Immobilized enzyme technology for debittering citrus fruit juices

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 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.

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 gl^-1) in the fermentation medium further increased naringinase production (8.9 IUml^-1) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.

Molecular characterization and enzymatic hydrolysis of flavanoid extracted from citrus waste

The citrus fruit processing industry generates substantial quantities of waste rich in phenolic substances, which is a valuable natural source of polyphenols (flavonoids) such as naringin and its disposal is becoming a major problem. In the US alone, the juice processing of oranges and grapefruit generates over 5 Mt of citrus waste every year. In the case of India, about 2.15 Mt of citrus peel out of 6.28 Mt of citrus fruits are produced yearly from citrus juice processing. In case of Australia, about 15-40% of citrus peel waste is generated by processing of citrus fruit (0.85 Mt). Thus Isolation of functional compounds (mostly flavanoids) and their further processing can be of interest to the food and pharmaceutical industry. This peel is rich in naringin and may be used for rhamnose production by utilizing α-L-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. We recently purified recombinant α-L-rhamnosidase from E. coli cells using immobilized metal-chelate affinity chromatography (IMAC) and used it for naringin hydrolysis. The purified enzyme established hydrolysis of naringin extracted from citrus peel and thus endorses its industrial applicability for producing rhamnose. Infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from citrus peel waste.

Hydrolysis of citrus peel waste with recombinant rhamnosidase for bioenergy generation

Large amounts of Citrus peel (rich in poly-phenolic compounds) are generated as a by-product of the juice processing industry. Development of alternative, higher valued products utilizing peel waste from grapefruit, oranges, Valencia and other citrus fruit would benefit citrus juice processors by providing them with means to profitably process their peel waste and to avoid environmentally hazardous dumping. Citrus peel waste [CPW, comprised of peel, membranes and juice vesicles] contains a high level of polyphenols and has been used for the production of animal feed, single-cell protein, fibre, enzyme(s), immobilization support & bio-sorbent for heavy metal removal. Naringin (a major tri-hydroxy flavonoid glycoside) is available in large amounts in citrus peel, processed juice and can be extracted from citrus peel waste1. The extracted naringin is further hydrolysed by rhamnosidase to produce D-rhamnose for the production of ethanol and other fermentation products. We have produced a recombinant enzyme2 that has the ability to catalyse the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. We have recovered important sugar “D-rhamnose” from the processed waste which would be utilized for ethanol production3. This presentation will summarize current efforts to develop an enzymatic treatment which would facilitate the economical processing of citrus waste for bioenergy generation.

Effects of trunk-shaker harvester and ethephon on plant water status, leaf gas exchange, and yield of citrus cultivated under Mediterranean conditions

This work was aimed to study whether the application of ethephon as an abscission agent and mechanical harvest using a trunk shaker have any effect on plant water status, leaf gas exchange, and yield of mandarin and orange trees cultivated under Mediterranean conditions. The experiment was performed from 2008 to 2011 in five commercial orchards where parameters related to the plant water status and leaf gas exchange were measured before the application of ethephon, at harvest time and at different occasions after harvest. In addition, the effects of ethephon dose on yield in the current and subsequent seasons were also evaluated. Results showed that ethephon applications and mechanical harvest did not detrimentally affect plant water status in any of the cultivars studied. Furthermore, either had no effect or had a short temporal decrease effect on leaf gas exchange depending on the cultivar studied although with no consequences for the fruit yield obtained during the current season. Increasing ethephon doses led to fruit yield reductions in the mandarin ‘Orogrande’ trees in subsequent seasons. When trunk-shaker and ethephon applications were combined, however, yields from the late-maturing orange significantly decline in subsequent seasons. Overall, results show that using a trunk shaker is a viable technique to mechanically harvest citrus trees destined to both fresh and industry market and can be considered as an alternative to the traditional manual harvest usually performed under Mediterranean conditions. However, its use cannot be recommended for late-maturing oranges, such as the ‘Navel Lane Late’ in which mature fruit and fruitlets coexist in the tree at the time of harvest.

Development of disease caused by Phytophthora cinnamomi in mature Xanthorrhoe australis

Over the past 30 years, heathland and open forest communities in south-eastern Australia dominated by Xanthorrhoea australis R.Br. have been severely affected by disease caused by Phytophthora cinnamomi Rands. The disease has caused a sharp decline in numbers of individuals within populations of X. australis; however, the etiology of the disease is unclear. The characteristics and disease symptoms induced by P. cinnamomi were analysed within nine mature X. australis plants that had been removed from the field. Seven plants showed typical disease symptoms that ranged from chlorotic leaves through to plant death. Plants showing disease symptoms had different numbers of infected roots, ranging from 0% in one dead plant, 40% infected roots in a plant showing yellowing of leaf tips and 67 and 86%, respectively, in two plants with severe chlorosis. There was variation within the roots, with some infected close to the stem while others were infected at more distal regions. Within stems of all plants, P. cinnamomi was difficult to isolate but was found in the desmium and stem apex and was associated with massive lesions within the central area of the stem. The symptoms of disease in X. australis are caused by a combination of damage to tissues of the roots and stem that may lead to a reduction in water and mineral transport throughout the plant.

Determination of synephrine in weight-loss products using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Adrenergic amines found in extracts of Citrus aurantium (bitter orange) evoke analytically useful chemiluminescence with acidic potassium permanganate in the presence of polyphosphates. From corrected chemiluminescence spectra, the wavelength of maximum intensity for these reactions was 680 ± 5 nm and, using flow injection analysis methodology, limits of detection for synephrine, octopamine, tyramine and hordenine were found to be between 1 × 10⁻⁹ and 1 × 10⁻⁸ M. We have applied this method of detection to the rapid determination of synephrine in dietary supplements using monolithic column chromatography.

Molecular identification of Staphylococcus xylosus MAK2, a new α-l-rhamnosidase producer

A bacterial strain, MAK-2, was isolated as a producer of α-l-rhamnosidase from a soil sample of Dehradoon, India. The strain was identified based on morphology, physiological tests and 16S rDNA analysis. The phylogenetic analysis based on the 16S rDNA sequence, identified the isolate as Staphylococcus xylosus, a nonpathogenic member of CNS (coagulase-negative staphylococci) family. The strain was capable of producing α-l-rhamnosidase by hydrolysing flavonoids thus confirming potential application in the citrus-processing industry.

Effect of Phytophthora cinnamomi on the habitat utilization of Antechinus stuartii in a Victorian forest

Phytophthora cinnamomi (Cinnamon fungus) is a pathogenic soil fungus which infects plant communities along the south-eastern coast of Australia, and the south-western corner of Western Australia. The symptoms of this disease include chlorosis, death of branches (ie. ‘dieback’), retarded growth and the eventual death of infected plants. This leads to devastating effects upon plant communities by altering both the structural and floristic characteristics of these communities. Small mammal species are dependent on specific features of their habitat such as vegetation structure and floristics. This thesis investigated alterations to the habitat of the insectivorous marsupial mouse, Antechinus stuartii, due to the presence of P. cinnamomi. The study was undertaken in an area of an open forest in the Brisbane Ranges, Victoria. Significant changes were found in both the floristic composition and structure of the vegetation at study sites infected with P, cinnamomi, compared to uninfected sites. The habitat utilization by A. stuartii of uninfected and infected vegetation was investigated using live trapping and radio-telemetric techniques. Capture rates were higher at sites uninfected by P. cinnamomi, and both male and females selected areas free from infection. Home range areas of males were significantly larger than those of females as assessed by telemetry. Both sexes spent a high proportion of time in areas dominated by Xanthorrhoea australis (Austral grass tree). There were significant relationships between the abundance of A. stuartii and the denseness of vegetation above 1 metre in height, and in particular, the proportion of cover afforded by X. australis. There were no significant differences in the cover of Eucalyptus spp. between uninfected and infected sites, but there were significantly more nest hollows in infected areas. The abundance of invertebrates was examined using pitfall traps. There were no significant differences in the abundance of the larger invertebrate taxa at infected and uninfected sites, but higher abundances of some micro-invertebrate groups in infected areas were recorded. The most likely factors considered to be influential in the habitat selection of A. stuartii were vegetation structure, and the presence of X. australis. To assess whether these factors were important the leaves of X. australis were removed with a brushcutter, to mimic the early effects of infection with P. cinnamomi. Animals did not respond to the alteration of vegetation structure in the short term (3-4 days). Longer-term experiments are required to assess the habitat utilization of A. stuartii at different periods following habitat manipulation. The implications of the presence of P. cinnamomi on the conservation of fauna are discussed. The destructive nature of the pathogen, and the slow rate of recovery from the disease, means that P. cinnamomi can be considered a threatening process to plant communities and the fauna that reside within that habitat. Future management of this disease within natural areas must therefore be cognisant of the potential of P. cinnamomi to significantly affect faunal as well as vegetative communities.

Fermentative production of rhamnosidase from Staphylococcus xylosus MAK 2 in a bioreactor for bio-energy generation

Increasing concern about the environment, food and feed shortages and hike in the price of petroleum have stimulated interest in new ways of producing biofuels. The interest is rapidly increasing towards converting agricultural wastes to commercially valuable products. Biofuels made from waste biomass can offer immediate and sustained greenhouse gas advantages. In this direction, we are focusing on Citrus processing waste, a byproduct of juice manufacture, which contains high amount of flavonoids and polysaccharides. There is a considerable industrial interest in the enzymatic transformation of flavonoids to hydrolysis products; that offers a pathway to bio-energy generation. Rhamnosidase of bacterial origin are very few and thus are potentially subject for research.

Staphylococcus xylosus, Gram positive cocci, a nonpathogenic member of CNS family, isolated from soil was used to produce α-L-rhamnosidase. This new strain, so far unknown for the production of α-L-Rhamnosidase, was identified and characterized as Staphyloccocus sp. through biochemical tests and 16S DNA sequence analysis. Effect of various medium and process parameters like pH, temperature, aeration and agitation rates and inducer concentration were studied. Further, the enzyme activity was enhanced by adding the inducer and divalent metal ion to the optimised fermentation medium. We have recovered important sugars “rhamnose” and “galacturonic acid” from the processed waste which would be utilized for ethanol production. This presentation will summarize current efforts to develop an enzymatic treatment which would facilitate the economical processing of citrus waste for bioenergy generation.

Immobilization of bacterial recombinant proteins

Recombinant α-L Rhamnosidase has several potential applications in citrus fruit juice processing industries. Immobilized recombinant α-L Rhamnosidase further provides an added advantage to this industrially important enzyme. Various techniques have been used to immobilize native rhamnosidase from fungal origin and applications were explored in great details by several workers. (Puri et al., 1996, 2000, 2001)

A recombinant rhamnosidase from a bacterial source was expressed in E.coli has been immobilized in calcium alginate beads (entrapment method). A batch bioreactor was created for the hydrolysis of naringin using immobilized recombinant α-L Rhamnosidase under shaking and stationary conditions and it was found to hydrolyze naringin effectively. The system was efficient to hydrolyze narigin under shaking conditions and was operationally stable up to 9 days. A high percent hydrolysis of naringin was achieved at pH 7.5 and 60˚C by immobilized rhamnosidase. Entrapped rhamnosidase was able to hydrolyze naringin content in kinnow juice repeatedly and this feature makes this technique economically suitable for debittering of fruit juices.