Microbial analysis of leafy vegetables in iceless cooling facility

Against the background that leafy vegetables stored under ambient conditions are influenced by environmental factors which may cause significant quality loss in terms of freshness, colour, texture and composition, the iceless cooler was used to evaluate microbial load of leafy vegetables. The samples were evaluated for bacterial (coliforms and mesophiles) and fungal (mold and yeast) loads. Results of the microbial test showed that Corchorus olitorius had highest mean microbial load with plate count of 6.7 x 104 CFU/g and Hibiscus sabdariffa had the least mean microbial load with plate count of 4.8 x 104 CFU/g after five days of storage. Corchorus olitorius show a significant increase of Escherichia coli, Staphylococcus aureus and Streptococci species after five days of storage. The ANOVA results showed that vegetables stored under ambient conditions were significantly different (p˂0.01) from those stored in the iceless cooler. The results also indicate that Amaranthus dubius recorded the highest mean weight of 1.94kg and maintained its freshness and colour for up to three days compared with Corchorus olitorius with the least weight of 1.84kg.


I. INTRODUCTION
Leafy vegetables such as Amaranthus dubius, Corchorus olitorius and Hibiscus sabdariffa are important traditional vegetables cultivated in the three Northern Regions of Ghana because of their health benefits (Amaglo & Nyarko, 2012). These vegetables contain high sources of proteins, carbohydrates, vitamins and minerals which are essential for a healthy human life (Buyukunal et al, 2015). However, leafy vegetables farmers in Ghana do not posses adequate hygienic production information and good agricultural practices (e.g., storage of vegetables) and thus, posing high potential public health risk (Adam et al., 2016). Knowledge and good skills of leafy vegetable storage is very important in order to help maintain the nutrient composition and texture. According to O'Connor-Shaw et al. (1996) microbial spoilage is a limiting factor for shelf life of leafy vegetables and fruit specie stored under controlled atmosphere conditions. Shelf life, including microbial spoilage results in 30-50% shrinkage of fresh-cut fruits and leafy vegetables (Warren, 2005). Contamination sources of fresh-cut fruits and vegetables include raw materials and contact with processing equipment. The microorganisms that exist on the surfaces of raw, whole produce appear to be the major source of microbial contamination and consequent spoilage of fresh-cut fruits and vegetables. Boyette et al. (1993) indicate that the microbial decay of fresh-cut lettuce is largely due to the growth of microorganisms originating from pre-harvest environments. Delaquis et al. (1999) indicate that the types of microorganisms found on shredded lettuce were highly associated with the microorganisms detected on lettuce before shedding. Microbial spoilage including off-flavor formation, slimy surface, wetness and soft rot, discoloration, and visual microbial growth/colonies has been used as a main or exclusive objective criterion to determine shelf life of fresh-cut products (O'Connor-Shaw et al., 1994; Sapers et al, 2001,). Jacxsens et al. (2003) reveal that green, yellow and red bell pepper was unacceptable by day 6 of storage under atmospheric condition of 7°C due to acidic flavor, water loss and texture change. Also, Allende et al. (2004) indicate that processed Lollo Rosso lettuce had a shelf life shorter than 7 days at 5°C due to high microbial counts and off-odor formation under MAP. It therefore suggests that leafy vegetables deteriorate quickly due to the active process of senescence and biochemical processes which change the original composition of the crop. The lack of appropriate vegetable storage techniques often times result in post-harvest loss (Adam et al., 2016). This is because storage of harvested products under controlled conditions is known to retard the growth of postharvest spoilage and pathogenic microorganism (Nguyen & Cardin, 1994). The reasons for the lack of adoption of modern storage technologies by vegetable farmers include huge cost of storage facilities coupled with unstable electricity power (Adam et al., 2016;Gogo et al., 2016). As an alternative to the modern technologies for storing fruits and leafy vegetables, Nicol et al. (1997) suggested the use of iceless coolers by farmers and traders who cannot afford to acquire improved storage technologies and equipment. The iceless cooler can be made up of a wooden frame with dimensions of 0.7m x 0.7m x 1.2m. The sides of the frame are covered with jute sacks. The top water trough is filled with water regularly to soak the jute sacks at the sides of the coolers. The iceless cooler also has a water trough at the base which collects excess water. The coolers are kept in an open shaded area where there is good flow of air current. Energy from the air current is used in evaporating the water at the sides of the cooler and also to preserve the vegetables. The arguments for the iceless cooler is that the equipment is less expensive and built on simple home-made technology which can keep fruits and vegetables for longer periods of time. The iceless cooling facility works on simple principles of evaporative cooling to conserve the quality and shelf life of leafy vegetables. However, there is very limited information regarding how changes in atmospheric composition affect spoilage microflora profile during iceless cooling storage of leafy vegetables. The main objective of this study was to assess the effects of iceless cooling facility on microbial development, quality and shelf life of traditional leafy vegetables.

II.
LITERATURE REVIEW The common types of leafy vegetables cultivated in Northern Region of Ghana include Amaranthus dubius, Hibiscus sabdariffa, Corchorus olitorius, bitter leaf, baobab leaves, lettuce, cabbage and asparagus. Amaranthus dubius (Aleefu) known as Amaranth is a dark green cosmopolitan genus of annual or short-lived perennial leafy vegetable in Africa, India, Bangladesh, Sri Lanka and the Caribbean. The vegetable forms an important source of plant protein (Babalola et al., 2010), which is very useful for treating fever. The vegetable also contains vitamins (A and C) and minerals (potassium, iron, magnesium and calcium) in the diet of people (Smith & Eyzaguirre, 2007;Tweneboah, 2000). Amaranth leaves in general are recommended as a good food with medicinal properties for young children, lactating mothers and for patients with fever, haemorrhage, anaemia, stomach ache (Schippers, 2002), constipation or kidney complaints. It is also an excellent nutritional food for AIDs patients especially when Amaranth grain porridge (1 cup) is combined with moringa leaf powder (1 table spoon) (Babalola et al., 2010). Corchorus olitorius (ayoyo) is a native plant of tropical Africa and Asia, and has since spread to Australia, South America and some parts of Europe. It is popularly used in soup preparation and folk medicine for the treatment of fever, chronic cystitis, cold and tumours. Scientists have indicated that drinking vegetable juice freshly made with Corchorus olitorius in an empty stomach can prevent and cure Ebola Virus Disease. It is also reported that Corchorus olitorius has the power to clear or open the natural ducts of the fluids and secretions of the body, as well as encourages lactation, purgative and tonic solving aches and pains, dysentery, enteritis, fever and tumors. Corchorus olitorius leaves are also rich sources of potassium, iron, copper, manganese, zinc and high energy values essential in human and animal nutrition. Hibiscus sabdariffa (Roselle) is an important annual crop grown successfully in tropical and sub-tropical climates. The plant is cultivated for its stem, fibre, edible calyces, leaves and seeds which are used in making various foods. The commercially important part of the plant is the fleshy calyx (sepals) surrounding the fruit (capsules). It is used for making wine, juice, jam, jelly, syrup, gelatin, pudding, cakes, ice cream and flavours and also dried and brewed into tea, among other things (Solomon, 2013). Hibiscus is used for treating loss of appetite, colds, heart and nerve diseases, upper respiratory tract pain and swelling (inflammation), fluid retention, stomach irritation, and disorders of circulation. It is also good for dissolving phlegm. According to Jonadet et al. (1990) Hibiscus can be used as a laxative and diuretic to increase urine output. Calyces of the red and dark red coloured type are extracted and sweetened to produce a refreshing drink while calyces and leaves of the green type are used for making vegetable stew (Babalola, 2001).

III. MATERIALS AND METHOD 3.1 Research design
The experimental design was a randomized complete block design. Nine samples of Corchorus olitorius, Hibiscus sabdariffa and Amaranthus dubius were bought from the Tamale central market to serve as control. Samples were also taken from the vegetables cultivated purposely for this research work. Harvested vegetables were weighed and kept inside the iceless cooler facility. Temperature readings and relative humidity inside and surroundings of the coolers were taken thrice a day at 9:00am, 12noon and 3:00pm. Daily records on colour change, freshness and weight loss of the vegetables taken.

Preparation of smears
A drop of physiological saline was placed on a clean grease free glass slide. A sterile inoculating loop was used to transfer a colony to the slide and emulsified to make a smear. The slides were left to air-dry before heat fixing, using the flame of a Benson burner to properly adhere the smear on the slides as well as preserve microorganisms and prevent smears from being washed from the slides. Smears were allowed to cool before staining.

Gram staining of cultures
Gram staining was done to help identify the gram status and morphology of organisms. The fixed smear was covered with crystal violet stain for 60 seconds and rapidly washed off with tap water. The smear was covered again with Lugol's iodine for 60 seconds and washed off with tap water. Acetone-alcohol solution was used to decolorise the stain for only 10 seconds and immediately washed off with tap water. The smear was covered again with neutral red stain (safranin) for 1 minute and washed off with tap water. The slides were placed in a draining rack for the smear to air dry. The smears were examined microscopically with oil immersion objective to report the gram status and morphology of bacteria.

Microbial load tests
Ten (10) grams of chopped vegetable each was weighed into each sampling bottle containing 90 millilitres (90 ml) of peptone water, making a dilution of 10 -1 . This was then agitated vigorously for two minutes to obtain homogeneity. One millilitre (1 ml) of the sample inoculum was pipetted from the 10 -1 dilution into a separate tube containing 9 ml of peptone water to make a dilution of 10 -2 . The liquids were carefully mixed by aspirating 10 times with a sterile pipette and again 1.0 ml transferred into another dilution tube containing 9 ml of dilution peptone water and mixed to give 10 -3 . These dilutions were then ready for inoculation.

Sample analysis
Sterile plates count agars were labeled 10 -1 , 10 -2 and 10 -3 and 0.1ml (100 µl) of the various dilutions inoculated. The inoculum was promptly spread on the surface of the plates using sterile glass spreader (Drigalsky spatulas). The plates were allowed to dry for 15 minutes before incubating in an inverted position at 35 o C -37 o C for 48 hours. Colonies were then counted and enumerated. All colonies in three plates corresponding to one dilution and showing between 30-300 colonies were counted. Averages of the replicates were calculated and multiplied by their dilution factor. This was then reported as the colony forming unit per gram (cfu/g). Overcrowded colonies were not counted and were reported as "Too Numerous to Count" (TNTC).

Data analysis
Using the Predictive Analytical Software version 21.0 and Student Edition of Statistix 9.0, the analysis was done to determine treatment effects on the quality parameters of the vegetables. The least significant difference at 5% and 1% level of significance were used to compare means of the vegetables kept under ambient conditions in the same vicinity with the cooling facilities for the same storage period of seven days. Microbial analysis on E. coli, Staphylococcus and Streptococcus was carried out at the Savannah Research Institute's laboratory in Tamale. Each day at 3pm, the weight of the samples were taken using a top pan scale. The weight loss was measured in percentages over the storage period of seven days. Results of microbial population for the different conditions and their interaction effects were subjected to analysis of variance (ANOVA) and coefficient of variation. Table 1 Table 3 presents the test of bacteria for the sampled vegetables. The findings show that there was no statistically significant difference between before storage and after storage of the leafy vegetables.

International Journal of Environment, Agriculture and Biotechnology (IJEAB)
Vol-3, Issue-1, Jan-Feb-2018  http://dx.doi.org/10.22161/ijeab/3.1.24  ISSN: 2456-1878 www.ijeab.com  Figures 1-3 show the rate of deterioration of leafy vegetables under ambient condition and in the iceless cooler facility. Figure 1 shows that Amaranthus dubius maintained its original weight of 2 kilograms from day zero through to the second day before losing 1.0% of its weight under the iceless cooler. By the fourth day of storage, its accumulated weight lost was 3% and by the fifth and sixth days of storage, the cumulative weight loss was 5.5% and 9.5% respectively. On the 7 th day, its cumulative loss was 15%. Amaranthus dubius reduced weight by 0.5% on first day of the storage and 2% on the second day under ambient condition. There was a sharp rise in cumulative weight loss of 22% on day four and 97% loss on the seventh day ( Figure 1).

Trends of deterioration of leafy vegetables
Corchorus olitorius maintained its original weight of 2 kilograms from day zero to day 2 before losing1.0% of its weight. By the fourth day of storage, there was a cumulated weight loss of 4% and on the fifth day an increase of weight loss up to 11.5% was recorded. By the sixth day, the cumulative weight loss rose to 20% ( Figure  2). However, under ambient conditions, the cumulative weight loss was 4% on the third day and by the fourth day, the cumulative weight loss increased to 29% and on the sixth day it recorded an increase of 57% ( Figure 2).
Hibiscus sabdariffa loss a cumulated weight of 2% on the third day under the iceless cooler facility. It also experienced a sharp increase of weight loss of 29% on the fourth day. Under ambient conditions, Hibiscus sabdariffa reduced weight by 1% a day after the storage which increased to 4% the second day. By the third and fourth days, the percentage weight loss was 29% and 57%, respectively.   Table 9 shows the freshness level of the leafy vegetables after five days of storage. The results show that Amaranthus dubius and Corchorus olitorius stored in the iceless cooler facility showed no significant differences (p˃0.01) in freshness rating.

Freshness rating for leafy vegetables
The results also indicate that there is no significant difference of freshness of the vegetables stored under ambient conditions.

V. DISCUSSION
The use of iceless cooling facility has a positive impact on the quality of leafy vegetables, even though there were increases of Escherichia coli count at all cases. There was a significant difference of total Escherichia coli count (p<0.01) between before storage and after storage of the leafy vegetables. This finding is similar to Abdullahi and Abdulkareem (2010) due to field contamination before vegetables are harvested. The study findings also indicate a significant difference (p<0.01) of deterioration of vegetables bought from the market and those cultivated for this study. Even though Hibiscus sabdariffa and Corchorus olitorius recorded higher counts, the variation in the data set for both market sourced vegetables and vegetables cultivated at home was 4.39. The findings further indicate that Amaranthus dubius deteriorated at a lower rate compared with Corchorus olitorius and Hibiscus sabdariffa. While Amaranthus dubius stayed in the cooler for more than 6 days and had not deteriorated beyond saleable weight (10% weight loss), Corchorus olitorius and Hibiscus sabdariffa could not stay such long. Under ambient conditions, it took only 3 days for all the vegetables to stay within saleable weight (5% -10%). The factors accounting for weight loss of leafy vegetable is water loss. According to Wilson et al. (1999), water loss is due to the temperature of the product and air velocity. The findings on leafy vegetable freshness indicate that Amaranthus dubius remained 100% fresh inside the iceless cooler facility for two days, while Hibiscus sabdariffa and Corchorus olitorius remained 100% fresh for only a day. Amaranthus dubius declined in freshness to 75% fresh for the next four days, while Hibiscus sabdariffa and Corchorus olitorius declined to 75% fresh for the next three days. This predisposed that Amaranthus dubius has a high potential of staying longer in the facility compared to the other leafy vegetables. Observation of leafy vegetables stored under ambient conditions show that they loss their freshness within the 1 st day and deteriorated to 50% in the 2 nd day of storage.

VI.
CONCLUSION Leafy vegetables are vital sources of minerals and vitamins for human health and development, hence, the need to conserve the quality and shelf life of vegetables through the use of iceless cooler facility. The iceless cooler facility has the tendency to retain freshness and colour of vegetables for a short while compared to vegetables stored under ambient temperature. The coefficient of variations confirms the efficiency of the iceless cooler facility which should be adopted by farmers and traders for the storage of leafy vegetables in order to reduce postharvest loss. Given the efficiency of the iceless cooler facility, it is prudent to use it for preserving the quality and shelf life of Amaranthus dubius, Corchorus olitorius and Hibiscus sabdariffa.