Carbohydrase-rich fungal supernatants as feed additives: exploring thermal stability and enzyme cocktail applications

MAJUL, LEONARDO M.; LEVIN, LAURA N.; CAMPOS, ELEONORA; WIRTH, SONIA ALEJANDRA; OMARINI, ALEJANDRA

Rosario

Workshop; V Encuentro y II workshop de la Red Argentina de Tecnología Enzimática; 2023

Red Argentina de Tecnología Enzimática

Animal production faces various challenges to achieve sustainability, including animal welfare associated with proper nutrition. Limitations of resources and environmental conservation demand new strategies to optimize the energy conversion of feed. In particular, the digestibility of feed can be improved by removing antinutritional factors present in plant-based ingredients (such as non-starch polysaccharides, NSPs) using enzymatic cocktails, mainly with xylanase, and beta-glucanase activities. In this study we aimed to produce in-house enzymatic cocktails rich in these activities and complementary galactosidases arabinofuranosidases and polymethylgalacturonases, to enhance the digestibility of plant feed ingredients such as maize-soybean. Based on previous results, three fungi were evaluated: Pycnoporus sanguineus BAFC 2126, Pleurotus sapidus DSMZ 8266, and Trametes trogii BAFC 463. Cultures were performed in 100 ml of liquid medium containing rice bran (RB), rice husk (RH), or wheat bran (WB) as carbon sources, supplemented with peptone, yeast extract, and micronutrients for 21 days at 28 ºC with agitation in 250 ml flasks. Samples of the culture supernatants were taken at 7, 14, and 21 days of incubation and stored at -20 ºC. The endoglucanase (EG), endoxylanase (EX), and polygalacturonase (PMG) activities were spectrophotometrically determined by incubating the supernatants with their respective substrates: carboxymethylcellulose 1%, xylan 1%, or apple pectin 0.1% and quantifying the enzymatic activity by the release of reducing sugars using the Somogyi-Nelson method. Additional activities such as arabinofuranosidase (ABF) and α-galactosidase (GAL) were quantified following the conversion of the substrates 4-Nitrophenyl-α-L-arabinofuranoside and 4-Nitrophenyl α-D-galactopyranoside to 4-nitrophenol at 430 nm. The highest titers of EX (0.9 U/ml) and EG (0.4 U/ml) were produced by T. trogii grown in RH, while P. sanguineus produced the highest PMG activity (3.06 U/ml) when grown on RB and the highest ABF (0.5 U/ml) and GAL (0.3 U/ml) activities by growth in WB, both at 14 days of culture. P. sapidus enzymatic titers were lower than the ones of T. trogii and P. sanguineus, except for EG activities produced in WB and RH that were similar to those produced by T. trogii in the same media. Therefore, we selected the supernatants of P. sanguineus in WB and T. trogii in RH, for further characterization. The optimal temperature and pH were determined as well as the thermal stability in a range between 40°C to 60 ºC. In both cases, the optimum temperature was found between 40°C and 50 ºC, and the optimum pH was between 4 and 5 for all activities. The enzymes from P. sanguineus showed high thermal stability, with a T1/2 > 2h at 60°C. On the other hand, the EG and EX of T. trogii showed a T1/2 of 10 min at 50°C, and a T1/2 > 2 h at 40°C, while for PMG the T1/2 was >2h at both temperatures. To further understand the enzymes involved in the observed activities, we will identify the most abundant proteins by mass spectrometry. Also, the activity of the enzymatic cocktails on feed components will be evaluated.