Reduction of Bioburden by Optimizing Gamma Dose for Enhancing Export Value of Pakistani Peaches

CONTENTS

List of figures

List of Tables

Abbreviations

Abstract

1 Introduction

2 Literature Review

3 Materials and Methods

4 Results

5 Discussions

References

LIST OF FIGURES

4.1 Impact of gamma irradiation on total bacterial count of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.2 Nutrient agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of one week

4.3 Nutrient agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of two weeks

4.4 Nutrient agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of three weeks

4.5 Impact of gamma irradiation on Gram negative Enterobacteriaceae count of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.6 MacConkey agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of one week

4.7 MacConkey agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of two weeks

4.8 MacConkey agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of three weeks

4.9 Impact of gamma irradiation on Salmonella-Shigella count of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.10 Salmonella-Shigella agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of one week

4.11 Salmonella-Shigella agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of two weeks

4.12 Salmonella-Shigella agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of three weeks

4.13 API 20E strip inoculated with Shigella sonnie before incubation

4.14 API 20E strip inoculated with Shigella sonnie after incubation and addition of reagents showing the positive and negative reactions

4.15 API 20E strip inoculated with non fermentor spp before incubation

4.16 API 20E strip inoculated with non fermentor spp after incubation and addition of reagents showing the positive and negative reactions

4.17 Impact of gamma irradiation on yeast and mold count of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.18 Potato dextrose agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of one week

4.19 Potato dextrose agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of two weeks

4.20 Potato dextrose agar plates showing the bacterial colonies isolated from the control and irradiated peaches kept at 4oC for period of three weeks

4.21 Microscopic examination of different yeast species isolated from peach surface

4.22 Impact of gamma irradiation on percentage decay of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.23 Percentage decay evaluation of control and irradiated peaches kept at 4oC for a period of two weeks

4.24 Percentage decay evaluation of control and irradiated peaches kept at 4oC for a period of two weeks

4.25 Percentage decay evaluation of control and irradiated peaches kept at 4oC for a period of three weeks

4.26 Impact of gamma irradiation on percentage weight loss of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

LIST OF TABLES

4.1 Impact of gamma irradiation on total bacterial count of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.2 Morphological and staining characteristics of bacterial colonies isolated on peach surface on nutrient agar during three weeks storage at 4oC

4.3 Impact of gamma irradiation on Gram negative Enterobacteriaceae count of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.4 Morphological characteristics of bacterial colonies isolated from peach surface on MacConkey agar during three weeks storage at 4oC

4.5 Impact of gamma irradiation on Salmonella-Shigella count of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.6 Morphological characteristics of bacterial colonies isolated from peach surface on Salmonella-Shigella agar during three weeks storage at 4oC

4.7 Colony morphology of Gram negative Enterobacteriaceae on unirradiated and radiated (0.25, 0.5 and 0.75kGy) samples for three weeks stored at 4oC

4.8 Identification of Enterobacteriaceae on the basis of test results of API 20E

4.9 Impact of gamma irradiation on yeast and mold count of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.10 Morphological characteristics of yeast and mold found growing on unirradiated (control) and irradiated (0.25, 0.5, 0.75kGy) peaches during three weeks of storage at 4oC

4.11 Impact of gamma irradiation on percentage decay of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.12 Impact of gamma irradiation on percentage weight loss of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

4.13 Impact of gamma irradiation on organoleptic properties of control and irradiated (0.25kGy, 0.5kGy and 0.75kGy) peaches kept at 4oC for three weeks

LIST OF ABBREVIATIONS

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ABSTRACT

Peach is the most commonly eaten stone fruit which grows in the temperate regions of the world. These are also among the most exported fruits in term of volume and value. Peaches, being perishable and susceptible to microbial spoilage, have a short shelf-life. The goal of this study was to determine the effect of gamma irradiation on the microbiological and organoleptic properties of peaches and secondly, to determine the effects of irradiation on shelf-life of the fruit. The fruit (local variety, No.4), at proper maturity stage was collected and irradiated with 0.25, 0.5, and 0.75kGy dose, stored under refrigerated (4oC) conditions for a period of three weeks. Total viable count, Gram negative Enterobacteriaceae count, Salmonella-Shigella count, yeast and mold count, loss in weight and decay percentage was evaluated after 7, 14 and 21 days of storage (4oC). Data obtained from microbiological and sensory evaluations of irradiated peaches was compared with the observations obtained from unirradiated (control) samples. Microbial evaluation of the fruits revealed the presence of Salmonella sonnie on SS agar and non fermentor spp. on macConkey agar. Ten types of yeasts were isolated on potato dextrose agar and analyzed microscopically. Studies showed total bacterial, yeast and molds significantly decreased with increasing dose level. The sensory evaluation of the fruits revealed that irradiation preserves the texture and appearance of fruit. The gamma-irradiation dose of 0.75kGy proved to be effective in reducing weight loss and significantly (p≤0.05) delaying the decaying of the fruit by 7 days under refrigerated (4oC) conditions. Statistical studies were carried out using Duncan’s multiple range test (DMRT) and means were separated by LSD at 5 %. It is hoped that this particular type of research will help in improving export quality of Pakistani peaches.

INTRODUCTION

Pakistan is the sixth most populous country, with an area covering 796,096 km². It is the 36th largest country in the world in terms of area. The climate of Pakistan varies from tropical to temperate conditions (UNIDO, 2010). There are four distinct seasons: a cool winter, a hot spring, the summer rainy season and the retreating monsoon period. Rainfall varies greatly from year to year. The diversity of climates in Pakistan allows a wide variety of trees and plants to flourish (Zaigham et al., 2009). The lush land makes agriculture as one of the four major drivers of growth. Pakistan produces 6,339,166 tons of fruits annually and this makes Pakistan to occupy 21st rank in the list of annual world food production. Area of Pakistan under fruits cultivation is 7,73,860 hectors and hence occupies 16th rank on the basis of area under fruit production (FAO, 2014). Main temperate fruits grown commercially in the country include apple, pear, peach, plum, cherry, strawberry and apricot. Horticulture is a vital agricultural sub-sector of Pakistan and performs an important role not only in refreshing the rural economy but also in enhancing food nutrition (UNIDO, 2010). The agriculture sector of Pakistan directly adds 21.4% to its GDP, provides 40% employment and 60% exports (MEFP, 2012). During 1990s, production of horticultural crop has increased from 11.3 million tons to 13.7 million tons. The share of fruits and vegetable shown by production analysis is 48.6% and 51.4% respectively (PHDEB, 2007). Horticulture industry has great potential in Pakistan as it provides enormous export quality, increases employment, improves the environment and helps in attainting nutritional stability (SMEDA, 2010). This sector adds nearly 12% to the National agriculture GDP of Pakistan (PHDEB, 2007). The current national horticulture exports are 400 Million USD (MEFP, 2012). Hence, this industry has shown possibilities to enhance earnings and lessen the hungriness and paucity from the society (SMEDA, 2010).

Peach (Prunus persica) is one of the important stone fruits (Jehan and Nazma, 2012). The seed of these plants are surrounded by a pit or stone due to which these are called as stone fruits (Ahmed and Dennison, 1981). The word prunus means temperate while persica means from Persia. Peaches belong to plant family Rosaceae and sub-family Amygdaloidae. Plant species of this genus are botanically termed as drupe (Brady, 1993).

The word drupe comes from Latin word druppa which means over-ripe olive. These fruits are valued worldwide for their taste and nutritional composition (Pizato et al., 2013) and rank 9th in important fruits of Pakistan (PHDEB, 2007). Peach skin is of two types; either smooth or velvety (Bajwa and Iqbal, 2006). The two important cultivated type of peaches produced globally are clingstone and free stone (Zeb and Khan, 2008). Peaches have the ability to grow on different types of soils. Mild summer temperature of 27-30oC is considered ideal for good flavor and fruit quality (PARC, 2007). Peaches originated from China and hence considered native to it (Jehan and Nazma, 2012). Peaches are an excellent source of vitamin A, vitamin B and ascorbic acid. These are comprised of 10-14 % sugars and 2 % proteins. In Pakistan, peaches are mainly cultivated in Khyber Pakhtunkhawa (KPK) and Baluchistan. Area of Pakistan under the cultivation of peaches is 15409 hectors in 2011-12 (NFS&R, 2013). On average, the amount of peach production in Pakistan annually is 60,000 tons (PARC, 2007). The amount of peach produced reached up to 83,670 tons in 2008. Peach season in Pakistan remains from May to September. More than 42 varieties of peach are grown in Pakistan (PHDEB, 2007). China is the largest producer of peaches with annual production of 11,528,801 tons (FAO, 2014).

Quality standards have established for the export of peaches to supply fresh product to the consumer. The minimum requirement for peach export according to (CODEXSTAN, 1981) is that it must be intact, clean, practically free from pests, free from damages caused by pests, free of abnormal moisture, smell and fruit stage must be such that it can tolerate transport and handling. Decay tolerance should not more than 2 % in total. Minimum weight of peach must be 85g while minimum diameter should be 56mm (EU, 2011). Packaging materials must be new, clean and of a proper quality, free of all foreign matter and stickers individually affixed to products should be removable (UNECE, 2011). Microbiological standards are available for ready-to-eat and precut fruits but it is noteworthy that there are no standards for raw fruits and vegetables as such (WAFMP, 2005). However, according to International Commission on Microbial Specification for Foods (ICMSF) and United State Subcommittee on Microbiological Criteria, the microbiological limit for fruits at ambient temperature is 1×10³ for Escherichia coli and 1×10⁴ for yeast & molds (Bell et al., 1997). Although Pakistan yields a large amount of crops, but total export quantity observed in 2005-06 was nearly 41 % less than production rate. The reason for this low export is the post-harvest losses and low shelf life of fruits (PHDEB, 2007). Only 2 % peaches were exported to international market in 2011-12 (NFS&R, 2013). On average, post-harvest loss ranges from 18%-31% of which 77% loss occurs in peach picking stage while 23% during transportation. More precise studies showed a post-harvest loss of 31 %, 22%, 24%, 18% and 22% in Variety 4, Variety 5, Variety 6, Variety 7 and Variety 8 respectively.

Peach fruit is difficult to store for a long period of time due to microbial growth and the hot and humid climatic conditions found in KPK. For the export of peach to the global markets, is required to have greater storage life and should be protected from the microbial diseases for a long period of time (Zaman et al., 2013). Different post-harvest techniques which are mostly recommended includes hydro cooling, dehydration, freezing, skin coating, low pressure storage, irradiation and canning (Khan, 2008). However, gamma radiations have shown certain advantages over the conventional methods. Gamma rays are produced by a radioactive source cobalt⁶⁰. Being highly penetrative and relatively inexpensive makes them a cost effective option for irradiation of food. These irradiations increases the food conservation and improves the hygiene, kills the disease causing pathogens, enhances the life span of fruits and helps to overcome quarantine barriers for the peach export from Pakistan (SMEDA, 2010). These radiations can be used to disinfect insects, to inhibit sprout, to delay ripening and reduce microbial load. Hazard Analysis and Critical Control Points (HACCP) also declared food irradiation as safe to use (Ganguly et al., 2012).

REVIEW OF LITERATURE

Peaches belong to plant family Rosaceae and sub-family Amygdaloidae.More than 5000 cultivars of peach are found across the world. The two important cultivated type of peaches produced globally are clingstone and free stone. Free stone varieties have the stone that quickly twists away from the fruit flesh while cling stone varieties have stone or pit firmly attached to the fruit. The former form is widely found in supermarkets; the latter is mainly used for canning.

2.1. ORIGIN AND DISTRIBUTION:

According to Khan (1990) peaches have originated from China and hence considered native to it. Dates back to the 10 century B.C. peaches were originated in China and later spread to Persia and Europe by way of the trade routes. The peach arrived in the New World in the early 1600s with the Spanish explorers. Currently, half of the peaches in the U.S. are grown in California, and the other half are grown in the southern states. True wild type peaches are only found in China; these are fuzzy, small and sour in taste. Peaches are considered as an ideogram of long life in China. In many Asian countries, the peach is very popular and is also featured in many folktales and considered to be a symbol of longevity. It was reported by CSU (2011) that approximately 80 % peaches produced annually are consumed fresh , remaining 20 % are dried, canned or processed to make peach drinks, juices, jelly and candies.

2.2. THE PEACH PLANT:

Edward (2011) reported that peach tree is about 15 feet in height. Peach tree has an average age of 15 years and it starts bearing fruits in 3-5 years age. The seeds are brown in color with average size of 1 - 3.2 cm. Skin of peach is of two types either; smooth (nectarine) or velvety (peaches) in different varieties. Ahmed and Dennison (1981) explained that peaches have the ability to grow on different types of soils. However, the well-drained, loamy soils having deep sub-soil are most appropriate and favorable. Peach tree cannot bear wet conditions. PARC (2007) narrated that mild summer temperature of 27-30 oC is considered ideal for good flavor and fruit quality.

2.3. NUTRITIONAL VALUE:

Bajwa and Iqbal (2006) reported that peaches are a nutritious addition to the diet. Peaches are free of sodium, saturated fat and cholesterol. These are an excellent source of Vitamin A, Vitamin B and ascorbic acid. These are comprised of 10-14 % sugars and 2 % proteins. Besides, they are also rich source of iron, calcium and phosphorus. Zeb and Khan (2008) explained that peaches are composed of more than 80 % water and provide good amounts of potassium. Peach skin is a very good source of vitamins and minerals. Peaches stand high among fruits in antioxidant and phytochemicals activity. According to USDA (2007) yellow peaches maintain heart and vision, it also enhances immune strength while white peaches have anti-cancer effects and maintain cholesterol level.

2.4. HEALTH BENEFITS:

Layne and Bassi (2008) explained that peaches are low in calories (100 g peach provides 39 calories) and contain no saturated fats. Nonetheless, they are packed with numerous health promoting compounds, minerals and vitamins. Fresh peaches are a moderate source of antioxidant, vitamin C. Vitamin-C has anti-oxidant effects and is required for connective tissue synthesis in the body. Consumption of foods rich in vitamin C helps the body develop resistance against infectious agents. Janick and Paull (2008) reported that fresh peaches are an also moderate source of vitamin A and ß-carotene. ß-carotene is a pro-vitamin, which converts into vitamin A in the body. Vitamin A is essential for vision. It is also required for maintaining healthy mucus membranes and skin. Consumption of natural fruits rich in vitamin A is known to offer protection from lung and oral cavity cancers. Peaches are rich in many vital minerals such as potassium, fluoride and iron. Iron is required for red blood cell formation. Potassium is an important component of cell and body fluids that help regulate heart rate and blood pressure. According to Tiffney (2009) peaches contain health promoting flavonoid poly phenolic antioxidants such as lutein, zea-xanthin and ß -cryptoxanthin.

2.5. PAKISTANI PEACHES:

According to MINFAL (2005) peaches are mainly cultivated in KPK and Baluchistan. About 64 % area of KPK, 36 % area of Baluchistan and 1.0 % area of the Punjab is under peach cultivation. Swat and Peshawar valleys are the central position for production of high quality and graded peaches. PHDEB (2007) reported that top 5 peach producing districts are Swat (60 %), Killa Saifullah (5.5 %), Quetta (5.07 %), Mustang (5.02%) and Pishin (3.54%). Others include Kalat, Peshawar, Mardan, Kohistan and in Pothwar area of Punjab. Khaliq (2013) researched that about 2,00,000 acres land is still there that can be used for fruit production and this will not only increase amount of peach and other fruits production in Swat but will also contribute to the local economy.

2.6. PEACH VARIETIES:

Jehan and Nazma (2012) explained that Pakistani peaches remain from May to September. More than 42 peach varieties are grown in Pakistan. From the survey of available literature, it is shown that Early Grande, Florida King, Loring, NJ241, Floridison, NJ238, Texas A6-69, 6-A, Flame Crest, Prime Rose 7, 8, 9 numbers, and Texas Y-55 cultivars are proposed to the agriculturists keeping in view the healthy growth of plants and their good quality. Zeb and Khan (2008) researched that about 65% of area in KPK under peach cultivation is occupied by 6A, Texas Y-55, and 7, 8, 9 number varieties while in Balochistan, Shah pasand, Shireen and Golden early varieties are mostly grown.

2.6. PEACH EXPORT:

PHDEB (2007) explained that peaches are picked and collected in cloth bags, than bulk collection is done within field. These are sorted and graded, packed in paper cartons or wooden crates. After that, these are transported to local and big markets using trucks. For export, these are further regarded and repacked. Precooling is done and then sent to foreign markets by shipment. NFS&R (2013) reported that in 2011, Pakistani peaches were exported to Afghanistan, Kuwait, UAE, UK with export quantity of 781665 Kg, 216 Kg, 3895 Kg, and 1000 Kg respectively. This makes Afghanistan the largest importer of Pakistani peaches. FAO (2014) documented that Russia is the biggest importer of peaches globally with annual import of 26, 62,20,376 kg.

2.7. POSTHARVEST LOSS:

Zaman et al. (2013) proclaimed that hot and humid climatic conditions allow the growth of certain micro-organisms. Therefore, storage life of peaches is limited due to the

changes in texture, appearance and microbial load on fruit surface. Pizato et al. (2013) explained that diseases may occur during the growing phase, at time of harvesting, during handling, storage, marketing, transport, or even after being purchased by the consumer. Sholberg and Conway (2004) researched that despite of supplying food safety measures to a high level, microbiological hazards occur. PHDEB (2007) proposed that the usage of refrigerated containers can help decreasing postharvest losses and enhance the shelf life of fruits. In this way, postharvest losses can be easily reduced up to 25%.

2.8. POST-HARVEST DISEASE OF PEACH:

Khan (2008) narrated that all living materials are suspected to be attacked by the parasites. Pathogens are widespread in air, soil or water. Some diseases are capable of penetrating through the unbroken fruit skin, while others may require an injury in order to cause the infection. Palou et al. (2009) publicised that fungi are commonly found to be attacking fruits while bacteria are postharvest pathogens of vegetables. Microbiological analysis of peaches revealed the presence of Monilinia fructicola, Penicillium expansum, Mucor piriformis, Botrytis cinerea, Geotrichum candidum, Alternaria alternata, and Rhizopus stolonifer on fruit surface . Brown rot is caused by several fungal including Monilinia fructicola, Monilinia laxa and Monilinia fructigena. Schnabel et al. (2006) explained that frequently occurring postharvest diseases of peach are sour rot which is caused by Geotrichum candidum and gray mold caused by Botrytis cinerea. Botryosphaeria dothidea is responsible for botryosphaeria fruit rot while Colletotrichum acutatum is the causative agent of anthracnose disease. Alternaria rot is caused by Alternaria alternata and blue mold is caused by Penicillium expansum. Trandafirescu and Botu (2009) researched that rhizopus rot is caused by Rhizopus stolonifer while Mucor piriformis causes mucor rot. Bacterial attack on peaches is caused by Pseudomonas morsprunorum f. sp. persicae and is a relatively new disease fruits. Peach dieback was mainly attributed to Pseudomonas syringa e.

2.9. FUNGAL PATHOGEN:

Layne and Bassi (2008) recounted that postharvest loss of stone fruits by decay-causing fungi is thought to be greatest complication of decay. The fungus Botrytis cinerea causes grey mold or Botrytis rot and this fungus is one of the chief pathogen of fresh peach fruit.

Khazaeli et al. (2006) explained that remarkable efforts are made in protecting the agricultural produce from Botrytis cinerea. In the recent years, the market size of US$ 15-25 million is spent for anti-Botrytis products. Layne and Bassi (2008) reported that Monilinia fructicola fungus (brown rot) is one of the main peach decay pathogen. Ziedan and Farrag (2008) documented that in order to minimize the problem of protecting peaches from pathogenic attack, information on life cycles of fungi, epidemiology, sanitation practices and pre/postharvest processes is required. Gupta et al. (2012) reported that three species of fungi were isolated to be the main causative agents of peach diseases. These were Rhizopus stolonifer, Aspergillus niger and Monilinia fructicola. Aspergillus is a fungal genus consisting of mold species found worldwide. Other properties of Aspergillus niger include food spoilage and production of toxic secondary metabolites.

Schnabel et al. (2006) researched that baby gold peaches are often used for the baby food production because the flesh has an orange color with apricot-like taste and remain firm during and after processing. These peaches are observed to be suffering from Phomopsis fruit rot caused by Phomopsis sp. Kim et al. (2010) reported that fungal infections were recorded to be caused by Botrytis cinerea, Penicillium expansum, Rhizopus stolonifer, Monilinia fructicola. According to Hindi et al. (2011) peaches have shown fungal decay in relation to local storage at shops and a number of Aspergillus spp. have been isolated including Aspergillus niger, Aspergillus nidulans, Aspergillus variecolour, Aspergillus fumigatus, Aspergillus candidus, Aspergillus tubingensis. Witthuhn et al. (2005) narrated that microbial content of high moisture commercial fruits showed that the total viable counts were the highest for nectarines with 100 CFU/g, while the counts of the clingstone peaches were only 40 CFU/g. Fungi species on clingstone peaches were 30 CFU/g and were found to be members of Aspergillus and Penicillium species.

Pizato et al. (2013) reported the presence of psychotropic microorganism on peach surface. Miteva et al. (2008) explained that during the storage of peaches at 3 ± 1oC for 12 days, increase was observed in peach microbial content reaching up to 7.17 log cfu/g. However Xu et al. (2008) documented that freeze dried peaches were observed to be having total aerobic plate count 8.1× 10² cfu/g while yeast and mold count was1.0 ×10¹

cfu/g. P. membranaefaciens was isolated from peach fruit surface in experimental analysis. Kim et al. (2009) reported that peaches were stored at 20oC temperature for 6 days and analyzed to be contaminated with both bacteria and fungi. Total aerobic bacteria were found to be 4.68 logs CFU/g while yeast and molds count was observed to be 4.67 logs CFU/g. Mengjun et al. (2011) reported that species of Monilinia fungi were isolated from peaches which were already decayed with brown rot. Isolated fungi including Monilinia mumecola and Monilinia yunnanen. Ziedan and Farrag (2008) reported that in Egypt, the main pathogens responsible for peach decay are Monilinia fructicola and Rhizopus stolonifer. Michailides (1991) naratted that twenty-six isolates of Mucor spp. were isolated from stone fruits, chiefly from nectarines and peaches and pathogenicity comparison was made. Candir et al. (2012) reported that six isolates from Chile peaches and thirteen from California peaches were obtained belonging to Mucor species. These include M.circinelloides, M. piriformis, M.racemosus and M.plumbeus. Others included M.hiemalis, M.genevensis.

2.10. BACTERIAL PATHOGENS:

Witthuhn et al. (2005) proclaimed that microbial analysis of high moisture containing fruits was performed and found to be members of the endospore-forming genus Bacillus. Lactobacilli were found to be 10 CFU/g for both nectarines and clingstone peaches. Schnabel et al. (2006) explained that Babygold peaches are susceptible to summer diseases like bacterial spot caused by Xanthomonas arboricola. Leff and Fierer (2013) publicized that a large number of bacteria harbor the surface of peach fruit. These were dominated by microbes belonging to the Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria phyla including the bacteria from families of Microbacteriaceae, Enterobacteriaceae, Rhodobacteriaceae, Flavobacteriaceae, Oxalobacteriaceae and Bacillaceae. Trandafirescu and Botu (2009) explained that bacteria Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas mors-prunorum f. sp. persicae, Erwinia amylovora and Erwinia herbicola species are found on peach fruit surface.

2.11. PEACH PESTS:

USDA (2011) reported that besides microbes, pests and flies are also responsible for causing peach fruit spoilage. Adult fruit fly Bactrocera zonata has also been reported to be a pest of stones fruits Codling moth and oriental fruit moth are the important pests of quarantine concern in peaches. Neven (2008) explained that flesh fly, Sacrophaga crassipalpis and Mediterranean fruit fly are main pests for peaches. According to UBC (2010) peach tree borer attacks peaches, apricots and other stone fruits. Peach twig borer larvae bore into the growing shoots making them to wilt and die. The caterpillars also attack the ripening fruit, these create holes and furrows at the end of stem. Watson and Gilman (1994) proclaimed that aphids are observed to cause distortion of new growths. Spider mites cause yellowing or stippling. Scales of several types infest Prunus spp. For control of scales, horticultural oil is used. Tent caterpillars eat the peach tree leaves but these can be controlled easily

2.12. IMPORTANCE OF QUARANTINE TREATMENT:

According to EU (2011) in order to meet the World Trade Organization (WTO) and quarantine regulations, pest and pathogen-free horticultural products are essentially required. Attractive appearance and texture of fruit, high nutritional value of the products with suitable market life is required. Minimum requirement for peach export according to is that it must be practically free from pests, free from damages caused by pathogens. Bajwa and Iqbal (2006) explained that major importance is being laid on the horticultural products, which are not contaminated with chemical residues. Zaman et al. (2013) researched that for increasing peach exportation from Pakistan, it is required to make peaches microbe free for a long period of time and enhance their shelf life at ambient temperature.

2.13. CURRENT TREATMENTS:

According to Bajwa and Iqbal (2006) quarantine treatments currently used are vapor heat, cold air, hot air, fumigation, hot water treatment etc. All the procedures have their own drawbacks. Vapor heat, hot air; hot water and cold air instantly cause an adverse effect the appearance and consistency of the horticultural product. Due to high energy consumption, they proved to be un-economical. At present, mostly methyl bromide is used as fumigant but it causes the depletion of ozone layer and has certain environmental effects, so their usage is phased out. Hence these have been banned, leading to limited export among different countries. Çandir et al. (2012) explained that in order to control the diseases, postharvest fungicides are also used but these have certain health and environment concerns. Treatment of high temperature forces increase the chilling injury in peach fruits

2.14. GAMMA IRRADIATION AND FOOD SAFETY:

NIFA (2005) reported that in 1976, the joint committee of FAO/IAEA/WHO recognized that irradiation can be used for food preservation Through food irradiation, the nutritive values of foods are maintained. Even if nutritional losses occur, it is negligible. Ganguly et al. (2012) explained that gamma radiation benefit to foods includes delay in the ripening process of fresh fruits, killing the fresh fruits insects. Gamma rays eradicate the fungi from the fresh fruits and also remove pathogenic bacteria from the foods. Bajwa and Iqbal (2006) suggested that in viewing the benefits of gamma irradiation, the food irradiation technology is considered as a perfect alternative to chemical fumigants and other or quarantine processes for peach export. NIFA (2005) researched that no matter how much a food is technically and scientifically safe, but the ultimate acceptance is made on whether the public likes to access it in the marketplaces or not. Tauxe (2001) reported that public acceptance of irradiated fruits was observed in a survey conducted by the Food Marketing Institute and at Food Net Sites, generally speaking about 50% of the population showed positive response to buy irradiated foods. Acceptance will be high if irradiated food costs less than non-irradiated food. The acceptance rate can enhance from 50% to 90% if customers are told that irradiation reduces harmful pathogens in food. Similar results were obtained when marketing irradiated products were tested. According to NIFA (2005) people mostly fear that irradiation is linked with radioactivity or toxic substances, that is why it becomes difficult to ensure them that irradiation is safe. It is a scientific fact that irradiation do not produce any toxic or radiolytic substances. Kener (2000) reported that food irradiation has been approved for NASA's Space Program and for Immuno-compromised hospital patient. WAFMP (2005) reported that for disinfection and delay ripening in fresh fruits, FDA has approved maximum dose as 1.0 kGy.

4.15. EFFECT OF GAMMA IRRADIATION ON PEACHES:

Gould (1996) reported that gamma irradiation dose of up 0.5 kGy is optimum for controlling Mediterranean fruit fly growth in peaches. NIFA (2005) narrated that a dose of 1.0kGy is enough to control the insect infestation in peaches and these irradiated products can be stored in good conditions for a period of up to 6 months. According to Xuetong (2009) dose of 1.0kGy is also suggested by United States Code of Federal McDonald et al. (2012) carried out an experiment by using six peach cultivars, which were exposed to irradiation at 0.29, 0.49, 0.66, 0.69 and 0.90 kGy and one act as control. Peaches were observed at the end of shelf life 13-27 days after harvesting. Irradiation was proved to be effective in insect control Irradiation on commercial scale did not badly affect life span but was observed to increase ripening. These were positively accepted by consumers. Consumers’ acceptability of irradiated peaches was more than unirradiated peaches. Statistical analysis was performed to find determinates of irradiation on peaches using linear mixed models.

Moy et al. (1983) reported that Mediterranean fruit fly Cerutitis capitata is disinfected by irradiation of peaches at 0.5kGy. The Medfly eggs did not hatch in irradiates peaches. Kim et al. (2010) researched that irradiation inhibits the growth of certain fungi including Monilinia and Rhizopus species. The impact of gamma rays was observed on peaches during storage for 6 days at 20 ± 3oC to inactivate the fungal pathogens. Decimal reduction doses were proved to be 0.14 kGy for Botrytis cinerea, 0.23 kGy Penicillium expansum, 0.16 kGy for Rhizopus stolonifer and 0.16 kGy for Monilinia fructicola. Dhaliwal and Salunkhe (1963) reported the effect of irradiation doses of 1.5, 2.0 and 2.5 kGy on the shelf-life and eating quality of Veteran peaches. These levels did not affect flavor, texture or color as evaluated by a taste panel, but were effective in controlling rot for four weeks. According to MOY (1977) the doses of 1.0 kGy and 3.0 kGy were effective in controlling fungus growth of species Alternaria, Rhizopus and Penicillium. It was also effective in storing the fruits for long period of time than in control. The gamma irradiations were effective in inhibition of brown rot control in peaches with minimum effective dose of 1.5kGy. Fields and White (2002) suggested that quarantine treatment at 0.3 kGy can be used as an alternative to methyl bromide.

Hussain et al. (2008) researched that irradiation treatment in range of 1.2-1.4 kGy is effective in increasing the shelf life of Elberta peaches by 6 days at room temperature and up to 20 days of increase at refrigerated temperature. Sommer et al. (1964) explained that gamma irradiation in highly useful in controlling the fungal growth on peaches. Kim et al. (2009) studied the effect of gamma irradiation on the microbiological properties of peaches during 6 day storage at 20±3oC. Total aerobic bacteria, yeasts and molds significantly decreased with increasing dose level. At dose of 1.5 kGy, the bacterial and yeast count was not detected within the limit of <10² log CFU/g while unirradiated peaches showed bacterial count of 4.68 log CFU/g and yeast & mold observed to be 4.78 log CFU/g after 6 days. Irradiation dose of 1.85 kGy inhibited brown rot for 10 days at 26 to 29oC.Unirradiated peaches were decayed within 5 days. Temur and Tiryaki (2013) reported that gamma irradiation dose inhibits decay was determined in apple, quince, and peach inoculated with P. expansum, Monilinia fructigena and Rhizopus stolonifer, respectively. Doses of 1, 2, 3 and 3.5 kGy did not inhibit decay on fruit, but infection was delayed for a certain period. Interval of time between infection and irradiation affected the growth response of Monilinia fructicola infections and the irradiation dose needed for it control.

Hussain et al. (2008) explained that irradiation significantly (p≤0.05) delayed the decaying of peaches under both storage conditions. Under ambient storage conditions, unirradiated samples were found to have started decaying after 3 days of storage, whereas no decay was observed in irradiated samples over the same storage period. Irradiated samples except those irradiated in the dose range of 1.0-1.4 kGy started decaying after 6 days of storage. Under refrigerated storage no decay was observed up to 20 days in sample irradiated to 1.0-1.4 kGy dose. The maximum dose required for the decay to be minimum was observed to be 1.2-1.4 kGy. Muhammad (2003) studied the response of gamma irradiation on the Early Grand peaches at KPK agricultural University Peshawar in 2000-02. Dose of 0.005, 0.01, 0.015, 0.02 kGy were used and positive results were obtained. In another experiment, peaches were exposed to doses 0.01, 0.02, 0.03 and 0.04 kGy. Maximum germination percentage, plant height, seedling diameter, number of leaves, number of nodes, number of branches, number of roots, root length and plant weight were observed. The higher radiation dose decreased the above parameters significantly than other treatments. Zaman et al. (2013) reported that gamma irradiation was given in combination with hot water treatment to increase the shelf life of peach fruit. The matured peaches fruits of variety Tex-A-6-69 were first dipped in hot water having temperature of 0, 40 and 60oC for a minute and then were subjected to 0.5 and 1.0 kGy doses of gamma radiation and stored in paper cartons at 25 ± 2 oC. The combine effect of hot water dipping treatment and irradiation for peach was study for the first time in Pakistan. The study was conducted to observe the effects on shelf-life and consumer liking. Laboratory scale irradiation showed no adverse effect on shelf life but it increased ripening. Thus irradiation was considered as a positive change by consumers.

MATERIALS AND METHODS

3.1. STUDY AREA:

All the research work was performed in the Department of Biotechnology, LCWU. However, irradiation process was carried out in Pakistan Radiation Services (PARAS).

3.2. SAMPLE COLLECTION:

Peaches (Variety No.4) were collected from local mandi in Lahore. The fruits were fresh, having uniform shape and size, firm texture and proper maturity.

3.3. SAMPLING:

Peaches were divided into four groups (one control group and three groups were specified for a particular dose). Weight of each group was determined .The samples were then packed in the polythene bags. Each bag was labeled with the different gamma dose (0.25kGy, 0.5kGy and 0.75kGy) and sent to PARAS for irradiation.

Abbildung in dieser Leseprobe nicht enthalten.

3.4. ENUMERATION OF BACTERIAL LOAD:

Nutrient agar was used for the enumeration of total viable count, MacConkey agar was used for enumeration of gram negative enterobacteriacae and Salmonella Shigella agar were used for determination of gram negative lactose non-fermenters (Harrigan,1998).

3.4.1 NUTRIENT AGAR:

For the preparation of nutrient agar solution, 14g of nutrient agar was weighed on weighing balance and dissolved in 500ml of distilled water. The solution was homogenized by constant stirring and was then autoclaved. Later on, it was cooled at room temperature. 15-20 ml of the media was poured in each sterilized petri plate. Petri plates were then allowed to solidify for 10-20 min (NEOGEN, 2011).

3.4.2. MACCONKEY AGAR:

26g of powder was weighed and dissolved in 500ml distilled water. The solution was homogenized by constant heating and mixing and was then autoclaved. It was then kept at room temperature to cool down 15-20 ml of the media was poured in each sterilized petri plate. Petri plates were then allowed to solidify for 10-20 min (NEOGEN, 2011).

3.4.3. SALMONELLA-SHIGELLA AGAR:

30g of powder was weighed and dissolved in 500ml distilled water. The solution was homogenized and sterilized by constant heating on hot plate stirrer. It was then kept at room temperature to cool down. 15-20 ml of the media was poured in each sterilized petri plate. Petri plates were then allowed to solidify for 10-20 min (NEOGEN, 2011).

3.5. MEDIA PREPARATION FOR FUNGAL ANALYSIS:

3.5.1. POTATO DEXTROSE AGAR

19.5 g of the powder was weighed on electric weighing balance and dissolved in 500ml of purified water. The solution was homogenized by constant heating and mixing and was then autoclaved. It was then kept at room temperature to cool down 15-20 ml of the media was poured in each sterilized petri plate. Petri plates were then allowed to solidify for 10-20 min (NEOGEN, 2011).

To, checks the sterility, all plates were placed in incubator for 24 hours at 37oC. Distilled water was sterilized by autoclaving. One peach was selected randomly from each bag. Sample was rinsed out in 100ml of distilled water in sterilized beaker .The samples were shaken for 5minutes and then 1ml of stock solution was added into test tube containing 9ml of sterilized water and serial diluted up to 10-³. For determination of total viable count, 0.1 ml aliquots from each dilution were transferred aseptically into sterile Petri dishes

3.6. INCUBATION:

These plates were then incubated at 37oC for 24 hour to check the result PDA. Plates were incubated at 30 C for 72-96 hours and growth pattern was studied (Harrigan, 1998).

3.7. COLONY COUNTING:

After 24 hours, the incubated petri plates were checked for the microbial growth. Colonies on the petri plates were counted on the basis of morphology and results were recorded in cfu\ml (Farag et al., 2013). Colonies were counted according to the formula given by (Jackie, 2011).

No. of colonies ×dilution factor × dilution spread = cfu /ml

3.8. PURIFICATION OF CULTURE ISOLATES:

Colonies were selected from differential media (MacConkey and Salmonella Shigella agar) and pure cultures were isolated by streaking. The colony characteristics and cellular morphology of culture isolates were examined by using these tests, (Gram staining, Endospore staining, and Motility determination)

3.8.1. GRAM STAINING:

Gram Staining was performed to differentiate the gram negative and gram positive bacteria. Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of cell wall). Gram-negative bacteria have walls with thin layers of peptidoglycan (10% of wall), and high lipid content. Due to the presence of thick peptidoglycan layer, gram positive bacteria retain crystal violet in their cell membrane in the form of crystal violet-iodine (CV-I) complex, due to which they are stained purple. Gram negative bacteria are stained pink due to safranin dye. Bacterial culture of 24 hours was used for the gram staining. The smear was prepared on a slide and heat fixed on flame for 1-2 seconds. Few drops of crystal violet were poured on smear and allowed to stand for 20 seconds. After that, it was washed off with water. Iodine drops were added on smear and allowed to react for 30 seconds. It was then rinsed with 95% ethyl alcohol for 10-20 seconds. Smear was counter stained with safranin for 20 second. To stop the decolorizing effect of alcohol, it was rinsed with water for few seconds, dried with blotting paper and observed under microscope at 10, 40 and 100X oil immersion (Brown, 2007).

3.8.2. ENDOSPORE STAINING:

Endospore staining was used to check for the presence of bacterial spores. The smear was prepared on a slide and heat fixed on flame for 1-2 seconds. It was covered with small

piece of paper toweling and saturated with malachite green. Smear was steamed over the boiling water for 5 minutes. Additional stain was added if stain boils off. After the slide was cooled sufficiently, paper toweling was removed and was rinsed with water for 30 seconds. It was counterstained with safranin for about 20 seconds. It was briefly rinsed with water to remove safranin. It was dried with bibulous paper and observed under microscope at 10, 40 and 100X oil immersion (Brown, 2007).

3.9. IDENTIFICATION OF FUNGAL SPECIES:

Fungal species were identified on the basis of morphology on PDA plates. For microscopic identification of yeast, smear was prepared and dried in air. It was than observed under 10, 40 and 100x magnifications. Molds were examined by staining with Lactophenol blue. Large drop of LCB was put onto the slide. Small quantity of the culture was taken with the help of forceps and transferred to the drop. Coverslip was placed on the slide gently to avoid entrapment of air bubbles. Slide was examined under 10, 40 and 100x magnifications (Pitt and Hocking, 2009).

3.10. WEIGHT LOSS DETERMINATION:

Percentage weight loss was determined weighing of samples periodically after each week Hussain et al. (2008). Five samples for each of three doses and control were used for each treatment. Weight loss was calculated from initial weight using the formula:

Weight loss (%) = (Wi-Ws/W)i ×100

Where,

Wi= initial weight; Ws=weight at sampling period.

3.11. DECAY PERCENTAGE:

Determination of decay percentage was done visually from known number of fruits (Hussain et al., 2013). Any fruit with fungal growth, extreme softness and brownish appearance was considered as decayed. Decay percentage was monitored under refrigerated temperature. Decay percentage was calculated as:

Decay percentage = (No of decayed fruits/Total number of fruits) ×100

3.12. DOSE OPTIMIZATION:

Both irradiated and unirradiated fruit samples were than stored at refrigerated temperature (4⁰ C) and weekly analyzed up to a period of three weeks. Effect of different doses was determined on the total viable microbial count. On the basis of these effects, dose was optimized.

3.13. STATISTICAL ANALYSIS:

Data were analyzed by 3 × 4 (weeks × treatment) arrangement using completely randomized design experiment. Five replicates of samples were tested per treatment and mean ± SEM values were reported. Analysis of variance (ANOVA) for the data was calculated using Co. Stat version 6.4. statistical analysis software. Means were separated by LSD at 5 % and were carried out using Duncan’s multiple range test (DMRT).Difference at p≤0.05 was taken to be statistically significant.

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