This is the published version Puri, Munish, Kaur, Aneet, Singh, Ram and Kanwar, Jagat 2008, Immobilized enzyme technology for debittering citrus fruit juices, in Food enzymes : application of new technologies, Transworld Research Network, Kerala, India, pp.91-103. Available from Deakin Research Online http://hdl.handle.net/10536/DRO/DU:30017028 Reproduced with the kind permission of the copyright owner Copyright: 2008, Transworld Research Network

Transworld Research Network 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India

Food Enzymes: Application of New Technologies, 2008: 91-103 ISBN: 978-81-7895-358-8 Editors: M. D. Busto and N. Ortega

Immobilized enzyme technology for debittering citrus fruit juices

Munish Puril, Aneet Kauri, Ram S. Singhl and Jagat R. Kanwa~ I Fermentation and Protein Biotechnology Lab., Department of Biotechnology Punjabi University, Patiala 147002, India; 21nstitute of Biotechnology, Deakin University, Victoria, Australia

Abstract There has been increased interest in the use of

immobilized enzymes in fruit juice industry for debittering of citrus fruit juices due to their high efficiency to remove bitter flavonoids. The structure of naringin, responsible for immediate bitterness, and of limonin, responsible for "delayed bitterness" has been discussed. This chapter also discusses various attempts that have been made to immobilize enzymes on an appropriate support so as to enable their use in

Correspondence/Reprint request: Dr. Munish Puri, Fermentation and Protein Biotechnology Lab., Department of Biotechnology, Punjabi University, Patiala 147002, India. E-mail: mpuri@pbLac.in

92 Munish Puri et al.

debittering of citrus fruit juices. These include physicochemical and enzyme biotechnological approaches which makes the fruit juice more acceptable and cost effective to the consumer. Despite of high volume of production of citrus fruits and fruit juices, suitable processes to produce non-bitter citrus juice by immobilized enzymes technology has not yet commercialized globally.

Abbreviations list CPG HPLC HEW LARL

: Controlled pore glass : High-performance liquid chromatography : Hen egg white : Limonoate-A-ring lactone

1. Introduction Citrus fruits are notable for their fragrance, vitamin C content and partly

due to flavonoids and limonoids contained in the rind, and most are juice­laden. The juice contains a high quantity of citric acid giving them their characteristic sharp flavour (1). The taxonomy of the genus is complex and the precise number of natural species is unclear, as many of the named species are clonally-propagated hybrids, and there is genetic evidence that even the wild, true-breeding species are of hybrid origin. Numerous natural and cultivated origin hybrids include commercially important fruit such as the orange, grapefruit, kinnow, tangerines, limes and some lemons (2).

Major commercial citrus growing areas include Southern China, the Mediterranean Basin (including Southern Spain), South Africa, Australia, the southernmost United States, parts of South America and Northern India. In the U.S., Florida, Texas, and California are major producers. United States of America are a very important market for both fresh citrus fruits and orange juice. Both Brazil and Florida dominate the world's juice market. Florida typically accounts for more than 90% of U.S. orange juice production (3). Global beverages brands like Coca-Cola and Pepsi-Cola have merged together with Tropicana, the most representative orange juice brand at the International level for major supplier of orange juice to the world (4).

During the recent years, consumer preferences are changing and they are becoming increasingly aware of the health and nutritive benefits of eating more fresh fruit and fruit juices. Citrus fruits are considered as one of the nutritious fruits because these are rich source of ~-carotene (vitamin A source), ascorbic acid (vitamin C) and folic acid. All these vitamins provide protection against fatal diseases like cancer and heart ailments (5). Many citrus fruits, such as oranges, tangerines, grapefruits, and clementines, are generally eaten fresh. Fresh citrus fruit juices are also very popular as breakfast beverage in

Immobilized enzyme technology for debittering 93

British, American and Continental breakfasts. More astringent citrus, such as lemons and limes are generally not eaten on their own. Lemonade or limeade are popular beverages prepared by diluting the juices of these fruits and adding sugar. A variety of flavours can be derived from different parts and treatments of citrus fruits. The fruit pulp can vary from sweet and tart to extremely sour. Citrus juices, rinds, or slices are used in a variety of mixed drinks. Commercially available citrus fruit products are juice, frozen citrus juice, squash, marmalade, cordial and concentrate, etc. However, fresh citrus juice is very common. Usually citrus juices are slightly bitter in taste but consumers are very sensitive to its bitterness. Hence the major thrust of research communities is to produce non-bitter citrus juice. The preservation of citrus juices is also very difficult because the citrus juice go highly bitter within few hours of extraction. Several efforts have been attempted to process citrus juice free from bitterness. A good amount of research in recent years has been done to develop the methods to de-bitter the citrus fruit juices and promoting debittering technology worldwide.

Most of this chapter deals with debittering of citrus fruit juices with which authors are most familiar. The processing of kinnow fruit, a hybrid of C. deliciosa and C. nobilis grown largely in Northern India, West Pakistan, Califronia and Arizona, USA is quiet identical in most ways to that of grapefruit.

Scientific mention of bitterness in literature was first made during 1857 in Java. Later two classes of chemical compounds namely flavonoids and limonoids were found responsible for bitterness in citrus juices. However, there is a difference between flavonoid and limonoid bitterness. The fruits containing high flavonoids are bitter even when consumed as fresh. The peel (rind) of the citrus fruit contains very high amount of flavonoids like naringin, neohesperidine, etc. making it highly bitter (6). Several other factors like storage temperature, acidic medium of the juice, etc. also playa vital role in the development of bitterness. This is known as "delayed bitterness" .

2. Bitterness in citrus juices It is experienced that bitterness of citrus fruit juice increased as a function

of duration of storage. This is due to naringin, the main bitter component of several citrus fruits (7). Naringin is responsible for "immediate" bitterness, and limonin is responsible for "delayed" bitterness in many citrus fruits (8).

2.1. Naringin Naringin (4, 5, 7-trihydroxyflavanone-7-rhamnoglucoside), the primary

bittering water-soluble component found in the fruit membrane and pulp (albedo) and which becomes extracted into fruit juices, is the 7-~-neohesperidoside

94

Naringilla<c C' .L.rbamnosidasc

{3-0-glucosidasc

Figure 1. Structure and putative hydrolysis of naringin.

Munish Puri et al.

of naringenin, the flavanone glucoside. Its concentration is a function of the degree of fruit maturity, with the least amount in ripened fruits. The typical concentrations of naringin in grapefruit juice are 400 mg/L (6). As the fruit matures, naringin is hydrolyzed by a-L-rhamnosidase to prunin and L­rhamnose. Prunin, which is 33 % less bitter as naringin, in tum is further hydrolyzed to naringenin and D-glucose with a further reduction in bitterness (Fig. 1).

Its hydrolysis with a concomitant decrease in bitterness is of industrial importance. Debittering can be achieved by treatment of the juice with an enzyme known as naringinase, which is an enzyme mixture containing both a-L-rhamnosidase (EC 3.2.1.40) and ~-D-glucosidase (EC 3.2.1.21) activities.

The presence of naringin (and hence the basis of bitterness) and the route of its degradation (and hence the mechanism of its debittering) in citrus fruit juices has been established through high-performance liquid chromatography (HPLC) analysis (9).

2.2. Limonin Limonin is the major limonoid found in most citrus fruit juices and is also

the major cause of delayed bitterness. It is a highly oxygenated triterpene derivative, comprised of furan ring and an epoxide group (10). It accounts for most of the delayed bitterness in grapefruit juice and Kinnow mandarin juice (7). The intact juice barely contains limonin; however, its nonbitter precursor, limonoate-A-ring lactone (LARL), is found to be present endogenously present in the cell cytoplasm, most probably at the neutral to slightly alkaline pH in membranous sacs (Fig. 2). As soon as these sacs get ruptured during the juice

Immobilized enzyme technology for debittering

o

y CH31 I I

Limonoare A ring la<:tone (LARL) (Nonhitter)

OH

Limol1oid ring Lactone hydrolase

Acidic pH

Limonill (Bitter)

Figure 2. Limonin structure and mechanism of delayed bitterness.

95

processing, the LARL encounters the net acidic pH of the juice, which gradually catalyses the closing of the ring to form limonin. Hence, the fresh citrus juice dose not taste bitter but turns highly bitter on storage (11).

3. Debittering of citrus fruit juices Bitterness in citrus fruits is one of the major problems in citrus fruit

juice industry and has a significant economic problem. To reduce bitterness in citrus juices below the threshold level for consumer acceptability, a number of physiochemical and enzymatic treatments have been devised. However, a majority of them are restricted to debittering grapefruit juice. Some of the physicochemical and enzyme biotechnological approaches have been described in this chapter to find out a solution to this problem.

3.1. Physicochemical approaches The fruit juice industry routinely removes phenols and flavonoids,

isoflavones and terpenes, from fruit juices by variety of debittering processes. Ever since the naringin and limonin discovered in grapefruit (c. paradise, Macf.) juice in 1965 and found to contribute significantly to its bitterness, a great deal of research efforts has been directed towards the understanding their anabolism, their role during the transition from raw to ripened fruit and their catabolism for improving juice palatability (12). These bitter components cause lesser acceptability of the finished products or commercially available fruit juices. Debittering improves the quality of the juices and concentrates and thus improving their commercial value.

Debittering performance can be enhanced by different factors such as adsorption technique (resins like polystyrene divinyl benzene, cyclodextrin polymer, Amberlite XAD-4), chemical treatments, blending with non-bitter

96 Munish Puri et al.

Table 1. Summary of physico-chemical processes to remove bitter phytonutrients from fruit juices.

Phytonutrient Typical compound Food Debittering process class Flavanones Naringin Grapefruit juice Adsoption to polymer

Naringenin Orange juice Passage through enzyme matrix

Flavones Tangeretin Passage through resins Nobiletin Passage through microbial

mass Sinensetin Accelerated ripining with

ethylene Triterpenes Limonin Use of cyclodextrin

. polymers

orange JUIce and sugars and even by enzymatic treatments using immobilized enzymes (Table 1).

Various degrees of debittering have been achieved with the uses of polyamides to selectively adsorption of significant quantities of limonin from the fruit juices (13). Passage through polystyrene DVB resins is also an effective way of debittering (14). Use of cyc10dextrin polymers is another very common process of debittering. Soluble ~- cyclodextrin has been used to reduce 58 % of the initial bitter taste of juice from grape fruit; Iyo Orange and C. natsudaidai (15), the reduced bitterness was due to the formation of an inclusion complex between k-cyclodextrin and naringin or limonin. Treatment of fruit juices with ethylene and even Carbon dioxide under pressure resulted in an average removal of naringin as well as limonin from orange juice (16).

Sekeroglu et at. (32), attempted immobilization of naringinase on celite by simple adsorption. The retained activity of celite-adsorbed naringinase was found to be 83 % at their optimum conditions. The activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60CC) for the hydrolysis of naringin.

Limitations The following are inherent limitations of the physico-chemical

techniques of debittering:

• These alter chemical composition of juices, either through chemical reactions or removal of nutrientslflavour/colour etc.

• These are non-specific in nature and therefore, inherently inefficient, introducing undesired or non-monitorable changes along with desirable ones.

Immobilized enzyme technology for debittering

e The chemicals used can not be recycled, thereby debittering and disposal of pollutants becomes cost prohibited.

• These methods were inherently inefficient or cumbersome, lacked reproducibility fetched less yields and partially lost desired nutrients in the process of removing bitter components.

97

These limitations of physiochemical approach lead to look for innovative techniques using enzyme biotechnological approaches to debitter the citrus fruit juices. With the successful commercial application of microbial biomass in fermentation processes and soluble/ immobilized enzymes, in the modification of starch/ penicillin, it appeared feasible and cost effective to develop comparable processes for fruit juice processing. For complete modification/degradation of debittering components, therefore several researchers have made concerted efforts to develop either soluble enzymes or microbial or immobilized enzyme capable of metabolizing the bitter components of citrus fruit juices.

3.2. Enzyme biotechnological approaches In later years different types of immobilized naringinase suitable for

industrial applications were used for debittering processes. The immobilization of enzymes used for debittering citrus fruit juices appears to offer several advantages in comparison with processes where soluble enzymes are used for clarification and debittering of fruit juices. Immobilization of biocatalysts have many advantages in large-scale processing, namely biocatalyst reuse, ease separation of biocatalyst from reaction media, continuous mode operation, prevention of contamination of the processed product, higher enzyme concentrations.

In order to control the naringin content in the citrus fruit juices naringinase has been immobilized on various insoluble carriers, such as hollow fiber devices, porous glass beads, DEAE-sephadex A-25, DEAE cellulose, tannin aminohexyl cellulose, on copolymers of styrene and maleic anhydride. However, besides the problem of enzyme stability, most of the carriers are expensive for industrial applications (17).

The naringinase of A. niger immobilized on copolymers of styrene and maleic anhydride has been used to hydrolyze naringin (18). In another case, naringinase immobilized on tannin-aminohexyl cellulose showed a half-life of 198 days at 25«: and 88 days at 37«: during debittering of Citrus natsudaidai juice (19). In the third case, naringinase immobilized on chitin with glutaraldehyde and sodium borohydride was inactivated during debittering of grapefruit juice (17). Glucose, fructose, and rhamnose were found to inhibit the enzyme, regardless of whether it was in the native or immobilized state. Both the source of the juice and the immobilization system used may affect the

98 Munish Puri et al.

stability of the enzyme preparation. In a hollow-fiber reactor for the hydrolysis of naringin in unclarified grapefruit juice, naringinase could be used for 4 h at 66 % efficiency (20). In further rigorous testing of this system, high levels (900 mg mel) of naringin in grapefruit juice were reduced to acceptably low values. The effects of operational parameters (flow rates, membrane surface area, temperature, and enzyme loading) on naringin hydrolysis to prunin and naringenin were reported (21).

Roitner et al. (22) immobilized the solid phase form of naringinase on controlled pore glass in an enzyme reactor for the conversion of naringin to prunin. The process was carried out in 0.1 M glycinelNaOH buffer at pH 12; 98 % conversion was achieved. The naringenin in the product stream was extracted with CHCh and the unreacted substrate in the aqueous phase was returned to the reactor. This was claimed to be the first problem-free process for the production of prunin.

The naringinase derived from Penicillium sp. has a high activity of a-L­rhamnosidase and a low activity of P-D-glucosidase. The enzyme immobilized on silicate and polysaccharides by using glutaraldehyde, is known to cleave anthraquinone and steroid glucoside in batch and continuous reactors. Immobilization of naringinase on silicate does not interfere with the analysis of the liberated L-rhamnose (23). Naringinase of Penicillium sp. when entrapped in cellulose triacetate fibers shows higher Km-values than in its soluble form (24). The fiber-entrapped enzyme has been used for hydrolyzing naringin in real and simulated grapefruit juice. This system was also used to simultaneously degrade limonin while hydrolyzing naringin (25). When grapefruit juice was debittered with this enzyme column, the sugar components, the total organic acids, and the turbidity levels remained unaltered. Moreover, the column could be regenerated by washing with warm water. It has afforded increased stability dUl1ng storage at 4CC without detectable leaching, wider temperature optima and easier recovelY for repeated operations. The covalent binding through glutaraldehyde increased thermal stability, continued recycling of naringinase without leaching and shorter (1 h) duration of debittering, have taken the cause of debittering of fruit juice a step further.

In another case, naringinase from Penicillium sp. was covalently linked to glycophase coated controlled pore glass (CPG) and used in debittering of fruit juice. The operational stability of this preparation was tested in a packed bed reactor. Theoretical models were used to predict the behavior of the system and good agreement with measured data were obtained (26).

According to one source (27), a system of co-immobilized amylase, polygalacturonase, and naringinase is superior for starch and pectin hydrolysis in fruit and vegetable juices relative to one in which the juice is treated with separately immobilized pectinase and amylase. In this work, the enzymes were co-immobilized by heating the enzymes and the silicate support suspension in

Immobilized enzyme technology for debittering 99

water, adding glutaraldehyde, and washing of the resulting solids. Caldini et al. (28), conducted kinetic and immobilization studies in fungal glucosidases for aroma enhancement in wine. The enzyme preparation of A. niger contained ~-glucosidase, a-arabinosidase, and a-rhamnosidase activities in a ratio considered suitable for aroma enhancement in wine making. The three activities were immobilized to a silianized bentonite solid carrier with glutaraldehyde with the aim of developing a continuous process for wine aroma enhancement. Puri et at. (29), worked on the use of alginate-entrapped naringinase for debittering of kinnow juice. Various alginate matrices were screened for the immobilization and a matrix obtained with 2 % sodium alginate was the optimal matrix system. A preparation that contained 30 units of naringinase hydrolyzed 82 % of initial naringin in 3 h. The immobilization broadened the pH optimum and improved the flexibility of the process for debittering juice of varying pH values. Immobilization also enhanced the thermal stability of the enzyme. The application of kinetic parameters optimized with pure naringin to kinnow juice resulted in 60 % debittering.

Immobilization of naringinase was also attempted by covalent binding on hen egg white beads (1 g HEW beads, 10 U of naringinase, 37CC, pH 4.0 and 48 h) through 1 % glutaraldehyde cross linking. Its leach proofing by glutaraldehyde cross linking and its application for debittering of Kinnow mandarin fruit juice was also investigated by our group. The efficiency of immobilization was 140 %, while soluble naringinase afforded 91 % efficacy for the hydrolysis of standard naringin under optimal conditions (5 U of HEW, pH 3.0, 60CC and 5 h). Its applicability for debittering Kinnow mandarin juice afforded 68 % debittering efficiency (19).

Later on to replace organic matrix with natural support, we explored the use of wood matrix for covalently immobilizing naringinase for debittering of kinnow juice (31). The wood chip matrix used was inexpensive and required one-step activation. The covalent binding of the enzyme to wood was through glutaraldehyde. The enzyme did not leach from the support. The system required a contact time of 5 h for debittering of the kinnow juice.

Recently, naringinase from Aspergillus niger, was immobilized into a polymeric matrix consisting of poly(vinyl alcohol) hydrogel, 95 % and 108 % of the added naringinase was actively entrapped in PV A cryogel. The entrapped naringinase was reused through six cycles retaining 36 % efficacy for the hydrolysis of naringin in simulated juice (33).

In another system, naringin hydrolysis was achieved by naringinase immobilized in k-carrageenan beads as a function of temperature (9-51 CC) and naringinase concentration (0.29-1.7 g L-1

) in grapefruit juice. The higher naringin conversion (95 %) was attained with naringinase concentrations higher than 800 mg L-1 and temperatures higher than 30°C. The results of this study were promising for a future application of enzymatic hydrolysis of naringin

100 Munish Puri et at.

immobilized in k-carrageenan, in grapefruit juice industry, maintaining juice properties (34).

Limitations On using immobilized cells/enzymes, certain limitations as listed below

needs immediate attention to provide suitable technology to citrus fruit processing industry.

• The operational stability of the immobilized naringinase was satisfactory for the naringin solution, but when it was used for debittering of fruit juice, the enzyme activity decreased in few cases. Entrapment of naringinase showed a number of disadvantages such as leakage, reduced half-life, diffusional limitations and delayed equilibrium attainment (17).

• Due to the use of unclarified juice, the permeability of limonin and naringin or their limited solubility under operational conditions seemed to be affected. Pretreatments of juices with pectinase may alleviate this drawback partially.

• Clogged columns, drop in pressure, etc. necessitated engineering inputs for rapid equilibrium and significant flow rates.

• Higher concentration of glutaraldehyde may pose toxic effects.

Advantages Immobilization of naringinase has made applicability of naringinase

possible in citrus fruit juice processing. The immobilization has afforded increased stability during storage without datable leaching, wider temperature optima and easier recovery for repeated operations. Mechanical strength through covalent binding, continuous recycling and shorter duration of debittering periods have taken the cause of debittering citrus juices a step further.

4. Conclusion From the foregoing survey of physicochemical/enzyme biotechnological

approaches, it is evident that currently there is neither a foolproof physicochemical nor a biotechnological approach available to debitter citrus fruit juices to palatable quality without affecting their natural characteristics. The comparative evaluation of available technologies on the basis of ease of operation, reproducibility, appetizing characteristics of the debittered juices, preservation of their nutritional characteristics, consumer acceptability, and economics of debittering shift the preference towards the development of enzyme biotechnological approaches vis-a.-vis traditional physico-chemical approaches so as to provide specific solution(s) to debittering citrus fruit juices. The immobilized enzyme leads to significant cost savings and can

Immobilized enzyme technology for debittering 101

easily be used in harsh juice processing conditions. It is anticipated that these techniques make fruit juice products and their respective beverage product more palatable and available to the consumer at reasonable prices.

Acknowledgment MP thanks Department of Biotechnology, Punjabi University Patiala, and

CSIR, New Delhi, India for providing financial assistance to carry out work on debittering of citrus fruit juices. AK gratefully acknowledges CSIR, New Delhi, for the award of a predoctoral fellowship from project grant CSIR 38(1133)/07/ EMR-1.

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