Characterisation of Some Rhizobium EPS and Their Biopreservative Potentials on Shelf Life Extension of Musa Species

1. INTRODUCTION

Microorganisms that produced Exopolysaccharides (EPS) can be isolated from variety of natural sources, including soil, water and biofilm, in both aquatic and terrestrial habitats. The most common commercially accessible EPSs consist of xanthan, gellen, alginate, cellulose and hyaluronic acid [1]. EPS are type of long-chain biopolymer made up of branched, recurring unit of sugars, as well as non-carbohydrate constituents that can be found connected to the exterior of cells or released into the environment [2]. EPS are slimy substances that may be defined in two forms based on their location either bounded to cell surface as cell-bound or secreted into surrounding media as released EPS [3]. The EPS potential to raise solution viscosity or cause gels development makes increases its usage in industrial product formulations in recent years [4].

Rhizobial are soil microorganisms with an extensive array of taxonomic, metabolic, and genetic diversity [5]. Nitrogen deficiency in rhizosphere results in legumes-bacteria partnership, which allows atmospheric di-nitrogen to be fixed. This association between the soil bacteria and legumes is based on a complicated molecular process that starts with the host plant roots secreting flavonoids that helps activate the Rhizobium regulatory gene products [6]. Rhizobial is the name given to this group of soil bacteria and majority of the bacteria found in legume symbionts belong to the proteobacteria class that include Mesorhizobium, Bradyrhizobium, Azorhizobium and Sinorhizobium [7]. Rhizobial can produce Lipopolysaccharides (LPS), exopolysaccharides and Cyclic Glucans (CG) among other surface polysaccharides, which are crucial for bacteria’s activity in free-living and symbiotic environments [8]. Until now, the only form of rhizobia EPS that is commercially available is Succinoglycan that is also called EPS1 that is isolated from Sinorhizobium meliloti [2].

2. METHODOLOGY

• Production, Extraction and Purification of EPS

EPS from selected Rhizobia was done using Yeast Extract Mineral Medium (YEMM) containing: 2% Sucrose, 2% Nitrogen, (glycine), 0.1% and Vitamin (biotin) 1µg/mL. The culture was incubated in a shaker for 144 hours and the enzymes that could degrade polymers were disabled by boiling using hot water bath [9] and left to chill to room temperature. 250 µg of 80% (w/v) trichloroactic acid was mixed with the samples stored at 4^oC so as to precipitate protein. Supernatant retrieved and precipitation of EPS was done by adding 2 volume of 100% of cold ethanol and stored overnight at 4^oC [10]. The precipitate retrieved at 10000g for 15 minutes was re-suspended in water. A dialysis tube was used to purify the unpurified EPS for 48 hours by changing the distilled water four times. All the analysis was duplicated while the uninoculated medium served as control.

• Determination of functional group of EPS produced by the Rhizobia: The functional group in the EPS was determined using Fourier Transform Infra-red (FT-IR) Spectroscopy.
• Determination of thermal stability of the EPS: The thermal stability of the EPS was determined using thermogravimetric Analysis (TGA). The TGA was done on 27.1 mg of the EPS using a PerkinElmer Diamond TGA instrument. The TGA on the x-axis signal is plotted against the reference EPS temperature, which is changed to percentage weight change on y-axis. EPS was positioned in a platinum crucible and heated at a linear rate of 20°C/min under nitrogen over a temperature range of 10°C to 980°C, with weight loss calculated.
• Determination of chemical composition of the EPS: The chemical compounds present in the EPS were determined using GC-MS analysis.
• Monosaccharide constituent of the EPS: To determine monosaccharide component of EPS from Rhizobial, every EPS was tested by RP-HPLC via 1-phenyl-3-methyl-5-pyrazolone (PMP) monomer chemical detection technique developed by Fu and O’Neill [11] and modified by Castellane et al. [10].
• Edible Coating Formulation: Ten grams of EPS dissolved in 100ml sterile distilled water to compose an EPS solution (1 percent, w/v).
• Coating Applications: Plantains and bananas were divided into two groups at random. The EPS treatment was given to one group, while the untreated control group was given to the other. Fruits were coated by immersing them twice in the film-forming solution (EPS) for 10 minutes each time. Fruits were left to naturally dry for 1 hour at 25°C before being stored for further research.
• Physical Parameters of Musa sp.
• Weight Loss: The selected plantains and bananas for each preparation were weighed at the start, right after covering and air-drying, and then every sampling day throughout storage. Weight loss was measured in terms of overall weight loss and daily weight loss. Weight loss = (Initial weight-Final weight)
• Chemical Parameters of Musa sp.
• pH: After checking for the weight loss, 10 g of grinded plantain and banana was suspended in 100 mL of water after being chopped into pieces and homogenized and filtered. The pH was measured with pH meter.
• Titratable Acidity (TA): The plantain and banana samples were cut and homogenized then 10 g of crushed plantain and banana were suspended in 100 mL distilled water and filtered. 0.1 N NaOH was used to titrate the acidity of the samples. The titratable acidity of the banana was measured in grams of citric acid per 100 grams of weight.
• Extraction and Evaluation of Total Sugar/ reducing sugar from Plantain and Banana mash
• The method of Kumar et al. [12] was employed for extraction and evaluation of total sugar / reducing sugar from plantain and banana mash.

4.0. DISCUSSION

Characterisation of EPS using FT-IR: FT-IR spectroscope is a valuable procedure that works on the standard that group of bonds vibrates at characteristic frequencies. It can be employed to identify functional groups and characterizing covalent bonding. Characterisation of EPS from Rhizobia using FT-IR showed the existence of functional groups such as hydroxyl, alkane, alkene, alkyl and amine group as well as stretches of C=O of alcohol group in the EPS. The presence of this band shows that the substance is a polysaccharide [13]. Lack of peaks in the range of 260- 290cm-¹obviously shows the lack of any protein and nucleic acids. The EPS contain large number of H-O stretching frequency with a broad absorption peak of 3539, 3395, 3538.66, 3537.11, 3537.09, 3389.58, 3416.92 and 3633.33cm^-1. The bands of some stretching vibration of carboxyl group (C=O) were seen and were due to associated water which indicates the presence of some organic substances such as the stretching of sugar (mannose or galactose) [14]. The relatively weak absorption peaks at 888.59, 883.82, 838.22, 839.61, 872.44, 887.08, 888.59 and 892.49 cm^-1 might be ascribed to C=C (alkene). Furthermore, the bands within 776.22 – 601.91 cm^-1 regions were assigned to the vibration of the C-H and C-I halo bands.

Characterisation of EPS using TGA: Thermal stability is a vital feature to consider for industrial use of EPS, particularly in food industry, because most food manufacturing and processing takes place at elevated temperatures [15]. According to Sri et al. [16] when EPS is subjected to diverse temperatures, the major measures that take place with the initial boost in temperature are gelatinization and swelling. Additional increase in temperature causes drying out and pyrolysis of the exopolysaccharide. The TGA of the EPS shows two stages of weight loss which occurred around 286.46 and 487.94, 262.32 and 498.9, 227 and 547.4, 244.45 and 394.24 and 268.68 and 494.75^oC. The large weight loss may be attributed to the breakdown of carbon. This primary weight loss may be related with the loss of moisture. Many reports recommended that initial loss of moisture is due to the carboxyl groups which are present in high level and are bound to water molecules [17]. Thus, the initial weight loss by EPS produced by the Rhizobial strains is due to the existence of elevated content of carboxyl groups. The difference between the degradation temperature values of various EPSs is ascribed to their varied structural composition.

Characterisation of EPS using GC-MS: GC-MS has been used to categorize the constituent volatile matter, long chain, branched chain hydrocarbons, alcohols, acids and esters [18]. The GC-MS chromatogram of all the EPS tested revealed the presence of some active compounds (pentadecanoic acid, octadecanoic acid, oleic acid and heptacosanoic acid).

Determination of monosaccharides constituent of Rhizobial EPS via HPLC: D-ribose had the highest concentration in the EPS produced from USDA 110 (EPSA) while rhamnose was observed to be the highest in USDA 532C (EPSB) and Rhizobium leguminosarumS2 EPS (EPSC) Mannose and arabinose had the least sugar concentration in USDA 110 and USDA 532C respectively. The sugar concentration varied when all the Rhizobial EPS were compared. Sugars such as rhamnose, arabinose, mannose, glucose and galactose were present in all the EPS samples with different concentration. D-ribose, xylose and Inositol was present in USDA 110 EPS. This work is in disparity with the results of Castellane et al. [10] who found that EPS obtained from Mesorhizobium huakuii, M. loti, M. plurifarium, Rhizobium giardinibv. Giardini, R. mongolense and Sinorhizobium (Ensifer) kostiense were primarily composed of glucose and galactose. Castellane et al [10] accounted that the EPSs from R. tropicis showed a related composition to rhizobia isolates, with glucose being the main sugar component accompanied by galactose and then glucuronic acid, rhamnose, and mannose, which were found in trace amounts. Numerous studies have reported that the EPS monosaccharide constituent may depend on the carbon source used for growth [19,20]. The complexities in their monosaccharide and non-carbohydrate compounds are responsible for their biotechnology properties which enable their potential use as gelling agents, emulsifiers [21,22], stabilizers, texture-enhancing agents and flocculation agents.

Effect of Rhizobial EPS coating on weight loss, pH, total sugar, reducing sugar and titratable acidity of Musa paradisiaca (Plantain), Musa sp (Apple Banana), Musa acuminata (Lady Finger) and Musa balbisiana (Cooking Banana) during storage was studied. Post-harvest spoilage is a main cost in food production and when polysaccharides based edible coverings are applied on fresh and minimally processed fruits and vegetables, by formation of a modified atmosphere condition help to trim down their respiratory rate. It’s enhanced mechanical handling property and additives carrying capacity [23]. In all the fruits preserved, the maximum weight loss was noticed in uncoated fruits (control) while the least weight loss was obtained in EPS coated fruits. The lowest pH was noticed in uncoated fruits while the highest pH was observed in covered fruit samples. The maximum titratable acidity was noticed in EPS coated fruits samples while the least titratable acidity was obtained in the non EPS coated fruit samples. The total sugar content steadily increased at day 10 of storage for both the covered and the uncovered fruit samples. The total sugar was noticed in EPS covered fruits samples while the least total sugar was obtained in the uncovered fruit samples. The outcome of EPS coating steadily increase the reducing sugar content among the fruits of the coated and the uncoated fruit samples. The rise in the reducing sugar with the advancement of ripening as well as storage time was due to derogation of starches. The utmost reducing sugar was observed in EPS coated fruits samples while the lowest reducing sugar was obtained in the uncoated fruit samples. In general, EPS coated fruits samples were observed to have longer shelf life than the uncoated samples.