Improvement of Lycopene Extraction with Polygalacturonase and Cellulase from Tomato Fruits(Lycopersicon esculentum MILL) DETERIORATED BY Aspergillus niger

By

ADEDEJI OLUWABUNMI MORADEKE

Presented To

Department of Biological Science

ABSTRACT

Lycopene is the carotenoid which gives tomatoes and other red fruits their characteristic colour. It is one of the most powerful antioxidants and singlet oxygen-quenching agents. Lycopene has been found to be of great medical importance, having various anticancer effects and it is able to ameliorate several other medical conditions. Lycopene had also been found to be very useful in the food industry where it is used as food additives, colourant and functional food. Freshly ripe tomato fruits of the Roma vf and Ibadan local varieties were allowed to deteriorate after infection with a 96-hr-old culture of Aspergillus niger. The extraction of cellulase and polygalactorunase produced during the deterioration of the tomato fruits was carried out ten days after incubation of the tomato fruits. The isolated enzymes were partially purified by a combination of ammonium sulphate precipitation followed by sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS PAGE). These partially purified enzymes were characterised and employed in the extraction of lycopene from tomato peels. The yield of lycopene was 45.25mg/kg and 45.86 mg/kg for enzymes extracted from the Roma vf and the Ibadan local varieties of tomato fruits respectively. The molecular weights of the enzyme fractions obtained from the partially purified fraction were approximately 32KDa and 96KDa. The activities of the enzymes were markedly affected by the substrate concentrations as well as the temperature and pH of the reactions. Optimum temperature of 300C and 250C was obtained for the polygalacturonase obtained from the Ibadan local variety and the Roma vf variety of tomato fruits respectively. Optimum pH values of 3.5 and 3.0 was obtained for polygalacturonase and cellulase respectively obtained from both varieties of tomato fruits. The Michealis Mentens constant (Km) which is also known as the dissociation constant is the substrate concentration at half maximum velocity. Calculated from the Lineweaver Burk plot, the apparent Km values for the hydrolysis of pectin by Aspergillus niger polygalacturonase fractions for the Ibadan local variety and the Roma vf variety were approximately 0.016mg/ml and 0.013 mg/ml respectively. The apparent Km values for the hydrolysis of carboxymethyl cellulase by Aspergillus niger cellulase fractions for the Ibadan local variety and the Roma vf variety were approximately 0.45mg/ml and 0.42 mg/ml respectively.
TABLE OF CONTENT

Title Page
Dedication  -  -  -  -  -  -  -  iii
Declaration  -  -  -  -  -  -  -  iv
Certification  -  -  -  -  -  - .. v
Acknowledgement  -  -  -  -  -  -  vi
Table of content  -  -  -  -  -  -  -  viii
List of figures  -  -  -  -  -  -  ix
List of tables  -  -  -  -  -  -  - - xiii
Abstract  -  -  -  -  -  -  - . xiv
Introduction  -  -  -  -  -  - - 1
Literature Review  -  -  -  -  -  - - 3
Materials and Methods  -  -  -  -  - - 16
Results  -  -  -  -  -  -  -  - . 22
Discussion and Conclusion  -  -  -  -  -  - . 52
References  -  -  -  -  -  -  - .. 58
Appendix  -  -  -  -  -  -  - - 69

LIST OF FIGURES

FIG    TITLE   PAGE

1  Structure of lycopene 6
2  Effect of temperature on the activity of the partially purified 27
Polygalaturonase obtained from the Ibadan local variety of tomato
Fruits infected by Aspergillus niger
3  Effect of temperature on the activity of the partially purified 28
cellulase obtained from the Ibadan local variety of tomato
Fruits infected by Aspergillus niger
4  Effect of temperature on the activity of the partially purified 29
Polygalaturonase obtained from the Roma vf variety of tomato
Fruits infected by Aspergillus niger
5  Effect of temperature on the activity of the partially purified 30
cellulase obtained from the Roma vf variety of tomato fruits
infected by Aspergillus niger
6  Effect of pH on the activity of the partially purified polygalacturonase 31
obtained from the Ibadan local variety of tomato fruits infected by
Aspergillus niger
7  Effect of pH on the activity of the partially purified cellulase 32
obtained from the Ibadan local variety of tomato fruits infected by
Aspergillus niger
8  Effect of pH on the activity of the partially purified polygalacturonase  33
obtained from the Roma vf variety of tomato fruits infected by
Aspergillus niger
9  Effect of pH on the activity of the partially purified cellulase obtained 34
from the Roma vf variety of tomato fruits infected by Aspergillus niger
10  Effect of substrate concentration on the activity of the partially 35
purified polygalacturonase obtained from the Ibadan local variety
of tomato Fruits infected by Aspergillus niger
11  Lineweaver-Burk plot for the hydrolysis of pectin by the   36
partially purified polygalacturonase obtained from the
Ibadan local variety of tomato fruits infected by Aspergillus niger
12  Effect of substrate concentration on the activity of the partially 37
purified cellulase obtained from the Ibadan local variety of tomato
fruits infected by Aspergillus niger
13  Lineweaver-Burk plot for the hydrolysis of carboxymethyl cellulose 38
by the partially purified cellulase obtained from the badan
local variety of tomato fruits infected by Aspergillus niger
14  Effect of substrate concentration on the activity of the partially 39
purified polygalacturonase obtained from the Roma vf variety of tomato
fruits infected by Aspergillus niger
15  Lineweaver-Burk plot for the hydrolysis of pectin by the   40
partially purified polygalacturonase obtained from the Roma vf
variety of tomato fruits infected by Aspergillus niger
16  Effect of substrate concentration on the activity of the partially 41
purified cellulase obtained from the Roma vf variety of tomato
fruits infected by Aspergillus niger
17  Lineweaver-Burk plot for the hydrolysis of carboxymethyl cellulose 42
by the partially purified polygalacturonase obtained from the
Roma vf variety of tomato fruits infected by Aspergillus niger
18  Absorption spectrum for water extracted lycopene 43
19  Absorption spectrum for lycopene extracted with the extracting 44
Solution made of hexane, ethanol and acetone in the ratio 2:1:1
20  Absorption spectrum for lycopene extracted with enzyme 45
obtained from the Ibadan local variety of tomato fruits.
21  Absorption spectrum for lycopene extracted with enzyme 46
obtained from the Roma vf variety of tomato fruits.


LIST OF TABLES
TABLE  TITLE PAGE

1    pH of tomato fruits with days of incubation   23
2    Partial purification table for polygalactorunase obtained 47
From the Roma vf variety of tomato fruits infected by
Aspergillus niger
3    Partial purification table for polygalactorunase obtained 48
From the Ibadan local variety of tomato fruits infected by
Aspergillus niger
4    Partial purification table for cellulase obtained from the 49
Roma vf variety of tomato fruits infected by Aspergillus niger
5   Partial purification table for cellulase obtained from the Ibadan 50
local variety of tomato fruits infected by Aspergillus niger
6 Lycopene content of tomato fruits after extraction with various 51
solvents


CHAPTER ONE
INTRODUCTION
The tomato is grown and eaten around the world. It is used in diverse ways, in the raw form in salads or processed into ketchup or tomato soup. Increased consumption of tomato and tomato products has been significantly associated with a reduced risk of prostate cancer in several epidemiological studies. Lycopene, which is the most abundant tomato carotenoid, had also been the primary focus of both in vitro and in vivo studies examining the relationship between increased intake of tomatoes and reduced risk of prostate cancer.
Lycopene is principally responsible for the characteristic red colour of ripe tomato fruits. It is also very important because of its antioxidant properties. Lycopene, has been the focus of considerable attention for its potential health benefits (Shi et al. 2002; Rao and Rao, 2004). Results from epidemiological and experimental studies support the view that lycopene may provide protection against cardiovascular disease and certain types of cancer (Giovannucci, 2005; Omoni and Aluko, 2005). Lavecchia and Zuorro (2011) reported the growing demand for
natural lycopene and therefore considerable interest has been directed to the possibility of obtaining lycopene from tomato processing waste. Tomato skins contain up to five times more lycopene than the pulp (Sharma and Le Maguer, 1996). However, the available solvent extraction technologies do not seem to allow a fast and economic recovery of the carotenoid. Fifty percent of the total lycopene was extracted from tomato processing waste using supercritical CO2 at 60°C and 30 MPa (Sabio et al., 2003). Similar results were obtained by Rozzi et al. (2002) with supercritical CO2  at 86 °C and 34.5 MPa. Low extraction efficiencies can be ascribed to the difficulty for the solvent to penetrate the compact tomato peel tissue
and solubilize the pigment, which is deeply embedded within the chromoplast
membrane structures (Harris and Spurr, 1969).
A large percentage of tomato fruits are lost to post harvest deterioration caused by
microorganisms. Fungi have been identified as the major microorganisms associated with spoilage of fruits and vegetables. Aspergillus niger is one of the fungi that had been associated with post harvest deterioration of tomato fruits (Ajayi et al, 2007).

This research was therefore designed to achieve the following objectives;
1.  To isolate cell wall degrading enzymes (cellulase and polygalacturonase) from tomato fruits deteriorated by Aspergillus niger.
2.  To partially purify the enzymes using ammonium sulphate precipitation and SDS PAGE.
3.  To characterise the partially purified enzymes.
4.  To employ the partially purified enzymes for improvement of lycopene
extraction from tomato wastes.

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