Phospholipase

A high-throughput serum NMR metabolomics platform was used to quantify 76 circulating lipid and metabolite measures in the three cohorts.²⁸ (link) This metabolomics platform provides simultaneous quantification of routine lipids, lipid concentrations of 14 lipoprotein subclasses and major subfractions, and further abundant fatty acids, amino acids, ketone bodies and gluconeogenesis-related metabolites in absolute concentration units (Supplementary Table S1, available as Supplementary data at IJE online). The platform has been applied extensively in epidemiological studies;^18–22 (link)^,24 (link)^,28 (link)^,29 (link) details of the experimentation and the analytical repeatability of the biomarker quantification have been described elsewhere.²⁸ (link)^,30 (link)^,31 (link) We also analysed 10 additional protein and hormonal biomarkers measured in at least two cohorts. These were high-sensitivity C-reactive protein (CRP), phospholipase activity, gamma glutamyl transferase (GGT), alanine aminotransferase (ALT), testosterone, sex-hormone binding globulin (SHBG), adiponectin, leptin, vitamin D and insulin;²⁴ (link) they were measured using conventional clinical chemistry and mass spectrometry as described in Supplementary Methods, available as Supplementary data at IJE online. Inclusion of these additional measures was selected a priori because suspected association with alcohol consumption and cardiovascular risk.

The virulence phenotypes were assessed by performing enzymatic tests for the expression of some soluble virulence factors. Overnight culture of the strains was evaluated for the following virulence factors expression: haemolysins, other pore forming toxins (lecithinase, lipase), proteases (caseinase, gelatinase), amylase and aesculin hydrolysis. Detection of haemolysin production was performed by spotting the fresh cultures on 5 % sheep blood agar medium and incubation at 37 °C for 24 h. The colorless area around the culture revealed the presence of haemolysis activity. For lipase production the strains were spotted on 1 % Tween 80 agar as a substrate and followed by incubation at 37 °C for 24 h. An opaque (precipitation) zone around the spot was registered as positive reaction; for lecithinase production, the cultures were spotted into 2.5 % yolk agar and incubated at 37 °C for 24 h. A clear zone around the spot indicated the lecithinase production. The protease activity (caseinase and gelatinase) was determined using 15 % soluble casein agar, respectively 3 % gelatin as substrate. The strains were spotted and after incubation at 37 °C for 24 h, a white precipitate surrounding the growth indicated casein proteolysis, and colorless area around culture due to the gelatin hydrolysis, indicated the positive reaction for gelatinase. Amylase was detected using agar with 1 % starch and hydrolysis was revealed after adding Lugol's solution (yellow ring around the culture, while the rest of the plate will be blue). For the aesculin hydrolysis the medium containing Fe ³⁺ citrate was used and inoculated by spotting, then incubated for 24 h at 37 °C. A black precipitate around culture due to esculetol released under the action of beta-galactosidase was considered positive reaction.
Evaluation of biofilm development on inert substrata was assessed by the microtiter method. Overnight bacterial cultures were grown in 96 multi-well plates containing Tryptic Soy Broth (TSB) for 24 h at 37 °C, then the plate was emptied and washed three times with phosphate buffered saline (PBS). The adherent cells were then fixed with cold methanol, stained with an alkaline 1 % violet crystal solution for 15 min, washed with water and resuspended in a 33 % acetic acid solution. The intensity of the suspension was spectrophotometrically assessed, the amount of adhered biomass being proportional to the absorbance value read at 492 nm.

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme Phospholipase from a genetically modified strain of A. oryzae strain NZYM‐PP.
Additional information was requested from the applicant during the assessment process on 13 July 2021 and 16 November 2021 and was consequently provided (see ‘Documentation provided to EFSA’).

For the detection of B. cereus toxin-encoding genes (Smase, sph; enterotoxin Bcet, bcet; enterotoxin FM, entFM; phosphatidylinositol-specific phospholipase; PI-PLC, plcA; cytotoxin K, cytK; NHEA, nheA; NHEB, nheB; and NHEC, nheC), PCR reactions were performed on bacterial genomic DNA. For each gene, primer pairs and amplification conditions were set as previously described [15 (link)].