PRODUCTION AND CHARACTERIZATION OF NARINGINASE OBTAINED FROM ASPERGILLUS NIGER IN SUBMERGED FERMENTATION SYSTEM USING NARINGIN EXTRACTED FROM LEMON PEELS AS CARBON SOURCE

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).

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