EFFECT OF UV-C LIGHT IN THE PRESERVATION OF RAW FERMENTED BEVERAGES

Abstract

The aim of this work was to check the efficacy of an annular ultraviolet reactor for stabilizing the growth of lactic acid bacteria (LAB), the main components of the microbiota of three raw fermented beverages, Boza, Ayran and Matsoni during cold storage. The ultraviolet (UV) dose of 260 mJ cm^sup -2^ caused a reduction of 2.6 and 1 log CFU mL^sup -1^ in the LAB loads of Boza and Ayran samples, respectively, whereas multiple UV treatments were required for Matsoni; they caused the decrease of 3 log CFU mL^sup -1^ in LAB counts. Twenty-two bacterial strains, belonging to 11 species, were identified in the natural microflora of the three beverages before and after the UV treatments. Only a minimal post-acidification process was registered up to day 30 in all the three beverages. This ultraviolet device is at disposal of beverage manufacturers, who could apply it for producing microbially-stable drinks with an extended shelf-life. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

The aim of this work was to check the efficacy of an annular ultraviolet reactor for stabilizing the growth of lactic acid bacteria (LAB), the main components of the microbiota of three raw fermented beverages, Boza, Ayran and Matsoni during cold storage. The ultraviolet (UV) dose of 260 mJ cm^sup -2^ caused a reduction of 2.6 and 1 log CFU mL^sup -1^ in the LAB loads of Boza and Ayran samples, respectively, whereas multiple UV treatments were required for Matsoni; they caused the decrease of 3 log CFU mL^sup -1^ in LAB counts. Twenty-two bacterial strains, belonging to 11 species, were identified in the natural microflora of the three beverages before and after the UV treatments. Only a minimal post-acidification process was registered up to day 30 in all the three beverages. This ultraviolet device is at disposal of beverage manufacturers, who could apply it for producing microbially-stable drinks with an extended shelf-life.

- Keywords: fermented beverages, lactic acid bacteria, mild technologies, molecular typing, post-acidification, ultraviolet thin film system -

INTRODUCTION

Many fermented foods are still produced around the world by empirical processes based on spontaneous fermentation of different raw materials.

Some raw fermented beverages produced in Eastern European Countries as Boza, Ayran and Matsoni have a very short shelf-life.

Boza is an ancient cereal-based fermented beverage originating in Mesopotamia 8000-9000 years ago and still produced in Turkey, Bulgaria, Albania and Romania under traditional processing conditions. Recent papers have investigated the production of bacteriocins from natural LAB occurring in Boza (TODOROV and DICKS, 2004; 2005; TODOROV, 2010) and their probiotic properties (TODOROV et al., 2008). However, some interactions among the autochthonous microorganisms could cause uncontrolled fermentations that lead to a variable product quality and a shelf-life less than two weeks under refrigeration condition (HANCIOGLU and KARAPINAR 1997; GOTCHEVA et al, 2000; GENÇ et al., 2002; BOTES et al., 2007). Currently, producer companies do not apply any preservation methods other than sórbate amendment.

Ayran is a yoghurt beverage produced in Turkey, traditionally manufactured by adding salted water to yoghurt at a level of 30-50% (ANONYMOUS, 1982). Quality of Ayran deteriorates during storage, due to acidity development and whey syneresis, thus limiting its shelf-life to 1 Ο-Ι 5 days at refrigeration temperatures (ANONYMOUS, 1982).

Matsoni is a Georgian bottle-fermented beverage based on pasteurized milk, back-slopped with an inoculum made up of thermophilic streptococci and different species of lactobacilli, leading to traces of alcohol and carbon dioxide (UCHIDA et al, 2007). The shelf-life of Matsoni is 5-7 days at refrigeration temperatures, after which the product becomes too sparkling and sour.

In these circumstances manufacturers need procedures suitable for stabilizing microbial loads and extending the shelf-life of these drinks without completely suppressing their inherent microorganisms, and yet still preserving their taste and texture. Moreover, these drinks are not suitable for severe procedures (heat, high pressure) and for the addition of preservatives and stabilizers, subjected to public concern and ever tighter legislative control.

Interest is being shown in alternative, so-called "physical" treatments such as UV-light as a result of increasing consumer demands for fresh-like quality food products (ZHU et al., 2005).

Many studies, carried out on disinfection of apple juice and microbial inactivation of beers and ciders using UV-light equipment, have shown the reduction of bacterial populations to undetectable limits (KOUTCHMA et al., 2004; GEVEKE, 2005; LU et al., 2010a; 2010b). Recently, LU et al (2011) set up a novel UV apparatus based on the Dean vortex technology for sanitizing milk highly contaminated by pathogenic bacteria. MANZOCCO et al. (2011) applied a UV-C light treatment to fresh-cut melons and showed that UV-light exposure could be a potential novel technology for surface decontamination of ready-to-eat foods.

For these reasons, a UV-C light system was applied in this study with the aim of preventing the outgrowth of lactic acid bacteria in raw fermented beverages as Boza, Ayran and Matsoni during their cold storage. Molecular analyses were performed on bacterial populations to understand how UV-C treatments affect the microbial composition of these beverages.

MATERIALS AND METHODS

Beverage samples

Boza was manufactured following the Turkish recipe reported by HANCIOGLU and KARAPINAR (1997) requiring 2% inoculum with a back-slopping starter obtained from the previous production. Boza samples were provided by Deniz Ticaret ?.ξ>. (Bursa, Turkey).

Ayran was produced by mixing home-made yoghurt and 0.5-1% NaCl solution according to the validated Turkish method of the Turkish Institute of Standards (ANONYMOUS, 1982). Ay-ran samples were provided by Aygm Sût ?.ξ>. (Konya, Turkey).

Matsoni was manufactured following the traditional Georgian recipe using cow's milk pasteurized at 90°C for ten min, cooled up to 45°C and inoculated with a 3% back-slopping obtained from the batch production of the previous day. Fermentation was carried out in glass bottles at 45°C until reaching the pH value of 4.6 (after 5-7 hours). Then, samples were placed in a cooling cell and stored at 6°C. Matsoni samples analyzed in this work were manufactured by Amaltea-Didube Milk (Tbilisi, Georgia).

All beverage samples were immediately transported under refrigerated condition to the laboratory where they were processed by the UV-system described below and then kept at 4°C for sixty days.

The UV-light system

The system consisted of a UV ozone-free lamp (LUCAS and AI^SHAMMA'A, 2003), which emitted energy at two wavelengths (254 and 185 nm) at high power levels (1 kW). The lamp was operated at a constant power of 30 W and was covered by a quartz sleeve situated in a stainless steel chamber. The liquid to be treated was placed into the liquid sample tank which held up to 3 L and had a liquid level indicator. As shown in Fig. 1, the liquid was flowed vertically from the sample tank into the UV reactor, as a thin film well exposed to the UV-light, and finally pumped to a receiver tank. The flow rate was set and altered by varying the gas pressure (P) within the liquid tank and allowing the liquid to flow through an adjustable needle valve. The gas pressure was fed from the laboratory source (5 bar) and was regulated to about 1 bar.

Cleaning of the system

The system was cleaned between each trial by running tap water 3 times through the instrument at a flow rate of 0.5 L min"1, then passing 2 L of 5% hypochlorite 3 times at room temperature for disinfection. Finally, sterile distilled water was passed through the system until microbial growth was no longer detected.

Single UV-C treatments

Two different doses of UV-light (66 mJ cm 2, flow rate 2 L min1 and 260 mJ cm 2, flow rate 0.47 L min1) were applied to 2-litre samples of Boza, Ayran and Matsoni 1 day after their production. During UV treatments, the beverage temperature did not undergo any appreciable changes.

Microbial loads, pH and total titratable acidity (TTA) were recorded for all samples immediately before, after UV treatment and during cold storage at 7, 14, 30 and 60 days. Each experiment was carried out in triplicate.

Serial UV-C treatments of Matsoni

As single UV doses resulted ineffective in decreasing microbial loads in Matsoni, multiple UV-C light treatments were applied to these drink samples. In particular, two different experiments were carried out. In the first experiment, samples were treated with three successive increasing doses of UV-light (87, 135 and 206 mJ cm 2), whereas in the second trial, six successive UV doses (266, 223, 238, 166, 231 and 240 m J cm2) were applied. Microbial loads were determined at the end of each treatment. Each experiment was carried out in triplicate. UV treatment of beverages did not cause any appreciable changes in temperature.

Microbiological analyses of beverage samples

Samples were collected from each beverage before and after UV treatment in glass vials and used immediately for counting the microbial viable cells. These samples were stored at 4°C for further analysis. Ten grams of each beverage sample were dispersed in 90 g of 2% sodium citrate solution, homogenised in sterile bags via a stomacher, and serially diluted in sterile 0.1% buffered peptone water. The appropriate dilutions were plated in triplicate on different media: Ml7 with 0.5% lactose (LM17, Oxoid S.p.A., Garbagnate, Milan, Italy) and de Man, Rogosa and Sharpe (MRS, Oxoid) agar for counting presumptive Gram positive cocci and rods, respectively; Violet Red Bile lactose Agar (VRBA Difco, Difco Laboratories, Detroit, MI) for enumerating total coliforms; Potato Dextrose Agar (PDA, Difco), supplemented with chloramphenicol (0.1 g L1) for isolating yeasts and moulds. LM 17 and MRS plates were incubated for 48 h under anaerobic conditions (AnaeroGene, Oxoid S.p.A.) at 30°C (for Boza sample) or 37°C (for Ayran and Matsoni samples), whereas VRBA plates were incubated at 37°C for 48 h and PDA plates at 25°C for 7 days under aerobic conditions. The microbial content of each experiment was analyzed in triplicate.

For each beverage sample, 10-15 well-isolated bacterial colonies, randomly picked up from MRS and LM 17 agar plates seeded with the highest sample dilutions, were inoculated in MRS and Ml7 broths, respectively, and incubated under appropriate conditions. All isolates, harvested by centrifugation (6,000 χ g for 5 min), were suspended in the same medium containing 20% glycerol, frozen at - 80°C and stored for further characterization.

Molecular analyses of microbial isolates

Total DNA for molecular analyses was extracted from bacterial isolates using the Wizard-Genomic DNA Purification kit (Promega, Madison, WI, USA), according to the manufacturer's instructions. DNA quantity and quality were determined by electrophoresis with known amounts of a molecular weight marker (Marker VI, Roche Diagnostics, Milan, Italy) as a standard.

Bacteria strain typing was performed using the "two-step RAPD PCR" protocol, as described by BARUZZI et al (2000), including control reaction mixtures lacking DNA template in each experiment. XD9 (MOSCHETO et al., 1998) and Cocí (COCCONCELLI et al., 1995) primers were used, respectively, for rod- and cocci-shaped LAB.

PCR amplifications were carried out in a Thermal Cycler 9700 (Perkin-Elmer, Alameda, CA, USA). Taq polymerase and deoxynucleoside triphosphates were purchased from Sigma-Aldrich S.r.l. (Milan, Italy), gel-filtration purified oligonucleotides from Sigma-Genosys Ltd. (Cambridge, UK), whereas DNA molecular weight markers were purchased from Qiagen SpA (Milan, Italy). The amplified fragments, separated by electrophoresis in 2.0% agarose gel, were sized using the Quantity One 4.3.1 software (Bio-Rad Laboratories S.r.l., Milan, Italy). When different isolates gave the same electrophoretic pattern, they were grouped. The ratio (P = n/t) between the number of colonies grouped for each strain (n) and total colonies, analyzed (t) from the same medium for each sample, represented the percentage (P) of the presence of that strain in the total population. The product of viable cell count value and the percentage (P5 of each isolate made it feasible to calculate the viable cell count for each strain. One isolate from each group was chosen as the representative strain and stored for further analyses.

Each representative strain from RAPD-PCR groups was identified by amplifying and sequencing the 16S rRNA gene, as previously reported (WIESBURG et a I, 1991). The DNA sequences were obtained using an ABI PRISM Big Dye Terminator Cycle Sequencing Kit ver3.1 (PE Applied Biosystems, Inc., Foster City, CA, USA) with both the forward and reverse primers being used. The reaction products were analyzed with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The Applied Biosystems software package (Sequencing Analysis ver. 3.3 and MT Navigator PPC ver. 1.0.2) was used for the analysis and comparison of DNA sequences. Taxonomic strain identification was performed, comparing the rDNA sequences of beverage isolates with the sequences present in the Basic BLAST Search, as described by ALTSCHUL et al (1997). Taxonomic identification of Lactobacillus plantarum, Lactobacillus paracasei Lactobacillus delbrueckii subsp. bulgaricus, Enterococcus faecium, Leuconostoc mesenteroides and Streptococcus thermophilus was confirmed by species-specific PCRs (LICK et al, 1996; SONG et al., 2000; TORRIANI et al, 2001; FURET et al, 2004; BENSALAH et al., 2006).

pH value and total titratable acidity determinations

The pH of beverage samples was determined by direct insertion of a pHmeter (Oakton Bench-top pH 510 meter, Cole-Parmer, Vernon Hills, Illinois, USA).

Total titratable acidity (TTA) was measured in the following way: 10 g of beverage sample were weighed in a 250 mL Erlenmeyer flask, and distilled water was added up to 50 mL including a few drops of Phenolphthalein. The mixture was titrated with 0.1 Ν NaOH according to the AOAC method no. 947.05 (AOAC 2000) and expressed as percentage of lactic acid in 100 grams of sample.

Statistical analysis

Statistical analysis of data was carried out using the Statistica 6 software package (StatSoft Italia, Vigonza, Italy). Unpaired two-tailed Student's t test was used to analyze statistical differences in incidence ratios of bacterial bio types and in the total viable cell counts of the treated beverage samples and controls with respect to each beverage, taking Ρ < 0.05 as significant. One-way analysis of variance (ANOVA) followed by the Duncan's multiple test range method was applied to verify whether there were significant differences (Ρ < 0.05) in pH and TTA mean values for each beverage before UV treatment and throughout its cold storage.

RESULTS AND DISCUSSION

The first practical applications of UV-C were in disinfection of surfaces and water sterilisation by setting up suitable UV equipment that are currently well accepted in the food industry (AL-SHAMMA'A et al., 2001; HOYER, 1998).

In the last 20 years, UV-light has been widely used for controlling foodborne pathogens as Escherichia coli Ol57:H7 and enterotoxigenic Staphylococcus aureus, Salmonella typhimurium and Listeria monocytogenes in pure culture suspensions, stainless steel and foods (MCKILLIP et al., 1998; KIM et al., 2002; KRISHNAMURTHY et al., 2004; YAUN et al., 2004).

A lab-scale UV apparatus was applied to three raw fermented beverages (Boza, Ayran and Matsoni) in order to stabilize the viable counts of microbial groups such as lactic acid bacteria (LAB) responsible for the phenomenon of beverage post-processing acidification during cold storage.

Microbiological and molecular analyses of untreated fermented beverages

Since UV-C light treatments were applied in order to reduce but not destroy the natural microflora of each beverage, it was necessary to monitor the dominant bacterial strains occurring before and after UV-C light treatments.

At the end of the manufacturing process, all beverage samples contained high microbial loads of lactic acid bacteria (7.8 log CFU/mL, on average); while fungi and yeasts were undetectable; during cold storage, bacterial counts in the untreated samples of the three drinks remained quite stable till day 60 (Fig. 2). The coliform counts in the three beverage samples were below the detection threshold.

Molecular analyses in the untreated Boza samples revealed complex and heterogeneous populations made up of nine different bacterial strains belonging to seven species (Table 1). These results are in accordance with those reported by other authors (GOTCHEVA, 2000; HANCIOGLU and KARAPINAR, 1997; BOTES etal., 2007).

Microbiological and molecular analyses of Ayran showed, in addition to the characteristic microflora of yoghurt, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, as previously reported (ANONYMOUS, 1982), even one single strain of Lactobacillus plantar-um (Table 2).

As concerns Matsoni, microbiological analyses allowed to ascertain that, in the untreated samples, Gram-positive cocci viable counts resulted dominant, accounting for more than 8 log CFU ml/1, followed by Gram-positive rods with more than 5 log CFU ml/1 (Fig. 2). Molecular analyses confirmed the results previously reported by UCHIDA et al. (2007), who, analysing 26 Matsoni samples collected throughout Georgia, found that the microbiota of this beverage was mainly made up of S. thermophilus and L. delbrueckii spp. bulgaricus. In addition, our molecular analyses revealed the dominance of S. thermophilus on L. delbrueckii subsp. bulgaricus strains in the samples (Table 3), confirming the results of the microbiological investigations (Fig. 2).

Effect of UV-C light treatment on beverages

The UV-C dose of 66 mJ cm"2 was ineffective on all beverages, whereas promising results were obtained treating Boza and Ayran samples with the UV-C dose of 260 mJ cm"2 and 0.47 L/min. A reduction of about 2 log CFU ml/1 was registered in both Gram-positive rods and cocci of Boza samples immediately after UV treatment (Fig. 2); the same values of microbial counts were registered at the end of cold storage.

Molecular analyses showed that some strains, occurring at high amounts before the UV-light treatment, were not isolated from the same sample after UV treatment; on the other hand, some new strains were recovered after UV-light treatment, suggesting they occurred as sub-populations in the untreated Boza samples (Table 1).

In Ayran samples, the 260 mJ cm 2 UV C dose caused a decrease of about 1 log cycle in both Gram-positive rods and cocci viable loads; lactic acid bacteria (LAB) counts remained quite stable till day 60 (Fig. 2). LU et cd. (2010b) registered a similar reduction value in total LAB by applying the 4.8 mJ cm*2 UV dose to beer samples, using a thin film apparatus equipped with quartz optical fibres. Molecular analyses of Ayran UV-treated samples revealed that LAB strains belonged to S. thermophilus and L. plantarum, whereas L. delbrueckii subsp. bulgaricus 401, recovered from the untreated samples, was not retrieved (Table 2).

Single UV doses were ineffective in decreasing microbial loads in Matsoni. GUERRERO- BELTRÀN and BARBOSA-CÁNOVAS (2004) found that milk solids may limit UV penetration and thereby decrease the efficiency of UV treatments. This behaviour could explain why one single UV-C treatment was more effective in reducing LAB loads in Boza than in Ayran. Thus, in the milk-based beverage Matsoni two different experiments were carried out: in the first one, samples were subjected to three successive UV-light treatments with increasing doses, whereas in the latter experiment six successive UV-C doses were applied to the drink samples, as previously described. As concerns the first UV experiment, Gram-positive rods were more resistant to UV-C treatment in comparison with cocci during cold storage (Fig. 2, BOZA and AYRAN). A reduction of more than 3.5 log CFU mL"1 was registered in Gram-positive cocci viable counts immediately after UV treatment; they increased by 1 log after one week of cold storage, decreasing to 2.8 log at day 60 (Fig. 2). The same UV-C dose caused a reduction of about 2.5 log CFU mL"1 in Gram-positive rod viable counts immediately after UV treatment; this bacterial population increased to over 4 log at day 15, decreasing to about 3 log at day 60 of cold storage (Fig. 2). Molecular analyses revealed that the UV-treated samples contained the same two strains of L. delbrueckii subsp. bulgaricus retrieved in the original sample, with one dominant biotype, suggesting that this latter strain was more resistant to UV treatment (Table 3); as concerns St thermophilus biotypes, the same RAPD-PCR pattern found in the untreated sample was retrieved as dominant, together with a second profile. In addition, one single Lactococcus lactis biotype was found after UV treatment (Table 3). The second UV-C experiment carried out on Matsoni samples caused the complete disappearance of LAB (data not shown); therefore this treatment was considered too drastic for the purpose of the present work.

Values of pH and total titratable acidity determinations of untreated and treated beverage samples

In accordance with the increase in microbial counts, pH decreased markedly while TTA increased in the untreated cold-stored Boza and Matsoni samples (Table 4). The marked decrease in pH in both untreated beverages during cold storage was probably due to the increase in TTA, which in turn depends on microbial fermentation (ROBINSON et al., 2006). Some bacterial species isolated from Boza as Leuconostoc lactis and Leuconostoc lactis subsp. cremoris displayed a psychrophilic behaviour that allowed them to grow at low temperatures and produce different types of organic acids (lactic and acetic acids) that have a direct influence on acidity and the final flavour of the beverage. ZORBA et al. (2003) reported the results of pH and TTA values in Boza during fermentation time; however, to the best of our knowledge, no reports on the analysis of these values during cold storage of this beverage are available so far.

Interestingly, in our work, pH and TTA in UV-treated Boza samples changed slightly during cold storage unlike the pH of the untreated samples (Table 4).

As in yoghurt, in the milk-based beverages Ay-ran and Matsoni heterofermentative bacteria as L. delbrueckii subsp. bulgaricus are responsible for the phenomenon of post-processing acidification, causing a dangerous drop in pH below 4.3, which destabilizes the casein clot and weaving of beverages in retail (ROBINSON et al., 2006).

No increase in TTA values was registered in all three UV-treated beverages after 2 months of cold storage. Thus, the phenomenon of beverage post-processing acidification did not occur up to day 30 in comparison with that of the untreated drinks. The best results were achieved in Matsoni, whose shelf-life is usually shorter than one week under refrigeration condition.

CONCLUSIONS

As an alternative to drastic treatments (heat, high pressure) and the addition of antimicrobials, here we used an annular UV-C system in which the fermented beverages were flushed as a thin film. This treatment did not affect the reciprocal relationships among LAB, even though reduced the microbial loads in the beverage samples. As these drinks usually contain viable microorganisms contributing to their typical quality and taste, special attention was paid to the choice of the appropriate UV doses in order to obtain a mild reduction but not the total inactivation of the beverage microbiota.

Promising results were obtained with Boza by applying a single UV-C dose of 260 mJ cmr2 that caused a suitable reduction in viable counts of both Gram-positive cocci and rods; then they remained stable till day 60 of cold storage. The same UV-C dose applied to Ayran was less effective, lowering bacterial loads by about 1 log CFU mL1; however, microbial counts did not increase during two months under refrigeration condition.

Interestingly, a significant reduction in bacterial populations was obtained in Matsoni after UV-C treatment stabilizing the acidity at least up to one month. Thus, the application of UV-C light by an annular device, as an alternative to more drastic methods, experienced in this study, could enable beverage manufacturers to produce microbially-stable drinks with an extended shelf-life.

ACKNOWLEDGMENTS

This work was supported by the Sixth Framework Programme of the European Commission (Horizontal Research Activities Involving SMEs - Co-operative Research Project FERBEV contract N°031918, www.ferbev.net). We thank DENIZTIC, AYGIN SUT and AMALTEA SMEs' entrepreneurs for their coopération in providing beverages and evaluating sensoiy desirability of UV-light treated beverages.

References

REFERENCES

Al-Shamma'aA.I., Pandithasl. and Lucas J. 2001. Low-pressure microwave plasma ultraviolet lamp for water purification and ozone applications: The future of technological plasmas. J. Phys. 34: 2775.

Altschul S.F., Thomas L. M., Schaffer ?., Zhang J., Zhang Z., Miller W. and Lápman D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389.

Anonymous. 1982. TS 3810 standard of Ayran. Ankara, Turkey: Turkish Institute of Standards, TSE, Ankara, Turkey.

AOAC. 2000. "Official Methods of Analysis" 17th Ed. Association of Official Analytical Chemists Method n. 947.05. In: W. Horwitz (Ed.). Gaithersburg, MD, USA.

Baruzzi F., Morea M., Matarante A. and Cocconcelli P.S. 2000. Changes in the Lactobacillus community during Ricotta forte cheese natural fermentation. J. Appl. Microbiol. 89: 807.

Bensalah F., Flores M.J. and Mouats A. 2006. A rapid PCR based method to distinguish between Enterococcus species by using degenerate and species-specific sodA gene primers. African J. Biotechnol. 5: 697.

Botes ?., Todorov S.D., Von Mollendorff J.W., Botha A. and Dicks L.M. 2007. Identification of lactic acid bacteria and yeast from Boza. Process Biochem. 42: 267.

Cocconcelli P.S., Porro D., Galandini S. and Senini L. 1995. Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci. Lett. Appl. Microbiol. 21: 376.

Furet J.P., Quénée P. and Tailliez P. 2004. Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR. Int. J. Food Microbiol. 97: 197.

Genç M., Zorba M. and Ova G. 2002. Determination of Theological properties of Boza by using physical and sensory analysis. J. Food Eng. 52: 95.

Geveke D.J. 2005. UV inactivation of bacteria in apple cider. J. Food Protect. 68: 1739.

Gotcheva V., Pandiella S.S., Angelov ?., Roshkova Z.G. and Webb C. 2000. Microflora identification of the Bulgarian cereal-based fermented beverage Boza. Process Biochem. 36: 127.

Guerrero-Beltràn J.A. and Barbosa-Cánovas G.V. 2004. Review: advantages and limitations on processing foods by UV light. Food Sei. Technol. Int. 10: 137.

Hancioglu O. and Karapinar M. 1997. Microflora of Boza, a traditional fermented Turkish beverage. Int. J. Food Microbiol. 35: 271.

Hoyer O. 1998. Testing performance and monitoring of UV systems for drinking water disinfection. Water Supply 16: 424.

Kim T., Silva J.L. and Chen T.C. 2002. Effects of UV irradiation on selected pathogens in peptone water and on stainless steel and chicken meat. J. Food Protect. 65: 1142.

Koutchma T., Keller S., Chirtel S. and Paiisi B. 2004. Ultraviolet disinfection of juice products in laminar and turbulent flow reactors. Innov. Food Sei. Emerg. Technol. 5: 179.

Krishnamurthy K, Demirci A. and Irudayaraj J. 2004. Inactivation of Staphylococcus aureus by pulsed UV-light sterilization. J. Food Protect. 67: 1027.

Láck S., Keller M., Bockelmann W. and Heller J. 1996. Rapid identification of Streptococcus thermophüus by primer-specific PCR amplification based on its lacZ gene. Syst. Appl. Microbiol. 19: 74.

Lu G., Là C. and Láu P. 2011. UV inactivation of milk-related microorganisms with a novel electrodeless lamp apparatus. Eur. Food Res. Technol. 233: 79.

Lu G., Là C., Uu P. Cu H., Xia, Y. and Wang J. 2010a. Inactivation of microorganisms in apple juice using an ultraviolet silica-fiber optical device. J. Photochem. Photobiol. B: Biol. 100: 167.

Lu G., U C., Uu P., Cu H., Yao Y. and Zhang Q. 2010b. UV inactivation of microorganisms in beer by a novel thin-film apparatus. Food Control 21: 1312.

Lucas J. and Al-Shamma'a A.I. 2003.Germicidal modified - atmosphere packaging for food products and water purification. British patent 407725 GB, October.

Manzocco L., Da Pieve S. and Maifreni M. 2011. Impact of UV-C light on safety and quality of fresh cut melon. Innov. Food Sei. Emerg. Technolog. 12: 13.

McKillip J.L., Jaykus LA. and Drake M. 1998. rRNA Stability in heat-killed and UV-irradiated enterotoxigenic Staphylococcus aureus and Escherichia coli 0157:H7. Appl. Environ. Microbiol. 64: 4264.

Moschetti G., Blaiotta G., Aponte M., Catzeddu P., Villani F., Deiana P. and Coppola S. 1998. Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism: powerful methods to differentiate Streptococcus thermophilus strains. J. Appl. Microbiol. 85: 25.

Robinson R.K, Lucey J.A. andTamine A.Y. 2006. Manufacture of yoghurt. In "Fermented Milks". A.Y. Tamime (Ed.), pp. 53-75. Blackwell Science Ltd, Oxford.

Song Y., Kato N., Uu C., Matsumiya Y., Kato H. and Watanabe K. 2000. Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Microbiol. Lett. 187: 167.

Todorov S.D. 2010. Diversity of bacteriocinogenic lactic acid bacteria isolated from boza, a cereal-based fermented beverage from Bulgaria. Food Control 21: 1011.

Todorov S.D. and Dicks L.M.T. 2004. Characterization of mesenteiicin ST99, a bacteriocin produced by Leuconostoc mesenteroides subsp. dextranicumST99 isolated from Boza. J. Ind. Microbiol. Biotechnol. 31: 323.

Todorov S.D. and Dicks L.M.T. 2005. Pediocin ST18, an anti-listeiial bacteriocins produced by Pediococcus pentosaceus ST18 isolated from Boza, a traditional cereal beverage from Bulgaria. Process Biochem. 40: 365.

Todorov S.D., Botes M., Guigas C., Schillinger U., Wiid I., Wachsman M.B., Holzapfel W.H. and Dicks L.M. 2008. Boza, a natural source of probiotic lactic acid bacteria. J. Appl. Microbiol. 104: 465.

Torriani S., Felis G.? and Dellaglio F. 2001. Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA Gene Sequence Analysis and Multiplex PCR Assay with recA Gene-Derived Primers. Appl. Environ. Microbiol. 67: 3450.

Uchida K, Urashima T., Chanishvili N., Arai I. and Motoshima H. 2007. Major microbiota of lactic acid bacteria from Matsoni, a traditional Georgian fermented milk. Anim. Sei. J. 78: 85.

Wiesburg G.W., Barns S.M., Pelletier D.A. and Lane D.J. 1991.16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173: 697.

Yaun B.R., Sumner S.S., Eifert J.D. and Marcy J.E. 2004. Inhibition of pathogens on fresh produce by ultraviolet energy. Int. J. Food Microbiol. 90: 1.

Zhu M., Du M., Cordray J. and Ahn D.U. 2005. Control of Listeria monocytogenes contamination in ready-to-eat meat products. Compr. Rev. Food Sei. F. 4: 34.

Zorba M., Hancioglu O., Genç M., Karapinar M. and Ova G. 2003. The use of starter cultures in the fermentation of boza, a traditional Turkish beverage. Process Biochem. 38:1405.

Paper received July 26, 2012 Accepted November 16, 2012