DE2458145A1 - PROTEIN AGGREGATION PROCESS - Google Patents

Description

PATENT ADVOCATE c

DR. ING. A. VAN DERWERTH I i,, DR. FRAN Z LE DE RE R

ItIIlII

(1934-1974) 8 MONKS 8O

LUClLE-GFtAHN-STR. 22 TEL. (089) -47 29 47

Munich, December 9, 1974 Q 707

UNILEVER N.V.

Burgemeester s'Jaco'bplein 1, Rotterdam, Netherlands

Process for the aggregation of proteins

The invention "relates to a new method using of polysaccharides to aggregate proteins.

Proteins and polysaccharides are two important classes of the natural Polymers. It has long been "known that their interactions, for example in food, are important * here charged polysaccharides are generally involved. It has now been found that certain polysaccharides surprisingly have strong effects on proteins. This is particularly surprising since polysaccharides are neutral. This phenomenon is explained in more detail below. The polysaccharides

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affect the solubility properties of proteins, and in general they increase their tendency to aggregate, in particular when heated. The aggregation can be monitored by optical methods. Often times, the aggregation of the Proteins that they precipitate or flocculate.

Polysaccharide mixtures which show this effect to a surprising extent are obtained by eliminating the branching of amylopectin or a maltodextrin with a low DE value (CDE = dextrose equivalent of less than 2?) Using alpha-1,6-glucosidase, which is free from alpha-1,4-glucosidase is obtained.

The invention therefore relates to a method in which the aggregation of a protein in an aqueous solution by Unbranched maltodextrin dissolved therein with a low DE value or amylopectin freed from branches is induced.

It is assumed that the polysaccharides which are responsible for this effect are essentially linear polyglucosides with 20 to 80 glucose units and in particular with 0 to 55 glucose units. The particular importance of these polyglucosides in z. B. the debranched amylopectin was demonstrated by fractionating the debranched amylopectin using standard gel column chromatography and measuring the number of glucose units in the polyglucosides in the particularly active fractions by the enzymatic method described by Manners et al. is described in Carbohydrate .research, J.2 (197Ό, 109 It is believed that polysaccharide mixtures in order to show the effect to a useful degree, at least two wt -.. ° / o by weight and preferably at least ten Ji " should contain linear polyglucosides with 30 to 55 glucose units.

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The invention also provides a method in which the aggregation of a protein in aqueous solution is induced by a dissolved polysaccharide mixture which at least 2 wt .-% linear polyglucosides containing 30 to Contains 55 glucose units.

Suitable polysaccharide mixtures can, as already described, by treating amylopectin or a maltodextrin obtained with a low DE value with an alpha-1,6-glucosidase will. Comparable mixtures can, at least in principle, by fractionating hydrolysates either from starch or from from starch fractions or by synthesis. .

Although polyglucosides are considered responsible within only a narrow range of the number of glucose units per molecule are considered for the remarkable effects observed, it was also found that saccharide mixtures which only contain a narrow range of polyglucosides which tend to be low in solubility in water. The presence a wide range of polyglucosides improves overall solubility. Since it is the polyglucosides in solution, which affect the proteins, it is advantageous to have other polyglucosides as well as the narrowly defined polyglucosides to have. Such mixtures of polyglucosides are made by removing the branches in amylopectin or maltodextrins with low DE-Vert according to all technically and economically easy to carry out methods of fractionation of hydrolysates of starch © or starch fraction obtained by synthetic methods.

The required amount of polysaccharide mixture depends on numerous factors including type and quantity of protein, the polysaccharide mixture used, the desired one

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Extent of the effect and the presence and amount of other ingredients, e.g. B. salts, from. Suitable amounts can be in can easily be determined by simple preliminary tests.

Temperature is a major factor in determining the extent to which aggregation occurs. A main result of the invention is to enable increased aggregation to occur at surprisingly low temperatures. In most cases, however, temperatures higher than ambient temperature, 17 ^° C., are required. The suitable minimum temperature can be determined in a simple manner by a simple experiment. It is of course known that heat brings about the aggregation of proteins and thus precipitation or flocculation, but surprisingly low temperatures can be used when using the process according to the invention. If z. B. proteins such as casein are present, the presence of the polysaccharide mixture causes the precipitation of proteins when heated. The aggregation of casein is usually insufficient to cause precipitation even when boiled in aqueous solution. Suitable temperatures for casein in the process according to the invention are as low as 50 ^° C.

The method according to the invention is of particular importance in food production where the acceptability of the Polysaccharides is essential. However, its advantages are not limited to this area. To the food, the properties of which can be influenced by using the method according to the invention generally include Aqueous fat emulsions which contain protein and which contain or may contain a dispersed gas phase. The invention is particularly applicable to such systems.

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Whipped cream and ice cream are examples of such aqueous fat emulsions which contain a dispersed gas phase. It is known that the quality of the end product, namely emulsions containing dispersed gas phase, can be influenced in a number of ways. For example, stabilizers can be added to prevent the coalescence of i'ett and to inhibit the growth of ice crystals in ice cream. Long chain polysaccharides are examples of such stabilizers. Emulsifiers can also be added. It was believed that these merely help by facilitating the emulsification of the i'ettes in the aqueous phase. However, it is now believed that their influence wesentlichster consists of at least some emulsifiers ·, the lumps of fat ^f J? Röpfchen to promote, this chunk of i'ettes leads to coalesce during or after breaking the emulsion. Although stabilizers may be present to an excessive coalescence to prevent from .Fett a particular i ^v IASS of such coalescing considered for good product properties as required.

It has been found that the application of the invention has a surprisingly strong effect on the stability of such aqueous i'ette emulsions that contain protein and a gas phase are exercised can be, especially if the gas phase after the Hitζebehnung, z. B. pasteurization, the protein-containing Emulsion was introduced. The protein, e.g. B. casein, can be made to aggregate and then precipitate. The aggregated or precipitated casein destabilizes the Jfett droplets by clumping together and finally ' are brought to coalesce. In this way the stability of the system is strongly influenced, especially if

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a gas is introduced. The amount of polysaccharide mixture, which is used according to the invention depends on numerous factors as described above. At a aqueous fat emulsion, it depends in particular on the desired stability properties. For example, is with ice cream an increase in the stability of the dispersed gas phase present when small amounts of the polysaccharide mixture are added as a result of an increased destabilization of the fat, but if more of the polysaccharide mixture is added and increased fat destabilization occurs, the stability of the product decreases.

1,6-glucosidases and their action on starch, starch fractions or other polysaccharides are described in a series of publications, e.g. U.S. Patents 3,532,602, 3,535,123, 3,556,942, 3,790,446, British Patents 1,313,422, 1,268,443, 1,313,421 and German Offenlegungsschrift 1,916,726. Suitable 1,6-glucosidases , especially if they are freed from excess amounts of other saccharidases, for use in the preparation of the polysaccharides used in the process according to the invention can be isolated from numerous sources including plants and in particular the following microorganisms (the publications indicate the isolation method): Aerobacter aerogenes U- 58, ATCC 9621 and ATCC 15050 (U.S. Patent 3,654,088), Cytophaga NCIB 9497 (British Patent 1,048,887 and U.S. Patent 3,790,446) and Pseudomonas amyloderamosa ATCC 21262, Escherichia intermedia ATCC 21073, Streptomyces diastatochromogenes Ii ¹ O 3337 , Actinomyces globisporus IiO 12208, Nocardia asteroides IFO 3384, Micromonospora melanospora IFO 12515 and Thermonospora viridis IFO 12207 (b ritical patent specification 1,313,422).

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A preferred source of alpha-1,6-glucosidase is Cytophaga NCIB 94-97. This is a species of the species Cytophaga manufactured by Glaxo Ltd. in the National Collection of Industrial Bacteria, Aberdeen, Scotland, was deposited under the number 94-97. She is in the British Patent 1,04-8,887 and U.S. Patent 3,530,738, and the production of an alpha-1,6-glucosidase This is referred to in US Pat. No. 3,790,446, An advantage of using Cytophaga NCIB 94-97 for the production of alpha-1,6-glucosidase lies in that this is formed practically free of other enzymes that act on amylopectin or amylopectin fragments. on in this way, amylopectin and maltodextrins with a low DE value can with negligible further degradation or concurrent degradation of any amylose or fractions of amylose present are freed from branches. For the sake of clarity. it should be noted that this freed of branches Amylopectin, which is used in the method according to the invention, is almost always produced from amylopectin which contains a small amount of amylose. However, the amylose only acts as a diluent. To a The highest possible yield of polyglucoside containing 20 to 80 glucose units contains, the amount of amylose should be as low as possible. Waxy corn starch is a preferred one Starting material as it contains a high proportion of amylopectin. They work with maltodextrins with a low DE value amylose present and the amylose fractions present also only as diluents. It is believed that Maltodextrins with a low DE value are made by liquefying a starch that is high in amylopectin has, as well as by subsequent hydrolysis under

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Use of amylase to get the desired DE value. As well as always the manufacturing method is l'ialtodextrins are with low DE value, however, products which are known per se and which are readily available commercially.

Another benefit of using Cytophaga JNCIB 94-97 is that it allows for the direct production of the desired saccharide mixture without isolation of the alpha-1,6-glucosidase can be used. This can be achieved by Cytophage NGlB 9 ^ -97 using amylopectin or a Low DE maltodextrin as the primary source of saccharide or even cultivated as the sole source of saccharide.

Alpha-1,6-ctlucosidases differ with regard to their Specificity. For example, pullulanase fialto sy I can be blunt Remove (short side chains.) of degraded amylopectin; the alpha-1,6-glucosidase produced by Oytophaga WCIB 9 ^ -97 cannot do this. As long as the enzyme or the enzyme system has alpha-1,6-glucosidase activity, i. H. Amylopectin or fialtodextrin with low DE of branches exempt and no other hydrolytic effect on amylopectin or fialtodextrin with low DE-Vert or the Owns products, it can be used. Differences in specificity can lead to weak differences in the End products. For example, some polyglucosides with stumps of Maltosyl units are expected after branching off with alpha-1,6-glucosidase from Cytophaga NClB 94-97. However, the presence of small amounts of such materials does not significantly affect the usefulness of the products.

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The invention is illustrated by the following examples' explained:

example 1

50 g Haltodextrin having a DE value of 12 was dissolved in 1 acetate buffer, pH = 5> 5 (.100 milliliters of sodium acetate, adjusted to pH = _5? 5 mxt 50 mM acetic acid) was dissolved. To this, crude 1,6-glucosidase extracted from Cytophaga NCIB 9497 was added to form an enzyme: substrate ratio of 1:50. The mixture was incubated at 57 ⁰ ^ 24 hours, was completed by the removal of the branches, then the product was freeze-dried.

Input of I ^v ialtodextrin_without branching_in_Eiscreme 12-DE-containing odextrin freed from branches was added to ice cream in an amount of 0.5% for a partial replacement of the sucrose. The ice cream mix was pasteurized at 70 ° C for 20 minutes and processed in the normal way in the freezer. The typical ice cream approach used was:

Water 65.56%

Milk Solids 9.48 %

Palm oil 9.45%

Sucrose 14.58 %

Locust bean gum 0.18%

42-DE corn syrup 1.92 %

Emulsifier 0.50 ⁰ A

Flavor / color 0.03%

0.50% freed from branches

Maltodextrin

The results of standard examinations on-maintenance the £ orm and on melting were:

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Retention
* Melting ice cream *

typical ice cream 53% 10 ml / h

typical ice cream,
which freed from branches
Contains maltodextrin ( according to the invention 70% 0.5 ml / h)

*) the investigation was carried out as in the
Dutch patent application
73 17 072 described, carried out.

Examples 2 and 3
Production of amylopectin
5 g amylopectin was dissolved in 1 1 of acetate buffer, pH = 5i5> by heating at 90 ⁰ C for 10 minutes. The solution was cooled and the enzyme was added, as described for maltodextrin freed from branches, with an enzyme: substrate ratio of 1:50 being set. The branches were removed at 37 ° ^C. for 24 hours. The product was freeze dried.

Input of ^ altodextrin without branches (example 2) _ and input of amylogekton without branches i.5 ^e i ^s Pi ^e .i_.52_i2; Simple cream

12-DE-maltodextrin freed from branches, which had been prepared as in Example 1, was added to a single whipped cream (18% fat) as a freeze-dried solid in an amount of 0.2 % . Of branches liberated amylopectin, which had been prepared as described above was added in an amount of 0.05 ° / °. The cream was pasteurized at 60 ⁰ C for 10 minutes, cooled to ⁰ ^ 5 and pitched in einem.üenwood mixer at 5 C.

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Results ^

Control - 200 ^ν / Ό serve after 30 minutes

Impact duration reached

Cream + maltodextrin freed from branches - Example 2 -

- 200 % impact was reached after 5 minutes of impact

Cream + amylopectin freed from branches - Example 3 -

- 200 % serve was reached after 5 minutes of impact.

In the control sample, the longer whipping produced a Worsening of the serve, while in the two examples the products were stable.

Example 4

Results similar to those obtained in Example 3 were obtained if amylopectin "freed from branches." was used, which was made as follows:

A culture medium whose composition is given below, was inoculated with Cytophaga .NCIB 9497 incubated at 30 ⁰ C for four days. A high yield of unbranched amylopectin was obtained by centrifuging the cells from the Cytophaga and freeze-drying the supernatant fluid.

The culture medium had the following composition in% by weight:

Amylopectin * 1

N 0.5

0.1

₄ 7H ₂ O 0.02

NaCl 0.01

CaCl ₂ .6H ₂ O 0.01

MnSO _4- TH ₂ O 0.00225

(NH ₄ ) ₆ Μο _? 0 ₂₄ .4H ₂ O 0.00036

Zn acetate.2H ₂ O 0.00020

CuSO ₄ .5H ₂ O 5og826 / Ü971 0.00012

U. 6H ₂ O 0.002

ultrapure water

(which contained NaOH,

about the total pH

to 7.4) to 100

*) Snowflake Specialty Starch 30200 (B9101) by Corn Products Corp.

Examples 5 to 7

In Table I are the aggregation of casein and soy protein, measured by light scattering and induced by low DE-Vert and through-branched maltodextrin amylopectin deprived of branches is shown.

Example 5 used 1 % casein plus 0.5% de-branched amylopectin; Example 6 used 1% casein plus 0.5% de-branched low DE maltodextrin. Control A contained 1% casein.

Example 7 contained 1 % soy protein plus 0.5% de-branched 12-DE-maltodextrin. Control B consisted of 1 % soy protein.

Table I.

Tem- % light scattering

pera

door Ex. 5 Ex. 6 Control A Ex. 7 Control B (C)

25th 42 42 42 47 3ö 35 45 48 41 50 38 40 52 Ö5 40 53 39 60 75 80 38 68 40 80 68 * 78 * 37 81 42

*) Reduced light scattering at elevated temperatures, probably due to the formation of larger aggregates.

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Example 8

The following Table II shows the effect of the branched low DE maltodextrin on whey protein possesses.

Tabel II % Light scatter Heating time
(Minutes; to 75 ⁰ G 8 control 0
5
10
20th E.g. 44
43
45
50 44
75
79
85

Example 8 consisted of 0.5% whey protein plus 0.5 de-branched 12-DE haltodextrin. The control consisted of 0.5% wheat protein.

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Claims (1)

Claims 1. A method for aggregating a protein dissolved in water, characterized in that the aggregation is induced by a dissolved polysaccharide mixture which has at least 2% by weight of linear polyglucosides which contain J> 0 to 5i> glucose units. 2. The method according to claim 1, characterized in that the polysaccharide mixture contains at least 10% linear polyglucosides. 5. The method according to claim 1 or 2, characterized in that that the polysaccharide mixture has the same linear constituents as an amylopectin freed from branches or has a low DE-free from branching dextrin. 4. A method for aggregating a dissolved water in the protein, wherein the aggregation is induced by a liberated branches of amylopectin or a liberated of branches i ^v laltodextrin low DE value. J ?. Method according to claim 3 or 4, characterized in that that the branched amylopectin or that Low DE-freed Haltodextrin, an amylopectin or a low-DE Üaltodextrin .DE is determined using an enzyme or enzyme system with alpha-1,6-glucosidase activity of Branches has been freed. 509826/0971 2458U5 6. The method according to claim 5, characterized in that the enzyme or enzyme system with alpha-1,6-glucosidase activity is free of any other enzyme that has hydrolytic activity on amylopectin, maltodextrin with a low DE value or products that have been freed from branching. 7. The method according to claim 5 or 6, characterized in that that the enzyme or enzyme system was produced by Cytophaga NCIB 94-97. 8. The method according to claim 7 »characterized in that the. amylopectin freed from branches or the low DE from branched üaltodextrin Growing Cytophaga NClB 9 ^ 97 using amylopectin or low DE naltodextrin as the primary or sole source of saccharide has been. 9. The method according to any one of the preceding claims, characterized in that the protein is casein, and that a Heating is used to aid induction of aggregation. 10. The method according to any one of the preceding claims, characterized characterized in that sufficient polysaccharide mixture is used to prevent precipitation or flocculation of the Bring about caseins. 11. The method according to any one of the preceding claims, characterized characterized in that the aggregation of the protein is carried out in the presence of emulsified fat. 509826/0971 12. Aqueous oil emulsion, characterized in that it has been produced by the method of claim 11. 15. Aqueous i'ett emulsion according to claim 12, characterized in that that a gas phase has been dispersed in it. 509826/0971