International Journal of Biotechnology and Biochemistry ISSN 0973-2691 Volume 7 Number 6 (2011) pp. 25–35 © Research India Publications http://www.ripublication.com/ijbb.htm
Microorganisms Associated with Volatile Metabolites Production in Soft Rot Disease of Sweet Pepper Fruits (Tattase) *1
A.D. Ibrahim, 3A. Sani, 1Manga S.B., 2A.A. Aliero, 1R.U. Joseph, 4 S.E. Yakubu and 1H. Ibafidon 1
Department of Microbiology, Faculty of Science, Usmanu Danfodiyo University, Sokoto, Nigeria 2 Department of Biological Sciences, Faculty of Science, Usmanu Danfodiyo University, Sokoto, Nigeria 3 Department of Microbiology, Faculty of Science, University of Ilorin, Ilorin, Nigeria 4 Department of Microbiology, Faculty of Science, Ahmadu Bello University Zaria, Nigeria *Corresponding Author E-mail: [email protected]
Abstract Microorganisms associated with the production of volatile metabolites in spoilt sweet pepper fruits have been isolated by standard plate count (SPC) and identified. The mean aerobic colony count range from 1.04 x 105 to 3.52 x 105 CFU/g. The highest mean value was obtained from Kasuwan Daji and the least man value from Kofar Gawo market all in Sokoto metropolis. The organisms isolated and identified include seven species of bacteria and three species of fungi. These include Bacillus megaterium, Bacillus licheniformis, Paenibacillus alvei, Enterobacter aerogenes, Proteus vulgaris, Staphylococcus auricularis, Yersinia enterocolitica and Aspergillus clavatus, Aspergillus flavus, and Fusarium poae respectively. GC-MS analysis revealed the presence of thirteen metabolites in the healthy ripe sweet pepper fruits. Endo-tricyclo [22.214.171.124. (2.6)] decane and Nonadecane were not detected in the spoilt sweet pepper fruits with the methyl group of 1,2,3- Trimethylbenzene shifted to position four to yield 1,2,4- Trimethylbenzene in the spoilt fruits. Octahydro - 4, 7- methanoindene and Nerolidyl acetate were unique to the spoilt fruits. This study suggests that these unique volatile metabolites could be exploited as biomarkers to discriminate diseases/pathogens or toxigenic fungi even when more than one disease is present thereby curbing post harvest loss and the risk of consuming mycotoxins after further validation.
A.D. Ibrahim et al Keywords: Aspergillus, Bacillus, Capsicum annuum, Fusarium, GC-MS, spoilage organisms, volatile metabolites.
Introduction Many economic plant produce are highly prone to microbial spoilage especially in storage. Bacteria and fungi are well known plant pathogens and therefore pose serious problems to farmers (Aboaba, 2007). Tomato and pepper fruit are commonly infected with soft rot bacteria but other vegetable fruits are also susceptible. Fruits are infected prior to harvest if wounded by insects or other means or the rot may progress from infected stems and branches into the fruit. Postharvest infection of fruit may occur through wounds made during harvesting, transit or storage periods or when warmer fruit are washed in cooler contaminated water (Kucharek and Bartz, 2000). Traditionally, fresh fruits and vegetables have not been considered high-risk foods in terms of causing food-borne illness, especially when compared with foods of animal origin (meat, dairy products and seafood’s). The general assumptions have been that the pH of fruits and vegetables are too low to support the growth of human disease-causing pathogens, and that the natural barriers of the fruits and vegetables would prevent microbes from entering and subsequently growing inside the food (Beuchat, 1996; Madden, 1992). Nonetheless, the relationship between fresh produce and so-called "travellers' diarrhoea" has frequently been observed (Kenny, 2002). Early detection of spoilage would be advantageous in reducing food loss because there may be interventions that could halt or delay deterioration, and on the other hand food that had reached the end of its designated shelf life but was not spoiled could still be used. Numerous methods for detection of spoilage have been devised with the goals of determining concentrations of spoilage microbes or volatile compounds produced by these microbes. However, many of these methods are considered inadequate because they are time-consuming, labour-intensive, and/or do not reliably give consistent results (Doyle, 2007). This study is aimed at isolating and identifying microorganisms associated with spoilt sweet pepper fruits in Sokoto metropolis; extract and identifying volatile metabolites associated with spoilt sweet pepper using GC-MS.
Materials and Methods Sample Collection Sixteen spoilt and four healthy intact ripe sweet pepper fruits were purchased from Kasuwan Daji, Mahauta, Kofar Gawo and Meat and Vegetable market in Sokoto metropolis. Samples were collected into polythene bags and brought to the research laboratory of Usmanu Danfodiyo University, Sokoto for the analysis. Isolation and count of microorganisms Bacteria were isolated by transferring an aliquot of 0.1 ml of a serially diluted (104) sample of spoilt sweet peppers onto molten nutrient agar plates and incubated at 37oC for 24 hours. The colonies that emerge were subculture continuously until a pure
Microorganisms Associated with Volatile Metabolites Production
culture is obtained. For fungi isolation an aliquot of the spoilt sweet pepper fruits was inoculated onto molten potato dextrose agar plates and incubated at room temperature for 7 days and was subculture continuously to obtain pure culture. Identification of Bacteria and Fungi The bacteria isolate were identified following series of biochemical test as described by Holt et al. (1994). Fungal colonies were studied by using Lactophenol Cotton Blue Mount (LPCB) as described by Oyeleke and Manga (2008). Extraction of volatile Metabolites Volatile compounds were extracted using general purpose solvent as described by Parliment (1997). Extraction of volatile compounds was done by direct solvent extraction method. Two gram of spoilt sweet pepper fruits and healthy ripe sweet pepper was weighed into a bottle and saturated with 20ml of diethyl ether. It was allowed to stand at room temperature for 24 hours, filtered using Whatman No. 1 filter Paper and the filtrate was collected in a sterile bottle, closed tightly before the GC-MS analysis. Gas chromatography mass spectrometry (GC-MS) analysis GC-MS analysis was performed using GC-MS-QP2010 plus (Shimadzu, Japan) equipped with flame ionization detector (FID). The injection was conducted in split less mode at 2500C for 3min by using an inlet of 0.75mm i.d to minimize peak broadening. Chromatographic separations were performed by using DB-WAX analytical column 30m 0.25mm, 0.25mm (J&W scientific, Folsom C.A) with helium as carrier gas at a constant flow rate of 0.8 ml/Min. The oven temperature was programmed at 60oC for 5min, followed by an increase (held for 5 min), and finally at 100C/min to 280oC (held for 10min). The temperature of the FID was set to 250oC. MS operating conditions (election impact ionization mode) were an ion source temperature of 200oC, ionization voltage of 70 eV and mass scan range of m/z 23450 at 2.76 scans/s. Identification and quantification of volatile Metabolites The chromatographic peak identification was carried out by comparing their mass spectra with those of the bibliography data of unknown compounds from the WILEY 6 library (Hewlett-Packard co., Palo Alto, CA) mass spectra database on the basis of the criterion similarity (SI)>800 (the highest value is 1,000). According to the method of (Wanakhachornkrai and Lertsiri, 2003) approximate quantification of volatile compounds was estimated by the integration of peaks on the total ion chromatogram using Xcalibur software (Vienna, VA). The results are presented as the peak area normalized (%).
Results Microorganisms associated with volatile metabolite production in sweet pepper has been isolated and identified. The aerobic colony count of spoilt sweet pepper fruits
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sold in Sokoto metropolis was evaluated and the result presented in Table1. From the result, the mean value ranges from 1.04 to 3.52 x 105 CFU/g. The highest mean value was obtained from Kasuwan Daji and the least from Kofar Gawo market. The bacteria microflora associated with spoilt sweet pepper fruits in Sokoto metropolis were isolated and identified. They include Bacillus megaterium, Bacillus licheniformis, Paenibacillus alvei, Enterobacter aerogenes, Proteus vulgaris, Staphylococcus auricularis, and Yersinia enterocolitica. The fungi microflora associated with spoilt sweet pepper fruits and there frequencies of occurrence are presented in Table 2. From the result only three fungi were isolated as follows:- Aspergillus clavatus, Aspergillus flavus, and Fusarium poae. Fusarium poae had the highest frequency (57%) while Aspergillus clavatus had the least occurrence of 14.5%. GC-MS analysis of the diethyl ether extract of healthy ripe and spoilt sweet pepper fruits was conducted to determine their volatile metabolite profile and the result presented in Table 3. The result revealed the presence of thirteen metabolites in the healthy ripe and spoilt sweet pepper fruits. Endo-tricyclo [126.96.36.199. (2.6)] decane and Nonadecane were not detected in the spoilt sweet pepper fruits with the methyl group of 1,2,3- Trimethylbenzene shifted to position four to yield 1,2,4Trimethylbenzene in the spoilt fruits. Octahydro - 4, 7- methanoindene and Nerolidyl acetate were unique to the spoilt fruits. Table 1: Aerobic colony counts obtained from spoilt pepper fruits in Sokoto metropolis. Sampling site Kasuwan Daji Mahauta Kofar gawo Meat and Vegetable market
Aerobic Colony Count x105 cfu/g Total Range 14.08 2.28 - 6.20 (1.85) 12.32 2.12 – 4.12 (0.83) 4.16 1.02 - 1.08 (0.52) 8.18 2.02 – 2.10 (0.13)
Mean (SD) 3.52 3.08 1.04 2.06
Table 2: Frequency of occurrence of fungi obtained from spoilt pepper fruits in Sokoto metropolis. Sampling site Kasuwan Daji Mahauta Kofar Gawo
Fungi Isolated Aspergillus flavus Fusarium poae Fusarium poae Fusarium poae
Frequency (%) 28.5
Microorganisms Associated with Volatile Metabolites Production
Aspergillus clavatus 14.5 Aspergillus flavus Meat and Vegetable market Fusarium poae
Table 3: Result of GC/MS analysis of healthy and spoilt sweet pepper fruits (Tattase). RT-1 (Min) Volatile Metabolites
Peak Area (%) Healthy Spoilt 3.82 Octane 10.05 7.09 6.34 Nonone 9.07 6.12 8.95 1,2,3- Trimethylbenzene (Hemellitol) 6.23 8.96 1,2,4- Trimethylbenzene (Pseudocumol) 3.62 9.65 Decane 11.09 7.18 12.34 Endo-tricyclo [188.8.131.52 (2.6)] decane 3.16 12.34 Octahydro-4, 7- methanoindene 1.89 12.97 Undecane 6.85 4.67 14.18 Benzocyclohezane (Tetralin) 4.21 1.02 14.75 Cyclopentacycloheptene (Azulene) 1.44 1.92 18.07 1,6 – Methano  annulene 6.56 11.04 21.42 Ethynyl- 2 – methyl- 1- propenyl) benzene 1.98 0.81 27.60 n- Henedecanoic acid 12.09 19.19 28.77 9- Octadecenoic acid (Z) 19.29 32.39 29.59 Nerolidyl acetate 3.06 30.38 Nonadecane 7.98 1 Retention time (RT) on DB-WBX column in GC-MS.
Discussion This study revealed that sweet pepper fruits sold in Kasuwan Daji and Mahauta had a high aerobic colony count of 3.52 and 3.08 x 105 cfu/g. This might be attributed to factors such as the environment, which include exposure of the pepper to air, soil; post production operations and personal hygiene of the food handlers (Ray and Bhunia, 2007; Jay et al., 2005; Beuchat, 1996). Exposure of the foods to air or dust at the point of sale is likely to increase the counts of the bacteria as virtually most of the bacteria are carried in aerosols by dust and air (Food and Drug Administration, 2009). Though, microbial quality limit is not applicable to fresh sweet pepper (Gilbert, et al., 2000), the types of organisms isolated implies that those who consume fresh sweet pepper fruits from these market may come up with gastrointestinal problems and other fatal disease due to mycotoxin. This results is in contrast to those of other workers in Nigeria who reported higher microbial load of 3.6 x 106 (Eni et al., 2010); 5.9 x 106 (Uzeh et al., 2009) and 1.60 x 107 cfu/g in their studies (Bukar et al., 2010); also contrary to those of other workers elsewhere who reported a lower bacteria population of 4.3 log10 CFUg-1 as initial microbial load of stored bell pepper (Tano et al., 2008);
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5.99 Log10 CFUg-1 at day 0 for ready to use (RTU) green peppers held at 4oC (Odumeru et al., 1997). The bacteria microflora associated with spoilt sweet pepper fruits in this study area are Bacillus megaterium, Bacillus licheniformis, Paenibacillus alvei, Enterobacter aerogenes, Proteus vulgaris, Staphylococcus auricularis, and Yersinia enterocolitica. The source of these organisms could probably from soil, the fruits themselves, the harvesting/ packaging containers, pepper handlers, air and dust (Jay et al., 2005). Sweet pepper fruits from these markets may not be safe for consumption as they may cause gastroenteritis because of the presence of Yersinia enterocolitica. Other studies on microorganisms associated with fruits and vegetables in Nigeria and elsewhere revealed similar bacteria microflora (Eni et al., 2010; Uzeh et al., 2009; Tambekar and Mundhada, 2006; Adebolu and Ifesan, 2001). However, these workers isolated other organisms such E. coli, Salmonella spp., Pseudomonas, Klebsiella Citrobacter fruendii and Micrococcus spp which were not detected in this study. Many researchers (Marchetti; et al., 1992; Garg et al., 1990; Magnusson et al., 1990; Carlin et al., 1989; Nguyen-the and Prunier, 1989; Brocklehurst et al., 1987; Manvell and Ackland, 1986) have determined that 80–90% of mesophilic bacteria in the aerobic plate counts of vegetables are Gram-negative rods, Pseudomonas spp., Enterobacter spp., or Erwinia spp. with pseudomonads prevailing over other genera. This result is in contrary to most of these authors as no Pseudomonas spp was isolated and the prevailing genera in this work is gram positive organism of the Bacillus genus. This probably is explained by the fact that Bacillus spp are able to overcome some of the intrinsic and extrinsic parameters that could have check their population due to their ability to form spores. The fungi microflora are Aspergillus clavatus, Aspergillus flavus, and Fusarium poae. This is not surprising considering the nutritional composition of vegetables which generally are capable of supporting the growth of molds, yeasts, and bacteria and, consequently, of being spoiled by any or all of these organisms (Jay et al., 2005). The moulds isolated in this study have been reported in similar studies as the major cause of spoilage in tomato (Ghosh, 2009) and other fruits (Battilani et al., 2008; Jay et al., 2005). The presence of these organisms in spoilt sweet pepper makes it unsafe for consumption as it is a source of potent mycotoxins which exhibits a wide spectrum of diseases which can even be fatal. Moulds are overwhelmingly present in postharvest diseases of fruits and vegetables and their populations have been reported in various types of fresh-cut fruits and vegetables (Tournas, 2005a; Nguyen-the and Carlin, 1994; Hagenmaier and Baker, 1998). Patulin (clavicin, expansin) and Cytochalasin E are important mycotoxin produce by Aspergillus clavatus (LopezDiaz and Flannigan, 1997; Davis and Diener, 1987). Aspergillus flavus produces aflatoxins AFB1 and AFB2; (Kurtzman et al., 1987). Fusarium poae is a producer of the mycotoxin culmorin and its derivatives (Thrane et al., 2004). The fatty acids detected in the spoilt and healthy fruits are n- Henedecanoic acid and 9- Octadecenoic acid (Z). The hydroxyl form of 9- Octadecenoic acid (Z) that is hydroxy fatty acids (HFA) have been described as multifunctional molecules that have a variety of applications (Bódalo et al., 2005), and they and their derivatives are used in cosmetics, paints and coatings, lubricants and in the food industry (Bódalo et
Microorganisms Associated with Volatile Metabolites Production
al., 2005). They are useful chemical intermediates in the synthesis of fine chemicals and pharmaceuticals (Bódalo et al., 2005). Moreover, some of them may protect plants against microbial infection, although the mechanism of these antimicrobial effects is poorly understood (Suzuki et al., 2005). Their sources could probably be from the fruit seed oils and microorganisms (Hayes, 1996). Their methyl ester forms along with azulene, pseudocumol and nonadecane have also been detected in the leaves of Senna surattensis. The importance of these esters has been describe to contribute to food aroma with the fact that esters with low carbon atoms are highly volatile at ambient temperatures and the perception thresholds are 10 times lower than their alcohol precursors (Izco and Torre, 2000; Nogueira et al., 2005). In addition to imparting a fruity floral character, esters can diminish or mask the sharpness of unpleasant free amino acid-derived notes (Yanfang and Wenyi, 2009). Azulene hydrocarbons were proved to posses many biological activities (El-Sawi and Sleem, 2009). Trimethylbenzene has been described as food aroma compound (Longo and Sanromán, 2006). Nerolidyl acetate may be responsible for the characteristic sweet greenish fruity aroma note displayed by the spoilt sweet bell pepper fruits. Neryl acetate, t-carvyl acetate and butyl acetate were detected in mango fruits and describe to impart sweet caramel fruity flora, sweet green tea fruity, and fruity aroma note (Mahattanatawee et al., 2005). Butyl acetate was also detected in guava fruits and bornyl acetate was detected in carambola fruits and was describe to impart sweet green aroma (Mahattanatawee et al., 2005). Forero et al. (2009), identified 140 steam volatile constituents chile pepper (Capsicum annuum L. var. glabriusculum) and reported that hexyl isopentanoate, hexyl 2-methylbutanoate, limonene, hexyl isohexanoate, (E)-2-hexenal, isopentyl isopentanoate and (Z)-3-hexenyl isopentanoate were the major steam volatile constituents of chile pepper at two ripening stages (green and red). They also reported that during fruit maturation, the majority of volatile compounds decreased. In general, green chile peppers have higher amounts of esters, with their fruity odour notes, than red fruits. The differences in the amount of total volatiles, is higher at the green stage and they concluded that the green stage is better in terms of its flavour than the red stage. The red stage, the microbial activities, the method of extraction and extraction solvent may be responsible for the few number of metabolites detected. The decease observed in most of the hydrocarbons may be attributed to the fact that the isolated organisms may have utilized them for their metabolism during spoilage. Similar observation has been reported by other worker (Jay et al., 2005).
Conclusion This study revealed high bacteria count of 3.52 x 105 cfu/g in spoilt sweet pepper fruits sold in Kasuwan Daji and also revealed that sweet pepper fruits sold in Sokoto Metropolis contain organisms such as Bacillus megaterium, Bacillus licheniformis, Paenibacillus alvei, Enterobacter aerogenes, Proteus vulgaris, Staphylococcus auricularis, Yersinia enterocolitica, Aspergillus clavatus, Aspergillus flavus, and Fusarium poae respectively. Hence spoilt sweet pepper fruits are not safe for consumption. The study revealed the presence of thirteen metabolites in sweet pepper
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fruits with Endo-tricyclo [184.108.40.206. (2.6)] decane and Nonadecane common to healthy ripe sweet pepper fruits while the methyl group of 1,2,3- Trimethylbenzene shifted to position four to yield 1,2,4- Trimethylbenzene in the spoilt fruits. Octahydro - 4, 7methanoindene and Nerolidyl acetate were unique to the spoilt fruits. This study suggests that these unique volatile metabolites could be exploited as biomarkers to discriminate diseases/pathogens or toxigenic fungi even when more than one disease is present thereby curbing post harvest loss and the risk of consuming mycotoxins after further validation.
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