Identification of cheese rancidity-related lipases in Aspergillus oryzae AHU 7139.

The adjunct product with enzymatic activity from Aspergillus oryzae is beneficial for flavor enrichment in the ripened cheese. However, an excessive lipolytic reaction leads to the release of volatile free fatty acids. Accordingly, a strong off-flavor (i.e., rancidity) has been detected when A. oryzae AHU 7139 is used. To identify the rancidity-related lipase from this strain, we evaluated the substrate specificity and lipase distribution using five mutants cultured on a whey-based solid medium under different initial pH conditions. The results showed a higher diacylglycerol lipase activity than triacylglycerol lipase activity. Moreover, an initial pH of 6.5 for the culture resulted in higher lipolytic activity than a pH of 4.0, and most of the activity was found in the extracellular fraction. Based on the gene expression analysis by real-time polymerase chain reaction and location and substrate specificity, five genes (No. 1, No. 19, mdlB, tglA, and cutL) were selected among 25 annotated lipase genes to identify the respective knockout strains. Because ΔtglA and ΔmdlB showed an outstanding involvement in the release of free fatty acids, these strains were applied to in vitro cheese curd experiments. In conclusion, we posit that triacylglycerol lipase (TglA) plays a key role as the trigger of rancidity and the resulting diglycerides have to be exposed to diacylglycerol lipase (MdlB) to stimulate rancidity in cheese made with A. oryzae AHU 7139. This finding could help screen suitable A.oryzae strains as cheese adjuncts to prevent the generation of the rancid-off flavor.

A study of lipolysis induced by adjuncts from edible Aspergillus sp. solid culture products on ripened semi-hard cheese.

BACKGROUND: Aspergillus sp. has been used in traditional Japanese fermented foods. Protease-containing culture products of A. oryzae have been applied as the adjunct enzyme source to enrich the flavor in ripened cheese. Although proteolysis was stimulated, the increase of free fatty acids (FFA) was recognized in some products. Since an excess amount of FFA accumulation can cause rancidity in cheese products, the assessment of lipase activity was considered to be essential for the cheese adjunct preparation. RESULTS: Although an equal lipase activity from the adjunct materials of A. kawachii NBRC 4308, A. luchuensis RIB 2604 and A. oryzae AHU 7139 was applied to semi-hard cheese, the FFA level was significantly higher in A. oryzae cheese than in the others. Furthermore, the profiles of volatile components were different in experimental cheeses. An in vitro study with experimental curds demonstrated that the high FFA might not depend on the lipase retainability on curds. On the contrary, the pronounced activation of the lipases occurred in A. oryzae after incubation with the curds. Moreover, incubation of the insoluble lipase that had been attached to the cells with skim milk curd extracts allowed the release of lipases from the cells into the medium with remarkable activation. CONCLUSION: A. oryzae AHU 7139 possessed a complex lipolytic system comprising extracellular and cell-binding lipases that were attributed to the increase in FFA in A. oryzae cheese. © 2022 Society of Chemical Industry.

Adjunctive application of solid-state culture products and its freeze-dried powder from Aspergillus sojae for semi-hard cheese.

BACKGROUND: Species belonging to the genus Aspergillus have been used in traditional Japanese fermented foods. Aspergillus sojae is a species responsible for strong proteolytic activity. Freeze-drying treatments followed by physical disruption enables the pulverization of the mycelia of A. sojae RIB 1045 grown in whey protein-base solid media. Intracellular proteases were extracted using this protocol to compare extracellular protease activity in terms of the reaction's pH dependence in the presence or absence of inhibitors. RESULT: With different sensitivities to inhibitors, intracellular and extracellular proteases showed the strongest activity under acidic conditions, which were considered suitable for cheese application. The raw culture product (CP) and its freeze-dried product (FDP) were mixed with cheese curds, prepared according to Gouda-type cheese-making methods, and were allowed to ripen for 3 months. Chemical analysis of the products showed 13.3% water-soluble nitrogen (WSN) in the control, which had received noncultured media, whereas 20.0% and 21.1% WSN was found in the CP and FDP experimental cheeses, respectively. Although these adjuncts significantly increased WSN, an insignificant difference was found between CP and FDP. Free fatty acids in all experimental cheeses were similar, showing that CP and FDP caused no rancid defects. CONCLUSION: The introduction of freeze-drying treatments accompanied by cell disruption resulted in a negligible effect in terms of WSN. However, the application of A. sojae can be beneficial when it comes to increasing the level of WSN compared with A. oryzae, as shown in our previous study. © 2020 Society of Chemical Industry.