Application of bacteriocins in the preservation of fruit juice

Bacteriocins (BCNs) are ribosomally synthesized polypeptides or proteins with antimicrobial activity, produced by different groups of bacteria. Many lactic acid bacteria (LAB) produce BCNs with broad spectra of inhibition. The antimicrobial activity of BCNs against spoilage organisms (SPOs) has raised considerable interest in their application in juice preservation. The objectives of the study were to: (i) isolate, identify and screen BCN producing bacteria for antimicrobial activity against spoilage bacteria and fungi, (ii) optimize production of BCN from selected producers and (iii) investigate the industrial application of the BCN as a preservative in fruit juice. Eleven LAB strains of BCN producers were screened for antimicrobial activity. BCNs from Lactobacillus plantarum and Pediococcus pentosaceus 34 were the most effective against juice spoilage bacteria and fungi. The effect of medium components on bacteriocin production in L. plantarum and P. pentosaceus 34 was also determined. Clementine:Valencia (1:1) juice was used for the first time as the growth medium for L. plantarum and P. pentosaceus 34. The BCN from L. plantarum showed the highest activity and was, therefore, chosen for juice fermentation studies. The identification of L. plantarum was confirmed by biochemical tests, polymerase chain reaction (PCR) and sequencing of the recA gene. The highest BCN activity was observed for L. plantarum grown in De Man-Rogosa-Sharpe (MRS) and a combination of all supplements (i.e. peptone, MnSO4.H2O, Tween 80, glucose and whey), followed by MRS and Tween 80, peptone, MnSO4.H2O and MRS alone. MRS was a better medium for BCN production than juice [Clementine:Valencia (1:1)]. Size exclusion chromatography (SEC) was used to isolate the active L. plantarum BCN fraction which corresponded to an approximate molecular weight of 3.2 kDa and was proteinaceous in nature. Plantaricin structural genes (plnEF, plnJ, plnK, plnN) were detected in the L. plantarum strain by PCR and sequenced, and were chromosomally encoded as no plasmids could be detected. This implies that the BCN from L. plantarum is most likely a type of class IIa plantaricin which is responsible for the broad inhibitory activity observed. For the industrial application studies, L. plantarum BCN-containing cell free supernatant (BCNsup) added to “Ready to Drink” (RTD) Clementine:Valencia (1:1) juice at concentrations of 3 600 - 500 000 ppm decreased growth of SPOs, Lactobacillus acidophilus and Streptococcus thermophilus. At 250 000 ppm, the L. plantarum BCNsup achieved 5.3 and 6.8 log reductions of the L. acidophilus, after 24 and 48 h, respectively, which is larger than the USFDA (2001) requirement of a 5 log reduction in SPO activity, for preservation of fruit juices. However, there was a decrease in the activity when the BCNsup was applied to industrial (Valor) RTD juice (mango-orange) at decreasing concentrations of 100 000, 50 000 and 25 000 ppm. Organoleptic tests showed that the BCN did not alter flavor or taste of the juice and did not cause toxicity or allergic reactions. A food safety risk assessment was conducted in order to determine the Critical Control Point(s) [CCP(s)] at which the BCN could be applied to control identified microbiological hazards, and a Hazard Analysis and Critical Control Point (HACCP) plan was developed. This is the first report on the optimisation of L. plantarum BCN production in juice [Clementine:Valencia (1:1)], followed by inoculation into RTD juice (mango-orange), including a HACCP plan for the application of the BCN as a preservative in juic