CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF STUDY

Avocado plant (Persea americana) that belongs to the family, Lauraceae and genus, Persea

originated from Guatemala and Mexico, but now grown throughout the tropics and sub-tropics

(Parameswaran and Murthin, 2014). The fruit and even the plant, is known as “Igba or apoka” in

Yoruba and as ube oyibo (loosely translated to ʻEnglish as ʻforeign pearʼ) in Ojoto and

neighboring Igbo speaking communities South East Nigeria. The fruit pulp is edible (Dreher and

Davenport, 2015). However, the seed is essentially discarded as solid agro waste, although

consumed in some countries including Nigeria, where it is milled and incorporated into foods

(owing perhaps to numerous ethno medicinal uses of avocado pear in the management of various

ailments including hypertension, diabetes, cancer and inflammation (Alhassan et al., 2012).

Avocado is an energetic fruit with high nutritional value and is considered a major tropical fruit,

since it is rich in protein and contains fat-soluble vitamins lacking in other fruits, including

Vitamins A and B, and median levels of vitamins D and E. It contains different oil levels in the

pulp, thus it is widely used in pharmaceutical and cosmetic industries, and for obtaining

commercial oils similar to olive oil, because of their similar fatty acid composition (Duarte et al.,

2016). In addition, this fruit has been recognized for its health benefits, especially due to the

compounds present in the lipid fraction, such as omega fatty acids, phytosterols, tocopherols, and

squalene (Duarte et al., 2016).

The seed and leaf extracts are traditionally used to treat hypertension (Ozoula et al., 2009). The

therapeutic value of Persea americana in some disease conditions has been scientifically

validated. The seed extract has been reported to lower blood pressure in normotensive and
hypertensive rat models (Imafidon and Amaechina, 2010; Arukwe et al., 2012). Other

morphological parts of the plant have been demonstrated to possess medicinal properties such as

anti-inflammatory, analgesic and antioxidant properties (Ojewole and Amabeoku, 2006).

Phytochemical analysis of leaves revealed the presence of flavonoids, saponins, alkaloids,

tannins, reducing sugars (Owolabi et al. 2010) and mineral compounds (Arukwe et al. 2012).

The most common meaning of fermentation is the conversion of a sugar into an organic acid or

an alcohol. Fermentation occurs naturally in many foods and humans have intentionally used it

since ancient times to improve both the preservation and organoleptic properties of food.

However, the term “fermentation” is also used in a broader sense for the intentional use of

microorganisms such as bacteria, yeast, and fungi to make products useful to humans (biomass,

enzymes, primary and secondary metabolites, recombinant products, and products of

biotransformation) on an industrial scale (Paulová et al., 2013).

1.1 AIM OF STUDY

The aim of the current study is to determine the effect of fermentation on alkaline treated Persea

americana seeds

Specific objectives include:

1. Processing of the Persea americana seeds

2. Alkaline treatment of the processed Persea americana seeds

3. Fermentation of the alkaline treated Persea americana seeds

4. Evaluation of the effect of fermentation on the alkaline treated Persea americana seeds
 CHAPTER TWO

LITERATURE REVIEW

2.1 AVOCADO PEAR (PERSEA AMERICANA)

The avocado tree originally from Mexico and Central America belongs to the Lauraceae family,

genus Perseal and comprises two subgenres: Persea and Eriodaphne. The avocado grown for

agricultural interests belong to two species of the genus Persea, being divided into three

botanical varieties: Persea americana Miller var. Drymifolia (Mexican species); Persea

americana Miller var. American (West Indian species); and Persea nubigena Miller var.

Guatemalensis (Guatemalan species) (Duarte et al., 2016).

2.1.1 Botanical Description of Persea americana

On average, the avocado tree can reach a height of 20 meters; However, when it grows in

commercial plantations, it is not grow more than 5 m, to facilitate phytosanitary control

practices, harvesting, pruning and foliar fertilization. This plant species has thick trunk and

elongated leaves, with several branches that generate dense foliage. It is considered a perennial

crop because it is grown throughout the year (Crop monograph, 2011).

The fruit is a drupe, pear shaped, light green to dark green and purple to black, rough skin with a

yellowish green flesh and a large central bone. There are approximately 400 varieties, so we can

find fruits of different shapes and weights, which may through 150 to 350 gr (Rodríguez and

Sánchez, 2005).

2.1.2 Taxonomical classification of Persea americana

Some aspects of the species related to systematic Persea americana Mill are (Téliz-Ortiz et al.,

2000):

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Laurales

Family: Lauraceae

Genre: Persea

Species: Persea americana Mill

2.1.3 Traditional Uses of Persea americana

This plant has been for long time, used traditionally to treat various ailments of the human body

in many parts of the globe. The root, bark, fruit, seed and leaf are used extensively in traditional

medicine in many tropical and subtropical countries for the treatment of various ailments

(Owolabi et al., 2005). Its seed is used in the treatment of diarrhea, dysentery, toothache,

intestinal parasites and for beautification (Pamplora and Roger, 1999). The leaves of Persea

americana have been popularly used in the treatment of diabetes in countries of Latin America

and Africa (Lima et al., 2012). The seed (crude or toasted) is employed in Mexican traditional

medicine to treat skin rashes, diarrhea, and dysentery caused by helminths and amoebas, for the

cure of infectious processes caused by fungi and bacteria, as well as for the treatment of asthma,

high blood pressure, and rheumatism (del Refugio et al., 2004). The bark, fruit, and leaf are used

in traditional medicine in South America, West Indies, and Africa to provide remedies for
various ailments (Morton, 1987). The fruit is employed as a vermifuge and remedy for

dysentery. The leaves decoction is also taken as a remedy for diarrhea, sore throat and

hemorrhage, and to stimulate and regulate menstruations (Morton, 1987). Odugbemi has also

documented the efficacy of the cotyledons of avocado pear seed for the treatment of

hypertension. The cotyledons are then cut into pieces, dried and grinded into powder. A dessert

spoonful in 200 ml of hot water taken after meals, gives relieve for the ailment (Odugbemi,

2006). In Cameroon, the leaves decoction is taken to treat toothache, high blood pressure,

diabetes, malaria; to stimulate uterine contractions and to relief painful menstruations. The leaves

and stem bark are also boiled together in water and the resulting liquid is taken to cure toothache,

malaria and typhoid fever. The fruit pulp is eaten to lower bad blood cholesterol, and to prevent

mental strain and cardiovascular diseases; while the aqueous extract of it seed is drunk against

intestinal worms. This tree planted around the house serves as air purifier.

2.1.4 Phytoconstituents and Nutrients of Persea americana

Phytochemicals are present in all the plant parts, at different concentration. The result for the

phytochemical constituents in Persea americana seed showed the presence of the following

compounds with their respective concentration: Alkaloids (2.92±0.028), Flavonoids

(4.76±0.053), Saponins (3.22±0.055), Steroids (1.58±0.05), Tanins (0.18±0.00), Phenol

(2.47±0.03). Idris et al also found the same phytoconstituents in this tree extracts (Idris et al.,

2009). Some of the general characteristics of flavonoid include potent water soluble super

antioxidants and free radical scavenger; they prevent oxidative cell damage, have strong

anticancer activity and protect against all stages of carcinogens (Salah et al., 1992). Flavonoids

in intestinal tract lower the risk of heart disease and inflammation (Omodamiro et al., 2016).

Isolated pure form of alkaloids and their synthetic derivatives are used as basic medicinal agents
for their analgesic and bacterial effects (Stray, 1988), antihypertensive, antiarrhythmic,

antimalarial and anti-cancer activities (Wink et al., 1998). Tannin rich medicinal plants are used

to heal a lot of illnesses; such as leucorrhoea, rhinorrhea and diarrhea. More recently, tannins

have gained medical interest, because of the high prevalence of deadly ailments such as AIDS

and numerous cancers (Blytt et al., 1988). In the dyestuff industry, tannins are useful as caustics

for dye and ink production. Also, in the food industry, tannins have proved usefulness in the

purification of wine, beer and fruit juices and also as coagulants in rubber production (Gyamfi

and Aniya, 2002). Saponins are responsible for antimicrobial, antifungal, anti-inflammatory,

anti-yeast and antidote activates. The function of saponins in plants generally serves as anti-

feedant and to protect the plant against microbes and fungi (Skene and Sutton, 2006). Phenols

have been extensively researched as disease preventives. Steroids are antioxidants in vitro, and

have a link with reproduction in humans (Rice-Evans et al., 1995). The nutrients present in one-

half of this tree fruit (68g), according to the NHANES analysis are given in Table 2.1. (ADA,

2009). The Avocado contains a significant amount of oil in comparison to other fruits. This

phytochemicals and nutrients present in Persea americana gives credence to the medicinal

benefits that this tree parts have been used for, in the past years as well as it nutritional value.
Table 2.1: The nutrients present in one-half of Persea americana fruit (68g) (ADA, 2009)

Analyte Quantity (g) Analyte Quantity (g) Analyte Quantity (g)

High- 6.7 g or 114 Vitamin C 6.0 mg Pantothenic 1.0 mg
monounsatur kcal acid
ated fatty
acid
Total sugar 0.2 g Vitamin E 1.3 mg Riboflavin 0.1 mg
Potassium 345 mg Vitamin K1 14 µg Choline 10 mg
Magnesium 19.5 mg Folate 60 mg Lutein/Zeaxa 85 µg
nthin
Sodium 5.5 mg Vitamin B6 0.2 mg Phytosterols 57 mg
Vitamin A 43 µg Niacin 1.3 mg Dietary fiber 4.6 mg

2.1.5 Pharmacological Activities of Avocado Pear

Over the years Persea americana has been reported to possess anti-malaria activity (Komlaga et

al., 2015; Tene et al., 2016), Anti-platelet and anti-thrombic activity (Rodriguez-Sanchez et al.,

2015), anti-convulsant effect (Ojewole and Amabeoku, 2006), Anti-viral activity (De Almeida et

al., 1998), anti-bacterial and anti-microbial activity (Jiménez-Arellanes et al., 2013; Han et al.,

2015), anti-oxidant activity (Ikpeme et al., 2014; Owusu et al., 2015), analgesic and anti-

inflammatory activity (Adeyemi et al., 2002), anti-hypercholesterolemic activity (Bartholomew

et al., 2007; Brai et al., 2007), anti-diarrheal activity (Odo et al., 2013), anti-anthritic and joint

diseases (Haider, 2012), anticancer activity (Ding et al., 2007), Hepatoprotective activity

(Mahmoed and Rezq, 2013; Brai et al., 2014), hypoglycemic and antidiabetic activity

(Thenmozhi et al., 2012; Marrero-Faz et al., 2014), Hypotensive activity (Djomeni et al., 2014),

vasorelaxant activity (Owolabi et al., 2005) and Wound healing activity (Nayak et al., 2008).
2.2 FERMENTATION

Fermentation is a metabolic process that produces chemical changes in organic substrates

through the action of enzymes. In biochemistry, it is narrowly defined as the extraction of energy

from carbohydrates in the absence of oxygen. In the context of food production, it may more

broadly refer to any process in which the activity of microorganisms brings about a desirable

change to a foodstuff or beverage (Hui, 2004).

2.2.1 Biochemical Overview of Fermentation

Fermentation reacts NADH with an endogenous, organic electron acceptor. Usually this is

pyruvate formed from sugar through glycolysis. The reaction produces NAD + and an organic

product, typical examples being ethanol, lactic acid, carbon dioxide, and hydrogen gas (H2).

However, more exotic compounds can be produced by fermentation, such as butyric acid and

acetone. Fermentation products contain chemical energy (they are not fully oxidized), but are

considered waste products, since they cannot be metabolized further without the use of oxygen.

Fermentation normally occurs in an anaerobic environment. In the presence of O 2, NADH, and

pyruvate are used to generate ATP in respiration. This is called oxidative phosphorylation, and it

generates much more ATP than glycolysis alone. For that reason, fermentation is rarely utilized

when oxygen is available. However, even in the presence of abundant oxygen, some strains of

yeast such as Saccharomyces cerevisiae prefer fermentation to aerobic respiration as long as

there is an adequate supply of sugars (a phenomenon known as the Crabtree effect) (Piškur and

Compagno, 2004). Some fermentation processes involve obligate anaerobes, which cannot

tolerate oxygen.
Although yeast carries out the fermentation in the production of ethanol in beers, wines, and

other alcoholic drinks, this is not the only possible agent: bacteria carry out the fermentation in

the production of xanthan gum.

Several products are generated from fermentation process. Examples include ethanol which is

produced from ethanol fermentation (Purves et al., 2003), lactic acid which is produced from

homolactic fermentation and hydrogen gas (Purves et al., 2003).

2.2.2 Modes of Operation

Most industrial fermentation uses batch or fed-batch procedures, although continuous

fermentation can be more economical if various challenges, particularly the difficulty of

maintaining sterility, can be met (Li et al., 2014).

2.2.2.1 Batch Fermentation

In a batch process, all the ingredients are combined and the reactions proceed without any further

input. Batch fermentation has been used for millennia to make bread and alcoholic beverages,

and it is still a common method, especially when the process is not well understood ( Cinar et al.,

2003). However, it can be expensive because the fermentor must be sterilized using high pressure

steam between batches (Li et al., 2014). Strictly speaking, there is often addition of small

quantities of chemicals to control the pH or suppress foaming (Cinar et al., 2003).

Batch fermentation goes through a series of phases. There is a lag phase in which cells adjust to

their environment; then a phase in which exponential growth occurs. Once many of the nutrients

have been consumed, the growth slows and becomes non-exponential, but production of

secondary metabolites (including commercially important antibiotics and enzymes) accelerates.

This continues through a stationary phase after most of the nutrients have been consumed, and

then the cells die (Cinar et al., 2003).

2.2.2.2 Continuous fermentation

In continuous fermentation, substrates are added and final products removed continuously (Li et

al., 2014). There are three varieties: chemostats, which hold nutrient levels constant; turbidostats,

which keep cell mass constant; and plug flow reactors in which the culture medium flows

steadily through a tube while the cells are recycled from the outlet to the inlet ( Schmid and

Schmidt-Dannert, 2016). If the process works well, there is a steady flow of feed and effluent and

the costs of repeatedly setting up a batch are avoided. Also, it can prolong the exponential

growth phase and avoid byproducts that inhibit the reactions by continuously removing them.

However, it is difficult to maintain a steady state and avoid contamination, and the design tends

to be complex (Li et al., 2014). Typically, the fermentor must run for over 500 hours to be more

economical than batch processors (Schmid and Schmidt-Dannert, 2016).