JPS59113848A - Purification of whey of concentrated whey protein - Google Patents

Abstract

PURPOSE:To obtain purified whey or concentrated whey protein useful as a protein source for baby, by adjusting the pH of the titled substance, adding ferric chloride to the substance, leaving the mixture to stand at a specific temperature, recovering the precipitate, and subjecting the precipitate to washing, dissolution in an acid, treatment with an ion exchange resin, etc. CONSTITUTION:The pH of whey or concentrated whey protein is adjusted and ferric chloride is added to the substance to obtain a mixture having a pH of about 3+ or -0.3 and a ferric chloride concentration of about 4+ or -0.9mM, and the mixture is left to stand at 26+ or -5 deg.C for >=2hr. The obtained precipitate is recovered, washed with water, and dissolved in an acid to adjust the pH to about 1.5+ or -0.2. The solution is made to contact with a cation exchange resin having low degree of crosslinking (e.g. Amberlite IR 112) to remove the iron, adjusted to about 6.8+ or -0.4pH, and subjected to the salt-removing operation such as dialysis in a dialyzing tube, etc. The obtained product is used as a protein source of nursery food.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides whey or whey protein concentrate (WheyT'r
The present invention relates to a method for purifying otein concentrate (hereinafter referred to as WPC) to the point where it can be used as a protein source in infant preparations.

In general, whey, which is a by-product during the production of cheese and casein, contains most of the water-soluble components in milk, excluding fat and casein. Lactose, the main component in whey, has been crystallized and separated from whey and used for food and medicinal purposes, but whey protein is a form that can only be used for limited purposes, although it is recognized for its high nutritional value. Minute 11! II
Only Io1 was collected. For example, the yield of 1111 was obtained by heat coagulation method, polyhydrochloride method, and the resulting product is insoluble and contains a wide variety of substances (phosphate radicals, iron salts), and cannot be used in foods. There are many restrictions on the use of protein as a source of protein, at least in infant preparations,9 which is the national situation.

The reason why whey or WPC is unsuitable as a protein source for gVA'lJ products for infants is the presence of a large amount of β-lactoglopurin. Parich WIi: (C
IinAIlergyl:369-580.197
1) Antibodies against β-lactoglobulin were frequently found in PCA reactions using monkeys, and Kleth
r R, (g) “VI, Freier 8. et+
I (C1in AIlprgl: 249-255°1'971 Qt In a group of infants sensitive to milk,
When examining serum gE antibodies against each milk protein, Shizuku frequently found IgH antibodies against β-lactoglobulin.
- It is known that it exhibits stronger allergenicity than lactalbumin.

In general, both casein and whey proteins in cow's milk represent foreign proteins to infants, and infants who are hypersensitive to milk proteins may have a strong allergic reaction to the intake of infant preparations; A large part of this is caused by globulin.

However, whey or HWPC contains a large amount of α-lactalbumin and immunoglobulin, which are useful protein sources for infants. Therefore, only β-lactoglobulin can be selectively removed from whey or WPC. If possible, not only would the whey protein component be similar to that in breast milk, but also the removal of β-lactoglobulin would be expected to be relatively less allergenic than regular whey. Many attempts have been made.

However, at the laboratory level, methods such as ion exchange resin, gel filtration, ammonium sulfate splitting, and fractional precipitation using carboxymethyl cellulose are possible, but no examples of successful production on an industrial scale have been seen.

However, in the past there have already been suggestions for the removal of β-lactoglobulin on an industrial scale. Specifically, Block et al.
(ArchaBiochem Bjophys 47
．． 88 1953) recovered whey protein as ferrilactin, it has been used in combination with heating (G, A!+mant
jaetal Can, Ir+St, Fond8,
Technol J, 7, 199 (1974)) in combination with polyphosphates (Jones S, B, , e
, tsI J, Aar, F (101((!hem.

20.229 (1972)), etc., and efforts have been made to improve the convergence. 12 years ago, it was not completed yet.

The present inventors have conducted many years of research to complete the purification of whey or WPC using ferric chloride, with the aim of industrially refining whey or WPC. They learned that only β-lactoglobulin can be isolated from

The present invention was completed by strictly combining many of these conditions, including adjusting the whey or whey protein concentrate and adding ferric chloride, and finally adjusting the pH'
f: about 3.0±0.3, ferric chloride concentration about 4.0±0
．． 9mM, leave it at about 26±5°C for about 2 days or more, and collect the resulting precipitate. ! It is the law.

The present invention also provides the PH of whey or whey protein concentrate.
After adjustment and addition of ferric chloride, the final pH was approximately 6.0.
±0.6, the ferric chloride concentration was set to approximately 4.0 ± 0.9 mM, and this was left at approximately 26 ± 5°C for approximately 2 hours or more, and the resulting precipitate was collected. was washed with water, dissolved in acid, adjusted to 1.5 pt + 2, and then brought into contact with cation-exchanged oil for 1!j degrees to remove Hq = min. #!jm is a method for producing whey or whey protein concentrate, in which the pH of the treated solution is then adjusted to about 6.8±0.4 and salt is removed.

And, the present invention is directed to the whey or WPCff obtained here.
This is a use of purified whey or whey protein concentrate, characterized in that it is used as a protein source for preparations for infants.

Generally, about 60% of the protein in whey or WPC is β-lactoglobulin, and about 20% is α-lactalbumin.
%, immunoglobulin is about 1696, but β-lactoglobulin is actually about 8 in one treatment of the purification method of the present invention.
．． Therefore, α-lactalbumin increased to 56% and immunoglobulin decreased to 23%.
．． This increases to 1%. Moreover, if the method of manufacturing the shank of the present invention is repeated, most of the β-lactoglobulin can be removed.

WPC refined in the present invention is a liquid obtained by concentrating whey, which is a by-product during the production of cheese and casein, and separating crystallized lactose. This WPC contains 90-98% protein, approximately 60% of which is β-lactoglobulin, approximately 20% (1-lactalbumin), and approximately 16% immunoglobulin. 1. Whey or WPC is usually neutral, so add an acid such as hydrochloric acid to it to make it approximately pH = 3, and then adjust the ferric chloride solution to a final concentration of about 4.0 to 0.9 mM. The pH of the mixture rises slightly at this point, so add an acid such as hydrochloric acid again to adjust the -1 level to about 6,0±.
It must be adjusted to a range of 0.5. Whey or W
When the - of PC becomes less than 2.7, the separation efficiency of β-lactoglobulin decreases rapidly, and even when the group becomes 6.6 or more, the separation efficiency of β-lactoglobulin deteriorates.

The separation efficiency of β-lactoglobulin is good when the ferrous chloride concentration is 4.0 mM. The concentration of diiron also has a very subtle effect, but preferably 5.0 to 3.
．． It is about 0mM, and the most preferable is 4.0mM.

pH (whey or W after adjustment and addition of ferric chloride)
The PC is left at about 26±5° C. for about 2 hours or more. The temperature of whey or hwpc may be set at around 26℃ from the beginning, but it is preferable to warm it up to 26℃±5 when left to stand.
℃, preferably 24 to 28℃ for 2 hours or more. If left for 2 hours or more, β-lactoglobulin will remain dissolved in the supernatant, and other effective proteins will precipitate, so centrifugation or decanting will be necessary. The precipitate can be easily recovered by cation.

The obtained precipitate is easily dissolved in an acid such as hydrochloric acid, so it is made into a solution, the pH is adjusted to about 1.5±0.2, and a low crosslinking degree such as Amberlite IR, 112 is used. Iron can be removed by passing the solution through a cation exchange resin and bringing it into contact. Since this treated solution still contains salt, the pH is adjusted to about 6.8±0.4, and if the solution is placed in a dialysis tube and dialyzed, the salt is also removed to its original form.

The purified whey paste WPC presented here contains only 8.8% β-lactoglobulin in protein and can be effectively used as a protein source in childcare preparations.

If this purified whey-spread WPC is subjected to the purification of the present invention again, the content of β-lactoglobulin will be lowered and it will become more effective;
A childcare preparation of 1 to 8% can be used satisfactorily and will not cause an allergic reaction in infants.

Next, test examples and examples of the present invention will be shown.

Test Example 1゜Whey pH affecting precipitation of whey protein with ferric chloride
Adjust the pH of the IVized whey from ptt2.5 to 50 with hydrochloric acid and sodium hydroxide, add 1M ferric chloride solution to a final concentration of 4mM, and adjust the pH to the desired -1
After correcting the temperature again and leaving it at room temperature for 2 hours,
Centrifuged at G for 15 minutes. The supernatant was analyzed by 8-polyacrylamide gel analysis and pneumophoresis. - From the electrophoresis chromatogram, it was found that at pH between 2.6 and 3.1, other whey proteins except β-lactoglobulin migrated to the precipitate compartment. ,, pH (in the range 3,4 to 4.5, β
- Lactocropurin is precipitated, but at the same time immunoglobulin is also partially precipitated. pl (for 4,5 or more,
At an iron concentration of 4 mM, there are only a few precipitated proteins, and when the iron concentration is increased, whey proteins precipitate, but selective precipitation is no longer observed.

Test Example 2゜Effect of whey p1 on precipitation of whey protein by ferric chloride
4 and the shadow of iron rod%li: Adjust the pH of each clarified Iε whey to 2.6 with hydrochloric acid and caustic soda.
~6.1, add 1M ferric chloride solution to final concentration of 3.
The concentration was adjusted to 4.5.6mM and left at 26°C for 2 hours, and the influence of the concentration was investigated within this pH range.

In order to quantitatively treat the selectivity of whey protein precipitation, separation efficiency was defined as follows, and optimal conditions were determined based on the separation efficiency.

5=Ss+8p S: Overall separation efficiency Ss = Abs(AB)X soluble 9 elements S
Town: Supernatant efficiency Sp = A11s(A-C)X insoluble nitrogen Sp: Precipitate efficiency A, B,! :, C are the ratios of α-lactalbumin in the supernatant and precipitate of untreated whey, iron salt-treated whey, respectively. Soluble nitrogen is the percentage of soluble nitrogen in the whey stock solution. Insoluble nitrogen 1
[111 minus soluble nitrogen) ξ-centage. The ratio of α-lactalbumin was determined by determining the polyacrylamide gel electrophoresis pattern using a tensitometer.
The results were calculated as follows and are shown in Figure 11.

As shown in Figure 7431, pH 2.9 iron concentration 4.0
The highest minute IL efficiency was achieved in mM. pl(3,0
In the range of ±0.1, 11 times the same separation efficiency can be obtained.6 Test Example 3゜Effect of temperature on precipitation of whey protein by ferric chloride: When - of whey is 3.2 or less Temperature does not affect protein precipitation between 4°C and 55°C, but in the pH range where β-lactoglobulin and immunoglobulin are precipitated at pH 3.4 or higher, the potency has a large effect on low temperature. It was confirmed that the amount of sediment increased as the temperature increased.

Test Example 4 Yield and protein composition of low β-lactodarobulin whey protein concentrate under optimal conditions: The pH of undesalted 717 clear 1 hiko stage cheese whey was set to 3.0, and 1M chloride was added to it. Ferric solution at final concentration of 4
Added so that it was 0mM and corrected ■ to a degree of 6.0, then room temperature 2
After standing for a period of time, the protein was separated. The nitrogen yield measured by the Kermer method was 28.7%.

The precipitated protein was dissolved in acid and then analyzed by anaphoresis after removing iron using an ion exchange resin. As a result, the protein composition was approximately as follows.

Low β-lactoglobulin WPC β-lactoglobulin 8.80 α-lactalbumin 56.0 Immunoglobulin
231 Bovine serum albumin 6.60
Others 5.5 Test example.

Removal of iron from low β-lactoglobulin WPC: low β-
Lactoglobulin WPC (hereinafter referred to as TJ o w-1gWP
C) is easily solubilized by acidic side with pH 1.8 or lower or neutral J lucalide with pH 5.5 or higher, and in the pi (region) where a precipitate is formed, the ionic strength is increased with salt etc. It gradually becomes solubilized, and more than 80 proteins become solubilized by adding 0.9N of salt.I, ow-
Ap, W P C with salt to pHt 4-1.6 K! 1 was added, stirred, filtered, and passed through a column of strongly acidic cation exchange resin (e.g., Rumberlite IR, -112) at a flow rate (s, v, t 3 ), resulting in untreated T,
rQv-1q WPC iron stand%), 1120mpFe-
In the case of R protein, 98 CH removal points 'r554 flow points are 113 OF! It takes about 3.1 to remove iron from proteins in
e and m fat are required.

Amberlite IR-1121d has a crosslinking degree of 2%, but when Lexin 10゛1 with a crosslinking degree of 8% was used, almost no iron was removed. This is presumably because the molecular weight of the dissolved Chichi protein mixture is large and cannot penetrate into the interior of the highly cross-linked resin.

Test-Example 6゜Release 1op・-1CI-WPC amino rafkn
hV: Liu ad chang (J, Bi
ol Chem 246: 2R42, 1971), iron removal low-pg W P C'&+o
- Hydrolyzed with t01oens ionicacid (11
0°G 24 hours) and Phoenix model M
It was analyzed using a 6800 amino acid analyzer. Tryptophan is 5 pieces (Ana!, Chen, 59:
1412.1967). The results are shown in Table-1.

As shown in Table 1, it has an excellent amino acid composition, but the chemical score t is 65.5, calculated based on the tentative comparative amino acid pattern for FAD/wlIO infants.
Met. Since infants require a higher amount of valine than school children and adults, it is understandable that the chemical score is lower than that of Ultrafiltered WPC.

Table 1 Amino Acid Composition G71 Protein Example 50e Clarified Kotteji Cheese Whey PLL was mixed with 780 ml of 3N hydrochloric acid to 3,10 ml, stirred, and 200 ml of ferric chloride 1M solution was added to the final 41 degrees 4.0 M M
And so. At this time, the pH was 2.82, and after adjusting the pH to 3.00 by adding 3N sodium hydroxide at a rate of 73.0 μl, the mixture was maintained at 25° C. for a filtration period. The precipitate was separated by centrifugation (10,000 g x 15 minutes). The weight of the precipitate was 610 g. The precipitate was dispersed twice using 150 g of deionized water, and centrifugation was repeated to wash out lactose and inorganic salts. 400 ml of sediment
After dispersing in 0ml of water, add 3N hydrochloric acid to make the pi (1.50), then rinse with a Whatman NQ 1 filter.
Amberlite IR, 112 column (φ6 diameter
, 0 cm x 90 (: line) at a flow rate of 8. The through iron was removed at V, t 5. The eluate from the column was immediately neutralized with 3N Nogu, placed in dialysis tubing, and dialyzed against tap water for 12 hours and then against deionized water for 24 hours.

The dialyzed fluid was concentrated (approximately 5 times) using a rotary evaporator, prayed carefully, and lyophilized to remove 87 g of iron.
gW P C Izhi.

Example 2゜The iron-free 1ow-JgWPC obtained in Example 1 and casein were used as protein raw materials, and soybean oil, lactose, various salts, and vitamins were added, and the balance and composition of each component was adjusted. After adjusting the pH before sterilization to 6.8, it is passed through a Claritaire, treated with active bacteria at 75℃ for 15 seconds or 95℃ for 15 minutes, concentrated and homogenized, and spray-dried to obtain the final product. Ta. This product was extremely suitable for preparing milk for infants.

[Brief explanation of the drawings] Figure 1 shows the pH and ferric chloride/Qlfm in Test Example 2.
Figure 1f is a diagram that examines the efficiency.

Claims (3)

[Claims] (1) Adjust the whey or whey protein concentrate and add ferric chloride, and finally pi (approximately 3.0 ± 0.6,
Whey or whey protein characterized by having a chloride concentration of about 4.0±0.9 mM, leaving it at about 26±5°C for about 2 hours or more, and collecting the obtained precipitate. Method for purifying concentrates. (2) 11 adjustments of whey or whey protein concentrate and addition of ferric chloride to final pH of approximately 3.0 ± 0.6 and ferric chloride concentration of approximately 4.0 ± 0.9 m M, and leave it at about 26±5°C for about 2 hours or more, collect the obtained precipitate, wash the precipitate with water once, dissolve it in acid, and adjust the pH.
was adjusted to approximately 1.5 ± 0.2, and brought into contact with a low crosslinking degree cation exchange resin to remove iron, and then the treatment liquid was adjusted to approximately 1.5 ± 0.2.
11. A method for purifying whey or whey protein concentrate, which comprises reducing the concentration to about 6.8±0.4 and removing salt. (3) Use of the purified whey or whey protein concentrate obtained according to claim 2 as a protein source for childcare preparations.