FI60103C - FOERFARANDE FOER BEHANDLING AV GENOM UPPHETTNING STERILISERAD MJOELK MED SULFHYDRYLOXIDAS I OCH FOER FOERBAETTRING AV MJOELKENS SMAK - Google Patents

Description

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Patent and Registration Authorities Ansökan utlajd och utUkrlftan publkarad 31.0Ö.Ö1 (32) (33) (31) Revoked Privilege— B * flrd pdoritet (71) Brik Pak Inc., 2775 Villa Creek Drive, Dallas, Texas 75231, USA ) (72) Harold E. Swaisgood, Raleigh, North Carolina, USA (US) (7U) Oy Kolster Ab (5I) Method for treating sterilized milk by heating with sulphhydryl oxidase to improve the taste of the milk - Förfarande för behanöling av genom upphettning steriliserad mjölk med sulh The present invention relates to a method of treating milk to improve its taste, which milk has been sterilized by heating it to about 120 ° C for about 4 seconds, which is sufficient to impart a boiled taste to the milk, wherein said heat-treated milk is contacted with a solid. with a sulfhydryl oxidase enzyme immobilized on the substance.

An enzyme in milk that catalyzes the oxidation of cysteine, glutathione (GSH), and sulfhydryl groups in milk proteins to the corresponding disulfides has been shown by Freidrick Kiermeier and Ernst Petz, Z, Lebensm, -Unters.-Forsch., Vol. 132, pp. 342-351 (1967) and Vol. 134, pp. 97-102 and 149-156 (1967)). This reaction was assumed to be 2 RSH + 1/2 O 2 -> RSSR + H 2 O 60105 so they called the enzyme sulfhydryl oxidase.

Kiermeier and Petz obtained an enzyme preparation from the whey fraction of skimmed milk. Their attempts to purify and isolate sulfhydryl oxidase failed. A method for isolating and purifying the sulfhydryl oxidase enzyme from milk has subsequently been invented. Sulfhydryl oxidase has been found to catalyze the oxidation of both small molecule compounds and sulfhydryl groups of proteins by oxygen. The enzyme, in both near-pure and immobilized form, has been found to be useful in treating milk to remove boiled flavor.

The method according to the invention is characterized in that the milk is contacted with the enzyme at a temperature of 20-45 ° C, preferably 30-35 ° C, that said solid to which the sulfhydryl oxidase enzyme is immobilized is sterilized together with the enzyme before the milk is treated, and sterilization is performed liquid or gas without a substantial rise in the temperature of the enzyme.

Isolation of sulfhydryl oxidase

In the first step, the untreated whole milk is defatted. The casein fraction obtained from the skimmed milk is coagulated with rennin. The whey is collected, cooled and poured into a half-saturated solution of ammonium sulfate to precipitate the enzyme fraction.

The resulting enzyme fraction is dissolved in a dilute, neutral buffer solution (e.g., phosphate buffer) to stabilize the enzyme.

The enzyme solution is then allowed to equilibrate (the enzyme dissociates quite slowly), preferably under cooling.

To separate the enzyme from the higher molecular weight material, the solution is treated, e.g., by centrifugation. Upon centrifugation of the solution, the enzyme is removed with the supernatant.

This is concentrated to a protein content of n, 2.5-3.5% e.g. by ultrafiltration. It has proved necessary to achieve a content of about 2.5% to increase the enzyme molecular size by recombination. The enzyme fraction is isolated e.g. by centrifugation to separate it from the lower molecular weight material. The specific activity of the enzyme fraction isolated in the form of a pellet is at least n, 1400 times higher than the activity of the enzyme in skimmed milk.

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3 60103

The specific activity of the enzyme can be improved by dissolving the above precipitate pellet in a neutral buffer solution at about twice the dilution (according to the volume of the liquids) and separating the enzyme from the resulting solution to give an enzyme preparation with a specific activity up to 3000 times higher than skimmed milk.

Isolated, near-pure sulfhydryl oxidase can be stored for long periods of time at about 4.4 ° C without significant loss of activity. However, it is most preferable to convert the enzyme to an immobilized form, as this can be easily isolated after completion of the reaction.

Immobilization of the enzyme is accomplished, e.g., by adsorbing the enzyme onto an insoluble carrier, retaining or enclosing it in gel matrices, attaching it to chemical insoluble supports, or crosslinking the enzyme molecule intramolecularly.

A preferred way to immobilize isolated sulfhydryl oxidase is to attach the enzyme to glass beads. A suitable method is described in Biochim. Biophys, Acta, pp. 243-256 (1974). Because immobilized sulfhydryl oxidase is sensitive to bacteria, heat, and pH, it is important that the enzyme be well protected during storage. Preferably, the immobilized sulfhydryl oxidase is stored refrigerated in a neutral buffer solution (phosphate buffer),

The following examples illustrate the invention.

Example 1

The crude sulfhydryl oxidase obtained from cow's milk was purified by the following method. For this isolation method, the concentration-dependent binding dissociation of the enzyme was chosen. Starting materials and reagents: whole cow's milk from afternoon milking (taken directly in glass containers and immediately cooled to 4 ° C and stored at this temperature), enzyme-grade sucrose and ammonium sulphate, manufactured by Schwartz / Mann, acrylamide,,, Ν'-methyleneib , N ', N'-tetramethylethylenediamine, 2-mercaptoethanol and sodium dodecyl sulphate, manufactured by Eastman (Rochester, NY),

Jit-> K '“60103 D-galactose, D-galactosamine, (grade I), L-fucose, N-acetyllineuramic acid, EDTA, o-dianisidine, GSH, DTNB (5,5'-dithiobis / 2-nitrobenzoic acid ) and glutathione reductase (type III) from Sigma (St. Louis, Mo), horseradish peroxidase (Worthington peroxidase D), ribonuclease A (crystallized 5 times) and yeast RNA from Worthington (Freehold NJ), and crystalline rennin from Pierce (Rockford, III).

All inorganic salts and buffer solution salts were analytically pure products of Baker.

The crude enzyme fraction was prepared as described by Kiermeier and Petz (see page 1). First, the whole milk was defatted by centrifugation at 4080 g for 30 minutes at 30 ° C.

Whey (fraction B) was obtained by coagulation with rennin: about 2 mg of rennin per 100 ml of skim milk was added and the reaction was allowed to proceed for 30 minutes at 30 ° C. Under these conditions, only the Phe 105-Met 106 bond of k-casein is hydrolyzed. The formed curd was removed by centrifugation at 16300 g for 45 minutes at 30 ° C, the whey was cooled and poured into half-saturated ammonium sulfate solution at 4 ° C. After standing overnight at 4 ° C and the precipitate (crude enzyme, fraction C) was removed by centrifugation (16300 g, 60 minutes, 4 ° C),

The crude enzyme (fraction C) was dissolved in 0.047 M sodium phosphate solution at pH 7.0 to a protein content of 3% and dialyzed against the same buffer at 4 ° C. This solution was diluted with buffer to a protein content of 0.15% and allowed to stand overnight at 4 ° C. The rapidly settling impurities (fraction D) were removed by centrifugation (2000 g, 30 minutes, 4 ° C) and the supernatant formed (fraction E) was concentrated at 4 ° C to a protein content of about 3% in an Amicon TCF-10 using an Amicon PM 10 membrane. The resulting solution was centrifuged (2000 g, 30 minutes, 4 ° C). The resulting precipitate pellet (fraction F) was sulfhydryl oxidase in almost pure form.

Example 2

The smaller molecular proteins of the final product of Example 1 were removed by dissolving the pellet in twice the volume of sodium phosphate buffer, standing overnight at 4 ° C to promote dissociation of 3λ 60 10 3, and centrifuging (2000 g, 30 minutes, 4 ° C). The resulting enzyme preparation (fraction H) was high purity sulfhydryl oxidase.

Determination of activity

The characteristics of the fractions obtained according to Examples 1 and 2 are shown in Table I. The activity was determined from the oxygen consumption rate using a vibrating platinum electrode and 0.8 mM GSH as substrate. One unit of activity is defined as one μM of oxygen consumed per minute in phosphate buffer at pH 7.0 at 35 ° C.

Two assay methods were developed, one based on the disappearance of sulfhydryl groups and the other on oxygen consumption. In the first method, the concentration of sulfhydryl groups was measured by reacting them with DTNB. In a typical assay, the reaction mixture contained 1.5 ml of 0.8 mM GSH in 0.047 M sodium phosphate at pH 7.0 (ionic strength 0.1) and 0.2 ml of enzyme solution. The control was otherwise similar, but the native enzyme was replaced with 0.2 ml of boiled enzyme solution. Incubated at 35 ° C, 0.3 ml samples were taken periodically and added to 9.7 ml of 0.017 M sodium phosphate pH at 8.0. 3 ml of the resulting solutions was taken and stirred in 20 volumes of 0.01 M DTNB in 0.047 M sodium phosphate, pH 7.0. The absorbance of this solution was measured after 2 minutes at 412 nm. The rate of the enzyme-catalyzed reaction was determined from the linear portion of the curve obtained as a function of sulfhydryl concentration over time,

In another assay method, the rate of oxygen consumption was measured using a Gilson Model K Oxygraph. In a typical assay, 0.2 ml of enzyme solution was added to 1.5 ml of 0.8 mM GSH equilibrated at 35 ° C in an electrode cell. Controls containing cooked enzyme did not consume oxygen. Enzyme reaction rates were calculated from the slopes obtained.

. t. I

6 60103

Table I

Purification of sulfhydryl oxidase from cow 's milk

Fraction Volume Total activity Specific activity Yield (ml) Ority (units) Visibility (units (%) _Units / mg N) _

Skimmed milk 1000 160.0 0.032 100

Renninhera 930 153.9 0.174 96

Crude enzyme (fraction C) 40 151.4 0.756 95

Precipitate obtained in the first centrifugation (fraction F) 80 96.0 47.1 60

The pellet obtained in the second centrifugation (fraction H) 24 65.1 103.8 41

Fraction F was significantly more active than fraction C. Fraction chromatography of fraction F yielded only the void eluting fraction. Determination of the protein eluting from the bands of the unstained gel showed that enzyme activity was present only at the upper end of the intermediate gel and at the interface between the intermediate gel and the separation gels. The proteins of fraction F on the separation gel were efficiently removed by diluting the solution slightly and centrifuging, only two bands could be detected by gel electrophoresis of fraction H, both of which were enzymatically active. In addition, "disc gel" electrophoresis of this fraction in sodium dodecyl sulfate showed only one stained protein band.

This purification method is described in Janolino and Swaisgood, "Isolation and Characterization of Sulfhydryl Oxidase from Bovine Milk", J. of, Biol, Chem., Vol. 250 (1975) No. 7, pp. 2532-2538.

It is apparent that iron forms an essential part of the enzyme. Upon treatment of the enzyme with EDTA, the activity completely disappeared, but could be restored by dialysis with 1 μM ferrous sulfate. In addition, atomic absorption and neutron activation analysis of several enzyme preparations showed that the enzyme contained 0.5 atoms of iron per subunit.

Contrary to the above conclusions of Kiermeier and Petz, it has now been found from stoichiometric studies of sulfhydryl oxidase-catalyzed reactions that the reaction is in fact catalyzed by "aerobic oxidase" as follows: 2 RSH + 02 - »RSSR + H202

The methods of Examples 1 and 2 have been repeated numerous times, in which case an enzyme preparation with a specific activity of more than 1400-3000 times higher than the activity of the enzyme in skimmed milk has always been obtained.

Example 3

The dependence of the activity of the purified enzyme obtained in Example 2 on temperature and pH was studied using GSH as a support. The maximum activity was observed at 35 ° C.

A symmetric clock curve for activity and pH was obtained, with a pH optimum of 6.8-7.0 and apparent pKa values of 5.5 and 8.1 on the ascending and descending arms of the curve, respectively. 0.2 ml of the enzyme-containing solution was added to 1.5 ml of 0.8 mM GSH equilibrated at 35 ° C in an electrode cell. Controls containing cooked enzyme did not consume oxygen. Enzyme reaction rates were calculated from the slopes obtained,

The molecular weights of the purified enzyme subunits obtained according to Example 2 were studied. In "disc gel" electrophoresis with polyacrylamide, the purified preparations of Example 2 had two zones showing activity, but in "disc gel" electrophoretic sodium dodecyl sulfate there was only one zone. Sulfhydryl oxidase was tested with a series of acrylamide concentrations. From the experiments performed with the purified enzyme, the apparent average molecular weights were 91,200 + 1200, 89,800 + 800, 89,000 + 400 and 89,000 + 1400 with acrylamide concentrations of 5.0, 7.5, 10.0 and 12.5%, respectively. Calibration curve at all concentrations , which represented the logarithm of molecular weight as a function of mobility, was almost straightforward. At the acrylamide concentrations of 10 and 12.5% »4, the values obtained with the separate enzyme preparation were quite consistent, and the average molecular weight of the subunit was found to be 8 60103 89 000 + 900. In all cases, a single protein band was observed with varying gel concentrations and enzyme amounts.

The chemical composition of the enzyme was also examined using amino acid and carbohydrate analysis. Amino acid analysis was performed by weighing freeze-dried samples of the purified enzyme into thick-walled annealing tubes and adding constant boiling (ca. 6-N) hydrochloric acid. The resulting solutions were cooled, deaerated, depressurized and sealed. The percentage moisture of the lyophilized samples was determined by drying to constant weight at 75 ° C under vacuum with phosphorus pentoxide. Hydrolysis was performed in a toluene bath at reflux for 16, 24, 48 and 72 hours and the hydrolysates were analyzed on a Beckman Model 116 amino acid analyzer.

Semi-cysteine was determined separately as cysteic acid after perura-fatty acid oxidation. Tryptophan was determined chromatographically after enzymatic pronase hydrolysis.

In the carbohydrate analysis, the total amount of hexose was determined by the phenol-sulfuric acid method. After one hour of hydrolysis at 80 ° C in 0.1 N hydrochloric acid, sialic acid was determined by the Aminoff method using standard N-acetylneutramic acid. Hexosamine was determined after 16 hours of hydrolysis in 2-N hydrochloric acid at 100 ° C, galactosamine was used as standard and values were reported as N-acetylhexosamine, Fucose (6-deoxy-I.-galactose) was determined by the thioglycolic acid-sulfuric acid method as fucose standard.

The results of the amino acid and carbohydrate analysis are shown in Table II. These values indicate that the majority (97%) of the enzyme consists of amino acid and carbohydrate moieties with 89% amino acid residues and 11% carbohydrates.

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10 60103

Amino acid and carbohydrate composition of purified sulfhydryl oxidase

Amino acid values are the averages of 16 ", 24-, 48- and 72-hour hydrolysis with the exception of the amino acids Thr, Ser, Val, Ile, Leu,

Tyr and Trp. Thr, Ser and Tyr values were obtained by extrapolation to zero for hydrolysis time and Vai, Ile and Leu values by extrapolation to infinity for hydrolysis time, Half-Cys values were corrected by assuming a cysteic acid yield of 95% after perforic acid oxidation.

Trp was determined after pronase digestion.

Example 4

The purified sulfhydryl oxidase obtained in Example 2 was immobilized by attaching the enzyme to glass beads. Succinate plate beads were prepared from N-aminopropyl glass beads (40-60 mesh, pore diameter 2000 A), manufactured by Corning Glass Works. The beads were washed with distilled water and equilibrated for 24 hours in 0.2 M phosphate buffer at pH 4.75. The gases were removed from the beads, to 0.5 g of beads was added crystalline 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) and allowed to react for 20 minutes at 25 ° C and pH 4.75. The beads were washed with distilled water and cold, neutral phosphate buffer solution to remove excess EDC. The isolated enzyme (0.1% w / v in phosphate buffer) was then contacted with succinylate beads for 16-24 hours, after which the beads were washed with 0.1 M phosphate buffer (pH 7). The isolated enzyme was covalently attached to the glass beads to give immobilized sulfhydryl oxidase. As mentioned above, the immobilized enzyme must be well protected during use by storing it in a cooled phosphate buffer solution at pH 7.

Example 5

The immobilized sulfhydryl oxidase is reactivated by contacting the inactivated enzyme with an aqueous solution of ferrous sulfate. As mentioned above, iron appears to form an essential part of sulfhydryl oxidase. Treatment of the enzyme with EDTA resulted in a complete loss of activity, as shown in Table IV. When dialyzed, 1 μM

In Fe solution, 70% of the original activity was restored. The activity of 2+ was also somewhat reversible with the cations Cu (30%) 2+ ...

and Mn (20%). The absorbance spectrum of the most common enzyme had a typical protein »1% 60103 protein maximum at 278 nm (E.j = 7.41), but no peaks at longer wavelengths. Thus, it is apparent that iron is not in the form of a heme prosthetic group. According to atomic absorption analysis, iron has 0.48 g atoms and copper about 0.15 g atoms per molecule (molecular weight 89,000). The neutron activation analysis of the separate preparation gave a value of 11.1, i.e. 0.3 μg iron / 38.5 mg sample, which is 0.46 g atoms of iron per molecule. Copper could not be determined by this method, and analysis of other metals showed only traces of zinc and cobalt. Thus, iron appears to be essential for the enzyme activity of sulfhydryl oxidase.

Table III

Effect of metal ions on sulfhydryl oxidase activity Assays were performed in phosphate at pH 7.0 and 35 ° C using 0.8 mM GSH as a medium. The stock enzyme solution was sampled, 1.0 nM EDTA was added and assayed. Other samples of the stock solution were treated with EDTA and then dialyzed against 1.0 mM solutions of various metal ions. Controls were prepared using boiled enzyme and dialysate containing metal ions. In both cases, no significant oxygen consumption could be detected.

Treatment Enzyme activity (oxygen consumption yjmoles / min)

Sulfhydryl oxidase enzyme (control) 0.368 + EDTA (1.0 mM) 0.012 + ferrous sulfate 0.256 + copper sulfate 0.109 + manganese (II) chloride 0.085 + cobalt chloride 0.021 + zinc sulfate 0.02 + molybdenum dibromide .

It follows from the above reasoning and examples that the separation method uses concentration-dependent attachment-dissociation properties to give an almost pure enzyme with an unusually high specific activity, 12,601 03

It is known that when milk is heated above 68 ° C for various lengths of time, it acquires a "boiled" taste. Today, milk is pasteurized or "ultra-pasteurized" by heat treating whole milk at 60 ° -74 ° C for 30 minutes to 2 seconds. Such treatment is necessary to kill disease-causing bacteria and to control the activity of fermentation bacteria present in untreated whole milk. But such treatment does not destroy all the bacteria in untreated whole milk, and because the milk has a limited shelf life, it must be cooled to a temperature of n, 4 ° C during transport, sale and storage.

Methods for preparing ultra-high temperature (UKL) sterilized milk are known. Such methods have been described e.g.

H. Burton, Dairy Science Abstr., 31, 287-297 (1969) and T. R. Ashton, J, Soc. Dairy Tech ,, Vol. 18, no. 2, pp. 65-83 (1965).

Although the UKL method for milk in the United States and Canada differs slightly from the European method, both treat milk at a temperature of at least 88 ° C for varying times, usually at about 149 ° C for about 4 seconds, unfortunately at such high temperatures (or lower, if treatment is prolonged) milk develops an undesirable and unpleasant “cooked” taste that generally makes milk unmarketable, especially in the U.S. Much has been studied about the nature of “cooked” taste and methods to eliminate it.As well as JT Hutton et al., J. of Dairy Science, 35, p. 699 (1952) and H. Burton, Proc. 15th International Dairy Congress 3, pp. 1729 (1952) speculate that the "boiled" taste of heated milk is due to the release of sulfhydryl groups in the milk.Later, Burton (Dairy Sci. Abstr. 31, pp. 295-1969) ) and others have questioned the role of free sulfhydryl groups in milk.

Prior to the present invention, no method was known for removing the "cooked" taste caused by heat treatment in UKL-sterilized milk and treated milk, respectively. In some cases, the intensity of the "cooked" taste may have been reduced but not eliminated by cooling,

In 1971, more than 20 billion kg of milk was sold in the United States, and the entire amount had to be heavily cooled. Although the obvious advantages of milk sterilization are the regulation of energy and fuel during the handling and transport of the milk and the extended shelf life of the product, which allows transport, sale and storage at ambient temperature, the "cooked" taste usually associated with such milk usually makes it unmarketable. Thus, it has now been found that the "cooked" taste associated with heat-treated milk can be eliminated in accordance with the invention,

The "boiled" taste of milk, which is created by heating the milk for a certain period of time, is removed by contacting the heat-treated liquid milk with an immobilized sulfhydryl oxidase enzyme.

As used herein, the term "cooked" flavor refers to the chalky or tasteless taste that occurs in milk when processed at a temperature generally above n, 68 ° C. Such milk can be unpleasant in both taste and smell, reminiscent of boiled cabbage. Although such a cabbage-like odor may disappear several days after the milk is processed, the tasteless chalky "cooked" taste that may develop into the taste of cooked cardboard is retained.

It has now been found that the taste of "cooked" in liquid milk is in fact due to the release of sulfhydryl groups in milk proteins and reactions after heat denaturation of milk. The sulfhydryl oxidase enzyme obtained and used from untreated whole milk catalyzes the conversion of sulfhydryls to disulfides. It is evident that the oxidation of sulfhydryl groups upon contact with immobilized sulfhydryl oxidase in whole milk removes the "cooked" taste.

The advantage of using an immobilized sulfhydryl oxidase catalyst is that the enzyme is obtained from whole milk, thus forming a natural component of the milk. In addition, the use of the enzyme in immobilized form means that no substances are added to the milk to be treated.

The invention is illustrated in more detail by the following examples.

Example 6

A biocatalytic reactor containing immobilized sulfhydryl oxidase was prepared by filling a 6 mm diameter glass column with 1.4 ml of sulfhydryl oxidase enzyme immobilized by attaching it to glass beads having a pore size of n, 2000 Å. milk flowing through the column and the layer of immobilized enzyme, * s>, Ψ (t, 14 Ä 60103 and exited after contact with the immobilized enzyme. The reactor could be connected directly to the UKL processing apparatus, whereby the flow through it was continuous.

Example 7

The cow's milk was heated at 149 ° C for about 4 seconds, after which it had a clear "boiled" taste. The milk was passed through the biocatalytic reactor described in Example 1 at 30 ° -35 ° C at a flow rate of about 40 ml / h. the unpleasant odor and the "cooked" taste had disappeared.

The percentage oxidation of sulfhydryl groups in heat-treated milk and the stability of the reaction described above are shown in Table IV.

Table IV

Stability of a UKH-sterilized skimmed milk sulfhydryl oxidase reactor

A reactor with a volume of 1.4 ml was operated at a flow rate of 40 ml / h at 35 ° C.

Date of use Date of use (h) Oxidation (%)

At the beginning 5 45

Fifth day 3 34

Tenth day 5 31

It is recommended to use glass beads with a pore size of more than about 700 A to immobilize the enzyme. Smaller sizes lead to unfavorable results and optimum activity is achieved with a pore size of about 2000 A.

Contact with milk and immobilized enzyme at high temperatures should be avoided as this may reduce the activity of the enzyme. A temperature of 20 ° -45 ° C, preferably 30 ° -35 ° C can be used without significant loss of enzyme activity.

The activity of the immobilized enzyme decreases in normal use of the present invention, and the enzyme can be reactivated by contacting it with a freshly prepared aqueous ferrous sulfate solution.

Contacting the heat-treated milk with the immobilized sulfhydryl oxidase enzyme may have other consequences. 15 601 03 than the removal of "boiled" taste alone, since such treatment also appears to prevent the development of other undesirable flavors and the destabilization of milk proteins. conventionally pasteurized, homogenized milk.

When a sulfhydryl oxidase enzyme is used in the treatment of sterile milk, the enzyme itself as well as the glass beads must be sterile. But the sulfhydryl oxidase enzyme cannot be sterilized by heating because the heat treatment sufficient to destroy the microorganisms also destroys the enzyme. It is also clear that many chemical sterilants react with the enzyme to chemically degrade it, and thus it is important to use a sterilization treatment that destroys all microorganisms but does not chemically alter the enzyme. Fortunately, a virtually unlimited time is available to sterilize the sulfhydryl oxidase enzyme and glass beads, so that relatively mild sterilants can be used. It has been found that sterilization of glass beads and the enzyme immobilized thereon can be performed with a sterilizing liquid or gas that does not affect the enzyme. Ethyl alcohol can be considered as suitable sterilizing liquids and ethylene oxide as suitable sterilizing gas. The sterilization treatment of the glass beads and enzyme is performed by keeping the beads in a sterilizing liquid, e.g. ethyl alcohol, for the time required for complete sterilization, e.g. it is also possible to keep the glass beads and the enzyme in a chamber containing sterilizing gas, e.g. ethylene oxide, for a suitable time before adding the glass beads to the milk reaction chamber, 'V ·,

Claims (3)

16 60103 A method of treating milk to enhance the taste of the milk sterilized by heating it to about 120 ° C for about 4 seconds, which is sufficient to impart a boiled taste to the milk, wherein said heat-treated milk is contacted with a sulfhydryloxyoxidase enzyme immobilized on a solid, that the milk is contacted with the enzyme at a temperature of 20-45 ° C, preferably 30-35 ° C, that said solid to which the sulfhydryl oxidase enzyme is immobilized is sterilized together with the enzyme before the milk is treated, and that the sterilization is carried out with liquid or gas the temperature of the enzyme substantially rises. Process according to Claim 1, characterized in that the sterilization is carried out by storing the enzyme immobilized on a solid in ethyl alcohol for 10 minutes to 5 hours. Process according to Claim 1, characterized in that the immobilized enzyme is stored in a state containing ethylene oxide gas before use, S ·