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.
Axel C, Zannini E, Arendt EK (2017). Mold spoilage of bread and its biopreservation: A review of current strategies for bread shelf life extension. Critical reviews in food science and nutrition 57 (16), 3528-3542
Cornea CP, Ciuc M, Voaides C, Gagiu V, Pop A (2011). Incidence of fungal contamination in a Romanian bakery: a molecular approach. Romanian Biotechnological Letters 16(1), 9.
De Clercq N, Van Coillie E, Van Pamel E, De Meulenaer B, Devlieghere F, Vlaemmynck G (2015). Detection and identification of xerophilic fungi in Belgien chocolate confectionery factories. Food Microbiology 46, 322-328
Regulation (EC) No 1333/2008 of the European Parliament and of the Council of 16 December 2008 on food additives. Official Journal of the European Union L 354, 16.
Frisvad JC, Larsen TO, Thrane U, Meijer M, Varga J, et al. (2011). Fumonisin and ochratoxin production in industrial Aspergillus niger strains. PLoS ONE 6(8), e23496.
Garcia MV, Bregâo AS, Parussolo G, Bernardi AO, Stefanello A, Copetti MV (2019). Incidence of spoilage fungi in the air of bakeries with different hygienic Status. International Journal of Food Microbiology 290 (2019) 254–261
Garofalo C, Zannini E, Aquilanti L, Silvestri G, Fierro O, Picariello G. and Clementi F. (2012). Selection of sourdough lactobacilli with antifungal activity for use as biopreservatives in bakery products. J. Agric. Food Chem. 60, 7719-7728.
Gerez CL, Torino MI, Rollán G, Font de Valdez G (2009). Prevention of bread mould spoilage by using lactic acid bacteria with antifungal properties. Food Control 20(2), 144-148.
Glass N, Donaldson G (1995). Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes. Applied and Environmental Microbiology 61(4), 1323-1330.
Kokkonen M, Jestoi M, Rizzo A (2005). The effect of substrate on mycotoxin production of selected Penicillium strains. International Journal of Food Microbiology 99, 207– 214
Le Lay C, Mounier J, Vasseur V, Weill A, Le Blay G, Barbier G, Coton E (2016). In vitro and in situ screening of lactic acid bacteria and propionibacteria antifungal activities against bakery product spoilage molds. Food Control 60, 247-255
Legan JD (1993). Mould spoilage of bread: the problem and some solutions. International Biodeterioration & Biodegradation 32, 33-53.
Legan JD & Voysey PA (1991). Yeast spoilage of bakery products and ingredients. Journal of Applied Bacteriology 70(5), 361-371.
Lund F, Filtenbory O, Westall S, Frisvad JC (1996). Associated mycoflora of rye bread. Letters in Applied Microbiology 23 (4), 213-217
Luz C, D’Opazo V, Mañez J, Meca, G. (2019). Antifungal activity and shelf life extension of loaf bread produced with sourdough fermented by Lactobacillus strains. J Food Process Preserv. 43, e14126.
Müller G (1997). Backwaren. In: Müller G, Holzapfel W, Weber H (Eds.), Mikrobiologie der Lebensmittel, Lebensmittel pflanzlicher Herkunft (1.Auflage, pp. 289–314). Hamburg: Behr’s Verlag.
Pitt JI & Hocking AD (2009). Fungi and food spoilage (3rd ed). Dordrecht; New York: Springer.
Reiss J (1973). Die Schimmelpilze des Brotes. Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene. Zweite Naturwissenschaftliche Abteilung: Allgemeine, Landwirtschaftliche und Technische Mikrobiologie 128 (7-8), 685-728
Rollàn G, Gerez CL, Dallgnol AM, Torino MI, Font G (2010). Update in bread fermentation by lactic acid bacteria. In: Méndez-Vilas A (Ed.), Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology (Vol 2, pp 1168–1174). Badajoz: Formatex.
Ryan LA, Zannini E, Dal Bello F, Pawlowska A, Koehler P, Arendt EK (2011). Lactobacillus amylovorus DSM 19280 as a novel food-grade antifungal agent for bakery products. Int. J. Food Microbiol. 146, 276-283.
Santos JLP, Chaves RD, Sant’Ana AS (2017). Estimation of growth parameters of six different fungal species for selection of strains to be used in challenge tests of bakery products. Food Bioscience 20, 62-66.
Schünemann C, Treu G, Creutz S, Meißner M (2016). Technologie der Backwarenherstellung: fachkundliches Lehrbuch für Bäcker und Bäckerinnen (11. überarbeitete Auflage). Alfeld/Leine: Gildebuchverlag.
Singh A & Singh AB (1994). Airborne Fungi in a Bakery and the Prevalence of Respiratory Dysfunction among Workers. Grana 33(6), 349-358.
Spicher G (1980). Zur Aufklärung der Quellen und Wege der Schimmelkontamination des Brotes im Grossbackbetrieb. Zentralblatt für Bakteriologie Parasitenkunde Infektionskrankheiten und Hygiene. 1. Abt. Original Reiheb Hygiene Betriebshygiene Praeventive Medizin 170, 508-528.
Varga J, Frisvad JC, Samson RA (2011). Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Studies in Mycology 69, 57-80.
Visagie CM, Houbraken J, Frisvad JC, Hong S-B, Klaassen CHW, Perrone G, … Samson RA (2014). Identification and nomenclature of the genus Penicillium. Studies in Mycology 78, 343-371.
Zusatzstoffverordnung, ZuV (2013). Verordnung des EDI über die zulässigen Zusatzstoffe in Lebensmittel vom 25. November 2013 (Stand am 1. Mai 2017).
Zannini E, Pontonio E, Waters DM, Arendt EK (2012). Applications of microbial fermentations for production of gluten-free products and perspectives. Appl. Microbiol. Biotechnol. 93, 473-485.