International Journal of Food Microbiology 191 (2014) 164–171

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Selection of functional lactic acid bacteria as starter cultures for the fermentation of Korean leek (Allium tuberosum Rottler ex Sprengel.) Jaesik Yang a, Yosep Ji a,b, Hyunjoon Park a,b, Jieun Lee a, Soyoung Park a,b, Soyoung Yeo a, Hyunkil Shin a, Wilhelm H. Holzapfel a,b,⁎ a b

School of Life Sciences, Handong Global University, Pohang, 791-708 Gyeongbuk, South Korea Graduate School of Advanced Green Energy and Environment (AGEE), South Korea

a r t i c l e

i n f o

Article history: Received 24 May 2014 Received in revised form 19 August 2014 Accepted 14 September 2014 Available online 19 September 2014 Keywords: Allium tuberosum Anti-oxidant capacity Lactic fermentation Korean leek

a b s t r a c t The purpose of this research was to find safe and suitable starter cultures for the fermentation of Korean leek (Allium tuberosum Rottler), also known as garlic chives or Oriental garlic. This traditional herb has several functional properties and a strong flavour; its leaves are used as food material. Eighteen strains of lactic acid bacteria (LAB) were isolated from well-fermented leek kimchi. Controlled fermentation of the leek leaves was conducted with 2 strains (Weissella confusa LK4 and Lactobacillus plantarum LK8), selected as potential starter cultures on the basis of their safety properties, and on the pH, total titratable acidity (TTA), and viable cell numbers [colony forming units (CFU ml−1)] achieved during the fermentation. Microbial dynamics was also followed during fermentation by using PCR-DGGE (Denaturing Gradient Gel Electrophoresis) on DNA level. To analyse bioactive compounds such as thiols and allicin (diallyl thiosulfinates), the total flavonoid and polyphenolic contents were determined by colorimetric methods. Functional properties were assessed on the basis of anti-oxidative capacities by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, and ferric reducing antioxidant power (FRAP). W. confusa LK4 rapidly increased during the first stage of leek fermentation, and was mainly responsible for accelerated fermentation during the early period in contrast to L. plantarum LK8, a stronger acid producer during the later stages of fermentation. After 48 h fermentation, leeks fermented with W. confusa LK4 showed the highest radical scavenging effects and reducing ability. The detectable amount of allicin of fermented leeks decreased relative to the change in pH, whereas the concentration of thiols significantly increased. Total flavonoid and poly-phenolic contents changed during fermentation and showed correlation with anti-oxidant effects. We therefore suggest the suitability of W. confusa LK4 as a potential starter culture for fermentation of leeks. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Leek (Allium tuberosum Rottler) belongs to the family Alliaceae that includes garlic and onion. It is one of the most commonly used vegetable ingredients in Korean dishes and, in addition, is often used for therapeutic purposes in traditional remedies for relieving conditions such as abdominal pain, diarrhoea, hematemesis, snakebite and asthma (Zhong, 1985). Several studies have reported beneficial properties of leek to be related to anti-oxidant (Lee et al., 2002; Yin and Cheng, 1998), anticarcinogenic (Park et al., 2002) and anti-hyperlipidemia effects (Son et al., 2012). However, the functional agents in the raw material are considered to be negatively influenced by different stress factors such as acid, heat and drying during processing, also including fermentation.

⁎ Corresponding author at: Graduate School of Advanced Green Energy and Environment (AGEE), Handong Global University, Pohang, 791-708 Gyeongbuk, South Korea. Tel.: +82 54 260 1314. E-mail address: [email protected] (W.H. Holzapfel).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.09.016 0168-1605/© 2014 Elsevier B.V. All rights reserved.

Yin and Cheng (1998) reported enhanced pro-oxidant activity in allium members after exposure to different stresses. Fermentation is widely accepted as a traditional means of food preservation; at the same time it can contribute to the sensory, safety and functional quality of a food (Bourdichon et al., 2011). Using starter cultures in food processing is a most promising method for maintaining consistent quality and safety of fermented products. This may be achieved by inoculating high numbers of viable starter cultures, thereby also effectively combating undesirable microorganisms during typical food fermentation processes (Holzapfel, 1997). With an increasing tendency towards more diversity in vegetable fermentations, benefits of both commercial and allochthonous starters have been reported for controlled lactic fermentation of vegetables and fruits. Selected autochthonous lactic acid bacterial (LAB) starter cultures, tailored for the specific plant matrix, were showed to be technologically superior, and, in addition, contributed to improvement of functional properties (Di Cagno et al., 2014). In Korea, various processes, in particular fermentation, are used for preparing leek with the purpose of reducing its strong odour and to

J. Yang et al. / International Journal of Food Microbiology 191 (2014) 164–171

maintain and/or increase its functional capacity. One paper reported on the anticarcinogenic and anti-mutagenic effect of leek kimchi (Jung et al., 2002), while changes in the antioxidant properties of green (European) leek leaves (Allium ampeloprasum var. porrum) have been studied by Štajner and Popović (2009) and Bernaert et al. (2013). Bioactive compounds of Egyptian leek (A. ampeloprasum var. kurrat) have also been reported (Abd El Rehem et al., 2013). The aim of this study was to develop a starter culture for leek fermentation and to determine its population dynamics, safety and beneficial impact in comparison to spontaneous fermentation as control. Weissella confusa LK4 and Lactobacillus plantarum LK8 were isolated from well fermented leek kimchi (pH 4.2) as candidate starter cultures. Safety of strains showing potential as starter cultures was assessed on the basis of biogenic amine production and resistance to antibiotics. In addition, their influence on the regulation of the microbial population during leek fermentation was investigated using Denaturing Gradient Gel Electrophoresis (DGGE) and determining viable plate counts. Bioactive compounds related to anti-oxidant and strong odours such as thiols, allicin, flavonoid and poly-phenolic contents were analysed (Ahn et al., 2005; Rabinkov et al., 2000; Yabuki et al., 2010), while the anti-oxidant capacity was determined by the DPPH and FRAP methods. 2. Materials and methods 2.1. Plant material (leek) An amount of 300 g leek powder, harvested and crushed in Kyungbuk Province, Republic of Korea, was obtained commercially online from Chamsaemi Food (http://www.chamsm.com/). 2.2. Starter culture candidates 2.2.1. Isolation, identification and culturing of strains 500 g of well-fermented leek kimchi was obtained from Juk-do traditional market in Pohang, Kyung-buk province, Republic of Korea. Supernatant was collected and plated on MRS agar after suspending 10 g of leek kimchi in 90 ml of 0.85% NaCl solution, and homogenised in a Stomacher 400 (Seward Co., London, United Kingdom) at 230 rpm for 3 min. Serial dilutions were made in 0.85% NaCl solution, and 0.1 ml aliquots were plated onto MRS agar (Difco Co., USA) followed by incubation at 30 °C for 48 h. In total, 18 colonies were randomly selected for isolation. They were purified by streaking on MRS agar, and identified by phenotypic tests and 16S rRNA gene sequencing, as Weissella

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viridescens (1 strain), W. confusa (2), Weissella cibaria (2), Lactobacillus alimentarius (3) and L. plantarum (4). The three pediococci and three rod-shaped strains were not further identified because they showed slow growth in MRS media (Table 1). Safety tests were performed on the basis of antibiotic resistance and determination of biogenic amine production, resulting in the final selection of one strain each of Weissella and Lactobacillus for further investigation. 2.2.2. Agar diffusion method for antibiotic resistance The tests were performed according to Clinical and Laboratory Standards Institute guidelines (CLSI), formerly NCCLS (NCCLS, 1997), as adopted by EFSA (2008). For the assessment of susceptibility to antibiotics of the isolated strains, serial two-fold dilution procedures in agar were used. Test strains were grown in MRS at 30 °C for 18 h and then diluted with the same fresh medium to a density 105 CFU/spot. Plates were incubated aerobically at 30 °C for 48 h and examined for growth. The minimum inhibitory concentration (MIC) was considered as the lowest concentration of an antibiotic that inhibits bacterial growth. 2.2.3. Detection of biogenic amine production For the detection of biogenic amine production, the procedures of Bover-Cid and Holzapfel (1999) were used. All strains were subcultured 2 times in MRS broth containing 0.1% of each precursor amino acid (LTyrosine disodium salt, L-Histidine monohydrochloride monohydrate, L-Ornithine monohydrochloride, and L-Lysine monohydrochloride, all obtained from Sigma Aldrich, Co., USA) in order to activate specific enzyme induction. Subsequently all strains were streaked out in duplicate on the decarboxylase medium and plates containing 1% of each amino acid (control without amino acid, and incubated at 30 °C for 2 days). 2.2.4. Identification of strains by 16S rRNA gene sequencing 16S rRNA of the selected strains was amplified by PCR using specific primers (27F 5′-AGA GTT TGA TCC TGG CTC AG-3′, 1492R 5′-GGT TAC CTT GTT ACG ACT T-3′). PCR products were sequenced bi-directionally at SolGent Co. (SolGent, Korea). The partial 16S gene sequences were compared with sequences in the NCBI (accession numbers: NR_040816.1 for W. confusa, and NR_075041.1 for L. plantarum). 2.3. Fermentation of leek 2.3.1. Fermentation Leek powders were fermented with the two selected bacterial strains. Briefly, 2 g (5%) of leek powder and 0.8 g (2%) of sodium

Table 1 Physiological properties and 16S rRNA gene sequencing of strains isolated from leek kimchi and analysis by BLAST. “+”: positive, “−”: negative, “X”: no data. Strain no.

Growth in MRS (30 °C, 37 °C)

Catalase test

Morphology

16S rRNA gene sequencing data (closest relative)

Similarity (%)

LK1 LK2 LK3 LK4 LK5 LK6 LK7 LK8 LK9 LK10 LK11 LK12 LK13 LK14 LK15 LK16 LK17 LK18

+ + + + + + + + + + + + + + − + + +

− − − − − − − − − − − − − − − − − −

Cocci in tetrads Cocci in tetrads Rod Rod Rod Cocci in tetrads Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod

X X Weissella viridescens (strain NRIC 1536) Weissella confusa (strain JCM 1093) Lactobacillus plantarum strain (NRRL B-14768) X Lactobacillus plantarum (strain WCFS1) Lactobacillus plantarum (strain WCFS2) Lactobacillus plantarum (strain WCFS3) Lactobacillus alimentarius 16S ribosomal RNA, complete sequence Lactobacillus alimentarius 16S ribosomal RNA, complete sequence X Lactobacillus alimentarius 16S ribosomal RNA, complete sequence X X Weissella cibaria (strain II-I-59) 16S ribosomal RNA, partial sequence Weissella cibaria (strain II-I-59) 16S ribosomal RNA, partial sequence Weissella confusa (strain JCM 1093) 16S ribosomal RNA, partial sequence

X X 99 99 99 X 99 99 99 99 99 X 99 X X 98 99 98

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chloride (NaCl, Merck, Darmstadt, Germany) solution were added to 40 ml distilled water in a 50 ml Falcon tube. 4 × 107 CFU/ml of each bacterial strain (washed 2× in 1 ml of 0.85% NaCl solution) or 1 ml of 0.85% NaCl solution (for spontaneous fermentation as control) was inoculated. After mixing, samples were incubated at 30 °C with shaking at the rate of 125 rpm. 2.3.2. pH, TTA and viable cell numbers 10 g of each fermented sample and 90 ml 0.85% NaCl solution were mixed for measuring pH and TTA. The pH was determined with an Orion 2Star bench top pH Meter (Thermo, USA). TTA was titrated up to pH 8.2 with a 0.1 N NaOH solution. Viable bacterial numbers were determined in the fermented samples by duplicate plating onto MRS agar (Difco Co., USA) and incubation at 30 °C for 48 h. Prior to plating, serial dilutions were made by homogenising 10 ml of each sample with 90 ml Ringer buffer in a Stomacher 400 (Seward Co., London, United Kingdom). 2.3.3. Determination of the lactic acid enantiomers produced Lactic acid enantiomers in samples were determined using D/L-lactic acid kits (R-, Biopharm, Darmstadt, Germany) according to the manufacturer's instructions. This is based on the oxidation of D(−)lactate and L(+)-lactate to pyruvate in the presence of NAD+, which in turn reduces to NADH by the corresponding enzymes D(−)-lactatedehydrogenase or L(+)-lactate-dehydrogenase. 2.4. Sample collection and storage Samples were collected time dependently and centrifuged at 4 °C and 5000 g for 15 min. Pellets were used to obtain DNA, and supernatants were filtered with Whatman No. 1 paper filter (GE Health Care, UK) and used to analyse bioactive compounds and anti-oxidant capacity. Filtered supernatants were stored at 4 °C. 2.5. PCR-DGGE Genomic DNA was obtained by means of the Qiagen DNA mini-kit (Qiagen, Venlo, Netherlands), following the product protocols. PCR amplification was carried out based on procedures described earlier (Janse et al., 2004; Smith and Osborn, 2009), while the PCR primer 357F-GC/ 518R (Muyzer et al., 1993) was used. A 35–50% denaturing gradient was used and the 8% polyacrylamide gel was 1.5 mm thick. Electrophoresis was performed at 60 °C and 60 V during a period of 16 h 30 min. For DGGE band sequencing, distinct bands were excised from the gel with a sterile scalpel blade (AILEE Co, Korea), washed in 70% ethanol, and then crushed into distilled water and stored 4 °C for 24 h. After amplification of Band DNA, PCR products were generated with 357F-GCM13R, 518R-AT-M13F primers (O'Sullivan et al., 2008)

2.6.2. Polyphenol and flavonoid contents The total poly-phenolic content of the sample was measured based on the procedures described by Singleton et al. (1999) with minor modification. Briefly, an aliquot of 20 μl samples was mixed with 100 μl Folin–Ciocalteu phenol reagent (Sigma Aldrich Co., St. Louis, MO, USA) and allowed to react for 3 min. Then 80 μl of a 2% Na2CO3 solution (Junsei chemical Co., Ltd., Japan) was added and allowed to react for 30 min at room temperature comparatively to a gallic acid (Sigma Aldrich Co., St. Louis, MO, USA) standard. Absorbance was measured at 725 nm using an automated microplate reader (Spectronano Star, BMG Labtech, USA). The results were expressed as mg gallic acid/l extract. The total flavonoid content (TFC) of the samples was determined using a modified colorimetric method described previously by Zhishen et al. (1999). Rutin hydrate (Sigma Aldrich Co., St. Louis, MO, USA) was used as a standard. Either extracts or standard solutions (0.1 ml) were mixed with distilled water (0.4 ml) and 0.5 ml of a 5% NaNO2 solution (Duksan Pure Chemical Co., Ltd., Korea). After allowing a reaction time of 6 min, the mixture was combined with 0.06 ml of a 10% AlCl3 (Sigma Aldrich Co., St. Louis, MO, USA) solution. 1 N NaOH (0.2 mL) was added to the mixture 5 min later. The absorbance of the solutions was then measured at 510 nm. The results were expressed as rutin equivalent (RE) (mg rutin/l extract). 2.7. Measurement of anti-oxidative activity 2.7.1. Radical scavenging activity (DPPH) The free radical scavenging activity of the methanol extract was measured using the method of Molyneux (2004). Briefly, a 0.2 M solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma Aldrich Co., St. Louis, MO, USA) in methanol was prepared, and 180 μl of this solution was added to 20 μl of the test sample or L-ascorbate standard (Sigma Aldrich Co., St. Louis, MO, USA). After 30 min incubation at ambient temperature, the absorbance was measured at 517 nm. The results were expressed as Vitamin C equivalent antioxidant capacity (VCEAC) in mg/l extract. 2.7.2. Reducing power (FRAP) FRAP (ferric reducing ability of plasma) was measured using the protocol of Benzie and Strain (1996). Briefly, the FRAP reagent was prepared as required by mixing 25 ml acetate buffer (300 mmol/l; pH 3.6), 2.5 ml of a 2,4,6‐tri(2‐pyridyl)‐1,3,5‐triazine (TPTZ) (Sigma Aldrich Co.) solution (10 mmol/l), and 2.5 ml of a FeCl3·6H2O (Sigma Aldrich Co., St. Louis, MO, USA) solution (20 mmol/l). 190 μl of the FRAP reagent was added to 10 μl of sample extract and was allowed to react for 30 min. The absorbance was measured at 593 nm. L-Ascorbate was used as a standard. 2.8. Statistical analysis

2.6. Analysis of bioactive compounds 2.6.1. Thiols and allicin Thiol and allicin contents of the sample were measured based on the colorimetric procedures described by Han et al. (1995) with modification. For analysing thiols, 100 μl of diluted samples in 50 mM HEPES buffer (pH 7.5), were added to 100 μl of 1.5 mM 5, 5′-dithios-bis-(2nitrobenzoic acid) (DTNB) and allowed to 10 min at room temperature. The absorbance was measured at 412 nm. To determine the allicin content, 100 μl of each sample was mixed with 250 μM of L-cysteine for reaction. After 10 min, 100 μl of the mixture was added to 100 μl of 1.5 mM DTNB and allowed to react for 10 min at room temperature. The absorbance was measured at 412 nm. Thiol and allicin contents were expressed as μM cysteine equivÞ ¼ Allicin contents μM alent/l of extract (Han et al., 1995): ½c‐ðb‐a 2 ðcysteine equivalent Þ(a: Thiols in samples, b: Thiols after reaction with L-cysteine, c: added L-cysteine).

Data from analytical determinations were at least the means of two independent experiments carried out in triplicate. One-way analysis of variance (ANOVA) was applied to experimental data. Differences with statistical significance were compared using Tukey's HSD test. All statements of significance were based on a probability of 0.05. 3. Results 3.1. Strain selection and identification For the selection of starter culture candidates, 18 strains were isolated from well-fermented leek kimchi (pH 4.2). They were purified by streaking on MRS agar, and by phenotypic and molecular procedures, identified as W. viridescens (1 strain), W. confusa (2), W. cibaria (2), L. alimentarius (3), and L. plantarum (4). Safety tests were performed by antibiotic resistance tests and determination of biogenic amine

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production. Among the 18 strains, 17 produced no biogenic amines, while W. cibaria strains showed slightly elevated resistance to Gentamicin and Ciprofloxacin, and one strain of L. plantarum (LK 5) to Chloramphenicol and three (LK 5, 7 and 9) to Ciprofloxacin. The L. alimentarius strains LK 10 and 13 showed only low Chloramphenicol resistance, but, based on pH, TTA, and viable cell numbers, did not show satisfactory growth in leek in preliminary experiments. Two strains, W. confusa LK4 and L. plantarum LK8, were selected on the basis of the safety tests and their high viable numbers during preliminary leek fermentation tests. They showed excellent growth, both in MRS and in the leek powder fermentation medium (data not shown). MIC values for antibiotic resistance of both strains were either equal to or lower than the breakpoint values suggested by Danielsen and Wind (2003) and also in line with EFSA (2008), with LK8 as the only L. plantarum strain not exceeding the breakpoint value for Ciprofloxacin. None of these strains produced any detectable biogenic amines in the screening test. Information on their identification, antibiotic resistance and biogenic amine formation is given in Tables 1 and 2. 3.2. Fermentation An 18 h culture of each of the selected two strains, W. confusa LK4 and L. plantarum LK8 in MRS broth, mixed with one volume of 0.85% NaCl was used for leek fermentation. During fermentation at 30 °C, pH, total titratable acidity (TTA) and viable cell counts were determined at specific time intervals (see Fig. 1). During spontaneous fermentation (SF) the pH was decreased after 24 h to pH 4.40 ± 0.04 (SEM) and showed minor change up to 48 h of fermentation. Inoculation with W. confusa LK4 and L. plantarum LK8 resulted in a stronger lowering of the pH from the outset and throughout the fermentation period, reaching pH 4.27 ± 0.03 (SEM) and pH 3.71 ± 0.01 (SEM), respectively, after 48 h (Fig. 1a). The total titratable acidity (TTA), determined by using a 0.1 N NaOH solution, of all samples increased over time. Inoculation with L. plantarum LK8 showed the strongest increase in TTA after 24, and continued for the 48 h period, while fermentation with W. confusa LK4 resulted in a gradual but constant increase of TTA throughout 48 h fermentation. Acid formation during spontaneous fermentation was lagging and slow during the first 12 h but was subsequently followed by a rapid increase in TTA (Fig. 1b). Viable cell (CFU/ ml) numbers of W. confusa LK 4 on MRS increased at a higher rate than those of L. plantarum LK8 during the first 6 h, and also remained at the level of ca. 8.5 log10 CFU/ml throughout, thus suggesting the superior adaptation of strain LK4 to the leek substrate. Following a slow initial growth rate, viable cell numbers in the spontaneous fermentation reached levels equal to LK 4 after 48 h at 30 °C (Fig. 1c). Lactic acid enantiomer production of each sample was determined after 48 h fermentation and compared with the TTA (Table 3).

Fig. 1. Concentration of D(−) and L(+) lactic acid in the leek samples after 48 h fermentation. Results are expressed as mean ± SEM (n = 3)

Table 2 Safety evaluation of selected strains based on antibiotic resistance [minimum inhibitory concentration (MIC) in μg of antibiotic/ml], and formation of biogenic amines. Strain and identification

Erythro-mycin

Gentamicina

Tetracycline

Chloramphenicol

Streptomycin

Ciprofloxacin

Biogenic amine

LK3 (W. viridescens) LK4 (W. confusa) LK5 (L. plantarum) LK7 (L. plantarum) LK8 (L. plantarum) LK9 (L. plantarum) LK10 (L. alimentarius) LK11 (L. alimentarius) LK12 (L. alimentarius) LK13 (L. alimentarius) LK16 (W. cibaria) LK17 (W. confusa) LK18 (W. cibaria) Suggested breakpoints acc. to EFSA (2008) Suggested breakpoints acc. to Danielsen and Wind (2003)

2 0.25 b0.25 0.5 1 0.25 0.25 b0.25 0.5 b0.25 2 1 1 1 4

128 128 128 128 128 128 64 64 128 128 256 256 256 16 128

64 8 16 32 16 32 8 8 4 8 64 64 64 32 64

4 4 32 8 16 4 4 8 2 2 16 16 16 8 16

128 128 128 128 32 64 64 32 64 64 256 256 256 n.r. N256

32 32 128 64 32 64 32 32 64 16 128 128 128 n.r. 32

– – – – – – – – – – – – –

n.r.: not required. a Possible interference of the growth medium.

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Table 3 Concentration of D(−) and L(+) lactic acid in the leek samples after 48 h fermentation. Results are expressed as mean ± SEM (n = 3). Samples

D(−) lactic acid

L(+) lactic acid

SF LK4F LK8F

1.424 ± 0.072 g/l 1.243 ± 0.055 g/l 5.498 ± 0.109 g/l

1.068 ± 0.118 g/l 1.386 ± 0.059 g/l 3.234 ± 0.116 g/l

SF: spontaneous fermentation, LK4F: fermentation with W. confusa LK4; LK8F: fermentation with L. plantarum LK8.

Table 4 M13-20F sequencing results of selected DGGE bands from bacterial fingerprints in Fig. 2. Band no.

Closest relative (NCBI accession no.)

Identity (%)

1, 2, 3, 5

Uncultured Weissella sp. clone B6B 16S ribosomal RNA gene, partial sequence (HQ897401.1) Weissella confusa strain FS044 16S ribosomal RNA gene, partial sequence (KC568546.1) Weissella confusa strain JCM 1093 16S ribosomal RNA, partial sequence (NR_040816.1) Lactobacillus plantarum strain KDLLL1-1 16S ribosomal RNA gene, partial sequence (KC454277.1)

100

4 6 7

100 100 100

3.3. Population dynamics PCR-DGGE was performed with DNA extract from fermented leek samples in each time (0, 6, 12, 24, 48 h). After 24 h spontaneous fermentation (SF), four bands were identified as uncultured Weissella sp. and another one corresponding to W. confusa. In leek fermented with W. confusa LK4, a band identified as W. confusa was detected from 6 h to 48 h. For L. plantarum LK8, a band identified as L. plantarum was detected at 12 h until 48 h; it increased in intensity with time (Fig. 2). Identification of selected DGGE bands from bacterial fingerprints is shown in Table 4.

3.4. Bioactive compounds

Samples fermented with L. plantarum LK8 (LK8F) contained the lowest allicin contents after 48 h fermentation, while for thiol contents the highest values were determined after 48 h fermentation. TPC values for LK8F showed a similar tendency as with W. confusa LK4 fermentation. Total flavonoid contents decreased significantly until 12 h fermentation, after which the contents increased by 126.11 (RE) mg/l. The final concentration of bioactive compounds in LK8F was 85.45% lower for allicin contents, but increases of 39.37% for thiol contents, 6.49% for TPC and 8.71% for TFC were measured when compared to spontaneous fermentation. 3.5. Anti-oxidant capacities

Bioactive compounds were determined by colorimetric analysis. Table 5 shows the changes in bioactive compounds of fermented leeks during fermentation. All bioactive compounds showed significant decrease during spontaneous fermentation, but with a significant reduction of allicin only detected after 24 h and 48 h of fermentation. On the other hand, thiols and total poly-phenolic contents (TPC) showed only a gradual decrease during the first 24 h of fermentation, with a reduction of 4.11% in the thiol content and 5.03% in TPC, as determined after 24 h. Total flavonoid contents decreased over time by 16.67% up to 48 h. In leeks fermented with W. confusa LK4 (LK4F), allicin contents decreased gradually during fermentation, showing a stronger, yet not differing statistically significantly when compared with spontaneous fermentation. Thiols and total poly-phenolic contents (TPC) were reduced significantly until 12 h fermentation to an average value of 281.66 mg of TPC (as GAE)/l, after which these increased up to a mean value of 358.43 up to 48 h fermentation. The highest value of total flavonoid contents (TFC) during fermentation was detected after 6 h fermentation after which it decreased significantly up to 12 h of fermentation, and remained fairly constant, within the range of variation, after 24 to 48 h. The final concentration of bioactive compounds in LK4F was 10.16% lower for allicin contents, but 17.70% higher for thiol contents, 7.32% for TPC, 11.03% for TFC, respectively, as compared to spontaneous fermentation.

To determine anti-oxidant capacities, DPPH radical scavenging activities and ferric reducing antioxidant power were measured, either as Vitamin C equivalent antioxidant capacity (VCEAC) or as vitamin C equivalent reducing power (VCERP). During spontaneous fermentation, DPPH radical scavenging activity was decreased by 9.23% over the fermentation period of 48 h. Fermentation with LK 4, and LK8 over 48 h resulted in an increase in DPPH radical scavenging activity values, respectively reaching 362.13 ± 48.28 and 368.68 ± 28.97 of VCEAC (in mg/l), corresponding to a 16.9% and 18.38% increase, compared to spontaneous fermentation (Fig. 3). The results of ferric reducing power (as VCERP) (Fig. 4) showed a similar tendency to those of DPPH activities. Spontaneous fermentation resulted in 17.37% lowering of the ferric reducing power, while inoculation with the strains LK4 and LK8 resulted in a fermentative increase over 48 h of the maximum values up to 416.59 ± 5.38 and 418.18 ± 6.57 (VCERP) (in mg/l), respectively, which corresponded to a 19.41% and 19.72%, respectively, compared to spontaneous fermentation. 4. Discussion The quality and interactive changes during spontaneous fermentation of a food condiment are neither predictable nor controllable. Appli-

Fig. 2. M13-20F sequencing results of selected DGGE bands from bacterial fingerprints in Fig. 2.

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Table 5 Changes in bioactive compounds during spontaneous fermentation of leek, compared to fermentation with W. confusa LK4 and L. plantarum LK8. Samples

Fermentation time (h)

Allicin (μM CE)

SF

0 6 12 24 48 0 6 12 24 48 0 6 12 24 48

416.69 449.15 429.60 233.67 169. 88 434.11 427.52 333.08 219.19 154.21 407.66 431.98 252.92 135.46 91.61

LK4F

LK8F

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Thiols (μM CE) 22.88ef 14.52f 10.86ef 8.40c 6.52b 38.47ef 11.77ef 23.04d 3.75c 16.10b 21.70e 11.51ef 17.83c 12.77b 3.09a

232.86 212.22 195.16 180.48 188.21 234.05 214.80 162.22 224.13 228.69 224.52 204.09 137.22 255.87 310.44

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

TPC (mg GAE/l) 9.70g 5.19efg 8.14cde 9.60bc 6.69cd 8.57gh 23.94efg 7.14b 17.10fg 5.05g 5.27fg 4.83def 5.74a 8.72h 18.45i

351.98 334.17 321.19 315.47 332.17 346.93 297.22 281.66 339.66 358.43 345.85 335.22 238.84 358.36 355.22

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

13.35fg 13.14def 15.04cde 11.70cd 9.39def 17.39fg 9.41bc 8.94b 13.26efg 21.49fg 13.28fg 8.58ef 10.46a 14.42g 12.41g

TFC (mg RE/l) 384.5 362.08 329.03 346.81 320.42 386.58 421.25 257.64 356.81 360.14 416.58 358.75 224.86 372.92 350.97

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

14.42de 27.27bcd 28.94bc 26.66bcd 14.46bc 18.42de 51.02e 50.60b 17.04cd 23.47cde 27.81e 33.00bcd 30.82a 37.89cde 21.86bcd

SF: spontaneous fermentation, LK4F: fermentation with W. confusa LK4; LK8F: fermentation with L. plantarum LK8; CE: cysteine equivalent; GAE: gallic acid equivalent; RE: rutin equivalent; TPC: total poly-phenolic content; TFC: total flavonoid content. Values represent mean ± SD (standard deviation); the same character in the same column indicates no significant difference (p b 0.05). ‘a’ represents the least value group.

cation of selected starter cultures can contribute to an improvement in flavour, safety and quality of a food, and also serves as some guarantee towards uniform production. The rationale for the selection of suitable starter cultures, may include considerations such as: (i) accelerated metabolic activities for reducing fermentation times (acidification or alcohol production), (ii) desirable sensory attributes, (iii) improved safety and reducing of hygienic and toxicological risks, and (iv) probiotic (functional or health beneficial) properties (Holzapfel, 2002). We isolated and purified lactic acid bacterial strains from wellfermented leek kimchi (pH 4.2), and, after preliminary identification by physiological tests, selected potentially suitable strains on the basis of safety tests including antibiotic resistance and biogenic amine production. To determine suitability of the strains for leek fermentation, we designed in vitro leek fermentations with selected strains and compared these with spontaneous fermentation on the basis of pH, TTA, and viable cell counts were conducted. Based on their performance, two strains, W. confusa LK4 and L. plantarum LK8, were found to be well adapted to leeks by the decrease in pH, lactic acid production, and their growth as determined by viable numbers over a period of 48 h. Compared to spontaneous fermentation, W. confusa LK4 showed accelerated growth and a more rapid rate of acid production during the first 12 h, while maintaining a “mild” pH of around 4.2 throughout the final fermentation period up to 48 h. By comparison, L. plantarum LK8 produced 3.4 times more lactic acid than LK4 and reduced the pH to 3.7 during the second part of the 48 h period. DGGE data showed

W. confusa and other Weissella spp. to dominate during spontaneous fermentation. Jung et al. (2012) reported that the proportion of Weissella strains in kimchi inoculated with starter was maintained at a higher level as compared to spontaneous kimchi fermentation without starter. Wouters et al. (2013) studied species diversity, community dynamics, and metabolite kinetics of spontaneous leek (Allium ampeloprassum var. porrum) fermentations. They reported the disappearance of Enterobacteriaceae, Pseudomonadaceae and yeasts after one week while strains of Leuconostoc mesenteroides, Lactobacillus sakei, L. plantarum, Lactobacillus brevis and Lactobacillus parabrevis were most frequently isolated. In our study, the levels of both W. confusa LK4 and L. plantarum LK8 were maintained effectively during the full fermentation period and no other undesirable microorganisms were detected. This contrasts to some extent with the report by Jung et al. (2012) on a stronger quantitative decrease in bacterial operational taxonomic units (OTUs) than in spontaneously fermented kimchi. Our data suggest the suitability of the two strains, LK4 and LK8, as potential candidates for starter cultures for leek fermentation, and are supported by their competitive behaviour and antagonistic activities against undesirable microbes. The presence of ‘abundant’ bioactive compounds, including allicin, thiols, poly-phenolic compounds and flavonoids, has been reported for different kinds of leek (Abd El Rehem et al., 2013; Bernaert et al., 2013; Bernhard, 1969; Jastrzebski et al., 2007; Soininen et al., 2014). Allicin (diallyl thiosulfinate) is responsible for the typical strong odour of

Fig. 3. DPPH radical scavenging activities in samples during fermentation of leek. VCEAC: Vitamin C equivalent antioxidant capacity; SF: spontaneous fermentation; LK4F: fermentation with W. confusa LK4; LK8F: fermentation with L. plantarum LK8; values represent mean ± SD (standard deviation). Values with different superscript letters are significantly different, p b 0.05.

Fig. 4. Ferric reducing antioxidant power of samples during fermentation of leek. VCERP: Vitamin C equivalent reducing power. SF: spontaneous fermentation; LK4F: fermentation with W. confusa LK4; LK8F: fermentation with L. plantarum LK8; values represent mean ± SD (standard deviation). Values with different superscript letters are significantly different, p b 0.05.

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leeks as well as their antimicrobial properties. In our study, fermentation, both with and without starter cultures, resulted in a significant decrease of allicin contents of leeks; this may be attributed both to its volatile nature (Yabuki et al., 2010) and to its utilisation by the lactobacilli involved in the fermentation (Filocamo et al., 2012). This result also indicated that fermentation improved the flavour of leek by reducing the strong odour related to allicin. Thiol compounds, including glutathione, cysteine, and poly-phenolic compounds, and flavonoids in leeks are responsible for the antioxidant effect (Ahn et al., 2005; Bernaert et al., 2013; Gorinstein et al., 2008; Rabinkov et al., 2000). During early fermentation until 12 h, a decrease in anti-oxidant related compounds was observed; however, in samples fermented with starter cultures these bioactive compounds were ‘regenerated’, and, in addition, a significant increase in thiol and poly-phenolic contents was documented. In our studies, the antioxidant potential of leek significantly increased during starter culture fermentation, and was indicated by values obtained for DPPH radical scavenging and ferric reducing activities. This result correlated with the modulation of bioactive compounds such as thiols, TPC and TFC during fermentation. On the other hand, spontaneously fermented samples showed significant decrease of ferric reducing power but with no significant change in DPPH radical scavenging activity. Factors such as heat and acid stress during processing of leeks have been reported to influence these changes (Yin and Cheng, 1998). Bernaert et al. (2013) reported that fermentation of green leek leaves for 21 days resulted in a 62% increase of the oxygen radical absorbance capacity (ORAC), whereas they could not detect any significant increase in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. While an increase of endogenous polyphenolic compounds (ferulic acid, astragalin, luteolin and naringenin) also resulted from fermentation, a series of polyphenolic compounds, not present in the fresh leek, have also been detected as a result of fermentation (Bernaert et al., 2013). In conclusion, this study provides information on the effects of selected starter cultures in leek fermentation. W. confusa LK4 reached high population levels early during leek fermentation, and, based on its high growth rates and adaptation to the substrate, may support accelerated fermentation in the critical early stages of the fermentation process. In addition, a mild pH range of around 4.2, once reached, was maintained throughout the fermentation period of 48 h. At this stage, leeks fermented with W. confusa LK4 showed a 16.9% increase in radical scavenging activity and 19.41% in reducing ability, compared to spontaneous fermentation. The detectable amount of allicin of fermented leeks decreased concomitantly with the change in pH, whereas the concentration of thiols significantly increased. The total flavonoid and polyphenolic contents changed during fermentation and showed correlation with anti-oxidant effects. On the other hand, L. plantarum LK8 showed fermentation ability in this substrate, albeit after a lag period compared to the development of W. confusa LK4. Moreover, L. plantarum LK8, induced increased antioxidant capacities. However, due to strong lactic acid production resulting in decrease of the pH b 4.0, the strong sour taste may have adverse sensory effects considering current consumer expectations of ‘mildly’ fermented foods. We therefore consider W. confusa LK4 most suitable as a potential starter culture for the fermentation of leeks. Acknowledgment “This research was supported by no. 20120056 of Handong Global University Research Grants”. References Abd El Rehem, F., Abd El Rehem, A., Mohammed Ali, R.F., 2013. Proximate compositions, phytochemical constituents, antioxidant activities and phenolic contents of seed and leaves extracts of Egyptian leek (Allium ampeloprasum var. kurrat). Eur. J. Chem. 4 (3), 185–190.

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