H.I.J. Udota* and E.E. Urua
Department Of Food Science And Technology, University Of Uyo, P.O. Box 4231, Uyo, Akwa Ibom State
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Juices stored under warm and cold conditions (27oC and 4oC) were comparatively analyzed for yeast and mould growth. Two commercially prepared canned orange and pineapple juice and laboratory prepared pasteurized ones were analysed. A total of 68 yeast isolates and 64 mould isolates were indentified using fungal plate count Agar. Fourteen different yeasts species were identified representing 11 genera while twenty-nine mould species were from 16 genera. Distributions of yeasts and mould species were considerably different depending on sample type. Eight different yeast and 13 mould species were isolated from canned pineapple (CP) juice while 4 yeast and 10 mould species were from pasteurized pineapple (PP). Among CP isolated were Saccharomyces cerevisiae, Candida utilis, C albicans Geotrichum candidum and Aspergillus versicolor while in (PP), C. albicans, C. itilis, S. cerevisiae and Diplodia spp were predominant. Eleven different yeasts and 13 moulds species were from canned orange (CO); S. cerevisiae 27%, G. candidum 17%, S. ruseus 14% and A. restrictus 11%. Eight yeast and 12 mould species were from pasteurized orange (PO); Pichia fermentans 18%, Byssochlamys nivea 13% and G. candidum 13%. In all, yeasts; S. cerevisiae 22%, G. candidum and C. pseudotropicalis 10% and moulds: B. nivea 9.5%, Monilia 7.8% and Alternaria sp 6.3% were predominant. High hygienic practices if enforced will significantly reduce the population of highly fermentative yeast and mycotoxin mould strains from processed juices and hence will be safe for consumption.
Moulds, Yeast, Orange, Pineapple-Juices
Although the internal tissues of healthy plants, fruits, vegetables and animals (meat) are essentially sterile (Ray 2004), raw and processed foods may contain different types of moulds, yeasts, bacteria and viruses. Microorganisms are contracted in food from both natural (including internal) and external sources to which food comes in contact with from the time of production and/or harvest until the time of consumption (Doyel 1989). Natural sources for foods of plant origin include the surfaces and openings (pores) of fruits, vegetables and grains. External sources are: air, soil, sewage, water, human, food ingredients, equipment, packages and insects. Microbial types and their populations in foods vary widely depending on the degree of sanitation used during processing and handling of the foods (Ray, 2004).
Fruit juices are rich medium that can encourage the growth of microorganism given favourable environmental factors. Studies from Cavadonga et al. (2002) and Ray (2004) show the extent to which citrus fruit juices are contaminated by bacteria, yeasts as well as mycotoxins produced by moulds.
It is important to keep food free of contaminantswhether physical, chemical or biological. In an effort to check this, Standard Organizations such as SON, (Standard, Organization of Nigeria), NAFDAC (National Agency for Food and Drug Administration and Control), NIS (Nigeria Industrial Standards) etc have been set up by government.
With so many fruit juices in different packaging materials entering the market in recent times, one wonders whether all have met the expected standards for consumption. This work therefore tries to ascertain microbial loads in two commercially prepared juices kin the market and compares this with a fleshly prepared one in the laboratory.
Also to identify the microorganisms so as to determine whether mycotoxin producing microorganisms are present after quality control measure
Sample Collection : Orange and pineapple fruits were purchased from Uyo main market. Samples of canned orange and pineapple juices were bought from supermarket in Uyo, Akwa Ibom State, Nigeria. The media and all other reagents were from the laboratory where all analyses were carried out. That is in the Department of Food Science and Technology, University of Uyo, Nigeria.
Sample Preparation : (Citrus reticukata) and pineapple (Ananas comosus) were each washed and peeled to expose the mesocarp which contains the juice. The juice was extracted using extractor machine. The extracted juice was filtered into McCartney bottles and pasteurized at 60-70°C for 15 minutes using water bath. The pasteurized juice was cooled down to room temperature before storing in the refrigerator at 10°C for subsequent analyses.
Media Preparation : A modified plate count Agar (PCA Oxoid, England) was prepared according to the manufacturer’s specification and as contained in Yousef & Carlstrom (2003). All media were sterilized by autoclacing at 121°C for 15 minutes and cooled to 55 °C before adding 2 mL of chloramphenicol (antibiotic) to 100 mL of molten PCA. This was thoroughly mixed before dispensing into sterile Petri dishes. About 20 mL of the sterilized antibiotic PCA were aseptically poured into a sterile Petri dish and allowed to set.
Enumeration and Isolation of Microorganisms : The juice was diluted using 10-fold dilution. Undiluted samples as well as diluted ones were plated. The juice (0.2mL each) from each sample type was dispensed into 3 plates containing antibiotic PCA. This process was repeated from 10-3 and 10-7 dilutions for each of the two laboratory prepared orange and pineapple juices as well as for the canned commercially prepared orange and pineapple juices. Spread plate method was applied to distribute the inoculum on the surface of the Agar medium. The inoculated plates were incubated invertedly at 25 °C for 5 days before counting the colonies that developed. Primary cultures were aseptically subcultured into a fresh medium and incubated for 5 days at 25 °C. Isolated colonies were subcultured again until pure cultures were obtained. The pure cultures were transferred onto slants in McCartney bottles and incubated. These were used for characterization.
Characterization of Isolates
Moulds: Subcultured plates were placed on the stage of a stereo-microscope and examined for structures of moulds as identified by Barnett and Hunter (1972). Lactophenol cotton blue dye was droped on a slide and using sterile inoculating loop moulds with spores were added to the dye on the slide and observed under the microscope. The spores, hyphae, mycelium and reproductive structures observed were recorded for identification. (Yousef and Carlstrom, 2003; Fawole and Oso 1998; Baron et al. 1994).
Yeasts : The pure isolated was picked from the bottle and placed in a drop of sterile water on a slide and smear was prepared and air dried. Crystal violet soln was used to stain the smearfor 60 seconds before observing under the microscope using objective at 100x (Yousef and Carlstrom, 2003).
Biochemical Tests : Sugar fermentation test was carried out to note sugar utilization ability of the yeast isolates.
Procedure : Nine grams of peptone water were suspended in 600mL distilled water. NaCl (0.85g) was added and stirred. In another beaker, 0.3g phenol red indicatolr was added to 6-0mL of distilled water stirred and filtered using cotton wool. One gram of each of glucose, lactose, maltose, sucrose, raffinose and galactose were measured into labelled beakers and 100 mL of basal medium (peptone water + 0.85g NaCl) were dispensed into each of the labelled beaker and stirred. Ten ml of the phenol red filtrate were also dispensed into each beaker and stirred.
Ten mL of the fermentation medium were accordingly dispensed into labelled test tubes containing Durham tube invertedly placed making sure that no air was inside. The test tubes were sterilized at 1kg/ cm2 pressure (121 °C) for 15 minutes in an autoclave.
Each of the isolates was aseptically inoculated into the test tubes and incubated at 25 °C for 3 days. A change in colour from red to yellow and air space in the filled Durham tubes indicated acid and gas production respectively. Negative results were further incubated up to 2 days.
Nitrate Reduction Test : This test was conducted to demonstrate the ability of enzyme nitrate reductase to reduce nitrate to nitrite. Test was done by inoculating a 24-h old culture into nitrate broth medium containing 0.5% NaNO3 and 1g peptone water in 100mL of water, which was sterilized and allowed to cool. Presence of nitrite crystals and gas production indicated a positive reaction and their absence indicated negative result.
Yeast Species in Orange and Pineapple Juice : A total of 68 isolates of yeast strains from CP 1, CP2, PP1, PP2, CO1, CO2, PO1 and PO2 were identified in this study (Table 1). Fourteen yeast species were identified representing 11 genera. G. candidum 14.7%, S. cerevisiae 21.1%, C. pseudotropicalis 10.3%, P. fermentans 7.4% and C. albicans 5.9%, were predominant among the isolates. R. mucilaginosa 2.9% was unique of preserved pasteurized freshly prepared orange juice. S. cerevisiae and C. pseudotrobicalis were the only species isolated from both pineapple and orange juices irrespective of the preservative measures.
PP2 had the least number of isolates while CO1 carried the greatest number of isolates; two and nine genera respectively. The results also show that more yeast strains were isolated from the canned fruit juices with orange having 42% of the total isolates. Laboratory prepared pineapple juice had the least number of isolates representing 13% of the total yeast isolates.
Table 3 reveals the mean values of yeast isolates from canned orange and pineapple juice 1.04 and 0.5; and standard deviation of 0.8957 and 0.4584 respectively explains that there was an average of 1.04 strains of yeasts species in every can containing orange juice with a significant deviation from the mean value of the contaminants (it can be higher in other cans). Similarly, pineapple juice (0.5 mean and SD 0.4584) reveals a low deviation of yeast strains from the mean showing that other cans may likely harbour similar levels of yeast strains.
In the laboratory prepared juices about double the mean quantity of yeast strains isolated from pineapple were got from orange juice. This suggests that oranges are more susceptible to contaminants than pineapple and more attention should be given while preparing.
Comparison of yeast strains in commercially and laboratory prepared juice (Table 3) shows that there exist a substantial or marked relationship of 0.5964 between the isolated in canned pineapple juice and the juice prepared in the laboratory when analyzed using Pearson’s correlation coefficient. In orange juice, 0.3566 shows a low correlation between the commercially and laboratory prepared conditions. Considering the mean and standard deviation, we can see that higher numbers of strains do actually grow in commercially than laboratory prepared fruit juices.
Moulds Species in Orange and Pineapple Juices: A total of 64 isolated of moulds were from CP 1, CP2, PP1, PP2, CO1, CO2, PO1 and PO2 in this study (Table 2). Twenty-nine species were identified representing 16 genera. Byssochlamys nivea (9.4%), G. candidum (7.8%), Monilia sp (7.8%) and Alternaria sp (6.3%) were common among the isolates. Aspergillus flavus represented only 2% of the total isolates. Monilia sp and Alternaria sp were the only species that were common in both pasteurized pineapple and pasteurized orange juice. Fruits are constantly liable to deterioration by microbes. Duckworth (1966) reported that spoilage is due to activity of moulds. Moulds isolates of commercially prepared juices were 56.25% of the total isolates and 43.75% of that prepared in the laboratory. This suggests that, preservatives used in the canned juice were not effective as to inhibit or destroy mould cells during production.
In either cases (orange and pineapple) there was an average of 0.31 moulds strain isolates. This is relatively low. Since most of the mould strains are very toxic to human, their cells in the fruit calls for a better preservative in our companies. A standard deviation of 0.4029 and 0.3809 in orange and pineapple juices respectively shows that more moulds strains may be present in subsequent can of the orange batch than in the pineapple juice. Averages of 0.0276 and 0.209 from laboratory prepared orange and pineapple juices respectively show that though pasteurized, moulds survived are mostly thermophilic and could grow to cause spoilage (Corpett and Brown, 1980; Baird- Parker, 1980; Olson and Nottingham, 1980). Using Pearson, 0.2407 and -0.08304 in orange and pineapple juice respectively, indicates a positive low relationship in mould species isolated from pasteurized and commercially prepared orange juices while that of pineapple shows a negligible negative correlation.
A total of 36 isolates of moulds and 42 isolates of yeasts strains were identified from canned orange and pineapple juices whereas 28 isolates of moulds and 26 isolates of yeasts strains were identified from laboratory prepared juices.
Quality control section of any food company should think safety and intensify efforts in her analyses at each line of production to aseptically monitor and reduce these contaminants that grow in juices.
Food agencies should intensify their effort in checking packaged products within 3 months.
Lastly, consumers should be consuming certified products to reduce incidences of contacting disease causing microorganisms.