Food Research Bulletin 2016/17 (2073/74 B.S.)

FOOD RESEARCH BULLETIN 2016/17 (2073/74 B.S.)

Government of Nepal Ministry of Agricultural Development Department of Food Technology and Quality Control Babar Mahal, Kathmandu, Nepal Phone: 4262369, 4262741, 4262739, 4240016, Fax: 4262337 Email: [email protected], [email protected] Website: www.dftqc.gov.np FFOODOOD RESEARCHRESEARCH BULLETINBULLETIN 2016/17 (2073/74 B.S.)

Editorial Board

Advisors Sanjeev Kumar Karn Dr. Matina Joshi (Vaidhya)

Editor in Chief Nawa Raj Dahal

Executive Editor Pratima Shrestha

Editors Bimal Kumar Dahal Shreeram Neupane Suraj Shrestha Kesharilaxmi Bajracharya

Ashok Gautam

PREFACE

The outcome of the research activities of the Department of Food Technology and Quality Control (DFTQC) has been published in the form of Food Research Bulletin. It is with the aim to provide information on the outcome of all the research programs and activities conducted within the entire network of Department of Food Technology and Quality Control (DFTQC) from central to regional level.

The research bulletin in hand has tried to cover the result of scientific research that were conducted in the fiscal year 073/74 B.S. (2016/2017). The scientific information published will be relevant and useful to the professionals, policy makers, development workers, institutions, and other interested individuals. It is hoped that these information play catalytic role in the development and promotion of science and technology in the field of food technology development, nutrition, and food safety and quality at large.

I would like to extend my sincere thanks to all the scientists and researchers of DFTQC who have contributed to achieve the goals destined for these scientific researches. Thanks goes to all editorial board members. Special effort made by Editor-in-Chief Nawa Raj Dahal and Executive Editor Pratima Shrestha in editing and bringing out this publication in this getup is highly appreciable.

……………………………. Sanjeev Kumar Karn Director General Department of Food Technology and Quality Control (DFTQC) Babarmahal, Kathmandu G.P.O. Box No. 21265, Babarmahal, Kathmandu

Government of Nepal 977-1-4262369 Tel. 977-1-4262741 Ministry of Agricultural Development 977-1-4240016 977-1-4262739 Department of Food Technology and Quality Control Fax: 977-1-4262337 E-mail: [email protected] Webpage:- www.dftqc.gov.np Our Ref. No. EDITORIAL Your Ref. No. Department of Food Technology and Quality Control (DFTQC) is the responsible government institution under the Ministry of Agricultural Development of Government of Nepal to implement Food and Feed Act and related laws so as to protect the right of consumers by assuring safety, quality and nutritional status of food and feed products available in the country. Besides these, the Department has also promotional mandate to support the commercialization and industrialization activity of agriculture as well as facilitation of trade by developing the appropriate food technology packages by assuring safe, quality and nutritious food in the market. In considering these two broad objectives, Government of Nepal is implementing various activities related to Food Technology, Food Nutrition and Food Safety/Quality Control activities on regular annual basis through this Department. This Research Bulletin covers the major outcome of the Research and Development activities made in the Fiscal year of 2073/74 B.S. (2016/17) by Department through various Divisions, Sections and Offices under it.Editorial board would like to thank all of our valued authors for their efforts in preparing and providing their valuable Review and Research Articles in the field of Food Safety, Technology, Nutrition and Quality Control. All editorial board members are highly thankful for their efforts on editing these manuscripts. Especial efforts made by Executive Editor Pratima Shrestha is highly appreciable for her untiring efforts in bringing out this publication in this get up. It is expected that this bulletin would be highly beneficial to all of our valued stakeholders including customers, food processors, export import personals and distributors as well as policy makers, academia, NGOs and INGOs. Editorial board would like to request you all to point out mistakes and weaknesses if any made while preparing and printing this Bulletin. Your valuable suggestions, comments and feedbacks are highly instrumental to improve the quality of the bulletin in the days to come.

………………….. Nawa Raj Dahal Editor-in-Chief and Senior Food Research Officer (Section Chief) Planning, Monitoring and Evaluation Section Department of Food Technology and Quality Control (DFTQC) Babarmahal, Kathmandu G.P.O. Box No. 21265, Babarmahal, Kathmandu

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TABLE OF CONTENTS

Status of Food Safety, Technology and Nutrition (FSTN) Sector in National Plan of Nepal Nawa Raj Dahal, Pratima Shrestha and Sanjeev Kumar Karn ...... 1 Food Quality Control System of Nepal: Existing Practices and Way Forward Nawa Raj Dahal and Sanjeev Kumar Karn ...... 16 Simultaneous Analysis of Commonly Used Artificial Sweeteners and Preservatives in Beverages by High Performance Liquid Chromatography Suraj Shrestha, Nibedita Chaudhary, Ranjana Chaudhary, Tayer Mahommad Miya, Shiva Sagar Chaudhary and Sanjeev Kumar Karn ...... 46 Encapsulation of Saccharomyces cerevisiae in alginate beads and its application for wine making Praksha Neupane, Smita Gurung, Saroj Khanal, Rojeena Shrestha, Huma Bokkhim and Sanjeev Kumar Karn ...... 62 Status of Pesticide Residues in Vegetables Entering into Kathmandu Valley Bimal Kumar Dahal, Man Bahadur Chetri and Matina Joshi Vaidhya ...... 74 Advanced Glycation End-products Inhibitory Activities of Crude Methanolic Extracts of Fennel and Fenugreek Seeds Nirat Katuwal and Hasta Bahadur Rai ...... 84 Availability of Sulphurdioxide in the Food Products Commonly Found in Nepal Hareram Pradhan, Ranjana Chaudhary, Tulsi Bhandari, Atish Ghimire, Krishna Prasad Rai and Sanjeev Kumar Karn ...... 91 Process Optimization and Quality Evaluation of Bara, an Indigenous Newari Food Pratima Shrestha ...... 101 Effect of Germination on Phytic Acid content of Amaranth seeds (Amarantus cruentus) Bijan Shrestha, Sanjay Bhandari and Pramod Koirala ...... 113 DFTQC, FRB 2016/17

A Study Report on Scenario of Consumer Awareness Activities in Nepal Bimal Kumar Dahal, Madan Kumar Chapagain and Matina Joshi Vaidhya ...... 121 Preparation of Tomato Caramel and Comparison with Plain Caramel Menash Shrestha, Anup Halwai and Raj Kumar Rijal ...... 132 Preparation of Tishyauri and Optimization of Flaxseed and Black gram Paste Ratio Rabindra Jha ...... 138 Preparation and Quality Evaluation of Chiraito (Swertia chirayita ) Incorporated White Bread Anup Halwai and Raj Kumar Rijal ...... 145 Effects of Stabilizers on Cloud Stability of Kiwifruit RTS Pushpa Lal Rai, Bimala Pokharel, Pramod Koirala, Sanjay Bhandari and Nirat Katuwal ...... 151 Preparation of Vanilla Flavored Chhurpi and its Quality Evaluation Kedar Poudel, Anup Halwai and Raj Kumar Rijal ...... 156 Effect of Different Treatments on Shelf Life of Oyster (Pleurotus) Mushroom Collected from Lalitpur, Nepal Saroj Khanal, Praksha Neupane, Smita Gurung, Shreeram Neupane and Huma Bokkhim ...... 160 Preparation of Soymilk Incorporating Mango Pulp Vivek Banjara ...... 164

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Status of Food Safety, Technology and Nutrition (FSTN) Sector in National Plan of Nepal

Nawa Raj Dahal*, Pratima Shrestha and Sanjeev Kumar Karn Department of Food Technology and Quality Control, Babarmahal, Kathmandu *Corresponding author: [email protected] Abstract:

Nepal initiated its development through written periodic plan around six decades ago. Development of Agriculture sector (as strategic plan) started before two decades with20 year’s Agriculture Prospective Plan (APP) 1995 to 2015. After termination of APP, Government of Nepal initiated subsequent strategic plan called Agriculture Development Strategy (ADS 2015-2035). In addition, ten years agriculture project named Prime Minister Agriculture Modernization Project (PMAMP 2016-2025) also launched by GON as a supporting project of ADS recently. This article briefly reviews the status of Food Safety, Food Technology and Food Nutrition Sector mentioned in National Plan document of Nepal with especial reference to ADS and PMAMP programs.

Keywords: ADS, Food safety, Nutrition, PMAMP, Technology

Introduction: Agriculture sector has been playing vital role in Nepalese economy, contributing approximately more than one third of GDP and employing nearly two third of country's population. There are about 21.6% of Nepalese living below the absolute poverty line, the massive growth in agriculture sector is must to increase income, alleviate poverty and uplift the living standard of Nepalese people. The ultimate goal of agriculture (Both from plant and animal source) is Food and Food is necessitated for human existence. Consuming nutritious and safe food is indispensable for good health. It makes people healthy, stronger and resourceful. This contributes to economic and all-round development of the country. According to FAO/WHO, people all over the place have the fundamental rights to access to food which is of good quality, safe and nutritious. Safe food is one which is handled properly at all steps of production to consumption and is unlikely to root illness or injury (World Food Summit, 1996). In this connection, safe & nutritious food is the major concern of public and the development of Food Safety, Technology and Nutrition is major goal of Nation so as to ensure adequate, safe and nutritious food to the public on regular basis for all time.

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With realization of all these facts, Government of Nepal is giving high priority on the development of Agriculture Sector (Livestock also a part of agriculture sector) through Periodic Plans from Six decades back. Development of Agriculture sector (as strategic plan) started before two decades with 20 year’s Agriculture Prospective Plan (APP) 1995 to 2015.After termination of APP, Agriculture Development Strategy (ADS 2015 to 2035), a 20 year vision for agriculture development in Nepal was launched in Nepal on 26 July 2015. It has appeared as a succeeding plan to Agriculture Perspective Plan (APP 1995/96-2014/15). The strategy includes a 20 year Action Plan and Roadmap and a rationale based on the assessment of the current and past performance of agriculture sector with main vision of "A self-reliant, sustainable, competitive, and inclusive agricultural sector that drives economic growth and contributes to improved livelihoods and food and nutrition security leading to food sovereignty". Ten years agriculture project (2016-2025) named Prime Minister Agriculture Modernization Project (PMAMP) also launched by GON as a supporting project of ADS recently. The major objective of this article is to explore the status of Food Technology, Technology and Nutrition Sector in National Policy and Plan with especial reference to two major Agriculture Strategic Plans named ADS and PMAMP recently launched by Government of Nepal. Periodic Plans Nepal initiated its development through written periodic plan around six decades ago. First Five Year Periodic Plan was documented in 1956 and it lasted up to 1961. Similarly, second Three Year Periodic Plan, 1962-1965, Third Five Year Periodic Plan, 1966 to 1971, Forth Five year Periodic Plan, 1971 to 1976, Fifth five year periodic plan 1976 to 1981, Sixth Five Year Periodic Plan1981 to 1986, Seventh Five Year Periodic Plan 1986 to 1991, Eighth Five year Periodic Plan 1993 to 1998 (Two Years 1991 to 1993 were plan-less period), Ninth Five year Periodic Plan 1998 to 2003, Tenth Five Year Periodic Plan 2003 to 2008, Eleventh Three year Periodic Plan 2008 to 2011, Twelfth Three Year Periodic Plan 2009 to 2012 and Thirteenth Three Year Periodic Plan, 2012 to 2015 were launched respectively. At this time, Fourteenth Three year Periodic Plan 2016 to 2018 is underway in the implementation. The concept of Food Safety, Technology and Nutrition in Nepal was initiated by first periodic plan 1956 to 1961 (61 years ago) mentioning the fact of exporting ghee of around 10 Million NRs (~ 100 Thousand USD of that time) to Tibet. At that period, there appeared a problem on export due to lack of proper processing skill of ghee. Similarly, problem of water adulteration in milk sold in Kathmandu Valley and need of testing the adulteration was also mentioned in the plan document (GON - 1956). Second Periodic Plan focused on minimizing the loss of food commodities (preserving the nutrition) so as to eradicate the hunger problem. In addition, this plan had also given priority to set the standard (and to establish a small well equipped lab

DFTQC, FRB 2016/17 for testing) of ghee, milk and oil so as to prevent the adulteration in the market. This fact indicates that plan document of GON has given the priority on Food Safety, Technology and Nutrition sector already from six decades back. On analyzing periodic plans, Third Periodic Plan to Seventh Periodic Plans were more or less the continuation of Second periodic Plan. Eighth periodic Plan focused on regulation of imported food products at custom points. Ninth periodic plan given more focus on nutrition including separate chapter on food based nutrition. Tenth Periodic Plan emphasizes on Agreement on the application of Sanitary and Phytosanitary (SPS) measures and on Laboratory Accreditation as effective tools for improving Food Safety, Technology and Nutrition sector to support the commercialization and industrialization on agriculture sector. Eleventh Plan has given additional priority to Food Security adding a separate chapter on Food Security (Food Safety, Technology and Nutrition again the major Part of Food security) and mentioning the provision of extension of organization network of DFTQC to district level to support implementation of Food Security program in the Nation (GON 2011). Twelfth and Thirteenth Plan seems more or less continuation of the Tenth and Eleventh Plan. Latest Fourteenth Periodic Plan highly emphasized this sector adding separate new chapter on Food security and Food Nutrition along with the regular program on the regulation of food export & import, control on the use of pesticides & veterinary drugs (so as to minimize their ultimate residues on food) and grading of hotels restaurants to maintain the food safety on major food outlets in the country. Constitution and National Policy Recent Constitution of Nepal (2015) mentioned about Food Security and Food Sovereignty as well as Consumer Right as fundamental right of all citizens. Clause 36 states that every citizen have the right to food (Clause 36a); every citizen will have the right for food security (Clause 36b) and every citizen will have the right for food sovereignty (Clause 36c). Similarly, Clause 44 of the constitution mentioned about the Right of the consumers that every citizen will have the right to consume quality goods and services (Clause 44a) and every citizen will have the right of getting compensation who suffer from non-quality goods and services (Clause 44b) (GON, 2015). This shows that latest constitution of Nepal 2015 also given high priority for food safety, technology and nutrition sector (this sector as a major part of Food Security). On analyzing the provisions on policy matters, although the Nation has not issued the separate policy on Food Safety, however, the certain issues of Food Safety Technology and Nutrition have been addressed through policy documents related to Agriculture. National Agriculture policy 2004 has included the provision of safeguarding the health of consumers by setting quality standard of food, quality control, quality certification and food quality regulation for supporting food security and poverty reduction (GON, 2008a). Similarly, on Agri-business promotion policy 2006 mentioned the provision on import substitution and export promotion

DFTQC, FRB 2016/17 by developing the agriculture based food industry, by improving the quality certification system (GON, 2008b). Agriculture Development Strategy (ADS) ADS has come as a subsequent plan of APP. APP was mostly focused on pre-harvest side i.e., agriculture production. Specifically, Food Safety, Technology and Nutrition Sector was very much limited in APP document. However, ADS Document has significant room for this sector. ADS has set vision to accelerate agricultural sector growth through four strategic components related to governance, productivity, profitable commercialization, and competitiveness while promoting inclusiveness (both social and geographic), sustainability (both natural resources and economic), development of private sector and cooperative sector, and connectivity to market infrastructure (e.g. agricultural roads, collection centers, packing houses, market centers), information infrastructure and ICT, and power infrastructure (e.g. rural electrification, renewable and alternative energy sources). The acceleration of inclusive, sustainable, multi-sector, and connectivity-based growth is expected to result in increased food and nutrition security, poverty reduction, agricultural trade competitiveness, higher and more equitable income of rural households, and strengthened farmers’ rights. ADS has set four main outcomes. These are: 1) Improved governance, 2) Higher productivity, 3) Profitable commercialization and 4) Increased competitiveness. Among these outcomes, Food Nutrition Sector is included in component 1, i.e. Improved Governance and Food Safety and Technology sector is included in component 4, i.e. Increased Competitiveness. Outcome, Output and Activities related to Food Safety, Technology and Nutrition is given in Table 1. Table1: Outcome, Output and Activities related to Food Safety, Technology and Nutrition Outcome/Output Activities 1. Improved Design a targeted national food and security program Governance Implement a targeted national food and security program 1.9 Improved food and Implement Agriculture and Food Security Project (AFSP) nutrition security of Implement Food and Nutrition Security Plan of Action most disadvantaged (FNSP) groups and rights to Coordinate with ongoing food and nutrition security food* projects Review of national programs on food and nutrition security Strengthen the capacity of the central and district food security coordination mechanisms Promote formulation of rights to food and food sovereignty Legislation

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4. Increased Promote establishment of Agribusiness Incubators Competitiveness 4.2 Growth of innovative small and medium agribusiness enterprises* 4.3 Growth of food and Strengthen capacity to promote exports and negotiate agricultural products more favorable trade agreements exports** 4.4 Enhanced food Legislative measure for a modern food act safety and quality** Formulation and promulgation of food safety and quality standards Adopt legislation on accreditation of standards certification bodies Adopt legislation on accreditation of national laboratories for food safety and quality certification Strengthen and upgrade laboratories to international accreditation Institutional capacity building (Physical and Human resource) of SPS and quarantine (Plant, food and animal) system Establish regional laboratories and district offices for DFTQC Form Food Agency with authority under food act Establish capacity for pest risk assessment, SPS management and surveillance (Food Sector to be considered). Adopt One Health Approach and Strengthen animal health surveillance, diagnostic, and response capacity (Food Sector to be considered). Coordination and reporting to subcommittee on Food and Nutrition Security and Food Safety of the NADSCC (National ADS Coordination committee) Note: * Flagship program, **Core program (Source: GON, 2016) The ADS has categorized three different types of programs: 1) Core Programs, 2) Flagship Programs, and 3) Other Programs. The Core Programs are those programs which are being implemented mostly through existing agencies already in place. In case of DFTQC, the regular programs which are being implemented are included in core programs (Table 2). The main objective of core programs conducted by DFTQC are to 1) Maintaining safety and quality of food and feed products in the country by implementing updated food and feed act and 5

DFTQC, FRB 2016/17 regulations, 2) Promoting entrepreneurship by developing and disseminating appropriate technologies and 3) Improving nutritional status of the people through food-based approaches. Table 2: Core programs being conducted by DFTQC Programs Activities Food/Feed Quality Food Inspection (Industry, Market and Hotel/Restaurants) Control and compliance Feed Inspection and Sampling Licensing of food/feed industries Food/feed standardization and harmonization Communication on the SPS related rules, regulations and standards Export import certification of food Creation of consumer awareness activities Food Technology Research and development activities in food processing Development and technologies Dissemination Providing consultant services to food industries Conduction of various training on different areas of food processing, packaging, and post-harvest operations Food/Feed Analysis Laboratory Analysis of various foods and food products for their quality standard Analysis of feeds and feed ingredients Analysis of food and feed for their microbiological quality, contaminants and additives Food Nutrition Development, updating and publication of food composition tables Conduction of food and nutritional surveys Conduction of training on food and nutrition Conduction of research on various aspects of food consumption and nutrition Publication of posters, pamphlets, books/booklets on food and nutrition Creation of awareness on food and nutrition through radio and television programs Note: These programs are being conducted by DFTQC and Its Offices recently. DFTQC has One Central office at Kathmandu, five regional offices at Biratnagar, Hetauda, Bhairahawa, Nepalgunj and Dhangadhi, four quarantine laboratories at Kakarvitta, Birgunj, Mahendranagar and Tatopani and One Apple Processing Centre at Jumla (Total 11 Offices). (Compiled from Source: GON, 2016 and DFTQC Regular Program of FY 2017/18)

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The Flagship Programs on the other hand require different management structure in view of the innovative and multisector nature of their activities and those programs are implemented by joint effort of various institutions collectively to achieve the goal. Other Programs are those that are currently implemented but are not part of the currently formulated Flagship or Core Programs. Ministry of Agricultural Development will lead the implementation of Flagship programs in high priority.

ADS Flagship Programs (FANUSEP Program) The ADS envisages some prioritized national programs around which could be mobilized sufficient consensus, resources, and effective management. These prioritized national programs will be referred to as “ADS Flagship Programs”. The flagship programs are: 1. Food and Nutrition Security Program (FANUSEP) 2. Decentralized Science, Technology, and Education Program (DSTEP) 3. Value Chain Development Program (VADEP) 4. Innovation and Agro-entrepreneurship Program (INAGEP) Among the four flagship programs, Food and Nutrition Security Program (FANUSEP) is directly linked to DFTQC (Recently Deputy Director General of Food Quality Control Division is assigned as Flagship Manager of FASUSEP program) whereas DFTQC will partly be involved to implement other three flagship programs.FANUSEP aims at improving food and nutrition security of the most disadvantaged groups. It consists of three subprograms: 1) Nepal Food Security Project (NAFSP), currently been finalized as part of the Global Agriculture and Food Security Program (GAFSP); 2) Food and Nutrition Security Action Plan (FNSP), currently been finalized with assistance of FAO; and 3) new national food and nutrition security project to be designed and implemented to complement NAFSP and FNSP. One of the major impacts of ADS is to ensure food and nutrition security, especially among the disadvantaged groups. The food and nutrition security (FNS) dimension is underlying the overall strategy and is present in all components and many activities of ADS. FNS is a multisector concept; agriculture is however, a major determinant of food security out of many. The vision for agriculture sector in Nepal implies that higher agricultural growth is reflected not only in additional income but also in the availability, access, and utilization of more nutritious food, particularly of those who are currently food insecure. The indicators and targets of FNS in ADS are reported in Table 3. They include:

1) Food poverty (proportion of people below poverty line and unable to meet calorie requirement of 2200kcal/person/day); 7

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2) Stunting (children under 5 years of age below a certain norm for height for age); 3) Underweight (children under 5 years of age below a certain norm for weight for age) and 4) Wasting (children under 5 years of age below a certain norm for weight for height); and a chronic energy deficiency of women in reproductive age, measured by a low Body Mass Index (BMI).

Table 3: Indicators and targets of Food and Nutrition Security Indicators Situation Target Target Target Long (2015) Short term Medium term term (20 years) (5 years) (10 years) Food Poverty% 27.6 16 11 5 Stunting% 37.4 29 20 8 Underweight% 30.1 20 13 5 Wasting% 11.3 5 2 1 Women in reproductive 18.1% 15% 13% women 5% women with age with chronic energy women women with low BMI low BMI deficiency with low with low BMI BMI (Source: GON, 2016) The commonality of the subprograms of FANUSEP is to target the poor, the disadvantaged groups and the geographically disadvantaged areas (e.g. Karnali). The program promotes interventions that improve productivity, livelihoods, and nutritional practices of targeted beneficiaries including pregnant and lactating women farmers. The ADS in general and FANUSEP in particular aligns with the Multisector Nutrition Plan (MSNP) a five year program already approved by the GON.All outcomes, outputs and activities of ADS contribute to improve FNS, either directly or indirectly. ADS addresses the food and nutrition security needs of the most disadvantaged rural population (including lactating and pregnant women, Janajatis, Dalits) and groups in disadvantaged regions such as Karnali. Governance in ADS refers to the capacity of government to design, formulate and implement policies and discharge functions. All the activities contributing to FNS are organized under one national flagship program named Food and Nutrition Security Program (FANUSEP). ADS through its FANUSEP program manager will coordinate with ongoing food and nutrition security projects. ADS supports strengthening capacity of the central and district food security coordination mechanism at national and district level. It also promotes rights to food and food sovereignty legislation included in new constitution and monitor their implementation. Productivity has impact on FNS by: 1) Increasing the volume of food production in Nepal in a sustainable way through higher productivity and sustainable use of natural resources and 2)

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Reducing vulnerability of farmers through improved food/feed/seed reserve systems and early warning system, improved preparedness and response to emergencies, and climate smart agricultural practices. Profitable commercialization has an impact on food and nutrition security by: 1) Increasing income of farmers, 2) Improving access to markets and 3) Reduction of post-harvest losses. Similarly, Competitiveness has impact on food and nutrition security by: 1) Improving food safety, 2) Relying upon trade for a more diversified diet and 3) Accelerating the growth of micro, small, and medium agro enterprises including those headed by women, youth, disadvantaged groups and individuals on disadvantaged regions.

Monitoring and Evaluation of ADS Monitoring and Evaluation is one of the main outputs of Governance component. It will not only identify progress towards vision, but it will also indicate possible problems. For monitoring and evaluation purpose, indicators have been set for each output. The indicators set for the outputs related to Food Safety, Technology and Nutrition are given in Table 4. Table 4: Monitoring Indicators related to Food Safety, Technology and Nutrition Output Indicators 1.9 Improved food and nutrition No. of beneficiaries from disadvantaged groups security of most disadvantaged Income of beneficiaries groups and rights to food Nutritional outcomes (stunting, wasting, undernutrition) 4.2 Growth of innovative small No. of agro-enterprises developed by different and medium agribusiness groups. enterprises Value of output of agro-enterprises developed by different groups 4.3 Growth of food and Value of exports of food and agricultural agricultural products exports products Value of imports of food and agricultural products 4.4 Enhanced food safety and Percentage of exports that are certified quality Percentage of domestic production that is certified. Occurrences of food safety incidents Rejections of exported products due to SPS compliance issues (Source: GON, 2016)

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Prime Minister Agriculture Modernization Project (PMAMP) Government of Nepal (GON) has launched a Mega Project named Prime Minister Agriculture Modernization Project (PMAMP) as a subsequent project of ADS which is prepared and being implemented with National Vision and National Investment mobilizing internal institutional human resources. It is known as a purely GON project (there is not involvement of Donors). This project has a timeframe of 10 years and has launched from this year 2017. The main vision of the project is agriculture modernization with development of agriculture industries and to promote the agriculture trade both in domestic and in international market. The basic design of the project includes the categorization of agriculure production areas into four major categories i.e. 1) Pocket, 2) Block, 3) Zone and 4) Super zone. Pocket, Block, Zone and Super zone consists of land area of 10, 100, 500 and 1000 hectare respectively. Target of the project is to develop 15000 pockets, 1500 blocks, 300 zones and 21 super zones in this period of ten year from 2100 pockets, 150 blocks, 30 zones and 7 super zones which were targeted for current year 2017. Yearly development plan of pocket, block, zone and super-zone is given in Table 5. The objective of this project is to develop pocket to Block, Block to Zone and Zone to Super zone with gradual improvement in agriculture sector. The major activities proposed in those sectors are given in Fig 1. Role of DFTQC (Food Safety, Technology and Nutrition) while implementing PMAMP project is concerned more on top side (Super-zone and Zone) where post-harvest processing activity exist (Fig 1).

Table 5: Development plan of Pocket,Block, Zone and Super-zone in PMAMP Project FY Pocket Block Zone Super-zone Total 2017/18 2100 150 30 7 2287 2018/19 1433 150 30 1 1614 2019/20 1433 150 30 1 1614 2020/21 1433 150 30 1 1614 2021/22 1433 150 30 1 1614 2022/23 1433 150 30 2 1615 2023/24 1433 150 30 2 1615 2024/25 1433 150 30 2 1615 2025/26 1434 150 30 2 1616 2026/27 1434 150 30 2 1616 Total 15000 1500 300 21 16821 (Source: GON, 2017)

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Super-zone

(Production of processed food from agriculture industrialization,

Research and Development, human resoruce development of agriculture, Marketing of processed food, Food Quality Control)

Zone

(Marketing of raw materials for agriculture industry, Primary processing, Storage, collection and development of resource centre)

Block

(Farm Registration, Productivity increment, Agriculture entrepreneurship development, demonstration and technology transfer)

Pocket

(Primary Production and Agriculture Technology Extension)

Fig 1: Stages in Development of Agriculture sector as aimed by PMAMP Project (Source: GON, 2017)

For modernization in Agriculture Sector, PMAMP strategies linked to DFTQC is Quality Control and assurance in Food Safety. Under this strategy, there is provision of mobilizing mobile laboratory facilities in pocket, block, zone and super zone areas to maintain quality of the production in such area so that food safety is ensured which ultimately contributes to food and nutrition security. Moreover, it also targets to establish internationally recognized laboratories, Rapid Bioassay Pesticide Residue laboratories in major market center to monitor pesticide residue levels, implementation of food safety standards like GAP, GMP, HACCP, RMP etc. and certify the food and agricultural production system, which opens the door for our products to global market.

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At present (FY 2017/18), 7 super zones and 30 zones are identified as per the criteria set up, commodity and Districts for zone and super-zone are presented in Table 6 (column 1, 2 and 3). To conduct the assigned programs based on this criteria, DFTQC has identified responsible offices Table 6 (column 4) and selection of commodities for Research and Development Table 6 (column 5) so as to conduct the program effectively overcoming gaps and overlaps of program. Table 6: Identification of Commodity, District and Responsibility in Super zone/Zone Province Superzone Zone Responsibility Commodity for R&D 1. Paddy Large Cardamom (Panchthar), RFTQCO, Paddy and (Jhapa) Fish (Morang), Maize Biratnagar Large (Khotang), Vegetables Cardamom (Dhankuta), Ginger (Sunsari), Turmeric (Sunsari), Mandarin (Solukhumbu, Udaypur) 2. Fish (Bara) Paddy (Sarlahi), Vegetables RFTQCO, Fish and (Rautahat, Parsa), Mango Hetauda Mango (Saptari) 3. Potato Vegetables (Chitwan), Maize DFTQC, Potato and (Kavre) (Dhading), Potato (Bhaktapur, Babarmahal Junar Nuwakot), Junar (Sindhuli) 4. Vegetables Vegetables (Palpa), Maize RFTQCO, Vegetables (Kaski) (Gulmi, Parbat), Paddy Bhairahawa and (Kapilvastu), Mandarin Mandarin (Syanja), Fish (Rupandehi) 5. Maize Ginger (Surkhet, Salyan), RFTQCO, Maize, (Dang) Turmeric (Surkhet, Salyan), Nepalgunj Ginger and Maize (Banke), Paddy Turmeric (Bardiya, Pyuthan) 6. Apple --- APC, Jumla Apple (Jumla) 7. Wheat Potato (Dadeldhura), Olive RFTQCO, Wheat and (Kailali) (Bajura), Paddy (Kanchanpur) Dhangadhi Olive Total Superzone Zone: 30 Offices: 7 Commodity : 7 : 14 (Compiled from Source: GON, 2017 and DFTQC PMAMP Program of FY 2017/18)

As a first year of this project, DFTQC (along with its offices) is now implementing PMAMP program. The program is basically focused on infrastructure, facility and capacity development of food analysis (Centre, Regional and Quarantine Labs), R&D activities for

DFTQC, FRB 2016/17 development of suitable food processing packages (for the development of food processing industries for agriculture modernization), Survey and baseline data collection from all stages of food chain to improve the food safety and nutrition situation for seven commodities has been assigned in all seven super zones including awareness and capacity development activities of Human Resource from concerned stakeholders group.

Inter-relationship between ADS and PMAMP PMAMP focuses clearly on increasing the production and productivity of agricultural sector via development of necessary technical infrastructure for mechanization in production, processing and marketing of agro products. It has been developed as a supporting project for implementation of ADS. It contributes to all four flagship programs of ADS. The inter- relationship between ADS flagship programs and PM-AMP is shown in Fig 2.

• PMAMP • PMAMP • Value chain development • Establishment of of 18 main crops commercial development • Establishment of agro service centres industries • Establishment of small and • Additional value chain medium enterprises development as per • Subsidy to targeted group necessity • Import of new technology • Development of local crop pockets and value chain VADEP INAGEP GOVERNANCE

PROFITABLE COMPETITIVENESS COMMERCIALIZATION ADS

DESTEP FANUSEP

• PMAMP • PMAMP • Establishment and operation • Increament of production of agriculture education, PRODUCTIVITY and productivity of main extension and research centres crops, Food safety and in blocks, zones and Quality control,promotion superzones of local crops

Fig 2: Inter-relationship between ADS Flagship Program and PMAMP (Note: FANUSEP: Food and Nutrition Security Program;DSTEP: Decentralized Science, Technology, and Education Program; VADEP: Value Chain Development Program; INAGEP: Innovation and Agro-entrepreneurship Program) (Source: GON, 2017)

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On analyzing the inter-relationship (Fig 2), it is clear that PMAMP contributes to all four ADS Flagship programs. In case of Food and Nutrition security program (FANUSEP), it is aimed to conduct the programs that favor overall increase in production and productivity of agricultural stuffs in all areas (pocket, block, zone and super zone) and Development of Food Processing Packages including Food safety and quality control activities. In addition PMAMP program also aimed for the promotion of local crops with product diversification for changes in food habit of consuming locally available foods as well as export promotion of processed foods leading to food and nutrition security both at local/national and international market. Concluding remarks The ultimate goal of agriculture (Both from plant and animal source) is Food and Food without safety and without required nutritional property is actually not called the Food. Latest National Plan (14th Periodic Plan) of Agriculture mentioned two major objectives of Agriculture, i. e. Food Security and Nutrition Security which indicates the importance of Food Safety, Technology and Nutrition to improve the existing agriculture practice to commercial agriculture. ADS and PMAMP are the two major Agriculture Strategies and Programs launched by GON recently to achieve those objectives. Priority of Nation on Food safety, Technology and Nutrition is increasing continuously in the days to come both from the fundamental right perspectives of constitution and from agriculture modernization/export promotion/import substitution perspective of agriculture. However, National policy, Plan and Program on Food safety, Technology and Nutrition are now scattered in several branches within agriculture/livestock sector. In this scenario, there is an urgent need of streamlining this sector by formulating the separate national policy. Formulation of Proper Plan and programs (overcoming existing gaps/overlaps) with adequate network and infrastructure covering all stages of Food Chain and then effective implementation are subsequent vital tools. Food Safety, Technology and Nutrition sector is now of course the prioritized future direction of Nation.

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References GON (1956), First Periodic Plan. 1956 to 1960. Published by National Planning Commission, Government of Nepal. GON (2008a). National Agriculture Policy, 2004 (RASTRIYA KRISHI NITI, 2061 B.S.). In Policy, Acts, Regulations and Orders Related to Agriculture. Published by Ministry of Agriculture and Cooperatives, Government of Nepal. GON (2008b). Agriculture Business Promotion Policy, 2007 (KRISHI BYABASAYA PRABARDHAN NITI, 2064 B.S.). In Policy, Acts, Regulations and Orders Related to Agriculture. Published by Ministry of Agriculture and Cooperatives, Government of Nepal. GON (2011). Golden Jubilee Souvenir of Department of Food Technology and Quality Control (KHADYA PRABIDHI TATHA GUNA NIYANTRAN BIBHAG KO SWARNA MAHOTSAB SMARIKA). Published by Published by Department of Food Technology and Quality Control under the Ministry of Agricultural Development, Government of Nepal. GON (2015), Constitution of Nepal 2072 B.S., Published by Ministry of Law, Justice, Constituent Assembly and Parliamentary Affairs, Nepal. GON (2016). Agriculture Development Strategy (ADS) 2015 to 2035 Part 1, Published by Ministry of Agricultural Development, Government of Nepal GON (2017). Project Document of Prime minister Agriculture Modernization Project (PMAMP), Published by Ministry of Agricultural Development, Government of Nepal World Food Summit (1996). Rome Declaration on World Food Security, 13-17, November, 1996, Rome, Italy.

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Food Quality Control System of Nepal: Existing Practices and Way Forward# Nawa Raj Dahal* and Sanjeev Kumar Karn Department of Food Technology and Quality Control (DFTQC), Babarmahal, Kathmandu

*Corresponding author: [email protected]

#Paper presented on 15th Asian Newtork for Quality Congress jointly organized by Asian Nework for Quality (ANQ) and Newtork for Quality, Productivity and Competitiveness Nepal (NQPCN) on 20-21 September, 2017 at Kathmandu. Abstract Department of Food Technology and Quality control (DFTQC) is responsible agency of Government of Nepal for regulation of Food Quality as per provisions given in Food Act 1966 and Food Regulation 1970. This paper highlights the major provisions on legislations and existing practices of food quality control in the country. Gap analysis of Food Quality Regulation and way forward to improve the existing practice has been reviewed in this article. Keywords: Food Act, Food Safety, Gap Analysis, Quality control System Introduction Food is necessary for human existence. Consuming nutritious and safe food is indispensable for good health. It makes people healthy, stronger and resourceful. This contributes to economic and all-round development of the country. According to FAO/WHO, people all over the place have the fundamental rights to access to food, which is of good quality, safe and nutritious. Safe food is one, which is handled properly at all steps of production to consumption and is unlikely to root illness or injury (World Food Summit, 1996). Thus, the quality and safety of food is the major concern of public. "We are what we eat" is an old proverb. Our nutritional status, health, physical and mental faculties depend on the food we eat and how we eat it. Access to good quality food has been man's main endeavor from the earliest days of human existence. Safety of food is a basic requirement of food quality. Food quality can be considered as a complex characteristic of food that determines its value or acceptability to consumers. Besides safety, quality attributes also include nutritional value; organoleptic properties such as appearance, colour, texture, taste; as well as functional properties (Desrosier and Desrosier, 1998). Health of the consumers is the subject of the public concern from the aspect of Human rights and of course, food is the major public issue. A Bullet can kill a Person at a time but unsafe food may kill thousands of people at a time. This shows the importance of food quality. 16

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Similarly, loss from the consumption of unsafe food is very high, which is not analysed in Nepal yet, however it is estimated that loss of around thirty five thousand million US dollar due to the consumption of unsafe food as a survey conducted at 1997 in USA (GON, 2011). So,it is very important to assess and analyze the status of food quality control mechanism in the country. Historical background on origin of concept of food quality in Nepal is shown by first periodic plan 1956 to 1961 (61 years ago) mentioning the fact of exporting ghee of around 10 Million NRs (~ 100 Thousand USD of that time) to Tibet. However, there appeared a problem on export due to lack of proper processing skill of ghee. Similarly, problem of water adulteration in milk sold in Kathmandu Valley and need of testing the adulteration was also noted on the plan document (GON - 1956). These fact actually initiated the concept of Food Quality Regulation in Nepal. This paper wishes to highlight the major provisions on legislations and existing practices of food quality control system in the country with especial reference to gap analysis and way forward to improve the existing food quality control system in Nepal. The area of this paper is concerned basically on four pillars (Legislation, Inspection, Information education and communication and lastly the Laboratory Surveillance) of Food Quality Control as per Codex criteria. Legislation Constitution and Policy Aspect Constitution of Nepal (2015) mentioned Food Security and Food Souverienity as well as Consumer Right as fundamental right of all citizens. Clause 36 states that every citizen have the right to food (Clause 36a); every citizen will have the right for food security (Clause 36b) and every citizen will have the right for food sovereignty (Clause 36c). Similarly, Clause 44 of the constitution mentioned about the Right of the consumers that every citizen will have the right to consume quality goods and services (Clause 44a) and every citizen will have the right of getting compensation who suffer from non-quality goods and services (Clause 44b) (GON, 2015). Similarly, on analyzing the provisions on policy matters, although the Nation has not issued the separate policy on Food Safety and Quality, however, the certain issues of Food Safety and quality has been addressed through the other policy documents related to Agriculture. National Agriculture policy 2004 has included the provision of safeguarding the health of consumers by setting quality standard of food, quality control, quality certification and food quality regulation for supporting food security and poverty reduction (GON, 2008a). Similarly on Agri-business promotion policy 2006 mentioned the provision on import substitution and export promotion by developing the agriculture based food industry, by improving the quality certification system (GON, 2008b).

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Legislative framework for Food Quality Control Food Act, 1967 and Food Regulation, 1970 are the major legislation for the quality control of food. Indirectly, Standards Weights and Measure Act, 1968 and its Regulation, 1979; Plant Protection Act, 1972, Feed Act, 1976 (Indirectly related to Food Quality), Pesticide Regulation Act, 199,Breast feeding substances (Sales and Distribution control) Act, 1992 and Rules, 1994; Environment Protection Act, 1996; Consumer Protection Act, 1997 and Regulations, 1998; Slaughterhouse and Meat inspection Act, 1998 and Regulation 2000 and Iodized Salt (Production, Sale and Distribution) Act, 1999 are related to food quality which are mandatory. Nepal Standards (Certification Mark) Act, 1980 and Rules, 1994 also deals with the food quality but this act is voluntary which provides the NS Certification Mark to those who applies to receive NS Mark to their products. List of the existing legislation is given in Table 1. Table 1: Existing Legislation on Food Quality Control in Nepal Acts/Regulation Responsible Remarks Institutions Food Act, 1967 and Regulation, 1970 DFTQC Mandatory Standard Weights and Measures Act, 1968 and NBSM Mandatory Regulation, 1979 Plant Protection Act, 1972 DOA Mandatory Feed Act, 1976 (Indirectly related to Food Quality) DFTQC Mandatory Nepal Standards (Certification Mark) Act, 1980 and NBSM Voluntary Rules, 1994 Pesticide Regulation Act, 1991 DOA Mandatory Breast feeding substances (Sales and Distribution DH/DFTQC Mandatory control) Act, 1992 and Rules, 1994 Environment Protection Act, 1996 MOSTE Mandatory Consumer Protection Act, 1997 and Regulations, 1998 DC Mandatory Slaughterhouse and Meat inspection Act, 1998 and DLS Mandatory Regulation 2000 Iodized Salt (Production, Sale and Distribution) Act, DH/DFTQC Mandatory 1999 (Source: GON 1967; GON 1970; GON 1972; GON 1976; GON 1979; GON 1980; GON 1991; GON 1992; GON 1994a/b; GON 1996; GON 1997; GON 1998a/b; 1999; GON 2000) [Note: DFTQC- Department of Food Technology and Quality Control under the Ministry of Agricultural Development; NBSM- Nepal Beaureu of Standards and Metrology under the Ministry of Industry; DOA- Department of Agriculture under the Ministry of Agricultural Development, DH- Department of Health under the Ministry of Health; MOSTE- Ministry of 18

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Science , Technology and Environment; DC- Department of Commerce under the Ministry of Commerce and Supplies; DLS- Department of Livestock Services under the Ministry of Livestock Development.] Among the Acts, Food Act is the major act regarding the Food Quality Control in Nepal. Food Act, 1967 is to prevent the health and welfare of people by prohibiting the adulteration and malpractices in food and by maintaining the proper quality standards in food.Standard Weights and Measures Act, 1968 is to protect the right of people by regulating the weight and measures of consumable products.Plant Protection Act, 1972is to prevent the import and expansion of the hazardous contagious pests and diseases related to plant and plant product.Feed Act, 1976is to promote the fair trade practices of feed by maintaining the quality and safety in feed products. Provision on Food Act-1967 Food Act 1967 has been enforced for the first time in Nepal at 1967 (2023 B.S.) so as to make legal provisions to prevent any undesirable adulteration in food or subtraction or extraction of any natural quality or utility from food and to keep on proper standards of food, for the purpose of maintaining the health and convenience of the general public (GON, 1967). In this sense, the Food Act 1967 can be taken as the first as well as major act related to food quality control. Food Act 1967 was come into force in Nepal in 1967 September 9 (2023.5.24 B.S.). This act has been amended three times at the date of 1970 April 5, 1992 June 3 and 1992 October 22 (GON, 1967). The major subjects given on provisions in Food Act 1967 are as follows. Definition of Food, Adulterated Food and Substandard Food Prohibition on Production and Sale of adulterated food or substandard food Provision of License to be obtained Provision of Punishment Provision of Liability of Offense committed by firm or body corporate Provision of Power to set the quality standard of food Provision of Food Testing Laboratory. CDO as a Case Trying Authority and Provision of Appeal Provision of Powers to frame Rules Food Act has defined food, adulterated (means Dusit in Nepali) and Substandard (means Nyuna-Gunastar in Nepali). Food means any unprocessed, semi processed, processed or produced food or drinking substance which the human being generally consumes and drinks,

DFTQC, FRB 2016/17 and includes any spices, food additives, color or flavor to be used in any food or drinking substance. Adulterated food means any food which is so rotten, decayed or kept or prepared in a dirty or filthy or poisonous condition that it is injurious to health; or the food of which some or all parts have been so made of any diseased or disease carrying animal, bird or injurious vegetation as to render it unfit for consumption by human being; or the food which is likely to be injurious to health because of the fact that any food additive, preservative, inner or outer mixed chemical compound or pesticide level exceeds the prescribed upper limit. Substandard food means any food in which the quantity of the main ingredients of which has been so lowered of with which any other food has been so mixed that its original/natural quality is substandard, provided, however, that if the food with which two or more food substances have been mixed clearly contains the names and quantities of such substances and is not injurious to health, such food shall not be considered as a sub-standard food; or the food of which standard is lower that the quality standard fixed or the minimum required prescribed in the rules or orders framed or issued under the act or of which standard exceeds the maximum standard, if any prescribed under the Food Act 1967. There is the prohibition on Production and Sale of adulterated food or substandard food. No person shall produce, sell, distribute, export or import any adulterated food or substandard food or hold such food for any of such purposes. No person shall sell or distribute any food by lying or misleading that food to be another food or any food of lower standard to be of higher standard. The prescribed authority may, if s/he suspects that any food is an adulterated food or sub-standard food, seal such food, hand over its custody to the owner of that food and receive a receipt thereof from him/her withhold it. After the food withheld pursuant to sub section 1 has been finally decided or held to be an adulterated food or substandard food, Nepal Government nay confiscate such food, by order of the prescribed authority, taking into account of the quantum of guilt, nature of guilt and injury likely to be resulted from it. Any person intending to produce, sell, store or process the prescribed food shall contain the license as prescribed. Notwithstanding anything contained in sub-section 1, into the case of a sealed food, a retailer having obtained the deed of guarantee as prescribed firm the licensed producer or wholesaler shall not be required to obtain the license. The provision of Punishment is summarized in Table 2.

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Table 2: Provision of Punishment on the violation of Food Act, 1967. S.N. Description of Violation Punishment 1 Any person who produces, sells, Fine from NRs 1,000.00 to 2000.00, for the first exports or imports the sub- instances, with a fine from NRs 2000.00 to standard food 5000.00 for each instances from the second instance onwards, or with imprisonment for a term from six months to one year or both. 2 Notwithstanding anything Fine from NRs 50.00 to 200.00 for the first contained in S.N. 1, if an instances and from NRs 200.00 to NRs 500.00 for itinerant seller or vendor who the second instances, or with imprisonment for a sells milk, curd or other food term not exceeding three months and with a fine without opening a shop violates from there months to six months or with both for this Act or the rule or order each instances from the third instance onwards. framed or issued under this act 3 Any person who produces, sells, Fine from NRs 5000.00 to 10000.00 or with exports or imports the imprisonment for a term from one year to two adulterated food years or with both. 4 If, after consuming any Fine from NRs 10000.00 to 25000.00 and adulterated food, any person is imprisonment for a term not exceeding three likely to die or dies or suffers an years; and such producer or seller has to provide irreptabable bodly damage or a compensation in a sum from NRs 25000.00 to likely to suffer such damage, 100,000.00 to the person affected from that adulterated food or his heir. 5 Any person who violates any Fine not exceeding NRs 1000.00. provision of this act or any matter contained in a rule or order framed or issued under this act, other than the matters as referred above. (Note: 1USD~100 NRs) (Source: GON, 1967) On analyzing the provision of punishment when the Food Act was enforced in 1967, the amount now very low at present date of 2017 after 50 years of enforcement of Food Act. Provision on Food Regulation-1970 In exercise of the powers conferred by Food Act 1967, Government of Nepal has framed Food Regulation 1970 at the date of 1970 August 31 (2027.5.15 B.S.). The regulation has been amended five times at the date of 1973 June 1, 1975 October 22, 1991 July 1, 1998 March 23

DFTQC, FRB 2016/17 and 2006 Septerber 30 respectively (GON, 1970). The major provisions mentioned in the Regulation are on the following subjects. • Central Food Laboratory and its procedures • Public Analyst, his qualifications, functions and duties • Food Inspector, their Qualification, Function, Duties and Power • Provision of Sampling and Analysis of Food • Custody of Food and its release if not contaminated • Filing of Cases • Re-analysis of food Sample • Food Standard Setting Committee and Formulation of Food Standard • Label of Packed Food • Matters to be followed by Food Seller and prohibition of sale of adulterated, sub standard or hazardous food • Use of color and preservative in food • Provision of License • Powers to seize license and restrict production • Right to enforce Directive Directives and Working Guidelines Some directives and working guidelines have been developed under Food Act 1967 and Food Regulation 1970, which are mentioned in Table 3. Table 3: Developed Directives/Working Guidelines for Food Quality Regulation Directive/Working Guideline Status Directives on Export-Import Inspection and Approved by Ministry in 2063 B.S. Quality Control Certification System in Nepal - 2063 Directives for production, processing and Approved by Ministry in 2074 B.S. distribution of Meat products, 2074 Directives on Regulation of Food Export/Import, Approved by Ministry in 2074 B.S. 2074 Working guideline for Food Fair Festivals, 2074 Approved by Ministry in 2074 B.S.

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Working guideline on Subsidy for food Approved by Ministry in 2074 B.S. processing entrepreneurs managed by female individuals/groups- 2074 Working guideline on Categorization of Approved by Ministry in 2074 B.S. Hotel/Restaurants including food business based on Food Safety Standards, 2074 Guideline for production of process drinking Approved by Ministry in 2074 B.S. water, 2074 Directives for Food recall Working guideline, Proposed draft submitted to Technical 2074 Committee Directives for Food Industry licensing, 2074 Proposed draft submitted to Technical Committee Directives on regulation of Dietary Supplements, Proposed draft submitted to Technical 2074 Committee Working guideline for management of Cases Preliminary draft submitted to filing accordance to Food Act 2023, 2074 Technical Committee (Source: GON 2016/17) Mandatory Food Standards (Technical Regulations) Setting the mandatory food quality standard i.e. food standardization is the first step on regulation of quality of food, which provides the legal ground for food quality regulation. Although, there are thousands of food products available in the market, Government of Nepal, till date, has formulated (Table 4) the mandatory food standards for only 111 (as generic standard) food products (GON, 2012). Table 4: Generic Standard developed by Government of Nepal Food Group Commodity Milk and Milk Cow milk, Buffalo milk, Ghee, Processed milk, Evaporated milk, Products Evaporated skimmed milk, Sweetened condensed milk, Skimmed Sweetened Condensed milk, Partially skimmed Sweetened condensed milk, Butter, Cream, yoghurt, Infant milk food, Infant food, whole milk powder, Skimmed milk powder and Paneer (17) Fats and Oil Mustard oil, Imported rapseed oil, Soybean oil, Palm oil, Palm kernel products oil, Palmolein, Groundnut oil, Coconut oil, sesame oil, Corn oil, sunflower oil, olive oil, Safflower oil, Refined vegetable oil, Vegetable ghee and Bakery shortening (16)

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Fruits and Fruit juice, tomato juice, fruit syrup, fruit squash, fruit beverage, Vegetable tomato sauce, Jam, Pectin mixed jam, marmalade, chutney, canned Products fruit cocktail, canned pineapple, canned orange segment, canned pears, canned lapsy, canned relish and pickle (17) Spices Products Cardamom Capsule, Cardamom seed, Cardamom powder, Dried ginger, Ginger powder, Turmeric Rhizome, Turmeric powder, Cumin seed, Cumin powder, Pepper seed, pepper powder, Chillies, Chillies powder, Coriander seed, Coriander powder, Fenugreek, Cinnamone whole, Ajowan seed, Whole clove, Spice powder, Cinnamon powder and Fennel (22) Tea, Coffee Tea, Coffee powder, Soluble instant coffee powder and green tea (4) Salt Common salt and iodized salt (2) Cereal grains and Food grain, whole wheat flour (atta), wheat flour (maida), Semolina, their products bread, biscuit, stick noodles, instant noodles, whole green gram, split green gram, dehusked split green gram, Red gram, whole black gram, split black gram, whole Bengal gram, split Bengal gram, whole lentil, dehusked lentil, Besan, wheat grain, maize grain, fortified whole wheat flour, fortified wheat flour, corn flakes and Rice (25) Processed Mineral water (1) Drinking Water Sweets Sugar, Mishri and honey (3) Confectionary Sugar boiled confectionary, lozenges and chewing gum/bubble gum (3) Meat Luncheon meat (1) Total 111 commodities (Source: GON, 2012).

It is obvious that Government of Nepal need to give high priority for formulation of food quality standards which are not covered in food standardization. However, Government of Nepal, has formulated the horizontal standards on some of the safety parameters (Table 5). Horizontal standard means the common standard which can apply to all food commodities of the similar food group. Horizontal standards on some of the preservatives (sulphur dioxide, benzoic acid, nitric acid and sorbic acid) as well as some of the toxic heavy metals (Lead, copper, arsenic, tin, zinc, cadmium, mercury, chromium and Nickel) on specific foods are included by Government of Nepal.

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Table 5: Horizontal Standard developed by Government of Nepal Parameters Initiatives

Food Additives Food Color, Preservatives, Stabilizers, Firming agents, Acidity regulators, Emulsifiers, Anti-caking agents, Anti-oxidants and monosodium glutamate are included.

Food Pesticide residues, aflatoxin, heavy metals (Lead, Copper, Arsenic, Contaminants Tin, Zinc, Cadmium, Mercury, Chromium, Nickel), radioactivity and microbiology are included

Food Labeling Labeling requirements include the name of the food product; name and address of the manufacturer; name of different ingredients used; weight and volume of the product; name of preservatives and colors used, batch no.; date of manufacturing and expiry date; Label should be written in Nepali or English or both and any other language may be used. (Source: GON, 2012) Responsible Institution for Food Quality Regulation Government of Nepal has established Food Department under the Ministry of Agriculture for implementing Food Act and Regultion in 1961 (2018 B.S.) within the Singhadarbar premises. With revision of name of this Department as various forms afterwards, now it is named as the Department of Food Technology and Quality Control in 2000 (2057.03.06 B.S.). In 1967, the department was renamed as Food Research Laboratory and at that time, Food Act was also enforced. In 1970, Food Regulation was enforced and at the same time Food Grain Standardization Testing Laboratory was established at Bhadrapur of Jhapa district under the support of USAID. Pilot plant unit was established at Kathmandu for the food processing and research and task began for the standardization of foods like ghee, oil and milk. In 1972, Food Research Laboratory was again renamed as Food Research unit of Food and Agriculture Market Service Departement under the Ministry of Food, Agriculture and Irrigitaion. In 1978, Eastern Food Laboratory was established at Dharan which was later shifted to Biratnagar at 1996. In 1980, the Food Research unit was again renamed as Central Food Research Laboratory under the Ministry of Agriculture and at the same time, food quality regulation, insection and certification work was initiated in Nepal. In 1992, It was again renamed as Food Directorate under the Department of Agriculture Development and at that time more regional offices were also set up with the establishment of Regional Food Laboratories at Hetauda, Pokhara (Later transferred to Bhairahawa at 2004), Nepalgunj and Dhangadhi as well as Apple 25

DFTQC, FRB 2016/17 processing Center at Jumla. In 1995, Food directorate was renamed as Central Food Research Laboratory under the Ministry of Agriculture and at the same time Food Act was aslo enforced to all of the 75 districts. Afterwards only at 2000, the Central Food Research Laboratory was again renamed as Department of Food Technology and Quality Control (DFTQC) with Departmental Portfolio. At 2004, when Nepal was assessed to World Trade Organization (WTO) as a WTO member, the importance of this department also highlighted by the government with establishment of four Food Quarantine Laboratories (At Kakarvitta, Birgunj, Mahendranagar and Tatopani), one SPS Enquiry Point at Kathmandu, one Custom Food Inspection Unit at Tribhuvan International Airport, and twenty District Food Inspection units at Jhapa, Sunsari, Saptari, Siraha, Udayapur, Dhanusha, Mahottari, Sarlahi, Chitawan, Bara, Parsa, Rautahat, Nawalparasi, Kapilbastu, Kaski, Tanahu, Bardiya, Surkhet, Dang and Kanchanpur. In this way, the structure of this department has been extended to 32 places including 11 offices, 20 district units and one Food Inspection unit at Tribhuvan International Airport. Food Act 1967 and Food Regulation 197 are the major act related to food quality.However lack of Food Safety policy, very old Food Act and Regulation and the provisios reflected in these Act and Regulation, large number of other acts directly or indirectly related to Food Quality with Gaps and overlaps among the acts, lack of umbrella act for regulation of consumable goods, scattered institution for individual acts, their independent implementation strategy and lack of coordination among the institutions are the major challenges of food quality regulation system in Nepal. Furhtermore, the existing capability of the regulation implementing institution was found limited due to incompatible legislative provision, limited structural network, limited human resources and due to limited financial resources allocated. Organizational Network of DFTQC DFTQC has one Central office in Kathmandu - Three Divisions (Food Quality Control Division, Central Food Laboratory, Food Technology Development and Training Division), One National Nutrition Programme, One SPS National Enquiry Point, One Planning, Monitoring and Evaluation Section, One Administration Section, One Finance Administration Section, One Legal Section; Five Regional Offices at Biratnagar, Hetauda, Bhairahawa, Nepalgunj and Dhangadhi; Four custom laboratories at major custom points - Kakarvitta, Birgunj, Mahendranagar and Tatopani; Twenty District Food Inspection Units at Jhapa, Sunsari, Saptari, Siraha, Udayapur, Dhanusha, Mahottari, Sarlahi, Bara, Chitwan, Parsa, Rautahat, Nawalparashi, Kapilbastu, Kaski, Tanahu, Bardiya, Surkhet, Dang, Kanchanpur, One Food Inspection unit at Tribhuvan International Airport and One Apple Processing Center at Jumla. On analyzing the existing structural network, it can be found that the existing network

DFTQC, FRB 2016/17 of DFTQC is only confined to the urban area, all districts are not still covered by DFTQC network. Altogether, there are 237 posts of sttaffs within DFTQC Structure at central, regional and district levels. There are 137 posts for central office and 62 posts in five regional offices .i.e., Biratnagar, Hetauda, Bhairahawa, Nepalgunj and Dhangadhi. Similarly there are 13 posts at four food quarantine labs i.e. Kakarvitta, Birgunj, Mahendranagar and Tatopani; 6 posts at Apple processing center Jumla. Ninteen districts of terai and inner terai region,Jhapa, Sunsari, Saptari, Siraha, Udayapur, Dhanusha, Mahottari, Sarlahi, Chitwan, Parsa, Rautahat, Nawalparashi, Kapilbastu, Kaski, Tanahu, Bardiya, Surkhet, Dang, Kanchanpur consists one/one food inspector in each districts at CDO offices and one Food Inspector at Food inspection unit at Tribhuvan International Airport. Inspection Inspection is second pillar of Food Quality Control. As per the inspection protocol mentioned in Food Act, Food inspector (an authentic person deputed by the Government for investigation), deputed by Department of Food Technology and Quality Control and its regional offices, will initiate the investigation. The investigation is initiated generally by three ways: (i) Regular investigation as per the approved programme of DFTQC and its regional offices, (ii) Complaints received by Consumer/Consumer Forum for inspection, sample collection and further actions and (iii) Request received by other agencies for inspection, sample collection and further actions. Firstly, Food inspector inspects market, hotel, restaurants and food industry whether the guidelines provided by DFTQC for maintaining the quality are being implemented or not. After the inspection, Food inspector will collect the suspicious food sample from the respective premises and forward the collected sample to the Public Analyst (an authentic person deputed by Government for the analysis food; generally Public analyst work at the Laboratories of DFTQC and its regional offices). The public analyst will analyze the food sample as per the quality standards set by the government and he/she will submit the analysis report to the respective food inspector clearly mentioning that whether the quality of the sample found within the quality standards set by the government or adulterated or substandard. In case of adulterated or substandard sample, the food inspector will begin the investigation. (During the investigation, he/she will enquiry to all of the respective entrepreneurs in the food chain as well as other investigations about the facts how the food has been adulterated or 27

DFTQC, FRB 2016/17 substandard and who may be the guilty on the case. He/she after his investigation will prepare the report in a file and submit the file to the Government Layer for approval to go ahead to file the case at CDO office. Government Layer, after receiving the file from the inspector, will review all of the documents and forward his decision to the food inspector whether the procedures followed by the food inspector and the evidences attached in the file are complete to case the file. After getting approval from the Government Layer, the food inspector will prepare the Accuse Letter mentioning the respective person who is liable for the punishment and the extent of the punishment, clearly mentioning the provisions as per the law. The prepared case is then filed to the CDO office. CDO, then, will again review the file and issues his decisions for the punishment. If any person who is not satisfied with the decision made by the Chief District Officer, may file an appeal in the appellate court within thirty-five days after the date when the decision was made. In this way, the regulation process end as per the existing Food law (GON, 2010). This way the inspection and further action will be terminated as per the Food Act 1967. Besides the inspection by food inspector as per the Food Act, other inspections mechanisms also exist in the country as governed by other legislative provisions (Mentioned in legislation part above). For this, multiple other agencies (Department of Consumer Welfare Protection, Chief District Officer, Police, Nepal Bureau of Standard and Metrology, Department of Health, Local government) are also involved in food inspection on individual as well as on joint inspection basis. The Composition of Joint Inspection Team /Rapid Response Inspection includes Food Inspector, Representative from Local Authority, Representative from Police, Representative from Customer Associations, Representative from Local Govt., Representative from Department of Agriculture, Representative from Department of Livestock and even the Media Personnel for the publicity of the fact regarding inspection to the public. Information, Education and Communication Information, Education and Communication activities related to food quality (safety and nutritional aspect) is being conducted by multiple agencies in Nepal. These includes 1) DFTQC: Consumer Education Section for conducting the program + 5 Regional offices + 4 FQLs + One APC, Jumla. 2) DOCS: 3) NBSM: 4) DOA: Farmers education on the use of Pesticides 5) DLS: Farmers education on the use of vet drugs/antibiotics 6) DOH: 7) Consumer Associations (Mandate of CA is Consumer education) 8) Local government 9) Manufactures and Suppliers by themselves and 10) NGOs/INGOS The general medium of IEC being used are: 1) Printing form, Magazines, Daily newspapers, leaflets, Posters, booklets, Books, Flex, Banner, Hoarding Board 2) Oral programs, Radio, FM

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3) Visual display: TV, Display 4) Food Fair programs 5) Food Safety day programs 6) Consumer Meetings 7) Interaction programs 8) Workshops and 9) Trainings (To reporters, Producers, Distributors, homestay, government staffs and other related stakeholders). Among the various stakeholders, the information education and communication materials disseminated by DFTQC for fiscal year 2017/18 was reported in total of 876 times through FM, radios and Television. Laboratory Surveillence Laboratory surveillance activities for food analysis is being provided by Central Food Laboratory of DFTQC including its 5 regional laboratories of its reginal offices (Biratnagar, Hetauda, Bhairahawa, Nepalgunj and Dhangadhi), 4 food quarantine laboratories (Kakarvitta, Birgunj, Mahendranagar and Tatopani). Among the various laboratories of DFTQC, Central Food Laboratory CFL) has been accreditated on ISO 17025 International standards for analysis and testing of total 27 chemical parameters (Actual Parameters 21) on different food commodities by National Accreditation Borard (NABL) of India at september 12, 2012, the details of food commodities and parameters accreditated is presented in Table 6. Table 6: Existing Scope (Chemical discipline) of Lab Accreditation of CFL, DFTQC Commodities Parameters Fats and Oils Free fatty acid, Refractive index, Acid value, Peroxide value (4) Fruits and vegetables Total soluble solids (TSS), Acidity (2) Ginger and Cardamom Volatile oil (1) Tea and coffee Total ash, Water etracts (2) Instant noodle, Biscuit, Infant food Moisture, Proteins (2) Honey Moisture, Acidity (2) Processed water pH, Hardness, Alkalinity, Chloride content, Calcium, Copper, Iron, Magnesium, Zinc (9) Skimmed/whole milk powder, Milk fat, Moisture, Protein, Total Ash, Fat (5) condensed milk (3) Commodities of total 8 groups Total Parameters (27), Actual Parameters (21) (Source: GON, 2016/17). CFL again extended the scope of analysis for total 17 microbiological parameters (Actual Parameters 4) on 2016 by the same organization, NABL of India (Table 7).

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Table 7: Existing Scope (Biological discipline) of Lab Accreditation of CFL, DFTQC Commodities Parameters Fruits and vegetable products (17) Total Bacterial Count, Yeast and Mould Count, Coliform count Cereals and Bakery products (8) Total Bacterial Count, Yeast and Mould Count, Coliform count Water (1) Total Bacterial Count, Yeast and Mould Count, Coliform count, Escherichia coli Milk and Milk Products (17) Total Bacterial Count, Yeast and Mould Count, Coliform count, Escherichia coli Meat and Meat products Total Bacterial Count, Yeast and Mould Count, Coliform count, Escherichia coli Commodities of total 5 groups Total Parameters (17), Actual Parameters (4) (Source: GON, 2016/17).

DFTQC has taken the policy of accreditation of CFL for all food safety parameters. To achieve this goal, the department has given top priority with allocation of necessary facility and logistics required for the task. In this connection, DFTQC has approved the plan of extention of scope of accreditation on total 47 parameters covering chemical contaminants including pesticide residues, food additives, micotoxins, heavy metals and other contaminants, the details of scope extension plan is given in Table 8.

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Table 8: Plan of CFL for extension of scope on Lab Accreditation for FY 2017/2018 Commodities Parameters Fruits and vegetables Sulphur dioxide (SO₂), Benzoic Acid (2) Products Tea and coffee Caffeine (1) Cereals and cereals products Total aflatoxin, Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, (Instant noodles, Biscuits, Aflatoxin G2, Zinc, Calcium, Magnesium, Iron (9) snacks etc.) Honey Hydroxyl methyl furfural (HMF) (1)

Meat products Sodium nitrite (1)

Processed drinking Water Lead, Cadmium, Arsenic, Mercury (4) Fruits and vegetables Organochlorine pesticides Alpha-BHC,SS; Gamma-BHC (Lindane), SS; Beta- BHC,SS; Delta-BHC,SS; Heptachlor,SS; Aldrin,SS Isomer B; SS Heptachlorepoxide; Gamma-chloradane,SS; Alpha- chlordane,SS; SS Endosulfan I (alpha); 4,4’-DDE,SS; Dieldrin,SS; Endrin,SS; SS Endosulfan II (beta); 4,4’- DDT,SS; EndrinAldehyde,SS; EndosulfanSulfate,SS; 4,4- DDT,SS; Endrin Ketone SS; Methoxychlor, SS (20) Organophosphorus pesticides O,O,O- Triethylphosphorothioate; Thionazin; Sulfotep; Phorate; Dimethoate; Disulfoton; Methyl Parathion; Parathion; Famphur (9) Commodities of total 7 Total Parameters (47), Actual Parameters (47) groups (Source: GON, 2016/17) Status on Quality of Food On assessing the current food quality situation in Nepal, based on the annual publication of responsible organization, it is noticed that around 10 to 12 % of the foods available in the market are found adulterated and substandard (Table 9).

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Table 9: Total samples collected, substandard samples and cases filed by DFTQC

S.N Status 2012/13 2013/14 2014/15 2015/16 2016/17 1 Total Samples 2834 2808 3064 3675 3604 Collected* 2 Substandard** 317 326 282 379 458 Samples 3 Percentage of 11.2 11.6 9.2 10.3 12.7 Substandard** 4 Cases Filed 297 284 220 204 263 Note: *Indicates Food Samples Collected by Food Inspector with respect to investigation on Market and Industry Status on Food Quality. ** includes both adulterated and substandard samples. [Source: (GON, 2012/13); (GON, 2013/14); (GON, 2014/15); (GON, 2015/16) and (GON, 2016/17)] Although it is not the national status conducted scientifically (only the outcome of the samples collected by Food inspectors in some defined market during inspection), it can be noticed that situation of food quality is poor in Nepalese market. Food industries and hotel restaurants are also inspected and monitored on regular basis (Table 10). Table 10: Industry, Hotel Restaurants Inspection and Total Number of Samples Analyzed S.N Status 2012/13 2013/14 2014/15 2015/16 2016/17 1 Industry Inspection* 1468 1206 1452 950 1400 2 Hotel Restaurant 1176 1745 2005 1989 1064 Inspection 3 Total Number of 27344 33646 36737 43056 49089 sample analyseed [Source: (GON, 2012/13); (GON, 2013/14); (GON, 2014/15); (GON, 2015/16) and (GON, 2016/17)] On an average around 40 to 50 thousands samples received annually in laboratories (One Central Food Lab, Five regional Labs, four quarantine labs and one apple processing centre Jumla lab) under the Department of Food Technology and Quality Control, the trend of receiving the samples in laboratories in increasing yearly which is also reflected in Table 10 This indicates increasing awareness of stakeholders on food safety and quality issues.

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On assessing the status of food industry licensing , which is the major responsibility of the producer for maintaining the quality standard of food products supplied in the market, it is observed that only around one thousand food producing company are covered within the regulatory framework (Table 11). Table 11: Situation of Licensing, Renewal and Recommendations for Food Industries for Fiscal Year 2016/17 S.N Status Biratna Hetaud Kathma Bhaira Nepalg Dhanga gar a ndu hawa unj dhi 1 Lisencing, New 134 84 118 85 48 48 Issue 2 Renewal 262 287 458 196 181 128 3 Recommendation 150 266 327 126 93 51 s [Source: (GON, 2016/17) It seems that Nepalese food processing entrepreneurs also need to be sensitize on food licensing process, Governent of Nepal and Food producing enterprizes both simultaneously work together for assuring the quality of food sold in the market. Hotel restaurant categorization approach was initiated by DFTQC from 2012 and till 2017, 747 hotels and restaurants were categorized as per the food safety prespective (Table 12). Among 747 hotels and restaurants, only 20 found good as per the food safety standards. This seems food hygiene and safety situation of hotels and restaurants need major concern to improve the food safety situation in Nepal in the days to come. Table 12: Situation of Hotel Restaurant Categorization S.N Category FY 2016/17 Till date 1 Good 20 106 2 Average 41 455 3 Low 17 185 4 Bad 1 1 Total 79 747 (Source: GON, 2016/17) Budget Investment on Food Regulation Budget allocated for the responsible organization for implementing food quality control programmes, i.e. DFTQC, and revenue collection by DFTQC was analysed. While assessing the national budget allocated, it is noticed that government of Nepal allocating Agriculture Development budget of around 2% of National budget (2.4% for fiscal year 2017/18) (Table 33

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13). Agriculture Development budget means budget allocated including three major departments of the Ministry, i.e. Department of Agriculture, Department of Livestock Services and DFTQC. Table 13: National Budget Allocated for Food Quality Control S.N Description 2013/14 2014/15 2015/16 2016/17 2017/18 1 National Budget* 517,240,0 618,100,0 819,468,8 1,048,921 1,278,994 00 00 84 ,354 ,855 2 Budget for Agriculture Development (also includes livestock development budget) Budget allocated* 21,403,12 23,283,17 26,682,58 35,903,82 30,396,67 7 8 0 2 5 Share of total 4.1% 3.8% 3.3% 3.4% 2.4% National budget 3 Budget for Food Quality Control Budget allocated* 144,484 139,231 213,973 312,987 304,717 Share of total 0.028% 0.022% 0.030% 0.026% 0.024% National budget Share of total 0.67% 0.60% 0.80% 0.87% 1.00% agriculture budget Note: * Budget in thousand Nepalese Rupees. (1 NRs ~ 100 USD). [Source: (GON, 2013/14a); (GON, 2014/15a); (GON, 2015/16a); (GON, 2016/17a); (GON, 2017/18a) It can be noticed that Government of Nepal has allocated only 0.024% of National budget on food quality control activities. This is only around 1% of total Agriculture budget (FY 2017/18). This shows that investment on food quality control activities is comparatively low and Government of Nepal may give high priority on food quality control activities.

On the other hand, by assessing the revenue collection, it is observed that DFTQC is collecting revenue accounting around 7% (for FY 2016/17) of the budget allocated (Table 14). The revenew is mainly collected as analysis fee of food samples and on issuing and renewing food license.

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Table 14: Revenew Collected from Food Quality Control Activities

S.N Description 2012/13 2013/14 2014/15 2015/16 2016/17 1 Revenew 14,699 20,610 20,767 20,065 22,894 collected* 2 Share on the 16.1% 14.3% 14.9% 9.4% 7.3% budget Note: * National figure on collected revenew in thousand Nepalese Rupees (1 NRs ~ 100 USD). [Source: (GON, 2012/13; GON, 2013/14); (GON, 2014/15); (GON, 2015/16); (GON, 2016/17)

Although DFTQC is not the responsible institution of revenue collection (its main mandate is to assure the quality and safety of food for protecting the health of consumers), it is noticed that there is a possibility of increasing the figure of revenue collection in coming days, as the number of food samples in the laboratory is increasing and government of Nepal will enforce food regulation extending the structure of DFTQC to the local level.

Gap Analysis and Way Forward Legislation Although food quality control activities have been initiated before six decades, the effectiveness of those activities need to improve on various aspect. Among them, legislation is the major one. The Gaps on legislation and way forward to address those respective gaps are given in Table 15. Government of Nepal is to formulate Food Safety policy, is to revise old food act and old food regulation. Although, formulation of standard of food product is a technical task, it needs modern analytical services to analyse all of the quality and safety parameters as well as scientific survey on food products available in the market. DFTQC (an authentic organization of Government of Nepal to set up the national mandatory standards) is to focus its activities to add more products annually on this regulatory framework. At present time, DFTQC regulation protocol is to collect sample and to proceed legal actions with lengthy legislative procedure. However, promotional activites, to adopt code of good manufacturing practices by the producers itself is the common practice to produce safe and quality food products. DFTQC now is to adopt international risk based system promoting food industries to follow international healthy manufacturing practices in the days to come. The obligations of WTO especially SPS and TBT obligations are the major obligations regarding

DFTQC, FRB 2016/17 food quality and safety. In this regard, Government of Nepal need to concentrate its priority to fulfill those obligations.

Table 15: Gaps on Legislation and Way Forward Gaps Way Forward Initiatives taken by GON Overlaps (Gaps as well) One Umbrella Act to in legislation by regulate consumable different legal goods and services and documents to different one institution to control institutions (DFTQC, the quality of Food. DOCSMD, NBSM, Assignment of clear CDO, Police, Consumer mandate of regulation to associations, Local all related institutions. governments) (Food Quality- DFTQC Food Price + Non Food commodities and services – DOCS, Food Quantity (Wt./Volume)– NBSM Assurance of Good Correlation of Mandate and Expertise. Lack of Food Safety Formulation of Food Draft in Ministry at final Policy till date. Safety Policy to stage for submission to streamline the regulation Cabinet for approval Food Act of Five Amendment of Food Draft submitted to Ministry. decades back not Act 1967 compatible with Modern Food Safety Principles Penalty provision in the Amendment of Food Draft submitted to Ministry. Food act at the time of Act 1967 50 years ago now very minimal due to time value of money plus other legal perspectives Food Regulation To be amended On Draft stage at Department Food Standards Setting of standard for Setting new standards and all food products and revision as per the regular harmonization as per the program codex standard 36

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Gaps Way Forward Initiatives taken by GON Directives, Working All required Directives, Seven Directives approved Guidelines, Code of Working Guidelines and and four other are on Practices Code of practices are to pipelines (Table 3) be formulated and approved by Ministry. Food Export import Formulation of export On Draft stage at Regulation import regulation Department Lack of Directive on To be approved by Draft submitted to Ministry. GAP Certification Ministry Lack of directive on To be formulated and Draft submitted to Ministry. RMP Implementation approved by Ministry and certification Note: DFTQC- Department of Food Technology and Quality Control, DoCSMD - Department of Commerce and Supply Management, NBSM- Nepal Bureau of Standards and Metrology, CDO- Chief District Officer)

Inspection As Inspection system in a part of legislation, various gaps are also observed on food inspection system and those respective gaps are analysed as given in Table 16 with way forward to address those gaps. Table 16: Gaps on Inspection and Way Forward

Gaps Way Forward Initiatives taken by GON Overlaps (Gaps as well) Assignment of clear on Inspection in mandate of regulation to accordance to all related institutions. duplication of Assurance of Good assignments by different Correlation of Mandate legal documents to and Expertise different institutions (Food Quality- DFTQC, (DFTQC, DOCSMD, Food Price + Non Food NBSM, CDO, Police, commodities and services – Consumer associations, DOCS, Food Quantity Local governments) (Wt./Volume) – NBSM Traditional inspection Risk based inspection Priority on the development and sampling from Statistical Sampling and of Directives, Working suspected lot as well as scientific transportation guidelines and code of end product testing and storage mechanism practice to address the issue mechanism. (Table 3). 37

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Gaps Way Forward Initiatives taken by GON of samples before timely delivery to lab. System certification (GAP/ GHP/ GMP/ HACCP/ FSMS/ RMP etc) Lack of structure on Extension of structure O & M as per Federal district level upto Local level. System Number of Food Assignment of Food O & M as per Federal Inspectors low and Inspectors in each System capability to be updated Municipality Absence of Food Food Inspection with inspector during Food expert. Assurance inspection (esp in food of Good Correlation of manufacturing Mandate and Expertise premises) creating (Food Quality- DFTQC, misinformation due to Food Price + Non Food the lack of expertise and commodities and misleading the mandate services – DOCS, Food of inspection Quantity (Wt./Volume) – NBSM Inspection tools Good Inspection (Sampling tools, Practice in scientific Inspection Action plan, way. Assurance of Good approved and complete Correlation of Mandate Inspection format, and Expertise (Food harmonization of Quality- DFTQC, Food inspector’s capability to Price + Non Food produce the commodities and reproducible result of services – DOCS, Food inspection outcomes to Quantity (Wt./Volume) be improved. – NBSM Note: DFTQC- Department of Food Technology and Quality Control, DoCSMD - Department of Commerce and Supply Management, NBSM- Nepal Bureau of Standards and Metrology, CDO- Chief District Officer) DFTQC is now working at only around 3% capacity of inspection i.e. only 31 food inspectors all over Nepal and food analyst are only 40 pesonnels i.e around only 16% capacity of lab analysis (Table 17). But international norms of food inspectors is that One inspector for around 30,000 (Thirty thousand) populations. Based on this criteria and taking consideration of total population of Nepal, as per the census 2011 (i.e. 26620809 Populations), Nepal Government 38

DFTQC, FRB 2016/17 need to appoint 887 food inspectors all over the country for effective regulation on quality of food. Table 17: Supply and Demand of Food Inspectors/ Food Analysts Posts Existing posts Requirement Total posts 237 around 1300 Food Inspectors 31 (3% of standard) 887 Food Analysts 40 (16% of standard) 250 (16000 samples annually) (30000 samples annually) Note: 1. Required posts for food inspectors are calculated based on the international requirements of one food inspector for 30,000 populations and considering the population census 2011 recently carried out by Central bureau of statistics (26620809 populations). 2. Required posts of food analyst was calculated as per the requirement of one analyst for analysis of one samples at three days considering 30,0000 samples to be analyzed annually. Information, Education and Communication (IEC) IEC is one of the main pillars of food quality control. The scale of consumer awareness and knowledge of consumers on food quality, safety and nutrition are the key factors for effective IEC activity. The gaps on existing IEC activities and way forward to address those respective gaps are given in Table 18.

As quality of food is the major concern of all of the citizens, there is the responsibility of all of the stakeholders (including consumers, producers, distributors and sellers, traders) on IEC. So all of the sectors of this broad chain is to be aware on the matter of food quality and their individual responsibility for making food safe. To achieve this objective, DFTQC, is conducting consumer awareness activities with its annual programmes, However, it is realized that Governemnt of Nepal need to conduct awareness programmes massively scaling up the recent activities including stakeholders in the broader periphery.

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Table 18: Gaps on IEC and Way Forward Gaps Way Forward Initiatives taken by GON Scale of IEC Program Government focus to narrow, not reached to increase scale of all consumers program and budget effectively. allocation. Consumer associations to focus on their main mandate of IEC. Program scattered (each IEC program is to be institutions going on streamlined with high their own way, priority OVERLAPS/GAPS Lack of expertise on IEC Development of material development Appropriate and specific and Dissemination. IEC material with (Wrong information) specific target group in Interpretation to good communicable consumers by those manner. persons who are not Dissemination of subject matter specialist. Information by e.g. Case of Nitrite, respective MSG experts/authorities Low priority of local Local government to Appointment of Peoples government on IEC give priority representation at local level

Laboratory Surveillance Food Analysis data from Laboratory are the basis for differentiating quality food from unsafe or adulterated or low quality or bad quality of food. The data genereated are basic scientific and legal tools for initiating and improving the quality control system. It is often said that “quality control without a good laboratory is like a tiger without teeth” which supports the importance of laboratory surveillance for food quality control. Although there are several labs in government and private sectors, there are several areas for improvement in this sector. In this connection, related Gaps and way forward to address those respective gaps are given in Table 19. Regarding the human resource for laboratory analyst, DFTQC is now working at only around only 16% capacity (including all Labs, 1 Central Lab, 5 Regional Labs, 4 Food quarantine Labs) analysis (Table 17). Considering the requirement of three days for complete analysis of one sample by one food analyst, it is observed that Government of Nepal need to appoint 250

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Food analysts (considering 30,000 samples to be analyzed annually) for effective and timely analysis of food samples received in the laboratory. Regarding the recognition on its analytical capability by international standard, Central Food Laboratory of DFTQC is recently accreditated for 27 chemical and 13 microbiological parameters (Table 6 and 7), the process of accreditation for safety parameters is on the way (Table 8), and it is expected that Central Food Laboratory of DFTQC will be accreditated for all safety parameters by coming 5 years. The process of accreditation needs to extend from Central to Regional Labs and ultimately to Quarantine labs too in the days to come. Table 19: Gaps on Laboratory Surveillence and Way Forward Gaps Way Forward Initiatives taken by GON Lack of Reference Food Establishment of Reference Concept and Directives on Lab (Lack of Food Lab. Reference Food Lab on draft coordination and stage. regulation of all government and private labs) Facilities for testing Facility development and CFL accreditated for 27 chemical contaminants, addition of scope of all chemical parameters (Table residues not fully safety parameters on 6) on 2012. and for 17 developed, Lab service accreditation plan. Plus microbiological parameters is not compatible to Strengthening Regional and (Table 7) on 2016 with customer demand Quarantine labs. Priority plan for accreditation for pesticide residues, heavy metals and other contaminants and toxins (Table 8). Testing Methodology of Development of Reference samples not harmonized Testing Manual and Method among the labs, Validation mandatory to all Validation of Methods labs. GLP mandatory to all labs. Reporting format of Harmonization of Reproting analysis of sample not format to all labs harmonized among the labs.

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Opportunities of Quality Control Although there are various gaps for effective quality control system, Nepal also has many opportunities to cope with the challenges faced in this regard. As a member of WTO, Nepal has opportunities for strengthening capacity for the regulation of export and import and increased interest of development partners in technical assistance and other support. Increasing awareness among the policy makers, political leaders on the importance of food quality and safety and continuous advocacy by Consumer Associations on the need of strengthening the capacity of government in the area of food quality and safety are other strengths. Opportunity of expansion of its structure on three tier of government system of federal system (i.e. Federal, Province and Local Level) during the restructuring on Federal system and increased concern and initiation of local governments especially municipalities for ensuring quality and safety of food products are supportive fact for improving the existing system. Similarly increased international and regional efforts for increasing the capacities of national competent authority are additional areas of strength on food quality control system of Nepal.

Concluding Remarks

Quality, Safe and nutritious food is the basic right to all consumers and it is the responsibility of the Nation to assure these aspects. For the purpose of public health protection (as well as to promote food trade) Government of Nepal had formulated Food legislation and assign responsibe institution to perform the regulation task five decades back. However there are several challenges and gaps for effective quality control system at this moment. Adoption of system approach (GAP, GVP, GMP, GLP, HACCP, RMP, FSMS etc) in all steps of food chain starting from the beginning seems now indispensable for sustainable improvement and effective quality control system. It is expected that Integrated single institution for food quality control (covering all steps of farm to fork) with adequate facility and effective tools would be the best option for effective quality control for food sector in Nepal.

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GON (2013/14). Annual Bulletin, 2070/71 B.S. Published by Published by Department of Food Technology and Quality Control under the Ministry of Agricultural Development, Government of Nepal. GON (2013/14a), RedBook ((2013/14). Statement of Expenditure 2070/71 B.S. Published by Ministry of Finance, Government of Nepal. GON (2014/15). Annual Bulletin, 2071/72 B.S. Published by Published by Department of Food Technology and Quality Control under the Ministry of Agricultural Development, Government of Nepal. GON (2014/15a), RedBook ((2014/15). Statement of Expenditure 2071/72 B.S. Published by Ministry of Finance, Government of Nepal. GON (2015), Constitution of Nepal 2072 B.S., Published by Ministry of Law, Justice, Constituent Assembly and Parliamentary Affairs, Nepal. GON (2015/16). Annual Bulletin, 2072/73 B.S. Published by Published by Department of Food Technology and Quality Control under the Ministry of Agricultural Development, Government of Nepal. GON (2015/16a), Red Book ((2015/16). Statement of Expenditure 2072/73 B.S. Published by Ministry of Finance, Government of Nepal. GON (2016/17). Annual Bulletin, 2073/74 B.S. Published by Published by Department of Food Technology and Quality Control under the Ministry of Agricultural Development, Government of Nepal. GON (2016/17a), Red Book ((2016/17). Statement of Expenditure 2073/74 B.S. Published by Ministry of Finance, Government of Nepal. GON (2017/18a), Red Book ((2017/18). Statement of Expenditure 2074/75 B.S. Published by Ministry of Finance, Government of Nepal. World Food Summit (1996). Rome Declaration on World Food Security, 13-17, November, 1996, Rome, Italy.

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Simultaneous Analysis of Commonly Used Artificial Sweeteners and Preservatives in Beverages by High Performance Liquid Chromatography Suraj Shrestha*, Nibedita Chaudhary, Ranjana Chaudhary, Tayer Mahommad Miya, Shiva Sagar Chaudhary and Sanjeev Kumar Karn Department of Food Technology and Quality Control, Babarmahal, Kathmandu * Corresponding author: [email protected]

Abstract: Beverages contain many ingredients like water, sugar, preservatives, etc. Many countries set the maximum permitted level of the preservatives and sweeteners used in food and beverages. Hence, it is very necessary to monitor the level of these chemicals in beverages. A simple, precise and accurate High Performance Liquid Chromatography with UV detection method was optimized and validated for simultaneous analysis of Acesulfame-K, Saccharin, Aspartame, Caffeine, Benzoic acid and Sorbic acid at 220 nm. Altogether 27 samples (4 carbonated beverages, 22 fruit based ready to serve beverages and 1 water based flavored drink) were analyzed. In carbonated beverages, all analytes were detected except Saccharin and Sorbic acid. In fruit based ready to serve beverage, all analytes were detected except Saccharin. Similarly, in water based flavored beverage, only Benzoic acid and Sorbic acid were detected.

Keywords: Artificial Sweetener, Beverage, High Performance Liquid Chromatography, Method validation, Preservatives

1. Introduction Beverages such as soft drink, diet soft drink, flavored juice, fruit based ready to serve drink etc. are common drinks consumed by people. The major ingredients of these beverages are water, fruit concentrate, sweetener, acid, flavor, and color additives. Artificial sweeteners are ingredients used to sweeten the foods as substitute of table sugar (sucrose). These sweeteners are many times sweeter than table sugar thus smaller amounts of sweeteners are needed to achieve the same level of sweetness as sugar in food (Anonymous).

Generally, sucrose is used as a sweetener in beverages but nowadays due to special dietary requirements of health concerns about obesity and dental caries, these artificial sweeteners are used as an alternative of sucrose. In these days people prefer beverages containing these high intensity sweeteners in place of table sugar (sucrose) because they do not contribute calories or only contribute a few calories to the diet.

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High amount of water, vitamins and minerals content in beverages leads to an attractive environment for micro-organisms. Hence preservatives are generally added in these beverages to inhibit the growth of micro-organisms. The choice of preservatives depends upon the ingredients, pH and packaging of beverages (Anonymous, 1995). The most commonly used preservatives in beverages are Benzoic Acid, Sorbic Acid, their salts and Sulphur dioxide and sulphites. Sorbic acid shows antifungal properties, it inhibits growth of molds and yeasts while Benzoic acid prevents bacterial growth (Dong and Wang, 2006; Lino and Pena, 2010). Many countries have their own legislative body for food safety and quality control. In Nepal, the Legislative body for food safety and quality control is Department of Food Technology and Quality Control (DFTQC). It regulates the use of preservatives in many foods but there is no regulation regarding use of artificial sweetener and its maximum permitted level till now. It is very necessary to regulate the use of artificial sweeteners and its maximum limit due to its increasing popularity. Simultaneous determination of these commonly used artificial sweeteners and preservatives in beverages is very important for quality control, food safety purpose and analysis time saving purpose. In this research, three most commonly used artificial sweeteners (Acesulfame-K, Saccharine and Aspartame), two preservatives (Benzoic acid and Sorbic acid) and Caffeine were chosen. There are many methods for analysis of these sweeteners and preservatives. But in this research, High Performance Liquid Chromatography with UV detection was used. All these analytes (sweetners and preservatives) were separated by Liquid Chromatography while detected by UV detector. The major objective of this research was to monitor the level of artificial sweeteners and preservatives used in beverages found in Nepali market. Another objective was to strengthen the capacity of Central Food Laboratory (CFL), DFTQC, in the field of food additives testing facility.

2. Materials and Methods 2.1 Reagents and chemicals Individual standards of AcesulfameK, Saccharin, Aspartame, Caffeine, Benzoic acid and Sorbic acid were purchased from Sigma Aldrich.HPLC grade water, Acetonitrile and Methanol were purchased from Merck. All other chemicals used were analytical standard unless and otherwise stated.

2.2 Preparation of Phosphate Buffer Solution (12.5 mM, pH 3.98)

1.70 g of Potassium dihydrogen orthophosphate (KH2PO4) was taken in 1 L volumetric flask, dissolved in HPLC grade water and volume was made up to the mark with water. The pH of the solution was adjusted to 3.98 with Phosphoric acid. It was filtered through 0.45 μm filter in vacuum filtration unit and sonicated for 5mins.

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2.3 Preparation of Carrez Solution No. 1

15 g of Potassium hexacyanoferrate (II) (K4[Fe(CN)6].3H2O) was dissolved in 100 mL of water.

2.4 Preparation of Carrez Solution No. 2

30 g of Zinc sulfate (ZnSO4.7H2O) was dissolved in 100 mL of water.

2.5 Standard and Calibration Curve Preparation Standard and calibration curve preparations involved following steps

2.5.1 Preparation of Individual Stock Solution 25 mg of each standard were transferred into separate 25mLvolumetric flask and dissolved in water. If complete dissolution was not seen, small amount of methanol was added for complete dissolution. After complete dissolution, volume was made up to the mark with water to get 1000 ppm each.

2.5.2 Preparation of Mixed Intermediate Solution 1 mL each of stock solution of Acesulfame-K, Saccharin, Caffeine and Benzoic acid, 2 mL stock solution of Sorbic acid and 3 mL stock solution of Aspartame were transferred into a 10 mL class-A volumetric flask and volume was made up to the mark by water to get solution of mixed standard containing 100 ppm of Acesulfame-K, Saccharin, Caffeine and Benzoic acid, 200 ppm Sorbic acid and 300 ppm Aspartame.

2.5.3 Preparation of Calibration Standard Solutions From mixed intermediate solution (2.5.2), six calibration standards were prepared in the range of 1 ppm each of Acesulfame-K, Saccharin, Caffeine and Benzoic acid, 2 ppm Sorbic acid and 3 ppm Aspartame to 100 ppm of Acesulfame-K, Saccharin, Caffeine and Benzoic acid, 200 ppm Sorbic acid and 300 ppm Aspartame.These calibration standards were injected to HPLC system for construction of calibration curve. All the above working standards were stored in refrigerator maintained at temperature 2-8 C.

2.6 Sampling Sampling was done based on random sampling technique. 27 beverage samples were collected from different places between Baisakh 2074-Asar 2074. The samples were given unique code from R1 to R27 and categorized into three categories: carbonated beverages, fruit based ready to serve beverages and water based flavored beverages. Samples were stored in refrigerator at 2 to 8 C.

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2.7 Sample Preparation Sample preparation was based on internationally accepted BS EN 12856:1999 method [5]. The collected samples were removed outside from refrigerator and left for some time for attenuation of room temperature. Sample preparation involved different process depending upon the physical nature of beverages.

2.7.1 Clear beverage samples (e.g. carbonated drinks, lemonades, and water based beverages) The sweeteners and preservatives in clear beverage samples were diluted with water and injected in HPLC system. The dissolved gas in carbonated beverage samples were removed by sonicating into sonicator for 20 mins. Sonication of other than carbonated beverages was not required. 5 mL of sample was taken in a 25 mL volumetric flask and water was added up to the mark. Flask was shaken properly. An aliquot of sample was filtered through a syringe membrane filter of pore size 0.45 μm and taken in a HPLC vial for injection.

2.7.2 Cloudy beverage samples (e.g. fruit juices) 5 mL of homogenized sample was taken in a 25 mL volumetric flask. 0.5 mL Carrez solution No. 1 and 0.5 mL Carrez solution No. 2 were added. Water was added up to the mark and flask was shaken vigorously. The flask was allowed to stand at room temperature for 10 mins. It was filtered through the filter paper discarding first 2.5 mL filtrate. An aliquot of filtrate was filtered through a syringe membrane filter of pore size 0.45 μm and taken in a HPLC vial for injection.

2.8 Instrumentation Analyses were performed by using Shimadzu Prominence HPLC system equipped with CBM 20 Alite controller, DGU-20 A5 Prominence online degasser, LC -20 AD binary pumps, SIL -20 A prominence Auto sampler ,CTO-20 prominence column oven and SPD 20AUV detector (Shimadzu Co.,Kyoto, Japan). The chromatographic system was controlled and operated by Shimadzu Lab solution software of version 5.60SP2.

2.9 Method Optimization The analytical part of this research was based on BS EN 12856:1999 method. But some parameters were optimized for better peak resolution and short analysis time.

2.10 Chromatographic Conditions

Column : Agilent ZORBAX Eclipse Plus C18 (150 mm×4.6 mm, 5μm)

Mobile Phase A : 12.5 mM Phosphate buffer pH 3.98 with Phosphoric acid

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Mobile Phase B : Acetonitrile

Elution Mode : Gradient

Flow Rate : 1.0 mL/Min

Column Temperature : 40°C

Detector : UV detector

Wavelength : 220 nm

Detector Cell Temperature : 40°C

Injection Volume : 10 μL

Run Time : 20 Mins.

2.11 LC Gradient Time Program The samples were analyzed in gradient elution mode and the optimized LC gradient time program is given in Table 1.

Table 1: Optimized LC gradient time program LC Time Program Time Module Command Value 0.01 Pumps Pump B Conc. 10 5.00 Pumps Pump B Conc. 15 16.50 Pumps Pump B Conc. 15 16.51 Pumps Pump B Conc. 10 20.00 Controller Stop

2.12 Method Validation Validation of the method was performed. System suitability, selectivity, linearity, range, precision (in terms of repeatability), accuracy (in terms of recovery), limit of detection (LOD) and limit of quantification (LOQ) were determined during validation studies.

2.13 Calculation and Expression of Results The individual analyte in sample was identified by comparing the corresponding retention time of peak of the individual standard and sample in the same chromatographic condition while the amount of each analyte in sample was calculated by comparing the peak area with the peak 50

DFTQC, FRB 2016/17 area of standard solution. This was automatically calculated by Shimadzu Lab solution software by making calibration curve. There was a linear relationship between the concentration and the area of the peak.

Concentration of each analyte in sample (mg/L or ppm)

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2.14 Statistical Analysis MS Office Excel 2013 was used for data analysis.

3. Results and Discussion 3.1 Optimization of method parameters This research was based on BS EN 12856:1999 method. It provided different combinations of Phosphate buffer (different concentration and different pH) and acetonitrile as suitable mobile phase. But all the combinations were isocratic one and run time was very long (more than 50 mins). Similarly, the peaks of Benzoic acid and Sorbic acid were not fully resolved. The recommended mobile phase combinations were tried. Firstly, 12.5 mM Phosphate buffer adjusted to pH 3.5 and Acetonitrile in the ratio of 90:10 was tried but run time was found very long. Hence, method optimization was performed considering the run time and resolution of all target analyte. When the polarity of mobile phase decreased by increasing the ratio of Acetonitrile then the run time was found to be shorter than that of 90:10 but the early eluted peaks of Acesulfame -K and Saccharin and lately eluted peaks of Benzoic acid and Sorbic acid were not resolved properly. To shorten the run time and good resolution of lately eluted peaks gradient elution was tested but it was very difficult to resolve Benzoic acid and Sorbic acid peak with higher concentration of Acetonitrile in mobile phase. Many combinations of Phosphate buffer and Acetonitrile combination and gradient program was tried but good resolution of Benzoic acid and Sorbic acid were not obtained. Finally, the pH of phosphate buffer was adjusted to 3.98 and gradient program (as shown in table 1) was carried out then all the target analytes were fully resolved within 15 minutes. The run time was set to 20 minutes to give complete equilibration time for repeated injections (batch analysis).Thus, by optimization of some parameters; this method resolved all the target analytes within less than half time of the original method. The Chromatogram of original method and optimized method are given in Figure 1A and 1B respectively.

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Fig 1A: Chromatogram of Original method (where,1 phenylalanine, 2 acesulfame-K , 3 theobromine, 4 saccharin, 5 aspartylphenylalanine , 6 diketopiperazine, 7 caffeine, 8 aspartame, 9 vanilline and 10 sorbic acid + benzoic acid)

Fig 1B: Chromatogram of standard mixture in Optimized method

3.2 Validation of Method Internationally accepted standard method do not require complete validation if it is used without any modification. Although it was internationally accepted standard method, some parameters were modified during optimization process and hence validation of method had been performed. There are many International guidelines for method validation like ICH, US FDA, AOAC, USP, IUPAC, etc. For this method, ICH (International Conference on Harmonization) and US FDA guideline were used for the method validation. System Suitability, Selectivity, Linearity, Range, LOD, LOQ, Precision, Recovery were performed (Anonymous, 2005; Anonymous 2001) .

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3.2.1 System Suitability Test System Suitability test was performed after optimization of method. In system suitability test different important parameters were tested which are listed in Table 2.

Table 2: Parameters tested in system suitability test

Acesulfame - Benzoic Sorbic Acceptance Parameter Saccharin Caffeine Aspartame K acid acid Criteria

Resolution - 2.44 8.88 7.56 6.73 3.47 ≥ 2

No. of Theoretical 2050 2310 5801 8017 6232 5475 ≥ 2000 Plate

Tailing 1.68 1.79 1.53 1.52 1.20 1.37 ≤ 2 Factor

3.2.2 Specificity/ Selectivity There was no any interfering peaks were observed in the blank matrix on retention time of standard solution which indicated the present method was selective (Figure 2).

9 m V 80 70 60 50 40 30 20 10 00 0.0 2. 5. 7. 10. 12. 15. 17. mi 0 5 0 5 0 5 0 5 Fig 2: Chromatogram of research sample R25 (Blank matrix)

3.2.3 Linearity and Range Linearity of method was tested by injecting six different concentrations of calibration standard solution prepared in 2.5.3 and making calibration curve automatically by Shimadzu Lab solution software using Least squares linear regression analysis. Linearity was evaluated by regression coefficient of calibration curve (r2) as well as residual analysis. The residual analysis was performed in MS Excel. The range of each target analyte was tested from low 53

DFTQC, FRB 2016/17 concentration (at LOQ level) to high concentration. The Linearity data and range are listed in Table 3 whereas calibration curve are given in Figure 3.

Table 3: Linearity and range data of target analyte

Acesulfame - Benzoic Sorbic Acceptance Parameter Saccharin Caffeine Aspartame K acid acid Criteria Regression 0.9999 0.9999 0.9999 0.9999 0.9999 0.9999 ≥ 0.996 Coefficient (r2) mean % -1.25 -2.19 1.46 2.02 0.27 0.05 < ± 20% residuals Range of % -10 to -14.7 to -2.16 to -2.38 to -1.71 to -3.05 to _ residual 1.94 1.54 7.3 13.8 1.76 2.27

Calibration 1-100 1-100 1-100 3-300 1-100 2-200 _ Range (ppm)

Area(x1,000,000) Area(x1,000,000) 6 6 5.0 2.5

4.0 2.0

3.0 1.5 5 5

1.0 2.0

4 4 0.5 1.0 3 3 2 2 1 1 0.0 0.0 0.0 25.0 50.0 75.0 Conc. 0.0 25.0 50.0 75.0 Conc.

FigFig 3A: 4A: Calibration Calibration Curve Curve of of Acesulfame-K Acesulfame-K Fig 3B: Calibration Curve of Saccharin

Area(x1,000,000) Area(x1,000,000) 6 6

3.0 2.0

2.5 1.5 2.0

5 5 1.5 1.0

1.0 4 0.5 4 0.5 3 3 2 2 1 1 0.0 0.0 0.0 25.0 50.0 75.0 Conc. 0 50 100 150 200 250 Conc.

Fig 3C: Calibration Curve of Caffeine Fig 3D: Calibration Curve of Aspartame

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Area(x1,000,000) Area(x1,000,000) 6 6 3.5 3.0

3.0 2.5 2.5 2.0 2.0 5 5 1.5 1.5 1.0 1.0 4 4 0.5 0.5 3 3 2 2 1 0.0 1 0.0 0.0 25.0 50.0 75.0 Conc. 0.0 25.0 50.0 75.0 100.0 125.0 150.0 175.0 Conc.

Fig 3E: Calibration Curve of Benzoic acid Fig 3F: Calibration Curve of Sorbic acid

3.2.4 LOD and LOQ

Limit of Detection (LOD) and Limit of Quantification (LOQ) were tested from calibration curve and verified by spiking same concentration of target analyte in blank matrix considering the recovery between 80-120 % and % relative standard deviation (% RSD) ≤ 20 % in different preparations. According to ICH guideline, LOD and LOQ can be determined on the basis of standard deviation of response and slope of calibration curve.

LOD =3.3ϭ/s and LOQ = 10ϭ/s Where,s = Slope of calibration curve and ϭ = S.D. of response (Standard deviation of the y- intercept). The standard deviation of Y-intercept was calculated using MS Excel. LOD and LOQ determined of this method are listed in Table 4.

Table 4: LOD and LOQ of the target analytes Benzoic Sorbic Parameter Acesulfame - K Saccharin Caffeine Aspartame acid acid LOD (ppm) 0.3 0.3 0.3 1 0.3 0.7 LOQ (ppm) 1 1 1 3 1 2

3.2.5 Precision and Accuracy

Precision of the method was divided in to three parts: System precision (injection repeatability), Intraday precision (intraday repeatability) and Intermediate precision (Interday precision). For injection repeatability, standard solution from same vial was injected six times and %RSD of retention time, peak area and peak height were measured. The injection repeatability was found to be within the acceptance criteria. The injection repeatability data were given in Table 5. 55

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Table 5: Injection repeatability of the target analytes Parameter Acesulfame- Saccharin Caffeine Aspartame Benzoic Sorbic K acid acid % RSD of 0.08 0.08 0.28 0.23 0.11 0.12 RT

% RSD of 0.20 0.17 0.11 0.07 0.70 0.35 Peak Area % RSD of 0.43 0.28 0.53 0.21 0.72 0.71 Peak Height

Intraday precision (intraday repeatability) and Intemediate precision (inter day repeatability) were performed. The intraday precision, intermediate precision data are listed in Table 6 while recovery data are listed in Table 7.

Table 6: Precision data of target analytes Analyte Intraday Precision (% Intemediate Precision (% RSD) RSD) Low Medium High Low Medium High Conc. Conc. Conc. Conc. Conc. Conc. Acesulfame-K 1.19 0.25 0.08 0.93 0.19 0.68

Saccharin 2.61 0.32 0.05 2.07 0.46 0.48

Caffeine 1.11 1.17 0.25 0.94 3.27 0.42

Aspartame 3.39 1.58 0.49 7.16 1.03 0.47

Benzoic acid 4.50 1.37 0.03 4.50 0.94 0.15

Sorbic acid 3.44 1.84 0.20 3.24 1.66 0.98

Acceptance criteria ≤20% ≤ 15% ≤ 15% ≤20% ≤ 15% ≤ 15%

The accuracy of the method was obtained by recovery studies. Recovery data of spiked samples analyzed in intraday precision were used in the calculation of Accuracy of method. The intraday precision, intermediate precision data are listed in Table 6 while recovery data are listed in Table 7.

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Table 7: Accuracy (recovery) data of target analytes Analyte Spiked level (each % Mean Minimum Maximum level in triplicate) recovery % % (ppm) (n=3) recovery recovery 1 85 Acesulfame-K 85 98 10 95 50 98 1 80 Saccharin 10 96 80 97 50 97 1 101 Caffeine 10 98 98 100 50 99 2 73 Aspartame 20 100 73 102 100 102 1 76 Benzoic acid 10 85 76 87 50 87 2 99 Sorbic acid 20 99 99 105 100 105 Acceptance >70 <120 Criteria All these data were found to be within the acceptance limit of validation guidelines. Mean recovery was found between 70 %-100 % while mean precision was found below 20%.

3.3 Sample Analysis

27 beverage samples (4 carbonated beverages, 22 fruit based beverages and 1water based flavored beverage) were analyzed with the above optimized and validated method. Each sample was analyzed in triplicate. Chromatogram of one sample is given in figure 4.

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Fig 4: Chromatogram of research sample R2

The number of positive sample, mean value, range and samples exceeding maximum permitted level (MPL) of target analytes found in samples are listed in Table 8. In carbonated beverages, 75% (3 out of 4) samples were found to contain Acesulfame-K, Aspartame and Benzoic acid with mean concentration 138 ± 53.37 ppm, 176±31.69 ppm and 107±2.11 ppm respectively. Caffeine was detected in 100% samples (4 out of 4) with mean concentration 428±203 ppm.

No carbonated beverage sample was found to contain Saccharin and Sorbic acid. In fruit based ready to serve beverages, Acesulfame-K, Aspartame, Caffeine, Benzoic acid and Sorbic acid were detected in 18% (4 out of 22), 23 % (5 out of 22), 5% (1 out of 22), 18% (4 out of 22) and 18% (4 out of 22) samples with mean concentrations 180 ± 122 ppm, 87±60 ppm, 11±1.18 ppm, 125±20 ppm and 185±157 ppm respectively. Saccharin was not detected in any fruit based ready to serve beverages. Similarly, in water based flavored beverage, only Benzoic acid and Sorbic acid were detected in 100% sample (1 out of 1) with mean concentration 13±0.06 ppm and 265±5.69 ppm respectively.

There is no technical regulation (standard) set for maximum permitted level of artificial sweetener in carbonated beverage, fruit based ready to serve beverage and water based flavored beverage in Nepal till now. Thus the obtained results cannot be compared with the standard. But there is technical regulation(standard) set for maximum permitted level of Benzoic acid and Sorbic acid in fruit based ready to serve beverages (Food Standard of Nepal, 2073).The obtained results were compared with this standard and it was found that 4.5% of fruit based ready to serve beverage research samples contain more Benzoic acid than maximum permitted limit (120 ppm) while 9% of fruit based beverage research samples contain more Sorbic acid than maximum permitted limit (50 ppm).

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Table 8: Number of positive sample, mean value, range and samples exceeding MPL of target analytes found in sample

Acesulfame-K Saccharin Aspartame Caffeine

No. of >Maxim Sample sample um Positive Mean ± Positive Mean Positive Mean Positive Mean (n) Range Range Range Range permitte samples s.d. samples ± s.d. samples ± s.d. samples ± s.d. (ppm) (ppm) (ppm) (ppm) d level % (n) (ppm) % (n) (ppm) % (n) (ppm) % (n) (ppm) (MPL) % (n) Carbonated 138 ± 176±3 135- 428 ± 107- Beverages 4 75 (3) 67-177 0 (0) _ _ 75 (3) 100 (4) _ 53.37 1.69 208 203 635

Fruit based

ready to 180 ± 11 ± 22 18 (4) 22-285 0 (0) _ _ 23 (5) 87±60 27-166 5 (1) 10-12 _ serve 122.49 1.18

beverages Water based 1 0 (0) _ _ 0 (0) _ _ 0 (0) _ _ 0 (0) _ _ _ beverage

Benzoic Acid Sorbic acid No. of Sample sample Positive Mean ± s.d. Range >MPL (150 Positive Mean ± Range > MPL (n) samples % (ppm) (ppm) ppm) % (n) samples % (n) s.d.(ppm) (ppm) (50 ppm) % (n) (n)

Carbonated Beverages

4 75 (3) 107±2.11 103-109 _ 0 (0) -- _ _

Fruit based ready to serve beverages 22 18 (4) 125±20 113-179 4.5 (1) 18 (4) 185±157 46-381 9 (2)

Water based flavored 1 100 (1) 13±0.06 13-13 _ 100 (1) 265±5.69 259-270 beverage _

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4. Conclusion Many beverages contain artificial sweetener and preservatives. National and International legislative agencies set acceptable body intake (ADI) and maximum permitted level (MPL) of these sweeteners and preservatives. Hence, it is very important to monitor their level in beverages, whether the level agrees with current legislation or not. A simple, precise and accurate HPLC method with UV detection methodwas optimized and validated. The optimized method required shorter run time and found good resolution of every target analytes. This method can be used as routine analysis. The validated method was used to analyze 27 beverage samples. It was found that that 4.5% of fruit based ready to serve beverage research samples contain more Benzoic acid than maximum permitted limit (120 ppm) while 9% of fruit based beverage research samples contain more Sorbic acid than maximum permitted limit (50 ppm). But there is no technical regulation (food standard) for maximum permitted limit of artificial sweetener in carbonated beverages, fruit based drinks and water based flavored drink. Hence technical regulation of artificial sweetener in such beverages is very necessary for effective monitoring the level of these sweeteners.

Acknowledgements Authors are very grateful to Director General of Department of Food Technology and Quality Control for providing valuable suggestions. Authors would like to thank all colleagues who involved in collection of beverage samples. Last but not the least, special thanks goes to Ms. Balkumari Sharma for providing the electronic copy of BS EN 12856 method.

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References

Food additives and ingredients. Food and Drug Administration. Available at: https://www.fda.gov/food/ingredientspackaginglabeling/foodadditivesingredien ts/ucm397716.html

European Parliament and Council Directive 95/2/EC (1995) on food additives other than colours or sweeteners. Official Journal of the European Communities L61, 18.3.95, 1–40.

Dong, C., & Wang, W. (2006). Headspace solid-phase microextraction applied to the simultaneous determination of sorbic acid and benzoic acid in beverages. AnalyticaChimicaActa, 562, 23-29.

Lino, C.M., & Pena, A. (2010). Occurrence of Caffeine, Saccharin, Benzoic acid and Sorbic acid in soft drinks and nectars in Portugal and subsequent exposure assessment. Food Chemistry, 121,503-508.

BS EN 12856:1999.Foodstuffs Determination of acesulfame-K, aspartame and saccharin. High performance liquid chromatographic method.

Validation of Analytical procedures: Text and Methodology Q2(R1) (2005). ICHHARMONISED TRIPARTITE GUIDELINE.

Guidance for Industry Bioanalytical Method Validation (2001). US FDA.

Food Standard of Nepal (2073). Department of Food Technology and Quality Control, Nepal, 90-92.

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Encapsulation of Saccharomyces cerevisiae in alginate beads and its application for wine making

Praksha Neupane, Smita Gurung, Saroj Khanal, Rojeena Shrestha, Huma Bokkhim* and Sanjeev Kumar Karn Department of Food Technology and Quality Control, Babarmahal, Kathmandu *Corresponding author: [email protected] Abstract

A study was carried out on encapsulation of wine yeast (Saccharomyces cerevisiae) and its uses for red and white wine making as compared to free yeast. Rehydrated active dry yeast was encapsulated in a 2% sodium alginate solution, cross linked with different molar concentration of CaCl2 solution (0.1, 0.2, 0.3,0.4 and 0.5M) for 30 minutes and assessed for yeast leakage density. The molar concentration with minimum leakage (0.2M) was used to cross-link alginate for yeast encapsulation. Colony count (CFU/ml) was analyzed for both free yeast (FY) and encapsulated yeast (EY) so as to equilibrate the rate of yeast pitching in wine fermentation. Physico-chemical properties; total soluble solids (T.S.S.), acidity and pH of red and white grapes used for wine making was analyzed and were found to be 16.5oBx, 0.397% and 3.92 for white grapes while for red grapes the value were 19.1oBx, 0.635% and 3.2 respectively. T.S.S. and acidity were examined every alternate days for 2 weeks for both wines prepared from FY and EY. In both wines, a gradual reduction in T.S.S. was noted while the change in acidity was not straight forwarded as it showed a pattern of an increase and decrease in alternate cycle finally stabilizing after 12 days. The final T.S.S. of wines was not significantly different for FY and EY but higher values were noted for red wine (7.11-7.23) than for white wine (6.1-6.2). Similar trend was noted for final acidity for both wines (0.826 - 0.840%). Though, no effect of yeast type on alcohol production was noted, the average alcohol content of red (13.22%) and white (7.54%) wine were found to be significantly different. However, wine prepared from EY had less turbidity than wine from FY. So, from this study it was concluded that encapsulating wine yeast does not affect its fermenting capability but will aid in wine clarification.

Keywords: Alginate, Encapsulation, Wine

62 DFTQC, FRB 2016/17

1. Introduction

Wine can be defined as an alcoholic beverage obtained from grape juice, primarily by the participation of enzymatic activities originating either in the grape or in a microorganism. Winemaking involves two principal operations: first, preparation of the grape must to tailor its composition and to maintain the qualities of the grape at harvest and second, conducting microbial fermentation through rational exploitation of the biochemical activities of yeasts and lactic acid bacteria. (Diviès et.al., 1994). Wine making is highly associated with biotechnology owing to the traditional nature of must fermentation. Nowadays, there have been considerable developments in wine making techniques affecting all phases of wine production but more importantly the fermentation process. It is well-known that the transformation of grape must by microbial activity results in the production of wine. An upsurge of interest in yeast immobilization for alcoholic beverages production has been taking place recently. This is mainly due to the numerous advantages that cell immobilization offers including enhanced fermentation productivity, feasibility of continuous processing, cell stability and lower costs of recovery and recycling and downstream processing (Kourkatous et al., 2004). Among the available techniques for immobilizing living cells, entrapment in calcium alginate beads has been frequently used for the immobilization of S. cerevisiae (Colagrande et al., 1994). Entrapment in Ca- alginate is a very simple process and alginate has the benefits of being non-toxic to the cells being immobilized, and it is an accepted food additive (Blandino et.al., 1999). The use of immobilized cells in wine making is a rapidly expanding research area with potential advantages as compared to free cell systems. The purpose for using immobilized cells in wine making is to increase fermentation productivity, to improve quality through low temperature fermentation or to produce sparkling wines (Tsakiris et al., 2004). Yeast performance in alcoholic fermentation depends directly on yeast activity which can be seen as a function of cell viability as well as the physiological state of viable cells.

Clarification of wine has always remained as a technological challenge in quality wine making. In the context of Nepal, different clarifying agents are effectively being used but no sufficient study has been done regarding the application of immobilized yeast in wine clarification. So, this work aims at preparing encapsulated yeast in convenient and economical way and to apply it for wine making. Further, this work would be useful in evaluating the efficacy of

63 DFTQC, FRB 2016/17 encapsulated yeast in improving the clarity and other quality parameters of wine as compared to clarifying agent and free yeast systems. Moreover, the result of this work could be beneficial to wine industries of Nepal in improving the quality of wine conveniently and economically.

2. Materials and Methods 2.1 Raw material collection

Wine ADY (Active Dry Yeast), sodium alginate, pectinase enzyme, CaCl2, fruits, sugar and all required chemicals were bought from local market of Kathmandu.

2.2 Rehydration of Active dry yeast 1 g of ADY was rehydrated into 10 ml final volume at 37 °C for 30 min in accordance with the manufacturer's specifications.

2.3 Encapsulation Calcium alginate capsules was prepared by using a simple one-step process similar to that described by Nigam et al. (1988). Sodium alginate was dissolved in hot water (40±5 ºC) to prepare sodium alginate solution of concentration 3.0%. Sodium alginate solution was then mixed with activated yeast suspension using magnetic stirrer to obtain uniform yeast alginate suspension having 2% sodium alginate. Droplets of alginate yeast suspension (5 ml) were then dropped through sterile syringe into 30 ml of (0.1, 0.2, 0.3, 0.4, 0.5M) CaCl2 solution. The CaCl2 solution was maintained under constant stirring (330 rev/min) using a magnetic stirrer. A dropping height of 10 cm was used to ensure the formation of spherical droplets. The gelation time was kept for 30 min and cell leakage efficiency was evaluated for varied concentration of CaCl2 solution. A cross-linking time of 30 min was adopted according to the finding of Bokkhim et al. (2016) as longer time led to higher leaching of active components into the cross-linking solution. Finally, the formed beads were rinsed with distilled water to remove excess calcium chloride. All of the above procedures were carried out at room temperature.

64 DFTQC, FRB 2016/17

2.4 Viability count of Yeast 1 g of calcium alginate beads loaded with microbial cells was mechanically crushed and homogenized with 9 cm3 of distilled water in a Stomacher Blender to obtain complete and homogeneous dispersion of cells. The Saccharomyces cerevisiae cell density and viability was calculated by spreading cell dilutions on Yeast Extract-Peptone-Dextrose (YPD) agar medium. The plates were incubated at 28 °C for 48 h and the Colony-forming Unit (CFU) was counted.

2.5 Cell leakage determination The cell leakage determination was evaluated by measuring the cell density in different CaCl2 solution after recovering the beads after 30 min of cross linking. (Callone et. al, 2008)

2.6 Physico-chemical analysis of Juice The total titrable acidity was assessed by titration with standardized sodium hydroxide. The pH value was measured using a digital pH meter. Total soluble solid (T.S.S.) was measured as ºBrix (ºBx) using hand refractometer.

2.7 Wine making Red wine was prepared by following standard procedure as mentioned by Sacchi et al. (2005). Cleaned red grapes were de-stemmed, crushed and sulfited at the rate of 75 ppm. TSS of must was adjusted to 25 ºBx by adding sugar. Also pectolytic enzyme was added at the rate of 0.01g/kg. Must was then pitched with yeast at the rate of 0.3 g/L free cell and left for fermentation at room temperature till residual sugar decreased to a constant value. After completion of primary fermentation, the clear wine was siphoned away from lees and kept for a week to settle further which was afterward racked, bottled and aged.

Likewise, white wine was prepared as mentioned by Pacock et al. (2011). Grapes were cleaned and juiced by using juicer. The juice was sulphited at the rate of 75 ppm T.S.S. maintained at 25 ºBx and then pasteurized at 72 ºC for 1 min. The settled juice was separated by drawing off and pectolytic enzyme was added at the rate of 0.01 g/kg. From here forth, pitching and fermentation until aging was done similar as in red wine making mentioned above. TSS, residual sugar and alcohol content were determined each day and fermentation kinetics was studied for free yeast (FY) and encapsulated yeast (EY).

65 DFTQC, FRB 2016/17

2.8 Analysis of wine TSS and acidity was determined every 2 days and fermentation kinetics was studied for FY and EY. TSS was determined by a hand refractometer and acidity by titration method as per Ranganna (2003). Ethanol content was determined by specific gravity method and free SO2 was determined as per AOAC Official Method 990.28.

3. Results and Discussions 3.1 Optimization of molar concentration of Calcium chloride Activated yeast suspension (1 g/10 mL) was mixed with 3% sodium alginate solution to achieve a final mixture of 2% sodium alginate and cross linked with different molar concentration of CaCl2 solution for 30 minutes. The encapsulated yeast was recovered and the left over CaCl2 solution was incubated at 28 °C for 48 h and the CFU was counted. The result obtained is shown in Figure 1. The result shows that highest leaked cell density was observed in 0.5 M concentration of CaCl2 solution which was found to be 78.2 CFU/mL CaCl2 solutions. Minimal leakage was found at 0.2 molar concentrations of CaCl2 solutions which was only

16.36 CFU/mL CaCl2 solution. Thus, 0.2 M of CaCl2 was used for cross linking the sodium alginate beads throughout the research for the formation of encapsulated yeast.

Effect of molar concentration of CaCl2 on cell lekage from beads 120 100 80 60

CFU/mL 40 20 0 0 0.1 0.2 0.3 0.4 0.5 0.6 Molar concentration of CaCl 2

Fig 1: Number of cells leakage in different molar concentration solution

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Lotfipour et al., (2012) had also observed that when concentrations of CaCl2 were varied in the range 1.3–5.5% w/v and the concentration of the sodium alginate solution was ﬁxed at 1.0% w/v, the use of concentrated CaCl2 solutions signiﬁcantly reduced the percentage of Lactobacillus acidophilus that diffused out of the capsules. Similarly, the result of this study is also in accordance with

Hariyadi et al., (2014) findings who reported that CaCl2 concentration of 0.1M didn’t form microsphere but form irregular shaped gelling sheets when forming ovalbumin loaded alginate microspheres using aerolisation techniques.

3.2 Microbial count of encapsulated yeast and free yeast. Viable Cell count of Active dry yeast and encapsulated yeast was done by YPD agar medium and result obtained is shown in Table 1.

Table 1: Total Colony Count of free and encapsulated yeast

S.N Yeast type CFU/g 1 Free yeast 3.26 × 109 2 Encapsulated yeast 4.91× 108

Live yeast cells were more than 10 fold less in per gram beads as compared to free yeast. Also, according to Shi et al, (2013) the cell loading on encapsulated yeast can be affected by various factors such as nozzle size, polymer concentration, hardening time in calcium chloride, initial cell concentration. So, CFU/g beads was found and equilibrated with free yeast cell count so that similar yeast concentration could be used for wine preparation with FY and EY. In our study, 1 g. active dry yeast was equal to 6.65 g. beads of EY in terms of live yeast cell count.

3.3 Physiochemical properties of white and red grapes TSS, acidity and pH of red and white grapes used for wine making in this study was analyzed and results obtained are tabulated in Table 2. Table 2: TSS, acidity and pH of white and red grapes Parameters White grapes Red Grapes T.S.S. 16.5 o Bx 19.1 o Bx Acidity (% Tartaric acid) 0.397% 0.635% pH 3.92 3.2

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Red grapes used were of high TSS and acidity as compared to white grapes used in this study. According to Joshi et al (2013), physico-chemical properties of wine vary according to the variety and environmental conditions of the region in which the grapes are grown. Higher T.S.S. of grape juice is associated with higher alcohol content of wine and acidity of wine juice influences the taste and flavor of thus formed wine. The wine with a pH of 3.2 will have bright fruit flavors, but it will also be thin, acidic and aggressive on the palate. On the other hand, the wine at 4.0 will be softer and rounder than the wine at 3.2, but also less vibrant. Also, for red wine fermentation ideal acidity is 0.6 - 0.7% and ideal pH is 3.4 to 3.7 and T.S.S. is 22 to 25 oBx. Likewise, he mentioned for white wine fermentation, optimum acidity of must is 0.6 to 0.9, pH is 3.2 to 3.5 and T.S.S. is 17-24%. In our study, we maintained the T.S.S. of must to 25 oBx for red wine and and 23 oBx for white wine by adding sugar. However, acidity wasn’t maintained though red wine must was in optimum level as mentioned but white wine must wasn’t adjusted which is limiting in this study.

3.4 TSS profile of red wine and white wine prepared with FY and EY TSS profile of red and white wine produced by using FY and EY was observed for 14 days of fermentation (Figure 2 & 3).

TSS profile of red wine for FY & EY

20 EY FY

T.S.S. of red wineT.S.S. of red 10

5 02468101214 Days of fermentation

Figure 2: TSS profile of red wine for free and encapsulated yeast

It is evident from the figures that TSS profile of red and white wines produced by FY and EY were quiet similar. TSS decreased steeply for a week and then

68 DFTQC, FRB 2016/17 leveled off thereafter. A constant TSS of approx. 6 and 7 oBx for white and red wine were achieved after 12 days of fermentation. It was observed that the rate of decrease in TSS was slightly faster for FY compared to EY in the first week of white wine fermentation. But this did not affect the efficiency of fermentation process as both FY and EY achieved the same final TSS.

TSS profile of white wine for FY & EY 25

20 FY EY 15

10 T.S.S. of white wine

5 0 2 4 6 8 10 12 14 Days of fermentation

Figure 3: TSS profile of white wine for free and encapsulated yeast

3.5 Acidity profile of red wine and prepared from FY and EY The total acidity profile of red wine prepared from free and encapsulated yeast was measured for 14 days and result obtained is shown in the Figure 4. The pH affects flavor, aroma, color, tartrate precipitation, carbon dioxide absorption, malolactic fermentation, stability, ageablity, and fermentation rate.

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Acidity profile of red wine for FY & EY 1.1 1 0.9 0.8 0.7 F Y 0.6

Acidity of red wine 0.5 024681012 Days of fermentation

Figure 4: Acidity profile of red wine for free and encapsulated yeast

It was observed from the figure that the acidity increased initially, decreased midway, increased thereafter before finally getting stabilized at final acidity of 0.8% as tartaric acid. The result is in accordance with the finding of Joshi et al. (2013) who reported that in wine making there is initial increase in acidity due to the metabolization of nitrogen by the yeast as nitrogen acts as buffer against the titrable acidity of the must. However, when alcohol is produced the acidity start to decrease.

3.6 Alcohol content of red and white wine with FY and EY

Alcohol content of white and red wine produced by FY and EY was determined and the result obtained is shown in Figure 5.

It was observed that wine produced by FY and EY wasn’t significantly different for both red wine and white wine. However, yeast in both conditions shows higher fermentability in red wine compared to white wine for similar production conditions. This could be due to the difference in composition of juice used for wine making. According to Sacchi et.al (2005), the nitrogen content of juice greatly influences the fermentation characteristics of yeast.

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Alcohol content of wine with FY and EY 16 a a 14 12 b 10 b 8 Free yeast 6 Encapsulated yeast 4 Alcohol content (%) 2 0 Red White Wine type

Figure 5: Alcohol content of red and white wine with FY and EY

4. Conclusions From this study, it was found that wine yeast Saccharomyces cerevisiae could be effectively encapsulated by using 2% sodium alginate solution and crosslinking with 0.2M CaCl2 solution for 30 minutes. There was no significant difference in T.S.S. and acidity profile of wine with the use of EY as compared to FY. Also, EY was equally efficient in alcoholic fermentation in wine. However, visually a more clear white wine was obtained with the use of EY.

Acknowledgements: The author would like to acknowledge Ms. Ratna Shakya and Mr. Bijan Shrestha, Food Research Officers from the Microbiology laboratory in Central Food Laboratory under DFTQC for their support in assessing the viability count of yeast during the experiment.

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References

AOAC (1994). No.990.28, Official methods of analysis. Washington, DC: Association of Official Analytical Chemistry. Blandino. A, Macias, M., & Cantero, D (1999). Formation of calcium alginate gel capsules: influence of sodium alginate and CaCl2 concentration on gelation kinetics. Journal of Bioscience and Bioengineering: 688-696. Bokkhim, H., Bansal, N., GrØndahl, L., & Bhandari, B. (2016). Characterization of alginate-lactoferrin beads prepared by extrusion gelation method. Food Hydrocolloids, 53: 270-276. Colagrande, O., Silva, A., & Fumi, M.D. (1994). Recent applications of biotechnology in wine production. Rev. Biotechnology Progress, 10(1): 2–18. Callone, E., Campostrini, R., Carturan, G., Cavazza, A., & Guzzon, R. (2008). Immobilization of yeast and bacteria cells in alginate microbeads coated with silica membranes: procedures, physico-chemical features and bioactivity, Journal of Materials Chemistry, 18: 4839–4848. Diviès, C., Cachon, R., Cavin, J-F., & Prévost, H. (1994). Immobilized Cell Technology in Wine Production. In critical reviews in biotechnology, CRC press, 14(2): 135-153. Hariyadi D. M, Hendradi, E., Purwanti, T., Fadil, F. D. G. P., & Ramadani, C. N. (2014). Effect of crosslinking agent and polymer on the characteristics of ovalbumin loaded alginate microspheres. International journal of pharmacy and pharmaceutical science, 6(4): 469-474. Joshi, V., Rao, B. S., & Reddy, R. S. (2013). Studies on the physicochemical properties of wine in different varieties of grapes. The Asian Journal of Horticulture, 8(1): 174-178. Kourkoutasa, Y., Bekatoroua, A., Banat, I. M., Marchant, R., & Koutinas, A. A. (2004). Immobilization technologies and support materials suitable in alcohol beverages production: a review. Food Microbiology, 21(4): 377–397. Lotfipour, F., Mirzaeei, S., & Maghsoodi, M. (2012). Evaluation of the effect of CaCl2 and alginate concentration and hardening time on the characteristics of Lactobacillus acidopillus loaded alginate beads using response surface analysis. Advanced pharmaceutical bulletin 2(1): 71-78. Nigam, S.C., Tsao, I-Fu, Sakoda, A., & Wang, H.Y. (1988). Techniques for preparing hydrogel membrane capsules. Biotechnology Technique 2(4): 271- 276.

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Pocock, K. F., Salazar, F. N., & Waters, E. J. (2011). The effect of bentonite fining at different stages of white winemaking on protein stability. Australian Journal of Grapes and Wine Research, 17(2): 280-284. Ranganna, S. (1986). Proximate Constituents. Handbook of Analysis and Quality Control for Fruit and Vegetable Products (2nd Ed.) (pp. 1-30). New Delhi: Tata McGraw-Hill Publishing Company Limited. Sacchi, K. L., Bisson, L. F., & Adams, D. O. (2005). A review of the effect of winemaking techniques on phenolic extraction in red wines. American Journal of Enology and Viticulture, 56: 197-206. Shi, L-E., Li, Z-H., Zhang, Z-L., Zhang, T-T., Yu, W-M., Zhou, M-L., & Tang, Z-X. (2013). Encapsulation of Lactobacillus bulgaricus in carragenan-locust bean gum coated milk microspheres with double layer structure. LWT-Food Science and Technology, 54: 147-151 Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., & Kailasapathy, K. (2000). Encapsulation of probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in yogurt. International Journal of Food Microbiology, 62(1-2): 47-55. Tsakiris, A., Sipsas, V., Bekatorou, A., Mallouchos, A., & Koutinas, A.A. (2004). Red wine making by immobilized cells and influence on volatile composition. Journal of Agricultural Food Chemistry, 52(5):1357-1363.

73 DFTQC, FRB 2016/17

Status of Pesticide Residues in Vegetables Entering into Kathmandu Valley

Bimal Kumar Dahal1*, Man Bahadur Chetri2 and Matina Joshi Vaidhya1

1Department of Food Technology and Quality Control, Babarmahal, Kathmandu

2Rapid Pesticide Testing Laboratory, Plant Protection Directorate, Hariharbhawan, Kathmandu

*Corresponding author: [email protected]

Abstract

One hundred and thirty-one samples from different 19 varieties of vegetables were collected randomly from the carrying vehicles entering to Kathmandu valley through 3 entry points viz. Balaju, Jagati and Thankot. They were tested at Rapid Pesticide Testing Lab located at Kalimati Fruits and Vegetable Market Development Board, Kalimati by Rapid Bioassay of Pesticide Residues (RBPR) method for enzyme inhibition by carbamates and organophosphates. Two samples of cauliflower entering through Jagati and one sample of cauliflower entering through Thankot were found sub-standard. Similarly, two samples of tomato entering through Jagati and one sample of tomato entering through Thankot were found sub-standard. Out of these 6 sub-standards, one sample of tomato was found to be in quarantine range while three samples of cauliflower and two samples of tomato were found in the disposable range. All six substandard vegetable samples were due to enzyme inhibition by organophosphates. Out of three sub-standard cauliflower samples, two were from Kavre and one was from Dhading. In the same way, three sub-standard tomato samples were one sample each from Sindhupalchok, Kavre and India.

Keywords: Carbamate, Enzyme inhibition, Organophosphates, RBPR method, Quarantine

1. Introduction

The chemical pesticides form an important means to control pests since long time. Small-scale farmers have become heavily dependent on chemical pesticide usage to achieve high production of fruits and vegetables. The uncontrolled use

74 DFTQC, FRB 2016/17 of these chemicals during agricultural production may cause food safety issues. In view of increasing public concern on food safety, most Asian countries are now making considerable efforts to reduce pesticide residues in food, particularly on fruits and vegetables (http://www.fftc.agnet.org).

Organophosphate pesticides are one group of insecticides commonly used for agricultural purposes. Some of the documented health effects in adults are cancer, respiratory illnesses, and liver and renal injuries. However, these pesticides can be more harmful to children than to adults because children breathe more air and consume more food and beverage per pound of body weight than adults do (Lizardi et al., 2008). The extensive use of carbamate pesticides in modern agriculture has raised serious public concern regarding the environment and food safety. Due to their broad spectrum of biological activity, carbamates can be used as insecticides, fungicides, nematocides, acaricides, molluscicides, sprout inhibitors or herbicides. Contamination of fruits and vegetables may result from treatment as well as from conditions such as improper use of pesticides, residues from preceding treatments in the soil and cross- contamination. The major concerns are their toxic effects such as interfering with the reproductive systems and foetal development (Morais et al, 2012).

To access the pesticide residues in the agriculture produce, the Rapid Bioassay of Pesticide Residues (RBPR) was developed in Taiwan in 1985. Compared to the standardized instrument/chemical analysis used by many developed countries, RBPR has both advantages and disadvantages. For instance, some of its advantages include: less-cost, simple and rapid operation; and no intensive training and sophisticated instruments requirement. However, it is less accurate/precise compared to chemical analysis, and is not applicable to all the chemical pesticides (http://www.fftc.agnet.org). RBPR has been adopted by Republic of Korea, Vietnam, Philippines, Panama and many southeast Asian countries (Kao et al, 2010). This method has been successfully adopted in Nepal as a supplement to sophisticated chemical analysis for the routine residue test of large amounts of samples.

The main objective of this research work was to find out the status of pesticide residues on vegetables entering into Kathmandu valley. Limitations of this research were limited samples, time and limitations of RBPR testing methods as well.

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2. Materials and Methods

2.1 Materials and Methods

Samples of different varieties of vegetables were collected randomly from the carrying vehicles entering to Kathmandu valley through 3 entry points viz. Balaju, Jagati and Thankot. Personnel from Department of Food Technology and Quality Control (DFTQC) and Plant Protection Directorate (PPD) were jointly involved in this mission from Mangsir 2073 to Jestha, 2074 B.S. During this period, 15 times monitoring was done and 131 samples of different 19 varieties of vegetable were collected. The detail of sample collection has been shown in Table 1.

Table 1: Detail of sample collection S.N. Date Entry No. of Crop variety Source point samples 1 19/08/2073 Thankot 5 Potato, onion etc. Bhairawa, Parsa, India 2 23/08/2073 Jagati 12 Potato, cauliflower, Kavre, tomato, cabbage, onion Sindhupalchok etc. 3 23/10/2073 Jagati 10 Tomato, potato, Kavre cauliflower etc. 4 24/10/2073 Thankot 8 Onion, potato, cabbage, Chitwan, Parsa, tomato, cauliflower etc. Morang, India 5 8/11/2073 Jagati 8 Potato, cauliflower, Kavre green leafy vegetable etc. 6 9/11/2073 Balaju 8 Broccoli, cauliflower, Nuwakot green peas, potato, cabbage etc. 7 10/11/2073 Thankot 10 Carrot, cauliflower, Chitwan, Dhading broccoli, pumpkin, cabbage, tomato etc. 8 25/12/2073 Jagati 10 Green peas, potato, Kavre, Dolakha, tomato, cabbage etc. Sindhupalchok, Dhanusa 9 28/12/2073 Thankot 11 Cabbage, tomato, green Nuwakot, beans, cucumber etc. Dhading, India 10 5/1/2074 Jagati 10 Cucumber, tomato, Kavre green leafy vegetable, cauliflower etc. 11 13/01/2074 Jagati 10 Tomato, potato, Kavre, Bhaktapur cauliflower, pumpkin etc.

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12 14/01/2074 Thankot 9 Tomato, potato, Dhading, Parsa, cauliflower, gourd etc. Chitwan, India 13 17/01/2074 Balaju 9 Cauliflower, cabbage, Nuwakot potato, tomato etc. 14 7/2/2074 Balaju 8 Green beans, tomato, Nuwakot green chillies, cauliflower etc. 15 14/02/2074 Thankot 3 Cabbage, green leafy Dhading vegetables etc. Total 131

Weight of each sample was approximately 1 Kg. They were kept under refrigerator at about 10Ԩ. The samples were tested within 12 hours of collection at Rapid Pesticide Testing Lab located at Kalimati Fruits and Vegetable Market Development Board, Kalimati by Rapid Bioassay of Pesticide Residues (RBPR) method.

2.2 Data Analysis

The data were analysed for range and weighted average by applying Microsoft Excel program.

3. Results and Discussion

3.1 Pesticide residues in different vegetables entering to Kathmandu valley

Results of pesticide residue in 131 samples from different 19 varieties of vegetable have been presented in Table 2. The results have been expressed in terms of percentage enzyme inhibition by carbamate and organophosphate. Three samples of cauliflower and three samples of tomato were found substandard. The weighted average for enzyme inhibition by carbamate and organophosphates in cauliflower were found to be 6.56 and 16.36 respectively. Similarly, the weighted average for enzyme inhibition by carbamate and organophosphorus in tomato were found to be 6.39 and 18.85 respectively. This indicates the majority of cauliflower and tomato samples were under low risk range.

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Table 2: Pesticide residues in different vegetables entering to Kathmandu valley

S. Range of Inhibition % Crop N n Source N. Carbamate Organophosphate

1 Bittergourd 2 0 0.13-14.62 (7.37) 5.38-9.4 (7.39) Chitwan, Dhading

2 Broccoli 2 0 2.18-9.72 (5.95) 1.56-2.58 (2.07) Nuwakot Dhading Dhading, Bhaktapur, 3 Cabbage 12 0 0.33-14.88 (3.54) 0.95-21.21 (7.30) Nuwakot, Kavre, Chitwan 4 Carrot 2 0 2.25-3.1 (2.67) 2.41-12.08 (7.2) Kavre, Chitwan

5 Cauliflower 21 3 0.3-26.76 (6.56) 1.17-66.85 (16.36) Kavre, Dhading

6 Coriander 2 0 1.81-2.90 (2.36) 2.4-8.08 (5.24) Nuwakot, Dhading

7 Cowpeas 2 0 2.8-3.56 (3.18) 8.95-13.06 (11.0) Dhading. Chitwan

8 Cucumber 4 0 2.11-19.03 (7.25) 5.41-30.20 (14.98) Kavre, Nuwakot Kavre, Nuwakot, 9 Green Beans 5 0 4.17-6.63 (5.37) 6.09-34.78 (16.72) Chitwan, Morang 10 Green Chilli 2 0 3.60-3.87 (3.73) 4.22-11.78 (8.0) Nuwakot, Kavre Green leafy Nuwakot, Dhading, 11 6 0 0.18-6.35 (4.59) 2.70-31.84 (6.41) vegetable Kavre, Dolakha 12 Green Peas 2 0 2.37-11.18 (6.77) 3.49-6.15 (4.82) Kavre, Nuwakot Guard (Sponge, 11.22-23.61 13 2 0 1.01-5.76 ( 3.38) Parsa, Chitwan pointed) (17.41) 14 Onion 3 0 2.04-13.86 (8.25) 1.05-22.62 (11.78) Kavre, India Kavre, Nuwakot, 15 Onion leaves 5 0 3.71-5.82 (4.84) 4.86-25.49 (10.45) Dhading Kavre, Sindhupalanchok, 16 Potato 31 0 0.61-15.65 (5.51) 0.37-24.45 (5.97) Nuwakot, Dhading, Bhairawa, India Nuwakot, Dhading, 17 Pumpkin 8 0 1.47-28.16 (6.20) 1.99-16.52 (9.85) Kavre 18 Radish 3 0 1.78-7.63 (5.72) 2.64-6.20 (3.88) Nuwakot, Kavre Kavre, 19 Tomato 17 3 0.49-31.87 (6.39) 5.78-57.56 (18.85) Sindhupalanchok, India Total 131

Note: 1. N = total numbers of samples, n = sub-standard samples, figures in parenthesis indicates weighted average. 2. According to RBPR method, Inhibition % ൏ 35 indicates consumable and inhibition ൐35 indicates substandard.

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3.2 Sub-standard vegetables entering to Kathmandu valley

Enzyme inhibition in sub-standard tomatoes and cauliflower has been shown in Table 3.

Two samples of cauliflower entering through Jagati and one sample of cauliflower entering through Thankot were found sub-standard. In the same way, two samples of tomato entering through Jagati and one sample of tomato entering through Thankot were found sub-standard. One sample of tomato was found to be in quarantine range and three samples of cauliflower and two samples of tomato were found in the disposable range.

Table 3: Sub-standard vegetables entering to Kathmandu valley

Entry Inhibition % Quantit Point S.N. Date Crop Source y (Kg) Carbamat OP e Thanko 1 10/11/2073 Cauliflower 1200 5.55 63.82 t Dhading Thanko 2 24/10/2073 Tomato 750 1.26 46.55 t India Jagati 3 23/10/2073 Tomato 3175 14.30 36.27 Kavre Jagati 4 23/10/2073 Cauliflower 3600 2.57 66.85 Kavre Jagati Sindhupalancho 5 23/08/2073 Tomato 2500 31.87 57.56 k Jagati 6 23/08/2073 Cauliflower 1800 26.76 52.42 Kavre Note: 1. Inhibition % below 35 indicates consumable, 35-45 for quarantine and more than 45 disposable 2. OP indicates for organophosphate.

3.3 Effect of sampling time on enzyme inhibition

The effect of sampling time on percentage enzyme inhibition in cauliflower has been shown in Fig 1. Twenty-one samples of cauliflower were collected in different dates from Mangsir 2073 to Jestha 2074. Out of 21 samples, three were found sub-standard due to organophosphate inhibition, one sample each from Mangsir, Magha and Falgun. The enzyme inhibition by organophosphate was found to be in irregular trend till Falgun. After Falgun, it was found decreasing. It may be due to the use of fewer amounts of organophosphate pesticides, or following the adequate waiting period, or washing of cauliflower before or during transportation. In case of enzyme inhibition by carbamate, it was found

79 DFTQC, FRB 2016/17 to be below 10% after Mangsir. This may be due to the use of fewer amount of carbamate in the field.

80.00 Inhibition % Carbamate 70.00 Inhibition % Organophosphate 60.00

50.00

40.00

30.00

20.00 Percentage enzyme inhibition Percentage enzyme 10.00

0.00

Sampling date

Fig 1: Effect of sampling time on percentage inhibition in cauliflower.

Effect of sampling time on percentage inhibition in tomato has been shown in Fig 2.

70.00 Inhibition % Carbamate 60.00 Inhibition % Organophosphate

50.00

40.00

30.00

20.00 Percentage inhibition 10.00

0.00

Sampling date

Fig 2: Effect of sampling time on enzyme inhibition in tomato.

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Seventeen samples of tomato were collected in different dates from Mangsir 2073 to Jestha 2074. Out of 17 samples, three were found sub-standard due to organophosphate inhibition, one sample each from Mangsir, Magha and Falgun. The enzyme inhibition by organophosphate was found to be in irregular trend till Chaitra. After Chaitra, it was found decreasing. It may be due to use of fewer amounts of organophosphate pesticides, or following the adequate waiting period, or washing of tomato before or during transportation. In case of enzyme inhibition by carbamate, it was found to be below 10% after Magha. This may be due to the use of fewer amount of carbamate in the field.

3.4 Effect of sample source on enzyme inhibition

The effect of sample source on enzyme inhibition in cauliflower has been shown in Fig 3. Twenty-one samples of cauliflower grown at Bhaktapur, Dhading, Kavre, Nuwakot and Parsa; were collected from Mangsir 2073 to Jestha 2074. Out of 21 samples, three were found sub-standard due to organophosphate. Two sub-standard samples were from Kavre and one was from Dhading.

The effect of sample source on enzyme inhibition in tomato has been shown in Fig 4. Seventeen samples of tomato grown at Chitwan, Dhanusha, Kavre, Nuwakot, Sindhupalchok and India; were collected from Mangsir 2073 to Jestha 2074. Out of 17 samples, three were found sub-standard due to organophosphate, one sub-standard sample each from Kavre, Sindhupalchok and India.

80.00 Inhibition % Carbamate 70.00 Inhibition % Organophosphate

60.00

50.00

40.00

30.00

Percentage inhibition 20.00

10.00

0.00

Sample source

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Fig 3: Effect of sample source on enzyme inhibition in cauliflower

70.00 Inhibition % Carbamate 60.00 Inhibition % Organophosphate

50.00

40.00

30.00

20.00 Percentage inhibition

10.00

0.00

Sample source

Fig 4: Effect of sample source on enzyme inhibition in tomato

4. Conclusions

Pesticide residues in the vegetables specially in cauliflower and tomatoes seem to be serious issue. This study concludes that 14.3% of cauliflower and 17.7 % of tomato samples entering to valley were found to be sub-standard. These figures shall further decrease at the time of consumption. Application of Good Agriculture Practices, raising awareness to farmers and consumer could be the suitable measures to reduce the level of pesticide residue in fruits and vegetables.

Acknowledgements

This research was conducted under the approved program (fiscal year 2073/074) for Department of Food Technology and Quality Control (DFTQC), The authors want to acknowledge Mr. Sanjeev Kumar Karn, Director General, DFTQC for his inspiration and financial support for this work. Also, the authors want to extent acknowledgement to Dr. Dilli Ram Sharma, Program Director, Plant Protection Directorate for supporting us in testing vegetable samples.

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Acknowledgement also goes to all sr. Officers and officers who are involved for sample collection for this work.

Abbreviations

DFTQC = Department of Food Technology and Quality Control, PPD = Plant Protection Directorate RBPR = Rapid Bioassay of Pesticide Residues

References

Kao C.H., Hsieh Y.S., Chiang M.Y. and Huang Y.B. Residue Control by Using Rapid Bioassay of Pesticide Residues for Market Inspection and Farm Education. Paper presented at AARDO Workshop on Technology on Reducing Post-harvest Losses and Maintining Quality of Fruits and Vegetables pp 72-82, 2010.

Lizardi P.S, Rourke M..K. and Morris R.J., 2008. The Effects of Organophosphate Pesticide Exposure on Hispanic Children’s Cognitive and Behavioral Functioning. Journal of Pediatric Psychology 33(1) pp. 91–101, 2008.

Morais S., Dias E. and Pereira M.L., Carbamates: Human Exposure and Health Effects pp 21-38, Chapter - January 2012, ResearchGate. http://www.fftc.agnet.org. Food and Fertilizer Technology Center for Asia and Pacific Region, Taiwan. Web http://www.fftc.agnet.org, viewd on 27/7/2017. http://www.dftqc.gov.np. Department of Food Technology and Quality Control, Kathmandu, Nepal. http://ppdnepal.gov.np. Plant Protection Directorate, Ministry Of Agricultural Development, Nepal.

83 DFTQC, FRB 2016/17

Advanced Glycation End-products Inhibitory Activities of Crude Methanolic Extracts of Fennel and Fenugreek Seeds

Nirat Katuwal* and Hasta Bahadur Rai

Regional Food Technology and Quality Control Office, Bhairahawa

*Corresponding author: [email protected]

Abstract Advanced glycation end-products (AGEs) are implicated with diabetes related complications, atherosclerosis and aging. Several pharmacological agents such as aminoguanidine have been developed for inhibiting the formation of AGEs. However, their side effects are severely limiting their applicability. This situation calls the need for finding effective and safe agents. In this regard, seeds of fenugreek and fennel, which are claimed traditionally to be antidiabetic and being used in antidiabetic formulation in Ayurveda system of medicine, selected for the study. The dry seeds were powdered and steeped in 80% methanol for 12 h and filtered. The filtered extracts were analysed for total phenol content, DPPH free radical scavenging activities, glyoxal inhibitory activities and AGEs inhibitory activities. The latter two analysis were made on glucose-BSA model. Total phenol content, IC50 concentration for DPPH inhibition, glyoxal content in glucose-BSA model and AGEs inhibitory activity of Fennel seed extract were 76.51 ± 2.93 mg GAE/ g dry extract, 16.09 ± 0.13 μg/mL, 0.12 ± 0.003 mM and 24.21 ± 0.39 % respectively. Similarly, total phenol content, IC50 concentration for DPPH inhibition, glyoxal content in glucose-BSA model and AGEs inhibitory activity of Fenugreek seed extract were 86.05 ± 4. 36 mg GAE/ g dry extract, 14.94 ± 0.11 μg/mL, 0.13 ± 0.007 mM and 15.75 ± 0.68 % respectively.

Keywords: AGEs, DPPH inhibition, Fennel Seeds, Fenugreek seeds, Glyoxal 1. Introduction Studies have suggested that advanced glycation end-products (AGEs) are associated with diabetic complications, atherosclerosis and aging (Araki et al., 1992; Kume et al., 1995; Makino et al., 1995). The formation of AGEs is a part of normal metabolism, but if excessively high levels of AGEs are reached in tissues and the circulation they can become pathogenic (Ulrich and Cerami, 2001). The pathologic effects of AGEs are related to their ability to promote oxidative stress and inflammation by binding with cell surface receptors or cross-

84 DFTQC, FRB 2016/17 linking with body proteins, altering their structure and function (Eble et al., 1983; Schmidt et al.,1999; Vlassara, 2001). Several pharmacological agents such as aminoguanidine have been developed for inhibiting the formation of AGEs. However side effects and potential toxicity make their application less practical (Ho et al., 2010; Thornalley, 2003). This situation calls the need for finding effective and safe agents. In this regard finding AGEs inhibitors from foods seems ideal. Previous works (Dongare et al., 2012; Krishnaswamy, 2008) suggest that Fennel seeds and Fenugreek seeds possess antidiabetic properties. Moreover, some formulations used in Ayurveda for treatment of diabetic complications contains fenugreek seeds. However, the reports on AGEs inhibitory activities of these seeds are scarce. Therefore, this study attempts to evaluate AGEs inhibitory activities of Fenugreek seeds and Fennel seeds. 2. Materials and methods 2.1 Materials Fennel seeds and fenugreek seeds were procured from local market. Chemicals were purchased from the local suppliers. 2.2 Methods 2.2.1 Preparation of plant extracts The plant materials were dried, powdered and steeped in methanol for 12 h. Then the extracts were filtered and stored under refrigerated condition before analysis. 2.2.2 Preparation of glycated materials Glycated materials were prepared by the method described by Chompoo et. al. (2011) with slight variation. The reaction mixture containing 10 mg/mL bovine serum albumin, 1000mM glucose, 0.5 mg/mL of test sample extract and 50 mM phosphate buffer (pH 7.4) to a total volume of 4mL was incubated at 60 °C for 24 h. A control containing all the reaction mixtures except the sample was also subjected to the same condition before measurement. 2.2.3 Inhibition of α-dicarbonyl compounds formation Inhibition of α-dicarbonyl compounds formation was determined as per Chompoo et. al. (2011). Briefly, 1 mL aliquots of glycated materials were incubated at room temperature for 1 h with a reaction mixture containing 0.5 mL of Girard-T stock solution (500 mM) and 8.5 mL of sodium formate (500 mM pH 2.9). Absorbance were measured at 290 nm and glyoxal contents were

85 DFTQC, FRB 2016/17 calculated using a standard curve for glyoxal prepared using 40 % glyoxal solution in similar way. 2.2.4 Determination of anti-AGEs activity The anti-AGEs activity of the plant extracts were determined by the method described by Ahmad et al. (2016) with slight modifications. Briefly, 1 mL extracts with a concentration of 0.5mg/mLwere incubated with BSA (10 mg/mL) and glucose (1000 mM) in 50 mM phosphate buffer (pH 7.4) at 60 ˚C for 24 h. The fluorescence (excitation 360 nm and emission 450 nm) due to the formation of AGEs will be measured by fluorimeter. The AGEs inhibition will be calculated as follows: % AGEs inhibition = [C-(A-B)] x 100/C Where A, B and C represent the fluorescence of test sample (glucose, BSA, sample extract and phosphate buffer), test sample without BSA (glucose, sample extract and phosphate buffer) and control (glucose, BSA and phosphate buffer) respectively incubated at 60 °C for 24 h. 2.2.5 Determination of total phenolic content Total phenolic contents of the extracts were determined by Folin-Ciocalteu colorimetric method as described by Ho et al. (2010). Briefly, 0.1 ml of diluted extract and 0.5 ml of Folin-Ciocalteu phenol reagent was taken in test tube and then 0.4 ml of 7.5% Na2CO3 was added. The resulting solution was incubated for 1 h at room temperature and solution absorbance was measured at 765 nm. Gallic acid was taken as standard and the results was expressed as mg of gallic acid equivalents (GAEs) per g of the dried extracts. 2.2.6 Determination of DPPH radical scavenging activity DPPH free radical scavenging activities of extracts was determined by method described by Vignoli et al. (2011) with slight variation. Different dilutions of the extracts were made using 80% methanol. Then 1 mL of the extract was mixed with 2 mL of 0.1 mM DPPH solution. The absorbance was read at 517 nm after 30 min incubation in the dark. Finally, percentage scavenging activity was determined using following equation

% scavenging activity = (Ac-As) ൈ 100 /Ac

Where Ac is the absorbance of control and As is the absorbance of test sample.

The IC50 value was determined as the concentration required to give 50% scavenging activity.

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2.3 Statistical analysis All experiments were conducted in triplicate. The data were analysed by one- way ANOVA using IBM SPSS 20 (IBM Corporation, Marlborough, MA, USA). In case of significant difference, Tukey’s HSD test, at 5% level of significance, was used to separate means. 3. Results and Discussion Table 1 shows total phenol contents, DPPH free radical scavenging activities, glyoxal contents and advanced glycation end-products inhibitory activities of crude methanolic extracts of Fenugreek seeds and Fennel seeds. Table 1: Properties of crude methanolic extracts of Fenugreek and Fennel seeds* Parameters/Sample Fenugreek seed Fennel seed extract extract Total phenol contents (mg GAE/g 86.05a± 4.36 76.51b ± 2.93 dry extract) IC50 concentration for DPPH 14.94a ± 0.11 16.09b ± 0.13 inhibition (μg/mL) 1/IC50 concentration for DPPH 0.067a ± 0.0005 0.062b ± 0.0005 inhibition Glyoxal content (mM) 0.13a ± 0.007 0.12b ± 0.003 AGEs inhibition by 0.5 mg/mL 15.75a ± 0.68 24.21b ± 0.39 extract (%) *Values are the means of three determinations and the figures in the parentheses are their standard deviations. Figures in the rows with different alphabets in the superscripts are significantly different at p=0.05 Fenugreek seed extracts had significantly higher total phenol contents and resulted in higher glyoxal content in glucose-BSA model compared to those of Fennel seed extract (p<0.05). On the other hand, Fenugreek seed extract had significantly lower IC50 concentration for DPPH inhibition and showed lower inhibitory activity against AGEs in glucose-BSA model compared to those of Fennel seed extract. Further, a weak positive correlation was observed between total phenol content and 1/IC50 concentration for DPPH inhibition (r=0.75). This suggests that antiradical (or antioxidant) activities of the extracts can be attributed to their phenol contents.

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Antioxidants are reported to protect cells from the damage of Reactive Oxygen Species (ROS). ROS have been implicated in the induction and complications of diabetes mellitus, age-related eye disease, and neurodegenerative diseases such as Parkinson's disease. Moreover, the initiation, promotion, and progression of cancer, as well as the side-effects of radiation and chemotherapy, have been linked to the imbalance between ROS and the antioxidant defense system (Lobo et al., 2010). In our results, Fennel seed extract had higher AGEs inhibitory activity compared to Fenugreek seed extract while Fenugreek seed extract had higher antioxidant activity compared to Fennel seed extracts. Similar to our findings, there are inconsistent reports regarding the relation of anti-AGE activity with antioxidant activity. Strong correlations between anti-AGE activity and antioxidant activity were reported for some plant extracts while weak or even no correlations were reported for the some other plant extracts (Chen et al., 2011; Harris et al., 2011; Ramkissoon et al., 2012). This probably can be due to several reactions steps involved in the formation of AGEs and these reaction steps may require different reaction activators, promoters etc. 4. Conclusions The results suggest that both Fenugreek and Fennel seeds can inhibit formation of AGEs and based on AGEs inhibitory activity Fennel seed appears to be more effective compared to Fenugreek seed in alleviating diabetes related complications. References

Acharya, E. and Pokhrel, B. (2006). Ethno-Medicinal Plants Used by Bantar of Bhaudaha, Morang, Nepal. Our nature.4, 96-103.

Ahmad, R., Ahmad, N., Naqvi, A. A., Exarchou, V., Upadhyay, A., Tuenter, E., Foubert, K., Apers, S., Hermans, N. and Pieters, L. (2016). Antioxidant and antiglycating constituents from leaves of Ziziphus oxyphylla and Cedrela serrata. Antioxidants.5 (9).

Araki, N., Ueno, N., Chacrabarti, B., Morino, Y. and Horiuchi, S. (1992). J. Biol. Chem.267, 10211-10214. [Cited in K. Ikeda, T. Higashi, H. Sano, Y. Jinnouchi, M. Yoshida, T. Araki, S. Ueda and S. Horiuchi. (1996). Nε- (Carboxymethyl)lysine Protein Adduct Is a Major Immunological Epitope in Proteins Modified with Advanced Glycation End Products of the Maillard Reaction. Biochem. 35, 8075-8083].

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Chompoo, J., Upadhyay, A., Kishimoto, W., Makise, T. and Tawata, S. (2011). Advanced glycation end products inhibitors from Alpinia zerumbet rhizomes. Food Chem.129, 709–715.

Duraipandiyan, V., Ayyanar, M. and Ignacimuthu, S. (2006). Antimicrobial activity of some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India. BMC Complementary and Alternative Med.6 (35).

Eble, A. S., Thorpe, S. R. and Baynes, J. W. (1983). Nonenzymatic glycosylation and glucose-dependent cross-linking of proteins. J Biol Chem.258, 9406–9412. [Cited in J. Uribarri, S. Woodruff, S. Goodman, W. Cai, X. Chen, R. Pyzik, A. Yong, G. E. Striker and H. Vlassara. (2010). Advanced Glycation End Products in Foods and a Practical Guide to Their Reduction in the Diet. J Am Diet Assoc.110 (6), 911–16].

Ho, S.-C., Wu, S.-P., Lin, S.-M. and Tang, Y.-L. (2010). Comparison of anti- glycation capacities of several herbal infusions with that of green tea. Food Chem.122, 768–774.

Kume, S., Takeya, M., Mori, T., Araki, N., Suzuki, H., Horiuchi, S., Kodama, T., Miyauchi, Y. and Takahashi, K. (1995). Am. J. Pathol.147, 654-667. [Cited in K. Ikeda, T. Higashi, H. Sano, Y. Jinnouchi, M. Yoshida, T. Araki, S. Ueda and S. Horiuchi. (1996). Nε-(Carboxymethyl)lysine Protein Adduct Is a Major Immunological Epitope in Proteins Modified with Advanced Glycation End Products of the Maillard Reaction. Biochem. 35, 8075-8083].

Limbu, D. K. and Rai, B. K. (2013). Ethno-medicinal practices among the limbu community in Limbuwan, eastern Nepal. Global J Human Social Sci.13 (2).

Makino, H., Shikata, K., Hironaka, K., Kushiro, M., Yamasaki, Y., Sugimoto, H., Ota, Z., Araki, N. and Horiuchi, S. (1995). Kidney Int.48, 517-526. [Cited in K. Ikeda, T. Higashi, H. Sano, Y. Jinnouchi, M. Yoshida, T. Araki, S. Ueda and S. Horiuchi. (1996). Nε-(Carboxymethyl)lysine Protein Adduct Is a Major Immunological Epitope in Proteins Modified with Advanced Glycation End Products of the Maillard Reaction. Biochem. 35, 8075-8083].

Muthenna, P., Akileshwari, C., Saraswat, M. and Reddy, G. B. (2012). Inhibition of advanced glycation end-product formation on eye lens protein by rutin. British J. of Nutri. 107, 941–949.

Rahmatullah, M., Noman, A., Hossan, M. S., Rashid, M., Rahman, T., Chowdhury, M. H. and Jahan, R. (2009). A survey of medicinal plants in two areas of Dinajpur district, Bangladesh including plants which can be used as functional foods. American-Eurasian J Sustainable Agri.3 (4), 862-876.

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Ruderman, N., Williamson, J. and Brownlee, M. (1992). "Hyperglycemia, Diabetes, and Vascular Disease". Springer. New York.

Schmidt, A. M., Yan, S. D., Wautier, J. L. and Stern, D. (1999). Activation of receptor for advanced glycation end products: A mechanism for chronic vascular dysfunction in diabetic vasculopathy and atherosclerosis. Circ Res.84, 489–497. [Cited in J. Uribarri, S. Woodruff, S. Goodman, W. Cai, X. Chen, R. Pyzik, A. Yong, G. E. Striker and H. Vlassara. (2010). Advanced Glycation End Products in Foods and a Practical Guide to Their Reduction in the Diet. J Am Diet Assoc.110 (6), 911–16].

Singh, R., Barden, A., Mori, T. and Beilin, L. (2001). Advanced glycation end- products: a review. Diabetologia.44, 129-146.

Thornalley, P. J. (1996). Pharmacology of methylglyoxal. Gen Pharmacol.27, 565-573. [Cited in R. Singh, A. Barden, T. Mori and L. Beilin. (2001). Advanced glycation end-products: a review. Diabetologia. 44, 129-146].

Ulrich, P. and Cerami, A. (2001). Protein glycation, diabetes, and aging. Recent Prog Horm Res.56, 1-21. [Cited in J. Uribarri, S. Woodruff, S. Goodman, W. Cai, X. Chen, R. Pyzik, A. Yong, G. E. Striker and H. Vlassara. (2010). Advanced Glycation End Products in Foods and a Practical Guide to Their Reduction in the Diet. J Am Diet Assoc.110 (6), 911–16].

Vlassara, H. (1996a). Advanced glycation end-products and atherosclerosis. Ann Med.28, 419–426. [Cited in S.-Y. Goh and M. E. Cooper. (2008). The role of advanced glycation end products in progression and complications of diabetes. J Clin Endocrinol Metab. 93 (4), 1143-1152].

90 DFTQC, FRB 2016/17

Availability of Sulphurdioxide in the Food Products Commonly Found in Nepal

Hareram Pradhan* , Ranjana Chaudhary, Tulsi Bhandari, Atish Ghimire, Krishna Prasad Rai and Sanjeev Kumar Karn

Department of Food Technology and Quality Control, Babarmahal, Kathmandu

*Corresponding author: [email protected]

Abstract Sixty-one food samples were collected from the different part of Nepal for the determination of SO2 content. The samples were characterized as fruit juices, squashes, jam/jelly/marmalade, dried products, paau/titaura/candy, pickles, meat products and wine. Modified-Monier-Williams method was used for the determination of SO2. Recovery test was performed for the method. SO2 was found in the range from 0 to 2114.7 ppm in the samples. Maximum amount of

SO2 was found in dried product that is raisins (2114.7 ppm) whereas there was no presence in meat products. Four samples including two orange squash and two dried products (raisins) were found to exceed the maximum limit of mandatory standard of Nepal Government. In general, SO2 was present below the maximum limit in all the food categories except squash and raisins products.

Keywords: Food preservatives, Mandatory standard, Modified-Monier- Williams method, Recovery, Sulphurdioxide

1. Introduction Food preservatives are the compounds used during food processing for the preservation of food products till they are consumed. Different kinds of preservatives are used in different kind of processed foods. Class I preservatives such as salt, sugar, vinegar and other acids, oil, spices, etc. are used for the preservation of different foods. There is no limit for the usage of these preservatives in the food products. Class II preservatives such as sulphurdioxide, nitrite, benzoate, sorbate are commonly used as in food products. The use of these preservatives is strictly limited up to certain level because of its health effects.

Among these, sulphurdioxide (SO2) is mostly used in fruit juice, jam, jelly, marmalade, sugar, dried foods, sausage with raw meat, wines, beer, pickles etc for the preservation purpose or as bleaching agent. This compound is also used 91 DFTQC, FRB 2016/17 in other processes as oxygen scavenger, reducing agent and enzyme inhibitor

(Fazio and Warner, 1990). SO2, in its gaseous or liquid form, or dissolved in water to form sulfurous acid, or in the form of its neutral or acid salts is widely used as a chemical preservative of food products (Joslyn and Braverman, 1954).

In the context of Nepal, SO2 in the form of KMS is usually used by small and medium scale entrepreneurs during the production fruits and vegetables juice/dried products. However, there are only few industries which have technical personnel to monitor the usage of those chemicals and most of them don’t have well equipped laboratory to determine the SO2in their products before they go to the market. Till now, there is no any study conducted to find out how much SO2 is available in the commonly used food product in our daily life. The usage of this compound is strictly regulated as it has negative effect on health if the amount exceeds the recommended daily intake (USDHHS, 1998).

Government of Nepal (GON) also has regulatory standard for the usage of SO2 in different food products as shown in table 1. During the processing of food, potassium metabisulphite (KMS) is used as source of SO2 as it is difficult to inject the gaseous form in the food products.

Table 1: Mandatory limits for the use of SO2 in different food products as per GON Food Products Standard (Maximum limit in part per million, ppm) Fruit juice concentrate 1500 Dried apricots, peaches, apple, pears, ginger 2000 Dried raisins 750 Squash, cordials, fruit syrups, barley water 350 Jam, Jelly, Marmalade 40 Corn syrup 450 Ready to serve beverages 70 Fruits or vegetables pickles 250 (Source: Minimum mandatory standard for food and feed products, DFTQC, 2073)

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2. Materials and Methods 2.1 Materials 2.1.1 Collection of samples Samples were collected from different parts of country such as Ilam, Dhankuta, Morang, Hetauda, Sindhuli, Chitwan, Dhading, Kavrepalanchowk, Kathmandu,

Lalitpur, Bhaktapur, Pokhara, Gorkha. Only those samples in which SO2 could be used according to Nepal standard were collected. Samples were categorized in eight classes viz. fruit juices, squashes, jam/jelly/marmalade, dried products, paau/titaura/candy, pickles, meat products, wine. 2.1.2 Reagents Aqueous hydrochloric acid solution (4N), methyl red indicator (0.25% in water), standard titrant (0.01N NaOH), 3% hydrogen peroxide solution (Prior to use, it was neutralized with 0.01N NaOH using methyl red indicator to yellow color), high purity nitrogen gas. 2.1.3 Apparatus Distillation apparatus: Apparatus was assembled as shown in the figure 1(b). Due to unavailability of three necks round bottom flask, two necks round bottom flask was used as shown in the figure. 2.2 Method

2.2.1 Determination of SO2 Qualitative test was performed first followed by quantitative test, Modified- Monier-Williams (AOAC, 2016), if detected positive.

2.2.2 Qualitative analysis 25 gm of sample was taken in a 200 ml flask. If the sample was solid, it was dissolved with small amount of water. Few pieces of sulphur free granulated zinc and approximately 10 ml of 4N HCL was added. Lead acetate paper was wrapped around the mouth of the flask. Observation of black color in the paper indicates the presence of SO2.

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2.2.3 Quantitative test

Quantitative determination of SO2 was done by Modified-Monier-Williams Method (AOAC 2016) as shown in the figure below. Duplicate analysis was performed for each sample.

(a) (b) Figure 1: Modified-Monier-Williams method (AOAC, 2016)

2.2.3.1 Preparation of sample Solid sample: 50 gm of sample was weighed in conical flask and mixed with 100ml of ethanol water (5+95, v/v) and chopped in small pieces. Liquid sample: 50 gm of sample was weighed in conical flask and mixed with 100ml of ethanol water (5+95, v/v). 2.2.4 Procedure Apparatus was assembled according to the figure 1(b). 400ml of deionized water was taken in flask C. 90ml of 4M HCl was introduced to the flask. N2 was started at the rate of 15-20 bubbles per min to flow from the side tube of the

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flask.Prepared sample was introduced through the side A, mouth of the flask. To

the vessel G, around 50 ml of neutralized H2O2 was placed and another tip of the bubbler F was dipped in the vessel G. Heating mantle was turned on to boil the

content. Boiling was done for 1.7h. Collected H2O2 in vessel G was immediately titrated with standardized NaOH to the end point. ૜૛Ǥ૙૜࢞ࢂ࢈࢞ࡹ࢞૚૙૙૙ Calculation: SO2, μg/g(ppm)= ࢃࢋ࢏ࢍࢎ࢚

Where, 32.03= milliequivalent weight of SO2; Vb= volume (ml) of NaOH of molarity M required to reach the end point; 1000=factor to convert milliequivalents to microequivalents; weight= weight, g, of test portion introduced in the flask b. 3. Results and Discussion 3.1 Sample collection Sixty one samples were collected from sixteen different districts of Nepal. It was found that the sampeles were manufactured in sixteen different districts of Nepal. Collected samples were categorized as shown in the figure 2.

3.2 SO2 content

SO2 content in different food products are shown in the Table 2. It was found

that SO2 was present in almost all categories of food samples. Highest amount was found in dried products followed by squash whereas any of the collected

meat products contained no SO2. Four samples including two orange squash and

two raisins had exceeded the maximum limit of SO2 as given by the Government

of Nepal. Considerable variation in the amount of SO2 was found in the same product within different brands. This indicates that those industries have done sulfitation at different level. Machado et al. (2008) had also found wide range of sulphurdioxide and differences between different brands.

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14 13 Collected sample 12 10 9 9 8 7 7 6 6 5 5 4 2 0 No of samples Squash Juice Dried Pickles Titaura/ Meat Jam/Jelly Wine Products Paaun/ Mada products

Figure 2: Category of collected samples

Table 2: SO2 content in different category of food products Sub No. of Lowest Highest Mean Commodity standard Samples SO2, ppm SO2, ppm SO2, ppm products 2 Squash 7 0 516 195.62 0 Juice 9 0 10.3 3.14 2 Dried products 13 0 2114.7 363.46 0 Pau/Titaura/Candy 9 0 24 4.96 0 Meat products 5 0 0 0 0 Jam/ Jelly 6 0 8.6 4.82 0 Wine 7 0 26.93 4.72 0 Pickles 5 0 14.6 3.97

The range of SO2 in squash is 0 to 516 ppm. Out of 7 samples of squashes, two products had exceeded the maximum limit of SO2. Three brands of squash viz. Druk, Sagarmatha and Takura were available in the market. It was also found that lesser amount of KMS was added in lemon squash than in orange squash. Pulpy, Frooti and Real brand juices were collected. Among the nine juice products, six products had shown negative result from qualitative analysis. Two

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juice products had contained <10ppm of SO2 and one product had contained

10.3ppm. This indicates that the amount of SO2 present may comes either from addition of small amount of KMS during production or from the residue present in different food ingredients like sugar. However, there was not mentioned anything about added SO2 as preservatives in the label of juices.

Widest range of SO2 was found in dried products. Dried products included raisin, pomelo, fish, anjeer, khurpani, maseura. Nepal Government has different standard for different dried product as shown in Table 1. Dried fish and dried pomelo had shown negative result in qualitative test. However, all other eleven dried products had contained SO2 ranging from 5.8 to 2114.7 ppm. Two raisin products were found to be substandard as per Nepal standard (<750 ppm). Five kinds of pickles with Paincho, Tasty, Pokhreli brand were collected. Three samples had shown negative result during qualitative test, one sample had contained < 10ppm and one sample contained 14.6ppm. Since, mustard seeds are widely used during the production of pickles, trace amount of SO2, may come from sulphide containing compounds. This indicates that although it is allowed to add SO2 in pickles, most of the industries either don’t add KMS or add it in very small amount. Titaura, paau, candies were also collected for the analysis. However, only one out of nine samples had contained > 10 ppm SO2 (24.01ppm). Rest of the samples contained either no or less than 10 ppm of SO2. This residual amount of SO2 may come from other ingredients like sugar. Further, paau, titaura and similar products contain black salt contains sulphide, bisulfate and bisulfite salts. These trace amount of sulfur containing compounds also contributes for SO2 during determination. Chicken salami, chicken sausage, ham, buffalo sausage, buffalo salami were collected from the local market for the analysis. SO2 was not detected in any of the five products. This indicates that KMS is not used for the production of meat products. Jam and marmalade samples had contained less than 10 ppm of SO2. This residual amount of SO2 may come from other ingredients like sugar or fruit concentrate used during production. Seven different brands of wine produced in Nepal were collected for the analysis. Four products had shown negative result in qualitative test, two products less

DFTQC, FRB 2016/17 than 10 ppm and only one product had contained 26.93 ppm. This indicates that fewer brands are using SO2 as preservative in wine. The final results are shown in the figure 3. 3.3 Recovery Test

79.9% of the added SO2 was recovered from sample. Lower recovery was due to oxidation or formation of complex with the food matrix and air as sulphite is highly reactive. It is mentioned that sodium hydroxymethyl sulphonate (HMS), which is bisulphite addition product of formaldehyde, structurally similar to some combined forms of sulfites in food, is useful for the spiking in the test materials. Recoveries of ≥ 80%from food matrixes spiked at 10 μg/g are recommended to ensure accurate analysis (AOAC 2016). Due to unavailability of HMS, KMS was used for the recovery test. This indicates that the recovery was satisfactory because KMS is more reactive than HMS.

Figure 3: SO2 contains in different food categories

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4. Conclusions Modified-Monier-Williams method was used for the quantitative determination of SO2. 61 food samples were collected from the sixteen different districts of

Nepal. SO2 was found in the range from 0 to 2114.7 ppm. Maximum amount of

SO2 was found in raisins whereas no SO2 was found in meat products. Pickles, jam/jelly and juices had content small amount of SO2. This amount of SO2 might come from the sulphurous compounds naturally present food ingredients. Four samples were found to exceed the maximum limit of Nepal standard. In general,

SO2 was present below the maximum limit in all the food categories except squash and raisins products. The recovery of sulphurdioxide from this method was found to be 80 %. Acknowledgement First of all, we would like to thank DFTQC for allocating fund for research programs to CFL and secondly, to CFL for believing in our capacity to conduct research work in sulphurdioxide. We would also like to pay our deepest gratitude to our Director General, Sanjeev Kumar Sir, for his valuable advice throughout our work. Similarly, we are also thankful to our laboratory chief, senior food research Officers: Mahipal Ram Baidya, Bimala Neupane, Kamal Prasad Regmi, Shiv Sagar Chaudhary, Neera Malakar, and Hemanta Kumar Parajuli for their continuous support to improve the quality of our research work. We are also grateful to our other colleagues in the laboratory who were with us to cheer up during our tedious lab work. References AOAC (2016). Official Methods of Analysis, 20th Edition, Association of Official Analytical Chemists, Washington, DC. DFTQC (2073). Minimum mandatory standard for food and feed products. Fazio, T., Warner, C. R. (1990). A review of sulphites in foods: analytical methodology and reported findings. Food Additives and Contaminants, 7(4): 433-454. FSSAI (2016). Manual of methods of analysis of food, food additives. Joslyn, M. A., and Braverman, J. B. S. (1954). The Chemistry and Technology of the pretreatment and preservation of fruits and vegetable products with sulfurdioxide and sulfites. Advance in Food Research, 5, 97-160.

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Machado, R. M. D., Toledo, M. C. F., Vincente, C. A. S. A. E., (2008). Analytical determination of sulphites in fruit juices available on the Brazilian market. Brazilian Journal of Food Technology, vol 11, No. 3, p. 226-233. USDHHS (1998). Toxicological profile for Sulfur dioxide, US Department of Health and Human Services.

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Process Optimization and Quality Evaluation of Bara, an Indigenous Newari Food

Pratima Shrestha

Department of Food Technology and Quality Control, Babarmahal, Kathmandu

Corresponding author: [email protected]

Abstract Bara, also known as wo, is traditional Nepalese food indigenous to Newar community of Kathmandu valley. It is shallow fried circular patties of black gram with spices and salt. The major objectives of the work were to formulate the recipe, optimize the process parameters and quality evaluation of bara. According to the survey, the batter formulation was cumin powder 1 %, ginger paste 1 %, asafoetida 0.1 %, salt 1.1 % and water 20ml/100g soaked and dehulled black gram. Soaking of black gram for 10 h, 2 min whipping of batter and roasted mustard seed oil as frying medium was found optimum for preparation of bara. Bara fried at 175˚C for 6 min was found to be best among all time-temperature combination studied. Lab prepared bara was superior to traditionally prepared bara. The weight, thickness, diameter and oil uptake of bara were found to be 55.87 g, 12.9 mm, 80.75 mm and 27.23 % respectively. The moisture content, crude protein, crude fat, crude fiber, total ash, total carbohydrate and calorific value of bara were 57.78 %, 7.76 %, 9.04 %, 1.8 %, 2.01 %, 21.61 % and 206.08 Kcal/100g. Calcium and iron content (per 100 g bara) were found to be 348.65mg and 68.37 mg respectively. Tannins, oxalates and phytates in the product (per 100g dry matter) were found to be 0.117 g, 0.0037 g and 428.67 mg respectively.

Keywords: Bara, Indigenous, Microbiology, Shallow frying

1. Introduction: Wo is one of the most important indigenous products of Nepal, typical to Newar community of Kathmandu valley. It is popular as bara throughout the country. It is circular, 8-14 mm thick patties made by shallow frying of dehulled black gram paste with spices and salt. The word wo means silver in Newari language and this name was probably given due to resemblance of colour of black gram paste with silver (Chakhhun, 2016). It is an important dish of Samaybaji. It is

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DFTQC, FRB 2016/17 generally prepared in special occasions like Dashain, Tihar, Sithinakha (Dewali of Newar community), Indra jatra, Janai purnima etc. as an offering to Gods and Goddesses. It is indispensible item in nhenbu and ghasu (ritual process of Newar community performed on 7th and 11th day of funeral respectively). It is also prepared as sagun in various cultural events like marriage ceremony, rice feeding ceremony, Bratabandha, Bel-bibaha, Suryadarshan, Janku, Mata-tirtha aausi, Kushe aausi, birthday ceremony and many more. It is also served as special dish to guests along with Samaybaji and Raksi. It is very delicious and energy rich food item liked by many people of all ages (Shresthaa, 2014). Wo is believed to have originated since 12th century when goddess Taleju bhawani was worshipped. It is believed that the first use of wo was made in tantric puja as an offering to gods and goddesses. Wo made for this purpose was made from black gram and salt only. Later on, people started using different spices and pulses. Wo is used both as sagun and as one of the ingredients of Samaybaji. Wo symbolizes wind/air when used as sagun whereas it represents one of the elements of panchabali when used with samaybaji (Chakhhun, 2016). There has not been any scientific study of the product and no written documents are available for this indigenous product of our country. Still, it is not commercialized. So, a detailed study of this product and optimization of its production process for a consistent quality of the product is the fundamental requirement. This could help to improve the quality of the product and its commercialization. The general objective of the work was to optimize process parameters and quality evaluation of bara, an indigenous food of Nepal. 2. Materials and Methods 2.1 Materials All the raw materials were purchased from local market. 2.2 Methods 2.2.1 Formulation of recipe: Survey of bara was conducted on 50 people of age group 40-85 years at different places of Kathmandu valley among the native Newar community following questionnaire method According to the survey, the batter formulation was cumin powder 1 %, ginger paste 1 %, asafoetida 0.1 %, salt 1.1 % and water 20 ml/100g

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DFTQC, FRB 2016/17 soaked and dehulled black gram. The procedure for preparation of bara is shown in figure 1. 2.2.3 Optimization of process parameters The overall optimization process was carried out in four stages. The optimum process from first step was used to optimize the second step and so on. Sensory evaluation was carried out using nine point hedonic rating involving 10 semi- trained panelists in each stage to find the optimum process condition. On the first stage of optimization, optimum soaking time was determined among 6 h, 8 h, 10 h and 12 h soaking of black gram. On the second stage, optimum whipping time of batter was determined among whipping time of 0 min, 1 min, 2 min and 3 min. Selection of frying medium was done on the third stage of optimization. Frying medium used were roasted mustard seed oil, raw mustard seed oil, sunflower oil and soybean oil. On the last stage of process optimization, optimum frying time temperature combination was determined among frying combination of 165˚C/5min, 165˚C/6 min, 165˚C/7 min, 175˚C/5min, 175˚C/6 min, 175˚C/7 min, 185˚C/5min, 185˚C/6 min and 185˚C/7 min.

Black gram

Cleaning

Soaking in water 6-12 hours

Complete dehulling by rubbing using hands to remove hull

Draining

Wet grinding in silauta

Fine paste

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Adding ginger paste, cumin powder, asafoetida, salt and water

Whipping/Foaming

Shallow frying in hot oil

Bara

Fig 1: Flowchart for the preparation of bara Source:Shresthaa (2014)

2.2.4 Analysis of optimized product Weight of the product was measured using electronic balance. The thickness of product was measured using electronic balance. The diameter of product was measured by measuring its circumference. Moisture, crude protein, crude fat, ash content and crude fibre content were determined as described by Ranganna (2007). Fat uptake of the product was determined on fat and moisture free basis as described by Katawal and Subba (2011). Carbohydrate content was determined by difference method. Calcium and Iron content were determined as per Ranganna (2007). Calorific value was determined by calculation method. Microbiological analysis was carried out using AOAC (1990). 2.3 Data analysis The experiment was conducted with three replications. Experimental data were analyzed using Microsoft Excel and for ANOVA using GenStat Discovery Edition 3 (Seventh-Edition, Version 7.2.2.222). The means were compared by L.S.D. method at 5% level of significance.

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3. Results and Discussion 3.1 Optimization of soaking time Different samples were prepared by soaking black gram in water of pH 6.8 at room temperature for different time (A= 6 h, B= 8 h, C= 10 h and D = 12 h) with all other parameters remaining constant. The samples were subjected for sensory evaluation. The mean of sensory score given to each quality attribute for all samples was calculated and a bar diagram was plotted which is shown in fig 2. The statistical analysis showed that different soaking time had significant effect (p<0.05) on the sensory attributes of bara. Sample C got highest mean score in terms of colour, taste and overall acceptability was selected as best sample. Shresthab (2016) also found that soaking time of 10 h was sufficient to make furaula (a deep fried traditional snack made from green gram and black gram) of good quality. Therefore, soaking time of 10 hours was selected as optimum.

A B C D

10 a a a c c b b a a b a a a a a a b c c 8 a 6 4 2 0 Mean sensory score Colour Smell Texture Taste Overall acceptability Sensory attributes

Fig 2: Effect of soaking time on sensory attributes of bara Similar alphabet above the bar for each parameter indicate that samples are not significantly different (p<0.05). Vertical error bars indicate standard deviation. 3.2 Optimization of whipping time Four different samples were prepared by using different whipping time (A= 0 min, B=1 min, C = 2 min, D= 3 min) with all other parameters remaining constant. The samples were subjected to sensory analysis. The mean of sensory 105

DFTQC, FRB 2016/17 score given to each quality attribute of each sample was calculated and a bar diagram was plotted which is shown in figure 3. The statistical analysis showed that different whipping time had significant effect (p<0.05) on the sensory attributes of bara.

A B C D

10 a b b b b b a a b b c a a a a a a a a ab 8 6 4 2 Mean sensory score 0 Colour Smell Texture Taste Overall acceptability Sensory attributes

Fig 3: Effect of whipping time on sensory attributes of bara Similar alphabet above the bar for each parameter indicate that samples are not significantly different (p<0.05). Vertical error bars indicate standard deviation. Whipping incorporates air into the batter, which gives light and spongy texture to the fried products, a desirable quality attribute of bara. Sample C got highest mean score in terms of smell, taste and overall acceptability. Even though sample D got highest score in terms of texture, it was not significantly different from sample C. Hence, sample C was selected as best sample. Therefore, whipping time of 2 min was selected as optimum. 3.3 Selection of frying medium Four different samples were prepared using different frying medium (A= raw mustard seed oil, B = roasted mustard seed oil, C = soybean oil and D = sunflower oil) keeping all other parameters constant. Sensory evaluation of the samples was done. The mean sensory score of each quality attributes for each of the samples was calculated and a bar diagram was plotted. Fig 4 shows the bar diagram of the mean sensory score for each quality attributes.The statistical

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DFTQC, FRB 2016/17 analysis showed that different frying medium had significant effect (p<0.05) on the sensory attributes of bara. Based on the statistical analysis, sample B was found to be best among 4 samples. Sample B got highest mean score in terms of colour, smell, taste and overall acceptability. It may probably be due to development of flavoring compounds on roasting of mustard seeds. Mustard oil is considered one of the healthiest edible oils as it has low amount of SFAs and a high amount of MUFA and PUFA fatty acids, which are good for health. Compared to other oils, mustard oil has several benefits. Furthermore, the ω-6: ω-3 ratio of mustard oil is near ideal 6: 5. Several clinical studies have also found that mustard oil may be the best for heart health (Mishra and Manchanda, 2012). Therefore, oil obtained from roasted mustard seed was selected as the best oil for making bara.

A B C D 12 b 10 b a b c a abab b b ac c c bc c a a a 8 a a 6 4 2 Mean sensory score 0 Colour Smell Texture Taste OA Sensory attributes

Fig 4: Effect of frying medium on sensory attributes of bara Similar alphabet above the bar for each parameter indicate that samples are not significantly different (p<0.05). Vertical error bars indicate standard deviation. 3.4 Optimization of frying time temperature combination Nine different samples were prepared using different time temperature combination with all other parameters remaining constant (A=165˚C/5min, B=165˚C/6 min, C=165˚C/7 min, D=175˚C/5min, E=175˚C/6 min, F=175˚C/7

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DFTQC, FRB 2016/17 min, G=185˚C/5min, H=185˚C/6 min and I=185˚C/7 min).The mean sensory score of each quality attributes for each of the samples was calculated. Fig 5 shows a bar diagram of mean sensory score of each quality attributes for each of the samples.

A B C D E F G H I d d 10 d dde ce eef ef d d c c c bbc bc b b c c c c bcc c d bc b ab ab a ab ad bd b ab b ab 8 a a aab a a 6 4 2 0 Mean sensory score Colour Smell Texture Taste Overall acceptance Sensory attributes

Fig 5: Effect of time-temperature combination on sensory attributes of bara

Similar alphabet above the bar for each parameter indicate that samples are not significantly different (p<0.05). Vertical error bars indicate standard deviation. The statistical analysis showed that different frying conditions had significant effect (p<0.05) on the sensory attributes of bara. From the sensory evaluation, sample E got highest mean score in terms of smell, taste and overall acceptability. Even though sample F got highest score in terms of colour and texture, statistical analysis showed that it was not significantly different from sample E. Hence, sample E was selected as best among nine samples. Therefore, optimum frying time temperature combination was found to be 175˚C for 6 min. 3.5 Comparison of traditionally and lab prepared bara Lab prepared (Sample A) and traditionally prepared (Sample B) bara were subjected for sensory evaluation. The mean of sensory score given to each quality attribute of each sample was calculated and a bar diagram was plotted which is shown in fig 6.

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A B a a a a a a 10 b b a b 5 0

Mean sensory score Colour Smell Texture Taste Overall acceptance Sensory attributes

Fig 6: Comparison of sensory attributes of traditionally and lab prepared bara Similar alphabet above the bar for each parameter indicate that samples are not significantly different (p>0.05). Vertical error bars indicate standard deviation. Based on the statistical analysis of the sensory data, lab prepared bara were superior to traditionally prepared bara in terms of colour, texture and overall acceptance. This may be due to control of frying time and temperature and adequate whipping of batter during bara preparation. Although, traditionally prepared bara got high mean score compared to lab prepared bara in terms of taste and smell, there was no significant difference (p>0.05). Hence, comparatively lab prepared bara is superior to traditionally prepared bara. 3.6 Physical analysis of bara Weight, thickness and diameter of bara was found to be 55.87 g, 12.9 mm and 80.75 mm respectively. The analytical result of physical parameters is given in table 1. Table 1: Physical analysis of bara Parameter Value Weight 55.87±1.85g Thickness 12.9±0.03mm Diameter 80.75±0.35mm The values obtained are mean of three determinations ± standard deviation.

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3.7 Chemical composition of bara on wet basis The chemical composition of bara (wet basis) is given in table 2.The moisture content of bara was found out to be 57.78 %. This is due to the shallow frying of the batter. The crude protein and crude fat content of bara was found to be 7.76 % and 9.04 % respectively. The protein content is due to the protein present in the main ingredient, i.e. black gram while the fat content is due to the presence of frying oil. The oil uptake of the product was found to be 27.23 %. The crude fiber content and total ash content was found to be 1.8 % and 2.01 % respectively. The calorific value of the product was found to be 206.08 Kcal. This calorific value is due to the presence of carbohydrate, fat and protein in the product. Calcium and iron contents were found to be 348.65 mg/100g and 68.37 mg/100g respectively. Similar results were found for furaula made from green gram and black gram (Shresthab, 2016). Table 2: Chemical composition of bara Parameters Value per 100 gram Moisture content (g) 57.78±0.24 Crude protein (g) 7.76±0.23 Crude fat (g) 9.04±0.05 Crude fiber (g) 1.8±0.03 Total ash (g) 2.01±0.01 Carbohydrate (g) 21.61±0.27 Energy 206.08 Kcal Calcium (mg) 348.65±0.72 Iron (mg) 68.37±0.39 The values obtained are mean of three determinations ± standard deviation.

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3.8 Microbiological analysis of bara Table 3 shows Total Plate count (TPC), E. coli and yeast and mold count of bara soon after frying as received by microbiological assay.

Table 3: Microbiological analysis of the product Parameters Value (cfu/g) TPC 25 E. coli Nil Yeast and mold Nil The product was found to be free from pathogenic E. coli and yeast and mold. The TPC count is also in low level. Therefore, the product can be considered safe from microbiological point of view. 4. Conclusion: On the basis of survey and analysis, soaking time, whipping time, frying medium and frying time-temperature combination were optimized to be 10 h, 2 min, roasted mustard oil and 175˚C for 6 min for the preparation of bara. Bara, thus prepared, was found to be nutritious , hygienic as well as sensorily acceptable. Acknowledgements: Author would like to thank Prof. Dr. Surendra Bahadur Katawal for his valuable suggestions during the work. Special thanks to Microbiology laboratory of Department of Food Technology and Quality Control and Central Campus of Technology, Hattisar, Dharan for the laboratory services.

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References AOAC. (1990). "Official Methods of Analysis" (15th ed.). Arlington, VA, USA.Association of Official Analytical Chemists. Chakhhun, K. L. (2016). Personal communication [Interview]. 1 July, 2016 Mishra, S. and Manchanda, S. C. (2012). Cooking oils for heart health. J. Preventive Cardiology.1 (3), 123-131. Ranganna, S. (2007). "Hand book of Analysis and Quality control for fruit and vegetable products" (4th ed.). Tata McGraw- Hill Publishing company Ltd. New Delhi. Shresthaa, R. (2014). Personal communication [Interview]. 28 November, 2014. Shresthab, S. (2016). Preparation, Recipe And Process Optimization and Quality Evaluation Of Furaula, An Indigenous Nepali Food. B. Tech Dissertation. Tribhuvan Univ., Nepal.

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Effect of Germination on Phytic Acid content of Amaranth seeds (Amarantus cruentus)

Bijan Shrestha1*, Sanjay Bhandari2and Pramod Koirala2 1Department of Food Technology and Quality Control, Babarmahal, Kathmandu. 2Regional Food Technology and Quality Control Office, Biratnagar. *Corresponding author: [email protected] Abstract The effect of germination on the level of phytic acid of amaranth grain grown in the Far-Western Region of Nepal was studied. Phytic acid level in the amaranth grain was in the range of 2171.48 ±22.84 mg/100g. The grains were soaked for 5 h and germinated to 12, 24, 48 and 60 h and phytic acid content was evaluated. Phytic acid content was significantly (P<0.05) reduced during soaking and germination. After germination for 60 h, 39% reduction in the phytic acid level was observed in the amaranth grain.

Keywords: Amaranth, antinutritional factors, germination, phytic acid

1. Introduction Amaranth is an ancient plant belonging to the family Amaranthaceae, which is believed to have originated from central and southern America. Amaranth is a hardy, wild, fast-growing cereal-like plant with a seed yield of about 3 tons per hectare in 3–4 months (Singhal and Kulkarni, 1988). It has been considered a ‘pseudo cereal’ because of its similarity with other cereals, and a ‘dual crop’ since both leaves and seeds of the plant can be consumed as vegetables and cereal respectively. The crop has the potential to address the nutritional needs of vulnerable people because of its high content of protein, essential fatty acids and micronutrients (Muyonga, et al., 2008). Studies have reported that amaranth species are rich in vitamins A, B6, C, riboflavin, thiamine and folates and essential minerals (Cheruiyot, 2011). The lysine content is given as the main reason for the high protein quality of amaranth, since the grain contains more of this essential amino acid than cereal grains (Saunders and Becker, 1983; Teutonico and Knorr, 1985; 113

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Stikic et al., 2012). However, the biological value of raw amaranth grain is not high as suggested by its amino acid pattern. The reasons for this effect may be due to the presence of inherent antinutritional factors in amaranth seeds. The antinutrients that are present in the amaranth seeds are saponins, phytic acid and toxic alkaloids that affect the nutritional properties of the seeds by lowering starch digestibility, protein and micronutrient absorption (Repo-Carrasco et. al., 2003; Valencia, 2004; Martínez-Villaluenga et. al., 2006). Lorenz and Wright (1984) also reported higher level of phytic acid and phenol contents in some varieties of amaranth seeds. The majority of ingested phytate is undergraded during transit through the gastrointestinal tract. One phytate molecule can bind up to six divalent cations, and the metal could possibly bridge at least two phytate molecules, depending on the redox state (Graf andEaston, 1990). Phytic acid is a powerful inhibitor of iron-driven hydroxyl radical formation because it forms a catalytically inactive iron chelate. In context of Nepal, leaves of amaranth are consumed, while its nutritious grain is left underutilized. General consumption pattern of the grain comprises puffing the grain in dry heat, shaping into a ball shaped confectionery with jaggery and as a topping on anarasa, an indigenous Nepalese food. DFTQC (2015) has developed a number of delicacies for the proper consumption and utilization of the nutrients in the grain. However, no study has been done to eliminate the inherent phytic acid present in the grain. This study is focused on the germination of amaranth grain to reduce phytic acid content of the grain. 2. Materials and Methods 2.1 Materials The raw material for the study was collected from the local market of Far- Western Development Region. The grains were Amaranthus cruentus as identified by National Agriculture Genetic Resources Centre (NAGRC), Nepal Agricultural Research Council (NARC). The collected grains were then cleaned and sorted to remove dust, foreign matters and damaged ones. The glasswares, chemicals and equipments was used as per available in Regional Food Technology and Quality Control Office, Biratnagar.

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2.2 Methodology 2.2.1 Germination The seeds were washed thoroughly and soaked in distilled water (seed-to-water ratio of 1:5, w/v) for 5 h at room temperature. The water was strained and the steeped seeds was spread over trays with the thickness of the soaked seeds being around 0.5-1.5 cm and covered by a muslin cloth. Germination was carried out for 60 h and samples were harvested at 12 h interval. During the course of the process, the drying out of the grain seeds was prevented by moistening the muslin cloth, by spreading the water. 2.2.2 Determination of changes in phytic acid content Phytic acid of the samples was determined as per the method described by Sadasivam and Manickam (2009). 2.2.2.1 Preparation of Sample 2 g finely powdered sample was extracted with 50 ml 3% trichloroacetic acid (TCA) for 45 min with occasional shaking. It was then centrifuged and 10 ml of the supernatant was taken in a test tube. 4 ml of FeCl3 solution (583mg FeCl3 in 100 ml 3% TCA) was blown rapidly into the supernatant. The contents were heated in boiling water bath for 30 min and centrifuged for 10-15 min. The supernatant was carefully decanted and precipitate was washed twice by dispersing in 25 ml 3% TCA along with heating in boiling water bath and centrifuge. The final precipitate was dispersed in few ml of water and 3 ml of 1.5 N NaOH was added. The volume was approximately made to 30 ml with water and then boiled for 30 min. The solution was filtered hot quantitatively through Whattman No. 2. The precipitate was washed with 60-70 ml hot water and the filtrate was discarded. The precipitate was then dissolved from the filter paper with 40 ml hot 3.2 N HNO3 into 100 ml volumetric flask. The filter paper was washed several times with water and washings were collected in the same volumetric flask and finally the volume was made upto mark. 2.2.2.2 Preparation of Standard Solution

439.2 mg of Fe(NO3)3 was dissolved in 100 ml distilled water. From this stock solution, 25 ml was taken and volume was made to 250 ml using distilled water.

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2.2.2.3 Spectrophotometric measurements 2 ml, 4 ml, 6 ml, 8 ml and 10 ml of the working standard was taken in a tube and 4ml of 1.5 N KSCN was added in each tube. The total volume was made to 20 ml by adding distilled water. Absorbances of standard solutions were measured at 480 nm using Cary 60 UV-VIS spectrophotometer (Agilent Technologies, model no. G6860A) and calibration curve was drawn (Fig. 1). Similarly, 1 ml of prepared sample solution was taken instead of the standard solution and absorbance was measured. μg of iron present in the test from the standard curve was obtained and the phytate content was calculated as Ɋ ͳͷ ൬ ൰ ൌ ͳͲͲ Ǥ ሺሻ

1.2

1 y = 0.002x - 0.2447 R² = 0.993 0.8

0.6

Absorbance 0.4

0.2

0 0 100 200 300 400 500 600 700 ug of Iron

Fig. 1 Calibration curve

2.3 Statistical Analysis The experiment was conducted in triplicates and the data were analyzed by IBM SPSS Statistics version 20. The values were analyzed for its significance using LSD (Least square difference) at 5% level of significance.

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3. Results and Discussion The phytic acid content of the raw, soaked and germinated amaranth grains was analysed and are presented in Table 1. The phytic acid content of the amaranth grain reduced with germination time (Fig. 2). Table 1: Effect of soaking and germination on phytic acid content of amaranth grain

Treatment Phytic acid (mg/100g) Raw 2171.48 ± 22.84a Soaked 1768.86 ± 25.81b 12 h germination 1691.04 ± 13.61c 24 h germination 1592.27 ± 19.13d 36 h germination 1475.71 ± 21.63e 48 h germination 1328.50 ± 14.95f 60 h germination 1322.16 ± 10.99f Results are mean±SD of three independent determinations. Values in a column with different letters are significantly different (P<0.05). Raw amaranth grain contained 2171.48±22.84mg/100g of phytic acid and the amount decreased significantly (p<0.05) on soaking and subsequent germination. Phytic acid were significantly reduced by 18%, 22%, 26%, 32%, 38% and 39% respectively, after soaking, 12, 24, 36, 48 and 60 h of germination. However, ANOVA (p<0.05) showed that there was no significant difference between the phytic acid content of the samples germinated for 48 h and 60 h. Gamel et al. (2006) has reported 22% reduction in the phytic acid content after 48 h of germination of the amaranth grain. Similarly, Azeke et al. (2011) have also observed reduction in the phytic acid content in cereals like rice, maize, corn, wheat and sorghum.

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2200

1800

1400

phytic acid phytic (mg/100g) 1000 Raw Soaked Gr12 Gr24 Gr36 Gr48 Gr60

germination time (hr)

Fig. 2: Reduction of phytic acid during soaking and germination of amaranth grain

The decrease in the level of phytic acid during soaking may be attributed to leaching the acid out into soaking water under the concentration gradient (Abd El Rahaman et al., 2007). Other researchers have reported a decrease in the level of phytic acid during germination due to phytase activity in the germinating grains (Larsson and Sandberg, 1992). Because germination is mainly a catabolicprocess that supplies important nutrients to the growing plant through hydrolysis of reserve nutrients, reduction in phytic acid was expected as phytic acid is a source of phosphorus and cations during germination (Colmenares De Ruiz' and Bressani, 1990). 4. Conclusions Soaking and subsequent germination of the amaranth grain reduced the phytic acid significantly. Since phytic acid may be one ofthe factors responsible for reducing mineral bioavailability, itsreduction during germination may enhance the nutritional qualitywith respect to mineral bioavailability of amaranth.

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References Abd El Rahaman, S.M., El Maki, H.B., Idris, W.H., Hassan, A.B.,Babiker, E.E., El Tinay, A.H. (2007). Antinutritional factorscontent and hydrochloric acid extractability of minerals in pearlmillet cultivars as affected by germination. Int. J. Food Sci. Nutr.58, 6–17. Azeke, M.A., Egielewa, S.J., Eigboggo, M.U. and Ihimire, I.G. (2011). Effect of germination on the phytase activity, phytate and total phosphorus contents of rice (Oryzasativa), maize (Zea mays), millet (Panicum miliaceum), sorghum, (Sorghum bicolor) and wheat (Triticum aestivum). J. Food Sci Tech.48(6), 724- 729. Cheruiyot, N. (2011).Determination of levels of some vitamins in Amaranthus hypochondriacusand Amaranthus cruenthus leaves and grains from selected areas of Kenya. Master Thesis. Kenyatta Univ., Kenya. Colmenares De Ruiz, A. S. and Bressani, R. (1990). Effect of Germination on the Chemical Composition of Amaranth Grain. J. Cereal chem. 67, 519-522. DFTQC (2015). “Information on nutrients present in the food commodities available at Mid and Far-Western hilly regions of Nepal”. Department of Food Technology and Quality Control, Nepal. Gamel, T. H., Linssen, J. P., Mesallam, A. S., Damir, A. A. and Shekib, L. A. (2006). Seed treatments affect functional and antinutritional properties of amaranth flours. J. Sci. Food Agric. 86, 1095–1102. Graf, E., Easton, J.W.(1990). Antioxidant function of phytic acid. Free Radic. Biol. Med.8, 61–69. Larsson, M., Sandberg, A.S., (1992). Phytate reduction in oats duringmalting. J. Food Sci.57, 994–997. Lorenz, K. and Wright, B. (1984). Phytate and tannin content of amaranth. J. Food Chem14(27), 34. Martínez-Villaluenga, C. Frías, J. and Vidal-Valverde, C. (2006). Functional lupin seeds (Lupinusalbus L. and Lupinusluteus L.) after extraction of a- galactosides. J. Food Chem.98(2), 291-299. Muyonga, J. H., Nabakabya, D., Nakimbudgwe, D. N. and Masinde, D. (2008). Efforts to promote Amaranth production and consumption in Uganda to fight malnutrition. [Report]. Department of Food Science and Technology. Makerere Univ., Kampala. Pp. 1-9.

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Repo-Carrasco-Valencia, R. Espinoza, C. and Jacobsen, S. (2003). Nutritional value and use of the andean crops quinoa (Chenopodium quinoa) and kañiwa (Chenopodium pallidicaule). Food Rev. Int. 19(1-2), 179-189. Sadasivam, S. and Manikam, A. (2009). In: “Biochemical Methods”, pp. 5-201. New Age International Pvt. Ltd., New Delhi. Saunders, R. M. and Becker, R. (1983). Amaranthus. In: “Advances in cereal science and technology”. (Pomeranz Y. Eds.) St. Paul, Minn, USA: American Association of Cereal Chemistry. Singhal, R. S. and Kulkarni, P.R. (1988). Amaranth as underutilized resource. Int. J. Food Sci Technol.23,125–139. Stikic, R. Glamoclija, D. Demin, M. Vucelic-Radovic, B. Jovanovic, Z. Milojkovic-Opsenica, D. Jacobsen, S. and Milovanovic, M. (2012). Agronomical and nutritional evaluation of quinoa seeds (Chenopodium quinoa Willd.) as an ingredient in bread formulations. J. Cereal Sci.55(2), 132-8. Teutonico, R. A. and Knorr, D. (1985). Amaranth: composition properties and applications of a rediscovered food crop. J. Food Technol. 39, 44-50. Valencia, S. A. C. (2004). Encyclopedia of Seed Science, Quinoa.

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A Study Report on Scenario of Consumer Awareness Activities in Nepal

Bimal Kumar Dahal*, Madan Kumar Chapagain and Matina Joshi Vaidhya

Department of Food Technology and Quality Control, Babarmahal, Kathmandu

*Corresponding author: [email protected]

Abstract Right to safe and nutritious food is the fundamental right of consumer. Apart from Food Act and Regulation, there are more than 10 prevailing laws that also help to ensure safe and nutritious food in Nepal. In the fiscal year 2073/074, Department of Food Technology and Quality Control (DFTQC) conducted several programs such as food sample collection 2864 (numbers), case filing 257 (numbers), surveillance visit of food industries 1400 (times), surveillance visit of hotel restaurant and sweet shops 2873 (times), consumer awareness message dissemination 1736 (times) and food festival celebration 6 (numbers) in different regions. Also, 8 consumer organizations conducted 213 meetings in different places involving around 17, 350 participants. The ongoing efforts of consumer related programs by different agencies not only helped to understand the issues of adulteration, malpractices and cheating in food business, but also to involve consumers in the management of safety and quality of food by themselves. Consumer awareness programs conducted by government and consumer organizations are not sufficient. Frequencies and their radius should be increased in the coming days. Managing safety and quality in food is a multi- sectoral and multi-disciplinary approach. Therefore, all stakeholders in the food chain starting from production, handling, storage, processing and distribution should have vital role to play for supplying safe and wholesome food to the consumers.

Keywords: Consumer awareness, consumer rights, food chain, food safety, food control system.

1. Introduction On March 1962, former US President John F. Kennedy presented a speech to the United States Congress in which he inscribed four basic consumer rights, later called the Consumer Bill of Rights. The four basic rights are right to safety, right to be informed, right to choose and right to be heard. On his declaration he said, 121

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“Consumers by definition include us all. They are the largest economic group, affecting and affected by almost every public and private economic decision. Yet they are the only important group… whose views are often not heard”. The basic rights of consumer are as follows. a. The right to safety: The assertion of this right is aimed at the defense of consumers against injuries caused by products and implies that products should cause no harm to their users if such use is executed as prescribed. The right was further formalized in 1972 by the US federal government through the Consumer Product Safety Commission. b. The right to be informed: This right states that businesses should always provide consumers with enough appropriate information to make intelligent and informed product choices. Product information provided by a business should always be complete and truthful. Consumers should be informed against misleading information in the areas of financing, advertising, labeling, and packaging. c. The right to choose: The right to free choice among product offerings states that consumers should have a variety of options provided by different companies from which to choose. The government has to take many steps to ensure the availability of a healthy environment open to competition through legislation including limits on concept ownership. d. The right to be heard: This right has the ability of consumers to voice complaints and concerns about a product in order to have the issue handled efficiently and responsively. On April 9, 1962, United Nations Organization expanded these rights through the United Nations Guidelines for Consumer Protection into eight rights. They are right to satisfaction of basic needs, right to safety, right to information, right to choose, right to be heard, right to redress and compensation, right to consumer education and right to healthy environment. Different consumers’ organizations and regulatory bodies have adopted these rights as a charter and started recognizing March 15 as World Consumer Rights Day. 2. History of consumer rights in Nepal In 1438 BS, the King Jayasthiti Malla declared some progressive economic programs to prevent any undesirable cheating in weights and volume measurements. He had started the use of standard weights measuring system, using Mana, Pathi, Pau, Dharni, etc. during buying and selling. The King Ram

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Shah (1663-1693) BS, had declared some rules to replace different traditional bamboo devices eg. Dhungra, Dala and Aari and started system of using Metallic Mana, Pathi, Pau, Dharni with a Chaap, the government mark on them for standard weights measuring system. The first written law of Nepal Muluki Ain (General Code) 1910 BS had addressed some consumer right issues. It stated that all citizens had rights to choose their profession except some professions prohibited by the government. It also had provisions for standard weights and volume measurements. The use of government seal or sign on the weight and volume measuring device was mandatory. The culprit who did not follow the provision would be punished. After establishment of democracy in the country in 2007 BS, many laws and regulations had been enacted to protect consumer rights. Some examples are Black Marketing Act 2008, Nepal Prevention of Hoarding of Food Stuffs Act 2008, Essential Goods Protection Act 2012, Essential Services Operation Act 2014 and Essential Commodities Control (Authorization) Act, 2017. The Panchayat System also had enforced some acts and regulation in favor of consumer rights such as Nayan Muluki Ain (The New General Code) 2020, Food Act 2023, Standard Weights and Measure Act 2025, Black-marketing and Some Other Social Offenses and Punishment Act 2032, Feed Act 2033, Drugs Act 2035, Nepal Standards (Certification Mark) Act 2037, Seeds Act 2045. Some of those Acts are still working. After restoration of democracy in 2047 BS, economic liberalization was started in Nepal. The constitution of 2047 addressed some fundamental rights of citizens such as right to freedom, right to education, right to health, etc. The constitution 2047 opened new dimensions in the field of consumer rights. New acts and regulations were enforced, such as The Pesticides Act 2048, Mother’s Milk Substitutes (Control of Sale and Distribution) Act 2049, Environment Protection Act 2053, Consumer Protection Act 2054, Iodized Salt (Production, Sale and Distribution) Act 2055, Animal Slaughterhouse and Meat Inspection Act 2055, Good Governance (Management and Operation) Act 2064, Right to Information Act 2064, Joint Market Inspection Directive 2069 etc. 3. Food Control System in Nepal Food is the basic consumable to all citizens. Right to safe and nutritious food product is the fundamental right of consumer. To achieve this right, effective

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DFTQC, FRB 2016/17 food control system is necessary. Food control means a mandatory or regulatory activities enforced by national or local authorities in favor of consumer. These activities are enforced to ensure that all foods during production, handling, storage, processing and distribution are safe, wholesome and fit for human consumption. The components of food control system are laws and regulations, implementing organization with clear mandate, inspection and laboratory services and information, education, communication and training to all stakeholders across the food chain. Food control is a multi-disciplinary and multi-sectoral function. It is also known as farm to fork concept. All stakeholders in the food chain shall have the vital role in a single mission of food safety and quality. Concerned acts and regulations should be properly implemented to achieve safe and nutritious food to the consumers. Table 1 presents some laws and regulations provisioned for managing food control system in Nepal. Table 1: Some laws and regulations provisioned for food control system in Nepal S.N. Laws and Regulations Implementing Organization 1 Food Act 2023, Food Rule 2027 Department of Food Technology and Quality Control (DFTQC) 2 Consumer Protection Act 2054 Department of Supply Consumer Protection Rule 2056 Management and Protection of Consumers Interest (DSMPCI) 3 Animal Slaughterhouse and Meat Department of Livestock (DOLs), Inspection Act 2055 and District Veterinary Office Slaughterhouse and Meat Inspection Rule 2057 4 Nepal Standard (Certification Mark) Nepal Bureau of Standard and Act 2037 Metrology (NBSM) 5 Animal health and livestock Service DOLs, District Veterinary Office Act 2055 6 Brest Feeding Substance (Sales and Ministry of Health, DFTQC Distribution Control) Act 2049 7 Iodized salt (Production, Sale and Ministry of Health, Salt Trading Distribution) Act 2055 Corporation and DFTQC 8 Pesticide Act 2048 Department of Agriculture (DOA) 9 Local self-governance Act 2055 Local level eg; Metropolitan city, sub-metropolitan city, rural municipality, etc. 10 Joint Market Inspection Directive All concerned regulatory body 2069

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4. Department of Food Technology and Quality Control in Managing Food Safety and Quality Table 2 shows the activities of DFTQC in managing food quality and safety. In the fiscal year 2073/074, DFTQC has strategically increased the surveillance by increasing the number of inspection, monitoring and samples, and also filed about 257 cases to them who were out of standard. Celebration of food festivals and conduction of consumer awareness training programs were also part of the activities which involved too many people. In this way, DFTQC has been engaged in managing food quality, development and dissemination of food processing technologies as well as food and nutrition programs. Apart from execution of food laws and regulations, role of DFTQC also has been seen in awareness programs among producers, traders as well as consumers. The ongoing effort of the DFTQC is to achieve the goal of assurance of wholesome, safe and nutritious food to the consumer is deemed to be successful by thorough involvement of stakeholders especially in consumer awareness activities. Table 2: Food Control and awareness activities conducted by DFTQC S.N. Important Activities Progress of Progress of fiscal year fiscal year 2072/73 2073/74 1. Sample collection and testing 2120 2864 (number) 2. Sub-standard standard samples 208 365 (number) 3. Surveillances visit and 1137 1400 monitoring food industries (times) 4. Case filing (number) 206 257 5. Hotel, restaurant and sweet 1812 2873 shops inspection (times) 6. Food industry license issuance 1597 2031 and renewal (number) 7. Food processing, nutrition 981 904 training (participants number)

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8. Consumer awareness activities - 949 (number of participants) 9. Consumer awareness message 1017 1736 dissemination (times) 10. Food festival celebration (times) 4 6

On the auspicious occasion of ninth food safety day, DFTQC has conducted a mass consumer awareness meeting, in collaboration with some consumer organizations as in table 3 on the date 23/11/2073 at different places of Kathmandu valley. Such awareness activities have been useful to understand the issues of adulteration, malpractices and cheating in food business. Table 3: Consumer awareness programs in coordination with some consumer organizations S. Name of Consumer Organization Place of Meeting No. of N participa . nts 1. Consumer Forum Nepal Sanothimi, 115 Bhaktapur 2. Forum for Protection of Consumer Sudal, Bhaktapur 107 Rights Nepal 3. National Consumer Forum Sankhamul, 100 Lalitpur 4. Nepal Consumer Women and Children Kapan, 104 Conservation Forum Kathmandu 5. Consumer Rights Investigation Forum Jarankhu, 100 Kathmandu 6. Public Justice Consumer Forum Jorpati, 103 Kathmandu 7. Federation of Goods and Consumer New Baneshor, 100 Service Protection Nepal Kathmandu 8. Consumer Eye Nepal Koteshwar, 220 Lalitpur Total 949

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5. Consumer organizations in awareness campaign After the restoration of democracy in 2047 BS, consumer’s voices were started to rise in print as well as electronic media. Consumers started to unite and form some organizations. The Upabhokta Manch Nepal (Consumer Forum Nepal) is supposed to be the first consumer organization in Nepal. Many consumer organizations are working in awareness campaign and creating awareness to the consumers. Some consumer organizations engaged in consumer awareness activities are presented in Table 4. Table 4: Consumer organizations engaged in consumer awareness activities S. Name and address of Area Responsible N consumer person . organization 1 Consumer Forum Awareness activities on food Mr. Harendra Nepal, safety, quality and consumer Bahadur Bhrikutimandap, rights Shrestha Kathmandu 2 Sewa Nepal, New Awareness activities on Ms. Kalyanee Baneshwor women and children rights, Shah Kathmandu household crime and discrimination 3 Forum for Protection Awareness activities on food Mr. Jyoti of Consumer Rights safety, quality and consumer Baniya Nepal, Hanumansthan, rights Kathmandu 4 National Consumer Awareness activities on food Mr. Premlal Forum, Anamnagar, safety, quality and consumer Maharjan Kathmandu rights 5 Nepal Consumer Awareness activities on food Ms. Apsara Women and Children safety, quality and consumer Koirala Conservation Forum, rights Chabahil, Kathmandu 6 GoGo (Good Empowering citizen to Mr. Kedar Governance) improve public service Khadka Foundation, delivery, to fight corruption, Anamnagar advocate for human rights Kathmandu and humanitarian needs 7 Consumer Rights Awareness activities on food Mr. Madhabha Investigation Forum, safety, quality and consumer Timsina Balaju, Kathmandu rights

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8 Public Justice Awareness activities on food Mr. Sahadev Consumer Forum, safety, quality and consumer Gautam Kathmandu rights 9 Federation of Goods Awareness activities on food Mr. Romlal and Consumer Service safety, quality and consumer Giri Protection Nepal, rights Kathmandu 1 Consumer Eye Nepal, Awareness activities on food Ms. Bimala 0 Anamnagar, safety, quality and consumer Khanal Kathmandu rights 1 Federation for Good Supporting for capacitating Mr. Om 1 Governance (FEG) governance systems, Lamichhane Nepal, New Baneshor, empowering citizen to Kathmandu improve public service delivery, to fight corruption, advocate for human rights

The authors have collected reports, news on media and taken interview with the representatives of some consumer organizations. Based on this study, activities conducted by some consumer organizations during the fiscal year 2073/074 have been presented in Table 5. Table 5: Activities conducted by some consumer organizations (fiscal year 2073/074) S.N. Name of Consumer Meeting with Meeting Approx. organization consumer conducted number of districts participan (times) (No.) ts 1. Consumer Forum 25 14 2000 Nepal 2. Forum for Protection 32 12 1600 of Consumer Rights Nepal 3. National Consumer 24 17 2300 Forum 4. Nepal Consumer 69 18 4300 Women and Children Conservation Forum

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5. Consumer Rights 8 5 1250 Investigation Forum 6. Public Justice 6 5 900 Consumer Forum 7. Federation of Goods 28 23 3000 and Consumer Service Protection Nepal 8. Consumer Eye Nepal 21 10 2000 Total 213 - 17, 350

Consumer organizations have conducted different consumer awareness activities at different districts such as Taplejung, Ilam, Jhapa, Morang, Sunsari, Saptari, Sarlahi, Dhanusha, Bara, Parsa, Makawanpur, Chitwan, Nawalparasi, Rupandehi, Kapilvastu, Dang, Banke, Kailali, Kanchanpur, Ramechhap, Rasuwa, Dhading, Nuwakot, Kathmandu, Lalitpur, Bhaktapur, Tanahu, Gorkha, Lamjung, Kaski, Syangya, Baglung, Parbat, Palpa, Pyuthan, Gulmi, Rukum, Daldeldhura etc in collaboration with Ministry of Supply, DFTQC and Department of Supply Management and Protection of Consumers Interest (DSMPCI). More than 17,300 people have participated in these programs. Most of these consumer organizations are found working in the area of consumer rights, food safety and security, safety on LP Gas operation. They have conducted 213 meetings in different places of 38 districts involving around 17, 350 participants. Some organizations have presented memorandum to Prime- minister, Parliamentary Committee and National Human Rights Commission for the improvement of present food safety and environmental situation. Some organizations have distributed awareness pamphlets and posters at different districts as mentioned above. 6. Conclusion of Consumer Meetings The major issues raised during the meeting are about the date expired foods, sale of low quality foods, stale foods and the participants have asked for punishment to the culprits. They have also blamed that feed products are heavily contaminated with antibiotics and growth hormones. They have asked for effective regulation. Some participants have blamed for not having regular

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DFTQC, FRB 2016/17 monitoring and awareness programs have been urbanized. These programs should be conducted in rural areas too. Some participants have asked to raise the frequencies of awareness programs. They also have asked for effective enforcement of existing laws and regulations. Farmers should be educated about pesticides and their usage. Some participants have praised for conducting fruitful awareness programs in their home town and asked for increasing frequencies of such programs. 7. Way forward We all are consumers. Managing safety and quality in food is a multi-sectoral and multi-disciplinary approach. Therefore, all stakeholders in the food chain starting from production, handling, storage, processing and distribution should have vital role to play for supplying safe and wholesome food to the consumers. Also, effective monitoring and implementation of existing laws and regulation across the food chain is necessary. Extension of consumer education and awareness campaign also help to protect consumers from different malpractices taking place in food markets. Consumer awareness programs conducted by government and consumer organizations are not sufficient. Their frequencies and radius should be increased in the coming days.

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References Annual Bulletin 2072/073, published by Department of Food Technology and Quality Control, Kathmandu, Nepal. Baniya J., Implementation of Consumerism in Nepal (in Nepali), Upabhokta Hit Samrakshyan Manch Nepal, New Baneshor, Kathmandu, 2071. Dahal, B., Good Governance and Food Safety Regulation (in Nepali), BAS Souvenir, Published by Bageshwari Ashal Shasan Club Central Office, Nepalgunj, 2073. Dahal, B., Study Reports on Activities of some Consumer Organizations in Nepal. Draft document. Press meet document issued by DFTQC during Yearly Progress Review Workshop (fiscal year 2073/074), date 10 August, 2017, Babarmahal, Kathmandu, Nepal. http://www.lawcommission.gov.np, Website of Nepal Law Commission, New Baneshor, Kathmandu, Nepal. http://consumernepal.org, Website of Forum for Protection of Consumer Rights, Nepal. http://www.sewanepal.org, Website of Sewa Nepal, New Baneshwor Kathmandu, Nepal. http://gogofoundation.org, Website of Good governance foundation, New Baneshor, Kathmandu, Nepal. http://www.crif.org.np, Website of Consumer Rights Investigation Forum, Balaju, Kathmandu. http://fegnepal.org, Website of Federation for Good Governance (FEG) Nepal, New Baneshor, Kathmandu. http://www.dftqc.gov.np. Website of Department of Food Technology and Quality Control, Kathmandu, Nepal.

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Preparation of Tomato Caramel and Comparison with Plain Caramel

Menash Shrestha*, Anup Halwai and Raj Kumar Rijal

Regional Food Technology and Quality Control Office, Hetauda

*Corresponding author: [email protected]

Abstract Seasonal flux of low cost tomatoes can be incorporated in caramel to enhance the nutritional value of the product. The main objective was to optimize the concentration of tomato juice to obtain a final product similar to plain caramel and perform sensorial comparison between the optimized product and plain caramel prepared from the similar recipe just excluding the tomato juice ingredient. Tomato caramel prepared from 44% by weight tomato juice (5°Bx) was selected having highest sensory score in terms of color, flavor, texture and overall acceptance. No significant different was found between the tomato caramel and plain caramels as shown by t-test of mean sensory scores between them. Similarly, plain caramel was prepared using the same formulation but excluding tomato juice. Both caramels were sent to sensory analysis and compared in terms of color, flavor, texture and overall acceptance.

Keywords: Caramel, Preservation, Tomato caramel 1. Introduction Tomato (Lycopersicum esculentum) not only aids to taste and color to the product but also enhances vitamins and mineral profile (Sethi and Maini, 1982). With the advent of commercial farming of tomatoes round the year, Nepalese market has never seen the shortage of tomatoes and is flushed during the seasonal harvest (Shrestha, 1992). Raw tomatoes will spoil over time due to bacteria, yeasts and molds. Preserving tomatoes in oil is currently not recommended (Andress, 2012). During the period of tomato season, its price is low due to high availability. Since tomatoes are basically used only as vegetable ingredient large quantities of them are wasted in countries like Nepal. Efficient utilization of them is of primary importance. Fresh tomatoes because of their high water content, soft texture and high respiration rate (30 mg CO2/kg/hr at 20°C) are highly perishable commodities.

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So they cannot be stored for long period at room temperature as they become over ripe and soft, and bacterial soft rot and fungal decay can be serious problems. On the other hand at lower temperature, chilling injury and Alternaria rot become problems (Colloch et al.,1968). Tomatoes can be preserved by canning, drying, freezing, or pickling. They can also be used in creating fruit spreads like jams, jellies and marmalades and also preserving as confections like toffee, caramel, etc. (Lal et al., 1998). Tomatoes can be preserved in the form of caramel. The importance of tomato caramel is to reduce bulk by removing moisture, thereby saving the storage space, packaging and transportation costs, to impart new taste and nutrition to the caramel and to improve the storage life of the product (due to low moisture, high sugar and acid content). Use of tomatoes in preparation of tomato caramel leads to preservation of tomatoes as well as develops new taste in the confectionary market. 2. Materials and methods 2.1 Materials Fresh and uniformly ripe Avinash- 2 variety tomatoes (Lycopersicum esculentum), sugar, table butter (fat content 81%) and standardized and pasteurized milk (milk fat 3% and milk SNF 8%) were bought from local market of Hetauda. Clear, regular grade 42 DE glucose syrup was obtained from RFTQCO, Hetauda. 2.2 Methods Fresh and uniformly ripe tomatoes were washed and cleaned and then cut into pieces and blended in blender. The obtained pulp was sieved and TSS and acidity were determined. Sieved tomato juice was heated in vessel for some minutes to o inactivate peroxidise enzyme system. TSS of the pulp was adjusted to 5 Bx by adding sugar. Tomato caramel was prepared by slight modification in the method and recipe given by Srivastava and Kumar (2002). The ingredients used were tomato juice, sugar, glucose syrup, milk and butter. Different products were prepared by varying the concentrations ofsugar, glucose syrup, milk, butter and tomato juice. Three products were prepared by varying the tomato juice concentration (40, 44 and 48% by weight), other ingredients remaining constant. Recipe used in optimizing tomato juice concentration is given in Table 1. The best tomato

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DFTQC, FRB 2016/17 caramel was compared with plain caramel of similar formulation excluding the tomato juice. Table 1: Recipes made for tomato juice variations Ingredients Product A Product B Product C

Sugar, g 125 125 125 Standardized pasteurized milk, g 65 65 65 Glucose syrup (42 DE), g 70 70 70 Table butter, g 20 20 20 *Tomato juice Variations (%) 40 44 48 o (5 Bx) Weight,g 200 220 224

2.3 Sensory analysis The prepared samples were evaluated for color, flavor, texture and overall acceptance on a 9-point hedonic rating scale by semi-trained staffs of RFTQCO, Hetauda. The data were subjected to statistical analysis and optimum tomato juice concentration was determined. Plain caramel was also subjected to sensory evaluation for comparison with tomato caramel. 2.4 Data analysis The data were subjected to statistical analysis and the scores given by the panelists were analyzed by two way analysis of variance (ANOVA) at 5% level of significance using statistical software GENSTAT Release (Version 12.1.0.3338, twelfth edition). The data of the mean sensory scores between the final tomato caramel and plain caramel were subjected to t-test. 3. Results and discussion The effect of varying the tomato juice concentration on sensory quality of tomato caramel is given in Figure 1. The mean sensory score for color, flavor, texture and overall acceptance of tomato caramel prepared from tomato juice concentration of 44% was highest and significantly different (p>0.05) than other two.

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9 8.4 8.1 7.9 8.2 8 6.66.5 6.6 6.5 7 6.3 6.1 6.4 6.4 6 5 40% 4 44%

Mean score 3 48% 2 1 0 Color Flavor Texture Overall Quality attributes

Fig 1: Effect of variation of tomato juice concentration on sensory quality of tomato caramel Sensory analysis of the final product and plain caramel were carried out in terms of color, flavor, texture and overall acceptance by using 9 point Hedonic rating scale. The mean sensory scores of the caramels are given in Table 2. Table 2: Mean sensory scores of tomato caramel and plain caramel Attributes Mean Sensory Score Tomato Caramel Plain Caramel Color 8.1 7.6 Flavor 8.1 8.2 Texture 8.3 8.2 Overall acceptance 8 8

The data of the mean scores between the final product and plain caramels were subjected to t-test and the result is given in Table 3. After performing a two-tail test (inequality), if t-stat < -t Critical two-tail or t-stat > t Critical two-tail, we reject the null hypothesis. This is not the case -2.776 < 0.807 < 2.776. Therefore, we do not reject the null hypothesis. The observed difference between the sample means (8.125 - 8) is not convincing enough to say that the mean sensory scores between the tomato caramel and plain caramel differ significantly.

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Table 3: t-Test: Two-Samples (assuming unequal variances)

Variable 1 Variable 2

Mean 8.125 8

Variance 0.015833 0.08

Observations 4 4

Hypothesized Mean Difference 0

Df 4

t Stat 0.807573

P(T<=t) one-tail 0.23231

t Critical one-tail 2.131847

P(T<=t) two-tail 0.464621

t Critical two-tail 2.776445

4. Conclusion Seasonal flux of low cost tomatoes can be utilized to prepare tomato caramel with high nutritional value whose sensory quality can be as satisfying as that of plain caramel. On the basis of statistical analysis it was found that tomato caramel prepared with 44% by weight of tomato juice (5°Bx) was superior (p>0.05) than tomato caramel prepared with 40 and 48% by weight of tomato juice. The mean sensory scores between such tomato caramels did not differ significantly from plain caramel.

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References Andress, E. L. (2012). Effect of temperature on the chemical composition and antioxidant activities of three tomato cultivars. Ph.D. Thesis. National Center for Home Food Preservation., Ireland. Colloch, Mac and Worthington. (1968). Market disease of tomatoes, peppers and egg plant. 28-74. "USDA, Agriculture Handbook". Lal, G., Siddappaa, G. S. and Tandon, G. L. (1998). Preservation of Fruits and Vegetables. Indian Council of Agricultural Resarch. New Delhi. Sethi, V. and Maini, S. B. (1982). Appropriate Technology for Reducing Post Harvest Losses in Fruits and Vegetables. Indian Food Packer,43(2):42-46. Shrestha, T. N. (1992). Achievement of vegetables research on technology generation and recommendation for future R & D in Nepal. 25-57. Srivastava, R. P. and Sanjeev, K. (2002). Fruit and Vegetable Preservation rd "Principles and Practices" 3 edition. International Book Distributing Company. Lucknow.

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Preparation of Tishyauri and Optimization of Flaxseed and Black gram Paste Ratio

Rabindra Jha

Regional Food Technology and Quality Control Office, Dhangadhi

Corresponding author: [email protected]

Abstract The growing trend of heart disease among all of us can be well reduced by the regular consumption of omega – 3 fatty acid; the cheapest source of which can be flaxseed (Linum usitassimum). Since omega fatty acid are very prone to oxidation, the major objective of the work was to make easy accessible of omega fatty acid without oxidation and with great taste. Tishyauri (indigenous to Terai belt of Nepal), a dried product of flaxseed with black gram paste was formulated with different proportions of flaxseed and black gram paste. The best formulation after sensory rating was found to be one with 94% flaxseed and 6% black gram paste. The mean sensory score were found to be 7.88, 7.12, 7.12, 7.18 and 7.25 respectively for appearance, color, flavor, overall acceptance and texture.

Keyword: Flax seed, Omega-3 fatty acid, Tishyauri

1. Introduction Flaxseed (Linum usitassimum), also commonly called linseed and Aalash (in Nepal), has a long history of use as a food, medicine, and textile fibre. The Latin name means “very useful”. Hippocrates used flax to treat abdominal pain. Originally cultivated in Mesopotamia, the use of flax has been documented as far back as 3000 BC (Cunnane & Thompson, 1995). This Flax differs in genera from the native New Zealand flax (Phormium tenax and Phormium cookianum) which was given the common name “flax” by settlers in reference to its use as a source of fibre for weaving (Cooper& Cambie, 1991). 1.1 Health and nutritional benefits The health benefits of flaxseed are mainly due to its alpha-linolenic acid (ALA), fibre and lignin content. Table 1 shows the nutritional composition of flaxseed. The high omega-3 and protein content make flaxseed a unique and superior to other fibre supplements and food ingredients.

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1.1.1 Alpha-linolenic acid (Omega-3) Dietary advice concerning fats and fatty acids has been consistent over the last 50 years. The main thrust has been to reduce trans and saturated fatty acids and replace them with unsaturated fatty acids. It is unfortunate that we have seen a subsequent increase in the intake of linoleic acid (omega-6) in response to this advice. There is an optimal ratio of omega-6 to omega-3 in the human diet. Oils such as flaxseed, walnut, and canola help to maintain this balance. Whilst it is true that very little ALA converts to the long chain polyunsaturated omega-3 found in marine oils, it does have beneficial effects itself (Fitzpatrick, 2011). The benefits of ALA are seen at intakes as low as 1g/day and 2g/day is recommended for a cardio protective effect (Rodriguez-Leyva et al., 2010). 2g of ALA is found in 30 g of flaxseed fiber. The upper limit for linoleic acid (omega-6) intake is around 7g/day giving a favorable ratio of omega-6: omega-3 of 3.5:1 when 2g/day omega-3 is consumed. This approximates the healthy ratio seen in the traditional Japanese diet. This ratio is associated with reduced risk of cardiovascular disease, osteoporosis, rheumatoid arthritis and cancer (Simopoulos, 2002). There is a growing movement globally to restrict the level of omega-6 fatty acids in the human diet and replace with mainly monounsaturated fatty acids. The major problem of incorporating high levels of ALA in our diet is that the fatty acid is extremely unstable and is prone to oxidation, giving rise to undesirable flavour in food products. Ground whole flax seed itself is unstable due to its high content of free oil. However, defatted flax seed meal is much more stable and resistant to oxidation. When stored under inert gas flaxseed fiber is reported to have a shelf life of two years. Table 1: Nutritional composition of flaxseed Parameters Quantity per 100 gm Energy 1635 KJ Protein 32 g Total Fat 10 g Saturated 0.4 g Monounsaturated 1.5 g Polyunsaturated 7 g Omega 3 (ALA) 5 g Total Carbohydrate 43.6 g Sugar 1.4 g

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Dietary Fiber 39 g Soluble fiber 8 g Insoluble fiber 31 g Lignans 500-1000 mg Data averaged from analyses undertaken by an independent NZ laboratory. 1.1.2 Dietary fiber Flaxseed meal is high in fiber, a significant amount of which is soluble (20%), in the form of gums and mucilage. In addition to accounting for the laxative effect of flax meal, soluble fiber is known to have potent cholesterol lowering qualities, therefore reducing a major risk factor for cardiovascular disease (Singh, Mridula, Rehal, & Barnwal, 2011). In Canada, where a great deal of research has been carried out, there is now an approved health claim for the use of ground flaxseed to lower cholesterol (Health Canada, 2014). Insoluble dietary fiber reduces insulin resistance, is useful in treating constipation and helps maintain overall bowel health. Increased stool bulk, normalized bowel transit time, healthy gut flora, and production of short-chain fatty acids such as butyrate are all positive effects on the bowel of a high fiber diet (Cummings & Mann, 2012). Low fiber diets are associated with many chronic diseases including inflammatory bowel disease, heart disease, obesity, diabetes and colorectal cancer (Cummings & Mann, 2012). The fiber content of flax meal makes it an ideal addition to a balanced diet aimed at reducing the risk of these chronic diseases. 1.1.3 Lignans Flaxseed is the richest food source of plant lignans. Lignans are polyphenolic compounds classed as phytoestrogens. The plant lignans found in flaxseed are converted to the enterolignans, enterodiol and enterolactone by gut bacteria. Sometimes referred to as mammalian lignans, enterodiol and enterolactone exhibit antioxidant, weak estrogenic and anti-estrogenic activities and may prevent formation or reduce the size of estrogen-dependent cancers such as breast cancer (Linus Pauling Institute, 2010). Population studies have shown an association between lignin consumption and a lower risk of cardiovascular disease, although it is unclear at this time as to whether lignans are responsible for this effect or whether the effect is due to other constituents found in high lignin foods (Peterson et al., 2010).

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1.2 Culinary attributes Flaxseed meal is gluten free and has a pleasant nutty flavour. The protein content, combined with the gelling/binding properties of the soluble fiber found in flaxseed meal, make it ideal for incorporating into gluten free baked goods, or as a gluten free thickening agent. One tablespoon of flaxseed meal combined with three tablespoons of water and allowed to gel can even be used as a substitute for an egg in baked goods. The flake is quite good combined with hot or cold breakfast cereals in the morning and is a good source of fiber. It has several applications as a dietary supplement: isolated encapsulated lignans supplements, fiber supplement (bulk laxative with a demulcent action) and as a component of protein powder blends. It can also be used as food ingredient. Breads and other baked goods such as cookies and muffins including gluten free products. The incorporation into bread results in an improved texture and crumb structure. It can also be used to make healthy functional snack foods such as high protein energy bars. The objective of the work was to formulate and evaluate sensory characteristics of Tishyauri (indigenous flaxseed product of Terai region) prepared from different formulation of flaxseed and black gram. 2. Materials and methods Raw material mainly flaxseed (Aalash) and black gram were purchased from local market of Dhangadhi. The procedure for making Tishyauri is as shown in Figure 1. 2.1 Formulation Five different formulations were prepared as specified in Table 2 to prepare Tishyauri (a flaxseed dried product) for optimization of flaxseed and black gram paste ratio. Table 2: Different formulations of Tishyauri Proportion (flaxseed: black gram paste) Sample code 98:2 A

96:4 B 94:6 C

92:8 D

90:10 E

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Black gram Flaxseed

Winnowing Proper sun drying

Soaking overnight Winnowing (complete removal of dust)

Mashing to remove outer skin

Steeping

Grinding

Semisolid black gram paste

Mixing in different proportion as

Addition of 0.5% salt

Mixing properly

Giving flat circular shape (3 cm diameter) with hand

Sun drying (to moisture content below 8%)

Finished product (Tishyauri)

Sensory evaluation after frying in sunflower oil Fig 1: Procedure for making Tishyauri

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2.2 Optimization and data analysis For optimization of formulation, 9 point hedonic rating (Ranganna, 2008) was followed. After frying tishyauri, 10 semi-trained panelists (Staffs of RFTQC) were asked to rate the product for appearance, color, flavor, texture and overall acceptance (1 being dislike extremely, 5 being neither like nor dislike and 9 being like extremely. The data were analyzed for ANOVA using Genstat Release 12.1. 3. Result and discussion The sensory evaluation of fried Tishyauri was carried out. Mean sensory scores obtained are presented in Table 3. Table 3: Mean sensory scores of different formulations Sensory A B C D E LSD attributes

c b a d e Appearance 5.50 ±0.53 7.12 ±0.83 7.88 ±0.83 4.75 ±0.70 3.7 ±0.70 0.72

b ab a c d Color 6.12 ±0.64 6.62 ±0.51 7.12 ±0.83 5 ±0.76 3.8 ±0.83 0.78

Flavor 6.18b±0.74 6.22b±0.74 7.12a±0.83 4.62c±0.74 3.88c±0.83 0.77

Texture 5.34c±0.64 6.28b±0.52 7.25a±0.70 5c±0.76 3.62d±0.92 0.80 Overall 6b ±0.76 6.36b±0.64 7.18a±0.92 5.25c±0.88 4d±0.76 0.74 acceptance Values presented are mean values ± SD of 10 sensory evaluations. Mean values with different superscripts within a row are significantly different (P < 0.05). A=2% black gram beans paste, B=4% black gram beans paste, C=6% black gram beans paste, D=8% black gram beans paste, E=10% black gram beans paste. The hedonic rating revealed that sample C (flaxseed to black gram paste ratio 94:6) got the high sensory score in all sensory attributes and was found to be significantly different (p < 0.05) in all sensory attributes. 4. Conclusion and recommendation Since flaxseed has high nutritional value, the important being the omega-3 fatty acid which is very prone to rapid oxidation. So the formulation of flaxseed product can make easy access of omega-3 fatty acid to the consumer without prior oxidation. Flaxseed can be utilized as tishyauri using the proportion of flaxseed: black gram paste 94:6.

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References Cooper, R., & Cambie, R. (1991) New Zealand’s economic native plants. Auckland, NZ: Oxford University Press. Cummings, J., Mann, J. (2012). Carbohydrates. In J. Mann & S. Truswell (Eds.) Essentials of human nutrition (4th ed.). New York, NY: Oxford University Press. Cunnane, S., & Thompson, L. (1995). Flaxseed in human nutrition. Champaign, IL: AOCS Press. Fitzpatrick, K. (2011). Health benefits of flaxseed. In E. Hernandez & M. Hosokawa (Eds.) Omega-3 Oils: Applications in functional foods. (pp 213-264). Urbana, IL: AOCS Press. Health Canada. (2014). Summary of Health Canada’s assessment of a health claim about ground whole flaxseed and blood cholesterol lowering. Retrieved from: http://www.hc-sc.gc.ca/fn-an/label-etiquet/claims-reclam/assess- evalu/flaxseed-graines-de-lin-eng.php Linus Pauling Institute. (2010) Lignans. Retrieved from: http://lpi.oregonstate.edu/infocenter / phyto-chemicals/lignans/ Peterson, J., Dwyer, J., Adlercreutz, H., Scalbert, A., Jacques, P., &McCullough, M. L. (2010). Dietary lignans: physiology and potential for cardiovascular disease risk reduction. Nutrition Reviews,68(10), 571-603. doi: 10.1111/j.1753- 4887.2010.00319.x Rodriguez-Leyva, D., Dupasquier, C., McCullough, R., & Pierce, G. (2010). The cardiovascular effects of flaxseed and its omega-3 fatty acid, alpha-linolenic acid. The Canadian Journal of Cardiology,26(9), 489-496. Simopoulos, A., (2002). The importance of the ratio of omega-6/ omega-3 essential fatty acids. Biomedicine and Pharmacotherapy. 56(8),365-379. Singh, K., Mridula, D., Rehal, J., & Barnwal, P. (2011). Flaxseed: a potential source of food, feed and fiber. Critical Reviews in Food Science and Nutrition, 51(3), 210-222. doi: 10.1080/10408390903537241

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Preparation and Quality Evaluation of Chiraito (Swertia chirayita ) Incorporated White Bread

Anup Halwai* and Raj Kumar Rijal

Regional Food Technology & Quality Control Office, Hetauda *Corresponding author: [email protected] Abstract The research was carried out to develop a white bread incorporated with chiraito (Swertia chirayita) powder which has medicinal value. Five samples of white bread were formulated varying chiraito content as 0.10, 0.20, 0.30, 0.40 and 0.00%, other ingredients remaining constant. Sensory analysis report revealed that 0.10% chiraito incorporation in bread was best among other samples. However, control sample without chiraito scored highest sensory mark. Similarly, sample containing 0.30% chiraito was found to be least accepted from sensory evaluation point of view.

Keywords: Bread, Chiraito, Sensory analysis

1. Introduction Bread is one of the most widely consumed food products in the world (Selomulyo and Zhou, 2007). The impacts of various ingredients on sensory and nutritional quality of bread have been widely studied (Barcenas and Rosell, 2005; Plessas et al., 2005). Bread is a baked product of aerated dough. The primary ingredients of which are wheat flour, yeast, salt and water. Two other ingredients are often added, fat to increase softness and keeping quality, and sugar to increase sweetness. Additives are also added for various reasons, for example to improve fermentation, moisture retention, volume, crumb structure and to prevent mold growth (Flynn and James, 1980). There is abundant amount of chiraito found in hilly regions of Nepal. However, there has not been proper utilization of this plant in food products. Flavinoid, Chiraitin and other component found in chiraito can of a great importance if it is added in a daily diet. There are only few researches carried out in S. chirayita and very limited study includes the extraction of biotic components. Also, bitterness property of chiraito needs a serious attention so as to explore appropriate food application with health benefits of chiraito. In order to

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DFTQC, FRB 2016/17 encourage the utilization of bitter chiraito and to exploit the different medicinal uses contained within, this research was selected. Therefore, this research work would be important from economic point of view as it enhances farmers to uplift their economic status by growing and selling chiraito with higher value and also for food product diversification and industrial application. Chiraito (Swertia chirayita) is a natural herb belonging to the family Gentianaceae (Kumar et al.,2007). Chiraito, which is available in the hilly regions of Nepal, is exported to India every year in a very low price. Following points can be considered as a statement of a problem:- Planned marketing of chiraito is lacking Unawareness of people about its biotic components No detailed study has been expedited as a herbal resources for industrial application Processing and preservation technique of chiraito is not available Therefore, to promote the status of this under-utilized herb, product diversification has a greater role. This work is focused to develop a product from wheat flour incorporated with chiraito powder which is competent with medicinal value. The general objectives of the study were preparation of bread from wheat flour incorporated with chiraito and chemical, physical and sensory evaluation of thus prepared product. Specifically, objectives of the study were: Product diversification and utilization of the under-utilized beneficial herb chiraito Proximate analysis of the prepared product Nutritional analysis of the prepared product Evaluation of significant presence of chiraitin in final product Nutrient addition 2. Material and Methods 2.1 Material Wheat flour, chiraito powder is main ingredient along with other ingredients like sugar, salt, shortening, dry yeast and water.

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2.2 Methods: 2.2.1 Preparation of Bread: The flowchart for the preparation of chiraito incorporated bread is shown in Figure 1. 2.2.2 Bread recipe and sample formulation: Recipe used for making bread is shown in Table 1. Different samples A, B, C, D and E were formulated incorporating 0.1%, 0.2%, 0.3%, 0.4% and 0% chiraito powder respectively. Table 1: Recipe for making bread Ingredients Quantity Wheat flour 10 kg Water 6 L Salt 180 g Yeast 115 g Dough improver 100 g Calcium propionate 25 g Butter fat 500 g Sugar 1.5 kg Milk powder 50 g

Shifted wheat flour

Mixing (flour, salt, sugar solution, yeast, chiraito powder, water)

Dough making

Primary fermentation (90 minutes at 30-32˚C)

Knock back

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Dividing and primary moulding

First proofing (27˚C for 10-15 minutes)

Final moulding and panning

Final proofing (37˚C for 30 min)

Baking (230˚C for 40 min)

Cooling (at room temperature for 2 hrs)

Slicing

Packaging (in LDPE bags)

Fig 1: Flowchart for preparation of bread

2.3 Sensory evaluation and statistical analysis Sensory evaluation parameters like color, flavor, taste, texture, and overall acceptability of bread samples and controlled sample was carried out by 10 semi trained panelists using 9-point hedonic rating scale. 3. Results and Discussion Weight of the bread and bread volume of each sample is given in Table 2. Table 2: Weight and bread volume

Sample Dough (g) Bread (g) Volume (cm³) A 430 420 1816.50 B 570 540 2695.00 C 570 545 2315.25 D 250 230 1120.00 E 570 540 2268.00

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Table 3: Chemical analysis of bread samples

Sample A Sample B Sample C Sample D Sample E Parameters (%) 0.10% 0.20% 0.30% 0.40% 0.00% Moisture Content 34.93 36.02 37.29 36.40 37.38

Crude Fat 3.77 8.79 2.50 5.22 1.28

Crude Protein 6.352 12.780 8.626 11.907 5.221

Crude Fiber 0.17 0.25 0.13 0.025 0.00

Total Ash 1.32 1.24 1.24 1.22 11.91

Acid Insoluble Ash 0.011 0.012 0.000 0.020 0.000

Alcoholic Acidity 0.104 0.102 0.09 0.104 0.108

Carbohydrate 53.342 40.804 50.121 45.1 44.101

Chemical analysis data of all white bread samples was carried out which is shown in Table 3. 3.1 Sensory evaluation The mean sensory score obtained by each sample is shown in Figure 2.

9.0 Sample A Sample B Sample C Sample D Sample E

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 Mean Sensory Score 0.0 Color Texture Taste Flavor Overall Acceptibility Sensory attributes Fig 2: Sensory analysis of samples

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From the sensory analysis graph, it was found that sample A containing 0.10% chiraito in the bread was best among other sample. However, sample E without chiraito was a reference sample that scored the highest sensory mark. Similarly, Sample C containing 0.30% chiraito was found to be least acceptable while carrying out sensory test. 4. Conclusion: Chiraito can be incorporated in bread upto 0.1% without compromising its sensory sttributes.

References: AOAC. (2005)."Official methods of analysis", 18th edition, Association of Official Analytical Chemists, Washington DC Barcenas, M.E., and Rosell, C.M. (2005). Effect of HPMC on the microstructure, quality and aging of wheat bread. Food Hydrocolloids 19: 1037 – 1043 Kumar V, Abbas A K, Fausto N, Mitchell R N. (2007). Robbins Basic Pathology. 8th ed. Saunders Elsevier. Pearson, E. H., Krik, R. S. and Swayer, R. (1981).Chemical analysis of foods, Churchill Livingstone, New York. Ranganna, S. (1986).Manual analysis of fruits and vegetable products.2nd edition. Tata McGra-Hill Publication Co., New Delhi. Selomulyo, V.O., and Zhou, W. B. (2007). Frozen bread dough: Effects of freezing storage and dough improvers. Journal of Cereal Science 45: 1 – 17.

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Effects of Stabilizers on Cloud Stability of Kiwifruit RTS Pushpa Lal Rai*, Bimala Pokharel, Pramod Koirala, Sanjay Bhandari and Nirat Katuwal Regional Food Technology and Quality Control Office, Biratnagar

*Corresponding author: [email protected]

Abstract Cloud loss of kiwifruit RTS (ready to serve) beverage severely limits its marketability. Therefore, this work attempts to study the effect of hydrocolloid stabilizers (xanthan gum, pectin, mixture of xanthan gum and pectin) and thermal treatment on cloud stability of kiwifruit RTS beverage. For this, kiwifruit RTS beverages were prepared by using these hydrocolloid stabilizers and thermal treatment, stored for 2 months and analyzed for cloud stability, colour, flavour, body and overall acceptability by sensory evaluation. Sensory analysis showed that mixture of xanthan gum and pectin prevented cloud loss while pectin alone and thermal treatment were not satisfactory. The result suggests that use of stabilizers can enhance marketability of kiwifruit RTS beverage.

Keywords: Cloud stability, Kiwifruit, RTS, Thermal treatment

1. Introduction Kiwifruit (Actidinia spp.) plant is a shrub which bears egg-shaped fruit (Adhikari, 2014). The fruit is indigenous to china but it was first developed commercially in New Zealand. Nowadays, the fruit is cultivated widely over different parts of the world (Razavi and Parvar, 2007). The fruit is rich in vitamin C and the bioavailability is comparable to that of synthetic vitamin C (Carr et al., 2013). It is good source of potassium, folate, polyphenol and antioxidant contents (Li, 2008; Recio-Rodriguez et al., 2015). Moreover, it is reported that kiwifruit may also be a source of vitamin E and K (Li, 2008). Regarding its consumption, it is consumed either fresh or after processing into products such as jam, jelly, marmalade, nectar etc(Razavi and Parvar, 2007). Kiwifruit is newly introduced in Nepal but is very rapidly emerging as a commercial fruit crop in the mid-hills of the country (Adhikari, 2014). Ilam and Dolakha are the popular site for Kiwifruit farming (ICIMOD, 2012; Manandhar,

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2015). Besides farming, kiwi processors manufacturing Kiwi jams, jelly, RTS (ready to serve) beverage etc have also been evolved. Among these products RTS is susceptible to quality loss by cloud loss phenomenon. The problem is reported by the processors and requested training for solution of the same. To date, two approaches have been used for eliminating cloud loss in fruit juice beverages viz.; thermal stabilization and use of stabilizers. There for this study will be focused at stabilization of kiwifruit RTS by thermal stabilization and use of stabilizers.

2. Materials and Methods 2.1 Materials Kiwifruit was procured from the local farmers of Ilam district. Pectin, xanthan gum, citric acid, sodium hydroxide and other chemicals were purchased from local suppliers of Biratnagar. Glassware required were purchased from local suppliers of Biratnagar. 2.2 Methods Kiwifruit RTS were prepared by using the recipe shown in Table 1. Table 1: Recipe of kiwifruit RTS beverage Component Quantity Kiwifruit juice 1 kg Water 5 kg Sugar 1.3 kg Citric Acid 25 kg

Four different types of stabilizers were used in the prepared kiwifruit RTS as shown in Table 2.

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Table 2: Types of stabilizers RTS Stabilizer A Pectin (0.5%) B Xanthan gum (0.5%) C Pectin + Xanthan gum (0.25% + 0.25%) D Thermal treatment (boiling for 5 min)

2.3 Sensory analysis Sensory evaluation was carried out by using 5 point hedonic rating test (1 = poor and 5 = excellent) as per Ranganna (1986). Semi-trained panelists consisting of staff of regional food technology and quality control office were asked to rate the samples in terms of cloud stability, colour, flavour, body and overall acceptability. 2.4 Statistical analysis The data were subjected to two way ANOVA using IMB SPSS version 20 (IBM Corporation, Marlborough, MA, USA). Upon significant difference, the means were separated by using Tukey’s HSD test at 5% level of significance. 3. Results and discussion The sensory scores for different RTS beverages are presented in Fig. 1. Mean sensory scores for cloud stability and body of RTS beverages were significantly different (p<0.05) according to types of stabilizers while those for colour and flavour were same (p>0.05). RTS beverage stabilized with xanthan gum, and mixture of xanthan gum and pectin had higher scores for cloud stability compared to RTS beverages stabilized with pectin alone and thermal treatment. Mean scores for body was higher in case of RTS stabilized with mixture of xanthan gum and pectin followed by RTS beverages stabilized by xanthan gum, pectin and heat treatment respectively. Similarly, overall acceptability score was higher for RTS beverage stabilized by xanthan gum plus pectin followed by RTS beverage stabilized by xanthan gum. RTS beverages stabilized by pectin and heat treatment had statistically similar but lower overall sensory scores.

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A B C D a a b 4.5 a a c a a a 4 a a a a a 3.5 3 ac b b 2.5 b b 2 c 1.5 1 0.5 0 Cloud stability Colour Flavour Body Overall Mean sensory score Acceptability Sensory Parameter

Fig 1: Sensory scores of RTS beverages Bars with same letters for any sensory parameter are not significantly different at p=0.05. A, B, C and D represents RTS beverages stabilized by xanthan gum, pectin, pectin and xanthan gum, and heat treatment (boiling) respectively. As xanthan gum and mixture of xanthan gum and pectin had no deteriorative effect on colour and flavour of RTS, they can be used satisfactorily for cloud stabilization. However, comparatively lower score for body in case of RTS stabilized by xanthan gum alone was due to gel like consistency of RTS. Therefore, mixture of xanthan gum and pectin within the concentration used in this study appears to be best for cloud stabilization of kiwifruit RTS. Similar to our findings, Ping et al. (1994) reported best cloud stability in fruit nectar with mixed use of stabilizers. 4. Conclusion Use of pectin and thermal treatment are not satisfactory for cloud stabilization of kiwifruit RTS. However, mixture of xanthan gum and pectin can be used for cloud stabilization without any deterioration in other sensory qualities of kiwifruit RTS. Abbreviations RTS: Ready to serve ANOVA: Analysis of variance HSD: Honestly significant difference

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References Adhikari, B. H. (2014). "Production Technology for Kiwi". NARC. Carr, A. C., Bozonet, S. M. and Vissers, M. C. M. (2013). A Randomised Cross- Over Pharmacokinetic Bioavailability Study of Synthetic versus Kiwifruit- Derived Vitamin C. Nutrients.5, 4451-4461. ICIMOD. (2012). "Kiwi farming gaining popularity in Ilam". Li, T. S. C. (2008). "Nutritional and Therapeutic Values Vegetables and Fruits". CRC Press Taylor & Francis Group. Manandhar, R. (2015). Kiwi grows in popularity among Dolakha farmers. The Kathamndu Post. Ping, S., Jianchu, C., Hairon, X. and Hoacheng, L. (1994). A study on the technology for fruit composite nectar. J of Zhenjiang Agri. Univ. Razavi, S. M. A. and Parvar, M. B. (2007). Some Physical and Mechanical Properties of Kiwifruit. International Journal of Food Engineering.3 (6). Recio-Rodriguez, J. I., Gomez-Marcos, M. A., Patino-Alonso, M. C., Puigdomenech, E., Notario-Pacheco, B., Mendizabal-Gallastegui, N., Fuente, A. C., Otegui-Ilarduya, L., Maderuelo-Fernandez, J. A., Laso, A. C., Agudo-Conde, C. and Garcia-Ortiz, L. (2015). Effects of kiwi consumption on plasma lipids, fibrinogen and insulin resistance in the context of a normal diet. Nutrition Journal.14 (97).

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Preparation of Vanilla Flavored Chhurpi and its Quality Evaluation

Kedar Paudel*, Anup Halwai and Raj Kumar Rijal Regional Food Technology and Quality Control Office, Hetauda *Corresponding author: [email protected]

Abstract Chhurpi is a traditional cheese, mainly produced in the Himalayan region of Nepal, Sikkim, Darjeeling, Bhutan and Tibet. Chhurpi is a popular cottage cheese prepared by the Himalayan native people by following their traditional knowledge and they preserve this proteinaceous food for the winter season for the preparation of healthy food and sell in the mountain and terai region of the country. This study explores the chhurpi preparation in the other places of Nepal also. This study aims to prepare vanilla flavored hard chhurpi which make it better competitor in the market. Different concentration of vanilla flavor was used to prepare the chhurpi. Based on sensory and chemical evaluation, product containing 0.4% vanilla flavor was found to be best.

Keywords: Cheese, Chhurpi, Vanilla flavoured chhurpi

1. Introduction Chhurpi is a traditional Cheese, mainly produced in the Himalayan region of Nepal, Sikkim, Darjeeling, Bhutan and Tibet. There are two varieties of Chhurpi- soft variety and hard variety. Soft variety may be consumed with side dish with rice, where as hard variety is chewed like betel nuts. Here our study is concerned with the preparation of hard one. Chhurpi is prepared in a local dairy or at home from butter milk. Very few industries in Nepal used to prepare chhurpi. The butter milk is boiled and fermented and the solid mass that is obtained, then separated from the liquid and wrapped and hung in a thin cloth to drain out water. It is soft chhurpi. Hard chhurpi is made applying pressure to the fermented solid mass of butter milk in a cheese pressure machine. To make hard Chhurpi the fermented solid mass of buttermilk is separated and kept in a soft clothes or jute bag. Then the bag is kept under pressure in cheese pressure machine. It is better to make the

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DFTQC, FRB 2016/17 environment cool nearly nearly10 degree centigrade. Low temperature stops further fermentation of the product so that unnecessary degradation of the product is controlled. After nearly 48 hours of pressure, the product is taken out and cut into small cuboidal pieces and hung over fire or solar dryer. After two days of sun drying, the pieces are dried in the hot air oven at 45 degree centigrade till the cheese become hard. Then chhurpi is packed in appropriate plastic bag. The objective of the study was to prepare flavoured chhurpi and to conduct the laboratory analysis of prepared product. 2. Materials and Methodology

2.1 Materials required Raw materials required for preparation of chhurpi were procured from local market. Chemicals were obtained from local suppliers. 2.2 Methodology The method used for preparing vanilla flavored churppi is as shown in Figure 1. Milk

Cream Separation

Pasteurization

Addition of citric acid

Separate solid mass in muslin cloth

Addition of vanilla flavor at the rate of 2 g/kg

Keep in jute bag

Keep in cheese pressure machine under pressure

Slicing in cubical shape

Drying in hot air oven

Chhurpi

Fig 1: Flowchart for preparation of vanilla flavored chhurpi

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3. Results: Sensory evaluation was carried under varying vanilla flavor percentages which is expressed in the Table 1. Table 1: Vanilla flavor percentage Vanilla 0.2% 0.3% 0.4% 0.5% Preference % 30 20 50 10 Based on sensory evaluation, 0.4% vanilla flavor incorporation was found to be best. The laboratory analysis of chhurpi is shown in Table 2. According to sensory and chemical evaluation, product containing 0.4% vanilla flavor was found to be best. Some proximate analysis were carried out. However, microbiological analysis is further necessary to assure the quality of the final product. Our analysis confirmed that chhurpi is very proteinaceous product, long preserved due to low moisture and fat. Moreover, vanilla flavor can be added to chhurpi, which make it better and competitive to the market. Table 2: Laboratory analysis of vanilla flavored chhurpi Vanilla flavor 0.2% 0.3% 0.4% 0.5% Moisture % 6.10 6.14 6.20 6.2 Acidity % 0.48 0.50 0.49 0.50 Protein % db 61.16 61.18 61.20 61.20 Fat % 7.1 7.0 6.91 6.94

4. Conclusion: Sun-dried hard cheese is a unique food stuff particularly found in the Hilly region of Nepal and Himalayan region. It is supplied in different city of Nepal. Chhurpi is a popular cottage cheese prepared by the Himalayan native people by following their traditional knowledge and they preserve this proteinaceous food for the winter season for the preparation of healthy food and sell in the mountain and terai region of the country. This study explores the chhurpi preparation in the other places of Nepal also. Here the preparation of chhurpi can encourage local people to make chhurpi.

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References Shrestha S.R., Pradhananga M., Das S.K.L., Utilization of Milk By-Product (Buttermilk) in the Preparation of Chhurpi and its Quality Evaluation. Prajapati J.B., Nair B.M. The history of fermented foods. Farnworth R. (2003) (Ed.), Handbook of fermented functional foods, CRC Press, New York. Tiwari S.C., Mahanta D. Ethnological observation on fermented food products of certain tribes of Arunachal Pradesh. Tamang J.P. (2010) Himalayan fermented foods: microbiology, nutrition, and ethnic values, CRC Press, Taylor and Francis, Boca Raton. Tomar S.K., Singh R.., Gupta S.C., Arora D.K., Joshi B.K.D. (2009) Kumar Phenotypic and genotypic characterization of lactobacilli from Churppi cheese. Singh R.K., Singh A., Sureja A.K. (2007). Traditional foods of Monpa tribe of West Kameng, Arunachal Pradesh. Raj A., Sharma P. (2015). Fermented milk products of Ladakh. McCormick F. (2012) Cows, milk and religion: the use of dairy produce in early societies.

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Effect of Different Treatments on Shelf Life of Oyster (Pleurotus) Mushroom Collected from Lalitpur, Nepal

Saroj Khanal*, Praksha Neupane, Smita Gurung, Shreeram Neupane and Huma Bokkhim

Department of Food Technology and Quality Control, Babarmahal, Kathmandu

*Corresponding author: [email protected] Abstract Mushrooms are fruiting bodies of macro-fungi and cultivated throughout the world. Freshly harvested mushroom are very sensitive and has a very short shelf life due to high moisture content and active enzymes. This research aimed to study the effect of drying (sun drying vs mechanical drying) and of three different treatment conditions on the shelf life of oyster mushroom. Results showed that, sun dried mushrooms had better appearance (more white in color) and rehydration ratio (1:4) compared to mechanically dried mushroom (1:6). Furthermore, a combination of pre-treatment of mushroom by dipping in 0.5% KMS followed by blanching in 0.1 % CA and steeping in 10% brine solution containing 0.2% AA and 200 ppm of SO2 gave the best shelf life compared to other treatment conditions.

Keywords: Drying, Moisture content, Oyster mushroom, Shelf life 1. Introduction Mushrooms are the fruiting bodies of macro-fungi. They include edible, medicinal and poisonous species. Edible mushrooms once called the “food of the gods” are still treated as a garnish or delicacy. The extractable products from medicinal mushrooms, designed to supplement the human diet is considered an enhancement for health and fitness rather than food (Parkashet al. 1986; Kumar et al. 2013). Commercial mushroom cultivation is popular among Nepali farmers. Mushroom farming is increasingly becoming attractive to small farmers near urban centers as it requires very less time to grow and harvest and also can be done with least investment. Commercially cultivated mushrooms in Nepal include Oyster mushroom, White button mushroom and Shitake mushroom (Poudel and Bajracharya, 2011).

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Oyster mushroom is regarded as the most efficient users of the straw and yield highly nutritious fruiting bodies. However, high moisture content and short shelf life are major hurdles for their mass production (Martiniz-sotoet al. 2001). Drying, canning, steeping and freezing are some methods that are found to be suitable for their preservation (Srivastava et al. 2009). The aim of this work is to investigate the effect of drying and steeping on shelf life of mushroom. 2. Material and Method 2.1 Materials The following materials were used for the research. a) Biological: Oyster Mushroom (fresh from farm, Bhaisepati, Lalitpur) b) Chemical: Common salt, Acetic acid (AA), Citric acid (CA) , Potassium meta-bisulphite (KMS) c) Instruments: Weighing balance, Pipette, Measuring cylinder, Steamer, Bowls, packaging plastic, plastic bottles, Mechanical dryer and knives. 2.2 Method 2.2.1 Drying and rehydration effects of mushroom The fresh mushrooms were cut into two equal halves with the sharp knife and divided into two equal parts. One part was subjected to mechanical drying at 45 °C in a cabinet dryer while another half was sun dried. The dried mushrooms were observed for their appearance and rehydration ratio.

2.2.2 Steeping effect on shelf life of mushroom: Fresh mushroom from farm was washed in cold water, pretreated and then steeped in three different concentration of steeping solution as shown in Table 1. Sterile water was taken as a control. The shelf life of the product was observed based on visual examination of the growth of molds and fungi. The products was observed every two days. Table 1: Different treatment conditions Treatment Pre-treatments Composition of Steeping solution Dipping in Stem blanching in Salt Acetic SO2 0.05% KMS 0.1% Citric acid (NaCl) acid (10 minutes) Control Yes Yes - - - T1 Yes Yes 10% 0.2% 200 ppm T2 Yes Yes 5% 0.2% 200 ppm T3 No Yes 5% 0.2% 200 ppm

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3. Results and Discussion 3.1 Drying and rehydration effects on mushroom The mushroom product obtained from sun drying was found to be better in acceptance (more white in color ) and had better rehydration ratio (1:4) compared to mechanically dried product with slight brown appearance and rehydration ratio of 1:6. The reason behind this might be due to low temperature drying in sun drying (approx 25°C) compared to cabinet drying (45°C). 3.2 Steeping effect on shelf life of mushroom Shelf life estimation was done based on the appearance of visible mold growth on dried mushroom (Figure 1).

Time taken for visible mold growth of Oyster mushroom 8 6 6

4 3

2 1

Time (Months) 0.34 0 Control T1 T2 T3 Treatments Fig 1: Time taken for visible mold growth of mushroom for different treatments The best treatment was found to be treatment-1 with 10% salt, 0.2% AA & 200 ppm SO2. The final product was found to be stable for six months without visual growth of of molds and fungi. However, the control and other treatments (T2 and T3) underwent fermentation and water emission early. Based on this research it can be stated that, 10% salt along with 0.2% acetic acid and 200 ppm SO2 was found to be optimum treatment condition for mushroom preservation. 4. Conclusion: The present study was an approach to study mushroom preservation technique. Based on this preliminary research it can be stated that, sun drying was effective compared to cabinet drying method in terms of external appearance and rehydration ratio. Steeping test of mushroom revealed that fruiting bodies of Oyster mushroom can be stored maximum up to 6 months without any noticeable changes (without visual growth of fungi and molds) by using treatment-1.

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Further study is required in evaluating the storage conditions and shelf life of the final product. References: http://www.forestrynepal.org/images/publications/Mushroom_Cultivation_in_ Nepal.pdf Khaskheli, G.S., Zheng, W, Sheikh, S.A., Khaskheli, A, Liu, Y,Wang, Y and Huang, W (2015) Effecting of Processing Techniques on the Quality and Acceptability of Auricularia auricula Mushroom. Journal of Food and Nutrition Research, 3(1): pp. 46-51 Kumar, A., Singh, M. and Singh, G.(2013) Effect of different pretreatments on the quality of mushrooms during solar drying. Journal of Food science and technology 50(1) pp. 165-170 Martinez-Soto, G., Ocana-Camacho, R. and Paredes-Lopez, O. (2001) Effect of pretreatment and drying on the quality of Oyster mushrooms (Pleurotus ostreatus) Drying Technology 19 (3&4): pp. 661-672 Poudel, S. and Bajracharya, A. (2011) Prospects and Challenges of Mushroom Cultivation in Nepal: A case study of Lakuri Bhanjyang ,Lalitpur Prakash, T.N., Tejaswini, M and Ramana, R. (1986) Mushroom a promising crop for future. SBM Farm News.pp.5-7 Srivastava, B., Singh, K.P. and Zimik, W.(2009) Effects of Blanching Methods on Drying Kinetics of Oyster Mushroom. International Journal of Food Engineering 5(4): Article 2

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Preparation of Soymilk Incorporating Mango Pulp Vivek Banjara Regional Food Technology and Quality Control Office, Nepalgunj Corresponding author: [email protected] Abstract The study was conducted to prepare soymilk incorporating mango pulp by using various combinations. Four blends of soymilk and mango pulp were prepared. Each sample was analyzed for chemical analysis and sensory qualities were evaluated by sensory panelist. From the results of chemical analysis, significant difference was in terms of TSS, percentage acidity, pH, fat percentage, ash percentage, total sugar percentage, reducing sugar percentage and ascorbic acid. The data on effects in overall acceptability suggested that the beverage prepared by 1:1 proportion of soya milk and mango pulp was liked by judges as maximum score was recorded for all the sensory parameters as compared to the rest of the combination. Keywords: Mango, Soymilk 1. Introduction Soybeans are considered to be complete source of protein. A complete protein is one that contains significant amounts of all the essential amino acids that must be provided to human body because of the body's inability to synthesize them (Henkel, 2008). Mango is one of the famous tropical fruits. Because of its delicate flesh and unique flavor, it is very popular among people. As a result, it is known as "the king of tropical fruit". Mango fruit contains various kinds of nutrients. The content of vitamin A is particularly high in mango, which is rare in other fruits. Mango is not only very nutritious, but also has a lot of health-care effects on human body. Particularly mango is selected for the purpose of making beverage because of its high production and also has unique flavor and taste and imparts its characteristics properties to the beverage. Mango beverage has excellent properties in terms of taste and nutritional value compared to any other fruit beverage (Anon, 2010). Mango pulp is rich in carbohydrates, minerals, vitamin- C, starch, pectins, carotenoids, but lacks in proteins, fat and some essential amino acids. Soybean on the other hand is excellent source of good quality proteins and

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DFTQC, FRB 2016/17 fat and holds a great promise for developing proteinacious food products (Chauhan and Sharma, 1996). Hence, the mango pulp can be supplemented with protein from soymilk for development of new protein-enriched product. A combination of mango pulp with soymilk will certainly help in the preparation of more nutritious mango beverages. Soymilk is a beverage made from soybeans with stable emulsion of oil, water, and protein. It is produced by soaking dry soybeans and grinding them with water. Soy milk contains about the same proportion of protein as cow's milk (Anon, 2009a). Soy based processed products are becoming more popular because of their nutritional and potential health benefits. Irrespective of inherent processing hurdles, advan ced techniques in processing of soybean recognized its nutraceutical importance for prevention and treatment of certain chronic diseases, including cancers, atherosclerosis, osteoporosis, and kidney disease (Messina, 1995). The fundamental constraint in processing of soybean is bitter taste development associated with the conversion of isoflavone, glucosides, to aglucones via the action of ᵝ-glucosidases (Matsura et.al., 1989). Soymilk, as a base for production of beverages, remained deprived of commercial exploitation because of its low acceptability associated with unpleasant beany flavor, astringent and bitter aftertaste. Traditionally processed soymilk is stable emulsion of oil, water, protein, resembling dairy milk in appearance and composition. Fortification of mango pulp in soymilk improves the nutritional as well as thereapeutic value of beverage. Soymilk based fruit juice beverage would offer several distinct nutritional advantages over the plain fruit beverage to the consumer. Mango pulp is added to soymilk to enhance its vitamin A, C and mineral contents. It also provides sweetness and masks the beany flavor of soymilk to some extent (Lee at.al., 1990). Therefore, the attempts were made in the present investigation to develop delicious and nutritionally enriched soymilk incorporating mango juice, as role of the value added products with better acceptability. 2. Material and Methods Soybean and ripe Dashera mangoes were procured from local market of Nepalgunj. Other essential food grade ingredients such as sugar and citric acid

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DFTQC, FRB 2016/17 were purchased from a local market. The reagents used for chemical analysis were of analytical grade. 2.1 Methods 2.1.1 Preparation of soybean milk Soybean of cultivar was used for the preparation of soymilk. The soybean was first cleaned to removed stones, other seeds, wood etc. The whole soybean was rinsed thoroughly to remove dust particles adhered on the surface. Then, soybean was soaked in water for 10 hours. The soaked soybean was dehulled to separate husk. The separated soybean was then cooked 150oC for 15 min to inactivate the enzyme which cause beany flavor. The soybean was grinded in grinding machine with (1:10) of water. The blend obtained was refined with the help of muslin cloth. After refining, it was bottled and sterilized at 121oC for 15 mins. The beany flavor was minimized with help of addition sugar. Thus, the soymilk was stored in refrigeration temperature. 2.1.2 Extraction of mango pulp Selection of undamaged and ripe mango sample from the lot was done. Mangoes were selected according to the color and freshness. After selecting, the mango was washed with water. Manual peeling was done to remove inedible parts of mango with the help of knife. Only pulp was taken from whole mango. After cutting and removing the seed, mango pulp was pulped with help of pulper into fine and smooth size with addition of water in equal proportion (1:1). The pulp was then pasteurized to 85oC for10 minutes in 200 ml bottle. The pasteurized pulp was filled in bottles and stored in refrigerated temperature of about 2-3days. 2.1.3 Preparation of soymilk incorporated with mango pulp Standard combination of soymilk and mango pulp on the basis of their respective proportions (P1-80:20, P2-70:30, P3-60:40, T4-50:50) were formulated. TSS of all the beverages was adjusted to 14◦ Brix with cane sugar. The prepared products were then evaluated for various physico-chemical and sensorial characteristics. The TSS, moisture, protein, fat, sugars, acidity, pH, ascorbic acid and ash content of the soymik, soymilk incorporated with mango pulp were analyzed by using AOAC (2005) methods. The sensory qualities of the product like color, appearance, flavor, taste, mouth feel and overall acceptability were evaluated by semi-trained panel of 10 judges on 9 point Hedonic scale (1-extremely dislike, 9- extremely like).

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3. Results and Discussion 3.1 Standardization of Soymilk incorporated with mango beverage The data on proximate analysis of soymilk and mango juice presented in Table 1 helped in justifying their contribution as base ingredient (soy milk) and supplementary ingredient (mango juice). Table 1: Proximate composition of soymilk and ripe mango pulp. Parameters Soy Milk Mango pulp TSS(◦) 7.2 16.1 pH 6.81 4.21 Acidity (%) 0.13 0.19 Fat (%) 3.1 0.21 Moisture (%) 92.91 85.31 Ash (%) 0.33 0.49 Reducing Sugar 0.55 17.81 Ascorbic Acid - 24.90 (mg/100g)

The data show that mango pulp contained 16.1 ◦ Bx TSS, 0.55 reducing sugar and 24.9 mg/100g of ascorbic acid, whereas soymilk contained 3.1% fat, which were found most feasible for production of acceptable beverage. It indicated that the soymilk was richer in protein and fat and ash content and poorer in vitamin C, whereas mango pulp was richer in vitamins, minerals and sugars.The data on effect of mango pulp on nutritional constituents of soymilk incorporated with mango pulp is presented in Table 2.

Table 2: Effect of mango pulp on nutritional constituents of soymilk incorporated with mango pulp

Blends of Soy TSS Acidity pH Fat Ash Total Reduci Ascorbic milk and (oBx) (%) (%) (%) sugar ng acid mango pulp (%) sugars (mg/ (%) 100g) P1(80:20) 14.1 0.137 6.05 0.81 0.20 9.23 1.503 7.33 P2(70:30) 14.0 0.159 5.76 0.78 0.57 9.56 1.836 7.90 P3(60:40) 13.9 0.200 5.56 0.67 0.98 10.03 2.427 9.47 P4(50:50) 14.0 0.241 5.22 0.62 1.63 10.7 2.45 9.77 SE 0.037 0.021 0.156 0.04 0.225 0.285 0.208 0.531 CD(p,0.05) 0.102 0.057 0.434 0.112 0.624 0.793 0.577 1.476

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It revealed that a standard drink prepared by blending of soymilk and mango pulp in an equal proportion P4 was found better with respect to the various physico- chemical constituents followed by P3, P2 and P1 combinations respectively. The increase in the protein, fat, total sugars, reducing sugars and the ascorbic acid contents in the blend of soymilk and mango pulp at 50:50 (P4) was significant (p<0.05) than the other blends 80:20 (P1), 70:30 (P2) and 60:40 (P3). This could be obviously due to increased amount of these constituent in mango juice. The beverage prepared by adequate monitoring of soymilk and mango pulp leading to notified combinations were subjected to sensory evaluation to study the overall acceptability of product (Table 3). Table 3: Sensory evaluation of soymilk incorporated with mango juice Blends of Color and Flavor Taste Mouth Overall soymilk appearance feel Acceptability and mango pulp P1(80:20) 5.00 4.00 4.00 4.66 5.00 P2(70:30) 5.66 4.67 6.00 6.33 5.06 P3(60:40) 6.66 5.50 6.50 6.40 6.00 P4(50:50) 7.86 6.50 7.30 6.50 7.83 SE 0.558 0.483 0.629 0.393 0.542 CD(p<0.05) 1.550 1.342 1.748 1.091 1.506

The data on effects of combination on overall acceptability suggested that the beverage prepared by 1:1 proportion of soymilk and mango pulp was liked by judges on the basis of maximum score recorded for almost all the sensory parameters as compared to the rest of combinations (P1- 4:1, P2- 2.5:1, P3- 1.5:1). This might be associated with the decreasing concentration of soymilk contribution in reduction of unpleasant beany flavor of soybean as a limiting factor of acceptability of the beans. Moreover, increasing the proportion of mango pulp in comparison with soymilk improved the flavor, taste, color, and overall acceptability of the beverage. The treatment P4 (50:50) specified by equal contribution of both soymilk and mango juice recorded better results with respect to the various sensory characteristic during the storage period.

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4. Conclusions It can be concluded that soymilk can be found successful for development of soymilk based mango beverages with optimum sensory characteristics. The nutritious beverages with better quality could be developed by addition of soymilk up to certain extend. The beverage prepared by blending soymilk and mango pulp in equal proportion (50:50) was found better in enhancing almost all the sensorial quality parameters as compared to other combinations. Soymilk based mango beverage has excellent color and flavor which has masked the beany flavor of the soymilk and thus highly appreciated.

References A.O.A.C. 2005. Official Methods of Analysis, 18th Edn. Association of Official Analytical Chemists, Washington, DC Anonymous (2010a). Mango. Available at:http://en.wikipedia.org/wiki/Mango. [Accessed at 7th February, 2011]. Chauhan, S.K. and Sharma, R.C. (1996). Effect of supplementation of soymilk with apricot for beverage preparation. Beverages Food world, 23, pp. 37-38 Henkel, J. (2008). Soy: Health Claims for Soy Protein, Question About Other Components.Food and Drug Administration Messina M. J. (1995) Modern applications for an ancient bean: Soybeans and the prevention and treatmentof chronic disease. The Journal of Nutrition.125:567 569. Matsura M., Obata A. and Fukushima D. (1989) Objectionable flavor of soymilk developedduring the soaking of soybeans and its control. Journal of Food Science 54:602-605.DOI: 10.1111/j.1365-2621.1989.tb04662.x Ranganna, S. (2001). Handbook of analysis and quality control for fruits and vegetables product. 2nd Ed. Tata McGraw Hill Publ. Company Ltd., New Delhi

Lee S.Y., Morr C.V. and Seo A. (1990) Comparison of milk based and soymilk based yogurt. Journal of Food Science. 55: 532-536.

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