cryo-extraction des cires des produits céréaliers (sorgho ...€¦ · coupled to mass...
TRANSCRIPT
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
ANNEX 4: ANOVA table and mean table of the comparison the means of percent
sorghum wax extracted by liquid nitrogen with 1 minute rest ......................................... 82
ANNEX 5: ANOVA table and mean table of the comparison the means of percent
sorghum wax extracted by liquid nitrogen with 1, 3, 5 minutes rest ................................ 83
ANNEX 6: ANOVA table and mean table of the comparison the means of percent
sorghum wax extracted by liquid nitrogen with 1 minutes rest ........................................ 84
ANNEX 7: ANOVA table and mean table of the comparison the means of percent rice
wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest .......................................... 85
ANNEX 8: ANOVA table and mean table of the comparison the means of percent rice
wax extracted by liquid nitrogen with 1, 3, 5 minutes of rest .......................................... 86
ANNEX 9: ANOVA table and mean table of the comparison the means of percent wheat
wax extracted by liquid nitrogen with 1 minute of rest .................................................... 87
ANNEX 10: ANOVA table and mean table of the comparison the means of percent
wheat wax extracted by liquid nitrogen with 3 minutes of rest ........................................ 88
ANNEX 11: ANOVA table and mean table of the comparison the means of percent
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.
Superscripts (1) to (7) indicates ratios that could not be calculated since amount obtained
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
Table A-15. Anova of percentage of rice wax extracted by liquid nitrogen with 1, 3, 5
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
minutes rest time) .................................................................................................................. 46
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
minutes rest time) .................................................................................................................. 49
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
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
7
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).
8
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
9
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
10
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
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).
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.
Superscripts (1) to (7) indicates ratios that could not be calculated since amount obtained
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
compounds Name of compounds
Retention
time
(min)
gram compound/ 100gram
wax Sorghum wax
n-
hexane
Liquid
N2 Rsorghum
Standard Pentadecanoic acid 15.51 0 0 ---
Acids
Hexadecanoic acid 16.42 0.45 0.37 1.22
Octadecanoic acid 18.16 1.17 0.61 1.92
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
Triacontanediol 25.86 13.21 3.91 3.38
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.
Superscripts (1) to (7) indicates ratios that could not be calculated since amount obtained
with n-hexane was zero.
Class of
compounds Name of compounds
Retention
time (min)
gram compound/ 100gram wax
Wheat wax
n-
hexane
Liquid
N2 Rwheat
Standard Pentadecanoic acid 15.51 0 0 ---
Acids
Hexadecanoic acid 16.42 2.53 0.33 7.67
Octadecanoic acid 18.16 1.89 0.13 14.54
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)
Triacontanediol 25.86 13.966 0.04 349.15
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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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
Mean Square F Value Pr > F
Model 6 0.02508136 0.00418023 11.83** <.0001
Error 14 0.00494862 0.00035347
Corrected
Total
20 0.03002998
(**) significatif (α<0.01)
Table A-4. Percent of sorghum wax extraction by n-hexane
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
Time extraction (minute) Mean of wax (%)
5 0.1982d
15 0.2453c
30 0.2539bc
45 0.3003a
60 0.2755abc
90 0.2843ab
120 0.3058a
81
ANNEX 3: ANOVA table and mean table of the comparison the means of percent wheat
wax extracted by n-hexane.
Table A-5. Anova of percentage of wheat wax extracted by n-hexane
Source DF Sum of
Squares
Mean Square F Value Pr > F
Model 6 0.01470204 0.00245034 12.21** <.0001
Error 14 0.00281047 0.00020075
Corrected
Total
20 0.01751252
(**) significatif (α<0.01)
Table A-6 . Percent of wheat wax extraction by n-hexane
Time extraction (minute) Mean of wax (%)
5 0.0353c
15 0.0651b
30 0.0979a
45 0.1039a
60 0.1081a
90 0.1089a
120 0.1086a
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
82
ANNEX 4: ANOVA table and mean table of the comparison the means of percent sorghum
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
Mean Square F Value Pr > F
Model 32 0.00058860 0.00001839 21.07** <.0001
Error 66 0.00005761 0.00000087
Corrected
Total
98 0.00064622
(**) significatif (α<0.01)
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
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
83
ANNEX 5: ANOVA table and mean table of the comparison the means of percent sorghum
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
Mean Square F Value Pr > F
Model 32 0.00022841 0.00000714 11.41** <.0001
Error 66 0.00004128 0.00000063
Corrected
Total
98 0.00026969
(**) significatif (α<0.01)
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
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
84
ANNEX 6: ANOVA table and mean table of the comparison the means of percent sorghum
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
Mean Square F Value Pr > F
Model 32 0.00051282 0.00001603 15.68** <.0001
Error 66 0.00006744 0.00000102
Corrected
Total
98 0.00058026
(**) significatif (α<0.01)
Table A-12. Percent of rice wax extracted by liquid nitrogen with 1 minute of rest
Time
extraction
(minute)
Rice Wax (%) 1 minute rest
1 cycle 2 cycles 3 cycles
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
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
85
ANNEX 7: ANOVA table and mean table of the comparison the means of percent rice wax
extracted by liquid nitrogen with 1, 3, 5 minutes of rest
Table A-13. Anova of percentage of rice wax extracted by liquid nitrogen with 1, 3, 5
minutes of rest
Source DF Sum of
Squares
Mean Square F Value Pr > F
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
1 cycle 2 cycles 3 cycles
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
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
86
ANNEX 8: ANOVA table and mean table of the comparison the means of percent rice wax
extracted by liquid nitrogen with 1, 3, 5 minutes of rest
Table A-15. Anova of percentage of rice wax extracted by liquid nitrogen with 1, 3, 5
minutes of rest
Source DF Sum of
Squares
Mean Square F Value Pr > F
Model 32 0.00068325 0.00002135 33.63** <.0001
Error 66 0.00004190 0.00000063
Corrected
Total
98 0.00072515
(**) significatif (α<0.01)
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
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
87
ANNEX 9: ANOVA table and mean table of the comparison the means of percent wheat
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
Mean Square F Value Pr > F
Model 43 0.00243387 0.00005660 66.84** <.0001
Error 88 0.00007452 0.00000085
Corrected
Total
131 0.00250839
(**) significatif (α<0.01)
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
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
88
ANNEX 10: ANOVA table and mean table of the comparison the means of percent wheat
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
Mean Square F Value Pr > F
Model 32 0.00219680 0.00006865 69.22** <.0001
Error 66 0.00006546 0.00000099
Corrected
Total
98 0.00226226
(**) significatif (α<0.01)
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
1 cycle 2 cycles 3 cycles
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
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.
89
ANNEX 11: ANOVA table and mean table of the comparison the means of percent wheat
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
Mean Square F Value Pr > F
Model 32 0.00171002 0.00005344 83.66** <.0001
Error 66 0.00004216 0.00000064
Corrected
Total
98 0.00175218
(**) significatif (α<0.01)
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
The means in the column with the same letter superscript are not significantly different
(P<0.05) by Duncan's Multiple Range test.