polish journal of food and nutrition sciences http://journal.pan.olsztyn.pl Pol. J. Food Nutr. Sci. e-mail: [email protected] 2010, Vol. 60, No. 4, pp. 369-373

YEAST OCCURRENCE IN HERRING PRODUCTS AND PROCESSING ENVIRONMENT AND THEIR BIOCHEMICAL PECULIARITIES

Algimantas Paškevičius, Regina Varnaitė

Institute of Botany, Laboratory of Biodeterioration Research, Vilnius, Lithuania

Key words: , species, fish, contamination

The aim of this work was to ascertain contamination of raw herring, the processed products and manufacturing environment, and to evalu- ate biochemical peculiarities of dominant species of the isolated yeasts, which could have influenced the product quality. A total of 36 yeast strains were isolated from herring products, raw material and their production environment during the manufacturing process. These yeasts were identified as 8 species belonging to Saccharomyces, , Pichia, Rhodotorula, Debaryomyces, Yarrowia yeast genera. Saccharomyces cerevisiae and Candida glabrata yeasts dominated in herring products. Candida blankii, Debaryomyces hansenii var. hansenii, Yarrowia lipolytica, and C. glaebosa yeasts were isolated from raw herring. The studies on contamination of the production room air, equipment, dishes, packing and other surfaces showed that the majority of samples were infected by Saccharomyces cerevisiae and Candida glabrata yeasts that were also found in the processed herring production. Research on enzymatic activity showed that yeasts Pichia membranifaciens and Debaryomyces hansenii var. hansenii exhibited proteolytic activity, while Yarrowia lipolytica – lipolytic activity. Based on the preliminary study results of assimilation and fermentation of carbon sources as well as proteolytic and lipolytic activities, this could have had the negative influence upon the product quality.

INTRODUCTION ria of the genera Listeria and Pseudomonas and representatives Microorganisms present in food products cause bio- of the Enterobacteriaceae family [Balasubramanian, 1992; chemical processes, which often determine product quality, McMeekin et al., 1992; Gram & Huss, 1996; Gram & Dal- and change their nutritious properties and flavor. Some sap- gaard, 2002; Bledsoe, 2003; Farben & Peterkin, 2000; Papado- rophytic microorganisms can be the reason of food product poulou et al., 2007; Khan et al., 2007]. Yeasts of the Candida, deterioration. Due to reproduction of pathogenic microorgan- Rhodotorula and Saccharomyces genera of various species are isms in raw material or food products, the food can become found, too. The presence of various species of fungi belonging unsuitable for consumption. Therefore, permanent micro- to genera Aspergillus, Eurotium, Penicillium, Fusarium, Cla- biological control of raw material and products is required. dosporium was also revealed on the samples of smoke-dried It is essential to control microbiological processes in food or dried salted fish in various countries. The amount of fungi products, to ascertain factors promoting the cause of these was 3.0×102 – 4.8×104 cfu/g [Wheeler et al., 1996; Spencer processes and to determine microorganisms able to change & Spencer, 1997; Jonsyn & Lahai, 2006; Adebayo-Tayo et al., the product quality and pose health risk to consumers. 2008]. Fish and its products can spoil especially rapidly. Psy- In the course of processing microorganisms from freshly chrophilic microorganisms can grow in raw fish when the raw caught fish easily spread, invade mechanically damaged fish material is stored at 0–4oC temperature. Live fish can also be tissues and deteriorate them causing rotting and other spoil- infected with microorganisms. Microorganisms are mostly age processes. In order to prevent rapid spoilage of fish, vari- found in mucous membrane between exuviae, on branchiae ous technologies are applied: quick freezing, gradual freezing surface and in the alimentary tract. From 102 to 107 colony and other methods. The culinary properties of fish change forming units (cfu) of microorganisms per 1 cm2 of fish exuvi- the least when fresh fish and processed raw material are fro- ae and branchiae surfaces can be recorded. In the alimentary zen quickly. In such cases microorganisms on fish are being tract, there were 104–108 cfu/g of microorganisms [Svičiulis et killed or go into anabiosis – a resting state – and mass deg- al., 1999]. Amounts of microbial propagules in a fish organ- radation processes are subdued, as well as spoilage processes ism depend on clearness of a water basin, seasons and many are slowed down or even completely stopped. Nevertheless, other environmental factors. Usually higher amounts of mi- as environmental conditions change, e.g. the temperature croorganisms are detected in fish caught in March–April and slightly increases, the degradation processes in raw material July–November. Many authors indicate that the main agents can start again because fluids, leaked out of fish due to au- of microbiological taints of fish and their products are bacte- tolysis of fish cells, are very suitable medium for the reproduc-

Author’s address for correspondence: Algimantas Paškevièius, Laboratory of Biodeterioration Research, Institute of Botany, Žaliuju Ežeru 49, Lt-08406 Vilnius, Lithuania; tel.:37052711723; e-mail: [email protected]

© Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences 370 A. Paškevičius & R. Varnaitė tion and growth of microorganisms. Therefore, storage time were grown for 4–5 days in an incubator at +25±1°C tem- of defrost fish should be reduced to the technically possible perature. Experiments were run in 3 replications. Yeast counts minimum [Levin & Witkowski, 1991; Hansen et al., 1996; were assessed according to the international ISO 7954 [1987] Narkevičius, 2004]. standard. The aim of this work was to ascertain yeast contamina- Identification of yeasts was performed according to mac- tion of raw herring, the processed products and manufactur- ro- and micromorphological properties of the isolated ing environment, and to evaluate biochemical peculiarities cultures, their biochemical and physiological peculiarities fol- of dominant species of the isolated yeasts, which could have lowing the manuals for yeast identification [Kreger-van Rij, influenced the product quality. 1984; Kurtzman & Fell, 1998]. Physiological characteristics were analysed by perform- MATERIALS AND METHODS ing fermentation and assimilation tests. Fer- mentation of was tested in Durham tubes with The samples were collected at the fish processing enter- D­‑glucose, D-galactose, maltose, sucrose, lactose, raffinose prise (Lithuania) where production taints of microbiological and trehalose. Tubes were incubated at +25±2°C and ob- origin arose. The expiry dates having been unexpired, the taste, served every few days during four weeks. odor and appearance taints of producible fish products were The carbohydrate assimilation was tested using Yeast Ni- observed. trogen Base (Difco, USA) with 33 carbon compounds: hexo- The samples for microbiological analysis were taken from ses (D-glucose, D-galactose, L-sorbose), pentoses (D-xylose, one processing technological process starting from raw fish L-rhamnose, L-arabinose, D-arabinose, D-ribose), disaccha- from Iceland and Norway and ending with ready products. rides (sucrose, maltose, trehalose, melibiose, lactose, cello- The products studied were: herring fillets, herring fillets in oil, biose), trisaccharides (raffinose, melezitose), polysaccharides smoke-flavored herring fillets, lightly salted herring fillets and (inulin, soluble starch), glycosides (α-Methyl-D-glucoside, lightly salted herring fillets in oil. Furthermore the air of pro- arbutin, salicin), alcohols (, glycerol, erythritol, ribitol, duction rooms, various surfaces and worker overalls were galactitol, D-mannitol, D-glucitol, m-inositol), organic acids analysed. The samples were collected at a production time. (succinate, citrate, DL-lactate, D-gluconate). Altogether 25 samples were studied. The assimilation of nitrogen compounds was performed Herring raw material and products samples were taken on Yeast Carbon Base (Difco, USA) with potassium nitrate by cutting off 3 pieces of 100 g from different fish sections and potassium nitrite. Yeast growth was examined after and put into sterile containers. The fish pieces (10 g) were 5 days. chopped, stirred for 10 min and dilutions were made. Next, To detect lipolytic activity of the yeasts, following media

1 mL of outwashs was spread on solid medium for yeast iso- were used (%): tributyrate – 1; KH2PO4 – 0.25; NaH2PO4 – lation. Yeast amount is presented as colony forming units per 0.25; (NH4)2 SO4 – 0.1; MgSO4 x 7H2O – 0.05; yeast extract – gramme (cfu/g). 0.05; agar – 1.5; Victoria Blue – 0.01; (pH 7-8). Yeast lipolytic The sampler of microorganisms (“Krotov 818”, Russia) activity was measured applying the method of Watanabe et was used for collection of airborne yeasts. Particles together al. [1977]. with the air were sucked through a tiny slit and directed at In order to detect yeast proteolytic activity, 2% agar with the surface of a solid medium in a Petri dish where they ad- 1.5% of sterile milk was used. A yeast culture was inoculated hered. While collecting, a Petri dish with the medium rotated using the streak method. Inoculates were cultivated for 5 days at 60–100 rpm velocity. This ensures equal distribution of par- at +25±1°C temperature. Visually clear zones formed around ticles onto the medium surface. The flow rate was 25 dm3/min, a streak were treated as a positive result. and sampling time – 2 min. Every sample was taken in 3 rep- Yeast detection frequency (A) was calculated according lications [Nevalainen et al., 1993]. Yeast amount is presented to formula: A= b/c × 100%, where b – number of analyses, as colony forming units per cubic meter (cfu/m3). when yeast species was detected; c – total amount of analyses. Herring brine samples were taken into sterile vessels. For The obtained data were processed using Microsoft Excel inoculation the water suspensions of the investigated micro- XP (mean, standard deviation). organisms were prepared. Herring brine samples were spread on media in 1-mL doses. Yeast amount is presented as colony RESULTS AND DISCUSSION forming units per 1 mL (cfu/mL). Swabs from equipment, work clothes and other surfaces The investigation showed (Table 1) that the number of yeasts were taken from 100 cm2 workspaces with sterile cotton swabs in the herring products made up from 1.3 to 12×103 cfu/g. and put into sterile tubes. The swabs were washed in 10 mL The highest number of yeasts was isolated from smoke- of sterile physiological solution and diluted if necessary. Next, flavoured herring fillets (12.0×103 cfu/g). Research of yeast 1 mL of outwashs was spread on solid medium for yeast iso- species composition showed that Saccharomyces cerevisiae lation. Yeast amount is presented as colony forming units per Meyen ex E. C. Hansen and Candida glabrata (Anderson) S. 1 cm2 (cfu/cm2). A. Meyer & Yarrow (Yarrow and Meyer) species dominated Yeasts were isolated onto Yeast extract-dextrose-chloram- in the tested herring products and their detection frequency phenicol-agar of the following composition (g/L): yeast (A) reached even 80%. Yeasts of the Debaryomyces hansenii extract – 5.0; dextrose (C6H12O6) – 20.0; chloramphenicol (Zopf) Lodder & Kreger-van Rij var. hansenii, Yarrowia lipoly-

(C11H12Cl2N2O5) – 0.1; agar – 20.0; and water – 1 L. Yeast tica (Wickerham, Kurtzman & Herman) van der Walt & von Occurrence of yeasts in herring products 371

Table 1. Cell number and diversity of yeast species in herring products and raw material. products can be contaminated with yeasts of different genera. Yeast species frequently isolated from fish and shellfish reflect Cell number species prevalent in water from which they are taken. Com- Isolation source –x±SD Isolated yeast species monly the red-pigmented yeasts (Rhodotorula glutinis, Rh. 3 (cfu/g ×10 ) mulcilaginosa) dominate as well as other basidiomycetous Herring products yeasts (Cryptococcus albidus, Trichosporon cutaneum) and as- Heavily salted 0 Not detected comycetous yeasts (Candida zeylanoides, C. tropicalis, Yarowia herring fillets lipolytica) [Kobatake et al., 2008; Papadopoulou et al., 2007]. Saccharomyces cerevisiae, Can- Lightly salted Das et al. [2007] showed that the count of yeasts in raw fish 1.3±0.1 dida glabrata, Pichia membrani- herring fillets 3 faciens depended on fish species and varied between 1.0×10 cfu/g 1 Herring fillets Candida glabrata, Saccharomyces in Batashi (Clupisoma atherinoides) and 7.0×10 cfu/g in Rui 2.1±0.2 in oil cerevisiae (Labeo rohita). Saccharomyces cerevisiae, Can- The investigation revealed that the number of yeasts Smoke-flavoured 12.0±0.6 dida glabrata, Rhodotorula mul- herring fillets in the indoor air of the manufacturing premises was low; cilaginosa in the range from 0 to 1.3×102 cfu/m3 (Table 2). Candida Debaryomyces hansenii var. han- Lightly salted senii, Yarrowia lipolytica, Sac- glabrata and Saccharomyces cerevisiae species yeasts domi- herring fillets 8.5±0.5 charomyces cerevisiae, Candida nated in the air of the manufacturing premises. The detection in oil glabrata frequency of these yeasts was 57.1% and 42.8%, respectively. Raw material Debaryomyces hansenii var. hansenii and Rhodotorula mul- Herring fil- cilaginosa species yeasts were isolated rarely from the air lets brought in 0.2±0.02 Candida blankii of manufacturing premises and their detection frequency was summer from 14.3%. Yeasts were not detected in both control (the outdoor Norway air) and the ventilation air. Herring fil- lets brought in Debaryomyces hansenii var. han- The results of yeast contamination on equipment 0.1±0.01 autumn from senii, Yarrowia lipolytica in the manufacturing premises, tools, working clothes and Norway other surfaces showed (Table 3) that majority of the samples Herring fillets were contaminated with yeasts Saccharomyces cerevisiae and brought in sum- 0.2±0.02 Candida glaebosa Candida glabrata that were also found during other stages mer from Island of processing and in the ready products. The detection fre- quency of Saccharomyces cerevisiae species yeasts reached even Arx, Pichia membranifaciens (E. C. Hansen) E. C. Hansen and 85.7% and that of Candida glabrata – 57.1%. Yeasts amount on Rhodotorula mulcilaginosa (Jörgensen) F. C. Harrison species the equipment and other surfaces reached from 0 to 50.0 cfu/ were rarely isolated (A=20%), from these products. cm2. The highest contamination with yeast was detected on Aiming to determine the reasons of the product contami- the used package – 50.0 cfu/cm2. High numbers of yeasts nation with yeasts, raw material and brine were investigated (20.0 cfu/cm2) were found in samples from working clothes. (Table 1). The raw material was also contaminated with The analysis of species composition of yeasts showed yeasts. The yeast species composition in different raw mate- that Saccharomyces cerevisiae and Candida glabrata prevailed rial batches varied. Candida blankii H. R. Buckley & van Uden in samples taken from various surfaces. Yeasts of these spe- yeasts dominated in samples of raw material brought from Norway in the summer, while Debaryomyces hansenii var. han- Table 2. Cell number and species composition of yeasts isolated from senii and Yarrowia lipolytica prevailed on the same raw mate- the indoor air of the manufacturing premises. rial delivered in the autumn. Candida glaebosa Komagata & Nakase yeasts dominated in raw material from Island. It was Cell number Sampling site –x±SD Isolated yeast species estimated that though the detection frequency of Debaryomy- (cfu/m3 ×102) ces hansenii var. hansenii and Yarrowia lipolytica species yeasts Preparation Debaryomyces hansenii var. 1.3±0.2 was 20% in herring raw material, they were detected in ready room hansenii products. Yeasts amount was considerably lower in her- Raw herring 0.2±0.1 Candida glabrata ring raw material than in ready products and reached from processing room 3 Ripening cham- 0.1 to 0.2×10 cfu/g. 0.2±0.1 Saccharomyces cerevisiae The research results showed that on the surface of her- ber No 1 Ripening cham- ring brine Candida glabrata yeasts dominated. This species 0.8±0.2 Candida glabrata ber No 2 of yeasts was not isolated from herring raw material but in fi- Saccharomyces cerevisiae, Can- nal production its detection frequency reached even 80%. Packing room 1.3±0.1 dida glabrata, Rhodotorula mul- A presumption could be made, therefore, that yeasts could cilaginosa get from production environment air or the mechanisms and Container wash- Saccharomyces cerevisiae, Can- 0.6±0.09 other surfaces present in the manufacturing premises. ing room dida glabrata There is very little data about yeast incidence in herring Ventilation air 0 Not detected Outdoor air raw material and products. Our results coincide with the re- 0 Not detected sults of other authors showing that raw fish and processed fish (control) 372 A. Paškevičius & R. Varnaitė

Table 3. Cell number and species composition of yeasts isolated from Table 4. Frequency of strains within yeast species isolated from raw equipment and other devices. herring and herring products able to produce extracellular proteolytic (ExP) and lipolytic (ExL) enzymes. Cell number – Sampling site x±SD Isolated yeast species Yeast species ExP ExL (cfu/cm2) Saccharomyces cerevisiae 0/4a 0/4 Raw herring Saccharomyces cerevisiae, Can- defrosting 20.0±2.0 Pichia membranifaciens 1/1 0/1 dida glabrata chamber Candida glabrata 0/5 5/5 A bath after 10.0±1.0 Saccharomyces cerevisiae disinfections Debaryomyces hansenii var. hansenii 2/2 0/2 Washing-up Yarrowia lipolytica 0/2 2/2 10.0±1.0 Saccharomyces cerevisiae brush Candida blankii 0/1 0/1 Saccharomyces cerevisiae, Rho- Floor broom 9.0±0.5 dotorula mulcilaginosa, Candida Candida glaebosa 0/1 0/1 glabrata Rhodotorula mulcilaginosa 0/1 0/1 Laminate before 0 Not detected and after heating a number of positive reaction of strains over the total number of strains Saccharomyces cerevisiae, Can- examined. Working clothes 20.0±2.0 dida glabrata Containers after Saccharomyces cerevisiae, Can- 50.0±3.0 use dida glabrata Numerous works of researches from different countries indicate that yeast show a wide spectrum of assimilative and fermentative abilities of carbon sources. Yeasts can grow on cies were widespread in both the indoor air of manufactur- fish products under conditions of high NaCl concentration ing premises and products. On the ground of preliminary and various pH [Gram & Huss, 1996; Betts et al., 1999; Palu- results of the conducted research it is possible to state that dan-Muller et al., 2002]. the manufacturing environment influenced more the inci- Research on enzymatic activity showed (Table 4) that dences of yeasts in herring products than raw material from yeasts Pichia membranifaciens and Debaryomyces hansenii which they were produced. var. hansenii exhibited proteolytic activity, Candida glabrata Despite that food processing enterprises usually have and Yarrowia lipolytica – lipolytic activity. The obtained results the hazard analysis and critical control point system, never- confirm the opinion that Yarowia lipolytica possesses high li- theless, such measures not always ensures the complete safety polytic and proteolytic activities. Proteolytic psychrotrophic of food products in respect of microbial contamination. Mi- yeasts are widely distributed in raw seafood and are able croorganisms can occur on raw material from the outside to grow under low temperature (from 0 to 5°C), therefore, – water, air, equipment, workers, and from these sources even- they can exert a negative influence on the quality of fish prod- tually microorganisms can get on food products and cause ucts [Kobatake et al., 2008]. spoilage [Willinghan et al., 1996; Langsrud et al., 2003]. The preliminary results of research showed that yeasts, Biochemical peculiarities of yeasts isolated from the her- alike other microorganisms, can infect the herring products ring products and during their processing did not show a wide and cause the taints of microbiological origin. On purpose spectrum of fermentation of carbon sources. It was assessed to estimate the reasons of yeast infection it is necessary to as- that yeasts Saccharomyces cerevisiae and Pichia membranifa- certain yeast incidence in raw material and on various techno- ciens fermented glucose intensively, while glucose fermenta- logical process stages in which the product or its components tion by Candida glabrata, Debaryomyces hansenii var. hansenii interact mostly with manufacturing environment. and Candida blankii yeasts was weak. Only Saccharomyces cerevisiae fermented galactose, sucrose and maltose. It should CONCLUSIONS be mentioned that the tested yeasts did not fermented lactose. Yeasts Saccharomyces cerevisiae exhibited the widest fermen- A total of 36 yeast strains were isolated from herring prod- tation spectrum of carbon sources. Meanwhile, Yarrowia li- ucts, raw material and their production environment during polytica, Candida glaebosa and Rhodotorula mulcilaginosa did the manufacturing process. These yeasts were identified as not ferment the tested carbon sources. 8 species belonging to Saccharomyces, Candida, Pichia, Rho- Investigation of carbon source assimilation showed that dotorula, Debaryomyces and Yarrowia yeast genera. if yeasts ferment a certain carbon source, they are also able It was assessed that Saccharomyces cerevisiae and Candida to assimilate it. Many of the tested yeasts intensively assimi- glabrata yeasts prevailed in herring products. Candida blankii, lated galactose, sucrose, maltose, citric and succinic acids. Debaryomyces hansenii var. hansenii, Yarrowia lipolytica, and C. Debaryomyces hansenii var. hansenii and Candida blankii were glaebosa yeasts were isolated from raw herring. Investigation able to assimilate starch, whereas only Candida blankii assim- of yeast contamination of the air in the premises as well as con- ilated inositol. 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