SOLD BY: Abstract
Vegetables and fruits that have been physically altered from their original states but remain in fresh states are considered minimally processed. Minimally processed fruits and vegetables are good media for growth of microorganisms, they habour potential pathogens. Four fruits and three vegetables (water melon, pineapple, paw-paw, Orange, Pumpkin leaf, Spinach and Cabbage) were assessed for microbial loads. The microbial loads were: water melon, 9.5 x103; Pineapple, 4.53 x103; Pawpaw, 5.52 x103, Orange, 3.46 x102; pumpkin leaf, 8.47×103; spinach,8.41 x103;and cabbage, 9.32 x 103. After washing with tap, distilled and salted water, the microbial loads were reduced significantly. Washing with salt water proved to have the highest effect in reducing the microbial loads to: water melon, 4.5 x101; Pineapple,6.14 x 101; Pawpaw, 2.68 x 101, Orange, 2.2 x101; pumpkin leaf, 3.36 x101; spinach,3.56 x 101;and cabbage,4.1 x 101 . Also, microorganisms isolated were mostlySalmonella spp. Shigella spp. and E. coli. Sensitivity tests were conducted using some selected antibiotics in other to treat some of the disease that can arise as a result of the consumption of the contaminated food but Ciprofloxacin, Gentamacine, Streptomycin and Chloramphenical were the most sensitive.
CHAPTER ONE
1.1 INTRODUCTION
Vegetables and fruits that have been physically altered from its original state but remain in its fresh state are considered minimally processed. Fresh fruits and vegetables are an important source of nourishment and a vital ingredient in healthy and balanced diets. Minimally processed fruits and vegetables are products that undergo some industrial or manual processing (such as peeling, slicing, chopping, shredding, trimming, washing or packaging) which make them ready for consumption. However, in this dicing step occurs the release of internal cellular fluid rich in nutrients, which allow microorganisms to multiply rapidly increasing the initial microbial load and thus reducing considerably the shelf life of these products. Packaging in modified atmospheres, together with refrigeration, slows down air flow and consequently the respiration of the vegetables. Bacterial growth is also inhibited, and the shelf-life of the product is enhanced (Fallik and Aharoni, 2004).
What the consumer perceives as the most appealing attribute of this products include: their fresh-like appearance, taste and flavor, in addition to convenience.
There are few factors that increase the risk associated with minimally processed fruits and vegetables above that of their non-processed counterparts. First, the more a product is handled and the longer between production and consumption, the more opportunity there is for contamination of the product. Secondly, most minimally processed or ready-to-eat products are meant to be taken home and eaten with no further washing or processing needed. The lack of any step that will kill or reduce the level of contamination before consumption increases the risk of consumer’s exposure to pathogens. Thirdly, in processing the peel or rind is often disrupted by cutting or peeling. The peel or rind can act as an effective barrier to microbial or chemical contaminants, so when it is disrupted, it leaves the fruits more vulnerable to contamination. (S.Demelo Silva, 2001).
1.2 OBJECTIVES
The aim of this project work is to assess the microorganisms in minimally processed fruits and vegetables and to provide a systematic review of the potential food safety risks from pre-processing to sale in the market as well as to proffer solution on better ways of prevent contamination and ways of decontamination.
1.3 SCOPE AND LIMITATION
The scope of this project is to assess micro organisms in minimally processed fruits (citrus, pineapple, water melon, and paw-paw) and vegetables (cabbage, spinach, pumpkin) in the local market of Nasarawa with special interest on Samolnella spp Shigella dysenteriae and Escherichia coli and to also perform an antibiotics susceptibility test in order identify the various antibiotics that can be used to combat the various diseases caused by these micro organism. The major limitation is the cost involved in running the analysis and the constraint involved in maintaining an aseptic condition throughout the period of the analysis.
1.5 JUSTIFICATION
The complete execution of this project work (assessment of microorganisms in minimally processed fruits and vegetables) will be of relevance in the production of micro-organism free fruits and vegetables for the teaming population as well as better ways of decontaminating and packaging the products.
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 INTRODUCTION
Fresh fruits and vegetables are considered for the sign of novel health and good source of nutritional supplement for the human body. Minimal processing helps to firstly keep the produce fresh and supply it in a convenient form, and secondly, extend shelf life and to facilitate its distribution to consumers. (Francis G.A 1999).
Removing the skin from the surface or altering the size leads to the loss of nutrients, rupture of cells accelerate the enzymatic reactions, rapid microbial growth, colour changes, textural changes and weight loss resulting poor quality of the produce. Minimally processing fruits and vegetables results in the release of internal cellular fluid, rich in nutrients, which allows microorganisms to multiply rapidly increasing the initial microbial load and thus reducing considerably the shelf life of these products.The Centres for Disease Control and Prevention (CDC, 2009) revealed that an estimated of 12.3% of total food borne diseases outbreaks from 1990 to 2007 were related to fruits and vegetables.
2.2 HEALTH AND NUTRITIONAL BENEFITS OF FRUITS AND VEGETABLES
Eating vegetables provides a lot of health benefits – people who eat more of vegetables and fruits as a part of an overall diet are likely to have a reduced risk of chronic disease. Vegetables provide nutrient vital for health and maintenance of the body, it may reduce the risk of heart disease, obesity, and type 2 diabetes.
2.3 METHODS OF PRESERVING MINIMALLY PROCESSED FRUITS AND VEGETABLES (FRESH CUTS)
The following are more natural ways of keeping the quality of minimally processed fruits and vegetables:
· Temperature
· Essential oil
· Modified atmospheres
· Packaging
TEMPERATURE:
Is one of the most effective factors, controlling freshness and the decay of the fresh cuts products and maintains good quality control (the cool chain) is the first factor in extending shelf-life of high quality fresh cuts products. Low storage temperature is more effective in delaying the ripening and microbial growth while preserving sensory quality of fresh cuts mango slices.
ESSENTIAL OILS:
Methyl Jasmonate (Mj, C 13 H 2003) is natural occurring substance that is commonly found in plant tissues and is a part of the defence mechanism in plant which makes it more resistant to temperature changes and to attacks by insects, bacteria and fungi.
PACKAGING AND MODIFIED ATMOSPHERE
Packaging fresh-cut fruits products in polymeric films that help in creating a suitable passive modified atmosphere can also be an effective supplement to proper temperature management in maintaining their quality. However, oxygen level can get too low if the wrong poly bag is used and this can lead to off-flavor development as a result of anaerobic respiration. This challenge is to select a packaging material which will have low enough oxygen to assist in quality retention without leading to off-flavor development.
2.4 EFFECTS OF WASHING
The lack of an effective antimicrobial treatment at any step from planting to consumption means that the pathogens introduced at any point may be present on the final food product. Washing and rinsing some types of fruits and vegetables can prolong shelf-life by reducing the number of microorganism on the surface. Infiltration may also be enhanced by the presence of suffectants and when the temperature of the fruit or vegetable is higher than the temperature of a water suspension of cell.
By subjecting bacteria to a salty environment, it keeps them from growing. Some bacteria however, have adapted to living in salty environments, such as Staph. Salt will kill off bacteria from the fruits by creating a hypertonic environment.
2.5 KIRBY-BAUER ANTIBIOTIC TESTING (DISK DIFFUSION ANTIBIOTIC SENSITIVITY TESTING)
This is a test which uses antibiotics –impregnated wafers to test whether bacteria are affected by antibiotics. In this test, wafers containing antibiotic are placed on agar plates where bacteria have been placed, and the plate is left to incubate. If an antibiotic stops the bacteria from growing or kills the bacteria, there will be an area around the wafer where the bacteria have not grown enough to be visible. This is called the zone of inhibition. This information can be used to choose appropriate antibiotics to combat a particular infection. (Mohanty A et al; 2010)
CHAPTER THREE
MATERIALS AND METHOD
3.1 MATERIALS
Material used include: Minimally processed; Pineapple, Paw-paw, Orange, Water- melon, Spinach, Cabbage, Pumpkin Leaf.
3.2 SAMPLING SITES
Sampling sites is carefully selected based on proximity to the targeted population because they are the key people of interest, Samples were collected within the local market of Nasarawa town in Nasarawa state.
3.3 SAMPLES COLLECTION
Five samples of each of the minimally processed fruits (pineapple, paw-paw, water-Melon and orange) and five samples of each of the vegetables (spinach, cabbage and popkin leave) were aseptically collected from the local market (Nasarawa market).
3.4 SAMPLE PREPARATION
The samples were prepared based on four different parameters, viz:
· Washed with salt water
· Washed with tap water
· Washed with distilled water
· Unwashed
At each stage the fruits and vegetables were well blended and these were serially 6 fold diluted (10-1, 10-2, 10-3, 10-4, 10-5, 10-6,). Dilutions are made depending on cell density. The sixth serial dilutions (10-5) were taken for analyzing the total microbial count based on each of the parameters stated above
3.5 PREPARATION OF MEDIA
All media used were prepared from commercially available powder, and manufacturer’s instructions were clearly followed. Sterilization of these media was achieved by autoclaving at 15 lb/sq inch and 1210c for 15 minutes. Pipettes and other glassware were packed in canisters and sterilized in the hot air oven at 1600c for 90 minutes. Aseptic conditions were maintained throughout the period of sample analysis to prevent contamination of samples and media.
3.6 DETERMINATION OF TOTAL BACTERIAL COUNT
Each of the samples obtained from four various parameters (that is the unwashed, the washed with salt water, the washed with tap water and the washed with table water) were analyzed for the presence of micro-organism by the plate count method. The last two dilutions (10-5, 10-6) were inoculated in five replicates on the plate count agar (PCA) using the pour plating method. Nutrient Agar media was prepared and used for the total plate count. Total bacteria count was determined after incubation at 370C for 24hours.
3.7 ISOLATION OF MICROORGANISM
Sample was collected from the incubated plate of the bacterial count and smeared into a new plate containing MacConkey agar which was prepared based on the manufacturer’s instructions. The plates were then incubated at 37oC for 24hrs. Morphological characteristics of colonies (size, shape, elevation, form, pigmentation and capacity) developed after incubation on MacConkey agar plate were carefully studied and recorded. Five to ten suspected colonies of E. coli, Salmonella and Shigella were picked up from the plate observed from the different colour they exhibit due to their lactose fermenting characteristics and subcultured onto fresh MacConkey plate to get pure culture.
Suspected salmonella isolates were confirmed using Salmonella-Shigella Agar, shigella on the other hand was confirmed with Xylose Lysine Deoxycholate Agar, while E-Coli were confirmed with Eosine Methyline Blue Agar and series of Biochemical test were conducted.
3.8 ENUMERATION OF MICROBES:
Microorganism Agar to be used for isolation analysis
Shigella dysenteriae Xylose Lysine Deoxycholate agar medium (Oxoid)
Escherichia coli, Mac conkey Agar
Salmonella Spp Salmoella-Shigella Agar (SSA).
3.9 ANTIMICROBIAL SUSCEPTIBILTY TEST
The isolate were screened for antimicrobial susceptibility, using the agar disk diffusion method by Kirby-Bauer. The following antibiotics were used: Ciprofloxacin (5µg), Gentamicin (10µg), Streptomycin (10µg), Tetracycline (30µg), Chloramphenicol (30µg), Amoxycillin(25µg), Erythromycin (5µg), Oxacillin (5µg), and Sulphamethoxazole/Trimethoprim (25µg),. The isolates were uniformly streaked on Nutrient Agar plate and the antibiotic impregnated disc was applied onto the inoculated plates using sterile forceps. The plates were then incubated at 370c for 24hrs, after which clear zones of inhibitions were measured in millimeters (mm) using transparent rulers. The results were interpreted using the clinical and Laboratory standards institute (CLSI) criteria.
CHAPTER FOUR
DATA PRESENTATION, ANALYSIS AND RESULT
4.1 MICROBIAL PLATE COUNT OF THE MINIMALLY PROCESSED FRUITS AND VEGETABLES.
Total plate count = No of colonies× dilution factor(cfu) (cfu/ml)
ml of sample(ml)
Serial dilution used =10-1 10-2 10-3 10-4 10-5 10-6
Dilution factor used = 10-5
Ml of sample used = 1ml (used for all the samples)
Total number of plate count is summarized in the table below:
4.2 Concentration of Salt Water Used For the Preparation Of Sample D
· Paw paw, water mellon and pineapple: 3g of salt to 200ml of water used to wash 30.5g of each of them
· Pumpkin Leaf: 4g of salt to 200ml of water used to wash 3leaves of popkin
· Spinach: 5g of salt to 500ml of water used to wash 3leaves of spinach
· Citrus: 3g of salt to 100ml of water was used to was the citrus
A summary of all the mean values of the plate count of the various fruits and vegetables assessed.
Fruits/ Vegetable | MEAN VALUES | ||||||
Unwashed | Washed with tap water | Washed with distilled water | Washed with salt water | ||||
Water melon | 9.5×103 | 8.0×103 | 5.3×103 | 4.5×101 | |||
Pineapple | 4.53×103 | 3.8×103 | 3.71×103 | 6.14×101 | |||
Paw-paw | 5.52×103 | 4.52×103 | 3.41×103 | 2.68×101 | |||
Citrus | 3.46×102 | 2.98×102 | 2.8×102 | 2.2×101 | |||
Pumpkin leaf | 8.47×103 | 6.37×103 | 4.4×103 | 3.36×101 | |||
Spinach | 8.41×103 | 6.48×103 | 4.52×103 | 3.56×101 | |||
Cabbage | 9.32×103 | 7.36×103 | 5.32×103 | 4.1×101 | |||
4.3 STATISTICAL ANALYSIS
Groups | Count | Sum | Average | Variance | ||
UNWASHED | 7 | 46096 | 6585.1429 | 11190612 | ||
WASHED WITH TAP WATER | 7 | 36828 | 5261.1429 | 6989789.1 | ||
WASHED WITH DISTILLED WATER | 7 | 26940 | 3848.5714 | 2995481 | ||
WASHED WITH SALT WATER | 7 | 275.4 | 39.342857 | 175.24952 | ||
ANOVA | ||||||
Source of Variation | SS | Df | MS | F | P-value | F crit |
Between Groups | 167758627 | 3 | 55919542 | 10.562786 | 0.0001283 | 3.0087866 |
Within Groups | 127056343 | 24 | 5294014.3 | |||
Total | 294814970 | 27 | ||||
HYPOTHESIS:
Ho: unwashed = washed with tap water = washed with distilled water= washed with salt water.
Ha: unwashed ≠ washed with tap water ≠ washed with distilled water ≠ washed with salt water
COMMENT: Since the calculated F-value is higher than the table F-value we reject the null hypothesis and accept the alternate hypothesis and conclude that the microbial load of the unwashed, washed with tap water, washed with distilled water and washed with salt water are not equal
4.4 Results Discussion
The following results were obtained from the isolation of salmonella. Shigella and E.coli.
A total of 56 samples of the fresh fruits and vegetables were analyzed for the prevalence of Salmonella spp, Shigella and Escherechia Coli using Mac Conkey Agar, Eosine Methylene blue Agar and Salmonella Shigella Agar. The result obtained is as shown in the table below.
Biochemical Characteristics and Possible Identification of Isolates from the Fruits and Vegetable Samples.
Isolates | Morphology | Oxidase | Gram reaction | Motility | H2S | Glucose | Mannitol | Indole | Urease | Citrate | Probable Identification |
WD1 | Rods | – | – | + | – | + | – | + | – | – | E.coli |
WD2 | Rods | – | – | – | – | + | + | – | – | – | Shigella spp. |
WS1 | Rods | – | – | + | + | + | + | + | + | + | Salmonella spp |
WS2 | Rods | – | – | + | + | + | + | + | + | + | Salmonella spp |
WT1 | Rods | – | – | + | + | + | + | + | + | + | Salmonella spp |
WT2 | Rods | – | – | – | – | + | – | + | – | – | Shigella spp |
WT3 | Rods | – | – | + | – | + | – | + | – | – | E.coli |
UW1 | Rods | – | – | + | + | + | + | + | + | + | Salmonella sp |
UW2 | Rods | – | – | – | – | + | + | – | – | + | Shigella spp |
KEY:
WD1 = Washed with distilled water 1; WD2= Washed with distilled water 2 ; WS1= Washed with salt water 1; WS2= Washed with salt water 2; WT1= Washed with tap water 1; WT2= Washed with tap water 2; WT3= Washed with tap water 3; UWT1= Unwashed 1; UW= Unwashed 2
4.4.1 Morphology of Salmonella spp. ,Shigella spp., and E. coli on Various Selective and Differential Media after 24hrs incubation at 37 ºC
Salmonella appear colourless with no clear transparence on Mac Conkey Agar plate. Shigella appear colourless but with a clear transparence on Mac Conkey Agar plate. E. Coli appear pink-red on Mac Conkey Agar plate
`4.5 ANTIMICROBIAL SUSCEPTIBILITY OF THE ISOLATES
The antibiotic susceptibility profile showed that, virtually all the isolated organisms were resistant to one or multiple antibiotics.
Gentamicin, ciprofloxacin and streptomycin were the most sensitive antibiotics whereas oxacillin, sulphamethoxazole/trimethoprim, chloramphenicol, amoxicillin, tetracycline, and erythromycin were the least sensitive.
CHAPTER FIVE
CONCLUSION AND RECOMMENDATION
5.1 CONCLUSION
Minimally processed products are fruits or vegetables that have been trimmed, peeled, and cut into 100% usable product that is bagged or pre-packaged to offer consumers high nutrition, convenience and flavour while still maintaining its freshness. Minimally processing gives additional value to fruits and vegetables in terms of convenience and time savings but are so prone to infestation of microorganisms such as Salmonella spp, Shigella spp and E. Coli. The results obtained have indicated that the microbiological quality of minimally processed fruits and vegetables that is, pineapple, water mellon, pawpaw, orange, spinach, popkin and cabbage sold within our locality here is poor .Hence to ensure the safety of these products in the future, it is critical that food safety control measures are effectively implemented.
5.2 RECOMMENDATION
With regards to the findings of this study, the following recommendations can aid in reducing microbial load of ready to eat products
· ASEPTIC HANDLING: This involves handling practices that is completely free from any form of disease causing contaminants or preventing contacts with microorganisms. Prevention of microbial contamination of fresh produce is favored over reliance on corrective actions once contamination has occurred.
· WATER QUALITY: The quality of water used in handling of fruits and vegetables can either add or reduce the microibial load of the minimally processed fruits. Minimize the potential of microbial contamination from water used with minimally processed fruits and vegetables.
· PROPER PACKAGING OF MINIMALLY PROCESSED FRUITS AND VEGETABLES: There is need for adequate packaging of minimally processed fruits and vegetables with impermeable packaging materials that are clean and microbial free immediately after it is minimally processed
REFERENCES
Anjezaeoku, M. L, Miranda, H., Roland B. (2011)“Micro biological examination of frozen fruits and vegetables sold in Tirana markets”, J.Int.Environmental Application &Science, Vol. 6(4):518-520.
Falik, E.and Aharoni, Y. (2004). Post Harvest Physiology, Pathology and Handling of Fresh Produce. Lecture Notes. International research and Development course on Postharvest biology and Technology. The volcani Centre Israel. 30pp.
Francis, G.A., Thomas, Cand O’beirne, D (1999). The microbiological safety of minimally processed vegetables. International journal of Food Science and Technology 34(1): 1 – 22.
Mohanty, A, (2010). Phusiochemical and Antimicrobial Study of polyherbal Pharmacieglobal, vol 4 (04), page 1-3
Demelo, S.,and Pereira da Costa, R.M.N(2010). “Microbial quality of minimally processed perolapine applegrown under good agricultural practices system”, 3rd International symposium on tropical and subtropical fruits, International Society for Horticultural Science,
No comments:
Post a Comment