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Impacts of Water, Extraction Procedure and Origin on Anthocyanins and Volatile
Compositions of Hibiscus Extracts and Freeze-Dried Hibiscus
Oumoule NDIAYE
Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University
in partial fulfillment of the requirements for the degree of
Master of Science in Life Science
In
Food Science and Technology
Committee Members
Sean F. O’Keefe
Susan E. Duncan
Mady Cisse
August 18, 2016
Blacksburg, Virginia
Keywords: Hibiscus, Calyces, Anthocyanin, Hibiscin, freeze-dried, Volatiles
Copyright 2016 Oumoule Ndiaye
Impacts of Water, Extraction Procedure and Origin on Anthocyanins and Volatile
Compositions of Hibiscus Extracts and Freeze-Dried Hibiscus
Oumoule NDIAYE
ABSTRACT
There has been a lot of interest in Roselle (Hibiscus sabdariffa L.), called Bissap in
Senegal, hibiscus recently because of consumer interest in nutraceutical products.
However, beverages made from hibiscus have a short self-life due to anthocyanin and
flavor degradation. The purpose of our study was first to assess the impacts of water,
extraction procedure and origin on the anthocyanins of hibiscus extracts and secondly, to
examine the impacts of freeze-drying on the anthocyanins and the volatiles compositions
of hibiscus extracts. For the first experiment, a 2x3 factorial design was used with
hibiscus calyces from Senegal and Egypt for the factor origin, distilled water and
reformulated Dakar (Senegal) water for the second factor water, and then cold and hot
extraction procedures were applied. For the second experiment, Senegalese hibiscus was
extracted with hot and cold water and one part of each extract was freeze-dried. For both
objectives, a ratio of 1:15 w/v (1 kilogram of calyces for 15 liters of water) were used.
The time-temperature was 98ºC / 30 min for hot and 22 ºC / 4 hours for cold extractions.
The anthocyanins were determined using high performance liquid chromatography
(HPLC). And the volatiles were measured using headspace-solid phase microextraction
and gas chromatography-mass spectrometry (HS-SPME-GCMS). Origin and temperature
as well as their interaction had significant effects on the anthocyanin contents, with
respective p-values of 0.0036 and 0.0025 and 0.0002. Freeze-drying showed no effect on
the anthocyanins in cold extracts. In contrast, a significant difference between the hot
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extract and its freeze-dried product was observed with a p-value of 0.0013. For the flavor
compounds, the aroma profiles were different between cold and hot extracts and their
instant powders. Globally the results of this study can help in the optimization when
processing hibiscus derivatives.
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Impacts of Water, Extraction Procedure and Origin on Anthocyanins and Volatile
Compositions of Hibiscus Extracts and Freeze-Dried Hibiscus
Oumoule NDIAYE
GENERAL AUDIENCE ABSTRACT
Roselle (Hibiscus sabdariffa L.), called Bissap in Senegal, is an annual shrub of tropical
countries, locally used as a nutritious functional beverage. It has been associated with
many health benefits and is also used in many industrial applications, hence its growing
interest among entrepreneurs. However, beverages made from hibiscus have a short self-
life due to degradation of anthocyanins and flavors. The purpose of our study was first to
assess the impacts of water, extraction procedure and origin on the anthocyanins of
hibiscus extracts. Senegal and Egypt hibiscus were used to prepare beverages with
distilled water and reformulated Dakar (Senegal) water with cold and hot extraction
temperature. Secondly, we examined the impact of freeze-drying on the anthocyanins and
the volatile compositions of hibiscus extracts. Senegalese hibiscus was extracted in hot
and cold conditions one part of each extract was freeze-dried. The result of this
investigation show that origin and extraction temperature have significant effects on the
anthocyanins contents. As expected, freeze-drying as no effect on the anthocyanins for
cold extract. In contrast, significant differences were seen between the hot extract and its
freeze-dried products. The aromas profiles were different when comparing cold and hot
extracts to their respective instant powders as well as between the hot and cold extracts.
The results of this study show freeze-dried hibiscus has volatiles and anthocyanins
similar to non dried, suggesting that freeze drying is an option for stabilizing hibiscus.
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DEDICATION
To
Living memories of my mother Ndiéme DIOUF and my dad Aliou NDIAYE a strong and
humanitarian soul whose unconditional love and daily life inspires wherever I am and in
whatever I do and allows me to set higher targets.
My brother Amadou, I am really grateful to you, you have been my inspiration; Thank
you for your great support and continuous care.
My friend Bijieck Jiecnyal for your support and everything.
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ACKNOWLEDGEMENTS
I would like to express the deepest appreciation to my advisor Professor Sean
O`Keefe who continuously and convincingly conveyed a spirit of adventure to reseach
and excitement in regards to teaching. His guidance and persistent help have made this
work easy for me.
I would like to thank my committee members Professor Susan E. Duncan and Dr.
Mady Cisse for their help, support guidance and understanding.
My special thanks to my sponsors: USAID/ERA (Education & Research in
Agriculture) & OIRED who funded my studies and research at Virginia Polytechnic
Institute and State University (Virginia Tech) in the USA and also the Senegalese
Institute of Food Technology for giving me the opportunity to be one of their candidates
applying for and getting the USAID/ERA scholarship.
I am sincerely grateful to the Senegalese Students of Blacksburg and the Food
Science and Technology (FST) Department staff and graduate students.
Finally, I acknowledge those who helped or supported me in finishing this thesis
specially Bijiek Jieknyal for your support, advice and encourage and everything you have
done for me.
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TABLE OF CONTENTS
ABSTRACT ....................................................................................................................... ii GENERAL AUDIENCE ABSTRACT ........................................................................... iii DEDICATION.................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................. v
TABLE OF CONTENTS ................................................................................................ vi CHAPTER I ...................................................................................................................... 1
INTRODUCTION ..................................................................................................................... 1
REFRENCES ............................................................................................................................. 3
CHAPTER II ..................................................................................................................... 5 LITERATURE REVIEW ......................................................................................................... 5
2.1 Overview of hibiscus ............................................................................................................. 5
2.2 Origin and distribution .......................................................................................................... 5
2.3 Botanical characteristics and diversity .................................................................................. 6
2.4 Physico-chemical characteristics of Hibiscus sabdariffa calyces ......................................... 8
2.5 Anthocyanins in the calyces .................................................................................................. 9
2.5.1 Structure, composition, and properties of the anthocyanins ............................................... 9
2.5.4 Effect of water, oxygen and enzymes ............................................................................... 12
2.6. Aroma compounds ............................................................................................................. 12
2.7 Microbiological quality ....................................................................................................... 14
2.8 Production of hibiscus and commercialization. ................................................................... 14
2.9 Main uses, consumption, trends, and health benefits .......................................................... 15
REFERENCES .......................................................................................................................... 17
CHAPTER III ................................................................................................................... 24 MATERIALS AND METHODS .............................................................................................. 24
3.1 Plant material and water used for the extraction ................................................................. 24
3.2 Aqueous preparation and freeze-drying process. ................................................................ 25
3. 3 Experiment I: Effects of Origin Water and Temperature on the Anthocyanins and Volatile
of Hibiscus. ................................................................................................................................ 26
3. 4 Experiment II: Effects of freeze-drying on anthocyanins and volatiles of instant Hibiscus
powder obtained from Hot and Cold Extracted juice. ............................................................... 27
3.6 Brix measurement ................................................................................................................ 29
3.7 Total anthocyanins and hibiscidin measurement by HPLC analysis ................................... 29
3.8 Aroma compounds analyses by SPME and GC-MS analysis ............................................. 29
REFERENCE ............................................................................................................................ 31
CHAPTER IV.................................................................................................................. 32 RESULTS AND DISCUSSIONS............................................................................................ 32
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Results Experiment I ................................................................................................................. 32
4.1 Effects of Origin, Water and Temperature on Total Anthocyanin Contents ....................... 32
Results Experiment II ................................................................................................................ 41
4.3 Effect of Freeze-Drying on Total Anthocyanins and Hibiscin Contents using Hot Extraction
Procedure ................................................................................................................................... 41
4.4 Effect of Freeze-drying on Total Anthocyanins and Hibiscidin Contents using Cold
Extraction Procedure ................................................................................................................. 43
4.5 pH and the Brix in hibiscus extracts .................................................................................... 44
4.6 Effects of freeze-drying on the volatiles of Instant hibiscus powder obtained from cold
extraction. .................................................................................................................................. 45
4.7 Effects of freeze-drying on the volatiles of instant hibiscus powder obtained from hot
extracted juice ............................................................................................................................ 48
REFERENCES ........................................................................................................................ 53
CHAPTER V ................................................................................................................... 55 CONCLUSIONS ...................................................................................................................... 55
APPENDICES ........................................................................................................................... 57
APPENDIX A – 1 ................................................................................................................. 57
APPENDIX B– 1 ................................................................................................................... 60
APPENDIX C – 1 .................................................................................................................. 60
APPENDIX D – 1 ................................................................................................................. 61
APPENDIX E – 1 .................................................................................................................. 61
APPENDIX F – 1 .................................................................................................................. 61
1
CHAPTER I
INTRODUCTION
In tropical countries, research investments have focused mainly on agricultural production.
Because of this, product development and food processing are often not well developed (Nkem
Khumbah & Melvin, 2014); this has negative impacts, particularly on the economy. People who live
in large cities rely heavily on imported, processed food. According to Van Wyk 2015, in recent years
research has been conducted on many African medicinal plants in order to find the best ways to
promote growth in their use and sales. Among the targeted species, Hibiscus sabdariffa, also called
Roselle is considered as a functional foods (Van Wyk 2015). Hibiscus has numerous health benefits,
such as lowering blood pressure, anticancer activity, and bactericidal properties. Consequently,
consumer demand for this nutraceutical product has increased worldwide (Chiou, & Langrish, 2007;
Hopkins, Lamm, Funk, & Ritenbaugh; Patel 2014; Sindi, Marshall, & Morgan, 2014; Camelo-
Méndez, Ragazzo-Sánchez, Jimenez-Aparicio, Vanegas-Espinoza, Paredes-López, & Del Villar-
Martinez, 2013; Cisse, Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a; Rodrigues, 2010.
Similarly, studies conducted by Wong, Yusof, Ghazali, & Che Man, 2002; Gonzalez-Palomares,
Estarrón-Espinosa, Gómez-Leyva, & Andrade-González, 2009; and Duangmal, Saicheua, &
Sueeprasan, 2008 found Roselle to be valuable to pharmaceutical, cosmetic and food industries.
Some researchers have considered hibiscus to be one of the most important botanical products in the
global market (Cisse, Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a; FAO, 2004). Lastly, research
has shown that Roselle contributes to the development of the rural economy and strengthens food
security. Observers see Roselle as an alternative to some food crops like peanut in Senegal (Cisse,
Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a). However, some studies have suggested that
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beverages made from hibiscus flowers main edible (the calyces) have a short self-life, due to
anthocyanin and flavor degradation (Sarni-Manchad & Cheynier V. 2006). This makes it very
difficult to ship, transport, and commercialize hibiscus products. Therefore, finding ways to obtain
ready-to-use and easy-to-export products from hibiscus extracts that are safe, have good qualities,
great consumer acceptability and acceptable shelf-life can improve product accessibility and
availability throughout the world. A lot of studies have been conducted on Hibiscus sabdariffa in
order to end up with products that are successful in the market place. For instance, Duangmal,
Saicheua, & Sueeprasan, (2008) have investigated the stability of anthocyanins in freeze-dried
Roselle using either maltodextrin or trehalose as a stabilizer. They found that the addition of these
products retarded total anthocyanin degradation. The effect of the temperature on spray drying of
Roselle extracts (using ethanol as solvent) has also been investigated by Gonzalez-Palomares,
Estarrón-Espinosa, Gómez-Leyva, & Andrade-González, (2009). They reported a significant effect
of spray drying temperature on the volatiles. However, further investigations are needed in order to
improve hibiscus calyces transformation, commercialization and consumption worldwide. The
purpose of this study is to investigate the effects of origin, water, and temperature on the
anthocyanin contents of hibiscus extracts and also to determine the effect of freeze-drying on
anthocyanin contents and aroma compositions of hibiscus instant powder obtained from hibiscus
extracts. The results of this current work will help determine factors relevant to processing hibiscus
instant powder.
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REFRENCES
Nkem Khumbah and Melvin P.Foote. (2014). Africa Needs Science, Not Aid. The New York
Time (http://www.nytimes.com/2014/08/01/opinion/africa-needs-Science-not-aid.html see on June
4th 2016).
Van Wyk, B.E. (2015). A review of commercially important African medicinal plants).
Chiou, D., & Langrish, T. A. G. (2007). Development and characterization of novel
nutraceuticals with spray drying technology. Journal of Food Engineering, 82(1), 84-91.
Hopkins, A. L., Lamm, M. G., Funk, J. L., & Ritenbaugh, C. (2013). Hibiscus sabdariffa L.
in the treatment of hypertension and hyperlipidemia: a comprehensive review of animal and human
studies. Fitoterapia, 85, 84-94.
Patel, S. (2014). Hibiscus sabdariffa: An ideal yet under-exploited candidate for nutraceutical
applications. Biomedicine & Preventive Nutrition, 4(1), 23-27.
Sindi, H. A., Marshall, L. J., & Morgan, M. R. (2014). Comparative chemical and
biochemical analysis of extracts of Hibiscus sabdariffa. Food chemistry, 164, 23-29.
Camelo-Méndez, G. A., Ragazzo-Sánchez, J. A., Jimenez-Aparicio, A. R., Vanegas-
Espinoza, P. E., Paredes-López, O., & Del Villar-Martinez, A. A. (2013). Comparative study of
anthocyanin and volatile compounds content of four varieties of Mexican roselle (Hibiscus
sabdariffa L.) by multivariable analysis. Plant foods for human nutrition, 68(3), 229-234.
Cisse, M., Dornier, M., Sakho, M., Ndiaye, A., Reynes, M., & Sock, O. (2009a). Le bissap
(Hibiscus sabdariffa L.): composition et principales utilisations. Fruits, 64(03), 179-193
Wong, P. K., Yusof, S., Ghazali, H. M., & Che Man, Y. B. (2002). Physico-chemical
characteristics of roselle (Hibiscus sabdariffa L.). Nutrition & Food Science, 32(2), 68-73.
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Gonzalez-Palomares, S., Estarrón-Espinosa, M., Gómez-Leyva, J. F., & Andrade-González,
I. (2009). Effect of the temperature on the spray drying of roselle extracts (Hibiscus sabdariffa
L.). Plant foods for human nutrition,64(1), 62-67.
Duangmal, K., Saicheua, B., & Sueeprasan, S. (2008). Colour evaluation of freeze-dried
roselle extract as a natural food colorant in a model system of a drink. LWT-Food Science and
Technology, 41(8), 1437-1445.
Food and Agriculture Organization of the United Nations (FAO) HIBISCUS: Post-
Production Management for Improved Market (Access, 2004)
Sarni-Manchad P. & Cheynier V. 2006. Les polyphénols en agroalimentaire, Éd Tec & Doc.,
Coll. Sci. & Techn. Agroaliment., Lavoisier, Paris, 398 p. [ Cheynier, V., & Sarni-Manchado, P.
(2006). Les polyphénols en agroalimentaire. Paris: Lavoisier Tec & Doc, 50-59.]
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CHAPTER II
LITERATURE REVIEW
2.1 Overview of hibiscus
Hibiscus sabdariffa, an herbaceous perennial shrub belonging to the Malvaceae family, is known by
different names. Some of its vernacular names include Roselle, Bissap in Senegal, Jamaican sorrel,
Indian sorrel, karkadeh or karkadé in North Africa, Oseille de Guinée, thé rose d’Abyssinie, Roselle
or sorrel sour tea in English, ngai ngai in Central Africa, currant Christmas in the Caribbean, and
Jamaica floras in Central America (Nyarko, Bayor, Craigon, & Suleimana, 2006; Cisse, Dornier,
Sakho, Diop, Reynes, Sock, 2009b; Juliani, Welch, Wu, Diouf, Malainy, & Simon, 2009;
McClintock, & El Tahir, 2011; Ali, Wabel, & Blunden, 2005; FAO 2004).
Hibiscus sabdariffa L is cultivated largely in tropical and subtropical areas of both hemispheres.
Hibiscus sabdariffa has several varieties and is used in various fields due to its biochemical
composition and growing consumers interest in nutraceutical products but also because it can be
used for many industrial applications and, as a consequence, generates income (Da-Costa-Rocha,
Bonnlaender, Sievers, Pischel, & Heinrich, 2014; Camelo-Méndez, Ragazzo-Sánchez, Jimenez-
Aparicio, Vanegas-Espinoza, Paredes-López, & Del Villar-Martinez, 2013; McClintock, & El
Tahir, 2011; Cissé, Bohuon, Sambe, Kane, Sakho, & Dornier, 2012). In Senegal, the production
and exportation of hibiscus has expanded due to an increased demand at local and international
levels. But certified seed supply and better management in production and processing would increase
interest in commercialization of hibiscus (Cissé, 2010 2012; McClintock, & El Tahir, 2011).
2.2 Origin and distribution
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The origin of Roselle or Sorrel (Hibiscus sabdariffa) is not exactly known. According to
some authors, it is native to West Africa (El Naim, & Ahmed, 2010; Ataogye, 2012). It was
eventually brought to other parts of the world like Asia, Central America and the United States of
America during slave trade (El Naim, & Ahmed, 2010). However, according to other authors,
Hibiscus sabdariffa came from India (Jade Senegal http://www.jade.sn/bissap/botanique.htm;
Nzikou, Bouanga-Kalou, Matos, Ganongo-Po, Mboungou-Mboussi, Moutoula, & Desobry, 2011 ;
Atta, Sarr, Diallo, Bakasso, Lona, & Saadou, 2013). Roselle is now cultivated in many tropical and
subtropical regions of the world. But the variety altissama is rare in Africa. In Senegal, Hibiscus
sabdariffa was introduced in the 19th century (Kerharo, & Adam, 1974; Cisse, Dornier, Sakho,
Diop, Reynes, & Sock, 2009b; Ataogye, 2012, Atta, Sarr, Diallo, Bakasso, Lona, & Saadou, 2013;
FAO, 2004; McClintock & El Tahir, 2011; Akanbi, Olaniyan, Togun, Ilupeju, & Olaniran, 2009).
Currently in Senegal, hibiscus is grown in many regions (Kaolack, Fatick, Diourbel, Thies, Saint-
Louis, Louga and Ziguinchor). The varieties grown, including Vimto, Koor, Thai and CLT 92. This
helps farmers have an alternative to the production of traditional crops, especially peanut, which is
declining nowadays. (Cisse, Dornier, Sakho, Diop, Reynes, & Sock, 2009b; Reynes, & Sock,
2009b).
2.3 Botanical characteristics and diversity
According to some authors, more than 200–300 species of hibiscus can be found in both
tropical and subtropical areas. Hibiscus has simple leaves that can be regular or lobed. The two
botanical kinds based on the color of its fibers are: the red type and the green one (or white). The red
and green (or white) depends on the presence or absence of anthocyanins (McClintock, & El Tahir,
2011; Omobuwajo, Sanni, & Balami, 2000; Cisse, Dornier, Sakho, Diop, Reynes, & Sock, 2009 and
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Jade Senegal http://www.jade.sn/bissap/botanique.htm). Except for Hibiscus sabdariffa, the other
species are used for ornamental purposes and according to Kerharo, & Adam (1974) and Morton
(1987), it has two botanical varieties: the variety altissima and the variety sabdariffa L. The variety
altissima grows tallest; it can reach three up to four meters in height, is not much branched, without
fleshy fruits, and its use is justified by its fibers. Hibiscus sabdariffa L. variety sabdariffa is a
vascular herb, with alternated leaves and fleshy fruits. It belongs to the family of Malvaceae
(Morton, 1987; Cisse, Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a). The variety sabdariffa L
has two botanical types differing in the color of its fibers. The hibiscus flowers are solitary, axillary,
form loose fake cobs and are hermaphrodites. They self-pollinate with the reproduction cycle lasting
is about 120 to 165 days. (Cisse, Dornier, Sakho, Diop, Reynes, & Sock, (2009 b). Roselle can be
produced as a rain field crop, which requires the seed to be sown early in the rainy season, but also
in market gardening. Roselle is drought resistant and the production of the calyces requires
temperatures higher than 17°C, and at least 13 hours of daylight. The fruit is a globoid cap, covered
by the calyx and contains kidney-shaped brown to dark brown seeds. The fruits have two parts: the
calyx representing about 6g and the part that envelops the seeds weighing about 5g (McClintock, &
El Tahir, 2011; Cisse, Dornier, Sakho, Diop, Reynes, & Sock, 2009b;
http://www.jade.sn/bissap/botanique.htm; Wong, Yusof, Ghazali, & Che Man, 2002; Cisse, Dornier,
Sakho, Ndiaye, Reynes, & Sock, 2009a).
Some characteristics of certain varieties of hibiscus found in Senegal were given by Cisse, Dornier,
Sakho, Diop, Reynes, & Sock, 2009b. The Vimto characterized by dark red color, amount of
anthocyanin that can reach 10 to 15 grams per kilogram and the vitamin C content 0.5 gram per kilo.
The "Koor" more acidic than the Vimto variety produces less anthocyanin but might have more
organic acids. The Thaï variety first identified in Thailand has high fiber content. The Thaï have
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been introduced Senegal by l’ASNAPP-Sénégal (l’antenne sénégalaise de l’Agribusiness in
Sustainable Natural African Plan Products) and fondation Éducation-Santé. The ''CLT 92 " variety is
characterized by blue-purple or dark red pigments and was brought to Senegal by VAPROVET
(Valorisation des Produits Végétaux). Other red varieties of Hibiscus sabdariffa include
"Bambara","Burkina", and "Violette," also called ordinary (Cisse, Dornier, Sakho, Diop, Reynes, &
Sock, 2009b).
2.4 Physico-chemical characteristics of Hibiscus sabdariffa calyces
The calyces constitute the main portion of the fruit with more than 54.11± 2.34% in terms of
percentages, and the rests consist of seeds (Wong, Yusof, Ghazali, & Che Man, 2002). Hibiscus
calyces are the main edible part of this plant. Their composition can vary, depending on several
factors such as the variety of hibiscus, its genetic composition, the area where the calyces are
produced, the harvesting conditions, the amount of rainfall, the nature of the soil, the temperature,
and so on (Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, 2014; Kerharo, & Adam,
1974; and Morton 1987).
Many authors have studied physicochemical characteristics of Roselle and its components and
Rodrigues 2010 has given a summary of nutritional composition of fresh hibiscus calyces.
Other components such as organic acids (succinic, oxalic, tartaric, and malic), with succinic and
oxalic representing the main ones (76%) (Wong, Yusof, Ghazali, & Che Man, 2002; Babalola,
Babalola, & Aworh, 2001; Dafallah, al-Mustafa 1996). Sugars, mainly glucose, fructose and sucrose,
were also reported, as were ascrobic acid, β-carotene, and lycopene (Wong, Yusof, Ghazali, & Che
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Man, 2002) and protocatechuic acid (Dickel, Rates, & Ritter, 2007 and Herrera-Arellano, Flores-
Romero, Chavez-Soto, & Tortoriello, 2004).
Tableau 1. Amino acid composition (mg/g of dried mater) of Hibiscus sabdariffa calyces. Calices
Morton 1987
Ala Arg Asp Cys Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val
3.70 3.60 16.30 1.30 7.20 3.80 1.50 3.00 5.00 3.90 1.00 3.20 5.60 3.50 3.00 - 2.20 3.80
2.5 Anthocyanins in the calyces
2.5.1 Structure, composition, and properties of the anthocyanins
The richness in anthocyanins is one of the major characteristics of hibiscus calyces. Anthocyanins
are molecules with a C15 skeleton, classified as flavonoid derivates, and are water soluble. The
chromophores of the anthocyanins, the aglycones, constitute their basic structure. The molecule has
two aromatic rings and one heterocycle which consists of carbon and oxygen. Anthocyanins are able
to absorb visible light and therefore can be quantified by measuring their visible spectra. They are
also natural pigments present in fruits, vegetables flowers, etc. They are responsible for a wide range
of colors in many species ranging from blue, red, purple to pink. This coloring power makes them a
good substitute to the artificial dyes. They are used in food industries for color ranging from red to
violet (Delgado-Vargas, Jiménez, & Paredes-López, 2000; Gradinaru, Biliaderis, Kallithraka,
Kefalas, & Garcia-Viguera, 2003; Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall, 2011;
Castaneda-Ovando, de Lourdes Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal,
2009; Cisse, Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a; Wang, Wang, Lin, Chu, Chou, &
Tseng, 2000).
The epidermal cell of the plant contains the vacuole where these pigments are localized (Harborne,
1967). Four types of anthocyanins were identified by Francis (1973) Many researchers have shown
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that anthocyanins are present at high concentrations in Hibiscus sabdariffa calyces and, according to
Wong and others 2002, its content is 2.52 gram per 100g expressed as delphinidin-3-glucoside. The
delphinidin-3-sambubioside (also known as delphinidin-3-xylosylglucoside or hibiscin) was 71.40%
of the total anthocyanins and the rest was constituted by the cyanidin-3-sambubioside or cyanidin-3-
xylosylglucoside or gossypicyanin and delphinidin-3-glucoside (Wong, Yusof, Ghazali, & Che Man,
2002).
Researchers have shown these two main anthocyanin component are quickly absorbed in rats’
gastrointestinal tract, hence their good bioavailability (Wang, Wang, Lin, Chu, Chou, Tseng, T. H.
(2000).
Figure 1. Chemical structures of main anthocyanins.
https://www.researchgate.net/profile/Michael_Heinrich/publication/262937428/figure/fig1/Chemical
-structures-of-main-anthocyanins.png.
2.5.2. Effect of pH on the anthocyanins
At very low pH, the anthocyanin color is red, but this decreases strongly at higher pH. Color
changes in the anthocyanins are due to chemical equilibrium that occurs depending on the four
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various forms that anthocyanin/aglycones present. According to Gradinaru, the copigmentation
(association of an anthocyanin chromophore with the planar electronically saturated part of the
copigments) is responsible for the greater absorbance that can be observed at visible range. In many
papers, the hibiscus pH extracts turn color around 2.4. According to Gradinaru, at neutral or less
acidic pH, the anthocyanins are colorless; therefore, their degradation is pH dependent (Gradinaru,
Biliaderis, Kallithraka, Kefalas, & Garcia-Viguera, 2003; Wong, Yusof, Ghazali, & Che Man, 2002;
Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, 2014; Cisse, Dornier, Sakho, Diop,
Reynes, & Sock, 2009b).
2.5.3 Effect of temperature on the anthocyanins
Many studies have shown that high temperature can affect the anthocyanins color, lifespan,
etc. As stated by Gradinaru et al temperature had a major influence on the degradation kinetics”.
Treatments by heat and storage at high temperature lead to anthocyanin degradation and color
changes going from red to brown. The only positive effect that hot extraction can have on hibiscus
calyces are that an increase from 30 to 100◦C can sxtract more anthocyanins and sugar but also
contribute to neutralization of microorganisms such as total microorganisms’ flora, fecal coliforms
and yeasts/molds. Thermal treatment such hot extraction and pasteurization (90◦C for 5 min)
negatively impacts the physicochemical characteristics of hibiscus calyces. At high temperature, the
pigment–copigments interaction is weakened (Cissé, Bohuon, Sambe, Kane, Sakho, & Dornier,
2012; Gradinaru, Biliaderis, Kallithraka, Kefalas, & Garcia-Viguera, 2003; Sarni-Manchad &
Cheynier 2006; Brouillard, Dangles, & Harborne, 1994.
Light has two opposite effects on anthocyanins: in vivo within the plant itself, light promotes the
biosynthesis of the anthocyanins but in vitro in the extracts or other derivatives of hibiscus, light
12
accelerates the degradation of the anthocyanins. Generally, the anthocyanins are unstable when
exposed to UV radiation or visible light or to other ionizing radiation. Their photo-oxidation gives
the same decomposition as those obtained by heating.
2.5.4 Effect of water, oxygen and enzymes
Some authors have shown that anthocyanin breakdown occurs from contact with water.
Anthocyanins degradation requires water availability. When anthocyanin degradation due to high
water activity occurs, it can lead to the formation of aglycones or chalcone (Erlandson, & Wrolstad,
1972; Gradinaru, Biliaderis, Kallithraka, Kefalas, & Garcia-Viguera, 2003; Markakis, Jurd, (1974).
Oxygen is believed to be one of the main agents that destroy anthocyanins. Under its effect, whether
directly (oxidation) or through oxidized elements, phenolic pigments are altered. This result in
formation of colorless or brown pigments (Gradinaru, Biliaderis, Kallithraka, Kefalas, & Garcia-
Viguera, 2003) have shown that when exposed to high relative humidity without a good barrier that
is impermeable to oxygen, faster anthocyanin degradation is observed.
The phenolic pigments can be degraded by proteins that have enzymatic activities such as
galactosidases and peroxidases (Cisse, Dornier, Sakho, Diop, Reynes, & Sock, 2009b)
2.6. Aroma compounds
Several studies have revealed that hibiscus contains volatile compounds, but the findings
differ a lot in term of amount and also in term of characteristics of those volatile profiles. Many of
the authors have mentioned that difference can be related to factors such as heat treatment, variety,
and extraction methods used to isolate the volatile (solvent used, time, solute concentration)
(Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall, 2011; Ramírez‐Rodrigues, 2010; Chen,
13
Huang, Ho, & Tsai, 1998; Gonzalez-Palomares, Estarrón-Espinosa, Gómez-Leyva, & Andrade-
González, 2009. By using cold (22 ◦C for 4 h) and hot (98 ◦C for 16 min) extraction on both fresh
and dried calyces and using GC-MS, Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall,
(2011) identified a total of 32 aroma compounds. They found that each of the extracts presented a
different volatile profile even though in all four extracts, thirteen volatiles were found identical. The
32 compounds were divided into two groups; one constituted by 15 volatiles compounds never
determined in in earlier studies and the second group by components such as limonene, linalool, α-
terpinol, eugenol, and furfural that were previously reported by Chen, Huang, Ho, & Tsai in 1998
and Salvador Gonzalez-Palomares in 2009.
In 1998, Chen, Huang, Ho, & Tsai reported the presence of 37 compounds including fatty acid
derivatives, sugar derivatives, phenolic derivatives and terpenes. They found lipid derivative (C6)
and 5-methyl 2-furfural, respectively, dominant in fresh and dried Roselle. The α-terpineol, linalool
oxide, and limonene were significantly reduced by drying. According to the authors, terpenes
derivative with fragrance are responsible of the Roselle flavor (Chen, Huang, Ho, & Tsai, 1998).
Salvador Gonzalez-Palomares et al have used SPME-GC-MS to identify volatiles in hibiscus
products. In their experiment, they have used fresh Roselle, ethanol as an extracting solvent and have
applied seven spray drying temperatures (150, 160, 170, 180, 190, 200 and 210 °C). In the Roselle
extract, 20 volatiles (terpene, esters, aldehydes and phenolic derivatives) compounds were reported.
In the powder, 14 volatile compounds were reported; ten were identical to those found in the extract
but with smaller amounts. The appearance of 4 other volatiles (furfural, cis-linalool oxide, furanic
linalool oxide Z and E, and eugenol) were justified by spray drying temperature, which causes
chemical degradations during the process, leading to their formation. They reported that the
alteration of sugar was responsible for the furfural, chemical destruction of and fatty acid generated
14
the cis-linalool oxide and the furanic linalool oxide Z and E and that the spray drying process
temperature leads to the eugenol (Gonzalez-Palomares, Estarrón-Espinosa, Gómez-Leyva, &
Andrade-González, 2009).
2.7 Microbiological quality
According to Cisse et al, microbiological problems are, in general, not encountered in
Hibiscus sabdariffa calyces or its derivatives such as the juices. This is due to the lower pH (Cisse,
Dornier, Sakho, Diop, Reynes, & Sock, 2009b)
2.8 Production of hibiscus and commercialization.
Pre-harvest operations include planting seeds at the beginning of the rainy season. The
calyces are considered ripe when the flowers have dropped but before the seed pods is dried and
opened. This stage determined the harvesting period of the fresh calyces. Calyces and seedpods are
separated by hand, or by using a metal tool. Drying under the sun is the mostly used, but also drying
tunnels or using air dryers can be used but should not be conducted under temperature higher than
43OC. To avoid contamination (insects, birds, rodents and dust) during harvesting, drying and
storage, special care is required (http://www.fao.org/3/a-av006e.pdf).
Studies have shown that countries around the world such as China, Thailand, Mexico, Egypt,
Senegal, Mali, Chad, Sudan, Tanzania and Jamaica produce and commercialize hibiscus; but for
most, the production is for local consumption. Market prices of hibiscus fluctuate a lot depending on
factors such as the oversupply, online transactions that occur with increases in demand of calyces,
amount of rainfall but also to climate and quality control problems. The main importers of hibiscus
calyces are Germany and the United States. Hibiscus calyces of best quality has red color with a
15
good sour-fruity taste and are clean. For commercial purposes, the calyces can be dried or frozen and
packaged. Senegal dried hibiscus calyces sold in Europe are filled in balls that can weighing 175 lbs
(80 kg). Senegalese farmers also supply Adina for Life the producer of Hibiscus Lemon Bissap
located in California, USA McClintock, & El Tahir, 2011.; FAO 2004; Ramírez‐Rodrigues, 2010).
2.9 Main uses, consumption, trends, and health benefits
Hibiscus is used for domestic consumption and in traditional medicine. Due to some of its
characteristic such as red color, refreshing properties, nutritious functional beverage with health
benefits and its applications in food, pharmaceutical, cosmetic and wine industries, interest has
grown in its production. It is used as a non-alcoholic beverage (hot, cold and with sugar), tea,
vegetable infusion, for jellies, jam, syrup, cocktails, etc. The refreshing sour tasting juice called
“Bissap” in Senegal (mainly used during hot weather and the month of Ramadan), da bilenni’ (Côte
d’Ivoire, Mali, Burkina Faso) is sold in urban markets. In African countries and in West India, it is
used mainly as a vegetable, but also as a beverage whereas in Europe, the calyces are utilized to
make extracts for flavoring liqueurs. The Food and Drug Administration in the United States allows
hibiscus extracts in alcoholic beverages. In the market place, ready to drink beverages using hibiscus
as a main ingredient are being manufactured and some of them are Hibiscus Lemon Bissap, Cnita
Aguas Fresca, Squish Hibiscus Presse and Simply Hibi. The leaves and green calyces are more used
as vegetable in sauces mixed with some ingredients and in Senegal a condiment called ‘bëkëj’ made
for the leaves or green calyces is eaten with a rice dishes. (Da-Costa-Rocha, Bonnlaender, Sievers,
Pischel, & Heinrich, 2014; McClintock, & El Tahir, 2011; FAO 2004; Cisse, Dornier, Sakho,
Ndiaye, Reynes, & Sock, 2009a; Wong, Yusof, Ghazali, & Che Man, 2002; Chiou, & Langrish,
2007., Ramírez‐Rodrigues, et al 2010). Many researchers have shown that the growing interest in
16
hibiscus and its derivatives is due to its health or medical benefits and its basic nutritional value. The
health benefits associated with hibiscus are multiple. It can lower the blood pressure; Herrera-
Arellano, Flores-Romero, Chavez-Soto, & Tortoriello, (2004) found that Hibiscus sabdariffa can
lower the systolic blood pressure on average from 139. to 124 mm Hg and the diastolic from 91 to
80 mm Hg. Other scientists have also reported that daily consumption of hibiscus tea like in diet may
lower the blood pressure (McKay, Chen, Saltzman, & Blumberg, 2010).
Some researchers have also found that many plants are popularly associated with weight loss. They
reported this “Seven species present pre-clinical data that indicate a potential role in the control of
certain conditions which are associated with obesity, such as hyperlipidemia”. Among those species
figured Hibiscus sabdariffa. It is believed to have no side effects. (Dickel, Rates, & Ritter, 2007 and
Hopkins, Lamm, Funk, & Ritenbaugh (2013) reported that antioxidant properties of hibiscus might
be related to Tocopherol or Vitamin E and acid ascorbic or Vitamin C. Much more health benefits
were reviewed by Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, (2014) and authors
such as Hopkins, Lamm, Funk, & Ritenbaugh, (2013) reported properties such as anticancer activity,
antiaging, antiparasitic and bactericidal, anti-inflammatory properties, but also effects, on diabetes,
nephro- and hepato-protective, renal/diuretic effect, anti-cholesterol, antispasmodic effect on uterus
and intestine smooth muscles etc. Some of these effects can be related to strong antioxidant
activities, inhibition of α-glucosidase and α-amylase, inhibition of angiotensin-converting enzymes,
and direct vaso-relaxant effect or calcium channel modulation (Da-Costa-Rocha, Bonnlaender,
Sievers, Pischel, & Heinrich 2014). Hibiscus bioactive elements such as organic acids, anthocyanins,
polysaccharides and flavonoids provide it with all these pharmacological properties (Eggensperger
& Wilker, 1996; Müller & Franz, 1990).
17
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24
CHAPTER III
MATERIALS AND METHODS
This work was done in the FST and HABB1 laboratories in Food Science and Technology
Department of Virginia Polytechnic Institute and State University, Virginia, United States of
America.
3.1 Plant material and water used for the extraction
The raw materials used for this study are dried calyx of Hibiscus sabdariffa L "Vimto"
variety purchased from Senegal, West Africa. The calyces were produced in some villages located in
the Department of Nioro, region of Kaolack in Senegal. The production of the calyces is done under
the supervision of qualified persons or non-governmental organizations. They were dried and packed
under the direction of APROVET, that helps the local producers of hibiscus. The samples were
purchased in May 2015. The packaging material used are large plastic bags designed so that
moisture, light, and insects cannot easily penetrate and alter calices. Another sample of Hibiscus
calyces was obtained from Egypt in March 2016. The samples were stored at room temperature in
Senegal and also in the USA. Hibiscus samples were cleaned twice. One at the Institute of Food
Technology lab in Dakar to remove contaminants and seeds. A second time at the laboratory of Food
Science and Technology Department in HABBI (Human and Agricultural Biosciences Building I),
Virginia Tech.
Distilled water (purchased in Kroger store) and formulated Dakar (Senegal) were used for juice
extraction. To formulate Dakar Water, major elements such as NaCl, MgSO4, CaCl2, NaHCO3, and
CaCO3 were added to distilled water based on their respective proportion given by OMS (OMS or
Human Health Organization water quality bulletin of the urban area of Dakar). Thus the following
25
proportions were added to volume of 6L of distilled water. Table salt (NaCl) 1.9g; epsom salt
(MgSO4) 0.7g; calcium (chloride CaCl2) 0.5g; baking soda (NaHCO3) 0.1g and chalk (CaCO3) 0.5g.
After addition of these elements, the pH was adjusted to the pH of Dakar water given by the same
source (OMS or Human Health Organization water quality bulletin of the urban area of Dakar).
3.2 Aqueous preparation and freeze-drying process.
As the calices are quiet large, before extraction they were ground in order to increase their
surface area with the water facilitating the juice extraction process. For this purpose, a Ninja brand
grinder was used. A total of two tests was performed and for both types of extraction carried out in
this study, a mass ratio of 1:15 (one kilogram of calyces per 15 liters of water) was used. Cold
extractions were done at room temperature (around 22º C) and the extraction time was set for 4
hours. A time temperature of 30 minutes for 98 º C was applied for all hot extractions. For the latter,
the Roselle calyces were always added at the boiling water and the temperature was monitored using
a thermometer.
Pots, bucket and jars and tanks were used when extracting higher volumes but to carry out smaller
extractions just lab materials such as beakers of one liter or more and funnel filter were used. Then,
all macerates were filtered using gravity filtration. In some cases, the macerate was filtered using a
stainless steel sieve of 1mm diameter in order to remove the bigger residues before performing the
finest filtration which was done using a cloth filter of 25 µm pore size.
For each test, after filtration, the parameters pH, Brix, anthocyanin content and aroma compounds of
the extract solution were immediately measured. Then the extracts solution was frozen in a -80º C
freezer (model 88600A Thermo Fisher Scientific Ashville, NC USA). The frozen extracts were then
freeze-dried for 72 hours in a Freeze-dryer (LABCONCO Kansas City, Missouri USA). At the end
26
of the freezing-drying process, the dried and instant hibiscus powder was immediately collected in
plastic bags then completely covered with aluminum foil in order to avoid the effect of the of
moisture, and the light. Anthocyanins and aroma compounds of this freeze-dried hibiscus were later
determined.
3. 3 Experiment I: Effects of Origin Water and Temperature on the Anthocyanins and Volatile of
Hibiscus.
This test consisted of the evaluation the effect of origin of hibiscus, the water used and the
extraction temperature as well as their interactions with anthocyanins and aroma compound of
hibiscus extracts and their freeze-dried products.
To do so, a 23 (two, x three) factorial experimental design was conducted in triplicate. The two levels
of each parameter being respectively for origin Egyptian vs. Senegalese; distilled water vs. Dakar
water for water and cold (22º C / 4 hours) vs. hot (98 º C / 30 mn) for temperature. The Table 6
below gives a summary of the different combinations of the three variables (source, water and
temperature). After filtration, pH, brix, anthocyanins and volatiles were measured and the rest of the
extracts was used for freeze-drying for process. After this step, anthocyanins and volatiles in the
instant powder were determined in order to compare these values to those of the fresh product.
Analyses of the experimental results were performed using Analysis of Variance (ANOVA), mean
comparisons using Tukey’s HSD and a Slice test with a level of significance of 5% (α=0.05. The
software JMP® Pro 11.0.0 statistical analysis software (SAS, Cary, NC, USA) used was.
27
Table 6. summary of the different combinations of the three variables (source, water and
temperature) for test 2.
Source Water Temperature
Egyptian Hibiscus Distilled Cold ( 22 ºC / 4 hours)
Egyptian Hibiscus Distilled Cold ( 22 ºC / 4 hours)
Egyptian Hibiscus Distilled Cold ( 22 ºC / 4 hours)
Egyptian Hibiscus Distilled Hot (98ºC / 30 mn)
Egyptian Hibiscus Distilled Hot (98ºC / 30 mn)
Egyptian Hibiscus Distilled Hot (98ºC / 30 mn)
Egyptian Hibiscus Dakar Water Cold ( 22 ºC / 4 hours)
Egyptian Hibiscus Dakar Water Cold ( 22 ºC / 4 hours)
Egyptian Hibiscus Dakar Water Cold ( 22 ºC / 4 hours)
Egyptian Hibiscus Dakar Water Hot (98ºC / 30 mn)
Egyptian Hibiscus Dakar Water Hot (98ºC / 30 mn)
Egyptian Hibiscus Dakar Water Hot (98ºC / 30 mn)
Senegalese Hibiscus Distilled Water Cold ( 22 ºC / 4 hours)
Senegalese Hibiscus Distilled Water Cold ( 22 ºC / 4 hours)
Senegalese Hibiscus Distilled Water Cold ( 22 ºC / 4 hours)
Senegalese Hibiscus Distilled Water Hot (98ºC / 30 mn)
Senegalese Hibiscus Distilled Water Hot (98ºC / 30 mn)
Senegalese Hibiscus Distilled Water Hot (98ºC / 30 mn)
Senegalese Hibiscus Dakar Water Cold ( 22 ºC / 4 hours)
Senegalese Hibiscus Dakar Water Cold ( 22 ºC / 4 hours)
Senegalese Hibiscus Dakar Water Cold ( 22 ºC / 4 hours)
Senegalese Hibiscus Dakar Water Hot (98ºC / 30 mn)
Senegalese Hibiscus Dakar Water Hot (98ºC / 30 mn)
Senegalese Hibiscus Dakar Water Hot (98ºC / 30 mn)
3. 4 Experiment II: Effects of freeze-drying on anthocyanins and volatiles of instant Hibiscus
powder obtained from Hot and Cold Extracted juice.
28
To investigate the effect of freeze-drying on hibiscus derivatives, hot and cold extraction
were conducted in three replicates each. Senegalese Hibiscus” Vimto” variety and distilled water
was used for this purpose and a ratio of 1:15 as mentioned above was applied. The following table is
a summary of the two processes.
Table 5. Extraction mode, ratio and time/temperature for test 1.
Factors Cold Extraction Hot Extraction
Samples Senegalese Hibiscus “Vimto” Senegalese Hibiscus “Vimto”
Ratio 1:15 1:15
Time/ Temperature 4 hours / 22 º C 30 mn / 98 º C
For each of the processes, after extraction, filtration was performed and then pH, brix, anthocyanins,
and volatiles measured immediately and then the process of freeze-drying initiated. The
anthocyanins and volatiles were determined on both instant powders obtained from freezing drying.
To evaluate the effect of freeze-drying on the dried products, comparison of the results from each
fresh and those from its instant powder (or freeze-drying product) was made. High-performance
liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC–MS) were used
respectively, for anthocyanins and volatile compounds measurements. All data were analyzed by
performing Analysis of Variance (ANOVA) and a level of 5% (α = 0.05) as a criterion of statistical
significance was taken into account to determine whether a variable or variables interaction have
effect or not. JMP® Pro 11.0.0 statistical analysis software (SAS, Cary, NC, USA) was used to
perform the analyses. Follow up statistical analyses were conducted on all significant effects by
performing mean comparisons using Tukey’s HSD and Slice tests.
29
3.5 pH measurement
For both tests, after extraction the pH values was measured (Fisher Scientific™ accumet™
Research AR25, Fisher Scientific, Pittsburgh, PA, US). The pH meter was calibrated using Buffer
solutions of pH 4.00 and pH 7.00.
3.6 Brix measurement
Brix measurement was performed using a Refractometer ATAGO portable.
3.7 Total anthocyanins and hibiscidin measurement by HPLC analysis
Anthocyanins are some of the compounds dissolved in n hibiscus solutio extracts. They are
constituted of elements but here we have only measured the total anthocyanins and the hibiscidin.
This was analyzed using high-performance liquid chromatography (HPLC). The specifications of
HPLC are: Agilent Technologies model 1260 series HPLC system (Santa Clara, CA), a column
25cm x 0.45cm i.d. 5µ C – C18C2 Luna column was obtained from Pheromenex (Torrex, CA) was
used and the mobile phases were composed of two solutions: solution A (99% distilled water, 1%
acetonitrile) and mobile phase B (99% distilled water, 1% acetic acid) with a flow rate of 1.0ml/min
and the Column temperature was set and maintained at 40°C.
3.8 Aroma compounds analyses by SPME and GC-MS analysis
The aroma compounds of the hibiscus juice were extracted by headspace solid-phase micro-
extraction (HS-SPME) and analyzed with gas chromatography-mass spectrometry (GC-MS). The
method described by Sheibani was used. For the extraction of the volatiles, a Shimadzu GCMS-
QP2010 Ultra equipped with an AOC-5000 Plus SPME auto-sampler (Shimadzu Scientific,
Columbia, MD, USA) was used as well as for injection into the GC-MS and identification. The
30
extraction injection and identification procedure followed the method described by Sheibani,
Duncan, Kuhn, Dietrich, Newkirk, & O'Keefe, (2015). The only difference being the column
specification. A Shimadzu SH Rxi-5MS column with 30.0m of length, 0.25µm of thickness and a
diameter of 0.25mm was used here.
31
REFERENCE
Sheibani, E. (2014). Effects of water chemistry and panning on flavor volatiles and catechins in teas
(Camelia Sinensis). Ph.D Dissertation (Advisor: Sean F. O’Keefe). Virginia Polytechnic
Institute and State University, Blacksburg, VA. 152 pp.
Sheibani, E., Duncan, S. E., Kuhn, D. D., Dietrich, A. M., Newkirk, J. J., & O'Keefe, Sean F. (2015).
Changes in flavor volatile composition of oolong tea after panning during tea processing.
Food Science & Nutrition, n/a-n/a.
OMS or Human Health Organization water quality bulletin of the urban area of Dakar
32
CHAPTER IV
RESULTS AND DISCUSSIONS
Results Experiment I
4.1 Effects of Origin, Water and Temperature on Total Anthocyanin Contents
A 2x3 factorial experimental design was applied to evaluate the effects of origin, water, and
temperature on hibiscus anthocyanins. Based on the standard curve, the determination of total
anthocyanins (in µg/mL) is shown in Table1.
33
Table 1. Total Anthocyanin contents of the samples
Origin Water Temperature
(°C)
Total Anthocyanins
(mg/mL)
Egypt Hib Distilled water Cold Ext 1659.21
Egypt Hib Distilled water Cold Ext 1547.47
Egypt Hib Distilled water Cold Ext 1567.99
Egypt Hib Distilled water Hot Ext 1247.09
Egypt Hib Distilled water Hot Ext 1192.40
Egypt Hib Distilled water Hot Ext 1341.04
Egypt Hib Dakar water Cold Ext 1622.59
Egypt Hib Dakar water Cold Ext 1488.21
Egypt Hib Dakar water Cold Ext 1485.79
Egypt Hib Dakar water Hot Ext 1208.51
Egypt Hib Dakar water Hot Ext 1336.19
Egypt Hib Dakar water Hot Ext 1299.40
Senegal Hib Distilled water Cold Ext 1293.64
Senegal Hib Distilled water Cold Ext 1129.38
Senegal Hib Distilled water Cold Ext 1268.25
Senegal Hib Distilled water Hot Ext 1292.71
Senegal Hib Distilled water Hot Ext 1168.41
Senegal Hib Distilled water Hot Ext 1384.03
Senegal Hib Dakar water Cold Ext 1422.99
Senegal Hib Dakar water Cold Ext 1275.43
Senegal Hib Dakar water Cold Ext 1273.50
Senegal Hib Dakar water Hot Ext 1391.51
Senegal Hib Dakar water Hot Ext 1237.39
Senegal Hib Dakar water Hot Ext 1441.61
A 0.05 criterion of statistical significance was used to determine whether a variable or variable
interaction has an effect or not. As shown in the JMP output in Appendix A1, the factors Origin and
Temperature were statistically significant with respective p-values of 0.0036 and 0.0025 as well as
their interaction Origin*Temperature with a p-value of 0.0002. The null hypotheses test regarding
Origin and Temperature as well as their interaction total anthocyanin contents were that there are no
treatment effects and that all treatment means were equal. As there is evidence of significant effect,
those null hypotheses were rejected. Therefore, Multiple Comparisons (post hoc tests) were
34
performed to make inferences about the individual pairs of treatment means to see which pairs of
treatment means do and do not differ significantly. A useful graphic, the LS Means Plot provides a
good way to illustrate how the group mean varies and how this interaction between Origin and
Temperature evolved. Least Squares Means being defined as a linear combination from a linear
model; (http://webpages.uidaho.edu/calsstatprog/sas/workshops/glm/lsmeans.htm). The LS Means
Plots for each main effect (Water, Origin and Temperature) as well as the interaction
Origin*Temperature plot are shown in Figure 4.1 below for a better understanding of factors
affecting the anthocyanin extraction.
Figure 1. LS Mean plot for Water (A), Origin (B), Temperature (C) and the interaction
Origin*Temperature plot (D).
As can be noticed, the plot A shows an almost straight line indicating that the amount of anthocyanin
stayed the same whether distilled water or Dakar water was used. This means that the quality of the
water did not affect the total anthocyanin contents in this case. In contrast, the plots B and C
A B
C D
35
respectively for the variable source and temperature display lines that have clear trend. A decrease of
the amount of total anthocyanins is noticed in hot extraction samples. In figure D, the two-lines of
the LS Means Plot cross over each other illustrating the interaction between Source*Temperature.
The printed line trend shows a reduction in the total anthocyanins content in Egyptian hibiscus from
cold to hot conditions but for the Senegalese hibiscus, a slight difference was noticed between cold
and hot conditions. Globally, among the overall effects, the only evidence of significant two-way
interactions was between Source and Temperature. Thus, the follow up analysis focused on that
interaction. For more details on this interaction, a pairwise comparisons on the marginal means was
performed. A Tukey's HSD “honest significant difference” procedure was used for this purpose, a
considering an error rate at a maximum of 0.05. A Tukey’s HSD is defined as a “pairwise
comparisons technique that uses the Studentized range distribution to construct simultaneous
confidence intervals for differences of all pairs of means”. In this test, different letters stand for
significant difference, (Chapter3, Lecture Analysis of Variance, Stat 5606: Biometry II, Spring 2015
Yili Hong).
Table 2. Results of the Tukey HSD for the interaction Source*Temperature on total anthocyanins of
the Egyptian and Senegalese hibiscus
Level Connecting
letter
Least Sq Mean
Egypt Hib,Cold Ext A 1561.88
Senegal Hib,Hot Ext B 1319.28
Senegal Hib,Cold Ext B 1277.20
Egypt Hib,Hot Ext B 1270.77
(Levels not connected by same letter are significantly different.)
The outcome of Tukey`s HSD test shows two different groups that are significantly different because
they are not connected by the same letter. On one hand, it shows the total anthocyanins obtained
36
with the Egyptian hibiscus juice cold extract (letter A) is different from the other samples (Senegal
Hib,Hot Ext, Senegal Hib,Cold Ext, and Egypt Hib,Hot Ext). These samples are connected by the
same letter B and with slight difference in their means indicating that they are not significantly
different from one another. The Least Square Mean of Egypt Hib,Cold Ext (1561.9 mg/L) is much
higher compared to the Least Square Mean obtained while carrying out hot extraction on the same
product (1270.8 mg/L); thus the variable temperature really has an effect on the anthocyanin
contents. Both time and temperature are important and we chose times for extractions based on
previous work. As the factors source and temperature were statistically significant as well as their
interaction Source*Temperature, a Slice test was performed in order to be able to see how each
factor evolves in this interaction upon the level the other factor. An interpretation given of a Slice
test is: ‘Comparisons among the levels of A depend on the level of B they are performed at; or
Comparisons among the levels of B depend on the level of A they are performed at”. The technique
of slicing is a procedure for which the correct terms are simple effects or simple main effects.
(http://www.ats.ucla.edu/stat/sas/library/SASSlice_os.htm accessed on July 28th 2016).
The JMP output in Appendix X shows the results of the different Slice tests indicating as can be
noticed that only Egyptian Origin has a significant effect (p-value<.0001*) and also only cold
temperature (p-value <.0001*) has significant effect on the total anthocyanin contents. However, as
we do not have a wide range of samples from Egypt to test, firm conclusion on the concentration in
Egyptian hibiscus is not possible with our data.
37
4.2 Effects Origin, Water and Temperature on Hibiscidin contents
Hibiscus contains many kinds of anthocyanins and among them we have the delphinidin-3-
sambubioside (also known as delphinidin-3-xylosylglucoside or Hibiscidin), which constitutes the
majority (around 70%) of the anthocyanins of hibiscus. (Francis, 1973; Wang et al., 2000). That is
why its amount was evaluated in this study in order to see how it would evolve in regard to the
experiments we have carried out. The results are displayed in the following Table 3.
38
Table 3. Hibiscidin content of the samples based on the different combinations
Source Water Temperature(°C) Hibiscidin mg/L
Egypt Hib Distilled water Cold Ext 913.15
Egypt Hib Distilled water Cold Ext 820.51
Egypt Hib Distilled water Cold Ext 822.47
Egypt Hib Distilled water Hot Ext 674.95
Egypt Hib Distilled water Hot Ext 638.83
Egypt Hib Distilled water Hot Ext 731.47
Egypt Hib Dakar water Cold Ext 838.21
Egypt Hib Dakar water Cold Ext 785.41
Egypt Hib Dakar water Cold Ext 778.58
Egypt Hib Dakar water Hot Ext 648.28
Egypt Hib Dakar water Hot Ext 726.92
Egypt Hib Dakar water Hot Ext 711.70
Senegal Hib Distilled water Cold Ext 705.83
Senegal Hib Distilled water Cold Ext 607.50
Senegal Hib Distilled water Cold Ext 711.61
Senegal Hib Distilled water Hot Ext 767.19
Senegal Hib Distilled water Hot Ext 686.98
Senegal Hib Distilled water Hot Ext 838.91
Senegal Hib Dakar water Cold Ext 777.18
Senegal Hib Dakar water Cold Ext 711.18
Senegal Hib Dakar water Cold Ext 681.76
Senegal Hib Dakar water Hot Ext 815.22
Senegal Hib Dakar water Hot Ext 734.81
Senegal Hib Dakar water Hot Ext 843.70
In Appendix B1, it can be noticed that at significance level of α = 0.05, only the interaction
Origin*Temperature has a significant effect on the hibiscin content of the samples with a p-value of
0.0001*. As for the total anthocyanins, the only significant two-way interaction was between Origin
and Temperature. Therefore, the next part of the analysis focused on that interaction between those
two variables. Here also, a Least Squares Means Plot was used for the visualization
Origin*Temperature interaction.
39
Figure 2. LS Means Plot of the interaction Source*Temperature
As can be observed in this plot, the interaction between the two variables is demonstrated by the two
lines that are not parallel, they overlap. For pairwise comparisons on the marginal means, a Tukey's
HSD test was applied as well considering an error rate at a maximum of 0.05.
Table 4. Outcome of Tukey HSD test for the interaction Source*Temperature effect on hibiscin
contents
As for Tukey`s HSD, levels not connected by same letter are significantly different; it can be noticed
that there are three different groups from this Table. Similarly to the results for the total anthocyanin
contents, the amount of hibiscin obtained in the combination Egypt Hib,Cold Ext (group A)
826.39±21.68 µg/mL and in Egypt Hib,Hot Ext (group C) 688.69±21.68 µg/mL are significantly
different. For the Senegalese hibiscus variety, the reverse situation was observed; there was no
significant difference between the amount of hibiscin got from the combination Senegal Hib,Hot Ext
and Senegal Hib,Cold (level being connected by the same letter).
Globally, for both components total anthocyanin and hibiscin, the interaction between Source and
Temperature has a significant effect on their amounts, characterized by a decrease. Additionally, the
Level (Combinations) Connecting letters Least Squares Mean
Egypt Hib,Cold Ext A 826.39
Senegal Hib,Hot Ext A B 781.13
Senegal Hib,Cold Ext B C 699.18
Egypt Hib,Hot Ext C 688.69
40
variable Temperature has also an effect on total anthocyanin contents whether hibiscus form Egypt
or hibiscus from Senegal was used and also whether distilled water or reformulated water was used
for the extraction. This decrease in the amount of total anthocyanins observed for hot extraction was
expected based on the results from previous studies. Earlier studies have shown that the anthocyanin
contents in hibiscus extracts varies depending on factors such as variety, extraction temperature,
production area, and rainfall. According to Gradinaru et al (2003), temperature contributes to the
degradation kinetics. This is in agreement Ramirez Rodrigues 2010 who, while evaluating the hue
tint (HT), (measurement of color degradation in anthocyanin containing products) in cold and hot
extracts of hibiscus, noticed that extracts obtained with cold water had lower HT values than those
obtained with hot water. This finding allowed her to conclude “temperature had an effect on hibiscus
extract color, and thus anthocyanins”. A lower HT is desirable as it indicates less anthocyanin
degradation.
Contrary to the temperature, the water chemistry did not affect the amounts of anthocyanins. The
results were almost the same whether distilled water or Dakar water was used and whatever was the
combination with the other parameters. This indicates that the hibiscus juice or concentrates
produced by small producers, mainly groups of female entrepreneurs are of good quality in terms of
eventual risk or concerns coming from the water. Hence purchasing hibiscus concentrate from those
individuals for exportation or to make hibiscus derivatives such as juice instant powder other
functional foods is a better way to promote activities around this tropical plant and as a consequence,
generate more income and reduce poverty in some rural areas.
41
Results Experiment II
4.3 Effect of Freeze-Drying on Total Anthocyanins and Hibiscin Contents using Hot Extraction
Procedure
To assess the effect of freeze-drying on total anthocyanins and hibiscin contents, Senegal
hibiscus (Vimto variety) was used and a hot extraction was conducted using three replicates. Each
extract was then divided into two parts and one part was freeze-dried. Anthocyanin contents (total
anthocyanins and hibiscin contents) were quantified by using high performance liquid
chromatography (HPLC) and the visible absorbance wavelength of 520nm on the extracts and also
on the powder obtained by freeze-drying the extract. Each experimental unit was analyzed three
times and the three replications were averaged. The Tables 5 and 6 below show, respectively, the
results for the total anthocyanins and the hibiscin contents of the samples.
Table 5. Total anthocyanin in hot and Table 6. Hibiscin contents in hot freeze-
dried hibiscus extracts and freeze-dried hibiscus extracts
determined by HPLC determined by HPLC
The data were analyzed using a One-Way ANOVA. The results showed a significant difference
between the hot extract and its freeze-dried product (p-value = 0.0013) in term of anthocyanin
content. The following plot illustrates better this significant difference between the fresh extract and
the freeze-dried extract.
Product Total Anthocyanins (mg/L)
E1 1462.0 ± 20.1
E2 1425.4 ± 20.1
E3 1458.1 ± 20.1
F E1 1333.9 ± 14.3
F E2 1348.1 ± 14.3
F E3 1319.5 ± 14.3
Product Hibiscin(mg/L)
E1 815.9 ± 15.4
E2 790.2 ± 15.4
E3 788.4 ± 15.4
F E1 732.6 ± 10.4
F E2 746.1± 10.4
F E3 753.0 ± 10.4
42
Figure 4.3 Normal Quantile Plot illustrating the difference between the hot extract and its freeze-
dried powder.
Likewise, as for the total anthocyanins, evidence of significant difference was also noticed in
hibiscidin contents between the hot extract and its freeze-dried powder. The One-Way ANOVA gave
a p-value of 0.0013*. This result, (1448.5 ± 20.1 for the fresh extract and 1333.9 ± 14.3 for the freeze-
dried powder was not expected because among the steps (freezing and freeze-drying) applied to get
the freeze-dried powder from the extract, none is known to have an effect on the anthocyanins of
hibiscus. This result might be due to the fact that hot extraction makes extracts less stable over the
time. Or perhaps, parts of the total anthocyanins was lost during the time it takes to obtain the freeze-
dried product. Earlier studies have also reported this. Cisse, Vaillant, Kane, Ndiaye, & Dornier, 2012
have demonstrated that procedures including heat and/or pasteurization make the extract less stable
and induced color changes during storage. Duangmal, Saicheua, & Sueeprasan 2008 have also
demonstrated, using CIELAB system to evaluate the color stability of freeze-dried Roselle
anthocyanins, that the total anthocyanins were responsible for the changes in Chroma. When
maltodextrin and trehalose were used as stabilizers, they noticed that anthocyanin degradation was
slowed.
43
4.4 Effect of Freeze-drying on Total Anthocyanins and Hibiscidin Contents using Cold Extraction
Procedure
The effect of freeze-drying on total anthocyanins and hibiscin content was also investigated on
Senegal hibiscus (Vimto variety). The same process (extraction, freeze-drying and measurements
using HPLC) were used except that cold extraction was realized in two replicates instead of three.
Total anthocyanins and hibiscin contents were quantified by using a HPLC with visible absorbance
spectra at 520nm. The results of the total anthocyanins and the hibiscin content are displayed in
Table 4.7 and Table 4.8
Table 5. Total anthocyanin in cold and Table 6. Hibiscin contents in cold freeze-
dried hibiscus extracts and freeze-dried hibiscus extracts
using HPLC using HPLC
As expected, results show no significant effect of freeze-drying on the anthocyanins in the cold
extract. The p-value (0.0564) is slightly greater than the criterion (0.05) taken as reference here. The
average amount of anthocyanins in the fresh extract (1466.4 ± 04) did not differ much from the
amount in the freeze-dried powder (1278.8 ± 65.8).
Also for the hibiscin content, a comparison was also made and no significant effect was observed
(p-value = 0.4457). The amount of hibiscin (848.4 ± 31.6) was close to that of the freeze-dried
powder (819.7± 29.2). These results can be justified by the fact that no heat was applied during all
the processes of extracting the juice and its as freeze-drying, which was conducted in cool
conditions.
Samples Total Anthocyanins (µg/mL)
C 1466.1 ± 0.4
C 1466.7 ± 0.4
F C 1232.2 ± 65.8
F C 1325.3 ± 65.8
Products Hibiscin (µg/mL)
C 826.1 ± 31.6
C 870.7 ± 31.6
F C 799.1 ± 29.2
F C 840.4 ± 29.2
44
4.5 pH and the Brix in hibiscus extracts
For all experiments carried out in this study, pH and Brix measurements were recorded just
after extraction. The pH values were constant for all extracts (cold, hot and the 2 x 3 factorial design
combining water, source and temperature) with values around 2.4. This can be explained by the fact
that that hibiscus calyces are very acidic. Also the ratio 1:15 used for all the extraction procedure,
did not provide much water that could have made easy some chemical reactions such as protonation
that could affect the pH. For the Senegalese hibiscus extracts, the Brix results, on average were 4.1
for the hot and 3.4 for the cold. In the 2 x 3 factorial experiment also, the same phenomenon was
observed. As can be seen in the Table 4.9 below, the soluble extract was less when cold extract was
made and higher in hot, despite the presence of the two other variables (temperature and source). So
whatever the combined variables were, still the highest were for hot and the lowest values for the
cold. The highest amount of dry matter in the hot extract can be explained by the fact that when
extraction is carried using high temperatures, the heat makes easy the disintegration of some soluble
particles and this increase their quantity in the juice.
45
Table 4.9 Brix in the 2 x 3 factorial experiment on Egyptian and Senegalese hibiscus
Source Water Temperature Brix
Egypt Hib Distilled water Cold Ext 3.9
Egypt Hib Distilled water Cold Ext 3.9
Egypt Hib Distilled water Cold Ext 3.8
Egypt Hib Distilled water Hot Ext 4.4
Egypt Hib Distilled water Hot Ext 4.4
Egypt Hib Distilled water Hot Ext 4.5
Egypt Hib Dakar water Cold Ext 3.8
Egypt Hib Dakar water Cold Ext 3.8
Egypt Hib Dakar water Cold Ext 3.8
Egypt Hib Dakar water Hot Ext 4.4
Egypt Hib Dakar water Hot Ext 4.6
Egypt Hib Dakar water Hot Ext 4.5
Senegal Hib Distilled water Cold Ext 3.5
Senegal Hib Distilled water Cold Ext 3.5
Senegal Hib Distilled water Cold Ext 3.6
Senegal Hib Distilled water Hot Ext 4.2
Senegal Hib Distilled water Hot Ext 4.3
Senegal Hib Distilled water Hot Ext 4.3
Senegal Hib Dakar water Cold Ext 3.7
Senegal Hib Dakar water Cold Ext 3.7
Senegal Hib Dakar water Cold Ext 3.7
Senegal Hib Dakar water Hot Ext 4.6
Senegal Hib Dakar water Hot Ext 4.5
Senegal Hib Dakar water Hot Ext 4.5
4.6 Effects of freeze-drying on the volatiles of Instant hibiscus powder obtained from cold
extraction.
Gas Chromatography - Mass Spectrometry (GC-MS) was used to identify the aroma
compounds present in the cold hibiscus extract and its freeze-dried powder. Mass spectra were
compared to NIST and Wiley libraries. Also, hydrocarbon standards (C5, C6, C7, C8, C10, C12,
C14, C16, C18, C20, C22) were run to calculate Kovats Indexes, also known as LRI, Linear
46
Retention Index, to facilitate identification of the aroma compounds. The identification of the
different flavor compounds was facilitated by consulting online databases Flavornet at
http://www.flavornet.org/flavornet.html and the Pherobase at http://www.pherobase.com/. Twenty
components were identified in the fresh, cold extract while only eight were identified in its freeze-
dried powder as can be observed in Table 4.10 and Table 4.11. This big difference in aromas
composition content between the two products can be justify by the different in soluble solids
present in the both product while running the GC-MS analyses. After freeze-drying the amount of
instant powder weighed to reconstituted solution did not generate the same amount of soluble solid
content as that of the fresh extract. This limiting factor may be the reason of the small amount of
aroma in the instant powder.
47
Table 10. Volatiles in cold Table 10. Volatiles in cold and
hibiscus extracts freeze-dried hibiscus extracts
determined by using GC-MS determined by using GC-MS Aroma compounds identified in
Cold Extract
alpha-terpineol
1-Octen-3-one
5-Methyl-2-Furancarboxaldehyde
(E)-2-Nonenal
(E-)-2-Octenal
6,10-dimethyl-5,9-Undecadien-2-one
5-6-methyl-Hepten-2-one
5-Hydroxymethylfurfural
Decanal
Eucalyptol
Furfural
Glycerol 1,2-diacetate
Heptanal
Hexanal
Acetic acid
Nonanal
Octanal
Oxime-, methoxy-phenyl-
2-methoxy-3-(2-propenyl)-Phenol
2-Decanone
Among the volatiles, three (furfural, hexanal and nonanal) were found in the cold extract and its
instant powder. The comparison of their amount of these three volatiles present in each product was
realized by comparing their peak areas. From the One Way ANOVA analysis, a significant
difference was noticed between the two products. Their comparison showed p-values that are lower
than 0.05 for all of the three volatiles allowing to conclude there is evidence of significant difference
of their concentration in the cold extract and its freeze-dried powder. The following Table 4.12
combines the different volatiles in both fresh cold extract and its instant powder as well as the p-
values of the JMP output.
Aroma in Freeze-dried Cold Extract
5-Methyl-2-Furancarboxaldehyde
Furfural
Hexanal
Nonanal
Acetic acid
2-Methyl-Benzaldehyde
Dodecane
Hexanoic acid, hexyl ester
48
Table 10. Volatiles in cold and freeze-dried hibiscus extracts determined by using GC-MS
Volatiles names Cold Extract
Freeze-dried
Cold Extract p-value
alpha-Terpineol X
1-Octen-3-one X
5-methyl-2-Furancarboxaldehyde X X
(E)-2-Nonenal X
(E)-2-Octenal X
6,10-dimethyl-5,9-Undecadien-2-one X
6-methyl-5-Hepten-2-one X
5-Hydroxymethylfurfural X
Decanal X
Eucalyptol X
Furfural X X 6.13E-05
Glycerol 1,2-diacetate X
Heptanal X
Hexanal X X 0.0037
Nonanal X X 0.0047
Octanal X
Oxime-, methoxy-phenyl-_ X
Phenol, 2-methoxy-3-(2-propenyl)- X
2-Decanone X
Acetic acid X X
2-Methyl-Benzaldehyde X X
Dodecane X X
Hexanoic acid, hexyl ester X X
4.7 Effects of freeze-drying on the volatiles of instant hibiscus powder obtained from hot extracted
juice
Similar to the cold extracts, aroma compounds present in the hot hibiscus extracted juice and
its freeze-dried powder were identified using a GC-MS. Twenty-seven compounds were identified in
the fresh hot extract and twenty-three in its instant powder. Tables 4.12 and Table 4.13 below show
those different aroma components.
49
Table 12. Volatiles in hot Table 13. Volatiles in hot
hibiscus extracts determined freeze-dried hibiscus extracts
by using GC-MS determined by using GC-MS
Aroma in Hot Extract
alpha-Terpineol
5-Methyl-2-Furancarboxaldehyde
(E)-2-Nonenal
(E)-2-Octenal
6,10-dimethyl-5,9-Undecadien-2-one
6-methyl-5-Hepten-2-one
5-Hydroxymethylfurfural
Decanal
Eucalyptol
Furfural
Heptanal
Hexanal
Nonanal
Octanal
Oxime-, methoxy-phenyl-
2-methoxy-3-(2-propenyl)-Phenol
Acetic acid
Hexanoic acid, hexyl ester
1-Nonanol
1-Octen-3-ol
(E,Z)-2,6-Nonadienal
(E)-2-Heptenal
(E)-2-Tridecenal
tert-butyl-Benzene
Benzeneacetaldehyde
D-Limonene
Furyl hydroxymethyl ketone
The results show that many all the aroma compounds detected in the hot extract were also detected
in the instant powder obtained by from freeze-drying the same extract. Statistical analysis was
performed by a One Way ANOVA to do the comparison of their respective areas in each product.
The results are shown in the Table 4.14 below.
Aroma compounds in freeze-dried Hot
Extract
alpha-Terpineol
5-Methyl-2-Furancarboxaldehyde
(E)-2-Nonenal
(E)-2-Octenal
6,10-dimethyl-5,9-Undecadien-2-one
6-methyl-5-Hepten-2-one
5-Hydroxymethylfurfural
Decanal
Furfural
Hexanal
Nonanal
Octanal
Oxime-, methoxy-phenyl-_
Phenol, 2-methoxy-3-(2-propenyl)-
Acetic acid
(E,Z)-2,6-Nonadienal
(E)-2-Heptenal
Benzene acetaldehyde
(E,E)-2,4-Nonadienal
Dodecanal
Nonanoic acid
Butanoic acid
Dimethyl phosphine
50
Table 10. Volatiles in hot and freeze-dried hibiscus extracts determined by using GC-MS and the p-
values of the Volatiles present in both of the products
Volatiles names Hot Extract
Freeze-dried
Hot Extract p- values
alpha-Terpineol X X 0.0276
5-Methyl-2-Furancarboxaldehyde X X 0.5598
(E)-2-Nonenal X X 0.0199
(E)-2-Octenal X X 0.0109
6,10-dimethyl-5,9-Undecadien-2-one X X 0.5345
6-methyl-5-Hepten-2-one X X 0.38785
5-Hydroxymethylfurfural X X 0.2160
Decanal X X 0.5269
Eucalyptol X
Furfural X X 0.9153
Heptanal X
Hexanal X X 0.0563
Nonanal X X 0.0103
Octanal X X 0.0093
Oxime-, methoxy-phenyl- X X 0.00689
2-methoxy-3-(2-propenyl)-Phenol X X 0.2140
Acetic acid X X 0.0249
Hexanoic acid, hexyl ester X
1-Nonanol X
1-Octen-3-ol X
5-methyl-2-Furanone X
(E,Z)-2,6-Nonadienal X X 0.8452498
(E)-2-Heptenal X X
(E)-2-Tridecenal X
tert-butyl-Benzene X
Benzeneacetaldehyde X X 0.3440548
D-Limonene X
Furyl hydroxymethyl ketone X
2-ethyl-1-Hexanol
X
(E,E)-2,4-Nonadienal
X
2-Furancarboxylic acid
X
Dodecanal
X
Nonanoic acid
X
51
Many of the aroma components detected in the samples and the procedure used in this study have
been identified in previous studies. Among the aroma compounds we observed, the following were
previously reported (hexanal, heptanal, limonene, octanal, nonanal, 1-octen-3-ol, acetic acid,
decanal, (E)-2-nonenal (Ramirez‐Rodrigues & others, 2011; Gonzalez-Palomares & others- 2009
and Chen, Huang, Ho, & Tsai, 1998).
As mentioned earlier in the literature review, many authors have found that differences in the
number of aroma compounds in the hibiscus or dried hibiscus can be related to factors such as heat
treatment, variety, and extraction methods used to isolate the volatile (solvent used, time, solute
concentration) (Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall, 2011;
Ramírez‐Rodrigues, 2010; Chen, Huang, Ho, & Tsai, 1998; Gonzalez-Palomares, Estarrón-Espinosa,
Gómez-Leyva, & Andrade-González, 2009).
Table 4.15 summary of all the aroma compounds found in the four different samples, the calculated
Kovats Indexes and similarity in the literature
Compound Cold Extract
Freeze-dried cold Extract
Hot Extract
Freeze-dried hot Extract
LRI (DB5) Odor description
Identied in the literature
Acetic acid X X X X 660 alkaline C
Hexanal X X X X 822 truffle, earth, nut AC
Furfural X X X X 838 ABC
Oxime-, methoxy-phenyl- X X X 898
sulfur, onion, meat
Heptanal X X 903 fat, citrus, rancid AC
(E)-2-Heptenal X X 959 AC 5-Methyl-2-furancarboxaldehyde X X X X 967 cooked meat
1-Octen-3-ol X 980 phenol C
1-Octen-3-one X 981
6-Methyl-5-hepten-2-one X X X 988 earth, herb C
Octanal X X X 1005 AC
52
tert-butyl-Benzene X 1030 citrus, mint
D-Limonene X 1035 Citrus, lemon
Benzeneacetaldehyde X X 1048 sweat
(E)-2-Octenal X X X 1060 green, nut, fat C
2-Methylbenzaldehyde X X 1086 roast Furyl hydroxymethyl ketone X 1086 roast
Nonanal X X X X 1106 AC
Hexanoic acid, hexyl ester X X X 1109 green, flower
(E,Z)-2,6-Nonadienal X X 1156
(E)-2-Nonenal X X X 1162
cucumber, fat, green C
1-Nonanol X 1172 flower C
Dodecane X X 1192 fruit, phenol
alpha-Terpineol X X X 1198 fruit, fat ABC
Decanal X X X 1207 BC
(E,E)-2,4-Nonadienal X
1217 fat, wax, green C
5-Hydroxymethylfurfural X X X 1226
Nonanoic acid X X 1263 mint, sweet
(E)-2-Tridecenal X 1264
Glycerol 1,2-diacetate X 1347 2-Methoxy-3-(2-propenyl)-phenol (eugenol) X X 1365 Turpentine, cloves
Dodecanal X 1411 green, spearmint 6,10-Dimethyl-5,9-undecadien-2-one X X X 1457
KI = Kovats Index.
A = Components earlier identified by Chen, Huang, Ho, & Tsai, 1998
B = Components earlier identified by Gonzalez-Palomares, Estarrón-Espinosa, Gómez-Leyva, &
Andrade-González, 2009
C = Components earlier identified by Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall,
2011
53
REFERENCES
http://webpages.uidaho.edu/calsstatprog/sas/workshops/glm/lsmeans.htm accessed on July
2016
Chapter3, Lecture Analysis of Variance, Stat 5606: Biometry II, Spring 2015 Yili Hong).
http://www.ats.ucla.edu/stat/sas/library/SASSlice_os.htm accessed on July 28th 2016.
Gradinaru, G., Biliaderis, C. G., Kallithraka, S., Kefalas, P., & Garcia-Viguera, C. (2003).
Thermal stability of Hibiscus sabdariffa L. anthocyanins in solution and in solid state: effects of
copigmentation and glass transition. Food Chemistry, 83(3), 423-436.
Rodrigues, M. M. R. (2010). Processing Hibiscus Beverage Using Dense Phase Carbon
Dioxide. University of Florida
Cissé, M., Bohuon, P., Sambe, F., Kane, C., Sakho, M., & Dornier, M. (2012). Aqueous
extraction of anthocyanins from Hibiscus sabdariffa: Experimental kinetics and modeling. Journal of
Food Engineering, 109(1), 16-21.
Duangmal, K., Saicheua, B., & Sueeprasan, S. (2008). Colour evaluation of freeze-dried
roselle extract as a natural food colorant in a model system of a drink. LWT-Food Science and
Technology, 41(8), 1437-1445.
online databases Flavornet at http://www.flavornet.org/flavornet.html accessed on July 8th
2016
Pherobase at http://www.pherobase.com/. on July 8th 2016
Ramirez‐Rodrigues, M. M., Plaza, M. L., Azeredo, A., Balaban, M. O., & Marshall, M. R.
(2011). Physicochemical and phytochemical properties of cold and hot water extraction from
Hibiscus sabdariffa. Journal of food science, 76(3), C428-C435.
54
Gonzalez-Palomares, S., Estarrón-Espinosa, M., Gómez-Leyva, J. F., & Andrade-González,
I. (2009). Effect of the temperature on the spray drying of roselle extracts (Hibiscus sabdariffa
L.). Plant foods for human nutrition,64(1), 62-67.
Chen, S. H., Huang, T. C., Ho, C. T., & Tsai, P. J. (1998). Extraction, analysis, and study on
the volatiles in roselle tea. Journal of Agricultural and Food Chemistry, 46(3), 1101-1105
55
CHAPTER V
CONCLUSIONS
This work focused on evaluating the effect of parameters such water, heat and origin on
hibiscus extracts and on instant powder obtained by freeze-drying part of the extracts. Parameters
such as anthocyanins as well as volatile components were determined. For the anthocyanin contents,
the variable temperature has a significant effect whether hibiscus was from Egypt or from Senegal
and also whether distilled water or reformulated water was used for the extraction. The anthocyanin
amount was higher when hot extraction was carried out. The variable Origin also impacted the
anthocyanin contents which was much more present in the Egyptian hibiscus. Although we do not
have evidence that this is true for all Egyptian hibiscus. Evidence of significant interaction effect
was noticed between these two variables on the anthocyanin content showing higher amount when
the combination Egyptian variety and cold extraction was applied. Contrary to the variables
temperature and source, the water chemistry did not affect the amounts of anthocyanins. The results
were almost the same whether distilled water or Dakar water (reformulated) was used and whatever
was the combination with the other parameters. There was no significant effect of freeze-drying on
the anthocyanins in the Cold Extract as expected. But in contrast to this, a difference was noticed in
anthocyanin contents between the hot extract and its freeze-dried powder. This result, was not
expected because among the steps (freezing and freeze-drying) applied to get the freeze-dried
powder from the extract, none is known to have an effect on the anthocyanins of hibiscus. This result
might be due to the fact that hot extraction makes extracts less stable over the time. Earlier studies
have also reported that anthocyanin were less stable Cisse et al 2012, Duangmal, Saicheua, &
Sueeprasan 2008). The pH was almost the same in all extracts. The soluble extract was less when
56
cold extract was made and higher in hot despite the presence of the two other variables (temperature
and source). This can be explained by the fact that heat makes easy the disintegration of some
soluble particles and this increase their quantity in the juice. GC-MS analysis of the volatiles showed
different profiles when comparing cold and hot extracts to their respective instant powder as well as
when comparing hot to dry extract. But many of the aroma compounds were found in both hot and
cold extraction. Globally, the results of this study can help in the optimization when processing
hibiscus derivatives such as juices, concentrates and instant powder in regards of the growing
interest worldwide in functional food product like hibiscus. The latter is a ready to use and easy to
commercialize and would be a good alternative for industrial as far as hibiscus has many potential
applications in the food, pharmaceutical, cosmetic industries, etc.
Expanding these investigations on hibiscus powder obtained from spray-drying and monitor the self-
life of both dried products over the time when stored at specific temperatures in future work can help
in activities carried around hibiscus. Likewise, the evaluation of consumer acceptability on the
different products through sensory evaluation tests also need to be done.
57
APPENDICES
APPENDIX A – 1
Figure A1 – Effect Tests of Effects of Source, Water and Temperature on the total anthocyanins content of
the samples
Least Squares Means Table Level Least Sq
Mean
Std Error
Egypt Hib,Cold Ext 1561.8828 34.717462
Egypt Hib,Hot Ext 1270.7760 34.717462
Senegal Hib,Cold Ext 1277.2046 34.717462
Senegal Hib,Hot Ext 1319.2830 34.717462
LS Means Plot
Appendix A – 2 Figure A2 – Least Squares Means Table and least square means plots of the
interaction Source*Temperature on total anthocyanins content
58
Appendix A – 3 Figure A3 – Slice source =Egyptian Hib
Appendix A – 4 Figure A4 – Slice source =Senegal Hib
Appendix A – 5 Figure A5 – Slice Temperature (°C)=Cold Ext
59
Appendix A – 6 Figure A6 Slice Temperature (°C)=Hot Ext
60
APPENDIX B– 1
Source Nparm DF Sum of
Squares
F Ratio Prob > F
Source 1 1 1813.124 0.6429 0.4344
Water 1 1 743.056 0.2635 0.6148
Temperature 1 1 4660.211 1.6523 0.2169
Source*Water 1 1 5343.255 1.8945 0.1877
Source*Temperature 1 1 72369.683 25.6591 0.0001*
Water*Temperature 1 1 950.796 0.3371 0.5696
Source*Water*Temperature 1 1 2401.854 0.8516 0.3698
Figure B1 – Effect Tests of Effects of Source, Water and Temperature on the Hibiscidin content
Least Squares Means Table Level Least Sq
Mean
Std Error
Egypt Hib,Cold Ext 826.39169 21.681117
Egypt Hib,Hot Ext 688.69694 21.681117
Senegal Hib,Cold Ext 699.18278 21.681117
Senegal Hib,Hot Ext 781.13879 21.681117
LS Means Plot
Figure B2 – Least Squares Means Table and least square means plots of the interaction
Source*Temperature on Hibiscidin content
APPENDIX C – 1 Analysis of Variance
Source DF Sum of
Squares
Mean Square F Ratio Prob > F
Product 1 19723.813 19723.8 64.7981 0.0013*
Error 4 1217.555 304.4
C. Total 5 20941.368
Means for Oneway Anova
Level Number Mean Std Error Lower 95% Upper 95%
E 3 1448.54 10.073 1420.6 1476.5
F E 3 1333.87 10.073 1305.9 1361.8
Std Error uses a pooled estimate of error variance
Figure C1 – Results of Total Anthocyanin the Senegalese Hot extraction and its freeze-dried powder.
61
APPENDIX D – 1
Analysis of Variance Source DF Sum of
Squares
Mean Square F Ratio Prob > F
Product 1 4414.9735 4414.97 25.7004 0.0071*
Error 4 687.1460 171.79
C. Total 5 5102.1195
Means for Oneway Anova Level Number Mean Std Error Lower 95% Upper 95%
E 3 798.148 7.5672 777.14 819.16
F E 3 743.896 7.5672 722.89 764.91 Std Error uses a pooled estimate of error variance
Figure D1 – Results of Hibiscidin of the Senegalese Hot extraction and its freeze-dried powder
APPENDIX E – 1
Analysis of Variance Source DF Sum of
Squares
Mean Square F Ratio Prob > F
Samples 1 35211.772 35211.8 16.2452 0.0564
Error 2 4335.026 2167.5
C. Total 3 39546.798
Means for Oneway Anova Level Number Mean Std Error Lower 95% Upper 95%
C 2 1466.43 32.920 1324.8 1608.1
F C 2 1278.79 32.920 1137.1 1420.4
Std Error uses a pooled estimate of error variance
Figure E1 – Results of Total Anthocyanin the Senegalese Cold extraction and its freeze-dried powder
APPENDIX F – 1
Analysis of Variance Source DF Sum of
Squares
Mean Square F Ratio Prob > F
Products 1 822.0836 822.084 0.8872 0.4457
Error 2 1853.2054 926.603
C. Total 3 2675.2890
Means for Oneway Anova Level Number Mean Std Error Lower 95% Upper 95%
C 2 848.420 21.524 755.81 941.03
F C 2 819.748 21.524 727.14 912.36 Std Error uses a pooled estimate of error variance
Figure F1 – Results of Hibiscin of the Senegalese Cold extraction and its freeze-dried powder
62