Micronutrients in Moringa Oleifera and Their Potential in Food Fortification

Micronutrients in Moringa oleifera and their Potential in Food Fortification

Yee Kei Kiki Chan

A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Department of Chemical Engineering and Applied Chemistry University of Toronto

Micronutrients in Moringa oleifera and their Potential in Food Fortiﬁcation

Yee Kei Kiki Chan Master of Applied Science Chemical Engineering and Applied Chemistry University of Toronto 2018

Moringa oleifera is frequently endorsed for its high micronutrient content relative to other vegetables, but reported data on Moringa’s nutritional value are inconsistent with common nutritional claims. A comparative analysis on the macronutrient and micronutrient content of Moringa leaves and pods was conducted. Moringa contains multiple nutrients but its iron content is similar to that in spinach and its vitamin A content is lower than carrots on a dry basis. Nevertheless, Moringa’s abundance in micronutrient deficient regions makes it applicable as a natural fortificant. Bouillon cubes were identified as a suitable food vehicle for the incorporation of Moringa. Bench-scale cold extrusion processing was conducted to explore the feasibility of creating Moringa-fortified bouillon cubes. Nutritional characterization confirmed that supplemental fortificants would be required to enhance the nutritional value and stability of Moringa-fortified bouillon cubes. Flavours of Moringa may either be enhanced or suppressed depending on the application.

ii To gung gung (1924-2016)

iii Acknowledgements

I would like to express my gratitude towards my co-advisors, Professor Levente Diosady and Professor Yu-Ling Cheng. Thank you for mentoring me, for teaching me eﬀective ways to frame and approach research problems (I am still working on this), and for the incredible opportunities you’ve given me at UofT and beyond. I would also like to thank my committee members, Professor M.G. Venkatesh Mannar and Professor Radhakrishnan Mahadevan, for their helpful insights on this thesis topic.

Heartfelt thanks to friends and family who eagerly listened to numerous recounts of small vic- tories and encouraged me to grow with every challenge presented.

To Mom and Dad: Thank you for your limitless love and support. Thank you for teaching me to be grateful and to count my blessings. Thank you for taking care of everything else so I could focus on whatever I chose to do.

To Nickie: Thank you for the inside jokes and for cheering me up always. Stay close, BB :)

To Elisa, Kiruba, Segun, Juveria, Azadeh, Folake and Rahul: Thank you for celebrat- ing with me when things went well and for urging me to keep going when things went wrong. I’m so lucky to get to work (or have worked) alongside you all at the Food Engineering lab.

To Jonathan: Thank you for showing me a glimpse of God’s love on earth. Thank you for keeping me grounded, for being a great friend and companion. Marrying you is one of the best, if not the best, decision I’ve made in my life. I love you so much.

And lastly...

Everything good within me comes from God. Without Him, I am nothing and all my works are empty.

Beloved, let us love one another, for love is from God, and whoever loves has been born of God and knows God. Anyone who does not love does not know God, because God is love. In this the love of God was made manifest among us, that God sent his only Son into the world, so that we might live through him. In this is love, not that we have loved God but that he loved us and sent his Son to be the propitiation for our sins. — 1 John 4:7-10

iv Contents

Dedication iii

Acknowledgements iv

Contents v

List of Tables vii

List of Figures viii

Acronyms x

1 Background 1 1.1 Global Food Insecurity and Micronutrient Deficiencies...... 1 1.2 Nutritional Content and Requirement Standards...... 3 1.3 Food Fortification...... 3 1.3.1 Existing Fortification Interventions...... 4 1.3.2 Fortificants...... 6 1.4 Moringa oleifera as a Natural Food Fortificant...... 6 1.5 Scope of Thesis...... 8 1.6 Objectives...... 9

2 Moringa oleifera Nutritional Characterization 10 2.1 Introduction...... 10 2.2 Methods...... 10 2.2.1 Materials and Sample Preparation...... 11 2.2.2 Proximate Analysis...... 11 2.2.3 Mineral Content...... 12 2.2.4 Vitamin Content and Protein Quality...... 12 2.3 Results and Discussion...... 12 2.3.1 Proximate Analysis...... 12 2.3.2 Mineral Content...... 14

v 2.3.3 Vitamin content...... 22 2.3.4 Protein Quality...... 24 2.4 Summary...... 25

3 Addressing Micronutrient Deficiencies using Moringa oleifera 26 3.1 Introduction...... 26 3.2 Existing Fortification Interventions using Moringa oleifera ...... 26 3.2.1 Yogurt...... 27 3.2.2 Bread...... 27 3.2.3 Cookies and Extruded Snacks...... 27 3.2.4 Region-specific Dishes...... 28 3.2.5 Raw Meat and Fruit Juices...... 29 3.3 Overcoming Technical Challenges...... 29 3.4 Food Vehicle Options...... 30 3.4.1 Consumption Coverage...... 32 3.4.2 Shelf-life...... 32 3.4.3 Ease of Adding Other Micronutrients...... 33 3.4.4 Public Health Concern...... 33 3.5 Proof of concept for Moringa-fortified bouillon cubes...... 33 3.5.1 Commercially Available Bouillon Cubes...... 34 3.5.2 Specifications...... 35 3.5.3 Experimental Design...... 37 3.5.4 Processing...... 38 3.5.5 Characterization Methods...... 41 3.5.6 Results...... 42 3.6 Summary...... 47

4 Future Work and Conclusions 48 4.1 Future Work...... 48 4.1.1 Taste, Aroma and Colouring Compounds...... 48 4.1.2 Encapsulation and Coating Processes...... 49 4.1.3 Alternative Binders and Excipients...... 49 4.1.4 Supplemental Fortiﬁcants...... 49 4.1.5 Manufacturing Process Selection and Optimization...... 50 4.1.6 Consumption Patterns and Consumer Preferences...... 50 4.2 Conclusions...... 51

5 Appendix 53

Bibliography 68

vi List of Tables

1.1 Common micronutrient deﬁciency symptoms and aﬀected vulnerable population groups...... 2 1.2 Nutritional content standards, RDA and NRV...... 3 1.3 Production yields and harvest periods for perennial and annual Moringa oleifera 7

2.1 Moringa samples for experimental analyses...... 11 2.2 Proximate compositions of Moringa leaves and pods. Rows marked dashes (-) in the reference column were experimentally determined in this project...... 13 2.3 Moisture content, dry mass and protein content in Moringa pods skin and ﬂesh. 13 2.4 Dietary Reference Intakes for select minerals (19-50 years old) [1]...... 15 2.5 Mineral content in Moringa leaves and pods. Rows marked with dashes (-) in the reference column were measured in this project...... 15 2.6 RDA for select vitamins (19-50 years old) [2]...... 23 2.7 Vitamin content in Moringa leaves and pods (literature values)...... 23 2.8 Protein quality and amino acid scores expressed as percentage of WHO Adult requirement in Moringa leaves and pods...... 24

3.1 Evaluation of food vehicles for incorporating Moringa ...... 31 3.2 Comparison of NRV and RDA values for minerals, vitamins and protein..... 35 3.3 Physical speciﬁcations for fortiﬁed bouillon cubes...... 36 3.4 In-barrel moisture content in screening experiments...... 40 3.5 In-barrel moisture for mixture design formulations...... 40 3.6 %NRV of iron, zinc, vitamin A, folate and protein per 3.3g serving...... 42 3.7 Mass, hardness, water activity and disintegration results for extruded cubes... 44 3.8 Hunter L*ab values for extruded cubes...... 46

5.1 Essential amino acids (mg/g protein) in Moringa leaves and pods (literature values) 54 5.2 Mineral content of extrudates per 3.3g serving or Moringa-fortiﬁed cubes.... 54

vii List of Figures

2.1 Protein content in Moringa leaf and pod samples measured in this study..... 14 2.2 Iron content comparisons in Moringa leaf samples. Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the RDA for individuals between 19 and 50 years old...... 16 2.3 Mineral content comparisons in Moringa leaf samples (calcium, magnesium, potassium and sodium). Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the minimum RDA or AI for individuals between 19 and 50 years old...... 17 2.4 Mineral content comparisons in Moringa leaf samples (zinc, copper and manganese). Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the minimum RDA for individuals between 19 and 50 years old...... 18 2.5 Iron content comparisons in Moringa pod samples. Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the RDA for individuals between 19 and 50 years old...... 19 2.6 Mineral content comparisons in Moringa pod samples (calcium, magnesium, potassium and sodium). Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the minimum RDA or AI for individuals between 19 and 50 years old...... 20 2.7 Mineral content comparisons in Moringa pod samples (zinc, copper and manganese). Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the minimum RDA for individuals between 19 and 50 years old...... 21 2.8 Percentage of total minerals in the skin of Moringa pods...... 22

3.1 Ingredient composition in a commercial bouillon cube...... 34 3.2 Simple axial mixture experimental design for proof of concept...... 37 3.3 Block ﬂow diagram for forming Moringa-fortiﬁed bouillon cubes...... 38 3.4 (left) Cold extrusion setup. Die attached to single Archimedes screw attachment; (right) Stainless steel extrusion die with 20*20mm square opening...... 39

viii 3.5 Iron content in mixture design formulations. Increasing bubble sizes correspond to higher levels of iron; numbers correspond to mean iron content per cube (mg/3.3g serving). Iron NRV = 14mg/d; benchmark was 15% NRV = 2.1mg.. 43 3.6 Hardness in mixture design formulations. Increasing bubble sizes correspond to increasing hardness; numbers correspond to mean hardness in newtons. The range for acceptable hardness is 5-50N...... 45 3.7 Formation of cracks (top face) after drying suggesting uneven extrusion pressure. Sample of cube made with 1:1 ratio of Moringa leaves and binder...... 45

ix Acronyms

AI Adequate Intake

AOAC Association of Oﬃcial Analytical Chemists

ANOVA Analysis of variance

ASTM American Society for Testing and Materials

DFE Dietary Folate Equivalent

DRI Dietary Reference Intakes

FAO Food and Agriculture Organization of the United Nations

FDA United States Food and Drug Administration

GC-MS gas chromatography-mass spectrometry

GDP Gross Domestic Product

HSD Honest Signiﬁcant Diﬀerence

ICP-AES Inductively coupled plasma - atomic emission spectroscopy

IoM Institute of Medicine

LMIC Low and middle-income country

MSG Monosodium glutamate n.d. Not determined

NRV Nutrient Reference Value

PPP Purchasing Power Parity

RAE Retinol Activity Equivalent

RDA Recommended Dietary Allowance

x SDG Sustainable Development Goal

UL Tolerable Upper Intake Level

WHO World Health Organization

xi Chapter 1

Background

This chapter presents an overview of global micronutrient deﬁciencies and approaches that have been investigated or implemented to address this global challenge. The background provided on Moringa oleifera and its potential in addressing micronutrient deﬁciencies leads to the motivation and scope of this thesis.

1.1 Global Food Insecurity and Micronutrient Deﬁciencies

Definitions of food security have evolved as the understanding of the relationship between food and well-being deepened. Up until the 1970s, food security referred to a nation’s ability to balance its caloric supply and demand, which overlooked numerous intricacies of access, distribution, and nutrition [3,4]. Since 1974, the Food and Agriculture Organization of the United Nations (FAO) has defined food security as “A situation that exists when all people, at all times, have physical, social and economic access to sufficient, safe and nutritious food that meets their dietary needs and food preferences for an active and healthy life” [5]. The inclusivity of this updated definition allows food insecurity to be examined at all stakeholder levels—from individuals to communities to economies—and as a multifaceted global challenge. The explicit reference for adequate nutrition points directly to the need to address micronutrient deficiencies, commonly called “hidden hunger”, which affects both developing and developed nations, albeit unequally [6]. The importance of addressing global micronutrient deficiencies is also highlighted by the United Nations in Sustainable Development Goal (SDG) #2: End hunger, achieve food security and improved nutrition and promote sustainable agriculture [7]. In this thesis, food security is viewed as a prerequisite for nutrition security. One of the main determinants for nutrition security is adequate micronutrient intake. Although it is possible that nutrition security could exist without food security (e.g. taking micronutrient supplements without adequate caloric intake), such cases are rare in low and middle income countries; it is more typical that households would need to be able to satisfy caloric requirements before being able to consider the nutrition within the foods consumed. The relation between food security and nutrition security continues to be debated in literature [8]. Nevertheless, differing opinions

1 Chapter 1. Background 2 in definitions do not negate the importance of addressing global micronutrient deficiencies. Micronutrient deficiencies affect individuals, communities and economies alike, and are linked to the perpetuation of the poverty cycle, where constant burdens of fatigue and disease lead to the inability to work and earn wages for survival [9]. Although significant improvements have been observed for caloric deficiencies since the year 2000, micronutrient deficiencies remain prominent and widespread [5]. A conservative estimate is that symptomatic micronutrient deficiencies affects two billion people globally, as asymptomatic cases of mild deficiencies are difficult to diagnose and quantify [3, 10, 11]. Public health indicators such as anemia, stunting, and night blindness suggest that micronutrient deficiencies are most prevalent in African and South Asian regions [5, 12].

Deficiencies in iron, vitamin A, iodine, folate, zinc, and vitamin B12 [13, 14] are most prevalent. Symptoms, signs and complications of micronutrient deficiencies are most prominent in pregnant women in whom reproductive health and fetus development is impeded, and in children in whom growth retardation is observed. Table 1.1 summarizes the impacts of common micronutrient deficiencies and demonstrates the importance of adequate micronutrient intake for health [14–16]. Economic impact estimates range from 2% Gross Domestic Product (GDP) loss associated with iron deficiency [14] to 5% overall GDP loss associated with vitamin A, iodine and iron deficiencies [17]. The World Health Organization (WHO) promotes three main strategies for increasing the intake of micronutrients: diversification, supplementation and fortification [10]. Public health organizations mandate and implement combinations of these strategies in parallel to form an integrated approach for addressing micronutrient deficiencies. Those living in low and middle income countries are most affected by food insecurity since poorer regions experience food shortages, and are unable to balance micronutrient intakes through diet diversification [6, 18].

Table 1.1: Common micronutrient deficiency symptoms and affected vulnerable population groups Micronutrient Essential for functioning of Symptoms Indicating Moderate to Severe Deficiency Most Affected Population Groups Goiter Pregnant women Iodine Thyroid hormones Mental retardation Newborns and infants Pregnancy complications Vegetarians Chronic fatigue Women of child-bearing ages Iron Hemoglobin and myoglobin Heart failure Children Pica Vegetarians Blindness Newborns Vitamin A Eyes and immune system Stunted growth Pregnant women Frequent infections Children Chronic fatigue Pregnant and lactating women Folate Amino acid synthesis Neural tube defects Newborns and infants Stunted growth People with alcoholic dependence Frequent infections Gastrointestinal disease patients Zinc Immune system and amino acid synthesis Stunted growth Vegetarians Loss of appetite Pregnant and lactating women Chronic fatigue Elderly Vitamin B12 Amino acid synthesis Heart failure Pregnant and lactating women Numbness in limbs Vegetarians Chapter 1. Background 3

1.2 Nutritional Content and Requirement Standards

Nomenclature for the reference values for nutritional requirements and benchmarks differ depending on the region and application. Two different types of reference values are used in this thesis: Recommended Dietary Allowance (RDA) and Nutrient Reference Value (NRV). In general, RDA is used by health professionals, such as dieticians, to assess the nutritional requirements of an individual; whereas NRV is used to benchmark the level of micronutrients in a food product (Table 1.2). Accordingly, Chapter2( Moringa oleifera Nutritional Charac- terization) of this thesis will reference RDA values, and Chapter3 (Addressing Micronutrient Deficiencies using Moringa oleifera) of this thesis will reference NRV for benchmarking.

Table 1.2: Nutritional content standards, RDA and NRV Recommended Dietary Allowance Nutrient Reference Value (RDA) (NRV) Originator Institute of Medicine Codex Alimentarius Commission Methodology Sufficient intake for 97-98% of the target population group Sufficient intake for 97.5% of the entire population Value Format mg/d; values split into different population groups according to age and sex mg/d; single value for entire population Application Health impact; nutritional requirements Benchmarking of fortification programs; nutrition labelling and claims

1.3 Food Fortiﬁcation

Food fortification is the process of adding nutrients to food, which may be mandated or performed on a voluntary basis. Mandatory food fortification programs are implemented in regions to address micronutrient deficiencies of public health concern, whereas voluntary fortification is often performed so that a health claim may be made for marketing purposes. Food fortification programs are proven to be effective for reducing the prevalence of several micronutrient deficiencies relatively quickly, sustainably, and economically [19–21]. Industrial fortification of various staple foods and condiments has been successful in reme- diating micronutrient deficiencies and have considerable benefits [11]. First, staple foods and condiments are widely consumed by significant portions of the population, so high household coverage can be achieved. Second, staple foods and condiments that are centrally produced by a small number of large manufacturers allow the quality and level of fortification to be controlled and monitored. Finally, minimal behavioural changes in dietary habits are required as long as there are no changes in organoleptic properties of fortified foods is detectable to the consumer. Significant challenges associated with food fortification are: determining the appropriate fortification level, cost, stability of fortificants—particularly due to possible interactions among multiple fortificants, and maintaining organoleptic properties. Considering the varying micronutrient requirements within a population and even within a single household, it is challenging to determine the amount of fortificant for a food vehicle that is appropriate for all potential consumers [20, 21]. If fortification levels are set too low, the fortified food will have little to no observable effect on reducing the targeted micronutrient deficiencies. If fortification levels Chapter 1. Background 4 are set too high, the excessive micronutrient intake may become toxic and detrimental to an individual’s health. Although fortification is considered a cost-effective intervention strategy, additional processing still inevitably increases the cost of a food product. This is especially burdensome for the poorest of the poor, who are extremely price sensitive and are likely to buy lower quality, inadequately fortified versions of the same food [10, 21]. In the development of food vehicles fortified with multiple micronutrients, undesirable interactions leading to reduced bioavailability and stability are common technical challenges. Similarly, maintaining the orig- inal organoleptic properties are also challenging due to interactions between the food vehicle and fortificant and is sometimes a major barrier for the commercialization of fortified food [10]. There are also challenges in quality control and cost containment for fortified foods. The level of fortified foods is likely to be higher and more consistent when fortification is implemented for food vehicles with a few key manufacturers as opposed to food with a great number of smaller scale manufacturers [22]. The level of food fortification is also more consistent in a mandatory environment where appropriate monitoring systems are in place [23, 24]. Additionally, large manufacturers are more likely able to reach economies of scale, and to contain additional costs associated with fortification processes.

1.3.1 Existing Fortiﬁcation Interventions

Salt Fortiﬁcation

Salt was among the first food vehicles to be commercially fortified in the 1920s to reduce goiter occurrences in Switzerland and the United States [25, 26]. Salt is considered an ideal food vehicle for fortification as it is universally consumed with minimal variations in consumption levels amongst households of different socioeconomic statuses [27, 28]. Salt iodization is the preferred method for controlling iodine deficiency disorders and there has been ongoing research to fortify salt with other micronutrients [27]. Implementation trials of double fortified salt with iodine and iron has demonstrated promising results in reducing iron deficiency anemia in low and middle-income countries [29, 30]. To produce double fortified salt, a premix of ferrous particles are agglomerated with the aid of binders in cold extrusion, encapsulated, and blended with iodized salt [31, 32]. Encapsulation provides both a colour-masking layer that covers the red-brown colour of the ferrous particles and a physical isolation barrier for stability. Other approaches to fortify salt with multiple micronutrients are also under development. Attempts were made to add vitamin A to produce triple fortified salt but were not commercial- ized due to technical barriers in stability [33] and high energy requirement of the production processes [34]. A variety of projects are ongoing to fortify salt with combinations of folic acid, B vitamins and zinc [35, 36]. Chapter 1. Background 5

Sugar Fortiﬁcation

The most common micronutrient added to sugar is vitamin A, and fortification has been implemented in numerous Central American countries [37]. Cold water soluble vitamin A palmitate beadlets adhere to the surface of sugar crystals and are coated with a vegetable oil-antioxidant mixture to form a premix. The vegetable oil fixes the palmitate beadlets onto the sugar crystals, while the antioxidant prevents the oil from becoming rancid and enhances stability of vitamin A palmitate. Lastly, the premix is blended with non-fortified sugar at 1:1000 ratio [38, 39]. A trial study conducted on iron fortified sugar resulted in increased iron stores in semirural Guatemalan populations [40], but no industrial feasibility studies have been published at this time.

Flour Fortiﬁcation

A staple food for many nations, flour is widely commercially fortified with an extensive range of micronutrients added at varying levels [41]. WHO provides guidelines on the fortification of flour with iron, folic acid, vitamin B12, vitamin A and zinc [42]. In many countries, flour is enriched with B vitamins to compensate for micronutrient losses during the flour milling process. A dry powder premix of micronutrients is added after the milling step and blended with flour to give fortified flour [43].

Oil Fortiﬁcation

Oil and its derivatives are ideal vehicles for the incorporation of fat-soluble micronutrients, namely vitamins A, D, E and K [44]. Edible oils are primarily fortiﬁed with vitamin A and, to a lesser extent, vitamin D due to nutritional needs and technical feasibility [44]. There have been marked successes in the fortiﬁcation of oil with vitamin A. Oil and vitamin A can be simply blended together without changing processing conditions, thus requiring minimal additional costs and technical expertise [10]. Oxidative degradation reduces the stability of vitamin A in oil, which could be countered by addition of antioxidants [44, 45]. Addition of minerals to oils has also been attempted with limited success.

Rice Fortiﬁcation

Similar to flour fortification, rice has also been fortified with a wide range of micronutrients [46]; but unlike flour, preferences on the type of rice used and organoleptic features of cooked rice differ significantly between cultures. This poses unique challenges in the standardization of fortification processes [10]. WHO recommends the fortification of iron, vitamin A and folic Chapter 1. Background 6 acid into rice [47]. In practice, rice is often enriched with B vitamins as they are lost during rice milling processes that remove the outer germ and bran layers. In rice fortification, fortified kernels are formed using coating or extrusion processes which are subsequently blended with regular rice kernels at a 0.5-2% ratio [46].

1.3.2 Fortiﬁcants

As shown in subsection 1.3.1, the use of synthetic fortificants for fortifying staple food vehicles is well studied. Pure micronutrients are used (with a food vehicle) to form a fortified premix with a high concentration of fortificant. The premix is then blended with non-fortified version of the food vehicle to give the appropriate level of fortification. A major advantage of using synthesized fortificants is greater control over the level of fortification, provided that the micronutrient compounds are of high purity and the fortification process is designed and operated appropriately. Many plants species contain a variety of micronutrients but have high moisture content, making them extremely perishable and low in micronutrient content on a per volume basis. Plant species, especially green leafy vegetables, may also contain antinutritional compounds such as oxalates, saponins and phytates that inhibit the absorption of nutrients for humans [48]. To increase the shelf-life, concentrations and bioavailability of the micronutrients, dehydration and blanching may be used to form “packages” containing micronutrients that could be used to enrich food vehicles [48–51]. These “packages” are natural fortificants. In addition to increasing the nutritional value of food vehicles, the components within the matrix of natural fortificants may improve storage stability. Natural fortificants allow the micronutrients in plants to be readily available year-round and reduce wastage from food spoilage. Ranawana et al. [49] showed that the fortification of bread using freeze-dried vegetable powders increased the nutritional value and enhanced the storage stability of bread samples. Similarly, Duthie et al. [50] also reported an increase in oxidative stability of meats with the addition of vegetable powders. On the other hand, Joshi and Mathur [51] demonstrated that the sensory properties of Western and Central Indian dishes remained acceptable when 10% w/w of vegetable leaf powders were added to the recipes.

1.4 Moringa oleifera as a Natural Food Fortiﬁcant

Moringa oleifera is a plant species that grows abundantly in tropical and subtropical regions and is one of 13 species classified under the Moringaceae family [52]. In this thesis, Moringa oleifera is referred to as ‘Moringa’. The time period from seed sowing to the harvest of Moringa fruits is six months, which is relatively short. Moringa fruits are also pods containing seeds and are typically 20-75cm long and weigh 90-150g, while its leaves 25-45cm long and are made oblique leaflets of 1cm in length. Moringa trees are also known as “drumstick trees” with Chapter 1. Background 7 reference to the pods’ resemblance to the musical instrument. Moringa is a multifunctional plant with uses in food, medicine [53], wastewater treatment [54, 55] and biofuels [56]. Various parts of Moringa are edible including its leaves and immature pods. India is the largest producer of Moringa and cultivates trees spread over 38,000 hectares with an annual production of Moringa pods of 1.1-1.3 million tonnes [57]. The production of Moringa leaves vary greatly depending on cultivation conditions (e.g. tree spacing, weather, and varietal) and yields up to 650 tonnes per hectare are found in literature [58]. Perennial Moringa trees require close to one year of growth before first harvest and produce fewer pods in the first two years of cultivation compared to annual Moringa trees (Table 1.3). Harvesting periods of Moringa are constrained by environmental factors such as monsoons, which could cause flowering Moringa trees to shed their blooms. A study conducted by the Tamil Nadu Agricultural University showed that Moringa trees purposefully grown to yield harvest during conventional off-seasons produced noticeably smaller and fewer pods [58].

Table 1.3: Production yields and harvest periods for perennial and annual Moringa oleifera Perennials Annuals Production Yield First two years: 80-90 250-400 (pods/year/tree) Year 4-5: 500-600 Harvest period(s) March-June (primary) six months after seed sowing; (in tropical climates) September-October 2-3 months of harvest

Moringa leaves are reported to be high in nutritional content [59] but have distinct astringent and grassy flavours, while immature Moringa pods are comparatively lower in nutritional content [60] and have mild sweet and crisp flavours likened to asparagus. Moringa leaves is presented in both academic and non-academic sources as containing more iron than spinach, more vitamin A than carrots, and more calcium than milk on an equivalent weight basis [61, 62]. A broad range of values exist in literature with regards to the nutritional content in Moringa leaves, ranging from high to very high nutrition [52, 59–63]. Although variations are expected in biological materials, the inconsistent values reported for Moringa on its micronutrient content lead to uncertainties in the nutritional value of Moringa. Furthermore, despite growing interest from health and agriculture communities on Moringa few regions currently include it as a regular food source. It is speculated that the astringent taste of Moringa leaves is not generally pleasing and Moringa pods are less nutritious and require more effort to eat due its fibrous outer skin. These challenges suggest that there are opportunities in increasing the consumption of Moringa as a means to alleviate micronutrient deficiencies. Given Moringa’s nutritive properties and abundance within the tropical region, which coin- cides extraordinarily well with regions most burdened by food insecurity, a number of campaigns have been launched to introduce Moringa as part of a diversification strategy. However, the inclusion of new foods into people’s diets is often slow and challenging since it requires substantial behaviour change. The diversification approach also does not address the key limitation of the undesirable astringent or bitter taste in Moringa leaves. Consumption of fresh leaves and Chapter 1. Background 8 pods is limited due to their high moisture content, which also limit the micronutrient intakes from the vegetables. Although Moringa trees may be easily grown in the tropical belt, the poor are often unable to grow Moringa for their own consumption as they are restricted to smaller living spaces. Moreover, the availability of fresh Moringa is seasonal and constrained by harvest yields. In India, seasonal availability causes the price of Moringa pods to range from Rs.5/kg during peak harvest season (March to August) to Rs.15-20/kg (September to October) to Rs.60/kg during off-season (November to February) [58]. According to the lower middle-income International Poverty Line of Rs.57/day (PPP$3.20 with 2017 PPP conversion factor), Moringa is unaffordable to the poor for at least four months of the year [64, 65]. The poor often are the most likely to be affected by food insecurity and the highly variable pricing of Moringa is a major barrier in allowing its nutrients to be consistently accessible. Further- more, fresh Moringa leaves and pods are extremely perishable even with cold storage, which only extends the vegetable’s shelf-life to two weeks. Cold storage is usually unavailable to the poorest or lowest socioeconomic segments of a population. The fortification approach was considered as an alternative way to promote the utilization and consumption of Moringa and its nutrients while minimizing behavioural changes in people’s dietary habits. Processing options to exclude undesirable organoleptic properties could be explored, so that changes to the food vehicle are undetectable. Processing Moringa into a natural fortificant will concentrate and preserve its micronutrients so that a greater amount may be consumed at each meal. The removal of moisture will preserve Moringa’s nutrients so that they are available year-round without the need for cold storage and reduce post-harvest food losses during peak harvest seasons.

1.5 Scope of Thesis

Several priorities were identified for this project. It was hypothesized incorporating Moringa as a natural fortificant would increase the nutritional value of the selected food vehicle and supplement the micronutrient requirements of undernourished individuals. To test this hypothesis, an understanding of the nutritional content of Moringa was required. Due to the broad range of values reported on Moringa’s nutritional content [52, 59, 60, 63], the extent of Moringa’s nutritive value was unclear, particularly in comparison to other green leafy vegetables. Sub- sequent to gaining an understanding of Moringa’s nutritional content, its suitability as a food fortificant could be evaluated, and the viability of including Moringa as a natural fortificant examined.

This thesis seeks to answer the following research questions:

1. What are the nutritional contents of Moringa leaves and immature pods?

2. Could Moringa be used as a natural food fortiﬁcant for industrial fortiﬁcation? Chapter 1. Background 9

3. What is a promising fortiﬁcation application for Moringa?

1.6 Objectives

The objectives of this project were as follows:

1. Determine the nutrient content in Moringa leaves and pods.

2. Evaluate the potential of incorporating Moringa as a natural food fortiﬁcant.

3. Explore the feasibility of a promising food fortiﬁcation application for Moringa. Chapter 2

Moringa oleifera Nutritional Characterization

In this chapter, the methods used to determine the nutritional content in Moringa leaves and pods are described; the results are then presented as evidence to evaluate the potential of Moringa as a natural fortiﬁcant.

2.1 Introduction

A key question in this dissertation is Moringa’s potential for addressing food insecurity as a natural food fortificant. Several studies have suggested that Moringa contains considerable levels of several micronutrients [61, 66, 67]. However, there are inconsistencies in the reported nutritional content in Moringa leaves and limited literature data are available on the nutritional content in Moringa pods. Moringa is primarily presented as a vegetable that is high in protein and iron in literature [66, 68]. Proximate composition, mineral content, vitamin content and protein quality were analyzed for this project to provide a comprehensive overview of the nutritional content in Moringa. Proximate analysis was used to determine the amount of moisture, crude protein, crude lipids, crude fibre, ash and carbohydrates within Moringa leaves and (whole) pods. Additionally, the crude protein content and mineral content in the skin and flesh components of Moringa pods were determined. Measured values were compared with data available in literature. The vitamin content and protein quality in Moringa leaves and pods were not experimentally measured for this thesis, but literature data were evaluated.

2.2 Methods

Detailed procedural descriptions referenced in this section are found in the Appendix.

10 Chapter 2. Moringa oleifera Nutritional Characterization 11

2.2.1 Materials and Sample Preparation

Moringa leaf and pod samples were obtained from various regions (Table 2.1). Fresh Moringa leaf samples from Chennai and Coimbatore and fresh pod samples from Delhi were couriered from their place of origin. Fresh samples were freeze-dried, ground into powder and sieved through a Taylor No. 35 mesh (500 µm). Dried samples were stored at 4◦C prior to analysis.

Table 2.1: Moringa samples for experimental analyses Time elapsed before cold storage Region Samples obtained Acquired from (for fresh samples only) Chennai, India Fresh leaves Cultivator (direct) ∼24 hours Coimbatore, India Fresh leaves and pods Cultivator (direct) 30 hours Leaf powder; Leaves: Costco.ca; Leaves: N/A; Delhi, India Fresh pods Pods: Vegetable market in Delhi Pods: ∼24 hours Kerala, India Fresh pods Vegetable market in Toronto 1 hour* Ibadan, Nigeria Shade-dried leaf powder Cultivator (direct) N/A *From supermarket to cold storage

The skin and flesh of Moringa pods were separated by carefully cutting the outer skin (green) until the inner flesh (cream/white) was exposed. The separation of skin and flesh was done on raw immature Moringa pods. The separated components were freeze-dried, ground into powder, and analyzed for their protein and mineral content. A limitation to this method of separating the skin and flesh of Moringa pods is that it does not perfectly mirror the way the vegetable is consumed in existing eating and cooking habits. For example, in South Indian cuisine, Moringa pods are cooked whole and eaten in a similar way to artichokes: small whole sections of the pods are chewed to extract the flesh and the skin is expelled after thorough mastication. The separation method used for this thesis does not take into account the extraction of nutrients from the skin of Moringa pods from chewing. This separation method also does not take into account the leaching of nutrients into the liquids during cooking. Variabilities in mastication and cooking are difficult to control, and the separation method described in this thesis was used to balance between results reproducibility and representing existing eating habits.

2.2.2 Proximate Analysis

Moisture content was determined using a standard oven drying method (ASTM 4442-16). Crude protein was determined using Kjeldahl analysis with a protein conversion factor of 6.25. Crude lipid content was determined using Soxhlet extraction using hexanes as the extraction solvent. Crude fibre was determined using weak acid-base digestion (AOAC 978.10). Ash content was determined using a muffler furnace at 575±25◦C (ASTM 1755-01). Non-fibre carbohydrates were determined by difference (100% - crude protein - crude lipids - crude fibre - ash). Compo- sition values were normalized to a moisture content of zero, and dry basis values were reported for comparison. Chapter 2. Moringa oleifera Nutritional Characterization 12

2.2.3 Mineral Content

Microwave digestion was used to dissolve metals in nitric acid. The digested sample was ﬁl- tered through a 0.45µm microﬁlter, diluted to the appropriate concentration within detectable ranges, and analyzed using ICP-AES. The minerals analyzed were calcium, magnesium, potassium, sodium, zinc, copper, manganese and iron. These minerals were analyzed based on their nutritional importance. Dried spinach was chosen as a benchmark for comparison as it has reasonable amounts of the analyzed minerals. The mineral content of dried spinach was calculated by using standard reference mineral content values for raw spinach from the USDA Food Composition Database [63] and were presented on a moisture-free basis. RDA values for adults between 19 and 50 years old recommended by the Institute of Medicine (IoM) were used.

2.2.4 Vitamin Content and Protein Quality

Moringa’s vitamin content and amino acid compositions were compiled using data available in literature. Protein quality was determined using the essential amino acid requirements for adults according to WHO [69]. Levels of vitamin A, B1,B6, folate (B9), B12 and C reported in literature were examined. Standard reference values of foods that are considered sources of these vitamins were used to illustrate the relative levels of each vitamin in Moringa. The maximum RDA value for adults between 19 and 50 years old recommended by the National Institutes of Health were used.

2.3 Results and Discussion

2.3.1 Proximate Analysis

Measured values for this project for crude protein, ash, crude fibre and non-fibre carbohydrates (Table 2.2) were consistent with values found in literature. Ash content in leaves were higher than in (whole) pods, which aligned with literature expectations of higher mineral content in Moringa leaves, which is further discussed in subsection 2.3.2. Whole Moringa pods contain a greater percentage of crude fibre than leaves, as expected, due to the presence of the outer protecting skin layer. The measured values for crude lipid content in leaves were noticeably higher than literature values. The most likely explanation for the discrepancies is natural variation between the samples used for this project and in other studies. Possible confounding results due to experimental errors—extraction of chlorophyll and residual solvent—were ruled out as the chlorophyll content in Moringa leaves is lower than 1% w/w [70] and the mass of extracted oil remained constant after prolonged solvent evaporation (72 hours). The difference between measured and literature values for crude lipid content in Moringa pod samples were considered reasonable as it could be attributed to natural variations [71, 72]. Chapter 2. Moringa oleifera Nutritional Characterization 13

Table 2.2: Proximate compositions of Moringa leaves and pods. Rows marked dashes (-) in the reference column were experimentally determined in this project. Composition (dry basis %) Source Ref. Moisture Crude Protein Crude Lipid Ash Crude Fibre Non-ﬁbre carbohydrates Leaves India [52] 75 26.8 6.8 9.2 3.6 53.6 Mexico [60] N/A 22.42 4.96 14.6 30.97 27.05 Niger [73] 6.2 26.65 11.3 8.96 8.42 44.67 Nigeria [74] 6.46 29.66 3.3 20.6 5.71 40.72 Tanzania [75] 7.07 36.39 4.83 9.1 13.57 36.1 USA [63] 78.66 44.05 6.56 10.59 9.37 29.43 Chennai, India - 80.6±0.34 29.1±0.42 14.8±1.58 10.8±0.56 N/A 45.4ˆ Delhi, India - 8.2±0.09 22.8±0.68 15.3±0.11 13.6±0.22 13.6±1.54 34.8

Pods India [52] 86.9 19.08 0.76 15.27 36.64 28.24 Mexico [60] N/A 19.34 1.28 7.62 46.78 24.98 Tanzania [75] 8.67 22.62 2.78 9.03 29.31 36.25 USA [63] 88.2 17.8 1.69 8.22 27.12 45.17 Kerala, India - 87.4±0.23 14.7±0.23 5.3±0.27 5.3±0.55 29.5±0.27 45.2 ˆInsuﬃcient sample material. Only total carbohydrates value available.

Samples with lower than 10% moisture content in Table 2.2 were pre-dried. For example, the moisture content for Moringa leaves from Delhi was lower as the samples were already in powder form. All composition values were normalized to a moisture-free basis. Further comparisons of measured protein content between Moringa leaf and pod samples (Table 2.2) showed that Moringa leaves have higher protein content than Moringa pods. The protein content in Moringa leaves and pods was found to range from 21% to 33% and 15 to 19% respectively, which matched expectations from literature (Figure 2.1). The protein contents in the skin and ﬂesh components of Moringa pods were examined. Re- sults showed that half of the protein in Moringa pods was contained within the skin (Table 2.3), which is often discarded as it is ﬁbrous and cannot be thoroughly masticated. Therefore, the existing cooking and eating habits for Moringa pods could result in the loss of up to half of the protein content in Moringa pods.

Table 2.3: Moisture content, dry mass and protein content in Moringa pods skin and ﬂesh Coimbatore, India Delhi, India Number of whole pods analyzed 3 4 Moisture content (%) Skin 80.4±1.96 N/A Flesh 88.2±1.93 N/A Dry Mass (g) Skin 4.95±0.48 5.01±1.13 Flesh 4.15±0.59 4.45±1.30 Protein content (%) Skin 17.3±3.71 14.0±1.47 Flesh 21.6±3.69 16.3±1.41 % of total protein content Skin 48.8 49.5 Flesh 51.2 50.5 Chapter 2. Moringa oleifera Nutritional Characterization 14

Figure 2.1: Protein content in Moringa leaf and pod samples measured in this study

2.3.2 Mineral Content

Figures 2.3 and 2.4 show the mineral content comparisons for Moringa leaves for datasets obtained through literature review and determined experimentally within this study. The benchmarks (dashed lines) indicate the minimum RDA or AI value for individuals between 19-50 years old (Table 2.4). Moringa leaf samples from Chennai (India), Coimbatore (India) and Ibadan (Nigeria) were experimentally analyzed in this study. Overall, the mineral content values that measured experimentally in this study matched expectations from literature considering the effects of natural variations (Table 2.5 and Figure 2.2). The iron content in Moringa leaves showed particularly large variability ranging from 8.3 to 110mg/100g. Moringa leaves are presumed to be high in iron and are reported to have up to 25 times more iron than spinach leaves [61, 76–78]. However, the analysis conducted for this study does not support this presumption. Using the iron content in Moringa leaf samples from Ibadan, Nigeria (110mg/100g) and in dried spinach (32mg/100g) [63], Moringa leaves contain 3.4 times more iron than spinach leaves. While Moringa leaves may be a good source of iron (Table 2.5), the analysis indicated that the iron levels are in the same order of magnitude as spinach. A possible explanation for this inconsistency and differing conclusions may be that comparisons were made between values reported for inequivalent moisture content. The reported iron content of spinach is 2.71mg/100g for fresh spinach with a moisture content of 91.4%. By simply taking the highest literature value for the iron content in dried Moringa leaves in Table 2.5, Moringa leaves would appear to have 18 times more iron than spinach—similar to the reported 25 times difference. Values for magnesium, sodium, copper and manganese were consistent amongst all samples. Chapter 2. Moringa oleifera Nutritional Characterization 15

Table 2.4: Dietary Reference Intakes for select minerals (19-50 years old) [1] Dietary Reference Intakes (mg/d) Calcium Magnesium Potassium Sodium Zinc Copper Manganese Iron RDA/AI RDA RDA AI AI RDA RDA RDA RDA Males 1000 400-420 4700 1500 11 0.9 2.3 8 Females* 1000 310-320 4700 1500 8 0.9 1.8 18 *non-pregnant and non-lactating

Table 2.5: Mineral content in Moringa leaves and pods. Rows marked with dashes (-) in the reference column were measured in this project. Mineral content (mg/100g) Source Ref. Calcium Magnesium Potassium Sodium Zinc Copper Manganese Iron Leaves Nigeria [74] 4.4E+02 2.4E+02 1.3E+03 N/A N/A 3.1E+00 N/A 8.3E+00 Pakistan [79] 2.3E+03 1.0E+01 2.1E+03 2.7E+02 2.6E+00 9.5E-01 7.7E-01 2.1E+01 South Africa [59] 3.7E+03 5.0E+02 1.5E+03 1.6E+02 3.1E+00 8.3E-01 N/A 4.9E+01 Tanzania [75] 6.3E+02 3.4E+02 3.6E+03 8.1E+02 3.2E+00 6.7E-01 N/A 1.2E+01 USA [63] 8.7E+02 2.0E+02 1.6E+03 4.2E+01 2.8E+00 4.9E-01 5.0E+00 1.9E+01 Chennai, India - 2.4E+03 4.7E+02 2.3E+02 6.1E+01 3.2E+00 2.1E+00 4.6E+00 6.5E+01 Coimbatore, India - 2.0E+03 7.7E+02 2.4E+02 4.0E+01 1.3E+00 5.6E-01 3.5E+00 2.4E+01 Ibadan, Nigeria - 2.1E+03 3.4E+02 2.4E+02 3.3E+00 9.1E-01 1.7E+00 7.7E+00 1.1E+02

Pods India [52] 3.0E+01 2.4E+01 2.6E+02 N/A N/A 3.1E+02 N/A 5.3E+00 Pakistan [79] 1.6E+02 9.6E+00 2.1E+03 2.1E+02 2.1E+00 2.7E+00 4.0E+00 1.6E+01 USA [63] 2.5E+02 3.8E+02 3.9E+03 3.6E+02 3.8E+00 7.1E-01 2.2E+00 3.1E+00 Kerala, India - 1.6E+02 2.4E+02 2.2E+02 1.9E+00 1.7E+00 9.8E-01 N/A 3.2E+01 Coimbatore, India - 2.2E+02 1.8E+02 5.9E+01 1.3E+00 2.1E+00 5.6E-01 N/A 5.2E+00 Delhi, India - 9.4E+01 1.8E+02 1.8E+02 N/A 1.8E+00 3.7E-01 8.9E-01 3.2E+00

Variations for calcium and potassium content in Moringa leaves may also be attributed to the nutrients available in the soil. Moringa leaves appear to have substantially lower amounts of magnesium, potassium, sodium, zinc and manganese compared to dried spinach leaves. Moringa leaves are low in sodium, potassium and zinc as the mineral content levels are below Adequate Intake (AI) and RDA thresholds (Figure 2.3 and Figure 2.4). Select samples of Moringa leaves contain suﬃcient copper and manganese per 100g to reach RDA thresholds (Figure 2.4). In general, Moringa pods were found to contain lower levels of individual minerals as compared to Moringa leaves. Fewer sources reported on the mineral content of Moringa pods, so limited comparisons could be drawn between literature values and values determined experimentally in this study. With the exception of the values for pod samples grown in Kerala, the values for iron content in Moringa pods were consistently below 18mg/100g (Figure 2.5). The iron content in Moringa pods is approximately 56% of that in dried spinach leaves. Two Moringa pod samples (Pakistan [79] and Kerala) had iron content levels over the RDA threshold for adult males. The values measured by this study for calcium were consistent with literature values. A Chapter 2. Moringa oleifera Nutritional Characterization 16

Figure 2.2: Iron content comparisons in Moringa leaf samples. Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the RDA for individuals between 19 and 50 years old. Chapter 2. Moringa oleifera Nutritional Characterization 17

Figure 2.3: Mineral content comparisons in Moringa leaf samples (calcium, magnesium, potassium and sodium). Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the minimum RDA or AI for individuals between 19 and 50 years old. Chapter 2. Moringa oleifera Nutritional Characterization 18

Figure 2.4: Mineral content comparisons in Moringa leaf samples (zinc, copper and manganese). Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the minimum RDA for individuals between 19 and 50 years old. Chapter 2. Moringa oleifera Nutritional Characterization 19

Figure 2.5: Iron content comparisons in Moringa pod samples. Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the RDA for individuals between 19 and 50 years old. substantial deviation was observed between the copper content reported by Ramachandran et al. [52] for samples originating from India and all other literature and measured values, which were consistent with each other. Moringa pods contain lower amounts of magnesium, zinc and potassium than Moringa leaves. Moringa pods contain low amounts of calcium, magnesium, potassium, sodium and zinc relative to the minerals’ AI and RDA levels (Figures 2.6 and 2.7. The calcium, magnesium and potassium contents for Moringa pod samples from the United States are approximately 10 times higher than that in samples from India (Figure 2.6). These differences in mineral content are influenced by a wide array of variables including plant varietal type, climate, soil nutrients and rainfall. The skin of Moringa pod samples contained 34-69% of the total minerals within the whole pods (Figure 2.8). The distribution of minerals in the skin and flesh of Moringa pods were variable in the two samples tested. Figure 2.8 shows that discarding the skin of Moringa pods would result in a loss of at least a third of the total minerals and further reduce the nutritional value Moringa pods. Chapter 2. Moringa oleifera Nutritional Characterization 20

Figure 2.6: Mineral content comparisons in Moringa pod samples (calcium, magnesium, potassium and sodium). Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the minimum RDA or AI for individuals between 19 and 50 years old. Chapter 2. Moringa oleifera Nutritional Characterization 21

Figure 2.7: Mineral content comparisons in Moringa pod samples (zinc, copper and manganese). Samples marked with asterisks (*) were experimentally determined in this study. Dashed lines indicate the minimum RDA for individuals between 19 and 50 years old. Chapter 2. Moringa oleifera Nutritional Characterization 22

Figure 2.8: Percentage of total minerals in the skin of Moringa pods

2.3.3 Vitamin content

Moringa leaves contain reasonable levels of vitamin A and C, but are generally low in B vitamins. Moringa leaves contain higher levels of vitamins than Moringa pods. Literature values on the vitamin content of Moringa leaves and pods varied greatly (Table 2.7), and it was not always clear if the values presented were on a wet or dry basis. However, since standard analytical procedures for vitamins are based on fresh material, it was assumed that the literature values were applicable for fresh Moringa leaves and pods. RDAs for the vitamins examined are shown in Table 2.6 as a reference level for comparison. 100g of fresh Moringa leaves were reported to have at least 42% of the RDA for vitamin A. Moringa leaves reportedly contain more vitamin A than carrots [78], but standard reference values showed that fresh carrots contain 835µg RAE/100g and dehydrated carrots contain 3423µg RAE/100g [63], both of which are higher than four out of five of the reported vitamin A literature values for Moringa leaves. A limited number of literature sources reported on the amount of B vitamins in Moringa leaves and pods. Two literature values were found on the level of vitamin B1 and one literature value was found on the amount of folate (vitamin B9) and vitamin B12 in Moringa leaves and pods. For vitamin B1, literature values ranged from 5-22% of the RDA in Moringa leaves while Moringa pods were reported to have 4% of the RDA in 100g. The sole literature value found on the amount of vitamin B6 indicated that 0.1g of Moringa leaves had sufficient levels Chapter 2. Moringa oleifera Nutritional Characterization 23 of the vitamin to fulfill the RDA for an adult, whereas Moringa pods contained low amounts of the vitamin and would require 1kg to satisfy the same RDA [63]. Literature values for Moringa leaves and pods suggested that both contained approximately 10% of the RDA for folate (vitamin B9) per 100g of material. The amount of folate in Moringa leaves and pods is substantially lower than that of spinach, which contains 194µg of folate for an equivalent mass of material. Neither Moringa leaves or pods contain vitamin B12, which matched expectations as vitamin B12 is most abundantly found in animal food products, such as eggs and shellfish. Literature values for the amount of vitamin C varied from 50-244% of the RDA in Moringa leaves and 72-157% in Moringa pods for 100g of the material. Moringa pods appear to contain more vitamin C than Moringa leaves on average. Notably, comparisons for vitamin C content between Moringa and kiwifruit (92.7mg/100g or 103% of the RDA) shows that Moringa contains a marked amount of vitamin C.

Table 2.6: RDA for select vitamins (19-50 years old) [2] Vitamin A Vitamin B Vitamin B Vitamin B Vitamin B Vitamin C Source 1 6 9 12 (µg RAE/day) (mg/day) (mg/day) (µg DFE/day) (µg/day) (mg/day) RDA 700-900 1.1-1.2 1.3 400 2.4 75-90

Table 2.7: Vitamin content in Moringa leaves and pods (literature values) Vitamin A Vitamin B Vitamin B Vitamin B Vitamin B Vitamin C Source Ref. 1 6 9 12 (µg RAE/100g) (mg/100g) (mg/100g) (µg DFE/100g) (µg/100g) (mg/100g) Leaves India [52] 565 0.06 N/A N/A N/A 220 Nigeria [74] 436 N/A N/A N/A N/A N/A South Africa [59] 1542 N/A N/A N/A N/A N/A Tanzania [75] N/A N/A N/A N/A N/A 48 USA [63] 378 0.26 1200 40 0 52

Pods India [52] 9.2 0.05 N/A N/A N/A 120 Tanzania [75] N/A N/A N/A N/A N/A 65 USA [63] 4 0.053 0.12 44 0 141 Chapter 2. Moringa oleifera Nutritional Characterization 24

2.3.4 Protein Quality

The compositions of amino acids per 1g of protein and WHO requirements are presented in Appendix A. The bolded column for each sample in Table 2.8 indicate the limiting amino acid, which is the amino acid present in the lowest quantities based on the optimal ratio suggested by WHO [80]. The associated percentage for the limiting amino acid is also the amino acid score. Lysine and the sum of methionine and cysteine are potential limiting amino acids in Moringa leaves and pods. These results matched expectations as lysine and the sum of methionine and cysteine are limiting amino acids in many plant proteins [80]. From this ﬁnding, it may be inferred that Moringa leaves and pods require multiple food types to complement its amino acid composition for better protein quality. Literature values on the in vitro protein digestibility for fresh Moringa leaves were low, with values reported at 31.83% and 57.22% [81, 82]. Cooking or processing Moringa leaves will likely increase the vegetable’s protein digestibility with the trade-oﬀ of lower bioavailability in micronutrients (e.g. vitamins) that are prone to thermal or oxidative destabilization.

Table 2.8: Protein quality and amino acid scores expressed as percentage of WHO Adult requirement in Moringa leaves and pods . Bolded numbers indicate protein scores. % of Adult Requirement/g protein Methionine Phenylalanine Source Ref Histidine Isoleucine Leucine Lysine Threonine Tryptophan Valine + cysteine + tyrosine Leaves India [52] 85% 307% 77% 78% 3% 5% 110% 12% 161% Ethiopia [83] N/A 105% 105% 84% 100% 123% 159% N/A 105% Mexico [60] 156% 99% 99% 113% 21% 120% 115% N/A 97% Niger [73] 116% 87% 86% 72% 88% 154% 118% 204% 85% Nicaragua [68] 117% 87% 89% 71% 80% 146% 101% 340% 82% USA [63] 85% 98% 88% 78% 78% 143% 117% 157% 102%

Pods Ethiopia [83] N/A 104% 100% 76% 188% 100% 150% N/A 100% Mexico [60] 151% 117% 108% 63% 46% 81% 163% N/A 125% Chapter 2. Moringa oleifera Nutritional Characterization 25

2.4 Summary

Moringa leaves and pods contain multiple micronutrients, and the level of micronutrients appeared comparative, rather than superior, to other foods consumed in a vegetarian diet. How- ever, the abundance of Moringa in regions with high prevalence of micronutrient deficiencies remains to be a benefit in using it as a way to address food insecurity. While some literature values were outliers and could potentially be attributed to incorrect reporting units, variations were mainly attributed to natural variations in the growing conditions. Fortified foods containing Moringa leaves and pods will likely need to be further enriched using supplemental fortificants to provide a complete package of micronutrients. Moringa leaves generally contain more protein, lipids and ash than Moringa pods, whereas Moringa pods contain more fibre. Variabilities between measured and literature values for the lipid content in Moringa leaves were attributed to natural variations. Moringa leaves contain higher levels of minerals than Moringa pods. Moringa leaves are often featured as a vegetable with high levels of iron, but comparisons with spinach leaves indicated that Moringa has either less or comparable amount of iron for an equivalent moisture-free mass. This finding is contrary to the belief that Moringa contains significantly more iron than other green leafy vegetables. Variations in the level of calcium, magnesium, zinc, copper and potassium were attributed to natural variations as a result of the availability of nutrients in the soil where literature and experimental samples were grown. In the limited data found on Moringa’s vitamin content, Moringa leaves were shown to contain noticeably higher levels of vitamins than pods with the exception of vitamin C. It was unclear whether Moringa leaves or pods contained more vitamin C, as the range of literature values were similar. Literature comparisons suggest that Moringa contains less vitamin A than an equivalent dry mass of carrots. Both Moringa leaves and pods contain only trace amounts of vitamin B1 and folate relative to the RDA of both vitamins. Moringa leaves contain significantly more vitamin B6 than pods. However, further analysis is recommended as only one literature source was found to report on the amount of vitamin B6 in Moringa. Both Moringa leaves and pods were reported to contain no vitamin B12, which matched expectations as vitamin B12 is typically found in animal food products. Based on the amino acid compositions available in literature, Moringa leaves and pods are limited by either lysine or the sum of methionine and cysteine. This suggests that the consumption of Moringa leaves and pods needs to be complemented by multiple other food types to provide an optimal ratio of essential amino acids. Chapter 3

Addressing Micronutrient Deﬁciencies using Moringa oleifera

This chapter summarizes studies that have attempted to use Moringa as a natural food fortificant and the accompanying challenges on undesirable organoleptic changes in the fortified foods reported in those studies. Different food vehicle options were proposed in this study and evaluated followed by an exploratory evaluation of the feasibility of incorporating Moringa into bouillon cubes to increase their nutritional content.

3.1 Introduction

Although Moringa contains lower levels of micronutrients than that presumed in literature, the abundance of Moringa in regions with high prevalence of food insecurity could still be leveraged to address micronutrient deficiencies. As discussed in Chapter1, Moringa-fortified foods should fit into the existing ecosystem of interventions and strategies implemented to reduce micronutrient deficiencies. Considering the nutritional content of Moringa documented in Chapter2 and the most prevalent micronutrient deficiencies discussed in Chapter1, the fortification of micronutrients naturally occurring in Moringa—iron, vitamin A, zinc and folate, was examined in this study. Iodine and vitamin B12 fortification will be will be explored in future work, as discussed in Chapter4 of this thesis.

3.2 Existing Fortiﬁcation Interventions using Moringa oleifera

Existing studies have evaluated the viability of using Moringa as a natural fortificant by an- alyzing the sensory acceptance, rheological and physical characteristics of the fortified food items. The rheological and physical characteristics affect both the sensory acceptance as well as the products’ manufacturability. Moringa’s antioxidant and antimicrobial properties have also been studied as a food preservative. The subsections below discuss these studies in further

26 Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 27 detail.

3.2.1 Yogurt

Hekmat et al. [84] and Hassan et al. [85] both concluded that a fortification level of 0.5% w/w was optimal for balancing the sensory properties with the nutritional value in fortified yogurt. At fortification levels exceeding 0.5% w/w, fortified yogurt samples had a noticeable green colour and marked undesirable flavours [84, 85]. Kuikman and O’Connor [86] evaluated the acceptability of yogurt jointly fortified by Moringa leaf powder and other fruits and vegetables. Yogurt fortified with Moringa (17.09g per 1L of yogurt) and banana puree (250mL per 1L of yogurt) was similarly preferred compared to the unfortified control, while yogurt fortified with only Moringa was least preferred [86]. However, given that a standard serving of yogurt is 170g [63], only 2.9g of Moringa leaves would be consumed per serving of Moringa-banana fortified yogurt. Based on the nutritional content of Moringa presented in Chapter2, this small amount of Moringa leaf powder will have minimal effect on micronutrient consumption.

3.2.2 Bread

The effects of replacing wheat flour with 1-5% w/w Moringa leaf powder or 5-15% w/w Moringa seed flour as a means to fortify bread have also been reported [74, 87]. The preference for bread samples decreased as the fraction of Moringa leaf powder increased [74]. This was attributed to the green colouring and herbal flavours in Moringa leaves. On the other hand, bread samples fortified with up to 10% w/w of debittered Moringa seed flour were found to have a distinct taste that was still within the acceptable range [87]. The inclusion of debittered Moringa seed flour did not have significant impact on the rheological characteristics of the resulting fortified flour blends [87]. Overall, Moringa-fortified bread had lower protein content and higher iron content than commercially prepared whole-wheat bread [63, 74, 87]. Considering that a slice of commercially prepared whole-wheat bread is 32g and a typical serving is two slices of bread [63], bread fortified with debittered Moringa seed flour would provide 2.7mg of iron, which is equivalent to 15% and 34% of the RDA for adult females and males respectively [10]. An attempt to add 10% w/w raw Moringa seed flour or defatted Moringa seed flour to wheat flour was deemed unacceptable to sensory panelists, who found the resulting fortified breads to be too bitter [87].

3.2.3 Cookies and Extruded Snacks

Debittered Moringa seed flour and Moringa leaf powder have been separately added to wheat flour used to prepare cookies [87, 88]. Sensory characteristics were noticeably altered in cookies fortified with over 20% debittered Moringa seed flour [87] and with over 10% Moringa leaf powder [88]. Moringa-fortified cookies were bitter and had surface cracking patterns that Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 28 were noticeably different to unfortified samples [87, 88]. The surface cracking pattern is a key organoleptic attribute used for evaluating the sensory acceptability of cookies, and is described by the width of the cracks and the “islands” formed in between the cracks. The islands formed in Moringa-fortified cookies were considered too large when compared to unfortified cookies [87, 88]. Cookies fortified with debittered Moringa seed flour had a dominant nutty flavour and cookies fortified with over 10% w/w Moringa leaf powder were green, bitter and had a gritty mouthfeel [88]. There were no noted negative impact on the rheological characteristics of the fortified wheat flour [87, 88]. Liu et al. [89] produced extruded snacks made with oat-flour blends that contained 15-45% w/w Moringa leaf powder. No significant differences in preference were observed between the fortified and unfortified snacks, possibly due to the vegetable oil and flour coating that was applied onto the extruded snacks [89]. Uncoated extrudates with higher amounts of Moringa leaf powder had a more noticeable green colour. In the same study, it was observed that the addition of Moringa leaf powder impacted the extrusion characteristics such as expansion, phase transition and pasting properties. The increase in fibre content from Moringa leaves resulted in a reduction in overall extrudability [89]. Based on a serving size of 28g, the extruded snacks made with the oat-flour blend with 45% w/w Moringa leaf powder had 1.2g of fibre, 3.8g of protein, 2.0mg of beta-carotene and 3.6mg of iron [89]. The iron content in the extruded snacks is equivalent to 20% and 45% of the RDA for adult females and males. Using a conversion of 12µg beta-carotene per 1µg RAE [90], the extruded snack contained 170µg RAE, which is equivalent to 20% of the RDA for vitamin A for adults.

3.2.4 Region-speciﬁc Dishes

A study conducted by the Central Food Technological Research Institute in India showed that chutney powder containing 8.1% w/w of Moringa leaf powder was well accepted because sensory characteristics associated with Moringa were masked by other spices within the chutney powder [70]. In West Africa, Moringa leaf powder was added to stiff dough ‘amala’ at up to 2.5% w/w [91]. Amala is a Nigerian dish made out of yam, plantain or cassava flour and is typically eaten with thick soup dishes. With the addition of Moringa, it was found that the amala dishes became dark green in colour which negatively impacted acceptability [91]. Amalas enriched with 2% w/w or lower of Moringa leaf powder score comparatively to the unfortified sample. Changes to the pasting properties, water absorption capacity and bulk density of the fortified amalas due to the addition of Moringa were found to be minimal [91]. The iron content of amala containing 2% w/w of Moringa leaf powder was 2.88mg/100g, which was a 0.45mg/100g increase from the unfortified sample [91]. Up to 25% w/w of Moringa leaf powder was added to ‘ogi’, a fermented cereal pudding eaten in West Africa [92]. Strong preferences for the unfortified samples were observed, and were attributed to undesirable colour changes and leafy tastes in the Moringa-fortified samples. Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 29

Glover-Amengor et al. [93] conducted a study on the acceptability of adding 2-3% w/w Moringa leaf powder into nine dishes on a kindergarten lunch menu in Ghana. The overall acceptability of the fortified dishes were rated by a group of kindergarteners on a pictorial hedonic scale out of five, and all dishes received scores over four (highly acceptable). The same group of participants were supplemented with 2g of Moringa leaf powder in their school lunches three times a week for two weeks, and it was concluded that students found the fortified lunches acceptable as they fully consumed the portions provided [93].

3.2.5 Raw Meat and Fruit Juices

Aqueous Moringa leaf extract was shown to inhibit lipid and myoglobin oxidation in raw beef, resulting in the preservation of the red colour in raw meats [94], which is preferred by consumers as it indicates freshness. Moreover, the microbial count in raw beef treated with aqueous Moringa leaf extract was found to be consistently lower than untreated samples throughout a storage period of 12 days [94], suggesting that Moringa could potentially prolong the shelf-life of raw meats by preventing microbial spoilage. 2% aqueous Moringa leaf extract added to fresh guava juice was shown to be a promising microbial inhibitor [95]. The addition of aqueous Moringa leaf extract added no noticeable odours and did not impact the sensory preference of the juice samples [95].

3.3 Overcoming Technical Challenges

Literature data has repeatedly demonstrated that the major barriers hindering the acceptance of Moringa-fortified foods are their undesirable bitter, leafy flavours and intense green colours, both of which originate from the plant’s leaves [96]. The following three methods are proposed by the author to address the barriers for using Moringa as a natural fortificant:

1. Mask undesirable sensory characteristics

2. Isolate desirable chemical components

3. Remove undesirable components

Masking of undesirable sensory characteristics may be accomplished by overpowering undesirable characteristics with desirable characteristics from other foods or by creating a physical barrier on the natural fortificant. Conveniently, the flavours in Moringa pods, which are sweeter and generally considered more pleasant, could be leveraged to mask the less desirable flavours in Moringa leaves. Moringa pods are also lighter in colour and could serve to lessen the intensity of the dark green colours associated with Moringa leaves. Moreover, some attention should also be placed on the foods that are consumed with a prospective food vehicle, as they could also contribute to the masking of undesirable sensory characteristics. For instance, a vegetable stew would be more amenable to masking unusual textures than a glass of water. Encapsulation and Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 30 coating techniques could also be used to create a physical barrier around the Moringa particles or fortified foods. Desirable chemical components could be isolated by chemical (e.g. solubilities) or mechani- cal means (e.g. pressure). Desirable chemical components includes nutritional components and compounds that contibute to the stability of the nutritional value of Moringa. The isolation of specific compounds from its natural matrix could have both favourable and unfavourable impacts on the stability and bioavailability of the nutritive components in Moringa. On one hand, bioavailability could improve as the antinutritional components are no longer present to inhibit the absorption of nutritional components. On the other hand, a nutritional component may be stabilized and made bioavailable by the presence of another component in the natural plant matrix. An example of a potential interaction within Moringa is between its vitamin A compounds and phenolic content. Vitamin A compounds are susceptible to oxidation and phenolic compounds are known antioxidants. The coexistence of both groups of compounds may allow the vitamin A content to be stabilized. Existing literature has documented the isolation of antioxidant and antimicrobial compounds in Moringa using rudimentary liquid-liquid extraction processes [68, 94, 95]. These studies have successfully extracted water-soluble compounds on a lab scale. Removal of undesirable sensory components could be accomplished by extracting undesirable compounds from the natural matrix or by altering the chemical structure of a compound to deactivate its undesirable properties. Similar to the isolation of desirable compounds, interactions between different compounds within a natural matrix are complex and the extraction of undesirable compounds could lead to unfavourable results. Although sensory characteristics are essential to a product’s acceptance, the primary intent of food fortification, especially in this application, is to address micronutrient deficiencies. Hence, the nutritional impact of removing specific compounds associated with undesirable sensory characteristics should be closely monitored and understood. The removal of bitter flavours from Moringa seeds on a lab scale have been described by Ogunsina et al. [87] and the resulting fortified food products with Moringa seed flour had a nutty flavour. Although all three approaches are feasible, to focus the scope and depth of the research conducted for this thesis, only the taste masking approach was pursued. Taste masking is the most direct approach. Additionally, taste masking does not require additional solvents, heating or pressure in fortification, and would thus avoid increases in manufacturing costs that can ultimately hinder adoption.

3.4 Food Vehicle Options

Four food vehicles were evaluated in this study: bouillon cubes, spice mixes, sauces and snack mixes. The proposed food vehicles were chosen as they all have the ability to mask undesirable sensory properties in Moringa. Bouillon cubes, spice mixes and sauces are added to dishes Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 31 with existing textures and colours. Flavours and colours in spices mixes and sauces may also be used to overpower undesirable sensory properties in Moringa leaves. Dried snack mixes, such as the Bombay mix, are seasoned with spices that would aid in masking the bitter and astringent ﬂavours of Moringa leaves. The range of consumer-accepted colours and textures for snack mixes is broad, and would aid in masking undesirable colour changes caused by the addition of Moringa leaves. A decision matrix approach was used to evaluate various foods as vehicles for Moringa forti- ﬁcation. As shown in Table 3.1, four factors were considered and weighed equally: consumption coverage, shelf life, ease of adding other micronutrients, and public health concerns. Each factor was allocated a score between 1 and 5 for each candidate vehicle. Scores assigned were used as a relative comparison amongst the four food vehicles. Higher scores for consumption coverage (A), shelf-life (B) and ease of adding other micronutrients (C) are preferred, as it indicates high consumption coverage and levels, longer shelf-life, and easy incorporation of other micronutrients. A lower score for public health concerns (D) is preferred as it indicates low public health concern. The overall score was calculated by summing the scores for consumption coverage, shelf-life and ease of adding other micronutrients, and subtracting the score for public health concerns (i.e. (A+B+C)-D). Of the vehicles evaluated, bouillon cubes had the highest overall score and were found to be most suitable for this application. The details of the evaluations are described in the subsections below.

Table 3.1: Evaluation of food vehicles for incorporating Moringa Consumption coverage Shelf-life Ease of adding other micronutrients Public health concern Overall (A) (B) (C) (D) (A+B+C)-D Bouillon Cubes 5 4 4 2 11 Spice Mixes 3 4 3 1 9 Sauces 3 2 4 2 7 Snack Mixes 2 4 3 4 5 Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 32

3.4.1 Consumption Coverage

Food vehicles with high consumption coverage across the entire population are ideal for industrial food fortification. More specifically, food vehicles should be consumed at consistent levels between different socioeconomic levels. This increases the likelihood of the fortified food reaching individuals of low socioeconomic status, who are more likely to be affected by food and nutrition insecurity. Out of the four food vehicles evaluated, bouillon cubes were assessed to have the highest consumption coverage. Household coverage assessments conducted in West Africa showed that bouillon cubes have at least 95% coverage across households of all socioeconomic statuses [97, 98]. The low unit price for bouillon cubes allows them to be consumed at similar levels regardless of a household’s socioeconomic status [97–99]. Although there are different types of commercially available bouillon cubes (e.g. vegetable, beef, chicken), the ingredients of each type are similar, and specific flavourings generally constitute 10% of the whole cube. Unlike salt, which is universally used, the consumption and use of spices and sauces vary extensively depending on individual taste preferences, which are influenced by variables such as culture and region. The organoleptic properties of fortified spice mixes and sauces would need to be tailored for a specific context, which could limit the extent of its reach. Variabilities in the consumption of spices and sauces also make it difficult to gather meaningful consumption data on these food vehicles. Nonetheless, the development of Moringa-fortified ‘sambar’ spice mixes have been suggested in literature [58]. Sambar is South Indian vegetable stew made with lentils and could include Moringa pods. As Moringa leaves and pods are already commonly eaten around South India, the inclusion of Moringa leaves is promising for that specific context. Data on the consumption of snack mixes are not available publicly as they are not captured in dietary recall surveys. Household coverage of snack mixes may also be low for households of low socioeconomic status as resources are typically prioritized towards food required for meals, and snack mixes may be considered a luxury for those with limited financial resources.

3.4.2 Shelf-life

Food vehicles with long shelf-lives are preferred as it allows the micronutrients within the fortiﬁed food vehicle to be readily available year-round. Furthermore, food vehicles that are shelf-stable are more practical for households of low socioeconomic status, who may not have access to cold storage. Out of the four food vehicles evaluated, bouillon cubes, spice mixes and snack mixes scored similarly and highest. Bouillon cubes, spice mixes and snack mixes are all generally shelf-stable. Bouillon cubes have shelf-lives of up to two years, while dried spice mixes may be used as long as their ﬂavours and aromas are considered potent enough by the consumer. Dried snack mixes have long shelf lives provided that the moisture content is kept low during storage. Conversely, the shelf-lives of sauces vary widely, depending on the ingredients within the condiment. For example, soy sauces, which contain 3300-5500mg Na/100g [63], has a shelf life of two years, whereas ketchup, which contains 20-907mg Na/100g [63], may only be stored Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 33 for a month without cold storage.

3.4.3 Ease of Adding Other Micronutrients

Moringa-fortified foods would likely require supplemental fortificants to enhance their nutritional value as well as increase the stability and bioavailability of micronutrients. Ideally, food vehicles should have both hydrophobic and hydrophilic components to incorporate both water- soluble and oil-soluble micronutrients. Of the four food vehicles evaluated, bouillon cubes and sauces scored highest as they both typically contain water and oils. The incorporation of additional fortificants into spice mixes and snack mixes is likely feasible, but cannot be determined specifically as the constituents vary greatly between different types of spice and snack mixes. Some snack or spice recipes contain only either water-soluble or oil-soluble components.

3.4.4 Public Health Concern

Fortiﬁed vehicles should have low or no public health concerns. The allocated public health concern score for spice mixes is lowest as an increased consumption of spices is not harmful for health. Bouillon cubes and sauces are typically high in sodium, and excessive sodium intake leads to increased risks of heart diseases and other health aliments. Similarly, snack mixes are often high in sodium and sugar content as they are designed to satisfy sugary or savory cravings. Furthermore, fortiﬁcation of snack mixes is deemed to be inappropriate by the FDA as it may “mislead consumers, causing them to substitute snacks for naturally nutrient dense foods” [100].

3.5 Proof of concept for Moringa-fortiﬁed bouillon cubes

Following the evaluation of various options discussed in Section 3.4, bouillon cubes were determined to be the most suitable for the incorporation of Moringa out of the food vehicles evaluated. The main advantages of incorporating Moringa into bouillon cubes are two-fold: the micronutrients in Moringa could increase the nutritional content of bouillon cubes, which currently has negligible to no nutritional benefits; and the inclusion of Moringa in bouillon cubes would increase the utilization of a locally-available edible plant. By using Moringa as a natural fortificant, post-harvest food loss would also be reduced in places where Moringa is already consumed. Consequently, a proof of concept was conducted to explore the feasibility of making Moringa-fortified bouillon cubes. In this thesis, the direct incorporation of Moringa leaf and pod powder was explored as a preliminary step for developing Moringa-fortified bouillon cubes. Moringa pod powder was included in this proof of concept as it has desirable sensory characteristics that could be leveraged to increase the acceptance of the fortified bouillon cubes. Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 34

3.5.1 Commercially Available Bouillon Cubes

The general composition of bouillon cubes is displayed in Figure 3.1[101–103]. The majority of the ingredients (salt, MSG, fat, ﬂavourings) in the recipe are included to enhance the taste of the bouillon cubes. Binders and fat components are included to maintain the shape and integrity of the bouillon cubes. Reported consumption levels vary between 1 and 4.8g/day per person [97–99] or 20g per dish for six people [104] in the Central and West African region.

Figure 3.1: Ingredient composition in a commercial bouillon cube

In 2012 and 2015, Nestléand Unilever respectively launched iron-fortified bouillon cubes in Central and West Africa [105, 106]. Nestlé’s‘Maggi’ cubes sold in Central and West Africa contain between 15% NRV (Nutrient Reference Value) to a maximum of 20% of Tolerable Upper Intake Level (UL) for iron per 3.3g serving [104]. The Codex NRV and UL for iron is 14mg/day and 45mg/day respectively [1, 107]. The serving size of 3.3g per person was determined by Nestlé’sinternal consumer research [104]. This fortification level was set based on the resulting sensory properties and production costs. At fortification levels above 15% NRV, Nestlédetermined that the changes to sensory properties were unacceptable to consumers [104]. Nestléalso determined that a fortification level of 30% NRV would result in incremental product costs that could not be absorbed through cost savings in packaging and processing [104]. 15% NRV aligns with the Codex Alimentarius definition of foods that are a “source of” a specific nutrient [108]. Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 35

3.5.2 Speciﬁcations

Nutritional Speciﬁcations

Globally, the most prevalent micronutrient deficiencies are iodine, iron, zinc, vitamin A, folate and vitamin B12 . The severity of these micronutrient deficiencies varies amongst regions depending on the local diet. For Moringa-fortified bouillon cubes, it follows that the micronutrients that are available in Moringa’s natural matrix should be examined first, followed by investigations on supplementation of the remaining micronutrients using additional fortificants. Micronutrients that are naturally available in Moringa in appreciable amounts are iron, zinc, vitamin A and folate. As discussed in Section 1.2, NRV is used for benchmarking the nutritional content in food products and food fortification programs, while RDA is used to assess the nutritional requirements for a specific target population group. Hence, NRV was used to benchmark the nutritional content of the fortified bouillon cubes. Differences in the NRV and RDA may be attributed to the literature reviewed by the respective organizations to establish the reference values (Ta- ble 3.2). A benchmark of 15% NRV for all micronutrients per 3.3g serving was used as an initial target. This benchmark is in alignment with Codex guidelines [108] and commercially available fortified bouillon cube products [104].

Table 3.2: Comparison of NRV and RDA values for minerals, vitamins and protein RDA* NRV Nutrient [1,2, 109] [107] Minerals Calcium 1000 1000 Magnesium 310-420 300 Potassium 4700 N/A Sodium 1500 N/A Zinc 8-11 15 Copper 0.9 N/A Manganese 1.8-2.3 300 Iron 8-18 14

Vitamins Vitamin A (µg RAE/d) 700-900 800 Vitamin B1 (mg/d) 1.1-1.2 1.2 Vitamin B6 (mg/d) 1.3 1.3 Vitamin B9 (µg DFE/day) 400 400 Vitamin B12 (µg/d) 2.4 2.4 Vitamin C (mg/d) 75-90 60

Macronutrients - Protein 46-56 50 *non-pregnant and non-lactating adults, 19-50 years old. Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 36

Physical Speciﬁcations

Physical characteristics of fortified bouillon cubes would ideally be the same as those in unfortified bouillon cubes. Physical specifications are required for user acceptance, transportation and storage (Table 3.3). All characteristics with the exception of water activity were considered in relation to the user’s familiarity and habits when using bouillon cubes. This project aimed to form small-sized bouillon cubes (3-4g). Minimum hardness is required for transportation and maximum hardness pertains to the users’ habits in crumbling cubes by hand before adding them during food preparation. Upper water activity thresholds are needed to ensure a shelf-stable food product.

Table 3.3: Physical specifications for fortified bouillon cubes Specification Function Benchmark Mass User acceptance ‘Small’ size: 3-4g ‘Large’ size: 10-13g User acceptance ‘Small’ size: 20 x 20 x 20mm Shape Transportation ‘Large’ size: 26 x 25 x 10mm Storage User acceptance Hardness 5-50N [102, 103] Transportation Food safety < 0.6 to inhibit enzymatic degradation and; Water activity Storage < 0.8 to inhibit microbial growth [110] Quick: < 30 seconds in boiling water [101] Disintegration time User acceptance Regular: < 3 minutes in boiling water [103]

Organoleptic Speciﬁcations

Food fortification should preferably be as undetectable as possible, so the organoleptic (taste, smell, sight, texture, sound) characteristics of fortified bouillon cubes should be the same or closely resemble the characteristics of the unfortified bouillon cubes. The colour, shape and size contribute to the appearance of the cubes. For Moringa-fortified bouillon cubes, it is expected that the taste and mouthfeel will be partially masked by the food they are consumed or prepared with. To focus the efforts of this proof of concept, the nutritional and physical characteristics were primarily concentrated upon as organoleptic characteristics could be optimized in future work. Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 37

3.5.3 Experimental Design

A simple axial mixture design (Figure 3.2) was used to explore the eﬀects of Moringa leaf powder, Moringa pod powder and binder (semolina) in extrudates. Semolina was used as it is commonly used in extrusion applications and is low cost. The wholesale pricing of semolina ranges from US$200-500/ton according to online wholesalers (Alibaba.com). The apices and edges corresponded to 100% and 0% of a speciﬁc ingredient respectively. Formulations were numbered 1-10 for quick reference.

Figure 3.2: Simple axial mixture experimental design for proof of concept Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 38

3.5.4 Processing

The processing of ingredients to form Moringa-fortiﬁed bouillon cubes is shown in Figure 3.3 and described below.

Figure 3.3: Block ﬂow diagram for forming Moringa-fortiﬁed bouillon cubes Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 39

Materials and Dry Ingredients Preparation

Fresh Moringa leaves from Coimbatore, India, and pods from Delhi, India, were used. The nutritional properties of the Moringa leaf and pod samples were described in Chapter2. Durum semolina (Brand: Unico) was procured from a local supermarket in Toronto, Canada. Moringa leaﬂets were stripped from their petioles and washed, while Moringa pods were washed then cut into 2-3” sections. Moringa leaves and pods were freeze-dried for 72 hours, milled and sieved through a Tyler No. 35 mesh (500µm). Dry ingredients were stored in sealed glass containers at 4◦C.

Extrusion Processing

Dry ingredients were blended and hydrated using a benchtop mixer (Model no.: KitchenAid KSM95TB). A benchtop cold extruder was assembled by ﬁtting a 20*20mm die to a single Archimedes screw attachment (Model no.: KitchenAid FDA) which was then attached to the motor of the aforementioned benchtop mixer (Figure 3.4). After extrusion, extrudates were manually cut to lengths of 20mm to give 20*20*20mm cubes and air-dried for 48 hours at 35◦C in a dehydrator (Model no.: Excalibur 2400).

Figure 3.4: (left) Cold extrusion setup. Die attached to single Archimedes screw attachment; (right) Stainless steel extrusion die with 20*20mm square opening

A series of screening experiments using semolina, 1:1 ratio blends of binder and commercial Moringa leaf powder (Brand: Yupik), and 1:1 ratio blends of binder and Moringa pod powder were conducted to determine the appropriate in-barrel moisture for extrusion (Table 3.4). For all combinations, blends with 17% in-barrel moisture were too dry and the dry ingredients did not cohere with each other after extrusion. The blend with 29% in-barrel moisture was found Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 40 to be suﬃcient for the extrusion of semolina. At 38% in-barrel moisture, extrudates made with only semolina deformed substantially at the outlet, and cross-sections of the extrudates did not exit uniformly as the dough was partially sticking to the sides of the die. For blends with 1:1 ratios of binder and Moringa leaf or pod powder, 38% in-barrel moisture was needed for blends to be extruded successfully.

Table 3.4: In-barrel moisture content in screening experiments Blend ratio Mass of dry ingredients (g) Volume of water added (mL) In-barrel moisture (%) Successfully extruded? (L:P:B) 0:0:1 150 30 17% No 0:0:1 150 45 29% Yes 0:0:1 150 60 38% No 1:0:1 150 30 17% No 1:0:1 150 45 29% No 1:0:1 150 60 38% Yes 0:1:1 150 30 17% No 0:1:1 150 45 29% No 0:1:1 150 60 38% Yes

The screening experiments showed that different in-barrel moisture content is required for different formulation blends. This is likely due to the different water absorption capacities for the three ingredients. Thus, for formulations in the mixture experimental design, dry ingredients were initially hydrated to 29% in-barrel moisture and the consistencies of the blends were qualitatively checked to match the blends that were successfully extruded in the screening experiments. If a blend was found to be too dry, an amount of water equivalent to 10% of the mass of the dry ingredients was added and the blend’s consistency was checked again. The process of adding water at 10% mass increments was repeated until the blend reached the appropriate consistency. Table 3.5 shows the in-barrel moisture used for each formulation. 1% w/w vegetable shortening (Brand: Crisco) was blended into all formulations for lubri- cation during extrusion. Hydrated blends were left in ambient conditions for 30 minutes for equilibration prior to extrusion. The motor rotational speed was set to setting number 4 for all formulations.

Table 3.5: In-barrel moisture for mixture design formulations Formulation Blend ratio (L:P:B) Mass of dry ingredients (g) Volume of water added (mL) In-barrel moisture (%) 1 1:0:0 100 70 41% 2 0:1:0 80 80 50% 3 0:0:1 150 60 29% 4 1:1:0 100 100 50% 5 0:1:1 150 90 38% 6 1:0:1 150 90 38% 7 1:1:1 120 96 44% 8 4:1:1 90 81 47% 9 1:4:1 90 81 47% 10 1:1:4 180 108 38% Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 41

3.5.5 Characterization Methods

Nutritional Content

The iron and zinc contents of the dry ingredients and formulations 7, 9 and 10 were determined experimentally. 10 minerals were analyzed, but only iron and zinc results are discussed in this section. See Appendix A for mineral content results on calcium, magnesium, potassium, sodium, zinc, copper, manganese, lead and selenium. Measured mineral contents were compared with values calculated using the mineral content of the individual dry ingredients and blend ratios. One sample t-tests were conducted between the measured and calculated values of every mineral and formulation to confirm that they were not statistically different (p = 0.05). Out of the 30 one sample t-tests conducted (three formulations measured, 10 minerals per formulation), only the measured value for selenium formulation 7 (1:1:1 blend ratio) was statistically different from its calculated value. The statistical difference for the selenium measurement is likely a cumulative result of experimental inaccuracies from weighing, dilutions and calibration. Given that 29 out of 30 measured values were not statistically different from their theoretical values, it was deduced that the mineral content of each formulation could be calculated using the mineral content of the dry ingredients and blend ratios. Vitamin A and folate contents were calculated using the blend ratios of each formulation and the standard reference values for Moringa leaves and pods available from the USDA Food Composition Database. Crude protein content was experimentally measured to determine the extrudates’ suitability as a protein supplement. Experimental methods used for determining the mineral and protein content were described in Chapter2.

Physical Characteristics

The mass of dried extrudates for all formulations was obtained and recorded. The shape of the samples was predetermined by the 20*20mm die used during extrusion. Hardness was measured using a tablet hardness tester (Model no.: Erweka TBH250). Water activity was measured using a benchtop water activity meter (Model no.: AQUALAB 4TE). Disintegration was determined by a binary test on the presence of particles with diameters larger than 2mm (Tyler No. 10 mesh) and hard cores after 20 minutes in 80◦C water with constant vertical agitation. This method was adapted from the US Pharmacopeia disintegration test (701) for tablets. Commercially available vegetable stock cubes (Brands: Knorr and Aurora) were obtained from a local supermarket in Toronto, Canada, and tested for their disintegration rate to give an additional reference data point. With the exception of water activity measurements, all measurements were triplicated. Only one measurement was obtained per formulation for water activity due to a shortage of extruded samples. One-way ANOVA tests were performed for mass and hardness results to determine the difference in means between all formulations. If means were shown to be different, Tukey Honest Significant Difference (HSD) tests were performed to determine the statistical significances of Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 42 the differences.

Colour

Photographs of dried extrudates were taken in a photobox under consistent indoor ﬂuorescent lighting. The same white balance was used for all photographs. The Hunter L*ab colour scale was used to quantify the colours of the extrudates.

3.5.6 Results

Nutritional Content

Levels of iron, zinc, vitamin A and protein increased with increasing Moringa leaf content (Table 3.6 and Figure 3.5). As expected, the nutritional content of the ingredients was unchanged after extrusion as no additional heat or pressure was applied during processing. The level of folate increased as the amount of semolina increased since semolina has more folate (72 µg DFE/100g) than Moringa leaves and pods (40 and 44 µg DFE/100g respectively). All formulations failed to reach the 15% NRV per 3.3g serving benchmark for iron, zinc, vitamin A, folate and protein (Table 3.6). Bioavailability considerations aside, 1.30 mg of iron would need to be added per serving (3.3g) to supplement cubes made solely with Moringa leaf powder (Figure 3.5). Quantitative bioavailability data for Moringa leaves have not been published in literature. However, it is known that Moringa leaves also contain oxalic acid [111], which is also present in spinach and is a likely inhibiting factor for iron absorption in humans [112]. Thus, it may be deduced that the iron bioavailability in Moringa leaves is likely less than 100% and cubes would need to be supplemented with over 1.30mg of iron to reach the 15% NRV benchmark. The upgrading of micronutrient content may be accomplished by using natural fortiﬁcants derived from other species or by using synthetic fortiﬁcants.

Table 3.6: %NRV of iron, zinc, vitamin A, folate and protein per 3.3g serving %Codex NRV* per 3.3g serving Formulation Blend ratio (L:P:B) Iron Zinc Vitamin A Folate Protein 1 1:00:00 5.7% 0.3% 1.6% 0.3% 2.1% 2 0:01:00 0.7% 0.4% 0.0% 0.4% 0.9% 3 0:00:01 0.8% 0.3% 0.0% 0.6% 0.9% 4 1:01:00 3.2% 0.3% 0.8% 0.3% 1.5% 5 0:01:01 0.8% 0.3% 0.0% 0.5% 0.9% 6 1:00:01 3.3% 0.3% 0.8% 0.5% 1.5% 7 1:01:01 2.4% 0.3% 0.5% 0.4% 1.3% 8 4:01:01 4.1% 0.3% 1.0% 0.4% 1.7% 9 1:04:01 1.6% 0.4% 0.3% 0.4% 1.1% 10 1:01:04 1.6% 0.3% 0.3% 0.5% 1.1% Fe NRV = 14mg/d; Zn = 15mg/d; Vitamin A = 800mg RAE/d; Folate = 400µg DFE/d; Protein = 50g/d Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 43

Figure 3.5: Iron content in mixture design formulations. Increasing bubble sizes correspond to higher levels of iron; numbers correspond to mean iron content per cube (mg/3.3g serving). Iron NRV = 14mg/d; benchmark was 15% NRV = 2.1mg

Physical Characteristics

Results on the physical characteristics of extrudates are shown in Table 3.7. Extrudates made with at least 67% semolina by weight were significantly denser. It was observed that doughs with more semolina were stickier due to the formation of gluten bonds and semolina-only doughs tended to adhere more strongly to the sides of the extrusion die compared to other formulations. The adhesion of the semolina doughs required a greater amount of force for extrusion, which led to increased compression and denser extrudates. Extrudates from all formulations had masses greater than 4g, which was the benchmark set in Subsection 3.5.2. As extrudate density is a function of in-barrel moisture, and the moisture content varied between formulations in this study, quantitative conclusions could not be drawn on the effect of moisture on the extrudability in the formulations tested. Hence, further investigation is required to examine the effects of in-barrel moisture on the extrudate bulk density for this system. Extrudates composed of at least 50% Moringa pods (Formulations 4, 5 and 9) appeared to be significantly harder than extrudates made with other formulations (Figure 3.6). Further investigation is required to understand this result. A possible explanation for this result is that the fibre content in Moringa pod powder contributed to the structural integrity of the extrudates. Extrudates formed with only Moringa leaf powder (formulation 1), only semolina (formulation 3), or with a 1:1 ratio of Moringa leaf powder and semolina (formulation 6) were within the benchmarked range for hardness. These formulations had the lowest hardness Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 44

Table 3.7: Mass, hardness, water activity and disintegration results for extruded cubes Blend ratio Disintegration Formulation Mass (g) Hardness (N) a (L:P:B) w (# of tests passed, n=3) 1 1:0:0 4.2 ± 0.3a 29 ± 16a 0.14 2 2 0:1:0 4.3 ± 1.0ab 75 ± 14ab 0.17 3 3 0:0:1 6.3 ± 0.7c 17 ± 11ab 0.52 0 4 1:1:0 5.1 ± 0.2abcde 360 ± 57c 0.14 2 5 0:1:1 5.7 ± 0.3cde 190 ± 39b 0.16 0 6 1:0:1 5.4 ± 0.1bcde 37 ± 15ab 0.16 0 7 1:1:1 5.3 ± 0.2abcde 93 ± 15ab 0.16 3 8 4:1:1 4.7 ± 0.2abde 97 ± 36ab 0.16 1 9 1:4:1 4.5 ± 0.2abd 374 ± 127c 0.16 3 10 1:1:4 5.7 ± 0.1ce 136 ± 56ab 0.17 0 Values within a column with the same superscript letters are not signiﬁcantly diﬀerent (p = 0.05) measurements and extrudates were breakable by hand. It is also worth noting that hardness does not equate to crumbliness, which is an organoleptic property and described as the ease of which a material breaks into smaller portions. The lower measured hardness may also be due to the development of cracks in the centre of the cube after drying (Figure 3.7). These cracks were parallel to the direction of extrusion and were likely formed due to an uneven compression force from the extruder screw. It was speculated that the uneven compression force was due to the unsteady operation of the extrusion system, caused by intermittent material loading, which was done to prevent excessive torque on the rotating gears within the bench-top mixer. The water activity for all formulations was below 0.6, which suggested that enzymatic and microbial degradation were inhibited within the extrudates. These results were expected as formulations were dried over a prolonged period (48 hours). Both brands of commercially available bouillon cubes passed all three replicates of the disintegration test and were completed dissolved within three minutes. On the other hand, only extrudates made with 100% Moringa pods fully dispersed within 20 minutes. Formulations that passed two or three replicates of disintegration tests were considered acceptable (n=3). Extrudates from other formulations that passed the disintegration test did not have a hard core, but a noticeable solid portion of the extrudate remained at the end of each test. The presence of solid chunks after 20 minutes would likely be unacceptable for the consumer. These unfavourable results were caused by the water insoluble starch particles in semolina.

Colour

Extrudates with 50% or more Moringa leaf powder were darker in colour (lower L* values) than extrudates of other formulations (Table 3.8). This result matched expectations as Moringa leaf powder is dark green. Extrudates were less green when they were made with more semolina, which matched expectations as semolina is yellow. All formulations had an obvious green colour, and deviated from the usual yellow-brown colour of bouillon cubes. Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 45

Figure 3.6: Hardness in mixture design formulations. Increasing bubble sizes correspond to increasing hardness; numbers correspond to mean hardness in newtons. The range for acceptable hardness is 5-50N.

Figure 3.7: Formation of cracks (top face) after drying suggesting uneven extrusion pressure. Sample of cube made with 1:1 ratio of Moringa leaves and binder. Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 46

Table 3.8: Hunter L*ab values for extruded cubes Formulation Blend ratio (L:P:B) L* a b 1 1:0:0 14.1±1.57 -2.3±0.14 7.40±0.52 2 0:1:0 42.0±2.90 -4.0±1.74 20.9±1.08 3 0:0:1 46.6±2.09 -1.2±0.55 17.7±1.83 4 1:1:0 15.7±3.13 -1.7±0.38 8.30±1.36 5 0:1:1 37.3±2.75 -3.1±0.83 17.7±1.21 6 1:0:1 13.0±0.73 -2.0±0.41 6.4±0.77 7 1:1:1 19.8±4.59 -4.1±1.52 10.8±2.63 8 4:1:1 12.4±3.13 -2.8±1.27 5.9±1.69 9 1:4:1 25.3±1.43 -2.0±5.49 14.6±0.85 10 1:1:4 21.9±4.43 -4.1±1.2 12.4±1.97 L*: 0 = dark, 100 = light a: -128 = green, 128 = red b: -128 = blue, 128 = yellow Chapter 3. Addressing Micronutrient Deficiencies using Moringa oleifera 47

3.6 Summary

A vast amount of work remains for the development of Moringa-fortified bouillon cubes. Rudi- mentary insights were obtained from the exploratory work described in this chapter. Additional fortificants will be required to upgrade the nutritional content in Moringa-fortified bouillon cubes. The flavours and colours of Moringa may either be enhanced or suppressed in flavouring cubes, depending on regional preferences. In select regions where Moringa is already widely consumed and preferred, the distinct flavours of Moringa may be enhanced to encourage further consumption. Conversely, Moringa’s flavours and colours may be suppressed when they are not preferred. Given the nutritional and physical characterization results determined in this thesis and sensory preferences observed in literature, the formulation with equal parts (i.e. 1:1:1) of Moringa leaf powder, Moringa pod powder and binder is the most promising out of the 10 formulations tested. Cubes with equal parts of all ingredients had high nutritional content relative to other formulations and passed all three replicates of the disintegration tests. Cubes with a 4:1:1 (L:P:B) ratio were lower in hardness but only passed one out of three times when tested for disintegration. Cubes with 100% Moringa leaf power yielded the highest nutritional content and were within the acceptable hardness range, but their dark green colours would be highly unacceptable to consumers. The undesirability of green colours in foods may be because of associations with mold and the degradation of food. Semolina was chosen for this proof of concept as it is commonly used as a binder in extrusion processes and has low unit cost. However, the starch in semolina is water-insoluble and unfavourable for the disintegration of bouillon cubes. Semolina provided a great deal of structural integrity within extrudates, but most of the formulations were too hard to be crumbled by hand. Bouillon cubes are commonly hand-crumbled during food preparation and deviations away from the typical crumbliness could reduce the user acceptability. The inclusion of semolina contributed to the dough’s elasticity during extrusion, which improved extrudability. As expected, Moringa leaf and pod powder provided dark and light green pigments to the extrudates respectively. Concerns regarding undesirable mouthfeels and tastes were not addressed in this proof of concept, but should be examined in depth in future work. Process- ing characteristics involving dough rheology and in-barrel moisture content were not assessed quantitatively and should also be examined in detail in the future. Chapter 4

Future Work and Conclusions

This chapter presents recommendations on future work for the development of fortiﬁed bouillon cubes and summarizes the research conducted in this thesis.

4.1 Future Work

4.1.1 Taste, Aroma and Colouring Compounds

The taste and aroma of a specific food is the result of complex interactions amongst chemical compounds within the food and with sensory receptors in an individual. Qualitatively, Moringa leaves have a distinct bitter taste [66] and Moringa pods have mild sweet flavours [58]. However, to date, quantitative analyses have not been published on the identification of biochemical interactions that contribute towards the taste, aroma and colouring of Moringa leaves and pods. Identification of biochemical interactions responsible for Moringa’s taste, aroma and colour is fundamental to manipulation of organoleptic properties and therefore the acceptance of Moringa-fortified foods. Compounds that contribute to undesirable sensory characteristics (e.g. bitterness) could be removed, whereas concentrated fractions of compounds that contribute to desirable sensory characteristics could be formed by extraction or synthesis. Concentration of desirable compounds is also pertinent to the development of other food products in regions where Moringa is widely consumed and preferred. Biochemical mechanisms for different taste sensations have been documented [113]. This knowledge may be combined with literature chemical characterizations for Moringa to form initial hypotheses on possible biochemical interactions responsible for the distinct tastes in Moringa leaves and pods. Aromas are mainly results of interactions between volatile compounds and olfaction receptors; and colours are a consequence to the light absorbance of chemical compounds. As taste could be affected by a broad range of compounds, from salts to amino acids to alkaloids, the analytical methods used for the identification of these compounds would be similarly diverse. Volatile compounds may be identified using gas chromatography-mass spectrometry (GC-MS) and colouring compounds may be identified using spectrophotometry.

48 Chapter 4. Future Work and Conclusions 49

The identiﬁcation of taste, aroma and colouring compounds and their biochemical interactions will commence in September 2018 with a three-month research exchange at the Tamil Nadu Agricultural University starting in November 2018.

4.1.2 Encapsulation and Coating Processes

As mentioned in Chapter3, an alternative method for masking undesirable sensory characteristics in Moringa is to utilize physical barriers such as those formed through coating and encapsulation processes. Encapsulation processing may also further extend the shelf life and stability of micronutrients in Moringa. Coatings used for microencapsulation should be selected based on their ability to enable the encapsulated Moringa particles to blend in with the particles of the food vehicle without limiting the bioavailability of the micronutrients. However, encapsulation and coating processing also increases particle size, which could limit the amount of micronutrients for fortiﬁcation. Considerations for potential coatings include texture, colour and release mechanisms.

4.1.3 Alternative Binders and Excipients

Alternative binders should be evaluated for future work. Binders should not change the organoleptic properties of bouillon cubes. Two approaches to ensure that organoleptic properties are unchanged are to use water-soluble binders or to reduce the particle size of binders. Binder particles should be either be water-soluble or be contained within a water-soluble matrix so that fortiﬁed bouillon cubes readily disintegrate under cooking conditions. Binder particles could be made small enough so that they are not visually and textually detectable in the food that bouillon cubes are consumed with. Other excipients such as aids for disintegration and dissolution may also explored. The inclusion of other excipients may improve the assimilability of fortiﬁed bouillon cubes, but will likely increase the manufacturing costs. Excipients are commonly used in pharmaceutical applications and transferrable knowledge may be obtained by studying literature on the formulation of pharmaceuticals.

4.1.4 Supplemental Fortiﬁcants

Moringa-fortiﬁed foods will require supplementation from other fortiﬁcants, natural or synthetic, to supplement appropriate levels of iodine, iron, zinc, vitamin A, folate and vitamin

B12. Fortificants derived from animal products are not considered for this application as people who do not consume animal food products, such as vegetarians, would be excluded from consuming the resulting Moringa-fortified food product. Vegetarians are susceptible to prevalent micronutrient deficiencies, so their acceptance of the resulting products should be carefully considered (see Section 1.1). Chapter 4. Future Work and Conclusions 50

Natural fortificants from other plant species may be used to complement the micronutrient content in Moringa. For example, seaweed is considered a source of iodine and many dairy products are considered sources of vitamin B12 [114, 115]. Clearly, the inclusion of dairy products would exclude those who do not consume animal-derived food products, but vitamin B12 does not naturally occur in appreciable levels in plant-based food products. The inclusion of other natural fortificants may reduce post-harvest food losses for other edible plant species. Synthetic fortificants may also be added to efficiently upgrade micronutrient content, and fortification technologies using synthetic fortificants are well-established in other food vehicles (see Subsection 1.3.1). However, the incorporation of synthetic fortificants may lead to unintended chemical interactions that will reduce the stability of micronutrients within the fortified food.

4.1.5 Manufacturing Process Selection and Optimization

The identification and optimization of suitable manufacturing processes will be required for the commercialization of the product. Cold extrusion was trialed in this thesis as a proof of concept for Moringa-fortified bouillon cubes given its ease of operation and equipment availability. For future work, the effects of different extruder designs and processing conditions could be considered. The effects of heat on the micronutrient content in fortified bouillon cubes is of particular interest, as vitamins destabilization through oxidation may be exacerbated under heated oxy- genated conditions. Extrudability may also be improved in future work by increasing the fat content in the dough and by varying extrusion process conditions. In this proof of concept, in-barrel moisture was adjusted to improve extrudability, but it would be more appropriate to vary the dough fat content in future work as extrusion is heavily dependent on the rheological characteristics of the dough. Manufacturing processes involving tablet presses may also be investigated for the production of fortified bouillon cubes. Tablet presses are commonly used for manufacturing existing commercially available bouillon cubes.

4.1.6 Consumption Patterns and Consumer Preferences

Limited public data are available on the consumption patterns and preferences for bouillon cubes. A clear understanding of the prevailing consumption patterns and preferences for bouillon cubes is required before the development of fortified bouillon cubes. It is also conceivable that existing bouillon cubes consumption patterns may not be equitable for Moringa-fortified bouillon cubes as the marketed product may deviate away from conventional products. For this proof of concept, a serving size of 3.3g/person/day was used to enable simple comparisons with existing commercial products, but the consumption level of Moringa-fortified bouillon cubes may be different as Moringa may impart specific flavours and fortified bouillon cubes may contain a different concentration of salt. The effects of these variables on consumption patterns may be explored in future work to gain insights on the acceptability of Moringa-fortified bouillon cubes. Chapter 4. Future Work and Conclusions 51

4.2 Conclusions

The Moringa-fortiﬁed bouillon cube is a unique approach for incorporating the micronutrients in Moringa into a staple condiment. Moringa-fortiﬁed bouillon cubes allow the micronutrients in Moringa to be shelf-stable. Bouillon cubes are suitable for incorporating a wide variety of micronutrients as its matrix contains both water-soluble and oil-soluble components. The sensory properties of Moringa may either be enhanced or suppressed to suit regional preferences. In either case, the amount of Moringa used is small, but would still increase the utilization of Moringa as a source of micronutrients.

The objectives of this thesis and the associated work completed are described below.

1. Determine the nutrient content in Moringa leaves and pods.

The macronutrient and micronutrient contents in Moringa from multiple regions were determined using a combination of experimental methods and literature review. Micronutrient content in Moringa was compared to vegetables that are considered sources of the speciﬁed micronutrients. For an equivalent dry mass, Moringa generally has similar or lower levels of micronutrients relative to other foods consumed in a vegetarian diet, except in the case for vitamin C. Moringa leaves contain 8.3-110mg Fe/100g, compared to spinach leaves which have 32mg Fe/100g. Moringa leaves and pods were found to have high levels of vitamin C relative to kiwifruit. The vitamin C content in Moringa leaves and pods are 48-220mg/100g and 65-141mg/100g respectively, compared to 93mg/100g in kiwifruit. In terms of protein quality, foods rich in lysine or the sum of methionine and cysteine could be used to complement the essential amino acids in Moringa leaves and pods. Lastly, the skin of Moringa pods contain a considerable fraction of macronutrients and micronutrients, and discarding the skin would result in a substantial loss of micronutrients within Moringa pods.

2. Evaluate the potential of incorporating Moringa as a natural food fortiﬁ- cant.

Literature studies demonstrated that the bitter tastes and green colours were major barriers for the acceptance of Moringa-fortiﬁed foods. Food vehicles that are able to mask the undesirable sensory properties of Moringa leaves were considered in this thesis. Bouillon cubes, spice mixes, sauces, and snack mixes were evaluated on their suitability for incorporating Moringa based on their consumption patterns, shelf-life, capacity to include additional fortiﬁcants (aside from Moringa), and health impact. Bouillon cubes were determined to be best suited for this application. Chapter 4. Future Work and Conclusions 52

3. Explore the feasibility of a promising food fortiﬁcation application for Moringa.

A bench-scale cold extrusion process with a single screw configuration was used to form Moringa- fortified bouillon cubes. 10 formulations with varying amounts of Moringa leaf powder, Moringa pod powder and semolina (binder) were tested. The nutritional content, physical characteristics and colour were determined for the extrudates. None of the extrudates met the nutritional content benchmark, which confirms that Moringa-fortified bouillon cubes require supplemental fortificants to provide sufficient levels of micronutrients. The physical characteristics of the extrudates were also examined to provide preliminary insights for future development. Generally, the physical characteristics of extrudates were significantly different when compared to conventional bouillon cubes. Although semolina is commonly used for extrusion and provides structural integrity to extrudates, an alternative binder should be explored to allow fortified bouillon cubes to fit into existing user habits. Chapter 5

Appendix

53 Chapter 5. Appendix 54

Appendix A: Supplemental Data Tables

Table 5.1: Essential amino acids (mg/g protein) in Moringa leaves and pods (literature values) % of Adult Requirement/g protein Methionine Phenylalanine Source Histidine Isoleucine Leucine Lysine Threonine Tryptophan Valine + cysteine + tyrosine WHO Adult Requirements 15 30 59 45 22 38 23 6 39 Leaves India [52] 18 128 63 49 1 3 35 1 87 Ethiopia [83] n.d. 39 76 46 27 57 45 n.d. 50 Mexico [60] 31 40 78 68 6 61 35 n.d. 50 Niger [73] 30 45 87 56 33 100 47 21 57 Nicaragua [68] 23 34 68 41 23 71 30 26 41 USA [63] 21 48 84 57 28 89 44 15 65

Pods Ethiopia [83] n.d. 30 56 32 39 36 33 n.d. 37 Mexico [60] 10 16 29 13 5 14 17 n.d. 22

Table 5.2: Mineral content of extrudates per 3.3g serving or Moringa-fortiﬁed cubes Mineral content (mg/3.3g serving) Blend Ratio (L:P:B) Calcium Magnesium Potassium Sodium Zinc Copper Manganese Iron Lead Selenium 1:00:00 65.8 25.3 7.9 1.3 0.04 0.02 0.11 0.8 0.04 0.04 0:01:00 3.1 5.9 5.8 4.1 0.06 0.01 0.03 0.1 0 0 0:00:01 0.2 2 0.7 0 0.04 0.01 0.04 0.12 0 0 1:01:00 34.5 15.6 6.8 2.7 0.05 0.02 0.07 0.45 0.02 0.02 0:01:01 1.7 4 3.3 2.1 0.05 0.01 0.03 0.11 0 0 1:00:01 33 13.6 4.3 0.7 0.04 0.01 0.08 0.46 0.02 0.02 1:01:01 23 11.1 4.8 1.8 0.05 0.01 0.06 0.34 0.01 0.01 4:01:01 44.4 18.2 6.3 1.6 0.04 0.02 0.09 0.57 0.03 0.02 1:04:01 13.1 8.5 5.3 3 0.05 0.01 0.04 0.22 0.01 0.01 1:01:04 11.6 6.5 2.7 0.9 0.05 0.01 0.05 0.23 0.01 0.01 Chapter 5. Appendix 55

Appendix B: Detailed Experimental Procedures

Ash Determination

Following the recommendations laid out in ASTM E1755.33855

Apparatus

• Ceramic ashing crucibles with lids

• Furnace

• Analytical balance (sensitive to 0.1mg)

Procedure

1. Label crucibles as 1, 2, and 3. Weigh crucibles (without lids) and record as Wc.

2. Weigh 0.5-1g of sample with ashing crucible and record to the nearest 0.1mg. Repeat

twice more for triplicates, tare balance to each crucible’s weight. Record weights as W1 (sample + crucible). Cover crucibles with lids.

3. Place covered crucibles into furnace and heat to 575±25◦C for ﬁve hours.

4. Remove crucibles from furnace, allow to cool for one hour. Weigh crucibles and record as

W2 (ash + crucible).

W1 − WC Ash Content = W2 − WC Chapter 5. Appendix 56

Moisture Determination

Following recommendations laid out in ASTM D4442.12075, Method A, oven-drying.

Apparatus

• Aluminum weighing boats

• Drying oven

• Analytical balance (sensitive to 0.1mg)

Procedure

1. Preheat drying oven at 103±2◦C.

2. Label weighing boats as 1, 2, and 3. Record weights of weighing boats as Wc.

3. Tare balance to weighing boat and weigh out 1-10g of sample. Record weight as W1 (sample + weighing boat). Repeat twice more for triplicates, tare balance to each weighing boat.

4. Place samples into drying oven for 24 hours.

5. Reweigh dried samples and record as W2 (dried sample + weighing boat).

W1 − WC Moisture Content = W2 − WC Chapter 5. Appendix 57

Crude Fibre Determination

Apparatus

• 6 x 500mL round ﬂat-bottomed ﬂasks

• 3 x 50mL coarse fritted glass crucible

• 1000mL Buchner ﬂask

• Flat rubber seal

• Rubber stopper

• Condenser

• Boiling stones

• Heater

• Buchner funnel

• Filter paper (ﬁtted for Buchner funnel)

• 3 x ceramic crucible

• Analytical balance

Reagents

• 1.25 w/w% H2SO4 (1L)

• 1.25 w/w% NaOH (1L)

• Distilled water (2L)

Procedure

1. Weigh fritted glass crucible and round bottom ﬂask and record as WC and WR respectively.

2. Weigh out 2-3g of defatted sample (to remove fat, see Crude Lipid Determination protocol)

into round ﬂat-bottomed ﬂasks. Record mass as W1.

3. Add 200mL of near-boiling 1.25 w/w% H2SO4 into ﬂask and place on heater. Connect with condenser and reﬂux for 30 minutes.

4. Assemble decantation setup with Buchner ﬂask, Buchner funnel and ﬁlter paper. Flow near-boiling water through funnel to warm it and decant liquid through funnel. Turn on water supply to induce vacuum. Chapter 5. Appendix 58

5. Decant acid-digested sample through Buchner funnel. Wash solids from ﬂat-bottomed ﬂask using four portions of 40-50mL of near-boiling water.

6. Transfer filter residue into a clean flat-bottomed flask using near-boiling 1.25 w/w% NaOH. Place on heater, connect with condenser and reflux for 30 minutes.

7. Assemble fritted glass crucible decantation setup with crucible, ﬂat rubber seal and Buch- ner ﬂask. Flow near-boiling water through crucible to warm it.

8. Decant liquid through fritted glass crucible. Wash reside with minimum near-boiling water. Increase vacuum as needed to maintain ﬁltration rate.

9. Wash residue in crucible once with 25-30mL near-boiling 1.25 w/w% H2SO4, and then with two portions of 25-30mL near-boiling water.

◦ 10. Dry crucible with residue overnight at 110 C. Cool in desiccator and weigh (W2).

◦ 11. Ash for two hours at 550 10 C cool in desiccator and weigh (W3).

W2 − W3 Crude Fibre Content = W1 − WR Chapter 5. Appendix 59

Crude Lipid Determination (Soxhlet Method)

Apparatus

• 500ml round ﬂat-bottomed ﬂasks

• Glass stoppers

• Soxhlet Extraction chambers

• Condenser with cooling water

• Thimble

• Beakers

• Boiling stones

• Drying Oven

• Rotavapor with Erlenmeyer vacuum setup

• Metal tongs

• Buchi round collection ﬂask

• Rotavapor collection ﬂask clamp

• Analytical balance

Reagents

• Hexanes

Procedure (triplicates per sample) Lipid Extraction

1. Label three round flat-bottomed flasks with 1, 2, and 3. Place 4-5 boiling stones for each flask. Record weight of each flask with the boiling stones.

2. Add 300 50ml of hexane into each ﬂask in the fumehood. Use glass stoppers to seal the ﬂasks and prevent evaporation of hexane.

3. Add 10 1g of sample into each thimble (1, 2 and 3). To make weighing easier, place at thimble into beaker so that it is upright for adding and weighing out the sample. Record weight of sample. Chapter 5. Appendix 60

4. Fold a piece of qualitative filter paper in half three times (to get eighths), cut a small hole at the tip of the folded filter paper. Fold the filter paper into a cone shape and place at the opening of the thimble. The top (flat edge) of the filter paper cone should be aligned with the top edge of the thimble. Repeat two more times so that all thimbles are fitted with a filter paper cone at the top.

5. Connect extraction chambers with the opening of the round ﬂat-bottomed ﬂasks.

6. Drop the correspondingly numbered thimble into the top of each extraction chamber-ﬂask assembly.

7. Attach each setup to the condenser unit. Use Teflon tape to seal (i) the connection between the condenser and the extraction chamber and; (ii) the connection between the extraction chamber and the round flat-bottomed flasks. The Teflon tape is for leak-detection.

8. Turn on the cooling water. Allow condenser to ﬁll and be circulated with cooling water.

9. Turn on heaters and turn dials to 60◦C. Wait for solutions to boil (10-15 minutes). Ensure that the top caps remain on the condenser unit (place back as soon as possible on if the pressure pushes a cap oﬀ)

10. Allow distillation to run for 24 hours. At the end of the distillation, turn oﬀ heaters and allow to cool for 15-20 minutes. The ﬂasks should be warm at the touch.

11. Remove extraction chambers and round bottom-ﬂasks and place into fumehood. Tip the setup to allow as much hexane as possible to drain through the siphon tubes and into the ﬂasks. Careful not to tip the setup too far and cause hexane to spill.

12. Using metal tongs, remove thimbles from extraction chamber and place in beaker. Remove and dispose of ﬁlter paper cones. Allow hexane to drain into beaker.

13. Disconnect extraction chambers from flasks. Pour remainder of hexane, including that drained from the thumbles, into flasks. Seal opening of flasks with glass stopper. Lay extraction chambers flat in fumehood to vent the small amount of hexane remaining.

14. Turn on drying oven to 100◦C.

Solvent Recovery

1. Turn on rotavapor to 40◦C check that the water level is appropriate. Obtain bottle for recovered solvent.

2. Assemble rotavapor setup by connecting the vacuum Erlenmeyer ﬂask to the water tap and securing the stopper the Erlenmeyer ﬂask. Ensure the vacuum release at the top of the rotavapor condenser is in the closed position. Chapter 5. Appendix 61

3. Connect round collection ﬂask to the outlet of the rotavapor condenser using clamp (turn wheel to tighten). Turn on cooling water to rotavapor condenser.

4. Turn on water tap to induce vacuum. Check that a vacuum is forming by placing a gloved-palm at the inlet of the condenser (where a round ﬂat-bottomed ﬂask would be connected). There should be suction.

5. Attach round flat-bottomed flask containing sample to condenser. Gently pull flask to check that the vacuum formed is sufficient to hold flask in place. If not, either wait for vacuum to form or increase water tap flow rate to increase suction.

6. Using the lever on the left, slowly lower the sample into the water bath (set at 40◦C). Check that (i) the connection between the condenser and the flask does not touch the edge of the water bath; (ii) the sample is sufficiently lowered so that the liquid level within the flask is slowly below that of the water bath and; (iii) the sample is gently boiling (i.e. level of liquid stays within the spherical portion of the flask) and reduce the flow of water for the vacuum water tap to reduce (lower flow) or increase (greater flow) the vacuum.

7. Turn on rotor and set to setting 4. Adjust vacuum and rotor setting according to boiling and liquid level.

8. When ﬂow of hexane into collection ﬂask has slowed, or stopped, turn rotor to setting 6. Allow solvent recovery to continue until no bubbles or foam is present in the sample. The remaining liquid are lipids.

9. Stop rotor and release vacuum by turning off water tap and turning the vacuum release (top of the condenser) to the open position. Keep one hand holding the neck of the sample flask at all times. Remove sample and seal flask with glass stopper.

10. Repeat steps 18-23 for the other two samples. Stop solvent recovery when the collection ﬂask is approximately 75% full. Pour recovered hexane into bottle for recovered solvent (to be done in fumehood then resume solvent recovery.

11. Pour all recovered hexane into bottle for recovered solvent (to be done in fumehood).

12. Disconnect vacuum ﬂask, empty out water collected in the Erlenmeyer ﬂask (if any).

13. Unseal ﬂasks and dry in pre-heated oven for one hour.

14. After drying, allow flasks to cool sufficiently before weighing the flask with the boiling stones and lipids. Calculate oil content using the formula below.

mass of oil Crude Lipid Content = mass of sample before extraction Chapter 5. Appendix 62

Crude Protein Determination (Kjeldahl Method)

Apparatus

• 4 x Buchi 300mL Kjeldahl tubes

• 4 x tube metal clamps

• 4 x tube rubber O-rings

• 2 x tube rack

• Analytical balance

• Nitrogen-free weighing paper

• Kjeldahl digester

• Digestion tubes manifold

• Aspirator connection (black cap and Teﬂon O-ring)

• Glass wool

• Timer

• 4 x conical ﬂasks

• Paraﬁlm

• Kjeldahl tubes tongs

• Kimwipes

Reagents

• Kjeldahl tablets

• Concentrated H2SO4 (100mL)

• 0.1N H2SO4 (30-100mL)

• 4 w/w% H3BO3 (500mL)

• 32 w/w% NaOH (5L)

• Sher indicator (10mL)

• distilled water (5L)

Procedure (triplicates per sample) Solutions Preparation Chapter 5. Appendix 63

1. 1-2 days prior to conducting experiment, check that all reagents are available at suﬃcient amounts. Prepare or purchase solutions as needed.

Digestion

1. Label the Kjeldahl tubes B (blank), 1, 2, and 3. Hold Kjeldahl tubes in rack.

2. Calculate the mass of sample to be tested by determining the expected protein content according to literature values. Back calculate the expected the titration volume so that it is between 3 to 17mL.

3. Fold a piece of weighing paper into a small envelop shape. Drop into Kjeldahl tube for the blank sample (tube labelled ‘B’).

4. Weigh out sample to 0.1mg accuracy according to mass calculated in step 1. Record mass. Fold weighing paper in half, place on analytical balance and tare. Once the desired amount of sample has been weighed out, fold weighing paper to envelop the sample. Drop the folded weighing paper with the sample into a Kjeldahl tube. Repeat for the remaining replicates.

5. Bring Kjeldahl tubes to a fumehood. Add four Kjeldahl tablets and 25mL of concentrated sulphuric acid into each tube. Add Kjeldahl tablets ﬁrst to minimize potential splashing hazards when adding concentrated sulphuric acid.

6. Assemble digestion setup. Put rubber O-rings onto the tube connections, ensuring that the smaller diameter side of the O-ring faces downward. Attach manifold to the four Kjeldahl tubes. Use metal clamps to secure the tubes and manifold together. Check that the tubes are straight and properly connected to the manifold.

7. Move the tubes-manifold digestion setup into the Buchi digester with the threaded side of the manifold facing towards and closest to the water tap. Assemble aspirator connection (black cap with Teﬂon ring). Connect the aspirator to the threaded end of the manifold.

8. Cut a small piece of glass wool big enough to plug the unthreaded end of the manifold. Roll into a small spherical shape and insert into manifold.

9. Turn on water supply to induce vacuum.

10. Turn on digester to setting 4. Digest sample according to the settings and times listed in the table below. Proceed to the next steps while waiting for digestion to complete.

11. Label conical ﬂasks B, 1, 2, and 3. Add 60mL of boric acid into each conical ﬂask.

12. Add four drops of Sher indicator into each flask. Each conical flask must have the same number of drops to give comparable titration results. Cover flasks with parafilm and set aside. Chapter 5. Appendix 64

Setting Time (minutes) 4 20 6 10 10 30 Total 60

13. Upon completion of digestion, remove manifold-tubes setup from digester and place back into a tube rack without turning oﬀ vacuum (water supply) so that the sulphuric acid fumes continue to be removed. (CAUTION: HOT SURFACE. USE HEAT PROTECT- ING GLOVES). The resulting digested solutions should be clear and light green. Allow the remaining fumes to be aspirated for 15-20 minutes.

14. Bring tubes into a fume hood. Allow digested solutions to cool down for 20-30 minutes. While waiting, ﬂush aspirator setup with 2L of tap water.

Distillation

1. Turn on the Buchi distillation unit and cooling water supply to the unit (open tap fully). Set timer to two minutes and wait for a loud beeping sound indicating that the machine is ready to be used. Press ‘Start’ to flush distillation unit. Remove Kjeldahl flask with tongs (CAUTION: HOT SURFACE). Remove and set aside conical flask used for flushing.

2. Remove manifold from tubes. Allow drops of acid (if any present) to drain into tube. Place in sink and soak with Sparkleen detergent.

3. Slowly add 50mL of distilled water into one of the digested samples. It is recommended to start with the blank sample. Gently mix water and digested sample (mostly acid) in a swirling motion. Turn tubes towards the back of fumehood when adding water as a safety precaution for potential liquid level rises due to violent reactions.

4. Attach Kjeldahl tube to distillation unit. Place corresponding conical ﬂask with its contents at the collection end.

5. Adjust timer on distillation unit to ﬁve minutes. Record start time in distillation unit log book.

6. Press ‘Reagent’ to ﬁll Kjeldahl tube with 32% sodium hydroxide until sample solution turns basic (opaque brown). Press ‘Start’ to begin distillation. While waiting for distillation to complete, repeat step 18 (addition of distilled water).

7. Remove Kjeldahl tube and collection flask after distillation is complete (loud beeping sound). Rinse tubing on both ends (tube and conical flask) into the tube/flask. Clean up any spills immediately by flushing the area with distilled water and wiping dry with Kimwipes. Chapter 5. Appendix 65

8. Repeat steps 19-22 for all four samples.

9. Using blank sample (conical ﬂask labelled ‘B’) and a standard, titrate samples with 0.1N sulphuric acid. Record titration volume.

g −3 mol (titration vol, in mL)(14 )(0.1 H2SO4) %N = mol N L mass of sample, in g

Protein content = %N ∗ conversion factor Chapter 5. Appendix 66

Mineral Determination

Apparatus

• Gloves

• Beaker

• Disposable pipette(s)

• 50ml Falcon tubes

• Spatula(s)

• Weighing paper

• Weighing balance (to 0.1mg accuracy)

• MARS6 Microwave Digester

• MARSXpress microwave digestion vessels (liner, plug and cap)

• MARSXpress Torque Tool

Reagents

• HNO3

• QC4 (ICP multi-element standard, sold by ANALEST in the Department of Chemistry, UofT)

Procedure

1. Weigh out 0.2-0.5g of sample onto weighing paper to 0.1mg accuracy. Record mass.

2. Transfer sample into microwave digestion liner (MARSXpress, 75ml). Reweigh weighing paper to get the remaining mass of sample on paper. Record remainder weight and discard weighing paper.

3. Add 10ml nitric acid into liner. Try to wash down portions of the sample that are on the inner walls of the liner.

4. Seal microwave digestion liner with plug and cap. Hand tighten cap and then use the torque tool to ensure the correct torque has been applied. NOTE: correct torque ensures that the vessel vents properly during digestion.

5. Repeat steps 1-3 for all samples. Chapter 5. Appendix 67

6. Load sealed vessels onto microwave digestion carousel/turnable according to the recom- mend distribution by the manufacturer. Ensure the distribution of vessel is balanced. Fill and seal vessels with 10ml nitric acid as counter balance if needed.

7. Load carousel into microwave digester.

8. Select the preset ‘Food’ method under ‘One Touch Methods’. Press start.

9. Once digestion is complete, remove liners one at a time from the carousel. Remove the cap using the torque tool. Do this in a fumehood as there will be fumes.

10. Transfer digested sample into a 50ml falcon tube. Rinse the plug and inside of the liner with deionized water. Careful to keep the total solution volume below 25ml.

11. Make up digested samples to 25ml in volumetric ﬂasks. Filter digested samples through 0.45-micron microﬁlters. Keep digested samples for a maximum of one week. Dispose of sample accordingly after experiments.

12. Dilute digested samples according expected literature values so that concentrations fall between 0.01ppm and 100ppm.

13. Prepare calibration solutions using QC4 at 0ppm (distilled water), 0.01ppm, 0.1ppm, 1ppm, 10ppm and 100ppm.

14. Analyze samples according operating manual for ICP-AES. Bibliography

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