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Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 08 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.808.010 Natural Micro Flora on Edible Raw Vegetables and Fruits J.H. Ameena Sulthana*, Suvarna, V. Chavannavar, J.S. Anusha and Neekshita Shetty Department of Agricultural Microbiology, GKVK, UAS, Bangalore, India *Corresponding author ABSTRACT Keywords Lactic acid bacteria, Guava, Amla, Radish, Carrot, Coriander, Penicillium sp., Fusarium sp., Cephalosporium sp., Rhizopus sp., Aspergillus niger and Aspergilus flavus Article Info Accepted: 04 July 2019 Available Online: 10 August 2019 Association of lactic acid bacteria (LAB) on the edible plant surfaces is well known. These are beneficial organisms, to exploit their properties i.e. association of LAB on surfaces of raw edible fruits and vegetables a study was conducted. Six samples viz., guava, amla, radish, carrot, coriander and fenugreek were chosen for research studies. LAB were isolated by standard plate technique. Spoilage bacteria and molds were isolated from same spoiled fruits and vegetables by standard plate count method. Spoilage moulds viz., Penicillium sp., Fusarium sp., Cephalosporium sp., Rhizopus sp., Aspergillus niger and Aspergilus flavus. 30 LAB isolates were isolated. The highest LAB population was present on carrot’s surface i.e. (214 x 103 CFU/g) followed by coriander (98.66 x 103 CFU/g) and guava (48.08 x103 CFU/g). important to know the natural micro flora on these edible raw fruits and vegetables, which in turns to help us to know the native micro flora may be beneficial to human health or neutral or causes deterioration of these fruits and vegetables. Introduction Fruits and vegetables supply vitamins, minerals, dietary fibers that function as antioxidants, phytoestrogens and antiinflammatory agents. Vegetables are rich in vitamin A, vitamin C, fiber, folate and potassium. Folate helps in the formation of red blood cells. The healthiest choices are fresh fruits as fruits are naturally low in fat, sodium and calories, and rich in potassium, fiber, vitamin C and folate. Fiber in fruit helps to protect against heart disease and lower cholesterol. Raw fruits and vegetables harbor some of the microorganisms on their surfaces which may be friends and also foes. It is So keeping in mind the importance of above factors the objectives of this study was designed to enumerate the friendly bacteria which in turn beneficial for human health that are to isolate and to characterize the native lactic acid bacteria from edible plant surfaces. Other microorganisms are foes means, microorganisms which are responsible for spoilage. Based on this other objective was 83 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 designed to isolate the spoilage microorganisms from the fruits and vegetables. The edible raw fruits and vegetables were chosen based on the consumption pattern of people living in Karnataka state, India. They are fruits such as amla and guava, vegetables such as coriander leaves, fenugreek leaves, radish and carrot. Identification of lactic acid bacteria The bacterial isolates were examined for colony morphology (Becking, 1974) included pigment, margin, elevation, position in the agar medium. Different samples of raw fruits and vegetables were collected for isolation of LAB from different locations viz., horticulture garden, market and food outlets at locality of yelahanka in Bangalore district, Karnataka state. Samples included amla, guava, radish, carrot, fenugreek leaves and coriander leaves. Cell morphology of the isolated LAB isolates was studied by simple staining and identified through various biochemical tests such as Gram staining (Harrigan and McCance, 1998), Catalase test (Balazevic and Ederes, 1975), Motility test (Tittsler and Sandholzer, 1936), Endospore staining (Murray et al., 1994), Acid and gas production (Seeley and Vandemark, 1970, Hydrolysis of gelatin (Ewing, 1966), Starch hydrolysis (Yazdanparast, 1993), Lipid hydrolysis (Aneja, 2012), Exopolysaccharide production (Paulo et al., 2012). Isolation and purification of lactic acid bacteria Isolation and purification of spoilage molds and bacteria Fruits and vegetables were gently washed. The peels of fruits and vegetables were peeled; 10g of sample was weighed and added to 100ml sterile water blank. It was subjected to serial dilution up to 10-7 dilutions. Lactic acid bacteria (LAB) were isolated by standard plate count technique using MRS medium. (De Man, Rogosa and Sharpe, 1960) (Fig. 1). The same spoiled fruits and vegetables were selected and infected parts were removed by cutting and it was added to 100ml sterile distilled water. It was further diluted up to 10-7 dilution. Materials and Methods Sample collection The 10-2 and 10-3 diluents were used for isolation of spoilage molds, 10-6 and 10-7 diluents were used for isolation of spoilage bacteria. (Chaudhary and Dhaka, 2016) purification of spoilage molds and bacteria was done on PDA medium. The molds were purified by selecting an isolated mold colony plug with the help of cork borer and placed on PDA medium. Total thirty isolates were isolated, five isolates from each sample. The colony morphology resembling to LAB were isolated, transferred to MRS broth and incubated at room temperature for four days. A loop of LAB culture from MRS broth was streaked on MRS agar medium (Fig. 2). The inoculated Petri plates were incubated at room temperature for four days and observed for single isolated colony. The single colony isolated was streaked on MRS slants for preservation. LAB cultures were maintained on MRS slants and sub culturing was done after six months. Spoilage bacteria were purified by selecting a single colony from streaked plate and streaked again on nutrient agar slants. The cultures were preserved and sub cultured once in 3 months. 84 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 Our results generally demonstrated that the lactic acid bacterial population was highest on the surfaces of carrot. Enumeration of LAB were done from raw edible fruits and vegetables where the same work was already done by Jalali et al., they also notified that higher densities of LAB were found on carrots tomato, soybeans, radish and lettuces. Characterization of spoilage molds and bacteria Identification of spoilage molds (Leck, 1999) A loop of fungal mycelia was transferred on clean slide consisting of water drop. Cotton blue (2 drops) was added to it and was covered with cover slip. The slide was observed for characteristics fruiting bodies of mold, arrangement of conidia. Identification isolates of lactic acid bacterial LAB isolates were characterized morphologically and biochemically for identification up to generic level. LAB colonies were white colored with varied sizes like medium, small and bold (Table 1). It was found that majority of LAB colonies were round and submerged, the results were in congruent with Ali et al., Biochemical characterizations of LAB isolates were also carried out (Table 2). In Gram staining test, all the 30 isolates were Gram positive. Some isolates were rods and some were cocci. These isolates were tested for catalase enzyme production and motility. All the isolates were non-motile and catalase negative. The LAB isolates were tested for lipid hydrolysis, clear transparent zone around the streaked colony indicates positive for the test. LAB isolates (27) could hydrolyze lipid. Isolates were subjected to gelatin hydrolysis test. Liquefied nutrient gelatin stabs were recorded as positive for this test and stabs with solidified gelatin were recorded as negative. In present study, all the nutrient gelatin stabs were not liquefied indicating that all the 30 isolates were negative for gelatin liquefaction. Further, these results were in concurrent with Pal et al., (2005). Characterization of spoilage bacteria Spoilage bacteria were examined for morphological characteristics such as colony pigment, margin, elevation and position in the agar medium. Cell morphology was also studied by simple staining. Various biochemical test were performed such as Gram staining, catalase test, endospore staining, gelatin hydrolysis, starch hydrolysis, lipid hydrolysis Citrate utilization test (Vaughn et al., 1950), Casein hydrolysis (Aneja, 2012), Oxidase test (Tarrand and Groschel, 1982) Methyl red and VogesProskauer tests (Clarke and kirner, 1941), H2S production (Aneja, 2012), Indole production test (Aneja, 2012), Urease test (Seeliger, 1956) in order to identify them upto generic level. Results and Discussion Enumeration of lactic Acid (LA) bacteria was carried out by standard plate count method using De Man, Rogosa and Sharpe (MRS) medium. Results related to enumeration are presented in figure 3. LAB population was the highest in carrot (214.0x 103 cfu/g), followed by coriander (98.66 x 103 cfu/g), guava (48.08 x 103 cfu/g), fenugreek (38.1 x 103 cfu/g), amla (34.9 x 103 cfu/g) and the least was observed in radish (22.7 x 103 cfu/g). The starch hydrolysis was also carried out as one of the biochemical test the results are in conformation with findings of Olympia et al., (1995), they showed that several strains of LAB could hydrolyze starch such as 85 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 Streptococcus equines, S. bovis, Lactobacillus amylovorous and Lactobacillus plantarum. Exopolysaccharide production was also one of the biochemical tests done in order to know the capacity of the isolates to produce exopolysaccharide. Precipitation formation in absolute alcohol indicates exopolysaccharide production. Out of 30 isolates 27 isolates shown positive results to this test. Similar results were reported by Welman and Maddox (2003). LAB such as Streptococcus thermophilus, Lactobacillus helviticus and Lactobacillus delbrueckii subsp. bulgaricus produced Exopolysaccharides. They also worked on enhancement of EPS production in these organisms. Results of all the above tests done indicated that isolates belonged to LAB genera. LAB may be classified as homofermentative or heterofermentative based on their byproducts of sugar fermentation. A test was conducted to know acid and gas production. Change in color from purple to pink indicated positive for acid production test. Production of air bubble in Durham’s tube indicated positive results for gas production. Out of thirty isolates, twenty-two were homofermentative and eight isolates were heterofermentative. Results are tabulated in Table 3. Fig. 3: Pure culture colonies of LAB Fig.1: Lactic acid bacterial colonies om amla 86 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 Fusarium sp. Penicillium sp. Aspergillus niger Rhizopus sp. Aspergillus flavus Cephalosporium sp. Fig.4: Spoilage molds isolated and their microscopic view 87 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 Table.1 Colony characteristics of LAB isolates of fruits and vegetables Sl. No. Source 1. Radish 2. Carrot 3. 4. 5. 6. Guava Amla Coriander Fenugreek Isolates R-1 R-2 Color White White Size Medium Bold Shape Round Oval Position Submerged Submerged R-3 R-4 R-5 Ct-1 White White White White Medium Small Small Medium Round Round Round Round Submerged Submerged Surface Submerged Ct-2 White Small Round Submerged Ct-3 White Bold Round Submerged Ct-4 White Small Irregular Submerged Ct-5 White Medium Round Surface G-1 White Medium Round Submerged G-2 White Small Round Surface G-3 White Small Round Submerged G-4 White Bold Round Surface G-5 White Medium Irregular Submerged Am-1 White Bold Round Surface Am-2 White Small Round Submerged Am-3 White Medium Round Surface Am-4 White Small Round Submerged Am-5 White Medium Round Submerged Co-1 White Small Round Submerged Co-2 White Medium Round Submerged Co-3 White Bold Irregular Submerged Co-4 White Bold Surface Surface Co-5 White Bold Oval Submerged Fg-1 White Small Round Surface Fg-2 White Small Round Submerged Fg-3 White Small Round Surface Fg-4 White Bold Round Surface Fg-5 White Bold Oval Submerged 88 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 Table.2 Cell morphological characteristics of LAB isolates Source Radish Carrot Guava Amla Coriander Fenugreek Reference strain Isolates Shape Motility Rod Gram reaction + R-1 R-2 R-3 - Catalase test - Lipid hydrolysis + Gelatin liquefaction - Starch hydrolysis + EPS production + Rod Cocci + + - - + + - + + + + R-4 R-5 Rod Cocci + + - - + + - + + + + Ct-1 Ct-2 Ct-3 Ct-4 Ct-5 G-1 G-2 G-3 G-4 G-5 Am-1 Am-2 Am-3 Am-4 Am-5 Co-1 Co-2 Co-3 Co-4 Co-5 Fg-1 Fg-2 Fg-3 Fg-4 Fg-5 L.. plantarum Rod Rod Rod Cocci Rod Rod Cocci Rod Rod Cocci Rod Cocci Rod Cocci Cocci Cocci Rod Cocci Rod Rod Cocci Cocci Rod Rod Rod Rod + + + + + + + + + + + + + + + + + + + + + + + + + + - + - + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Note: Present (+); Absent (-), EPS- Exo polysaccharide production, L. plantarum- Lactobacillus plantarum. 89 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 Table.3 Acid and Gas production by LAB Sl. No 1. Source Radish 2. Carrot 3. Guava 4. Amla 5. Coriander 6. Fenugreek 7. Reference strain Isolates Acid production Gas production Homo fermentative Hetero fermentative R-1 + - + - R-2 + - + - R-3 + + - + R-4 + + - + R-5 + - + - Ct-1 Ct-2 Ct-3 Ct-4 Ct-5 G-1 G-2 G-3 G-4 G-5 + + + + + + + + + + + + + - + + + + + + + + + + - Am-1 Am-2 Am-3 Am-4 Am-5 Co-1 Co-2 Co-3 Co-4 Co-5 + + + + + + + + + + + + - + + + + + + + + + + - Fg-1 Fg-2 Fg-3 Fg-4 Fg-5 Lactobacillus plantarum + + + + + + + + + + + + - + + 90 Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 Table.4 Spoilage molds isolated from fruits and vegetable Sl. No. 1. Source Spoilage molds Radish Aspergillus niger 2. Carrot Aspergillus flavus 3. Guava Rhizopus sp. 4. Amla Penicillium sp. 5. Coriander Fusarium sp. 6. Fenugreek Cephalosporium sp. Colony characteristics Microscopic observation Black in colour Globose shaped conidia Yellow to greenish Globose shaped colony conidia Grey color colony later Round sporangium produces black spores Initially white colour Conidia in long chains later turns to dark green brush like head with branched sterigmata Wooly, white to pink. Sickle shaped macroconidia Cottony pure white Conidia held in mass colony at tip of conidiophore Table.5 Biochemical characterization of spoilage bacteria isolated from raw fruits and vegetables Sl. No. 1 2 Isolates Gram staining Shape 3 4 H2S Production Indole production Starch hydrolysis Citrate utilization Nutrient gelatin test Lipid hydrolysis Casein hydrolysis Oxidase test MR VP Catalase test Urease test Endospore staining 5 6 7 8 9 10 11 12 13 14 15 RAD Short rods - CAR Cocci GUA + Rods AML + Cocci FEN + Rods - COR Short rods - - - + - + + + + + + + - + - + - - + + - + - - + + - + + + + + + + + + - + + - + + 91 - Int.J.Curr.Microbiol.App.Sci (2019) 8(8): 83-94 Table.6 Assimilation of different carbon sources by spoilage bacterial isolates of fruits and vegetables Sl. No. 1 2 3 4 5 6 Isolates RAD GUA AML COR FEN CAR Glucose AG A A AG A A Sucrose AG A A AG A AG Lactose AG A AG AG Note: AG: Acid and gas production A: Acid production -: No acid or gas production test and results are congruent with Chaudhary and Dhaka (2016) work. They isolated eleven bacterial isolates, caused spoilage in different fruits. Biochemical test such as carbohydrate utilization, indole production, MRVP, citrate utilization, catalase, oxidase, urease production, H2S production, starch hydrolysis, casein hydrolysis and gelatin liquefaction were done Assimilation of different carbon sources by spoilage bacterial isolates of fruits and vegetables was given in Table 6. Isolation of spoilage molds from fruits and vegetables Spoilage molds were isolated from each spoiled fruit and vegetable. Six spoilage molds were isolated and their microscopic characteristics are tabulated in table 4. The observations revealed the identity of spoilage molds- Rhizopus sp. (guava), Penicillium sp., (amla), Aspergillus niger (radish), A. flavus (carrot), Fusarium sp. (coriander) and Cephalosporium sp. (fenugreek). The results are in line with Abdullah et al., (2016). They isolated spoilage molds from spoiled fruits. The most common spoilage molds isolated and identified were Penicillium expansum, Colletotrichum musae, Aspergillus niger and Penicillium glabrum. Results lead to thinking that, consumption of raw carrot not only provides β- carotene but also serves as a sink for lactic acid bacteria to render the health benefits. LAB present on various habitats, phyllosphere is one among them. Fruits and vegetables consumption in the form of salads helps in ingestion of live LAB which protects from intestinal pathogens in turn helps in restoring good health. Further, spoilage organisms were isolated from same fruits and vegetables. Spoilage bacteria (SB) isolates were characterized morphologically and biochemically up to generic level. Isolates were coded in short forms as CAR isolate (carrot), RAD (radish), FEN (fenugreek), COR (coriander), AML (amla) and GUA (guava). Majority of SB isolates were rods in shape, only two of them were cocci. Isolates were subjected to various biochemical test (Table 5). Spoilage molds such as Aspergillus niger, A. flavus, Rhizopus sp., Fusarium sp., Penicillium sp. and Cephalosporium sp. were isolated. They were identified based on the conidial structure, arrangement of spores. Spoilage bacteria were also isolated. These informations will help us in preventing these spoilage microorganisms so that it will be Similar for identification of spoilage bacteria certain test were done where, all the above 92