Cryo-extraction des cires des produits céréaliers (sorgho ...€¦ · coupled to mass...

CRYO-EXTRACTION DES CIRES DES

PRODUITS CÉRÉALIERS

(SORGHO, BLÉ ET RIZ BRUN)

MÉMOIRE

Thi Can Tho PHAM

Maîtrise en Sciences et technologie des aliments

Maître ès Sciences (M.Sc.)

Québec, Canada

© Thi Can Tho PHAM, 2014

iii

Résumé court

La cire est un lipide naturel et possède beaucoup d‟applications intéressantes dans

l‟industrie cosmétique et pharmaceutique. Nous visons à étudier l‟extraction des cires du

sorgho, du blé et du riz brun par l‟azote liquide comme méthode „verte‟. Ces céréales ont

été immergées dans l‟azote liquide de 1 à 3 cycles à différentes durées et différentes du

temps entre les cycles. Nos résultats ont montré que l'extraction de grains de céréales par

l'azote liquide est réalisable. En comparant avec la méthode utilisant n-hexane, la quantités

de cires extraites par l'azote liquide était de 5, 7.5 et 9.3 fois plus faible mais le temps

d‟extraction était de 2.3, 5.5 et 11.25 fois plus court pour le blé, riz et sorgho,

respectivement. Selon l‟analyse par SEM, les cires superficielles des grains ont pu être

extraites par l‟azote liquide. La composition des cires a été identifiée par GC-MS et GC-

FID.

v

Short Abstract

Wax is a natural lipid and has many interesting applications in cosmetics and

pharmaceuticals. The purpose of this study is to use liquid nitrogen as a “green method” to

extract waxes from rice, sorghum and wheat. These cereals were treated in liquid nitrogen

in 1 to 3 cycles with different time intervals and different time between the cycles. The

results showed that waxes could be extracted with liquid nitrogen. When compared to the

traditional method (n-hexane), the extracted amounts of waxes with liquid nitrogen were 5,

7.5 and 9.3 times lower but the extraction time was 2.3, 5.5 and 11.25 times shorter for

wheat, rice and sorghum, respectively. SEM microphotographs depicted that superficial

waxes was extracted from grains by liquid nitrogen. GC-MS and GC-FID confirmed that

extracted waxes had similar compositions in both extraction methods. This study points out

a new friendly-environmental way to extract waxes from cereals.

vii

Résumé long

L‟extraction des cires végétales est fréquemment réalisée en utilisant des solvants tels que

l‟hexane, le cyclohexane, le méthanol, etc. Ces solvants sont très efficaces pour dissoudre

les lipides. Cependant, ils sont aussi inflammables, très volatils et toxiques. Récemment,

des recherches ont montré que les fluides cryogéniques peuvent aussi servir à cette fin. Par

exemple, l‟immersion cyclique des fruits entiers dans l‟azote liquide (bleuet, argousier,

raisin vert) a permis de diminuer les cires cuticulaires jusqu‟à 50%. Le but de ce travail est

donc d‟étudier l‟immersion dans l‟azote liquide comme méthode „verte‟ pour extraire la

cire des grains.

Du sorgho, du blé et du riz brun ont été immergés dans l‟azote liquide en 1 à 3 cycles à

différentes durées. L‟impact du temps de repos entre les cycles (1-5 minutes à température

ambiante) sur le rendement d'extraction a aussi été analysé. La performance d‟extraction a

été comparée à celle par le n-hexane avec la méthode reflux. La microscopie électronique à

balayage (SEM) a permis de visualiser l'effet de l'immersion dans l'azote liquide et dans

l‟hexane sur la surface des grains. La chromatographie en phase gazeuse couplée à un

spectromètre de masse (GC-MS) et à un détecteur à ionisation de flamme (GC-FID) a été

utilisée pour la détermination de la composition chimique des extraits obtenus par les deux

méthodes.

Les résultats ont montré que le rendement d‟extraction le plus élevé était de 0.026% (g pds

cire/100g pds grain) (riz-11minutes d'extraction), 0.032% (sorgho-4 minutes) et 0.025%

(blé-13 minutes). En comparaison, le rendement des cires extraites par le n-hexane (70oC) a

été de 0.196% (riz-60 minutes d'extraction), 0.298% (sorgho-45 minutes) et 0.126% (blé-30

minutes). Pour le blé et le riz, une augmentation significative du rendement a été observée

pour des temps de repos entre 3 et 5 minutes entre les cycles par rapport à celui de 1

minute.

viii

Les résultats de GC-MS et GC-FID nous ont confirmé que de la cire a bien été extraire par

l‟azote liquide et le n-hexane, dont la composition est similaire dans les deux cas.

Ces résultats montrent que la méthode d‟extraction par l'azote liquide est réalisable malgré

son rendement plus faible que celle par l'n-hexane mais avec un temps d‟extraction plus

court. L‟azote liquide n‟est pas toxique et c‟est un solvant « vert » qui peut être éliminé du

produit très facilement par simple évaporation, sans laisser aucune trace problématique.

L'utilisation de l'azote liquide comme solvant d'extraction s'avère une méthode prometteuse

pour la séparation des composés cireux des grains. La qualité des céréales traitées par

l‟azote liquide demeure inchangée donc elle pourra être considérée comme une étape

secondaire dans le processus de polissage.

ix

Extended Abstract

Wax extraction from plants is conventionally carried out by using organic solvents such as

n-hexane, cyclohexane and methanol. Although, those solvents are very effective in

dissolving lipids, they are flammable, toxic and highly volatile. Recent studies show that

cryogenic fluids could also be used for wax extraction. For instance, cyclic immersion of

whole fruits (blueberry, seabuckthorn, grapes) in liquid nitrogen has significantly reduced

cuticular waxes up to 50%. So, the main aim of this work is to use liquid nitrogen as a

“green method” to extract waxes from cereals.

Several approaches were used to evaluate the extraction method by liquid nitrogen.

Sorghum, wheat and brown rice were treated in liquid nitrogen in 1 to 3 cycles with

different time intervals. The impact of rest time between cycles (1-5 minutes at room

temperature) on the extraction yield was also reported by weighing waxes. The extraction

efficiency of the method was compared to that of n-hexane which was referred as the

control one. The surface of the grains treated by liquid nitrogen and n-hexane was

visualized by the scanning electron microscopy (SEM). Finally, the gas chromatography

coupled to mass spectrometry (GC-MS) and flame ionization detector (GC –FID) was used

to determine the composition in the extracts resulted from the two methods.

Our results showed that the highest extraction efficiency was 0.026% (g cire/100g grain)

(rice - 11 extraction minutes), 0.032% (Sorghum - 4 minutes) and 0.025% (wheat -13

minutes). Compared to the extraction by n-hexane method, its efficiency was 0.196% (rice

– 60 extraction minutes), 0.298% (sorghum - 45 minutes) and 0.126% (wheat - 30

minutes). For wheat and rice, a significant increase in yield between 3 and 5 rest minutes

with respect to the one of 1 minute was observed.

The results of GC-MS and GC –FID confirmed that the waxes were extracted out of the

grains by both liquid nitrogen and n- hexane. The wax composition of the extract is similar

in both cases.

x

These results conclude that the wax extraction method by liquid nitrogen is feasible despite

it extracts waxes less than that of n-hexane, but the method benefits for the shorter

extraction time. Liquid nitrogen is not toxic and is a "green" solvent which can be easily

removed from the products by spontaneously simple evaporation leading no leftover

residues. Therefore, using liquid nitrogen as the extraction solvent might be a promising

way for separating waxes from waxy grains. The quality of grain processed by liquid

nitrogen remains unchanged, thus it may be considered a sub-step in the milling process.

xi

Table of contents

Résumé court ..................................................................................................................... iii

Short Abstract..................................................................................................................... v

Résumé long ..................................................................................................................... vii

Extended Abstract ............................................................................................................. ix

Table of contents ............................................................................................................... xi

List of tables .................................................................................................................... xiii

List of figures ................................................................................................................... xv

Remerciements ............................................................................................................... xvii

CHAPTER 1. INTRODUCTION ...................................................................................... 1

CHAPTER 2. LITERATURE REVIEW ........................................................................... 5

2.1. Rice (Oryza sativa L.) .................................................................................................. 5

2.1.1. Production of rice ...................................................................................................... 6

2.1.2. Structure and composition of rice grain .................................................................... 6

2.2. Wheat (Triticum aestivum) .......................................................................................... 9

2.2.1. Wheat production .................................................................................................... 10

2.2.2. Structure and composition of wheat ........................................................................ 10

2.3. Sorghum (Sorghum bicolor L.) .................................................................................. 11

2.3.1 Sorghum production ................................................................................................. 12

2.3.2 Structure and composition of sorghum. ................................................................... 13

2.4. Rice, wheat and sorghum waxes and their applications ............................................. 15

2.4.1. Wax generalities: types and composition ................................................................ 15

2.4.2. Cereal wax applications .......................................................................................... 18

2.5. Extraction methods of lipid ........................................................................................ 19

2.6. Extraction methods of cereal waxes ........................................................................... 21

2.7. Liquid nitrogen and its potentials for wax extraction ................................................ 22

2.7.1. Properties of liquid nitrogen .................................................................................... 22

2.7.2. Potential of wax extraction by liquid nitrogen ........................................................ 23

CHAPTER 3. HYPOTHESIS AND OBJECTIVES ........................................................ 25

3.1. Hypothesis .................................................................................................................. 25

3.2. Objectives ................................................................................................................... 25

3.2.1 General objective ..................................................................................................... 25

3.2.2 Specific objectives ................................................................................................... 25

CHAPTER 4. MATERIALS AND METHODS .............................................................. 27

4.1. Materials ..................................................................................................................... 27

4.2. Methodology .............................................................................................................. 27

4.2.1. Determination of moisture content .......................................................................... 29

4.2.2. Wax extraction by liquid nitrogen........................................................................... 29

4.2.3. Wax extraction by n-hexane .................................................................................... 31

4.2.4. Calculation of wax yield ......................................................................................... 32

4.2.5. Examination of wax by scanning electron microscope (SEM) ............................... 32

4.2.6. Analysis of wax composition .................................................................................. 33

4.3. Statistical analysis ...................................................................................................... 34

CHAPTER 5. RESULTS AND DISCUSSION ............................................................... 35

5.1. Extraction of cereal waxes with n-hexane ................................................................. 35

xii

5.2. Extraction of cereal waxes with liquid nitrogen ........................................................ 38

5.3. Impact of number of immersion cycles and rest time with liquid nitrogen ............... 41

5.3.1. Sorghum.................................................................................................................. 41

5.3.2 Rice .......................................................................................................................... 41

5.3.3 Wheat ....................................................................................................................... 47

5.4. Comparison between two methods of wax extraction ............................................... 50

5.5. Color of sorghum, rice and wheat waxes .................................................................. 50

5.6. Scanning electron microscopy (SEM) ....................................................................... 52

5.7. Composition of waxes - Gas chromatography .......................................................... 56

CHAPTER 6. CONCLUSION ......................................................................................... 69

References ........................................................................................................................ 71

ANNEX 1: ANOVA table and mean table of the comparison the means of percent rice

wax extracted by n-hexane. .............................................................................................. 79

ANNEX 2: ANOVA table and mean table of the comparison the means of percent

sorghum wax extracted by n-hexane. ............................................................................... 80

ANNEX 3: ANOVA table and mean table of the comparison the means of percent wheat

wax extracted by n-hexane. .............................................................................................. 81


sorghum wax extracted by liquid nitrogen with 1 minute rest ......................................... 82


sorghum wax extracted by liquid nitrogen with 1, 3, 5 minutes rest ................................ 83


sorghum wax extracted by liquid nitrogen with 1 minutes rest ........................................ 84


wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest .......................................... 85


wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest .......................................... 86


wax extracted by liquid nitrogen with 1 minute of rest .................................................... 87


wheat wax extracted by liquid nitrogen with 3 minutes of rest ........................................ 88


wheat wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest ................................ 89

xiii

List of tables

Table 2.1. World production of rice (Source FAO, 2012) ...................................................... 6

Table 2.2. Composition of brown, milled and parboiled rice (source: Adair, 1972) .............. 8

Table 2.3. White rice and by-products from paddy milling process (Source: Kahlon, 2009) 8

Table 2.4. The production of wheat (Source FAO, 2012) .................................................... 10

Table 2.5. The production of sorghum (Source FAO, 2012) ................................................ 13

Table 2.6. Composition of the whole kernel and its part for sorghum (moisture-free basis)

(Wall et Charles, 1970) ......................................................................................................... 14

Table 2. 7. The most common substance classes of cuticular waxes (Luka et al., 2009) ..... 16

Table 2.8. Properties of nitrogen (Source Afsset, 2008) ...................................................... 23

Table 5.1. Fitted parameters for kinetics constants Wmax and b from Eqn. (5.1) – hexane

extraction (r2 is the coefficient of determination) ................................................................. 36

Table 5.2. Fitted parameters for kinetics constants Wmax and b from Eqn. (5.1) – liquid

nitrogen extraction (r2 is the coefficient of determination) ................................................... 40

Table 5.3. Comparison of liquid nitrogen and n-hexane extraction methods ....................... 50

Table 5.4. Classes of compounds present in the rice wax extracted with their retention time.

Superscripts (1) to (7) indicates ratios that could not be calculated since amount obtained

with n-hexane was zero. ........................................................................................................ 64

Table 5.5. Classes of compounds present in the sorghum wax extracted with their retention

time. Superscripts (1) to (7) indicates ratios that could not be calculated since amount

obtained with n-hexane was zero. ......................................................................................... 65

Table 5.6. Classes compounds present in the wheat wax extracted with their retention time.


with n-hexane was zero. ........................................................................................................ 66

Table A-1. Anova of the percentage of rice wax extracted by n-hexane .............................. 79

Table A-2. Rice wax extraction by n-hexane ....................................................................... 79

Table A-3. Anova of the percentage of sorghum wax extracted by n-hexane ...................... 80

Table A-4. Percent of sorghum wax extraction by n-hexane ............................................... 80

Table A-5. Anova of percentage of wheat wax extracted by n-hexane ................................ 81

Table A-6 . Percent of wheat wax extraction by n-hexane ................................................... 81

Table A-7. Anova of percentage of sorghum wax extracted by liquid nitrogen with 1 minute

of rest .................................................................................................................................... 82

Table A-8. Percent of sorghum wax extracted by liquid nitrogen with 1minute of rest ...... 82

Table A-9. Anova of percentage of sorghum wax extracted by liquid nitrogen with 1, 3, 5

minutes of rest ....................................................................................................................... 83

Table A-10. Percent of sorghum wax extracted by liquid nitrogen with 1, 3, 5 minutes of

rest ......................................................................................................................................... 83

Table A-11. Anova of percentage of rice wax extracted by liquid nitrogen with 1 minute of

rest ......................................................................................................................................... 84

Table A-12. Percent of rice wax extracted by liquid nitrogen with 1 minute of rest ........... 84

Table A-13. Anova of percentage of rice wax extracted by liquid nitrogen with 1, 3, 5

minutes of rest ....................................................................................................................... 85

Table A-14. Percent of rice wax extracted by liquid nitrogen with 3 minutes of rest .......... 85


minutes of rest ....................................................................................................................... 86

xiv

Table A-16. Percent of rice wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest . 86

Table A-17. Anova of percentage of wheat wax extracted by liquid nitrogen with 1 minute

of rest .................................................................................................................................... 87

Table A-18. Percent of wheat wax extracted by liquid nitrogen with 1 minute of rest ....... 87

Table A-19. Anova of percentage of wheat wax extracted by liquid nitrogen with 3 minutes

of rest .................................................................................................................................... 88

Table A-20. Percent of wheat wax extracted by liquid nitrogen with 3 minutes of rest ...... 88

Table A-21. Anova of percentage of wheat wax extracted by liquid nitrogen with 1, 3, 5

minutes of rest ...................................................................................................................... 89

Table A-22. Percent of wheat wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest

.............................................................................................................................................. 89

xv

List of figures

Figure 2. 1. Rice plant and rice grain ...................................................................................... 5

Figure 2.2.Structure of rice grain (from Encyclopaedia Britannica, http://

www.britannica.com) .............................................................................................................. 7

Figure 2.3.Wheat plant and wheat grain ................................................................................. 9

Figure 2.4. Struture of wheat grain (from Encyclopaedia Britannica,

http://www.britannica.com) .................................................................................................. 11

Figure 2.5. Sorghum plant and sorghum grain ..................................................................... 12

Figure 2.6.Structure of sorghum grain (Earp.C.F et al., 2003) ............................................. 13

Figure 2.7. Structure of cuticule (lipidlibrary.aocs.org) ....................................................... 15

Figure 4.1. Proposed methodology, liquid nitrogen extraction on the left and n-hexane on

the right ................................................................................................................................. 28

Figure 4.2. Description of wax extraction in liquid nitrogen. ............................................... 30

Figure 4.3. Illustration of immersion process in liquid nitrogen .......................................... 30

Figure 4.4.Illustration of reflux system used for wax extraction by n-hexane

(www.chem.wisc.edu) .......................................................................................................... 32

Figure 5.1. Extraction kinetics of sorghum, rice and wheat wax by reflux extraction using

n-hexane. ............................................................................................................................... 36

Figure 5.2. Extraction kinetics of sorghum, rice and wheat wax by immersion in liquid

nitrogen. ................................................................................................................................ 39

Figure 5.3. Impact of number of immersion cycles on wax yield (%) for sorghum (1 minute

rest time) ............................................................................................................................... 43

Figure 5.4. Impact of rest time on wax yield (%) for sorghum (2 cycles with 1, 3 or 5

minutes rest time) .................................................................................................................. 43

Figure 5.5 . Rice wax yield from extraction with liquid nitrogen (1 minute rest time) ........ 44

Figure 5.6. Rice wax yield from extraction with liquid nitrogen (3 minutes rest time) ....... 44

Figure 5.7. Rice wax yield from extraction with liquid nitrogen (2 cycles with 1, 3 or 5


Figure 5.8. Wheat wax yield from extraction with liquid nitrogen (1 minute rest) .............. 48

Figure 5.9. Wheat wax yield from extraction with liquid nitrogen (3 minute rest) .............. 48

Figure 5. 10 . Wheat wax yield from extraction with liquid nitrogen (2 cycles with 1, 3 or 5


Figure 5.11. Wax extracted with n-hexane: sorghum (a), wheat (b), rice (c). Sorghum wax

extracted by liquid nitrogen (d) ............................................................................................ 51

Figure 5.12. Surface visualization by SEM of the sorghum grain: fresh (a), treated by n-

hexane (b) and treated by liquid nitrogen (c) ........................................................................ 53

Figure 5.13. Surface visualization by SEM of the rice grain: fresh (a), treated by n-hexane

(b) and treated by liquid nitrogen (c) .................................................................................... 54

Figure 5.14. Surface visualization by SEM of the wheat grain: fresh (a), treated by n-

hexane (b) and treated by liquid nitrogen (c) ........................................................................ 55

Figure 5.15. GC-MS Chromatogram of rice wax extracted by n-hexane ............................. 57

Figure 5.16.GC-MS Chromatogram of rice wax extracted by liquid nitrogen ..................... 57

Figure 5. 17. GC-MS Chromatogram of sorghum wax extracted by n-hexane .................... 58

Figure 5.18. GC-MS Chromatogram of sorghum wax extracted by liquid nitrogen ............ 58

xvi

Figure 5.19. GC-MS Chromatogram of wheat wax extracted by n-hexane ......................... 59

Figure 5.20. GC-MS Chromatogram of wheat wax extracted by liquid nitrogen ................ 59

Figure 5.21. GC-FID Chromatogram of rice wax extracted by n-hexane............................ 61

Figure 5.22. GC-FID Chromatogram of sorghum wax extracted by liquid nitrogen ........... 61

Figure 5.23. GC-FID Chromatogram of sorghum wax extracted by n-hexane .................... 62

Figure 5.24. GC-FID Chromatogram of sorghum wax extracted by liquid nitrogen ........... 62

Figure 5.25. GC-FID Chromatogram of wheat wax extracted by n-hexane ........................ 63

Figure 5.26. GC-FID Chromatogram of wheat wax extracted by liquid nitrogen ............... 63

xvii

Remerciements

J'aimerais remercier à ma directrice, Madame la professeure Cristina Ratti qui est très

gentille et sympathique. Je suis vraiment très chanceuse d'avoir travaillé avec elle. Merci de

ses conseils, ses supports, sa patience et ses encouragements tout au long de mes études et

aussi dans ma vie. Je me sens de temps en temps coupable quand je n'ai pas pu accomplir

bien des taches au laboratoire.

J'aimerais consacrer mes remerciements à Monsieur le professeur Paul Angers, mon co-

directeur pour ses supports, ses suggestions, sa disponibilité et ses encouragements

précieux durant mes études de la maitrise.

Merci beaucoup à Monsieur Ronan Corcuff de m'avoir permis d'utiliser le système de Gas

Chromatography-Mass spectrometry et de m‟aider à faire des analyses des composants de

la cire par cette technique.

Merci également à Monsieur Richard Janvier qui m'a aidé à générer des photos par la

microscopie électronique.

Merci bien Céline Paquin, Jocelyne Giasson et Diane Gagnon pour leurs supports

techniques et leurs conseils au laboratoire.

Merci beaucoup à mon mari qui m'accompagne dans la vie, a supporté et encouragé tout au

long de ma maitrise. Je pense toujours à mon fils de son courage pendant le combat avec

son cancer. C'était lui qui m'a donné la force afin de surmonter toutes les difficultés dans la

vie. Merci beaucoup à ma famille, surtout ma mère et ma belle-mère qui sont venues à

Québec m'aider quand ma vie est dans le besoin et dans la difficulté. Merci à tous mes

amis à Québec et au Vietnam pour leur encouragement pendant mes études. Finalement, Je

remercie le CRSNG pour le soutien financier de ce projet.

xix

To my lovely son, who has been given me the belief and the motivation to realize my dream.

1

CHAPTER 1. INTRODUCTION

Cereals are members of the grass family. They are among the important foods for humans

and have been used for thousands of years. Production of cereals in the world is more than

2500 million tonnes in the year 2013 (FAO, 2013). Wheat, rice and sorghum are in the

group of major cereals of the world and provide high of the world‟s dietary energy supply.

Rice is very popular in Africa and especially in Asia and sorghum is especially well known

and accepted in Africa while wheat is mostly planted in North America and Europe. Bread

wheat (Triticum aestivum), Asian rice (Oryza sativa L.) and Sorghum (Sorghum bicolor L.)

are the mostly widely cultivated in the world among of other varieties. In 2010, world

production of wheat, rice and sorghum paddies was 651 million tonnes, 672 million tonnes

and 57 million tonnes, respectively (FAO, 2012).

The surface of cereal kernels is covered by a layer containing wax, called bran, a natural

lipid biochemically synthesized during growth. Bran is highly hydrophobic so it is used as a

natural protector against water and moisture loss, even against microorganisms. The major

components of cereal wax are esters of long chain fatty acids and long chain fatty alcohols

or sterols, hydrocarbons, acylglycerides, free fatty acids and free alcohols (Ohnishi et al.,

1985; Vali et al., 2005; Kim, 2008). These waxes have been considered as a by-product or

waste material from oil production and are usually removed by refining processes called

winterization before the oils are marketed (Kim, 2008).

Nowadays, cereal waxes get increased attention since they can have many interesting

applications. In cosmetic industries, for instance, they play an important role in the

production of creams, crayons and lipsticks because of their ability for making skin smooth

and soft. Also, waxes can form a protective film to prevent the loss of skin moisture.

Moreover, waxes of these grains are used in pharmaceutical industries, thanks to the

significant presence of policosanols (Lui et al., 2005; Wang et al., 2007; Irmak et al., 2006;

2

Chen et al., 2008; Dunford et al., 2010). This component, which is a mixture of long chain

saturated alcohols, is believed to lower the bad cholesterol, to treat gastric and duodenal

ulcers and especially to exhibit substantial anti-inflammatory activity (Vali et al., 2005).

Waxes also have other potential applications in the food, polymer and leather industries.

To extract waxes from cereals, many methods using organic solvents have been utilized.

Due to their intrinsic hydrophobicity, waxes have mainly been extracted by non-polar

solvents or mixtures of solvent like chloroform-methanol (Ohnishi et al., 1986), benzene

(Avato et al., 1990) and hexane (Hwang et al., 2002; 2004; 2005). For the further purpose

of separating the wax from the extract, two ways have been proposed. The first one consists

of evaporation of the solvent from the mixture containing waxes and mixing the wax

extract with other polar solvents (acetone or alcohol), followed by precipitation of waxes

and filtration. The second one, in crystallizing the waxes by incubation of the extraction

mixture at -18oC, and then the crystallized waxes are collected by filtration (Hwang et al.,

2002). Although solvent wax extraction methods are simple, and economical require no

complex equipment, they are not considered to be environmental-friendly, with potential

harm to human health.

Recently, research focused on taking advantage of liquid nitrogen to pre-treat whole fruits

prior to drying. Ketata et al. (2012) showed that liquid nitrogen could decrease wax content

on the surface of blueberries, resulting in shortening the drying process. Similarly,

blueberries, seabuckthorn fruits and green grapes treated with liquid nitrogen showed a

decrease in drying times for different drying methods (vacuum, hot air and freeze-drying).

The surface of these fruits was observed by scanning electron microscopy before and after

liquid nitrogen pre-treatments, pointing out the disappearance of their epicuticular wax,

which was mainly responsible for slowing down drying (Thromas et al., 2010; 2011).

These studies strongly support the fact that liquid nitrogen could be used to extract waxes

from cereal grains like wheat, rice and sorghum. In addition, liquid nitrogen is proved to be

non-toxic, colorless, odorless, tasteless, inflammable and inert. Especially, it vaporizes

itself at room temperature so there would be no leftover residue in extracted waxes neither

in grains. Therefore, the aim of this research is to study the possibility of extracting waxes

3

from wheat (Triticum aestivum), rice (Oryza sativa L.) and sorghum (Sorghum bicolor L.)

by immersion in liquid nitrogen as well as to determine composition of the extract.

5

CHAPTER 2. LITERATURE REVIEW

2.1. Rice (Oryza sativa L.)

Rice is one of the world's important cereals, serving as a staple food for a large section of

the population, especially in Asia and Africa. It has been under intensive cultivation in Asia

for over 4 000 years and has since spread across the world, where almost a third of the

population depends on rice for vital nutrition (Grist, 1975). Among the 40 000 different rice

varieties, Asian rice (Oryza sativa L.) and African rice (Oryza glaberrima) are the most

popular cultivated in the world (Grist, 1972). They belong to genus Oryza and there are

only small differences between these species, mainly in grain size and glume pubescence.

Oryza sativa L. is divided into four groups: indica, japonica, brevindica and brevis.

However, the sub-species brevindica and brevis are not generally recognized. Japonica

varieties are usually cultivated in dry fields, in temperate East Asia, upland areas of

Southeast Asia and high elevations in South Asia, while indica varieties are mainly lowland

rice, grown mostly submerged, throughout tropical Asia (Figure 2.1). The grain of japonica

is shorter and broader than the grain indica. Japonica is sticky, and it tends to soften

rapidly after certain time of cooking while Indica is non-sticky and usually can be

overcooked (Grist, 1975).

Figure 2. 1. Rice plant and rice grain

http://en.wikipedia.org/wiki/Oryza_sativa


http://en.wikipedia.org/wiki/Oryza_glaberrima

6

2.1.1. Production of rice

The world production of rice is very large and it has risen from 519 million tonnes of rice

paddy in 1990 to 672 million tonnes in 2010 (FAO, 2012; table 2.1). Although the

production is large, only 5-6% of produced rice is traded internationally. Asian countries

produce most of the rice in the world. In 2010, Thailand, Vietnam and India were the three

largest exporters of rice. They accounted for nearly 70% of the world rice exports.

Table 2.1. World production of rice (Source FAO, 2012)

Year

Production (metric tonnes)

World Asian

countries

African

countries

American

countries

North American

countries

1990 518 568 263 477 692 981 12 697 109 22 655 617 7 080 000

2000 599 355 455 545 546 464 17 476 517 32 032 396 8 657 820

2010 672 015 587 607 328 408 22 855 318 37 170 221 11 027 000

2.1.2. Structure and composition of rice grain

Rice has different names with different nutrition values depending on where it is positioned

in the processing line. To understand better the description of rice and its different names,

Figure 2.2 shows a schematic cross section of a grain of rice, with its main components.

Rice harvested from the field is called paddy which is enclosed by the hull, or husk. Milling

usually removes both the hull and bran layers of the rice. In the rice milling process, first

the outermost layer, the hull, is removed to produce brown rice. This process is the least

damaging to the nutritional value of the rice and avoids the unnecessary loss of nutrients

that occurs with the further processing. Brown rice would be considered a whole grain. It is

a source of thiamine, niacin, riboflavin, iron, and calcium. Rice that is milled to remove the

bran as well is called white rice. White rice has greatly diminished its nutrient content from

brown rice. When white rice forms a major portion of the diet (lacking of important

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nutrients), there is a risk of „beriberi‟, a disease resulting from the deficiency of thiamine

and minerals.

Figure 2.2.Structure of rice grain (from Encyclopaedia Britannica, http://

www.britannica.com)

In parts of India and some countries of West Africa, parboiled rice is popular. Parboiled

rice is a term indicating that rice paddy has been subjected to a steaming or parboiling

process. This makes nutrients from the outer husk, especially thiamine, move into the grain

itself. The rice is then dried, and can be milled as usual or used as brown rice. The milled

parboiled rice is nutritionally superior to the standard milled rice. Table 2.2 presents the

composition of brown, milled raw and milled parboiled rice (Adair, 1972).

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Table 2.2. Composition of brown, milled and parboiled rice (source: Adair, 1972)

Component Brown rice Milled raw rice Milled parboiled rice

Moisture (%)

Calories (/100g)

Protein (%)

Fat (%)

N-free extract (%)

Fiber (%)

Ash (%)

Thiamine (mg/100 g)

Riboflavin (mg/100 g)

Niacin (mg/100 g)

12.0

360.0

7.5

1.9

77.4

0.9

1.2

0.34

0.05

4.7

12.0

363.0

6.7

0.4

80.4

0.3

0.5

0.07

0.03

1.6

10.3

369.0

7.4

0.3

81.3

0.2

0.7

0.4

…

3.5

Rice processing releases many by-products. Firstly, the by-products of milling, including

bran and rice polish (finely powdered bran and starch resulting from polishing), are used as

livestock feed. Secondly, oil is processed from the bran for both food and industrial uses.

Thirdly, broken rice is used in brewing, distilling, and manufacturing starch and rice flour.

Fourthly, hulls are used for fuel, packing material, industrial grinding, and fertilizer

manufacture. Finally, straw is used for feed, livestock bedding, roof thatching, mats,

garments and packing material. Table 2.3 presents an estimation of the percentage of

different types of rice and by-products in a paddy milling processing operation (Kahlon,

2009).

Table 2.3. White rice and by-products from paddy milling process (Source: Kahlon, 2009)

Composition White rice Broken rice Bran Hulls

Wt (%) 56-58 10-12 10-12 18-20

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2.2. Wheat (Triticum aestivum)

Wheat is one of the oldest and the most extensively grown among grain crops. It emerged

as a crop about 10 000 years ago and became a major component of most diets of the world

because of its agronomic adaptability, ease of storage, nutritional goodness and the ability

of its flour to produce a variety of palatable, interesting and satisfying foods (Feldman,

2001). Wheat origin is in southwest Asia and it is now cultivated worldwide. Wheat

belongs to the genus Triticum, and the most widely cultivated in the world is common

wheat or bread wheat (Triticum aestivum) (Figure 2.3) (Pomeranz, 1978). Bread wheat is a

hexaploid species and constitutes about 90% of the wheat grown worldwide, 93% of the

wheat produced in the United States and 95% in Canada (Wrigley C.W., 2009). It has high

economic value because of the kernel hardness and high protein content. Common wheat

may be of either winter or spring growing habit and may have either red or white kernels

(Shellenberger, 1978). Besides of Triticum aestivum, Triticum durum is the second most

widely cultivated wheat. It belongs to tetraploid species and is used primarily for pasta

production. Triticum monococcum, Triticum dicoccum, and Triticum spelta are species of

wheat cultivated in limited quantities in the world (Wrigley C.W., 2009).

Figure 2.3.Wheat plant and wheat grain

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2.2.1. Wheat production

As shown in Table 2.4, the world production of wheat is very large and has been stable

between 1990 and 2010. According to the statistics of FAO, the major wheat production

comes from Asian, European and North American countries. More than 80% of the wheat

production is consumed within source countries and mainly as human food, while 20%

(more than 100 million tonnes) enters into international trade annually. For that reason,

wheat becomes the most-traded cereal in the world (Wrigley C.W., 2009).

Table 2.4. The production of wheat (Source FAO, 2012)

Year

Production (metric tonnes)

World Asian

countries

European

countries

American

countries

North American

countries

1990 592 310 517 203 062 087 233 143 757 127 159 034 106 392 000

2000 585 690 370 254 524 075 183 599 454 110 863 548 87 174 900

2010 650 881 002 292 441 446 201 149 388 112 690 549 83 269 400

2.2.2. Structure and composition of wheat

Figure 2.4 shows the cross section of a wheat grain with its main structural components.

Wheat grains are generally oval shape although different wheat species have grains that

range from almost spherical to long, narrow and flattened shapes. The grain is usually

between 5 and 9 mm in length and between 35 and 50 mg in weight. When the husk is

removed, the brown grain is exposed. It contains 2-3% germ, 13-17% bran and 80-85%

endosperm (Šramková et al., 2009).

11

Figure 2.4. Struture of wheat grain (from Encyclopaedia Britannica,

http://www.britannica.com)

Most of wheat constituents have important applications and nutritional values. The bran is

made up of several layers and acts as a protective covering. It is an important by-product in

milling processing. Wheat bran is rich in vitamins B, antioxidants, minerals and also a

source of fibre. It is used in feed-stock and adds fibre to foods, many recipes are available

today that use wheat bran. Wheat germ is an especially rich source of α- and β- tocopherols

and oil produced from it provides more vitamin E activity than most other oils (Piironen et

al., 2009). Wheat germ is added to baked goods and casseroles and boosts the nutritional

value of the food. In addition to vitamins and minerals, wheat germ provides us with

naturally occurring antioxidants. The major component of wheat is flour and starchy

endosperm and most bread are made with wheat flour, including many breads named for

the other grains they contain like most rye and oat breads (Ponte, 1978).

2.3. Sorghum (Sorghum bicolor L.)

Sorghum (Figure 2.5) is the fifth important crop in the world coming after wheat, rice,

maize and barley (FAO, 2012). It is a member of grass family and difficult to classify

because of its genetic variability. This cereal has different names like: milo, jowar, kafir

corn, guinea corn and cholam (Raiph, 2003).

12

Sorghum is used for several purposes. It provides a major source of food and feed in many

countries, especially Africa, the United States, Asia and Latin America. In the United

States, sorghum grain is used primarily for animal feed, but also is used in the food

industry. In Africa, sorghum is the fourth main source of energy after cassava, maize, yam

(Henley et al., 2010). Worldwide, haft of sorghum production is used for human

consumption. It is also used to produce biofuels like ethanol and co-productions. Sorghum

is a source of alcoholic beverages in many countries (Moench et al., 1970). Nowadays,

sorghum is known like a gluten-free food for people who have been the diagnosed with

celiac disease (Liu et al., 2012). Sorghum is also high in antioxidants and policosanols,

group of long chain alcohols, so it might help lower the risk of cancer, diabetes and heart

diseases (Henley, 2010).

Figure 2.5. Sorghum plant and sorghum grain

2.3.1 Sorghum production

Table 2.5 shows the production of sorghum in the world. The world production of sorghum

has been stable for 2 decades, about 56 million metric tons in 1990 and 57 million metric

tons in 2012. From 1990 to 2012, the production of sorghum in Africa increased from 11

million tons to 23 million tons while in Asia, it decreased a half, from 18 million tons to 9.5

million tons (FAO, 2012). The weather of Europe is not suitable for the growth of sorghum

and also the habit of consumers is not familiar with sorghum. The annual production in this

13

area is less than 1 million ton. In the United States, sorghum is widely used for livestock

feed and the production has been very stable from the year 1990 until now.

Table 2.5. The production of sorghum (Source FAO, 2012)

Year

Production (metric tons)

World Africa

countries

American

countries

Asian

countries

Europe

countries

1990 56 807 007 11 980 273 24 643 325 18 571 513 665 155

2000 55 856 128 18 412 620 23 244 861 11 317 149 762 047

2012 57 004 922 23 312 557 21 170 356 9 502 615 775 888

2.3.2 Structure and composition of sorghum.

Figure 2.6.Structure of sorghum grain (Earp.C.F et al., 2003)

Sorghum grain has different colors from pale yellow through various shades of red and

brown to a deep purple brown. Figure 2.6 shows the cross section of a sorghum grain. Like

most other cereals, sorghum grain has three major components: the pericarp (outer layer),

the endosperm (storage tissue) and the germ (embryo). The pericap, endosperm and germ

contribute for about 6, 84 and 10%, respectively, of the grain weight.

14

The pericarp is composed of three tissues: epicarp, mesocarp and endocarp (Earp and

Rooney, 1982). The epicarp is generally covered with a thin layer of wax. The endosperm

is the main part of the grain. It contains starch granules, protein, group B vitamin, and it is

also a good source of carbohydrates. The germ contains two parts: embryonic and

scutellum. Table 2.6 presents the composition of the different parts of sorghum grain. The

composition of sorghum is also different depending on the varieties and growing conditions

and it also varies during sorghum plant development.

Table 2.6. Composition of the whole kernel and its part for sorghum (moisture-free basis)

(Wall et Charles, 1970)

Components Whole

grain

Bran Horny

endosperm

Starchy

endosperm

Germ

Proportion of kernel (%)

Ash (%)

Ether extract (%)

Protein (%)

Crude fiber (%)

Nitrogen-free extract (%)

Starch (%)

100

1.89

3.47

13.99

1.93

78.72

68.52

5.5

3.07

4.33

7.08

15.36

70.16

1.60

54.7

0.56

0.15

15.11

0.69

83.49

72.24

28.7

0.71

0.28

8.91

0.81

89.29

82.5

11.1

9.46

19.92

20.84

9.11

40.67

1.53

15

2.4. Rice, wheat and sorghum waxes and their applications

2.4.1. Wax generalities: types and composition

Figure 2.7. Structure of cuticule (lipidlibrary.aocs.org)

Wax is a fat-like material which exists on the surface of plant material as well as on the

surface coating of insects or animal skin. According to the origin of wax, it can be

classified into four groups: animal and insect waxes (beeswax, spermaceti and lanolin);

vegetable wax (bayberry, carnauba, cane sugar, jojoba, sorghum, rice bran and wheat bran);

petroleum and mineral wax (paraffin, ceresin, petrolatum and microcrystalline) and

synthetic wax (polyethylene, carbowax, polarwax, syncrowax, acrawax and stearone) (Li.,

1999).

The compositions of wax vary from one to another based on their sources. They typically

consist of several components, including wax esters, wax acids, wax alcohols, and

hydrocarbons. For this reason, organic solvents are conventionally used to extract wax and

wax-like materiels. Among them, wax esters of long-chain fatty acids and long-chain fatty

16

alcohols are the major components which has interesting biological characteristics such as

to reduce cholosterols in humains (Christie W., 2011). The Table 2.7 is an example of

vegetable wax composition. It shows that long chain hydrophobe substances are present in

vegetable waxes. The carbon number varies greatly from 16 carbons for fatty acid and up to

70 carbons for esters (Luka et al., 2009).

Table 2. 7. The most common substance classes of cuticular waxes (Luka et al., 2009)

Substance class Chemical formula Range of chain lengths Major homologues

Acids CH3-(CH2)n-COOH C16-C32 C24, C26, C28

Aldehydes CH3-(CH2)n-CHO C22-C32 C26, C28, C30

Alcohols CH3-(CH2)n-CH2OH C22-C32 C26, C28, C30

Alkanes CH3-(CH2)n-CH3 C21-C35 C29, C31

Secondary

alcohols

CH3-(CH2)n-CHOH-

(CH2)n-CH3 C23-C33 C29, C31

Esters CH3-(CH2)n-COO-

(CH2)n-CH3 C36-C70 C40, C42, C44

Wax in grains is a type of vegetable (plant) wax and exists on the surface of grains. It is

believed to help grains to control water evaporation, to maintain moisture and to protect

grains from micro-organisms (Hwang et al., 2004). The amount of wax in grains depends

both on the type of grains and their variety (Imark and Dunford, 2005). Figure 2.7 shows us

that vegetable waxes are from two types of waxes: epicuticular waxes and intracuticular

waxes. Epicuticular waxes are present in the outer surface of the grain coating whereas

intracuticular waxes are inside the grain coating layer. Epicuticular wax is rich in alkanes

while intracuticular wax is rich in primary alcohols and triterpenoids. (Buschhaus et al.,

2007). Cereal waxes have not been much studied in the literature despite the large

worldwide production of these crops. They are usually obtained as by-products of the rice

and wheat bran oil industries or by-products of the milling processing industries. Their

properties are similar to carnauba wax and they could be used as a replacement of hard

vegetable wax (Huston, 1972; Jones J., 2007).

17

Based on its composition, rice bran wax is classified into soft and hard wax. Both wax

types are recovered from rice bran crude oil with melting points of 79.5oC (hard wax) and

74oC (soft wax). The hard wax consists of 64.5% fatty alcohols, 33.5% fatty acids and 2%

hydrocarbons. Soft wax includes 51.8% fatty alcohols, 46.2% fatty acids and hydrocarbons

(Orthoefer et al., 2005; Kim et al., 2008).

According to the research done by Vali et al. (2005), the major components of rice bran

wax are wax esters and they account for 93-94% of total wax esters with even carbon

numbers between C44 and C62. Saponification of wax esters will result in the formation of

C24-C40 fatty alcohols and C16-C24 fatty acids. Less than 7% of the total wax esters have odd

carbon numbers of C45-C61 (combination of C16-C24 fatty acids and C25- C39 fatty alcohols).

The compositions of even number fatty alcohols are triacontanol (C30) 24-27%;

dotriacotanol (C32) 17-20%; octacosanol (C28) 12-17%; hexacosanol (C26) 5-7%;

tetracosanol (C24) 2-5%. The major compositions of FA are lignoceric acid (24:0) : 60-67%

and behenic acids (22:0): 17-20%. These results are found to be similar to the studies done

by Liu et al. (2008) and Orthoefer et al. (2005).

The average composition of wheat bran wax is similar to that from rice wax. However, up

to now there are few reported studies about the composition of wheat bran wax (Ohnishi et

al., 1986). Thin-layer-chromatography (TCL) of waxy esters in wheat showed that the

contents of acylsterols, shorter alkylesters, hydrocarbons and longer alkylesters of the

neutral lipids are 7.4, 0.5, 0.4 and <0.1%, respectively. The ratio of alkanes, alkenes and

squalene is approximately 79:9:12. The main alcohols of the crude longer alkylesters are

tetracosanol (C24) (39.9%), hexacosanol (C26) (20.2%), docosanol (C22) (18.7%) and

octacosanol (C28) (9.1%) whereas fatty acids are palmitic (19.8%), arachidic (17.1%),

behenic (15.0%) and stearic (9.1%) (Ohnishi et al., 1986). Similarly, the research of

Dunford et al. (2009) states that the major components of long chain fatty alcohols in wheat

bran wax are docosanol (C22), tetracosanol (C24), hexacosanol (C26), and octacosanol (C28).

Hwang et al., 2002 reported that 0.16-0.3% wax could be extracted from sorghum grain

depending on the type of solvent used for extraction. The melting point of this extracted

18

wax varied from 77 to 85oC. They are composed of hydrocarbons, wax esters, aldehydes,

alcohols and acids with the long hydrocarbon chain. The composition of sorghum wax

varied according to the sources of Sorghum and extraction methods. Another research

reported that long chain lipids extracted by hot hexane and hot ethanol generated the yield

of 0.2-0.3% (w/w) and the extract contained policosanols 37-44%, aldehydes 44-55% and

acid 4-5% (Hwang et al., 2004). The main alcohols are octacosanol (C28) (43.7%),

triacontanol (40.9%) and hexacosanol (C26) (8.2%). Cai et al., 2013 also confirm that the

major components of sorghum wax were long chain fatty acid, primary alcohols, aldehydes

and alkanes. However, the yields of fatty acids, primary alcohols and aldehydes are

36.39%, 9.3%, 18.38% (w/w), respectively. Among them, compounds with 30 carbon in

length were largest part in the sorghum wax.

2.4.2. Cereal wax applications

Among active components in cereal wax, long chain aliphatic primary alcohols (C24-C34),

known as policosanols, are the important components in pharmaceutical industries.

Policosanols are popularly used as drugs. Originally, they are extracted from cane sugar,

yams and beeswax but today they are also found and extracted from whole grains,

especially in sorghum grains (Hwang et al., 2002; 2004; 2005), rice grains (Wang et al.,

2007; Cravotto et al., 2004; Vali et al., 2005; Liu et al., 2006) and wheat grains (Irmak et

al., 2005; 2007; Dunford et al., 2009). According to the research of Hargrove et al. (2004),

5-20mg/day of mixed fatty alcohols (C24-C34) can lower LSD-Cholesterol by 21-29% and

raise high-density lipoprotein HDL-Cholesterol by 8-15%. Another research showed that

the ingestion of policosanol 5mg/day for 8 weeks reduces serum total cholesterol by 12%

and LDL-Cholesterol levels by 17%. It also raises HDL-Cholesterol and improves

LDL/HDL ratios in those with elevated blood lipids (Jones J., 2007). Policosanols are

believed to be safe in animal studies and even in human studies (Jones J., 2007).

Many waxes such as white wax have been used for cosmetic and pharmaceutical industries.

Sabale et al. (2009) showed that ointment base made with rice bran wax had better

spreadability (the average spreadability was 16.8±2.4 g.mm-1

.s-1

) than the standard base

19

(average spreadability 10.13±1.66 g.mm-1

.s-1

). These authors indicated that ointment

prepared with rice bran wax base also showed better diffusibility than the one prepared with

standard base. They believed then that rice bran wax could be further used as an oleaginous

ointment base as far as their pharmaceutical properties are concerned in contrast to the

traditional available costly bases. In addition, the Food and Drug Administration (FDA)

allows rice bran wax to be directly added in foods and it is also considered as a safe

ingredient in cosmetics.

Cereal bran wax has been extensively applied in cosmetics preparation. They are used to

prepare lipsticks, creams and mascaras. They are believed to be able to efficiently remove

the anti-wrinkle, restructure the healthy skin and also make the skin smooth and soft. Wax

can form a protective film so it prevents the loss of skin moisture (Ito et al., 2003). In

addition, the cereal wax is similar with carnauba wax so it can be used in place of or with

carnauba wax in car, shoes and floor polishes (Lotchte-Watson et al., 1999)

2.5. Extraction methods of lipid

A large number of lipid extraction methods have been developed and described at

laboratory scales. Among them, soxhlet extraction method is one of the most commonly

used, in which organic solvents are used to solubilize lipids from solid materials. As the

lipids are hydrophobic compounds, they are usually extracted by hexane, benzene,

chloroform, light petroleum ether and acetone (Hwang et al., 2002). Up to now, Soxhlet

apparatus is widely used in worldwide laboratories and has become the standard and

reference method for most of lipid analysis in foods when hexane is used as the solvent.

However, the method has several disadvantages such as time-consuming operation, unsafe

and flammable solvents and unfriendly aspect of environment and especially hazardous

residues remaining in final products. In order to reduce the extraction time, derivative

methods of Soxhlet such as microwave-integrated or assisted Soxhlet and Soxtec have been

introduced. The main difference is to equip the Sohxlet system with a microwave and/or a

heating device as described by (Virot et al., 2007) but the extraction process principle

remains similar.

20

Another extraction method, known as supercritical fluid extraction, for food lipids has

recently and widely documented for many reasons. The extraction principle replies on the

unique ability of a substance to diffuse through solids like a gas, and dissolve materials like

a liquid. Carbon dioxide and water are the two commonly used substances to extract

hydrophobic and hydrophilic materials, respectively. Supercritical fluid extraction methods

using CO2 (SC-CO2) was used to extract many food-based and medicine-based sources

since CO2 can easily become gas at ambient condition (Lang et al., 2001). In addition,

Gomez et al. (1995) reported that SC-CO2 and Soxhlet extraction yielded similar recovery

of grape seed oil. With the distinct properties of low viscosity and high diffusivity of CO2,

it is believed to penetrate more quickly into porous solid food products and render mass

transfer more easily, resulting in the extraction process more efficiently (Sahena et al.,

2009). Moreover, thanks to the ease to change the properties of the supercritical fluids, via

minor changes in temperature or pressure, the extracted compounds may be remarkably

selected. For example, Song et al., (1992) claimed that a vidoline component present in the

leaves of Cathanranthus roseus was successfully isolated by SC-CO2 from more than 100

alkaloid compounds. Finally, the SC-CO2 is believed to be suitable for extracting thermally

labile compounds as the method is conducted at low temperature. SC-CO2 was used to

extract, for instance, the ginger oils with more protected natural compounds than the

distilling method at high temperature (Bartley and Foley, 1994). For those advantages of

SC-CO2 over extraction methods using solvent like hexane except for its requirement of

more sophisticated equipment, it is likely that SC-CO2 can be the chosen method to extract

high value compounds present in lipids e.g. compounds with medicinal activity.

Also, liquid CO2 has been used as a solvent to extract essential oils and lipids. This method

is different from the SC-CO2 at the fact that the liquid CO2 method is performed at lower

temperature and pressure than SC-CO2. In the liquid form, CO2 is reported to be a good

solvent to extract lipids (Sahena et al. 2009). For example, it was used by Spricigo et al.,

(1999) to extract lipids from nutmeg. According to those authors, this method was ideal to

fractionate the essential oils since the extraction operation was at mild condition (at

ambient temperature). Interestingly, no toxic residues remaining in final products were

found, and the process was environmentally friendly. The essential oil of black pepper and

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21

clove oil extracted by liquid CO2 were also published by Ferreira et al. (1993) and Guan et

al. (2007), respectively. They reported that liquid CO2 could efficiently extract essential oil

with high yield and up to 96% wt% in the case of black pepper oil. Therefore, liquid CO2 is

promising solvent to extract lipids or more precisely to fractionate essential oil from total

lipids of plant-based materials.

As lipids are present in all biological material including foods, many methods attempting to

extract more efficiently and intactly these lipids has been described and developed. Each

method possesses its own distinct advantages and disadvantages as well as specific

purposes (e.g selectivity or yield). Any novel the lipid extraction method would need to be

validated and compared to a reference method. In the food industry, Soxhlet extraction

method using the solvent n-hexane has been frequently used as the standard and reference

method.

2.6. Extraction methods of cereal waxes

Cereal wax is a type of lipid so the method extraction has the same principle with the

method extraction of lipid. Rice and wheat bran waxes are mostly obtained during bran oil

extraction in the dewaxing step of refining process. Other extraction methods from whole

grain cereals have also been reported. Because the surface waxes are very hydrophobic,

they may be extracted by non-polar solvents. The wheat grains were then dipped in

chloroform-methanol solution (2:1, v/v) for three times and the optimal ratio of grain and

solvent is 1:4 (w/v). Then, grains were immersed twice into three volumes of water-

saturated butanol. After that, the extracts were washed with water, the solvent was

evaporated and the mixture was dried to obtain the wax (Ohnishi et al., 1986). The result

shows that the yield of total lipids was 2.7% with the major components were acylsterols,

alkylesters, alkanes, alkenes, alcohols and hydrocarbons. Another method was described by

Sariava (1995), in which petroleum ether was used to extract wax from the whole sorghum

kernel. After filtration to remove the grain from solvent, this solution was stored at low

temperature and the precipitated wax was collected. Similarly, the report of Hwang et al.

(2002) showed that the sorghum wax could be extracted by refluxing them in organic

22

solvents (hexane, chloroform, light petroleum ether) for an extended time. The wax was

then separated from the mixture by two ways. The first one comprises evaporating the

solvent from the mixture containing waxes, mixing the wax extract with other polar

solvents (acetone or alcohol) followed by filtration to separate the precipitated wax. The

second consisted in crystallizing the waxes by incubation of the extraction mixture at -

18oC, then the crystallized wax were collected by filtration. Hwang et al. (2004 and 2005)

applied the previous described method to extract wax from selected cereals of Korean

origin (grain sorghum, brown rice, purple rice, wheat and maize). These grains were

refluxed in hexane for 30 minutes, the ratio of hexane and grains are 1:1 (v/w). These

mixtures were then filtered at low temperature and precipitated wax was obtained. The wax

yield varied from 0.2-0.3% depending on the type of grain and the solvents. The

compositions of the wax obtained by this Hwang are acids, aldehydes and alcohols.

Although solvent wax extraction methods are simple and economical, requiring no complex

equipment, they are not considered to be environmental-friendly, with potential harm to

human health due to solvent residues.

2.7. Liquid nitrogen and its potentials for wax extraction

2.7.1. Properties of liquid nitrogen

Nitrogen is a non-toxic, colorless, odorless and tasteless gas that constitutes 78% of the air

atmosphere. It is inert, nonflammable, and it does not support combustion. At -195.8oC

(boiling point) and atmospheric pressure, nitrogen becomes a colorless liquid. The liquid to

gas expansion ratio of nitrogen is 1:694 at 20 °C so liquid nitrogen boils to fill a volume

with nitrogen gas very quickly. The transition between liquid phase and solid phase occurs

at -210oC (melting point) and atmospheric pressure. In industry, liquid nitrogen is produced

in large quantities by fractional distillation of liquid air (Afsset, 2008). Table 2.8 shows

several properties of nitrogen (Afsset, 2008).

http://en.wikipedia.org/wiki/Fractional_distillation

http://en.wikipedia.org/wiki/Liquid_air

23

Table 2.8. Properties of nitrogen (Source Afsset, 2008)

Chemical symbol N2

Molecular Weight (g/mol) 28.01

Boiling point -195.8oC

Melting point -210oC

Gas density 1.2506 kg/m3(P=1.013 bar and T= 0

oC)

Liquid density 808.607 kg/m3(P=1.013 bar and T= -195.8

oC)

Solubility in water 0.0234 vol/vol (P=1.013 bar and 0 °C)

Triple point T =-210oC and P=12.5 kPa

Critical point T= -146.9oC; P=3399 kPa

2.7.2. Potential of wax extraction by liquid nitrogen

A few studies revealed the possibility of using liquid nitrogen to extract waxes from fruits.

For example, the research done by Ketata et al. (2012) showed that three 8 and 10 seconds

immersions of whole highbush and lowbush blueberries in liquid nitrogen increased their

coefficients of water loss to 55.39%, 49.23% and the coefficient of sugar gain to 63.46%

and 68.81% during further osmotic dehydration, respectively, when compared to non-

treated berries. Observed by scanning electron microscopy, their epicutilar wax seemed to

be gradually disappearing along with the increase in the number of immersion cycles.

After three cycles, the thickness of epidermis decreased more than 80% for highbush

blueberries and 55% for lowbush blueberries. Similarly, drying times for different drying

methods (vacuum, freeze and convective drying) are markedly shortened for whole

blueberries, seabuckthorn fruits and green grapes pretreated with liquid nitrogen, when

compared to non-treated samples (Thromas et al., 2010; 2011). The thickness of the

epidermis of the fruits (determined by optical microscopy) decreased 38% for seabuckthorn

fruits and 22% for green grapes after 5 immersion cycles in liquid nitrogen. Also, dewaxing

of the fruit surface was observed by scanning electronic microscopy. These reported

findings strongly support the fact that liquid nitrogen could be used to extract waxes from

other vegetable surfaces like cereal grains.

25

CHAPTER 3. HYPOTHESIS AND OBJECTIVES

3.1. Hypothesis

Wax from cereal grains (rice, wheat and sorghum) can be extracted by cyclic immersions of

different time intervals in liquid nitrogen.

3.2. Objectives

3.2.1 General objective

To assess the potential of a novel „green‟ process, by use of liquid nitrogen, for rice,

sorghum and wheat wax extraction as an alternative to traditional extraction methods, by n-

hexane.

3.2.2 Specific objectives

- To determine the optimal conditions (duration, number of immersion cycles) for

extracting the wax of rice, sorghum and wheat grains with liquid nitrogen.

- To compare the impact of the two extraction methods (liquid nitrogen and n-hexane) on

the surface of grains by scanning electron microscope (SEM).

- To analyse the composition of extracted rice, sorghum and wheat waxes.

- To evaluate the effectiveness of liquid nitrogen extraction in comparison to n-hexane

extraction in terms of yield of extracted wax and composition.

27

CHAPTER 4. MATERIALS AND METHODS

4.1. Materials

Brown rice (Oryza sativa L.) was bought from Carl Garrich Lone Pine Enterprises, United

State. Sorghum (Sorghum bicolor L.) and wheat (Triticum aestivum) were purchased from

Farinart, Quebec, Canada. These cereals were dried at 45oC in a ventilated oven, Fisher

338F, Canada for 24 hours to about 10% of moisture (Hwang et al., 2002) and stored in

plastic bags under vacuum conditions until used. Liquid nitrogen was obtained from

Praxair, Canada. N-hexane and Whatman paper were bought from Fisher Scientific,

Canada. (Trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane and standard

pentadecanoic acids (C15) were purchased from Sigma-Alrich Co., st. Louis, USA.

4.2. Methodology

Figure 4.1 shows a schematic diagram of the proposed methodology for this research.

Detailed methods and further information on each step follows.


28

Grains (rice, sorghum or wheat)

Drying to about 10% moisture

Storing the grains in vacuum condition

Immersion in liquid nitrogen Extraction by hexane (Cycles from 1 to 4 and 0.5 to 10 minutes) (5, 15, 30, 45, 60, 90 and 120 minutes)

Liquid nitrogen removal Precipitation of wax at -18oC

Crude waxes Filtering with Whatman paper No 42

Weighing Drying at temperature ambient

Storage at -18oC Weighing

Storage at -18oC

Wax composition analysis

Figure 4.1. Proposed methodology, liquid nitrogen extraction on the left and n-hexane on

the right

29

4.2.1. Determination of moisture content

Moisture content of the grains was determined according to the standard method in an oven

(AOAC, 1980). The samples were first weighed (W1) with a Mettler Toledo balance

(Model AB104-S, Greinfesee, Switzerland) and then placed in a vacuum oven (Fisher

scientific 281A) in the presence of P2O5. The vacuum oven was set at 50oC under gauge

pressure of 25 mmHg. After being dried to constant weight, they were transferred to

desiccators for 15 minutes to cool down to the room temperature. Finally, the dried samples

were rapidly weighed (W2) again, and their moisture content was calculated by the formula

(4.1).

( )

(4.1)

Where: MC (%): Moisture content, wet basis (%)

W1: Initial grain mass (kg)

W2: Dried grain mass (kg)

4.2.2. Wax extraction by liquid nitrogen

Wax extraction was carried out according to the figures 4.2 and 4.3 which illustrate the

immersion process of the grains (rice, sorghum and wheat) in liquid nitrogen. 100 g of

grains in a metal strainer was submerged into 500 ml of liquid nitrogen (Figure 4.2) with

various durations of immersion (from 0.5 to 10 minnutes) and numbers of cycles (ranging

from one to four). One cycle is defined as the period where the samples were in the liquid

nitrogen (Timm) and out of liquid nitrogen (Trest) (Figure 4.3). After each immersion in

liquid nitrogen with the temperature -195.8oC (TliqN2), the samples were retained for one

minute at room temperature (Tamb). Once the nitrogen presence in treated samples was

extensively evaporated, the amount of extracted wax was determined by weighing the

aluminum foils wrapping the container before and after the extraction (Figure 4.2). The

wax were collected and stored at -18oC until analyzed.

30

When the rest time (Trest) was studied, the grains were immersed for 0.5 to 10 minutes in

liquid nitrogen and followed by 1, 3, 5 minutes in ambient temperature (Tamb).

The experiment was triplicated for each cycle and each immersion duration.

Figure 4.2. Description of wax extraction in liquid nitrogen.

Figure 4.3. Illustration of immersion process in liquid nitrogen

Tamb

TliqN2

0 Tcycle

Trest Timm

Timposed 1 cycle 2 cycles

31

The extraction time was calculated by the equation (4.2)

( ) (4.2)

Where:

Ncycle: The number of immersion cycle of grains in liquid nitrogen.

Timm: Immersion time (minute).

Trest: The length of time when treated samples remain at ambient temperature until they are

subject to the second treatment (minute).

Text: Extraction time (minute)

4.2.3. Wax extraction by n-hexane

A conventional solvent-based wax extraction method was conducted for comparison

purposes. We used the method described by Hwang et al. (2005) for wax extraction of

Korean cereal grains with some modifications. 50 g of grain was mixed with n-hexane in a

round flask, placed in a device with reflux system (Figure 4.4) and extracted for 5, 15, 30,

45, 60, 90 and 120 minutes. The proportion of n-hexane and the sample was 2:1 (v:w), and

the temperature in the extraction chamber was fixed at 68-70oC, corresponding to the

boiling point of n-hexane at the atmospheric pressure. Once the extraction was finished, the

bottom flask containing extracted wax was placed in a freezer at -18oC for about 8 hours.

Then, it was filtered through a Whatman No. 42 paper filter under vacuum to collect the

wax. The wax was then dried under the hood at room temperature for 24 hours to a constant

weight or all the solvent was evaporated. The amount of crude wax was measured and

stored at -18oC for the future analysis. Three repetitions for each extraction time were

conducted.

32

Figure 4.4.Illustration of reflux system used for wax extraction by n-hexane

(www.chem.wisc.edu)

4.2.4. Calculation of wax yield

The amount of wax for each extraction was calculated by the following formula (4.3)

(Watson et al., 1999):

( ) ( )

( )

( ) (4.3)

MC (%): percent moisture content of the grains, as determined in point 4.2.1.

4.2.5. Examination of wax by scanning electron microscope (SEM)

The fresh and treated grains of cereals (rice, sorghum and wheat) for wax extraction by n-

hexane and liquid nitrogen were placed onto the observation support of the scanning

electron microscope (JEOL, JSM6360LV, Tokyo, Japan) and metalized with palladium in

an argon plasma chamber. The grain surfaces were then captured by the microscope under

the accelerating potential of 15kV and the magnification by 3000 times.

33

4.2.6. Analysis of wax composition

The wax composition was analyzed by Gas chromatography- mass spectrometry (GC-MS)

for qualitative analysis and Gas chromatography- Flame ionization detector (GC-FID) for

quantitative analysis. Preparation of samples was performed as previously described by

Athukorala et al. (2010).

The wax (minimum of 3 mg) obtained from n-hexane and liquid nitrogen extractions was

trimethylsilylated with 200 µl of bis-(trimethylsilyl)-trifluoroacetamide having 1%

trimethylchlorosilane (TMCS) and 100 µl of toluene, at 75oC during 30 minutes. The

reaction mixture was allowed to cool down to room remperature. Then it was re-dissolved

in toluene for analysis. GC-MS analysis was then conducted with an Agilent 6890/5973

GC-MS network gas system (Agilent technologies, Wilmington, DE) equipped with a

splitless injector and a network mass selection detector. The high temperature capillary

column employed was a DB-17HT (30 m x 0.25 mm I.D., 0.15 µl film thicknesses, J&W

Scientific, Folsom, CA). Helium was used as the carrier gas (1.3 ml/min). The temperature

of the injector was 300oC. The initial oven temperature of 50

oC (1 minute) was increased at

10oC/min to 280

oC, then at 5

oC/min to 350

oC (hold at this temperature for 10 minutes). The

Agilent 5973 quadrupole mass spectra was operated in the electron ionization (EI) mode at

70 eV, a source temperature of 280oC, quadrupole at 150

oC, in the scan range m/z 42 to

550. Data were collected with the Agilent enhanced Chemstation software (standard MSD

version) and search against the NIST (v.02) and Wiley (v.138) libraries (Palisade Corp.,

Newfield, NY). Compounds were identified by comparing their spectra with the library and

the spectra of authentic standards.

The standard acid pentadecanoic C15 was used to quantify the composition extracted. Wax

samples were exactly prepared as described above for GC-MS analysis except that 0.125

g of acid pentadecanoic was added into each l of the analysing samples. We used the

system Gas chromatography- Flame ionization detector (GC-FID) to analyse these samples.

Hydrogen was used instead of helium with a split injector. The quantity and percentage of

34

chemical components was calculated based on the quantity of the standard C15 (Equation

4.4 and 4.5).

(4.4)

Where:

A: Area of a compound in the peaks GC-FID

ASD: Area of pentadecanoic acid (C15) in the peaks GC-FID

Wcompound: Weight of a compound (µg)

(4.5)

Where:

P: Concentration of a compound in the wax (%)

Wcompound: Weight of a compound (µg)

Wwax: Weight of wax (µg)

4.3. Statistical analysis

The experiments were repeated three times and the data were analyzed using the analysis of

variance (ANOVA). Significant differences among treatment means and comparison of the

efficiency of two extraction methods were analyzed by the test Duncan's multiple-rang test

(P<0.05). The data were analyzed by SAS statistical software version 9.2 (SAS Institute,

USA).

35

CHAPTER 5. RESULTS AND DISCUSSION

5.1. Extraction of cereal waxes with n-hexane

This experiment was conducted by refluxing n-hexane through the cereal grains (rice,

sorghum and wheat) at normal boiling point (approx.70oC) during times ranging from 5 to

120 minutes. The kinetics of wax extraction is presented in Figure 5.1. This Figure shows a

typical extraction kinetics curve, with an overall exponential trend approaching to an

asymptotic limit. Equilibrium is reached when the concentration in the extract becomes

independent of the extraction time and the extraction curve levels off asymptotically

reaching the upper limit. This curve can be represented mathematically by the following

expression (Equation 5.1):

t b

tW (%)Wax max

(5.1)

Where Wmax and b, are kinetics constants; Wmax could be interpreted as the equilibrium yield

of extracted wax and b, as a „response time‟ for extraction. These two constants can be

determined by fitting Eqn. (5.1) to experimental data through non-linear regression with

Sigmaplot v. 11. Results of the fitted parameters are shown in Table 5.1, and predicted

curves are presented in Figure 5.1 together with experimental data. Eqn. (5.1) represents

conveniently experimental data as can be observed from Figure 5.1 as well as from the high

r2 values (Table 5.1).

36

Table 5.1. Fitted parameters for kinetics constants Wmax and b from Eqn. (5.1) – hexane

extraction (r2 is the coefficient of determination)

Product Wmax (%) b (min) r2

Sorghum 0.298 2.772 0.984

Rice 0.196 1.446 0.996

Wheat 0.126 11.620 0.987

time (minutes)

0 20 40 60 80 100 120 140

Wa

x (

%)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Sorghum

Rice

Wheat

Figure 5.1. Extraction kinetics of sorghum, rice and wheat wax by reflux extraction using

n-hexane.

37

From Figure 5.1, it can be observed that sorghum is the grain that presents the highest wax

amount to be extracted, followed by rice and finally, wheat. This is in agreement with

Hwang et al., (2004&2005). Also, extraction kinetics for sorghum and wheat indicate that

their wax is readily available for extraction on the surface and macro pores since their

curves have a quick increase in the beginning. Wheat wax extraction curve, on the other

hand, leads to the impression that equilibrium cannot be properly reached, leveling off

more slowly than in the other two cases, like if wax distribution in this grain was deposited

in sites of markedly different sizes.

For sorghum, the optimal extraction time was found to be 45 minutes, with equilibrium

yield corresponding to 0.298% (Wmax from Table 5.1). This is in agreement with the wax

amount extracted by Hwang et al. (2002 & 2004), which is 0.16-0.3% during 30 minutes

hexane reflux, but is higher than the results found by Lotchte-Watson and Weller (1999),

which for the same reflux duration they obtained 0.16-0.2% of wax.

For rice, equilibrium of wax extraction was reached at times higher than 60 minutes with a

maximum yield of 0.196% (Wmax from Table 5.1). This yield is higher than the one

obtained by Hwang et al. (2004) and Kim et al., (2008) which were 0.033% and 0.025-

0.056%, respectively for waxes extracted from rice with n-hexane.

For wheat wax, its maximum yield is 0.126% (Wmax from Table 5.1) after equilibrium at 30

minutes of extraction. This amount is higher than the yield found by Hwang et al. (2004)

(0.01% of wax in 30 minutes reflux with n-hexane).

The quantity of wax obtained depends on the classes, the maturity of plant and the region

(Bianchi et al., 1978; Imark et al., 2007; Asikin et al., 2012). In the research of Hwang et

al, (2004, 2005) they used the Korean cereals (purple color sorghum, soft red winter wheat

and brown rice japonica type) which are different from the American grains used in this

study (bicolor Sorghum, hard red winter wheat and brown short-grain rice). This may

explain the higher yield of waxes is this study compared to Korean cereals (Hwang et al.,

2004).

38

5.2. Extraction of cereal waxes with liquid nitrogen

Figure 5.2 shows the results of immersion in liquid nitrogen (1 cycle) on the extraction of

waxes from sorghum, rice and wheat. As can be observed from this Figure, similarly as was

found with hexane extractions (Figure 5.1), also with liquid nitrogen the amount of wax

extracted from sorghum was higher than for rice and the lowest amount correspond to

wheat.

39

time (minutes)

0 2 4 6 8 10

wa

x (

%)

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Sorghum

Rice

Wheat

Figure 5.2. Extraction kinetics of sorghum, rice and wheat wax by immersion in liquid

nitrogen.

40

The rapid extraction trend shown in Figure 5.2, may suggest that extraction with liquid

nitrogen could only extract readily available wax-like materials, without a deep penetration

into the cutin.

Table 5.2 presents parameters Wmax and b from the fitting of Eqn (5.1) to experimental data

on liquid nitrogen extraction. Predicted curves are presented in Figure 5.2 together with

experimental data. The model given by Eqn. (5.1) represents conveniently experimental

data on liquid nitrogen extraction as can be observed from Figure 5.2 as well as from the

high r2 values (Table 5.2).

Table 5.2. Fitted parameters for kinetics constants Wmax and b from Eqn. (5.1) – liquid

nitrogen extraction (r2 is the coefficient of determination)

Product Wmax (%) b (min) r2

Sorghum 0.032 0.329 0.995

Rice 0.022 0.479 0.959

Wheat 0.015 0.181 0.977

For sorghum, time to reach equilibrium after immersion in liquid nitrogen (1 cycle) was 4

minutes with an equilibrium yield of 0.032%, for rice is 8 minutes with 0.022% and for

wheat, 3 minutes with 0.015% (Figure 5.12 and Table 5.2). Comparing equilibrium yields

obtained with 1 cycle immersion in liquid nitrogen (Table 5.2) to those with hexane

extraction (Table 5.1), it can be noted that wax yields obtained from 1 immersion in liquid

nitrogen extraction are 8 to 9 times lower than hexane extraction. However, the extraction

response time b is 3 to 64 times higher for liquid nitrogen.

The impact of cycle numbers and rest time of liquid nitrogen extraction were studied to

search for an increase in wax extraction yields. It is also because the cereal grains were

broken when immersed longer than 10 minutes in liquid nitrogen at -195.8oC. To increase

the immersion time of the grains we cycled grain immersions; an immersion cycle includes

grain immersion in liquid nitrogen, followed by rest time. In addition, changes in

41

temperature between the temperature of liquid nitrogen and the ambient temperature might

provoke thermal shock on the surface of the grain, making wax be extracted more easily.

5.3. Impact of number of immersion cycles and rest time with liquid

nitrogen

5.3.1. Sorghum

Among cereal waxes, the wax of sorghum is the most studied. The quantity of wax in

sorghum is higher than in rice and wheat (Hwang et al., 2002 & 2004). Figure 5.3 presents

the results of wax yield after immersion of sorghum in liquid nitrogen during 0.5 to 10

minutes, 1 minute rest time. The highest wax yield obtained was 0.033% with 3 cycles and

6 minutes of immersion (Text = 20 minutes). However, this result is not significantly

different from the yield obtained for 1 cycle and 4 minutes immersion (0.032%, Table 5.2).

The impact of rest time was not significant (5% level) on wax extraction yield from

sorghum. Figure 5.4 shows an example of results for 2 cycles and the rest time varying

from 1 to 5 minutes.

In conclusion, optimal parameters for wax extraction from sorghum with liquid nitrogen are

1 cycle and 4 minutes immersion (wax yield = 0.032%, Text=4 minutes, table 5.2). As

explained earlier, this percentage is only about 10% of the yield obtained with n-hexane

extraction, but the time extraction is shorter.

5.3.2 Rice

Figures 5.5 and 5.6 show the impact of immersion cycles on wax extraction yield for 1 and

3 minutes rest time, respectively. In general, it was observed that increasing the number of

cycles helps increasing wax yield, but only at lower immersion times. On the other hand, at

equilibrium, the impact of cycle numbers was not significant.

42

Time required to reach the equilibrium with one cycle immersion was found to be 8

minutes. Five (5) minutes immersion time was required to reach equilibrium with 2 and 3

cycles (thus, total extraction time is = 13 minutes). The equilibrium extraction yield in the

case of 2 or 3 cycles was not significantly different.

43

Figure 5.3. Impact of number of immersion cycles on wax yield (%) for sorghum (1 minute

rest time)

Figure 5.4. Impact of rest time on wax yield (%) for sorghum (2 cycles with 1, 3 or 5

minutes rest time)

44

Figure 5.5 . Rice wax yield from extraction with liquid nitrogen (1 minute rest time)

Figure 5.6. Rice wax yield from extraction with liquid nitrogen (3 minutes rest time)

45

The highest wax yield obtained was 0.024% (2 cycles 9 minutes with 1 rest minute).

However, the yield in the case of 1 cycle 8 minutes is not significantly different. Thus, it

can be concluded that the optimal liquid nitrogen conditions for wax extraction from rice

are 1 cycle and 8 minutes immersion giving 0.022% wax yield. This yield is only 11% of

the yield obtained by n-hexane.

Rest time was found to have a significant increase on rice wax yield. Figure 5.7 shows the

yields of extracted wax with liquid nitrogen having a rest time of 1, 3 and 5 minutes (2

cycles). There is a significant increase in yield between 3 and 5 minutes resting time with

respect to 1 minute. However, there is not a significant increase between 3 and 5 minutes

resting times.

From Figure 5.7 it can be seen that the treatments with 2 cycles and 4 minutes immersion

(both 3 and 5 minutes of rest time) were at equilibrium. The maximum yield obtained was

0.026% with 2 cycles 4 minutes (3 minutes rest).

Other experiments done with 3 minutes rest time but 3 cycles (instead of two), showed

similar results than those obtained for 2 cycles. Thus, it can be concluded that the optimal

time for extraction with liquid nitrogen is 11 minutes (2 cycles 4 minutes with 3 minutes of

rest time (Text=11 minutes) giving a maximum yield of 0.026%. This yield is only 13%

when we compare to the wax extracted by n-hexane. However, the extraction time by liquid

nitrogen is only 11 minutes while the time extraction by n-hexane is 60 minutes.

46

Figure 5.7. Rice wax yield from extraction with liquid nitrogen (2 cycles with 1, 3 or 5

minutes rest time)

47

5.3.3 Wheat

Figures 5.8 and 5.9 present the percentages of wheat wax extracted by liquid nitrogen with

1 minute and 3 minutes rest time, respectively, and several cycles. As can be seen from

these results, number of cycles has a significant impact of wax yield, especially 3 and 4

cycles with respect to 1 and 2 in the case of 1 minute rest time (Figure 5.8), and 2 and 3

cycles with respect to 1 cycle in the case of 3 minutes rest time (Figure 5.9). Wax yield

increases up to 2/3 of their value in the optimal conditions.

The maximum yield is 0.026% with 3 cycles, 9 minutes immersion time and 1 minute rest

time (Text=30 minutes). However, this result is not significantly different from the one of 3

cycles, 8 minutes (26 minutes) and 4 cycles 5 minutes (23 minutes).

The impact of rest time on wax yield was also evaluated for wheat. Figure 5.20 presents

these results found for 2 cycles. As can be seen, there is not a significant difference

between wax yield obtained with 3 and 5 minutes rest time, but there is between 3 - 5

minutes and 1 minute rest time.

The optimal yield of wheat wax extracted with liquid nitrogen was found to be 0.025% for

2 cycles 5 minutes immersion and 3 minutes rest time (total extraction time 13 minutes).

This yield is 25% of the wheat wax yield extracted by n-hexane reflux.

48

Figure 5.8. Wheat wax yield from extraction with liquid nitrogen (1 minute rest)

Figure 5.9. Wheat wax yield from extraction with liquid nitrogen (3 minute rest)

49

Figure 5. 10 . Wheat wax yield from extraction with liquid nitrogen (2 cycles with 1, 3 or 5

minutes rest time)

50

5.4. Comparison between two methods of wax extraction

Table 5.3 shows the optimal extraction yield and equilibrium time for the two methods

studied in this work. Yields and extraction times are different depending on the grains

under study. The wax yield is 5 to 9.3 times higher when using n-hexane for extraction.

However, extraction time with this solvent is more than 2.3 to 11.25 times the one required

to perform liquid nitrogen extraction.

In general, it can be concluded that extraction with n-hexane reflux gives higher yields but

it takes longer than when using liquid nitrogen.

Table 5.3. Comparison of liquid nitrogen and n-hexane extraction methods

5.5. Color of sorghum, rice and wheat waxes

The color of extracted wax is shown in Figure 5.11. As can be seen from this Figure, the

color of wax extracted with n-hexane varies according to the type of grain. These colors are

between red and pale green. These colors are in accordance to published information by

Vali et al. (2005) and Hwang et al. (2002).

Grains

Yield of wax extracted

(g wax/ 100 g grain)

Y1/Y2

Time extraction (minutes)

T1/T2 n-hexane

(Y1)

Liquid

nitrogen

(Y2)

n-hexane

(T1)

Liquid

nitrogen

(T2)

Rice 0.196 0.026 7.5 60 11 5.5

Sorghum 0.298 0.032 9.3 45 4 11.25

Wheat 0.126 0.025 5.0 30 13 2.3

51

Figure 5.11. Wax extracted with n-hexane: sorghum (a), wheat (b), rice (c). Sorghum wax

extracted by liquid nitrogen (d)

(a)

(c) (d)

(b)

52

However, wax extracted by liquid nitrogen was always grey-brown, independent from the

grain under study. This observation pointed out that possibly the compounds extracted by

these two methods could be different. Liquid nitrogen might be more appropriate to extract

only surface epicuticular waxes, whereas n-hexane may extract both epicuticular and

intracuticular waxes, in addition to other colored co-products. The wax colors could explain

why the yields are much higher with n-hexane extraction method. Thus, it could be

interesting to visualize how the surface of grains treated by these two methods changes

after extraction in order to support the fact that liquid nitrogen could extract only

epicuticular waxes.

5.6. Scanning electron microscopy (SEM)

Scanning electron microscopy was used to capture the changes accounted in the surface of

sorghum, rice and wheat grains after extraction with n-hexane and liquid nitrogen, and to

compare them to the initial fresh state.

Figures 5.12 to 5.14 illustrate the surface of sorghum grain, rice grain and wheat grain,

respectively. It can be observed from Figures 5.12 (a), 5.13 (a) and 5.14 (a) that, in fresh

state, sorghum grain has more wax on its surface when compared to rice and wheat,

respectively. This is in agreement with previously presented results for n-hexane extraction

as well as those by Hwang et al. (2004).

Prior to treatments, a considerable amount of wax are present on the surface of the grain

Figure 5.12 to 5.13 (a). However, after the extraction, the surface of grains present lower

amounts of wax for both methods, n-hexane and liquid nitrogen (Figures 5.12 to 5.13 (b),

(c), respectively). Comparing the impact of extraction methods on the surfaces of grains, it

can be concluded that liquid nitrogen can extract most of the surface wax (epicuticular

waxes), however n-hexane presents more penetration into the cuticle (intracuticular waxes).

This result is in agreement with the higher wax yield found previously in the case of

sorghum, rice or wheat.

53

Figure 5.12. Surface visualization by SEM of the sorghum grain: fresh (a), treated by n-

hexane (b) and treated by liquid nitrogen (c)

(b)

(c)

(a)

54

Figure 5.13. Surface visualization by SEM of the rice grain: fresh (a), treated by n-hexane

(b) and treated by liquid nitrogen (c)

(b)

(c)

(a)

55

Figure 5.14. Surface visualization by SEM of the wheat grain: fresh (a), treated by n-

hexane (b) and treated by liquid nitrogen (c)

(a)

(b)

(c)

56

5.7. Composition of waxes - Gas chromatography

Wax composition was analyzed as described by Athukorala et al. (2010). Standard acid

pentadecanoic C15 was used to determine quantitatively the composition of extracted wax.

Figures 5.15 to 5.20 show the gas chromatography-mass spectrometry (GC-MS)

chromatograms of waxes from rice, sorghum and wheat, extracted with n-hexane and liquid

nitrogen, respectively. The main composition of wax in these chromatograms consists in

acids, alcohols, alkanes and sterols. Wheat and rice waxes are abundant in acids while

sorghum wax is rich in alcohols. Some peaks, however, could not be identified because of

the lack of compound information of the system library. In agreement to the findings in the

present work, Athukorala et al. (2009, 2010) and Hwang et al. (2004) reported that the

main composition of vegetable wax is formed of acids, alcohols, alkanes and sterols. In

contrast, Bianchi et al. (1979), showed the presence of aldehydes in plant waxes. In our

result, aldehydes could not be determined in the waxes, because of the insufficient

information in the library. Hwang et al. (2002) also was not able to determine aldehydes in

the compositions of waxes extracted from sorghum grain. In many previous studies, esters

were noticed in large quantities in grain waxes, but were not found in our results. There

might be two reasons for this discrepancy: one could be the lack of information in the GC-

MS system library as stated earlier, and the other could be related to the range of retention

times for these analysis, which could be out of the one used in this work due to low

volatility of these compounds. After 40 minutes in the retention time, some peaks appeared

but they could not being completed due to the insufficient duration of the present tests.

57

Figure 5.15. GC-MS Chromatogram of rice wax extracted by n-hexane

Figure 5.16.GC-MS Chromatogram of rice wax extracted by liquid nitrogen

Time (min)

Time (min)

58

Figure 5. 17. GC-MS Chromatogram of sorghum wax extracted by n-hexane

Figure 5.18. GC-MS Chromatogram of sorghum wax extracted by liquid nitrogen

Time (min)

Time (min)

59

Figure 5.19. GC-MS Chromatogram of wheat wax extracted by n-hexane

Figure 5.20. GC-MS Chromatogram of wheat wax extracted by liquid nitrogen

Time (min)

Time (min)

60

After determining some of the compounds present in the waxes, the system Gas

chromatography-Flame ionization detector (GC-FID) was used to quantity the amount of

these compounds in the samples (using the same column, same conditions as for the GC-

MS and with changing a split injector and the detector to an FID). The amount of each

chemical compound was calculated as explained in Materials and Methods by comparing

them with the quantity of the standard C15 (Equation 4.4 and 4.5). These chromatograms are

illustrated in Figures 5.21-5.26. As can be seen, chromatograms for each extraction method

are similar in terms of compound type; however the abundance is generally much higher

for the extraction with n-hexane than with liquid nitrogen. However, it should be pointed

out that many other compounds seem to be present at higher retention times, which could

not be determined in this study.

Table 5.4; 5.5 and 5.6 compile the concentration (gram compound/100 grams wax) of

compounds detected with this study for each extraction method, together with the ratio of

concentrations between waxes extracted with hexane versus liquid nitrogen extraction. If

the R coefficient is higher than one, it indicates that n-hexane extracted higher amounts

than liquid nitrogen. In Table 5.4; 5.5 and 5.6 are marked in grey the compounds that were

extracted in higher quantities by liquid nitrogen.

For sorghum, wax extracted by n-hexane is abundant in policosanols (octacosanol-

11,97g/100g wax extracted, triacotanol-12.32 g/100g wax extracted) and triacontanediol-

13.21 g/100g wax extracted. Hwang et al. (2004) also showed that octacosanol and

triacontanol were the highest amount among the primary alcohols extracted by n-hexane

(70oC, 30 min). The major alkanes found are hexacosane, tetracosane and nonacosane.

Among them, hexacosane is the most abundant with 2.19 g/100g extracted. Fatty acids

from C16 to C18 are also present in the wax with high quantity. Samples extracted by liquid

nitrogen show the similar chemical compositions but the quantity is much less. The

presence of octacosanol, triacotanol, triacontanediol is about 1/3 when compared with the

method using n-hexane. (octacosanol- 3,3 g/100g wax, triacotanol- 3,0g/100g wax,

triacontanediol- 3.91 g/100g wax). Sorghum wax compounds extracted with liquid nitrogen

were only higher for eicosanoic acid and stigmasterol.

61

Figure 5.21. GC-FID Chromatogram of rice wax extracted by n-hexane

Figure 5.22. GC-FID Chromatogram of sorghum wax extracted by liquid nitrogen

62

Figure 5.23. GC-FID Chromatogram of sorghum wax extracted by n-hexane

Figure 5.24. GC-FID Chromatogram of sorghum wax extracted by liquid nitrogen

63

Figure 5.25. GC-FID Chromatogram of wheat wax extracted by n-hexane

Figure 5.26. GC-FID Chromatogram of wheat wax extracted by liquid nitrogen

64

Table 5.4. Classes of compounds present in the rice wax extracted with their retention time.


with n-hexane was zero.

Class of

compounds Name of compounds

Retention

time (min)

Gram compound/ 100gram wax

Rice Wax

n-

hexane Liquid N2 Rrice

Standard Pentadecanoic acid 15.51 0 0 ---

Acids

Hexadecanoic acid 16.42 4.78 1.48 3.22

Octadecanoic acid 18.16 8.70 1.83 4.75

Octadecadienoic acid 18.33 4.23 0.36 11.75

Tricosanic acid 21.59 0.03 0.02 1.5

Docosanic acid 21.22 --- 0.05 ----

Azelaic acid 14.6 0 0.18 0/0.18(1)

Eicosanoic acid 19.73 0.29 0.08 3.63

Alkanes

Hexadecane 22.9 0.06 0 (2)

tetracosane 22.6 0.64 1.89 2.95

nonacosane 22.65 0 0.08 0/0.08(3)

Alcohols

Octacosanol 24.138 0.14 0.059 2.37

Triacontanol 25.56 0.53 0.123 4.3

Triacontanediol 25.86 0.39 0.127 3.07

Stigmasterol 26.32 0.02 0.02 1.00

Beta-sitosterol 26.61 0.28 0.05 5.6

65

Table 5.5. Classes of compounds present in the sorghum wax extracted with their retention

time. Superscripts (1) to (7) indicates ratios that could not be calculated since amount

obtained with n-hexane was zero.

Class of


Retention

time

(min)

gram compound/ 100gram

wax Sorghum wax

n-

hexane

Liquid

N2 Rsorghum


Acids



Octadecadienoic acid 18.33 1.00 0.31 3.22

Tricosanic acid 21.59 0.73 0.19 3.84

Docosanic acid 21.22 0.27 0.14 1.93

Azelaic acid 14.6 0 0 ---

Eicosanoic acid 19.73 0 0.04 0/0.04(4)

Alkanes

Hexadecane 22.9 2.19 0.61 3.59

tetracosane 22.6 0.17 0.07 2.43

nonacosane 22.65 0.45 0.19 2.36

Alcohols

Octacosanol 24.138 11.97 3.31 3.62

Triacontanol 25.56 12.32 3.01 4.09


Stigmasterol 26.32 0 0.05 0/0.05(5)

Beta-sitosterol 26.61 1.23 0.20 6.15

66

Table 5.6. Classes compounds present in the wheat wax extracted with their retention time.


with n-hexane was zero.

Class of


Retention

time (min)

gram compound/ 100gram wax

Wheat wax

n-

hexane

Liquid

N2 Rwheat


Acids



Octadecadienoic acid 18.33 4.59 0.03 153

Tricosanic acid 21.59 0.07 0.03 2.33

Docosanic acid 21.22 0.21 0.02 10.5

Azelaic acid 14.6 0 0.02 0/0.02(6)

Eicosanoic acid 19.73 0.14 0.02 7

Alkanes

Hexadecane 22.9 0.052 0.04 1.3

tetracosane 22.6 0.13 0.04 3.25

nonacosane 22.65 0.18 0.04 4.5

Alcohols

Octacosanol 24.138 0.046 0.07 0.66

Triacontanol 25.56 0 0.07 0/0.07(7)


Stigmasterol 26.32 0.17 0 (9)

Beta-sitosterol 26.61 0.21 0.03 7

67

The most abundant compounds in rice wax are fatty acids, the method using n-hexane

resulted in 8.7 g octadecanoic acid /100g wax, 4.78 g hexadecanoic acid /100g wax, and

4.32 g octadecanoic acid /100g wax. Similarly to the results found for sorghum wax, when

liquid nitrogen was used for extraction, the composition was similar to the compounds

extracted by n-hexane, but it was found in smaller quantity. Rice wax compounds

extracted with liquid nitrogen were only higher for azelaic acid and alkanes such as

tetracosane and nonacosane (Table 5.4).

There were found little amounts of octacosanol and triacontanol present in wheat wax

extracted by liquid nitrogen but no triacontanol was found in wheat wax extracted by n-

hexane (this last result is in agreement with the research performed by Hwang et al., 2002).

Also, as found for rice wax, liquid nitrogen extraction was able to extract azelaic acid from

wheat wax (no azelaic acid was obtained from extraction with n-hexane). Azelaic acid is

used to treat mild to moderate acne, both comedonal acne and inflammatory acne (Liu et

al., 2006) and is industrially produced by the ozonolysis of oleic acid.

69

CHAPTER 6. CONCLUSION

The extraction of cereal grains (rice, sorghum and wheat) by cyclic immersion in liquid

nitrogen is feasible. The optimal conditions in terms of number of cycles and extraction

total time were: 2 cycles/ total 11 minutes for rice, 1 cycle/ total 4 minutes for sorghum,

and 2 cyles/ total 13 minutes for wheat. The yield obtained at these conditions was 5 to 9.3

times lower than by using n-hexane. However, the extraction time with liquid nitrogen is

2.3 to 11.25 times shorter and this solvent is non-toxic to the environment. In addition, the

method is simple and the quality of grain treated by liquid nitrogen cereals remained

unchanged.

Color observations of extracted wax samples showed that compounds obtained with n-

hexane reflux have co-products extracted together with waxes. Also, SEM photos of the

surfaces of grains after treatments indicated that n-hexane penetrates deeper in the cuticle

than liquid nitrogen, which correlates well with yield results, and may a led to the

conclusion that liquid nitrogen is only effective to extract waxes from the grain surface.

Qualitative and quantitative analysis of compounds found in wax, pointed out the presence

of acids, alcohols, alkanes and sterols in waxes extracted by n-hexane or liquid nitrogen,

but the quantities obtained with n-hexane were much higher. Hexane can extract all the

policosanols found in sorghum and rice wax samples. On the other hand, only liquid

nitrogen extraction was able to extract azelaic acid from wheat and rice wax.

Future research on quantitative and qualitative compound determination from waxes

extracted by n-hexane and liquid nitrogen is required to obtain the whole composition of

extracted material. It is also suggested to compare the wax extracted by n-hexane or n-

pentane at low temperature with wax extracted by liquid nitrogen to examine extraction

selectivity as well as extraction yield.

The extraction of sorghum, wheat and rice waxes by cyclic immersion in liquid nitrogen is

a new and interesting green alternative to conventional extraction methods. On one hand,

70

no hazardous organic solvents were used in the process and, in addition, no leftover toxic

residue was present in the product. This can contribute to reduce solvent-related

environmental damages and represent no potential harm to human health.

The proposed liquid nitrogen wax extraction method could be considered as a secondary

step in the milling process: after removing their hulls, grains would be immersed in liquid

nitrogen to extract the wax followed by further milling to separate bran from grains.

Finally, the fact that waxes from cereal (sorghum, rice and wheat) grains can be extracted

by liquid nitrogen may likely result in the potential extraction of other wax-containing

compounds.

The results obtained in this research on wax extraction by immersion in liquid nitrogen

could be extrapolated to different plant or animal sources or to other applications, i.e. to

replace the winterization process in order to improve the quality of oils.

71

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79

ANNEX 1: ANOVA table and mean table of the comparison the means of percent rice wax

extracted by n-hexane.

Table A-1. Anova of the percentage of rice wax extracted by n-hexane

Source DF Sum of

Squares

Mean Square F Value Pr > F

Model 6 0.00425879 0.00070980 18.26** <.0001

Error 14 0.00054428 0.00003888

Corrected

Total

20 0.00480307

(**) significatif (α<0.01)

Table A-2. Rice wax extraction by n-hexane

Time extraction (minute) Mean of wax (%)

5 0.1556e

15 0.1720d

30 0.1835c

45 0.1868bc

60 0.1988a

90 0.1940abc

120 0.1959ab

The means in the column with the same letter superscript are not significantly different

(P<0.05) by Duncan's Multiple Range test.

80

ANNEX 2: ANOVA table and mean table of the comparison the means of percent sorghum

wax extracted by n-hexane.

Table A-3. Anova of the percentage of sorghum wax extracted by n-hexane

Source DF Sum of

Squares


Model 6 0.02508136 0.00418023 11.83** <.0001

Error 14 0.00494862 0.00035347

Corrected

Total

20 0.03002998


Table A-4. Percent of sorghum wax extraction by n-hexane




5 0.1982d

15 0.2453c

30 0.2539bc

45 0.3003a

60 0.2755abc

90 0.2843ab

120 0.3058a

81


wax extracted by n-hexane.

Table A-5. Anova of percentage of wheat wax extracted by n-hexane

Source DF Sum of

Squares


Model 6 0.01470204 0.00245034 12.21** <.0001

Error 14 0.00281047 0.00020075

Corrected

Total

20 0.01751252


Table A-6 . Percent of wheat wax extraction by n-hexane


5 0.0353c

15 0.0651b

30 0.0979a

45 0.1039a

60 0.1081a

90 0.1089a

120 0.1086a



82


wax extracted by liquid nitrogen with 1 minute rest

Table A-7. Anova of percentage of sorghum wax extracted by liquid nitrogen with 1

minute of rest

Source DF Sum of

Squares


Model 32 0.00058860 0.00001839 21.07** <.0001

Error 66 0.00005761 0.00000087

Corrected

Total

98 0.00064622


Table A-8. Percent of sorghum wax extracted by liquid nitrogen with 1minute of rest

Time

extraction

(minute)

Sorghum Wax (%) 1 minute rest

1 cycle 2 cycles 3 cycles

0.5 0.0240l 0.0254

kl 0.0245

l

1 0.0241l 0.0267

jk 0.0299

defg

2 0.0266jk

0.0286ghi

0.0296efgh

3 0.0278ij 0.0290

fghi 0.0298

defg

4 0.0306bcdef

0.0280hij

0.0296defgh

5 0.0304cdefg

0.0302cdefg

0.0320abc

6 0.0308bcdef

0.0296defgh

0.0327a

7 0.0303cdefg

0.0311abcde

0.0326a

8 0.0297defg

0.0313abcde

0.0323ab

9 0.0304cdefg

0.0313abcde

0.0327a

10 0.0304cdefg

0.0314abcd

0.0327a



83


wax extracted by liquid nitrogen with 1, 3, 5 minutes rest

Table A-9. Anova of percentage of sorghum wax extracted by liquid nitrogen with 1, 3, 5

minutes of rest

Source DF Sum of

Squares


Model 32 0.00022841 0.00000714 11.41** <.0001

Error 66 0.00004128 0.00000063

Corrected

Total

98 0.00026969


Table A-10. Percent of sorghum wax extracted by liquid nitrogen with 1, 3, 5 minutes of

rest

Time

extraction

(minute)

Sorghum Wax (%)

2 cycles - 1 minute

realx

2 cycles - 3 minute

realx

2 cycles - 5 minute

realx

0.5 0.0254l 0.0272

ijk 0.0279

hijk

1 0.0267kl

0.0269jk

0.0278hijk

2 0.0286efghi

0.0272ijk

0.0285fghi

3 0.0290defgh

0.0282ghij

0.0310abc

4 0.0280ijkh

0.0300abcdef

0.0308abc

5 0.0302abcd

0.0301abcde

0.0300abcdef

6 0.0296cdefg

0.0297abcdef

0.0298abcdef

7 0.0311abc

0.0300abcdef

0.0307abc

8 0.0313ab

0.0302abcd

0.0299abcdef

9 0.0313ab

0.0299abcdef

0.0308abc

10 0.0314a 0.0302

abcd 0.0301

abcdef



84


wax extracted by liquid nitrogen with 1 minutes rest

Table A-11. Anova of percentage of rice wax extracted by liquid nitrogen with 1 minute of

rest

Source DF Sum of

Squares


Model 32 0.00051282 0.00001603 15.68** <.0001

Error 66 0.00006744 0.00000102

Corrected

Total

98 0.00058026


Table A-12. Percent of rice wax extracted by liquid nitrogen with 1 minute of rest

Time

extraction

(minute)

Rice Wax (%) 1 minute rest


0.5 0.0164k 0.0176

jk 0.0191

ij

1 0.0167k 0.0177

jk 0.0211

defgh

2 0.0167k 0.0187

ij 0.0211

defgh

3 0.0188ij 0.0189

ij 0.0218

bcde

4 0.0187ij 0.0197

fghi 0.0218

bcde

5 0.0194hij

0.0224abcd

0.0232abc

6 0.0201efghi

0.0227abcd

0.0242a

7 0.0197ghi

0.0215cdefg

0.0232abc

8 0.0223abcd

0.0216cdef

0.0233abc

9 0.0224abcd

0.0236ab

0.0230abcd

10 0.0230abcd

0.0232abc

0.0236ab



85


extracted by liquid nitrogen with 1, 3, 5 minutes of rest


minutes of rest

Source DF Sum of

Squares


Model 32 0.00081182 0.00002537 19.05** <.0001

Error 66 0.00008791 0.00000133

Corrected

Total

98 0.00089973

(**) Significatif (α<0.01)

Table A-14. Percent of rice wax extracted by liquid nitrogen with 3 minutes of rest

Time

extraction

(minute)

Rice Wax (%) 3 minutes rest


0.5 0.0164k 0.0182

ijk 0.0181

ijk

1 0.0167jk

0.0212fgh

0.0224 def

2 0.0167jk

0.0224def

0.0210fgh

3 0.0188i 0.0236

bcde 0.0221

efghi

4 0.0187 ij 0.0256

ab 0.0256

ab

5 0.0194hi

0.0256ab

0.0238abcde

6 0.0201ghi

0.0243abcde

0.0249abc

7 0.0197hi

0.0247abc

0.0235bcde

8 0.0223def

0.0245abcd

0.0241abcde

9 0.0224def

0.0251abc

0.0244abcd

10 0.0230cdef

0.0260a 0.0244

abcd



86


extracted by liquid nitrogen with 1, 3, 5 minutes of rest


minutes of rest

Source DF Sum of

Squares


Model 32 0.00068325 0.00002135 33.63** <.0001

Error 66 0.00004190 0.00000063

Corrected

Total

98 0.00072515


Table A-16. Percent of rice wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest

Time

extraction

(minute)

Rice Wax (%)

2 cycles - 1 minute

realx

2 cycles - 3 minute

realx

2 cycles - 5 minute

realx

0.5 0.0176k 0.0182k 0.0222fgh

1 0.0177k 0.0212gh 0.0214gh

2 0.0187jk

0.0223fgh 0.0209hi

3 0.0189jk 0.0236ef 0.0222fgh

4 0.0197ij 0.0256abcd 0.0245cde

5 0.0224fg 0.0256abcd 0.0255abcd

6 0.0227fg 0.0243de 0.0257abcd

7 0.0215gh 0.0247bcde 0.0260abc

8 0.0216gh 0.0245cde 0.0261ab

9 0.0236ef 0.0251abcd 0.0251abcd

10 0.0232ef 0.0260abc 0.0264a



87


wax extracted by liquid nitrogen with 1 minute of rest

Table A-17. Anova of percentage of wheat wax extracted by liquid nitrogen with 1 minute

of rest

Source DF Sum of

Squares


Model 43 0.00243387 0.00005660 66.84** <.0001

Error 88 0.00007452 0.00000085

Corrected

Total

131 0.00250839


Table A-18. Percent of wheat wax extracted by liquid nitrogen with 1 minute of rest

Time

extraction

(minute)

Wheat Wax (%) 1 minute rest

1 cycle 2 cycles 3 cycles 4 cycles

0.5 0.0120t 0.0135

rst 0.0188

k 0.0192jk

1 0.0124st 0.0140

qrs 0.0196

ijk 0.0206hij

2 0.0138rs 0.0142

pqr 0.0196

ijk 0.0209ghi

3 0.0152mnopqr

0.0159lmnop

0.0213fgh

0.0213fgh

4 0.0150nopqr

0.0168lm

0.0215fgh

0.0234cde

5 0.0157lmnopq

0.0166lmn

0.0225efg

0.0242abcd

6 0.0157lmnopq

0.0168lm

0.0228def

0.0248abc

7 0.0147opqr

0.0169lm

0.0236bcde

0.0251abc

8 0.0142pqr

0.0172l 0.0247

abc 0.0255a

9 0.0142pqr

0.0171l 0.0257

a 0.0252ab

10 0.0142pqr

0.0164lmno

0.0255a 0.0252ab



88


wax extracted by liquid nitrogen with 3 minutes of rest

Table A-19. Anova of percentage of wheat wax extracted by liquid nitrogen with 3 minutes

of rest

Source DF Sum of

Squares


Model 32 0.00219680 0.00006865 69.22** <.0001

Error 66 0.00006546 0.00000099

Corrected

Total

98 0.00226226


Table A-20. Percent of wheat wax extracted by liquid nitrogen with 3 minutes of rest

Time

extraction

(minute)

Wheat Wax (%) 3 minute of rest


0.5 0.0120i 0.0190f 0.0176f

1 0.0124hi 0.0219de 0.0208e

2 0.0138gh 0.0224de 0.0226d

3 0.0152g 0.0233bcd 0.0236bcd

4 0.0150g 0.0236bcd 0.0231cd

5 0.0157g 0.0248abc 0.0238abcd

6 0.0157g 0.0246abc 0.0246abc

7 0.0147g 0.0252ab 0.0250abc

8 0.0142gh 0.0252ab 0.0257a

9 0.0142gh 0.0252ab 0.0248abc

10 0.0142gh 0.0252ab 0.0250abc



89


wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest

Table A-21. Anova of percentage of wheat wax extracted by liquid nitrogen with 1, 3, 5

minutes of rest

Source DF Sum of

Squares


Model 32 0.00171002 0.00005344 83.66** <.0001

Error 66 0.00004216 0.00000064

Corrected

Total

98 0.00175218


Table A-22. Percent of wheat wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest

Time

extraction

(minute)

Wheat Wax (%) 2 cycles

1 minute rest 3 minutes rest 5 minutes rest

0.5 0.0135k 0.0190

i 0.0171

j

1 0.0140k 0.0219

g 0.0205h

2 0.0142k 0.0224

fg 0.0194hi

3 0.0159j 0.0233efg 0.0239cde

4 0.0168j 0.0236def 0.0241

bcde

5 0.0165j 0.0248

abcd 0.0254

abc

6 0.0168j 0.0246

abcde 0.0252

abc

7 0.0169j 0.0252

abc 0.0255

ab

8 0.0172j 0.0252

abc 0.0251

abc

9 0.0171j 0.0252

abc 0.0256

ab

10 0.0164j 0.0252

abc 0.0257

a



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