Technical Field

This Invention relates to whey protein concentrate and to food products, such as Ice cream utilizing whey protein concentrate as an Ingredient. In this application, the term "1ce cream" covers full fat 1ce cream, reduced fat 1ce cream, low fat Ice cream and non-fat 1ce cream.

Background Art Whey Is a by-product when cheese Is produced from milk. After suitable pre-treatment well known to persons skilled 1n the art, milk 1s generally treated with a suitable culture to produce curd which Is subsequently separated from the remaining liquid, namely dairy whey, and used to make cheese. It 1s known that whey contains useful proteins, generally known as dairy whey proteins. It 1s also known that the principal proteins 1n such whey are β-lactoglobulln and -lactalbumln. Other proteins Include serum derived 1mmunoglobul1ns. Proteose peptones are also present.

Large quantities of w ey are produced as a by-product of cheese production, and various uses for such whey have been developed over the years, mainly as a food Ingredient. Such whey usually contains about 12% protein

by weight on a total solIds basis. It has become conventional to further treat the whey to provide a product containing at least about 30% protein by weight on a total solids basis, which product Is known as whey protein concentrate (WPC). It is usually whey protein concentrate rather than the original fluid whey or dry total whey solids which 1s used as a food Ingredient. A well known process for extracting the proteins from the whey Involves heat treating the whey at an add pH of about 4.5 so as to denature the proteins which then precipitate and are separated from the liquid medium by centrifugatlon. However, in this process, a significant proportion of the proteins are not denatured and consequently, are lost in the centrifugatlon step.

Buhler et al, in U.S. 4,265,924 and 4,291,067 disclose a process aimed at Improving the protein yield by increasing the amount of protein denaturatlon and hence precipltatable protein which can then be recovered. The claimed process Involves denaturing the proteins present in the whey to an extent of from 35%-70%; removing the non-fat whey constituents from the other contents by ultrafiltration and subjecting the proteins in the retentate to a further heat treatment to effect as complete as possible denaturatlon of the proteins. The objective Is to denature all heat denaturable proteins in the whey and

this 1s reflected 1n the severe heat treatment required, such as using a temperature of 95°C-100 ^β C for a period of from 10 to 30 minutes or a temperature of 120°C-160°C for from 5 to 120 seconds. This results 1n a coagulated product containing particles.

Another problem with such prior process, and this 1s referred to In Buhler, is that the high temperature used especially 1n combination with add pH's have a deleterious effect on the fat present and the organoleptic properties of the resulting protein product. This would preclude Its use in many applications.

Whey protein concentrate has been used as an Ingredient 1n 1ce cream production, namely 1n the production of full fat Ice cream or reduced fat ice cream and has been proposed as an Ingredient in low and non-fat 1ce cream (sometimes called low and non-fat frozen dairy dessert), see for example U.S. Patent 4,840,813 (Greenberg et al.).

However, a major obstacle to such use, particularly 1n low and non-fat ice cream, has been the fact that the whey protein concentrate tends to cause coagulation of the ice cream mix while 1t 1s being pasteurized, with the result that 1ce cream production has to be shut down to enable the

coagulated material to be removed. This 1s because, 1n the past, the whey protein concentrate used has been whey protein concentrate of a conventional kind, namely with at least most, and preferably all, of its protein In the natural state, I.e. undenatured. However, undenatured whey protein can also cause problems, e.g. undesirable gelling during use.

Since persons skilled In the art may interpret the meaning of denaturatlon and the manner 1n which denaturatlon should be measured 1n different ways, and the value of that characteristic 1s most Important In the present context, percentage denaturatlon 1n this application as applied to the present invention means the percentage denaturatlon when calculated in accordance with the optical based methodology described (unless otherwise stated as "PM" which is defined by reference herein).

Disclosure of Invention

It has now been discovered that the problem of coagulation in Ice cream production using whey protein concentrate can be substantially overcome if the denaturation of the whey protein in the whey protein concentrate is controlled during its production so as to be at least about 50% but less than 90% relative to the said

proteins in the raw milk and more preferably from about 60 to about 80%, and more preferably 65 to 75%, when measured by the method described at the end of this specification.

Below about 50%, the prior art problem of coagulation during 1ce cream production arises. Above about 90%, the 1ce cream product may have a somewhat sticky, gummy mouth feel which may be unacceptable to some consumers.

In one aspect the present Invention provides a process wherein ultraflltered whey containing substantially undenatured whey protein Is subjected to a controlled heating regimen comprising heating at a temperature of less than 90 ^β C for a period of time sufficient to heat denature not less than about 50% but not more than 90% of said heat denaturable protein to produce a whey protein product.

In many Instances, non-ultraf1ltered whey 1s readily available and hence In another aspect therefore, the present Invention provides a process for preparing a whey protein product comprising: a) subjecting a whey comprising substantially undenatured whey proteins and lactose to an ultraflltration step to'form a retentate containing whey proteins and a permeate containing a major part of the lactose; and

b) subjecting the retentate to a controlled heating regimen comprising heating at a temperature of less than 90 ^C C for a period of time sufficient to heat denature not less than about 50% but not more than about 90% of said heat denaturable proteins to produce a whey protein product.

In yet another aspect, the present Invention provides a process for preparing whey protein concentrate comprising pasteurizing raw milk with resultant denaturatlon of some whey protein, forming curds in said milk, removing the curds from the remaining whey, subjecting the whey to an ultraflltratlon step to remove lactose as permeate, subjecting the ultrafHtered whey retentate to heat treatment to denature further whey protein to cause a total of at least about 50% but not more than 90% of the whey protein to be denatured relative to the raw milk, and concentrating the heat treated whey to produce whey protein concentrate.

Control of the heating regimen or heat treatment, that 1s the temperature and associated time period, 1s very Important if the product having the desired organoleptic properties is to be achieved. A temperature lower than about 75°C has been found unsuitable bearing in mind that heating periods of greater than say 60 seconds, preferably

30 seconds are to be avoided. Consequently, 1t is preferred that the ultraflltered whey be treated at a temperature of from about 75 to 85°C, especially 78 to 82 ^β C for a period of from 5 to 30 seconds. It has been found that a temperature of about 80 ^β C ±0.5 for a time period from 10 to 20 seconds 1s advantageous. The heat treatment may be effected 1n any suitable equipment for example, plate or coll heat exchanger or the equivalent. The specific characteristic of the equipment are a factor 1n determining the optimum temperature/time regimen to be used.

It will be appreciated that the temperatures used to denature the whey proteins according to the present Invention are low compared to prior process and hence the regimen is more moderate or gentle resulting In a

"tempered" denatured protein product. Hence, 1n this specification "temperately denatured" means the controlled denaturatlon of ultraflltered partially del.actosed substantially undenatured proteins at a temperature of not more than 90 ^β C, and preferably, more than 75 ^β C.

Further, the product may be concentrated and hence be a "WPC". It may be used in a liquid or slurry form or dried by usual techniques such as spray drying. The dried product 1s readily redlspersed in water with no loss of the desired characteristics.

It is preferred that not less than 60% but not more than 80%, and especially about 65 to 75%, and most preferably 68 to 72% of the heat denaturable proteins, have been denatured 1n the process.

The starting whey substrate following the ultrafiltration step and prior to heat treatment preferably has a total solids content of from 5 to 15%, especially 7 to 11% and advantageously 8 to 10%.

In a further aspect the present Invention provides a whey protein product comprising temperately denatured whey protein, which 1s denatured to not less than about 50% and to not more than about 90% based on a total amount of heat-denaturable proteins contained In raw milk.

The protein product of the Invention 1n dry form preferably contains from 30 to 65% whey proteins, partially temperately denatured as detailed herein. Lactose content will generally be In the range of from about 25 to 55%.

The product of the present Invention 1s a partially denatured whey protein useful in a variety of food applications where its fat-substitution and organoleptically pliant and bland characteristics may be used to advantage.

Because of the complex nature of the protein content of whey protein concentrate, the reason for the success of the present Invention 1s not clearly understood. It had previously been believed that the whey protein concentrate used 1n Ice cream production should Initially have at least most of Its protein 1n the undenatured state 1n order to produce acceptable Ice cream.

It 1s possible, although this 1s just postulation, that the advantages are connected with the relative degree of denaturatlon of different proteins, such as β-lactoglobul1n and < -lactalbum1n, when the denaturatlon Is as specified 1n the present Invention. However, the ratio of β-lactoglobul1n to - -lactalbumln 1s not affected by being treated according to the present Invention.

It may be advantageous to use raw milk as the basic starting material since the Invention may be carried out 1n a cheese making plant where the whey 1s produced. However, 1t may be convenient 1n other Instances to use earlier produced whey provided 1t 1s of the desired quality.

Although the type and composition of starting whey substrate may vary, being a natural product and produced by a variety of processes, 1t is extremely important that it be of a high quality If the desired product 1s to be

achieved.

The whey should preferably be fresh, substantially uncoloured and preferably has been passed through fine savers. Advantageously, It may be a by-product of the production of brick, Cheddar or farmer's cheese but preferably mozzarella.

Preferred characteristics of the whey are as follows: a) a minimum unadjusted pH of at least 6, not more than 6.5, preferably from 6.25 to 6.35; b) a titratable addlty of from 0.10 to 0.20%, preferably 0.13 to 0.15%; c) must not contain a significant amount of non-heat denaturable rennet which has been found to produce off flavours; d) only heat denaturable enzymes may be present, and especially, no non-heat denaturable llpase may be present, this emanating for example, from parmesan cheese production; e) hydrogen peroxides, bacterlcldes, antifoaming or de-foaming agents or titanium dioxide should be excluded.

Further, it 1s most preferred that the whey, upon

being removed from the drain table during cheese production, be cooled to and maintained at 6 ^β C or below, prior to Its being processed according to the present Invention to form the whey protein product. 5

In summary, careful control of the starting whey substrate 1s extremely Important to achieving the protein product of the present Invention.

10 It 1s possible to use a starting whey substrate which 1s a combination of two or more types of whey for example, a mixture of mozzarella and Cheddar whey.

It will be appreciated that a small proportion of 15. the heat denaturable proteins in whey may be, and usually are, denatured during production of the whey from raw milk (herein "raw milk" means untreated milk from which a specific substantially undenatured whey protein 1s derived). Typically at most 10% or 15% but 1n extreme 20 cases possibly 20% of the said whey proteins relative to raw milk are denatured. This figure 1s obtained theoretically since the described optical method of evaluation is not readily applicable in the presence of significant amounts of casein as are present in raw milk. 25 However, because of the importance of controlling the characteristics of the starting whey and the heating

regimen of the whey according to the present invention, 1t is preferred that such pre-treatment denaturatlon be kept to a low value for example less than 15% and preferably less than 10% and if possible less than about 5%. For comparison purposes although there is no direct correlation between the two methods, it may be noted that about 15% denaturatlon measured by the optical method amounts to less than about 5% when measured by alternative precipitation methods ("PM") of evaluation (refer S.J. Rowlands 1938, Determination of Nitrogen Distribution of Milk, 3. Dairy Research p.42-26 for the "PM" method.) These denaturation figures may be typical for commercially available WPC's which may be processed according to the present Invention.

The whey protein product of the present Invention may be used in the production of full fat or reduced fat ice cream or 1n the production of low or non-fat 1ce cream and other food products such as yoghurt, sour cream, white sauces, salad dressing, pudding, milk shakes, soft serve ice cream, mayonnaise and other applications where a protein content 1s required, such as cheese, etc. It 1s essentially a functional heat "tempered" product in contrast to prior art undenatured or denatured whey protein products. It has improved organoleptic properties as well as a reduced tendency to cause excessive gelling or form lumps or the like when admixed with other food components,

e.g. during the production of 1ce cream.

In this specification all percentages 1n formulations, etc. are by weight unless stated otherwise.

Brief Description of Drawings

Preferred embodiments and examples of the invention will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 1s a schematic view of a process for producing cheese and also whey protein concentrate In accordance with the invention, Figure.2 Is a schematic view of a process for making 0% fat 1ce cream 1n accordance with the Invention,

Figure 31s a schematic view of a. process for making 1% (by weight) fat 1ce cream 1n accordance with the

Invention, and

Figure 41s a schematic view of a process for making ice cream with 7% (by weight) and higher amounts of fat in accordance with the invention.

Mode For Carrying Out The Invention

Referring to Figure 1, which shows the preparation of whey protein concentrate In accordance with a preferred embodiment of the invention, raw milk at a temperature of from about 3 to about 6 ^β C is preheated in a preheating step 10 to a temperature of from about 43 to about 49 ^β C and then passed to a fat separation step 12 where some fat Is separated, the actual amount depending upon the type of cheese to be produced. The preheated fat-reduced milk 1s then pasteurized in a pasteurization step 14 at a temperature of about 73°C for about 20 seconds, with subsequent cooling to a temperature of from about 32 to about 38 ^β C. The pasteurized fat-reduced milk then passes to a curd forming step 16 where lactic culture Is Injected and rennet 1s added In known manner and the contents are cooked and cut to produce curd.

The resultant curd/whey slurry 1s pumped to curd removal step 18 where raw whey Is drained off at a temperature of from about 38 to about 41 ^β C. The curd is subsequently processed Into cheese, 1n this case mozzarella cheese, 1n any desired manner. At this stage, the protein in the whey is from about 5 to about 10% denatured, relative to the raw milk, most of the denaturatlon having occurred when the milk was pasteurized in the

pasteurization step 14.

The procedure from hereon was also followed using a mixture of 90% mozzarella and 10% Cheddar cheese whey with similar results.

The whey having a pH of about 6.1 solIds content of about 6% from curd removal step 181s pumped to pasteurization step 20 where further pasteurization occurs at a temperature of about 74 ^β C for about 30 seconds, with subsequent cooling to a temperature of from about 50 to about 52 ^β C. This treatment causes further denaturatlon of the protein such that the protein is then from about 10 to about 15% denatured relative to the raw milk. It will be appreciated that pasteurization steps are carried out for practical handling reasons and to ensure retention of whey quality. In other plant configurations they may not be needed.

The pasteurized whey 1s pumped to an ultrafiltration step 22 where the whey Is ultraflltered with a membrane having a nominal molecular weight cut-off of 5,000 (such as a K0CHXL-1000 ^TH by KOCH Membrane Systems Inc., Wilmington. MA, U.S.A. The permeate from ultrafiltration step 20 may be used as desired. Most of the lactose 1n the whey will be in the permeate.

The retentate, namely ultraflltered whey with a pH of 6.1 and about 9% total solids by weight, is pumped to a heat treatment step 24 where 1 Is subjected to treatment in a plate heat exchanger (made by APV) at a temperature of about 80 ^β C for about 17 seconds. Further and by far the most denaturatlon occurs during this stage such that the protein in the whey 1s from about 60 to about 80% denatured, (relative to that 1n raw milk). This specific time temperature regimen gave a product having a denaturatlon value of about 71% (which product gave a value in the order of 40% PM. The pasteurized ultraflltered whey proceeds to a concentration step 26 where evaporation 1s carried out at a temperature of about 69 ^β C under a vacuum of about 23 inches Hg to concentrate the total solIds content to from about 30 to about 32% by weight. After concentration step 26, the whey protein concentrate (WPC) 1s cooled to about 6°C 1n a cooling step 28, and may be used in Its liquid form. The product was also spray dried for use In Its dry form.

The following table details a number of whey protein concentrates of the present invention produced using the procedure generally as described above the products being in liquid form:

Lactose in all cases constituted about 50 to 55% of the total solids.

To determine the effects of a more severe heat treatment, the product denoted by "•" was diluted to a total solIds content of 15.39 and heated at 120 ^β C for 60 seconds. The resulting protein product was effectively completely denatured giving a protein denaturatlon value of >70% (PM) and had coagulated and contained readily discernible particles. It was clearly totally unacceptable under the criteria of the present invention.

The denaturatlon of the whey protein concentrate produced in accordance with the process described above can be controlled so as to be at a value in accordance with the invention by varying the temperature and/or time 1n the heat treatment step 24 within limits as described above.

The above general procedure was repeated but wherein some specific conditions were varied as given:

EXAMPLE Λ

Treatment Regimen 78 to 79°C/16 sees

Total Solids 40%

Drier Temperature 74 to 77 ^* C Protein Denaturatlon 30 to 40% PM

This gave a dry product.

EXAMPLE B

Treatment Regimen 79 to 80 ^β C/16 sees Total Solids 31 to 32%

Protein Denaturatlon 32 to 37% PM This product was used in its fluid form.

It may be noted that when Example A was repeated but with a temperature regimen of 74 ^β C for 16 seconds and a drier temperature of about 71°C, protein denaturatlon in the resulting dry product was only 12 to 18% (PM). (The difference in drier temperature was not found to be significant.)

EXAMPLE C

A starting 6% solIds whey was evaluated for percentage denaturatlon prior, and subsequent to, being subjected to the said heat regimen as follows: % Denaturatlon(PM)

Before the Ultrafiltration Step 0.95 Following the Ultrafiltration Step 5.12 * Following Heat Treatment at 80 to 81 ^β C 53.87

* This may be compared with use of a heat treatment at 74°C under otherwise similar conditions which resulted 1n the whey protein being denatured to a value of only about 11%.

EXAMPLE P

Further studies Indicate that small changes 1n temperature 1n the heating regimen are Important and, for example, have more effect than changes 1n total solids:

Further, the higher temperatures tend to produce products of lesser quality and hence temperatures of about 80°C are preferred. Denaturatlon values of greater than about 65% (PM) are therefore undesirable.

In a white sauce application, the product of the present Invention at say the 2% by weight level based on the total composition, replaces part of the butter or vegetable oil component as well as allowing reduced levels of starch, since the product assists In the creation of a smooth sauce product.

In yoghurt, again, the use of the product of the invention allows a reduction of fat content, and assists, via its gelling properties, in obtaining the desired "body" in the yoghurt.

A sample sour cream utilizing the WPC of the present Invention is as follows:

1% Fat Sour Cream

Processing:

1. Dry blend Ingredients.

2. Combine all ingredients and heat to 170 to 175 ^β F (77 to 79°C) for 20 minutes.

3. HTST process at 180 to 185°F (82 to 85°C) for 26 seconds. Homogenize at 1800 psi single stage.

4. Inoculate.

5. Break pH 4.7-4.75.

6. Shear at 15 ps1 higher than line pressure. Product should smooth out - no graininess should appear.

7. Package and cool .

8. Shelf life -35 days at 4 to 5 ^β C

As can be seen the denatured whey protein product of the present Invention can be used to advantage 1n many food applications, due 1n part to its ability to at least partially replace the fat or the like component and to assist 1n providing body, properties which are demanded by many food items.

Referring now to Figure 2, a process for preparing 0% fat 1ce cream 1n accordance with a preferred embodiment of the Invention Includes blending liquid sweetener, namely high fructose corn syrup, and water 1n a blending step 30. The resultant blend from blending step 30 1s then blended with a first dry blend 1n a blending step 32, the first dry blend comprising skim milk solIds, sweeteners, namely corn syrup solids and dry sugar, and bulking agents, namely tapioca starch and maltodextrin. The resultant blend from blending step 32 is blended with a second dry blend in blending step 34, the second dry blend

comprising stabilizers, namely guar gum, carrageenan, locust bean gum, micro-crystalline cellulose gum, carboxy-methyl cellulose gum and xanthan gum and emulsifiers, namely mono-diglycerides. The resultant blend of dairy Ingredients, sweeteners, bulking agents, stabilizers and emulsifiers from blending step 34 Is then blended 1n a blending step 36 with whey protein concentrate from the process described with reference to Figure 1 to form an ice cream mix.

The Ice cream mix from blending step 36 is pasteurized in a pasteurization step 38 at about 78 ^β C for about 10 minutes and 1s then homogenized 1n a two-stage homogenlzation step 40. The first stage 1s carried out at a pressure of about 2500 p.s.1. and the second stage is carried out at a pressure of about 800 p.s.1. The homogenized blend 1s then cooled in a cooling step 42 to about 4 ^β C, and the cooled blend 1s then aged for about 24 hours 1n an aging step 44.

The aged blend Is passed to a flavouring step 46 where appropriate flavouring is added, and the flavoured blend is frozen and whipped with an overrun (i.e. increase in volume due to air content) of from about 40 to about 80% in a freezing step 48 to produce 0% fat ice cream which is then extruded from the freezing step 48 at about -6 ^β C. The

1ce cream 1s then hardened 1n a hardening step 50 until a core temperature (In a two litre container) of about -18°C 1s reached, this being In about 2 hours.

By way of example, preferred ranges of the

Ingredients for 0% fat ice cream are as follows:

Per Cent SolIds By Weight Ingredients Of Total Mix From about 30 to about 40% protein by weight on a total solids basis -

60 to 80% Denatured

25 to 40% solids by weight 2 to 7 In a specific example of the Invention, the following ingredients for 0% fat ice cream were used:

Per Cent SolIds By Weight Ingredients Of Total Mix 35% protein by weight on a total solIds basis -

71% Denatured

31% solids by weight 7

The liquid blend is blended in a Lanco blender for two minutes at a speed of 1300 r.p.m. The first dry blend 1s then added slowly and blending 1s carried out for a further 5 minutes at the same speed. The second dry blend 1s then added and further blending 1s carried out for 5 minutes at the same speed. The speed 1s then reduced to 400 r.p.m., and the whey protein concentrate is added and further blending carried out for 2-3 minutes. The resultant blend 1s then treated In the manner described above with reference to Figure 2.

Referring now to Figure 3, a process for preparing 1% fat (by weight) 1ce cream 1n accordance with a preferred embodiment of the Invention Includes blending liquid sweeteners, a dairy fat source such as cream and/or butter fat and water 1n a blending step 52, the liquid sweeteners comprising liquid sugar, liquid corn syrup solids and high fructose corn syrup. The resultant blend from blending step 521s blended with a dry blend in a

blending step 54, the dry blend comprising skim milk solids and stabilizers, namely guar gum, carrageenan, locust bean gum and micro-crystalline cellulose gum. The resultant blend of dairy Ingredients (including fat), sweeteners and stabilizers from blending step 541s then blended in a blending step 56 with whey protein concentrate from the process described with reference to Figure 1 to form an ice cream mix.

The ice cream mix from blending step 561s pasteurized in a pasteurization step 58 at a temperature of about 82 ^β C for about 32 seconds and 1s then homogenized 1n a two-stage homogenlzation step 60. The first stage is carried out at a pressure of about 1800 p.s.i. and the second stage Is carried out at a pressure of about 700 p.s.1. The homogenized blend 1s then cooled 1n a cooling step 62 to about 4 ^β C. The cooled blend Is then aged for about 24 hours in an aging step 64.

The aged blend then passes to a flavouring step

66 where appropriate flavour Is added and the flavoured blend 1s frozen and whipped with an overrun of from about 40 to about 80% in a freezing step 68 to produce 1% fat ice cream which is then extruded from freezing step 68 at about -6 ^β C. The 1% fat ice cream is hardened in a hardening step 70 until a core temperature (in a two litre container) of

about -18 ^β C 1s reached, this being 1n about 2 hours.

By way of example, preferred ranges of

Ingredients for 1% (by weight) fat 1ce cream are as follows: Per Cent SolIds

By Weight Ingredients Of Total Mi

Liquid Blend Liquid Sugar 4 to 8

Liquid Corn Syrup Solids 2 to 6

Water to make up 100%

Cream/Butter Fat 0.5 to 1.5

High Fructose Corn Syrup 7 to 12 Pry Blend

Skim M1lk SolIds 1 to 10

Carrageenan 0.01 to 0.04

Guar Gum 0.01 to 0.1

Locust Bean Gum 0 to 0.05 Micro-crystall1ne Cellulose Gum 0 to 0.5

Per Cent SolIds By Weight Whev Protein Concentrate Of Total Mix

From about 30 to about 40% protein by weight on a total solids basis -

60 to 80% Denatured 25 to 40% solids by weight 2 to 7

In a specific example of the Invention, the following Ingredients for 1% (by weight) fat Ice cream were used:

The liquid blend is blended in a Lanco blender or about 5 minutes at a speed of about 1300 r.p.m. The

dry blend 1s then added and further blending carried out for about 5 minutes at the same speed. The speed 1s then reduced to about 400 r.p.m., the whey protein concentrate 1s added and further blending carried out for 2-3 minutes. The resultant blend 1s then processed 1n the manner described above with reference to Figure 3.

Referring now to Figure 4, a process for preparing 1ce cream with 7% (by weight) and higher amounts of fat in accordance with a preferred embodiment of the

Invention includes blending liquid ingredients and water 1n a blending step 72, the liquid Ingredients comprising liquid sugar, liquid corn syrup solids, whey solIds, milk solids non fat, and a dairy fat source such as cream and/or butter fat. The resultant blend from blending step 72 1s blended with a dry blend of stabilizers and emulsifiers 1n a blending step 74, the stabilizers being carrageenan, locust bean gum, guar gum and micro-crystalline cellulose gum, and the emulsifiers being polysorbate 80 and mono-d1glycer1des.

The blend of dairy Ingredients, sweeteners, stabilizers and emulsifiers from blending step 74 is then blended in a blending step 75 with whey protein concentrate from the process described with reference to Fig. 1 to form an ice cream mix. The 1ce cream mix from blending step 76

is pasteurized in a pasteurization step 76 at about 81 ^β C for about 32 seconds and is then homogenized 1n a two stage homogenlzation step 78. The first stage 1s carried out at a pressure of about 1500 p.s.1., and the second stage 1s carried out at a pressure of about 700 to 800 p.s.1. The homogenized blend Is then cooled 1n a cooling step 80 to about 4 ^β C, and the cooled blend 1s aged for about 24 hours In a aging step 82.

The aged blend is passed to a flavouring step 84 where appropriate flavouring 1s added, and the flavoured blend is frozen and whipped with an overrun of from about 30 to about 110% in a freezing step 86 to produce ice cream with 7% fat or higher (for example up to about 20% fat) which 1s then extruded from freezing step 86 at about -6 ^β C. The ice cream 1s hardened in a hardening step 88 until a core temperature On a two litre container) of about -18 ^β C 1s reached, this being 1n about 2 hours.

By way of example, preferred ranges of the

Ingredients for 1ce cream with 7% (by weight) fat or higher are as follows:

Per Cent SolIds By Weight Ingredients Of Total Mix Llguid Blend

Water to make up 100%

Sucrose Solids 6 to 12

Corn Syrup Solids 3 to 7

Total Fat 7 to 15 Whey SolIds 0 to 6

Milk Solids Non Fat 1 to 10

Drv Blend

Carrageenan 0.01 to 0.04

Locust Bean Gum 0 to 0.05 Guar Gum 0.04 to 0.1

Micro-crystalline Cellulose Gum 0 to 0.4

Polysorbate 80 0 to 0.1

Mono-d1glycer1des 0 to 0.25

Whev Protein Concentrate 2 to 9

(as in the previous examples)

In a specific example of the Invention, the following Ingredients were used for 7% (by weight) fat ice cream:

(as In the previous examples)

In a specific example of the Invention, the following Ingredients were used for 10% (by weight) fat 1ce cream:

(as 1n the previous examples)

CALCULATION QF PERCENTAGE PENATURATΣQN

The methodology for calculating the percentage denaturatlon of the whey protein concentrate will now be described.

In the broadest sense, denaturatlon of protein

refers to any conformational change 1n the three dimensional structure of a protein away from Its native state. For the purpose of this and 1n fact most methods which characterize denaturatlon, the conformational changes must result 1n a loss of solubility of the protein.

This method Involves measuring the protein which remains 1n solution after a mechanical separation of the precipitated (denatured) portion.

This 1s a comparative method 1n which a reference sample Is used as a point of "zero denaturatlon". In most cases, this reference will in fact be partially denatured to a degree which may or may not be known. What 1s being measured 1s the percent denaturation 1n the sample with respect to the reference.

Usually the denaturatlon of the sample 1n question Is associated with a processing step such as a high heat treatment. In this case, the reference could simply be the sample prior to high heat treatment.

The reference sample is centrifuged to separate out the precipitated proteins. The protein which remains in solution is quantified by UV spectroscopy.

The reference is then completely heat denatured and precipitated proteins are separated by centrifugatlon. Again, the protein which remains 1n solution 1s quantified by UV spectroscopy.

The sample In question Is then centrlfuged and the protein in solution 1s measured by UV spectroscopy. By comparing the spectroscopic data for the sample to the data for the undenatured and completely denatured reference, a relative percent denaturatlon can be calculated.

ULTRAVIOLET SPECTROSCOPY

The amount of UV radiation which a sample absorbs is a function of the concentration of the absorbing components within the sample. This relationship 1s linear and can be expressed 1n terms of the Beer-Lambert law.

A - be Where: A ■ Absorbance

■ Extinction Coefficient b » Path Length c = Concentration

The extinction coefficient ( ) is a constant for a given substance and the path (b) 1s a constant for a

given cuvette.

For this method, the absorbances of the aromatic amino acids, tyrosine and tryptophan 1n the region of 280 nm are used to characterize the concentration of protein In solution. β-lactoglobul1n and «»<-lactalbum1n contain these amino adds 1n different proportions.

Both tyrosine and tryptophan absorb 1n the 280 nm range. The broad peak which 1s seen 1n this region 1s therefore a composite of absorption peaks of these two amino acids. The two peaks can be viewed separately by looking at the first derivative of the wavelength scan.

Pure solutions of LA and βLG are used to determine the extinction coefficients of each of these proteins. Accurately prepared mixtures containing different ratios of the two proteins are used to determine composite extinction coefficients for blends.

PERCENT PENATURATIQN

Once protein concentration in the sample (C _samp - _|e ), the undenatured reference <C _zer0 ), and the completely denatured reference ^lιave been determined, the percent denaturatlon 1s determined by the

following equation.

% Denaturatlon - C ^(c _Z ero~ ^C saπιple ^{/ C} zero~ ^C 100% ^{] x 10} °

It will be appreciated that the fundamental basis for the degree of denaturatlon of the denatured protein products of the present Invention is the amount of undenatured whey proteins in the milk, from which the whey treated according to the present invention 1s produced. For convenience, since for example, it may not be possible to readily determine the content of undenatured whey proteins in the said milk, then the optical calculation may be used on the whey to be treated but a correction factor must be applied. If necessary, the above theoretical value may be used.

SAFETY CONSIDERATIONS

This method does not Involve any hazardous chemicals. Proper care should be exercised when using the superspeed centrifuge.

APPARATUS

1. Double Beam Scanning UV Spectrophotometer and Quartz Cuvettes (Shimadzu UV160U)

2. Superspeed Centrifuge and Tubes (approx. 25000 G)

3. Boiling Water-Bath

4. 250 ml volumetric flasks

5. Ice-Bath

6. Computer and Spectra-Calc, and RS-1 Software Packages

REAGENTS

1. Distilled Water

2. Purified •••< -lactalbumin (Sigma L-7269) 3. Purified β-lactoglobulin (Sigma L-0130)

PROCEDURE

1. Determination Of Extinction

Coefficient For °< -lactalbumin

a) Accurately prepare a minimum of 5 solutions (10 ml each) of pure LA ranging from 0.02 to 0.12% (w/w).

b) Set up the parameters of the UV spectrophotometer as follows:

Mode: Wavelength Scan

Wavelengths: 400 to 230 nm

Scanning Speed: Slow

c) Run a baseline correction on the instrument

using distilled water in the reference and sample holders.

d) Scan each solution of LA using distilled water as the reference.

e) Accurately record the peak absorbance In the 280 nm region for each sample. (Use Spectra-Calc to determine peak A) (See "CALCULATIONS" section for determination of )

2. Determination Of Extinction

Coefficient For β-lactoglobulin

a) Accurately prepare a minimum of 5 solutions (10 ml each) of pure BLG ranging from 0.04 to 0.20% (w/w).

b) Set up the parameters of the UV spectrophotometer as follows:

Mode: Wavelength Scan Wavelengths: 400 to 230 nm Scanning Speed: Slow

c) Run a baseline correction on the Instrument using distilled water in the reference and sample holders.

d) Scan each solution of BLG using distilled water as the reference.

e) Accurately record the peak absorbance 1n the 280 nm region for each sample. (Use Spectra-Calc to determine Peak A)

(See "CALCULATIONS" section for determination of )

3. Determination Of Composite Extinction Coefficients

a) Accurately prepare 0.1% (w/w) solutions (25 ml of each) of pure LA and BLG.

b) Using these solutions, accurately prepare a minimum of 6 composite samples of varying protein ratios.

c) Set up the parameters of the UV spectrophotometer as follows:

Mode: Wavelength Scan Wavelengths: 400 to 230 nm Scanning Speed: Slow

d) Run a baseline correction on the Instrument using distilled water 1n the reference and sample holders.

e) Scan each composite sample with distilled water as the reference. Using

Spectra-Calc, determine the maximum peak intensities of the two main first derivative peaks. These peaks will be at approximately 293 and 286 nm. (See "CALCULATIONS" section for the determination of composite )

Analysis Of Reference Sample (Zero Point)

a) Accurately dilute a portion of the reference sample to a solids level of 0.4%.

b) Centrifuge at room temperature for 20 minutes at approximately 25000 G.

c) Set up the parameters of the UV

spectrophotometer as follows:

Mode: Wavelength Scan Wavelengths: 400 to 230 nm 5 Scanning Speed: Slow

d) Run a baseline correction on the Instrument using distilled water in the reference and sample holders.

10 e) Scan the supernatant using distilled water in the reference cuvette.

f) Record the peak absorbance (280 nm) as well 15 as the peak intensities of the two main first derivative peaks. (Use Spectra-Calc software)

4b. Analysis Of Reference Sample 20 (100% Denaturatlon Point)

a) Fill about 5250 ml volumetrlcs with the reference sample.

25 b) Place the flasks into a boiling water bath and remove 1 every twenty minutes.

For Each Sample:

c) Cool in an ice-bath and use distilled water to bring the volume back to 250 ml.

d) Accurately dilute to 0.4% solIds and centrifuge for 20 minutes at approximately 25000 G.

e) Set up the parameters for the UV spectrophotometer as follows:

Mode: Wavelength Scan Wavelengths: 400 to 230 nm Scanning Speed: Slow

f) Run a baseline correction on the Instrument using distilled water 1n the reference and sample holders.

g) Scan the supernatant using distilled water 1n the reference cuvette.

h) Record the absorbance at 280 nm and the intensities of the two main first derivative peaks.

1) Continue testing samples until there Is no further decrease in the absorbance at 280 nm, I.e. after about 60 minutes.

Analysis Of Unknown Sample

a) Accurately dilute the sample to 0.4% solids.

b) Centrifuge for 20 minutes at 25000 G.

10 c) Set up the parameters of the UV spectrophotometer as follows:

Mode: Wavelength Scan 15 Wavelengths: 400 to 230 nm

Scanning Speed: Slow

d) Run a baseline correction on the Instrument using distilled water 1n the reference and 20 sample holders.

e> Scan the supernatant using distilled water 1n the reference cuvette.

25 f) Record the absorbance at 280 nm and the

Intensity of the two main first derivative peaks.

CALCULATIONS

1. Extinction Coefficient For=*• -lactalbumin

a) Plot a graph of peak absorbance as a function of concentration for the solutions of pure LA.

b) Using RS-1, fit a linear function to the data using the following format:

Absorbance « e x Concentration

c) LA « e

Extinction Coefficient For β-lactoglobu11π

a) Plot a graph of peak absorbance as a function of concentration for the solutions for pure BLG.

b) Using RS-1, fit a linear function to the data using the following format:

Absorbance = e x Concentration

c) BLG

3. Composite Extinction Coefficient

a) Plot a graph of the Ratio LA/βLG as a function of the ratio of the first derivative peaks <A293 _nι * _n /A286nm-' ^ ^{or eacπ} of the composite protein samples.

b) Using RS-1, fit a function to the data using the following format: LA/βLG « a ₊ [b x <A _293nπι /A _286nm )J ⁿ determine: a, b, and n

4. Concentration Of Reference Sample (Zero Point)

a) Calculate the ratio of the two main first derivative peaks.

^A 293nm ^/A 286nm

b) Use this value 1n the equation derived in step 3 above to determine R, the ratio

LA/βLG.

c) Calculate the soluble protein concentration, ^C ( _zer0 ). i ⁿ the undenatured reference sample as follows:

^C (zero) - ^A/ {π/ ⁽ R+D] x _βLG } + {Cl- ⁽ 1/R+1 ⁾ 3 x _LA }

5. Concentration Of Reference Sample (100%)

For the sample subjected to the longest heat treatment:

a) Calculate the ratio of the two main first derivative peaks.

^A 293nm ^/A 286nm

b) Insert this value Into the equation derived In step 3 above to determine R, the ratio

LA/βLG.

c) Calculate the soluble protein concentration, C -| ₀ o%). ^ ⁿ the undenatured reference sample as follows:

^c (ioo%) » ^A {π/ ⁽ R+ι> ^] x _βLG } + ui-α/R+i ^{) ]} x _LA }

6. Concentration Of Unknown Sample

a) Calculate the ratio of the two main first derivative peaks.

^A 293nm ^/A 286nm

b) Use this value in the equation derived in step 3 above to determine R, the ratio LA/βLG.

c) Calculate the soluble protein concentration, _(Samp]e , 1n the undenatured reference sample as follows:

^c (sample) " ^A/ (H/CR+l ^)] x _βL G> + Ul-d/R+l ^)] x LA)

7. Determination Of Degree Of Denaturatlon

a) Calculate the percent denaturatlon relative to the reference sample as follows:

% Denaturatlon -C< ^C ( _ze ro) ^"C (sample> ^{/ C} (zero) ^'C (100%) ^{)]x 10} °

As previously mentioned, the percentage denaturatlon specified 1n the present Invention is the percentage denaturatlon relative to raw milk from which the undenatured starting whey proteins originate.

Other embodiments and examples of the Invention will be readily apparent to a person skilled 1n the art from the foregoing description of preferred embodiments and examples, the scope of the Invention being defined in the following claims.