Isolation, Identification and Characterization of Pectinase Producers from Agro Wastes (Citrus sinensis and Ananas comosus)

When fruit is detached from the living plants it cannot repair its cells or defend its tissues against natural deterioration. There are many reasons of fruit destruction, but main cause of fruit spoilage is invasion by microorganisms such as mould, yeast and bacteria. Fruits contain high levels of sugars and nutrients and their low pH make fruits particularly desirable to fungal decay [1]. During postharvest handling, mechanical damage causes the release of enzymes present in the cell tissues. These enzymes break down the cellular material. Pectinases are the first enzymes to be secreted by fungal pathogens when they attack plant cell walls, weaken the plant cell wall and expose other polymers to degradation by hemicellulases and cellulases [2]. Pectinases are one of the most widely distributed enzymes in bacteria, fungi and plants insects, nematodes, plants, and protozoan [3]. Fungi are considered to be prospective enzyme producing sources.

Pectinases breaks down pectin and are of significant importance in the current biotechnological era with their all-embracing applications in fruit juice extraction and its clarification, scouring of cotton, degumming of plant fibers, waste water treatment, vegetable oil extraction, tea and coffee fermentations, bleaching of paper, in poultry feed additives, in alcoholic beverages and food industries [4]. Pectinases constitute approximately 10% of the total enzyme production in the world market and 25% of global sale in the food industry [5].

In the fruit juice industry, they are used for clarification of fruit juice by reduction in viscosity, increasing of juice yield by enzymatic liquefaction, and maceration of pulps, enhancing pigmentation by extracting more anthocyanin [6]. Microbial pectinases have been widely studied due to the requirement of highly productive strains and the cost-effective production of enzymes. In the present study, the pectinase was purified from a newly isolated Bacillus subtilis strain and this enzyme was characterized Identification and characterization of various pectinase producers have been published. There is paucity of information from the literature on combined action of two fruits and their mode of action. The aim of this study is to isolate and characterize based on primary and secondary screening, pectinase producers and how to optimize them from Agro-wastes, orange and pineapple

Reagents

The reagents and chemicals used in the study include: Pectin, K₂HPO₄, MgSO₄.7H₂O, NaCl, CaCl₂.2H₂O, FeCl₃.6H₂ O, yeast extract, Agar, urea, (NH4)2SO₄, MgSO₄, FeSO₄, and glucose, were all of analytical grade and were purchased from Hi Media and Sigma Chemicals, USA.

Collection of samples

The fresh oranges were plucked from a tree in Alagomeji, Lagos, Nigeria. The fresh pineapples were gotten from a pineapple plantation in Epe, Lagos. The oranges and pineapples which were intended to be rotten were purchased from Mushin market, Lagos, Nigeria. The rottenness of the oranges and pineapples was obtained by leaving the purchased fruits for 14 days on a flat surface in the laboratory.

Isolation of pectinolytic fungi

Fungi were isolated from the rotten fruits (oranges and pineapples) by using the modified pectin agar medium. In brief, two-fold dilutions of the spoiled samples were plated in pectin agar medium and incubated at room temperature for 6 days. The strains grown in the plate were subcultured into pectin agar medium separately and flooded with 1% cetrimide for primary screening. The pectinolytic fungi produced a clearing zone when exposed to cetrimide [7].

Secondary screening

The fungal strains which showed clearing zones in pectin agar medium were selected for secondary screening by estimating the polygalacturonase (PG) and pectin lyase (PL) production under solid state fermentation by using the modified medium [8].

Isolation of pectinolytic bacteria

The isolates of both the rotten and fresh pineapple were cultured and incubated at 35°C for 48 h. Thereafter, the distinct bacteria colonies were streaked on nutrient agar and incubated at 35 °C for 24 h. When Gram-stained films were examined, the isolates that appeared as Gram-positive rods was identified as Bacillus subtilis based on result from biochemical test as enumerated [9-10]. The Bacillus isolates were maintained in nutrient agar slants at 4°C.

Enzyme assays

Polygalacturonase activity was measured according to the method described by [11-13] One unit of enzyme activity has been defined as the amount of enzyme that releases one μ mol of galacturonic acid /min under the assay conditions [12-13]. Pectin lyase activity was measured by the reaction between unsaturated end products of pectin degradation and thiobarbituric acid. One unit of activity is the amount of enzyme causing a change in absorbance of 0.01 under the conditions of the assay [14].

Effect of inoculum age, inoculum size, and incubation time

Inoculum age was optimized by inoculating the medium (secondary culture) with inocula (primary culture) of varying age, namely 8, 12, 16, 20, and 24 h, and assaying the filtered broth for enzyme activity. To study the effect of inoculum size, (103-106CFU/ ml) inoculum was used to inoculate the production medium, and polygalacturonase activity was assayed in the broth. The effect of incubation time was studied by incubating the microorganism in production medium for different time intervals (24, 48, 72 and 96 h) and measuring the enzyme activity.

Effect of temperature and pH

Most favorable production temperature was studied by incubating the production medium at different temperatures (20, 30, 35 and 40 °C). Polygalacturonase activity was assayed by using the standard method [11]. For optimizing the production of pH, the production medium varying pH, namely, 4.0, 4.5, 5.0, 5.5, was used for enzyme production, and activity was measured.

Effect of nitrogen source

The effect of various nitrogen sources (Ca (NO₃)₂, (NH₄)_{2SO4, (NH4)2NO3, NH4Cl, NaNO3, KNO3, and Urea on the production of enzyme was studied by supplementing 0.1% w/v of these to the production media.}

Effect of D-galacturonic acid on polygalacturonase production

To study the effect of D-galacturonic acid on polygalacturonase production, it was added to the culture broth at a final concentration of 0.3, 0.6, 0.9, 1.2, and 1.5%w/v under aseptic conditions. The resulting extracellular polygalacturonase activities produced were measured by estimating the reducing groups produced (Figures 1-6).

Pectinolytic enzyme can be derived from different sources [15-17]. However, pectinase producing microorganisms have due advantage over other sources because they can be subjected to genetic and environmental manipulations to increase yield [18]. It has been reported that most Bacillus sp, enhances the production of pectinase [19]. This result indicates that orange and pineapple contain appreciable amount of pectin. The pectin was used as inducer for the production of pectinolytic enzymes. In this study, pectinase production by Bacillus subtilis and Aspergillus niger were compared. Our results show that both Bacillus subtilis and Aspergillus niger were able to produce pectinase. The isolates were later designated based on morphology as Bacillus subtilis, and Aspergillus niger are among the most studied pectinolytic fungi (Sukumaran et al., 2005; Favela-Torres et al., 2006). [20] also has reported that Bacillus sp, has produced higher yield of pectinase compared to most other microbes such as Aspergillus.