The Effect of pH and Chemical Preservatives on the Growth of Bacterial Isolates from Some Nigerian Packaged Fruit Juices

INTRODUCTION

Juice is a liquid naturally contained in fruit or vegetable tissue. Juice is prepared by mechanically squeezing or macerating fresh fruits or vegetables without the application of heat or solvents. For example orange juice is a liquid extract of one fruit of orange tree. Juice may be prepared in the home from fresh fruits and vegetables using variety of hand or juice extractor. Many commercial juices are filtered to remove fiber or pulp, but high pulp fresh orange juice is a popular beverage. Juice may be marketed in concentrated form, sometimes frozen, requiring the user to add water to reconstitute the liquid back to its original state. However, concentrates generally have a noticeably different taste than their comparable fresh squeezed versions. Other juices are reconstituted before packaging for retail sale. Common methods for preservation and processing of fruit juices include canning, pasteurization, freezing, evaporation and spray drying (Fasoyiro et al., 2005).

Popular juices include, but are not limited to apple, orange, grape fruit, pineapple, tomato, passion fruit mango, carrot, cranberry and pomegranate. It has become increasingly popular to combine a variety of fruit into single juice drinks. Popular blends include Cran-apple (Cranberry and apple) and apple and black currant. A demonstration of this trend is that prepackaged single fruit juices have lost market share to prepackaged fruit juice combination (Frazier and Westhoff, 1986).

However, fruit juices are nutritious which offer great taste and health benefits. The 2005 Dietary Guide lines for Americans (2005) recommended consumption of several cups per day of fruits and vegetable. Most fruit juices bought from grocery stores and supermarket shelves are pasteurized. This means that the liquid has been brought to a high temperature that kills harmful bacteria. However, a small percentage of fresh fruit juices are unpasteurized. This means that there is a chance that the product may contain bacteria harmful to our health. Most people can enjoy unpasteurized juice and drinks, however, for young children, the elderly and people with weakened Immune systems, the effect can be severe or ever deadly (Esteve et al., 2005).

Unpasteurized fruit and vegetable juice have posed serious public health risk in recent years. Seventy people including a child who died-become ill in 1996 after drinking unpasteurized apple juice Contaminated by a strain of Escherichia coli bacteria (Amato, 1999). In 1999 and 2000, unpasteurized orange sickened hundred people in United States and three Canadian provinces. The 1999 outbreak contributed to one death (Formanek, 2001) A 2005 study in Japan found out that up to 52% of commercial fruit juices sold in Japan were contaminated with thermostable acidophilic bacteria (Hara et al., 2005).

Bacteria are responsible for the contamination and spoilage of packaged fruit juices resulting in discoloration, abnormal flavour and odour rendering it unacceptable for human consumption. The bacterial strain that spoil fruit juices includes, Bacillus licheniformis, Aeromonas hydrophila, Bacillus circulans, Proteus morganii, Pseudomonas chlororaphis, Bacillus alvei, Pseudomonas cepacia and soon. Their presence may pose risks to consumer’s health and should not be taken for granted (Hatcher et al., 1992).

Fruit juices are well recognized for their nutritive value, mineral and vitamin content. They are beverages that are consumed for their nutritional value, thirst-quenching properties, and stimulating effect or for their medicinal values (Fawole and Osho, 2002). Contamination of fruit juice by bacterial may occur when the organism enters the processing plant or on the surface of the fruit having originate from soil untreated surface water, dust and decomposing fruit. The degree of contamination varies depending upon how the fruit was handled from the field and in the processing plant. Proper handling washing and sanitizing the fruit contribute materially to good product quality. The low PH of fruit juices greatly limits the number and the type of bacteria that can survive or grow at this low PH but some bacterial that their PH is lower than that of the fruit juice can grow at this condition (Ryu and Beuchat, 1998).

Yeast and moulds are also present and can grow when the juice is held at a temperature permitting their growth. Yeasts are primarily responsible for the spoilage of chilled juice that is not sterile and some can withstand the effect of chemicals used to preserve them (Sandeep et al., 2001). Most industrial juice concentrators use a high temperature evaporation (thermal accelerated short time evaporation) and microbes are generally killed during this process and the freezing process should kill many of the survivors though this process will preserve the ultimate survivor. Thus frozen concentrated fruit juice should have few if any microbes (Parish, 1997). It is possible to reduce the growth of bacterial in fruit juices by addition of some chemical preservatives, thus inhibiting abnormal flavor, odour and spoilage of fruit juices and possibly improving shelf-life and permitting preservation for longer period with maximum retention of its nutritive valve.

According to the department of Zoology, Andhra University, India (2004) contamination of ready-to-eat foods and beverages sold by street vendors and hawkers rendering them unacceptable for human consumption have become a global health problem. In tropical countries, fruit juices are common man’s beverages and are solid at all public places and roadside shops. However in view of their ready consumption, quick method of handling, cleaning and extraction they could often prove to be a public health threat. Improper washing of fruits add these bacteria to extract leading to contamination (Geldrich, 1974).

In addition, use of unhygienic water for dilution, dressing with ice, prolong preservation without refrigeration, unhygienic surroundings often with swarming house flies and fruit flies and air bone dust can also act as sources of contamination such juices have shown to be potential source of bacterial pathogen notably E. Coli, Salmonella, Shigella and Staphylococcus aureus (Buchannan et al., 1999). Although the infectious dose for these contaminating bacteria in fruit juices is not yet well established based on the standards provided for drinking water (APHA, 1998) the numbers required to cause illness could be low particularly with reference to faecal Coliforms and Streptococci.

In India, there is always a great demand for fresh vegetables and fruit juices. Being tropical in location hot weather continues for a greater part of the year (February-September) increasing the need for these commodities. While most restaurants and cafes serve juices in apparently hygienic conditions in the roadside shops and recreational areas and busy market places, their microbiological quality remains questionable (Sandeep et al., 2001). In these shops juices extracted by squeezing from a variety of fresh fruits namely oranges, grape pomegranate, apple, pineapple, watermelon, papaya, carrot and soon were served after considerable dilution with water and ice (Splittstosser, 1979).

Despite periodic quality control checks and closure of shops, out breaks of gastroenteritis caused by pathogenic E Coli, Salmonella and Shigella are not uncommon in these areas although a specific correlation has not been shown between out break of gastroenteritis and consumption of these juices (Kobayashi et al., 1963). In views of the high demand for fresh fruit juices during summer and over crowding of streets vended shop in many areas in the city rapid review of the street vended fruit juices was under taken with a view to assess their safety for human consumption and as possible sources of bacterial pathogens (Uljas and Ingram, 1998). Driven by the harm caused by the poor quality of most packaged fruit juices, this work was therefore aimed at; Isolation and characterization of bacterial pathogen from some Nigerian packaged fruit juices, determination of the antibiotic susceptibility profiles and evaluation of the effect of pH and chemical preservatives on the growth of the isolates.

MATERIALS AND METHODS
Collection of samples: A total of seven packaged fruit juices samples were purchased from different selling point in Ogbomoso South West Nigeria. The juices had at least 3 months to their expiry date from the period of analysis. The fruit juice samples were approved by the appropriate regulatory agency; which is the National Agency for Food and Drug Administration and Control (NAFDAC).

Isolation of microorganisms: 12.85 g of peptone was dissolve in 500 ml of distilled water, then a sterile pipette was used to dispense 9 ml of the prepared peptone in screw capped bottle and then sterilized for 15 min at 121^oC and allowed to cool. 1 ml of each packaged fruit juice sample was serially diluted and 1 m of an appropriate dilution was inoculated on sterile MacConkey, Nutrient agar, Salmonella/shigella agar and the plate was incubated for 24 h at 37^oC. After 24 h sterile wire loop was used to pick the isolate from the plate and was streaked on a freshly prepared nutrient agar then incubated for 24 h at 37^oC in order to get pure culture. The routine laboratory method of Cruickshank et al. (1975) was used to characterize different isolates. The isolates were identified using their macroscopic, cellular, physiological and biochemical characteristics.

Antibiotics susceptibility test: Sterile nutrient agar medium was poured into sterile petri dishes and allowed to solidify. A suspension of the isolated organisms was transferred into petri-dishes accordingly and swab over the entire plate, it was then incubated for 1 h at 37^oC and a forcep was used to transfer each sensitivity disc on the plate and incubated for 24 h at 37^oC. The antibiotics used included amoxyllin, ampicillin, tetracycline, ampicillin-cloxacillin, gentamycin, ofloxacin, augmentin, ciprofloxacin, cifloxacin, erythromycin, clindamycin, nitrofurantion, chloramphenicol, cotrimazole, norfloxacin.

Growth of isolate at different pH ranges: Nutrient broth was prepared and the pH was adjusted using 0.1 M phosphate buffer of different pH to adjust the pH of the broth to 3.0, 5.0, 7.0 and 9.0. It was then dispensed into screw-capped bottles and then sterilized in the autoclave at 121^oC for 15 min. After cooling, the various test isolates were inoculated into it and incubated at 30^oC for 48 h. Growth was detected by increase turbidity using Cecil 2031 (automatic) spectrophotometer. Uninoculated tubes serve as control. This test was done to detect the best pH that favours growth and metabolism as indicated by the increased turbidity (Schillinger and Lucke, 1989).

Evaluation of the effect of chemical preservatives on the growth of isolates
Growth of isolates in different concentration of benzoic acid: Nutrient broth containing 250, 750 and 1000 mg/l of benzoic acid was prepared and 10 ml of the broth was dispensed into sterile screw capped bottles and then sterilized for 15 min at 121^oC. After cooling, the bottles were inoculated with the test organisms and incubated for 24 h at 37^oC. Growth was detected using an automatic spectrophotometer. Increase in turbidity of the medium was recorded as positive for growth while a negative result shows no turbidity. Uninnoculated tubes serve as control.

Growth of isolates in different concentration of sodium chloride: Nutrient broth containing 2, 3, 4 and 5% (w/v) NaCl was prepared and sterilized at 121^oC for 15 min. 20 ml of the broth was the dispensed into sterile screw capped vials aseptically. After cooling, the tubes were inoculated with the test organisms and incubated for 24 h at 30^oC increased turbidity of the medium was recorded as positive for growth while a negative result shows no turbidity. Uninoculated tubes serve as control (Schillinger and Lucke, 1989).