PHYTOCHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF FOUR NIGERIAN SPICES


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TABLE OF CONTENTS

Title page
Table of contents
Abstract

CHAPTER ONE
1.1.      Introduction
1.2.      Statement of the Problem
1.3.      Justification for the Study
1.4.      Significance of the Study
1.5       Aims and Objectives

CHAPTER TWO
2.0 Literature Review
2.1. Spices
2.2. Phytochemicals
2.3. Classes of major phytochemicals, food sources and nutritional benefits
2.4. Polyphenols
2.5.      Flavonoids
2.6       Anthocyanins
2.7       Carotenoids
2.8       Ascorbic acid
2.9       Phytosterols
2.10 Phytoestrogens
2.11     Phytochemical metabolism in human
2.12     Lipid oxidation
2.13     Degenerative effects and suppression of lipid oxidation
2.14     Functions and mechanism of action of antioxidants in foods
2.15     Natural antioxidants
2.16     Antioxidant properties of spices and spice extracts
2.17     Assessment of antioxidant activity of antioxidant compounds and the degree of lipid  Oxidation in food  system
2.18     Major microorganisms of food poisoning
2.19     Important preservation techniques for preventing food poisoning from pathogenic microorganisms
2.20     Antimicrobial properties of spices and spice extracts
2.21     Choice of solvents for preparation of crude extracts from biological Materials
2.22     Biology and ecology of Tetrapleura tetrapetra (Schum & Thonn)
2.23     Nutrient composition of Tetrapleura tetrapetra
2.24     Food and medicinal uses of Tetrapleura         tetrapetra
2.25     Ecology, botany and distribution of Monodora tennifolia (Benth)
2.26     Chemical composition and uses of Monodora tennifolia (Benth)
2.27     Ecology, botany and distribution of African nutmeg (Monodora myristica Gaetn)
2.28     Chemical composition and uses of African nutmeg (Monodora myristica Gaertn)
2.29     Ecology, botany and distribution of Ocimum viride (Willd)
2.30     Chemical composition and uses of Ocimum viride

CHAPTER THREE
3.0  MATERIALS AND METHODS
3.1.      Materials
3.2       Preparation of spice extracts
3.3       Preparation and storage of cooked ground beef and pork patties
3.4       Proximate analysis
3.4.1    Moisture content
3.4.2    Crude protein content
3.4.3    ether extract
3.4.4    Crude fibre content
3.4.5    Total ash content
3.5       Energy value  
3.6       Digestion and analysis for minerals
3.7       Determination of vitamin composition
3.7.1    Determination of ascorbic acid (Vitamin C) content
3.7.2    Determination of niacin content
3.7.3    Determination of riboflavin content
3.7.4    Determination of thiamin content
3.8       Determinations of phytochemical composition
3.8.1    Determination of total phenol
3.8.2    Determination of total flavonoids
3.8.3    Determination of condensed tannin content
3.8.4    Determination of total anthocyanin content
3.8.5    Determination of carotenoid content
3.8.6    Determination of alkaloid content
3.8.7    Determination of phytate content
3.8.8    Determination of oxalate content
3.8.9    Saponin content determination
3.9       Determination of Antioxidant Properties of Spices
3.9.1    Determination of free radical scavenging activity
3.9.2   Measurement of reducing power of the crude extracts of spices
3.9.3    Determination of antioxidant activity of crude extracts of the spices by the Ferric  thiocyanate (CTC) method
3.10                 Determination of antimicrobial properties of the spices
3.10.1 minimum inhibitory concentration (MIC) determination
3.10.2  Preparation and inoculation of substrates
3.10.3  Bacterial strains and preparation of inoculants
3.10.4  Monitoring of survival and growth of pathogen population
3.11     Determination of Thiobarbituric acid (TBA) reactive substances in minced meat patties during storage
3.12                 Experimental design
3.13                 Statistical analysis

CHAPTER FOUR
4.0       RESULTS AND DISCUSSIONS
4.1       Proximate composition and energy value of spices
4.2       Mineral composition of the spices
4.3       Vitamin content of the spices
4.4 Yield of crude extracts of spices
4.5       Effects of different extraction solvents on non-phenolic phytochemical content of the spices
4.5.1    Alkaloid
4.5.2    Oxalate
4.5.3    Saponin
4.5.4    Phytate
4.6 Effects of different extraction solvents on phenolic phytochemical contents     of the spices
4.6.1 Total phenol content
4.6.2:  Condensed tannin content
4.6.3    Total flavonoid content
4.6.4    Total carotenoid content
4.6.5    Total anthocyanin content
4.7       Estimation of reducing power of spices
4.7.1    Reducing power of Ocimum viride
4.7.2    Reducing power of of Monodora myristica
4.7.3    Reducing power of Monodora trifolia
4.7.4    Reducing power of Tetrapleura tetrapetra
4.7.5    Comparative effect of extracting solvents on reducing power of the four spices
4.8       Inhibition of Linoleic acid peroxidation
4.9.1    Scavenging of 1,1 – diphenyl-2-picryl hydra zyl radical (DPPH) by the spices
4.9.2    DPPH radical scavenging activity of Ocimum viride
4.9.3    DPPH radical scavenging activity of Monodera myristica
4.9.4    DPPH radical scavenging activity of Monodora tenuifolia
4.9.5    DPPH Radical scavenging activity of Tetrapleura tetrapetra
4.9.6  Comparison of DPPH radical scavenging activities of solvent extract of spices
4.10     Inhibition of lipid peroxidation in cooked, ground meat patties by spices during storage
4.11  Comparison of Mean TBA values of meat patties treated with spices during storage
4.11.1  Mean TBA values for Ocimim viride
4.11.2  Mean TBA values for Monodora myristica
4.11.3  Mean TBA values for Monodora tenuifolia
4.11.4  Mean TBA values for Tetrapleura tetropetra
4.12     Antimicrobial activities of the spices against some selected food pathogens
4.12.1  Sensitivity of the three bacteria toward inhibitory activity of aqueous and ethanol   extracts of the spices
4.12.2  Growth of pathogens in control substrates
4.12.3  Antibacterial activities of of Monodora myristica against food borne Pathogens
4.12.4  Antibacterial activities of Monodora tenuifolia against food borne pathogens
4.12.5  Antibacterial activities of Ocimum viride against food borne pathogens
4.12.6  Antibacterial activities of Tetrapleura tetrapetra against food borne pathogens
4.12.7 Phytochemical composition of cooked spice-treated food extracts

CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS
5.1 CONCLUSION
RECOMMENDATIONS
References
Appendices



ABSTRACT

This study evaluated phytochemical composition, antioxidant and antimicrobial properties of four Nigerian spices, namely Ocimum viride (leaves), Monodora myristica (seeds), Monodora tenuifolia (seeds) and Tetrepleura tetrapetra (fruits). The spices were screened for phytochemical [alkaloid, saponin, oxalate, phytate, total phenol (TP), condensed tannin (CT), total flavonoid (TF) and total anthocyanin (TA)] contents and antioxidant activities in five different extracting solvents [distilled water, 95 % methanol, acetone / hexane (1 : 1, v/v), hexane / methanol / acetone (2 : 1 : 1, v/v/v) and acetone / water / acetic acid (70 : 29.5 : 0.5, v/v/v)] using standard methods. Antioxidant capacities of the extracts to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, reduce iron (iii) chloride (FeCl3), suppress linoleic acid ( LA) peroxidation in ferric thiocyanate (FTC) oxidizing systems and inhibit formation of thiobarbituric acid reactive substances (TBARS) in refrigerated (4OC, 14 days) spice-treated (0, 0.6. 1.2 and 2.0%, w/w basis) beef and pork patties were investigated. Aqueous extracts (10% w/w) of beef, rice and vegetables were treated with the spices (0, 2.5 and 7.5%, w/v), inoculated with the pathogens Escherichia coli, Salmonella typhii and Staphylococcus aureus

(5logCFU/ml), and periodically analysed, by enumerating surviving populations after 24h intervals, during 10 days of storage (4OC) to determine antimicrobial activities of the spices. Phytochemical contents differed significantly (p < 0.05) among the spices and among solvent extracts of the same spice. The highest phytochemical content was total Phenol which ranged from 2.13 garlic acid equivalent per100 g (GAE / 100 g) in M. myristica to 13.93 GAE / 100 g in T. tetrapetra while total anthocyanin content was the lowest and ranged from 0.00 GAE / 100g in M. tenuifolia to 0.06 GAE / 100g in M. myristica. The extracts of spices exhibited high degree of antioxidant and antimicrobial activities. The spices suppressed lipid peroxidation in cooked ground beef (from 2.58 to 0.76 mean thiobarbituric acid value) and pork patties (from 4.33 to 1.03 thiobarbituric acid value) in dose-dependent order during 14 days of storage. Spice extracts reduced Fe3+ to Fe2+, scavenged DPPH radical (78 – 93%) and inhibited LA peroxidation (46 – 95%) in dose-depend ent order. Methanol (95 %) extracts of M. myristica, M. tenuifolia and O. viride, and water extract of T. tetrapetra exhibited the highest (1.6 nm) reducing power while the acetone/water/acetic acid extracts exhibited the highest (93%) scavenging capacity of DPPH radical. Water extracts of O. viride and T. tetrapetra, methanol extract of M. tenuifolia and acetone/water/acetic acid extract of M. myristica had the highest inhibition of LA peroxidation. The four spices exhibited dose-dependent bactericidal effects against E. coli (from 42.25 to 0.00 x 106 CFU / ml), S. typhii (from 47.1 to 3.7 x 106 CFU / ml) and S. aureus subsp.aureus (from 48.95 to 0.00 x 106 CFU / ml). During storage, antimicrobial effects of the spices were more pronounced in food extracts than in nutrient broth and in rice and vegetable extracts than in beef extracts. Of the four pathogens, E. coli was most susceptible to these spices, followed by S. aureus subsp. aureus. Tetrapleura tetrapetra was the most potent of these spices against the pathogens, followed by O. viride. The antioxidant and antimicrobial properties exhibited by these spices increased with spice concentrations and occurred in the following decreasing order: T. tetrapetra > O. viride > M. myristica > M. tenuifolia.




CHAPTER ONE

1.1        INTRODUCTION

In Nigeria, a high proportion of the rural and urban population resort to natural food ingredients, particularly because of their availability. Spices are a large group of such natural ingredients, and include dried seeds, fruits, roots, rhizomes, barks, leaves, flowers and any other vegetative substances used in a very small quantity as food additives to colour, flavour or preserve food (Birt, 2006). Spices are fragrant, aromatic and pleasant. The bulk of the spices consist of carbohydrates such as cellulose, starch, pentosans and mucilage, and some amount of protein and minerals (Ogutimein et al., 1989). Only very small fractions of dry matter of the spices such as the phytochemicals are responsible for the flavouring, colourng, preservative and health-promoting characteristics (Cowan, 1999).


These phytochemicals are plant metabolites (Sofowurra, 1993) which act as natural defense systems for host plants, and also provide characteristic colour, aroma and flavour in specific plant parts. They are a group of non-nutrient compounds that are biologically active when consumed by human. Many phytochemicals are health-promoting and are of many disease-preventive (Rowland, 1999; Birt, 2006). Both epidemiological and clinical studies have proven that phytochemicals present in cereals, fruits and vegetables are mainly responsible for reduced incidence of chronic and degenerative diseases among populations whose diets are high in these foods (Shahidi, 1996). As a result there has been an increased search for phytochemical constituents that possess antioxidant and antimicrobial potency in recent time (Jayaprakasha and Jaganmohan, 2000, Birt, 2006). Typical phytochemicals with antioxidant and antimicrobial activities include polyphenols, phenolic acids and their derivatives, flavonoids, phospholipids, ascorbic acid, carotenoids and sterols. A number of exotic spices of international recognition with known phytochemical constituents have been proven to be good.....


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