Report Type: Final Technical Report Project Title: Enhanced Preservation of Fruits in South Asia IDRC Project Number: 106931 Research Organizations: University of Guelph (UG), Guelph, Canada; TamilNadu Agricultural University (TNAU), Coimbatore, India; Industrial Technology Institute (ITI), Colombo, Sri Lanka Locations of the Study: Canada – Guelph, Vineland; India- State of Tamil Nadu; Sri Lanka- Colombo Authors: Dr. Jayasankar Subramanian, University of Guelph, Guelph, Canada Dr. K.S.Subramanian, TamilNadu Agricultural University, Coimbatore, India Dr. Shanthi Wilson Wijeratnam, Industrial Technology Institute, Colombo, Sri Lanka Period covered: Mar1, 2012 – Sep 30, 2014 Date of Submission: 1
Transcript
Report Type: Final Technical Report
Project Title: Enhanced Preservation of Fruits in South Asia
IDRC Project Number: 106931
Research Organizations: University of Guelph (UG), Guelph, Canada; TamilNadu Agricultural University (TNAU), Coimbatore, India; Industrial Technology Institute (ITI), Colombo, Sri Lanka
Locations of the Study: Canada – Guelph, Vineland; India- State of Tamil Nadu; Sri Lanka- Colombo
Authors: Dr. Jayasankar Subramanian, University of Guelph, Guelph, Canada Dr. K.S.Subramanian, TamilNadu Agricultural University, Coimbatore, India Dr. Shanthi Wilson Wijeratnam, Industrial Technology Institute, Colombo, Sri Lanka
Period covered: Mar1, 2012 – Sep 30, 2014 Date of Submission:
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Table of Contents
List of Acronyms 3
Executive Summary 5
The Research Problem 6
Progress towards milestones 8
Synthesis of research results 13
Synthesis of results towards AFS Outcomes 20
Problems and Challenges 22
Recommendations 22
List of Annexes 24
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List of acronyms used in the project report- CIFSRF 106931- April 2014
Acronym Expansion
BDO Business Development Officer
CIFSRF Canadian International Food Security Research Fund
EFF Enhanced Freshness Formulation: a spray mixture developed by UG scientists
Post harvest losses in fruits contribute to several billion dollars worldwide. Decreasing the post harvest losses can significantly improve the economic return for the farmers. A large proportion of women are directly or indirectly involved in the production and post production related activities (e.g. harvesting, sorting, packing, vending and accounting) in countries like India and Sri Lanka. Preventing these losses will ensure increased availability of nutrient-rich fruits and enhance the food security in these countries, increase their farm activities and fruit production, thus improving the overall economic returns and enhancing food security. Scientists at University of Guelph have identified that hexanal, a natural compound that is produced by plants, can help to extend post harvest shelf life of fruits. This IDRC-CIFSRF project involving University of Guelph, Canada, Tamil Nadu Agricultural University, India and Industrial Technology Institute, Sri Lanka aims to reduce the post harvest losses using a two pronged approach. In the first approach we extend the fruit retention time on trees for mangoes and other stone fruits using hexanal based pre-harvest sprays. Later the harvested fruits are subjected to a nanotechnology based hexanal smart delivery system (HSDS). Preliminary analyses of socio-economic returns reveal that, growers using this technology can earn an increase of INR 6400 per ha (~100USD) and this increase helps the women in these farm families to have more spending capacity and powers. Results from this project could potentially influence the development of policies towards the use of nanotechnology in agriculture, especially in South Asia.
1. Field sprays were taken up in five farmer’s field in India and also involving premium varieties as suggested by the Project Advisory Committee (PAC) for at least two seasons. 2% hexanal formulation exhibits significant fruit retention of up to 15 days longer and also extends the post harvest shelf-life by up to 21 days in mango. In peach, nectarine and strawberry post harvest storage improved by 7-10 days.
2. Extensive studies have been carried out in biosafety of hexanal on beneficial organisms, natural enemies, honey bees, earth worm, and human cell lines and the results confirm that hexanal is safe to spray in the orchards without any deleterious environmental or ecological effects.
3. Banana fiber has been confirmed as a material of choice for developing nano-particle based packaging material. Further, cellulosic matrices (cellulose is the major component in most fibers) also provide an additional base for retaining and releasing hexanal slowly, thus providing us an additional nano-particle option for hexanal delivery.
4. Procedures for the encapsulation in electrospun fibers and controlled release of hexanal were established and tested with peach, which shows reduced membrane damage even after 3 weeks. A US letter patent has been filed for this invention (UG).
5. Two local patents, tenable within Sri Lanka has been filed for ‘Processing technology of papers or boards from banana fibers for sorption and slow releasing applications’ and ‘Hexanal impregnated fiber polymer composite board and ITI wax modified with antimicrobial agents’.
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6. In TamilNadu, reduction of post-harvest losses resulted in additional availability of quality fruits in the market, additional income of about Rs. 5600 (equivalent to 100 USD) per ha per year. Additional income was mostly spent on nutrition, education and health related expenses.
7. Capacity building at partner institutes was achieved at several levels including specific trainings both researchers, growers as well as training of graduate students.
8. Trainings to 45 Producers Group led to production of value added products of mango like mango bars, pickle, ready-to-serve drinks etc. Involvement of women in decision making has been improved as a result of these training sessions.
2. The Research Problem:
India and Sri Lanka have the ability to produce a lot of fruits with India being the second largest producer of fruits in the world with 67 million tons. However, the availability of fruits is just 50% or less of the daily requirements in these countries. This is primarily due to post harvest loss of fruits. It has been estimated that the post-harvest loss of fruits is almost 40%. If proper packaging materials are designed and fabricated, the losses can be prevented and the per capita availability can be improved. This would ensure nutritional security and enhancement of rural livelihood. Storage of fruits poses a formidable challenge for the growers all over the world and is a major drain in the economy of developing countries. Existing storage methods are either narrow focused, expensive or have limited success and often not adaptable to the common fruit grower/packer.
For perishable commodities, optimal packaging, storage, and distribution technologies are critical as improper handling results in a significant loss. This is especially prevalent in countries like India and Sri Lanka where distribution and food preservation systems are lacking. Our baseline survey on the key mango producing areas of India and Sri Lanka involving small, medium and large mango growers and packers also confirmed that a narrow window of harvest and lack of post harvest storage and packing facilities are the major problem to be addressed. Based on these premises, the current project was taken up in the mango and tender fruit growing regions identified in the map below.
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Post harvest storage loss in fruits and vegetables can be reduced by controlling a key enzyme, phospholipase D (PLD), that causes the membranes to collapse thus accelerating the storage loss. The phospholipase D inhibition technology developed at the University of Guelph (Paliyath and Murr, 2007 US patents # 6,514,914, 7, 198,811, Indian Patent #223077) uses hexanal to prevent spoilage in fruits very effectively. Hexanal is a GRAS compound that has been approved by Food and Drug Administration (FDA) and is often used as a food additive. It is often produced by plants as a defense response to biotic stress factors including herbivory. Nanotechnology is an emerging science that is exploited in an array of fields, but mostly industrial. Application of nanotechnology in agriculture and food is very appropriate in the context of huge post harvest losses in South Asia, particularly in perishables such as fruits. As is the case with any new technology, there are fears when the term nanotechnology is used in the media, especially in the food stream. One of the concerns that will be posed by the public and fueled by the media is to safety of products like hexanal and the use of nanoparticles in fruits such as mango. It is imperative that such cutting edge scientific research need to be translated and communicated in a manner that is appropriate for the grass root farmers, which admittedly is a challenge to the researchers. To address this situation, NGOs such as –Mysore Resettlement Development Agency (MYRADA) in India and Vidatha in Sri Lanka who can take the work to the farming community were involved.
Together all these technologies have provided ample ammunition to enhance post-harvest shelf life of fruits, which needs to be tested at a larger, industrial scale. Locally these problems need to be addressed at the grower level which will start the ‘benefit processes’ through increased returns
Study areas in the project in Sri Lanka, TamilNadu (India) and Ontario (Canada)
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in income and globally, nutrition rich food material such as fruits will be available to the public, especially in areas where nutrition security is of paramount importance. Expanding this work to other countries or continents as well as other perishable but nutritious fruits (phase 2 of this project) will address the problem. Thus we anticipate this research to provide new knowledge and directions towards the use of nanotechnology in agriculture and also provide valuable insight to policy makers to allay any societal fears on the use of this new technology in food and agriculture.
3. Progress towards milestones:
3.1. Project Inception Workshop TNAU/UG) (Achieved): The inception workshop was held successfully in spite of a number of hiccups along the way between April 24 and 27, 2012. Due to the unexpected political situation in TamilNadu and the reluctance of the administration to permit Si Lankan scientists over safety and security fears, the ITI team, unfortunately, could not attend the ceremonial inauguration held at TNAU, Coimbatore. However, rest of the sessions went on as planned in Mysore and the team had a great interaction. Dr. Sara Ahmed of IDRC, New Delhi presented the audience the vision of the IDRC-CIFSRF projects and how these projects reach the end users. Project lead Dr. Jayasankar Subramanian of UG presented the proposed research and the use of nanotechnology to improve post harvest shelf life of fruits. Grower reps from various regions of TamilNadu spoke about their problems in post harvest handling of mango and explained how improvement in post harvest handling will help them. After the inaugural ceremony the team went to Mysore for internal discussions. April 25 was earmarked for planning the outlay of the project and making adjustments in the milestones based on the discussions. The whole team was split into smaller sub sections each with a lead member and the sub sections came out with a research plan, milestones and how those milestones will be achieved. This was done by both TNAU and ITI separately but with a collective, common goal. The team was ably helped by Drs. Sara Ahmed and Susan Robertson in the deliberations as well as inputs during the presentations. Ms. Reena Prasad of IDRC presented the budget planning and accounting aspects of the grant and stressed the need to keep track of the budget as well gave an overview of financial reporting, which is done annually for our project. On April 26 the whole team was divided into subgroups based on the six different fields [nanotechnology/material science, horticulture, plant pathology, post-harvest/food technology, socio-economics, biosafety] and the teams had their group meeting which was facilitated by IDRC personnel to make sure that all are having the same goal. After these discussions several mutually beneficial networks emerged. Since there was an overwhelming interest about hexanal and its uses, at the suggestion of Dr. Ahmed, an impromptu presentation was made by Dr. Gopi Paliyath which was followed by detailed discussions. On April 27, ITI and TNAU had their independent group meetings facilitated by UG members and then a collective meeting between the teams before departure. At the end it was generally agreed upon to facilitate training in nanotech department for 1-2 material science people from ITI, explore the possibility of a training session in genomics/ quantitative RealTime Polymerase Chain Reaction (q Rt-
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PCR) for the TNAU and ITI personnel involved by UG, Dr. Sekar from TNAU to help ITI people in formulating socio-economic studies and TNAU nano team to help analyze samples for ITI based on needs. The inception workshop was well covered in the local media including TV.
3.2. Recruitment of key personnel completed and project advisory committee formed
(TNAU/ITI/UG) (Achieved): All the students, post-docs and researchers that was enlisted in the proposal and amended during the inception workshop have been recruited in all three centers. A project advisory committee was also formed for the three centers and the committee met at Colombo as a whole and at the respective centers periodically.
3.3. Agreement on intellectual property (IP) issues, future plan (UG/TNAU/ITI) (Achieved):
University of Guelph took the lead on developing a mutually agreeable IP Memorandum of Understanding (MoU) which was executed by all three partnering institutions eventually..
3.4. Socio-economic analysis of participating growers (Achieved): A survey has been completed in
major mango growing regions of TamilNadu and based on the mango growing regions of the state, three regions have been identified viz., Krishnagiri, Theni and Kanyakumari which comprises of 70, 25 and 5% of mango growing respectively. From each region target growers have been identified for research trials. Similarly in Sri Lanka target farms [since no spray studies to be done there] and the varieties have also been identified and fruits have been procured from them for research studies at ITI. A total of 550 respondents from the three mango growing regions [400, 120 and 30] have been approached for the socio-economic analysis and baseline survey in TamilNadu. Among these 200 respondents have already been collected and the rest are in progress. The anticipated completion could not be accomplished as the survey itself was started only in June after recruitments of both respondents as well as associates to work on the project. Further the cropping period also coincided with the summer months and the geographical area of collection is fairly large to accomplish within three months. It is anticipated that the rest will be collected before the next report. In Sri Lanka 75 respondents were identified and the survey was completed.
3.5. Identification and quantification of nano-fibres resources required (TNAU) (Achieved):
Developing nanomaterials from coconut fibers met with some unexpected road blocks as the fiber was too elastic to grind to the desired nano-level. Any further hard grinding resulted in clumping which was detrimental for hexanal loading. However, banana fibers were made to desired sizes that were used for downstream studies. Interestingly varietal differences have been observed with the ultrastructure of these fibers. Fibers from Sri Lankan banana varieties have also been used for production of nanofibers. Hexanal incorporation into banana fibers has been done successfully by TNAU and the detailed results on retention and release are presented in the research results. Further, UG team has also developed procedures for the encapsulation in electrospun fibers and controlled release of hexanal.
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3.6. Development of protocols for pre-harvest sprays (TNAU/UG) (Achieved): A specific
formulation of hexanal dubbed as Enhanced Freshness Formulation [EFF] that contains 0.02% v/v hexanal - was demonstrated to both TNAU and ITI researchers. After studying the ripening pattern of mango, it is recommended that a spray approximately 30 days before harvest date [which varies with varieties and region] and a second spray 15 days before harvest were used. This protocol was successfully used during the project and works well against mango and tender fruits. Uncorroborated observations from the growers include – darkening of foliage in the sprayed mango trees at the end of the season], a significant reduction in certain pests [nut weevil] and diseases [anthracnose incidence], which is all good if sustained and true. The only negative observation that was brought to us was the lingering strong, but fruity smell, in the sprayed farms for 2-3 days after spray.
3.7. Training of MYRADA personnel and fruit growers completed and (TNAU) (Achieved): The
project coordinator for MYRADA, Mr. Vijay Prakash and field coordinators were trained on household data collection for the Execution of Baseline Survey and the survey was completed. Based on the survey results, intervention villages [42 target villages comprising 3 districts have been finalized] were identified. From these 42 villages, a total of 37 producer groups comprising 723 members [384 are women] have been formed. Growers participating in the trials have been trained on the spraying schedule and identification of the correct stage for each variety. A major problem encountered in this is the influence of ‘middle-men’ who indirectly control the small farmers.
3.8. Pre-harvest trials using hexanal formulation (Achieved): In India, sprays were taken up in 5
farms in the three major mango growing regions and two research stations. The trials involved 3 varieties -Neelum, Bangalora and Banganapalli- of commercial importance. The spray experiments and the results are detailed in Annex 4. In short, 2 sprays at 30 and 15d before harvest works well, results in extended fruit retention, more luster and shine to fruits, extended post harvest shelf life and reduced incidence of anthracnose. Growers are very happy with this spray. In Canada, 2% EFF spray was taken up on 1 peach variety in three locations [2 farms + 1 research station] and 2 peach and 2 nectarine varieties in the research station and three strawberry varieties as well. Results are similar to those observed in mango with fruit retention and firmness after storage being the most important criteria.
3.9. Evaluation of quality/storage parameters in the trail sites (Achieved): Both in India and
Canada fruits that were sprayed with hexanal formulations exhibited positive traits that are bound to translate as profits for the growers. The major parameters that consistently worked well is the retention of fruits for a longer time, increased fruit firmness, reduction in acidity in both mango (India) and tender fruits (Canada). Details of the data collected and analyses are furnished in Annex 5. Overall
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these results suggest the benefits of using hexanal containing EFF spray that should translate into increase in profit margins for the growers.
3.10. First annual project review meeting and report [ITI/UG] (Achieved): The first
review/monitoring meeting was held at Colombo, Sri Lanka between January 28-Feb 2 at the Galle Face hotel. The meeting was attended by researchers from all institutes, members of Project Advisory Committee (PAC) and BDOs from all three institutes. The meeting was preceded by a Stake holders meeting organized by ITI which was presided by the acting High Commissioner of Canada, HE Mr. R. McDougall. There were four talks presented by members of the team for the stakeholders followed by some interesting discussions. A report on the meeting highlights were submitted in the 12 month report.
3.11. Agreement on intellectual property (IP) issues, future plan (UG/TNAU/ITI) (Achieved):
University of Guelph took the lead on developing a mutually agreeable IP Memorandum of Understanding (MoU), which was eventually executed by all three partners.
3.12. Toxicology evaluation of hexanal on various products (TNAU/UG) (Achieved):
Biosafety of hexanal was studied extensively at various tropic levels to address the reviewers’ remarks. Safety tests were done against beneficial microorganisms (bacteria and fungi), natural enemies (predators and parasites), honey bees, earthworms and human cell lines. Overall results of this work confirmed that hexanal at the concentrations recommended (or even above the recommended dose) is safe to all these tropics and thus to the environment and ecology. A graduate student (female) who worked on this project completed her M.Tech. degree in TNAU. The detailed results will be used to bring out a Compendium, which could serve as a baseline document for policy makers.
3.13. Evaluation of IPR potential for new products that may emerge from this work
(UG/TNAU/ITI) (Achieved): Three areas emerged as having potential for IPR and progress has been made in securing these inventions. In Guelph, a US provisional patent entitled, “Methods to Encapsulate and Control Release of Volatile Organic Compounds for Increasing Shelf-Life of Perishable Products” (Mihindukulasuriya et al., 2014; US provisional patent # 61/927,650; Jan 15, 2014) has been filed. This invention describes the use of nano-fibers loaded with hexanal that were derived through electro-spinning and can release hexanal at a steady state over longer period of time, thus helping the storage life of perishable fruits and vegetables. In Sri Lanka, 2 local patents have been filed for ‘Processing technology of papers or boards from banana fibers for sorption and slow releasing applications’ and ‘Hexanal impregnated fiber polymer composite board and ITI wax modified with antimicrobial agents.
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3.14. Initial studies on product development – prototype packaging with HSDS and modified wax formulation (TNAU/ITI) (Achieved*): In TNAU, a paper board matrix was developed by blending of agro-wastes. The paper board after hexanal was tested. The results indicated that 600 ppm for 12 hrs extends the shelf life of mango fruits (variety Banglora) by 7 and 18 days under ambient and cold storage conditions, respectively. In Sri Lanka, six new bio waxes were developed and tested along with the field spray treatments. The hexanal and EFF incorporated wax formulations were found to be less effective in controlling post harvest disease compared with the new ITI bio wax formulations C2, C5 and C6. Of these the C6 formulation was the most cost effective and has been selected for further commercial development. Details are provided in Annex 6.
3.15. Second annual project review meeting (Achieved): The second annual monitoring and
review meeting was held at Guelph in April 2014 and the project results were presented.
3.16. Socio-economic analyses of measurable increases in incomes of participating growers (Achieved): In TamilNadu, Reduction of post-harvest losses resulted in additional availability of quality fruits in the market, thereby generating an additional income of about Rs. 5600 (equivalent to 100 USD) ha-1 year -1. The hands-on trainings imparted to the women members of the 45 Producers Group led to production of value added products of mango like mango bars, pickle, Ready-to-serve etc. This activity resulted in additional household income generation of approximately 10%. The additional income is utilized for nutrition (purchase of nutrition rich and quality food stuffs), education (providing children high quality education in private schools) and health related expenses. Further, the involvement of women in decision making has been improved as a result of these training sessions. The details are provided in Annex 7.
3.17. Large scale production of HSDS packages with Industry partners (Not Achieved):
Looking back, obviously this is an ambitious milestone. At this stage we have the prototypes developed for hexanal delivery systems (TNAU) and hexanal fortified bio-wax and banana waste based cardboard dividers (ITI) and some are being tested. Although private industries are definitely interested in these technologies, industry needs much more data before going on commercial production scales and thus this milestone needs to be carried over to the scale up proposal.
3.18. Quality, biochemical and Sensory analyses of fruits treated with hexanal (Achieved): In
all the three centers and all crops worked on, fruits that were sprayed with hexanal formulations exhibited positive traits that are bound to translate as profits for the growers. The major parameters that consistently worked well is the retention of fruits for a longer time, increased fruit firmness, reduction in acidity in both mango (India, Sri Lanka) and tender fruits and strawberry (Canada; Annex 8) ; In addition, increase in sugar content, increase in carotenoids and increase in TSS were also observed in mango after treatment with EFF. Details of the data collected and analyses are furnished in
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Annex 5. Overall these results suggest the benefits of using hexanal containing EFF spray that should translate into increase in profit margins for the growers. It must be admitted that the sensory analyses could not done in a thoroughly scientific manner; however, ‘off-hands’ sensory evaluations with farmers and farm workers all point out to the treatments being better for the overall quality of the fruits.
4. Synthesis of research results:
4.1. The overall objective of the Project is to reduce post harvest losses of fruits, in particular mangoes. To meet this objective, two different studies –pre-harvest spraying of hexanal based formulations on the fruit trees before harvest or dipping the harvested fruits in hexanal based formulations-were taken up in both India and Canada [for peaches and strawberries] .
4.1.1. Progress and Results in India: Pre-harvest spray studies were conducted in multiple sites during three seasons. Hexanal as 2% EFF formulation helps in extending the harvesting window of mango fruits in the tree itself to the tune of 10-14 days. This directly contributes to overall post harvest storage of mangoes in a given season and is also very helpful to small farmers as they normally do not have storage facilities. In addition, hexanal treated mango trees remained green, retained fruit firmness and exhibited lesser incidence of diseases. Further, there was also significant improvement in quality parameters such as reduction in acidity, increase in sugar and carotenoid content. Based on the comments from PAC during the first monitoring and review meeting, EFF spray was extended to a premium variety (Himampasand) as well and the results were consistent with earlier observations.. Further our studies also show that it results on an average an increase of ~INR5600 (~USD100) per ha in the returns, which is substantial. Unexpectedly, hexanal treated mango trees remained green, retained fruit firmness besides lesser incidence of diseases. Growers who are involved the project is extremely happy with the observations so far and several unintended benefits (e.g. more presence of beneficial insects, lesser weevil incidence to name a few) are also being reported by them. However, these observations need to be scientifically confirmed, which is beyond our aim at this stage and perhaps will be determined during the second phase. The laboratory studies have shown that the spores and mycelia of anthracnose causing fungus (Colletotrichum gloeosporioides) and stem end rot fungus (Lasiodiplodia theobromae) are definitely affected, indicating a potential ‘green fungicide’ role for hexanal. Further it was also confirmed that hexanal is not intervening with other beneficial bacteria such as Pseudomonas fluorescens (Pf1) and Bacillus subtilis (EPCO-16). Details of the work done on the post harvest diseases are provided in Annex 9. Detailed assessment on biosaftey has been carried out and the results of this work are provided in Annex 10. The data on the impact of hexanal spray on natural enemies and honey bees has shown absolutely no observable ill-effects. The work carried out on the bio-safety of hexanal will be compiled separately in due course and provided as a baseline document for policy makers when hexanal will be taken up at commercial operations.
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Overall, the results have shown that the hexanal formulation has a definite effect on extension of shelf life of mangoes.
Table 1. Shelf life of Mango fruits in response to hexanal as 2% EFF spray (Days)
Varieties Control 2% hexanal spray Increased in Shelf life (Days)*
(* Increased in days as compared to control fruits under ambient conditions)
4.1.2. Progress and Results in Canada: Pre harvest spray of hexanal formulation was done on three peach, two nectarines and one strawberry cultivar in Vineland and Niagara region for two seasons. In general fruit ripening was slightly delayed in the trees/bushes that received the treatment. Both peach and nectarine retained the fruits much longer in the sprayed trees than the unsprayed controls (Fig.1). Pre-harvest sprayed fruits retain firmness much better than the controls (Fig.2). Post harvest dipping treatments on peach and nectarines revealed that the dip treated fruits retained firmness much higher than untreated fruits even after 4 weeks, as in pre-harvest spray treatments. However, the response of nectarines is much better than peaches for post-harvest dip treatment. Details on the post harvest dip treatments are provided in Annex 8. Hexanal treatment improved the firmness and retention of all three strawberry cultivars tested and the details are provided in Annex 11.
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4.1.3. Progress and Results in Sri Lanka: Further to clarifications on the hexanal formulation, the original ITI bio-wax is further studied for its suitability to be used as a Hexanal carrier along with other new formulations that have been developed during the period under review. Six new bio waxes were developed and tested along with the field spray treatments. Some of these wax formulations included the UoG, EFF formulation and .02% hexanal respectively. The most effective wax formulation was then selected for further testing based on results from respective storage trials. The quality parameters tested were, % weight loss, fruit firmness, brix, pH, acidity, color and sensory analysis. All tests were carried out in duplicate for both TJC and KK mango varieties. The hexanal and EFF incorporated wax formulations were found to be less effective in controlling post harvest disease and extending storage life of TJC and KK variety mangoes compared with the new ITI bio wax formulations C2, C5 and C6. The post harvest disease control observed in fruits treated with these waxes may be attributed to the respective cinnamon extracts incorporated into these waxes. Of these the C6 formulation was the most cost effective and may be selected for further commercial development.
4.2. Specific Objective 1. Develop hexanal impregnated nano fibre delivery systems for fruit using cellulosic waste (banana fibre or coconut coir in particular)
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Left: Effect of hexanal formulation (2%EFF) spray on retention of fruits in Left:Virtue- Control; Right
Fig.2. Firmness of Virtue peach (left) and Fantasia nectarine (right) after spraying with 2%EFF. Red –treated and Blue –control. X-axis Days after harvest; Y axis- Pressure in Newtons
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The first steps towards this objective are: i) to identify the correct stage of the fiber that can be used for impregnation, ii) characterization of the fiber using electron microscopy to ensure that the fiber has expected characteristics for absorbing, retaining and releasing the hexanal in a sustained manner and iii) actual impregnation of hexanal into the fibers. Studies were carried out with these sub-targets in mind and considerable progress has been made. A synopsis of the research results for this objective is furnished below.
As previously reported (first annual report) fibers from several indigenous crops were characterized for their ability to hold hexanal and it was narrowed down to coconut and bananas. Results from current research show that banana fibers are better than coconut as grinding coconut fiber to nano size is quite challenging. Further when they are sized down to nano particles, they have a tendency to agglomerate quickly. Interesting varietal differences have been observed in banana fiber as well (Annex 12). Studies on hexanal loading and characterization were conducted with banana and coconut fibers. The hexanal loaded in the fibres were extracted using methanol and the loading pattern was studied in Gas Chromatography-Mass Spectrometry (GC-MS), a sensitive technique that can accurately measure the amount of the compound we are loading/ testing. The data revealed that there is variability among banana fibres derived from different cultivars and banana fibers in general have the tendency to retain more hexanal than coconut fibers. Similarly hexanal loading is also affected by biopolymer choice and the retention and release of hexanal has been significantly improved. Further, these studies have also shown the temperature sensitive nature of hexanal and the capacity to retain hexanal at for much longer times at ambient [20C] and cold storage, as shown in Fig 3.
Several new basic studies were initiated in using the cellulose from banana for developing nanoparticles for hexanal delivery. Briefly, studies were undertaken to synthesis macro, micro and nano-fibers from two banana varieties. The fibers were extracted by mechanical coming process and
Fig.3. Hexanal release is sustained in a continuous but steady fashion even after 60 h when hexanal coated biopolymers are used as carrier.
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the resultant fiber is referred as “Macro”. The macro-fibers were dried in the hot air oven at 65oC for 8 hrs and dried fibers were ground for 5 minutes using Wiley mill. These fibers were sieved through 90 mesh sieve and the product is referred as micro-fibers (0.5-2.0 mm). The micro-fibers were further subjected to cryo-grinding to achieve the fibers in nano-dimension. We also extracted micro-fibers using chemical method. Macro-fibers were treated with 2% sodium hydroxide (fiber and liquid ratio 1:10) in an autoclave and kept under 20 lbs pressure for a further of 1 hr. The fibers were removed from the autoclave and were washed in water till it was free of alkali. The bleaching was repeated 6 times. The resultant fiber is referred as “micro-fibers”. After bleaching, fibers were treated with 11 % oxalic acid in an autoclave and the step was repeated 8 times. The nanofibrils were suspended in water and stirred with a mechanical stirrer @ 8000 rpm for about 4 hrs until the fibers are dispersed uniformly. The suspension was kept in oven @ 90o C till it was dry. The three forms of fibers have been characterized using electron microscopy and X-ray diffraction (XRD). The XRD pattern suggests that relative intensity has reduced from 67% to 37% for the macro and nano-fibers, respectively. A significant improvement has been made in encapsulating hexanal in a polymer based system and by using this system hexanal can be retained for much longer periods. Based on the extensive work done on these encapsulated polymers a US patent has been filed (Mihindukulasuriya, S. D. F., Lim, L.-T., Paliyath, G., Subramanian, J., & Sullivan, J. A. (2014).Methods to Encapsulate and Control Release of Volatile Organic Compounds for Increasing Shelf-Life of Perishable Products. US provisional 61/927,650).
4.3. Specific objective 2. Incorporate a hexanal smart delivery system into biodegradable packaging films for individual fruits
Hexanal impregnation and release pattern studies were carried out at as pure form or with additives viz., castor oil and silicon oil in cellulosic matrices at varied doses over a period of 12 hours time. The filter paper (cellulosic) and paper board matrices used to assess the release pattern of hexanal were characterized using sophisticated instruments to gain insight of hexanal release pattern (Fig. 4). Among the two matrices, hexanal release observed during 12 hours after loading was higher in filter paper than in paper board. Irrespective of the doses, hexanal mixed with the castor oil resulted in higher hexanal vapor load than silicon oil mix. The electro micrograph obtained from the SEM revealed that the microfibers were arranged randomly in both the matrices with an average size of 13.93 and 16.26 µm diameters in paper board and filter paper respectively while the latter exhibited micro pits of 3.67 µm diameter intermittently in between fibrillar structures. Additional tests suggested that the composition of matrix and hexanal did not interact with each other as the release was observed to be uniform in all the matrices at 0 hours after loading (HAL). However at 12 HAL, the vapor load was higher where castor oil mixed hexanal than silicon oil which was superior to pure hexanal.
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Paper board MatrixCellulosic Matrix
Fig.4. FT-IR Spectra of Cellulosic Matrices
Blue circles indicate the hexanal spectra
Overall, the investigation has demonstrated that hexanal mixed castor oil impregnated in cellulosic matrix can retain and release hexanal to attain effective vapor concentration in the confined package environment. The preliminary studies have revealed that the composition of hexanal and castor oil could be effective to regulate the volatilization of the hexanal in the confined environment using filter paper as matrix. Admittedly, this work did not progress as we expected mainly due to high volatile nature of hexanal which was a challenge for impregnation; however, we anticipate completing this work and translating into commercial stream in the phase 2.
4.4. Specific objective 3. Develop a hexanal smart delivery system using a bio wax developed at the Industrial Technology Institute, Sri Lanka.
In Sri Lanka, the original ITI bio-wax is further studied for its suitability to be used as a Hexanal carrier along with other new formulations that have been developed during the period under review. Reformulation into hexanal modified bio-wax will be followed thereafter. The optimum micro and nano emulsion conditions at which the encapsulated Hexanal express highest shelf life preservation will then be evaluated for the further investigations (Fig .5.).
Fig 3. Various biowax formulations for hexanal impregnations [left] and dividers prepared from banana fibers that will be impregnated with hexanal
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In order to achieve the above objective, ITI researchers proceeded to investigate the possibility
of incorporating the patented EFF formulation to their own patented bio-wax formulation. While the ITI wax was able to reduce moisture loss and lower respiration rate of mango, loss due to disease was not reduced by the original ITI formulation. EFF is reported to reduce disease and therefore if incorporated to the wax formulation, it would make the formulation more effective in minimizing loss. However, it was not possible to incorporate EFF directly to the ITI wax as the ingredients of the EFF formulation were not compatible with those of the ITI wax formulation. It was therefore necessary to develop a bio-wax formulation compatible with EFF ingredients (hexanal) in order to harness the beneficial (storage life extension) capabilities of hexanal. Six EFF and Hexanal compatible bio-wax formulations that had necessary characteristics for application onto fruits were selected for laboratory scale testing on two major commercial varieties- TJC and Karutha Kolumbaan (KK) respectively. The study entailed a series of 16 storage trials in total which included fruits from field spray treatments. Details of the trials are summarized and presented in Annex 6.
Two of the tested bio-wax formulations ( Formulation C5 and C6) reduced post harvest loss of mangoes stored over 14 days at 13°C and 85% RH. Marketability of fruits was increased by 20 percent when fruits were subjected to the above wax treatments. There was no statistically significant difference in loss reduction between the two formulations. The C6 formulation reduced incidence of disease probably due to the anti microbial properties of the cinnamon bark oil incorporated in to this formulation. However, C5 formulation, which had both EFF and cinnamon bark oil, showed both reduced incidence of disease and glossier fruits than was the case with C6. A Patent has been applied from the Sri Lankan National Intellectual Property Office (NIPO) on “Processing technology of edible wax formulation incorporated with natural anti-senescence and anti-microbial agents for postharvest preservation of Mango”. Scaled up trials will be done in phase 2 of the project to fine tune the formulations and assess cost effectiveness of the treatments prior to commercialization.
A novel polysaccharide based aqueous formula was developed to encapsulate hexanal into banana paper boards by a simple dip and dry treatment. Hexanal releasing patterns from the treated banana paper boards were studied over a time period of two weeks using Gas Chromatography. Laboratory trials showed that the 2% and 3% hexanal impregnated banana paper boards delayed ripening and reduce disease incidence in mangoes for 14 days.
In order to further reduce the cost, Hexanal was incorporated into fiber-polymer composite material. Storage trials were conducted with different Hexanal concentrations in fiber-polymer composite material (0.8%, 1.7%, 2.5%, 3.4%, 5%, and 6.7%). It was observed that the 2.5 % Hexanal incorporated board has the ability to delay the ripening. Development of banana paper board with smooth texture was completed. A Patent has been applied from the Sri Lankan National Intellectual
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Property Office (NIPO) on “Processing technology of papers/boards from banana fiber for sorption and slow release applications”. Improvements of flexibility & strength will be carried out during the next phase of the project to be used as a wrapper instead of plastic sleeve.
5. Synthesis of results towards AFS Outcomes: 5.1. New technologies and/or farming systems and practices. Results from the use of hexanal formulations as pre-harvest spray is getting popular among the growers in India and Canada and already measurable changes in income can be seen in spite of the small area that was sprayed. Fruit retention and slowing down of post harvest ripening is consistent and the growers express great interest in extending this technology. As a bonus, significant reduction in fungal diseases as well as increase in beneficial insect population is seen in sprayed farms. This innovative technology will definitely help to increase food production by reducing the post harvest losses. A US Provisional patent has been applied for the technology on developing hexanal loaded nanofibers using electrospinning as detailed in the report. Use of hexanal spray increased fruit retention time by up to 2 weeks in mango and 5-7 days in peach and nectarines. Fruits treated with hexanal as a pre-harvest spray or post harvest dip exhibited extended shelf life, fewer incidences of post harvest diseases and extended marketability. 5.2. Engagement of Canadian researchers with Southern researcher organizations. The use of hexanal to increase fruit retention and post harvest shelf life is a technology from University of Guelph that is being brought to developing countries through this project. Appropriate training and on developing the correct formulation for spray as well as dip treatment of mango was provided by UG scientists. Guidance on how to plan experiments so that the outcomes can be maximized was provided in small sub groups with UG scientists taking the lead in each group as appropriate. Researchers from both TNAU and ITI visited UG for advanced training and were also helped in translating the work into useful publications. UG scientists also offered their services to suggest appropriate research work for the graduate students who were hired in the project. UG also helped TNAU to form their first ever Research Ethics Board and from now on all the projects involving human subjects (not restricted to IDRC projects alone) need to be approved by their REB. UG also conducted a genomics workshop in TNAU which was attended by IDRC as well as non-IDRC researchers in TNAU and the feedback was great. 5.3. Research groups. The team in each country generally operates in small sub groups, based on the research involved. For instance in Canada- Production; post-harvest and material groups, though there is a bit of overlap. Our research is supported by tender fruit growers and one of the growers is in the PAC as well. In India six sub-groups are operating each with their own identified targets. These include- Production Horticulture, Nanoscience, Food science and engineering, Plant Pathology, Biosafety and Social studies. These research groups are ably supported by state mango growers’
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association as well as their regional chapters. The president of the association himself is a member of the PAC and provides valuable field based inputs from the growers’ perspective. In Sri Lanka, there is Post-harvest, materials and social studies group. 5.4. Food processing and storage. It is becoming clear that the results of this project will increase the availability of mango due to longer fruit retention and post harvest storage that will enable India and Sri Lanka to potentially increase their marketability including export markets, which in turn could increase food security. Further retention of fruits in the trees as a result of hexanal sprays will give growers a bit more control on their harvesting times and thus they can adjust it based on the market demands. 5.5. Risk-mitigation. Whenever a new chemical (hexanal in our case) or a new technology (nanofibers) is introduced there is always a concern of risk and questions on how to address it will arise. Our risk mitigation approaches include extensive biosafety studies using both hexanal as well as nanofibers. As detailed in the report, our studies on the biosafety of hexanal using cell cultures or honeybees have turned out every encouraging and no adverse effect has been observed. These results will help to fast track the use of hexanal in the field as a spray/dip formulation.
5.6. Access to resources. It is anticipated that the key resources such as hexanal formulation will be made available in the near future for farmers once the policy papers are progressed. Thereafter access to this technology will be available to everyone interested. Private entrepreneurs have already expressed interest in licensing such formulations.
5.7. Income generation. As noted earlier, an increase of INR 5600 (~100 CAD) per ha is being realized by the growers who experimented with the spray. When these fruits will be processed and packaged in a larger scale significant increases are expected. It is also worthy to note that most of the additional income is spent on women and children welfare by these families.
5.8. Policy options. A policy decision is likely with respect to the use of hexanal as a safe chemical for using in orchards, based on our research. It may be quicker in India and Sri Lanka than in Canada.
5.9. Gender. Although this project does not directly address these questions, several of the researchers including one PI involved in this project are women. Since this is a more application based research both genders can do the work equally. Extension of harvest periods lends to the availability of fruits for longer time which can translate to more sales at farmgate, which are often done by women in India and Sri Lanka. This can provide them better access to income as well control of the said income. Similarly extended harvest times will help the women folk engaged in harvesting and sorting to do less, but more profitable work on those areas reducing the drudgery on one time harvest of too many fruits
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and sorting them quickly. As noted earlier, additional income generated through the extended sales are mostly used for women and children welfare and nutrition needs.
5.10. Environment. Synthetic chemical sprays are often among the most conspicuous environmental pollutants and the high levels of pesticide use in modern agricultures aggravate the situation. In such a backdrop using hexanal that is produced by the plant and has been proven to be safe compound will be a refreshing entry with huge benefits both to the growers and the environment. In addition to its role in reducing post harvest losses through membrane stability, hexanal is also showing signs of fungicide activity in the studies conducted both in Asia and Canada in this project. Thus hexanal could double up its role in future contributing further to environmental sustainability. Use of nanoparticles is sometimes deemed as an additional pollutant; however, these particles are already there and we just started noticing about them. Further in this project we are using nanoparticles derived from plants and thus are biodegradable. To our knowledge we are the first group to use biodegradable nanoparticles and this should be a major contributor to environmental sustainability.
6. Problems and Challenges: The project was conducted smoothly except for things beyond anyone’s control such as weather in Canada or political unrest/volatility in TamilNadu. Since this project is largely field and crop based we are -like any farmer- heavily dependent on weather. We did have our issues as the spring came considerably early in 2012 and hence the whole peach crop was lost. Similarly mango is prone to a malaise called erratic bearing that is largely controlled by weather. These are things beyond anyone’s control. In spite of these uncertainties the project went well and we are happy where we are now and only hope to better these results in the years to come. One of the challenges is to find a window for annual review meetings, in between teaching and crop season and thus our project does not have the luxury of holding meetings like most other projects do. We have to operate within a short window and use that window effectively. The only delay is the large scale production of HSDS packaging which is a lofty goal and will be addressed in the scale up proposal. There were concerns raised in the media about the nanoparticles and their impact on health etc. based on the media coverage of this work. However, these were addressed effectively when we were asked to since we are using bio nanoparticles and they are largely contained. 7. Recommendations: Since the team faced some minor issues when it came to travel for meetings and such, we suggest that a small paragraph be added in the contract itself to depute the concerned Pi or researcher for other meetings where the team could and the project could benefit. For instance the reluctance of administration to send PI/researcher [Deserving ones only] from partner institutes [not in Canada
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though] for other unplanned meetings of significance was an issue. Such things can be at least partially addressed by incorporating it in the contract. Having annual technical report instead of 6 monthly reports, for project of such technical nature like ours is a welcome change as otherwise we [at least the PI] spend a large amount of time in the reporting itself. Although IDRC permits vertical/horizontal transfer of funds within the grant, partnering institutes had major issues with it as we had in the last year or so. Thus an emphasis to this in the central contract is suggested so even when the central administration/or head changes at these partnering institutes, a valid contract can help the respective PIs to enforce such changes in the budget.
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Annexes Annex 1: Monitoring AFS Expected Outcomes Annex 2: Digital File formats supported by IDRC library Annex 3: AFS Research Output and Title and Abstract page Annex 4: Pre-Harvest trials in Mango (India) Annex 5: Quality, biochemical and sensory analyses of fruits treated with hexanal Annex 6: Bio-wax prototype development Annex 7: Socio-economic studies in India Annex 8: Hexanal effects on strawberry quality Annex 9: Effect of hexanal on post-harvest fungi of mango Annex 10: Biosafety and toxicology studies on hexanal Annex 11: Effect of preharvest hexanal spray in strawberry Annex 12: Banana nano fiber development and quality analysis Annex 13: Acronyms used in the project Annex 14: Patents and publications from ITI Annex 15: Mango consumption pattern in rural Sri lanka Annex 16: TNAU activity report Mar 2014-Sep 2014 Annex 17: Dissemination Workshop-India -July 2014 Annex 18: Loss assessment in mango-Sri Lanka
