Total fungal count

7. TREATMENT COMPARISON OF KUMQUAT FRUIT USING THE

7.6 Results and Discussion

7.6.6 Microbiological

7.6.6.3 Total fungal count

these studies appear to be more than the reductions reported in Tables 7.13 and 7.14. This can be expected as the treatments and the type of citrus fruit differ. The author was unable to find published literature pertaining to the microbial load on kumquat fruit.

Based on the results for the CC, it can be deduced that P. digitatum-inoculated kumquat fruit treated with anolyte water × 30s + HWT × 30s × 53°C + B13 and P. italicum- inoculated kumquat fruit treated with anolyte water × 30s + HWT × 30s × 53°C + B13 carried the lowest CC immediately after treatment on Day 0A and the end of the 21-day storage period.

Table 7.15 Population dynamics of the total fungal count (log CFU.g^-1) on the surface of Penicillium digitatum-inoculated kumquat fruit over a 21-day storage period subjected to different integrated pre-packaging treatments

Treatment Storage Period (Days)

0B 0A 7 14 21

Anolyte water × 30s + HWT × 20s × 53°C + B13 (1) 6.1^bcd 5.9^ab 6.3^de 6.5^ef 6.5^ef Anolyte water × 30s + HWT × 30s × 53°C + B13 (2) 6.1^bcd 5.8^a 6.4^def 6.6^fg 6.6^fg Anolyte water × 30s + HWT × 20s × 60°C + B13 (3) 6.1^bcd 6.0^abc 6.2^cd 6.4^def 6.5^ef Anolyte water × 30s + HWT × 30s × 60°C + B13 (4) 6.1^bcd 6.1^bcd 6.4^def 6.4^def 6.5^ef

TW × 30s (5) 6.1^bcd 6.1^bcd 6.5^ef 6.7^gh 6.8^hi

No treatment (6) 6.1^bcd 6.3^de 6.6^fg 6.8^hi 6.9ⁱ

Significance

Treatment (A) *

Storage Period (B) *

AB *

CV (%) 2.5

NS, *, ** Non-significant or significant at P≤0.05 or P≤0.001, respectively. Means within a column followed by the same letter(s) are not significantly different from each other according to Duncan’s Multiple Range Test (P≤0.05), (n=3). CV, Coefficient of variation; 0B, Day 0 before treatment application; 0A, Day 0 after treatment application; HWT, hot water treatment; TW, tap water; s, dipping time in seconds; +, ‘combined with’. Each treatment was allocated a treatment number within brackets

Table 7.15 presents the changes in the total fungal count (FC) of P. italicum-inoculated kumquat fruit. This count also includes the yeast present on the fruit surface. The treatment, storage period and the interaction of both these factors were found to have a significant (P≤0.05) influence on the FC. Treatment 4 reduced the FC from 5.9 log CFU.g^-

1 on Day 0B to 5.7 log CFU.g^-1 on Day 0A. However, on Day 21 Treatment 2 produced fruit with the lowest FC of 6.6 log CFU.g^-1. No significant difference between Treatments 3 and 4 were noted. Sporulation and fungal formation were observed only on the control treatment by Day 14.

Table 7.16 Population dynamics of the total fungal count (log CFU.g^-1) on the surface of Penicillium italicum-inoculated kumquat fruit over a 21-day storage period subjected to different integrated pre-packaging treatments

Treatment Storage Period (Days)

0B 0A 7 14 21

Anolyte water × 30s + HWT × 20s × 53°C + B13 (1) 5.9^c 5.7^ab 5.9^c 6.6^fg 6.8^gh Anolyte water × 30s + HWT × 30s × 53°C + B13 (2) 5.9^c 5.7^ab 5.9^c 6.5^f 6.6^fg Anolyte water × 30s + HWT × 20s × 60°C + B13 (3) 5.9^c 5.7^ab 5.7^ab 6.6^fg 6.7^g Anolyte water × 30s + HWT × 30s × 60°C + B13 (4) 5.9^c 5.6^a 5.6^a 6.6^fg 6.7^g

TW × 30s (5) 5.9^c 5.8^bc 6.0^cd 6.7^g 6.9^hi

No treatment (6) 5.9^c 6.0^cd 6.1^de 6.8^gh 7.0ⁱ

Significance

Treatment (A) *

Storage Period (B) *

AB *

CV (%) 4.5

NS, *, ** Non-significant or significant at P≤0.05 or P≤0.001, respectively. Means within a column followed by the same letter(s) are not significantly different from each other according to Duncan’s Multiple Range Test (P≤0.05), (n=3). CV, Coefficient of variation; 0B, Day 0 before treatment application; 0A, Day 0 after treatment application; HWT, hot water treatment; TW, tap water; s, dipping time in seconds; +, ‘combined with’. Each treatment was allocated a treatment number within brackets

Due to the manner in which the kumquat is consumed, it is of special concern to determine the microbial load on the fruit surface as consumption of contaminated fruit can pose a food safety threat and hazard to human health (Gultie and Sahile, 2013). Enumeration of aerobic microorganisms is a common indicator of the quality and probable shelf life of raw fresh foods (Stannard, 1997). However, yeast or mould counts are more relevant indicators for commodities of low pH.

In addition to the beneficial effect of heat treatments on kumquat fruit, anolyte water may further improve the microbial quality of fruit (Palma et al., 2013; Kassim et al., 2016).

Chapters 4 and 5 clearly indicated that anolyte water possesses sufficient free chlorine to reduce fungal growth. The active compound of anolyte water is the hypchlorous acid (HOCL) (Acher et al., 1997; Whangchai et al., 2010). The HOCL is capable of oxidising microbial cell nucleic acid and proteins, thereby lethally damaging the cells. Kim et al.

(2000) and Riondet et al. (2000) suggested that the oxidation-reduction potential (ORP) was also a contributor toward microbial inactivation due to changes in the metabolic fluxes and ATP production. The addition of a third treatment of a biocontrol agent

supplemented the germicidal action of the anolyte water and hot water in a preventative manner. The inability of the biocontrol agent to provide a curative treatment is due to this antagonist being unable to penetrate the fruit tissue to the location of the pathogen (Abraham et al., 2010). Therefore, each treatment has a specific mode of action as a disinfectant, curative and preventative.

Many fruits are capable of withstanding exposure to hot water temperatures of 50–60°C for up to 10 min (Lurie, 1998). However, the exposure times and associated temperatures required to kill those bacterial cells, which are commonly associated with foods, are in excess of 10 minutes and 50–60°C, respectively. E. coli requires a treatment time of 20- 30 minutes at 57.3°C and Streptococcus thermophiles requires an exposure time and temperature of 70-75°C (Thakur et al., 2000). Hsu and Beuchat (2012) stated that bacteria do not play as an important role in the spoilage of fruit as moulds and yeasts, which are capable of inducing appreciable spoilage of fruit. This is due to the inherent acidity associated with many fruit as well as the presence of bactericidal substances that are able to destroy certain kinds of bacteria. However, once ingested, bacteria can have detrimental effects on human and animal health (Leff and Fierer, 2013). Therefore, it is of importance to determine the effects of the combined treatments identified in this study on the total aerobic and coliform counts in addition to the more dominant P. digitatum and P. italicum fungal pathogens affecting citrus fruit.

According to Tables 7.11 to 7.16, there was not a drastic reduction in the APC, CC and P. digitatum and P. italicum counts between days 0B and 0A. This could be attributed to the spore cells remaining dormant on the fruit surface due to the applied treatments.

However, once the slurries, which were obtained from the fruit surface (Section 7.4.6), were plated onto the nutrient-rich media, the spores began to sporulate giving rise to large microbial population numbers. However, a reduction in the population for APC, CC and P. digitatum and P. italicum was observed between Day 0B and 0A, alluding to the effectiveness of the combined treatments as anolyte water only and hot water only are not sufficient to eradicate these microorganisms.

Based on the results, it can be deduced that Treatment 2, anolyte water × 30s + HWT × 30s × 53°C + B13, resulted in the least FC present on the fruit surface on Day 0A for P.

digitatum-inoculated kumquat fruit. Treatments 1, 3 and 4 led to the lowest FC at the end

of the 21-day storage period. For P. italicum-inoculated kumquat fruit anolyte water × 30s + HWT × 30s × 53°C + B13 developed the least FC immediately after treatment on Day 0A and the end of the 21-day storage period.