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.

Ultraviolet vision and mate choice in the guppy (Poecilia reticulata)

Ultraviolet (UV) vision is well documented for many species of vertebrates. UV cues are known to be used in foraging, navigation and in mate choice. We conducted a series of behavioral experiments to investigate the role of UV perception in mate choice in both female and male guppies (Poecilia reticulata). In our experiments the visual appearance of potential mates was manipulated using either UV transmitting (UV+) or UV blocking (UV-) filters. Female guppies significantly preferred UV+ males. Male guppies tended to prefer UV- females, but their preferences were marginally nonsignificant. Further experiments investigating the role of luminance, indicate that UV wavelengths are probably being used for color discrimination rather than for detecting differences in brightness. These experiments raise the possibility that UV is used in mate assessment in different ways by male and female guppies. This may reflect the different strategies that the two sexes have in order to maximize reproductive success. To our knowledge, these are the first data showing that UV is used by any fish species in mate selection.

Predicting the direction of ornament evolution in Trinidadian guppies (Poecilia reticulata)

Sexual selection is thought to be opposed by natural selection such that ornamental traits express a balance between these two antagonistic influences. Phenotypic variation among populations may indicate local shifts in this balance, or that different stable ‘solutions’ are possible, but testing these alternatives presents a major challenge. In the guppy (Poecilia reticulata), a small freshwater fish with male-limited ornamental coloration, these issues can be addressed by transplanting fish among sites of varying predation pressure, thus effectively manipulating the strength and nature of natural selection. Here, we contrast the evolutionary outcome of two such introductions conducted in the Trinidadian El Cedro and Aripo Rivers. We use sophisticated colour appraisal methods that account for full spectrum colour variation and which incorporate the very latest visual sensitivity data for guppies and their predators. Our data indicate that ornamentation evolved along different trajectories: whereas Aripo males evolved more numerous and/or larger orange, black and iridescent markings, El Cedro males only evolved more extensive and brighter iridescence. Examination of the El Cedro experiment also revealed little or no ornamental evolution at the control site over 29 years, which contrasts markedly with the rapid (approx. 2–3 years) changes reported for introduction populations. Finally, whole colour-pattern analysis suggested that the greatest visual difference between El Cedro introduction and control fish would be perceived by the two most salient viewers: guppies and the putatively dangerous predator Crenicichla alta. We discuss whether and how these evolutionary trajectories may result from founder effects, population-specific mate preferences and/or sensory drive.

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.

Geographic variation in allometry in the guppy (Poecilia reticulata)

Variation in static allometry, the power relationship between character size and body size among individuals at similar developmental stages, remains poorly understood. We tested whether predation or other ecological factors could affect static allometry by comparing the allometry between the caudal fin length and the body length in adult male guppies (Poecilia reticulata) among populations from different geographical areas, exposed to different predation pressures. Neither the allometric slopes nor the allometric elevations (intercept at constant slope) changed with predation pressure. However, populations from the Northern Range in Trinidad showed allometry with similar slopes but lower intercepts than populations from the Caroni and the Oropouche drainages. Because most of these populations are exposed to predation by the prawn Macrobrachium crenulatum, we speculated that the specific selection pressures exerted by this predator generated this change in relative caudal fin size, although effects of other environmental factors could not be ruled out. This study further suggests that the allometric elevation is more variable than the allometric slope.

The effects of familiarity and group size on mating preferences in the guppy, Poecilia reticulata

In recent years, it has become evident that frequency dependence in the attractiveness of a particular phenotype to mates can contribute to the maintenance of polymorphism. However, these preferences for rare and unfamiliar male phenotypes have only been demonstrated in small, controlled experiments. Here, we tested the preference for unfamiliar mates in groups of six to 96 individuals over 13 days, in the guppy (Poecilia reticulata). We observed individual behaviour in situ to test whether fish discriminate two unfamiliar individuals among many familiar ones. We found that unfamiliar males and females were preferred over the familiar fishes in all groups and that this effect decayed over time. Increasing group sizes and levels of sexual activity did not hamper the preference for unfamiliar mates, providing further support for the role of frequency dependent mate choice in the maintenance of trait polymorphism in natural populations.

Testing the interactive effects of carotenoids and polyunsaturated fatty acids on ejaculate traits in the guppy Poecilia reticulata (Pisces: Poeciliidae)

Using the polyandrous livebearing guppy Poecilia reticulata, this study revealed no main effects of carotenoids in the diet on ejaculate traits, but significant main effects of polyunsaturated fatty acids (PUFAs) on sperm viability and weak but significant interacting effects of both nutrients on sperm length. Collectively, these findings not only add evidence that PUFAs are critical determinants of sperm quality, but also provide tentative evidence that for some traits these effects may be moderated by carotenoid intake.

Dynamic social networks in guppies (Poecilia reticulata)

One of the main challenges in the study of social networks in vertebrates is to close the gap between group patterns and dynamics. Usually scan samples or transect data are recorded to provide information about social patterns of animals, but these techniques themselves do not shed much light on the underlying dynamics of such groups. Here we show an approach which captures the fission-fusion dynamics of a fish population in the wild and demonstrates how the gap between pattern and dynamics may be closed. Our analysis revealed that guppies have complex association patterns that are characterised by close strong connections between individuals of similar behavioural type. Intriguingly, the preference for particular social partners is not expressed in the length of associations but in their frequency. Finally, we show that the observed association preferences could have important consequences for transmission processes in animal social networks, thus moving the emphasis of network research from descriptive mechanistic studies to functional and predictive ones.

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.