WO1989011227A1

WO1989011227A1 - Inhibition of enzymatic browning

Info

Publication number: WO1989011227A1
Application number: PCT/US1989/002165
Authority: WO
Inventors: Petros S. Taoukis; Theodore P. Labuza; Shao-Wen Lin; Janet H. Lillemo
Original assignee: Regents Of The University Of Minnesota
Priority date: 1988-05-20
Filing date: 1989-05-18
Publication date: 1989-11-30

Abstract

A process for inhibiting oxidative darkening of foods by treating the food with a protease effective to inhibit oxidative darkening of the food. A composition for inhibiting oxidative darkening and a kit for preparing the composition.

Description

INHIBITION OF ENZYMATIC BROWNING BACKGROUND OF THE INVENTION 1. Field of the Invention The subject invention relates to extending the shelf life of foods, and more specifically the use of proteolytic enzymes to inhibit oxidative darkening or browning of plant tissues, fruit tissues, or shellfish.

2. Description of the Prior Art Enzymatic browning or oxidative darkening is a phenomenon which occurs in many types of foods. Such browning is desirable in certain types of foods such as apple juices, black tea, raisins, and dates. However, in a majority of fresh foods, such darkening is particularly objectionable to the consumer. Notable examples of foods subject to objectionable rapid browning include shrimp, lettuce, peaches, apples, and potatoes. Browning is most severe when the natural structure of the food is subjected to disorganization such as results from cutting, peeling, comminuting, pureeing, pitting, pulping, freezing, etc. Browning is generally accompanied by a change in other Organoleptic properties of the food product. By organoleptic properties is meant those properties of food capable of detection by human senses, especially appearance, and by other organoleptic properties is meant especially the taste and texture of the foods. Once such browning has occurred, the commercial value of the foods is dramatically diminished. Browning is also perceived by consumers to accompany a loss of nutritive value.

The general mechanism of browning has been understood for many years and is useful as a model in understanding known methods. Ortho-diphenol derivatives (o-diphenols) , also known as catechols, are catalytically oxidized by polyphenol oxidase (PPO) (monophenol, dihydroxy phenylalanine: oxygen reductase, E.C.I.14.18.10). PPO also was previously known as catecholoxidase. The products of the reaction are quinones. Quinones in turn polymerize to form dark, high molecular weight polymers. The molecular weight of the quinone polymers correlates with an increasingly brown product. The polymerization reactions are non-enzymatic secondary reactions.

PPO is present in high concentrations in foods which are particularly sensitive to oxidative browning, for example mushrooms, potatoes, apples, bananas, tea leaves, coffee, and peaches.

Many approaches to preventing or at least inhibiting oxidative browning are well known. By preventing is meant a complete elimination of browning. By inhibition is meant any significant slowing of the rate of browning up to and including preventing of browning. In one example, a variety of peach, the Freestone, has been developed which is unusually low in PPO. The variety has a better shelf life than traditional peach varieties. The improved shelf life is compatible with a model in which the rate of quinone production is severely limited by low PPO concentration.

Older methods include blanching of fruit. Blanching may be explained in terms of the model as effective because of thermal denaturation of PPO. However, since PPO is relatively heat stable enzyme. Thus with, apple, four to five minutes at boiling temperatures are generally required to inactivate the enzyme. Several disadvantages accompany the blanching method. For example, blanching is relatively energy intensive, destructive of vitamins, and tends to alter the microstructural properties of the food product being treated.

Another method is altering the pH of the food product. The effectiveness of pH alteration may be explained in terms of the model as effectively reducing enzyme activity, thereby inhibiting production of quinones. Common agents employed to alter the pH are citric acid, malic acid, or phosphoric acid, or buffer solutions of these acids.

Altering the pH entails certain disadvantages which limit the usefulness of the method. For example, pH levels of about pH 3 (a pH at which PPO is relatively inactive) are generally incompatible with maintenance of organoleptic properties, especially taste and texture, of many foods. Similarly, increasing the pH to approximately pH 8-9, through the use of phosphate buffers, also tends to alter the organoleptic properties of many foods.

Another approach is the use of chelating agents, such as EDTA, which may be explained in terms of the model as functioning by effectively "tying up" essential metal ions necessary for enzymatic activity. Alternatively, reducing agents, such as ascorbic acid and salts of ascorbic acid, may be used to remove oxygen. Oxygen removal may be explained in terms of the model as eliminating the oxidant.

Another approach is to enzymatically alter the o-phenols by methylation. Success of enzymatic methylation is compatible with a model in which the substrate for PPO has been eliminated. (See Finkle, 197 Nature 902-903 (1963); U.S. Patent 3,126,287.) Substrate elimination may also be achieved by a ring-cleaving enzyme, such as protocatechuate 3,4 dioxygenase. See Kelly, 20 F. Fd. Agric. 629-32 (1969).

Sulfite salts have been used in the food and beverage industry for centuries. For example, sulfur dioxide was employed to disinfect wine vessels since

Roman times. Sulfite salts are highly effective in controlling enzymatic browning of foods. Additionally, sulfite salts serve as bleaches, and antimicrobials. Based mainly on such a longstanding history of use, sulfites were considered safe additives for food. Recently, however, sulfite salts have been implicated as initiators of dangerous asthmatic reactions in a small subset of the asthmatic population, estimated at between 180,000-720,000 individuals in the United States.

In many food uses, the fate of sulfites is such that ^* most sulfite is oxidized to harmless sulfate. One major exception to the general rule, however, is lettuce which has been treated with sulfite. In the case of lettuce, the majority of sulfite remains as free, inorganic sulfite. Thus many asthmatic reactions which have been reported are attributed to ingestion of sulfite treated lettuce. A recent study, "Status Summary: Sulfites as Food Ingredients," Food Technology June 1986, estimated the level of sulfites in salad bar lettuce as high as between 400 to 950 ppm. Dried fruits, excluding raisins and prunes, had levels of approximately 1200 ppm, dried potatoes had levels of from 35 to 90 ppm. Fresh shrimp had levels up to approximately 40 ppm. frozen potatoes approximately 20 ppm and fresh mushrooms approximately 13 ppm. An acceptable daily intake for adults has been estimated at approximately 42 mg. of S0₂ per day. However, some sulfite-sensitive individuals have tolerance thresholds as low as 3 mg. A single 100 gram portion of lettuce treated with sulfite might include from 40-95 mg. of sulfite.

SUMMARY OF THE INVENTION The present invention includes a process of inhibiting oxidative darkening of foods by treating of the food with a protease which is effective to inhibit oxidative darkening of the food. The present invention also includes a kit comprising a soluble protease effective to prevent oxidative browning and a container for storing the soluble protease prior to use.

DESCRIPTION OF THE INVENTION We have surprisingly discovered that enzymatic oxidative darkening can be inhibited by treatment of a food with a protease. By treatment is meant contacting the food or a disorganized surface of the food with the protease or a solution of the protease by any application method including dusting, mixing, sprinkling, dipping, spraying, immersing or soaking, etc. The mechanism, while not yet proven, is believed to be hydrolysis and thereby permanent inactivation of the food enzymes, for example, PPO, responsible for oxidative darkening. Some of the proteases effective to prevent oxidative darkening are well known in the food industry. One effective protease, papain, has been used as a meat tenderizer for many years. However, the use of papain or other proteases to inhibit or prevent oxidative browning has not been previously reported.

The invention includes the use of a protease to inhibit oxidative darkening. By enzymes is meant proteins which catalyze chemical reactions. PPO is one example of an enzyme. Proteases are a subgroup of enzymes which catalyze hydrolysis of proteins into peptides and/or amino acid residues. It is generally believed that hydrolysis of an enzyme will reduce or eliminate the catalytic activity of the enzyme.

It is believed that an effective protease of the invention acts to hydrolyze and therefore inactivate the enzyme or enzymes responsible for enzymatic browning. Presumably, polyphenol oxidase (PPO) or similar enzymes in fruit, vegetables, and shellfish is responsible for enzymatic browning and is the target of protease treatment in the method. Effectively, inactivation of PPO prevents or inhibits the oxidation of O-diphenols to quinone compounds. Since quinones are the precursors of high molecular weight polymers responsible for darkening of foods, completely eliminating the enzyme activity of PPO is a highly effective method to prevent enzymatic oxidative browning. In theory, any protease which will hydrolyze and thereby inactive PPO will inhibit the browning reaction. However, in order for the treated food to remain a wholesome product, it is preferred that the protease applied to the food be safe for subsequent human consumption. Thus, enzymes commonly available for use in the food industry are particularly preferred. One well known enzyme, papain, is currently considered as "generally recognized as safe" (GRAS) by the FDA. (See 21 C.F.R. § 184.1585.) Additionally, other proteases which may be isolated from common foods are believed useful. Ficin, isolated from fig, and actinidin (E.C.3.4.22.14) isolated from kiwi fruit, represent two examples.

Papain and ficin have been found to inhibit enzymatic browning of apples and potatoes, respectively. Ficin also inhibits or prevents black spot formation in shrimp. The results obtained are comparable to the inhibition of browning provided by sulfite treatment. Not all proteases, however, are capable of satisfactorily inhibiting browning. For instance, only when apple and potato are stored at 24^βC can bacterial protease exhibit satisfactory inhibitory results. Bromelain cannot inhibit the browning effect except in the case of an apple stored at 4^βC. Fungal protease appeared to accelerate the browning of apples stored at 24'C. In general, testing for the effectiveness of a particular protease requires treating a desired food of solid, puree or liquid form by exposure to the dry protease or a solution of the protease. Instrumental measurement or visual observation of the treated food in comparison to an untreated sample reveals the effectiveness of the particular protease. Similarly, amounts or concentrations of the protease necessary for effective inhibition may be determined by comparison to an untreated sample of the food.

The upper limit of useful concentrations or amounts of protease with which a food is treated is believed to be controlled primarily by the market price of the protease. The lower limit is believed to be controlled by the economics of exposure times, since lower protease concentrations are generally believed to require longer exposure to be effective.

The invention is further explained by the following examples which are intended to aid under- standing rather than limit the invention. In Examples 1 and 2, solid foods are sliced or cut to provide surfaces with disorganized natural structures which are particularly susceptible to oxidative darkening.

Example 1; The Inhibition of Enzymatic Browning in Apple

The proteases used were obtained from Enzyme Development Corp., New York, NY 10121: specifically Enzeco Bromelain, Enzeco Neutral Bacterial Protease, Enzeco Purified Papain, Enzeco Ficin, and Enzeco Fungal Protease were used. All other chemicals used were AR grade.

The activities of the proteases used were approximately 100 Milk Clot Units per milligram for ficin, bromelain, and papain; 150 Northrup Units per gram for bacterial protease; and 100,000 H.U.T.

Hemoglobin Units per gram for fungal protease.

A series of 2 percent weight/volume enzyme solutions was prepared by mixing each enzyme with 125 ml 0.1 molar citric buffer (pH 4.5). The citric buffer and 0.5 percent NaHS0₃ solution (in the same buffer) were used as controls.

Apples (Red Delicious) were cored and cut, under water (at about 10*C) into about 5 mm thick pieces of about 4 cm diameter, and soaked for about 5 minutes in the different solutions. Duplicate samples for each treatment were put in Petri dishes and stored at 24^* or 4^βC in a desiccator saturated with water vapor so as to avoid surface dehydration of the samples. Untreated duplicate samples were stored similarly as a control.

The color changes of the samples were measured by Tristimulus reflectance colorimetry, using a Minolta Chroma eter CR-200, at the initial and subsequent time intervals.

Careful visual observation of the apple slices indicated that a normalized L value of 0.975 cannot be distinguished from the initial slice. Slight browning corresponds to a normalized L value of about 0.95 and is subjectively considered as the lowest acceptable result. An extremely brown apple slice has a normalized L value of 0.85. The normalized L values are presented in Table I, with Lo being an initial L measurement.

The normalized results demonstrate that papain treatment can prevent enzymatic browning of apple about as well as sulfite treatment at both room temperature (24'C) and refrigerated temperatures (4°C). In ' contrast, fungal protease actually accelerates browning of apple slices at room and refrigerated temperatures relative to untreated and citric acid buffered (pH 4.5) apple slices. Ficin shows a notable ability to inhibit enzymatic browning at a wide range of temperatures. Bacterial protease shows a limited ability to prevent enzymatic browning at room temperature within short time periods (for example, up to about 6 hours) . Bromelain was generally ineffective at room temperature. At refrigerated temperatures, bromelain treatment was somewhat effective at inhibiting enzymatic browning. TABLE I

Apple Pieces at 24^β< -~

L/Lo at various times

Tnsatment 3 Hr. 6 Hr. 10 Hr.

1. No Treatment 0.930 0.920 0.910

2. Citric Acid pH 4.5 0.930 0.920 0.910

3. Sulfite 0.990 0.990 0.970

4. Papain 0.990 0.980 0.980

5. Ficin 0.970 0.960 0.930

6. Bacterial Protease 0.970 0.960 0.960

7. Bromelain 0.940 0.930 0.920

8. Fungal Protease 0.930 0.900 0.860

Apple Pieces at 4^βC

L/Lo at various times

Tr<satment 10 Hr. 20 Hr. 30 Hr.

1. No Treatment 0.950 0.940 0.930

2. Citric Acid pH 4.5 0.950 0.940 0.940

3. Sulfite 0.996 0.995 0.985

4. Papain 0.990 0.990 0.985

5. Ficin 0.980 0.975 0.970

6. Bacterial Protease 0.960 0.950 0.950

7. Bromelain 0.970 0.970 0.970

8. Fungal Protease 0.960 0.940 0.920

Example 2: The Inhibition of Enzymatic

Browning in Potato

A procedure similar to Example 1 was employed to measure inhibition of browning in white baking potato slices. The pH of the citric buffer was 5.2 rather than pH 4.5 as in Example 1. As indicated in Table II, normalized L values of the potato samples were measured at various time intervals. Careful visual observation of potato slices indicated that, as with apple slices, a normalized L value of about 0.97 cannot be distinguished from the initial slice. Slight browning corresponds to a normalized L value of about 0.95 and is subjectively considered as the lowest acceptable result.

The results demonstrate that ficin is as effective or nearly as effective as sulfite treatment in inhibiting enzymatic browning of potatoes at room and refrigerated temperatures. Bacterial protease was also effective at room temperature. Papain showed a limited effect. Bromelain and fungal protease were generally ineffective in inhibiting browning.

TABLE II

Potato Pieces at 24^β C

L/Lo at various times

Tnsatment 3 Hr. 6 Hr. 10 Hr.

1. No Treatment 0.750 0.580 0.500

2. Citric Acid pH 5.2 0.920 0.820 0.750

3. Sulfite 1.000 0.990 0.990

4. Papain 0.990 0.940 0.900

5. Ficin 0.990 0.975 0.975

6. Bacterial Protease 1.000 0.980 0.970

7. Bromelain 0.930 0.900 0.770

8. Fungal Protease 0.950 0.900 0.790

Potatoι Pieces at 4*< C

L/Lo at various times

Tnsatment 20 Hr. 40 Hr. 60 Hr.

1. No Treatment 0.750 0.720 0.720

2. Citric Acid pH 5.2 0.940 0.890 0.860

3. Sulfite 0.990 0.990 0.990

4. Papain 0.980 0.960 0.940

5. Ficin 1.000 1.000 0.990

6. Bacterial Protease 0.960 0.940 0.900

7. Bromelain 0.970 0.940 0.890

8. Fungal Protease 0.970 0.930 0.850

Example 3: The Inhibition of Black Spot Formation on Shrimp by Ficin.

De-headed fresh shrimp, Florida Key Pink, were provided by the Singleton Seafood Company (Key West, FL 33040) and were certified to be free from any additives or chemical treatments by Singleton Seafood Company. The shrimp were treated in the solutions as described in Example 1, except for the pH of the buffer, which was 5.2 for shrimp. Untreated shrimp were also included as a control. The samples, in four replicates for each treatment, were put in Petri dishes and stored at 24°C, 4^βC, and under frozen condition (-25^βC). At the initial time and at subsequent desired time intervals, the samples were visually inspected for identification and enumeration of black spots. Subsequent to treatment, no new black spots were formed. Therefore, the ficin treatment effectively prevents black spot formation in shrimp. For example, the results of 4^βC storage presented in Table III demonstrate the effectiveness of ficin in preventing black spot formation.

TABLE III Shrimp After Storage at 4°C Percent Shrimp w/ Black Spots

Treatment 0 Hr. 24 Hr. 72 Hr.

1. No Treatment <. 2 38 68

2. Citric Acid pH 5.2 < 2 52 66

3. Sulfite <2 12 39

4. Ficin <2 <2 <2

The invention also includes a convenient kit for use in the food industry. The kit includes a container with an effective soluble protease. Within the container, the protease is protected from moisture and other environmental conditions which reduce protease activity during storage. When a food worker desires to prevent oxidative darkening of solid foods, the worker opens the container and mixes the protease with water or buffer. For liquid food or purees, the protease can alternatively be added in a dry form. Preferably, the kit also includes instructions on preparing the protease solution. Direct application of powdered enzyme to foods is also effective. The instructions suggest the most appropriate concentration and warn against frothing during mixing. Additionally, the instructions include appropriate methods of application and duration of treatment time to achieve an effective treatment of the food. Effective application methods include dipping, misting, dusting, mixing or spraying or any other method in which the food is contacted by an effective amount of the protease for a time period effctive to inhibit darkening.

The kit may also include a suitable dry buffer composition. When mixed with water, preferably tap water, the dry buffer composition dissolves to provide a pH buffer compatible with the food to be treated and compatible with protease activity. Because time and efficiency of preparation are highly important in the food industry, the inclusion of a buffer composition requiring only addition to a measured quantity of water is desirable. In a particularly preferred embodiment of the kit, the buffer composition and protease are packaged together. One problem with protease solutions is the tendency to self hydrolyze, thus often causing a rapid loss in activity. Therefore, the freshness of the solution is desirable in efficient use of the protease. The kit of this invention is particularly suited to preparation of fresh protease solutions and freshness can be emphasized in the accompanying instructions. The problem of self hydrolysis also indicates that the distribution of prepared solutions to the food industry is generally a less preferred embodiment of this invention. Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A process for inhibiting oxidative darkening in foods, comprising the step: treating the food with a protease effective to inhibit oxidative darkening of the food.

2. The process of claim 1, wherein the food is a solid food, a puree, or a liquid food.

3. The process of claim 1, wherein the food includes fruit.

4. The process of claim 3, wherein the fruit is apple.

5. The process of claim 1, wherein the food includes vegetable.

6. The process of claim 5, wherein the vegetable is a tuber.

7. The process of claim 6, wherein the tuber is a potato.

8. The process of claim 1, wherein the food includes shellfish.

9. The process of claim 1, wherein the shellfish is a shrimp.

10. The process of claim 1, wherein the protease is chosen from the group consisting of: papain, bromelein, actinidin, and ficin.

11. The process of claim 1, wherein the treating step includes applying an amount of protease, the amount effective to inhibit oxidative darkening.

12. The process of claim 11, wherein the protease is dissolved in a solution and the applying step includes: mixing, dipping, misting, sprinkling or spraying of the food with the solution.

13. The process of claim 11, wherein the protease is a powder and the applying step includes dusting the food with the powder, mixing the food with the powder, or dissolving the powder in the food.

14. A composition for use in inhibiting oxidative darkening of a food product by comprising a protease solution having a concentration of the protease permitting application of the protease to the food product in an amount effective to inhibit oxidative darkening of the food product.

15. The composition of claim 14, wherein the protease is chosen from the group consisting of: papain, bromelain, actinidin, and ficin.

16. The composition of claim 14, further comprising: a pH buffer compatible with the food product and the protease.

17. A food product treated with an effective amount of a protease effective to inhibit oxidative darkening.

18. A kit for preparing a protease solution effective to prevent oxidative darkening in foods, comprising: a soluble protease effective to prevent oxidative darkening in the food; and a container suitable for storage of the soluble protease prior to use.

19. The kit of claim 18, further comprising: a set of instructions for effective treatment of the food with the protease solution.

20. The^kit of claim 18, further comprising: a set of instructions for preparing the protease solution from the soluble protease.

21. The kit of claim 18, wherein the protease is papain and the food is apple.

22. The kit of claim 18, wherein the protease is ficin and the food is potato.

23. The kit of claim 28, wherein the protease is ficin and the food is shrimp.

24. The kit of claim 28, further comprising a suitable buffer composition.