Antimicrobial Effect of Lime Juice Treatment on the Shelf-Life of Zobo Drink

Zobo drink is an indigenous non-alcoholic drink made from hot water roselle calyces of Hibiscus sabdariffa can be used as natural preservative to prolong the shelf- life of zobo drink for a period 5 days under normal room temperature.


INTRODUCTION
Zobo drinks are non-alcoholic local beverages made from differet varieties of dried petals, acidsucculent aqueous extract of calyx of rosette, Hibiscus sabdariffa, which is an annual herb that is widely cultivated in India and Africa [1]. Zobo is the local name for Hibiscus sabdariffa plant in Hausa (northern Nigeria). There is increase in demand for zobo drinks due to its low price, nutritional and medicinal properties [2]. The nonalcoholic drink (zobo) is quite popular especially in northern Nigeria and is usually served chilled at various social gathering [3]. This drink have been found to be rich in anthocyannins, vitamins, natural carbohydrate, protein and other antioxidants [4,5] and also minerals [6]. The drink is used in curing minor stomach ailments, sore throat and strengthening the heart among other uses [1,5]. The leaves of the plant are also used as vegetables and the seeds are source of oil [7]. Zobo drink if well prepared and packaged will compete favourably with most of the nonalcoholic beverages available in the country, considering the increasing acceptance, socioeconomic potentials and ready source of protein, vitamin C and other minerals.
The greatest limitation for large-scale production of zobo drinks is the rapid deterioration of the drink. Its shelf-life is approximately 24 hours following production, if not refrigerated. Microorganisms associated with the dried calyces, the production processes and other factors may contribute to its spoilage. Apart from the fact that most chemical preservatives may have adverse effect on humans, they are expensive and usually not affordable by the local people that produce this drink. Moreover, refrigeration may not be available. There is the need for alternative source of preservative that is natural and human-friendly, affordable and readily available. Fresh lime contains flavonoids, ascorbic acid and hydrocarbons that give it the antimicrobial properties [8]. Therefore, lime juice can be used to improve the shelf-life of zobo drink. This work was aimed at investigating the effect of lime juice on the microbial quality of zobo drink with a view to improving the shelf-life of the drink.

Sample Preparation
Zobo drink was prepared in Abia State Polytechnic Microbiology Laboratory under proper hygienic conditions. The dried calyses were boiled with distilled water for about 10 -15 minutes from which the embedded pigments were extracted. After extraction, the filtrate was allowed to cool and sweetened with granulated sugar and spiced with ginger.

Microbiological Analysis
One milliliter of zobo drink sample was put in 9 mL of sterile distilled water in sterile test tube, shaken and then serially diluted. From the appropriate dilution, 0.1 mL was inoculated separately on nutrient agar, MacConkey agar and Potato dextrose agar plates and spread evenly using sterile bent glass rod. Each experiment was carried out in duplicates to get a mean standard value of the colony forming units (cfu/mL) on the plates. The inoculated nutrient agar and MacConkey plates were incubated at 37ºC for 24 hours while the Potato dextrose agar plates were incubated at room temperature for 5 -7 days. After the period of incubation, the colonies on the plates were counted and recorded as colony forming unit per milliliter (cfu/mL) and coliform respectively [9]. Each of the bacterial colonies on both Nutrient agar and MacConkey agar plates was sub-cultured and pure culture obtained. Isolates were identified by carrying out tests which include Gram staining, spore staining and biochemical test which include catalase, coagulase, oxidase, citrate utilization, indole, urease, methyl red, Voges-Proskauer and sugar fermentation [10].
Fungal isolates observed on Potato dextrose agar plates were identified by their cultural characteristics such as color, morphology and growth pattern of mycelia [9].

Antimicrobial Analysis of Lime Juice
The lime fruits were washed with sterile water and cut open with a sterile knife and the juice pressed out aseptically. The crude extracts were obtained by filtration [11]. Sterile cork borer of about 6mm was used to make wells on prepared sterile molten Nutrient agar plates in triplicates. 1.0 mL of lime juice at different concentrations of 100%, 75%, 50%, 25%, 12.5% and 6.25% were aseptically introduced into each well. The setup was allowed to stand for about 5 minutes before incubation. The antimicrobial activities of the lime juice in each of the wells was determined by measuring the diameter of the zones of inhibition in millimeter [12].

Treatment of Zobo Samples with Lime Juice
Some lime fruits were surface-sterilized with 70% ethanol and peeled using a pre-sterilized knife. The fruits were squeezed aseptically into a 100ml sterile conical flask [13]. Ten-fold dilutions of the zobo drink were obtained and 0.1, 0.5, 1.0, 1.5 and 2.0 mL of lime juice concentration were added respectively to each test tube of the tenfold dilution. The mixtures were allowed for 6hours and then for 24 hours at room temperature. From each treatment, 0.1 mL was inoculated into duplicate plates of Nutrient agar and MacConkey agar at 37ºC and Potato Dextrose agar at room temperature. The colonies were counted and recorded as colony forming units per milliliter (cfu/mL -1 ) after 24 hrs for a period of 5 days [14].

Study Questionnaire
Twenty individuals were randomly selected to evaluate the taste and appearance of the drink after treatment with lime juice for the period of 5 days. Table 1 shows the microbial counts of zobo drink prepared under proper hygienic conditions. The result showed an average aerobic bacterial count of 0.  Table 2. A total of three bacterial isolates were identified which include Bacillus spp, Staphylococcus spp and Lactobacillus spp, Penicillium spp was the only fungal isolate identified (Table 3). Table 4 shows the antimicrobial effect of lime juice on the different isolates. Staphylococcus spp was the most susceptible isolate with 22.0 mm as the mean zone of inhibition while Penicillium spp was the least susceptible with 14.2 mm zone of inhibition at 100% concentration respectively. At 12.5% concentration of lime juice, Penicillium spp was resistant. Table 5 shows the total microbial counts of Zobo drink after treatment with lime juice. The result showed that lime juice at 50-100% concentration had no growth at room temperature for a period of 3 days and a period of 5 days at 75-100% concentration. Table 6 shows the result of the questionnaire on the taste of Zobo drink treated with lime juice after 5 days. A total of 17(94.4%) claimed the drink tasted "good" while 1(5.6%) claimed the drink tasted normal at 100% concentration respectively. In all concentration, none reported "sour" taste. Table 7 shows the result of the questionnaire on the appearance of Zobo drink treated with Lime Juice after 5 days. Out of 18 individuals that reported on the appearance, 4(22.2%) claimed the appearance was good, 13(77.1%) attested that appearance was normal at 100% concentration respectively. In all the concentrations, none reported "poor in appearance".

DISCUSSION
In the quest toward zero import dependence and food security in Nigeria, the food drink industry has come under scrutiny, as many imported drinks (especially "energy drinks") have almost no food value, contain harmful or even carcinogenic chemicals and have been shown to aggravate certain diseases such as diabetes and high blood pressure [15]. This has precipitated research into locally produced drinks such as zobo drink. Zobo drink has been shown to possess medicinal/pharmaceutical properties including anti-diabetic, antihypertensive and antiinflammatory properties [16].
Despite these obvious advantages, the leap from locally marketed product to large scale production is still improbable due to its poor shelf life [17]. This is due to high microbial proliferation that is presently associated with zobo drink sold in Nigeria. Zobo drink when left for more than 24 hours at room temperature turn sour. This may be as a result of microbial fermentation. This fermentation process has been found to cause loss of taste and nutritional value, increased rate of browning, offensive odour and presence of cloudy materials at the bottom of the containers [18].
In the present study, the antimicrobial effect of lime juice on the shelf-life of zobo drink was investigated. The result obtained revealed that lime juice has significant effect in prolonging the shelf-life of zobo. This observation is supported by the findings of [19] and Ibukun et al. [20] on the antimicrobial effect of lime juice on different microbial genera. The result obtained from the treatment of zobo drink with lime at 50-100% concentration showed no growth at room temperature for a period of 3 days and a period of 5 days at 75-100% concentration. The result obtained from the questionnaire carried out on the treatment of zobo drink with lime juice showed that lime juice had no negative effect on the taste and appearance of zobo drink after 5days. This is also supported by the work carried out by Nwachukwu et al. [21] who observed that lime juice reduced the bacterial load of Zobo drink. Oboh et al. [22] had earlier reported that the bioactive component of lime juice was Phydroxybenzoate with citric acid (pH 2.54). The antibacterial activity of P-hydroxybenzoic acid esteri (Parabens) highlighted by Hugo and Russell [23] showed a wide range of activities against microorganisms. Thus, they serve as preservatives in foods, toiletries and in some pharmaceutical preparations such as emulsions, creams and lotions. This finding from the study therefore, recommends the addition of lime juice at appropriate concentration to help prolong the shelf life of the drink, where refrigeration is not available.

CONCLUSION
The greatest limitation for large-scale production of zobo drinks is the rapid deterioration of the drink. Preservation of zobo drink with chemical preservatives may be expensive and unaffordable to the local people that produce this drink and may also have some adverse effect on humans. There is a need for an alternative source of preservative that is natural and humanfriendly, affordable and readily available. In the present study, the effect of lime juice in prolonging the shelf-life of zobo drink was investigated. The result revealed that lime juice can preserve zobo drink by reducing the microbial load. However, good environmental and personal hygiene should be maintained when processing. Additives and packaging materials should also be properly sterilized. This study therefore recommends the use of lime juice for the preservation of zobo drink to increase the shelf-life.