Traditionally produced sauerkraut as source of autochthonous functional starter cultures

Microbiological Research 169 (2014) 623–632

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Traditionally produced sauerkraut as source of autochthonous functional starter cultures Jasna Beganovic´ a , Blaˇzenka Kos a,∗ , Andreja Leboˇs Pavunc a , Ksenija Uroic´ a , ˇ skovic´ a Mladen Jokic´ b , Jagoda Suˇ a Laboratory for Antibiotic, Enzyme, Probiotic and Starter Cultures Technology, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia b Division of Molecular Medicine, Rud¯er Boˇskovi´c Institute, Bijeniˇcka c. 54, 10 000 Zagreb, Croatia

a r t i c l e

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Article history: Received 16 July 2013 Received in revised form 27 September 2013 Accepted 30 September 2013 Available online 8 October 2013 Keywords: Autochthonous lactic acid bacteria Industrial sauerkraut fermentation S-layer proteins Functional starter cultures

a b s t r a c t Spontaneous sauerkraut fermentation was performed at industrial scale in “Prehrana Inc.”, Varaˇzdin, in order to select autochthonous lactic acid bacteria (LAB) which were evaluated according probiotic criteria and tested for their capacity as probiotic starter cultures. At the end of the spontaneous sauerkraut fermentation, total LAB counts reached 9.0 × 105 CFU/ml. This underlines that the need for addition of the well characterised probiotic cultures, in appropriate viable cell counts, would be valuable in probiotic sauerkraut production. Phenotypic characterisation through API 50 CHL and SDS-PAGE of cell protein patterns revealed that Lactobacillus plantarum is predominant LAB strain in homofermentative phase of fermentation. Autochthonous LAB isolates SF1, SF2, SF4, SF9 and SF15 were selected based on the survival in in vitro gastrointestinal tract conditions. RAPD fingerprints indicated that the selected autochthonous LAB were distinct from one another. All of the strains efficiently inhibited the growth of indicator strains and satisfied technological properties such as acidification rate, tolerance to NaCl and viability during freeze-drying. Strains Lb. paraplantarum SF9 and Lb. brevis SF15, identified by AFLP DNA fingerprints, have shown the best properties to be applied as probiotic starter cultures, because of their highest adhesion to Caco-2 cells and expression of specific, protective S-layer proteins of 45 kDa in size. With addition of these strains, probiotic attribute of the sauerkraut will be achieved, including health promoting, nutritional, technological and economic advantages in large scale industrial sauerkraut production. © 2013 Elsevier GmbH. All rights reserved.

1. Introduction Spontaneous fermentations, such as the one occurring during sauerkraut production, rely on autochthonous lactic acid bacteria (LAB) present on the raw substrate. Characterisation and control of these autochthonous microorganisms are essential for the sensory quality and the safety of the spontaneously fermented sauerkraut. The fermentation processes involve mixed cultures of LAB, yeast and fungi, and as such traditional fermented foods are a plentiful source of starter microorganisms, with some of them exerting even probiotic characteristics. Still, the research of these vegetables, especially sauerkraut as source for probiotic microorganisms, is rather scarce compared with their dairy counterpart (Tamminen

∗ Corresponding author at: Department of Biochemical Engineering, Laboratory for Antibiotics, Enzymes, Probiotic and Starter Cultures Technology, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10 000 Zagreb, Croatia. Tel.: +385 1 4605 125; fax: +385 1 4836 424. E-mail address: [email protected] (B. Kos). 0944-5013/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.micres.2013.09.015

et al. 2004; Sánchez et al. 2005; Madrau et al. 2006; Kos et al. 2008; Leboˇs Pavunc et al. 2011; Leboˇs Pavunc et al. 2012). In most cases, probiotic LAB strains incorporated in the products, other than fermented dairy, are exogenous and the lack of the knowledge about the interactions between these strains and the present microflora makes technical feasibility unpredictable. Nowadays there is an increasing consumer demand for the non-dairy-based probiotic products and drinks (Leroy and De Vuyst 2004; Hajduk et al. 2009; Beganovic´ et al. 2011a). Functional starter cultures with probiotic properties offer additional advantages, compared to classical starter cultures and represent an interesting approach of improving the fermentations and achieving tastier, safer and healthier ˇ skovic´ et al. 2010). products (Leroy et al. 2006; Suˇ Although there is a great evidence for the characterisation of the novel starter cultures for the cheese and sausages fermentations, still there is a lack of a commercial functional starter cultures suitable for the fermentation of sauerkraut and other vegetables (Tamminen et al. 2004; Klingberg et al. 2005; Beganovic´ et al. 2010). The main challenge in developing such starter cultures is to improve safety, but also to preserve the typical

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sensory quality of traditional fermented products. The development of the probiotic starter cultures from autochthonous LAB of traditional fermented sauerkraut can be an optimal alternative, as these strains are well adapted to the sauerkraut environment, and compete well against present undesirable microorganisms. Therefore, the aim of the present study was to isolate autochthonous LAB, during spontaneous fermentation of the high quality Croatian white cabbage Brassica oleracea var. capitata cultivar Varaˇzdinski, with probiotic characteristics, which could be applied as functional starter cultures in the probiotic sauerkraut production. In vitro screening of autochthonous LAB strains through probiotic criteria, including survival in simulated gastrointestinal tract (GIT) conditions, autoaggregation, coaggregation, antibacterial activity and adhesion capacity to human intestinal epithelial cells, was undertaken to achieve probiotic bacterial strains for sauerkraut as functional product. Selected potential probiotic strains were also investigated for their technological characteristics such as tolerance to high NaCl concentrations and survival during freeze-drying with an addition of different cryoprotectants.

2. Materials and methods 2.1. Fermentation trails Spontaneous sauerkraut fermentations were carried out in processing plant “Prehrana Inc.”, Varaˇzdin, Croatia. The white cabbage heads (Brassica oleracea var. capitata cultivar Varaˇzdinski) were prepared by removing the outer leaves and were manually transferred in the tanks. NaCl solution 4% (w/v) was applied on white cabbage heads and all together was pressed tightly and covered with a plastic film. Brine samples from each fermentation tanks (n = 3), for analysis, were collected during fermentation on days 4, 10, 18, 24, 32 and 40 after start of fermentation. The decrease in pH of the sauerkraut brine was measured by pH metre (Metrohm, Herisau, Switzerland). Titratable acidity in the brine samples was determined by titration with a standard solution of 0.1 M NaOH using phenolphthalein as an indicator. The concentration of the lactic acid in the sauerkraut brines and cell-free cultures supernatants of LAB was evaluated by commercial d- and l-lactic acid determination kit (Test-Combination d-lactic acid/llactic acid UV method; Boehringer Mannheim GmbH, Mannheim, Germany).

2.2. Bacterial strains Autochthonous isolates were recovered from the brines sampled during the course of spontaneous sauerkraut fermentation. For microbial enumeration appropriate decimal dilutions were prepared and plated, in triplicate, on de Man Rogosa Sharp agar (MRS, Biolife, Milano, Italy) incubated at 30 ◦ C for 48 h; and at 37 ◦ C for 48–72 h. Approximately, 6–9 randomly selected colonies from the highest sample dilution were grown in MRS-broth and maintained at −80 ◦ C in MRS-broth (Biolife, Milano, Italy) supplemented with 15% (v/v) glycerol. Lactobacillus rhamnosus GG and Escherichia coli 3014 were used in adhesion experiments as reference strains. Indicator strains: Staphylococcus aureus 3048, S. aureus K-144, E. coli 3014, Salmonella enterica serovar Typhimurium FP1, Bacillus subtilis ATCC 6633 and B. cereus TM2, used for the screening of the antimicrobial activity of selected autochthonous isolates are from the Culture Collection of the Laboratory of Antibiotics, Enzymes, Probiotics and Starter Cultures Technology, Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia.

2.3. Initial selection and identification of autochthonous LAB isolates from spontaneous sauerkraut fermentation Autochthonous isolates were examined for their cell morphology, Gram stain reaction, spore formation and catalase activity using 3 g/l of H2 O2 . Single colonies were evaluated for their acidifying activities using a 744 pH metre (Metrohm, Herisau, Switzerland) and for lactic acid production according to the method described by Beganovic´ et al. (2011a, 2011b). For the phenotypic species-specific identification, dominant LAB isolates from sauerkraut were further subjected to API 50 CHL identification system (BioMérieux, Marcy l Etoile, France) according manufacturers’ instructions. 2.4. SDS-PAGE of surface protein extracts Surface proteins extraction was performed according to method of Kos et al. (2003). Electrophoresis was performed using a power supply PowerPac HCTM (Hercules, Biorad, USA) at a constant voltage of 200 V. Proteins were stained by 0.1% (w/v) Coomassie brilliant blue R-250 (Sigma, Steinhaim, Germany) in 50% (v/v) methanol and 7% (v/v) acetic acid. The excess stain was washed out with 7% (v/v) acetic acid. Low Molecular Weight Calibration Kit for SDS Electrophoresis (GE Healthcare, UK), containing 6 proteins ranging in size from 14 400 to 97 000 Da, was used as a molecular weight marker. Gel images were digitised with HP Scanjet 3800 Photo Scanner. 2.5. RAPD analysis Chromosomal DNA from selected autochthonous strains was extracted as previously described by Leboˇs Pavunc et al. (2009). Total DNA concentration and purity was determined by UV spectrophotometer (BioSpec-nano, Shimadzu Biotech, Kyoto, Japan). The extracted DNA was then used as a template in subsequent PCR amplifications, which were performed in a total volume of 50 ␮l in a DNA thermal cycler Mastercycler® (Eppendorf, Hamburg, Germany). PCR mixtures contained 10–100 ng chromosomal DNA; 50 pmol oligonucleotide ISS1rev: 5 -GGATCCAAGACA-ACGTTTCAAA-3 ; 50 pmol of each NTP (Fermentas, EU); 1.5 mmol l−1 mM MgCl2 ; 5 ml 10× Taq buffer and 0.5 U Taq polymerase (Boehringer GmbH, Mannheim, Germany) (Veyrat et al. 1999). PCR products were electrophoresed in 1.2% agarose (Eurobio, Les Ulis, France) gels with 1× TAE buffer, afterwards stained with ethidium bromide (0.5 ␮g ml−1 ) and visualised by UV transilluminator MiniBisPro (DNR Bio-Imaging Systems LTD, Jerusalem, Israel). 2.6. DNA fingerprinting using AFLPTM Identification of the isolated autochthonous LAB was performed using amplified fragment length polymorphism (AFLPTM ) method provided by Belgian Co-ordinated Collections of Microorganisms/Laboratory for Microbiology (BCCM/LMG, University of Ghent) identification service. Purified total DNA was digested using two restriction enzymes TaqI (hexacutter) and EcoRI (tetracutter). Adaptor 5 -CTCGTAGACTGCGTACC-3 3 -CTGACGCATGGTTAA-5 was used with restriction enzyme EcoRI, and adaptor 5 GACGATGAGTCCTGAC-3 3 -TACTCAGGACTGGA-5 with restriction enzyme TaqI. PCR primers EO1 (5 -GACTGCGTACCAATTCA-3 ) and T11 (5 -GTTTCTTATGAGTCCTGACCGAA-3 ) were specially hybridised with the adaptor ends of the restriction fragments. PCR products were separated according to their length using an ABI PRISM® 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Fragments that contain an adaptor specific

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for restriction halfsite created by the six bp-cutter are visualised due to the 5 -end labelling of the corresponding primer with the fluorescent dye FAM. The resulting electrophoretic patterns were normalised and subjected to a band pattern recognition procedure using the GeneMapper v. 4.0 software (Applera, Norwalk, CT, USA). Normalised tables of peaks, containing fragments of 20–600 base pairs, were transferred to BioNumericsTM v. 5.1 software (Applied Maths, Ghent, Belgium). For numerical analyses, data interval was delineated between the 40- and 580-bp bands of the internal size standard. DNA profiles of strains were compared with the reference profiles of the LAB taxa (including bifidobacteria) from BCCM database. Clustering of the patterns was done using the Dice coefficient and the UPGMA algorithm. 2.7. Characterisation of the potential probiotic strains 2.7.1. Survival of autochthonous isolates in simulated gastrointestinal tract conditions Simulated gastric and small intestinal juices were prepared according to Kos et al. (2000). Briefly, simulated gastric juice was prepared by dissolving pepsin (3 g/l) in sodium chloride solution (0.5%) adjusting the pH to 2.0 with concentrated HCl. Simulated intestinal juice was prepared by suspending pancreatin (1 g/l) and bile salts (3 mg/ml oxgall) in sodium chloride solution (0.5%) and pH was adjusted to 8.0 with 0.1 M NaOH. Both gastric and intestinal juices were prepared fresh. Washed cell suspensions of autochthonous isolates (0.5 ml) were added to 4.5 ml of tempered (37 ◦ C) simulated gastric juice, mixed well and incubated for 2 h at 37 ◦ C. The bacterial cells were separated by centrifugation and added in simulated intestinal juice tempered at 37 ◦ C and incubated for 4 h at 37 ◦ C with periodical shaking. Surviving bacteria after each time point were enumerated by pour plating method on MRS agar at 37 ◦ C for 48 h. Initial populations for all strains were approx. 9.0 ± 0.50 [mean ± standard deviation (SD)] log CFU/ml. All enumerations were carried out using the standard serial dilution method in physiological solution, and plated on MRS agar at the appropriate temperature. 2.7.2. Antibiotic susceptibility testing Antibiotic susceptibility of bacterial isolates was tested using the agar disc diffusion method. The 12 different antibiotics were tested in order to cover all the known chemical and functional groups of antibiotics: ampicillin, bacitracin and vancomycin as inhibitors of cell wall synthesis; azithromycin, erythromycin, gentamicin, clindamycin, chloramphenicol, streptomycin and tetracycline as inhibitors of protein synthesis; rifampicin, as inhibitor of nucleic acid synthesis, in concentrations indicated in Table 2. Oxoid (Basingstoke, United Kingdom) susceptibility test disks of each of the antibiotic tested were applied to MRS agar plates inoculated with autochthonous isolates. After incubation at 30 ◦ C for 24 h under anaerobic conditions the results (average of 3 readings) were expressed as sensitive (S), intermediate (I) and resistant (R). 2.7.3. Testing of autoaggregation and coaggregation ability The autoaggregation assay and coaggregation with pathogenic bacteria were performed according to Kos et al. (2003). 2.7.4. Adhesion of autochthonous isolates to Caco-2 epithelial cell lines Bacterial adherence to the Caco-2 epithelial cell line, kindly – Boˇskovic´ Institute, Zagreb, Croatia, was tested supplied by Ruder essentially as described by Goh et al. (2009) with slight modifications. The Caco-2 cells were routinely grown in a 95% air-5% CO2 atmosphere in minimum essential medium supplemented with 20% (v/v) inactivated fetal bovine serum (56 ◦ C; 30 min),

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0.10 mM nonessential amino acids, and 1.0 mM sodium pyruvate. All reagents used in maintenance of Caco-2 cells were obtained from Gibco-Invitrogen Corp., Carlsbad, CA. Monolayers of Caco-2 cells were seeded at a concentration of 2.0 × 105 cells/ml and dispensed into each well of a 24-well tissue culture plate (Greiner, Kremsmünster, Austria). The culture medium was replaced every 2 days, and all adherence assays were performed after 7 days of incubation in a 5% CO2 incubator. Briefly, 10 ml mid-log-phase bacterial cells (optical density at 620 nm, 0.6–0.9) grown in MRS broth were harvested by centrifugation at 4000 × g for 10 min to eliminate any effect of low pH or extracellular proteins in culture supernatants and were washed twice with phosphate buffered saline (PBS, pH 7.4). Caco2 monolayers were washed twice with PBS and treated with 1 ml of bacterial suspension at a concentration of ca. 4.0 × 108 CFU/ml, followed by incubation for 1.5 h at 37 ◦ C. After incubation, the monolayers were washed five times with PBS to remove unbound bacteria and then incubated with 0.05% (v/v) Triton X-100 (Sigma, Germany) for 10 min. To determine the number of viable bacteria, adhered to Caco-2 cells, appropriate dilutions in physiological solution were inoculated on MRS agar and colony counts were performed. Adhesion percentage was calculated with the following equation: (Adhered strains/strains added to the well) × 100. Experiments were performed in triplicate. Lb. rhamnosus GG and E. coli 3014 were tested as an example of the high and the low adhesive strain, respectively. 2.7.5. Antimicrobial activity Antimicrobial activity of the autochthonous sauerkraut isolates was tested by agar spot assay described by Shillinger and Lücke (1989) and by turbidimetric method described by Beganovic´ et al. (2011b). 2.7.6. Tolerance to NaCl Tolerance to NaCl was examined in MRS broth containing from 4.0% to 6.0% (w/v) NaCl in steps of 0.5%. Strain growth in MRS broth, without any addition of NaCl, was used as control. The absorbance (A620 ) reached at the end of incubation at 30 ◦ C was read on Microplate Reader (LKB 5060-006, Vienna, Austria). The results were expressed as ratio A620 in modified MRS broth against A620 in standard MRS broth. 2.7.7. Survival during freeze-drying Late exponential phase bacterial cells grown in MRS medium were freeze-dried using a 10% skim milk (Dukat, Croatia) as cryoprotective agent, while suspension of bacterial cells in phosphate buffer (pH 7.0) was used as control. Sucrose (Zagreb, Kemika, Croatia) and prebiotics inulin (Difco, Detroit, USA) and sorbitol (Zagreb, Kemika, Croatia) were also tested as possible cryoprotectans. The bacterial cultures were centrifuged for 15 min at 3300 × g. The supernatants were discarded and the cells were washed twice and resuspended in 5 ml of phosphate-buffer saline (pH 7.0) with or without addition of skim milk. The suspensions were frozen at −80 ◦ C overnight. Freeze-drying of the frozen cultures was performed in Christ Alpha 1-2 LD freeze drier (Martin Christ, Osterode, Germany) during 45 h. Viable cells count (CFU/ml), both before and after freeze-drying, were determined, by colony formation on MRS agar using the standard pour-plating method. Each experiment was conducted in triplicate. 2.8. Statistical analysis Data were expressed as means of three independent trials ± standard deviation (SD). Data were subjected to a one-way analysis of variance. Statistical analysis was made by Statistica 9.0

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Table 1 Identification of autochthonous strains, isolated during the course of spontaneous sauerkraut fermentation, by API 50 CHL, acidifying ability, lactic acid production after overnight growth in MRS medium and survival in simulated gastrointestinal tract conditions. The percentage value, % ID, shows the similarity of the tested strain with an API database strain. Isolate

API 50 CHL identification

% ID

% Lactic acid

pH

Survival in simulated GIT conditions (log CFU/ml)

SF1 SF2 SF3 SF4 SF5c SF6a SF7 SF8 SF9 SF10 SF11 SF12c SF13 SF14 SF15 SF16b SF18b SF19b SF21b SF22 b SF23b SF24b SF25b

Lb. plantarum Lb. plantarum Lb. plantarum Ln. mesenteroides ssp. mesenteroides/dextranicum Lb. crispatus Lb.plantarum Lb. plantarum Lb. pentosus Lb. plantarum Ln. mesenteroides ssp. mesenteroides/dextranicum Lb. plantarum No valid identification Lb. plantarum Lb. plantarum Lb. brevis Lb. plantarum Lb. plantarum Lb. plantarum Lb. plantarum Lb. plantarum Lb. plantarum Lb. plantarum Lb. plantarum

99.9 99.9 85.0 78.2 Unacceptable Profile 66.4 99.9 84.1 99.7 99.6 99.9 Unacceptable Profile 74.8 97.1 99.9 97.1 94.7 99.9 99.9 99.9 99.9 99.9 99.9

3.015 2.997 2.745 1.790 1.071 1.859 2.340 1.827 1.724 1.228 2.840 0.855 1.986 2.052 3.168 2.439 1.936 2.250 2.313 2.993 3.114 2.876 2.646

3.61 3.85 4.09 3.91 4.13 3.94 4.05 4.25 3.89 4.30 3.92 4.21 4.05 3.85 3.92 3.99 3.97 3.77 3.92 3.77 3.94 3.85 3.87

5.82 5.44 2.75 5.04 4.72 5.98 3.15

a b c

6.40 3.88 4.65 0.00 3.55 4.19 6. 91 3.23 2.99 3.79 3.73 3.88 3.69 3.58 3.66

SDS-PAGE of whole cell proteins revealed that electrophoretic pattern of strain SF6 was similar to the one of strain SF9 (data not shown). SDS-PAGE of whole cell proteins revealed that electrophoretic patterns of strains SF16-SF25 were similar. Profile was defined as unacceptable, when identification was below threshold value.

software (StatSoft Inc., Tulsa, OK). A P value of <0.05 was considered to indicate a significant difference. 3. Results 3.1. Screening of autochthonous LAB sauerkraut isolates Spontaneous sauerkraut fermentations were carried out in local plant “Prehrana Inc.” in Varaˇzdin, and autochthonous LAB strains were isolated during sauerkraut fermentation. According to microbiological analysis, at the end of the fermentation, viable LAB counts reached 9.0 × 105 colony forming unit (CFU/ml) (Fig. 1), the level close but lower than minimum recommended daily dose necessary to obtain healthy effects as probiotic formula (106 –108 CFU/ml). For further research, in initial selection, emphasis was put on the autochthonous isolates that were well adapted to the sauerkraut 7

7

pH

6

Lacc acid(g/L) 6

pH

4 5 3 2

Total LAB (log CFU/mL) log CFU/ml

5

4

1 0

3 4

10

18 24 32 Time (days)

40

Fig. 1. pH changes, lactic acid concentrations and total lactic acid bacteria (LAB) counts grown on MRS agar determined in sauerkraut brine sampled from the tanks during large scale industrial spontaneous fermentation performed in “Prehrana Inc.”, Varaˇzdin, Croatia. *Standard deviations of independent triplicate samples are calculated.

environment by selecting strains prevalent in fermentation, with fast acidifying ability and high lactic acid concentration production, which resulted in selection of 23 autochthonous, Gram-positive, asporgeneous, catalase – negative sauerkraut isolates. According to the API 50 CHL sugar fermentation profiles, Leuconostoc mesenteroides was detected among the colonies isolated during early, heterofermentative phase of fermentation, while Lb. plantarum was the most frequently isolated species in the sauerkraut brine further on, as fermentation proceeded (Table 1). In order to observe the diversity of the autochthonous isolates, presumptive LAB, the whole cell protein patterns obtained by SDS-PAGE of the SDS soluble proteins prepared from the overnight cultures, were compared (data not shown). The whole-cell protein fingerprint obtained for the isolates SF6 and SF9, designated by API 50 CHL as Lb. plantarum, corresponded to each other (Table 1). In addition, whole cell protein pattern obtained for the strains SF16–SF25, which were also designated by sugar fermentation profiles as Lb. plantarum corresponded to each other (Table 1). A dominant protein band was observed in a protein profiles of a strains SF6 and SF9 as well as in SF15. In this research, survival at extreme gastrointestinal tract (GIT) conditions at high viable cell count was taken as main selection criterion for strains which were later evaluated for their probiotic properties. Viable counts of SF1, SF2, SF4, SF6, SF9 and SF15 revealed as the highest, after their exposure to the simulated gastric and small intestine juice and were found to be above 5 log CFU/ml, after 5 h exposure, to simulated GIT conditions (Table 1). These strains were further examined for the presence of cell surface proteins by SDS-PAGE which revealed that SF9 and SF15 strains possess a potential S-layer protein, with molecular weight of approximately 45 (Fig. 2).

3.2. Probiotic properties of selected autochthonous LAB For typing purpose, Random amplified polymorphic DNA (RAPD) was used for discriminating among selected strains which

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Table 2 Antibiotic susceptibility profiles of the five selected autochthonous LAB isolates. Antibiotic

Concentration

Ampicillin Azithromycin Bacitracin Erythromycin Gentamicin Clindamycin Chloramphenicol Rifampicin Streptomycin Tetracycline Vancomycin

10 ␮g 15 ␮g 10 units 15 ␮g 120 ␮g 2 ␮g 30 ␮g 5 ␮g 10 ␮g 30 ␮g 30 ␮g

Sauerkraut autochthonous LAB isolate SF1

SF2

SF4

SF9

SF15

S I I S S S S S R S R

S S I S S S S S R S R

S S S S S S S S I S S

S S I S S S S S R I R

S I I S I I S S R S R

R: resistant; I: intermediate; S: susceptible.

Fig. 2. SDS-PAGE surface protein profiles of selected autochthonous LAB isolates; lane 1, Lb. plantarum D6; lane 2, SF1; lane 3, SF2; lane 4, SF4; lane 5, SF9; lane 6, SF15; S-low molecular weight protein standards.

resulted in distinct RAPD patterns with variations in the number of bands and fragment size (Fig. 3). According to antibiotic resistance criteria proposed for safety use of bacteria by EFSA (2008) selected autochthonous strains were tested for their susceptibility against 12 different antibiotics. Antibiotic susceptibility testing, by disc diffusion revealed, that none of the 5 strains exhibited phenotypic resistance to the most of tested antibiotics (Table 2). In further experiments, the level of indicator strains inhibition by selected strains was determined. The percentages of inhibition obtained by cell free culture supernatants were strain depended and varied in the range of 10–90% (Fig. 4). For the competitive exclusion of pathogens from the intestinal epithelium, besides antimicrobial properties, an ability of probiotic to coaggregate with pathogens is desirable, whilst autoaggregation represents a first

step for adhesion. Hence coaggregation and autoaggregation abilities of the autochthonous isolates were tested which appeared to be strain-specific. Strain SF15 showed the best autoaggregation ability among the selected strains and coaggregated the best with E. coli 3014 and S. enterica serovar Typhimurium FP1, among all strains tested, while in comparison, coaggregation of SF9 with the same strains was lower (Fig. 5 and Table 3). Fig. 6 represents the adhesion capacity of the selected autochthonous LAB to Caco-2 cell lines. Strain SF9 and especially SF15 demonstrated high adhesion capacity compared to the well-recognised adherent Lb. rhamnosus GG. Other three strains adhered to Caco-2 cells moderately compared to low adhesive E. coli 3014. A common formulation used for probiotic storage and incorporation in foods is freeze-dried culture, hence selected autochthonous strains were freeze-dried with different cryoprotectants. Addition of cryoprotectants supported cell survival after

Fig. 4. Antimicrobial activity of the culture supernatant of strains SF1, SF2, SF4, SF9 and SF15 against indicator strains 1 – S. aureus 3048 2 – S. aureus K-144, 3 – E. coli 3014, 4 – S. enterica serovar Typhimurium FP1, 5 – B. subtilis ATCC 6633 and 6 – B. cereus TM2. 90

autoaggregation (%)

80 70

SF15

60

SF2

50

SF9

40

SF4

30

SF1

20 10 0

Fig. 3. RAPD DNA patterns of five selected autochthonous isolates. Lane S contained the DNA ladder size standards; lane 1: SF1; lane 2: SF2; lane 3: SF4; lane 4: SF9; lane 5: SF15.

0

1

2

t (h)

3

4

5

Fig. 5. Autoaggregation abilities determined for selected autochthonous bacterial isolates SF1, SF2, SF4, SF9 and SF15.

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Table 3 Coaggregation percentage of autochthonous strains SF1, SF2, SF4, SF9 and SF15 with the representatives of pathogen bacteria S. enterica serovar Typhimurium FP1 and E. coli 3014, after 4 h of incubation. Pathogen

Coaggregation (%)

FP1 3014

SF1

SF2

SF4

SF9

SF15

0 0.89 ± 0.66

0 ± 0.10 4.95 ± 1.10

0 7.47 ± 2.69

7.11 ± 3.89 1.37 ± 0.70

8.67 ± 2.16 27.92 ± 2.84

FP1: S. enterica serovar Typhimurium FP1; 3014: E. coli 3014.

Table 4 Survival of the potential probiotic strain cells after freeze-drying with an addition of different cryoprotectans. Initial cell counts were determined before freeze-drying and were calculated by plate counts on approximately 1011 CFU/ml. Autochthonous LAB isolate

Final log CFU/ml

SF1 SF2 SF4 SF9 SF15

PBS

Inulin

Sucrose

Sorbitol

9.11 (0.11) 10.11 (0.06) 8.22 (0.14) 9.45 (0.21) 10.01 (0.02)

10.55 (0.08) 10.78 (0.06) 8.70 (0.03) 10.11 (0.11) 10.79 (0.07)

10.35 (0.04) 10.56 (0.12) 9.64 (0.09) 9.84 (0.07) 10.19 (0.09)

9.75 (0.02) 10. 72 (0.07) 7.08 (0.32) 9.79 (0.13) 10.67(0.11)

Standard deviations of independent triplicate samples are shown in brackets.

freeze drying equally well, as viable cell counts (CFU/ml) of strains were not decreased after the freeze-drying (Table 4). In addition, strains SF2 and SF15, appeared to be highly resistant to NaCl at even 6.0% (w/v) (Fig. 7). Selected autochthonous probiotic strains were subjected to AFLP cluster analysis with all available profiles of reference strains

in BCCM/LMG ‘LAB’ database, which identified the strain SF9 as Lb. paraplantarum and SF15 as Lb. brevis (Fig. 8).

4. Discussion 4.1. Screening of autochthonous LAB sauerkraut isolates

Adhesion capacity (%)

35 30 25 20 15 10 5 0 SF1

SF2

SF4

SF9

SF15

LGG

E.coli

Fig. 6. The adhesion capacity of selected autochthonous isolates SF1, SF2, SF4, SF9 and SF15 to Caco-2 cell line, expressed as the percentage of adhered bacteria (CFU/ml) in the relation to the added bacteria. Lb. rhamnosus GG and E. coli 3014 were tested as an example of the high and the low adhesive strain, respectively.

Fig. 7. Effect of the different NaCl concentrations (4.0–6.0%) on the growth capacity of autochthonous lactic acid bacteria isolates during overnight growth in MRS broth.

Recently published literature is mostly focused on the characterisation of either the cheese or sausage microflora and development of the functional starter cultures for their processing, while less is known about sauerkraut as a source of probiotic starter cultures. Several authors characterised the natural sauerkraut microflora or beneficial effects obtained by an implementation of the starter cultures, originating from the sources other than sauerkraut, in cabbage fermentation, in order to produce sauerkraut of the improved quality in shorter fermentation course and with an application of lower NaCl concentrations (Wiander and Ryhanen 2005; Johanningsmeier et al. 2007; Plengvidhya et al. 2007; Beganovic´ et al. 2011a). The objectives of this research were isolation of LAB during spontaneous sauerkraut fermentation and in vitro investigation of their functional and technological characteristics as autochthonous probiotic starter cultures. Namely, sensory quality of sauerkraut produced in plant “Prehrana Inc.”, Varaˇzdin from autochthonous cabbage cultivar “Varaˇzdinski” was desirable due to the observed leaves’ firmness and crunchiness what implies that the amount of total lactic acid formed and added NaCl levels were satisfactory, and the sauerkraut was evaluated as milder-flavoured. In addition, oxidative changes were not observed according to the sensory evaluation of cabbage heads which were with natural light coloured leaves. Microbiological quality of analysed sauerkraut suggested that this product could be a valuable source of beneficial ˇ MSc Thesis 2009). Application of microorganisms (Spoljarec, selected autochthonous LAB strains as probiotic starter cultures, at high viable cell count, is a critical prerequisite to exert health benefits to the host (Klingberg et al. 2005). These underlines that the need for addition of the well characterised probiotic cultures, in appropriate viable cell counts, would be valuable in probiotic sauerkraut production (FAO and WHO 2006; Beganovic´ et al. 2011a). Moreover, an increase in LAB counts during spontaneous fermentation would be accompanied by faster decline of pH due to the production of lactic acid compared to spontaneous

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Fig. 8. BioNumerics 5.1 software-generated AFLPTM DNA fingerprints from: (a) ID 13940t2 (SF9), BCCM reference strains of Lb. plantarum and phylogenetically related species; (b) ID13941t2 (SF15), reference strains of Lb. brevis from BCCM and phylogenetically related species, and a dendrogram of the cluster analyses of these profiles.

fermentation performed without addition of probiotic starter cultures, what is important from technological perspective. Based on the API carbohydrate fermentation profiles, Lb. plantarum dominated among isolated autochthonous strains. Lb. plantarum is characterised as the most acid tolerant species, predominant in the late, homofermentative phase of sauerkraut fermentation (Johanningsmeier et al. 2007; Plengvidhya et al. 2007). Ln. mesenteroides subsp. mesenteroides is heterofermentative LAB that initiates sauerkraut fermentations by lowering the pH with lactic and acetic acid production while reducing the oxygen level through production of CO2 . This bacterium has a major impact on the flavour and quality of fermented cabbage products. Ln. mesenteroides is a facultative anaerobe requiring complex growth factors and amino acids. Under microaerophilic conditions, heterolactic fermentation is carried out. Glucose and other hexose sugars are converted to equimolar amounts of d-lactate, ethanol and CO2 via a combination of the hexose monophosphate and pentose phosphate pathways while other metabolic pathways include conversion of citrate to diacetyl and acetoin (Breidt 2008). Although quick to predominate, Ln. mesenteroides rapidly dies off, as the fermentation proceeds, due to its sensitivity to acid conditions. After three days of domination of Ln. mesenteroides, the pH decreases,

which is succeeded by Lb. plantarum and other LAB which complete the fermentation. Analyses of soluble whole-cell protein patterns was shown mostly consistent with the physiological results, as it confirmed that Lb. plantarum became the predominant species during the progress of spontaneous fermentation. Lb. plantarum synthesises large amounts of the lactic acid from remaining carbohydrates, thus lowering pH in the final stage of fermentation and becomes the most prevalent group of LAB in sauerkraut because of its inherent acid tolerance and active F0 F1 -ATPase that reguˇ skovic´ et al. 2010). lates intercellular pH (Delgado et al. 2005; Suˇ Both Lb. brevis and Lb. plantarum strains are typical species that are present in a second phase of spontaneous fermentation, after Ln. mesenteroides, which is typical strain in the first phase of fermentation (Tamminen et al. 2004). Acid tolerance is also one of the most important properties during the selection of potential probiotic strains. Indeed, survival of the microorganism in rigorous gastric environment conditions at high viable cell counts which enables them to reach the small intestine and colonise the host, thereby rendering their benefits to host, is one of the most important selection probiotic criteria. Strains SF1, SF2, SF4, SF6, SF9 and SF15 survived simulated gastric and small intestine condition in the highest CFU/ml. As whole cell protein profile showed that some

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of these strains are with similar electrophoretic patterns, further research was limited on SF1, SF2, SF4, SF9 and SF15 strains which were evaluated as potential probiotic strains based on the best survival rates achieved after incubation in simulated GIT conditions. SDS-PAGE analysis of cell surface proteins revealed that SF9 and SF15 strains possess a potential S-layer protein of approximately 45 kDa. These proteins were recovered in the LiCl extracts. This is the first study, to our knowledge, that reports on the presence of potential S-layer proteins in Lb. brevis and Lb. paraplantarum strains isolated from sauerkraut. Particularly, the presence of the potential S-layer in these strains should be studied in detail for their functional role in probiotic properties. Previously, we have reported on functional role of S-layer protein in the probiotic activity of Lb. helveticus M92, primarily in adhesion ability, Salmonella exclusion and immunomodulation in mice intestinal tract (Kos et al. 2003; Frece et al. 2005; Beganovic´ et al. 2011b). Hence, as S-layer was identified in several probiotic lactobacilli, there is increasing scientific evidence that some probiotic properties of bacteria, such as adhesion, aggregation or pathogens inhibition are related to the occurrence of particular types of S-layers. Moreover, detection of S-layer expressing Lactobacillus strains present in complex sauerkraut microbiota could be fast and effective approach to screen for probiotic sauerkraut. 4.2. Probiotic properties of selected autochthonous LAB Several aspects, including safety, functional and technological characteristics, have to be taken into consideration during probiotic selection (Mattila-Sandholm et al. 2002). Characterisation of a strain provides an indication of its presumed safety and meets requirements for exact information on the nomenclature of the strain. Traditional phenotypical identification of Lactobacillus strains, based mainly on fermentation sugar profiles (API CHL 50), is still on a routine base widely applied, but does not provide reliable identification because the different strains of same species cannot be distinguished. Therefore, in order to provide reliable strain discrimination and comparison RAPD method and AFLP were employed. For typing purpose, RAPD technique is reported to be simple and fast to perform, providing good levels of strains discrimination. Hence, RAPD was used for discriminating among selected strains. Although LAB have a long history of safe use in fermented products and are generally recognised as safe (GRAS), the antibiotic resistance pattern of the potential probiotic strains must be determined to limit the possibility of an application of the strains containing transferable antibiotic-resistance genes. Namely, absence of antibiotic resistance is considered as a safety prerequisite for the selection of a probiotic strain (EFSA, 2008). Beneficial strains characterised as functional starter cultures or probiotics must be tested for the presence of antibiotic resistance as there is a possibility that the resistance is transferred to food or intestinal pathogens. These 5 strains were sensitive to most of the tested antibiotics but displayed phenotypic resistance to vancomycin with exception of strain SF4. This antibiotic inhibits the final stages of peptidoglycan assembly by binding to d-alanine – d-alanine terminal residues of cell wall precursors present on the bacterial cell surface. Many Lactobacillus strains carry intrinsic resistance to this drug, owing this to the presence of d-ala-d-lactate in their peptidoglycan instead of d-alanine – d-alanine which is a target of the antibiotic (Bernardeau et al. 2008). Intrinsic resistance in Leuconostoc sp. and especially in lactobacilli to this glycopeptide antibiotic is attributed to the synthesis of modified cell wall peptidoglycan precursors that terminate in lactate (Delgado et al. 2005). This type of resistance is not considered questionable since it is chromosomally encoded and therefore not transmissible unlike inducible, transferable mechanisms observed in other bacteria.

Tested strains have also shown resistance to streptomycin, for which lactobacilli poses high natural resistance (Daniels and Wind 2003). Finally, selected strains SF1, SF2, SF3, SF9 and SF15, with regard to their antibiotic resistance, are considered safe to be used as probiotic. Antimicrobial activity of probiotic strains may contribute to the quality improvement of fermented foods, which is achieved through the control of spoilage and pathogenic bacteria, thus extending shelf-life and improving sensory quality (Kos et al. 2008, 2011; Beganovic´ et al. 2011b). Antagonistic activity, essential in order to prevent the infection of undesirable bacteria, of five potential probiotic strains was determined against the representatives of food pathogens. Antibacterial activity was found against Grampositive and -negative potential pathogens. It is well established that strong antimicrobial activity of LAB is mediated by lactic acid production. Non-dissociated form of lactic acid triggers lower internal pH of the bacterial cell, which causes collapse in electrochemical proton gradient in sensitive bacteria, hence having a ˇ skovic´ et al. 2010). Inhibibacteriostatic or bactericidal effect (Suˇ tion of the pathogens offers a possibility of biopreservation, based on effective competitive exclusion of pathogens by probiotic, which results in extended storage life and enhanced safety of food brought by autochthonous microflora. Coaggregation of probiotic strains with pathogens as well as their antimicrobial activity promotes pathogen exclusion, which is of importance for the recovery of the misbalanced (aberrant) specific microenvironment microflora (Kos et al. 2003; Collado et al. 2007; Beganovic´ et al. 2011b). Hence, coaggregation of the selected strains with common food pathogens, as well as their autoaggregation ability, which is considered as a prerequisite of the adhesion, were investigated. Coaggregation abilities of Lactobacillus species might enable them to form a barrier that prevents colonisation of pathogenic bacteria, while autoaggregation, could be involved in adhesion, which is known to be a prerequisite for the colonisation of the GIT by probiotic strains in high viable cell count. Ability to survive the acidic and bile challenges in GIT and to antagonise enteropathogens is advantageous for probiotic bacteria to adhere to the luminal epithelium. Adhesion to the intestinal epithelium is also another criterion to be fulfilled by probiotic culture preventing immediate washout of the strain by peristalsis. Adhesion is believed to be a requirement for the realisation of probiotic effects, as it is the first step required for colonisation of GIT and important prerequisite for competitive exclusion of enteropathogens and immunomodulation of the host (Kos et al. 2003; Buck et al. 2005; Beganovic´ et al. 2011b). The adhesion mechanisms are not completely understood, however, there is evidence that bacterial cell-surface associated proteins, e.g. S-layer proteins in lactobacilli, could be involved in adhesion through autoaggregation (Kos et al. 2003; Frece et al. 2005; Mobili et al. 2009; Beganovic´ et al. 2011b). As potential surface layer proteins were detected in surface proteins profiles of strains SF9 and SF15, further characterisation of these proteins will be undertaken in order to explain their functional role in specific probiotic characteristics, such as adhesion to intestinal epithelial cells. In addition, technological properties that are considered important for vegetable fermentations, such as technology of probiotic starter cultures preparation, salt tolerance, and specific biochemical properties, must be taken into account in the initial probiotic starter screening. Overall, important prerequisites for the use of probiotics are that they are able to survive and maintain their health-promoting properties throughout the technological process. Nowadays, starter cultures are mostly applied freeze-dried. Freeze-drying of the cultures causes cellular stress, which affects the culture recovery and growth (Sánchez et al. 2005). In order to establish if freeze-drying affects the survival of selected strains, their viability was determined after freeze-drying. Besides PBS

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and sucrose, common prebiotics inulin and sorbitol were tested as potential cryoprotectants. It can be concluded that viabilities of bacteria after freeze-drying were shown to be strain specific and dependent on protective agents. An appropriate selection of these factors is essential for obtaining maximum survival of bacterial cells which is of great importance for the industrial development of the starter culture formulation. The addition of the salt is one of the critical points in sauerkraut fermentation as it influences the type and extent of microbial ˜ growth and sensory properties of the final product (Penas et al. 2010; Beganovic´ et al. 2011a). Most of the strains were similarly inhibited by NaCl, with the exception of SF15 which was more resistant. Here it should be emphasised that in our experiment the lowest NaCl concentration tested (4%, w/v) represents the maximum level of NaCl usually added during sauerkraut fermentations, hence indicating that selected strains could be applied in sauerkraut fermentations. Moreover, as these stains are shown to be antimicrobial, their application in sauerkraut fermentation could reduce the level of NaCl applied in fermentations, which is, besides of economical, also of ecological importance. Finally, in the context of development of probiotic starter culture it is very important to be able to identify specifically particular probiotic LAB strains from food samples by molecular identification methods. AFLP method is rapid and reliable tool for molecular fingerprinting in order to identify and distinguish various strains of the same species as well as pulsed-field gel electrophoresis (PFGE) and PCR typing techniques based on 16S rRNA genes sequences (Ventura and Zink 2002; Di Cagno et al. 2010). Therefore, computer-generated profiles and dendrograms of the cluster analyses of AFLP DNA fingerprints from reference strains of the subspecies of Lb. paraplantarum and Lb. brevis were obtained and compared with autochthonous strains isolated and characterised in this work, Lb. paraplantarum SF9 and Lb. brevis SF15.

5. Conclusions Nowadays consumers demand for more natural and traditional products, with reduced amount of chemically synthesised preservatives, because of their unique characteristics which are related with the artisanal production and specific organoleptic profiles. Thus, there is a need to protect these products and promote their value. Therefore, in this study, two potential probiotic starter cultures from autochthonous LAB sauerkraut population were preliminary characterised. Besides their potential as probiotics, these strains are promising to be applied as mix functional starter cultures in sauerkraut production with aim to improve safety and quality of the product, while preserving the typical sensory characteristics of traditional sauerkraut, and thus bringing additional functional value to the final product. Moreover their application offers besides health promoting even nutritional, technological and economic advantages of sauerkraut fermentation on large scale industrial production.

Acknowledgments The authors are grateful for the financial support from The Ministry of Science and Technology of Republic Croatia (Projects: “Probiotics, prebiotics and functional starter cultures” – 0580581990-2007 and “Production and application of probiotic ˇ and Prehrana Inc., cultures” TP 5039). We thank Marina Spoljarec Varaˇzdin, Croatia for her cooperation and providing the sauerkraut samples during the course of spontaneous fermentations, used in this study.

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