Effect of spices on the microbiological quality of Hibiscus sabdariffa (zobo) drink and molecular characterization of the associated spoilage organisms

Hibiscus sabdariffa drink (zobo) was preserved with three concentrations of cloves (Syzygium aromaticum), ginger (Zingiber officinale) and mixture of cloves and ginger, respectively while the control had no spice. The bottled H. sabdariffa drink was stored at 28 ± 2°C for 16 days. The total bacteria and fungi counts were determined every 48 h during storage. Bacterial and fungal growths were not detected in H. sabdariffa drink containing 0.25 and 0.45% concentrations of either of the spices through the period of storage. The following organisms which were isolated from the control and associated with H. sabdariffa drink spoilage were inhibited by the spices at 0.25 and 0.45% concentrations: Stenotrophomonas rhizophila, Pseudomonas azotoformans, Leucobacter aridicollis, Leucobacter komagatae, Pantoea gaviniae, Staphylococcus auricularis, Aspergillus niger, Ascomycota species, Candida parapsilosis, Fusarium solani, and Penicillium chermesinum.


INTRODUCTION
Hibiscus sabdariffa (sorrel) drink commonly called zobo in Nigeria and known in many parts of the world with different names is a refreshing drink made from H. sabdariffa (sorrel) plant, obtained by extracting the calyx of the plant's flower usually with hot water with addition of sweeteners and flavourings.However, despite the increasing popularity, the greatest limitation for largescale production of the drink is its rapid rate of deterioration and its shelf life is approximately 24 h after production if not refrigerated (Onuoha and Fatokun, 2014;Izah et al., 2016).
There have been different researches on elongating the shelf life of H. sabdariffa drink with the use of chemical preservatives.However, much success has not been achieved and the growing concern about chemical preservatives has recently led to the search of natural antimicrobials (Arques et al., 2008).Now, a variety of spices are being researched, for their potential in elongating the shelf life of H. sabdariffa drink.
Among spices possessing antimicrobial property are  (Izah et al., 2016).Ginger obtained from the underground stems or rhizomes of Zingiber officinale (Rosc.), a herbaceous tropical perennial belonging to the family Zingiberaceae has been found to possess antimicrobial activity (Umaru et al., 2014), for instance in relation to Staphylococcus, Salmonella, Streptococcus, and E. coli (Aziz et al., 2015;Ofongo-Abule and Ohimain, 2015).Also, from previous studies, cloves (Syzygium aromaticum) a member of the Myrtaceae family has been found to have broad spectrum of activity against numerous bacteria and fungi (Casterton et al., 2005;Davidson, 2005).
Research is being intensified on the search for preservative method for H. sabdariffa drink and as a contribution towards this, it is imperative to molecularly characterize the organisms associated with H. sabdariffa drink spoilage and determine the effect of the different spices on these organisms.

Collection of samples
H. sabdariffa calyx (the dark red variety), granulated sugar, dried cloves and fresh ginger were obtained from the open market in Ilishan, Remo Ogun State, Nigeria.

Preparation of zobo samples
The drink was produced by soaking the H. sabdariffa calyx along with the different spices in hot water for 1 h (Ilondu and Iloh, 2007) after which it was filtered with already sterilized sieve.Spices added were finely grated ginger, ground cloves, and mixture of ginger and cloves.Three different concentrations (0.05, 0.25, and 0.45%) of each spice were added separately and percentage concentration of sugar added to the drink was 9% (Mohammed and Ismail, 2014).
The different samples were dispensed into previously sterilized plastic bottles and were pasteurized in a batch process at 72°C for 5 min (Ukwuru and Uzodinma, 2010;Braide et al., 2012).The samples were stored at ambient temperature of 28 ± 2°C, while bacterial and fungal counts were determined every 48 h for 16 days.

Isolation and enumeration of microorganisms
The total number of viable microorganisms (colonies) developed were enumerated and expressed as colony forming unit per millilitre (CFU/mL) by the methods of Sanders (2012) and they were isolated for molecular characterization.

Molecular characterization of the associated microorganisms
Pure cultures of each of the bacteria and fungi isolates were plated on nutrient agar and potato dextrose agar, respectively.Gram staining was done to identify Gram-positive and Gram-negative bacteria.The method described by Onasanya et al. (2003) was adopted for DNA extraction after which sequencing was done and the sequenced data was trimmed by removing the unknown data using CLC software.The cleaned data was copied into National Center for Biotechnology Information (NCBI) website and the microorganisms were identified by comparing their sequence with those in the sequence database using Basic Local Alignment Search Tool (BLAST).

RESULTS
Results of the total bacterial counts of the stored zobo samples are as shown in Table 1.The bacterial count of zobo without spice (the control) ranged from 0.5×10 1 to 1.3×10 3 cfu/mL from 2nd day of storage to the end of storage while the bacterial count of zobo containing 0.05% concentration of the different spices was between 1.1 ×10 1 and 6.2×10 2 cfu/mL from the 4th day of storage to the end of storage.There was no bacterial growth in zobo containing 0.25 and 0.45% concentrations of the spices.
Table 2 shows the results of total fungal counts of the stored samples.The fungal count for zobo without the spices (control) ranged from 0.7×10 1 to 0.6×10 3 cfu/mL from the 4th day of storage to the end of storage.While the fungal count of zobo samples containing 0.05% concentration of the spices was between 0.2×10 1 and 1.7×10 2 from the 4th day of storage to the end of storage, there was no fungal growth in zobo containing 0.25 and 0.45% concentrations of the spices.
Microorganisms isolated from the control whose growth was inhibited by the spices at 0.25 and 0.45% concentration are shown in Table 3.There were six bacteria and five fungi.

DISCUSSION
The anti-microbial properties of ginger and cloves used in zobo production were evaluated.Bacterial growth was observed in the control (zobo with only H. sabdariffa) on the 4th day (Table 1) and fungal growth on the 6th day (Table 2).This observation is supported by the reports of Egbere et al. (2007), Ukwuru and Uzodinma (2010), Braide et al. (2012) and Nwokocha et al. (2012) for pasteurized zobo.
Preservation by 0.05% concentration of the spices was not pronounced, however, it reduced microbial growth and mixture of cloves and ginger was more inhibitory than the individual spices (Carlsen et al., 2010).This finding is in agreement with the report of Ukwuru and Uzodinma (2010).Spice concentrations of 0.25 and 0.45% of cloves, ginger and mixture of cloves and ginger were able to preserve the zobo for the 16 days period of storage (Tables 1 and  2) as no growth was detected at these levels of spice concentration.This observation corroborates the report of Ogiehor et al. (2008) who found that 0.2% of ginger retarded the total viable bacteria and fungi count for 10 days.Ukwuru and Uzodinma (2010) also reported inhibitory effect of cloves and ginger on spoilage microorganism of zobo drink.

Bacteria Fungi Gram -ve, Stenotrophomonas rhizophila strain PCAˍˍ 13
Aspergillus niger strain SCSGAF0145 Gram -ve, Pseudomonas azotoformans strain +Y74 Ascomycota species strain dd08057 Gram +ve, Leucobacter aridicollis strain BIHB 353 Candida parapsilosis strain LEMI 7687 Gram +ve, Leucobacter komagatae strain 37A Fusarium solani strain CBS 121450 Gram -ve, Pantoea gaviniae strain A18/07 Penicillium chermesinum strain NRRL 2048 Gram +ve, Staphylococcus auricularis strain E WAX 1 Further in the study, microorganisms associated with the drink spoilage and inhibited by cloves and ginger were identified (Table 3).The organisms were isolated from the control through the 16 days period of storage.In agreement with the finding of this research, the following genera of microorganisms Staphylococcus, Leuconostoc, Aspergillus, Penicillium, and Fusarium have been reported to be associated with zobo beverage during storage (Akinyosoye and Akinyele, 2000;Ogiehor and Nwafor, 2004;Ezeigbo et al., 2015).Omemu et al. (2006) confirmed the presence of Aspergillus species in zobo and Penicillium species on the calyx, Pseudomonas and Staphylococcus species on calyx and retail zobo, and Candida species on calyx and stored zobo.Egbere et al. (2007) and Doughari et al. (2007) also reported the presence of Aspergillus niger in association with progressive spoilage of the drink.These reports and other findings reported the presence of other organisms that were not detected in the present findings.
Stenotrophomonas rhizophila, Pantoea gaviniae and Ascomycota species reported in this finding were not mentioned in previous reports and also there are some variations in the microorganisms reported by these different reports.This could be attributed to the method used for the identification.All these reports depended on biochemical method of identification which is based on characteristics observed in known and reference strains.This reference strain has predictable biochemical and physical properties under optimal growth conditions.However, these characteristics are not static and can change with time as a result of certain conditions like stress or evolution (Ochman et al., 2005).Effectiveness of cloves, ginger and mixture of cloves and ginger against the isolated microorganisms in the stored zobo is corroborated by earlier reports on the antimicrobial activities of cloves and ginger.Several studies demonstrated antifungal (Pawar and Thaker, 2006;Park et al., 2007) and antibacterial effects of clove (Fu et al., 2007;Agaoglu et al., 2007).
Ginger (Z.officinale) extracts were found to be effective against Gram-positive and Gram-negative pathogenic bacteria at 8 to 10% (v/v) (Pattaratanawadee et al., 2006;Okiki et al., 2015).Furthermore, synergistic effect observed with the mixture of cloves and ginger is being supported by earlier reports of Abdalla et al. (2007) and Rota et al. (2008).These findings confirm the antimicrobial properties of the spices used and their effectiveness against micro-organisms associated with spoilage of zobo.

Conclusion
Addition of ginger and cloves to zobo at a concentration of 0.25% and above using this method of production ensured storability at 28 ± 2°C for at least two weeks.

Table 1 .
Total bacteria count of zobo drink produced with 5% H. sabdariffa, cloves, ginger and mixture of cloves and ginger stored at ambient condition.

Table 2 .
Total fungi count of zobo drink produced with 5% H. sabdariffa, cloves, ginger and mixture of cloves and ginger stored at ambient condition.

Table 3 .
Microorganisms inhibited by the spices.