Microbial Contamination of Date Rutab Collected from the Markets of Al-Hofuf City in Saudi Arabia

The microbial contamination of 60 samples from six date cultivars in the rutab stage purchased from different retail outlets in AL-Hofuf City, Saudi Arabia was studied. All samples were found contaminated with aerobic mesophilic bacteria at loads in the order 102 to 105 cfu/cm2 with some significant differences among varieties that can be attributed to differences in the weather conditions during rutab season. Also all samples, except only one, were contaminated with molds and yeasts at loads in the order 102 to 103 cfu/cm2. Potentially pathogenic Staphylococcus aureus was detected in 57 samples and A. flavus/parasiticus in 13 samples, while coliforms were detected in 39 samples.


Introduction
Date (Phoenix dactylifera L.) is mainly produced in Middle Eastern and North African countries. It is considered an important subsistence food crop in most of these countries, in addition to its high cultural and religious significance. Date fruit production has constantly increased worldwide over the last four decades from 1.8 million tons in 1963 to 6.7 millions in 2003 [1]. Saudi Arabia, with about 830 thousand tons annual production, ranks as the third largest date producer in the world [1]. Rutab (an Arabic name) is a stage of maturity in which the date fruit can be consumed as human food. It is a fresh product that contains 35 to 40% water and 45 to 48% sugars (dry matter basis) [2]. Because of its high moisture content, rutab is highly susceptible to microbial growth and spoilage, especially when poor hygienic practices during handling lead to heavy microbial contamination. Microbiological studies on dates are limited, especially in the main countries of production [3][4][5][6][7][8][9]. No published work on the microbial contamination of rutab was found.
This present study was undertaken to investigate the microbial contamination of rutab from different date cultivars grown in the Gulf Region in Saudi Arabia.

Rutab Samples.
The rutab samples were purchased from 10 different retail outlets in Al-Hofuf City and represented the most popular six date cultivars grown in the region: Khulas, Um-Ruhaim, Hilali, Shahal, Tiar, and Megnaz. A total of 60 samples were collected at weekly interval, 10 samples from each cultivar at each sampling time, and microbial analysis was carried out on the same day.

Microbiological Analysis.
Four fruits from each rutab sample were transferred into sterile stomacher bags, 50 mL sterile peptone water (CM0009, Oxoid, Basingstoke, UK) added, and the fruits washed manually for 2 minutes in this water, then aliquots (1.0 or 0.1 mL) were plated in duplicate as 10-fold dilutions in peptone water (surface area of the 4 fruits was then measured). Three replicates were analyzed for each sample and the average microbial loads calculated and reported as colony-forming units (cfu) per cm 2 . Aerobic mesophilic bacteria were enumerated on plate count agar (CM0325, Oxoid) incubated at 30 • C for 2 to 3 days. Coliforms were determined on violet red bile agar plates (VRBA, CM0107, Oxoid) incubated at 37 • C for 24 hours. Round, 2 The Scientific World Journal

Results and Discussion
All rutab samples from the six date varieties tested were found contaminated with aerobic mesophilic bacteria at loads in the order 10 2 to 10 5 cfu/cm 2 ( Table 1). The levels of contamination with these microorganisms are indices of the general microbiological quality and give idea about the expected shelf lives of the foods concerned. The highest levels of contamination with these bacteria were found in Hilali.
Five samples from this variety had loads of 1.6 × 10 5 to 2.3 × 10 5 , one sample a load of 3.8 × 10 4 , and 4 samples 2.9 × 10 3 to 6.9 × 10 3 cfu/cm 2 . Shahal samples had generally lower loads of contamination than Hilali, with one sample having 2.6 × 10 5 , 6 samples 1. The level of Hilali contamination was significantly higher than that of Khulas, Um Ruhaim, Tiar, and Megnaz; it was also higher than the level of Shahal, but the difference was not significant. On the other hand, the level of contamination of Shahal was higher than that of Khulas, Um Ruhaim, Tiar, and Megnaz, but the difference was not significant. The levels of contamination of Khulas, Um-Ruhaim, Tiar and Megnaz were quite similar ( Table 1). The differences in the levels of contamination with aerobic mesophilic bacteria among these rutab samples may be attributed to differences in environmental temperatures during rutab season. Khulas, Um Ruhaim, Tiar, and Megnaz are early maturing varieties and they were sampled and analyzed by the end of August where the temperatures in Saudi Arabia were above 40 • C, thus the spread of mesophilic bacteria is expected to be limited. Shahal matures in mid-August and was sampled and analyzed mid-September, where the temperatures start to drop below 40 • C, while Hilali is a late maturing variety and its samples in this study were collected and analyzed in October where the temperatures were in the thirties, and hence the mesophilic bacteria are supposed to dominate in the environment. Almost all samples were contaminated with varying loads of molds and yeasts (Table 2), which are generally spoilage organisms, although some of them could be pathogenic. Shahal and Hilali showed the highest levels of contaminations with molds and yeasts. One Shahal sample had 1.9 × 10 4 , 2 samples 2.3 × 10 3 and 4.6 × 10 3 , and 7 samples 1.7 × 10 2 to 7.8 × 10 2 cfu/cm 2 . One Hilali sample had 1.1 × 10 4 , 3 samples 1.2 × 10 3 to 5.9 × 10 3 , and 6 samples 1.1 × 10 2 to 4.7 × 10 2 cfu/cm 2 . Two Um Ruhaim samples had 1.8 × 10 3 and 2.0 × 10 3 , and 8 samples 2.0 × 10 2 to 6.9 × 10 2 , cfu/ cm 2 , while 3 Khulas samples had 1.0 × 10 3 to 1.2 × 10 3 , 6 samples 2.1 × 10 2 to 4.8 × 10 2 cfu/cm 2 , and one sample  undetectable level. In case of Tiar, only one sample showed a level of contamination of 1.5 × 10 3 and the contamination of the other 9 samples was in the range 1.3 × 10 2 to 3.3 × 10 2 cfu/cm 2 . The contamination of Megnaz samples was 1.4 × 10 3 in one sample and 1.3 × 10 2 to 5.2 × 10 2 cfu/cm 2 in 9 samples. Contamination of Khulas, Um Ruhaim, Tiar, and Megnaz with molds and yeasts was generally lower than that of Hilali and Shahal, but the differences were not significant ( Table 2). Microbial contaminations in the order 10 5 cfu/g·cm 2 ·mL can be regarded as high since signs of microbial spoilage start to appear in most foods at loads of about 10 6 cfu/g·cm 2 ·mL [11]. Rutab with such high microbial contamination levels is expected to have a short shelf life if kept at room temperature. This high level of contamination was mainly found in Hilali where 5 of the 10 samples tested contained more than 10 5 cfu/cm 2 (Table 1).
With respect to pathogenic microorganisms, all samples of Khulas, Um-Ruhaim, Shahal, Hilali, and Megnaz and 7 samples of Tiar were found contaminated with the potential food poisoning bacterium Staphylococcus aureus ( Table 3). The highest level of contamination with this bacterium was in Shahal, followed by Um Ruhaim, then Khulas but the differences between the three were not significant. The level of contamination of Hilali and Megnaz was significantly lower than that of Shahal, Um Ruhaim, and Khulas and the level of contamination of Tiar was still lower than all other samples with significant difference from all of them (Table 3). Rutab in Saudi Arabia is harvested manually; thus it seems that contamination of the date fruits with this bacterium came mainly from workers, and differences in the degree of contamination may be attributed to differences in the hygienic status of these workers. Staphylococcus aureus can infect and grow in wounds on the hands of workers inevitably caused during harvesting by the sharp and strong ends of the leaves of the palm tree.
Coliforms were found in 39 out of the 60 samples analyzed, that is, 65% of the samples studied (Table 4). Their rate of occurrence was similar in 5 cultivars ranging between 6 and 8 contaminated samples, while in Khulas only 4 samples were found contaminated. The loads were relatively low reaching the range of 10 2 cfu/g in only 4 samples. The source of contamination was most likely the workers who harvested rutab manually. No Escherichia coli O157 was detected among these coliforms.
The potential mycotoxin producing molds A. flavus and A. parasiticus were found in 13 out of the 60 samples analyzed, that is, 21.7% contaminated samples (results not