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Title:
METHOD AND AGENT FOR INHIBITING QUALITY-REDUCING PROCESSES IN FOOD PRODUCTS
Document Type and Number:
WIPO Patent Application WO/1998/016117
Kind Code:
A1
Abstract:
The present invention relates to a method for preventing or inhibiting quality-reducing processes in a food product, comprising: applying in a suitable manner casein and/or a hydrolysate and/or one or more fragments thereof as inhibitor of metal-containing enzymes during the preparation, processing or preserving of the food product. The metal-containing enzymes are for instance lipoxygenase, polyphenoloxidase or peroxidase.

Inventors:
Wichers, Harm Jacob (Breehoven 74, SN Bennekom, NL-6721, NL)
Blok, Robert Isaac Claus (Adriaen Beyerkade 172, XV Utrecht, NL-3515, NL)
Application Number:
PCT/NL1997/000570
Publication Date:
April 23, 1998
Filing Date:
October 14, 1997
Export Citation:
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Assignee:
INSTITUUT VOOR AGROTECHNOLOGISCH ONDERZOEK (ATO-DLO) (Bornsesteeg 59, PD Wageningen, NL-6708, NL)
Wichers, Harm Jacob (Breehoven 74, SN Bennekom, NL-6721, NL)
Blok, Robert Isaac Claus (Adriaen Beyerkade 172, XV Utrecht, NL-3515, NL)
International Classes:
A23B7/154; A23C15/20; A23L3/3526; (IPC1-7): A23B7/154; A23C15/20; A23J1/20; A23L3/3526
Domestic Patent References:
WO1995023516A1
Foreign References:
EP0522212A1
EP0191311A2
Other References:
VARDA KAHN: "Effect of proteins, protein hydrolyzates and amino acids on o-dihydroxyphenolase activity of polyphenol oxidase of mushroom, avocado, and banana" JOURNAL OF FOOD SCIENCE, vol. 50, no. 1, 1985, CHICAGO US, pages 111-115, XP002035431
Attorney, Agent or Firm:
Van Someren, Petronella Francisca Hendrika Maria (Arnold & Siedsma, Sweelinckplein 1, GK The Hague, NL-2517, NL)
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Claims:
CLAIMS
1. Method for preventing or inhibiting quality reducing processes in a food product, comprising of applying in suitable manner case in and/or a hydrolysate and/or one or more fragments thereof as inhibitor of metalcontaining enzymes during the preparation, processing or preserving of the food product.
2. Method as claimed in claim 1, characterized in that the metalcontaining enzyme is lipoxygenase, polyphenoloxidase or peroxidase.
3. Method as claimed in claim 1 or 2, characterized in that the hydrolysate is obtained by chemical and/or enzymatic hydrolysis of casein or a caseincontaining product.
4. Method as claimed in claim 1, 2 or 3, characterized in that the food product consists of vegetables, fruit, salads or dairy products.
5. Method as claimed in any of the claims 14, characterized in that the qualityreducing processes consists of the development of "off flavors", texture changes, browning or loss of nutritional value.
6. Casein and/or a hydrolysate and/or fragments thereof for use as agent for inhibiting or preventing qualityreducing processes in food products.
7. Agent for inhibiting or preventing quality reducing processes in food products, comprising casein and/or a hydrolysate and/or fragments thereof.
8. Food product to which casein and/or a hydrolysate and/or fragments thereof have been added.
Description:
METHOD AND AGENT FOR INHIBITING QUALITY-REDUCING PROCESSES IN FOOD PRODUCTS The present invention relates to a method for preventing or inhibiting quality-reducing processes in a food product.

Food products such as vegetables, fruit, salads and dairy products, such as butter, are rather quickly subject to quality reduction for instance due to the occurrence of so-called "off flavors", browning, loss of nutritional value or texture changes. The present consum- er is becoming increasingly critical and makes different demands than formerly in respect of aroma, color, taste, texture, appearance and storage life of particular food products. Whereas for instance green beans used to become relatively soft after processing (boiling or preserving), the consumer nowadays requires a product with more "bite". So-called non-natural additives, which were used regularly in the past, are nowadays also viewed with distrust. The demands made of particular food products and their processing are therefore subject to extensive changes. This also requires modifications in the produc- tion and processing of food products.

The changing production and processing of food products involves its own specific problems. Preserved green beans can again serve as an example. For a product with more "bite" shorter cooking times are required. The result however is that determined enzymes, which were once "boiled to death", now remain active, whereby in the case of for instance green beans so-called "off flavors" are formed. Such "off flavors" are undesirable, but are unavoidably linked to the shorter cooking time.

Another example is the browning of vegetables and fruit such as mushrooms, apples and lettuce. The changing lifestyle of consumers results in a demand for products which will keep increasingly longer but which also preserve an attractive appearance and retain their aroma and taste. Without some form of processing however, this cannot be achieved.

A known problem with fats and fat-containing products such as butter is that they rapidly become rancid. An adequate solution for this has still not been found.

The above mentioned problems have in common that they are caused wholly or partially by enzymes, and then generally by metal-containing enzymes. Known examples are lipoxygenase, which is responsible inter alia for the forming of "off flavors" and is involved in fats becoming rancid, and polyphenoloxidase, which plays a part in the browning. Peroxidases, which result in the formation of free radicals, can also cause quality reduction.

It is now the object of the present invention to provide a method and agent for inhibiting or preventing such processes, which can moreover be deemed "natural".

Surprisingly, it has now been found that casein is capable of inhibiting the activity of metal-containing enzymes.

The invention accordingly provides a method for preventing or inhibiting quality-reducing processes in a food product, comprising of applying in suitable manner case in and/or a hydrolysate and/or one or more fragments thereof as inhibitor of metal-containing enzymes during the preparation, processing or preserving of the food product.

In addition to casein, hydrolysates or isolated casein fragments can also be used. Casein is a relatively large protein. Hydrolysates can be prepared chemically or enzymatically. Chemical hydrolysates consist of casein cleaved in random manner, while in enzymatic hydrolysis casein is cleaved by proteases localized at specific sites. After hydrolysis the casein fragments formed in this manner can be mutually separated. The fragments with the most inhibiting activity can be used separately or in combination.

In vegetables and fruit the enzymes for inhibiting are usually situated in the cells. In the treatment of food products, which consist substantially of intact cells, complete casein will be able to penetrate much less easily into the cells to carry out its inhibiting action. Advantageously therefore, a hydrolysate or casein fragments are used in such cases.

Casein is a mixture of phosphoproteins, which occurs naturally in for instance milk and dairy products and is therefore a "natural" agent. There are four subtypes, namely aS1, aS2, and . All subtypes of casein are capable of inhibiting metal-containing enzymes, in particular lipoxygenase. Furthermore, casein is heat-stable, whereby the activity is not lost due to sterilization or pasteurization treatments.

The agent according to the invention therefore consists of pure casein, a hydrolysate, separate frag- ments or combinations of one or more of these three. The agent may optionally be a composition which comprises suitable excipients and/or additives in addition to the active ingredient. Such excipients or additives may for instance be required to improve membrane passage, for further optimization of the inhibiting process, an in- creased solubility of the agent or an improved emulsifi- cation of the agent.

The agent according to the invention can be added directly, for instance in dry form or in solution, to the food product and mixed therethrough, such as for instance in salads. In the case of preserves it can be added to the "brine" (the liquid in a jar or tin of (sterilized) product).

The invention is illustrated hereinbelow with reference to a number of examples. Lipoxygenase is used herein as model enzyme. The examples are not however intended to limit the invention thereto.

EXAMPLES EXAMPLE 1 In vitro inhibition of lipoxvqenase by casein or case in hydrolysate For this enzyme assay, wherein in vitro inhibition of the enzyme activity by casein (hydrolysates) is measured, use is made of soya bean Lipoxygenase type-l (optimum pH 9). The activity of the lipoxygenase is determined in oximetric manner. The speed of oxygen consumption is in linear relation to the enzyme activity.

Used as substrate for the enzyme is a solution of 0.032 M linoleic acid and 0.25% (v/v) Tween 20 in 0.05 M Na2B407 buffer (pH 9). Control measurements are performed by mixing 2.83 ml 0.05 M Na2B4O7 buffer (pH 9) and 150 jil substrate in a reaction cell at a temperature of 300C.

Subsequently added thereto is 20y1 0.2 mg/ml lipoxygenase in 0.05 M Na2B407 buffer (pH 9), whereafter the oxygen consumption is recorded using an oxygraph.

Inhibition measurements are performed by replacing a part of the Na2B407 buffer in the reaction cell with a solution of casein or a casein hydrolysate in the same buffer. Hydrolysates are produced as described in Example 2.

The table below shows the degree of inhibition of the enzyme after inhibition with different types of casein. Inhibition is shown in relation to a standard measurement without casein.

Type of casein Inhibition (W) casein (10 mg/ml) 37,9 casein (50 mg/ml) 56,4 dephosphorylated casein (10 mg/ml) 31,9 dephosphorylated a-casein 46,2 (10 mg/ml) -caseine (10 mg/ml) 43,9 dephosphorylated -casein 36,2 (10 mg/ml) K-casein (10 mg/ml) 57,9 Trypsin digest (10 mg/ml) 25,1 11 Chymosin digest (10 mg/ml) 25,8 I 11 Esprion 580 hydrolysate (50 mg/ml) 66,2 B5pr1nTM 300 hydrolysate (50 mg/ml) 49,9

Esprion is a hydrolysate of isolated whey protein As shown by the above data, the enzyme activity is clearly inhibited in reaction mixtures to which casein or a hydrolysate thereof is added.

EXAMPLE 2 Preparation of hydrolysates Hydrolysates of casein are prepared by dissolving 1.25 g casein at room temperature while stirring in 25 ml 100 mM Tris buffer, pH 7.8 with 10 mM CaCl2. 1% (w/w) trypsin and 0.5 ml 2% sodium azide is added thereto while stirring. After a chosen time period (for instance 24 hours) the reaction is stopped by precipitation with 25 ml 5% trichloroacetic acid (TCA).

After centrifugation the supernatant is dialyzed for several days against demineralized water and subsequently

suitable buffer. The degree of hydrolysis is determined using SDS-PAGE or HPLC.

A second possible method of performing the hydrolysis is by dissolving 1.25 g casein at room temperature while stirring in 25 ml 100 mM phosphate buffer pH 6.5. 1% (w/w) chymosin and 0.5 ml 2% sodium azide is added thereto while stirring. After a chosen time period (for instance 24 hours) the reaction is stopped by precipitation with 25 ml 5% trichloroacetic acid (TCA). After centrifugation the supernatant is dialyzed for several days against demineralized water and subsequently free-dried. The dry matter can then be dissolved in a suitable buffer. The degree of hydrolysis can be determined using SDS-PAGE or HPLC.

The hydrolysis can also be performed using other enzymes, such as for instance papain or clostrypain, in a buffer with a suitable pH. The described methods are only examples which can be modified depending on the situation.

If desired, the mixture can be separated into the separate fragments using for instance HPLC, FPLC, GPC or column chromatography. These can then be tested separately for their lipoxygenase-inhibiting activities.

EXAMPLE 3 Inhibit ion of quality-reducing processes in potato salad To portions of 50 g potato salad, prepared with mayonnaise, was added 1-10 g casein or casein hydrolysate per kilogram. Mayonnaise contains among other things linoleic acid, which is sensitive to the effect of lip- oxygenase. Potatoes are in this case the source of lip- oxygenase.

The salad was subsequently stored for 1 week at 40C. As control, potato salad without casein or hydrolysate was also stored under the same conditions.

At the end of the storage time the salad with casein was found to display much fewer taste deviations

than the salad without casein, which was clearly demonstrated by testing with a sensory panel.

EXAMPLE 4 Inhibition of quality-reducing processes in green beans Green beans (500 g/l water) were evacuated in water containing 30 g/l potassium caseinate. They were then steeped for 1 hour in water at 650C. This step is necessary to obtain a crisp bean but also activates the lipoxygenase present in the bean, whereby a rancid taste may occur. The green beans were subsequently placed in glass jars and sterilized for 25 minutes at 1180C. The jars were stored at 150C for 1 month. As control, green beans without any caseinate received a similar treatment.

At the end of the storage time it was found that the green beans which were treated with caseinate tasted less rancid than green beans without caseinate.

This was clearly demonstrated by testing with a sensory panel.