IE60982B1 - Method for removing beta-lactoglobulin from bovine milk whey - Google Patents

Description

METHOD FOR REMOVING β-LACTOGLOBULIN FROM BOVINE MILK WHEY Field of the Invention The present invention relates to a method for producing an improved bovine milk whey. Particularly, it relates to a method for producing a bovine milk whey which has a reduced amount of p-lactoglobulin and, more particularly, to a method for removing/3-lactoglobulin from bovine milk whey by means of adsorption on a cation exchange cellulose, i.e», a cellulose cation exchanger.

Description of Prior Art Upon production of cheese, most of water-soluble components contained in bovine milk, except fat and casein, transfer to the resulting milk whey. Lactose abundantly contained in the milk whey can be easily recovered therefrom by crystallization. Such a recovered lactose has been utilized for foods and chemicals.

However, the remaining milk whey obtained after the removal of lactose has been used only as a material for foods, in most cases, as a low-lactose whey podwer, and in some 2o cases, as a demineralized low-lactose whey powder or ultrafiltrated whey protein concentrate (WPG).

Milk serum proteins have not been isolated and utilised individually on a commercial scale, except such special cases as the selective isolation of lactoferrin from cow's milk whey. - 2 It is the present state of the art that proteins contained in bovine milk whey have not been utilized in a highly effective manner, so as to make the best of the characteristics of various proteins contained therein.

One reason therefor is that the milk serum proteins contained in bovine milk whey include a large quantity of ^-lactoglobulin (which may hereinafter be referred to as ”/3-Lg). y3-Lg is a protein which is not present in human milk. It is therefore disadvantageous to use milk serum proteins containing /?-Lg as a protein source for. e.g. 9 modified milk powders (infant formulas) for feeding infants since yd-Lg may act as an allergen for some babies, even though the use of the proteins is advantageous from the viewpoint of the improvement of efficiency in the utilization of proteins by infants.

In generals the proteins of human milk and cow's milk are much different in the content of caseins; the former contains 2.5 g/l and the latter 27.3 g/l.

On the other hands the contents of milk serum proteins, other than caseins, are not so much different. (6,.4 g/l in human milk and 5.8 g/l in cow’s milk). The protein composition of human and cows milks, the comparison thereof showing the difference in character between both, is shown in Table 1. ~ 3 = Table 1: Character of Proteins in Human and Cow’s Milks* Human Milk (g/1) Cow’s Milk (g/1) Casein 2.5 27.3 Milk Serum Proteins 6.4 5.8 5 g-Lactalbumin 2.6 . 1.1 Lactoferrin 1.7 Trace 0-Lactoglobulin --- 3.6 Lysozyme 0.5 Trace Serum Aluburain 0.5 0.4 10 Immunoglobulin A 1.0 0.03 Immunoglobulin G 0.03 0.6 Immunoglobulin M 0.02 0.03 [Note] * According to Hambraeus et al.

As shown in Table 1, 0-Lg is not contained in human milk, whereas cow’s milk contains 0~Lg ac & concentration as high as 3.6 g/1.

In, che production of modified milk powders for feeding infants, there have been used casein-free wheys or whey protein concentrates, in order to make the composition 2o of the milk powders as closer as possible co chat of human milk. Although the content ratio of casein and milk serum proteins contained in such modified milk powders is closely similar to that in human milk, there still remains the problem that the kinds - 4 of the milk serum proteins are different.

If β-Lg contained in cow's milk can be selectively removed, the composition of milk serum proteins can be rendered more similar to that of human milk.

In addition to this, it can be expected that the allergyinducing properties of the milk serum proteins will be weakened.

Various attempts have been made to isolate or remove β-Lg from milk whey. Known methods and their problems are as follows. 1, Coprecipitation method using polymeric.polyelectolytes (J. Hidalgo et al.. J. Dairy Sci., 54, 1270 (1970) and N. Melachouris, J. Agr. Food Chem., 20, 798 (1972)] Xn this method, β-Lg is removed by allowing it to coprecipitate with polymeric polyelectolytes through adjustment of the pH and the concentration of polymeric polyelectolytes added, thereby causing no reactions with G“lactalubumin (hereinafter referred to as c-La”).

This method suffers from the disadvantage that a trace amount of polymeric polyelectolytes used for the coprecipitation remains in the β-Lg free whey obtained. 2. Thermal separation method [R. J. Pearce, Aust. J. Dairy Technol., 38. 144 (1983)] This method utilizes the phenomenon chat, a-La coagulates more readily than β-Lg when heated, e.g., at a temperature of 55 °C or above and at a pH in the range of .from 4.2 co 4.6. - 5 In this method, however, the separation of 0/-La and y?-Lg can be effected insufficiently, and the solubility of the resulting milk whey proteins may become lower because of denaturation which may be caused by the heating treatment.

In addition» the β-Lg recovered by this method is inferior in such functional properties as solubitility, foaming properties, gelling properties, etc., and therefore has a limited application. 3» Ion exchange chromatography [P. J. Skudder, Chemistry and Industry J., 7, 8lO (1983)] Like the method of the present invention, a silicabased diethylaminoethyl ion exchanger (Spherosil QMA manufactured by Rhone-Poulene) is used in this method.

However, the ion exchanger Is not only expensive but also incapable of effecting the separation to a sufficient degree.

BRIEF SUMMARY OP THE INVENTION The present Invention relates to a method for effectively removing β-Lg from bovine milk whey.

There is provided by the present invention a method for removing Lg from bovine milk whey, which comprises adjusting milk whey’s pH value, demineralization rate and protein concentration to 4.3 to 4.6, 60 to 90$ by ash weight (ash content) and 0.5 to 1.5$ by weight, respectively, and - 6 then making the resulting milk whey contact a cation exchange cellulose.

Bovine milk whey from which ^-Lg is to be removed by the method of the present invention is selected from the group consisting of sweet wheys, acid wheys, whey powders, and whey protein concentrates, the whey powders and whey protein concentrates being produced from the sweet or acid wheys.

In the method of the present invention, /-Lg will be adsorbed on a cation exchange cellulose, and the adsorbed jd-Lg may be eluted by an alkaline solution.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the ratio of unadsorbed (K-La and /?-Lg in the pH range of from 4,1 to 4.9, which was obtained in Test Example 1. Fig. 2 is a graph showing the quantities of unadsorbed rt-La and/-Lg in the protein concentration range of from 0 to 5„0% by weight in Test Example 2. Fig. 3 is a graph showing the ratio of unadsorbed oJ-La and jS-Lg in the protein concentration range of from 0 to 5·θ$ by weight in Test Example 2. Fig. 4 is a graph showing the amounts of unadsorbed s a graph showing the ratio of unadsorbed Ci-La and fi-Lg in the ash content range of from 0 to 0.440% by weight, the ash contents corresponding to the amounts added of the ultrafiltrated rennet whey permeate powder in Test Example 3. Fig. 6 is a graph showing the elution curves of unadsorbed CC-La and yS-Lg obtained when rennet whey was passed through a cation exchange cellulose column in Test Example 4.

Fig. 7 is a graph showing the elution curves of unadsorbed Οί-La and y9-Lg in the case where the ultrafiltrated two-fold concentrated rennet whey was passed through a cation exchange cellulose column in Test Example 4. Fig. 8 is a graph showing the elution curves of unadsorbed ci-La and /?-Lg in the case where the ultrafiltrated two-fold concentrated reduced rennet whey was passed through a cation exchange cellulose column in Test Example 4. Pig. 9 is a graph showing the elution behavior of unadsorbed ©.'-La and y^-Lg of a Goada cheese whey in Test Example 5» Fig. 10 is a graph showing the elution behavior of unadsorbed Ci-La and /3-Lg of the 92% demineralised Goada cheese whey in Test Example 5· Fig· H is a graph showing the elution behavior of unadsorbed (X-La and y$-Lg of the 70% demineralised Goada cheese whey in Test Example 5· DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In the present invention, bovine milk whey is treated so as to have a pH value of from 4.3 to 4.6, a demineralization rate of from 60 to 90% by weight (ash - 8 content) and a protein concentration of from 0.5 to 1.5% by weight, and the thus treated milk whey is then brought into contact with a cation exchange cellulose to selectively remove /?-Lg alone from the whey, the β-Lg being adsorbed on a cation exchange cellulose.

In order to effect the selective adsorption of zS-Lg on the cation exchange cellulose, it is highly important for bovine milk whey to have a pH value of from ^-3 to ^.6, a demineralization rate of from 60 to 90% by weiht (ash content) and a protein concentration of from 0»5 to 1.5% by weight. If these conditions are not fulfilled, the removal by adsorption of y9-Lg will become lower, and hence the object of the present invention will not be attained. The cation exchange cellulose to be used in the present invention is one type of cellulose ion exchangers, and is prepared by introducing such dissociative cation-exchange groups as carboxymethyl (CM) groups, sulfopropyl (SP) groups, or carboxyl groups, etc into cellulose molecules. The cation exchange celluloses to be used in the present invention include carboxymethyl cellulose (CMC), sulfopropyl cellulose (SPC), carboxy cellulose, and the like. In general, they are available on the market. and commercial cation exchange celluloses may be used.

In the method of the present invention, the cation exchange cellulose (hereinafter referred to as CEC) can - 9 be used either by a column method or by a batch method, in the same manner as in ordinary ion exchange resins.

A continuous method can be more advantageous than a batch method with respect to the efficiency of operation.

In the case where a column method is employed. /$-Lg can be adsorbed and removed by simply passing-the cow’s milk whey, whose pH value has been adjusted to 4,3 to 4.69 demineralization rate to 60 to 90% by weight (ash content) and protein concentration to 0.5 to 1,5% by weight, through the CEC column. The adsorbed /F-Lg can be eluted by passing a solution of an alkali» such as sodium hydroxide and potassium hydroxide, through the column. The washed column can be used repeatedly for treating cow’s milk whey.

The present invention will further be illustrated by way of examples.

In the following test examples, the two major proteins contained in cow’s milk whey, namely cY-La and ^-Lgs were monitored. Serum albumin and immunoglobulins were neglected since, as already known * the pore sise of CEC is too small to adsorb the large molecules of serum albumin or immunoglobulins and hence CEC is hardly capable of adsorbing them. The samples used in the test examples had the compositions shown In Table 2.

CM © ρ-I Λ Cd Ε^ •ο © ω ν m •Η © »—ϊ 0) (ΰ jz ο ω c cd •Η Ό > 6·? ε «0 β) Ο Φ Ο 33 I Ο Ο 3= © © CM OT •pi © pH © rt 33 b o © (3 rt •rl Ό > &·? ε rt Φ CM © o JC O' ra o ΓΟΟ d CM ιη M3 M3 F—1 Ο CM CM CM m GO CM o « m ® o ra © cd © OT m MO p“4 Ό © >1 ω O' in cd © © ω ® o o 3= a 33 3: Ο sQ o Ό © •P Ό rt φ >» tl © P ed 33 pH Ό S •p| rH so ra fc O £ © -P rt fc φ © © b 1 o 3 C •P o S3 ra S3 pH 5 o © © 3 Eh o 03 03 ra sO σ' r- CM d cn d M3 © CM co CM s0 CM 1 M3 CM CM *<Γ m r* m CM ra in O' P** Γ— 1 ra ra cn o 1 CM *—1 CM cm oo Γ*. r-J CO ω ΜΓ CM Ο Ο Γd η Ό ω 4-> χ} rt ω >> β -ρ © Ρ ri 33 «η xj ί-ι 2: •Η Η -Ρ <ρ ο C ·Ρ rt fc. α> © β I Ο β -ρ Ο S3 S3 pH ϊ ο © & Η α κ CM ιη ρ—I ιη γC0 m Ο M3 -S’ M3 ΗΤ <£> ιη σ» Mien Ο CM ο Γο cn ra £4 Φ © P > ri S o b © -6.¾ e* *e-r p*l s ω •P p fc -P ed rt © a> ti $3 S P C pH © G eo ra 03 PU M Γη ο Γ— ιη θ' M3 Ό in en r» sO in SO O' V—9 cn CM © O ra cn m e^—4 o r* CM CM Ο Γ— ό cn in ra a-nd CM O' P*·» © ® o MS’ cn in ¥—9 o CM O' O' ra B—} © σ © Ι-ι Η M3 CM CO M3 CM cn 4-) © c c © es ra in O' ra m in s « m o ra o ra in ra m m cn CM cr O' r— P-l o rd cn /’s w G •rt Φ 4-4 •rt V=d o CO 44 £ r-4 GO 0 44 C trh*U u o o ω h a < S'? GO ε rt «9* Z-\ p- — ir? S'? eo eo ε ε ΖΛ &>? oa ε M-Z Z=— z—' 8·? S'? CO oo ε ε eo ►J ι CS U eu Lf) in Test Example 1 (Test on optimum pH) To 20 ml of rennet whey containing (X-La and /?-Lg at a ratio, by weight, of ca. 30s70 was added 5 ml of CMC (CM-Cellulofine CH manufactured by Chisso Corp.; CMC's used in other Test Examples and Example described hereinafter were the same product). While stirring, 2N HCI was added thereto to adjust its pH to a prescribed value. The resulting mixture was filtered by using Toyo Filter Paper No. 2, and unadsorbed proteins contained in the filtrate were analyzed by high performance gel filtration liquid chromatography, using TSK 2000 SW column manufactured by Toyo Soda Manufacturing Co., Ltd.

There was obtained an elution curve shown in Fig. 1.

As shown in Fig. 1, the maximum ratio of unadsorbed cr-La to unadsorbed /9~Lg (this ratio and other ratios described hereinbelow are based on weight) is attained at pH 4.5» at which the ratio Ci-La §y3-Lg was about 61 = 39- Both Oi-La and /3-Lg were adsorbed in large quantities below pH 4.1 and both of them adsorbed only slightly above pH 4.9· Test Example 2 (Test on optimum protein concentration) Wheys containing proteins at various concentrations were prepared by admixing a whey protein isolate (hereinafter referred to as WPI) having a protein content of 93-25$ with a powder of the permeate obtained by ultrafiltration of rennet whey (hereinafter referred to as ultrafiltrated rennet whey permeate powder). The behavior of adsorption of the proteins was examined by the batch method as in Test Example 1 (at pH 4.5). The amount of the ultrafiltrated rennet whey permeate powder added thereto was fixed at 5%The amounts of unadsorbed La and yg-Lg increase monotonously with an increase in the concentration of proteins contained in the samples. Results obtained are shown in Fig. 2. The ratio (X-Las^-Lg decreased sharply when the protein concentration exceeded 1%, the protein concentration meaning the total concentration of the proteins contained in the whey prepared. The ratio of unadsorbed (X-La to unadsorbed /3-Lg was reached to the maximum in the protein concentration range of from 0=5 to 1.0%, in which the ratio Ci-La ;y3-Lg was from 75-25 to 78s 22. In Fig. 3 is shown a curve that indicates the results of this test.

Similar results were obtained when SPC (Indion SP manufactured by Phoenix Chemicals. New Zealand; SPC in Test Example 5 was the same product) was employed.

Test Example 3 (Test on the influence of concentration of minerals) There were prepared 20 ml each of WPI solutions having a protein concentration of 1% and containing from 0 to 5/° of the ultrafiltrated rennet whey permeate powder, and the solutions were subjected to the same batch treatment as in Test Example 1 to examine the behavior of elution of the proteins, i.e., (X-La and y6*~Lg, (at pH 4.5). - 13 The amounts of unadsorbed (X-La and /d’-Lg increased with an increase in the amount of the ultrafiltrated rennet whey permeate powder added. The ratio #-Las^-Lg reached to the maximum when the permeate powder was added in the range of concentration of from 1 to 2%. In other words, the ratio &-Lasβ-Lg had the maximum value in the ash content range of from 0.096 to 0.182%. When the ultrafiltrated rennet whey permeate powder was added at concentrations less than 1%. both OC-La and yS-Lg were equally adsorbed, and the ratio of OC-La to β-Lg decreased.

The influence of the addition of the ultrafiltrated rennet whey permeate powder on the adsorption of (X-La and yS’-Lg (batch method) Is shown In Fig. 4, and the relationship between the ratio (Z-Las^-Lg and the ash content (%) corresponding to the quantity of the ultrafiltrated rennet whey permeate powder added is shown in Fig. 5Test Example 4 (Test on the influence of ultrafiltration) Into each of glass columns having a diameter of 43 mm and a height of 180 mm was charged 100 ral of throughly washed CMC. After the charging, the pH value of the CMC was adjusted to 4.5 with 6N HCI.

Into the columns were supplied, respectively, (i) rennet whey, (ii) a two-fold concentrated rennet whey prepared by ultrafiltrating rennet whey (hereinafter referred to as ultrafiltrated two-fold concentrated rennet whey) - 14 the two-fold concentration meaning that the volume of the retentate (concentrate) obtained by the ultrafiltration is half of its original volume, and (Iii) a reduced whey prepared by diluting the ultrafiltrated two-fold concentrated rennet whey with an equal amount of water (hereinafter referred to as ultrafiltrated two-fold concentrated reduced rennet whey).

In the case of the ultrafiltrated two-fold concentrated rennet whey (which had a protein concentration of 1.52% in spite of the fact that it had an ash content almost at the same level as that before the concentration), the elution of unadsorbed pf~Lg started earlier than in the case of the rennet whey, and the whey amount used and the separation ability became smaller.

On the other hand, in the case of the ultrafiltrated two-fold concentrated reduced rennet whey, the beginning of the elution of unadsorbed $-La and /9-Lg was later than in the case of the rennet whey, and the ratio of unadsorbed ¢6-La to unadsorbed y5-Lg was greater than in the case of the rennet whey. These results can be attributed to the demineralization.

In Fig. 6 are shown the elution curves of unadsorbed (Y-La and yJ-Lg in the case where the rennet whey was passed through the column. The relative protein concentration (%) in eluate shown in the ordinate of Fig. 6 indicates - 15 the ratio in percentage of each concentration of the proteins (i.e., (X-La and /?-Lg) contained in the eluate to that of the same proteins contained in the rennet whey to be applied to the column. This definition is applied similarly to Figs. 7 to 11; for example, in the case of Fig. 7 the ultrafiltrated two-fold concentrated rennet whey to be applied to the column corresponds to said rennet whey.

In Fig. 7 are shown the elution curves of unadsorbed Ci-La and β-Lg in the case where the ultrafiltrated two-fold concentrated rennet whey was passed through the column.

In Fig. 8 are shown the elution curves of unadsorbed 0(-La and /?-Lg in the case where the ultrafiltrated two-fold concentrated reduced rennet whey was passed through the column.

Test Example 5 (Test on the Influence of electrodialysis) Goada cheese wheys were subjected to a similar test as in Test Example 4.

When a Goada cheese whey (pH 4,,5) was applied to the column, y£~Lg unadsorbed was eluted in large qunatities at around the eluate volume of 500 ml, and the separation could not be effected sufficiently. On the other hand, in the case of a Goada cheese whey demineralized by 92% by using ion exchange resins (SKI-B and SA 20A-P manufactured by Mitsubishi Kasei Corp., hereinafter referred to as ”92% demineralised Goada cheese whey”), both #-La and yff~Lg were adsorbed, and the ratio of unadsorbed i%~La to unadsorbed y$-Lg contained in the eluate was not so much different from - Ιό that of Ct-La to ^-Lg contained in the original 92% demineralized Goada cheese whey, that is to says no substantial separation could be effected.

In the case of a Goada cheese whey demineralized by 70% by means of electrodialysis (hereinafter referred to as 70% demineralized Goada cheese whey)» yS-Lg was selectively adsorbed, and the ratio of unadsorbed (X-La to unadsorbed ^-Lg contained in the eluate markedly increased in comparison with that of cc-La to β-lig in the original 70% demineralized Goada cheese whey. The concentration of unadsorbed ^-Lg started to increase when the eluate, volume reached to ca. 3*000 ml. This is presumably because the cation exchange groups contained in the CMC were saturated with the adsorbed /3~Lg.

The ratio £/-La Lg in 4soOO ml of eluate was 7*5=1» In Fig. 9 is shorn the elution behavior of unadsorbed (/-La and y^-Lg in the Goada cheese whey (by the column method). In Fig. 10 is shown the elution behavior of unadsorbed oi-La and /?-Lg in the 92% demineralized Goada cheese whey (by the column method). In Fig. 11 is shown the elution behavior of unadsorbed C-5-La and yd-Lg in the 70% demineralized Goada cheese whey (by the column method).

On the basis of the results in the above tests, there was obtained the finding that milk whey must be treated so as to have a pH value of from 4.3 to 4.6, a demineralization rate of 60 to 90%, preferably from 70 to 80%, and a protein - 17 25 concentration of from 0.5 to 1.5%» preferably from 0.5 to 1.0%, and then brought into contact with CMC, in order to effectively remove y9-Lg from the milk whey. Substantially the same results were obtained in cases where several types of SPC were used.

Example Into a vinyl chloride column having a diameter of 4-5 cm and a height of 50 cm (manufactured by Shoe! Seisakusho Co., Ltd.) was charged 1? liters of CMC, and the pH value was adjusted, while being stirred, to 4-5 with 6N HCl. Thereafter, the 70% demineralized Goada cheese whey whose pH value has been adjusted to 4.5 with oN HCl was passed through the column at a rate of from 25 to 30 l/nr.

After 660 kg of the whey had been passed through the column, the CMC was washed with 80 liters of water.

After the washing, the pH value of the CMC was adjusted, while being stirred, to 8.5 with oN NaOH, for eluting the whey proteins adsorbed on the CMC. Thereafter, 80 liters of water was passed through the column to recover the adsorbed proteins.

The 70% demineralized Goada cheese whey, the first eluate containing unadsorbed ci-La and ^-Lg, and the second eluate (alkaline) had the respective compositions as shown in Table 3In the first eluate, the ratio OG-Las^-Lg was 3°3^sl« and contents of immunoglobulins and non-protein nitrogens remained almost unchanged.

The alkaline eluate containing desorbed /3-Lg, etc contained very small quantities of serum albumin and ci-La. However, almost no immunoglobulins and non-protein nitrogens were contained therein.

Claims (5)

1. What is claimed is;

1. A method for removing y3-lactoglobulin from bovine milk whey, which comprises adjusting milk whey’s pH value, demineralization rate and protein concentration to 4.3 to 5 4,6, 60 to 90% by weight (ash content) and 0.5 to 1.5% by weight, respectively, and then causing the resulting milk whey to contact a cation exchange cellulose.

2. A method for removing ^-lactoglobulin as defined in claim 1. wherein said bovine milk whey is at least one 10 selected from the group consisting of sweet wheys, acid wheys, whey powders, and whey protein concentrates, the whey powders and whey protein concentrates having been prepared from the sweet wheys or acid wheys.

3. = A method for removing yS-lactoglobulin as defined 15 in claim 1. wherein β-lactoglobulin adsorbed on the cation exchange cellulose by said contact is eluted with an alkaline solution.

4. A method according to claim 1 for removing β-lactoglobulin from bovine milk whey, substantially as hereinbefore 20 described and exemplified.

5. Bovine milk whey whenever treated by a method claimed in a preceding claim.