JPH0119863B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides whey or whey protein concentrate (Whey
The present invention relates to a method for purifying Protein Concentrate (hereinafter referred to as WPC) to the point where it can be used as a protein source in infant preparations. Generally, whey, which is a by-product during the production of cheese and casein, contains most of the water-soluble components in milk except for 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. It was only separated and recovered. For example, the products obtained by recovery using heat coagulation methods, polyphosphate methods, etc. are insoluble or contain large amounts of inorganic salts (phosphate radicals, iron salts), and there are many restrictions on their use in food. can be,
At least its use as a protein source in infant preparations has been difficult. The reason why whey or WPC is unsuitable as a protein source for infant preparations is the presence of large amounts of β-lactoglobulin. Parich WE (Clin
Allergy 1: 369-380, 1971) used monkeys.
Antibodies against β-lactoglobulin were most frequently found in the PCA reaction, and Kletter B,
Gery I, Freier S.etal (Clin Allergy1:249-
255, 1971) was a group of infants sensitive to milk;
Examine serum IgE antibodies against various milk proteins, and
- It is known that β-lactoglobulin exhibits stronger allergenicity than casein and α-lactalbumin, as IgE antibodies against lactoglobulin are most often found. In general, both the casein and whey proteins of cow's milk represent foreign proteins for infants, and infants who are hypersensitive to milk proteins may have a strong allergic reaction to the intake of infant preparations; -This is largely due to lactoglobulin. However, whey or WPC contains large amounts of α-lactalbumin and immunoglobulin, which are useful protein sources for infants. Therefore, if only β-lactoglobulin can be selectively removed from whey or WPC, not only will the whey protein component be similar to that in breast milk, but also β-lactoglobulin can be removed selectively from whey or WPC.
- Lactoglobulin-removed whey is expected to be relatively less allergenic than regular whey, and many attempts have been made to remove β-lactoglobulin. However, at the laboratory level, methods such as ion exchange resin, gel filtration, ammonium sulfate splitting, and fractional precipitation using carboxymethyl cellulose are also possible.
There are no examples of successful production on an industrial scale. However, there have already been suggestions for the removal of β-lactoglobulin on an industrial scale. That is, regarding the precipitation of whey proteins by ferric chloride.
Block etal (Arch.Biochem Biophys47, 88
Since G. Ammantia et al. (1953) recovered whey protein as ferrilactin, it has been used in combination with heating (G. Ammantia et al.
Can.Inst.Food S.Technol J.7, 199 (1974)) Combination with polyphosphate (Jones SB, etal J.Agr.
Improvements such as Food Chem. 20, 229 (1972)) have been investigated, and efforts have been made to improve yields. However, it was not yet completed. The present inventors aimed to industrially purify whey or WPC by using ferric chloride to purify whey or WPC.
After many years of research to perfect the purification of WPC, we learned that only β-lactoglobulin can be separated from whey or WPC when a wide variety of conditions are met. The present invention was completed by strictly combining many of these conditions, and by adjusting the pH of whey or whey protein concentrate and adding ferric chloride, the final pH was adjusted to approximately 3.0±0.3. Purification of whey or whey protein concentrate, characterized in that the ferric chloride concentration is about 4.0±0.9mM, this is left at about 26±5°C for about 2 hours or more, and the obtained precipitate is collected. It is the law. Then, the present invention adjusts the pH of whey or whey protein concentrate and adds ferric chloride, and finally the pH
is about 3.0±0.3, and the ferric chloride concentration is about 4.0±0.9mM. This is left at about 26±5℃ for about 2 hours or more, the resulting precipitate is collected, and the precipitate is washed with water. death,
Dissolve in acid, adjust pH to approximately 1.5±0.2, contact with low cross-linked cation exchange resin to remove iron, then adjust the pH of the treatment solution to approximately 6.8±0.4, and perform salt removal treatment. This is a method for purifying whey or whey protein concentrate, which is characterized by the following. Generally, about 60% of the protein in whey or WPC is β
-Of lactoglobulin, α-lactalbumin accounts for approximately 20% and immunoglobulin accounts for approximately 13%, but β-lactoglobulin can actually be reduced to approximately 8.8% with a single treatment using the purification method of the present invention. Therefore, α-lactalbumin increases to 56% and immunoglobulin increases to 23.1%. Furthermore, if the purification method of the present invention is repeated, most of the β-lactoglobulin can be removed. WPC purified in the present invention refers to 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, of which approximately 60% is β-lactoglobulin and approximately 20%
is α-lactalbumin, and approximately 13% is composed of immunoglobulin. Since whey or WPC is normally neutral, it is mixed with an acid such as hydrochloric acid to bring the pH to approximately 3, and then a ferric chloride solution is added to a final concentration of approximately 4.0±0.9mM and stirred. . The PH of the mixture will rise slightly at this point, so add an acid such as hydrochloric acid again.
The pH must be adjusted to a range of approximately 3.0±0.3. When the pH of whey or WPC falls below 2.7, the efficiency of separating β-lactoglobulin sharply decreases, and even when the pH rises to 3.3 or higher, the efficiency of separating β-lactoglobulin deteriorates. When the ferric chloride concentration is 4.0 mM, the separation efficiency of β-lactoglobulin is best at a pH of 2.8 to 3.1, most preferably at a pH of 2.9. The concentration of ferric chloride also has a very subtle effect, but preferably 5.0~
The concentration is about 3.0mM, and the most preferred is 4.0mM. Whey or WPC after pH adjustment and addition of ferric chloride is left at about 26±5°C for about 2 hours or more.
The temperature of the whey or WPC may be set at about 26°C from the beginning, but preferably it is heated when it is left to stand and maintained at 26°C ± 5°C, preferably 24 to 28°C for 2 hours or more. If it is left to stand for 2 hours or more, β-lactoglobulin will remain dissolved in the supernatant, and other effective proteins will precipitate, so the precipitate can be easily recovered by centrifugation or decantation. The obtained precipitate is easily dissolved in acids such as hydrochloric acid, so make it into a solution, adjust the pH to about 1.5 ± 0.2, and pass it through a low crosslinking degree cation exchange resin such as Amberlite IR112. The iron content can be removed by contacting with the metal. This treated solution still contains salt, so if the pH is adjusted to around 6.8±0.4 and the solution is placed in a dialysis tube and dialyzed, the salt will be completely removed. The purified whey or WPC obtained here contains only 8.8% β-lactoglobulin in protein and can be effectively used as a protein source for infant preparations. If this purified whey or WPC is purified again according to the present invention, the content of β-lactoglobulin will further decrease and become more effective, but even with a single purification, the content has decreased from 60% to 8.8%, which is normal. Childcare preparations are sufficient and do not cause allergic reactions in infants. Next, test examples and examples of the present invention will be shown. Test example 1 Effect of whey pH on precipitation of whey proteins by ferric chloride: The pH of clarified whey was
Adjust the pH from 2.5 to 5.0, add 1M ferric chloride solution to a final concentration of 4mM, correct the desired pH again, leave it at room temperature for 2 hours, and then heat it at 10,000G for 15 minutes.
Minute centrifugation. The supernatant was analyzed by 8% polyacrylamide gel electrophoresis. From the electrophoretic chromatogram, it was found that whey proteins other than β-lactoglobulin migrated to the precipitate compartment between pH 2.6 and 3.1. In the pH range of 3.4 to 4.3, β-lactoglobulin precipitates, but at the same time immunoglobulin also partially precipitates. At PH4.5 or higher,
At an iron concentration of 4mM, the amount of precipitated protein is small, and as the iron concentration increases, whey protein precipitates, but selective precipitation is no longer observed. Test Example 2 Effect of whey pH and iron concentration on precipitation of whey protein by ferric chloride: The pH of each clarified whey was adjusted with hydrochloric acid and caustic soda.
2.6 to 3.1, add 1M ferric chloride solution to a final concentration of 3, 4, 5, 6mM at 26°C.
The effect of iron concentration was investigated in this PH range after leaving it for a while. 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. S=Ss+Sp S: Overall separation efficiency Ss=Abs(A-B)×% soluble nitrogen
Ss: Supernatant efficiency Sp = Abs (A-C) x insoluble nitrogen %
Sp: Precipitate efficiency A, B, and 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 is 100 minus the soluble nitrogen percentage. The ratio of α-lactalbumin was calculated as follows by determining the polyacrylamide gel electrophoresis pattern using a tensitometer. Separation efficiency = area of α-lactalbumin/area of α-lactalbumin + area of β-lactoglobulin The results are shown in FIG. As shown in Figure 1, PH2.9 iron concentration 4.0mM
The highest separation efficiency was obtained. Almost the same separation efficiency can be obtained within the pH range of 3.0±0.1. Test Example 3 Effect of temperature on precipitation of whey protein by ferric chloride: When the pH of whey is 3.2 or less, temperature does not affect the precipitation of protein between 4℃ and 55℃, but the PH
In the pH range where β-lactoglobulin and immunoglobulin of 3.4 or higher precipitate, it was confirmed that the effect of temperature is large and the amount of precipitation increases as the temperature decreases. Test Example 4 Yield and protein composition of low β-lactoglobulin whey protein concentrate under optimal conditions: The pH of undesalted clarified cottage whey was set to 3.0, and 1M ferric chloride solution was added to it at the final concentration.
After adjusting the pH to 3.0 again and leaving it at room temperature for 2 hours, the precipitated protein was separated. The nitrogen yield measured by Kjeldahl method was 28.7%. After dissolving the precipitated protein with acid, iron was removed using a cation exchange resin, neutralization was performed, and electrophoretic analysis revealed that the protein composition was approximately as follows. Low β-lactoglobulin WPC β-lactoglobulin 8.80 α-lactalbumin 56.0 Immunoglobulin 23.1 Bovine serum albumin 6.60 Others 5.5 Test example 5 Removal of iron from low β-lactoglobulin WPC: Low β-lactoglobulin WPC (hereinafter referred to as Low- Lg
WPC) is easily solubilized on the acidic side below PH1.8 or on the neutral or alkaline side above PH5.5, and even in the PH range where precipitates are formed, if the ionic strength is increased with salt etc. Solubilized in
More than 80% of the protein is solubilized by adding 0.9N of salt. Hydrochloric acid is added to Low-Lg WPC to adjust the pH to 1.4 to 1.6, and after stirring, the liquid is passed through a column of strongly acidic cation exchange resin (e.g. Amberlite IR-112) at a flow rate (SV1.3). Untreated Low-Lg
Iron content of WPC, 98% for 1120mgFe/g protein
If the removal point is the flow-through point, approximately 3.1 ml of resin is required to remove iron from 100 g of protein.
Amberlite IR-112 has a cross-linking degree of 2%, but when Lexin |0|, which has a cross-linking degree of 8%, was used, almost no iron was removed. This is presumably because the dissolved iron protein mixture has a large molecular weight and cannot penetrate into the highly crosslinked resin. Test example 6 Amino acid composition of iron-free low-Lg-WPC: Liu and chang (J.Biol Chem246:2842,
1971), the iron-free low-Lg WPC was
-Hydrolysis with toluensufonic acid (110℃ for 24 hours)
and analyzed using a Phoenix model M6800 amino acid analyzer. Tryptophan is Spies (Anal.
Chen. 39:1412, 1967). The results are shown in Table-1. As shown in Table 1, it has an excellent amino acid composition, but the chemical score calculated based on the preliminary comparative amino acid pattern for FAD/WHO infants was 65.5. It can be seen that infants require a higher amount of valine than school children and adults, so the chemical score is lower than that of Ultrafiltered WPC.

【table】

[Table] Example 1 PH of 50 clarified cheese whey was 3N
Adjust the pH to 3.10 with 780ml of hydrochloric acid and add 1M ferric chloride while stirring.
200ml of the solution was added to give a final concentration of 4.0mM. At this time, the pH was 2.82, and after adjusting the pH to 3.00 by adding 70 ml of 3N caustic soda, it was kept at 25°C for 3 hours. Centrifugation (10000
g×15 min) to separate the precipitate. The weight of the sediment is
It was 610g. Disperse the precipitate twice using deionized water from Step 5 and repeat centrifugation to remove lactose,
Inorganic salts were washed out. Immediately after dispersing the precipitate in 4000ml of water, add 3N hydrochloric acid to bring the pH to 1.50.
Passed through whatman No. 1 filter and passed through an Amberlite IR112 column (φ6.0cm in diameter x 90cm) at a flow rate.
The through iron was removed in SV1.3. The eluate from the column was immediately neutralized with 3N caustic soda, placed in a dialysis tube, 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 and then freeze-dried to obtain 87 g of iron-free low-Lg WPC. Example 2 The iron-free low-Lg WPC obtained in Example 1 and casein were used as protein raw materials, and soybean white oil, lactose, various salts,
After adding vitamins, adjusting the balance and composition of each ingredient, and adjusting the pH before sterilization to 6.8, pass it through a Clarity Ear and heat at 75℃ for 15 seconds or at 95℃.
The final product was obtained by sterilization for 15 minutes, concentration homogenization, and spray drying. This was extremely suitable for adjusting infant formula milk.

[Brief explanation of drawings]

FIG. 1 is a diagram comparing the separation efficiency affected by pH and ferric chloride concentration in Test Example 2.

Claims (1)

[Scope of Claims] 1 Adjust the pH of whey or whey protein concentrate and add ferric chloride until the final pH is about 3.0±0.3 and the ferric chloride concentration is about 4.0±0.9mM. about 26
A method for purifying whey or whey protein concentrate, which comprises leaving it at ±5°C for about 2 hours or more and collecting the resulting precipitate. 2. Adjust the pH of the whey or whey protein concentrate and add ferric chloride until the final pH is approximately 3.0±0.3 and the ferric chloride concentration is approximately 4.0±0.9mM, which is approximately 26
The precipitate obtained by leaving it at ±5℃ for about 2 hours or more was collected, washed with water, dissolved in acid, and PH
is 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 pH of the treated solution is adjusted to approximately 6.8 ± 0.4 for salt removal treatment. or a method for purifying whey protein concentrate.