Procedure for air investigations in industrial bakeries and identification of the isolated moulds

In five industrial bakeries, the air was tested at three different sampling locations (A, dough preparation; B, before or in the bread proofer; C, after the baking process or before packaging) for mould contamination using passive and active methods. Dichloran 18% glycerol (DG18) agar (Oxoid, CM0729), and dichloran Rose-Bengal chloramphenicol (DRBC) (ISO) agar (Oxoid, CM1148) were used for the analyses. For the passive method, a petri dish containing each nutrient medium was left open on a flat surface at the three sampling locations for 60 minutes. Active air samples were also taken at the same locations using a MAS-100 NT air sampler (MBV, Stäfa). In each case, both 100 and 500 litres of air were actively drawn in. Afterwards, the nutrient media were incubated at 25°C. for 5 days. The colony-forming units (CFU) on the petri dishes were then enumerated and the moulds were documented.

Between 5 and 20 loaves of bread from each bakery, which had been baked and packaged at the same time as the air samples were taken, were randomly selected and stored for 4 to 7 days at room temperature. The moulds that developed were transferred to DRBC and DG18 plates. With one exception, no preservatives were used in the selected loaves. All of the breads were made with wheat flour, sometimes with a small amount of rye flour, and in one case with corn flour.

Subsequently, the moulds in the air at sampling location C (100 l) and a selection of the moulds that had developed on the loaves during storage were isolated on malt extract agar [20 g/l malt extract broth (Biolife, 4016602), 15 g/l Agar Bacteriological (Biolife, 4110302)], purified and frozen at -80°C according to Visagie et al (2014). The aw and pH values of the breads were also determined.

Mould isolates from sampling location C in bakeries 1 (bread and air), 2 (bread and air) and 3 (bread), were then identified. The identification was done using a multiphasic approach: in the first step, the mould isolates were divided into groups, and in the second step examples from each group were selected and identified by sequencing the relevant genes. To group the moulds, their appearance on different nutrient media was macroscopically evaluated. Yeast Extract Sucrose Agar (YES), Czapek Yeast Autolysate (CYA), Creatine Sucrose Agar (CREA), Czapek Agar (CZ), MEA and DG18 were used for this. Each culture medium was prepared according to Pitt & Hocking (2009). Mould spore suspensions, prepared in 30% (w/v) glycerol (AppliChem, A1123), 0.05% (w/v) Agar Bacteriological (Biolife, 4110302), and 0.05% (w/v) Tween 80 (Sigma-Aldrich, P1754), were placed on the six culture media (three 5µl spots per plate) and incubated for 7 days at 25°C. Following the incubation period, the macroscopic assessment and classification of the moulds took place. For the identification, a range of moulds were also cultivated on MEA for 5 days at 25°C. The purity of the moulds was assessed visually and if no contamination was found, they were added to 20 ml bouillon [4 % (w/v) D(+)-glucose (Roth, X997.4), 0.5 % (w/v) Pepton Bacteriological (Biolife, 4122592), 0,3 % (w/v) Yeast Extract (Difco, 2636.3), and 0,3 % (w/v) Malt Extract (Biolife, 4116502)], and cultivated at 25°C and 150 rpm for 3 days. Using a Quick DNA Plant/Seed Miniprep kit (Zymo Research, D6020), the DNA was isolated from the mycelium that had developed according to the manufacturer's instructions. The DNA was diluted (1:100) with double deionized H2O for the PCR. The ITS region and the β-tubulin gene were amplified with the ITS1/ITS4 and Bt2a/Bt2b (Microsynth, Balgach, Switzerland) primer pairs, in accordance with Glass & Donaldson (1995) in a PCR cycler (Labcycler, SensoQuest GmbH). To evaluate the success of the PCR, gel electropheresis (1.5% agarose gel, 40 min, 90 V) was then performed. After the PCR, the DNA Clean and Concentrater-5 kit (Zymo Research, D4014) was used according to the manufacturer's instructions to purify the samples. The purified PCR products were sequenced by Microsynth (Balgach, Switzerland), and then the Basic Local Alignment Search Tool (BLAST) was used to analyse and identify the moulds.



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