Identification of Food Spoilage Microorganisms in Different Species of Dacryodes edulis

INTRODUCTION

Dacryodes edulis is a fruit tree native to Africa which has several common names such as safou, plum, atanga, ube, African pear, bush pear, African plum, nsafu, bush butter tree, or butterfruit [1]. The fruit has an ellipsoidal shape which varies in length from 4 to 12 cm. The skin of the fruit is dark blue or violet, whereas the flesh is pale to light green. The tree flowers at the beginning of the rainy season and bears fruits for 2 to 5 months after flowering [2]. While the African pear tree is used by traditionalist as herbal medicines to treat several health disorders such as fever, wound, dysentery, sores and skin diseases, studies now reveal that the extracts of from the fruit contains antioxidizing, diuretic, antimicrobial, anti-inflammatory, anti-hypertensive, anti-sickle-cell and anti-spasmodic properties [3, 4]. These immense properties of African pear are as a result of the broad range of chemical compounds present in the fruit such as saponins, terpenes, tannins, flavonoids, alkaloids.

It is apparent that Dacryodes fruits are high in trace elements, especially in potassium, calcium, magnesium and sodium. Potassium is the principal cation in the intracellular fluids of D. edulis [5, 6]. Dacrydes edulis is rich in minerals such as calcium, sodium, potassium and magnesium [7]. By consuming approximately half a kilogram of this fruit, an individual gets the recommended daily intake for sodium [8]. The fruit of D. edulis contains other nutrients such as different types of fatty acids (palmitic, oleic, linoleic and stearic acids) [9]. It is also composed of vitamins such as ascorbic acids [8], niacin and pyridoxine that could meet the daily requirements [10].

Spoilage microorganisms are microbes that act on food products and cause their degradation thereby producing odors and unpleasant tastes and textures[11]. The contamination of fruits by microorganisms is a major cause of spoilage in fruits. Microbial spoilage in pear represents significant economic loss. Globally, microbial spoilage of food after harvest accounts for twenty five percent of food loss [12]. 

Different microorganisms can contaminate fruits at any stage of handling and processing from the environment, handlers, processing equipment and storage conditions. Contact between spoilt and non-spoilt fruits can also cause contamination by microorganisms. Using the concept of Koch’s postulates which can be applied to other fields to determine the relationship between plant disease and microbe, not all microorganisms have the spoilage effect on the fruit. This brought about the idea of specific spoilage microorganisms which account for a relatively small number in fresh products but grow much faster than other microorganisms [13]. The involvement of some microorganisms is not due to their spoilage potential but as a result of the condition of storage [14]. Change in the oxygen condition to anaerobic or aerobic conditions could enhance the spoilage of the food and also determine the types of microorganisms responsible for the spoilage [15].

The major focus of this study was to detect bacteria and fungi responsible for spoilage of Dacryodes edulis fruits. To achieve this, we utilized a range of morphological characteristics on agar coupled with biochemical features to identify microorganisms associated with D. edulis from the healthy state to the spoilt state. The variation in the number of microorganisms before and after spoilage were noted.

2. material and methods

2.2 Study Design

This study employed a cross-sectional study design.This study was carried out in the Department of Medical Microbiology of the Rivers State University, Nkpolu-Oroworukwu, Port Harcourt, Nigeria.

2.3Sample Collectionand Exposure Conditions

A total of 8 local pear samples (consisting of 4 different species) were purchased randomly at mile 3 markets in Port Harcourt of Rivers State in Nigeria. They were taken to the medical microbiology laboratory of the Department of Medical Laboratory Science in Rivers State University for bacteriological analysis.Prior to storage, swab samples of the fruits were collected using a moist sterile swab and cultured. The fruits were kept in separate air-tight containers according to the speciesat room temperature for 8 days after which there was physical deterioration in the fruit. After noticeable deterioration in the fruit, the samples were also collected using a sterile swab stick and for suspected fungi isolates, an excised portion was cultured.

2.4Sample Culture and Microbial Isolation

Ten (10) ml of normal saline was pipetted into sterile test tubes with each labelled A, B, C, and D. Sterile swab was used to collect samples from pear and mixed into the normal saline in the test tubes. From the sterile test tubes 1 ml of suspension was pipetted into the first sterile bottle labelled 1 (1:100 dilution), mixed properly and this step was repeated up until the last bottle labelled 3 (1:10000 dilution). From the last bottle 100 µl of the suspension was pipetted into MacConkey agar, nutrient agar and sabouraud agar each and spread.After 24 hours of incubation, the colonies on the nutrient agar plates were counted and recorded in colony forming units per millilitre (cfu/ml). Colonies of lactose fermenting organisms on MacConkey plates and sabouraud agar plates were also counted and recorded. Discrete colonies were picked with a sterilized wire loop and transferred to a fresh agar plate to obtain a pure culture of organisms that can be used for further analysis. The identification of microorganisms was based on their cultural characteristics and tests such as Gram staining, colonial morphology, cell morphology and biochemical tests. The biochemical test included coagulase, oxidase, citrate, catalase, indole and motility tests.

2.5 Data Analyses

Data were represented as charts and tables using GraphPad prism. Comparison between groups was done using students t-test. Results were considered significant at a95% confidence interval.

3. results

3.1 Bacteria Load on D. edulis before and after Spoilage

Figures 2A to D represent bacteria isolates from the four species of D. edulis. The bars in each graph show the level of the microorganisms isolated before and after spoilage. There was no significant difference (p=.9854)between the bacteria isolates (pre-spoilage:178.5±75.2, post-spoilage 177.5±72.9 x10⁴cfu/ml) spoilage for species A and no significant difference (p=.3730, p=.3472 and p=.3443) respectively for species B (pre-spoilage: 226.8±71.9, post-spoilage: 208.3±77.1 x10⁴cfu/ml), C (pre-spoilage: 127.5±65.0, post-spoilage: 189.3±102.2 x10⁴cfu/ml), and D (pre-spoilage: 186.0±93.6, post-spoilage: 239.3±39.5 x10⁴cfu/ml)3.3Load and Prevalence of Bacterial Species Isolated from Dacryodes edulis

Table 1 shows the bacterial load and prevalence of bacteria species isolated from different species of Dacryodes edulis before and after spoilage. There was no significant difference (p=.7081, p=.1982, p=.9401) between the bacterial loads before spoilage, Bacillusspp, Staphylococcus aureus spp, Klebsiella spp (99.8±48.7, 41.0±12.7, 58.3± 41.9 ×10⁴cfu/ml) with prevalence of 50, 21, 29% and post-spoilage (85.8±52.1, 71.0±18.4, 56.3±29.1 ×10⁴cfu/ml) with prevalence of 40, 33 and 27% in species A respectively. Micrococcus spp and E. coli were not isolated from species A. There was no significant difference (p=.6862, p=.8849, p=.8219) between the level of bacteria namely Bacillus spp, Staphylococcus aureus, Klebsiella spp before spoilage (104.8±36.6, 111.0±8.5, 66.5± 26.1 ×10⁴cfu/ml) with prevalence of 37, 39, 24% and after spoilage (93.3±43.3, 107.5±29.0, 61.3±36.2) with prevalence of (36, 41, 23%) spoilage in species B respectively. Micrococcus spp and E. coli were not also isolated from species B. There was no significant difference (p=.3196, p=.5671, p=.3208, p=.3211) inBacillus species, Staphylococcus spp, Klebsiella spp and Micrococcus sppbetweenbefore (68.0±17.6, 47.5±36.1, 32.5± 22.3, 8.5±9.2 ×10⁴cfu/ml) with prevalence of (44, 30, 21, 5%) and after (100.3±56.8, 66.5±16.3, 6.3±37.6, 0.0±0.0×10⁴cfu/ml) with prevalence of (45, 30, 25, 0%) spoilage in species C respectively. E. coli was not isolated from species C. There was no significant difference (p=.8298, p=.9800) in the level ofBacillusspp and E. colibetween the pre-spoilage stage (114.5±92.6, 221.0±108.9×10⁴cfu/ml) with prevalence of (31, 59%) and thepost-spoilage stage (130.5±0.7, 223.5±61.5×10⁴cfu/ml) with prevalence of (27 and 47%) spoilage in species D respectively. Only Klebsiella spp was significantly lower (p=.0033) at pre-spoilage stage (36.5±4.9×10⁴cfu/ml) with prevalence of 10%compared to post-spoilage (122.5±4.9×10⁴cfu/ml) with prevalence of (26%) spoilage in species D. Staphylococcus spp and E. coliwere not isolated in species D.

3.4Level and Prevalence of Fungal Species Isolated from Dacryodes edulis

Table 2shows the specific fungal load and prevalence of fungal species isolated from different species of Dacryodes edulis before and after spoilage. There was no significant difference (p=.2580, p=.4226 p=.4226) between Yeast species, Fusarium spp, Aspergillus spp before spoilage (13.0±5.7, 1.50±2.1, 0.5±0.7×10³cfu/ml) with prevalence of 87, 10 and 3% respectively and after spoilage (6.0±2.8, 0.0±0.0, 0.0±0.0×10³cfu/ml) with prevalence of 40, 33 and 27%respectively in species A. Rhizopus spp was not isolated from species A. There was no significant difference (p=.0695, p=.4226) between Yeastspp,Aspergillus sppbefore (20.0±2.8, 0.0±0.0×10³cfu/ml) spoilage with prevalence of (100, 0%) and after (8.5±3.5, 1.5±2.1×10³cfu/ml) spoilage with prevalence of (85 and 15%) in species B respectively. Fusarium sppandAspergillus spp were also not isolated from species B. There was no significant difference (p=.1835, p=.6985) between yeast species and Rhizopusspp before spoilage (18.0±5.7, 1.0±1.4 ×10⁴cfu/ml) with prevalence of (95 and 5%) and after spoilage(8.0±4.2, 0.5±7.1 ×10⁴cfu/ml) with prevalence of (94 and 6%) in species C respectively.Fusarium spp and Aspergillus spp were not isolated from species C. There was no significant difference (p=.1243) between Yeast species before spoilage (30.5±13.4 ×10⁴cfu/ml) withprevalence of (100%) and after spoilage (6.0±1.4 ×10³cfu/ml) with prevalence of (100%) spoilage in species D.

4. discussion

Characterization of microbes responsible for fruit and vegetable spoilage at the species and strain level needs crucial consideration and has recently received considerable attention. This approach could potentially be essential in understanding meat spoilage, as different strains of the same species may exert various spoilage activities or be affected by different storage conditions [16]. Unfortunately, limited informationis available onthe microbes responsible for the spoilage of the fruit.

The results in Figure 1 show that there was no significant association(p>0.05) between the pre-spoilage and post-spoilage African pear for the four different species (A, B, C, and D). This is not consistent with thestudies [17, 18] that cultured species of D. edulis before and after spoilage and observed a (p<0.05) significant difference between the bacteria isolates before and after spoilage. The reason for these differences may be that the time allowed for spoilageto occur in our study was far shorter than that of Onuoha et al. [17] and Akusuet al. [18]. It may also be due to the fact that they worked on a larger sample size as opposed to this study.

Again, the results in Figures 2A, C and Dshow no significant relationship (p>0.05) between the before-spoilage and after-spoilage of African pear despite having higher levels of fungi colonization prior to spoilage. This is also not in line with otherstudies [17, 18]. They cultured species of D. edulis before and after spoilage and observed that significant difference (p<0.005) between the fungi isolates before and after spoilage. The only case with a significantly high level of fungi colonization prior to spoilage was in specimen B. Reasons for these discrepancies may be because the time kept for spoilage in this study was far shorter than that of Onuoha et al. [17] and Akusuet al. [18]. It may also be due to the fact that they worked on a larger sample size as opposed to this study. Another key factor could also be due to the species’ variation as all specimens represented different species.

In the distribution and prevalence of the isolated bacteria, there was no significant difference between the before and after spoilage of Bacillus spp, Staphylococcus spp,andKlebsiella sppfor species A of D. edulis. The highest prevalence (50 and 40% respectively) for both the before and after spoilage group was seen in Bacillus spp Micrococcus sppand E. coli were not isolated from species A.No significant difference was seen between Bacillusspp, Staphylococcusspp, Klebsiellaspp before spoilage and after spoilage in species B. The highest prevalence was seen in Staphylococcus aureus,Micrococcus spp and E coli were not isolated from species B.There was no significant difference between Bacillus species, Staphylococcusspp, Klebsiellaspp and Micrococcusspp before spoilage in species C respectively. The highest prevalence in species C is Bacillusspp. E. coli was not isolated from species C. There was no significant difference between bacillus species and E. coli before and after spoilage in species D respectively. The most prevalent organism is E. coli. E. coli and Klebsiella sppwere significant before and after. Staphylococcusspp and E. coli was not isolated in species D. Available literature on the different species of D. edulis is very little and carries very little information about isolated bacteria on different species of pear. However, it is likely that these results from this study where Bacillusspp and Staphylococcusspp are higher than theother organisms may be linked to where this pear was bought (marketplace), this will likely have been acquired there and continued to proliferate increasingly after spoilage. The reason why microorganisms were non-significant to each other may be as a result of far shorter spoilage time and also small sample size.

For the level and prevalence of isolated fungi, there was no significant difference between Yeastspp, Fusarium spp and Aspergillus spp before spoilage and after spoilage in species A respectively. The organism with the highest before and after spoilage was Yeastspp. There was no significant difference between Yeast spp, Aspergillus spp before spoilage and after spoilage in species B respectively. Fusarium sppand Aspergillus spp were also not isolated from species B. There was no significant difference in Yeast species and Rhizopus spp before and after spoilage in species C respectively. The organism with the highest prevalence was the Yeast. Fusarium sppand Aspergillus sppwere not isolated from species C. There was no significant difference between Yeast spp before and after spoilage in species D. Available literature on the different species of D. edulis is very little and carries very little information about isolated bacteria on different species of pear. However, these results obtained from this study where Yeastspp is higher than other organisms may be linked to where this species of pear were bought (marketplace), this will likely have been acquired there and continued to proliferate increasingly after spoilage. The reason why fungi were non-significant to each other may be due to a far shorter spoilage time and the small sample size used.

For improvement of the study, the use of different temperature conditions could be explored to see if variation in temperatures could also affect the presence of other kinds of microorganisms. An earlier study suggests that the presence of mesophilic bacteria significantly decreased after exposure to temperatures below -19^oC[19]. Again, the length of exposure could also have influenced the presence of microbes. For instance, fungi which is well known to cause fruit spoilage grow more slowly than bacteria. Hence, prolonged storage could have enabled the growth of other types of fungi besides those isolate[20].

5. Conclusion

Different species of bacteria and fungi were observed to grow on these species of Dacryodes edulis even. Colonies of Bacillus spp, Staphylococcus spp, Aspergillus spp, Yeast spp and Rhizopus spp are likely to be seen in spoilt African pear when cultured. Significantly higher levels of Klebsiellaspp were noted after spoilage. Only species B of D. edulis demonstrated significantly lower levels of fungi isolates after spoilage. This study shows these bacteria could contribute to the deterioration of pears.

AcknowledgEments

The authors wish to appreciate the Department of Medical Microbiology, Rivers State University for permission to use its facility to perform the study.

Competing interests

 Authors have declared that no competing interests exist.

Authors’ Contributions

This work was carried out in collaboration among all authors. Authors MTP, KBR and GLK designed the study, Author MTP and GLK performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. Authors KBR managed the analysis of the study and author GLK managed the literature searches. All authors read and approved of the final manuscript.

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