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.

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.

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.

Updates on naringinase : structural and biotechnological aspects

Naringinases has attracted a great deal of attention in recent years due to its hydrolytic activities which include the production of rhamnose, and prunin and debittering of citrus fruit juices. While this enzyme is widely distributed in fungi, its production from bacterial sources is less commonly known. Fungal naringinase are very important as they are used industrially in large amounts and have been extensively studied during the past decade. In this article, production of bacterial naringinase and potential biotechnological applications are discussed. Bacterial rhamnosidases are exotype enzymes that hydrolyse terminal non-reducing α-l-rhamnosyl groups from α-l-rhamnose containing polysaccharides and glycosides. Structurally, they are classified into family 78 of glycoside hydrolases and characterized by the presence of Asp567 and Glu841 in their active site. Optimization of fermentation conditions and enzyme engineering will allow the development of improved rhamnosidases for advancing suggested industrial applications.

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.

Anaerobic treatment of juice and fruit-processing effluents

Effluents from the juice and fruit processing industries have high organic matter content. Discharge of these effluents without appropriate treatment would therefore have a negative impact on the environment. High organic contents and low contamination levels make such effluents suitable for biological treatment, especially anaerobic digestion. In the latter process, significant amounts of digester gas can be produced, turning a waste stream into a source of renewable energy that can be used for electricity and heat production, leading to financial benefits.This paper investigates the feasibility of anaerobic digestion and the gas generation potential of five different effluents from the carrot-juice, orange-juice and sultana processing industries. Benefits are assessed in terms of digester gas production and organic matter reduction. The results show that the specific gas production ranges between 665 and 860 m3 per tonne of effluent treated (as organic dry matter). Furthermore, nearly 100% of the organic matter is converted into gas in the case of the carrot- and orange-juice processing residues, while a 84.5% reduction of the organic matter was found to be achievable in the case of the sultana wastes. While these results are promising, further testing will be required to validate them in a larger scale.

Socio-economic inequalities in women`s fruit and vegetable intakes: a multilevel study of individual, social and environmental mediators

Objective: This study employed a multilevel design to test the contribution of individual, social and environmental factors to mediating socio-economic status (SES) inequalities in fruit and vegetable consumption among women. Design: A cross-sectional survey was linked with objective environmental data. Setting: A community sample involving 45 neighbourhoods. Subjects: In total, 1347 women from 45 neighbourhoods provided survey data on their SES (highest education level), nutrition knowledge, health considerations related to food purchasing, and social support for healthy eating. These data were linked with objective environmental data on the density of supermarkets and fruit and vegetable outlets in local neighbourhoods. Results: Multilevel modelling showed that individual and social factors partly mediated, but did not completely explain, SES variations in fruit and vegetable consumption. Store density did not mediate the relationship of SES with fruit or vegetable consumption. Conclusions: Nutrition promotion interventions should focus on enhancing nutrition knowledge and health considerations underlying food purchasing in order to promote healthy eating, particularly among those who are socio-economically disadvantaged. Further investigation is required to identify additional potential mediators of SES–diet relationships, particularly at the environmental level.

Fruit and vegetable consumption and stroke : meta-analysis of cohort studies

Background
Increased consumption of fruit and vegetables has been shown to be associated with a reduced risk of stroke in most epidemiological studies, although the extent of the association is uncertain. We quantitatively assessed the relation between fruit and vegetable intake and incidence of stroke in a meta-analysis of cohort studies.

Methods
We searched MEDLINE, EMBASE, the Cochrane Library, and bibliographies of retrieved articles. Studies were included if they reported relative risks and corresponding 95% CIs of stroke with respect to frequency of fruit and vegetable intake.

Findings
Eight studies, consisting of nine independent cohorts, met the inclusion criteria. These groups included 257 551 individuals (4917 stroke events) with an average follow-up of 13 years. Compared with individuals who had less than three servings of fruit and vegetables per day, the pooled relative risk of stroke was 0·89 (95% CI 0·83–0·97) for those with three to five servings per day, and 0·74 (0·69–0·79) for those with more than five servings per day. Subgroup analyses showed that fruit and vegetables had a significant protective effect on both ischaemic and haemorrhagic stroke.

Interpretation
Increased fruit and vegetable intake in the range commonly consumed is associated with a reduced risk of stroke. Our results provide strong support for the recommendations to consume more than five servings of fruit and vegetables per day, which is likely to cause a major reduction in strokes.

Increased consumption of fruit and vegetables is related to a reduced risk of coronary heart disease: meta-analysis of cohort studies

Increased consumption of fruit and vegetables has been shown to be associated with a reduced risk of coronary heart disease (CHD) in many epidemiological studies, however, the extent of the association is uncertain. We quantitatively assessed the relation between fruit and vegetable intake and incidence of CHD by carrying out a meta-analysis of cohort studies. Studies were included if they reported relative risks (RRs) and corresponding 95% confidence interval (CI) of CHD with respect to frequency of fruit and vegetable intake. Twelve studies, consisting of 13 independent cohorts, met the inclusion criteria. There were 278 459 individuals (9143 CHD events) with a median follow-up of 11 years. Compared with individuals who had less than 3 servings/day of fruit and vegetables, the pooled RR of CHD was 0.93 (95% CI: 0.86–1.00, P=0.06) for those with 3–5 servings/day and 0.83 (0.77–0.89, P<0.0001) for those with more than 5 servings/day. Subgroup analyses showed that both fruits and vegetables had a significant protective effect on CHD. Our meta-analysis of prospective cohort studies demonstrates that increased consumption of fruit and vegetables from less than 3 to more than 5 servings/day is related to a 17% reduction in CHD risk, whereas increased intake to 3–5 servings/day is associated with a smaller and borderline significant reduction in CHD risk. These results provide strong support for the recommendations to consume more than 5 servings/day of fruit and vegetables.

Fresh kids: the efficacy of a health promoting schools approach to increasing consumption of fruit and water in Australia

The Fresh Kids programme utilized the Health Promoting Schools (HPSs) framework to design a whole-of-school, multifaceted intervention targeting specific behaviours to promote healthy eating and reduce the risk factors associated with childhood obesity. The aim of the programme was to evaluate the effectiveness of the HPS framework to increase fruit and water consumption among primary school-aged children over a 2-year period. The study design was an interrupted time series. Four primary schools in the inner west of Melbourne, Australia, participated in the programme intervention. Baseline data were collected using a lunch box audit to assess the frequency of children with fresh fruit, water and sweet drinks, either brought from home or selected from canteen lunch orders. The lunch box audit was repeated periodically for up to 2 years following programme implementation to assess the sustainability of dietary changes. Across all participating schools, significant increases between 25 and 50% were observed in the proportion of children bringing fresh fruit. Similarly, all schools recorded increases between 15 and 60% in the proportion of students bringing filled water bottles to school and reductions between 8 and 38% in the proportion of children bringing sweet drinks. These significant changes in dietary patterns were sustained for up to 2 years following programme implementation. Targeting key nutrition behaviours and using the HPS framework is an effective and simple approach which could be readily implemented in similar childhood settings. Effective strategies include facilitating organizational change within the school; integrating curriculum activities; formalizing school policy and establishing project partnerships with local community nutrition and dietetic services.

Evaluation of brief dietary questions to estimate vegetable and fruit consumption - using serum carotenoids and red-cell folate

Objective To evaluate responses to self-administered brief questions regarding consumption of vegetables and fruit by comparison with blood levels of serum carotenoids and red-cell folate.

Design A cross-sectional study in which participants reported their usual intake of fruit and vegetables in servings per day, and serum levels of five carotenoids (α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and lycopene) and red-cell folate were measured. Serum carotenoid levels were determined by high-performance liquid chromatography, and red-cell folate by an automated immunoassay system.

Settings and subjects Between October and December 2000, a sample of 1598 adults aged 25 years and over, from six randomly selected urban centres in Queensland, Australia, were examined as part of a national study conducted to determine the prevalence of diabetes and associated cardiovascular risk factors.

Results Statistically significant (P<0.01) associations with vegetable and fruit intake (categorised into groups: ≤1 serving, 2–3 servings and ≥4 servings per day) were observed for α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and red-cell folate. The mean level of these carotenoids and of red-cell folate increased with increasing frequency of reported servings of vegetables and fruit, both before and after adjusting for potential confounding factors. A significant association with lycopene was observed only for vegetable intake before adjusting for confounders.

Conclusions These data indicate that brief questions may be a simple and valuable tool for monitoring vegetable and fruit intake in this population.

Properties of ectin isolated from Lawulu (Crysophylum roxbergi G Don) and development of jam and fruit leather using Lawulu and pineapple

Lawulu fruit (Crysophylum roxberghi GDon) possess nutritional, medicinal and functional properties. However, it is less consumed due to its  characteristic off flavour. The present study was carried out to investigate the potential of utilizing lawulu fruit for isolation of pectin and to develop jam and fruit leather. Products were evaluated based on physico-chemical and sensory properties.

Pectin isolated fromf irm ripe lawuluf ruit using 0.1 M hydrochloric acid  followed by 96% ethanol precipitation yielded 7. 3% pectin on wet weight basis and 26.1% on dry weight basis. The isolated pectin contained 0.74% ash, 0.02% acetyl content and 7.85% methoxyl content with equivalent weight 993.5. These values were comparable with commercial high methoxyl pectin. In addition, Iawulu pectin at 1.5% concentration formed a gel within 12-14 min in the presence of 68% sucrose and 0.5% citric acid.

Jam was prepared by using Iawulu-pineapple ratio as 1:2, 1:1 and 2:1 respectively. The gel strength of jam (65⁰ Brix and pH 3.1) at 0.35% commercial high methoxyl pectin was comparable with commercial mixed fruit jam. Sensory evaluation indicated a significant preference (p<.05) for jam containing lawulu-pineapple ratio of 1:2 and 1.1 respectively overthe ratio of 2:1. With increased lawulu percentage both yellowness and lightness of jam increased significantly (p<0.05).

Fruit leather was prepared by changing lawulu-pineapple ratio as 1:2, 1:1 and 2:1 respectively with 20% sucrose, 0.3% citric acid, 0.05% pectin and 100 ppm potassium metabisulphite followed by drying at 65±1⁰C for 12-14 h. Sensory evaluation data revealed that changes in lawulu-pineapple ratio had no significant effect on taste, texture and overall quality of fruit leather.   However, significant preference (p<0.05) for colour was observed with increasing lawulu percentage. Both yellowness (b' value) and lightness (L'value) of fruit leather were sign[icantly increased (p<0.05) with increasing lawulu percentage.