Research Paper Level of Mycological Associates in Some Packaged Fruit
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Academia Journal of Medicinal Plants 7(5): 125-128, May 2019 DOI: 10.15413/ajmp.2019.0135 ISSN: 2315-7720 ©2019 Academia Publishing Research Paper Level of mycological associates in some packaged fruit juice sold in Anambra State Accepted 16th May, 2019 ABSTRACT The level of mycological associates of twelve brands of fruit juice (Five alive citrus burst, Dansa orange, Dansa apple, Dansa mango, Frutta apple, Frutta orange, Frutta mango, Chivita apple, Chivita pineapple, Chivita mango, Chivita orange and Five apple splash) sold in Anambra state were studied. In the study, standard mycological methods was adopted to determine the Total Viable Fungal Count (TVFC), characteristics and frequency of the fungal isolates respectively. The results showed that the mean fungal counts ranged from 1.0 × 102 to 6.0 × 102 cfu/ml and the yeast and moulds frequency were also observed. The isolated organisms were identified to be Aspergillus niger, Mucor, Rhizopus and Saccharomyces species. Aspergillus sp were the most predominant isolates in fruit juices (Chivita pineapple) studied. The study, thus suggest that such level of Aspergillus sp (mould) in Chivita pineapple is not safe for human consumption. The study therefore calls for continuous awareness to farmers during pre-harvest and post-harvest of the raw fruits before processing of these fruit juices and Onuoha T. adherence to good manufacturing practices so as to avoid contamination of some Department of Microbiology, Anambra packaged fruit juice sold in Anambra markets. State University, Uli, Nigeria. E-mail: [email protected]. Key words: Fruit juice, Aspergillus niger, Mucor, Rhizopus. INTRODUCTION Fruit juice is a liquid naturally present in fruit and nectar. vegetable tissues. It is defined generally as the extractable Juices are preserved by physical and chemical methods substance of cells or tissues (Hatcher et al., 2003). Juice is (Pao et al., 1996). The usual physical and chemical methods prepared by mechanically squeezing or crushing fresh fruit includes: canning pasteurization, freezing, evaporation and without the addition of heat or other soluble liquid. For the use of preservatives such as benzoic acid in the form of example, orange juice is the liquid extract from the fruit of sodium benzoate and citric acid (Hoover, 1997). Fruit juice orange tree. However, many fruit juices are diluted with has a level of standard defined as purity, which is 100% in water and sugar because of the sour taste of the liquid and many countries like United States of American (USA) and such fruits include lemon and lime (Hatcher et al., 2003). New Zealand etc (FDA, 1999). Juices are mostly consumed Juice may be prepared for household use from a variety for their health benefits because it is rich in vitamin C (Pao of fresh fruits using hand or electric juicers. Juices sold et al., 1996). commercially are filtered to remove fibre or pulp but The purity of fruit juice depends on the raw fruits, sometimes sold directly without filtration. Juice may also be processing mechanisms, package material and storage sold in its concentrated form and sometimes frozen conditions. The number of micro-organisms which initiate requiring the user to add water to reverse the liquid back to spoilage in processed fruit juices depends on the condition its original state (Salunkle and Kadan, 1996). Moreover, of the fruit used and the washing process (Frazier and fruit juices are of various types such as pure fruit juice, Westhoff, 2002). Many micro-organisms majorly mixed fruit juice, carbonated fruit juice, fruit puree and mycological associates-fungi (yeast and mold) which are Academia Journal of Medicinal Plants; Onuoha. 126 acid loving or acid tolerant can use fruit juice as substrate Enumeration of the fungal isolates and cause spoilage producing off odour and flavor, discolouration of the product and if the contaminants are The Total Viable Fungi Colonies (TVFC) formed were pathogenic could cause human illness after consumption counted using a colony counter and means expressed as (Parish and Higgins,1989). cfu/ml (Misilivec et al., 1992); that is, sample × number of The aim of this research was to determine the colony formed. mycological associates in packaged fruit juices sold in Anambra State. Characterization and identification of fungal isolates MATERIALS AND METHODS The identification of fungal colonies and characterization was based on the report of Barnett et al. (2000) using their Study area cultural, morphological and microscopic characteristics. Lactophenol blue was used to stain a wet mount Anambra State have a lot of supermarkets located in preparation of a discrete colony and viewed under ×10 and different localities in the city where fruit juice (Five alive ×40 objectives lens of binocular light microscope. citrus burst, Dansa orange, Dansa apple, Dansa mango, Frutta apple, Frutta orange, Frutta mango, Chivita apple, Chivita pineapple, Chivita mango, Chivita orange and Five RESULTS AND DISCUSSION apple splash) are sold for final consumption. The markets include Main market, Eke awka, Uli market, Nnewi market The results from this research showed that the twelve fruit and Ekwulobia market. The fruit juice (Five alive citrus juice samples gotten from markets in Anambra State had burst, Dansa orange, Dansa apple, Dansa mango, Frutta growth after 24 to 48 h of incubation at room temperatrure apple, Frutta orange, Frutta mango, Chivita apple, Chivita (28±2°C). The mean fungal counts isolated from the twelve pineapple, Chivita mango, Chivita orange and Five apple fruit juice samples ranged from 1.0 × 102 to 6.0 × 102 and splash) are usually transported from within the state or Chivita pineapple had the highest count as depicted in other neighboring western, eastern, southern or northern Table 1. However, four (4) colonies were identified to be part of the country. Aspergillus niger, Saccharomyces cerevisae, Mucor species and Rhizopus sp based on their cultural, morphology and microscopic characterization reaction (Table 2) according Sample collection to the resport of Cheesbrough (2000) and Barnett et al. (2000). The result in this study is line with the result of the Random sampling of fruit juice, Five alive citrus burst, previous study of Frazier and Westhoff (1986). Dansa orange, Dansa apple, Dansa mango, Frutta apple, Table 3 shows the fungal frequency of the isolates (mold Frutta orange, Frutta mango, Chivita apple, Chivita and yeasts), while the bar chart in Figure 1 also showed the pineapple, Chivita mango, Chivita orange and Five apple fungal occurrence with Aspergillus sp with the highest splash) were purchased from various markets in Anambra occurrence followed by Rhizopus sp., Saccharomyces and state. The samples have six months to the expiration date Mucor sp. The presence of these organisms (mold and from the date of manufacturing. However, samples were yeast) in fruit juice could be attributed to the ability of the provided to the analytical laboratory for analysis organisms to survive in low pH (acid loving), refrigeration (Cheesebrough, 2003). temperature and use it as a substrate for growth (Zarate et al., 2000). The results obtained in this study are in line with previous results of Hatcher et al. (2003), Oloke (2003) and Media and reagents Pao et al. (1996). They assessed fruit juice in New Zealand, Benin City and Addo Ekiti and observed that fruit juice The culture media used in this study were Sabouraud normally contain sugar such as glucose, sucrose and Dextrose Agar (SDA) (Oloke, 2000). The media was fructose and therefore provide an excellent substrate for sterilized by autoclaving and aseptically dispersed into these fungal isolates. However, processed and packaged sterile Petri dish after cooling to about 45°C. One (1 ml) of fruit juice usually contains little or no chemical sample were taken with calibrated pipette and dispensed in preservatives which inhibit microbial growth (Frazier and 9 ml of distilled water for each of the fruit juice making it Westhoff, 2002). Thus, these organisms can thrive due to (1:10) in three test tubes. 0.1 ml aliquot from 10-2 and 10-3 inadequate pasteurization or post-pasteurization, dilutions were spread onto the various plates in duplicates contamination during cooling, bulk storage, packaging and for determination of fungal growth. Chloramphenicol was subsequently cause spoilage. added to inhibit bacterial growth and incubated at room In addition, adherence to good manufacturing practices temperature 28±2°C for 2 to 5 days. and the use of low grade raw materials during processing is Academia Journal of Medicinal Plants; Onuoha. 127 Table 1: Mean fungal counts on sabouraud dextrose agar. Juice Mean counts (× 102 cfu/ml) FACB 1.0 × 102 DO 2.0 × 102 DA 2.0 × 102 DM 2.0 × 102 FrA 1.0 × 102 FrO 2.0 × 102 FrM 1.0 × 102 CA 1.0 × 102 CP 6.0 × 102 CM 2.0 × 102 CO 3.0 × 102 FAAS 2.0 × 102 Key: Cfu: Colony forming unit; FACB: Five alive citrus burst; DO: Dansa orange; DA: Dansa apple; DM: Dansa mango; FrA: Frutta apple; FrO: Frutta Orange; FrM: Fruta Mango; CA: Chivita apple; CP: Chivita pinapple; CM: Chivita Mango; CO: Chivita Orange; FAAS: Five alive apple splash. Table 2: Cultural, morphological and characteristics of fungal isolates. Characteristics F1 F2 F3 F4 Cultural Colour White fluffy/green Cream Grey White Morphological Microscopy Type of spore Conidiospores Ovoid cells Sporangiospores Sporangiospores Type of hyphae Septate Pseudohyphae Non-septate Septate Possible isolates Aspergillus niger Saccharomyces cerevisae Rhizopus sp. Mucor Key: F1 = First fungal isolate; F2 = Second fungal isolate; F3 = Third fungal isolate; F4 = Fourth fungal isolate. Table 3: Frequency of fungal isolates. Yeast sp Mold sp Juice Rhizopus Mucor Total Saccharomyces Aspergillus Five alive - 1 - - 1 Dansa orange 1 - 1 - 2 Dansa apple - 1 - 1 2 Dansa mango - - 1 1 2 Frutta apple - - 1 - 1 Frutta orange 1 1 - - 2 Frutta mango - - - 1 1 Chivita apple - - 1 - 1 Chivita pineapple 2 2 1 1 6 Chivita mango 1 1 - - 2 Chivita orange - 1 1 1 3 Five alive splash 1 1 - - 2 Total 6 8 6 5 25 Academia Journal of Medicinal Plants; Onuoha.