Fruit-peel-degrading Potential of Some Legumes-associated Bacteria

This research aimed at determining the digestive ability of nine legumes-associated bacteria on the peels of some fruits (orange, watermelon, plantain, banana, pineapple and pawpaw). The bacteria were cultivated separately on each peel for 18 hours at 28C; the amount of glucose released was quantified using Dinitrosalicylic acid reagent method. The results obtained showed that the bacteria degraded all the peels with the highest (0.297 mg/mL) and lowest (0.087 mg/mL) glucose concentrations produced by Rhizobium leguminosarum FUBO001 and Bonitrorhizobium winogradskyi FUBO004 in banana and pawpaw peels, respectively. The Bo. winogradskyi FUBO004 synthesized 0.101 mg/mL as minimum sugar amount in the former peel while Bradyrhizobium nigeriasis FUBO005 produced the highest glucose quantity (0.167 mg/mL) in the latter peel. The lowest amounts of glucose in orange, watermelon, pineapple and plantain peels produced by Bradyrhizobium nigeriasis FUBO003, Rhizobium nigeriasis, R. nigeriasis and Br. nigeriasis FUBO003 were 0.095, 0.132, 0.09 and 0.248 mg/mL respectively. In these peels, the highest amount of the reducing sugar made was 0.131 mg/mL by Br. nigeriasis FUBO005, 0.211 mg/mL by Br. nigeriasis FUBO005, 0.156 mg/mL by Bo. winogradskyi FUBO004 and 0.291 mg/mL by R. nigeriasis. These results suggest that the bacteria catabolized the fruit peels, reflecting their high potential in the conversion of the fruit peels to useful products.


Introduction
Fruits and vegetables are covered by a protective layer called peel Pranav et al., 2017). Based on thickness and tastes of fruit peels, the peels could be eaten as part of the fruits; but in some instances, fruit peels are discarded as wastes particularly when they have unpleasant tastes or constitute inedible portions as seen in banana (Pranav et al., 2017). After the consumption of the inner succulent part of a fruit or during its use in fruit juice production, fruit peels are considered as wastes in order to prevent contamination (Olukunle et al., 2007;Oladiji et al., 2010). Fruits and vegetables are responsible for about 22% of food losses and wastes along the supply chain (Santos et al., 2022). Solid wastes such as peel, core, unripe and over-ripe fruits, as high as 50% of raw materials, are generated by fruit processing industries (Lekhuleni et al., 2021).
Fruit peels consist of pectin and related substances occurring in the cell walls and middle lamellae of all higher plants (Dutta, 1981;Singh et al., 2003). These substances hold cells together in all plants (Okafor, 1987;Pretel et al., 2018). The cell walls of plants generally contain different components. However, the primary cell wall is majorly made up of eight polymers which are pectin existing in three forms containing D galacturonic acid, cellulose, three glycans having neutral sugars and structural proteins (Pretel et al., 2018). Wongkaew et al. (2021) reported that mango peel is a potential source of dietary fibre and depending on fruit varieties and methods of extraction, it contains 5 -11% pectin.
Peels are manually, mechanically or enzymatically separated from other parts of plants Pranav et al., 2017). The separation of plant's individual cells occurs by the degradation of pectin and similar polymers and hence loss of tissue coherence (Shigetaka, 1977;Kumar, 2015). The removal of fruit peels by enzymes is based on the principle of digestion of pectic substances present in the cell wall of the plants (Bruemmer et al., 1978;Berry et al., 1988;Ajayi and Boboye, 2012). Maceration is important industrially for the removal of the segment of fruit's membrane and this can positively affect the integrity of the fruit juice (Ben-Shalom et al., 1986;Boboye and Ajayi, 2012). Previous research efforts have shown that extraction of fruit juices using enzymes to peel fruits leads to higher yields and improvement of juice appearance (Kumar, 2015). Zerva et al. (2019), suggested and shown that maceration of plant tissues is carried out by many microorganisms. Pathogenic activity of many of these microorganisms limit their agricultural and industrial applications. It is important to search for non-pathogenic microbes which can be used for fruit-peel removal. Rhizobia are nonpathogenic soil bacteria that form nodules on the roots of legumes using the mechanism of intracellular infection. Based on this, the organisms are useful for the removal of fruit peels, particularly Rizhobium spp. CWP G34B . This research was proposed to screen some tropical rhizobia for their ability to catabolize peels of some Nigerian fruits.

Samples and Their Preparation
The fruits (orange, pineapple, watermelon, plantain, banana and pawpaw) from which peels were prepared for this study were obtained from different markets namely: "Sasha", "Oba", "Isinkan", "Agagu Road", "Mojere" and "Iloro" in Akure, Southwestern, Nigeria. The fruits were washed thoroughly in sterile water to remove any dirt and peeled using a sterilized knife, sun dried (between 30 o C and 40 o C) for 5 hours daily for 7 days. The samples were then ground using Marlex Electroline Blender and kept in the refrigerator maintained at 4 o C until needed.

Culture Media and Their Preparation
The culture media used in this study were nutrient agar, nutrient broth (Lab M, Topley House, England), basal medium and fruit-peel medium (FPM) (Composed in this Work). The first two media were prepared according to the manufacturer's specification. Basal medium containing 0.1% (w/v) NH 4 NO 3 , 0.5% (w/v) KH 2 PO 4 , 0.05% (w/v) MgSO 4 .7H 2 O and 0.01% (w/v) CaCl 2 .2H 2 O was prepared in 100 mL of distilled water at pH 5.6. The FPM was prepared with 10 mL of the basal medium and 1% (w/v) fruit peel. All media were sterilized by autoclaving at 121 o C for 15 minutes. Determination of the Fruit-Peel-Degrading Potential of the Bacteria

Cultivation of bacteria
The sterilized fruit-peel medium (FPM) was inoculated with 0.1 mL of the 24 hours old nutrient broth inoculum (prepared above) and incubated at 28 o C for 24 hours. The test was carried out in triplicates for each bacterial isolate. Uninoculated FPM was used as control. Each culture and the control were centrifuged at 3600 rpm for 15 minutes and the supernatants were used to assay for the quantities of glucose formed by the bacteria.

Measurement of Glucose Released from Fruit Peel
Standard glucose curve was prepared according to the Dinitrosalicylic acid (DNSA) reagent method (Bernfeld, 1955) as described by Boboye and Alao (2008). Amount of glucose released into the medium by each microbe was measured by subjecting the culture supernatant of individual bacterium to the DNSA method and the optical density (OD) measured at 540 nm was referred to the standard curve to obtain the concentration of glucose in the culture supernatant of each microorganism. Glucose standard curve value of the control (FPM) was subtracted from the glucose standard curve reading of each of the supernatants of the bacterial grown in FPM to obtain the actual glucose concentration.

Statistical Analysis of Data
The data collected were analysed using the analysis of variance (ANOVA) technique and expressed as a mean of value. Duncan Multiple Range Test was carried out to determine differences in the means using SPSS Software Package (Duncan, 1955) as applied by Ajayi and Boboye (2012).

Results
All the legumes-associated bacteria showed considerable (at 95% confidence limit) degradation ability on all the fruit peels used in this study. The amount of glucose released by the individual bacterium into the growth medium of each fruit-peel is shown in Figures 1 -6. The highest and lowest glucose concentrations of 0.297 mg/mL and 0.087 mg/mL were produced by Rhizobium leguminosarum FUBO001 and Bonitrorhizobium winogradskyi FUTABO004 in banana and pawpaw peels respectively ( Figures   1 and 2). In the banana peel, Bo. winogradskyi formed 0.101 mg/mL as the lowest glucose concentration ( Figure 1) and Bradyrhizobium nigeriasis FUBO005 produced 0.167 mg/mL as the highest quantity of the sugar in the pawpaw peel ( Figure 2).
In orange peel, Br. nigeriasis FUBO003 released the lowest (0.095 mg/mL) and Br. nigeriasis FUBO005 the highest (0.131 mg/mL) amounts of the sugar (Figure 3). Similarly, the concentrations of glucose made in watermelon by Rhizobium nigeriasis and Br. nigeriasis FUBO005 ranged from 0.132 mg/mL to 0.211 mg/mL ( Figure 4).
Rhizobium nigeriasis produced 0.09 mg/mL and Bonitrorhizobium winogradskyi FUBO004 made 0.156 mg/mL as the lowest and highest glucose concentrations in pineapple peel ( Figure 5).
Bradyrhizobium nigeriasis FUBO003 and R. nigeriasis catabolised plantain peel to release 0.248 mg/mL and 0.291 mg/mL of glucose as the lowest and highest concentrations respectively ( Figure 6).
It was observed that none of the concentrations of glucose released from plantain peel was below 0.2 mg/mL in contrast to other peels. Generally, Br. nigeriasis FUBO005 produced the highest amounts of glucose in the peels of three fruits (pawpaw, orange and watermelon) (Figures 2, 3  and 4). Comparatively, the bacterium made considerable glucose amounts of 0.253 mg/mL, 0.146 mg/mL and 0.290 mg/mL in banana, pineapple and plantain peels respectively (Figures 1, 5 and 6).

Discussion
The results of this study showed that all the bacteria produced glucose from the peels of the fruits (orange, watermelon, plantain, banana, pineapple and pawpaw) tested, meaning that the bacteria degraded the substrates. Generally, peels and plant outer coverings contain pectin and its derivatives which are made up of polymers of glucose, hence the glucose formed from the peels by these microbes. The difference in the amounts of glucose released, is an indication that the ability of the bacteria to degrade the peels differs. Those bacteria that made the lowest and highest amounts of the sugar have lesser and better peel degrading potentials respectively on the same peel in contrast to their counterpart microbes. This result is supported by the findings of some researchers like Kong et al. (2022). watermelon peels ( Fig. 1 and 4) in which 44.44% and 11.11% of the bacteria produced ˃0.2 mg/mL glucose respectively and hence, the banana peel is rated as the second best degraded after plantain peel. The higher glucose concentrations obtained in the plantain peel than in other peels (apart from banana) infers that any of the bacteria can be used to degrade plantain peel with the best being Rhizobium nigeriasis followed by Br. Nigeriasis FUBO005 and R. leguminosarum FUBO001.
The better catabolism of the plantain peel than other peels may be associated with their nutrient's composition. Relatively, all the peels are high in fibre content since they contain ˃5g/100g; fruit peels are commonly rich in fibres as proven by many researchers including Dias et al. (2020). Fibre is a structural carbohydrate and it aids digestion; the fibre concentrations in banana, pawpaw, orange, watermelon and pineapple peels have been reported to be 11.81 ± 0.06, 12.16 ± 0.06, 14.19 ± 0.01, 26.31 and 14.80 ± 0.01 g/100g dry weight respectively (Feumba et al., 2016). Similarly, Morais et al. (2017) reported the fibre concentrations of banana, pawpaw, watermelon and pineapple peels to be 20.1 ± 0.27 -23.5 ± 3.8, 16.7 ± 0.5 -18.7 ± 1.8, 32.3 ± 4.6 -37.4 ± 7.1, and 13.9 ± 1.1 -15.9 ± 2.4 g/100g dry weight respectively. For plantain peel, the total dietary fibre (TDF) is 64.33 g/100g (Arun et al., 2015) while from the studies of Emaga et al. (2007) on peels of five different varieties of plantain, the TDF varied from 32.9 to 49.9 g/100g. The higher fibre content of the plantain peel could have made it easier for the bacteria to penetrate this peel than other peels and thus breakdown the carbohydrate to the reducing sugar.

Conclusion
The fruit peels used in this study were catabolized by the legume-associated bacteria. This implies that the bacteria have potential for use in the peeling of fruits and for the production of enriched animal feeds. Based on the highest glucose quantities formed by the bacteria, Rhizobium leguminosarum FUBO001, Bonitrorhizobium winogradskyi FUBO004 and R. nigeriasis may be particularly useful in the removal of banana, pineapple and plantain peels while Bradyrhizobium nigeriasis FUBO005 is suitable for the removal of pawpaw, orange and watermelon peels.