Banana Pseudostem: properties nutritional composition and use as food
Jun Ma
A thesis in fulfillment of the requirements for the degree of Masters by Research
School of Chemical Engineering
Faculty of Engineering
September 2015
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Banana pseudostems are crop waste, which cause economic loss and environment issues after harvest. However, pseudostems are rich in dietary fibre and have health benefits. This study explored the chemical composition
(proximates, minerals, vitamins) as well as the digestibility and functionality of the carbohydrates. Dietary fibre was estimated using three methods namely the established AOAC method, Gas Chromatography and Nuclear Magnetic
Resonance. As fresh banana pseudostems have limited shelf life, drying the stems using cabinet dryer using several conditions such as, drying at 40 C and
50C with/ without blanching, were compared with regard to drying time, colour as well as quality of the dried product in terms of the retention of nutrients.
Drying at 50 C without blanching provided the whitest colour and shortest drying time. Thus the optimum condition for drying was established at 50 C without blanching based on nutrient retention. Musa balbisiana and Musa acuminata banana pseudostems were used in this study. There was no significant difference between protein, fat and carbohydrate content of banana pseudostems dried under different conditions. Moisture content was significantly higher in banana pseudostems dried at 40 C without blanching; ash content was significantly higher in pseudostems dried at 50 C without blanching.
According to the percentage of total dietary fibre and resistant starch, pseudostem dried at 40 C with blanching had the lowest digestibility. The neutral sugars in the non-starch polysaccharides were studied and compared
with commercially available natural dietary fibre supplements sold in Australia.
Pseudostems had higher ratio of soluble dietary fibre to insoluble dietary fibre, as compared to the commercial supplements. The main neutral sugars in the pseudostems were glucose, mannose and xylose, while those in the commercial supplements were xylose, arabinose and mannose, which had different functionality compared to pseudostem fibre. This study is the first to demonstrate that banana pseudostem is a potential dietary fibre supplement, which may bring health benefits to consumers and economic profits to the banana growers.
Table of Contents
1. Introduction ...... 1
2. Literature Review ...... 3
2.1 The banana plant ...... 3
2.1.1 Origin and world production ...... 3
2.1.2 Banana industry in Australia ...... 4
2.1.3 Taxonomy of banana ...... 5
2.2 Banana pseudostem ...... 6
2.2.1 Introduction of banana pseudostem ...... 6
2.2.2 Structure of banana pseudostem ...... 7
2.2.3 Nutritional value of banana pseudostem ...... 10
2.2.3.1 Proximate nutrients ...... 10
2.2.3.2 Minerals ...... 12
2.2.4 The utilization of the banana pseudostem ...... 13
2.3 Pretreatment ‐‐ Anti‐browning ...... 14
2.3.1 Causes of browning ...... 14
2.3.1.1 Enzymatic browning ...... 15
2.3.1.2 Non‐enzymatic browning ...... 17
2.3.2 Browning prevention methods ...... 19
2.3.2.1 Physical methods of prevention ...... 20
2.3.2.2 Chemical prevention ...... 23
2.4 Drying ...... 25
2.4.1 Drying principle ...... 25
2.4.2 Factors that affect drying rate ...... 27
i 2.4.2.1 Air properties ...... 27
2.4.2.2 Physical properties of food ...... 28
2.4.2.3 Other factors ...... 28
2.4.3 Drying models ...... 29
2.4.4 Effects of drying conditions ...... 30
2.5 Carbohydrates ...... 30
2.5.1 Carbohydrate chemistry ...... 31
2.5.1.1 Mono‐ and di‐saccharides...... 32
2.5.1.2 Oligosaccharides ...... 34
2.5.1.3 Polysaccharides ...... 34
2.5.2 Potential health benefits of carbohydrates ...... 38
2.5.2.1 Hyperlipidemia ...... 39
2.5.2.2 Diabetes mellitus ...... 40
2.5.2.3 Coronary heart disease (CHD) ...... 43
2.5.3 Physico‐chemical properties of carbohydrate ...... 44
2.5.3.1 Water holding capacity (WHC)...... 44
2.5.3.2 Swelling capacity (SWC) ...... 47
2.5.3.3 Viscosity ...... 47
2.5.3.4 Gelatinization ...... 48
2.5.4 Analytical methods for non‐starch polysaccharides ...... 50
2.5.4.1 Enzymatic – gravimetric method ...... 50
2.5.4.2 Enzymatic – chemical method ...... 51
2.5.4.3 Nuclear magnetic resonance spectroscopy (NMR) ...... 52
2.5.4.4 Near‐infared reflectance spectroscopy (NIR) ...... 52
2.6 B‐complex vitamins ...... 53
2.6.1Vitamin B3 (Niacin) ...... 53
2.6.2 Vitamin B9 (Folate) ...... 55
ii 2.6.3 Vitamin B1 (Thiamin) ...... 56
2.6.4 Vitamin B6 (Pyridoxine) ...... 58
2.6.5 Vitamin B2 (Riboflavin) ...... 58
3. Materials and Methods ...... 61
3.1 Raw material ...... 61
3.2 Sample Preparation and Drying ...... 62
3.2.1 Equipment ...... 62
3.2.2 Procedure of sample preparation ...... 62
3.2.3 Procedure of batch drying of banana pseudostem ...... 63
3.3 Nutrient analysis ...... 64
3.3.1 Moisture ...... 64
3.3.1.1 Equipment ...... 64
3.3.1.2 Procedure of moisture measurement ...... 64
3.3.2 Fat ...... 65
3.3.2.1 Equipment ...... 65
3.3.2.2 Chemicals ...... 66
3.3.2.3 Procedure of fat content measurement ...... 66
3.3.3 Proteins ...... 67
3.3.3.1 Equipment ...... 67
3.3.3.2 Procedure for protein analysis ...... 68
3.3.4 Ash ...... 68
3.3.4.1 Equipment ...... 68
3.3.4.2 Procedure of ash analysis ...... 68
3.3.5 Sugars ...... 70
3.3.5.1 Equipment ...... 70
3.3.5.2 Chemicals ...... 71
iii 3.3.5.3 Procedure for sugar extraction ...... 71
3.3.6 Starch ...... 72
3.3.6.1 Equipment ...... 72
3.3.6.2 Chemicals ...... 73
3.3.6.3 Procedure of total starch extraction by HPLC ...... 73
3.3.6.4 Chromatographic conditions for HPLC ...... 74
3.3.6.5 Procedure of starch digestibility analysis ...... 74
3.3.7 Dietary fibre ...... 79
3.3.7.1 Equipment ...... 79
3.3.7.2 Chemicals ...... 81
3.3.7.3 Procedure for dietary fibre analysis by Megazyme kit ...... 82
3.3.7.4 Procedure of dietary fibre analysis by solid state NMR ...... 85
3.3.7.5 Procedure of enzyme and acid hydrolysis of non‐starch polysaccharides ...... 86
3.3.7.6 Procedure of non‐starch polysaccharide analysis on solution state NMR ...... 88
3.3.7.7 Procedure of NSP analysis by GC ...... 89
3.3.8 Minerals ...... 93
3.3.8.1 Equipment...... 93
3.3.8.2 Chemicals ...... 93
3.3.8.3 Procedure of mineral analysis ...... 94
3.3.9 B‐complex Vitamins ...... 95
3.3.9.1 Equipment ...... 95
3.3.9.2 Chemicals ...... 96
3.3.9.3 Procedure of B‐complex vitamins analysis ...... 96
3.4 Physicochemical properties analysis ...... 100
3.4.1 Water holding capacity (WHC) ...... 100
3.4.1.1 Equipment ...... 100
3.4.1.2 Procedure of WHC analysis ...... 100
iv 3.4.2 Procedure for determining swelling capacity (SWC) and solubility analysis ....101
3.4.2.1 Equipment ...... 101
3.4.2.2 Procedure of SWC and solubility analysis ...... 101
3.4.3 Crystalline Index (CI) analysis ...... 102
3.4.3.1 Equipment ...... 102
3.4.3.2 Procedure of CI analysis ...... 102
3.5 Statistical analysis ...... 103
4. Drying of banana pseudostem ...... 104
4.1 Drying curve determination ...... 104
4.1.1 Effects of processing on the drying curve of Musa balbisiana banana
pseudostem ...... 104
4.1.2 Effects of species on the drying curve ...... 106
4.2 Effects of drying on the colour of banana pseudostem ...... 106
4.3 Water activity (aw) ...... 108
5. Effects of drying on the nutrient stability of banana pseudostem ...... 110
5.1 Proximate nutrients ...... 110
5.1.1 Moisture ...... 110
5.1.2 Ash ...... 112
5.1.3 Protein ...... 113
5.1.4 Fat ...... 113
5.1.5 Carbohydrates ...... 114
5.2 Micronutrients ...... 114
5.2.1 Minerals ...... 114
5.2.2 B complex vitamins ...... 118
5.2.2.1 Chromatography ...... 118
v 5.2.2.2 Method validation ...... 121
5.2.2.3 Quantitative analysis of the vitamins in the banana pseudostem ...... 124
6. Effect of drying on the carbohydrate digestibility of
banana pseudostem ...... 128
6.1 Sugars ...... 130
6.2 Starch ...... 135
6.2.1 HPLC Method ...... 136
6.2.2 Englyst Method ...... 137
6.2.3 Comparison between HPLC and Englyst methods for analysis of SDS, RDS and
RS of banana pseudostem ...... 143
6.3 Dietary fibre ...... 144
6.3.1 Enzymatic‐gravimetric method (Megazyme method) ...... 146
6.3.1.1 TDF ...... 147
6.3.1.2 IDF ...... 147
6.3.1.2 SDF ...... 148
6.3.2 NMR method ...... 149
6.3.2.1 Solid state NMR ...... 149
6.3.2.2 Solution state NMR ...... 151
6.3.3 Enzymatic‐chemical method (Englyst Method) ...... 154
6.3.3.1 Method validation ...... 154
6.3.3.2 NSP of banana pseudostem ...... 163
6.3.3.3 Comparison of NSP of banana pseudostem with commercial dietary fibre
supplement ...... 168
6.3.4 Dietary fibre method comparisons ...... 172
vi 7. Effect of drying on the physicochemical properties of
banana pseudostem ...... 176
7.1 Water holding capacity (WHC) ...... 178
7.2 Swelling capacity (SWC) and Solubility ...... 179
7.3 Crystallinity Index (CI) ...... 180
7.4 Pasting properties (RVA) ...... 184
8. Conclusions, recommendations and contributions ...... 189
8.1 Conclusions ...... 189
8.2 Recommendations ...... 191
8.3 Contributions to knowledge ...... 192
9. Reference ...... 193
10. Publications Derived from this Project ...... 221
11. Appendix ...... 222
vii List of Tables TABLE 2.1 THE TOP PRODUCERS OF BANANAS IN THE WORLD AND THE PRODUCTION IN AUSTRALIA IN 2012 ...... 4
TABLE 2.2 PRODUCTION BY STATE IN AUSTRALIA (TONNES) ...... 5
TABLE 2.3 PROXIMATE COMPOSITION OF BANANA PSEUDOSTEM ...... 10
TABLE 2.4 PROXIMATE COMPOSITION OF TENDER CORE OF BANANA PSEUDOSTEM FLOUR ...... 11
TABLE 2.5 MINERAL CONTENTS OF BANANA PSEUDOSTEM...... 13
TABLE 2.6 TYPICAL MODELS FOR DRYING SOLIDS ...... 29
TABLE 2.7 CLASSIFICATION OF THE PRINCIPAL DIETARY CARBOHYDRATES (CUMMINGS ET AL., 1997)...... 33
TABLE 2.8 PROPERTIES OF THE AMYLOSE AND AMYLOPECTIN IN STARCH ...... 36
TABLE 3.1 EQUIPMENT USED IN SAMPLE PREPARATION AND DRYING ...... 62
TABLE 3.2 EQUIPMENT USED FOR MOISTURE ANALYSIS ...... 64
TABLE 3.3 EQUIPMENT USED IN FAT CONTENT ANALYSIS ...... 65
TABLE 3.4 CHEMICALS USED FOR FAT ANALYSIS ...... 66
TABLE 3.5 EQUIPMENT USED FOR PROTEIN ANALYSIS ...... 67
TABLE 3.6 EQUIPMENT USED FOR ASH ANALYSIS ...... 68
TABLE 3.7 EQUIPMENT USED FOR SUGAR ANALYSIS ...... 70
TABLE 3.8 CHEMICALS USED FOR SUGAR ANALYSIS ...... 71
TABLE 3.9 EQUIPMENT USED FOR STARCH ANALYSIS ...... 72
TABLE 3.10 CHEMICALS USED IN STARCH ANALYSIS ...... 73
TABLE 3.11 STANDARD CONCENTRATION TABLE ...... 76
TABLE 3.12 EQUIPMENT USED IN DIETARY FIBRE ANALYSIS ...... 79
TABLE 3.13 CHEMICALS USED FOR DIETARY FIBRE ANALYSIS ...... 81
TABLE 3.14 CALIBRATION CONCENTRATION OF THE STANDARD SUGAR MIXTURE (MG/ L) ...... 91
TABLE 3.15 EQUIPMENT USED FOR MINERAL ANALYSIS ...... 93
TABLE 3.16 CHEMICALS USED FOR MINERAL ANALYSIS ...... 93
TABLE 3.17 EQUIPMENT USED FOR B‐COMPLEX VITAMINS ANALYSIS ...... 95
TABLE 3.18 CHEMICALS USED IN B‐COMPLEX VITAMINS ANALYSIS...... 96
TABLE 3.19 EQUIPMENT USED FOR WHC ANALYSIS ...... 100
TABLE 3.20 EQUIPMENT USED IN SWC AND SOLUBILITY ANALYSIS…………………………………………………….101
viii TABLE 3.21 EQUIPMENT USED FOR CI ANALYSIS ...... 102
TABLE 4.1 COLOUR OF DRIED GROUND BANANA PSEUDOSTEM (N= 3 ± SD) ...... 107
TABLE 4.2 WATER ACTIVITY (AW) OF MUSA BALBISIANA AND MUSA ACUMINATA ...... 109
TABLE 5.1 MEAN PROXIMATE CONTENTS OF MUSA BALBISIANA AND MUSA ACUMINATA...... 110
TABLE 5.2 MEAN MINERAL CONTENT (MG/100 G) OF MUSA BALBISIANA AND MUSA ACUMINATA ...... 115
TABLE 5.3 LINEARITY OF STANDARD CURVES AND SENSITIVITY FOR THE SEVEN B‐COMPLEX VITAMIN...... 121
TABLE 5.4 RESULT OF HPLC ANALYSIS OF B GROUP VITAMIN CONCENTRATIONS IN CERTIFIED REFERENCE MATERIAL
(BCR 485‐MIXED VEGETABLE) ...... 122
TABLE 5.5 INTER‐DAY (OVER A PERIOD OF 7 CONSECUTIVE DAYS) AND INTRA‐DAY (N=6) VALUES FOR THE B‐
COMPLEX VITAMINS ...... 124
TABLE 5.6 THE CONCENTRATION OF B‐COMPLEX VITAMINS (ΜG/G) IN BANANA PSEUDOSTEM DRIED IN DIFFERENT
CONDITIONS AND FROM DIFFERENT SPECIES ...... 126
TABLE 6.1 MEAN FRUCTOSE, GLUCOSE, SUCROSE AND TOTAL SUGAR CONTENT OF MUSA ACUMINATA PSEUDOSTEM
AND MUSA BALBISIANA PSEUDOSTEM (%, DWB) ...... 130
TABLE 6.2 RAPIDLY DIGESTIBLE STARCH, SLOWLY DIGESTIBLE STARCH, RESISTANT STARCH AND TOTAL STARCH
(ENGLYST METHOD & HPLC METHOD) OF MUSA ACUMINATA AND MUSA BALBISIANA PSEUDOSTEM (%, DB)
...... 135
TABLE 6.3 RDS, SDS, RS AND TS CONTENTS IN SOME CARBOHYDRATE‐ CONTAINING FOODS ...... 141
TABLE 6.4 RDS, SDS, RS AND TS CONTENTS IN SOME CARBOHYDRATE‐ CONTAINING FOODS ...... 142
TABLE 6.5 MEAN SDF, IDF, TF OF MUSA BALBISIANA PSEUDOSTEM AND MUSA ACUMINATA PSEUDOSTEM ...... 146
TABLE 6.6: CHEMICAL SHIFTS AND PORTION OF SUGAR FRACTIONS IN THE BANANA PSEUDOSTEM DIETARY FIBRE
EXTRACTION ...... 153
TABLE 6.7 GC DATA OF MONOSACCHARIDES IN STANDARD MIXTURE AND BANANA PSEUDOSTEM SAMPLES ...... 158
TABLE 6.8 CALIBRATION CONCENTRATION OF THE STANDARD SUGAR MIXTURE (MG/ L) ...... 159
TABLE 6.9 SENSITIVITY AND LINEARITY CHARACTERISTICS OF NEUTRAL SUGARS DETERMINATION IN THE PRESENCE
OF ALLOSE AS INTERNAL STANDARD (1 MG/ML) ...... 160
TABLE 6.10 INTER‐DAY (OVER A PERIOD OF 5 CONSECUTIVE DAYS) AND INTRA‐DAY (N=7) PRECISION AND
ACCURACY STUDY FOR THE DETERMINATION OF NEUTRAL SUGARS...... 162
TABLE 6.11 DISTRIBUTION OF NEUTRAL SUGARS (G/ 100 G OF DRY SAMPLE) IN THE SOLUBLE, INSOLUBLE AND
ix TOTAL FRACTIONS OF NSP FROM BANANA PSEUDOSTEM ...... 165
TABLE 6.12 INGREDIENTS IN COMMERCIAL DIETARY FIBRE SUPPLEMENTS ...... 171
TABLE 7.1 PHYSICOCHEMICAL PROPERTIES OF DRIED BANANA PSEUDOSTEM POWDER ...... 177
TABLE 7.2 PASTING PROPERTIES OF BANANA PSEUDOSTEM (BP40, BP40B, BP50, BP50B, BPA) MEASURED BY
RAPID VISCO ANALYSER ...... 185
x
List of Figures FIGURE 2.1 BANANA PSEUDOSTEM ...... 6
FIGURE 2.2 BANANA PLANT ...... 7
FIGURE 2.3 THE STRUCTURE OF BANANA PLANT ...... 8
FIGURE 2.4 THE INNER STRUCTURE OF BANANA PSEUDOSTEM ...... 9
FIGURE 2.5 TENDER CORE OF BANANA PSEUDOSTEM ...... 9
FIGURE 2.6 POLYPHENOL OXIDASE PATHWAY ...... 17
FIGURE 2.7 SIMPLIFIED MECHANISM FOR THE HYDROXYLATION AND OXIDATION OF DIPHENOL BY PHENOLOXIDASE17
FIGURE 2.8 OUTLINE OF THE CHEMICAL PATHWAY OF MAILLARD REACTION ...... 19
FIGURE 2.9 MOVEMENT OF MOISTURE ...... 26
FIGURE 2.10 DRYING CURVE ...... 27
FIGURE 2.11 BIOAVAILABILITY OF DIGESTIBLE AND NO‐DIGESTIBLE OLISACCHARIDES ...... 35
FIGURE 4.1 EFFECT OF PROCESSING ON THE DRYING RATIOS OF BANANA PSEUDOSTEM ...... 104
FIGURE 4.2 EFFECT OF SPECIES ON THE DRYING RATIOS OF BANANA PSEUDOSTEM ...... 106
FIGURE 5.1 CHROMATOGRAPHY OF STANDARD THIAMIN AND 5‐MTHF WITH PH ADJUSTMENT (A) AND WITHOUT PH
ADJUSTMENT (B) OF MOBILE PHASE A ...... 119
FIGURE 5.2 CHROMATOGRAPHY OF 7 B‐COMPLEX VITAMINS ...... 120
FIGURE 6.1 EFFECT OF DRYING CONDITION ON THE RATIO OF FRUCTOSE, GLUCOSE AND SUCROSE OF MUSA
BALBISIANA PSEUDOSTEM ...... 132
FIGURE 6.2 EFFECT OF BANANA SPECIES ON THE RATIO OF FRUCTOSE, GLUCOSE AND SUCROSE ...... 133
FIGURE 6.3 EFFECT OF DRYING CONDITIONS ON THE RATIO OF RAPIDLY DIGESTIBLE STARCH (RDS), SLOWLY
DIGESTIBLE STARCH (SDS) AND RESISTANT STARCH (RS) OF MUSA BALBISIANA PSEUDOSTEM ...... 138
FIGURE 6.4 EFFECT OF BANANA SPECIES ON THE RATIO OF RAPIDLY DIGESTIBLE STARCH (RDS), SLOWLY DIGESTIBLE
STARCH (SDS) AND RESISTANT STARCH (RS) ...... 140
FIGURE 6.5 PLOT OF HPLC METHOD VERSUS ENGLYST METHOD FOR TOTAL STARCH OF BANANA PSEUDOSTEM ... 143
FIGURE 6.6 TOTAL STARCH CONTENT OF BANANA PSEUDOSTEM DETECTED BY HPLC METHOD AND ENGLYST
METHOD ...... 144
FIGURE 6.7 A: 13C CP/MAS NMR SPECTRUM OF SOLUBLE DIETARY FIBRE OF DRIED BANANA PSEUDOSTEM
xi B: 13C CP/MAS NMR SPECTRUM OF INSOLUBLE DIETARY FIBRE OF DRIED BANANA PSEUDOSTEM ...... 149
FIGURE 6.8 13C CP/MAS NMR SPECTRA OF COMMERCIAL PECTINS ...... 150
FIGURE 6.9 CP/MAS 13C NMR SPECTRUM OF CELLULOSE ISOLATED FROM SWITCHGRASS FROM LITERATURE ..... 151
FIGURE 6.10 1H NMR SPECTROSCOPY OF BANANA PSEUDOSTEM TOTAL DIETARY FIBRE EXTRACTION ...... 152
FIGURE 6.11 CHROMATOGRAMS OF DERIVATISED MONOSACCHARIDES IN A STANDARD SOLUTION (A) AND BANANA
PSEUDOSTEM SAMPLE (B) ...... 156
FIGURE 6.12 CHROMATOGRAM OF (A) STANDARD WITH WRONG COMBINATION RATIO OF STANDARDS TO INTERNAL
STANDARD AND (B) SAMPLE WITH STALE AMMONIA SOLUTION ...... 157
FIGURE 7.1 XRD RESULTS OBTAINED FROM MUSA ACUMINATA (A), MUSA BALBISIANA (B) BANANA PSEUDOSTEM AND
METAMUCIL (C)...... 183
FIGURE 7.2 PASTING PROFILES OF BANANA PSEUDOSTEM ...... 184
xii
ACKNOWLEDGEMENTS
A journey is easier when you travel together. Throughout my two-year journey of experimental and writing processes, I have been accompanied and supported by many people. It is a pleasant aspect that I have now the opportunity to express my gratitude to all of them.
First, I would like to thank my parents for their financial support to ensure my excellent education abroad, their encouragement for me to learn new things and their mental support when I faced difficulties.
I would also like to express my deepest gratitude to my supervisors, Associate Professor
Jayashree Arcot and Dr. George Srzednicki, the School of Chemical Engineering,
Faculty of Engineering, UNSW. Their enthusiasm and integral view on research inspired my love for food science and research. Without their excellent guidance, support and mentorship, I would never have extended myself so far.
I thank Paul Nicholson, Sydney Royal Botanic Garden, Australia, for banana pseudostems supply. My sincere thanks to Mr. Lewis Adler for teaching me to use Gas
Chromatography and Dr. Maria Veronica Chandra-Hioe for teaching me to use High
Performance Liquid Chromatography. I thank them for their immense patience and valuable help on Chromatography method development.
I would like to acknowledge the lab manager Camillo for his assistance and my friends
Yiqing Zhao, Ji Liang, Xin Sun, Yannie Pan, Na Wang and Kitty Tang for their help, friendship and laughter in the labs. Finally, I would like to thank those closest to me who provide their emotional support, entertainment, time and advice.
xiii List of Abbreviations
5-MTHF 5-Methyltetrahydrofolate
ANOVA Analysis of Variance
Ara Arabinose aw Water Activity
BD Breakdown
BP40 Banana Pseudostem (Musa balbisiana) Dried at
40 C without Blanching
BP40B Banana Pseudostem (Musa balbisiana) Dried at
40 C with 3 min Blanching
BP50 Banana Pseudostem (Musa balbisiana) Dried at
50 C without Blanching
BP50B Banana Pseudostem (Musa balbisiana) Dried at
50 C with 3 min Blanching
BPA Banana Pseudostem (Musa acuminata) Dried at
50 C without Blanching
CHD Coronary Heart Disease
CP/MAS Cross Polarization Magic Angle Spinning
DF Dietary Fibre
FA Folic Acid
FID Flame Ionization Detector
Fuc Fucose
FV Final Viscosity
xiv Gala Galactose
GI Glycemic Index
GLC Gas Liquid Chromatography
Glu Glucose
GOD-PAP Glucose Oxidase
HDL High Density Lipoproteins
HPLC High Performance Liquid Chromatography
ICP-OES Inductively Coupled Plasma Optical Omission
Spectrometry
IDF Insoluble Dietary Fibre
IDL Intermediate Density Lipoproteins
IS Internal Standard
LDL Low Density Lipoproteins
LOD Limit of Detection
LOQ Limit of Quantification
LSD Least Significant Difference
Man Mannose
M Moisture content (dry basis)
Me Equilibrium moisture content (dry basis)
Mo Initial moisture content (dry basis)
NA Nicotinic Acid
NAM Nicotinamide
NIR Near Infrared Reflectance Spectroscopy
NMR Nuclear Magnetic Resonance Spectroscopy
xv NSP Non-starch Polysaccharide
POD Peroxidase
PPO Polyphenol Oxidase
PV Peak Viscosity
PYR Pyridoxine
RDI Recommend Daily Intake
RDS Rapidly Digestible Starch
Rha Rhamnose
RIB Riboflavin
RID Refractive Index Detection
RS Resistant Starch
RVA Rapid Visco Analyzer
RVU Rapid Visco Units
SAS Statistical Analysis System
SB Set Back
SDF Soluble Dietary Fibre
SDS Slowly Digestible Starch
SWC Swelling Capacity
TDF Total Dietary Fibre
TG Total Glucose
THF Tetrahydrofolate
THI Thiamin
TS Total Starch
TV Through Viscosity
xvi UV Ultraviolet
VLDL Very Low Density Lipoproteins
WHC Water Holding Capacity
Wf Final weight of the food material
Wo Initial weight of the food material
XRD X-ray Diffraction
Xyl Xylose
xvii 1. Introduction
Banana is a herbaceous plant of the family Musaceae. In terms of overall production, it is in the second place after citrus, accounting for about 16% of the world’s total fruit production (Deharveng et al., 1999). There are two wild species of banana, namely Musa acuminata and Musa balbisiana. Almost all modern edible parthenocarpic bananas come from these two species (Robinson
& Sauco, 2010). Therefore, these two species of bananas were chosen for this study. The stem of the banana plant, which is also called pseudostem produces a single bunch of bananas before dying and is replaced by a new pseudostem
(Anhwange et al., 2009). This crop generates a large amount of residue, due to the fact that each plant produces only one bunch of bananas. After the harvest, the bare pseudo-stem is cut and usually left on the plantation or burned, which could ultimately cause environment issues (Cordeiro et al., 2004). Thus the utilization of the banana waste—pseudostems has gained more attention in recent years. The banana pseudostem has been used as material for paper, furniture and forage (Buragohain et al., 2010; Umaz et al, 2005). Moreover, it has been reported that these banana waste materials are rich in minerals and nutrients, especially dietary fibre (Aziz et al., 2011). However, little is known on the composition of the pseudostem; its drying properties; effects of drying on composition; quality of dried product or its utilization in food manufacture and the characterization of fibre components. The banana pseudostem could potentially be used more in food rather than in other industries. The exploitation of waste banana pseudostems into products could significantly benefit the
1 environment and increase its economic value. This study focused on efficient utilization of banana pseudostem using drying techniques to achieve a dried product of high quality, nutrient composition and physico-chemical properties of fibre components.
2 2. Literature Review
2.1 The banana plant
2.1.1 Origin and world production
The origin of the banana plant is complex because of the nature of the banana’s taxonomic origins themselves. It is believed by the archeologists that the first domesticated banana was grown in New Guinea around 8,000 BC. From New
Guinea, the domesticated banana appears to have spread to the Philippines, and then radiated widely across the tropics. It took probably two millennia for the banana after domestication to arrive in India, Indonesia, Australia and
Malaysia. Plantains may have been grown in eastern Africa as early as 3,000
BC and in Madagascar by 1,000 BC. Buddhist literature records indicate that
Indian traders travelling through the Malaysian region had tasted the fruit and brought plants back with them in 600 BC. In 327 BC, when Alexander the Great and his army invaded India, he discovered banana crop and tasted the fruit in the Indian Valley. Then he introduced this new discovered crop to the Western world (De-Langhe, 1995).
By 200 AD banana had spread to China and grew only in the southern region of
China at that time. The Chinese never really popularised this fruit until the 20th
Century as they were considered to be a strange and exotic alien fruit. At about
650 AD, bananas began to be grown in Africa (Australian bananas, 2015).
Table 2.1 shows the production of banana worldwide and the production in
3 Australia. Foulkes et al. (1978) indicated that banana fruit occupied 25% of the total banana plant weight, while banana pseudostem occupied 61% of the weight (Pérez & Fujita, 1997). This means every year approximately 600,000 tonnes of banana pseudostem are produced in Australia. Additionally, in the top banana production country (India) 72 million tonnes of banana pseudostem are produced annually.
Table 2.1 The top Producers of bananas in the world and the production in Australia in 2012
Rank Country Production (Million of Tonnes) 1 India 29.7 2 Uganda 11.1 3 China 10.7 4 Philippines 9.2 5 Ecuador 8.0 6 Brazil 7.3 7 Indonesia 6.1 8 Colombia 5.1 9 Cameroon 4.8 10 Tanzania 3.9 … Australia 0.2
Source: Horticulture Australia (HAL, 2012) FAO, 2011
2.1.2 Banana industry in Australia
In comparison to the world banana production, Australian grown bananas do not contribute a major share. In Australia, bananas are grown in both tropical and subtropical regions. The banana industry in Australia is diverse in terms of the farming practices, the geographical location of banana farms, the size and
4 type of farms that grow bananas, the varieties of bananas grown and their flavour (Australian Bananas, 2015).
The distribution of banana production in Australia by state is shown in
Table 2.2. All states in Australia have banana production, but Queensland dominates the production. All fresh bananas available in Australia are grown locally. There are no imports due to stringent quarantine regulations. Thus, in order to solve the environmental issue caused by banana pseudostem, the studies on utilization of banana pseudostem is warranted in Australia.
Table 2.2 Production by state in Australia (tonnes)
Season NSW QLD WA NT Total
2009/10 10,749 279,805 *5,638 5,981 302,173
2008/09 *17,276 245,735 **5,886 **506 270,393
2007/08 *14,045 187,636 5,339 41 207,061
Source: Horticulture Australia (HAL) Notes: * Estimate has relative standard error of 10% to less than 25% and should be used with caution **Estimate has relative standard error of 35% to 50% and should be used with caution
2.1.3 Taxonomy of banana
Banana (Musa sp) belongs to the Musaceae family. Over 70 species of Musa were recognized by the World Checklist of Selected Plant Families, but only few species are edible. There are two wild species of banana, including Musa acuminata and Musa balbisiana. Almost all modern edible parthenocarpic bananas come from these two species (Valmayor et al., 1999). As a result, this
5 study focused on these two species. The hybrid of Musa acuminata and Musa balbisiana is named Musa paradisiaca (Valmayor et al., 1999).
2.2 Banana pseudostem
2.2.1 Introduction of banana pseudostem
Banana pseudostem (Fig 2.1 & Fig 2.2) is the stem of banana plant; it produces a single bunch of banana before dying and then is replaced by new pseudostem
(Anhwange et al., 2009). Since each plant produces only one bunch of bananas and cannot be used for the next harvest, this agricultural activity generates a large amount of residue (Cordeiro et al., 2004). It has been reported that banana is the second largest produced fruit in terms of quantity, contributing about 16% of the world’s total fruit production (Mohapatra et al., 2010).
Therefore, every year after harvesting, a large amount of bare pseudostem is cut and left behind as waste worldwide, which ultimately causes contamination of water sources as well as can affect the environment and health of living microorganisms (Aziz et al., 2011, Hossain et al., 2011).
Figure 2.1 Banana pseudostem
Source: Photograph taken by the author
6
Figure 2.2 Banana plant (Source: Photograph taken by the author) Hence, the exploitation of waste banana pseudostems into products could significantly benefit the environment and increase its economic value.
2.2.2 Structure of banana pseudostem
Figure 2.3 shows the structure of banana plant. The plant is normally tall and fairly sturdy, as a result is often mistaken for a tree. However, the trunk of the banana plant is actually a false stem or pseudostem (Stover & Simmond, 1972).
The pseudostem is normally 5 to 7.6 meters tall (varies from species to species) growing from a corm (Nelson et al., 2006). As shown in Figure 2.4, the pseudostem consists of a tender core and several outer sheaths. The tender core (Fig 2.5) inside the pseudostem carries the immature inflorescence until eventually it emerges at the top. Therefore most of the nutrients of the pseudostem are present in the tender core.
7
Figure 2.3 The structure of banana plant
Source: URL https://ferrebeekeeper.wordpress.com/tag/banana/
8 Outer sheaths
of banana Inner core of the
banana
Figure 2.4 The inner structure of banana pseudostem
Source: Photograph taken by the author
Figure 2.5 Tender core of banana pseudostem
Source: Photograph taken by the author
9 2.2.3 Nutritional value of banana pseudostem
2.2.3.1 Proximate nutrients
Banana pseudostem has very high content of dietary fibre. It has caught the attention of food scientists in recent years. It could be used more in food rather than in the feed industry. Aziz et al. (2011) and Bhaskar et al. (2011) researched the proximate composition of banana pseudostem. Tables 2.3 and
2.4 showed the proximate nutrient values from previous studies.
Table 2.3 Proximate composition of banana pseudostem
Nutrients Content (%)
Protein 2.5
Fat 1.7
Free sugar 3.4
Soluble dietary fibre 1.4
Insoluble dietary fibre 27.4
Starch 27.3
Ash 0.3
Moisture 15.1
Source: Bhaskar et al. (2011)
Note: All values are calculated on a dry weight basis
10 Table 2.4 Proximate composition of tender core of banana pseudostem flour
Nutrients Content (%)
Moisture 8.8
Fat 1.2
Protein 3.5
Ash 10.1
Crude fibre 19.5
Total carbohydrate 56.9
Source: Aziz et al. (2011)
Note: All values are calculated on a dry weight basis
The values from these two studies are considerably different to each other. One possibility is that some of the nutrients were underestimated from the study of
Bhaskar et al. (2011). For example, the ash value of that study was 0.3%. In contrast, Ho et al. (2012) reported that the total ash was 6.8% and mineral content including sodium, potassium, calcium, magnesium, phosphorus, iron, zinc and manganese was 3.1%. The total mineral value detected by the study was even more than 0.3%. Hence, it is reasonable to believe the ash measured in the study of Bhaskar et al. (2011) was underestimated. Another possibility is that the methods used in the two studies were different. The total carbohydrate obtained from the study of Bhaskar et al. (2011) was the sum of sugar, starch and dietary fibre, while the total carbohydrate was calculated as 100% minus moisture, fat, protein, ash and crude fibre in the study of Aziz et al. (2011). The study of Aziz et al. (2011) has some limitations. The crude fibre is part of total carbohydrate. Subtracting the crude fibre to calculate total carbohydrate
11 would underestimate the total carbohydrate value. The value of sugar, starch, dietary fibre could not be shown from this kind of calculation. The studies on the proximate nutrients of banana pseudostem are limited. Since carbohydrate is the main component of banana pseudostem, more accurate and detailed studies should be done on the carbohydrate composition to estimate the nutritional value of banana pseudostem.
Other factors such as species, maturation stages and measurement methods may all cause the differences in the nutrient values of the banana pseudostem.
For example, Saravanan and Aradhya (2011) claimed that total phenolics and total flavonoids in various solvent extracts of different banana pseudostem cultivars are different. They may range from 7.58 to 291 mg gallic acid equivalent and from 4 to 80 mg catechin equivalent, respectively.
Happi-Emaga (2007) stated that maturation of fruits involved increase in soluble sugar content and decrease in starch. Marlett & Vollendorf (1994) compared two methods, the AOAC method (Prosky et al., 1987) and Uppsala method
(Theander et al., 1990), to determine dietary fibre content in fruits. They found that the AOAC method always had greater concentration of dietary fibre than
Uppsala method, and the two data sets were significantly different.
2.2.3.2 Minerals
Minerals play an important role in maintaining proper function and good health in the human body (Bhat et al., 2010). Deficiency of minerals in the diet is always associated with increased susceptibility to infectious diseases due to the weakening of the immune system. The minerals of dried ground banana
12 pseudostem are shown in Table 2.5. The mineral content will be affected by maturation stage, species, and collection season and sample preparation
(Ho et al., 2012; Happi-Emaga et al., 2007; Lahav et al., 1985). For instance, with the maturation of the banana pseudostem, the Ca concentrations will increase, while the K will decrease, as a result of the presence of Ca in the tissues.
Table 2.5 Mineral contents of banana pseudostem
Minerals Content (mg/ 100 g dry sample)
Sodium 444.1
Potassium 944.1
Calcium 1335.3
Magnesium 255.0
Phosphorus 137.8
Iron 3.3
Zinc 8.1
Manganese 1.3
Source: Ho et al. (2012)
2.2.4 The utilization of the banana pseudostem
Nowadays, banana pseudostems are widely used in animal feeding, clothing and paper industry. Buragohain et al. (2010) claimed that banana pseudostem could be used as an important staple food for pigs in banana producing areas.
They can be used as feed material in both fresh and sun-dried forms and both whole or chopped forms. Starch extracted from banana pseudostem
13 could also be used to produce the glue used in the manufacture of cartons.
Umaz et al. (2005) reported that due to the fact that the fibre of banana pseudostems is widely recognized for its good qualities vs. synthetic fibres, it is used for making apparels, garments and home furnishing. Banana pseudostem has been seen as a kind of vegetable in some countries. For example, in India and Malaysia, the fresh tender core of banana pseudostem is cooked and consumed, whereas the consumption of banana pseudostem as food in
Australia is rare.
2.3 Pretreatment -- Anti-browning
Harvested banana pseudostem easily turns brown when it is exposed to air.
This browning reaction will change sensory properties and decrease nutritional quality of the banana pseudostem, hence, eventually reducing consumer acceptance. Since the browning reaction can affect the quality of food products mentioned above, it can cause considerable economic losses. Hence, in order to protect the original quality of banana pseudostem during processing and storage, the following paragraphs will review the causes of browning reactions and the prevention methods of these reactions in food processing.
2.3.1 Causes of browning
Browning reaction is one of the most important and common reactions that takes place during food processing and storage. There are three main causes of browning reactions in food processing—enzymatic browning, non-enzymatic browning and microorganism caused browning.
14 2.3.1.1 Enzymatic browning
It is estimated that enzymatic browning is the main cause in plant material and one of the most important colour reactions that affects the quality of fruits and vegetables, such as apple, banana and potato. After harvest, the cell wall of plant material is disrupted and the enzymes form brown or sometimes yellow, black or pink pigments (Caballero et al., 2003). Enzymatic browning is catalyzed by the enzymes polyphenol oxidases (PPOs), (Corzo-Martínez et al., 2012) and peroxidases (PODs) (Cano et al., 1997).
PPOs are a group of copper proteins, which are also referred to as phenoloxidase, phenolase, monophenol oxidase, diphenol oxidase and tyrosinase that are widely distributed from bacteria to mammals and are responsible for producing brown pigments, oxygen scavenging and act as a defense mechanism against plant pathogens. They make the tissues brown by catalyzing the oxidation of phenolics to quinones (Queiroz et al., 2008). PPOs are oxidoreductases that are able to oxidize phenol compounds. They act by employing oxygen as a hydrogen acceptor. The polyphenol oxidase pathway is shown in Figure 2.6. PPOs catalyze the hydroxylation to the o-position adjacent to an existing hydroxyl group of the phenolic substrate (monophenol oxidase activity) and oxidize diphenol to o-benzoquinones. Then the o-benzoquinones condense spontaneously with other o-benzoquinones, polyphenols and many other plant constituents, such as proteins and carbohydrates and eventually form high molecular weight polymers, such as melanoidin, which display brown and dark pigments in injured vegetable and fruit tissues (Mai & Glomb, 2013).
Figure 2.7 describes the simplified mechanism for the hydroxylation and
15 oxidation of diphenol by phenoloxidase. PPO, as a bi-functional enzyme, containing copper in its structure, has been described as oxygen and four electron-transferring phenol oxidase (Corzo-Martínez et al., 2012). In this process, it produces o-quinone.
Peroxidases are glycoproteins with a hematin compound as cofactor. The mechanism of peroxidase oxidation is to oxidize hydrogen donors at the expense of peroxides. Although they are highly specific for hydrogen peroxide, they accept a wide range of hydrogen donors, including polyphenols. Following equations show the overall catalyzed reaction of POD.
AH2 + H2O2 == A + 2H2O
2AH + H2O2 == AA + 2H2O2
Peroxidases are widely distributed, especially in plants, but it is implied that they generally appear to be little involved in enzymatic browning of fruits and vegetables following mechanical stress (Caballero et al., 2003). Thus, PPOs are the main enzymes that should be considered in enzymatic browning protection.
16 Figure 2.6 Polyphenol oxidase pathway (Source: Corzo-Martínez et al., 2012)
Figure 2.7 Simplified mechanism for the hydroxylation and oxidation of diphenol by phenoloxidase Source: Corzo-Martínez et al., 2012
2.3.1.2 Non-enzymatic browning
Non-enzymatic browning is one of the most complex reactions in food chemistry since a large number of food components are participating in this reaction through different pathways, which gives rise to a complex mixture of products
(Corzo-Martínez et al., 2012). The non-enzymatic browning is produced by heat treatment, including a wide number of reactions such as Maillard reaction, caramelisation, chemical oxidation of phenols and maderisation
17 (Manzocco, 2000). These reactions can promote nutritional changes such as loss of nutritional quality. For instance, they contribute to the destruction of essential amino acids, reduction of protein digestibility and amino acid availability. Maillard reaction is the most important non-enzymatic browning reaction. It occurs as a result of the reaction between free amino groups from amino acids, peptides or proteins and the carbonyl group of a reducing sugar. In this stage, the reaction loses a molecule of water and forms glycosylamines.
Then, the Amadori rearrangement produces Amadori compounds followed by breakdown to form degradation products—sugar reductones. Finally, nitrogen- containing brown polymers and copolymers known as melanoidins are formed
(Lee & Whitaker, 1995). Figure 2.8 shows the outline of the chemical pathway of Maillard reaction.
18
Figure 2.8 Outline of the chemical pathway of Maillard reaction
Source: Caballero et al., 2003
2.3.2 Browning prevention methods
Since browning, especially enzymatic browning affects the quality of numerous plant organs which are rich in oxidizable phenols such as fruits and vegetables and decrease their economic returns in the food industry, measures should be taken to protect both nutritional and economical value of foods subjected to browning. There are various methods that can be used for browning
19 prevention. They can be divided into two categories—physical methods and chemical methods. Various methods in these two categories will be reviewed in this section.
2.3.2.1 Physical methods of prevention
Although there are several methods to prevent enzymatic browning, they have the same principle, which is to reduce or inactivate the enzyme, and thus to prevent browning. As a prevention method, physical methods reduce or inactivate enzyme activity by controlling the external environment, such as temperature and oxygen level.
2.3.2.1.1 Temperature control
Freezing
Freezing (below -18°C) is estimated to block colour changes due to the fact that enzymes such as PPOs could be inactive under this condition. However, when the temperature rises, browning starts again, and will be even more visible if the cellular structures of the plant organ have been severely damaged by freezing, chemical peeling and slicing (Caballero et al., 2003). Cano et al. (1997) support this viewpoint by indicating freezing/thawing processes, which produced a significant increase in phenol levels due to cellular breakdown.
Blanching
Heat treatment or blanching is believed to be one of the simplest and most direct methods of enzyme inactivation and inhibition of colour deterioration, due to the fact that blanching could reduce the microbial load and inactivate
20 deleterious enzymes, such as PPOs and PODs. Castro et al. (2008) implied that thermal blanching treatments could progressively decrease PPO activities from 25% to 75% in green peppers. Additionally, Cano et al. (1990) demonstrated that blanching peeled bananas in boiling water produced significant inactivation of PPO and POD (96-100%). Bahçeci et al. (2005) also agreed that blanching could inactivate enzymes. They found that a blanching treatment at 90 °C for 3 minutes was able to inactivate 90% of the activities of
PODs in green beans.
There are two major methods that are widely used in the food industry for blanching. One is hot water blanching and the other is steam blanching. Hot water blanching is achieved by plunging vegetables or fruits into hot water to inactive enzymes, while steam blanching is accomplished by utilizing the heat from steam to inactive enzymes. Compared with hot water blanching, steam blanching uses less energy and water. Johnson (2011) illustrated that when steam blanching was applied directly to the food products, it used approximately half the amount of water vs. that used in water blanching. It could save approximately $4000- $8000 in energy cost by steam blanching one million lbs
(454 metric tons) of carrots or peas than water blanching. Moreover, steam blanching achieved better nutrient retention than water blanching. The nutrient loss of steam blanching is only one third of hot water blanching (Lee &
Whitaker, 1995). When food enters hot water, some soluble nutrients such as soluble vitamins may dissolve in the hot water and the nutrients in the hot water blanched food could be lost. Because steam blanching minimizes the leaching of soluble solids, this method could leave more nutrients and natural
21 sugars in food products, thus it could improve flavour retention and colour retention to produce a final product with better flavour, texture and colour than hot water blanching (Johnson, 2011).
However, blanching still has some disadvantages. Over-heating causes loss of sensory (texture, taste, flavour and colour) and nutritional quality attributes
(Castro et al., 2008). Jackson et al. (1996) blanched whole green bananas at
50, 60,70,80,90 and 100 °C for 2, 15 and 30 minutes, then fried peeled and sliced banana to make chips. They suggested blanching for 22 minutes at 69 oC as the optimized blanching condition for crispness of banana chips. If the blanching temperature is higher than 69 °C and time is longer than 22 minutes it would have negative effects on crispness. Castro et al. (2008) stated that ascorbic acid content decreased progressively as blanching conditions were more severe. The higher the temperature and longer treatment time, the lower the ascorbic acid content in the samples. The retained ascorbic acid content in green and red peppers was 45% and 30% respectively. Therefore, blanching temperature and time should be well controlled. The optimal temperature and duration time is the condition that could both inhibit enzyme activities and maintain the quality of food samples.
2.3.2.1.2 Other physical methods
Other techniques, such as high pressure, ultrasound and nitrogen preservation, are also used in the food industry to protect the colour of foodstuff. Palou et al.
(1999) indicated that peroxidase, catalase, phosphatase and PPOs, which cause browning, were resistant to pressures of 600-700 MPa at 25 °C.
22 Caballero et al. (2003) stated that browning can also be prevented by keeping the food products in low oxygen atmospheres. Nowadays, food industry uses the carbon dioxide or pure nitrogen atmospheres to protect freshness of fruits and vegetables against enzymatic browning. Between these two gases, nitrogen provides better protection of the original flavour and aroma. Segovia-
Bravo et al. (2012) measured damaged olives by keeping them in N2 atmosphere for 24 h. They found that the browning of bruised areas of the olives was very similar to that suffered by healthy olives maintained in the air, which meant the N2 atmosphere was able to prevent the browning. However, N2 atmosphere is not suitable for any food. There is evidence showing that treatment of bananas in N2 atmosphere is not effective (Segovia-Bravo et al.,
2012).
2.3.2.2 Chemical prevention
Chemical prevention utilizes the chemical reaction of compounds, which cause browning, and the functional group of some compounds, acting as antioxidant agents, enzyme inhibitors, acidulants, chelating agents or complexing agents, are used to inhibit browning (Queiroz et al., 2011). Some of the compounds could also prevent both enzymatic and non-enzymatic browning. Despite the fact that some compounds, such as sulfur dioxide, have significant effects on colour and flavour preservation, they cause safety issues and thus are forbidden in many countries. In the case of plant foodstuffs, the legislation in most countries allows only ascorbic acid and its derivatives, sodium chloride and, within stricter limits, citric acid (Caballero et al., 2003) to be used for this purpose. The browning inhibition effects of some chemicals and their
23 synergistic effects will be reviewed in the following paragraphs.
It has been reported that ascorbic acid showed strong inhibition of PPO, causing full enzyme inactivation even at low concentration. Ascorbic acid with its first oxidation product dehydroascorbic acid, which constitutes vitamin C, reduces o-quinones progressively as they are formed (Caballero et al., 2003).
Ünal (2006) proved that ascorbic acid at 0.2 mM and 0.8 mM resulted in 99% and 100% inhibition of banana PPO, respectively (Queiroz et al., 2011). Täufel and Voigt (1964) claimed that concentrations between 0.5 to 1% of sodium chloride had an inhibiting effect on the enzymatic browning of whole apples or apple pieces, whereas Pizzocaro et al. (1993) argued that sodium chloride in concentrations between 0.2 g/L to 1 g/L activated PPO and 1 g/L even increased PPO activity by about 90%.
The synergistic effect of different compounds for browning inhibition is stronger than that of single compounds. Pizzocaro et al. (1993) studied the synergistic effect of ascorbic acid and citric acid plus ascorbic acid and sodium chloride.
They discovered that both citric acid and sodium chloride increased the inhibiting effect of ascorbic acid. They concluded that compared with citric acid, sodium chloride is more efficient. Adding 0.5 g/L of sodium chloride instead of 2 g/L of citric acid to 10 g/L of ascorbic acid showed that the PPO inhibition was
100% instead of 87%.
24 2.4 Drying
Drying is one of the most effective methods used to preserve fruit and vegetables, because fruits and vegetables have high moisture and this method can remove the moisture in food by evaporation. The rate of microbial activity and other deteriorative reactions in fresh fruit and vegetables therefore can be slowed. Also, other important advantages include nutritive value (66-90% carbohydrate) maintenance and shelf life prolongation (Hui et al., 2006); reduction of transportation and storage costs and the convenience of use
(Fellows, 2002). Drying processes are also reported as causing modification to the physical properties of the fibre matrix and affect the hydration properties as well (Dhingra et al., 2012). Different drying conditions may also cause a difference in food quality. If the drying time is too long or the drying temperature is too high, the quality of the food including nutritional values, colour, taste etc. will be negatively affected. Thus, appropriate drying conditions should be chosen during food preservation. In this section, the principle of drying, drying curve, factors that will affect drying rate as well as effect of drying conditions will be presented.
2.4.1 Drying principle
Drying is a process that utilizes the dry air to evaporate bound water from inside the fresh food into the atmosphere. Breaking water bonds, releasing and transferring heating connected to phase change all need energy, including convective (warm air), contact (cooled surface), radiative (infrared rays), and excitation (microwave) energies (Hui et al., 2006). There are two basic
25 mechanisms involved in the drying process; one is the migration of moisture from the interior of the food to the surface and the other is the evaporation of moisture from the surface to the surrounding air. When hot air is blown over a wet food, water vapor diffuses through a boundary film of ambient air and is carried away by the moving air (Figure 2.9). A water vapour pressure gradient is established from the moist interior of the food to the dry air, thereby most moisture in food could be removed (Fellows, 2002).
Figure 2.9 Movement of moisture
(Source: Fellows, 2002) There are three periods characterizing for drying rates: initial rate period, constant rate period and falling rate period (Figure 2.10). Initial rate period is a short initial settling down period as the surface of the food heats up to wet bulb temperature. At this stage, the drying rate keeps increasing. Constant rate period is the stage where the water moves from the interior of the food at the same rate as it evaporates from the surface. This step stays till the critical moisture content is reached. The unbound water is removed from the food product. The evaporation is not dependent on the solid matrix and does not end
26 until water from the interior is no longer available at the food surface
(Geankoplis, 2003). Falling rate period is the last and the longest part of drying operation. The drying rate falls down slowly until the moisture content in the product reaches the humidity of the air in the dryer. This moisture content of the product is called equilibrium moisture content (Fellows, 2002).
Figure 2.10 drying curve
(Source: Goyal, 2013) 2.4.2 Factors that affect drying rate
2.4.2.1 Air properties
The properties of the air flowing around the food being dried are a major factor in determining the rate of drying. Air temperature, air humidity and air velocity are three key properties of air that affect the drying rate (Fellows, 2002). Low temperatures impede evaporation of moisture from food, while high
27 temperatures promote drying rate. Sacilik and Elicin (2006) studied the relationship between drying temperature and drying rate of 5 mm thickness apple slices. They stated that an increase of drying air temperature from 40 to
60 °C resulted in a decrease of drying time from 400 minutes to 240 minutes.
Meanwhile, Doymaz (2007) proved that the increase in the air temperature in the range 55 to 70 °C significantly increased the drying rate of tomatoes. High relative humidity retards evaporation, whereas high velocity of air speeds up drying, because fresh air passing over a wet food helps removal of moisture.
2.4.2.2 Physical properties of food
The drying rate is affected by different physical properties of food such as the size, composition, structure, and moisture content of food to be dried. For example, for foods that have the same volume, the one in small pieces and larger surface area has quicker drying rate than the one with small surface area as a result of shorter distance for moisture to travel through the food.
2.4.2.3 Other factors
Other factors such as blanching, freezing, osmotic pretreatment will also affect the drying rate. Agarry et al. (2005) concluded that freezing and increasing freezing time could significantly increase the drying rate and therefore decrease the drying time of potatoes. Similarly, Dandamrongrak et al. (2003) claimed that freezing and combined blanching and freezing could significantly increase drying rate. The drying times of samples subjected to freezing or subjected to blanching combined with freezing were 22.4 and 27.2 hours respectively. This is about half the drying time of untreated samples.
28 2.4.3 Drying models
Various drying models were developed for different foods owing to their difference in moisture content and transport phenomena during drying. Drying models are very useful in prediction of drying times under particular process conditions; therefore they are widely used in the drying process. Four typical models—Page’s model, Henderson and Pabis’ model, logarithmic’s model and two-term exponential model of drying solids and some equations for drying are described below (Table 2.6).
Table 2.6 Typical models for drying solids
Model Names Model Equation
Page MR = exp ( ktn)
Henderson and Pabis MR = a exp ( ktn)
Logarithmic MR = a exp ( ktn) + c
Two-term exponential MR = a exp ( ktn) + (1-a) exp ( kt)
(Source: Agarry et al., 2013)
Where a, c, n are empirical constants; k is drying constant; t is drying time; MR is moisture ratio
Moisture content (wet basis); equation (2-1)
Wo Wf MC (2-1) Wo
Where, Wo and Wf is the initial and final weight of the food material (g).
29 • Moisture ratio (MR); equation (2-2)
(2-2)
Where, M is Moisture content (dry basis)
Me is Equilibrium moisture content (dry basis)
Mo is Initial moisture content (dry basis)
Since the equilibrium moisture content (Me) is relatively small compared to moisture content of the sample at each moment (M) and initial moisture content
(Mo), the term (M-Me)/(Mo-Me) can be simplified as M/Mo.
2.4.4 Effects of drying conditions
Control of temperature and mass transfer in drying process is important to enhance product quality, such as colour and flavour. Although high drying temperature could improve the drying rate and decrease drying time in food processing, it also has an adverse effect on food quality. It has been reported that lower drying temperature should maintain original colour of fresh apple slices (Sacilik & Elicin, 2006). However, the studies on the influence of drying conditions on the contents of nutrients are limited. Therefore, more studies in this field should be done.
2.5 Carbohydrates
Carbohydrates, the main source of the human diet, represent the primary
30 energy source, contributing to nearly 55-70% of the total energy consumption
(Osorio-Diaz et al., 2002). Their nutritional energy value amounts to 17 kJ/g.
The function of carbohydrates in food includes sweetening, gel or paste-forming action, thickening agents and stabilizers. Moreover, the carbohydrates are precursors for aroma and coloring substances in the food industry, especially in thermal processing (Belitz et al., 2009). For instance, the Maillard reaction and caramelization both require carbohydrate as reagents. The non-digestible carbohydrates, such as the non-starch polysaccharides acting as bulk materials, are of importance to balanced daily nutrition. Food sources such as cereals, fruits, vegetables and legumes are all rich in carbohydrates.
2.5.1 Carbohydrate chemistry
Carbohydrates are major components of food with diversified structure and functionalities. Carbohydrates are commonly divided into 3 main groups, including mono- and di-saccharides, oligosaccharides and polysaccharides according to their molecular weight. However, the classification based on carbohydrate chemistry purely does not indicate a ready guide to their importance for health. As a result, physiological properties are also considered into the classification of carbohydrate. The 3 main groups are then subdivided on the basis of the types of monosaccharide that are consistent, with a view to keeping broadly similar physiological types together with the subgroup
(Cummings et al., 1997). This classification of food carbohydrates
(Table 2.7) not only presents the chemistry of the carbohydrates, but also implies their digestibility, which reflects their health properties of it.
31 2.5.1.1 Mono- and di-saccharides
Free sugars are the simplest form of carbohydrate, which are present in fruits and vegetables. Mono-saccharides, such as glucose and fructose, are the basic units to form other types of carbohydrates and cannot be further hydrolyzed.
According to the chain length of the mono-saccharides, they can be divided into pentoses (5 carbon atoms) and hexoses (6 carbon atoms). Disaccharides contain two monosaccharide units linked by glycosidic bonds. Sucrose is usually the most abundant disaccharide in the diets (Asp, 1995), which consists of a molecule of α-glucose and β-fructose. Mostly, mono- and disaccharides are rapidly digested and absorbed in the small intestine and provide a ready source of energy.
32 Table 2.7 Classification of the principal dietary carbohydrates (Cummings et al., 1997)
Major classes Sub‐groups (type of Physiology (DP) monosaccharide and or bonds) Sugars (1±2) (i) Monosaccharides Absorbed from small intestine Glucose, fructose Glucose and sucrose give rapid glycemic responses (ii) Disaccharides Sucrose, maltose, trehalose Absorbed Lactose Lactose is fermented in many populations (iii) Sugar alcohols Sorbitol, maltitol, lactitol Poorly absorbed and partly fermented Oligosaccharid (i) Malto‐oligosaccharides (a) Digestible ‐ digested and absorbed from small intestine and give rapid glycemic response es (3±10) (‐glucan) (b) Resistant ‐ Pass into the large intestine and may be fermented. (ii) Other oligosaccharides (NDO) Fructooligosaccharides Fermented. Some selectively stimulate growth of bifidobacteria in large bowel Polysaccharide (i) Starch (‐glucans) (a) Digestible ‐ varying rates of digestion and glycemic responses s (10)a (b) Resistant ‐ not absorbed in small bowel. May be fermented, and affect large bowel function (ii) Non starch polysaccharides (a) Cell wall‐contribute to regulation of carbohydrate digestion in small bowel. Fermented (NSP) mostly but dependent on cell wall structure; major determinant of large bowel function; provide physical structure to plant foods (b) Non cell wall ‐ fermented to a variable degree. Varying effects on carbohydrate and lipid absorption and in the large bowel DP = Degree of Polymerisation or the number of monosaccharide units which make up the molecule. For isolated (synthetic) oligosaccharides used as food ingredients DP refers to the average value; NDO = Non-digestible oligosaccharides. a) In practice the division between oligosaccharides and polysaccharides is best made on the basis of solubility in 80%v/v ethanol. This group will thus contain carbohydrates of DP greater than 10. If IUB-IUPAC maintain the current definition of oligosaccharides then it will be necessary to find a new name for this group, such as short chain carbohydrates. 33 2.5.1.2 Oligosaccharides
Oligosaccharides are saccharide polymers containing 3 to 9 simple sugar units and can be only partially digested by humans. The most commonly known oligosaccharides are mainly polymers of fructose and galactose. They are found in plant seeds, especially legumes such as beans and peas. According to the digestibility, oligosaccharides can be further divided into digestible oligosaccharides and non-digestibility oligosaccharides (Fig. 2.11). Digestible oligosaccharides are hydrolyzed in the upper part of the gastrointestinal tract and form monosaccharides. The monosaccharides are transported through the portal blood to the liver and eventually to the systemic circulation. The non- digestible oligosaccharides pass into the large bowel as they are eaten
(Delzenne and Roberfroid, 1994).
2.5.1.3 Polysaccharides
Polysaccharides are carbohydrates containing 10 or more monomeric residues, which can be further divided into starch and non-starch polysaccharides (NSP).
2.5.1.3.1 Starch
Starch is the major carbohydrate in the human diet, which occupies 80-90 % of all polysaccharides eaten (Cummings et al., 1997). The starches are storage polysaccharides and are defined as polymers of glucose linked with α-glucosidic linkage (Englyst et al., 1994).
34
Figure 2.11 Bioavailability of digestible and no-digestible oligosaccharides
Source: Delzenne and Roberfroid, 1994
35 According to the chemical structure, starch is a mixture of two large components in different proportions, including amylose and amylopectin. Table
2.8 summarizes the properties of the amylose and amylopectin fractions in starch. Amylose is a -1, 4-linked D-glucose. It contains 200-10,000 anhydroglucose units and this molecular size varies depending on the plant source and maturity. Amylopectin is starch, a branched polymer consisting of -
1, 6 linked D-glucose and its molecular weight exceeds 107 (Gallant et al.,
1992). Amylopectin dominates in the crystalline regions in the starch granule in comparison with the amorphous structure of amylose (French, 1984; Zobel,
1992). Most starches have amylose content of about 25% and amylopectin content of about 75% (Bemiller and Whistler, 1996). Starches that are made up of more than 99% amylopectin are waxy starches.
Table 2.8 Properties of the amylose and amylopectin in starch
Property Amylose Amylopectin
Molecular structure Linear (- 1,4) Branched (- 1,4 and -1,6)
Gels Stiff, irreversible Soft, reversible
Iodine colour Blue Red-purple
Digestibility, - 100% 60% amylase
Molecular weight (Da) 106 107 - 109
Source: Zobel, 1988
There are three crystalline structures of starch granules in the plant, namely A,
B and C, as distinguished by their X-ray diffraction patterns. A structure mostly
36 formed in cereal starches, where water molecules are located between starch molecules forming double helices. Potato and other tuber starches mostly have the B structure; the double helices are packed densely in a hexagonal pattern with water molecules only inside this structure. C pattern is mostly found in legume starches (Colonna et al., 1992).
In 1992, Englyst et al. further divided starch into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS), according to their digestibility in the intestine (Table 2.9).
Table 2.9 In vitro nutritional classification of starch (Source: Englyst et al., 1992)
Type of starch Example of occurrence Probable digestion in
small intestine
RDS Freshly cooked starchy food Rapid
SDS Most raw cereals Slow but complete
RS
1. RS1 Partly milled grans and Resistant
seeds
2. RS2 Raw potato and banana Resistant
3. RS3 Cooled, cooked potato, Resistant
bread and corn flakes
4. RS4 Commercially manufactured Resistant
starches
RDS = rapidly digestible starch; SDS = slowly digestible starch; RS= resistant starch; RS1 = physically inaccessible starch; RS2= resistant starch granules;
RS3 = retrograded starch; RS4 = chemically modified starch
37 It has been once assumed that starch hydrolyzed and absorbed more slowly than sugars, because they have larger molecular sizes than sugars. However,
RDS is rapidly digested and gives raise to blood glucose responses similar to or even greater than sugars (Wolever & Miller, 1995). On the other hand, SDS and
RS can slow gastric emptying; reduce the glycemic index and insulin responses. This character is important for diabetes (Cummings et al., 1997).
2.5.1.3.2 Non-starch polysaccharides (NSPs)
NSPs are linear β-glucans and a range of heteropolysaccharides without α- glucosidic linkage, which are mostly the plant cell walls. They are found in all cereals, vegetables and fruits (Blackwood et al., 2000; Englyst et al., 1994).
According to their solubility, NSP can be divided to soluble NSP and insoluble
NSP. The NSP cannot be digested in the small intestine because human digestive enzymes can only cleave α - (1→ 4) glucan bonds (Nielsen, 2010).
The NSP offers health benefits as well. They are fermented by microbes in the large intestine (Bhaskar et al., 2011). Due to their high water binding capacity,
NSP play an important role in providing bulk to gut contents and allow easy passage through the human intestine. Thus, they play a crucial role for the correct functioning of the digestive system (Nielsen, 2010; Kumar et al., 2012).
2.5.2 Potential health benefits of carbohydrates
It has been stated in recent decades that some types of carbohydrates, especially non-digestible carbohydrate has some benefits on the chronic diseases. According to World Health Organization (WHO), chronic diseases, such as heart disease, stroke, cancer, chronic respiratory diseases and
38 diabetes, are by far the leading causes of mortality in the world, representing
63% of all deaths. Non-digestible carbohydrate, such as resistant starch and dietary fibre may be the constituents that could protect human beings from these modern diseases. Dietary fibre has been shown to have numerous benefits, which are not easily disputed like improving intestinal function, cholesterol reduction and increasing microbial biomass. Other benefits of fibre and resistant starch include weight reduction, satiety, regulating glucose and insulin responses, improvement as well as modification of microbiota composition, inflammatory markers such as C-reactive protein, interleukin 6 and tumor necrosis factor alpha in obesity and diabetes (Cui & Roberts, 2009).
Long-term intake of the non-digestible carbohydrate could decrease the risk of some chronic disease, such as diabetes mellitus, obesity, coronary heart disease (CHD) and hyperlipidemia. The physiological effects of carbohydrates as well as their relationship to some diseases will be reviewed in this section.
2.5.2.1 Hyperlipidemia
Hyperlipidemia means abnormally high levels of any or all lipids and/ or lipoproteins in the blood, which may be physiological increase after meal.
People now pay more attention to this disease since it could cause other chronic diseases, such as coronary heart disease, stroke, peripheral vascular disease and atheroma. Pathological hyperlipidemia results from disordered metabolism of the lipoproteins with either excess production, altered clearance, or both excess production and altered clearance (Caballero et al., 2003).
Hyperlipidemia normally refers to high concentrations of the blood lipids and lipoproteins, cholesterol and triglyceride. Lipoproteins include very
39 low density lipoproteins (VLDL), low density lipoproteins (LDL), intermediate density lipoproteins (IDL) and high density lipoproteins (HDL).
It has been reported that the proper diet treatment can preserve and reduce the risk of hyperlipidemia. Although a high digestible carbohydrate intake, such as refined carbohydrate food intake and sugar intake may increase hypertriglyceridemia, however non-digestible carbohydrate may help to protect from hyperlipidemia. Kim et al. (2003) studied the effect of resistant starch on hyperlipidemia actions in diabetic rats. They found that the total plasma lipid and cholesterol concentrations were significantly lower in the resistant starch treated rats than the diabetic control ones. Furthermore, some forms of dietary fibre are also believed to have positive effect on lowering blood lipids, total cholesterol and LDL cholesterol (Leeds, 2005). Soluble dietary fibre like pectin, guar gum and gum may lower both serum cholesterol and triglycerides (Dhingra et al., 2012). Brown et al. (1999) studied the cholesterol lowering effects of dietary fibre. The results showed that intake of 2 to 10 grams of soluble dietary fibre every day was associated with small but significant fall in total cholesterol
[-0.045 mmol/L· g soluble fibre (95% CI: -0.054, -0.035)] and LDL cholesterol [-
0.057 mmol/L · g (95% CI: -0.070, -0.044)]. Leeds (2005) claimed that intake of food products providing more than 3 g of soluble fibre every day had a greater blood cholesterol lowering effect.
2.5.2.2 Diabetes mellitus
Diabetes mellitus is a metabolic disorder involving impaired glucose homeostasis due to either failure of secretion of the hormone insulin
40 (insulin-dependent or type I diabetes) or impaired responses of tissues to insulin
(non-insulin-dependent or type II diabetes). If untreated, the blood concentration of glucose rises to abnormally high levels (hyperglycaemia) after a meal and glucose is excreted in the urine (glucosuria). Prolonged hyperglycaemia may damage nerves, blood vessels, and kidneys, and lead to development of cataracts, so effective control of blood glucose levels is important
(Bender, 2009). Diabetes has been a growing public health problem worldwide.
In 1985 there were approximately 30 million people living with diabetes worldwide, this estimate increased to 171 million in 2000 and is projected to be around 366 million persons in 2030 living with this disease
(Cui & Roberts, 2009). In Australia, the prevalence of diabetes more than doubled from 1.5% to 5.1% of Australians from 1989 to 2008. A large body of evidence has demonstrated that the presence of diabetes doubles the risk of a wider range of vascular diseases. In addition, diabetes is moderately associated with death from cancers of the liver, pancreas, ovary, colorectum, lung, bladder and breast (Dong et al., 2011). Numerous reports claimed that dietary therapy remains a cornerstone of treatment and management of type II diabetes. Thus preventive foods are urgently needed to reduce the huge burden of diabetes.
2.5.2.2.1 Glycemic index (GI)
GI is the measurement of an individual’s glucose response to a known quantity of available carbohydrate from a test food, in comparison to that person’s glycemic response from a standard food, usually glucose or white bread. Low
GI is believed to indicate a corresponding low glucose and insulin response (Cui
& Roberts, 2009). The GI is considered as a quantitative
41 classification of carbohydrate foods based on the absorption rate of carbohydrate as reflected in the glycemic response (Wong & Jenkins, 2007). It has been proven to be a robust tool to determine the relative glycemic responses of both single and mixed meals (Miller, 1994). Thus, it has now been used as a nutrition strategy to prevent and manage chronic diseases.
Epidemiological studies implied that low GI diets may reduce the risk of coronary heart disease, diabetes and certain cancers
2.5.2.2.2 The effects of carbohydrates on diabetes
Consumption of digestible carbohydrates is considered with glycemia postprandial, while a high level of non-digestible carbohydrate consumption is believed to resist the raising of blood glucose after meal. One hypothesis indicated that the colonic fermentation by the resident microflora of non- digestible carbohydrates, including fructo-oligosaccharides, resistant starch,
NSP, pectins and gums, may affect glucose and lipid metabolism. The major nongaseous by-products of this anaerobic fermentation are short chain fatty acids, which are rapidly absorbed by the colonic epithelia with significant quantities of acetate and propionate entering the portal blood stream. The acetate may lower plasma nonesterified fatty acids levels. It has been implied that elevated plasma nonesterified fatty acids levels may contribute to the impairment of insulin action (Cho, 2001). Thus the effect of acetate to lower the level of plasma nonesterified fatty acid may prevent the insulin action from impairing.
Behall et al. (2006) stated that resistant starch has the effects to lower the
42 plasma glucose and insulin responses of both normal and overweight women.
On the one hand, some food scientists support the idea that insoluble dietary fibre could decrease the risk of type II diabetes. Meyer et al. (2000) studied the relations of baseline intake of dietary fibre with incidence of diabetes of 35988
Iowa women aged 55-69 for 6 years. The result showed that, whole-grain, total dietary fibre and cereal fibre had strong inverse associations with incidence of diabetes after adjustment, which indicated a protective role of total dietary fibre in the development of type II diabetes in older women. However, the study found that the soluble fibre had no significant association with type II diabetes; therefore fibre from fruit and vegetables was unrelated to diabetes risk. The research of Wolever (1990), who studied the relationship of dietary fibre in twenty-five foods and their GI, had similar results. The result of Wolever’s study showed that total dietary fibre was significantly related to GI and soluble fibre was not significantly related to GI.
On the other hand, some researchers claim that soluble fibres had greater effects on glycemic responses than do insoluble fibres. Chandalia et al. (2000) studied the effect of increasing the intake of dietary fibre on glycemic control in twelve men and a woman patient with type II diabetes. They found that an increase in the intake of total dietary fibre, which consisted predominantly of soluble fibre, significantly improved glycemic control and decreased the degree of hyperinsulinemia and lowered plasma lipid concentrations in patients with type II diabetes.
2.5.2.3 Coronary heart disease (CHD)
43 Coronary heart disease is the major cause of death in most western countries.
Healthy eating for CHD prevention is a hot issue in the latest decade. High consumption of dietary fibre, especially soluble fibre is believed to have inverse association with risk of coronary heart disease. Mechanisms of non digestible carbohydrates reducing the risk of CHD improve blood lipid profiles by lowering blood pressure and improving insulin sensitivity and fibrinolytic activity (Pereira et al., 2004).
During the past decades, there were numerous key epidemiological studies to prove that dietary fibre could prevent CHD. In 1999, Wolk et al. indicated that higher fibre intake, especially cereal dietary fibre intake, reduced the risk of
CHD in women. They studied 68782 women aged 37 to 64 years for ten years and concluded that women in the highest quintile of cereal fibre intake had a
34% lower risk of total CHD compared with those in the lowest quintile. Pereira et al. (2004) followed up 91058 men and 245186 women over 6 to 10 years and fed them each 10 g/d increment of energy-adjusted and measurement error- corrected total dietary fibre. They found a 14% decrease in risk of all coronary events and a 27% decrease in risk of coronary death by dietary fibre enhancement diet. Such examples could be multiplied indefinitely, such as studies by Pietinen et al. (1996) of Finnish men; studies of Ludwig et al. (1999) of young adults; studies of Lairon et al. (2005) of French adults etc.
2.5.3 Physico-chemical properties of carbohydrate
2.5.3.1 Water holding capacity (WHC)
WHC is defined as the amount of water that is retained by 1 g of dry
44 material under specified conditions of temperature, time soaking, and duration and speed of centrifugation (Elleuch et al., 2011). Dietary fibre holds water by adsorption and absorption. WHC has a significant effect on fecal output and stool hardness (Caballero et al., 2003). It is also an important functional property that has been widely studied in food, since they are associated with food quality. The WHC of fibre differs between fibre sources. Table 2.10 shows the water holding capacity of some foods and food by products. According to
Table 2.10, cereal dietary fibre presents lower water holding capacity than fibre from fruit by-products and algae. For WHC purposes, dietary fibre from fruit by- products and algae are good sources.
45 Table 2.10 Water holding capacity of some fibre
Sources of fibre WHC (g water/g)
Wheat bran 2.7
Coconut fibre 4.4
Defatted rice bran 4.9
Apple dietary fibre 6.3
Pear dietary fibre 6.8
Orange dietary fibre 7.3
Pea hulls 7.4
Lime peel 7.0-12.8
Sugarcane bagasse 7.5
Winter cabbage 9.7
Seedless grape fruit 9.7
Native banana pseudostem flour 10.7
Mango dietary fibre 11
Apple processing wastes 11.7
Asparagus by- products 11.4-20.3
Peach dietary fibre 12.6
Orange processing wastes 16.2
Tender core of banana pseudostem flour 18.3
Carrot dietary fibre 18.6
Algae 20.8
(Source: Elleuch et al., 2011; Aziz et al., 2011; Grigelmo-Miguel et al., 1999)
46 2.5.3.2 Swelling capacity (SWC)
The swelling capacity of dietary fibre is determined by the amount of liquid material that can be absorbed. Food materials high in dietary fibre are always associated with higher swelling capacity. For example, the swelling capacity of the tender core of banana pseudostem, whose dietary fibre content is 47.98, is
13.82 grams swollen granules per gram of dry matter (Aziz et al., 2011). The swelling capacity of dietary fibres is related to the chemical structure of the component polysaccharides and other factors such as particle size, pH, temperature (Elleuch et al., 2011). Raghavendra et al., (2006) claimed that with the reduction of the particle size of coconut milk residue from 1127 to 550 μm, the swelling capacity increased from 17 to 20 mL/g. They guessed that this might be due to the increase in theoretical surface area; total pore volume and structural modification.
2.5.3.3 Viscosity
Viscosity (g), which is the resistance to flow, is defined as the ratio of shear stress (C) to shear rate (c). It is one of the most important physical properties of
DF from both physiological and technological points of view. The viscosity of DF is determined by its structure (branched or linear; ferulic acid content), solubility, molecular weights, shape of the molecules, the presence and magnitude of charges of hydrated molecules and their concentration. Generally, as the molecular weight or chain length of fibre increases, the viscosity of fibre in solution increases. For example, long chain polymers, such as the gums (guar gum, tragacanth gum) bind significant amount of water and exhibit high solution viscosity. It is believed that water soluble fibre is the major fraction of
47 increasing the viscosity of a solution (Kumar et al., 2012; Elleuch et al., 2011).
DFs, which have the ability to form viscous solutions or gels, can change the rheology of the intestinal contents, and are known to produce local responses along the gastrointestinal tract. Dietary fibre interacts with the intestinal brush border and thickens the rate-limiting unstirred water layer of the mucosa reducing the efficiency of nutrient absorption through the intestinal wall.
Moreover, high digested viscosity delays gastric emptying and feed transit time with a resulting blood glucose lowering effect and stimulation of microbial growth in the intestine (Kumar et al., 2012). Since the characteristic of DFs is to delay gastric emptying, it is often considered as beneficial for human health.
Additionally, dietary fibres could affect the rheological behavior and enhance viscosity development when added into food products. Soukoulis et al. (2009) studied the enrichment effect of ice cream with dietary fibre. They concluded that dietary fibre significantly increased viscosity development when mixed with ice cream. They inferred that the enhancement is due to the contribution of insoluble fibres to the increase of total solids, which affects the three- dimensional conformation of the hydrated biopolymers. Lai et al. (2009) mixed dietary fibre into rice starch to detect its influence on the physicochemical characteristics of rice starch. They found that addition of dietary fibre (at 5% wt% on dry starch basis) into Taichung Sen 10 rice flours could increase peak viscosity from 382 to 567 RVU.
2.5.3.4 Gelatinization
Undamaged starch granules are tightly packed and generally insoluble in water
48 because of the collective strength of the hydrogen bonds binding the chains together (Coultate, 2002). Since the starch/ water system undergoes an order- disorder transition during heating, the textural and digestive properties of food products containing starch can be significantly affected by gelatinization
(Liu, Lelievre & Ayoung-Chee, 1991). Water begins to be imbibed when the temperature of the starch suspension is raised to its initial gelatinisation temperature. When the water is 30 °C, the starch granules are intact and absorb some water. When the temperature reaches 40 °C, more surface water is adsorption, hydrogen bonds start to loosen slightly and water may enter some starch granules. 50 °C temperature breaks hydrogen bonds, thus more water is adsorbed onto the surface and absorbed into granule. At this stage, some of the amylose can move into water around granules and granule structure further opens and swell. When the temperature is 60 °C, water forms H bonds with amylose and amylopectin. As a result, the amount of free water outside the granule decreases, the amount of amylose outside the granule increases and the granule expands. The starch solution becomes more translucent and viscous. Further increasing temperature to 65 °C causes loss of birefringence of the starch. Shorter amylose chains are more likely to leave granule and the granules become an amorphous network of starch and water. With the temperature raising closes 100 °C, granules may implode and fragment, hence the viscosity of the starch solution drops.
2.5.3.4.1 RVA
The starch viscosity behavior during gelatinization process can be measured using the Rapid Visco-Analyser (RVA). The RVA is a cooking, stirring
49 viscometer with ramped temperature and variable shear capability optimized for testing the viscous properties of starch, grain, flour and foods. It provides a viscograph that can assess the starch viscosity and gelatinization properties.
The RVA was developed by Newport Scientific, Australia, with the aim of offering starch analysis using smaller sample size as well as a choice of short and long test time (Haase et al., 1995). The RVA has been widely used in research into food products like wheat, potatoes, cereal, maize and rice (Haase et al., 1995; Almeida-Dominguez et al., 1997; Mijland et al., 1999; Yun and
Quail, 1999; Batey and Curtin, 2000).
2.5.4 Analytical methods for non-starch polysaccharides
2.5.4.1 Enzymatic – gravimetric method
The principle of enzymatic – gravimetric method is determining the dietary fibre of dried samples by enzymatic treatments for starch and protein removal
(Prosky et al., 1987). The residue of the enzymatic digestion is used for insoluble dietary fibre measurement after correction for protein and ash in the residue. Soluble dietary fibre is determined by precipitating supernatant fluid by aqueous ethanol.
Enzymatic –gravimetric method is one of the most widely used methods for dietary fibre analysis. It could provide the amount of soluble, insoluble and total dietary fibre in dried samples. However, the limitation of the gravimetric method is that hydrolyzed byproducts remain in solution. Salts such as Na and Ca in the buffers and samples are insoluble in alcohol, as a result, the amount of ash is underestimated and the polysaccharides are overestimated.
50 Moreover, this method is based on a definition of dietary fibre as the sum of indigestible polysaccharides and lignin. However, inulin or all resistant starches are not measured by this method, which appears to underestimate the value of non-starch polysaccharides (Englyst et al., 1995). Additionally, the sugar fractions in the dietary fibre cannot be identified in this method (Caprita &
Caprita, 2011).
2.5.4.2 Enzymatic – chemical method
Enzymatic - chemical procedure for dietary fibre measured as non-starch polysaccharide (NSP) has evolved from the principles laid down by Southgate in 1969 (Englyst et al., 1994). The enzymatic removal of starch and some protein in this method is similar to that in enzymatic – gravimetric method. After the enzymatic digestion, NSPs are measured by spectrophotometry, gas-liquid chromatography (GLC) and high-performance liquid chromatography (HPLC), as the sum of the constituent sugars released by acid hydrolysis. This method gives values for individual monosaccharides and uronic acids released from
NSP.
The enzymatic – gravimetric method is well established and robust.
Furthermore, it quantifies the sugar fractions in the NSP, which indicates the physic-chemical properties of the NSP. This method allows the separation of cellulose from non-cellulosic polysaccharides, and soluble from insoluble polysaccharides (Institute of Medicine, 2001). Therefore, this method provides considerable detail on the polysaccharide components of human foods. GC provides high separation capability, while HPLC gives inferior separation. GC is
51 based on selectivity by volatility and requires derivatization; however derivatization is not demanded for HPLC (Hell et al., 2014).
2.5.4.3 Nuclear magnetic resonance spectroscopy (NMR)
NMR is mainly applied for structure elucidation and structure confirmation. It utilizes the spin properties of certain atomic nuclei, the most prominent being 1H and 13C. The spins will align either parallel or anti-parallel to the magnetic field when exposed to a strong magnetic field. The strength of the magnetic field is correlated to a frequency in the radio wavelength spectrum and leads to the difference in energy. As a result, spins can be excited from the low to the high energy state by irradiation at the transitional wavelength. Other factors such as chemical environment of the nucleus and neighbouring spins can reinforce or weaken the magnetic field as well and thus give characteristic deviations in the excitation wavelengths. According to this principle, vital information about connectivity between atoms is rendered (Hell et al., 2014).
The advantages of NMR technology are that linkage pattern information is provided. It is non-destructive and all-encompassing. The disadvantage of this method is that impurities severely impact sensitivity (Hell et al., 2014).
Moreover, the signal of different sugar fractions may overlap, no unique signal can be used to identify and quantify the fractions in the dietary fibre.
2.5.4.4 Near-infared reflectance spectroscopy (NIR)
NIR technology involves measurement of sample absorptions in the electromagnetic regions of the spectrum from 750-2500 nm. Absorptions are calibrated to an analyzed component of the sample, allowing subsequent
52 prediction of the component in new samples. Since this method relies on the prediction of the component, reliable and accurate reference laboratory method and a mathematical model related the spectral data to the reference measurements are required (Kays et al., 1999).
NIR analysis is very rapid, requires little or no sample preparation, and there is no creation of chemical waste (Kays et al., 1999). However, this analysis can only detect relative comparisons and extensive calibration is required. It is more suitable for large scale determination of dietary fibre in the industry rather than property studies of dietary fibre in the research.
2.6 B-complex vitamins
Vitamins can be divided into two major groups, including water-soluble and fat- soluble vitamins. The major part of the water-soluble vitamins comprises the B- complex vitamins. Each member of the B-complex vitamins has a unique structure and performs unique functions in the human body
(Papadoyannis, 1990). Though a very small amount of the B-complex vitamins is required daily, they play very important roles in our health. Deficiency of the vitamins may cause diseases, such as pellagra, beriberi and megaloblastic anemia (Aminoff et al., 1999; Burch et al., 1950; Chindarkar et al, 2014). This chapter will review some of the B-complex vitamins, their requirements, excess and deficiency, sources from food matrix, bioavailability and stability. Moreover their analyses methods will also be reviewed.
2.6.1Vitamin B3 (Niacin)
53 Niacin (known as vitamin B3) is the generic descriptor for two vitamers (nicotinic acid and nicotinamide), which are colorless and odorless and both based on a pyridine ring bound to a carboxylic group or respectively to a carboxamide group (Rose-Sallin et al., 2001). The two compounds have similar absorption spectra in water with absorption maximum at near 260 nm. These two forms of niacin have equal biological activity and are easily inter-convertible. They are components of the co-enzymes to form NAD and NADP+ in humans
(Ball, 2005; Eitenmiller et al., 2007)).
Niacin is the most stable water-soluble vitamin as its biological activity is unaffected by air, oxygen, light, acid, alkali and heat in the dry state and in neutral aqueous solution (Combs, 2008; Ball, 2005; Eitenmiller et al., 2007).
Hence, in food processing, niacin would not be lost during storage, cooking and heating. However, since niacin is freely soluble in boiling water (Ball, 2005), it is subject to loss through leaching during food preparation. In particular, almost fifty percent of the available niacin is lost when combining soaking and cooking together (Eitenmiller et al., 2007).
Niacin exists in a variety of foodstuffs, such as cereal grains, mushrooms and chicken (CNF, 2010). However, its bioavailability in natural foods is often low.
For example, it has been found that natural cereal grains contain a significant amount of niacin, but 85% - 90% of the niacin is present in chemically bound form, which cannot be absorbed or utilized. Niacin often occurs in food, bound to polysaccharides on the carboxyl group, referred as niacytin, or combined with peptides via amide linkage between amino group and peptide as niacinogen. In order to accurately quantify niacin content in food, an acid or enzymic
54 hydrolysis is often used to liberate the nicotinic acid from the bound forms. This allows the determination of accurate total content of niacin, but at the same time can overestimate the available niacin from food (Ball, 2005; Eitenmiller et al., 2007).
The recommended daily intake of niacin is 14 and 16 mg Niacin equivalents for adult women and men respectively (NHMRC, 2006). Deficiency of niacin will cause pellagra, while a high dosage of niacin (3-6g/ day) can affect liver structure and function with hepatotoxic consequences.
2.6.2 Vitamin B9 (Folate)
Folate is the generic descriptor for folic acid and related compounds exhibiting the biological activity of folic acid. The folates from natural sources usually have a single carbon unit at N-5 and/or N-10 position. The single carbon units that may be transported and stored by folates can vary in oxidation state from the methyl (e.g 5-methyletrahydrofolic acid) to formyl (e.g. 5-formyltetrahydrofolic acid). Folate is sensitive to heat, oxygen, light, acid and alkali, but folic acid is stable. Thereby, folic acid is always used in fortified foods and supplementation of commercial products and in pharmaceutical formulations. The maxima UV absorption region of the folates are mostly 280 – 300 nm (Combs, 2012).
Animal organ meats like liver and kidney, avocado, nuts, whole grains, leafy green vegetables and peas are all food sources of natural folate. The naturally occurring folate in foods are tetrahydrofolate (THF), 5-methyltetrahydrofolate (5-
MTHF) and 10-formyltetrahydrofolate (Ottaway, 1993; Ruggeri et al., 1999). The primary form in fresh foods of polyglutamates is 5-MTHF. Under
55 storage conditions, it will slowly degrade to monoglutamates and be oxidized to less available folate (Ottaway, 1993). The bioavailability of folate in foods varies from extrinsic factors such as chemical and physical form of the vitamin and the concentration of the vitamin, food matrix and antagonists to the intrinsic factors including animal species of the food and health of small intestine (Combs, 2008;
Eitenmiller et al., 2007; Zempleni et al., 2013). Folic acid has 85% bioavailability, which is 50% more than other forms of folate as a result of its high resistance to thermal processing and degradation. By contrast, the folate from natural source found in foods such as THF and 5-MTHF has 75% degradation under cooking conditions (Ball, 2005; Eitenmiller et al., 2007;
Younis et al., 2009).
The recommend intake of folate is 400 μg/ day for adults, 600 μg/ day for pregnancy and 500 μg/ day for lactation (NHMRC, 2006). Folate deficiency will cause serious problems such as failure of DNA replication which leads to impaired cell division. The clinical manifestation of folate deficiency is megaloblastic anemia (Herbert and Zalusky, 1962). Moreover, folate deficiency leads to elevated homocysteine levels as a result of the lack of methylation of homocysteine to methionine, which increases the risk of heart disease. Also, folate plays an important role in B12 deficiency, since B12 induced anemia can be alleviated by treating with folic acid. If folate supplements are taken without
B12, an irreversible neurological damage may occur. Therefore, folate supplements always co-supplemented with B12 (Ball, 2005).
2.6.3 Vitamin B1 (Thiamin)
56 Free thiamin is unstable as its quaternary nitrogen will be cleaved to the thiol form in water (Combs, 2012). For this reason, the hydrochloride and mononitrate forms are used in commerce. Thiamin hydrochloride is a water- soluble vitamin, and is also known as Vitamin B1. It consists of a substituted pyrimidine ring linked by methylene group to a sulphur containing thiazole ring
(Combs, 2012). Aqueous solutions of thiamin are stable at an acidic pH; however, they are unstable in alkaline solutions and are sensitive to ultraviolet light and high temperature (Matarese & Gottschlich, 1998; Shils & Shike, 2006).
Thiamin plays an essential role in the supply of energy to the tissue in carbohydrate metabolism and in the metabolic links between carbohydrate, protein and fat metabolism. Following ingestion, absorption of thiamin occurs mainly in the jejunum, actively at low concentrations and passively at high concentrations. It is transported in blood in both plasma and red blood cells. If intake is high, only a small amount of the thiamin is absorbed and elevated serum values result in active urinary excretion (Davls et al., 1984).
Thiamin is found in a wide variety of foods at low concentrations. Cereal foods, lean pork, macadamia nuts, sunflower seeds are all good sources for thiamin
(Combs, 2008). The recommended daily intake of thiamin is 1.2 mg/day for adult men and 1.1 mg/day for adult women (NHMRC, 2006). The deficiency of thiamin will result in beri-beri, alcoholic neuropathy and encephalopathy, however, the excess of thiamin over 3 g/day will also lead to toxic headaches, irritability, insomnia, rapid pulse and even death.
57 2.6.4 Vitamin B6
Vitamin B6 is a group of compounds, which can be divided into free form and phosphorylated form, including pyridoxine, pyridoxal, pyridoxamine, pyridoxine
5’-phosphate, pyridoxal 5’-phosphate, pyridoxamine 5’-phosphate and 4- pyridoxic acid. Free form is mostly found in plants, while phosphorylated form is more commonly found in animal tissues. Free and phosphorylated B6 vitamers are white to off-white platelets or rod crystals, and the commercial preparations normally are hydrochlorides.
Good food sources of VB6 include meats, whole grain products, vegetables, nuts and bananas. More than 50% of vitamin B6 may be lost during cooking, storage and processing (McCormick, 2006). Different forms of Vitamin B6 have different stability. Pyridoxine is far more stable than the pyridoxal or pyridoxamine. As a result, plant foods lose the least Vitamin B6 during processing as they contain mostly pyridoxine.
Recommended daily intake of Vitamin B6 is 1.4 mg/day for adult people under
50; 1.7 mg/day for adult men over 50 and 1.5 mg/day for adult women over 50
(NHMRC, 2006). Deficiency of Vitamin B6 may result in increased urinary excretion of urea, xanthurenic acid, kynurenine and hydroxykynurenine and oxalic acid (Eastwood, 2013).
2.6.5 Vitamin B2 (Riboflavin)
Riboflavin is a yellowish compound present in most living organisms. It is an active part of the coenzymes of flavin mononucleotide and flavin adenine
58 dinucleotide that catalyze many oxidation-reduction reactions. The bioactive forms of riboflavin are the oxidized and reduced forms of flavin adenine dinucleotide (FAD and FADH2) and flavin mononucleotide (FMN and FMNH2)
(NHMRC, 2006).
Stability of riboflavin is affected by oxygen, water activity and other components.
For example, when oxygen is present during storage, destruction rate of riboflavin increases dramatically. Moreover, lower water activity increases the stability of riboflavin (Dennison et al., 1977). Riboflavin is relatively heat stable; however, it is very sensitive to light. Loss of riboflavin under light depends on the light intensity, exposure time, light wavelength, packaging materials and food processing methods.
Riboflavin is necessary for overall normal growth and development of the body, production and regulation of certain hormones, and formation of red blood cells
(Ajayi et al., 1993). Riboflavin helps cells to metabolize carbohydrates, lipids, and proteins and is crucial for the production of biological energy in the electron transport system. It is also essential for the maintenance of healthy vision, skin, hair, and nails. Riboflavin is involved in the utilization of neurotransmitters, which are implicated in emotional health including the development of depression.
Deficiency of riboflavin may cause minimum morbidity, but is associated with skin and mucous membrane issues, such as chelosis, angular, stomatitis and superficial interstitial keratosis of the cornea, nasolabial seborrhea (Eastwood,
2013). The recommend daily intake (RDI) of riboflavin for adult men under 70 is
59 1.3 mg/day and 1.6 mg/day for men over 70; the RDI for adult women under 70 is 1.1 mg/day and 1.3 mg/ day for women over 70 (NHMRC, 2006). Liver, milk, cheese, egg, some green vegetables and beer are all dietary sources of riboflavin.
60 3. Materials and Methods
3.1 Raw material
Fresh banana pseudostem from banana plants (Musa acuminata & Musa balbisiana) was used for the experimental work. The banana pseudostem was collected from the Royal Botanic Garden in Sydney. The banana pseudostem was refrigerated immediately after collection and was dried within few days.
61 3.2 Sample Preparation and Drying
3.2.1 Equipment
The equipment for sample preparation and drying is listed in Table 3.1.
Table 3.1 Equipment used in sample preparation and drying
Equipment Brand Model
1. Cabinet dryer Built by UNSW Chemical Engineering Workshop
2. Drying trays
3. Vegetable slicer DITO-SAMA TR21
Blade with 4 mm gap
4. Analytical balance ANR GR-200
5. Sterile knife
6. Chopping board
7. Desiccator
8. Aluminum containers
9. Brass sieve Aperture 1 mm
10. Mill FRITSCH (GERMANY) PULVERISETTE
11. Water bath GRANT INSTRUMENTS (UK) SB 3
12. Thermometer LIVINGSTONE
3.2.2 Procedure of sample preparation
The stored banana pseudo stem was taken out of the fridge and the outer layer of the pseudo stem was peeled off leaving the core of the pseudo stem. The tender core of the pseudo stem was cut transversally into 4 mm slices
62 using the slicer. Afterwards, the cut pseudo stem slices were separated into two parts. Half the quantity was blanched at 70 °C water for 3 minutes and another half was left unblanched.
3.2.3 Procedure of batch drying of banana pseudostem
The drying tray used for drying was first weighed. Afterwards, blanched and untreated samples were further divided into two parts separately. Samples were spread out neatly onto the drying tray. Tray of banana pseudo stems was weighed again before they were placed into a batch dryer. The temperature of the dryer was set at 40±2 °C and 50±2 °C, respectively. The weight of banana pseudo stem was recorded manually every hour until the weights were constant. Banana pseudo stem samples were taken out of the dryer and the final weight was recorded. The samples were placed immediately into a desiccator to cool down. From these recorded weights, drying curves of the samples were plotted on a graph with time in hours against weight loss in grams. Furthermore, the samples were ground in a 1 mm mesh and stored immediately in a sealed glass container.
The moisture ratio was calculated using equation (3-1).
M Me MR (3-1) Mo Me
63 3.3 Nutrient analysis
3.3.1 Moisture
3.3.1.1 Equipment
The equipment for moisture analysis is listed in Table 3.2.
Table 3.2 Equipment used for moisture analysis
Equipment Brand Model
1. Aluminum containers
2.Analytical balance ANR GR-200
3.Air oven LABEC
4.Desiccator
3.3.1.2 Procedure of moisture measurement
The initial moisture content of fresh and dried banana pseudo stem was determined according to AOAC method 934.01 (AOAC, 2006). The method uses the air oven method. Triplicates of fresh and dried banana pseudo-stem were sliced and placed in pre-dried and pre-weighed aluminum containers.
Then the samples in aluminum containers were dried in an air oven overnight at
105 ± 2 °C. After drying, the samples were taken out and placed into the desiccators to cool down and final weights of the samples were recorded. The moisture content of fresh and dried banana pseudo stem was determined.
The moisture contents (wet basis) were calculated using equation (3-2).
Wo- Wf MCwb= 100 (3-2) Wo
64 The moisture contents (dry basis) were calculated using equation (3-3).
Wo- Wf MCdb= 100 (3-3) Wf
3.3.2 Fat
3.3.2.1 Equipment
The equipment used for fat analysis is listed in Table 3.3.
Table 3.3 Equipment used in fat content analysis
Equipment Brand Model
1. Steam bath LABEC
2. Beakers 50 mL
3. Analytical balance ANR GR-200
4. Glass stirring rods
5. Measuring cylinders 10 mL and 25 mL
6. Mojonnier fat extraction flasks with glass stoppers
7. Erlenmeyer flasks 250 mL
8. Glass filter funnels
9. Glass beads
10. Air oven LABEC
11. Spatula
12. Watch glasses
13. Pledget cotton wool
65 3.3.2.2 Chemicals
The chemicals used for fat analysis are listed in Table 3.4.
Table 3.4 Chemicals used for fat analysis
Chemical Grade Manufacturer
1. Ethanol, absolute AR Grade Ajax Aps
2. Hydrochloric acid AR Grade Ajax Aps
3. Diethyl ether AR Grade Aldrich
4. Petroleum ether AR Grade Aldrich
5. Sodium chloride AR Grade Sigma
6. RM typical diet 1548a National Institute of Standards and
Technology (NIST)
3.3.2.3 Fat analysis
Total fat content in the banana pseudo stem samples was determined by acid hydrolysis method – AOAC method 954.02 (AOAC, 2006). Duplicates of each sample (2 g) were weighed into beakers. Blanks were run along with the samples. Ethanol and 7 N HCl were added and heated for 40 minutes while stirring frequently. The mixtures were transferred into a Mojonnier fat-extraction flask; 25 mL of diethyl ether were added and shaken vigorously for 1 minute.
Furthermore, 25 mL of petroleum ether was added into the flasks and inverted gently without shaking. The upper ether-fat layer was decanted off and filtered through a cotton pledget into pre-weighed Erlenmeyer flasks with glass beads.
The remaining liquid in the Mojonnier flasks were re-extracted twice with 15 mL
66 using both ethers followed by same procedure with decanting and filtered into same flasks and the funnels were washed thoroughly with 10 mL of mixture of the two ethers in equal volumes. Then, the ethers were slowly evaporated on a steam bath in the fume cupboard. The Erlenmeyer flasks containing the fat were dried in an oven at 100 ± 2 °C for 90 minutes; furthermore, the flasks were allowed to stand in air for exactly 30 minutes and weighed to determine the final fat content in the samples. A reference material obtained from NIST, US, typical diet 1548a was used for quality control.
The fat content was calculated using equation (3-4):
Wt of fat %Fat in sample = 100 Wt of sample (3-4)
3.3.3 Proteins
3.3.3.1 Equipment
The equipment used for proteins analysis is listed in Table 3.5.
Table 3.5 Equipment used for protein analysis
Equipment Brand Model
LECO TruSpec LECO S Model
Analyser
67 3.3.3.2 Protein analysis
Protein content of the samples was determined by using the LECO Analyser.
The percentage of nitrogen (N) in the samples was determined and the protein conversion factor, 6.25 was used to convert the nitrogen content into protein content.
The protein content was calculated using equation (3-5).
% Protein in sample = % Nitrogen x protein factor (3-5)
3.3.4 Ash
3.3.4.1 Equipment
The equipment used for ash analysis is listed in Table 3.6.
Table 3.6 Equipment used for ash analysis
Equipment Brand Model
Crucibles
Muffle furnace Ether MR 260
Analytical balance ANR GR-200
Aluminium containers
Desiccator
Spatula
Air oven LABEC
3.3.4.2 Procedure of ash analysis
68 The ash content of banana pseudostem was quantified by dry ashing method
942.05 (AOAC, 2006). The crucibles were prepared by washing with warm soapy water and rinsing in tap water. The crucibles were soaked in 7.5 M HNO3 overnight in a fume cupboard. The crucibles were then rinsed with deionized water and dried in an air oven at 110±2 °C, followed by conditioning in a muffle furnace at 530±2 °C overnight. Finally the crucible was cooled and stored in a desiccator prior to analysis.
The previously conditioned crucibles with lid were weighed. Duplicate of each sample (4 g) were weighed into the crucibles. Blanks were run along with the samples. Then the samples were charred on a hot plate. The crucible and lid were placed into the muffle furnace at 530 ± 2 °C overnight. If the ash was still grey, a few drops of 7.5 M Nitric acid was added to the cooled crucible and evaporated to dry on a hot plate in the fume cupboard and returned to the muffle furnace overnight again. At completion of ashing, the samples were taken out and placed into a desiccator to cool and then reweighed in order to determine the weight of ash.
The ash content was calculated using equation (3-6)
(3-6)
69 3.3.5 Sugars
3.3.5.1 Equipment
The equipment used for sugar analysis is listed in Table 3.7.
Table 3.7 Equipment used for sugar analysis
Equipment Brand Model
Analytical balance ANR GR-200
Beakers 100 mL
Glass stirring rods
Measuring cylinder 25 mL
Filter paper Whatman No.541 (15.0 cm)
Glass filter funnels
Round bottom, short neck flasks 250 mL
Air oven LABEC
Steam bath LABEC
pH meter TPS
Autosampler vials with crimp top seals to fit and crimper
PTFE filter 0.45 μm
NH2 column SUPELCO 15 cm, particle size 4.6 μm
Syringe 10 mL
HPLC system equipped with Shimadzu UFLC-1 refractive index detector
70 3.3.5.2 Chemicals
The Chemicals used for sugar analysis are listed in Table 3.8.
Table 3.8 Chemicals used for sugar analysis
Chemical Grade Manufacturer
Ethanol, absolute AR Grade Ajax Aps
Acetonitrile HPLC Grade Ajax Finechem
D- (+) glucose AR Grade Sigma
Fructose AR Grade Sigma
Sucrose AR Grade Sigma
3.3.5.3 Procedure for sugar extraction
Sugars were extracted with aqueous ethanol following the AOAC method
(AOAC, 2006). Switched on steam bath first. Duplicates of each sample (5 g) were weighed into each beaker. Whatman No.541 filter papers were also pre- weighed. A volume of 25 mL of boiling 85% ethanol was added and pH measured. Then, a few drops of 0.5 NaOH were added to increase the pH to
7.0+0.5. A volume of 25 mL boiling 85% ethanol was added to beaker containing food samples and placed it in a steam bath for a few minutes, covered with a watch glass and stirred frequently. The solution was then removed from the steam bath and filtered through the filter paper into a round bottom flask. The extraction was repeated three times with 25 mL of boiling
85% ethanol. Then the flask containing the ethanol solution was evaporated off on a rotary evaporator at 45 °C leaving behind aqueous solution of
71 approximately 3 mL. The aqueous solution was transferred into a 10 mL volumetric flask and made up to volume with milliQ water. Furthermore, the solution was passed through a 10 mL syringe and ultrafilter with PTFE membrane and filtered into HPLC sample vials.
3.3.6 Starch
3.3.6.1 Equipment
The equipment used for starch analysis is listed in Table 3.9.
Table 3.9 Equipment used for starch analysis
Equipment Brand Model
Analytical balance ANR GR-200
Air oven LABEC
PH meter TPS
Volumetric flasks 1 L
Autosampler vials
Beakers 100 mL
Adjustable pipette Thermo 100 μL, 1000 μL, 5 mL
HPLC system equipped with Shimadzu UFLC-1 refractive index detector
NH2 column SUPELCO 15 cm, particle size 4.6 μm
PTFE filter 0.45 μm
Syringe 10 mL
72 3.3.6.2 Chemicals
The chemicals used for starch analysis are listed in Table 3.10.
Table 3.10 Chemicals used in starch analysis
Chemical Grade Manufacturer
D- (+) glucose AR Grade Sigma
Ethanol, absolute AR grade Ajax Aps
Sodium acetate trihydrate Reagent grade Sigma
Thermostable α-amylase Megazyme
Amylogluosidase Megazyme
Benzoic acid AR Grade Sigma
Hydrochloric acid AR Grade Ajax Aps
Calcium chloride dehydrate AR Grade BDH
Glacial acetic acid AR Grade Ajax Aps
Sodium hydroxide solution AR Grade BDH
Starch AR Grade Ajax Aps
Acetonitrile HPLC grade Ajax Finechem
Invertase Sigma-Aldrich
Pancreatin Sigma-Aldrich
Pullulanase Novozymes
Glucose oxidase (GOD-PAP) Thermo scientific
3.3.6.3 Procedure of total starch extraction by HPLC
The residues from sugar extraction were further used for starch extraction. 350
73 mg residue of each sample was dissolved into 10 mL Milli-Q water. The dissolved samples were sealed with foil and placed into a boiling shaking water bath for 4 hours. 0.3 mL acetate buffer was added, followed by addition of 0.4 mL amyloglucosidase. The mixtures were shaken and incubated at 37 °C overnight. After incubation, the samples were added into 30 mL absolute ethanol and made up to 50 mL by absolute ethanol. The solution and precipitate were filtered through Whatman No. 541 filter paper. Aliquots of 25 mL were evaporated by a rotary evaporator at 45 °C until approximately 3 mL aqueous solution was left. Then the solution was passed through a 10 mL syringe and filtered with PTFE membrane into HPLC sample vials.
3.3.6.4 Chromatographic conditions for HPLC
Sugar and starch content was determined by using HPLC with Refractive Index
Detection (RID) (Shimadzu, UFLC-1, and Japan). Normal phase LC-NH2 column was used in the analysis. Acetonitrile and MilliQ water (87:13) was used as the mobile phase. The conditions were: 20 μL injection, isocratic mode, flow rate of 0.4 mL/min and temperature control of column set to 30 °C. Samples and standards were injected to determine the final weight of sugar and starch in the samples by comparing the peak heights of standard and samples.
3.3.6.5 Procedure of starch digestibility analysis
The digestibility of starch in banana pseudostem was determined using the methods described by Englyst et al. (1992) with slight modifications. The recipe for glucose measurement was modified. 100 μL samples mixed with 2 mL GOD-
PAP in Englyst method was replaced by 10 μL samples mixed with 270 μL
74 GOD-PAP
3.3.6.5.1 Reagents preparation
Enzyme solution 1
A sample of 0.1 mL of 6000 units/ mL amyloglucosidase was diluted with 4.2 mL of water to make a solution containing 140 units/mL amyloglucosidase. 3.0 g pancreatin was weighed into each of the four centrifuge tubes and each portion was suspended in 20 mL water. The pancreatin solution was stirred magnetically for 10 min and then centrifuged for 10 min at 1500g. 13.5 mL supernatant from each tube was pipetted out into another tube and mixed with 6 mL diluted amyloglucosidase and 4 mL invertase solution (3000 unit/ mL).
Sodium acetate buffer
13.6 g sodium acetate trihydrate was dissolved in 250 mL saturated benzoic acid solution, and made upto 1 L with water. pH was adjusted to 5.2 with 0.1 M acetic acid. To stabilize and activate enzymes, 4 mL 1 M CaCl2 was added into a liter of buffer.
Glucose standard solution
200 mg glucose (dried to constant weight) was weighed into a test tube to the nearest 0.1 mg. The volume was made up to 20 mL with sodium acetate buffer to give a 10 mg/mL solution. The following gradient table was used to build the standard curve.
75
Table 3.11 Standard concentration table
Concentration Volume of 10 mg/ mL glucose Volume of water added (mg/ mL) solution added (mL) (mL) 1.5 3 17
1.25 2.5 17.5
0.1 2 18
0.75 1.5 18.5
0.5 1 19
0.25 0.5 19.5
3.3.6.5.2 Measurement of free glucose (FG)
Dried ground banana pseudostem (500 mg) was mixed with 0.1 M acetate buffer and 51.5 cm diameter glass balls in a test tube and vortex mixed vigorously. The tubes were placed into a boiling water bath for 30 min. Tubes were then transferred to another water bath at 37 °C, by shaking vigorously again. When the temperature stablised, 0.2 mL invertase was added and the tubes were capped and immersed horizontally in the shaking water bath at 37
°C for 30 min.
The tubes were taken out of the water bath and manually shaken vigorously.
0.2 mL of the contents was removed into eppendorf tubes containing 1mL absolute ethanol and vortex mixed. They were then centrifuged at 1,500 × G for
5 min. 0.2mL supernatant was diluted in 1 mL water, and mixed well by inversion.
76 3.3.6.5.3 Measurement of RDS, SDS and RS fractions
Guar gum powder (50 mg) was added and 20 mL of 0.1 M acetate buffer was pipetted into each sample tube with 500 mg dried ground banana pseudostem and 51.5 cm diameter glass balls. The contents were mixed thoroughly to disperse the guar gum. The samples, standards and blank were placed in a water bath at 37 °C. Into each tube was added 5 mL enzyme solution 1. After 1 min the second sample was added. The tubes were capped and immersed horizontally in the 37 °C shaking water bath, securing firmly.
After 20 min, 0.2 mL of the hydrolysate was pipetted out into a labeled eppendorf tube containing 1 mL absolute ethanol and mixed well. The tube was placed back into the shaking water bath. After a further 100 min, a second 0.2 mL sample was removed from the same tube in the same way, but this time the tube was not placed back into the water bath. The portion taken after 20 min and 120 was designated G20 and G120 respectively.
3.3.6.5.4 Measurement of Total Glucose (TG)
The samples were vortex mixed after 120 min to break up large particles. The tubes were placed in a boiling water bath for 30 min. The tubes were then vortex mixed again and cooled in ice water for 15 min.10 mL of 7M KOH was added and mixed well. The tubes were immersed horizontally in the ice-water shaking bath (0 °C) for 30 min. Removed the sample tubes from the ice water and 0.2 mL of the contents were pipetted into eppendorf tubes containing 1 mL
0.5 M acetic acid. The contents were mixed well. 0.2 mL diluted amyloglocosidase (50 AGU/ml) was added, mixed and incubated for 30 min at
77 70 °C. The tubes were transferred into a boiling water bath for 10 min and cooled to room temperature. 0.2 mL sample was pipetted and diluted with 1 mL water. The tubes were centrifuged at 1,500 × G for 5 min to remove the precipitate.
3.3.6.5.5 Measurement of glucose
10 μL blank, samples and standards in duplicate were pipetted into labeled test tubes. 270 μL GOD-PAP reagent was added into each and mixed. Each mixture was pipetted into the microplate and incubated at 37 °C for 20 min. The absorbance of standards and samples were measured against reagent/ enzyme blanks at 510 nm in a microplate reader (Molecular Devices, SpectraMax M2,
Canada). The standard curve was drawn to calculate % glucose in samples.
TS= (TG – FG) * 0.9
RDS = (G20 – FG) * 0.9
SDS = (G120 – G20) * 0.9
RS = TS – (RDS + SDS)
78 3.3.7 Dietary fibre
3.3.7.1 Equipment
The equipment used for dietary fibre analysis is listed in Table 3.12.
Table 3.12 Equipment used in dietary fibre analysis
Equipment Brand Model
Beakers 400 mL and 600 mL
Fritted crucible
Heavy-walled filtering 1 L flask
Shaking water bath RATEK
Analytical balance ANR GR-200
Air oven LABEC
Desiccator
PH meter TPS
Pipettors 200 μL and 5000 mL
Cylinder 1 L
Magnetic stirrers and stirring bars
Spatulas
Muffle furnace ETHER MR 260
79 Vacuum pump
Centrifuge Thermo Scientific Multifuge X3R
Centrivap concentrator LABCONCO T310000
NMR
NMR Bruker Avance III 300 solid state
GC System Bruker Avance III HD 600 mHz
Agilent 7890A
80 3.3.7.2 Chemicals
The chemicals used for dietary fibre analysis are listed in Table 3.13.
Table 3.13 Chemicals used for dietary fibre analysis
Chemical Grade Manufacturer
1. Ethanol, absolute AR grade Ajax Aps
2. Acetone HPLC grade Ajax Finechem
3. α -Amylase, heat-stable Megazyme
4. Protease Megazyme
5. Amyloglucosidase Megazyme
6. Celite® AR Grade Sigma
7. Ethanesulfonic acid (MES) AR Grade Sigma
8. Tris (hydroxymethyl) AR Grade Sigma-Aldrich
--aminomethane (TRIS)
9. Hydrochloric acid AR Grade Ajax Aps
10. Termamyl AR Grade Novozyme
11. Pancreatin AR Grade Sigma
12. Pullulanase AR Grade Novozyme
13. Sodium acetate AR Grade Sigma
14. Monosodium phosphate AR Grade Sigma
15. Disodium phosphate AR Grade Sigma
16. Calcium chloride AR Grade Sigma
17. Benzoic acid AR Grade BDH
81 Table 3.13 Chemicals used in dietary fibre analysis (Cont’d)
Chemical Grade Manufacturer
18. L-Rhamnose AR Grade Sigma
19. L-(-)-Fucose AR Grade Sigma-Aldrich
20. L-(+)-Arabinose AR Grade Sigma-Aldrich
21. D-(+)-Xylose AR Grade Sigma-Aldrich
22. D-(+)-Mannose AR Grade Sigma-Aldrich
23. D-(+)-Galactose AR Grade Sigma-Aldrich
24. D-(+)-Glucose AR Grade Sigma-Aldrich
25. Allose AR Grade Sigma-Aldrich
26. Ammonia solution AR Grade Sigma-Aldrich
27. Octan-2-ol AR Grade Sigma-Aldrich
28. Sodium tetrahydroborate AR Grade Thermo Fisher Scientific
29. 1-methylimidazole AR Grade Sigma-Aldrich
30. Bromophenol blue AR Grade Sigma-Aldrich
31. Potassium hydroxide AR Grade Sigma-Aldrich
32. Galacturonic acid AR Grade Sigma-Aldrich
33. Sodium chloride AR Grade BDH
34. Boric acid AR Grade Sigma-Aldrich
35. 3,5-dimethylphenol AR Grade Ajax Finechem
3.3.7.3 Procedure for dietary fibre analysis by Megazyme kit
Total dietary fibre, which consists of soluble and insoluble dietary fibre in the samples, was determined by using the Megazyme recommended method suitable for cereal products, fruits and vegetables (Megazyme Total Dietary
82 fibre assay procedure, 2012). The method was based on AOAC method 991.43 and AACC method 32-07.01.
Duplicates of each sample (1 g) were weighed into beakers. Blanks were run along with samples. A volume of 40 mL of MES-TRIS blend buffer solution was added to each beaker. The beakers were placed on the magnetic stirrer with the addition of a magnetic stirring bar until all samples were completely dispersed in solution. Then the samples were incubated in order with three different enzymes. First, 50 μL of heat-stable α-amylase was added into the beakers, covered with foil and placed into the shaking water at 100 °C to incubate for 35 minutes. After incubation, the beakers were cooled to 60 °C and the sidewalls of the beakers were rinsed with distilled water. The second incubation was initiated after addition of 100 μL of protease solution. This time the incubation temperature was set to 60±1 °C and the time was 30 minutes. After second incubation, 5 mL of 0.561 N HCL was added into the beakers while stirring.
Finally in the last incubation, 200 μL of amyloglucosidase solution was added into the beakers and incubated in the shaking water bath at 60±1 °C again for
30 minutes. The samples were removed from the water bath after incubation and filtered. The crucibles containing Celite® was weighed and the Celite® was redistributed using 3 mL of distilled water and vacuum source. The enzyme mixture from above was filtered through the crucible into the filter flask using vacuum. The residue in the crucible was washed twice using 20 mL of distilled water preheated to 70 °C.
83 Insoluble dietary fibre
The filtrates were saved for determination of soluble dietary fibre. The residue in the crucibles was washed twice with 10 mL of 95% ethanol and acetone. Finally the crucibles were dried in the air oven at 103 °C overnight.
Soluble dietary fibre
The filtrates from above were transferred into pre-weighed beakers. Then 4 volumes of 95% ethanol preheated to 60 °C were added into the beakers. The beakers were left at room temperature for 60 minutes to allow for the formation of precipitate. After that, new crucibles containing Celite® were weighed and the
Celite® was redistributed using 15 mL of 78% ethanol and vacuum source. The precipitated enzyme digest was filtered through the crucible with vacuum and the residue in the crucibles was washed with 78% ethanol, followed by 95% ethanol and finally acetone. Again, the crucibles were dried in the air oven at
103 °C overnight.
Protein and ash determination
On the next day the crucibles were removed from the oven and cooled in the desiccators for approximately 1 hour. The crucibles were weighed and the weight of residue was obtained by subtraction. The residue from one duplicate sample was analysed for protein and then the second duplicate sample was analysed for ash. For protein, the residues were determined by Kjeldahl method. For ash, the crucibles containing residue were placed into the muffle furnace for 5 hours at 525 °C. Then they were taken out and cooled in the
84 desiccators and the weight was recorded
The dietary fibre content was calculated as equation (3-7):
R1 R2 p A B Dietary fibre (%)= 2 100 (3-7) m1 m2 2 where:
R1 = residue weight from m1; R2 = residue weight from m2;
m1 = weight of sample 1; m2 = weight of sample 2;
A = ash weight from R1; p = protein weight from R2; and
B= Blank, see equation (3-8).
BR1 BR2 B BP BA (3-8) 2
3.3.7.4 Procedure of dietary fibre analysis by solid state NMR
3.3.7.4.1 Sample preparation
Soluble and insoluble dietary fibre extraction methods were followed by the
Megazyme method described above. The dried residues of both soluble and insoluble fibre were used directly in NMR analysis.
3.3.7.4.2 Solid state NMR spectroscopy
13C CP/MAS NMR spectra of samples were measured using Bruker Avance III
300 solid state NMR operating at a spectrometer frequency of 75.4 MHz (2 ms contact time, recycle delay of 3 s, sweep width of 20 kHz, Spinning speed of
85 4 kHz).
3.3.7.5 Procedure of enzyme and acid hydrolysis of non-starch polysaccharides
3.3.7.5.1 Reagent preparation
Sodium acetate buffer (0.1 mol/mL, pH 5.2)
Dissolved 8.2 g of sodium acetate and made to 1 L with MillQ water. Adjusted to pH 5.2 with 0.1 mol/L acetate acid. In order to stabilize and activate the enzyme, 4 mL of 1 mol/L calcium chloride was added to the buffer.
Sodium phosphate buffer (0.2 mol/L, pH 7)
Adjusted 0.2 mol/L Na2HPO4 to pH 7 with 0.2 mol/L NaH2PO4.
Sulfuric acid (12 mol/L)
Accurately measured 280 mL of water into a strong 2 L beaker. Placed the beaker in a bowl of ice water in a fume cupboard and slowly added 390 mL of concentrated sulfuric acid with stirring.
Enzyme solution 1
Took 2.5 mL of Termamyl and made up to 200 mL with acetate buffer, mixed and kept it in a 50 °C water bath.
Enzyme solution 2
Placed 1.2 g of pancreatin into a 50 mL tube, added 12 mL of water, vortex mixed initially and then mixed for 10 min with a magnetic stirrer. Vortex mixed again, then centrifuged for 10 min at 1,500 × G. Took 10 mL of the supernatant,
86 added 2.5 mL of pullulanase and vortex mixed again.
3.3.7.5.2 Total, insoluble non-starch polysaccharides (NSP) extraction
The extraction of non-starch polysaccharides in banana pseudostem was determined using the methods described by Englyst et al. (1994) with minor modifications.
A sample aliquot of 300 mg sample was weighted into the glass tube with 2 mL dimethylsulfoxide and vortex mixed. Duplicates were immediately placed in a boiling water bath and were removed after 20 s, vortex mixed and immediately placed into the boiling water bath. This procedure was repeated until all the tubes were in the bath.
After 30 min, one tube was removed at a time, vortex mixed, uncapped and 8 mL of enzyme solution 1 (keep at 50 °C) was immediately added, the tube was capped and vortex mixed thoroughly, ensuring that no material adhered to the tube wall. Tubes were replaced in the boiling water bath for 10 min. The rack of tubes was transferred to a 50 °C water bath. After 3 min, the rack was removed and 0.5 mL of enzyme solution 2 was added to each tube and the contents were mixed thoroughly to aid distribution of the enzyme throughout the sample.
The tubes were replaced in the 50 °C shaking water bath and left for 30 min.
The rack of tubes was transferred to the boiling water and left for 10 min. The samples were placed in water at room temperature to cool.
For total NSP, 40 mL of absolute ethanol was added to each sample and they were mixed well by inversion. They were then placed in ice water for 30 min.
Samples were centrifuged at 1,500 × G for 10 min to obtain a clear
87 supernatant liquid. Removed as much of the supernatant as possible by pipette.10 mL of 85% ethanol was added to the samples and vortex mixed.
Samples were made up to 50 mL with 85% ethanol, mixed by inversion, centrifuged and the supernatant was removed as above.
For insoluble NSP, added 40 mL of sodium phosphate buffer and put in the boiling shaking water bath for 30 min. After cooling down the samples, centrifuged at 1,500 × G for 10 min to obtain a clear supernatant liquid.
Removed as much of the supernatant as possible by pipette.10 mL of water was added to the samples and vortexed. Then made to 50 mL with water, mixed by inversion. Repeated the procedure described above by using 50 mL of absolute ethanol.
For both treated total NSP and insoluble NSP, uncapped tube was placid in a beaker of water at 80 °C on a hot plate stirrer in the fume cupboard and the residue was stirred until dry to make sure all of the samples were free of acetone.
Added 5 mL of 12 mol/L sulfuric acid to the dried residue and immediately vortex mixed. Left the tubes in shaking water bath at 35 °C for 1 h to disperse the cellulose. Added 25 mL of water rapidly and vortex mixed. Placed into a shaking boiling water bath and left for 1 h. Cooled the tubes in tap water at room temperature.
3.3.7.6 Procedure of non-starch polysaccharide analysis on solution state NMR
The acid hydrolysed samples from 3.3.7.5 were neutralized with 1.5 g of
BaCO3, centrifuged and removed the precipitate. Concentrated the
88 supernatant liquid using centrivap and re-dilute with 1 mL D2O (100%).
1H NMR spectra of samples were measured using Bruker Avance III HD 600
MHz equipped with a 5 mm CPTCI 1H cryoprobe. HOD suppression was obtained by pulse program zgesgp with 90 degrees pulse. The solvent used was H2O and D2O.
Decomposition of 1H NMR spectra in the region of 5.50-5.00 ppm was pursued by library manager of Chenomx NMR Suite 7.6 (Chenomx Inc., Canada).
3.3.7.7 Procedure of NSP analysis by GC
3.3.7.7.1 Reagent preparation
Ammonia solution-sodium tetrahydroborate solution
Prepared 6 mol/ L ammonia solution containing 200 mg/ml of sodium tetrahydroborate (Prepare immediately before use).
Bromophenol Blue solution, 0.4g/L aqueous (MilliQ water)
3.3.7.7.2 Chromatographic conditions
For the separation of neutral sugars, a Supelco SP-2380 wide bore capillary column (30 m * 0.53 mm); a GC system with a flame-ionization detector
(Agilent, 7890A, USA) and auto-injector (Agilent, 7683B, USA) was used. The injector temperature and detector temperature were set to 275 °C and the column temperature was set to 220 °C. Helium was selected as the carries gas and the flow rate was 8 mL/min. A split/ splitless topered liner (Agilent, USA) was used for the injection. The injection volume was 1 L and a split injector
89 with a dilution factor of 30 was used.
3.3.7.7.3 Preparation of standard solutions
Standard stock solution and internal standard (allose) stock solution were separately prepared in 50% saturated benzoic acid. Made the internal standard solution by weighing 100 mg allose (dried to constant mass under reduced pressure with phosphorus pentoxide) to the nearest 1 mg. Diluted to 100 ml with
50% saturated benzoic acid to give a 1 mg/mL solution. The stock sugar solution was made by weighing (all sugars dried to constant mass under reduced pressure with phosphorus pentoxide), to the nearest 1 mg. Placed
0.65 g of rhamnose, 0.6 g of fucose, 5.938 g of arabinose, 5.563 g of xylose,
2.875 g of mannose, 3.525 g of galactose and 11.7 g of glucose in a 100 mL calibrated flask and diluted to volume with 50% saturated benzoic acid. All of the stock solutions were stored in a refrigerator covered with aluminum foil.
Working solutions for calibration were prepared daily by appropriate dilution in
50% saturated sugar mixture, which was used to build up the standard curves for each standard. The concentration of the standard is shown in the
Table 3.14.
90
Table 3.14 Calibration concentration of the standard sugar mixture (mg/ L)
Level Rha Fuc Ara Xyl Man Gala Glu
9 650 600 5938 5563 2875 3525 11750
8 520 480 4750 4450 2300 2820 9400
7 416 384 3800 3560 1840 2256 7520
6 347 320 3167 2967 1533 1880 6267
5 208 192 1900 1780 920 1128 3760
4 130 120 1188 1113 575 705 2350
3 52 48 475 445 230 282 940
2 26 24 238 223 115 141 470
1 13 12 119 111 58 71 235
Rha: Rhamnose; Fuc: Fucose; Ara: Arabinose, Xyl: Xylose; Man: mannose;
Gala: Galactose; Glu: Glucose
3.3.7.7.4 Method validation
The method described has been validated with respect to accuracy, within-day repeatability (n=7), between-day precision for five consecutive days, linearity and sensitivity. Linearity was studied by a series of mixed standards of the examined neutral sugars, covering the entire working range; nine concentration levels were used. Each solution was injected twice. Correlation coefficients for the sugars on the basis of plots of concentration ratio of external standard sugars, on the basis of plots of concentration ratio (external standard sugars
/internal standard sugar) against peak area ratio (external standard sugars/ internal standard sugar) were found to be >0.998.
91 The detection limits and quantitation limits were considered to be the quantities that produce a signal of peak height three times the size of the background noise.
3.3.7.7.5 Sample Preparation
The sample preparation for the GC analysis followed the method described by
Englyst et al. (1994). To prepare the standard sugar mixture, 0.2 mL of the sugar mixture solution and 0.5 mL of 2.4 mol/ L sulfuric acid were mixed.
Duplicate standard sugar mixtures at each concentration level were used for calibration.
0.5 mL of internal standard (1 mg/mL allose) was added to 1 ml of the acid hydrolysates from 3.3.7.5 or 1 mL of the standard sugar mixture and vortexed.
The tubes were placed in ice-water and 0.4 mL of 12 mol/L ammonia solution was added and vortex mixed. Approximately 7 drops of additionally ammonia solution were added to make sure the solution is alkaline. Approximately 5 L of the antifoam agent octan-2-ol and 0.1 mL of the ammonia solution-sodium tetrahydroborate solution were added and vortexed. The sample tubes were left in a 40 °C water bath for 30 min. 0.2 mL of glacial acetic acid was added and mixed again. 0.5 mL sample was transferred to a 50 mL glass tube and add 0.5 mL of 1-methylimidazole. 5 mL of acetic anhydride was added and vortex mixed immediately. 0.9 mL of absolute ethanol was added. After being vortex mixed, samples were left 5 min for reaction. 10 mL water was added, vortex mixed and left for 5 min. 0.5 mL Bromophenol Blue solution was added. The tubes were placed in ice water and 7.5 mol/ L potassium hydroxide was added; 5 min later,
92 a further 5 mL of 7.5 mol/L potassium hydroxide was added. The samples were mixed by inversion. The samples were centrifuged at 5000 × G for 5 min. Part of the upper phase was drawn by a pipette to the GC vial.
3.3.8 Minerals
3.3.8.1 Equipment
The equipment used for mineral analysis is listed in Table 3.15.
Table 3.15 Equipment used for mineral analysis
Equipment Brand Model
Microwave digester Milestone
Volumetric flask 100 mL
ICP-MS PerkinElmer Nexion
ICP-OES PerkinElmer OPTIMA 7300
Ion Chromatography
3.3.8.2 Chemicals
The chemicals used for mineral analysis are listed in Table 3.16.
Table 3.16 Chemicals used for mineral analysis
Chemical Grade Manufacturer
1. Nitric acid AR Grade Ajax Finechem
2. Hydrogen peroxide AR Grade Ajax Aps
3. Sodium hydroxide AR Grade BDH
4. Hydrochloric acid AR Grade Ajax Aps
93 3.3.8.3 Procedure of mineral analysis
Minerals in banana pseudostem were determined by using inductively coupled plasma optical emission spectrometry (ICP-OES). The samples were digested with nitric acid and hydrogen peroxide before being analysed by the ICP-OES.
94 3.3.9 B-complex Vitamins
3.3.9.1 Equipment
The equipment used for B-complex vitamins analysis is listed in Table 3.17.
Table 3.17 Equipment used for B-complex vitamins analysis
Equipment Brand Model
1. Beakers 100 mL
2. Pipettor 200 L, 1000 L, 5000 L
3. pH meter TPS
4. Duran Bottle 1L, 2L
5. Shaking water bath RATEK
6. Centrifuge Thermo Scientific Multifuge X3R
7.HPLC system Shimadzu UFLC-1 equipped with UV- visible diode-array detector (PDA)
8. Reversed-phase Phenomenex 150*4.6 mm, 2.6 μm
Kinetex Biphenyl 100A
Column
9.Strata C18-E SPE Phenomenex 55 μm, 70A cartridge
10. Syringe 10 mL
11. PTFE membrane 0.45 μm pore
95 3.3.9.2 Chemicals
The chemicals used for B-complex vitamins analysis are listed in Table 3.18.
Table 3.18 Chemicals used in B-complex vitamins analysis
Chemical Grade Manufacturer
1. Vitamin B1 AR grade Sigma-Aldrich
2. Vitamin B2 AR grade Sigma-Aldrich
3. Vitamin B6 AR grade Sigma-Aldrich
4. Vitamin B12 AR grade Sigma-Aldrich
5. Nicotinic acid Sigma-Aldrich
6. Nicotinamide AR Grade Sigma-Aldrich
7. Folic acid AR Grade Sigma-Aldrich
8. 5-methyltetrahydrofolate AR Grade Sigma-Aldrich
Hydrochloric acid AR Grade Ajax Aps
Sodium acetate AR Grade Ajax Finechem
Takadiastase AR Grade Sigma-Aldrich
Claradiastase AR Grade Sigma-Aldrich
Ammonia acetate AR Grade Ajax Finechem
Methanol HPLC Grade Thermo Fisher Scientific
3.3.9.3 Procedure of B-complex vitamins analysis
3.3.9.3.1 Chromatographic conditions
For the separation of the B-complex vitamins, a reversed-phase Kinetex
Biphenyl 100A (150*4.6 mm, 2.6 m) was used at ambient temperature. The method was followed with method of Chatzimichalakis et al. (2004) with
96 modification. The mobile phase of the HPLC system was delivered at a flow rate of 0.8 mL/ min, and consisted of A: 0.05 M CH3COONH4/CH3OH (99/1) and B:
H2O/CH3OH (50/50) at pH 5.0. A multi-step gradient was used, starting at an A:
B v/v composition of 99:1, and remaining isocratic for 8 min. This composition was changed linearly to reach 40% of solvent B after 18 min, to reach 55% of solvent B after 28 min, reach 100% of solvent B after 33 min and finally elution was performed isocratically for 8 min. A 10 min equilibration time was performed between injections.
Detection was performed with a photodiode array detector (PDA) at 280 nm.
The quantitation was performed at maximum wavelength for each vitamin as follows: 260 for nicotinic acid, 257nm for pyridoxine, 256 nm for nicotinamide,
270 nm for thiamin, 280 nm for folic acid, 290 nm for 5-methylfolate and 265 nm for riboflavin. Identification of resolved peaks in real samples has been executed by comparing their spectra with those derived from standard solutions.
The injection volume was 25 L.
3.3.9.3.2 Preparation of standard solutions
The aqueous stock and standard solutions of the B-complex vitamins were prepared with the mobile phase (mobile phase A: 0.05 M CH3COONH4/CH3OH
(99/1) and mobile phase B: H2O/CH3OH (50/50) at pH 5.0). The concentration of the stock solutions was 250 ng/ L, while the concentration of the working solutions ranges from 0.25 ng/ L to 50 ng/ L. The stock solutions were stored in a refrigerator and covered with aluminum foil in order to protect them from light. The working solutions were prepared fresh before determination.
97 3.3.9.3.3 Method validation
The method described has been validated with respect to accuracy, within-day repeatability (n=10), between-day precision for seven consecutive days, linearity and sensitivity. Linearity was studied by a series of mixed standards of the examined vitamins, covering the entire working range; fourteen concentration levels were used in the range 0.25-50 ng/ L. Each solution was injected three times. Correlation coefficients for the B-complex vitamins on the basis of plots of concentration (ng/ L) against peak area (maul) were found to be >0.996.
Accuracy was determined by replicate analysis of reference material (mixed vegetable, CRM4851). The detection limits and quantitation limits were assessed by the software Absolution.
3.3.9.3.4 Sample Preparation
The extraction procedure used in this study was based on a combination of acid digestion and enzymatic hydrolysis according to the study of Jakobsen (2008) and Lebiedzińska et al. (2007) with modification. One gram of sample or 1 mL of standard solution was mixed with 8 mL 0.1 N hydrochloric acid and boiled in the water bath for 20 min. After cooling down the sample, the pH of sample was adjusted to 4.5 with 2 N sodium acetate. A mixture of 25 mg takadiastase and
25 mg claradiastase was added. An enzyme blank was always prepared to correct for the addition of vitamins from the enzyme suspension. After 18 h enzyme incubation at 37 C, samples were heated at 100 C for 5 min to inactivate the enzyme. After cooling down, the samples were centrifuged for 10
98 min at 14,000 × G. The supernatant was kept for SPE.
3.3.9.3.5 Solid Phase Extraction (SPE)
The banana pseudostem consists of many components that cause chromatographic interferences with vitamins. As a result SPE with Strata C18-E
(55 m, 70A) cartridge was used for purification. The SPE method of Cho et al.
(2000) was used of the extraction of water soluble vitamins. The stationary phase was flushed with 10 mL methanol and 10 mL acidified Milli Q water (pH
4.2) to activate the stationary phase. The supernatant of the sample was loaded and eluted with 5 mL Milli Q water (pH 4.2) then 10 mL methanol at a flow rate of 1 mL/ min. The eluent was collected in a bottle covered with foil and evaporated to dryness. The residue was dissolved in 2 mL mobile phase. The solution was passed through a 10 mL syringe and filtered with PTFE membrane
(pore: 0.45 m) into the HPLC sample vials.
99 3.4 Physicochemical properties analysis
3.4.1 Water holding capacity (WHC)
3.4.1.1 Equipment
The equipment used for WHC is listed in Table 3.19.
Table 3.19 Equipment used for WHC analysis
Equipment Brand Model
Magnetic stirrers
Stirring bars
Centrifuge tubes 50 mL
Centrifuge Thermo Scientific Multifuge X3R
Analytical balance ANR GR-200
3.4.1.2 Procedure of WHC analysis
The water holding capacity was determined based on the standard methods
(Heywood et al., 2002). A sample of 250 mg dry banana pseudostem sample was weighted to nearest 0.1 mg and mixed with 25 mL distilled water in a weighted centrifuge tube. It was stirred with a magnetic stir bar at room temperature for 30 min and left for 30 min. It was then centrifuged at 3500 rpm for 30 min.
100 After that, the supernatants were decanted, each centrifuge tube was weighed then the WHC was calculated as g water of dry sample using equation (3-9).
wt of centrifuge tube after decanting wt of dry tube total sample wt WHC total sample wt (3-9)
3.4.2 Procedure for determining swelling capacity (SWC) and solubility analysis
3.4.2.1 Equipment
The equipment used for determining SWC is listed in Table 3.20.
Table 3.20 Equipment used in SWC and solubility analysis
Equipment Brand Model
Magnetic stirrers
Stirring bars
Centrifuge tubes 50 mL
Centrifuge Thermo Scientific Multifuge X3R
Water bath GRANT INSTRUMENTS SB 3
Analytical balance ANR GR-200
3.4.2.2 Procedure of SWC and solubility analysis
The determination of SWC and solubility were following the method suggested by Mestres et al. (1997). Dry banana pseudostem sample (1.0 g) was accurately weighed and quantitatively transferred into a clear dried test tube and re-weighed (W1). The sample was then dispersed in 50 mL of
101 distilled water using a stirrer. The resultant slurry was heated at the 95 °C for 30 min in a water bath.
Aliquots (5 mL) of the supernatant were dried to a constant weight at 110 °C.
The residue obtained after drying the supernatant represented the amount of starch solubilised in water. Solubility was calculated as g per 100 g of sample on a dry weight basis.
The residue obtained from the above experiment (after centrifugation) with the water it retained was quantitatively transferred to the clean dried test tube used earlier and weighed (W2).
Swelling of starch = W2 - W1/weight of starch
3.4.3 Crystalline Index (CI) analysis
3.4.3.1 Equipment
The equipment used for CI analysis is listed in Table 3.21.
Table 3.21 Equipment used for CI analysis
Equipment Brand Model
X-ray Diffraction System PANalytical X’pert MPD
3.4.3.2 Procedure of CI analysis
The CI of the banana pseudostem powder was measured by X-ray diffraction
(XRD). The X’Pert PRO Multi-purpose X-ray Diffraction System (MPD system) was used in this experiment. The banana pseudostem powder was packed in a
102 standard sample holder with 30 mm diameter and 2.5 mm depth. After packing, all of the samples were placed on the automatic multi-sample changer for X-ray diffraction. The XRD was operating at tension of 45 kV/ 40 mA. Scans were obtained from 4 to 55 degrees 2θ in 0.026 degree steps for 50.05 s per step.
To calculate the CI of banana pseudostem, individual crystalline peaks were extracted by a curve fitting process from the diffraction intensity profiles. A peak fitting program (Magic Plot; www.magicplot.com) was used, assuming Gaussian functions for each peak and a broad peak at around 21.5 °C assigned to the amorphous contribution. After subtracting the diffractogram of the amorphous from the diffractogram of the whole sample, the CI was calculated by dividing the remaining diffractogram area due to crystallinity by the total area of the original diffractogram.
3.5 Statistical analysis
Statistical analyses were conducted by using SAS (Statistical Analysis System) v9.4 software. One-way analysis of variance (ANOVA) was employed to assess difference among the carbohydrate contents at the different processing conditions. The significant differences between mean values were determined by the t-test (LSD) at a significance level of p< 0.05.
103 4. Drying of banana pseudostem
4.1 Drying curve determination
4.1.1 Effects of processing on the drying curve of Musa balbisiana
banana pseudostem
The drying curves show variations of the moisture ratio versus drying time for banana pseudostem (Musa balbisiana) dried under different treatments as shown in Figure 4.1. The moisture ratio is defined as the ratio of moisture content of the sample at each moment (M) and initial moisture content (Mo) of the sample (Agarry et al., 2013). The patterns of the curves obtained under 4 conditions, namely 40 °C (BP40B) and 50 °C (BP50B) with 3 minutes blanching at 70 °C and 40 °C (BP40) and 50 °C (BP50) with no pretreatment, show similar shapes.
Figure 4.1 Effect of processing on the drying ratios of banana pseudostem
At the initial stages of the drying process, the drying rates were high and with
104 the increase in time the drying rates decreased until the samples reached the equilibrium moisture content indicated by a stable weight. For a set of drying conditions (temperature and relative humidity) the end of the drying process was attained. The reason for this trend to occur is due to the migration of water out of the sample (Fellows, 2002). Initially, the surface of the samples heated up to wet bulb temperature and the free water moved out of the samples and evaporated, following a constant rate of drying. With increasing time, water moved from the interior of the material to the surface following a falling rate of drying. Figure 4.1 shows that drying at 50 °C without pretreatment exhibited the fastest drying rate. This drying temperature appears to be suitable for the industry as it can save both energy and cost. This is due to the fact that an increase in air temperature causes water to evaporate more rapidly from a wet surface and thereby produces a greater fall in surface temperature (Fellows,
2002). Hence, drying at 50 °C without pretreatment would be chosen as optimal condition for drying banana pseudostem.
Drying at 50 °C without pre-treatment was the optimal condition for Musa balbisiana pseudostem, and this condition was also selected to dry Musa acuminata pseudostem.
105 4.1.2 Effects of species on the drying curve
Figure 4.2 shows the drying curves of these two different species.
Figure 4.2 Effect of species on the drying ratios of banana pseudostem
According to the figure above, Musa balbisiana pseudostem has a higher drying rate than Musa acuminata pseudostem, which means the loss of moisture from
Musa balbisiana pseudostem per each unit of time is higher than that from
Musa acuminata pseudostem. Musa balbisiana pseudostem needs 4 hours to dry, while Musa acuminata pseudostem requires 6 to 7 hours to dry. This may indicate that there is less free water and more bound water in Musa acuminata pseudostem tissue than in Musa balbisiana pseudostem tissue.
4.2 Effects of drying on the colour of banana pseudostem
Table 4.1 shows the colour (L*, a*, b*) of BP40, BP40B, BP50, BP50B and
106 BPA. L* value reflects the lightness of the samples, the higher L* value is, the whiter the sample is. The a* values are indicative of the red and green colours, in which a positive a* value correlates with red, while a negative a* value indicates green colour. The b* values are indicative of yellow and blue colour, in which a negative b* value indicates blue and a positive b* value indicates yellow
(Ho et al., 2013).
Table 4.1 Colour of dried ground banana pseudostem (n= 3 ± SD)
L* a* b*
40 79.02e ± 0.15 1.02e ± 0.01 10.16a ± 0.05
40B 84.03b ± 0.25 1.22c ± 0.02 8.89c ± 0.15
50 86.44a ± 0.09 0.43d ± 0.03 8.22d ± 0.03
50B 80.57c ± 0.34 1.48b ± 0.03 9.48b ± 0.07
BPA 79.99d ± 0.36 1.91a ± 0.03 9.36b ± 0.05
Means within a column followed by different superscripts are significantly different (P<0.05).
All values are based on dry weight basis.
Significant difference (p<0.05) of colour (L*, a* and b*) was observed in Musa balbisiana pseudostem processed under different conditions. BP50 had the highest L* value and lowest b* value, while BP40 showed the lowest L* value and highest b* value. This result indicates that the banana pseudostem dried at
50 °C without blanching provided the whitest samples among the 4 conditions.
Banana pseudostem samples dried at 40 °C without blanching had brown colour. It is believed that blanching can inactivate enzyme and inhibit colour
107 deterioration. Blanching was proved to progressively decrease polyphenoloxidase (PPO) and peroxidase (POD) activities in green peppers and peeled bananas (Castro et al., 2008; Cano et al., 1990). However, blanching samples in this study didn’t provide a whiter colour than samples without blanching. One possibility is that the samples collected in this study were fresh.
There was several hours gap between collection and sample preparation. The oxidation was still at the beginning stage and there was not much browning effect on the samples. Despite the fact that BP50 provided the whitest colour,
Figure 4.1 also shows that BP50 had the highest drying rate, while BP40 had the lowest drying rate. Thus, in the case of drying and white colour preservation, drying banana pseudostem at 50 °C without blanching is suitable to be chosen as optimal condition for drying.
As for the colour of different banana species, Musa balbisiana pseudostem provided higher L* and lower b* value than Musa acuminata pseudostem, which indicated that Musa balbisiana pseudostems were whiter than those of Musa acuminata under the same processing conditions. There was significant difference in colour between the two banana species under the sample processing conditions (P < 0.05).
4.3 Water activity (aw)
Water activity differs from moisture content. Water activity measures the availability of free water in a food system that is responsible for any biochemical reactions, while the moisture content represents the water composition in a food system (Fazaeli et al., 2012). Water activity of dried food below 0.6 is
108 believed to be good for food preservation as no microbes can multiply at this condition (Sikorski, 2006). As shown in Table 4.2, all of the aw of banana pseudostem in this study were below 0.6. This means all of the drying methods applied in this study are acceptable for banana pseudostem preservation.
Table 4.2 Water activity (aw) of Musa balbisiana and Musa acuminata
aw
BP40 0.41
BP40B 0.51
BP50 0.40
BP50B 0.35
BPA 0.41
All values are expressed on dry weight basis
109 5. Effects of drying on the nutrient stability of banana pseudostem
5.1 Proximate nutrients
The result of the proximate analysis are shown in Table 5.1
Table 5.1 Mean proximate contents of Musa balbisiana and Musa acuminata
Moisture Protein Fat Ash Carbohydrate Total
(%) (%) (%) (%) (%) (%)
Musa balbisiana
BP40 6.2a 3.1b 3.2a 12.9c,d 66.3a 91.7
BP40B 5.0b,c 3.2b 4.0a 12.3d 66.2a 90.7
BP50 4.4d 3.4b 3.4a 14.0b 64.4a 89.6
BP50B 4.5c,d 3.4b 3.0a 12.4c 68a 91.3
Musa acuminata
BPA 5.8a,b 6.1a 2.8a 15.9a 62.7a 93.3
Means within a column followed by different superscripts are significantly different (P<0.05).
All values are based on dry weight basis and means of replicate determinations
5.1.1 Moisture
The moisture contents for samples subjected to different drying conditions:
BP40, BP40B, BP50, BP50B were 6.2%, 5.0%, 4.4% and 4.5% respectively
110 (Table 5.1). These values were lower than those reported in literature that determined the moisture content of banana pseudostem by using the same oven drying method. Both Aziz et al. (2011) and Bhaskar et al. (2011) reported higher moisture contents in dried banana pseudostem (8.82% and 15.1%). The reasons for this difference could be attributed to species, size of banana pseudostem pieces and drying conditions (temperature, relative humidity and time). Thicker pieces of cut banana pseudostem would take longer time to dry and the moisture content in these dried pieces would be higher than in thinner dried pieces. The banana species used in the studies of Aziz et al. (2011) and
Bhaskar et al. (2011) were different from this study, which may cause the difference in the moisture content. The banana species collected from Bhaskar et al. (2011) is Musa sp. var. elakki bale and from Aziz et al. (2011) was the hybrid of Musa acuminata and Musa balbisiana pseudostem. Samples dried at
50 °C had the lowest moisture content, while samples dried at 40 °C had the highest moisture content. This result was similar to the drying curve obtained at
50 °C without pretreatment which was the optimal condition for drying banana pseudostem. There was no significant difference between BP40B and BP50B
(P>0.05), but there was significant difference between BP40, BP50 and BP40B or BP50B. This result implies that temperature has no effect on the drying efficiency in blanched samples, but has important influence on samples without the blanching pretreatment. Higher temperature provides higher drying ratio.
The moisture content of dried Musa acuminata pseudostem was 5.8% compared with 4.4% of dried Musa balbisiana pseudostem. These two values were significantly different (P<0.05). This result correlates with the result
111 that drying at 50 °C without pretreatment has the highest drying rate. This may be caused by more bound water being present in the Musa acuminata pseudostem than in the Musa balbisiana pseudostem, making it more difficult to evaporate. As a result, more moisture was retained in dried Musa acuminata pseudostem.
5.1.2 Ash
As shown in Table 5.1, the ash content found in BP40, BP40B, BP50, BP50B and BPA samples was 12.9%, 12.3%, 14.0%, 13.4% and 15.9%, respectively.
All values reported in this study were on a dry weight basis. Significant increase in ash content was observed with the increase in drying temperature (P < 0.5).
No significant change in ash content was found between BP40 and BP40B (P >
0.5), but the ash contents of BP50 and BP50B were significantly (P<0.05) different. These results indicate that drying temperature has an effect on ash content of Musa balbisiana pseudostem, whereas blanching has no influence.
Additionally, the ash content of Musa acuminata pseudostem was significantly higher (P<0.05) than that of Musa balbisiana pseudostem. According to the results, the value of ash varied with different drying temperatures and banana species.
Ho. et al. (2012) and Aziz et al. (2011) reported lower ash contents, which were
6.75% and 10.08%. Higher ash content in this study indicates higher mineral contents. It is possible that the usage of different parts of the banana pseudostem resulted in different mineral contents. The pseudostem utilized in this study was the tender core. It is likely that minerals are concentrated in the
112 tender core of the pseudostem. Another possibility is that the banana species used in this study were different from those mentioned in literature.
5.1.3 Protein
The protein content of BP40, BP40B, BP50 and BP50B were 3.1%, 3.2%, 3.4% and 3.4%. All values are based on a dry weight basis. There is no significant difference (P>0.05) among these values. Temperature and blanching did not affect the protein values in Musa balbisiana pseudostem. Bhaskar et al. (2011) reported lower protein content in banana pseudostem, which was 2.5%. The differences in the values may be due to the various methods used to analyze protein. Aziz et al. (2011) showed similar protein in tender core of banana pseudostem flour with this study, which was 3.52%, but the protein content of the native banana pseudo-stem flour was only 0.89 in their study. The protein value of Musa acuminata pseudostem (6.1%) was significantly higher (P<0.05) than Musa balbisiana pseudostem (3.4%), which was nearly double the value of
Musa balbisiana pseudostem. However, compared with plain wheat flour
(NUTTAB, 2010), which was 10.8 g/100 g, the protein contents in both banana pseudostem species are low. It implies that dried banana pseudostem flour has a potential to make low gluten food, such as cake.
5.1.4 Fat
The fat contents of BP40, BP40B, BP50, BP50B and BPA were found to be
3.2%, 4.0%, 3.4%, 3.0% and 2.8%. There was no significant difference (P>0.05) between different drying treatments of Musa balbisiana pseudostem. The fat in
113 Musa acuminata pseudostem was slightly lower than in Musa balbisiana pseudostem, but there was no significant difference between these two species.
These values were higher than those reported by others for dried banana pseudostem, which had a fat content of 1.18% and 1.7% (Aziz et al., 2011 &
Bhasker et al., 2011).
5.1.5 Carbohydrates
The total carbohydrates in BP40, BP40B, BP50, BP50B and BPA were 66.3%,
66.2%, 64.4%, 68% and 62.7%. Although BP50B had the highest carbohydrate content, there was no significant difference (P >0.5) between all the banana pseudostem samples irrespective of the differences in pretreatments or the species. The details of the carbohydrate in banana pseudostem and the digestibility of it will be described in chapter 6.
5.2 Micronutrients
5.2.1 Minerals
The mineral contents in the two species are reported in Table 5.2. The mineral elements detected were potassium, phosphorus, sodium, calcium, magnesium, copper, iron, zinc, manganese and selenium. The predominant elements found in the dried banana pseudostem were phosphorus, potassium, calcium and magnesium, which are classified as macro-elements.
For Musa balbisiana, potassium was the most predominant (5374 mg/100 g,
5015 mg/100 g, 6091 mg/100 g and 5524 mg/100 g for BP40, BP40B, BP50,
114 BP50B, respectively) followed by Mg (197 mg /100 g, 200 mg/100 g, 255 mg/100 g and 233 mg/100 g for BP40, BP40B, BP50, BP50B, respectively), Ca
(115 mg/100 g, 130 mg/100 g, 129 mg/100 g and 145 mg/100 g for BP40,
BP40B, BP50, BP50B, respectively) and P (59 mg/100 g, 65 mg/100 g, 69 mg/100 g and 64 mg/100 g for BP40, BP40B, BP50, BP50B, respectively). For
Musa acuminata, potassium still dominates the macro-elements value, which contributes 6963 mg /100 g. The value of Mg in Musa acuminata was similar to that of Musa balbisiana (230 mg/100 g). Nevertheless, the Ca in Musa acuminata was 262 mg/100 g, which was almost double that of the Musa balbisiana.
Table 5.2 Mean mineral content (mg/100 g) of Musa balbisiana and Musa acuminata
Ca Cu Fe K Mg Mn Na P Se Zn
Musa balbisiana
40 115 0.5 8.5 5374 197 0.7 0.7 59 0.1 16.6
40 B 130 0.6 6.8 5015 200 0.5 13.2 65 0.1 11.1
50 129 0.4 9.4 6091 255 0.7 1.5 69 0.1 22.4
50 B 145 0.9 11.6 5524 234 0.7 13.6 64 0.1 13.8
Musa acuminata
BPA 262 0.5 8.5 6963 230.4 0.9 1.4 163 0.0 9.5
All values are based on a dry weight basis. All values are means of replicate determinations
However, the results obtained from the present study differed from those reported by Ho et al. (2012), who have reported that banana
115 pseudostem (Musa acuminata), which was the same species as in this study, had a higher amount of Ca (1335.33 mg/100 g of dry sample). The calcium content was almost 6 times that found in the present study. Meanwhile, the potassium content reported by Ho et al. (2012) was 944.12 mg/ 100 g, which was only about one sixth of that in the present study. The reason for this may be due to the difference in the maturity of banana pseudostem samples collected. Ca concentration increased with age (Ho et al., 2012). More mature pseudostems seemed to have been used in Ho et al. (2012) study compared to the present study. Therefore the calcium content in the study of Ho et al. (2012) was significantly higher and the potassium was lower than in this study.
Potassium accumulates in the stalk before efflorescence. Its concentration in the pseudostem decreases during the fruiting phase, because the bananas require substantial amounts for fruit development (Ho et al., 2012). At the maturation stage, the calcium concentrations increase due to competition with other cations, especially potassium (Ho et al., 2012). The potassium in the banana pseudostem found in this study was extremely high; it even exceeded the amount of banana fruit, which is considered as a high potassium food. The
K content of raw peeled Cavendish banana and ladyfinger banana are 346 and
322 mg/100 g (NUTTAB, 2010, wet weight basis), whereas the K of Musa balbisiana and Musa acuminata pseudostem in this study were both over 450 mg/ 100 g (wet weight basis). Banana pseudostem could be a good source of potassium. Na contents in blanched banana pseudostems were significantly higher (P<0.05) than in unblanched banana pseudostems, which were 13.2 mg/100 g and 13.6 mg/100 g in BP40B and BP50B compared with 0.7 mg/100
116 g, 1.5 mg/100 g and 1.4 mg/100 g in BP40, BP50 and BPA. This may be because the banana pseudostem was in contact with water when blanched and could have absorbed sodium from the blanching water.
In the case of micro-elements, the contents of Cu, Fe, Zn, Mn, and Se were found to be low in this study. Among these elements, Zn was the highest, which was 16.6 mg/100 g, 11.1 mg/100 g, 22.4 mg/100 g, 13.8 mg/100 g and 9.5 mg/100 g in BP40, BP40B, BP50, BP50B and BPA, respectively. Iron was the next highest with 8.5 mg/100 g, 6.8 mg/100 g, 9.4 mg/100 g and 11.6 mg/100 g,
8.51 mg/100 g in BP40, BP40B, BP50, BP50B and BPA, respectively. These values were higher than those described by Ho et al. (2012), which were 8.05 mg/100 g for Zn and 3.31 mg/100 g for Fe. The difference may be due to the difference in species. The pseudostems used in this study were Musa balbisiana and Musa acuminata, while the pseudostems used in the study of Ho et al. was the hybrid of Musa acuminata and Musa balbisiana, Compared with other dried fruits and vegetables, such as dried apple, apricot and tomato, the contents of Zn and Fe in Banana pseudostem were higher (NUTTAB, 2010).
The normal level of Mn in plants ranged from 2.0-50.0 mg/100 g (Ho et al.,
2012), whereas this study indicated that the Mn contents in banana pseudostem were lower than the normal range, which were 0.7 mg/100 g, 0.5 mg/100 g, 0.7 mg/100 g, 0.7 mg/100 g and 0.88 mg/100 g in BP40, BP40B, BP50, BP50B and
BPA, respectively. Overall, the Musa balbisiana banana pseudostems subjected to four drying treatments were high in minerals, which agree with the high ash content (15.9 g/ 100 g in Musa acuminata pseudostem and 14.0 g/ 100 g in
Musa balbisiana pseudostem) in banana pseudostem samples as
117 described in chapter 5.1.2. The total amount of minerals of Musa balbisiana detected in this study was 6579 mg/ 100 g and of Musa acuminata pseudostem was 7639 mg/ 100 g, which were nearly half the values of ash contents.
5.2.2 B complex vitamins
5.2.2.1 Chromatography
There are two operation modes for HPLC separations, including isocratic and gradient elution (Dong, 2006). Isocratic elution is a separation in which the mobile phase composition remains constant through the procedure. However, gradient elution is most frequently used in chemical separation, which elutes the mixture of compounds by changing the concentration of the mobile phase
(Klejdus et al., 2004).
The method used in this study was followed using the method of
Chatzimichalakis et al. (2004) with modification. The gradient elution using the mobile phase A: 0.05M CH3COONH4/CH3OH (99/1) adjusted to pH 5.0 and B:
H2O/CH3OH (50/50) was selected for the separation of seven water soluble vitamins on a reversed-phase Kinetex Biphenyl 100A (150*4.6 mm, 2.6 m) column. A multi-step gradient was used, starting at an A: B v/v composition of
99:1, and remaining isocratic for 8 min. This composition was changed linearly to reach 40% of solvent B after 18 min, to reach 55% of solvent B after 28 min, reached 100% of solvent B after 33 min and finally elution was performed isocratically for 8 min. A 10 min equilibration time was observed between injections. The pH of the mobile phase is an extremely critical factor for the separation of vitamins (Vinas et al., 1996; Landeghem et al., 2005). It can
118 impact the chromatographic selectivity, peak shape and retention (Dabre et al.,
2011). Without pH adjustment all the vitamins were separated except thiamin and 5-MTHF (Figure 5.1a). Therefore, the influence of the mobile phase pH within the range from 3.5 to 7.5 was studied, and it was found that thiamin and
5-MTHF were well separated at pH 5.0 (Figure 5.1b).
mAU(x100) 9.0 260nm1nm (1.00) 8.5 a 8.0 F
7.5 TH 5-M 7.0
6.5
6.0
5.5
5.0 in 4.5 Thi am 4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5 18.0 19.0 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 min
mAU(x100) 6.0 260nm,1nm (1.00)
5.5 F
5.0 H T -M
4.5 5
e b 4.0 in m ia h
3.5 T
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5 20.0 22.5 25.0 27.5 30.0 min
Figure 5.1 Chromatography of standard thiamin and 5-MTHF with pH adjustment (a) and without pH adjustment (b) of mobile phase A
The chromatographic conditions used in the developed method provided an excellent separation within approximately 43 min. A typical chromatogram is
119 shown in Figure 5.2. Retention times of examined vitamins were: 1. Nicotinic acid tR = 3.71 min, 2. Pyridoxine tR = 7.43 min, 3. Nicotinamide tR =11.79 min,
4. Folic acid tR = 16.83 min, 5. Thiamin tR = 19.51 min, 6. 5-MTHF tR = 23.66 min and 7. Riboflavin tR = 38.80 min.
p mA U
350
325 Riboflavin
300
275
250
225
200
175
150
125 5-MTHF
100 Nicotinic Acid
75 Folic acid Thiamine
50
25 Nicotinamide Pyridoxine 0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 min
Figure 5.2 Chromatography of 7 B-complex vitamins
120 5.2.2.2 Method validation
Linearity and sensitivity data are summarized in Tale 5.3.
Table 5.3 Linearity of standard curves and sensitivity for the seven B-complex vitamin.
Vitamin Linear range Correlation LOD LOQ
(ng/ L) coefficient (ng/ L) (ng/ L)
Nicotinic acid 0.25-50 0.9997 0.06 0.20
Pyridoxine 2.5-50 0.9997 0.17 0.58
Nicotinamide 1-50 0.9997 0.08 0.27
Thiamin 2.5-50 0.9996 0.11 0.38
Folic acid 1-35 0.9965 0.05 0.17
5-methylfolate 1-45 0.9997 0.16 0.54
Riboflavin 0.25-40 0.9984 0.04 0.13
The calibration graphs were constructed by plotting the peak area based on results corresponding to triplicate injections of seven vitamin standards against concentration (ng/ L). A satisfactory linearity was observed, as the correlation coefficient ranged from 0.9965 to 0.9997. The limit of detection (LOD) of the method used was calculated as three standard deviations of the background noise of the standard finally diluted in the same buffer as the banana pseudostem samples. The LODs for nicotinic acid, pyridoxine, nicotinamide, thiamin, folic acid, 5-methyfolate and riboflavin were 0.06, 0.17, 0.08, 0.11,
0.05, 0.16 and 0.04 ng/ L, respectively. The limit of quantification (LOQ) was calculated as ten times SD. The values for nicotinic acid, pyridoxine, nicotinamide, thiamin, folic acid, 5-methylfolate and riboflavin were 0.20, 0.58,
121 0.27, 0.38, 0.17, 0.54 and 0.13 ng/ L. LOD and LOQ proved to be comparable with previously published work (Chatzimichalakis et al., 2004).
Since 1989, the Community Bureau of Reference started a research program to improve the quality of vitamin analysis in food (Ollilainen et al., 2001). The accuracy of the method was estimated by analyzing certified material BCR 485
(mixed vegetable). The BCR reference material can be used to verify the accuracy of results and to monitor the performance of the method. The values expressed as mean, standard deviation and recovery are reported in Table 5.4.
Table 5.4 Result of HPLC analysis of B group vitamin concentrations in certified reference
material (BCR 485-Mixed vegetable)
Reference value Data obtained Recovery
(mg/ kg) (mg/ kg) (%)
Vitamin B1 3.07±0.34 2.82±0.15 91.86
Vitamin B6 4.80±0.80 5.10±0.18 106.25
5-MTHF 2.14±0.42 2.24±0.09 104.67
Values represent the mean of triplicate determination and standard deviations
(SD).
All values are reported on a dry weight basis.
The analytical results obtained for the reference material were comparable to the certified values and the recoveries ranged between 91.9% and 106.3%.
Moreover, the banana pseudostem samples were spiked with known amounts of vitamins at three concentration levels corresponding to the calibration ranges of the method.
122 The intra-day repeatability was evaluated based on 10 measurements of the same sample, independently prepared, within the sample set. The inter-day precision was determined by analyzing the same pseudostem samples during 7 consequent days. Validation results obtained for the vitamins analyzed are summarized in Table 5.5.
The RSD for the same day ranged between 0.6% and 2.5% for peak areas. The
RSD between days were slightly higher (4.2%-9.7%) than the intra-day RSD values. However, both of the RSDs of intra-day and inter-day experiments were below 10%. The recovery rate of the vitamins in the intra-day experiment ranged between 92.54% and 105.25%, while the recovery rate in the inter-day experiment ranged between 91.25% and 106.11%.
123 Table 5.5 Inter-day (over a period of 7 consecutive days) and intra-day (n=6) values for the B-
complex vitamins
Intra‐day (n=10) Inter‐day (n=7)
Vitamin Added Found RSD Recovery Found RSD Recovery
(μg) (μg) ±SD (%) (%) (μg) ±SD (%) (%)
Nicotinic 0.30 0.31±0.00 2.1 103.33 0.32±0.03 9.7 106.67 acid 0.40 0.42±0.01 2.4 105.25 0.41±0.04 9.3 102.51
0.50 0.51±0.00 1.6 102.58 0.53±0.04 7.7 106.11
Pyridoxine 4.00 4.21±0.08 1.9 104.25 4.23±0.32 7.6 105.73
5.00 4.89±0.12 2.5 97.81 4.85±0.26 5.4 96.40
6.00 6.04±0.07 1.2 100.67 6.07±0.29 4.8 101.17
Thiamin 0.80 0.74±0.01 1.1 92.54 0.73±0.04 6.0 91.25
1.00 0.93±0.01 1.3 93.18 0.94±0.04 4.6 93.98
1.20 1.16±0.02 2.0 96.67 1.14±0.05 4.2 95.00
Folic acid 0.80 0.78±0.01 1.0 97.50 0.77±0.06 7.9 96.24
1.00 1.04±0.02 2.2 104.31 1.03±0.09 8.4 103.15
1.20 1.18±0.01 0.9 98.35 1.17±0.07 6.2 97.53
5‐MTHF 1.70 1.77±0.02 1.4 104.12 1.79±0.10 5.6 105.29
2.10 2.09±0.01 0.6 99.52 2.12±0.17 8.1 100.95
2.60 2.64±0.02 0.8 101.54 2.66±0.13 4.8 102.31
All values are mean ± SD of replicate determinations.
All values are reported on a dry weight basis.
5.2.2.3 Quantitative analysis of the vitamins in the banana pseudostem
The chromatographic procedure described above was applied to the
124 determination of the seven B group vitamins in the banana pseudostem. The chromatograms were obtained by injecting the pseudostem samples and the peaks were identified by the following two steps. Firstly, by comparing the retention time obtained from the samples, standards and the samples spiked with the standards under identical conditions. Secondly, by comparing the UV- visible spectrum of both samples and standard peaks by using photo-diode array detector (Shimadzu, Japan). After identification of the vitamins and confirmation of the absence of a matrix effect, the compounds were quantified in the banana pseudostem samples. The data obtained are given in Table 5.6.
125 Table 5.6 The concentration of B-complex vitamins (μg/g) in banana pseudostem dried in
different conditions and from different species
NA NAM PYR THI FA 5-MTHF RIB
Different drying conditions
BP40 0.80±0.08 ND 10.11±0.20 2.05±0.10 ND ND ND
BP40B 0.24±0.03 ND 6.72±0.65 2.14±0.24 ND ND ND
BP50 0.68±0.04 ND 8.50±0.00 2.23±0.15 ND ND ND
BP50B 0.60±0.08 ND 9.13±0.63 2.13±0.19 ND ND ND
Different species
Musa balbasiana
0.68±0.04 ND 8.50±0.00 2.23±0.15 ND ND ND
Musa acuminata
0.17±0.03 ND 7.48±0.52 2.68±0.17 ND ND ND
NA = nicotinic acid; NAM = nicotinamide; PYR = pyridoxine; THI = thiamin;
FA = folic acid; 5-MTHF = 5-methyltetrahydrofolate; Rib = Riboflavin
Values represent the means of triplicate determinations with standard deviations (SD).
All values are reported on a dry weight basis.
ND: Not detected.
Nicotinic acid, pyridoxine and thiamin were found in banana pseudostem, while nicotinamide, folic acid, 5-methylfolate and riboflavin were not detected in the banana pseudostem samples of both Musa acuminata and Musa balbisiana. As shown in Table 5.6, the concentration ranges for nicotinic acid, pyridoxine and
126 thiamin of Musa balbisiana dried in different conditions were 0.24-0.8, 6.72-
10.11 and 2.05-2.23 μg/g respectively. Compared with dried fig (0.05 mg/100 g), currant (0.11 mg/100 g), litchi (0.01 mg/100 g), banana (0.18 mg/100 g) and mixed fruit (0.10 mg/100 g), banana pseudostem (0.205- 0.223 mg/ 100g) had higher thiamin content (NUTTAB, 2010 & USDA, 2007). Dried banana pseudostem powder contained higher pyridoxine (0.672-1.011 mg/100 g) than dried apricot (0.1 mg/100 g), plums (0.21 mg/100 g) and litchi (0.09 mg/100 g) and similar value with dried prunes (0.75 mg/100 g; NUTTAB, 2010 & USDA,
2007). The high content of pyridoxine in banana pseudostem may be associated with the high level of pyridoxine in banana fruit. There was 0.2 mg
/100 g pyridoxine in raw peeled Cavendish banana, whereas the pyridoxine values of other raw fruit reported in NUTTAB Database (2010) were all below
0.1 mg/100 g. The niacin content detected in banana pseudostem powder was comparatively less than in other dried vegetable and fruit. The niacin values of dried apricot, cranberry, fig, peach, apple, seaweed and mashed potato were
2.5, 0.9, 0.5, 1.5, 0.91, 12.8 and 0.4 mg/100 g, while only 0.024-0.08 mg/ 100 g niacin were found in dried banana pseudostem samples (NUTTAB, 2010 &
CNF, 2010). Compared with Musa balbisiana, Musa acuminata has less nicotinic acid and pyridoxine and similar value of thiamin.
127 6. Effect of drying on the carbohydrate digestibility of banana pseudostem
Foods contain a range of chemically distinct carbohydrate components, which have varied gastrointestinal and metabolic properties (Englyst et al., 2007). The bioavailability of food carbohydrates is the most important nutritional property, since it describes the utilization and biological effect of dietary carbohydrates. In recent decades, the traditional belief that carbohydrates such as starch are completely digested although slowly digestible, have been overthrown. It is believed that the digestibility of carbohydrates determines the place and form in which carbohydrates are absorbed. Different kinds of carbohydrates have different nutritional benefits to humans. Although rapid digestion and absorption of carbohydrates has benefits for some aspects of sports nutrition, this is not commonly considered desirable as the elevated glycemic response after a meal relates to several chronic diseases, especially with diabetes or with features of metabolic syndrome (Englyst & Englyst, 2005). Slowly digested and absorbed carbohydrate sources, such as some of the oligosaccharides and SDS, may reduce postprandial glucose surges as well as the risk of coronary heart disease and diabetes incidence. Carbohydrates that escape digestion in the small intestine and poorly metabolized like resistant starch, some of the oligosaccharides and dietary fibre will enter the colon and the fermentable carbohydrates will be salvaged as short-chain fatty acids in the colon and meanwhile may stimulate colonic microflora like bifidobacteria, which are beneficial to health (Wong & Jenkins, 2007). The non-digestible
128 carbohydrates can reduce the risk of cardiovascular disease, diabetes, obesity, hyperlipidaemia and hypertension (Anderson & Hanna, 1999; Scheppach et al.,
2001). The new knowledge about carbohydrate digestion and absorption completely changes the way people think about dietary carbohydrates.
This chapter will provide values of rapidly digestible, slowly digestible and non- digestible carbohydrate as well as discuss the health benefits of banana pseudostem with different drying conditions and species.
129 6.1 Sugars
The fructose, glucose, sucrose and total sugar contents of the pseudostems
(dried in different conditions) of Musa balbisiana pseudostem and of Musa acuminata pseudostem (dried at 50 °C) are presented in Table 6.1. All values are based on a dry weight basis.
Table 6.1 Mean fructose, glucose, sucrose and total sugar content of Musa acuminata
pseudostem and Musa balbisiana pseudostem (%, dwb)
Fructose (%) Glucose (%) Sucrose (%) Total sugar (%)
Different drying conditions
BP40 8.0 ± 0.58a 10.4 ± 0.45a 4.4 ± 0.15a 22.8 ± 1.18a
BP40B 3.1 ± 0.24b 6.9 ± 0.44b 4.9 ± 0.63a 14.9 ± 1.41b
BP50 6.8 ± 0.33a 9.3 ± 0.82a 3.9 ± 0.43c 20.0± 1.79a
BP50B 3.0 ± 0.35bc 4.8 ± 0.59c 4.5 ± 0.51a 12.3 ± 0.58c
Different species
Musa 6.8 ± 0.33a 9.3 ± 0.82a 3.9 ± 0.43c 20.0± 1.79a balbisiana
Musa 1.8 ± 0.25c 4.0± 0.52c 3.5 ± 0.34b 9.2 ± 1.14c acuminata
All values are mean ± SD of replicate determinations
Means within a column followed by different superscripts are significantly different (P<0.05).
All values are based on dry weight basis.
When Musa balbisiana pseudostem was subjected to blanching, a highly
130 significant decrease (P<0.05) in fructose from 8.0% to 3.1% was observed in samples dried at 40 °C and 6.8% to 3.0% in that dried at 50 °C. However, no significant change (P>0.05) was detected in fructose values of samples dried at different temperatures without blanching. Similar results were found in glucose and total sugar content as well. These results indicate that blanching affects the fructose, glucose and total sugar values in Musa balbisiana pseudostem, while temperature plays no role. Similar results were found in soybean seeds.
Saldivar et al. (2010) postulated that there was a significantly lower sugar content in soybean seeds, which were blanched in water, than those without water blanching. This could be attributed to some of the soluble sugars leaching into the blanching water. In order to prove this assumption, the sugar contents in the blanching water were detected by HPLC in the present study. A loss of
8.2 mg glucose/ 100 g fresh sample and 4.6 mg of fructose/ 100 g fresh sample was observed. However, the loss of sucrose has no correlation with temperature and blanching in this study. BP50 had the lowest sucrose content
(3.9%) compared with the other three drying conditions. The sucrose content of
BP40, BP40B and BP50B were 4.4%, 4.9% and 4.5%, respectively. There was no significant difference (P>0.05) among these three drying conditions. It is probably the result of the molecular size of sucrose. As sucrose is a disaccharide and its diffusivity and solubility is lower than that of monosaccharides, it is not as easy as glucose and fructose to leach into the water (Gamboa- Santos et al., 2012). Thermal destruction may be the cause of monosaccharide loss as well. Heat damages cytoplasmic and other membranes of the cell wall, as well as disrupts subcellular organelles and their contents
131 become free to interact within the cell (Fellows, 2002). Another possibility is that blanching stops the enzyme reactions in the banana pseudostem, which deters the degradation of starch to single sugars.
Glucose, sucrose and fructose are metabolized differently. It has been stated that fructose produces a much lower postprandial glycemic and insulin response than sucrose and glucose (Asp, 1995; Melanson et al., 2007). As a result, the ratio of glucose to fructose in the diet is of physiological interest, especially in diabetes. Fig. 6.1 shows the ratio of single sugars in Musa balbisiana pseudostem.
Figure 6.1 Effect of drying condition on the ratio of fructose, glucose and sucrose of Musa balbisiana pseudostem
All values are mean of triplicate determinations All values are expressed on dry weight basis. According to Figure 6.1, BP40 and BP50 have a similar single sugars ratio, and
BP40B and BP50B have a similar ratio. This implies that drying
132 temperature has no influence on the change of single sugars ratio in Musa balbisiana pseudostem, while blanching affects the ratio. The glucose ratio in all samples is close to each other. Through blanching, the percentage of sucrose increases and the proportion of fructose decreases. Fructose has lower postprandial glycemic and insulin response than sucrose, thus non-blanched samples containing higher fructose would be more desirable from a nutritional standpoint. With the same drying conditions, different banana species provided different sugar values and ratios of sugar fractions (Fig. 6.2).
Figure 6.2 Effect of banana species on the ratio of fructose, glucose and sucrose
All values are mean of triplicate determinations All values are expressed on dry weight basis. McDonald (1981) stated that species is one of the major factors influencing the water-soluble carbohydrates content. Compared with Musa acuminata (6.8% fructose, 9.4% glucose and 18.9% total sugar), Musa balbisiana (1.8% fructose,
133 4.0% glucose and 9.2% total sugar) has a higher value of fructose, glucose and total sugar. However, sucrose content in Musa balbisiana is similar to that in
Musa acuminata. The sugar values of the two banana species significantly differ
(P<0.05) from each other. The glucose to total sugar ratio in these two species is similar, but the fructose to total sugar ratio and sucrose to total sugar ratio was reversed in these two species.
The sugar content of the banana pseudostem is similar to the range of most vegetables. The fructose content of the banana pseudostem ranges from 0.1 g
/100 g to 0.7 g /100 g on a wet weight basis. This range of fructose is similar to that of most vegetables, such as raw artichoke (0.3 g /100 g), fresh raw green bean (0.1 g/100 g), fresh raw broccoli (0.2 g/100 g), raw celery (0.5 g/ 100 g), raw peeled garlic (0.6 g/100 g) and raw red skin peeled pumpkin (0.8 g/100 g)
(NUTTAB, 2010), but lower than most fruits like cherry (5.4 g/ 100 g), banana
(4.9 g/ 100 g), pineapple (2.1 g/ 100 g) etc. (CNF, 2010). The glucose value of the banana pseudostem is between 0.3 – 0.9 g / 100 g. It is similar to raw artichoke (0.4 g / 100 g), fresh raw green bean (0.4 g/ 100 g), raw celery (0.7 g/
100 g), raw peeled garlic (0.4 g/ 100 g), raw cherry tomato (1 g/ 100 g) and raw red skin peeled potato (0.9 g/ 100 g) (NUTTAB, 2010). The sucrose levels in the banana pseudostem ranged from 0.2 g/ 100 g to 0.5 g/ 100 g, which is similar value to artichoke (0.2 g / 100 g), fresh raw green bean (0.2 g/ 100 g), fresh raw broccoli (0.1 g/ 100 g), raw peeled garlic (0.5 g/ 100 g), raw peeled pumpkin
(0.2 g/100 g), as well as muscadine grapes (0.6 g/ 100 g), raw sweet cherries
(0.2 g/ 100 g) and green kiwifruit (0.2 g/ 100 g) (NUTTAB, 2010 & USDA, 2007).
134 6.2 Starch
Starch content of the banana pseudostem in this study has been detected by two methods, including HPLC method and Englyst method (1992). Results are shown in Table 6.2.
Table 6.2 Rapidly digestible starch, slowly digestible starch, resistant starch and total starch
(Englyst Method & HPLC Method) of Musa acuminata and Musa balbisiana
pseudostem (%, db)
RDS (%) SDS (%) RS (%) TS (%) TS (%)
(Englyst Method) (HPLC Method)
Musa balbisiana
BP40 9.1 0.6b 6.4 0.5b 7.4 0.3c 22.9 1.0c 24.3 2.1c
BP40B 9.7 0.0b 8.2 0.2a 14.2 0.1b 32.1 0.2b 32.8 2.0b
BP50 7.7 0.5b 6.2 0.3b 6.6 0.9c 20.5 0.9c 22.9 1.1c
BP50B 16.4 1.2a 8.9 0.3a 7.5 0.8c 32.8 0.5b 31.7 1.9b
Musa acuminata
BPA 15.0 0.4a 2.9 0.1c 17.9 1.1a 35.8 0.8a 37.6 0.0a
All values are means of duplicate determinations ± SD of replicate analyses.
Means within a column followed by different superscripts are significantly different (P<0.05). All values are based on a dry weight basis.
The HPLC method determined the total starch content of the banana pseudostem by digesting into glucose; while Englyst method not only detected the total starch content but also categorized starch further into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS),
135 which provides more information about the digestibility of starch. The starch contents in these two methods, as well as the comparison of these two methods, will be presented in this section.
6.2.1 HPLC Method
The total starch content of the blanched samples was 32.8% and 31.7% in comparison with 24.3% and 21.9% in samples without blanching. No significant differences (P>0.05) were observed in samples with the same blanching conditions (with blanching/ without blanching) except in samples dried at different temperatures. However, in each set of samples dried at the same temperature, the sample with blanching had a notably higher total starch content than that without blanching.
The result above indicates that the drying temperature had no effect on total starch content of Musa balbisiana pseudostem. With the same drying temperature, the blanching process seemed to preserve more starch in the sample. This may be as a result of the enzyme inhibition during blanching.
Harvested, bananas have the ability to convert starch into sugar, such as glucose and fructose (Dadzie &Orchard, 1997). The enzymes in the cell wall, including α-amylase, β-amylase and starch phosphorylase, catabolize the starch in the plant (Paliyath et al., 2009). Since the samples used in this study were banana pseudostems, it is probable that the samples have some similarity in properties to the banana fruit. Due to the heating effect of blanching, which can destruct enzymes as well as damage the cytoplasm and other membranes
(Grandison, 2006), the reactions degrading starch into sugar will be stopped by
136 blanching. By contrast, samples without blanching have enzymatic reactions that are likely to degrade starch further into glucose and fructose. As a result, the starch contents of BP40 and BP50 are significantly lower than BP40B and
BP50B, but the fructose and glucose contents of BP40 and BP50 are dramatically higher than BP40B and BP50B. This is one of the possibilities of why blanched samples have higher starch values, but lower glucose and fructose values. Further studies are required to prove that the enzymatic reactions take place in the banana pseudostem after harvest.
6.2.2 Englyst Method
The RDS, SDS, RS and TS contents of Musa balbisiana and Musa acuminata by Englyst method are presented in Table 6.2. All values are on a dry weight basis.
A highly significant increase (P<0.05) in RDS and SDS was noted in BP50B.
Compared with BP50, the RDS in BP50B rose over 100% from 7.7% to 16.4%.
The resistant starch in BP40B is significantly higher (P<0.05) than the other three drying conditions in Musa balbisiana pseudostem, and there is no significant (P>0.05) difference in resistant starch in the other three drying conditions. No significant difference (P>0.05) was observed in TS and SDS of samples detected by Englyst method with the same blanching conditions (with blanching/without blanching) but dried at different temperatures. However, in each group of samples with the same temperature, blanched samples had a dramatically higher total starch content than that without blanching. This trend in total starch was the same as that observed in using the HPLC method. A similar
137 RDS, SDS, RS ratio was found in BP40 and BP50, however, the ratio observed in BP40B and BP50B was different (Figure 6.3).
Figure 6.3 Effect of drying conditions on the ratio of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) of Musa balbisiana pseudostem
All values are mean of triplicate determinations All values are expressed on dry weight basis. This implies that blanching not only has influence on the total starch content, but also affects the ratio of RDS, SDS and RS in the banana pseudostem.
BP50B has the highest RDS portion, which is up to 50%, and lowest RS level, which is only 22.9%. In contrast, BP40B has the lowest RDS ratio (30.2%) and highest RS (44.2%). The RS level of BP40B is over 60% higher than
138 BP50B. According to the ratio of RS, SDS and RDS of the four samples, the order of the digestibility is BP40B < BP50 The effect of banana species on the fraction of RDS, SDS, RS and TS is shown in Table 6.2. Despite SDS of Musa acuminata being lower than Musa balbisiana, other fractions of starch and total starch content are higher than Musa balbisiana. RDS, SDS, RS and TS of both species have significant differences (P<0.05). 139 It has been found in Figure 6.4 that the ratio of RDS of both banana species is almost the same, but the ratio of RS of Musa acuminata is remarkably higher than Musa balbisiana and the ratio of SDS is considerably lower than Musa balbisiana. Therefore, the banana species has a great effect on the starch content of pseudostem. Figure 6.4 Effect of banana species on the ratio of rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS) All values are mean of triplicate determinations All values are expressed on dry weight basis. 140 Compared with other food sources, banana pseudostem has a higher level of RS. Table 6.3 shows the RDS, SDS, RS and TS content of some cereals, fruits and vegetables. Table 6.3 RDS, SDS, RS and TS contents in some carbohydrate- containing foods Food name RDS (%) SDS (%) RS (%) TS (%) Wholemeal bread 56 4 1 61 Corn flakes 73 2 3 78 Potato biscuit 23 17 15 55 Boiled potato (hot) 65 5 5 75 Boiled potato (cold) 53 11 10 74 Banana flour 3 15 57 75 Spaghetti (cooled) 33 42 4 79 Millet (boiled 20 min, 42 28 6 76 cold) Maize RS product 55 9 34 98 Peas (frozen, boiled 5 12 2 5 19 min) Bean flakes 27 16 6 49 Source: Englyst, 1992 As shown in the table, boiled potato (hot and cold), cold boiled millet and bean flakes have similar levels of RS as the banana pseudostem. The content of RS of the whole-meal bread, corn flakes and cooled spaghetti were lower than the pseudostem samples, while the RS value of potato biscuit, banana flour and maize RS products were higher than that in banana pseudostem. Banana 141 pseudostem is a good source of resistant starch, because though its RS content was not the highest, the ratio of RS and SDS was relatively higher than other food sources and the RDS is relatively lower (Table 6.4). Table 6.4 RDS, SDS, RS and TS contents in some carbohydrate- containing foods Food name RDS% SDS% RS% Wholemeal bread 91.8 6.6 1.6 Corn flakes 93.6 2.6 3.8 Potato biscuit 41.8 30.9 27.3 Boiled potato (hot) 86.7 6.7 6.7 Boiled potato (cold) 71.6 14.9 13.5 Banana flour 4.0 20.0 76.0 Spaghetti (cooled) 41.8 53.2 5.1 Millet (boiled 20 min, cold) 55.3 36.8 7.9 Maize RS product 56.1 9.2 34.7 Peas (frozen, boiled 5 min) 63.2 10.5 26.3 Bean flakes 55.1 32.7 12.2 Source: Englyst, 1992 The RS content of potato biscuit is nearly double that of the pseudostem, but RS is notably less and RDS is more than the pseudostem. Meanwhile, the fraction of RDS level of Maize RS product is more and the SDS is less than pseudostem in spite of the RS value of maize RS product which is 3 times that of banana pseudostem and the RS ratio is approximately the same as banana pseudostem. Among the foods displayed in the tables, only banana flour had 142 significantly higher content and ratio of RS to RDS than banana pseudostem. 6.2.3 Comparison between HPLC and Englyst methods for analysis of SDS, RDS and RS of banana pseudostem Partial least squares (PLS) were the regression method selected to relate total starch content using Englyst method in comparison to the HPLC method. Linear regression of Englyst-determined total starch against HPLC-determined total starch (Y = 0.97X -0.22) gave an intercept significantly different from 0.0 (P > 0.5), but the slope showed no significant difference from 1.0 (P < 0.5) and R2 of 0.94 (Fig. 6.5). Figure 6.5 Plot of HPLC method versus Englyst method for total starch of banana pseudostem The results obtained for total starch by HPLC and Englyst methods were similar, though these two methods were not perfectly related with each other. Thus, other in vitro methods, such as the Nutriscan artificial gut can be used to determine the RDS, SDS, RS and TS, to validate the two methods. Most of the 143 data obtained using the Englyst method was lesser than the HPLC method, except for BP50B (Figure.6.6). The difference is more likely due to analytical errors. Figure 6.6 Total starch content of banana pseudostem detected by HPLC method and Englyst method All values are means of triplicate determinations. All values are expressed on dry weight basis. 6.3 Dietary fibre Dietary fibre is composed of total dietary fibre (TDF), which includes both soluble (SDF) and insoluble dietary fibre (IDF). Moreover, dietary fibre is composed of different sugar fractions. The sugar contents within the soluble and insoluble fractions can be analysed to provide the total soluble 144 and insoluble fibre contents. Three methods, namely the Megazyme method based on AOAC method 991.43 (AOAC, 2006), NMR method and a GC method, were used in this study. 145 6.3.1 Enzymatic-gravimetric method (Megazyme method) The Table 6.5 illustrates the TDF, SDF and IDF of banana pseudostem with different drying conditions and species using Megazyme total dietary fibre kit (Megazyme International Ireland Ltd, Wicklow, Ireland). Table 6.5 Mean SDF, IDF, TF of Musa balbisiana pseudostem and Musa acuminata pseudostem IDF SDF TDF Banana pseudostem dried in different conditions BP40 15.6 0.1c 3.5 0.2c 19.2 0.1d,e BP40B 15.2 0.3c 3.3 0.2c 18.5 0.5e BP50 17.8 0.3c 3.6 0.1c 21.5 0.4d BP50B 20.3 0.3b 3.7 0.3c 24.0 0.0c Banana pseudostem with different species 17.8 0.3c 3.6 0.1c 21.5 0.4d Musa balbisiana 11.2 0.2d 3.3 0.2c 14.5 0.2f Musa acuminata Commercial-sale dietary fibre supplement Metamucil 84.58 0.5a 4.27 0.1b 88.85 0.4a Benefibre 0.98 0.1e 70.20 0.1a 71.18 0.1b Means within a column followed by different superscripts are significantly different (P<0.05). All values are based on a dry weight basis and means of replicate. 146 6.3.1.1 TDF The total dietary fibre contents of BP40, BP40B, BP50, BP50B and BPA were 19.2%, 18.5%, 21.5%, 24.0% and 15.89%. Drying at 50 °C with blanching had a significantly (P < 0.05) high dietary fibre content. There was no significant (P>0.05) difference between BP40 and BP50, as well as BP40 and BP40B, but there was a significant difference (P <0.05) between BP40B and BP50. From the result, both blanching and drying temperatures seemed to have effect on the content of total dietary fibre. Musa acuminata pseudostem gives a significantly lower value of total dietary fibre than Musa balbisiana. Species had great influence on the dietary fibre content of banana pseudostem. The total dietary fibre content in this study was similar to that of Bhaskar et al. (2011) (28.8%). All of the banana pseudostem samples contained much higher levels of fibre than dried bulgur, unprocessed oat bran, dried apple and sundried tomato (12.1%, 15.5%, 9.1% and 14.7% respectively) (NUTTAB, 2010). This result indicated that dried banana pseudostem has the potential to be used as a high-fibre substitute in food products. 6.3.1.2 IDF The dominant portion of fibre in all banana pseudostem samples was insoluble fibre. The content of insoluble fibre in BP40, BP40B, BP50, BP50B and BPA was 15.6%, 15.2%, 17.8%, 20.3% and 11.2%, respectively. BP50B has a notably high value of insoluble dietary fibre (P < 0.5), but no significant difference was observed between BP40, BP40B and BP50. The IDF of Musa acuminata pseudostem is only 2/3 of that of Musa balbisiana. The insoluble 147 dietary fibre in the banana pseudostem could promote intestinal regulation by providing fecal bulking and increase of microbial biomass as indicated through similar fibre containing foods (Jenkins et al., 2001, Cui & Roberts, 2009). Hence the banana pseudostem provides health benefits. 6.3.1.2 SDF Soluble dietary fibre in BP40, BP40B, BP50, BP50B were 3.5%, 3.3%, 3.6% and 3.7%, respectively. There was no significant difference in soluble dietary fibre between these four conditions. Though the total dietary fibre in banana pseudostem reported by Bhaskar et al. (2011) and Aziz et al. (2011) were both higher than in this study, the soluble dietary fibre content detected in this study is higher (1.40% and 1.89%, for Bhaskar et al., 2011 and Aziz et al., 2011, respectively). The dietary fibre analysis in this study implied that dried banana pseudostem shows a potential to be used as fibre replacement for bulgur and oat bran. In order to know the properties of the banana pseudostem and to use it for developing food products, further studies should be done to estimate the structure of the dietary fibre. It is notable that Benefibre, a commercial natural fibre supplement sold in the Australian market had a significantly high (p<0.05) content of soluble fibre (70.2%). As labeled, Benefibre contains 75% soluble dietary fibre, and this result is similar to the values obtained in the current study. 148 6.3.2 NMR method Solid state NMR and solution state NMR were used to explore the structure of dietary fibre in banana pseudostem samples. Since NMR is still a new technique to be used for such studies accurate quantification cannot be determined by NMR and hence only BP50 was used for this study. 6.3.2.1 Solid state NMR The solid state NMR technology had been used to determine the structure and characterization of plant cell wall polymers in apple, kiwifruit, lemon and maize (Irwin et al, 1984; Newman, Ha & Melton, 1994; Newman & Redgwell, 2002). The CP/MAS NMR spectrums of soluble dietary fibre of dried banana pseudostem are presented in Figure 6.7a. a b Figure 6.7 a: 13C CP/MAS NMR spectrum of soluble dietary fibre of dried banana pseudostem b: 13C CP/MAS NMR spectrum of insoluble dietary fibre of dried banana pseudostem According to the literature (Figure 6.8), the resonances of pectin at 176-168 ppm were assigned to C6 carbons of galacturonic units, while OCOCH3 carbons 149 of acetyl groups in acetylated samples were also situated in this region. The resonances arising from glycosidic bond carbons C1 and C4 were at 101ppm and 79 ppm, respectively. Figure 6.8 13C CP/MAS NMR spectra of commercial pectins The peaks at 67-72 ppm came from the other carbons of pyranoid ring. The resonance at 53 ppm represents methyl carbons of the methyl ester COOCH3, which can be used for the estimation of degrees of methylation. The resonance at 21 ppm represents methyl carbons of the acetyl ester OCOCH3, which can be used for the estimation of acetylation values (Synytsya et al., 2003). The chemical shift in the dried banana pseudostem soluble dietary fibre fraction was compared with the chemical shift of the commercial pectins in the literature as pectin is a major compound in soluble dietary fibre of dried banana pseudostem. However, the quantification of the pectin and the identification of other compounds in the soluble dietary fibre could not be elucidated using this 150 method. The CP/MAS NMR spectrums of insoluble dietary fibre of dried banana pseudostem are presented in Figure 6.7b. The chemical shifts of crystalline cellulose observed from the literature were the peaks at 105.5 ppm for C1, peaks at 84.9 and 89.4 for C4, peaks at 62.5 and 65.4 for C6, and peaks at 73.0 and 75.0 for C2,3,5 (Ratnayake et al., 2011). Compared with Figure 6.9, similar spectrum was observed in Figure 6.7b. As a result, the insoluble dietary fibre of banana pseudostem powder was dominated by signals assigned to cellulose. The weaker signal at 21 ppm in the figure was assigned to the acetyl group. By this method, it could be found that cellulose was the major compound in the insoluble dietary fibre fraction of banana pseudostem. However, the quantification of the cellulose and the identification of other compounds in the insoluble dietary fibre could not be elucidated using this method. Figure 6.9 CP/MAS 13C NMR spectrum of cellulose isolated from switchgrass from literature Source: Pu et al., 2013 6.3.2.2 Solution state NMR 1H NMR spectroscopy is a valuable technique of characterization and 151 classification of dietary fibre structural features. This study used the Chenomx NMR Suite library to fit the 1H NMR spectroscopy and to identify the sugar fractions in the banana pseudostem dietary fibre. Figure 6.10 showed the1H NMR spectroscopy of banana pseudostem total dietary fibre extraction. Figure 6.10 1H NMR spectroscopy of banana pseudostem total dietary fibre extraction Black peaks represented spectroscopy detected by NMR and red peaks were spectroscopy fitted by Chenomx NMR Suite. As shown in Table 6.6, the main component of banana pseudostem dietary fibre was glucose, which occupied 58.7% of the sugar fraction, followed by xylose (9.2%). 152 Table 6.6: Chemical shifts and portion of sugar fractions in the banana pseudostem dietary fibre extraction Arabinose Galactose Glucose Fucose Xylose Chemical shift for 5.22 5.18 5.15 5.13 5.11 calculation (ppm) 5.21 5.17 5.14 5.12 5.10 Portion (%) 0.52 3.46 58.7 0.81 9.17 Other components included galactose, fucose and arabinose (Table 6.6). Although the 1H NMR can detect the sugar fractions in the dietary fibre and give a high signal to noise ratio in a short experimental time, which reflects the properties of the dietary fibre within several minutes, this method has its drawbacks. It suffers from severe signal overlaps due to its short chemical shift ranges (12-0 ppm). As a result, no unique signals can be selected for calculation. Moreover, some of the sugar fractions in the sample cannot be confirmed, due to the limitation of the library. For example, in Figure 6.10 the sugar fractions between 4.4 ppm to 4.2 ppm cannot be identified. To sum up, NMR is a good instrument to be used to identify neutral sugar compounds in dietary fibre in a short time, but it is still a newly developed technique, which cannot be used for accurate quantification of the neutral sugar contents of dietary fibre. Hence for quantification of the fibre components, a gas chromatographic method was used. 153 6.3.3 Enzymatic-chemical method (Englyst Method) Dietary fibre was determined as non-starch polysaccharides by GC-FID. The procedure involved: 1) enzymic hydrolysis of starch; 2) precipitation of NSP in ethanol; 3) acid hydrolysis of the NSP to neutral sugars; 4) conversion of aldito acetate derivatives of sugars to volatile compounds 5) measurement of the released constituent sugars by GC-FID; 6) measurement of uronic acid by spectrophotometry. The sample digestion was followed using Englyst method (1994). The GC analysis was by the same method with following modifications: 1) GC-FID was used instead of GLC; 2) One-point calibration was replaced by multi-point calibration; 3) Split injection was chosen for better separation. This method identified and quantified the individual constituent sugars. This method has also been described to be useful in studies where the relationship between intakes of NSP and health as well as the relationship between NSP and its physico-chemical properties (Englyst & Geoffrey, 1996; Kumar et al., 2012) can be evaluated. Method validation of the modified GC method and discussion of the NSP result of banana pseudostem are presented in the following sections. 6.3.3.1 Method validation Method validation is the process of proving that an analytical method is acceptable for its intended purpose (Green, 1996). In order to provide accurate and reliable data from GC for NSP analysis, the linearity, limits of detection and quantitation, precision and recovery were estimated in this study. 154 6.3.3.1.1 GC chromatography The GC-FID separation was carried out using a Supelco SP-2380 wide-bore capillary column (30m*0.53mm i.d.) at 210 °C for isothermal oven temperature and 275 °C for inlet and detector, which results in a good resolution and accurate determination of the individual sugars in the standard sugar mixture within 9 min. A typical chromatogram is shown in Fig 6.11, and the elution times and peak areas are listed in Table 6.7. The peaks for all neutral sugars were sharp and symmetrical. It must be noted that good separation of the peaks are related to 1) the ratio of the combination of the standard sugar mixture and internal standard (allose); 2) freshness of the ammonia solution used in sample derivation; 3) dilution factor of split injection. Fig 6.12 shows the chromatogram of standards with a wrong combination ratio (a) and with stale ammonia solution (b) as examples. 155 a b Figure 6.11 Chromatograms of derivatised monosaccharides in a standard solution (a) and banana pseudostem sample (b) 156 a b Figure 6.12 Chromatogram of (a) standard with wrong combination ratio of standards to internal standard and (b) sample with stale ammonia solution 157 Table 6.7 GC data of monosaccharides in standard mixture and banana pseudostem samples Standard mixture (Fig a) Real sample (Fig b) Peak Retention time Peak Peak Retention time Peak (min) area (min) area Rha 1 2.303 83.50 2 2.287 4.985 Fuc 2 2.445 90.88 3 2.429 4.729 Ara 3 3.329 852.16 6 3.299 109.172 Xyl 4 4.413 772.82 7 4.372 225.766 IS 5 5.747 615.66 8 5.701 678.966 Man 6 6.481 442.60 9 6.418 30.630 Gala 7 7.288 479.36 10 7.217 91.896 Glu 8 8.475 1455.62 11 8.392 974.567 Rha = L-Rhamnose; Fuc = L-(-)-Fucose; Ara = L-(+)-Arabinose; Xyl = D-(+)-Xylose; IS = Allose; Man= D-(+)-Mannose; Gala = D-(+)-Galactose; Glu = D-(+)-Glucose Allose was chosen as internal standard (IS) since it was eluted within the time that all sugars were eluted and it did not co-elute with any of the tested sugars. Furthermore, allose is not found in non-starch polysaccharide acid hydrolysates. Therefore, it will not interfere with the determination of neutral sugars. 158 6.3.3.1.2 Linearity The calibration curves were obtained by plotting the peak area ratio between the derivatives of neutral sugar standards and that of allose (IS) against their concentration ratio. The linearity was studied by preparing nine concentration levels of sugar mixture with 1 mg/ml internal standard as described in Table 6.8. The reasons for choosing the calibration concentration at these levels were: 1) the ratio of neutral sugars in each level was similar to the ratio in real food samples; 2) chromatography following this ratio of neutral sugars in each level provided excellent separation of the peaks; 3) the sugar values of test samples were in the middle levels of the calibration range. Table 6.8 Calibration concentration of the standard sugar mixture (mg/ L) Level Rha Fuc Ara Xyl Man Gala Glu 9 650 600 5938 5563 2875 3525 11750 8 520 480 4750 4450 2300 2820 9400 7 416 384 3800 3560 1840 2256 7520 6 347 320 3167 2967 1533 1880 6267 5 208 192 1900 1780 920 1128 3760 4 130 120 1188 1113 575 705 2350 3 52 48 475 445 230 282 940 2 26 24 238 223 115 141 470 1 13 12 119 111 58 71 235 The data were subjected to statistical analysis using a linear-regression model; the linear range, regression equation and correlation coefficients are shown in 159 Table 6.9. The calibration plots showed satisfactory linearity for all sugars as the coefficient of determination (R2) were all higher than 0.99, which ranged from 0.99816 to 0.99995. Table 6.9 Sensitivity and linearity characteristics of neutral sugars determination in the presence of allose as internal standard (1 mg/mL) Linear range Regression equationa Coefficient of LOD LOQ (mg/L) determination (mg/L) (mg/L) Rha 13-650 y=0.2473x+8.9352*10-5 0.99981 6.5 19.5 Fuc 12-600 y=0.3048x-5.3672*10-4 0.99971 6.0 18.2 Ara 119-5938 y=0.2775x+2.5804*10-3 0.99995 7.9 23.8 Xyl 111-5563 y=0.2818x+1.5095*10-4 0.99990 22.3 66.9 Man 575-2875 y=0.0272x+8.1250*10-4 0.99816 76.7 230.0 Gal 71-3525 y=0.2729x-3.1117*10-4 0.99964 14.1 42.3 Glu 235-11750 y=0.2464x-2.6697*10-5 0.99970 23.5 70.5 a In the regression equation y=ax+b, x refers to the concentration ratio of the neutral sugar to internal standard, y refers to the ratio of peak area; LOD, limit of detection; LOQ, limit of quantification 6.3.3.1.3 Limits of detection (LOD) and quantitation (LOQ) The limit of detection (LOD) and quantitation (LOQ) were determined by measuring the magnitude of the analytical background response, plus 3.3 and 10 fold of the mean background signal ratio, respectively. The values obtained are listed in Table 6.9. The LOD varied between 6.0 mg/L (Fuc) and 76.7 mg/L (Man). The non-starch polysaccharides had FID sensitivity level between 18.2 160 mg/L and 230.0 mg/L for optimal quantification. 6.3.3.1.4 Intra-day and inter-day precision In order to estimate the accuracy of the method, within-day repeatability and day-to-day precision were observed as indicated by the recovery and relative standard deviation (RSD). The data are presented in Table 6.10. The precision experiments were done by spiking with known amounts of neutral sugar mixtures at three concentration levels. The three concentration levels were chosen as approximately 80%, 100% and 120% of neutral sugar concentration of banana pseudostem sample. According to the RSD values obtained for the same sugar mixture sample, which was injected seven times repeatedly on the same day, the GC system presented a satisfactory response with a range of RSD values from 0.12 to 4.35%, indicating that the consistency of the monosaccharide level injected into the GC system was within 5%. The results show good repeatability for the optimized method. The inter-day precision was determined over five consecutive days in order to determine the accumulation of the random errors between different extractions and days. The RSD values between days range between 0.17% and 5.67%, which is less than 6%. As shown in Table 6.10, the recovery rate of the sugars in the intra-day experiment range from 97.39% to 102.73%, while the recovery rate in the inter- day experiment range between 96.74% and 104.71%. The recoveries of both inter-day and intra-day experiment are 100± 5%. This implies that the method is deemed to be accurate. 161 Table 6.10 Inter-day (over a period of 5 consecutive days) and intra-day (n=7) precision and accuracy study for the determination of neutral sugars. Intra‐day (n=7) Inter‐day (n=5) Added Found ± SD RSD Recovery Found ± SD RSD Recovery Rha 130 131.23 ± 1.90 1.45 100.95 136.13 ± 3.78 2.78 104.71 52 52.28 ± 1.73 3.31 100.54 53.32 ± 2.40 4.49 102.54 26 26.71 ± 0.68 2.56 102.73 26.61 ± 1.51 5.67 102.36 Fuc 120 119.52 ± 1.72 1.44 99.6 119.84 ± 1.08 0.90 99.87 48 46.96 ± 0.81 1.72 97.83 47.55 ± 0.78 1.63 99.06 24 24.19 ± 0.51 2.10 100.79 24.46 ± 0.44 1.79 101.93 Ara 1188 1185.32 ± 4.14 0.35 99.77 1181.9 ± 1.99 0.17 99.53 475 462.89 ± 6.34 1.37 97.45 459.52 ± 1.18 0.26 96.74 238 231.78 ± 0.28 0.12 97.39 235.28 ± 3.00 1.28 99.07 Xyl 1113 1129.22 ± 3.16 0.28 101.46 1132.60 ±3.51 0.31 101.81 445 434.64 ± 1.43 0.33 97.67 437.04 ± 1.16 0.26 98.21 223 219.78 ± 0.90 0.41 98.56 221.62 ± 1.68 0.76 99.61 Man 920 916.24 ± 25.28 2.76 99.59 913.46 ± 32.12 3.50 99.29 574 576.94 ± 11.42 1.98 100.51 557.51 ± 29.85 5.35 96.96 230 215.35 ± 9.37 4.35 93.63 215.25 ± 10.11 4.70 93.63 Gal 705 707.00 ± 8.70 1.23 100.28 701.95 ± 5.63 0.80 99.57 282 269.90 ± 3.02 1.12 95.71 262.69 ± 7.31 2.78 93.15 141 144.15 ± 1.95 1.35 102.23 136.3 ± 16.9 12.4 96.66 Glu 2350 2351.26 ± 3.53 0.15 100.05 2350.75 ± 4.03 0.17 100.03 940 922.31 ± 3.04 0.33 98.12 924.15 ± 3.81 0.41 98.31 470 456.03 ± 1.28 0.28 97.03 454.44 ± 1.96 0.43 96.69 Units for RSD and recovery were %. Values represent the mean of replicate determinations and standard deviations. All values are reported on a dry weight basis. 162 6.3.3.2 NSP of banana pseudostem 6.3.3.2.1 Soluble, insoluble and total NSP content The total NSP contents in banana pseudostems dried at different conditions were found to be in the range of 40.67-47.21 g/100g of dry sample (Table 6.11). There was no significant difference between pseudostems dried at different conditions (p>0.05). All samples analyzed contained less soluble NSP than insoluble fiber. The soluble NSP content in banana pseudostem dried at different conditions ranged from 10.71 to 17.47 g/100 g on a dry weight basis and insoluble NSP content varied from 26.37 to 36.50 g/100 g on a dry weight basis. Samples dried at 50 °C had significantly high insoluble content than samples dried at 40 °C. Although the value of total NSP detected by the GC method was higher than that detected by the AOAC method, the trend showed that drying at higher temperature had slightly higher total NSP using both methods. Samples dried without blanching had significantly high soluble content than blanched samples. Since NSP is a relatively stable chemical compound, drying and blanching did not have an effect on the total value of the NSP. However, through thermal treatment, the soluble and insoluble fractions may vary because they can be converted into more insoluble compounds. High temperature processing caused an increase in insoluble NSP and decrease in soluble NSP. The insoluble/ soluble NSP ratio varied from 1.5 to 3.4 and the soluble NSP content was found to be 22-40% of total NSP. According to Figuerola et al. (2005), fibre sources have an insoluble/ soluble dietary fibre ratio close to 1:2 which is suitable for use as a food ingredient. Furthermore, Grigelmo-Miguel et al. (1999) indicated that soluble/ insoluble ratio 163 was important for health properties and also for technological characteristics. A portion of 30%-50% of soluble dietary fibre and 70%-50% of insoluble dietary fibre is considered as a well-balanced proportion in order to obtain the physiological effects associated with both fractions. Banana pseudostems dried without blanching had a lower insoluble/soluble ratio than samples when blanched. Moreover, the pseudostems dried at higher temperatures had higher insoluble/soluble ratio. The insoluble/soluble ratio of BP40, BP40B, BP50, BP50B was 1.5, 2.5, 2.0 and 3.4 respectively. Although the ratio of all the samples was close to 2.0, drying at 50°C without blanching is the optimal condition for banana pseudostem considering the functionality of NSP. Additionally, as described in chapter 4 drying at 50 °C provided the whitest colour and used less drying time with less energy consumption. As a result, banana pseudostem dried at 50 °C without blanching has the potential to be used as a good source of dietary fibre supplement. The total NSP of Musa balbisiana and Musa acuminata dried at similar conditions (dried at 50 °C without blanching) did not show any significant difference (p>0.05), whereas, Musa acuminata had significantly higher soluble NSP and lower insoluble of NSP than Musa balbisiana (p<0.05). The soluble NSP content of Musa balbisiana and Musa acuminata were 33% and 41% respectively, which were both in the range of 30%-50%. 164 Table 6.11 Distribution of neutral sugars (g/ 100 g of dry sample) in the soluble, insoluble and total fractions of NSP from banana pseudostem Neutral sugars Total Uronic Total sugars acid NSP Rha Fuc Ara Xyl Man Gal Glu by GLC Banana pseudostem dried in different conditions BP40 Soluble NSP 0.13b±0.00 0.11b,c±0.00 1.22b±0.09 1.29b±0.11 3.24a±0.10 1.34a±0.03 9.23a±1.29 16.56a 0.91a±0.10 17.47a Insoluble NSP ND ND 1.30b±0.12 3.84d,e±0.03 3.91b±0.12 0.82c±0.08 16.17d±1.08 26.04d 0.33a±0.02 26.37d Total NSP 0.13b±0.00 0.11b,c±0.00 2.52b±0.02 5.13b,c±0.14 7.15c±0.02 2.16c±0.05 25.4b±0.24 42.60b 1.24a±0.12 43.84b BP40B Soluble NSP 0.12b±0.01 0.10c±0.01 0.74c±0.09 1.01b±0.10 3.21c,d±0.28 0.99c,d±0.19 4.35c±0.68 10.53b 0.98a±0.02 11.51b Insoluble NSP ND ND 1.98b±0.07 4.06c,d±0.05 4.11b±0.45 1.07b±0.00 17.64c±0.69 28.85c,d 0.31a±0.01 29.13c,d Total NSP 0.12b±0.01 0.10c±0.01 2.71b±0.16 5.07b,c±0.15 7.33c±0.73 2.06c±0.19 21.99c±1.66 39.38b 1.29a±0.03 40.67b BP50 Soluble NSP 0.14b±0.00 0.11b,c±0.00 1.07b,c±0.01 1.42b±0.07 3.26a,b±0.04 1.27a,b±0.02 7.25a,b±0.77 14.52a 0.92a±0.02 15.44a Insoluble NSP ND ND 1.71b±0.02 4.83b,c±0.13 4.52b±0.14 0.95b±0.02 19.18b±0.31 31.18b,c 0.27a±0.02 31.46b,c Total NSP 0.14b±0.00 0.11b,c±0.00 2.78b±0.02 6.25b±0.05 7.78c±0.09 2.22c±0.01 26.43a±1.03 45.71b 1.19a±0.04 46.90b BP50B Soluble NSP 0.14b±0.01 0.12b±0.01 0.99b,c±0.16 1.16b±0.12 3.77b,c±0.29 1.17b,c±0.16 2.48c,d±0.05 9.82b 0.89b±0.08 10.71b Insoluble NSP ND ND 1.79b±0.00 5.14b±0.14 4.37b±0.36 1.01b±0.00 23.98a±1.84 36.30b 0.20b±0.03 36.50b Total NSP 0.14b±0.01 0.12b±0.01 2.78b±0.16 6.30b±0.26 8.14c±0.65 2.18c±0.16 26.46a±2.64 46.12b 1.09b±0.11 47.21b Banana pseudostem with different species Musa Soluble NSP 0.14b±0.00 0.11b,c±0.00 1.07b,c±0.01 1.42b±0.07 3.26a,b±0.04 1.27a,b±0.02 7.25a,b±0.77 14.52b 0.92a±0.02 15.44b balbisiana Insoluble NSP ND ND 1.71b±0.02 4.83b,c±0.13 4.52b±0.14 0.95b±0.02 19.18b±0.31 31.18b,c 0.27a±0.02 31.46b,c Total NSP 0.14b±0.00 0.11b,c±0.00 2.78b±0.02 6.25b±0.05 7.78c±0.09 2.22c±0.01 26.43a±1.03 45.71b 1.19a±0.04 46.90b Musa Soluble NSP 0.15b±0.01 0.13a±0.00 1.04b,c±0.06 1.15b±0.11 5.53a±0.45 1.46a±0.07 7.45b±1.50 16.90a 0.63b±0.07 17.53a acuminata Insoluble NSP ND ND 1.69b±0.01 2.87e±0.02 4.69b±0.02 1.02b±0.00 14.84d±0.26 25.11d 0.32a±0.02 25.43d Total NSP 0.15b±0.01 0.13a±0.00 2.72b±0.05 4.02c±0.08 10.22b±0.46 2.48b±0.07 22.28c±1.14 42.00b 0.95b±0.09 42.95b Commercial-sale dietary fibre supplement (Metamucil) Soluble NSP 0.37a±0.06 0.04d±0.00 4.10a±0.57 8.39a±0.31 2.66d±0.20 0.84d±0.11 2.02d±0.44 18.42a 0.91a±0.72 19.33a Insoluble NSP 0.38±0.00 ND 20.94a±2.11 33.17a±1.40 20.78a±2.62 4.06a±0.15 11.85e±0.67 91.19a 0.36a±0.04 91.55a Total NSP 0.75a±0.06 0.04d±0.00 25.05a±1.54 41.56a±1.71 23.44a±2.82 4.90a±0.26 13.87c±0.51 109.61a 1.27a±0.81 110.88a 165 6.3.3.2.2 Sugar composition of soluble, insoluble and total NSP The contents of total sugars, uronic acid, sugar composition of the soluble, insoluble and total NSP isolated from banana pseudostem are depicted in Table 6.11. The total sugar content in the total, soluble and insoluble NSP of banana pseudostem dried at different conditions ranged from 39.38 to 46.12, 9.82 to 16.56 and 26.05 to 36.30 g/100 g dry sample, respectively. No significant difference in total NSP was observed in samples with different drying conditions (p>0.05). However, samples dried at higher temperature (500C) had significantly higher (p<0.05) insoluble NSP content than when dried at low temperature and samples without blanching had significantly high level of soluble NSP than samples subjected to blanching. The blanched samples contained significantly higher (p<0.05) insoluble glucose and lower soluble (p<0.05) glucose than unblanched samples, which caused the difference in the ratio of soluble/ insoluble NSP in banana pseudostem dried at different temperatures. The total uronic acid content varied from 1.09 to 1.29 g/100 g dry sample. Banana pseudostem dried at 50 °C with blanching had significantly (p<0.05) lower uronic acid than banana pseudostem dried using other conditions. For different banana pseudostem species, no significant difference (p>0.05) was observed in the total NSP content. However, significantly high insoluble NSP and uronic acid and low soluble NSP contents were detected from Musa balbisiana. Uronic acid contents in the soluble fraction were dramatically higher than that in the insoluble fraction in all samples. This may be because uronic acid is one of the most important components of pectin, which belongs to soluble fibre. 166 Glucose, Mannose and xylose were the dominant neutral sugars in the soluble, insoluble and total NSP fractions of the banana pseudostem samples followed by arabinose, galactose, rhamnose and fucose as shown in Table 6.11. Glucose occupied 53-60% of the total neutral sugar content in banana pseudostem, which had similar value with that estimated by NMR (58.7%). Higher amount of glucose in the insoluble NSP could be related to cellulosic cell wall polysaccharides or due to associated β-glucan type polysaccharides, which agrees with reports from other authors (Aziz et al., 2011; Cordeiro et al., 2004 & Aisah et al., 2013). Results from this study were similar to those from NMR, indicating that cellulose may be the predominant compound in dietary fibre of banana pseudostem. Cellulose is the main structural component of all plant tissues, which forms a network of cellulose microfibers (Knudsen, 2014). The cellulose microfibrils are remarkably distinctive features of the cell walls of all plants since it may be associated with water and matrix polysaccharides such as the (1-3, 1-4)-β-D-glucans, heteroxylans (arabino-xylans), and glucomannans (Kumar et al., 2012). β-glucan not only has the viscosity enhancement property, but also has an elastic gel network formation property. Wood & Webster (1986) stated that β-glucan can be utilized as thickening agents to modify the texture and appearance of food products. Besides physico-chemical properties, β-glucan has beneficial effects on human health. For example, it has cholesterol lowering effects (Kahlon et al., 1993) and positive effect on type II diabetes by flattening glucose and insulin responses of postprandial blood glucose (Ahmad et al., 2009). Mannose occupied 17- 19% of the total neutral sugars in banana pseudostem. High amount of 167 mannose and glucose may relate to the hemicellulose family such as mannans, and glucomannans which are both polysaccharides. Glucomannans are water soluble and have the ability to 1) lower glucose absorption from the intestine; 2) lower the total plasma cholesterol and triglycerides and control weight due to the satiation feeling produced by the filling of the intestine with mannan gel (Kumar et al., 2012). Xylose occupied 12-14% of the total neutral sugars in banana pseudostem, which was close to the results estimated by NMR (9.17%). High amounts of glucose and xylose could be due to neutral hemicellulose polysaccharide xyloglucans. Xyloglucans can be associated with the cellulose microfibrils (Knudsen, 2014). It has been reported that aqueous solutions containing 0.05% to 5% by weight of xyloglucan and 10% to 70% of glycerol were suitable to be applied on human mucous membranes, such as oral and vaginal mucous membranes, as moisturizing and softening agents or as pharmaceutical release systems (Kumar et al., 2012). Small amounts of arabinoxylan type of polysaccharides and pectic substances including arabinans, galactans and arabinogalactans may also exist in dried banana pseudostem. Pectic substances rhamnogalacturonans may be in minor amounts in banana pseudostem powder. 6.3.3.3 Comparison of NSP of banana pseudostem with commercial dietary fibre supplement Two commercial dietary fibre supplements were tested in this study. They were Metamucil natural granular powder (Procter & Gamble, USA) and Benefiber (Novartis, Australia) powder, which are the most common fibre supplements used by the Australian consumers. Since Benefiber dissolved 168 completely in water, nearly all of the NSP in Benefiber were removed with sugar and starch during the NSP precipitation procedure. As a result, the NSP value of the Benefiber could not be determined accurately by GC. Metamucil had a significantly higher insoluble and total NSP than banana pseudostem and similar soluble NSP contents as the banana pseudostem (Table 6.11). However, the insoluble/ soluble dietary fibre ratio of the Metamucil was 4.7, which was far more than the ratio of banana pseudostem (1.5-3.5). This difference in insoluble/ soluble dietary fibre ratio may be due to the different types of the NSP source. The main component of Metamucil is psyllium husk, which is extracted from a cereal, whereas banana pseudostem NSP is extracted from a horticultural plant. Figuerola et al. (2005) expressed that fibre extracted from fruits and vegetables contain a considerably higher proportion of soluble dietary fibre, while cereal fibre has more insoluble fibre. As described before, a food source whose insoluble/ soluble ratio is 2 can be considered as a good source for dietary fibre. The insoluble/ soluble dietary fibre of Banana pseudostem is more close to this ratio compared with Metamucil. If the dietary fibre of the banana pseudostem was extracted and developed as a dietary fibre supplement, it would be a better source of fibre with a well-balanced proportion between soluble and insoluble fraction. Banana pseudostem may be a good source of soluble dietary fibre in many food applications, such as in weight control diets due to its ability to retain water and increase the feeling of satiety as well as to decrease nutrient absorption time (Grigelmo-Miguel et al., 1999). Metamucil is a good source of intestinal regulation and stool volume enhancement, which are related to 169 the consumption of insoluble dietary fibre. Sugar composition of Metamucil was dramatically different from the banana pseudostem. The main neutral sugars in Metamucil were xylose (38% of the total neutral sugar), followed by arabinose (23%) and mannose (21%), while the dominant sugar compounds in banana pseudostem were Glucose (53- 60%), mannose (17-19%) and xylose (12-14%). Rhamnose and fucose were present in only trace amounts in both Metamucil and banana pseudostem powder. According to the sugar type in Metamucil, it is likely to be composed of high amounts of arabinoxylans, arabinans, mannans and glucuronoarabinoxylans; small amounts of xyloglucans and glucomannans; and minor amounts of cellulose, β-glucan, galactans, galactomannans and arabinogalactans. This agrees with Englyst and Englyst (2005) that xylose is found predominantly as arabinoxylans in cereal products. It has been reported that arabinoxylans have several physicochemical properties, including high viscosity, good water holding capacity and good emulsion ability (Kumar et al., 2012; Michniewicz et al., 1992). Additionally, Biliaderis et al. (1995) stated that water-extractable arabinoxylans can slow starch retrogradation to produce less firm breadcrumbs. 170 A survey was done on the popular dietary fibre supplements sold in the Australia market. As shown in Table 6.12, most of the dietary fibre supplements sold in the market were made from cereals and only one (Normafibe Natural fibre daily supplement) among the six was a fruit dietary fibre. Table 6.12 Ingredients in commercial dietary fibre supplements Brand-Name Ingredients NSP source type Metamucil Plantago psyllium husk Cereal Carusos Natural Health Oat bran, Linseed, Rice bran, Cereal Quick Fibre Plus Plantago psyllium husks, Wheat dextrin Benefiber Wheat dextrin Cereal Normafibe Natural fibre Sterculia Fruit daily supplement Bovit Psyllium Fibre Plantago psyllium husk Cereal Agiofibe Ispaghula husk, Plantago afra, Cereal Plantago ovate seeds As discussed in the previous paragraph, fruits and vegetables have more soluble dietary fibre and better insoluble/ soluble ratio than cereals. The fruit and vegetable based dietary fibre also contains different proportions of neutral sugars in NSP from cereal based NSP, which provide different functionality. Developing fruit and vegetable based dietary fibre supplements may meet the demand of consumers who may require it to improve satiety rather than only 171 being used for fecal bulking. Banana pseudostem is a by-product of banana fruit and its price is low. Based on its ideal NSP ratio, banana pseudostem has a good potential to be developed as a dietary fibre supplement, which may bring health benefits to consumers, growers and manufacturers alike. Isolation and purity of the NSP fraction in banana pseudostem should be the next step in producing a high quality fibre supplement. An appropriate method for extraction should be developed for the extraction. 6.3.4 Dietary fibre method comparisons Dietary fibre is one of the most important components in banana pseudostems. The use of banana pseudostems in food products depends on the characteristics of the dietary fibre. Three methods were used in this study to gain detailed information of dietary fibre from banana pseudostem. At first, enzymatic-gravimetric method (AOAC 991.43 / 985.29; AOAC, 2006) was used to measure the sum of indigestible polysaccharides in the banana pseudostem. In order to analysis the chemical structure and glucosidic linkages of the pseudostem fibre, NMR was used. Although NMR is a quick method for fibre identification, it is a comparatively new technique and has limitations. It can only be used to identify the main components in banana pseudostem dietary fibre but cannot be used to quantify the compounds accurately. Furthermore, due to the limitation of the library, some of the sugars cannot be identified. GC was used to measure polysaccharides that do not contain the 1-4 glucosidic linkages characteristic of NSP. GC method can quantify uronic acid and neutral sugars within the non-starch polysaccharides 172 component. The difference in the sugar composition and glycosidic linkages are important features in determining the physico-chemical properties of NSP (Englyst et al., 2007). The results from GC method were similar to that from the NMR method. However, results from the AOAC method using the total dietary fibre assay kit (K-TDFR, Megazyme International Ireland Ltd, Wicklow, Ireland) was lower than that obtained from the GC analysis. One possibility is that the GC and calibration method used in this study is different from that used in Englyst method (Englyst et al., 1994). Nine level of calibration were selected according to the NSP range of the banana pseudostem and commercial dietary fibre supplement used in this study, while Englyst method used one point calibration. It is reasonable to believe that the results gained from nine-level calibration curve are more accurate than one point calibration. The operational errors and instrument errors are likely to be eliminated in the multi-point calibration. Moreover, the multi-point calibration range was built according to the real samples; this kind of calibration method can reflect the results much closer to the real samples. From the study, it has been found that the results obtained from multi-point calibration were approximately 5% higher than those from one-point calibration in the calibration range used in this study. Moreover, the aim of the two methods is different, which causes the difference in the values. The aim of Englyst method is to measure polysaccharides that do not contain the -1, 4 glucosidic linkages characteristic of starch, while AOAC method (985.29 & 991.43; AOAC, 2006) is to measure the sum of indigestible polysaccharides and lignin. Englyst achieved the aim; however AOAC 173 method did not (Englyst et al., 2007). The AOAC method does not measure all components of dietary fibre as currently defined by CODEX Alimentarius (Alimentarius, 2010). Most resistant starch and all non-digestible oligosaccharides are not included which results in an underestimation of dietary fibre (Schweizer & Edwards, 2013). Both methods have their own benefits and disadvantages. AOAC method gives values of TDF, SDF and IDF with good reproducibility which provides enough information for routine analysis. However, it does not account for analysis based on the structure and functionality of the dietary fibre. Difficulties in the filtration step after enzymatic hydrolysis was seen in samples with good water holding capacity, swelling capacity and gelling capacity such as Metamucil. When 1 g of Metamucil was used for the analysis, the Metamucil formed a gel, which covered the celite layer and could not be filtered. Lowering the sample size to 0.1g sample still proved difficult to filter which caused the underestimation of the soluble fibres in samples with good gelling capacity. Englyst method (2007) on the other hand reflected a wide range of carbohydrate components and their diverse nutritional properties. Moreover, this method is not as time consuming as AOAC method when a large amount of samples need to be analyzed. Dozens of samples can be hydrolyzed within 1.5 days and 15 min is required for each sample on GC analysis. However, Englyst method has a drawback as well. For example, it cannot determine the value of dietary fibre samples with special processing, which makes the samples soluble in water, such as Benefiber. As the samples are easily dissolved, it would be removed as sugars and starch during enzyme 174 hydrolysis. Hence, the NSP content of these samples cannot be determined accurately by Englyst method. 175 7. Effect of drying on the physicochemical properties of banana pseudostem Crystallinity index (CI), water holding (WHC), swelling (SWC) and solubility (SC) capacities of banana pseudostem dried at different conditions and different species are presented in Table 7.1. These properties suggest some possibilities about the use of banana pseudostem powder as ingredients in food products. For example, high WHC implies that the powder can be used as functional ingredients to avoid syneresis and modify the viscosity and texture of some formulated foods (Grigelmo-Miguel et al., 1999). Moreover, the study of the physicochemical properties of the banana pseudostem can reflect its health benefits. For instance, high viscosity and solubility is able to increase fecal bulking and reduce intestinal transit time, which may improve intestinal peristalsis (Fleury & Lahaye, 1991; Gupta & Premavalli, 2011). The following section will discuss the physicochemical properties of the banana pseudostem as well as the influence of drying conditions and species on the physicochemical properties of the banana pseudostem powder. 176 Table 7.1 Physicochemical properties of dried banana pseudostem powder WHC SWC SC CI Different drying condition pseudostem BP40 10.2a,b ± 0.7 7.5b ± 0.5 34.2a ± 0.7 45.2c,d ± 0.4 BP40B 10.9a ± 0.4 6.3c ± 0.3 27.6b ± 0.3 36.5d ± 3.9 BP50 10.2a,b ± 0.4 7.0b,c ± 0.6 25.5b ± 1.2 54.1b ± 4.7 BP50B 9.0b ± 0.4 6.5c ± 0.6 26.5b ± 0.4 51.2b,c ± 4.5 Different species pseudostem Musa 11.0a ± 0.5 4.3d ± 0.5 25.2b ± 1.2 56.6b ± 1.6 acuminata Musa 10.2a,b ± 0.4 7.0b,c ± 0.6 25.5b ± 1.2 54.1b ± 4.7 balbisiana Commercial-sale dietary fibre supplement Metamucil 20.3c ± 0.6 38.2a ± 0.8 11.8c ± 0.2 73.4a ± 6.4 Unit for WHC is g water/ g of dry matter. Unit for SWC is g of swollen granules/ g of dry matter. Unit for Soluble Capacity is %. Means within a column followed by different superscripts are significantly different (P<0.05). All values are based on a dry weight basis and means of triplicate determinations. 177 7.1 Water holding capacity (WHC) WHC is defined as the amount of water that is retained by 1 g of dry matter under specified conditions of the temperature, time soaked and duration and speed of centrifugation (Fleury & Lahaye, 1991). It is an important functional property that has been widely studied in food, since it is associated with food quality. The range of WHC of banana pseudostem was 9.0-11.0 g water/g of dry matter. It has been reported that WHC of oat bran, rice bran, soy flour and wheat bran fibre were 2.10, 4.89, 4.79-6.74 and 5.03 g water/g of dry matter, respectively (Abdul-Hamid & Luan, 2000; Chen et al., 1988; Heywood et al., 2002). The WHC content of banana pseudostem detected in this study far exceeds those values from the literature. This implied that banana pseudostem powder was able to bind or entrap more water than oat bran, rice bran, soy flour and wheat bran. According to Table 7.1, there is no significant (p>0.05) difference in WHC between species (Musa acuminata & Musa balbisiana). BP50B had significantly lower value (p<0.05) than BP40B, however, this significant difference had not been observed between BP40 and BP50. Both drying temperature and blanching do not affect the WHC in this case. Mutamucil has significantly higher (p<0.05) WHC than pseudostem powder. This may be because of the high amount of arabinoxylan in the Mutamucil NSP. It has been stated that arabinoxylans, especially covalently cross-linked arabinoxylans, have good water holding capacity, which may hold up to 100 g water per 1 g polysaccharides (Kumar et al., 2012). Moreover, since 178 arabinoxylans are not bound to the cell walls, they can absorb about ten times their weight of water (Choct, 1997). According to Chau and Huang (2003), the proportion of cellulose in the NSP affected the WHC. Those foods containing more cellulose would have lower WHC than those foods containing more of other components, such as pectin, lignin and hemicellulose. Different ratios of cellulose, pectin, hemicellulose and lignin cause different cross-linked structural formation of polysaccharide molecules, which lead to different physicochemical properties. 7.2 Swelling capacity (SWC) and Solubility The SWC of Musa balbisiana pseudostem (7.0 g of swollen granules/g of dry matter) exceeded that of Musa acuminata pseudostem (4.3 g of swollen granules /g of dry matter). This indicated that banana species influenced the SWC of the pseudostem. This finding agrees with the study by Mei et al. (2010), who claimed that significant differences (p<0.05) were observed in sweet potato DF from 10 varieties. Blanched samples provided lower SWC than unblanched samples. Akter et al. (2010) and Aziz et al. (2011) stated that blanching led to the increase of SWC, however this was opposite to the finding in this study. One of the reasons is that other factors may also affect the SWC. For example if the ratio of amylopectin and amylose is low in blanched banana pseudostem powder, it may lead to the low SWC (Bello-Perez et al., 1999). Thus, further studies need to be done to detect the amylose and amylopectin ratio in banana pseudostem to prove this hypothesis. There is no significant difference (p>0.05) among BP40B, BP50 and BP50B, but BP40 has a 179 significantly higher value (p<0.05) of SWC than BP40B and BP50B. The range of SWC of banana pseudostem was 4.3 -7.5 g of swollen granules/g of dry matter. This value was similar to pea hulls and grape (5.2 g and 6.69of swollen granules/ g of dry matter) and lower than carrot insoluble fibre, coconut fibre and sugar beet fibre (18, 17 and 11 ml/g; Elleuch et al., 2011). The solubility of BP40 was significantly higher (p<0.05) than other samples relating to the SWC. The solubility was not affected by banana pseudostem species. Metamucil had significantly higher (p<0.05) SWC but lower solubility capacity than banana pseudostem. This may be because the Metamucil has the capacity of holding more water than the banana pseudostem. Banana pseudostem had better soluble/insoluble ratio of NSP than Metamucil, which is reflected in the high solubility capacity it had. 7.3 Crystallinity Index (CI) Crystallinity index (CI) can be defined as the percentage of the crystalline regions with respect to the total material. It is a valuable parameter to consider as it influences the physicochemical properties of the material (Lopez-Rubio et al., 2008). For example, the crystallinity of the banana pseudostem powder may influence the flowability, bulk density, ease of handling, dust forming, compressibility and surface activity (Rahman, 2009). XRD plus a peak-fitting program (Magicplot System, LLC) were used to predict the CI in banana pseudostem in different drying conditions and species in this study. Gaussian 180 functions were assumed for each peak and a broad peak at around 21.5. The patterns of XRD from Musa balbisiana dried at different conditions were similar, whereas the XRD patterns of banana pseudostem from different species of banana pseudostem were dramatically different (Figure 7.1). The sharp peaks in the figures represent crystalline area, while the humps represent amorphous regions. Both species of banana pseudostem had a mixed structure (coexistence of amorphous and crystalline structure). This mixed structure may occur by the structure of banana pseudostem itself or by grinding. As shown in the figures, most crystalline peaks of Musa balbisiana were in the range of 13 - 25° and 32.5 – 40° with semi-crystalline peaks. Despite these similar peaks, Musa acuminata had sharp crystalline peaks at 28°, 41° and 50°. According to Table 7.1, drying at 50 °C without blanching provided the highest CI, while drying at 40 °C with blanching showed lowest CI. Blanching has no effect on the CI. However, with the increase in drying temperature, the CI was positively affected. This may be attributed to the gelatinization of the starch in banana pseudostem. With the increase of temperature, the crystal structure of starch was destroyed gradually and more amorphous networks were formed. No significant differences (P>0.05) were observed in samples from different species. As shown in Figure 7.1 (c), Metamucil has more crystalline and less amorphous structure than the banana pseudostem. Moreover, the XRD pattern of the Metamucil was different from that of banana pseudostem. The 181 crystalline peaks of Metamucil were in the range of 7.5 – 27.5°. These differences may be caused by the different components of the two sources. Metamucil is pure dietary fibre, while banana pseudostem is a composite of dietary fibre, starch and other components. 182 Crystalline a Amorphous Crystalline b Amorphous c Figure 7.1 XRD results obtained from Musa acuminata (a), Musa balbisiana (b) banana pseudostem and Metamucil (c). 183 7.4 Pasting properties (RVA) The pasting properties study using the Rapid Visco Analyzer (RVA) showed that there were significant differences in pasting characteristics, including peak viscosity (PV); through viscosity (TV); breakdown (BD); final viscosity (FV) and set back (SB) between the banana pseudostem samples in different drying conditions and species (Table 7.2). The pasting profiles of the banana pseudostem in different drying conditions and different species were shown in Figure7.2. Figure 7.2 Pasting profiles of banana pseudostem 184 Table 7.2 Pasting properties of banana pseudostem (BP40, BP40B, BP50, BP50B, BPA) measured by Rapid Visco Analyser PV (RVU) TV (RVU) BD (RVU) FV (RVU) SB (RVU) PEAK TIME (min) PASTING TEMP (C) Different drying condition pseudostem BP40 219.5a± 8.9 115.6a ±3.6 103.6a ± 7.1 146.8a ± 4.1 31.6b ± 1.2 4.2d ± 0.0 73.9c ± 0.5 BP40B 169.8b,c±4.3 95.5b ± 8.4 74.3b,c ± 1.2 112.8b ± 7.3 17.3c ± 0.2 4.5b ± 0.1 80.0a ± 0.1 BP50 170.0b,c ± 4.8 89.6b ± 2.6 80.4b ± 5.5 108.9b ± 3.7 19.3c ± 1.5 4.3c ± 0.0 74.4c ± .02 BP50B 188.9b ± 3.9 116.4a ± 5.3 72.5b,c ± 1.2 153.5a ± 4.6 37.0a ± 0.9 4.3c ± 0.0 77.1b ± 0.5 Different species pseudostem Musa balbisiana 170.0b,c ± 4.8 89.6b ± 2.6 80.4b ± 5.5 108.9b ± 3.7 19.3c ± 1.5 4.3c ± 0.0 74.4c ± .02 Musa acuminate 157.3c ±3.5 100.3b ± 5.8 57.0c ± 0.2 117.8b ± 7.4 17.5c ± 1.8 4.7a ± 0.0 79.8a ± 0.2 Where: PV - Peak viscosity; TV – Through viscosity; FV - Final viscosity; BD – Breakdown; SB – Set back All values are means ± SD triplicate measurements Values in the same column with different superscripts are significantly different from each other (p< 0.05) 185 According to the figure, all of the differently treated banana pseudostem samples have similar pasting curve shape. Among banana pseudostem samples dried at different conditions, BP40 had the highest PV, BD and FV, while BP50B had the highest TV and SB. No significant difference (P > 0.05) was observed in the value of PV, TV, BD, FV and SB of BP40B and BP50. BP40B required longest time (4.5 min) to reach maximum viscosity. This might be due to the lower rate of absorption and swelling of starch granules, which is similar to the previous results as shown in Table 7.1. BP40B has the lowest swelling capacity (6.3 g of dry matter). BP40 had significant higher (p<0.05) PV and required the shortest period of time to reach the peak viscosity than banana pseudostem dried at other conditions. This result implies that BP40 has the highest swelling power, which agrees with the previous result (Table 7.1). The peak viscosity prior to disruption of the swollen granules is determined from the volume occupied by these swollen granules. Kim et al. (1999) reported that starch granules with high swelling capacity occupied more volume in the water suspension, thus the space between the granules became narrower and frictions among granules were likely to occur. BP40 was assumed to have less structure rigidity during migration of water into its starch granules during gelatinization. As a result, BP40, which observed strong swelling capacity, had the highest PV and is easy to reach maximum viscosity. This result suggests that BP40 would behave differently during cooking and processing compared with samples dried in the other three conditions. The soluble to insoluble NSP ratio is also significantly higher than other processed pseudostems, supporting this result of 186 a higher swelling capacity (Table 6.11; page 165). No significant difference (p > 0.05) was exhibited between PV of BP40B, BP50 and BP50B, and between different banana species. During the holding period of the viscosity test, the material slurries are subjected to high temperature (95 C) and mechanical shear stress, which further disrupt starch granules resulting in amylose leaching out and alignment (Ragaee et al., 2006). This period is commonly associated with a breakdown in viscosity (BD). It is a measurement of degree of disintegration of granules or paste stability (Newport Scientific, 1995). High value of breakdown is associated with high peak viscosities, which in turn is related to the swelling capacity of the granules during heating (Ragaee et al., 2006). As shown in table 7.2, BP40 had highest BD while BP40B, BP50 and BP50B showed no significant difference (p > 0.05) of BV. This result indicated that BP40 has poor paste stability when it is exposed to heat treatment at high temperature and mechanical stirring. Compared with Musa balbisiana, Musa acuminata had significantly lower (P < 0.05) BV. Musa acuminata pseudostem is more resistant against heat and shear force than Musa balbisiana. This indicates that Musa acuminata may have a potential as a food ingredient for food exposed to heat treatment. The SB is correlated (r2=0.94) to the FV and is related to the gelling ability or retrogradation of starch molecules. During cooling, re-association between starch molecules, especially amylose, will result in the formation of a gel structure and thereby viscosity will increase to a final viscosity (Ho et al., 2012). The low SB values implies low rate of starch retrogradation and syneresis. 187 BP40B and BP50 showed lowest SB (17.3 RVU and 19.3 RVU, respectively) followed by BP40 (31.6 RVU) and BP50B (37.0 RVU). This result indicated that BP50B had the highest amylose retrogradation. There were no significant differences (p > 0.05) in SB between Musa balbisiana and Musa acuminata. FV is an important indicator of the strength of the gel formed upon cooling and represents an important quality parameter (Cornejo-Villegas et al., 2010). FV of BP40B and BP50 are significantly lower (p < 0.05) than BP40 and BP50B. No significant difference (P > 0.05) of FV was observed between Musa balbisiana and Musa acuminata. 188 8. Conclusions, recommendations and contributions 8.1 Conclusions Two banana pseudostem species (Musa balbisiana and Musa acuminata) were used in this study together with 4 drying conditions in order to figure out the effect of species and drying conditions on the retention of nutrients, digestibility and functionality of carbohydrates in banana pseudostem. The results showed that drying at 50 C without blanching provided whitest colour and fastest drying rate. In terms of nutrient retention, drying at 50 C without blanching performed better than other conditions as samples dried at this condition were observed to have significantly (p<0.05) lower moisture content, higher ash content, and no significant difference (p>0.05) in protein, fat and carbohydrates compared with samples dried at other conditions. Therefore, drying at 50 C without blanching was considered as optimum condition for drying. This condition has been selected to drying both Musa balbisiana and Musa acuminata for comparing the effect of species on the nutrients retention, carbohydrate digestibility and functionality. Musa acuminata had significantly higher (p<0.05) moisture, protein and ash content than Musa balbisiana. There was no significant difference (p>0.05) between fat and carbohydrate content of pseudostems from different species. Considering the digestibility of carbohydrates in pseudostem, samples dried at 40 C with 3 min blanching had the lowest digestibility. As a result of the 189 percentage of total dietary fibre and resistant starch in samples dried at this condition were the highest. Musa acuminata had lower digestibility than Musa balbisiana. It has also been found that higher temperature (50 C) provided higher level of total dietary fibre content. Moreover, blanching affected the sugar and starch content in banana pseudostem. Higher starch and lower sugar contents were obtained in blanched samples. Additionally, an inverse relationship was observed between total starch and sugars in the pseudostem samples. In order to study the functionality of dietary fibre in the banana pseudostem, three methods were used and compared for determining the structure of dietary fibre, namely AOAC, NMR and GC methods. According to the result, cellulose was the main insoluble dietary fibre and pectin was the dominant soluble dietary fibre in the pseudostem. Throughout GC analysis, the neutral sugars that consisted of non-starch polysaccharides in the pseudostem were qualified and quantified. It has been identified that glucose, mannose and xylose were the main compounds in the pseudostem fibre. Commercial dietary fibre supplements (Metamucil) were compared with the pseudostem powder. The primary neutral sugars in the Metamucil were xylose, arabinose and mannose, which were different from that in the banana pseudostem and resulted in different functionality characters. The WHC, SWC, SC and CI of Musa balbisiana were in the ranges of 9.0-11.0 g water/g of dry matter, 4.3-7.5 granules/g of dry matter, 25.5-34.2 % and 36.5- 54.1 %, respectively. Musa balbisiana had significantly higher SWC (p<0.05) 190 than Musa acuminata. No significant difference (p>0.05) was observed in WHC, SC and CI between species. Banana pseudostem dried under different conditions had similar pasting curve shape. However, BP40 showed significantly higher (p<0.05) PV and BD, which indicated that BP40 had the highest peak viscosity and poor pasting stability. BP40B and BP50 had significantly lower (p<0.05) SB and FV, which implied lower starch retrogradation and syneresis. Musa acuminata revealed significantly lower (p<0.05) BV than Musa balbisiana, but no significant difference (p>0.05) in SB and FV was observed between these species. The water holding capacity, swelling capacity and crystallinity index of Metamucil were significantly higher than banana pseudostem (p<0.05) and the solubility capacity of Metamucil was significantly lower (p>0.05) than for banana pseudostem. 8.2 Recommendations Banana pseudostem is a good source of dietary fibre, it has potential to be developed as a plant based dietary fibre supplement, which differs from commercial dietary fibres sold in the market. Further research could be done on efficient extraction of non-starch polysaccharides from banana pseudostem. 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Appendix 2nd INTERNATIONAL CONFERENCE ON NATURAL FIBERS Please leave these first three lines blank for the Editors Please leave these first three lines blank for the Editors Please leave these first three lines blank for the Editors EFFECTS OF DRYING ON THE DIETARY FIBRE OF BANANA PSEUDOSTEM (MUSA BALBISIANA & MUSA ACUMINATA) Please leave this line blank Jun Ma, George Srzednicki(*) and Jayashree Arcot Food Science and Technology Group, School of Chemical Engineering, UNSW Australia, Sydney 2052 (*)Email: [email protected] Please leave these two lines blank Please leave these two lines blank ABSTRACT This study used two methods to identify the dietary fibre in banana pseudostem. The first method was using Megazyme (2012) to identify the total, soluble and insoluble fibre in banana pseudostem of two species, Musa balbisiana and Musa acuminata. Pseudostem samples were subjected to four drying treatments namely drying at 40 °C with or without blanching and drying at 50 °C with or without blanching. Banana pseudostem drying at 50°C with blanching resulted in significantly higher (p<0.05) insoluble and total dietary fibre than banana pseudostem content than other treatments. No significant difference was observed in soluble fibre of banana pseudostem between treatments or species. Musa balbisiana pseudostem showed significantly higher total and insoluble dietary fibre value (p<0.05) than Musa acuminata pseudostem. The second method was using enzyme and acid hydrolysis to extract dietary fibre and break down the links to release single sugars and then used solution state 600 MHz NMR 1H and solid state 13C CP/MAS NMR method to identify the composition and network of dietary fibre in banana pseudostem. The results of the study of fibre structure and value in the banana pseudostem characterise its physicochemical properties, nutritional value and thus provide information on the utilisation of banana pseudostem in the food industry that have potential to increase the nutritional and economical value of banana pseudostem. Keywords: Banana, Pseudostem, Dietary fibre, Drying, Blanching INTRODUCTION The stem of the banana plant, which is also called pseudostem, produces a single bunch of banana before dying and replaced by a new pseudostem (Anhwange et al., 2011). Due to this character, the crop generates a large amount of residue. It has been estimated that a few tonnes of banana pseudostem per hectare are produced every year (Aziz et al., 2011). After the harvest, the bare pseudostem is cut and usually left on the plantation or burned, which could ultimately cause environmental issues and economic loss (Cordeiro et al., 2004). In Australia, over 300,000 tonnes of bananas are produced annually and million tonnes of pseudostems are wasted (Horticulture Australia, 2014; Hossain et al., 2011). Thus, the utilisation of the banana waste - pseudostems and the processing methods to enhance their economic value has gained more attention in recent years. Nowadays, the banana pseudostems are being used as a raw material for paper, furniture and forage (Buragohain et al., 2010; Umaz et al., 2005). In some regions such as India and Malaysia, the fresh tender core of banana pseudostem is cooked and consumed, whereas the consumption of banana pseudostem as food in Australia is rare. Some studies also state that banana pseudostem is rich in minerals and nutrients especially dietary fibre (Cordeiro et al., 2004; Ho et al., 2012; Aziz et al., 2011; Bhaskar et al., 2011). However, the studies on the 2nd ICNF – From Nature to Market 1 222 223 224 225 226 227 228 229