternatively, an additional step of monitoring the rate of glycation by monitoring the production of furosine may be carried out before step iii) and the reaction 30 terminated once glycation has proceed to a desired level.

The whey protein-containing material may comprise one or more of the following: whey protein isolate (WPI), whey protein concentrate (WPC), whey powder, milk protein concentrate (MPC), milk protein isolate (MPI) and skim and whole milk powder.

WO 00/18249 PCT/NZ99/00163 _ The reducing sugar may comprise one or more of the following: lactose, glucose, galactose, maltose, fructose or their derivatives including glucose-6-phosphate, gluconic acid, or any oligosaccharide or saccharide conjugate that contains a reducing sugar.

Preferably, the glycated whey protein product has one or more enhanced functional properties selected from the group comprising enhanced heat stability, emulsifying activity, foamability, antioxidant activity and enterotoxin binding capacity.

In a further aspect the invention provides a process for enhancing the functional properties 10 of stored dairy powder products comprising the steps: i) adjusting the storage conditions for optimal glycation of whey protein contained in the dairy powder product; ii) allowing glycation to proceed for a time sufficient to provide a desired level of glycation; and iii) changing the storage conditions to inhibit further progression of the Maillard reaction into non-desirable browning.

The stored dairy powder product may comprise WPC, WPI, whey powder, MPC, MPI and skim or whole milk powders.

The conditions for optimal glycation may comprise: •25 a) the presence in the dairy powder product of a reducing sugar; b) a water activity of between 0.3-0.8 a^,; and c) temperature of between 30-75°C.

Preferably the pH of the dairy powder product is also between pH 6.0-8.0.

The reducing sugar may be inherently present in the dairy powder product or it may be added.

WO 00/18249 PCT/NZ99/00163 _ Preferably the reducing sugar is present in a ratio of 1.0g whey protein: 0.02-0.6g reducing sugar.

Preferably the reducing sugar is selected from one or more of lactose, glucose, galactose, maltose, fructose or their derivatives on any oligosaccharide or saccharide conjugate that contains a reducing sugar.

Preferably the time sufficient to provide a desired level of glycation in step ii) is between 1 hour and 80 days.

Preferably the conditions in step iii) which will inhibit further glycation include the reduction of temperature to below 20°C and/or the reduction of water activity to below 0.2aw. In this way, the stored dairy products treated according to this process and maintained under conditions which inhibit further glycation do not suffer from the phenomenon of 'browning' that occurs in stored dairy powder products due to extensive Maillard reaction.

Preferably the enhanced functional properties of the stored dairy powders includes one or more of the following: enhanced heat stability, emulsifying activity, antioxidant activity and enterotoxin binding capacity.

In a further aspect the invention provides a whey protein-containing dairy product wherein the whey protein has been glycated to a desired level according to the process of the invention to provide enhanced functional properties to the product.

The whey protein-containing dairy product may comprise WPI, WPC, whey powder, MPC, MPI, skim and whole milk products.

Preferably the enhanced functional properties of the dairy product include one or more of the following: enhanced heat stability, emulsifying activity, antioxidant activity and enterotoxin binding capacity.

Preferably the dairy product is used as an additive to improve the functionality and nutritional content of foodstuffs.

Preferably the product is used as an additive in clear acid beverages, especially in citric acid, phosphoric acid, lactic acid, malic acid and gluconic acid beverages where the glycated product improves the heat stability of the acid beverage formulations. The product may also be used as an additive in the formulation of low fat salad dressing where the improved emulsification properties are of particular use.

In a further aspect the present invention provides for the use of a glycated whey protein-containing material according to the invention in the production of an enhanced functional foodstuff.

In a further aspect the present invention provides for a foodstuff containing a glycated whey protein-containing material according to the invention.

Preferably the foodstuff is an acid beverage or low fat salad dressing.

In yet a further aspect the present invention provides a modified whey protein-containing material comprising a desired level of glycated whey protein having one or more enhanced functional properties.

The whey protein material may be selected from WPI, WPC, whey powder, MPI, and skim or whole milk powder. The functional properties are as defined above and the modified whey protein may be useful as a food additive as described above for the dairy product of the invention.

The invention will now be described with reference to the figures of the accompanying drawings and the following examples.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 and 2 show mass spectrometer time courses for the glycation of P-lactoglobulin in lactose supplemented WPI samples.

Figure 3 shows a thermogram of control and glycated whey protein-containing material.

Figure 4 shows the pH dependent emulsifying properties of glycated and control whey protein-containing material.

PCT/NZ99/00163 Received 8 August 2000 show acid heat stabilities of control and glycated whey protein-containing material. shows the mass spectrum of P-lactoglobulin in a WPI after glycation for 6 days at 40°C with a protein to lactose ratio of 1:0.1(w/w). shows mass spectra for P-lactoglobulin in a WPI after glycation for 18 h at 50°C with protein to lactose ratios of 1:0.1(w/w) and 1:0.4(w/w). shows mass spectra for P-lactoglobulin in a WPC after glycation for 0 and 2 days. shows mass spectra for a-lactoglobulin in a lactose supplemented WPI after glycation for 0 and 10 days. show mass spectra for P-lactoglobulin in WPC samples glycated by manipulating storage conditions. show mass spectra for P-lactoglobulin in WPC samples glycated by manipulating storage conditions. shows the distribution of glycoforms before and after lactosylation at 75°C and water activity (aw) of 0.79.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES 1. GLYCATION OF WHEY PROTEIN ISOLATES ) Two experiments were carried out using whey protein isolate as a whey protein source and 25 lactose as reducing sugar. The two samples of WPI had different levels of initial lactosylation due to different processing histories.

Preparation of samples WPI (49.91 grams. 94% protein) and lactose (18.92 grams, Analar BDH) were dissolved in 400 ml of MilliQ water (final ratio lactose to protein 0.4:1 w/w). The solutions were adjusted to 6.8 with NaOH IN. The solution was freeze dried. The dried material was incubated in four, 120 ml containers using a sealed microwave steamer dish containing Figures 5 and 6 Figure 7 Figure 8 Figure 9 Figure 10 Figures 11 & 12 I Figures 13-15 15 Figure 16 AMENDED GHELi IPEA/A.U WO 00/18249 PCT/NZ99/00163 - a saturated solution of potassium bromide (KBr) to give a constant aw of 0.79. The container lids were loosened for 24 hours to allow equilibration at the desired moisture content of 20% (Iglesias & Chirife, 1982). Incubation was carried out at 40°C in a temperature controlled room. Aliquots were removed at 3, 5, 7 and 10 days for mass spectral analysis. Once the target lactosylation level was obtained, the mixture was dissolved in 400ml MilliQ water and dialysed for 48h using a 10,000 molecular weight cut off dialysis tubing (Union Carbide) against distilled water. The dialysed solution was then ultrafiltered using a YM10 membrane (Amicon). The retentate was then dried. ■ Analysis of lactosylated WPI The nitrogen content was determined by the Dumas principle using a Leco FP-2000, Leco Corporation, St Joseph, Michigan, USA.

The progress of the glycosylation of the lysines of whey proteins by the early Maillard reaction was monitored by infusion flow injection electrospray ionisation mass spectometry (ESI-MS) using a Perkin-Elmer Sciex API 300, triple quadruple LC/MS/MS mass spectrometer (Sciex, Thomhill, Ontario, Canada) by the method described by Leonil et al (1997). Samples of glycated whey proteins were redissolved at 5mg/ml in distilled water and were then diluted 5 fold with 50% acetonitrile/0.5% formic acid, prior to infusion.

The number of reacted lysines in the protein was estimated by measuring furosine by an adaption of the method of Henle et al (1995). The level of the early Maillard Amadori compound lactulosyl-lysine was calculated by multiplying the furosine value by 2.25 (the factor 2.25 accounts for the yield of furosine from the Amadori product (Molnar-Perl & Pinter-Szakacs, 1986)). The amount of lysine in the product was estimated as 12 g/lOOg.

Table 1 shows the results for furosine measurements, and the equivalent percentage of reacted lysine amino groups calculated from the furosine results, for the two WPI samples. The controls showed that the two WPI samples had different initial levels of lactosylated lysines. This was due to the different processing histories of the samples. The results for the 240h (10 days) of lactosylation showed that the sample with the higher initial level of lactosylation (WPI-2) had a much higher percentage of lysines reacted at the end of the 240h incubation period. This indicates that the initial level of lactosylation of any whey protein-containing powder, to be used for controlled lactosylation, must be predetermined. /3 r/o^£.ty)A<s?^ oteeo Whey protein isolate is a rich source of lysine (approximately I2g/I00g) and although 43.6% of the lysines are blocked in WPI-2A, the product still has 7g/l00g of available lysine, a value very close to caseinate, which is considered a good source of lysine (8g/l00g).

Table 1 WPI lactosylation levels WPI-1 WPI-2 Oh Control 168h(7 days) lactosylation (WPI-1 A) 240h lactosylation (WPI-IB) Oh Control 240h lactosylation (WPI-2A) %Protein 94.42 93.79 . 92.51 93.20 87.40 Furosine (mg/lOOg protein) .11 904.26 1610.30 446.73 2660.00 %Blocked lysine 0.44 .90 27.94 7.80 43.60 The time course of lactosylation can be followed accurately by mass spectrometer analysis. Figure 1 shows the results for WPI-1 at Oh, 168h (7 days) and 240h (10 days). A and B refer to p-lactoglobulin variants A and B. At Oh very little lactosylation is detected and the major peaks are the native P-lactoglobulin variants A and B. After 7 days adducts of 1-4 lactosyl-lysines are seen (llac-41ac) with incremental mass increases of 324 Daltons (the molecular weight of lactose) to the molecular mass of the native P-lactoglobulin variants A and B (WPI-1 A). Some residual native P-lactoglobulin still remain at this time. After 10 days all the native (non-lactosylated) P-lactoglobulin has gone, and lactosylated derivatives of P-lactoglobulin variants A and B with up to 10 lysines blocked can be seen, with the mean distribution around 6 adducts (WPI-IB).

TENDED SHEET WO 00/18249 PCT/NZ99/00163 .

Figure 2 shows the mass spectrometer results for WPI-2 at 0 and 10 days. At time 0 WPI-2 shows several adducts of P-lactoglobulin, with up to 3 adducts detectable, unlike WPI-1. There is a clear correlation between the degree of glycation and the furosine analysis. After 10 days a similar pattern to WPI-1 is seen, with WPI-2 showing up to 8 5 adducts with the mean around 5 lactosylation sites.

Physico-chemical and functional properties of the lactosylated WPI Differential scanning calorimetry The thermal behaviour of WPI-1 was studied with a Perkin-EImer DSC-7 differential scanning calorimeter. Selected samples were desalted using Centricon micro concentrators with a 3000 molecular weight cut-off. Aliquots of 10|iL of a 10% protein solution (w/w), in simulated milk ultrafiltrate (Jenness & Koops, 1962) were taken and sealed in stainless steel capsules. The weight of the pans was measured before and after analysis. All analyses were performed in duplicate. The scanning temperature range was 30-115°C at a heating rate of 10°C/min. The onset and denaturation temperature as well as denaturation enthalpies were calculated from the thermogram using a Perkin-EImer Pyris DSC software data analyser.

Figure 3 shows the enhanced heat stability of the lactosylated WPI (WPI-IB) compared to the original WPI control (WPI-1). This is demonstrated by the higher onset (onset temp) and denaturation temperature (Td). Values for the onset temp, ^^25 Td, AT,7j (measurement of the cooperativity for protein aggregation) and AH (total energy required to denature the protein mixture) are shown in Table 2.

Table 2 DSC Results Sample Onset temp.(°C) Td(°C) ATM(°C) AH(J/g) WPI-1 (control) 72.81 77.53 .40 0.660 Lactosylated WPI (WPI-IB) 75.71 80.87 6.98 0.600 PCT /NZ99/00163 Emulsifying ability The emulsifying ability was tested using a Labplant jet homogenizer (Labplant, Huddersfield, UK) as outlined in the manufacturers handbook. A known mass of protein product was dissolved in 100ml of buffer at 55°C in a shaking water bath. The weight of protein used was sufficient to give a protein content in the final emulsion of 0.5wt%. The solutions were held at 55°C for 1 hour. At the same time a portion of soya oil was heated to 55°C. The buffers used were citric acid-disodium hydrogen phosphate (Mcllvaine) buffers at pH 3.6, 4.0 and 6.8. All emulsions were made up at 20wt% soya oil.

Another of the targeted functional properties that can be enhanced in a glycated product is the emulsifying ability. Figure 4 shows the mean d32 for the non-lactosylated (WPI-1) and the lactosylated WPI (WPI-1B) at the three pH values. A smaller value of d3,2 means that the protein is a better emulsifier. It is clear that there were large differences in the emulsifying ability of the two samples at pH 4.0. The emulsifying properties of whey proteins show a relatively complex pH dependence. A minimum is observed in the emulsifying properties at the isoelectric point (pi). This can be attributed to the minimum in protein solubility that occurs at that pH value. At pH values above and below the pi the emulsifying properties improve, although generally the emulsifying activity at pH values below the pi is lower than at pH values above the pi. The glycated whey protein isolate showed a marked improvement in emulsifying ability (smaller particle size) at pH 4.0. This is probably a result of a change in the pi caused by blockage of positively charged lysine side chains by reaction with sugar residues.

Acid/heat stability For the acid stability test, all samples were redissolved at 3% (w/v) protein in MilliQ water. Subsamples were then pH adjusted with 10%(w/v) solutions of either phosphoric or citric acid (the two most common acids used in clear acid beverage products). The standards and samples were studied over the pH range 3.6-4.2. Samples (pH adjusted) were heated at 80°C for 20 min. The clarity of the samples was measured before and after heating by monitoring absorption at 610mm.

WO 00/18249 PCT/NZ99/00163 - WPIs have been targeted for use in acid beverage formulations, where solution clarity at low pH, after heat treatment, is a prime requirement. WPI-1 A, WPI-IB and WPI-2A were tested for acid heat stability in comparison with their control WPIs (WPI-1 and WPI-2, respectively). This demonstrated the positive effect of 5 glycation on the acid heat stability of WPIs. Figure 5 shows the results for WPI-1 A and WPI-IB in heated systems adjusted with (a) citric acid and (b) phosphoric acid.

Figure 5 shows clearly that lactosylation has increased the acid stability of WPI-1 10 (as measured by absorbance at 610nm) in both (a) citric acid and (b) phosphoric acid (Figure 2). The more extensive the lactosylation (WPI-IB) the greater the improvement in acid stability. The increase in acid-heat stability was particularly large at pH 4.0 and pH 4.2. The pH 4.2 results shown for citric acid had reached the maximum turbidity measurable at 610nm (3.0) and the samples had 15 precipitated. The WPI used for the above experiments (WPI-1) had poor initial clarity in solution at neutral pH(A610nm=0.356). To fully investigate the benefits of lactosylation these experiments were repeated with WPI-2, a product that had good clarity at neutral pH(A610nm=0.061). These results are shown in Figure 6, which shows a dramatic improvement in the acid-heat stability of the lactosylated 20 sample at pH values 4.0 and 4.2 in both phosphoric and citric acids. The improvement at pH 4.0 is good enough to make this product acceptable for use in clear citric acid beverages at this pH, an application in which many WPIs have so far failed to work.

Glycation of WPI under other conditions The above experiments were carried out under one set of conditions. Other conditions for promoting glycation were examined with the reactions monitored by mass spectrometry. Figure 7 shows the results for the lactosylation of WPI-2 for 6 days at 40°C, with a ratio 30 of lactose to protein of 0.1:1 (w/w). The aw for this experiment was 0.57.

Figure 8 shows the mass spectrometer time course for two experiments using WPI-2 at 50°C. One experiment used a lactose to protein ratio of 0.7:l(w/w) and the other a lactose to protein ratio of 0.4:l(w/w). Increasing the temperature decreased the time required for 35 the glycation to 18 hours. The aw for this experiment was 0.6. 2 GLYCATION OF WPC Glycation of a standard 56% protein WPC (ALACEN 421, New Zealand Milk Products) was performed at 40°C under controlled conditions of aw (0.7). The WPC contained 34% 5 lactose, which gave a lactose:protein ratio of 0.6:l(w/w). The aw of the product was adjusted to 0.7 to carry out the glycation. The reaction was monitored by mass spectrometry.

Figure 9 shows the mass spectrometry results for the lactosylation of ALACEN 421. At 10 time 0 the produet already eontains lactosylated p-lactoglobulin derivatives, with up to 3 adducts detected. As before this is due to the processing history of the product. After two days a product with 8 adducts is produced with the mean around 4 or 5 adducts. This product could form the feed to a process to produce a lactosylated WPI with enhanced functionalities. 3 GLYCATION OF OTHER WHEY PROTEINS All of the above experiments present time course analysis of P-lactoglobulin. This is because it is the major whey protein and as such is the most important both qualitatively 20 and quantatively. P-lactoglobulin dominates the functional behaviour of WPIs and WPCs and so its glycation has the largest impact on the various functional properties. There is evidence that other whey proteins are also glycated. Figure 10 shows the mass spectrometer time course analysis of the lactosylation of a-lactalbumin in WPI-2(40°C, 10 days, 0.4 g lactose/g protein). Time 0 shows 1 adduct and indication of a second. ^^25 After 10 days the second adduct is clearly seen. Unfortunately a-lactalbumin is a naturally glycosylated protein containing complex oligosaccharide chains that result in a complex mass spectrometer trace. This hinders clear identification of other adducts.

The reaction conditions selected in the present invention enable the reaction to proceed 30 efficiently to produce a whey protein product having enhanced functional properties on a commercially viable scale. In particular, at high ratios of whey protein reducing sugar (eg 1.0:0.1), the sugar is completely consumed in the reaction and there is no need for removal of excess sugar. At the higher sugar levels (eg 1.0 of whey protein : 0.6 of sugar) the reaction proceeds rapidly, but the excess of sugar that remains after the desired 35 degree of glycation desired is achieved must be removed. If not the reaction will proceed to the latter stages of the Maillard reaction and undesirable properties will appear (browning, off flavours, lack of functionality etc). Outside of the stated range of water PCT/NZ99/00163 . activity (ie 0.3-0.8 aw) the reaction proceeds very slowly. Regarding the temperature range, it has been found that below 30°C the reaction proceeds very slowly and above 75°C other reactions can take place that are detrimental to protein functionality.

. PREPARATION OF GLYCATED DAIRY POWDERS.

Dairy powders (56 % WPC, 80% WPC, Skim Milk powder, Whole Milk powder and Milk Protein Concentrate) were stored at 30, 35 and 40°C under controlled water activities (aw) of 0.33, 0.72 and 0.79. Samples were removed at various time intervals between 4 h and 10 36 days for analysis. The giycation process was monitored by either mass spectroscopy, measurement of furosine (Table 3) or measurement of available amine.

Table 3. Furosine levels of Stored Dairy Powders Sample Storage Conditions Furosine (mg/100 g powder) Temp. a„ 0 (Control) 6 days 36 days Alapro 4850 °C 0.33 243 257 297 (MPC from NZMP) 0.72 243 352 514 0.79 243 377 679 °C 0.33 243 373 538 0.72 243 512 735 0.79 243 643 959 40°C 0.33 243 449 583 0.72 243 689 1010 0.79 243 858 1070 Alacen 421 °C 0.33 319 330 383 (56% WPC from NZMP) 0.72 319 522 1690 0.79 319 1110 2400 °C 0.33 319 239 744 0.72 319 1350 2590 0.79 319 1200 1850 40°C 0.33 319 419 886 0.72 319 576 1450 0.79 319 1810 2350 Alacen 392 °C 0.33 517 525 738 (80% WPC from NZMP) 0.72 517 595 955 0.79 517 589 1020 °C 0.33 517 618 920 0.72 517 779 1120 0.79 517 824 1040 40°C 0.33 517 733 913 • 0.72 517 880 1180 - 0.79 517 993 1110 Whole Milk Powder °C 0.33 48 47 76 0.72 48 138 407 0.79 48 111 346 °C 0.33 48 59 377 0.72 48 291 614 0.79 48 158 398 40°C 0.33 48 93 835 0.72 48 441 830 0.79 48 399 835* ;Skim Milk Powder ;30°C ;0.33 ;94 ;93 ;128 ;0.72 ;94 ;197 ;655 ;0.79 ;94 ;215 ;569 ;35°C ;0.33 ;94 ;89 ;424 ;0.72 ;94 ;411 ;900 ;0.79 ;94 ;323 ;719 ;40°C ;0.33 ;94 ;122 ;320 ;0.72 ;94 ;659 ;1100 ;0.79 ;94 ;442 ;894 ;* 24 day sample since 36 day sample unavailable . HIGH TEMPERATURE SHORT TIME GLYCATION TO PREPARE GLYCATED DAIRY POWDER DURING STORAGE Glycation of dairy powders is possible under a variety of conditions and can be controlled by manipulating the storage conditions of products containing reducing sugars. The examples given show a range of both storage conditons and powders. a) Glycation of a 56 % protein WPC (ALACEN 421) was carried out at 50 and 70°C with an aw of 0.8. Glycation was monitored by mass spectroscopy at time intervals between 30 minutes-and 24 h (Figures 11 and 12). b) Glycation of WPC samples (ALACEN 421, ALACEN 472, ALACEN 891 NZMP) were carried out at 75 °C under a controlled a,v of 0.8. Glycation was monitored by mass spectroscopy at time intervals between 30 minutes and 22 h (Figures 13-15).

It will be appreciated that it is not intended to limit the invention to the aforementioned examples only, many variations, such as might readily occur to a person skilled in the art being possible, without departing from the scope thereof.

INDUSTRIAL APPLICATION The present invention provides a process for producing a glycated whey protein product and glycated whey protein products per se which have enhanced functional properties useful as food additives to improve functionality and nutritional content of foodstuffs. Of particular industrial applicability is the use of the glycated whey protein product in clear acid beverage formulations.

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PCT/NZ99/00163 Received 8 August 2000

Claims (37)

CLAIMS:

1. A method of storing whey protein-containing dry powdered material comprising the steps: i) predetermining the initial level of glycation of the whey protein-containing dry powdered material; and either ii) increasing the glycation level of the whey protein-containing dry powdered material of step i) by subjecting the material to Maillard-type glycation reaction by storing the whey protein-containing dry powdered material under controlled temperature and moisture conditions for a sufficient time to produce a desired level of glycation without causing denaturation of the whey protein followed by termination of the Maillard-type reaction after a desired level of glycation has been reached by storing the whey protein-containing dry powdered material under controlled temperature and moisture conditions which inhibit the glycation reaction; or iii) inhibiting further glycation of the whey protein-containing dry powdered material of step i) by storing the material under controlled temperature and moisture conditions to inhibit the glycation reaction.

2. A method according to claim 1, wherein the whey protein-containing dry powder material inherently contains a reducing sugar at a ratio of lg whey protein: 0.02-0.6g reducing sugar.

3. A method according to claim 1 or 2, wherein the storage conditions to increase glycation comprise a water activity of 0.3-0.Ba^,, a temperature of 30-75°C for 18-240 hours.

4. A method according to claim 3, wherein the water activity is 0.6 aw .

5. A method according to claim 3, wherein the temperature is held at between 30°C and 50°C.

6. A method according to claim 1, wherein the reaction is terminated at the required degree of glycation in step ii) or further glycation is inhibited in step iii) by storing at a temperature of 20°C or below under conditions which will reduce the water activity of less than 0.20. 7.

A method according to claim 1, wherein the powdered material has a pH of between 6.0-8.0. PCT/NZ99/00163 Received 8 August 2000 -24-

A method according to any preceding claim whereby the level of glycation is precisely measured and monitored by mass spectrometry or any other method known in the art, to produce a product having the desired level of glycation.

A method according to claim 8, wherein glycation is measured by monitoring the production of furosine.

A method as claimed in any preceding claim, wherein the rate of glycation is measured by monitoring the production of furosine before the reaction is terminated once glycation has proceeded to a desired level.

A method according to claim 1, wherein the whey protein-containing dry powdered material is selected from the group consisting of one or more of the following: whey protein isolate (WPI), whey protein concentrate (WPC), whey powder, milk protein concentrate (MPC), milk protein isolate (MPI) and skim or whole milk powder.

A method according to claim 1, wherein the inherent reducing sugar is selected from a group consisting of one or more of the following: lactose, glucose, galactose, maltose, fructose or their derivatives including gluconic acid, and any oligosaccharide or saccharide conjugate that contains a reducing sugar.

A method according to claim 1, wherein the glycated whey protein product has one or more enhanced functional properties selected from the group consisting of enhanced heat stability, emulsifying activity, antioxidant activity and enterotoxin binding capacity.

A process for enhancing the functional properties of stored dairy powder products comprising the steps: i) adjusting the storage conditions for optimal glycation of whey protein contained in the dairy powder product; ii) allowing glycation to proceed for a time sufficient to provide a desired level of glycation; and iii) changing the storage conditions to inhibit further progression of the Maillard reaction into non-desirable browning.

A process according to claim 14, wherein the stored dairy powder product is selected from the group consisting of WPC, WPI, whey powder, MPC, MPI and skim or whole milk powders. PCT/NZ99/00163 Received 8 August 2000 -25 -

16. A process according to claim 14, wherein the conditions for optimal glycation comprise: a) the presence in the dairy powder product of a reducing sugar; b) a water activity of between 0.3-0.8 a^ and c) temperature of between 30-75°C.

17. A process according to claim 16, wherein the pH of the dairy powder product is also between pH 6.0-8.0.

18. A process according to claim 14, wherein the reducing sugar is inherently present in the dairy powder product.

19. A process according to claim 18, wherein the reducing sugar is present in a ratio of 1.0g whey protein: 0.02-0.6g reducing sugar.

20. A process according to any one of claims 16, 18 or 19, wherein the reducing sugar is selected from one or more of lactose, glucose, galactose, maltose, fructose or their derivatives and any oligosaccharide or saccharide conjugate that contains a reducing sugar.

21. A process according to claim 14, wherein the time sufficient to provide a desired level of glycation in step ii) is between 1 hour and 80 days.

22. A process according to claim 14, wherein the conditions in step iii) which will inhibit further glycation include the reduction of temperature to below 20° C and/or the reduction of water activity to below 0.2aw.

23. A process according to claim 14, wherein the enhanced functional properties of the stored dairy powders includes one or more of the following: enhanced heat stability, emulsifying activity, antioxidant activity and enterotoxin binding capacity.

24. A whey protein-containing dry dairy powder product selected from the group consisting of WPI, WPC, whey powder, MPC, MPI and skim or whole milk products, wherein the whey protein has been glycated to a desired level according to the method of any one of the preceding claims to provide enhanced functional properties to the product.

25. A whey protein-containing dry dairy powder product as claimed in claim 24, wherein the enhanced functional properties of the dairy product include one or more of the following: enhanced heat stability, emulsifying activity, antioxidant activity and enterotoxin binding capacity. -26-

26. A whey protein-containing dry dairy powder product as claimed in claim 24, suitable for use as an additive to improve the functionality and nutritional content of foodstuffs.

27. A whey protein-containing dry dairy powder product as claimed in claim 26, wherein the product is suitable for use as an additive in clear acid beverages.

28. A whey protein-containing dry daiiy powder product according to claim 27, wherein the clear acid beverage contains one or more of citric acid, phosphoric acid, lactic acid, malic acid and gluconic acid and wherein the glycated product improves the heat stability of the acid beverage formulations.

* 29. A whey protein-containing dry daiiy powder product according to claim 26, wherein the product is suitable for use as an additive in the formulation of low fat salad dressing wherein the improved emulsificationproperties are of particular use,

30. The use of a glycated whey protein-containing dry powdered material according to claim 24 or 25 in the production of an enhanced functional foodstuff.

31. A foodstuff containing a glycated whey protein containing dry powdered material according to claim 24 or 25.

32. A foodstuff according to claim 31, comprising an acid beverage or low fat salad dressing.

33. A process as claimed in claim 1 substantially as herein described with reference to any example thereof and/or the accompanying drawings.

34. A process as claimed in claim 14 substantially as herein described with reference to any example thereof and/or the accompanying drawings.

35. A whey protein-containing dairy powder product as claimed in claim 24 substantially as herein described with reference to any example thereof and/or the accompanying drawings.

36. The use as claimed in claim 30 substantially as herein described with reference to any example thereof and/or the accompanying drawings. INTELLECTUAL PROPERTY OFFICE OF N.Z.

37. A foodstuff as claimed in claim 31 substantially as herein described with - 2 MAY 2003 reference to any example thereof and/or the accompanying drawings. RECEIVED