5 bromo 4 chloro 3 indolyl β d galacto pyranoside x gal

A chromogenic method was used to screen the fungi for their β-galactosidase activity in Petri plate incubation. The growth medium contained 20% (v/v) malt extract (20 mL/L), lactose (20 g/L), peptone (1 g/L) and agar (20 g/L). After sterilization, the medium was supplemented with 0.5% (v/v) of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal; Thermo Fisher Scientific, Waltham, USA) solution (20 mg/mL in dimethyl-sulfoxide). A volume of 20 µL of spore suspension (10⁶ spores/mL) was added to each agar plate and the cultures were incubated for 10 days at temperatures depending on the culturing requirements of the tested fungus. Visual detection of the plates was performed daily, and the level of the β-galactosidase production was evaluated from the intensity of the blue color developed. The categories of no blue color (NC), light blue color (+), darker blue color (++), blue color (+++), dark blue color (++++) and deep dark blue color (+++++) were defined to evaluate the chromogenic test. Three biological replicates were performed with each tested strain. The qualitative chromogenic assay serves as a preliminary test to screen for enzyme production as reported in other similar studies [12 (link),21 ,22 ,23 (link),24 (link)].

The strains, their derivatives and plasmids used in this study are listed in Table 1. Lactococcus lactis strains were grown in M17 medium (Merck GmbH, Darmstadt, Germany) supplemented with 0.5% glucose (GM17) at 30°C. Escherichia coli strains (DH5α and EC101) were grown in Luria Bertani broth (LB) at 37°C with aeration. To each medium, agar (1.5%; Torlak, Belgrade, Serbia) was added for use as a solid medium. The following antibiotic concentrations were used: ampicillin, 100 μg/ml (E. coli); erythromycin, 10 μg/ml (lactococci) and 300 μg/ml (E. coli); chloramphenicol, 10 μg/ml (lactococci) and 35 μg/ml (E. coli). The 5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside (X-Gal) (Fermentas, Vilnius, Lithuania) was added to LB medium plates for blue/white color screening of colonies with cloned fragments at a final concentration of 80 μg/ml (lactococci) and 40 μg/ml (E. coli). Ortho-nitrophenyl-β-galactoside (ONPG) was used for the β-galactosidase assay (Sigma Chemical Co., St. Louis, MO, United States).
All strains carrying constructs were stored in growth medium containing 15% glycerol (Sigma Chemie GmbH, Deisenhofen, Germany) at −80°C.

Lactococcus raffinolactis BGTRK10-1 was isolated from autochthonous sweet kajmak produced from sheep milk without the use of starter cultures in a household of the Vlašić mountain region, central Bosnia and Herzegovina [17 (link)] (Table 1). Preliminary strain classification was done according to its fermentation ability using API 50CHL (Api System SA; Bio-Merieux, Montelieu-Vercieu, France), temperature of growth (30°C, 37°C, and 45°C), growth in the presence of salt (4% and 6.5%), and pH tolerance. Final taxonomic classification of BGTRK10-1 was performed by sequencing of amplified 16S rDNA using primers previously described [18 (link)]. The strain was grown in M17 medium (Merck GmbH, Darmstadt, Germany) supplemented with D-glucose (0.5% w/v) (GM17) at 30°C. Escherichia coli DH5α, HB101, and ER2523 strains were grown aerobically in Luria-Bertani (LB) broth at 37°C, unless otherwise specified. Solid medium was made by adding 1.75% (w/v) agar (Torlak, Belgrade, Serbia), to the liquid media. Antibiotics were used at the following concentrations: erythromycin 300 μg/ml and ampicillin 100 μg/ml for selection and maintaining of transformants. The 5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside (X-Gal) (Fermentas, Vilnius, Lithuania) was added to LB medium plates for blue/white colour screening of colonies with cloned fragments at final concentration of 40 μg/ml.