Samples of vodka and spirit drinks, with different alcohol content, were .... Studying the chemical composition of distilled spirits we noticed that glycerol, used as.
UNIVERSITATEA DIN CRAIOVA UNIVERSITY OF CRAIOVA
Seria: 9 Biologie 9 +RUWLFXOWXUă 9 7HKQRORJLDSUHOXFUăULL produselor agricole 9 Ingineria mediului
Vo l. XVII ( LIII ) - 2012
IDENTIFICATION OF MICROORGANISMS INVOLVED IN DISTILLED SPIRITS CONTAMINATION AND THEIR EVOLUTION DURING FINAL PRODUCT STORAGE $QFXĠD05RWDU 1, Cristina Anamaria Semeniuc1, Mihaela Duma 2, Nadejda Costru1, Marinela Curliuc1 Key words: Distilled spirits, contamination, moulds, storage, glycerol
ABSTRACT The aim of this study was to identify the microorganisms in distilled spirits and their evolution during storage of the final product using Standard microscopy and microbiology techniques. Because moulds were found in distilled spirits, selective media were used for TYMC determination. Strains of Penicillium, Oidium, Alternaria and Geotrichum were isolated from vodka and spirit drink samples. The initial presence of moulds in distilled spirits is a consequence of microaeroflora contamination. Glycerol used as ingredient offers protection against the effect of alcohol from distilled spirits. During the final product storage moulds consume glycerol, and no longer protected, are destroyed by the alcohol.
INTRODUCTION Distilled alcoholic beverages contain ethanol between 20 and 50% alcohol by volume, derived from the distillation of agricultural raw materials subjected to alcoholic fermentation. Due to the wide range of fermented vegetal raw materials and manufacturing technologies, a variety of distilled spirits with specific sensory characteristics are available, which are consumed either as an appetizer or as a dessert drinks (Banu et al., 1999; Cioltean, 2008, 2009). According to Standards, distilled spirits must be sterile, free of microorganisms and without risk to consumer health. The processing units are completely responsible to this aspect (Cioltean, 2008, 2009). In the manufacturing of natural distilled spirit, the extractive substances used for raw material processing can influence the taste and aroma of the product. Ethanol, the main ingredient of natural alcoholic beverages, can be obtained by distillation or by synthetic pathway (Iovu, 2005).Vodka represents a spirit drink which is produced from ethanol of agricultural origin. It has a minimum alcohol content of 37.5 % alcohol by volume. The most important raw materials used are potatoes, different kind of cereals or molasses. The flavorings added in spirit drinks can be allowed only if the type of spirit permitted it (is not allowed in the case of vodka) and must be specified on the label.
1 2
Faculty of Food Science and Technology, UASVM Cluj-Napoca, Romania Cluj-Napoca Sanitary Veterinary Agency
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The main aim of this investigation was to identify the species of mould involved in the contamination of distilled spirits from a local distillery, the cause of the appearance and their evolution during the final product storage. Table 1 shows the maximum levels of ethanol residues present in the two spirit drinks analyzed in our study (according to REG 110/2008). Table 1 The maximum levels of ethanol residues present in spirit drinks Maximum level of ethanol residues
Vodka
Methanol, mg/100 ml pure ethanol Esters, mg ethyl acetate/100 ml pure ethanol Aldehydes, mg acetaldehyde/100 ml pure ethanol Higher alcohols, mg 2-methyl-1-propanol/100 ml pure ethanol
10 1.3 0.5 0.5
Spirit drink (not flavored) 200 1.3 0.5 0.5
MATERIALS AND METHODS Sampling and experimental design Samples of vodka and spirit drinks, with different alcohol content, were aseptically collected from a local distillery (Table 1) and tested for TYMC at 30 and 60 days of storage. Sealed bottles from the warehouse were used for distilled spirits sampling. Each type of colony was confirmed by microscopy. TYMC test. The reference method (SR ISO 21527-1:2009) was used for enumeration of yeasts and moulds in distilled spirits samples. Media used were Dichloran Rose Bengal Chloramphenicol Agar, DRBC (Oxoid, Basingstoke, Hampshire, UK) and Dichloran 18% Glycerol Agar, DG-18 (Oxoid, Basingstoke, Hampshire, UK). Chloramphenicol (Oxoid Ltd., Basingstoke, Hampshire, UK) was added (100 mg/L) to the media as a selective agent to inhibit the growth of bacteria. Preparation and dilutions of distilled spirits and drinking water samples was in accordance with ISO 7218:2007, SR EN ISO 6887-1:2002 standards. For each sample was prepared two successive dilutions. Tree Petri dishes was used for each dilution. An aliquot of 0.1 mL of the diluted sample was inoculated using a sterile pipette into each Petri dish containing DRBC and DG-18 media. Then, the Petri dishes were sealed in plastic bags (to avoid contamination) and incubated at 25 ± 1°C for 5 days. After five days of incubation, the colonies are counted. The total number of yeasts and moulds is calculated using the formula specified in the standard: N=
¦c
[cfu/mL]
(V (n1 0.1 u n2 ) u d
where: ȈF-colonies sum; V-inoculum volume (mL); n 1 -Petri dishes number for first dilution; n 2 -Petri dishes number for second dilution; d-dilution.
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Macro- and microscopic characterization of colonies. Macroscopic characterization The following criteria were used: shape, edge aspect, size, colony profile, colour and colony type. Microscopic characterization Each type of colony was sampled using a microbiological loop and loaded on a glass slide, covered with sterile saline and a cover slip. The preparations were observed under a microscope (Motic AE31 microscope). RESULTS AND DISCUSSIONS In distilled spirit samples were identified different mould species: Penicillium, Oidium, Alternaria and Geotrichum. The predominant colonies belonged to Penicillium genus, subgenus Furcatum, with approximately 1 cm colony diameter, rough surface, green colour with a white border, circular shape and raised elevation (Fig. 1a). Figure 1b shows the microscopic image of Penicillium subgenus Furcatum with phialides that are ampulliform and shorter than their supporting metulae (Pitt and Hocking, 2009).
Figure 1a, b. Penicillium genus, subgenus Furcatum (macro- and microscopic images) Colonies of Alternaria genus were also identified. Different sized colonies with brown color, rhizoidal form and edges with convex profile and type R rough surface (Fig. 2a). Under the microscope, ovoid conidia with a short conical beak and pale brown color (Fig. 2b) were observed. This saprophyte mold can be present in the freshly harvested grains, being an indicator for their freshness (Dan et al., 2009).
Fig. 2a, b. Alternaria genus (macro- and microscopic images) 347
Also white flat colonies with rhizoidal edges, hilly profile, and type R rough surface were identified (Fig. 3a). The colonies belong to Oidium genus. Under the microscope, Oidium looks like a string of beads (with conidiophores arising from a hyphal cell) (Fig. 3b).
Fig. 3a, b. Oidium genus (macro- and microscopic images) The last type of colonies identified belonged to Geotrichum genus and were characterized by white color, rhizoidal and filamentous edges, bulging profile and type R rough surface (Fig. 4a). Under the microscope was noticed the presence of hyphae with reproductive structures completely fragmented at maturity (Fig. 4b). Geotrichum is a cosmopolitan fungus with a world wide distribution that can be isolated from soil and plants. (http://www.forensica.com/f/labservices/microbiology/library/geotrichum.asp). It is a common contaminant of equipment and for this reason is called "mold machines”. Geotrichum genus was identified in all distilled spirits samples.
Fig. 4a, b. Geotrichum genus (macro- and microscopic images)
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In our previous study (Rotar et al., 2012), microaeroflora from processing areas was identified as source of microbial contamination of distilled spirits. Table 1 shows TYMC in distilled spirits after obtaining (Rotar et al., 2012), at 30 and 60 days of storage. Table 1 TYMC (cfu/mL) in distilled spirits TYMC Sample After obtaining 30 d of storage Vodka 1, 40% v/v alcohol 7 cfu/mL 1.5 cfu/mL Vodka 2, 40% v/v alcohol 6.6 cfu/mL 1.3 cfu/mL Vodka 3, 37.5 % v/v alcohol 4.0 ufc/mL 2.5 ufc/mL Vodka 4, 37.5 % v/v alcohol 3.5 ufc/mL 2.1 ufc/mL Spirit drink, 26 % v/v alcohol 4.1 ufc/mL 0.5 ufc/mL Spirit drink, 26 % v/v alcohol 4.5 ufc/mL 0.5 ufc/mL
60 d of storage absent absent absent absent absent absent
A significant decrease of TYMC can be observed during distilled spirits storage. Studying the chemical composition of distilled spirits we noticed that glycerol, used as ingredient in distilled spirits, protects the moulds against the destroying effect of alcohol. Subsequently, glycerol (which is the nutritive substrate for moulds) is consumed during the final product storage; moulds are no longer protected, therefore are absent after 60 days of storage.
CONCLUSIONS The presence of moulds in distilled spirits is a consequence of microaeroflora contamination. In distilled spirits Penicillium, Oidium, Alternaria and Geotrichum genus were identified macro- and microscopic. Good hygiene of production areas is imposed. ACKNOWLEDGMENTS This work was supported by the USAMV-CN/1215/15/06.02.2012 Grant.
REFERENCES Banu C., Manualul inginerului în industria alimentDUӽ, Vol. I, vol II, Ed. 7HKQLFӽ, Bucureúti, 1999. Cioltean I., Fabricarea EӽXWXULORUDOFRROLFHGLQIUXFWH, Ed. Risoprint, Cluj-Napoca, 2008. Cioltean I., 0DWXUDUHDúLvQYHFKLUHDĠXLFLLúLUDFKLXULORU, (I), Agro Terra, 2009. Dan Valentina , Cristina Kramer, Gabriela Bahrim, Anca Nicolau, Margareta Zara, Memorator pentru mucegaiuri, Ed. Evrika, Braila, 2009. Iovu M., &KLPLHRUJDQLFӽ%XFXUHúWL2005. Pitt, J. I. and Ailsa D. Hocking, Fungi and food spoilage, 3rd ed, Springer, NY, USA., 2009. 5RWDU$QFXĠD M., Semeniuc Cristina Anamaria, Mudura Elena, Coldea Teodora, /D]ăU3RS&DUPHQ, Identification of microbial contamination sources in distilled spirits, 349
Bulletin of Universitu of Agricultural Science snd Veterinary Medicine Cluj-Napoca, 2012, vol 69 (2), pg. 380-386. SR ISO 21527-1:2009-Microbiology of food and animal feeding stuffs -Horizontal method for the enumeration of yeasts and moulds -- Part 1: Colony count technique in products with water activity greater than 0.95 SR EN ISO 6887-1:2002-Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 1: General rules for the preparation of the initial suspension and decimal dilutions (ISO 6887-1:1999) ISO 7218:2007-Microbiology of food and animal feeding stuffs-General requirements and guidance for microbiological examination Regulation (EC) No 110/2008 of the European Parliament and of the CounciL of 15 January 2008 on the definition, description, presentation, labelling and the protection of geographical indications of spirit drinks and repealing Council Regulation (EEC) No 1576/89 http://www.forensica.com/f/lab-services/microbiology/library/geotrichum.asp).
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