THE INFLUENCE OF INACTIVATED ASPERGILLUS NIGER AND PENICILLIUM SP. INDUCTION ON HYDROLYTIC ENZYME ACTIVITY AND ANTIFUNGAL ABILITY OF WICKERHAMOMYCES ANOMALUS

• Main Author: Altof, Raihana
• Format: Theses
• Language: Indonesia
• Subjects: Teknologi
• Online Access: https://digilib.itb.ac.id/gdl/view/87173
• Institution: Institut Teknologi Bandung

Mold, especially from the Aspergillus and Penicillium groups, have caused huge losses to the postharvest products as it could cause physical damage, rot, and mycotoxin contamination. Although synthetic fungicides are effective in inhibiting the growth of mushrooms, their use risk causing pathogen resistance, environmental pollution, and adverse effects on human health. Therefore, biocontrol agents such as Wickerhamomyces anomalus yeast were developed because of it’s antagonistic properties to fungal growth. It can inhibit mold through various mechanisms, one of which is the production of hydrolytic enzymes that degrade fungal cell wall. However, the effectiveness of this biocontrol agent is still not equivalent to that of a synthetic fungicide, since it has not reached more than 95% effectivity. The biomass of inactive mold was known to induce antagonist yeast to increase the production of fungal cell wall degrading enzymes. Therefore, this study aimed to test the influence of inactivated Aspergillus niger and Penicillium sp. induction on the extracellular chitinase, cellulase, and protease enzymes activity and the biocontrol ability of W. anomalus. Inactive fungal biomass was obtained by growing each mold in the Potato Dextrose Broth (PDB) for 5 days with 60 rpm agitation at room temperature. After that, the biomass was washed, centrifuged, freeze dried for 24 hours, grinded, and stored at -20 °C. Inactive fungal biomass were added to the PDB with concentration variations 0.2%, 0.5%, 1%, and 1.5% w/v, then sterilized at 121 °C for 15 minutes. W. anomalus was cultivated in induction media, and noninduction media was used as a control. Extracellular chitinase, cellulase, and protease activity was measured at 0, 2, 8, 14, and 24 hours of incubation using colorimetric methods with dinitrosalicylic acid reagent for chitinase and cellulase activity, as well as Folin Ciocalteu reagent for protease. The absorbance was converted into enzyme activity (unit/mL) based on the standard curve. Extracellular protein profiles were visualized through SDS-PAGE with a 12% (w/v) separating gel. The antifungal capability of W. anomalus supernatant to A. niger and Penicillium sp. spores germination were tested. We used variations of supernatant volume (20–100 ?L) against the spores suspension (104 spores/mL). Then it were incubated for 24 hours. Statistical analysis was performed using ANOVA two-way and Tukey tests for normal distributed data, or Kruskal-Wallis and Dunn tests for not normal distributed data.. The results showed that the induction of inactivated A. niger and Penicillium sp. increased extracellular cellulase activity at the 2nd and 8th hours of incubation. Based on the ANOVA one-way and Tukey test (p <0.05), variation of A. niger did not give a significant difference in the activity of extracellular cellulase, whereas variation of Penicillium sp. showed significant differences at concentrations of 1% and 1.5% (w/v) compared with 0.5% (w/v). The highest extracellular cellulase activity was obtained at the induction of 1.5% (w/v) A. niger (2.47 U/mL) and 1% (w/v) Penicillium sp. (2.25 U/ml). However, inactivated A. niger and Penicillium sp. induction showed no pattern to extracellular chitinase and protease activity with the changes of induction concentration and incubation time. SDS-PAGE results showed that induction of 1% and 1.5% A. niger (w/v) as well as Penicillium sp. 1% and 1.5% (w/v) produce protein bands with molecular mass ranging from 140 kDa suspected as ?-glucanase, 100–130 kDa suspected as chitinase subgroup C, 60–70 kDa suspected as endo ?-1,4-glucanase, 45–60 kDa suspected as beta-1,3-glucanase and aspartate acid protease, and 25–45 kD suspected as chitinase subgroup B. Antifungal tests showed that W. anomalus, which was induced by inactivated A. niger, decreased germination of A. niger spores up to 7% and Penicillium sp. spores up to 18% compared to non-induced yeast, whereas induction of inactivated Penicillium sp. decreased germination of Penicillium sp. spores up to 8% compared to non-induced yeast. This research showed that induction of inactivated A. niger and Penicillium sp. slightly increased the extracellular hydrolytic enzyme activity of W. anomalus. Different protein profiles were found between induced and non-induced yeast. Induction of inactivated A. niger in yeast supernatant slightly decreased the germination of A. niger and Penicillium sp. spores compared with non-induced yeast. Meanwhile induction of inactivated Penicillium sp. only slightly decreased the germination of Penicillium sp. spores compared with non-induced yeast. This result suggested the potential of inavtivated mold induction toward the antifungal ability of antagonist yeast. Further study was needed to understand molecular mechanisms and improve biocontrol agent effectiveness more efficiently.