Micronutrients in Moringa oleifera and their Potential in Food ......2.2 Iron content comparisons in...

Micronutrients in Moringa oleifera and their Potential in FoodFortification

by

Yee Kei Kiki Chan

A thesis submitted in conformity with the requirementsfor the degree of Master of Applied Science

Department of Chemical Engineering and Applied ChemistryUniversity of Toronto

c© Copyright 2018 by Yee Kei Kiki Chan

Abstract

Micronutrients in Moringa oleifera and their Potential in Food Fortification

Yee Kei Kiki ChanMaster of Applied Science

Chemical Engineering and Applied ChemistryUniversity 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 applica-

ble 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.

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To gung gung(1924-2016)

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Acknowledgements

I would like to express my gratitude towards my co-advisors, Professor Levente Diosady andProfessor Yu-Ling Cheng. Thank you for mentoring me, for teaching me effective ways to frameand approach research problems (I am still working on this), and for the incredible opportuni-ties 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 helpfulinsights 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 teachingme to be grateful and to count my blessings. Thank you for taking care of everything else so Icould 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 forkeeping 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 allmy works are empty.

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

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Contents

Dedication iii

Acknowledgements iv

Contents v

List of Tables vii

List of Figures viii

Acronyms x

1 Background 11.1 Global Food Insecurity and Micronutrient Deficiencies . . . . . . . . . . . . . . . 11.2 Nutritional Content and Requirement Standards . . . . . . . . . . . . . . . . . . 31.3 Food Fortification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.3.1 Existing Fortification Interventions . . . . . . . . . . . . . . . . . . . . . . 41.3.2 Fortificants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.4 Moringa oleifera as a Natural Food Fortificant . . . . . . . . . . . . . . . . . . . 61.5 Scope of Thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.6 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2 Moringa oleifera Nutritional Characterization 102.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.2.1 Materials and Sample Preparation . . . . . . . . . . . . . . . . . . . . . . 112.2.2 Proximate Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.2.3 Mineral Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.2.4 Vitamin Content and Protein Quality . . . . . . . . . . . . . . . . . . . . 12

2.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.3.1 Proximate Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.3.2 Mineral Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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2.3.3 Vitamin content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.3.4 Protein Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

3 Addressing Micronutrient Deficiencies using Moringa oleifera 263.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263.2 Existing Fortification Interventions using Moringa oleifera . . . . . . . . . . . . . 26

3.2.1 Yogurt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.2.2 Bread . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.2.3 Cookies and Extruded Snacks . . . . . . . . . . . . . . . . . . . . . . . . . 273.2.4 Region-specific Dishes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.2.5 Raw Meat and Fruit Juices . . . . . . . . . . . . . . . . . . . . . . . . . . 29

3.3 Overcoming Technical Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.4 Food Vehicle Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

3.4.1 Consumption Coverage . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.4.2 Shelf-life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.4.3 Ease of Adding Other Micronutrients . . . . . . . . . . . . . . . . . . . . . 333.4.4 Public Health Concern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

3.5 Proof of concept for Moringa-fortified bouillon cubes . . . . . . . . . . . . . . . . 333.5.1 Commercially Available Bouillon Cubes . . . . . . . . . . . . . . . . . . . 343.5.2 Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353.5.3 Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373.5.4 Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383.5.5 Characterization Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 413.5.6 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

4 Future Work and Conclusions 484.1 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

4.1.1 Taste, Aroma and Colouring Compounds . . . . . . . . . . . . . . . . . . 484.1.2 Encapsulation and Coating Processes . . . . . . . . . . . . . . . . . . . . 494.1.3 Alternative Binders and Excipients . . . . . . . . . . . . . . . . . . . . . . 494.1.4 Supplemental Fortificants . . . . . . . . . . . . . . . . . . . . . . . . . . . 494.1.5 Manufacturing Process Selection and Optimization . . . . . . . . . . . . . 504.1.6 Consumption Patterns and Consumer Preferences . . . . . . . . . . . . . 50

4.2 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

5 Appendix 53

Bibliography 68

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List of Tables

1.1 Common micronutrient deficiency symptoms and affected vulnerable populationgroups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2 Nutritional content standards, RDA and NRV . . . . . . . . . . . . . . . . . . . . 31.3 Production yields and harvest periods for perennial and annual Moringa oleifera 7

2.1 Moringa samples for experimental analyses . . . . . . . . . . . . . . . . . . . . . 112.2 Proximate compositions of Moringa leaves and pods. Rows marked dashes (-) in

the reference column were experimentally determined in this project. . . . . . . . 132.3 Moisture content, dry mass and protein content in Moringa pods skin and flesh . 132.4 Dietary Reference Intakes for select minerals (19-50 years old) [1] . . . . . . . . . 152.5 Mineral content in Moringa leaves and pods. Rows marked with dashes (-) in

the reference column were measured in this project. . . . . . . . . . . . . . . . . . 152.6 RDA for select vitamins (19-50 years old) [2] . . . . . . . . . . . . . . . . . . . . 232.7 Vitamin content in Moringa leaves and pods (literature values) . . . . . . . . . . 232.8 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 . . . . . . . . . . . . . . . 313.2 Comparison of NRV and RDA values for minerals, vitamins and protein . . . . . 353.3 Physical specifications for fortified bouillon cubes . . . . . . . . . . . . . . . . . . 363.4 In-barrel moisture content in screening experiments . . . . . . . . . . . . . . . . . 403.5 In-barrel moisture for mixture design formulations . . . . . . . . . . . . . . . . . 403.6 %NRV of iron, zinc, vitamin A, folate and protein per 3.3g serving . . . . . . . . 423.7 Mass, hardness, water activity and disintegration results for extruded cubes . . . 443.8 Hunter L*ab values for extruded cubes . . . . . . . . . . . . . . . . . . . . . . . . 46

5.1 Essential amino acids (mg/g protein) in Moringa leaves and pods (literature values) 545.2 Mineral content of extrudates per 3.3g serving or Moringa-fortified cubes . . . . 54

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List of Figures

2.1 Protein content in Moringa leaf and pod samples measured in this study . . . . . 142.2 Iron content comparisons in Moringa leaf samples. Samples marked with aster-

isks (*) were experimentally determined in this study. Dashed lines indicate theRDA 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 experimen-tally determined in this study. Dashed lines indicate the minimum RDA or AIfor individuals between 19 and 50 years old. . . . . . . . . . . . . . . . . . . . . . 17

2.4 Mineral content comparisons in Moringa leaf samples (zinc, copper and man-ganese). Samples marked with asterisks (*) were experimentally determined inthis study. Dashed lines indicate the minimum RDA for individuals between 19and 50 years old. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.5 Iron content comparisons in Moringa pod samples. Samples marked with aster-isks (*) were experimentally determined in this study. Dashed lines indicate theRDA 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 experimen-tally determined in this study. Dashed lines indicate the minimum RDA or AIfor individuals between 19 and 50 years old. . . . . . . . . . . . . . . . . . . . . . 20

2.7 Mineral content comparisons in Moringa pod samples (zinc, copper and man-ganese). Samples marked with asterisks (*) were experimentally determined inthis study. Dashed lines indicate the minimum RDA for individuals between 19and 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 . . . . . . . . . . . . . . . 343.2 Simple axial mixture experimental design for proof of concept . . . . . . . . . . . 373.3 Block flow diagram for forming Moringa-fortified bouillon cubes . . . . . . . . . . 383.4 (left) Cold extrusion setup. Die attached to single Archimedes screw attachment;

(right) Stainless steel extrusion die with 20*20mm square opening . . . . . . . . 39

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3.5 Iron content in mixture design formulations. Increasing bubble sizes correspondto 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 toincreasing hardness; numbers correspond to mean hardness in newtons. Therange 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

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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

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Chapter 1

Background

This chapter presents an overview of global micronutrient deficiencies and approaches thathave been investigated or implemented to address this global challenge. The background pro-vided on Moringa oleifera and its potential in addressing micronutrient deficiencies leads to themotivation 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 betweenfood and well-being deepened. Up until the 1970s, food security referred to a nation’s abilityto 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 theUnited Nations (FAO) has defined food security as “A situation that exists when all people, atall times, have physical, social and economic access to sufficient, safe and nutritious food thatmeets their dietary needs and food preferences for an active and healthy life” [5]. The inclusivityof this updated definition allows food insecurity to be examined at all stakeholder levels—fromindividuals to communities to economies—and as a multifaceted global challenge. The explicitreference for adequate nutrition points directly to the need to address micronutrient deficiencies,commonly called “hidden hunger”, which affects both developing and developed nations, albeitunequally [6]. The importance of addressing global micronutrient deficiencies is also highlightedby the United Nations in Sustainable Development Goal (SDG) #2: End hunger, achieve foodsecurity and improved nutrition and promote sustainable agriculture [7].

In this thesis, food security is viewed as a prerequisite for nutrition security. One of the maindeterminants for nutrition security is adequate micronutrient intake. Although it is possiblethat nutrition security could exist without food security (e.g. taking micronutrient supplementswithout adequate caloric intake), such cases are rare in low and middle income countries; it ismore typical that households would need to be able to satisfy caloric requirements before beingable to consider the nutrition within the foods consumed. The relation between food securityand nutrition security continues to be debated in literature [8]. Nevertheless, differing opinions

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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 diseaselead to the inability to work and earn wages for survival [9]. Although significant improvementshave been observed for caloric deficiencies since the year 2000, micronutrient deficiencies re-main prominent and widespread [5]. A conservative estimate is that symptomatic micronutrientdeficiencies affects two billion people globally, as asymptomatic cases of mild deficiencies aredifficult 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 andSouth Asian regions [5, 12].

Deficiencies in iron, vitamin A, iodine, folate, zinc, and vitamin B12 [13, 14] are mostprevalent. Symptoms, signs and complications of micronutrient deficiencies are most prominentin pregnant women in whom reproductive health and fetus development is impeded, and inchildren in whom growth retardation is observed. Table 1.1 summarizes the impacts of commonmicronutrient deficiencies and demonstrates the importance of adequate micronutrient intakefor 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 theintake of micronutrients: diversification, supplementation and fortification [10]. Public healthorganizations mandate and implement combinations of these strategies in parallel to form anintegrated approach for addressing micronutrient deficiencies. Those living in low and middleincome countries are most affected by food insecurity since poorer regions experience foodshortages, and are unable to balance micronutrient intakes through diet diversification [6, 18].

Table 1.1: Common micronutrient deficiency symptoms and affected vulnerable populationgroupsMicronutrient Essential for functioning of Symptoms Indicating Moderate to Severe Deficiency Most Affected Population Groups

Iodine Thyroid hormonesGoiter

Mental retardationPregnancy complications

Pregnant womenNewborns and infants

Vegetarians

Iron Hemoglobin and myoglobinChronic fatigue

Heart failurePica

Women of child-bearing agesChildren

Vegetarians

Vitamin A Eyes and immune systemBlindness

Stunted growthFrequent infections

NewbornsPregnant women

Children

Folate Amino acid synthesisChronic fatigue

Neural tube defectsStunted growth

Pregnant and lactating womenNewborns and infants

People with alcoholic dependence

Zinc Immune system and amino acid synthesisFrequent infections

Stunted growthLoss of appetite

Gastrointestinal disease patientsVegetarians

Pregnant and lactating women

Vitamin B12 Amino acid synthesisChronic fatigue

Heart failureNumbness in limbs

ElderlyPregnant and lactating women

Vegetarians


1.2 Nutritional Content and Requirement Standards

Nomenclature for the reference values for nutritional requirements and benchmarks differ de-pending on the region and application. Two different types of reference values are used inthis thesis: Recommended Dietary Allowance (RDA) and Nutrient Reference Value (NRV).In general, RDA is used by health professionals, such as dieticians, to assess the nutritionalrequirements of an individual; whereas NRV is used to benchmark the level of micronutrientsin a food product (Table 1.2). Accordingly, Chapter 2 (Moringa oleifera Nutritional Charac-terization) of this thesis will reference RDA values, and Chapter 3 (Addressing MicronutrientDeficiencies using Moringa oleifera) of this thesis will reference NRV for benchmarking.

Table 1.2: Nutritional content standards, RDA and NRVRecommended Dietary Allowance

(RDA)Nutrient Reference Value

(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 populationValue Format mg/d; values split into different population groups according to age and sex mg/d; single value for entire populationApplication 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 per-formed on a voluntary basis. Mandatory food fortification programs are implemented in regionsto address micronutrient deficiencies of public health concern, whereas voluntary fortificationis often performed so that a health claim may be made for marketing purposes. Food fortifi-cation programs are proven to be effective for reducing the prevalence of several micronutrientdeficiencies 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 andcondiments are widely consumed by significant portions of the population, so high householdcoverage can be achieved. Second, staple foods and condiments that are centrally produced by asmall number of large manufacturers allow the quality and level of fortification to be controlledand monitored. Finally, minimal behavioural changes in dietary habits are required as long asthere are no changes in organoleptic properties of fortified foods is detectable to the consumer.

Significant challenges associated with food fortification are: determining the appropriatefortification level, cost, stability of fortificants—particularly due to possible interactions amongmultiple fortificants, and maintaining organoleptic properties. Considering the varying micronu-trient requirements within a population and even within a single household, it is challengingto determine the amount of fortificant for a food vehicle that is appropriate for all potentialconsumers [20, 21]. If fortification levels are set too low, the fortified food will have little tono observable effect on reducing the targeted micronutrient deficiencies. If fortification levels


are set too high, the excessive micronutrient intake may become toxic and detrimental to anindividual’s health. Although fortification is considered a cost-effective intervention strategy,additional processing still inevitably increases the cost of a food product. This is especiallyburdensome for the poorest of the poor, who are extremely price sensitive and are likely to buylower quality, inadequately fortified versions of the same food [10, 21]. In the development offood vehicles fortified with multiple micronutrients, undesirable interactions leading to reducedbioavailability and stability are common technical challenges. Similarly, maintaining the orig-inal organoleptic properties are also challenging due to interactions between the food vehicleand 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. Thelevel of fortified foods is likely to be higher and more consistent when fortification is implementedfor food vehicles with a few key manufacturers as opposed to food with a great number of smallerscale manufacturers [22]. The level of food fortification is also more consistent in a mandatoryenvironment where appropriate monitoring systems are in place [23, 24]. Additionally, largemanufacturers are more likely able to reach economies of scale, and to contain additional costsassociated 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 goiteroccurrences in Switzerland and the United States [25, 26]. Salt is considered an ideal foodvehicle for fortification as it is universally consumed with minimal variations in consumptionlevels amongst households of different socioeconomic statuses [27, 28]. Salt iodization is thepreferred method for controlling iodine deficiency disorders and there has been ongoing researchto fortify salt with other micronutrients [27].

Implementation trials of double fortified salt with iodine and iron has demonstrated promis-ing results in reducing iron deficiency anemia in low and middle-income countries [29, 30]. Toproduce double fortified salt, a premix of ferrous particles are agglomerated with the aid ofbinders in cold extrusion, encapsulated, and blended with iodized salt [31, 32]. Encapsulationprovides both a colour-masking layer that covers the red-brown colour of the ferrous particlesand 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 productionprocesses [34]. A variety of projects are ongoing to fortify salt with combinations of folic acid,B vitamins and zinc [35, 36].


Sugar Fortification

The most common micronutrient added to sugar is vitamin A, and fortification has been imple-mented in numerous Central American countries [37]. Cold water soluble vitamin A palmitatebeadlets adhere to the surface of sugar crystals and are coated with a vegetable oil-antioxidantmixture 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 vitaminA 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 semiruralGuatemalan populations [40], but no industrial feasibility studies have been published at thistime.

Flour Fortification

A staple food for many nations, flour is widely commercially fortified with an extensive rangeof micronutrients added at varying levels [41]. WHO provides guidelines on the fortificationof flour with iron, folic acid, vitamin B12, vitamin A and zinc [42]. In many countries, flouris enriched with B vitamins to compensate for micronutrient losses during the flour millingprocess. A dry powder premix of micronutrients is added after the milling step and blendedwith 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 havebeen marked successes in the fortification of oil with vitamin A. Oil and vitamin A can be sim-ply blended together without changing processing conditions, thus requiring minimal additionalcosts and technical expertise [10]. Oxidative degradation reduces the stability of vitamin A inoil, which could be countered by addition of antioxidants [44, 45]. Addition of minerals to oilshas 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 cookedrice differ significantly between cultures. This poses unique challenges in the standardizationof fortification processes [10]. WHO recommends the fortification of iron, vitamin A and folic


acid into rice [47]. In practice, rice is often enriched with B vitamins as they are lost duringrice milling processes that remove the outer germ and bran layers.

In rice fortification, fortified kernels are formed using coating or extrusion processes whichare 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 vehiclesis well studied. Pure micronutrients are used (with a food vehicle) to form a fortified premixwith a high concentration of fortificant. The premix is then blended with non-fortified versionof the food vehicle to give the appropriate level of fortification. A major advantage of usingsynthesized fortificants is greater control over the level of fortification, provided that the mi-cronutrient compounds are of high purity and the fortification process is designed and operatedappropriately.

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 compoundssuch 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, dehydrationand blanching may be used to form “packages” containing micronutrients that could be used toenrich food vehicles [48–51]. These “packages” are natural fortificants. In addition to increasingthe nutritional value of food vehicles, the components within the matrix of natural fortificantsmay improve storage stability. Natural fortificants allow the micronutrients in plants to bereadily available year-round and reduce wastage from food spoilage. Ranawana et al. [49] showedthat the fortification of bread using freeze-dried vegetable powders increased the nutritionalvalue and enhanced the storage stability of bread samples. Similarly, Duthie et al. [50] alsoreported an increase in oxidative stability of meats with the addition of vegetable powders. Onthe other hand, Joshi and Mathur [51] demonstrated that the sensory properties of Westernand Central Indian dishes remained acceptable when 10% w/w of vegetable leaf powders wereadded 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 regionsand is one of 13 species classified under the Moringaceae family [52]. In this thesis, Moringaoleifera is referred to as ‘Moringa’. The time period from seed sowing to the harvest of Moringafruits is six months, which is relatively short. Moringa fruits are also pods containing seedsand are typically 20-75cm long and weigh 90-150g, while its leaves 25-45cm long and are madeoblique leaflets of 1cm in length. Moringa trees are also known as “drumstick trees” with


reference to the pods’ resemblance to the musical instrument. Moringa is a multifunctionalplant 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 thelargest producer of Moringa and cultivates trees spread over 38,000 hectares with an annualproduction of Moringa pods of 1.1-1.3 million tonnes [57]. The production of Moringa leavesvary greatly depending on cultivation conditions (e.g. tree spacing, weather, and varietal) andyields up to 650 tonnes per hectare are found in literature [58]. Perennial Moringa trees requireclose to one year of growth before first harvest and produce fewer pods in the first two years ofcultivation compared to annual Moringa trees (Table 1.3). Harvesting periods of Moringa areconstrained by environmental factors such as monsoons, which could cause flowering Moringatrees to shed their blooms. A study conducted by the Tamil Nadu Agricultural Universityshowed that Moringa trees purposefully grown to yield harvest during conventional off-seasonsproduced noticeably smaller and fewer pods [58].

Table 1.3: Production yields and harvest periods for perennial and annual Moringa oleiferaPerennials Annuals

Production Yield(pods/year/tree)

First two years: 80-90Year 4-5: 500-600 250-400

Harvest period(s)(in tropical climates)

March-June (primary)September-October

six months after seed sowing;2-3 months of harvest

Moringa leaves are reported to be high in nutritional content [59] but have distinct astrin-gent and grassy flavours, while immature Moringa pods are comparatively lower in nutritionalcontent [60] and have mild sweet and crisp flavours likened to asparagus. Moringa leaves ispresented 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 Moringaleaves, ranging from high to very high nutrition [52, 59–63]. Although variations are expected inbiological materials, the inconsistent values reported for Moringa on its micronutrient contentlead to uncertainties in the nutritional value of Moringa. Furthermore, despite growing interestfrom health and agriculture communities on Moringa few regions currently include it as a reg-ular food source. It is speculated that the astringent taste of Moringa leaves is not generallypleasing and Moringa pods are less nutritious and require more effort to eat due its fibrousouter skin. These challenges suggest that there are opportunities in increasing the consumptionof 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 campaignshave been launched to introduce Moringa as part of a diversification strategy. However, theinclusion of new foods into people’s diets is often slow and challenging since it requires substan-tial behaviour change. The diversification approach also does not address the key limitation ofthe undesirable astringent or bitter taste in Moringa leaves. Consumption of fresh leaves and


pods is limited due to their high moisture content, which also limit the micronutrient intakesfrom the vegetables. Although Moringa trees may be easily grown in the tropical belt, thepoor are often unable to grow Moringa for their own consumption as they are restricted tosmaller living spaces. Moreover, the availability of fresh Moringa is seasonal and constrainedby harvest yields. In India, seasonal availability causes the price of Moringa pods to rangefrom Rs.5/kg during peak harvest season (March to August) to Rs.15-20/kg (September toOctober) to Rs.60/kg during off-season (November to February) [58]. According to the lowermiddle-income International Poverty Line of Rs.57/day (PPP$3.20 with 2017 PPP conversionfactor), Moringa is unaffordable to the poor for at least four months of the year [64, 65]. Thepoor often are the most likely to be affected by food insecurity and the highly variable pricingof 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, whichonly extends the vegetable’s shelf-life to two weeks. Cold storage is usually unavailable to thepoorest or lowest socioeconomic segments of a population.

The fortification approach was considered as an alternative way to promote the utilizationand consumption of Moringa and its nutrients while minimizing behavioural changes in people’sdietary habits. Processing options to exclude undesirable organoleptic properties could beexplored, so that changes to the food vehicle are undetectable. Processing Moringa into anatural fortificant will concentrate and preserve its micronutrients so that a greater amountmay be consumed at each meal. The removal of moisture will preserve Moringa’s nutrients sothat they are available year-round without the need for cold storage and reduce post-harvestfood losses during peak harvest seasons.

1.5 Scope of Thesis

Several priorities were identified for this project. It was hypothesized incorporating Moringa asa natural fortificant would increase the nutritional value of the selected food vehicle and sup-plement the micronutrient requirements of undernourished individuals. To test this hypothesis,an understanding of the nutritional content of Moringa was required. Due to the broad rangeof values reported on Moringa’s nutritional content [52, 59, 60, 63], the extent of Moringa’snutritive 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 foodfortificant could be evaluated, and the viability of including Moringa as a natural fortificantexamined.

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?


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 NutritionalCharacterization

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

2.1 Introduction

A key question in this dissertation is Moringa’s potential for addressing food insecurity as anatural food fortificant. Several studies have suggested that Moringa contains considerablelevels of several micronutrients [61, 66, 67]. However, there are inconsistencies in the reportednutritional content in Moringa leaves and limited literature data are available on the nutritionalcontent 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 analyzedfor 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, thecrude protein content and mineral content in the skin and flesh components of Moringa podswere determined. Measured values were compared with data available in literature. The vitamincontent and protein quality in Moringa leaves and pods were not experimentally measured forthis 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 Moringaleaf samples from Chennai and Coimbatore and fresh pod samples from Delhi were courieredfrom their place of origin. Fresh samples were freeze-dried, ground into powder and sievedthrough 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

Region Samples obtained Acquired from Time elapsed before cold storage(for fresh samples only)

Chennai, India Fresh leaves Cultivator (direct) ∼24 hoursCoimbatore, India Fresh leaves and pods Cultivator (direct) 30 hours

Delhi, India Leaf powder;Fresh pods

Leaves: Costco.ca;Pods: Vegetable market in Delhi

Leaves: N/A;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 onraw 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 separatingthe skin and flesh of Moringa pods is that it does not perfectly mirror the way the vegetable isconsumed in existing eating and cooking habits. For example, in South Indian cuisine, Moringapods are cooked whole and eaten in a similar way to artichokes: small whole sections of thepods are chewed to extract the flesh and the skin is expelled after thorough mastication. Theseparation method used for this thesis does not take into account the extraction of nutrientsfrom the skin of Moringa pods from chewing. This separation method also does not take intoaccount the leaching of nutrients into the liquids during cooking. Variabilities in masticationand cooking are difficult to control, and the separation method described in this thesis was usedto 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). Crudeprotein was determined using Kjeldahl analysis with a protein conversion factor of 6.25. Crudelipid 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 wasdetermined using a muffler furnace at 575±25◦C (ASTM 1755-01). Non-fibre carbohydrateswere 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 reportedfor comparison.


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 detectableranges, and analyzed using ICP-AES. The minerals analyzed were calcium, magnesium, potas-sium, sodium, zinc, copper, manganese and iron. These minerals were analyzed based on theirnutritional importance.

Dried spinach was chosen as a benchmark for comparison as it has reasonable amounts ofthe analyzed minerals. The mineral content of dried spinach was calculated by using standardreference 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 50years 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 availablein literature. Protein quality was determined using the essential amino acid requirements foradults according to WHO [69]. Levels of vitamin A, B1, B6, folate (B9), B12 and C reportedin literature were examined. Standard reference values of foods that are considered sourcesof these vitamins were used to illustrate the relative levels of each vitamin in Moringa. Themaximum RDA value for adults between 19 and 50 years old recommended by the NationalInstitutes 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 higherthan in (whole) pods, which aligned with literature expectations of higher mineral content inMoringa leaves, which is further discussed in subsection 2.3.2. Whole Moringa pods containa greater percentage of crude fibre than leaves, as expected, due to the presence of the outerprotecting skin layer.

The measured values for crude lipid content in leaves were noticeably higher than litera-ture values. The most likely explanation for the discrepancies is natural variation between thesamples used for this project and in other studies. Possible confounding results due to experi-mental errors—extraction of chlorophyll and residual solvent—were ruled out as the chlorophyllcontent in Moringa leaves is lower than 1% w/w [70] and the mass of extracted oil remainedconstant after prolonged solvent evaporation (72 hours). The difference between measured andliterature values for crude lipid content in Moringa pod samples were considered reasonable asit could be attributed to natural variations [71, 72].


Table 2.2: Proximate compositions of Moringa leaves and pods. Rows marked dashes (-) in thereference column were experimentally determined in this project.

Composition (dry basis %)Source Ref. Moisture Crude Protein Crude Lipid Ash Crude Fibre Non-fibre carbohydratesLeavesIndia [52] 75 26.8 6.8 9.2 3.6 53.6

Mexico [60] N/A 22.42 4.96 14.6 30.97 27.05Niger [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 40.72Tanzania [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 29.43Chennai, 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

PodsIndia [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 24.98Tanzania [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 45.17Kerala, India - 87.4±0.23 14.7±0.23 5.3±0.27 5.3±0.55 29.5±0.27 45.2ˆInsufficient 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 inpowder 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. Theprotein content in Moringa leaves and pods was found to range from 21% to 33% and 15 to19% 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, theexisting cooking and eating habits for Moringa pods could result in the loss of up to half of theprotein content in Moringa pods.

Table 2.3: Moisture content, dry mass and protein content in Moringa pods skin and fleshCoimbatore, India Delhi, India

Number of whole pods analyzed 3 4Moisture content (%)Skin 80.4±1.96 N/AFlesh 88.2±1.93 N/A

Dry Mass (g)Skin 4.95±0.48 5.01±1.13Flesh 4.15±0.59 4.45±1.30

Protein content (%)Skin 17.3±3.71 14.0±1.47Flesh 21.6±3.69 16.3±1.41

% of total protein contentSkin 48.8 49.5Flesh 51.2 50.5


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 ob-tained through literature review and determined experimentally within this study. The bench-marks (dashed lines) indicate the minimum RDA or AI value for individuals between 19-50years old (Table 2.4).

Moringa leaf samples from Chennai (India), Coimbatore (India) and Ibadan (Nigeria) wereexperimentally analyzed in this study. Overall, the mineral content values that measured exper-imentally in this study matched expectations from literature considering the effects of naturalvariations (Table 2.5 and Figure 2.2). The iron content in Moringa leaves showed particularlylarge variability ranging from 8.3 to 110mg/100g. Moringa leaves are presumed to be high iniron 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 contentin 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 leavesmay be a good source of iron (Table 2.5), the analysis indicated that the iron levels are in thesame order of magnitude as spinach.

A possible explanation for this inconsistency and differing conclusions may be that com-parisons were made between values reported for inequivalent moisture content. The reportediron 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 inTable 2.5, Moringa leaves would appear to have 18 times more iron than spinach—similar tothe reported 25 times difference.

Values for magnesium, sodium, copper and manganese were consistent amongst all samples.


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 IronRDA/AI RDA RDA AI AI RDA RDA RDA RDA

Males 1000 400-420 4700 1500 11 0.9 2.3 8Females* 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 thereference column were measured in this project.

Mineral content (mg/100g)Source Ref. Calcium Magnesium Potassium Sodium Zinc Copper Manganese IronLeavesNigeria [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+01South 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+01USA [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+01Coimbatore, 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

PodsIndia [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+01USA [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+01Coimbatore, 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 thenutrients available in the soil. Moringa leaves appear to have substantially lower amounts ofmagnesium, potassium, sodium, zinc and manganese compared to dried spinach leaves. Moringaleaves are low in sodium, potassium and zinc as the mineral content levels are below AdequateIntake (AI) and RDA thresholds (Figure 2.3 and Figure 2.4). Select samples of Moringa leavescontain 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 com-pared to Moringa leaves. Fewer sources reported on the mineral content of Moringa pods, solimited comparisons could be drawn between literature values and values determined exper-imentally 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. TwoMoringa pod samples (Pakistan [79] and Kerala) had iron content levels over the RDA thresholdfor adult males.

The values measured by this study for calcium were consistent with literature values. A


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 individualsbetween 19 and 50 years old.


Figure 2.3: Mineral content comparisons in Moringa leaf samples (calcium, magnesium, potas-sium and sodium). Samples marked with asterisks (*) were experimentally determined in thisstudy. Dashed lines indicate the minimum RDA or AI for individuals between 19 and 50 yearsold.


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 linesindicate the minimum RDA for individuals between 19 and 50 years old.


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 individualsbetween 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 wereconsistent with each other. Moringa pods contain lower amounts of magnesium, zinc andpotassium 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 UnitedStates are approximately 10 times higher than that in samples from India (Figure 2.6). Thesedifferences in mineral content are influenced by a wide array of variables including plant varietaltype, climate, soil nutrients and rainfall.

The skin of Moringa pod samples contained 34-69% of the total minerals within the wholepods (Figure 2.8). The distribution of minerals in the skin and flesh of Moringa pods werevariable in the two samples tested. Figure 2.8 shows that discarding the skin of Moringa podswould result in a loss of at least a third of the total minerals and further reduce the nutritionalvalue Moringa pods.


Figure 2.6: Mineral content comparisons in Moringa pod samples (calcium, magnesium, potas-sium and sodium). Samples marked with asterisks (*) were experimentally determined in thisstudy. Dashed lines indicate the minimum RDA or AI for individuals between 19 and 50 yearsold.


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 linesindicate the minimum RDA for individuals between 19 and 50 years old.


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 thevitamin content of Moringa leaves and pods varied greatly (Table 2.7), and it was not alwaysclear if the values presented were on a wet or dry basis. However, since standard analyticalprocedures for vitamins are based on fresh material, it was assumed that the literature valueswere applicable for fresh Moringa leaves and pods. RDAs for the vitamins examined are shownin 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 vitaminA. Moringa leaves reportedly contain more vitamin A than carrots [78], but standard referencevalues showed that fresh carrots contain 835µg RAE/100g and dehydrated carrots contain3423µg RAE/100g [63], both of which are higher than four out of five of the reported vitaminA literature values for Moringa leaves.

A limited number of literature sources reported on the amount of B vitamins in Moringaleaves and pods. Two literature values were found on the level of vitamin B1 and one literaturevalue was found on the amount of folate (vitamin B9) and vitamin B12 in Moringa leaves andpods. For vitamin B1, literature values ranged from 5-22% of the RDA in Moringa leaveswhile Moringa pods were reported to have 4% of the RDA in 100g. The sole literature valuefound on the amount of vitamin B6 indicated that 0.1g of Moringa leaves had sufficient levels


of the vitamin to fulfill the RDA for an adult, whereas Moringa pods contained low amountsof the vitamin and would require 1kg to satisfy the same RDA [63]. Literature values forMoringa leaves and pods suggested that both contained approximately 10% of the RDA forfolate (vitamin B9) per 100g of material. The amount of folate in Moringa leaves and pods issubstantially lower than that of spinach, which contains 194µg of folate for an equivalent massof material. Neither Moringa leaves or pods contain vitamin B12, which matched expectationsas 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 Moringaleaves and 72-157% in Moringa pods for 100g of the material. Moringa pods appear to containmore vitamin C than Moringa leaves on average. Notably, comparisons for vitamin C contentbetween Moringa and kiwifruit (92.7mg/100g or 103% of the RDA) shows that Moringa containsa marked amount of vitamin C.

Table 2.6: RDA for select vitamins (19-50 years old) [2]

Source Vitamin A(µg RAE/day)

Vitamin B1(mg/day)

Vitamin B6(mg/day)

Vitamin B9(µg DFE/day)

Vitamin B12(µg/day)

Vitamin C(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)

Source Ref. Vitamin A(µg RAE/100g)

Vitamin B1(mg/100g)

Vitamin B6(mg/100g)

Vitamin B9(µg DFE/100g)

Vitamin B12(µg/100g)

Vitamin C(mg/100g)

LeavesIndia [52] 565 0.06 N/A N/A N/A 220

Nigeria [74] 436 N/A N/A N/A N/A N/ASouth 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 48USA [63] 378 0.26 1200 40 0 52

PodsIndia [52] 9.2 0.05 N/A N/A N/A 120

Tanzania [75] N/A N/A N/A N/A N/A 65USA [63] 4 0.053 0.12 44 0 141


2.3.4 Protein Quality

The compositions of amino acids per 1g of protein and WHO requirements are presented inAppendix 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 byWHO [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 Moringaleaves and pods. These results matched expectations as lysine and the sum of methionineand cysteine are limiting amino acids in many plant proteins [80]. From this finding, it may beinferred that Moringa leaves and pods require multiple food types to complement its amino acidcomposition for better protein quality. Literature values on the in vitro protein digestibility forfresh Moringa leaves were low, with values reported at 31.83% and 57.22% [81, 82]. Cookingor processing Moringa leaves will likely increase the vegetable’s protein digestibility with thetrade-off of lower bioavailability in micronutrients (e.g. vitamins) that are prone to thermal oroxidative destabilization.

Table 2.8: Protein quality and amino acid scores expressed as percentage of WHO Adult re-quirement in Moringa leaves and pods

. Bolded numbers indicate protein scores.% of Adult Requirement/g protein

Source Ref Histidine Isoleucine Leucine Lysine Methionine+ cysteine

Phenylalanine+ tyrosine Threonine Tryptophan Valine

LeavesIndia [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%

PodsEthiopia [83] N/A 104% 100% 76% 188% 100% 150% N/A 100%Mexico [60] 151% 117% 108% 63% 46% 81% 163% N/A 125%


2.4 Summary

Moringa leaves and pods contain multiple micronutrients, and the level of micronutrients ap-peared 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 deficienciesremains 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 incorrectreporting units, variations were mainly attributed to natural variations in the growing condi-tions. Fortified foods containing Moringa leaves and pods will likely need to be further enrichedusing supplemental fortificants to provide a complete package of micronutrients.

Moringa leaves generally contain more protein, lipids and ash than Moringa pods, whereasMoringa pods contain more fibre. Variabilities between measured and literature values for thelipid content in Moringa leaves were attributed to natural variations. Moringa leaves containhigher levels of minerals than Moringa pods. Moringa leaves are often featured as a vegetablewith high levels of iron, but comparisons with spinach leaves indicated that Moringa has eitherless or comparable amount of iron for an equivalent moisture-free mass. This finding is contraryto 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 tonatural variations as a result of the availability of nutrients in the soil where literature andexperimental samples were grown.

In the limited data found on Moringa’s vitamin content, Moringa leaves were shown tocontain noticeably higher levels of vitamins than pods with the exception of vitamin C. It wasunclear whether Moringa leaves or pods contained more vitamin C, as the range of literaturevalues were similar. Literature comparisons suggest that Moringa contains less vitamin A thanan equivalent dry mass of carrots. Both Moringa leaves and pods contain only trace amounts ofvitamin B1 and folate relative to the RDA of both vitamins. Moringa leaves contain significantlymore vitamin B6 than pods. However, further analysis is recommended as only one literaturesource was found to report on the amount of vitamin B6 in Moringa. Both Moringa leaves andpods were reported to contain no vitamin B12, which matched expectations as vitamin B12 istypically found in animal food products.

Based on the amino acid compositions available in literature, Moringa leaves and podsare limited by either lysine or the sum of methionine and cysteine. This suggests that theconsumption of Moringa leaves and pods needs to be complemented by multiple other foodtypes to provide an optimal ratio of essential amino acids.

Chapter 3

Addressing MicronutrientDeficiencies using Moringa oleifera

This chapter summarizes studies that have attempted to use Moringa as a natural food for-tificant and the accompanying challenges on undesirable organoleptic changes in the fortifiedfoods reported in those studies. Different food vehicle options were proposed in this study andevaluated followed by an exploratory evaluation of the feasibility of incorporating Moringa intobouillon 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 beleveraged to address micronutrient deficiencies. As discussed in Chapter 1, Moringa-fortifiedfoods should fit into the existing ecosystem of interventions and strategies implemented toreduce micronutrient deficiencies. Considering the nutritional content of Moringa documentedin Chapter 2 and the most prevalent micronutrient deficiencies discussed in Chapter 1, thefortification 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 infuture work, as discussed in Chapter 4 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 fooditems. The rheological and physical characteristics affect both the sensory acceptance as wellas the products’ manufacturability. Moringa’s antioxidant and antimicrobial properties havealso 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/wwas 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 greencolour and marked undesirable flavours [84, 85].

Kuikman and O’Connor [86] evaluated the acceptability of yogurt jointly fortified by Moringaleaf powder and other fruits and vegetables. Yogurt fortified with Moringa (17.09g per 1L ofyogurt) and banana puree (250mL per 1L of yogurt) was similarly preferred compared to theunfortified 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 beconsumed per serving of Moringa-banana fortified yogurt. Based on the nutritional content ofMoringa presented in Chapter 2, this small amount of Moringa leaf powder will have minimaleffect 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 Moringaseed flour as a means to fortify bread have also been reported [74, 87]. The preference for breadsamples decreased as the fraction of Moringa leaf powder increased [74]. This was attributed tothe green colouring and herbal flavours in Moringa leaves. On the other hand, bread samplesfortified with up to 10% w/w of debittered Moringa seed flour were found to have a distincttaste that was still within the acceptable range [87]. The inclusion of debittered Moringa seedflour did not have significant impact on the rheological characteristics of the resulting fortifiedflour blends [87]. Overall, Moringa-fortified bread had lower protein content and higher ironcontent than commercially prepared whole-wheat bread [63, 74, 87]. Considering that a sliceof 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 isequivalent 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 towheat flour was deemed unacceptable to sensory panelists, who found the resulting fortifiedbreads 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 wheatflour used to prepare cookies [87, 88]. Sensory characteristics were noticeably altered in cookiesfortified with over 20% debittered Moringa seed flour [87] and with over 10% Moringa leafpowder [88]. Moringa-fortified cookies were bitter and had surface cracking patterns that


were noticeably different to unfortified samples [87, 88]. The surface cracking pattern is a keyorganoleptic attribute used for evaluating the sensory acceptability of cookies, and is describedby the width of the cracks and the “islands” formed in between the cracks. The islands formedin 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 andcookies fortified with over 10% w/w Moringa leaf powder were green, bitter and had a grittymouthfeel [88]. There were no noted negative impact on the rheological characteristics of thefortified 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 thefortified and unfortified snacks, possibly due to the vegetable oil and flour coating that wasapplied onto the extruded snacks [89]. Uncoated extrudates with higher amounts of Moringaleaf powder had a more noticeable green colour. In the same study, it was observed that theaddition of Moringa leaf powder impacted the extrusion characteristics such as expansion, phasetransition and pasting properties. The increase in fibre content from Moringa leaves resultedin a reduction in overall extrudability [89]. Based on a serving size of 28g, the extruded snacksmade with the oat-flour blend with 45% w/w Moringa leaf powder had 1.2g of fibre, 3.8g ofprotein, 2.0mg of beta-carotene and 3.6mg of iron [89]. The iron content in the extruded snacksis equivalent to 20% and 45% of the RDA for adult females and males. Using a conversionof 12µg beta-carotene per 1µg RAE [90], the extruded snack contained 170µg RAE, which isequivalent 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 thatchutney powder containing 8.1% w/w of Moringa leaf powder was well accepted because sensorycharacteristics 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 eatenwith thick soup dishes. With the addition of Moringa, it was found that the amala dishesbecame dark green in colour which negatively impacted acceptability [91]. Amalas enrichedwith 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 fortifiedamalas due to the addition of Moringa were found to be minimal [91]. The iron content ofamala containing 2% w/w of Moringa leaf powder was 2.88mg/100g, which was a 0.45mg/100gincrease from the unfortified sample [91].

Up to 25% w/w of Moringa leaf powder was added to ‘ogi’, a fermented cereal puddingeaten in West Africa [92]. Strong preferences for the unfortified samples were observed, andwere attributed to undesirable colour changes and leafy tastes in the Moringa-fortified samples.


Glover-Amengor et al. [93] conducted a study on the acceptability of adding 2-3% w/wMoringa leaf powder into nine dishes on a kindergarten lunch menu in Ghana. The overallacceptability of the fortified dishes were rated by a group of kindergarteners on a pictorialhedonic scale out of five, and all dishes received scores over four (highly acceptable). The samegroup of participants were supplemented with 2g of Moringa leaf powder in their school lunchesthree times a week for two weeks, and it was concluded that students found the fortified lunchesacceptable 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 consumersas it indicates freshness. Moreover, the microbial count in raw beef treated with aqueousMoringa leaf extract was found to be consistently lower than untreated samples throughout astorage period of 12 days [94], suggesting that Moringa could potentially prolong the shelf-lifeof raw meats by preventing microbial spoilage.

2% aqueous Moringa leaf extract added to fresh guava juice was shown to be a promisingmicrobial inhibitor [95]. The addition of aqueous Moringa leaf extract added no noticeableodours 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 acceptanceof 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 proposedby 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 unde-sirable characteristics with desirable characteristics from other foods or by creating a physicalbarrier on the natural fortificant. Conveniently, the flavours in Moringa pods, which are sweeterand generally considered more pleasant, could be leveraged to mask the less desirable flavours inMoringa leaves. Moringa pods are also lighter in colour and could serve to lessen the intensityof the dark green colours associated with Moringa leaves. Moreover, some attention should alsobe placed on the foods that are consumed with a prospective food vehicle, as they could alsocontribute to the masking of undesirable sensory characteristics. For instance, a vegetable stewwould be more amenable to masking unusual textures than a glass of water. Encapsulation and


coating techniques could also be used to create a physical barrier around the Moringa particlesor 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 andcompounds that contibute to the stability of the nutritional value of Moringa. The isolationof specific compounds from its natural matrix could have both favourable and unfavourableimpacts on the stability and bioavailability of the nutritive components in Moringa. On onehand, bioavailability could improve as the antinutritional components are no longer present toinhibit the absorption of nutritional components. On the other hand, a nutritional componentmay be stabilized and made bioavailable by the presence of another component in the naturalplant matrix. An example of a potential interaction within Moringa is between its vitamin Acompounds and phenolic content. Vitamin A compounds are susceptible to oxidation and phe-nolic compounds are known antioxidants. The coexistence of both groups of compounds mayallow the vitamin A content to be stabilized. Existing literature has documented the isolationof antioxidant and antimicrobial compounds in Moringa using rudimentary liquid-liquid extrac-tion processes [68, 94, 95]. These studies have successfully extracted water-soluble compoundson a lab scale.

Removal of undesirable sensory components could be accomplished by extracting undesir-able compounds from the natural matrix or by altering the chemical structure of a compoundto deactivate its undesirable properties. Similar to the isolation of desirable compounds, inter-actions between different compounds within a natural matrix are complex and the extraction ofundesirable compounds could lead to unfavourable results. Although sensory characteristics areessential to a product’s acceptance, the primary intent of food fortification, especially in thisapplication, is to address micronutrient deficiencies. Hence, the nutritional impact of removingspecific compounds associated with undesirable sensory characteristics should be closely moni-tored and understood. The removal of bitter flavours from Moringa seeds on a lab scale havebeen described by Ogunsina et al. [87] and the resulting fortified food products with Moringaseed flour had a nutty flavour.

Although all three approaches are feasible, to focus the scope and depth of the researchconducted for this thesis, only the taste masking approach was pursued. Taste masking is themost direct approach. Additionally, taste masking does not require additional solvents, heatingor pressure in fortification, and would thus avoid increases in manufacturing costs that canultimately hinder adoption.

3.4 Food Vehicle Options

Four food vehicles were evaluated in this study: bouillon cubes, spice mixes, sauces and snackmixes. The proposed food vehicles were chosen as they all have the ability to mask undesirablesensory properties in Moringa. Bouillon cubes, spice mixes and sauces are added to dishes


with existing textures and colours. Flavours and colours in spices mixes and sauces may alsobe 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 andastringent flavours of Moringa leaves. The range of consumer-accepted colours and texturesfor snack mixes is broad, and would aid in masking undesirable colour changes caused by theaddition 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: consumptioncoverage, shelf life, ease of adding other micronutrients, and public health concerns. Each factorwas allocated a score between 1 and 5 for each candidate vehicle. Scores assigned were used asa 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 highconsumption coverage and levels, longer shelf-life, and easy incorporation of other micronutri-ents. A lower score for public health concerns (D) is preferred as it indicates low public healthconcern. 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 healthconcerns (i.e. (A+B+C)-D). Of the vehicles evaluated, bouillon cubes had the highest overallscore and were found to be most suitable for this application. The details of the evaluationsare described in the subsections below.

Table 3.1: Evaluation of food vehicles for incorporating MoringaConsumption coverage Shelf-life Ease of adding other micronutrients Public health concern Overall

(A) (B) (C) (D) (A+B+C)-DBouillon Cubes 5 4 4 2 11Spice Mixes 3 4 3 1 9Sauces 3 2 4 2 7Snack Mixes 2 4 3 4 5


3.4.1 Consumption Coverage

Food vehicles with high consumption coverage across the entire population are ideal for in-dustrial food fortification. More specifically, food vehicles should be consumed at consistentlevels between different socioeconomic levels. This increases the likelihood of the fortified foodreaching individuals of low socioeconomic status, who are more likely to be affected by foodand nutrition insecurity. Out of the four food vehicles evaluated, bouillon cubes were assessedto have the highest consumption coverage. Household coverage assessments conducted in WestAfrica showed that bouillon cubes have at least 95% coverage across households of all socioe-conomic statuses [97, 98]. The low unit price for bouillon cubes allows them to be consumedat similar levels regardless of a household’s socioeconomic status [97–99]. Although there aredifferent types of commercially available bouillon cubes (e.g. vegetable, beef, chicken), the in-gredients of each type are similar, and specific flavourings generally constitute 10% of the wholecube.

Unlike salt, which is universally used, the consumption and use of spices and sauces varyextensively depending on individual taste preferences, which are influenced by variables such asculture and region. The organoleptic properties of fortified spice mixes and sauces would needto be tailored for a specific context, which could limit the extent of its reach. Variabilities inthe consumption of spices and sauces also make it difficult to gather meaningful consumptiondata on these food vehicles. Nonetheless, the development of Moringa-fortified ‘sambar’ spicemixes have been suggested in literature [58]. Sambar is South Indian vegetable stew made withlentils and could include Moringa pods. As Moringa leaves and pods are already commonlyeaten 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 capturedin dietary recall surveys. Household coverage of snack mixes may also be low for households oflow 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 thefortified food vehicle to be readily available year-round. Furthermore, food vehicles that areshelf-stable are more practical for households of low socioeconomic status, who may not haveaccess to cold storage. Out of the four food vehicles evaluated, bouillon cubes, spice mixes andsnack mixes scored similarly and highest. Bouillon cubes, spice mixes and snack mixes are allgenerally shelf-stable. Bouillon cubes have shelf-lives of up to two years, while dried spice mixesmay 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 duringstorage. Conversely, the shelf-lives of sauces vary widely, depending on the ingredients withinthe condiment. For example, soy sauces, which contain 3300-5500mg Na/100g [63], has a shelflife of two years, whereas ketchup, which contains 20-907mg Na/100g [63], may only be stored


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 nutri-tional value as well as increase the stability and bioavailability of micronutrients. Ideally, foodvehicles should have both hydrophobic and hydrophilic components to incorporate both water-soluble and oil-soluble micronutrients. Of the four food vehicles evaluated, bouillon cubes andsauces scored highest as they both typically contain water and oils. The incorporation of addi-tional fortificants into spice mixes and snack mixes is likely feasible, but cannot be determinedspecifically 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 healthconcern score for spice mixes is lowest as an increased consumption of spices is not harmful forhealth. Bouillon cubes and sauces are typically high in sodium, and excessive sodium intakeleads to increased risks of heart diseases and other health aliments. Similarly, snack mixesare often high in sodium and sugar content as they are designed to satisfy sugary or savorycravings. Furthermore, fortification of snack mixes is deemed to be inappropriate by the FDAas it may “mislead consumers, causing them to substitute snacks for naturally nutrient densefoods” [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 de-termined to be the most suitable for the incorporation of Moringa out of the food vehiclesevaluated. The main advantages of incorporating Moringa into bouillon cubes are two-fold:the micronutrients in Moringa could increase the nutritional content of bouillon cubes, whichcurrently has negligible to no nutritional benefits; and the inclusion of Moringa in bouilloncubes would increase the utilization of a locally-available edible plant. By using Moringa asa natural fortificant, post-harvest food loss would also be reduced in places where Moringa isalready consumed. Consequently, a proof of concept was conducted to explore the feasibility ofmaking Moringa-fortified bouillon cubes. In this thesis, the direct incorporation of Moringa leafand pod powder was explored as a preliminary step for developing Moringa-fortified bouilloncubes. Moringa pod powder was included in this proof of concept as it has desirable sensorycharacteristics that could be leveraged to increase the acceptance of the fortified bouillon cubes.


3.5.1 Commercially Available Bouillon Cubes

The general composition of bouillon cubes is displayed in Figure 3.1 [101–103]. The majorityof the ingredients (salt, MSG, fat, flavourings) in the recipe are included to enhance the tasteof the bouillon cubes. Binders and fat components are included to maintain the shape andintegrity of the bouillon cubes. Reported consumption levels vary between 1 and 4.8g/day perperson [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, Nestle and Unilever respectively launched iron-fortified bouillon cubesin Central and West Africa [105, 106]. Nestle’s ‘Maggi’ cubes sold in Central and West Africacontain between 15% NRV (Nutrient Reference Value) to a maximum of 20% of TolerableUpper Intake Level (UL) for iron per 3.3g serving [104]. The Codex NRV and UL for ironis 14mg/day and 45mg/day respectively [1, 107]. The serving size of 3.3g per person wasdetermined by Nestle’s internal consumer research [104]. This fortification level was set basedon the resulting sensory properties and production costs. At fortification levels above 15% NRV,Nestle determined that the changes to sensory properties were unacceptable to consumers [104].Nestle also determined that a fortification level of 30% NRV would result in incremental productcosts 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 specificnutrient [108].


3.5.2 Specifications

Nutritional Specifications

Globally, the most prevalent micronutrient deficiencies are iodine, iron, zinc, vitamin A, fo-late and vitamin B12 . The severity of these micronutrient deficiencies varies amongst regionsdepending on the local diet. For Moringa-fortified bouillon cubes, it follows that the micronu-trients that are available in Moringa’s natural matrix should be examined first, followed byinvestigations 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 foodproducts and food fortification programs, while RDA is used to assess the nutritional require-ments for a specific target population group. Hence, NRV was used to benchmark the nutritionalcontent of the fortified bouillon cubes. Differences in the NRV and RDA may be attributedto 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 initialtarget. This benchmark is in alignment with Codex guidelines [108] and commercially availablefortified bouillon cube products [104].

Table 3.2: Comparison of NRV and RDA values for minerals, vitamins and protein

Nutrient RDA*[1, 2, 109]

NRV[107]

MineralsCalcium 1000 1000

Magnesium 310-420 300Potassium 4700 N/A

Sodium 1500 N/AZinc 8-11 15

Copper 0.9 N/AManganese 1.8-2.3 300

Iron 8-18 14

VitaminsVitamin A (µg RAE/d) 700-900 800

Vitamin B1 (mg/d) 1.1-1.2 1.2Vitamin B6 (mg/d) 1.3 1.3

Vitamin B9 (µg DFE/day) 400 400Vitamin B12 (µg/d) 2.4 2.4Vitamin C (mg/d) 75-90 60

Macronutrients - Protein 46-56 50*non-pregnant and non-lactating adults, 19-50 years old.


Physical Specifications

Physical characteristics of fortified bouillon cubes would ideally be the same as those in unfor-tified bouillon cubes. Physical specifications are required for user acceptance, transportationand storage (Table 3.3). All characteristics with the exception of water activity were consideredin relation to the user’s familiarity and habits when using bouillon cubes. This project aimedto form small-sized bouillon cubes (3-4g).

Minimum hardness is required for transportation and maximum hardness pertains to theusers’ habits in crumbling cubes by hand before adding them during food preparation. Upperwater activity thresholds are needed to ensure a shelf-stable food product.

Table 3.3: Physical specifications for fortified bouillon cubesSpecification Function BenchmarkMass User acceptance ‘Small’ size: 3-4g

‘Large’ size: 10-13g

ShapeUser acceptanceTransportationStorage

‘Small’ size: 20 x 20 x 20mm‘Large’ size: 26 x 25 x 10mm

Hardness User acceptanceTransportation 5-50N [102, 103]

Water activity Food safetyStorage

< 0.6 to inhibit enzymatic degradation and;< 0.8 to inhibit microbial growth [110]

Disintegration time User acceptance Quick: < 30 seconds in boiling water [101]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 orclosely resemble the characteristics of the unfortified bouillon cubes. The colour, shape and sizecontribute to the appearance of the cubes. For Moringa-fortified bouillon cubes, it is expectedthat the taste and mouthfeel will be partially masked by the food they are consumed or preparedwith. To focus the efforts of this proof of concept, the nutritional and physical characteristicswere primarily concentrated upon as organoleptic characteristics could be optimized in futurework.


3.5.3 Experimental Design

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

Figure 3.2: Simple axial mixture experimental design for proof of concept


3.5.4 Processing

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

Figure 3.3: Block flow diagram for forming Moringa-fortified bouillon cubes


Materials and Dry Ingredients Preparation

Fresh Moringa leaves from Coimbatore, India, and pods from Delhi, India, were used. Thenutritional properties of the Moringa leaf and pod samples were described in Chapter 2. Durumsemolina (Brand: Unico) was procured from a local supermarket in Toronto, Canada. Moringaleaflets were stripped from their petioles and washed, while Moringa pods were washed then cutinto 2-3” sections. Moringa leaves and pods were freeze-dried for 72 hours, milled and sievedthrough a Tyler No. 35 mesh (500µm). Dry ingredients were stored in sealed glass containersat 4◦C.

Extrusion Processing

Dry ingredients were blended and hydrated using a benchtop mixer (Model no.: KitchenAidKSM95TB). A benchtop cold extruder was assembled by fitting a 20*20mm die to a singleArchimedes screw attachment (Model no.: KitchenAid FDA) which was then attached to themotor of the aforementioned benchtop mixer (Figure 3.4). After extrusion, extrudates weremanually cut to lengths of 20mm to give 20*20*20mm cubes and air-dried for 48 hours at 35◦Cin 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 commercialMoringa leaf powder (Brand: Yupik), and 1:1 ratio blends of binder and Moringa pod powderwere conducted to determine the appropriate in-barrel moisture for extrusion (Table 3.4). Forall combinations, blends with 17% in-barrel moisture were too dry and the dry ingredients didnot cohere with each other after extrusion. The blend with 29% in-barrel moisture was found


to be sufficient for the extrusion of semolina. At 38% in-barrel moisture, extrudates made withonly semolina deformed substantially at the outlet, and cross-sections of the extrudates did notexit uniformly as the dough was partially sticking to the sides of the die. For blends with 1:1ratios of binder and Moringa leaf or pod powder, 38% in-barrel moisture was needed for blendsto be extruded successfully.

Table 3.4: In-barrel moisture content in screening experimentsBlend ratio

(L:P:B) Mass of dry ingredients (g) Volume of water added (mL) In-barrel moisture (%) Successfully extruded?

0:0:1 150 30 17% No0:0:1 150 45 29% Yes0:0:1 150 60 38% No1:0:1 150 30 17% No1:0:1 150 45 29% No1:0:1 150 60 38% Yes0:1:1 150 30 17% No0:1:1 150 45 29% No0:1:1 150 60 38% Yes

The screening experiments showed that different in-barrel moisture content is required fordifferent formulation blends. This is likely due to the different water absorption capacitiesfor the three ingredients. Thus, for formulations in the mixture experimental design, dryingredients were initially hydrated to 29% in-barrel moisture and the consistencies of the blendswere qualitatively checked to match the blends that were successfully extruded in the screeningexperiments. If a blend was found to be too dry, an amount of water equivalent to 10% ofthe mass of the dry ingredients was added and the blend’s consistency was checked again. Theprocess of adding water at 10% mass increments was repeated until the blend reached theappropriate 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 forequilibration prior to extrusion. The motor rotational speed was set to setting number 4 for allformulations.

Table 3.5: In-barrel moisture for mixture design formulationsFormulation 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%


3.5.5 Characterization Methods

Nutritional Content

The iron and zinc contents of the dry ingredients and formulations 7, 9 and 10 were determinedexperimentally. 10 minerals were analyzed, but only iron and zinc results are discussed in thissection. See Appendix A for mineral content results on calcium, magnesium, potassium, sodium,zinc, copper, manganese, lead and selenium. Measured mineral contents were compared withvalues 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 mineraland formulation to confirm that they were not statistically different (p = 0.05). Out of the30 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 differentfrom its calculated value. The statistical difference for the selenium measurement is likely acumulative result of experimental inaccuracies from weighing, dilutions and calibration. Giventhat 29 out of 30 measured values were not statistically different from their theoretical values, itwas deduced that the mineral content of each formulation could be calculated using the mineralcontent of the dry ingredients and blend ratios.

Vitamin A and folate contents were calculated using the blend ratios of each formulationand the standard reference values for Moringa leaves and pods available from the USDA FoodComposition Database. Crude protein content was experimentally measured to determine theextrudates’ suitability as a protein supplement. Experimental methods used for determiningthe mineral and protein content were described in Chapter 2.

Physical Characteristics

The mass of dried extrudates for all formulations was obtained and recorded. The shape ofthe samples was predetermined by the 20*20mm die used during extrusion. Hardness wasmeasured using a tablet hardness tester (Model no.: Erweka TBH250). Water activity wasmeasured using a benchtop water activity meter (Model no.: AQUALAB 4TE). Disintegrationwas 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 verticalagitation. This method was adapted from the US Pharmacopeia disintegration test (701)for tablets. Commercially available vegetable stock cubes (Brands: Knorr and Aurora) wereobtained from a local supermarket in Toronto, Canada, and tested for their disintegration rateto give an additional reference data point.

With the exception of water activity measurements, all measurements were triplicated. Onlyone measurement was obtained per formulation for water activity due to a shortage of extrudedsamples. One-way ANOVA tests were performed for mass and hardness results to determine thedifference in means between all formulations. If means were shown to be different, Tukey HonestSignificant Difference (HSD) tests were performed to determine the statistical significances of


the differences.

Colour

Photographs of dried extrudates were taken in a photobox under consistent indoor fluorescentlighting. The same white balance was used for all photographs. The Hunter L*ab colour scalewas 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 un-changed after extrusion as no additional heat or pressure was applied during processing. Thelevel 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). Allformulations failed to reach the 15% NRV per 3.3g serving benchmark for iron, zinc, vitaminA, folate and protein (Table 3.6). Bioavailability considerations aside, 1.30 mg of iron wouldneed 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 inliterature. However, it is known that Moringa leaves also contain oxalic acid [111], which isalso 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% NRVbenchmark. The upgrading of micronutrient content may be accomplished by using naturalfortificants 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 Protein1 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


Figure 3.5: Iron content in mixture design formulations. Increasing bubble sizes correspondto 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 madewith at least 67% semolina by weight were significantly denser. It was observed that doughswith more semolina were stickier due to the formation of gluten bonds and semolina-only doughstended 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, whichled to increased compression and denser extrudates.

Extrudates from all formulations had masses greater than 4g, which was the benchmarkset in Subsection 3.5.2. As extrudate density is a function of in-barrel moisture, and themoisture content varied between formulations in this study, quantitative conclusions could notbe 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 extrudatebulk density for this system.

Extrudates composed of at least 50% Moringa pods (Formulations 4, 5 and 9) appearedto be significantly harder than extrudates made with other formulations (Figure 3.6). Furtherinvestigation is required to understand this result. A possible explanation for this result isthat the fibre content in Moringa pod powder contributed to the structural integrity of theextrudates. 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


Table 3.7: Mass, hardness, water activity and disintegration results for extruded cubes

Formulation Blend ratio(L:P:B) Mass (g) Hardness (N) aw

Disintegration(# of tests passed, n=3)

1 1:0:0 4.2± 0.3a 29± 16a 0.14 22 0:1:0 4.3± 1.0ab 75± 14ab 0.17 33 0:0:1 6.3± 0.7c 17± 11ab 0.52 04 1:1:0 5.1± 0.2abcde 360± 57c 0.14 25 0:1:1 5.7± 0.3cde 190± 39b 0.16 06 1:0:1 5.4± 0.1bcde 37± 15ab 0.16 07 1:1:1 5.3± 0.2abcde 93± 15ab 0.16 38 4:1:1 4.7± 0.2abde 97± 36ab 0.16 19 1:4:1 4.5± 0.2abd 374± 127c 0.16 310 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 hardnessdoes not equate to crumbliness, which is an organoleptic property and described as the ease ofwhich a material breaks into smaller portions. The lower measured hardness may also be dueto the development of cracks in the centre of the cube after drying (Figure 3.7). These crackswere parallel to the direction of extrusion and were likely formed due to an uneven compressionforce from the extruder screw. It was speculated that the uneven compression force was due tothe unsteady operation of the extrusion system, caused by intermittent material loading, whichwas 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 andmicrobial degradation were inhibited within the extrudates. These results were expected asformulations were dried over a prolonged period (48 hours).

Both brands of commercially available bouillon cubes passed all three replicates of thedisintegration test and were completed dissolved within three minutes. On the other hand,only extrudates made with 100% Moringa pods fully dispersed within 20 minutes. Formulationsthat 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 hardcore, but a noticeable solid portion of the extrudate remained at the end of each test. Thepresence of solid chunks after 20 minutes would likely be unacceptable for the consumer. Theseunfavourable 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) thanextrudates of other formulations (Table 3.8). This result matched expectations as Moringa leafpowder 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.


Figure 3.6: Hardness in mixture design formulations. Increasing bubble sizes correspond to in-creasing hardness; numbers correspond to mean hardness in newtons. The range for acceptablehardness 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.


Table 3.8: Hunter L*ab values for extruded cubesFormulation Blend ratio (L:P:B) L* a b1 1:0:0 14.1±1.57 -2.3±0.14 7.40±0.522 0:1:0 42.0±2.90 -4.0±1.74 20.9±1.083 0:0:1 46.6±2.09 -1.2±0.55 17.7±1.834 1:1:0 15.7±3.13 -1.7±0.38 8.30±1.365 0:1:1 37.3±2.75 -3.1±0.83 17.7±1.216 1:0:1 13.0±0.73 -2.0±0.41 6.4±0.777 1:1:1 19.8±4.59 -4.1±1.52 10.8±2.638 4:1:1 12.4±3.13 -2.8±1.27 5.9±1.699 1:4:1 25.3±1.43 -2.0±5.49 14.6±0.8510 1:1:4 21.9±4.43 -4.1±1.2 12.4±1.97L*: 0 = dark, 100 = lighta: -128 = green, 128 = redb: -128 = blue, 128 = yellow


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. Additionalfortificants will be required to upgrade the nutritional content in Moringa-fortified bouilloncubes. The flavours and colours of Moringa may either be enhanced or suppressed in flavouringcubes, depending on regional preferences. In select regions where Moringa is already widelyconsumed and preferred, the distinct flavours of Moringa may be enhanced to encourage furtherconsumption. Conversely, Moringa’s flavours and colours may be suppressed when they are notpreferred.

Given the nutritional and physical characterization results determined in this thesis andsensory preferences observed in literature, the formulation with equal parts (i.e. 1:1:1) ofMoringa leaf powder, Moringa pod powder and binder is the most promising out of the 10formulations tested. Cubes with equal parts of all ingredients had high nutritional contentrelative to other formulations and passed all three replicates of the disintegration tests. Cubeswith a 4:1:1 (L:P:B) ratio were lower in hardness but only passed one out of three times whentested for disintegration. Cubes with 100% Moringa leaf power yielded the highest nutritionalcontent and were within the acceptable hardness range, but their dark green colours would behighly unacceptable to consumers. The undesirability of green colours in foods may be becauseof 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 extru-sion processes and has low unit cost. However, the starch in semolina is water-insoluble andunfavourable for the disintegration of bouillon cubes. Semolina provided a great deal of struc-tural integrity within extrudates, but most of the formulations were too hard to be crumbledby hand. Bouillon cubes are commonly hand-crumbled during food preparation and deviationsaway from the typical crumbliness could reduce the user acceptability. The inclusion of semolinacontributed to the dough’s elasticity during extrusion, which improved extrudability.

As expected, Moringa leaf and pod powder provided dark and light green pigments tothe extrudates respectively. Concerns regarding undesirable mouthfeels and tastes were notaddressed 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 assessedquantitatively 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 bouilloncubes 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 chemicalcompounds within the food and with sensory receptors in an individual. Qualitatively, Moringaleaves 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 biochemicalinteractions that contribute towards the taste, aroma and colouring of Moringa leaves andpods. Identification of biochemical interactions responsible for Moringa’s taste, aroma andcolour is fundamental to manipulation of organoleptic properties and therefore the acceptance ofMoringa-fortified foods. Compounds that contribute to undesirable sensory characteristics (e.g.bitterness) could be removed, whereas concentrated fractions of compounds that contribute todesirable sensory characteristics could be formed by extraction or synthesis. Concentration ofdesirable compounds is also pertinent to the development of other food products in regionswhere Moringa is widely consumed and preferred.

Biochemical mechanisms for different taste sensations have been documented [113]. Thisknowledge may be combined with literature chemical characterizations for Moringa to forminitial hypotheses on possible biochemical interactions responsible for the distinct tastes inMoringa leaves and pods. Aromas are mainly results of interactions between volatile compoundsand olfaction receptors; and colours are a consequence to the light absorbance of chemicalcompounds. As taste could be affected by a broad range of compounds, from salts to aminoacids to alkaloids, the analytical methods used for the identification of these compounds wouldbe similarly diverse. Volatile compounds may be identified using gas chromatography-massspectrometry (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 inter-actions will commence in September 2018 with a three-month research exchange at the TamilNadu Agricultural University starting in November 2018.

4.1.2 Encapsulation and Coating Processes

As mentioned in Chapter 3, an alternative method for masking undesirable sensory charac-teristics in Moringa is to utilize physical barriers such as those formed through coating andencapsulation processes. Encapsulation processing may also further extend the shelf life andstability of micronutrients in Moringa. Coatings used for microencapsulation should be selectedbased on their ability to enable the encapsulated Moringa particles to blend in with the par-ticles of the food vehicle without limiting the bioavailability of the micronutrients. However,encapsulation and coating processing also increases particle size, which could limit the amountof micronutrients for fortification. Considerations for potential coatings include texture, colourand release mechanisms.

4.1.3 Alternative Binders and Excipients

Alternative binders should be evaluated for future work. Binders should not change theorganoleptic properties of bouillon cubes. Two approaches to ensure that organoleptic prop-erties 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 matrixso that fortified bouillon cubes readily disintegrate under cooking conditions. Binder particlescould be made small enough so that they are not visually and textually detectable in the foodthat bouillon cubes are consumed with.

Other excipients such as aids for disintegration and dissolution may also explored. Theinclusion of other excipients may improve the assimilability of fortified bouillon cubes, but willlikely increase the manufacturing costs. Excipients are commonly used in pharmaceutical appli-cations and transferrable knowledge may be obtained by studying literature on the formulationof pharmaceuticals.

4.1.4 Supplemental Fortificants

Moringa-fortified foods will require supplementation from other fortificants, natural or syn-thetic, to supplement appropriate levels of iodine, iron, zinc, vitamin A, folate and vitaminB12. Fortificants derived from animal products are not considered for this application as peo-ple who do not consume animal food products, such as vegetarians, would be excluded fromconsuming the resulting Moringa-fortified food product. Vegetarians are susceptible to preva-lent micronutrient deficiencies, so their acceptance of the resulting products should be carefullyconsidered (see Section 1.1).


Natural fortificants from other plant species may be used to complement the micronutrientcontent in Moringa. For example, seaweed is considered a source of iodine and many dairyproducts are considered sources of vitamin B12 [114, 115]. Clearly, the inclusion of dairy prod-ucts 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 ofother 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 forti-fication technologies using synthetic fortificants are well-established in other food vehicles (seeSubsection 1.3.1). However, the incorporation of synthetic fortificants may lead to unintendedchemical 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 thecommercialization of the product. Cold extrusion was trialed in this thesis as a proof of conceptfor Moringa-fortified bouillon cubes given its ease of operation and equipment availability. Forfuture work, the effects of different extruder designs and processing conditions could be consid-ered. The effects of heat on the micronutrient content in fortified bouillon cubes is of particularinterest, as vitamins destabilization through oxidation may be exacerbated under heated oxy-genated conditions. Extrudability may also be improved in future work by increasing the fatcontent 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 tovary the dough fat content in future work as extrusion is heavily dependent on the rheologicalcharacteristics of the dough.

Manufacturing processes involving tablet presses may also be investigated for the produc-tion of fortified bouillon cubes. Tablet presses are commonly used for manufacturing existingcommercially available bouillon cubes.

4.1.6 Consumption Patterns and Consumer Preferences

Limited public data are available on the consumption patterns and preferences for bouilloncubes. A clear understanding of the prevailing consumption patterns and preferences for bouil-lon cubes is required before the development of fortified bouillon cubes. It is also conceivablethat existing bouillon cubes consumption patterns may not be equitable for Moringa-fortifiedbouillon cubes as the marketed product may deviate away from conventional products. For thisproof of concept, a serving size of 3.3g/person/day was used to enable simple comparisons withexisting commercial products, but the consumption level of Moringa-fortified bouillon cubesmay be different as Moringa may impart specific flavours and fortified bouillon cubes maycontain a different concentration of salt. The effects of these variables on consumption pat-terns may be explored in future work to gain insights on the acceptability of Moringa-fortifiedbouillon cubes.


4.2 Conclusions

The Moringa-fortified bouillon cube is a unique approach for incorporating the micronutrientsin Moringa into a staple condiment. Moringa-fortified bouillon cubes allow the micronutrientsin Moringa to be shelf-stable. Bouillon cubes are suitable for incorporating a wide variety ofmicronutrients as its matrix contains both water-soluble and oil-soluble components. The sen-sory 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 ofMoringa 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 deter-mined using a combination of experimental methods and literature review. Micronutrientcontent in Moringa was compared to vegetables that are considered sources of the specifiedmicronutrients. For an equivalent dry mass, Moringa generally has similar or lower levels ofmicronutrients relative to other foods consumed in a vegetarian diet, except in the case forvitamin C. Moringa leaves contain 8.3-110mg Fe/100g, compared to spinach leaves which have32mg Fe/100g. Moringa leaves and pods were found to have high levels of vitamin C rela-tive to kiwifruit. The vitamin C content in Moringa leaves and pods are 48-220mg/100g and65-141mg/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 theessential amino acids in Moringa leaves and pods. Lastly, the skin of Moringa pods containa considerable fraction of macronutrients and micronutrients, and discarding the skin wouldresult 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 barriersfor the acceptance of Moringa-fortified foods. Food vehicles that are able to mask the undesir-able sensory properties of Moringa leaves were considered in this thesis. Bouillon cubes, spicemixes, sauces, and snack mixes were evaluated on their suitability for incorporating Moringabased on their consumption patterns, shelf-life, capacity to include additional fortificants (asidefrom Moringa), and health impact. Bouillon cubes were determined to be best suited for thisapplication.


3. Explore the feasibility of a promising food fortification application forMoringa.

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, Moringapod powder and semolina (binder) were tested. The nutritional content, physical characteris-tics and colour were determined for the extrudates. None of the extrudates met the nutritionalcontent benchmark, which confirms that Moringa-fortified bouillon cubes require supplementalfortificants to provide sufficient levels of micronutrients.

The physical characteristics of the extrudates were also examined to provide preliminaryinsights for future development. Generally, the physical characteristics of extrudates weresignificantly different when compared to conventional bouillon cubes. Although semolina iscommonly used for extrusion and provides structural integrity to extrudates, an alternativebinder 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

Source Histidine Isoleucine Leucine Lysine Methionine+ cysteine

Phenylalanine+ tyrosine Threonine Tryptophan Valine

WHO Adult Requirements 15 30 59 45 22 38 23 6 39Leaves

India [52] 18 128 63 49 1 3 35 1 87Ethiopia [83] n.d. 39 76 46 27 57 45 n.d. 50Mexico [60] 31 40 78 68 6 61 35 n.d. 50Niger [73] 30 45 87 56 33 100 47 21 57

Nicaragua [68] 23 34 68 41 23 71 30 26 41USA [63] 21 48 84 57 28 89 44 15 65

PodsEthiopia [83] n.d. 30 56 32 39 36 33 n.d. 37Mexico [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 cubesMineral content (mg/3.3g serving)

Blend Ratio (L:P:B) Calcium Magnesium Potassium Sodium Zinc Copper Manganese Iron Lead Selenium1:00:00 65.8 25.3 7.9 1.3 0.04 0.02 0.11 0.8 0.04 0.040:01:00 3.1 5.9 5.8 4.1 0.06 0.01 0.03 0.1 0 00:00:01 0.2 2 0.7 0 0.04 0.01 0.04 0.12 0 01:01:00 34.5 15.6 6.8 2.7 0.05 0.02 0.07 0.45 0.02 0.020:01:01 1.7 4 3.3 2.1 0.05 0.01 0.03 0.11 0 01:00:01 33 13.6 4.3 0.7 0.04 0.01 0.08 0.46 0.02 0.021:01:01 23 11.1 4.8 1.8 0.05 0.01 0.06 0.34 0.01 0.014:01:01 44.4 18.2 6.3 1.6 0.04 0.02 0.09 0.57 0.03 0.021:04:01 13.1 8.5 5.3 3 0.05 0.01 0.04 0.22 0.01 0.011:01:04 11.6 6.5 2.7 0.9 0.05 0.01 0.05 0.23 0.01 0.01


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. Repeattwice 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 asW2 (ash + crucible).

Ash Content = W1 −WC

W2 −WC


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 weighingboat.

4. Place samples into drying oven for 24 hours.

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

Moisture Content = W1 −WC

W2 −WC


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. Connectwith condenser and reflux for 30 minutes.

4. Assemble decantation setup with Buchner flask, Buchner funnel and filter paper. Flownear-boiling water through funnel to warm it and decant liquid through funnel. Turn onwater supply to induce vacuum.


5. Decant acid-digested sample through Buchner funnel. Wash solids from flat-bottomedflask 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-boilingwater. 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 thenwith 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).

Crude Fibre Content = W2 −W3W1 −WR


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


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 eachflask. 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 theflasks and prevent evaporation of hexane.

3. Add 10 1g of sample into each thimble (1, 2 and 3). To make weighing easier, place atthimble into beaker so that it is upright for adding and weighing out the sample. Recordweight of sample.


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

7. Attach each setup to the condenser unit. Use Teflon tape to seal (i) the connection betweenthe condenser and the extraction chamber and; (ii) the connection between the extractionchamber 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). Ensurethat the top caps remain on the condenser unit (place back as soon as possible on if thepressure pushes a cap off)

10. Allow distillation to run for 24 hours. At the end of the distillation, turn off heaters andallow 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 thesetup to allow as much hexane as possible to drain through the siphon tubes and into theflasks. 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. Removeand dispose of filter paper cones. Allow hexane to drain into beaker.

13. Disconnect extraction chambers from flasks. Pour remainder of hexane, including thatdrained from the thumbles, into flasks. Seal opening of flasks with glass stopper. Layextraction 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 forrecovered solvent.

2. Assemble rotavapor setup by connecting the vacuum Erlenmeyer flask to the water tapand securing the stopper the Erlenmeyer flask. Ensure the vacuum release at the top ofthe rotavapor condenser is in the closed position.


3. Connect round collection flask to the outlet of the rotavapor condenser using clamp (turnwheel 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 agloved-palm at the inlet of the condenser (where a round flat-bottomed flask would beconnected). There should be suction.

5. Attach round flat-bottomed flask containing sample to condenser. Gently pull flask tocheck that the vacuum formed is sufficient to hold flask in place. If not, either wait forvacuum 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 theedge of the water bath; (ii) the sample is sufficiently lowered so that the liquid level withinthe 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 waterfor 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 boilingand 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. Theremaining 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 sampleflask 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 collectionflask 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 boilingstones and lipids. Calculate oil content using the formula below.

Crude Lipid Content = mass of oilmass of sample before extraction


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


• 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


1. 1-2 days prior to conducting experiment, check that all reagents are available at sufficientamounts. 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 contentaccording to literature values. Back calculate the expected the titration volume so thatit is between 3 to 17mL.

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

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

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

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

7. Move the tubes-manifold digestion setup into the Buchi digester with the threaded side ofthe 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 inthe 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 samenumber of drops to give comparable titration results. Cover flasks with parafilm and setaside.


Setting Time (minutes)4 206 1010 30

Total 60

13. Upon completion of digestion, remove manifold-tubes setup from digester and place backinto a tube rack without turning off vacuum (water supply) so that the sulphuric acidfumes continue to be removed. (CAUTION: HOT SURFACE. USE HEAT PROTECT-ING GLOVES). The resulting digested solutions should be clear and light green. Allowthe 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 isready 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 recommendedto start with the blank sample. Gently mix water and digested sample (mostly acid) ina swirling motion. Turn tubes towards the back of fumehood when adding water as asafety precaution for potential liquid level rises due to violent reactions.

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

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

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

7. Remove Kjeldahl tube and collection flask after distillation is complete (loud beepingsound). Rinse tubing on both ends (tube and conical flask) into the tube/flask. Cleanup any spills immediately by flushing the area with distilled water and wiping dry withKimwipes.


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

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

%N =(titration vol, in mL)(14 g

mol N)(0.1−3 molL H2SO4)

mass of sample, in g

Protein content = %N ∗ conversion factor


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 weighingpaper to get the remaining mass of sample on paper. Record remainder weight and discardweighing paper.

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

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

5. Repeat steps 1-3 for all samples.


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. Filland 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 thecap 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 linerwith deionized water. Careful to keep the total solution volume below 25ml.

11. Make up digested samples to 25ml in volumetric flasks. Filter digested samples through0.45-micron microfilters. Keep digested samples for a maximum of one week. Dispose ofsample accordingly after experiments.

12. Dilute digested samples according expected literature values so that concentrations fallbetween 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.

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https://ods.od.nih.gov/factsheets/VitaminB12-HealthProfessional/

https://ods.od.nih.gov/factsheets/VitaminB12-HealthProfessional/

Micronutrients in Moringa oleifera and their Potential in Food ......2.2 Iron content comparisons in...

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