Isolation of Bacteria From Orange Juice From Three (3) Commercial Company
Chapter One
AIMS OF THE STUDY
- To isolate bacteria from orange juice from three commercial companies
- To identify the bacterial types
- To determine the bacterial load of each bacterial type.
- To determine the bacterial loads of the orange juice from three commercial companies sampled.
CHAPTER TWO
LITERATURE REVIEW
Historical Perspective
The researchers Uchino & Doi (1967) initiated the interest in Alicyclobacillus after reporting the isolation of an aerobic, acidophilic, spore-forming bacterium from thermal waters from hot springs in Japan. Preliminary morphological and cultural studies showed the isolates to be closer related to B. coagulans than to other thermophiles. The bacteria grew over a pH range of 2.3 to 5.0 and over a temperature range of 45˚ to 71˚C. In 1971 this bacterial species was also isolated from hot springs in the USA (Darland & Brock, 1971). It was recommended that a new species of Bacillus should be created to accommodate these isolates as no other species of Bacillus possessed ω-alicyclic fatty acids and hopanoids in the bacterial membrane (Walls & Chuyate, 1998). It was named B. acidocaldarius and described as thermo-acidophilic, aerobic spore-formers containing ω-alicyclic fatty acids as the major membrane component.
In the years to follow, acidophilic spore-formers were isolated from various other environmental sources, including garden soil, forest soil, apple juice and water (Pontius et al., 1998), emphasising the fact that they can survive non-thermal conditions in the form of endospores (Hippchen et al., 1981). These bacteria are adapted to different climate zones around the world and can even be isolated from commercial juice products, showing their ability to survive from the environment into the retail market (Wisse & Parish, 1998; Eiroa et al., 1999).
The environmental isolates found in the years to follow the discovery of Uchino & Doi (1967) were shown to differ from B. acidocaldarius, regarding their growth requirements and biochemical characteristics (Walls & Chuyate, 1998). The optimum growth pH of these isolates was found to be between 2.0 and 5.0, while the optimum temperature for growth ranged from 22˚ to 62˚C. The fatty acid composition of the cellular membrane was analysed and also found to contain ω-alicyclic fatty acids. Sub-terminal or terminal endospores were seen to be contained in slightly swelled sporangia. These findings lead to the conclusion that a new species, different from
acidocaldarius was isolated (Deinhard et al., 1987). Another bacteria with the same growth requirements as B. acidocaldarius, but with ω-cycloheptane (instead of ω-cyclohexane) membrane fatty acids were later isolated (Poralla & König, 1983). Deinhard et al. (1987) described the two new species and named them B. acidoterrestris and B. cycloheptanicus.
The three species B. acidocaldarius, B. acidoterrestris and B. cycloheptanicus were sufficiently different from other Bacillus spp. to warrant reclassification into a new genus (Walls & Chuyate, 1998). The genus Alicyclobacillus was described in 1992 based on 16S ribosomal RNA (rRNA) gene sequence analyses, as well as the unique fatty acid profile of the bacterial membrane (Wisotzkey et al., 1992; Goto et al., 2002a).
Characteristics
There are currently eleven species recognised within the genus Alicyclobacillus (Table 1). Species of Alicyclobacillus are rod-shaped, non-pathogenic and Gram- positive (Fig. 1). They are referred to as thermophilic acidophilic bacteria (TAB) due to their ability to survive acidic conditions and elevated temperatures. The cell size of species of Alicyclobacillus ranges from 2.9 to 4.3 μm in length and 0.6 to 0.8 μm in width (Jensen, 1999). The colonies are described as circular, creamy white, flat, translucent to opaque and 3 to 5 mm in diameter (Walls & Chuyate, 1998; Chang & Kang, 2004). The endospores are located terminally or sub-terminally and the sporangium is not necessarily swollen. Isolates from A. acidoterrestris sporulate rapidly, usually within 24 h in liquid and solid media, as well as in fruit juice (Splittstoesser et al., 1994).
The unique and distinctive characteristic of Alicyclobacillus spp. is the presence of ω– alicyclic fatty acids as the major components of the cellular membrane (Silva & Gibbs, 2001; Goto et al., 2002a). The main membrane component in the species A. acidocaldarius, A. acidoterrestris, A. hesperidium and A. acidiphilus is ω-cyclohexane fatty acids (Fig. 2) (Deinhard et al., 1987; Jensen, 1999; Albuquerque et al., 2000; Matsubara et al., 2002; Chang & Kang, 2004). The fatty acid ω-cycloheptane are mainly present in the membranes of A. cycloheptanicus and A. herbarius (Poralla & König, 1983; Goto et al., 2002a; Chang & Kang, 2004). These fatty acids are believed to play an important role in the acid- and heat- resistance of Alicyclobacillus spp. (Wisotzkey et al., 1992). When they are tightly packed in a cyclohexane ring structure it may serve as a protective coating for the cell membrane, contributing to the survival of the cells under extreme conditions (Chang & Kang, 2004).
Growth temperature varies between the different species of Alicyclobacillus and might be influenced by the pH of the growth medium (Farrand et al., 1983). The temperatures supporting growth is reported to be between 20˚ and 70˚C, with optimum temperatures between 42˚ and 60˚C (Yamazaki et al., 1996; Jensen, 1999; Chang & Kang, 2004). Growth at extreme temperatures of 12˚ to 80˚C has also been reported, but at these temperatures growth initiation is extremely slow, usually taking several weeks (Deinhard et al., 1987). The optimum growth temperature fo acidoterrestris is between 35˚ and 55˚C, while A. acidocaldarius has a higher optimum growth temperature range of between 45˚ and 70˚C (Simbahan et al., 2004).
Pathogenicity was of concern in food products contaminated with Alicyclobacillus destined for human consumption. A study on the pathogenicity of acidoterrestris in fruit juice was conducted by Walls & Chuyate (2000) during which they injected spores into mice and fed inoculated juice to guinea pigs. No illnesses or deaths were reported and they concluded that A. acidoterrestris was not pathogenic at the levels tested. The risk of secondary growth of other pathogens such as Clostridium botulinum is not of concern, as growth of A. acidoterrestris does not affect the pH of the juice (Brown, 2000). Juice spoilage by Alicyclobacillus spp. has a major economical impact on the fruit juice industry, but there is no health risk involved in consuming fruit juice containing this bacterium or its spores.
An interesting and unusual characteristic of this genus is that when they are grown in an unbuffered, liquid medium containing amino acids they will produce ammonia from the amino acids, increasing the pH to inhibitory levels (Jensen, 1999). Growth is initiated at a of pH 6, but does not continue due to the increase in the pH of the growth medium (Splittstoesser et al., 1998).
Walker & Phillips (2005) found that the presence of headspace in the packaging affects the growth of A. acidoterrestris in fruit juice, but samples having 25, 50 and 75% headspace showed no significant differences in cell numbers. The presence or absence of headspace thus affected the growth of A. acidoterrestris, and not the percentage of headspace. Intermittent shaking was also found to increase growth of A. acidoterrestris in fruit juice at a suboptimal growth rate at 30˚C (Walker & Philips, 2005).
CHAPTER THREE
MATERIALS AND METHOD
Materials and methods:
Study place:
The laboratory works of this research was done in the microbiology research laboratory.
Study period:
This research work was carried out from October, 2015 to June, 2016.
Materials
Equipment
Laminar airflow cabinet (Model-SLF-V, vertical, SAARC group Nigeria)
Incubator (Model-0SI-500D, Digi system Laboratory Instruments Inc. Taiwan)
Vortex machine (Digi system Taiwan, VM-2000)
Autoclave machine (Model: WIS 20R Daihan Scientific Co. ltd, Korea)
Glass wares, laboratory distillation apparatus- fractional distillatory set up, microscope, pH meter petri-dishes, slants, micro-pipettes, Bunsen burner, hot plate, clamp stands, electric balance, etc.
Media
Different types of media were used for selective growth, enrichment culture, and indication of specific properties. Media preparation and sterilization were done according to the protocol and standard recipe.
CHAPTER FOUR
RESULT
Result
Although Orange juices are very common and potential for human health, but over their hygiene, safety and quality much concerns have been raised. Many Orange juice company have already started producing different Orange juices and many of them going to market their products in the market, but most of the companies have no concern about the quality of the juice products. On the other hand, while making Orange juices in home people only think about the nutritional benefits other than the quality of the juice. In present study, ten juice samples (Five commercially packed Orange juices such as three different branded mango juices, one litchi juice, one grape juice and five freshly homemade Orange juices such as orange, pomegranate, jujube, mango and orange juices) were examined for microbial analysis.
CHAPTER FIVE
DISCUSSION
Discussion:
In many developing countries including Nigeria, millions of people are widely consuming Orange juices in every season as it provides an affordable source of nutrients to them. Packed Orange juices being good in taste and available at low price and at the same time they are liked by the consumers. (Ohiokpehai, 2003). Many Orange juice company are producing and marketing different Orange juices, but most of the companies have no concern about the quality of the juice products. Most of them think commercially and they are only concerned about the marketing (with colorful advertisement) of their products. Though they might maintain a lot of hygiene in their factory to avoid contamination (which is a good sign) but in most of the factory they use preservatives or harmful chemicals to lower the microbial growth in juice. In long time effect these harmful preservatives and chemicals can cause a thousand times more powerful disease to human being than the microbes, or cause mutation inside our body which eventually kills people ten times faster than normal diseases. Factors which determine the colonization of juices by microorganisms include pH, redox potential, water activity, nutrients, structures, antimicrobial agents, temperature, relative humidity, and atmosphere (Raybaudi, 2009). In the present study the frequencies of occurrence of molds and yeasts were more as compared to bacterial genera which are attributed to low pH values and high sugar content (A. Rivas, 2006).
Colony morphology, phenotypic and biochemical traits of the isolates
From different medium after incubation of 24 hours, some different morphological characteristic showing colonies from nutrient agar, typical pink, circular, convex colonies from MacConkey Agar (considered as coliforms), black colonies from XLD agar (considered as Salmonella spp.), blue colonies from MFc agar (considered as fecal coliform) and yellow colonies from Mannitol Salt Agar (considered as Staphylococcus spp.) were initially isolated. Isolates from MacConkey- and MFc agar media were observed as Gram negative, single, short rods, compared to the characteristic of coliforms whereas isolates from MSA were Gram positive in a cluster arrangement which were typical for Staphylococcus spp. and from XLD agar media again some Gram negative rod were also observed compared to the characteristics of Salmonella spp. Based on the biochemical characteristics, isolates were confirmed as E. coli, Klebsiella spp., Salmonella spp., Staphylococcus spp., Serratia spp. and some other different organisms.
Total viable count (TVC)
Most of the Orange juice samples showed equal or much higher heterotrophic count than Gulf standard. The highest bacterial count (2.3×106 cfu/ ml) for freshly homemade Orange juice sample was found in a mango juice (sample FM), collected from Mirpur and the lowest count was (1.0×105 cfu/ ml) found in an orange juice (sample FO) collected from Dhanmondi area (Table 3). On the other hand, the highest total bacterial count (1.25×106 cfu/ ml) for packed Orange juice sample was found in a mango juice (sample CM2, Table 4) and the lowest count was observed to be 0.5 x103 cfu/ ml in another mango juice (sample CM1, Table 4). Variations in TVC of both types of Orange juices may be due to the unhygienic maintenance during preparing the juice. Rahman et al. (2011) reported that total viable bacterial count in most of the Orange juice samples was higher than the commercially packed juice, as the highest count was found as 2.4×104 cfu/ ml and 3.2×103cfu/ml in fresh and packed juice, respectively which was found to be lower than this study. Tasnim et al. (2010) also found the load of viable bacteria in processed juice samples within the standard limit in the average of 103 cfu/ml. Bagde and Tumane (2011) found that total bacterial counts in juice samples ranged between 2.0 x106 to 1.0 x 105 cfu/ ml in Nagpur, India. Munjur et al. (2014) reported that the highest bacterial load (3.7×108 cfu/ ml) for fresh Orange juice sample was found in a grape juice, which was found to be higher than this study.
Prevalence of coliforms and fecal coliforms
The presence of coliform in Orange juice is not allowed by safe food consumption standard (Andres et al., 2004). Most of the Orange juices in this study were found to be unfavorable for consumption because many of them showed the presence of coliforms (E. coli and Klebsiella spp.). The highest coliform count for fresh homemade Orange juice and packed juice samples were 1.75×104 cfu/ ml (sample FG, Table 3) and1.5×102 cfu/ ml (sample CM2, Table 4), respectively. And the highest fecal coliform count for fresh homemade Orange juice and packed juice samples were 5.75×103 cfu/ ml (sample FG, Table 3) and3.25×101 cfu/ml (sample CL, Table 4), respectively. In Nigeria, Ahmed et al. (2009) showed the presence of E. coli ranging from 43 to > 2400/ 100 ml in different types of vended squeezed Orange juices in Lagos city, Also Munjur et al. (2014) reported that the highest and lowest coliform count for fresh Orange juice samples were 8.2×106 cfu/ ml and 1.53×103 cfu/ ml respectively. In India, the Orange juices were heavily contaminated bycoli (Bagde and Tumane, 2011). Moreover, 3 fresh Orange juice samples (FP, FJ and FG) and 1 packed juice (CL) exhibited the presence of fecal coliform in the present study (Table 3 and 4).
Prevalence of Staphylococci spp.
A few reports have shown the prevalence of Staphylococci in Orange juice samples (Ahmed et al., 2009; Tambekar et al., 2009). Coagulase-positive Staphylococci may cause human disease through the production of toxins. Effective levels of toxin formation require a high number of microorganisms (approximately 105-106 micro-organisms per ml of food) (IDF, 1994). In this study, Staphylococci spp. was found in some tested samples. The highest total Staphylococcal count for homemade Orange juice sample (1.4×104 cfu/ ml) was found in a jujube juice (sample FJ, Table 3). On the other contrary, the highest total Staphylococcal count for packed Orange juice sample (2.5×103 cfu/ ml) was found in a mango juice (sample CM2, Table 4).
Prevalence of Salmonella spp.
In fruit samples there were no report about Salmonella prevalence in Orange juice samples. In this study, some Salmonella spp. were found in some tested samples but the number was very low. The highest Salmonella count for homemade Orange juice sample (3.5×104 cfu/ ml) was found in a grape juice (sample FG, Table 3). On the other contrary, the highest total staphylococcal count for packed Orange juice sample (2.5×102 cfu/ ml) was found in a litchi juice (sample CL, Table 4). Out of 10 Orange juice samples 3 had shown the growth of Salmonella.
Interestingly, coliform, fecal coliform and Salmonella were absent in three packed juice samples (samples CM1, CM3 and CG) in this study (Table 3), and hence these samples were considered to be safe. Notably, these samples were prepared under good sanitation practices and stored in appropriate storage conditions. Besides, the results of this study (Tables 3 and 4) showed the safer consumption of commercially packed juice than the fresh homemade juice. This might be due to the usage of automated machine directing aseptic processing as well as for the application of some preservatives. But some preservatives of higher concentrations can be harmful for our health (Bashar and Sabita, 2007). Therefore, further studies on the optimization of preservative concentrations should be performed.
Antibiotic susceptibility test
Rashed et al. (2012) used a new aspect on their investigation comparative to the previous related ones is the study of antibiogram of the pathogenic isolates found in the juice samples. They found the E. coli isolates highly resistant against ciprofloxacin (61%), nalidixic acid (71%) and ceftriaxone (57%). Klebsiella spp. showed higher resistance against ampicillin (74%), ciprofloxacin (86%), piperaciline (88%), amoxicillin (72%), ceftriaxone (97%) and nalidixic acid (61%). Staphylococcus spp. showed resistance against ampicillin (93%), piperaciline (75%), amoxicillin (92%) and vancomycin (63%). Such drug resistance properties may render these pathogens cause serious health hazards because of ineffective treatment of the sufferers by the commonly prescribed antibiotics.
In this study, E. coli showed resistance against Ampicillin, Clindamycin, Cloxacillin, and Nitrofurantoin; Pseudomonas spp. showed resistance against Amoxicillin, Doxycycline, Tetracycline and Trimethoprim/ Sulfamethozole; Salmonella spp. showed resistance against only Ampicillin and rest of the organisms Staphylococcus aureus, Bacillus spp. and Serratia spp. did not show any resistance to any antibiotics that were used.
The present research study has been carried out to investigate and compare the microbial quality of fresh homemade Orange juice collected from different houses and commercially packed juice sold in the local public shops. Where, in the study they exhibited the presence of E. coli, Salmonella spp., Staphylococcus spp., Bacillus spp, Serratia spp., Pseudomonas spp., Micrococcus spp, and some other species. Probiotic microorganisms may also be isolated from the packaged Orange juice. The average counts for bacteria of the packaged Orange juice samples examined were generally below the maximum allowable limit in foods to be marketed for consumption (103 cfu/ ml) but fresh homemade Orange juices were above the limit. However, the average ranges obtained for the bacteria indicated a public health concern as they showed counts far above this limit in both packed and fresh homemade juices. These high counts indicate heavy bacterial contamination of both the packaged and fresh homemade Orange juice during handling since they are liquid, which could have contributed to the development as well as multiplication of these contaminants. Also, contamination can occur within fruits and materials used for the production of the juice as well as poor sanitation, extraction, raw material contaminations (often from insect damage), lack of both proper heat sterilization and adequate quality control during processing of Orange juice. The study has also shown that these packaged Orange juices are not sterile and thus can favour the growth of microorganisms when conditions become favourable, which could pose a public health risk to their consumers.
Conclusion:
From the data presented in the current study, it could be hard to claim that, consumption of fresh homemade juice was safe than commercially packed juice because almost all types of fresh homemade and commercially packed juice samples collected from different areas of Lagos city were not satisfactory as Escherichia coli, Klebsiella spp., Enterobacter spp., and Staphylococcus spp., were found in large numbers from samples. There is a generalized belief among the people that, automated machines and some preservatives are used during processing of commercial Orange juices. Despite all these issues, a large number of coliforms, and Staphylococci count were detected from both commercially packed Orange juices and fresh homemade juice in this current study, which clearly indicates poor plant management and personnel hygiene. A combination of regular monitoring and proper training could be an appropriate choice in Orange juice industries to minimize the health risks. In addition to this, not only government authorized institution like BCSIR and BSTI but also some strongly active administrative organization like mobile court should be given more authorization to undertake precautionary investigations to check the microbial and chemical quality of Orange juices. Besides, government and non-government institutions should create public awareness about the contamination and adulteration of Orange juices more intensely with the help of social media. So that people can take initiative for increasing awareness among them for checking batch manufacturing date before consume juice products.
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