ABSTRACT
Naringin was extracted from lemon peels with extraction yield of 0.25%. Two assay methods were used to assay for naringinase activity, one is naringin disappearance assay method and the other is reducing sugars appearance assay method. The naringinase activities for the crude enzyme were found to be 157.7 µmol/min using naringin disappearance method and 493.64 µmol/min using reducing sugars appearance method. Ammonium sulphate saturation (50%) was found suitable for precipitation of naringinase with highest activity. After gel filtration a 32.84  fold  increase  was observed  with specific  activity  of 7279.37  µ/mg  using  naringin disappearance  assay method  and a 20.48 fold increase with specific  activity  of 14463.57 µ/mg using reducing sugars appearance assay method. The percentage yields were found to be 11.63%  using  naringin disappearance  assay method  and  7.38%  using reducing  sugars appearance assay method. The optimum pH and temperature were found to be 3.50 and 50OC, respectively  using  both the  naringin  disappearance  and reducing  sugar  appearance  assay method. The Michealis constant, Km and maximum velocity, Vmax obtained from Lineweaver- Burk plot of initial velocity data at different concentration of naringin were found to be 5.8 mg/ml and 1111.11 µmol/min using reducing sugars appearance assay method.
CHAPTER ONE
INTRODUCTION
Bitterness is the major limiting factor for commercial acceptance of processed citrus fruits
such as juice, wine and vinegar (Puri et al., 2010). Some bitterness in processed grape and lemon fruit products is acceptable to consumer but excessive bitterness is one of the major causes of consumer rejections to such products. Increased sales of these fruit products may result if the packaging of excessively bitter products is avoided. It would be advantageous for manufacturers to simultaneously remove bitterness from fruit juices (Vaidyanathan and Periasamy, 2010). The bitterness in citrus fruit is affected by limonin and naringin (which is said to be more bitter than quinine), which are flavonoids and are generally recognized as the two major bitter compounds responsible for making juice bitter (Pavithra et al., 2013). Naringin was first found in grapefruit (Pichaiyongvongdee and Haruenkit, 2009). It is present in grape, lemon, lime and orange fruits, and may cause interference during the citrus fruit juice processing and be the cause for the bitter taste (Saranya et al., 2009). Albedo, the fruit membrane, is one of the major parts containing naringin, flavonone glycoside, and when squeezed the naringin is extracted into the juice. Naringin is abundant in immature fruit but its concentration decreases as fruit ripens (Puri and Banergee, 2000). The naringin has sugar complexes (α-L-rhamnose and β-D-glucose) and an aglycone (Naringenin) part. The main objective of the industry is to process fruits at the lowest possible cost, maintaining organoleptic quality and stability of the finished products, with increased consumer acceptability (Ferreira et al., 2008). Numerous techniques used to reduce naringin, such as adsorptive debittering, chemical methods, poly-styrene divinyl benzene styrene resin treatment, and β-cyclodextrin treatment have limitations in altering nutrient composition either through chemical reactions or removal of nutrients, flavour and colour, etc (Puri and Banergee, 2000). A suitable debittering can be achieved by treating the juice with an enzyme known as naringinase (E.C.3.2.1.40), which directly hydrolyses naringin. This hydrolytic mechanism can considerably reduce the bitterness in citrus juice. The enzyme, naringinase (E.C.3.2.1.40), hydrolyses naringin (4,5,7-trihydroxy-flavonone 7-rhamnoglucoside) and produce a tasteless compound naringenin (4,5,7-trihydroxy-flavonone) by two steps of reaction (Vila-Real et al., 2007). Naringinase has two subunits of enzyme called α-L- rhamnosidase (E.C.3.2.1.40) and β-D-glucosidase (E.C.3.2.1.23). The enzyme α-L– rhamnosidase, which acts on sugar complex releases prunin and β-D-glucosidase, which acts
on prunin, releases naringenin (Ni et al., 2011). The reduction in bitterness as a result of the enzymatic process, controlling the quality and improving commercial value of lemon fruit and other juices and as the maintenance of health properties, increases the acceptance by the consumers. This makes enzymatic hydrolysis of naringin to still be an interesting application and a promising approach (Ferreira et al., 2008). These flavonoids, namely naringin and naringenin, from citrus, are functional chemicals with important properties in the fields of healthcare, food and agriculture. These products of enzymatic hydrolysis of naringin may be potentially useful as pharmacological agents such as anti-cancer, in the treatment or prevention of atherosclerosis, with a number of antigenic activities; as anti-thrombotic and vasodilator (Chen et al., 2003). Many microorganisms producing naringinase has been reported, Penicillium decumbens (Nourouzian et al., 2000), Candida tropicalis (Saranya et al., 2009) and Aspergillus niger (Puri et al., 2005).
1.1 Lemon Fruit
Fig. 1: picture of lemon fruit (Wikipedia, 2013)
According to Wikipedia (2013), the common lemon is scientifically classified as follows; Kingdom: Plantae
Division: Angiospermae Order: Sapindales Family: Rutaceae
Genus: Citrus
Species: C. limon
Lemon (Citrus limon) is a small evergreen tree native to Asia. It forms a spreading bush or a small tree, 3 to 6 meter (10 to 20 feet) high if not pruned. The trees are commonly grown in orchards, spaced 5 to 8 meter (16 to 26 feet) apart. Lemon trees usually bloom throughout the year, and the fruit is picked 6 to 10 times a year. Its young leaves have a decidedly reddish tint, later they turn green. In some varieties, the young branches of the lemon are angular and some have sharp thorns at the axils of the leaves. The flowers have a sweet odour and are rather large, solitary or in small clusters in the axils of the leaves. Reddish-tinted in the bud, the petals are white above and reddish purple below (Wood, 2003).
Lemon fruit is oval with a broad, low, apical nipple and 8 to 10 segments. The outer rind or peel is yellow when ripe and rather thick in some varieties, is prominently glandular-dotted. The white, spongy inner part of the peel, called the mesocarp, or albedo, is nearly tasteless and is the chief source of commercial grades of naringin and pectin. The pulp is decidedly acidic and the predominant acid present is citric acid, which may amount to 5 percent or more by weight of the lemon’s juice. Lemon seeds are small, ovoid, and pointed; occasionally, fruits are seedless. The fruit is used for culinary and non-culinary purposes throughout the world, primarily for its juice, though the pulp and rind are also used in cooking and baking. The juice of the lemon is about 5% to 6% citric acid, which gives lemons a sour taste. The distinctive sour taste of lemon juice makes it a key ingredient in drinks and foods such as lemonade (Craig, 2006).
1.1.1 Lemon Fruit Waste
Agro-industrial wastes are mainly composed of complex polysaccharides that might serve as good raw materials. In the production and utilization of lemon fruit into products such as juice, flavourings, lemon oil and perfumes, wastes are generated in the form of peels. These wastes are bio-transformed in the agro-industry as a nutrient for microbial growth and production of enzymes although many valuable byproducts can be produced from the rich waste. This bio-transformation results in the reduction of serious disposal problem (Gomez et al., 2013).
1.1.2 Phytochemical And Nutrtional Constituents of Lemon Fruit
Lemon fruit is rich in a variety of phytochemicals (Penniston et al., 2008) and nutrients (Table 1). Lemon fruit pulp is high in dietary fiber, vitamin C, alkaloid, phytosterol, phenols and provitamin A carotenoids (Penniston et al., 2008). Lemon contains essential vitamins like antioxidant vitamins A, C and E, vitamin B6, vitamin K and other B vitamins and minerals such as potassium, copper, zinc etc (Table 1) and amino acids (Wikipedia, 2013).
Lemon peels contain pigments that may have antioxidant properties. These include caroteniods such as the provitamin A compound, beta-carotene, lutein and phenols. Both mango pulp and peel contain cardiac glycosides and steroids (Penniston et al., 2008). In addition, lemon peels have been shown to be a rich source of volatile oil (Wikipedia, 2013).
Table 1: Nutrient and phytochemical contents of raw lemon (Citrus limon)
nutrition value per 100 g
Principle | Nutritive value | Percentage of RDA |
Energy | 29kcal | 1.5% |
Carbohydrate | 9.32g | 7% |
Protein | 1.10g | 2% |
Total fat | 0.30g | 1% |
Cholesterol | 0mg | 0% |
Dietary fiber | 2.80g | 7% |
Vitamins | ||
Folates | 11µg | 3% |
Niacin | 0.100mg | 1% |
Pantothenic acid | 0.190mg | 4% |
Pyridoxine | 0.080mg | 6% |
Riboflavin | 0.020mg | 1.5% |
Thamine | 0.040mg | 3.5% |
Vitamin C | 53mg | 88% |
Vitamin A | 22IU | 1% |
Vitamin E | 0.15mg | 1% |
Vitamin K | 0µg | 0% |
Electrolytes | ||
Sodium | 2mg | 0% |
Potassium | 138mg | 3% |
Minerals | ||
Calcium | 26mg | 3% |
Copper | 37µg | 4% |
Iron | 0.60mg | 7.5% |
Magnesium | 8mg | 2% |
Manganese | 0.030mg | 1% |
Zinc | 0.06mg | 0.5% |
Phyto-nutrients | ||
Carotene β | 3µg | – |
Carotene α | 1µg | – |
Crypto-xanthine β | 20µg | – |
Lutein-zeaxanthin | 11µ | – |
Lycopene | 0µg | – |
(Source: Wikipedia, 2013).
1.1.3 Benefits of Lemon Fruit
1.1.3.1 Industrial Use of Lemon Fruit
Lemons are the primary commercial source of citric acid prior to the development of fermentation-based processes (Hofrichter, 2010).
1.1.3.2 Use of Lemon as a Cleaning Agent
The juice of the lemon may be used for cleaning. A halved lemon dipped in salt or baking powder is used to brighten copper cookware. The acid dissolves the tarnish and the abrasives assist the cleaning. As a sanitary kitchen  deodorizer the juice can deodorize, remove grease, bleach stains, and disinfect; when mixed with baking soda,  it  removes stains from plastic food storage containers. The oil of the lemon’s peel also has various uses. It is used as a wood cleaner and polish, where its solvent property is employed to dissolve old wax, fingerprints, and grime. Lemon oil and  orange oil are  also used as a nontoxic  insecticide treatment. A halved lemon is used as a finger moistener for those counting large amounts of bills, such as tellers and cashiers (Ensminger and Nsminger, 2009).
This material content is developed to serve as a GUIDE for students to conduct academic research
PRODUCTION AND CHARACTERIZATION OF NARINGINASE OBTAINED FROM ASPERGILLUS NIGER IN SUBMERGED FERMENTATION SYSTEM USING NARINGIN EXTRACTED FROM LEMON PEELS AS CARBON SOURCE>
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