In situ reuterin production by Lactobacillus reuteri in dairy products

Food Control 33 (2013) 200e206

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In situ reuterin production by Lactobacillus reuteri in dairy products Susana Langa, José M. Landete, Izaskun Martín-Cabrejas, Eva Rodríguez, Juan L. Arqués*, Margarita Medina Dpto de Tecnología de Alimentos, INIA, Carretera de La Coruña Km 7, 28040 Madrid, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 November 2012 Received in revised form 20 February 2013 Accepted 26 February 2013

In situ reuterin production during the manufacture and storage of two dairy model systems elaborated from milk supplemented with an optimized concentration of glycerol (50 mM) and inoculated with a commercial starter and with different reuterin-producing Lactobacillus reuteri strains was investigated. L. reuteri was able to survive and to produce reuterin in cheese and yogurt models. The highest reuterin production was achieved by L. reuteri INIA P572 and INIA P579, which displayed reuterin concentrations up to 5.5 mM in cheese and up to 1.5 mM in yogurt. The addition of reuterin-producing L. reuteri and glycerol to milk reduced the viable counts of the cheese starter from day 10 onwards, while did not influence the counts of the yogurt starter, compared to control dairy models without L. reuteri. Strains L. reuteri INIA P572 and INIA P579 could be promising candidates in the development of bioprotective cultures to control pathogenic microorganisms in dairy products due to the potentially inhibitory concentrations of reuterin achieved in situ. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Reuterin Lactobacillus reuteri in situ reuterin production Bioprotection

1. Introduction Lactobacillus reuteri is a heterofermentative lactobacilli recognized as normal inhabitant of the human and animal gut (Oh et al., 2010; Reuter, 2001; Walter, 2008). It is also frequently found in fermented and probiotic foods (Casas & Dobrogosz, 2000; Stiles & Holzapfel, 1997; Vollenweider & Lacroix, 2004). L. reuteri as a food supplement is accepted and widely used to improve gastrointestinal health and has been granted qualified presumption of safety (QPS) by the European Food Safety Authority (EFSA) (Andreoletti et al., 2008). Consumption of 109e1010 living L. reuteri cells per day is considered safe and well tolerated, even in immune deficient individuals (Jones, Martoni, Di Pietro, Simon, & Prakash, 2012; Wolf, Wheeler, Ataya, & Garleb, 1998). Dairy products have been used as the main vehicles for probiotic lactobacilli. However, the number of studies about the addition of L. reuteri as adjunct to the starter culture is limited (De Angelis, Curtin, McSweeney, Faccia, & Gobbetti, 2002; Minervini et al., 2012; Sreekumar, Al-Attabi, Deeth, & Turner, 2009; Tungjaroenchai, White, Holmes, & Drake, 2004). Probiotic effects of L. reuteri have been proposed due to the ability of some strains to produce reuterin (b-hydroxypropionaldehyde; b-HPA) during anaerobic metabolism of glycerol (Rodríguez, Arqués, Rodríguez, Nuñez, & Medina, 2003;

* Corresponding author. Fax: þ34 91 3572293. E-mail address: [email protected] (J.L. Arqués). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.02.035

Talarico & Dobrogosz, 1989; Vollenweider, Evers, Zurbriggen, & Lacroix, 2010). Reuterin is an antimicrobial compound soluble in water, resistant to heat and stable over a wide range of pH values, that inactivates Gram-negative and Gram-positive bacteria (Axelsson, Chung, Dobrogosz, & Lindgren, 1989; Cleusix, Lacroix, Vollenweider, Duboux, & Le Blay, 2007; Vollenweider, Grasi, König, & Puhan, 2003). The HPA-system was first isolated, purified and identified as an equilibrium mixture of monomeric (b-HPA), hydrated monomeric (HPA-hydrate) and cyclic dimeric (HPA-dimer) forms of HPA (Talarico & Dobrogosz, 1989). Recently, it has been demonstrated that the aldehyde form of reuterin is the bioactive agent which causes an oxidative stress response by modifying thiol groups in proteins and small molecules (Schaefer et al., 2010; Vollenweider et al., 2010). Reuterin has been proposed as a potential food additive to prevent the growth of pathogenic and spoilage microorganisms. However, the efficacy of reuterin in foods can be limited by main environmental factors such as temperature, pH and salt, and specific studies should be performed. It has been described that reuterin keeps its activity at low pH, high NaCl concentrations (Rasch, 2002; Rasch, Métris, Baranyi, & Bjørn Budde, 2007) and also at refrigeration temperatures (Arqués, Rodríguez, Nuñez, & Medina, 2008a). Direct addition of reuterin to control food-borne pathogens such Salmonella sp., Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus has been investigated in milk and dairy products (Arqués et al., 2004; Arqués et al., 2008a; Arqués, Rodríguez, Nuñez, & Medina, 2008b; El-Ziney & Debevere,

S. Langa et al. / Food Control 33 (2013) 200e206

1998). Although reuterin as a food preservative is not legislated, the application of L. reuteri plus glycerol (registered in the European Union as food additive E 422) for production of reuterin in situ during the manufacture and storage of foods products is an alternative for reuterin addition in foods. In this work, we evaluate the ability of five selected strains of L. reuteri to survive and to produce reuterin in situ during the manufacture and storage of cheese and yogurt model systems. 2. Materials and methods 2.1. Bacterial strains and culture conditions The reuterin-producing L. reuteri INIA P569, INIA P570, INIA P571, INIA P572, INIA P573, INIA P577 and INIA P579 were selected from the INIA culture collection due to their high reuterin yields (Rodríguez et al., 2003). They were propagated in MRS broth (Oxoid, Unipath Ltd., Basingstoke, United Kingdom) at 37  C and anaerobic conditions (Anaerogen TM, Oxoid) and subcultured twice in sterile reconstituted milk supplemented with 0.3% yeast extract before used in dairy products manufacture. Commercial lactic cultures MA 016 (Larbus S.A., Madrid, Spain), that contains Lactococcus lactis subsp. lactis plus L. lactis subsp. cremoris strains, and YC-X16 e Yo-FlexÒ (Chr. Hansen, Hoersholm, Denmark), that contains a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, were prepared as specified by the manufacturers and kept at 20  C. E. coli K12 grown in Tryptic Soy Broth (TSB; Biolife, Milano, Italy) at 37  C for 24 h, was used as the indicator strain in reuterin activity assays. 2.2. Pulse field electrophoresis (PFGE) analysis The L. reuteri isolates were subjected to genotypic characterization by PFGE. Chromosomal DNA was prepared from the isolates in Certified Low Melt Agarose (Bio-Rad, Hercules, CA, USA) blocks. Macro-restriction of the genomic DNA was carried out at 25  C with ApaI (50 U/ml) or SmaI (20 U/ml) endonucleases (New England Biolabs, Ipswich, MA, USA). Electrophoresis was carried out at 15  C in a CHEF DR II apparatus (Bio-Rad Laboratories, Madrid, Spain) in 1% (wt/vol) Pulsed Field Certified Agarose (BioRad) with 0.5 TriseBorate-EDTA buffer (Sigma, St. Louis, MO, USA) at 6 V/cm with a 1e10 s linear ramp pulse time for 20 h. Low-Range PFG marker (New England Biolabs) were used as molecular size standards. The gels were stained with ethidium bromide (0.5 mg/ml), photographed under UV light, and analyzed with by using the Diversity Database program (Bio-Rad Laboratories).

201

2.4. Reuterin production in milk L. reuteri pulsotypes were individually inoculated with the commercial lactic cultures at 1% in reconstituted skim milk supplemented with 0, 25, 50, 100 or 250 mM of glycerol (Panreac Quimica, Barcelona, Spain). After 24 h at 37  C under anaerobic conditions, 1 ml of culture was centrifuged at 12,000g for 5 min and the supernatant was analyzed for reuterin production by dehydratation to acrolein according to the method of Smiley and Sobolov (1962). The resulting acrolein was measured by the colorimetric method of Circle, Stone, and Boruff (1945). Differences in absorbance measured at 490 nm in a Beckman DU 650 spectrophotometer (Beckman Instruments Inc., Fullerton, CA, USA) accounted for differences in reuterin production when compared against a blank without glycerol and using an acrolein standard (0e6 mM) for quantification (Fluka; SigmaeAldrich Quimica SA, Madrid, Spain). 2.5. Reuterin production in dairy products Dairy products were made from UHT semi-skimmed milk with 1.6% fat (Pascual, Aranda de Duero, Spain) in duplicate experiments carried out on different days. 2.5.1. Cheese model system In each experiment, six screw-capped flasks containing 200 mL of milk supplemented with 0.02% CaCl2 and 50 mM glycerol at 33  C were inoculated with 1% of the commercial mesophilic lactic culture MA 016. Five flasks were individually inoculated with 1% (approximately 106 cfu/ml) of a culture of the different L. reuteri strains. Milk without L. reuteri served as control. After addition of rennet (0.015 g/L, Maxiren 150, Gist-Brocades, Delft, The Netherlands), milk from each flask was distributed into seven sterile containers and held at 37  C for 2 h. The curds were kept at 25  C until 24 h and then stored for 30 d at a ripening temperature of 12  C under anaerobic conditions. 2.5.2. Yogurt model system In each trial, six screw-capped flasks containing 200 mL of milk at 40  C were supplemented with 5% skim milk powder and 50 mM glycerol. Comercial YC-X16 lactic culture was added at 0.4%. One flask without L. reuteri served as control, and the other five were individually inoculated with 1% (approximately 106 cfu/ml) of a culture of the different L. reuteri strains. Milk from each flask were distributed into 25-mL sterile containers and incubated at 40e 43  C to reach a pH value of 4.55e4.60 (approx. 5 h). The yogurts were stored at 6  C for 28 d under anaerobic conditions. 2.6. Microbiological analysis and pH

2.3. PCR assays for potential production of biogenic amines by L. reuteri Bacterial DNA from L. reuteri strains for partial histidine decarboxylase (hdc), tyrosine decarboxylase (tdc) or ornithine decarboxylase (odc) gene amplification was obtained according to the method proposed by Ruiz-Barba, Maldonado, and JiménezDíaz (2005). The strains were checked by specific PCRs using the primer pairs JV16HC/JV17HC for amplifying the internal part of hdc gene, P2-for/P1-rev for tdc gene, and 3/16 for odc gene, and conditions proposed by De las Rivas, Marcobal, and Muñoz (2005). DNA from histamine- and putrescine-producing Lactobacillus 30a (De las Rivas et al., 2005), and tyramine-producing Enterococcus faecalis INIA P289 (Rodríguez et al., 2012) were used as positive controls.

Cheese model was sampled in duplicate at days 1, 5, 10, 15, 20, 25 and 30. L. lactis counts from commercial starter were determined on duplicate plates of M17 supplemented with 0.5% glucose (GM17 medium) and incubated at 30  C for 24 h, and L. reuteri counts on duplicate plates of Rogosa agar (Biolife) incubated at 37  C for 48 h under anaerobic conditions. Yogurt samples were analyzed after 1, 3, 6, 9, 14, 21 and 28 d. Counts of S. thermophilus and L. delbrueckii subsp. bulgaricus were determined on duplicate plates of M17 and MRS agar after incubation at 30  C and 37  C, respectively, in aerobic conditions. L. reuteri counts were determined as above. The pH values were obtained in duplicate with a Crison penetration electrode (model 52-3,2; Crison Instruments S.A., Barcelona, Spain) by means of a Crison GPL 22 pH meter.

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2.7. Quantification of reuterin and antimicrobial activity in dairy products

INIA P570, INIA P572, INIA P577 and INIA P579 strains for further experiments.

For the determination of reuterin production by the different L. reuteri strains, dairy samples were homogenized and centrifuged (12000 g, 20 min, 4  C). The supernatants were filtered (0.22 mm, Millipore Corporation, Bedford, MA, USA) and used for quantifying the presence of reuterin on triplicate by the colorimetric assay previously described. The antimicrobial activity of reuterin-containing supernatants was determined after a modification of the method described by Chung, Axelsson, Lindgren, and Dobrogosz (1989), with E. coli K12 as the indicator strain. Supernatants were adjusted to pH 6.0 with 0.1 N NaOH and serially diluted. Absorbance at 600 nm was measured in a Multiskan Spectrum microplate reader (Thermo Fisher Scientific, Vantaa, Finland) and reuterin arbitrary units (AU/ ml) were defined as the reciprocal of the highest dilution which did show inhibition of the indicator strain.

3.2. Production of biogenic amines

2.8. Statistical analysis Data were subjected to ANOVA with the SPSS program 12.0 for Windows (SPSS Inc., Chicago, IL, USA). Significant differences were assessed by Tukey’s test at P < 0.01 using the same program. 3. Results 3.1. Differentiation of L. reuteri isolates by PFGE Seven isolates of L. reuteri were investigated. PFGE after digestion with ApaI or SmaI restriction enzymes generated multiple DNA fragments (Fig. 1). From the analysis of band patterns, five different pulsotypes were characterized. The five different reuterinproducing pulsotypes were represented by L. reuteri INIA P569,

None of the five L. reuteri strains analyzed harbored the hdc, tdc or odc genes responsible for the production of histamine, tyramine or putrescine (data not shown). 3.3. Reuterin production in milk All L. reuteri strains selected were able to grow in milk in the presence of commercial starters, reaching concentrations of 107108 cfu/ml when cultivated under anaerobic conditions for 24 h at 37  C in absence of glycerol (data not shown). The highest reuterin production was achieved in milk supplemented with 50e250 mM glycerol in all strains tested, with reuterin concentrations between 8.8 and 13.9 mM. Reuterin production was markedly reduced when glycerol concentration was 10 mM (Table 1). Maximum reuterin antimicrobial activity detected in supernatants from milk samples was 8 AU/ml in all the strains studied (data not shown). 3.4. Reuterin production in cheese The L. reuteri count in inoculated milk was on average 5.85 log cfu/ml. L. reuteri counts in all the cheese models manufactured with the commercial starter plus different reuterinproducing strains remained at approximately 5 log units during the first 15 d, and decreased to approximately 3 log units after 30 d (Table 2). Counts of commercial lactic culture in cheese were significantly influenced (P < 0.001) by inoculation of milk with reuterinproducing L. reuteri and by cheese age, after analysis of variance. In control cheese made from milk not inoculated with reuterin-

Fig. 1. Pulsed field gel electrophoresis (PFGE) band patterns of ApaI- (A) and SmaI-digested (B) L. reuteri genomic DNA. Starting from the left-hand side: Lane 1, low-range PFG marker; lane 2, L. reuteri INIA P569; lane 3, L. reuteri INIA P570; lane 4, L. reuteri INIA P571; lane 5, L. reuteri INIA P572; lane 6, L. reuteri INIA P573; lane 7, L. reuteri INIA P577; lane 8, L. reuteri INIA P579; lane 9, low-range PFG marker.

S. Langa et al. / Food Control 33 (2013) 200e206 Table 1 Reuterin production (mmol/l) by L. reuteri strains in milk with a commercial lactic culture (MA 016) at different concentrations of glycerol (0e250 mM) after incubation in anaerobic conditions during 24 h at 37  C. L. reuteri strains Glycerol

INIA P569

INIA P570

INIA P572

INIA P577

INIA P579

0 mM 10 mM 25 mM 50 mM 100 mM 250 mM

0.00a 1.94a 4.64ab 10.99b 11.22b 9.89b

0.00a 0,31a 6.21b 9.70b 9.39b 8.83b

0.00a 1.80a 11.21b 13.90b 12.82b 11.74b

0.00a 2.32a 9.61b 10.02b 9.84b 8.82b

0.00a 2.14a 12.01b 13.35b 13.22b 12.10b

Values with different superscripts in the same column indicate statistically significant (P < 0.01) differences.

producing L. reuteri, commercial starter counts reached 9.17 log cfu/ ml on day 1 and decreased to 6.55 log cfu/ml on day 30. L. lactis counts in cheese models made with different L. reuteri strains were significantly (P < 0.01) lower than in control cheese after day 10. On day 30, cheeses with L. reuteri INIA P569, INIA P570 or INIA P577 showed L. lactis counts 0.78e1.98 log units lower than those in cheeses without reuterin-producing L. reuteri. A higher inhibition of the starter culture was detected in cheeses manufactured with strains L. reuteri INIA P572 and INIA P579, where counts of L. lactis were 4.84 and 6.40 units lower, respectively, than in control cheese after 30 d at 12  C (Table 2). Values of pH were between 4.10 and 4.21 on day 1 and between 4.15 and 4.23 on day 30. Differences between pH values of control cheese and cheeses with reuterin-producing lactobacilli were 0.11 pH units or lower (data not shown). A preliminary sensory evaluation carried out by a number of trained panelists indicated that there were no abnormal odors in the cheeses made with the different reuterin-producing L. reuteri strains. The presence of reuterin was observed in all cheese models made with L. reuteri. The greatest concentrations of reuterin were found in cheeses with L. reuteri INIA P572 and INIA P579 on day 10, reaching levels of 5.71 and 5.52 mM, respectively. Cheese with L. reuteri INIA P570 exhibited a maximum of 2.46 mM of reuterin on day 5. Reuterin levels in cheeses made with L. reuteri INIA P569 or INIA P577 were lower than 1.50 mM throughout the storage period investigated. After 30 d, reuterin concentration in cheese models made with reuterin producers decreased to values between 2.85 and 0.08 mM (Table 3). Antimicrobial activity was detected in all cheeses made with the different L. reuteri strains at any time during the 30 d at 12  C, but not in cheeses made from milk without lactobacilli (Table 3). The Table 2 Log counts (cfu/ml) of L. lactis and L. reuteri strains in cheese model made with the commercial lactic culture (CLC) MA 016 and different reuterin-producing lactobacilli. Lactic cultures 1 d L. lactis

CLC CLC CLC CLC CLC CLC

þ þ þ þ þ

L. reuteri CLC CLC CLC CLC CLC CLC

þ þ þ þ þ

5d

10 d

15 d

20 d

25 d

30 d

P569 P570 P572 P577 P579

9.17a 9.15a 9.03a 9.14a 9.15a 9.09a

9.33a 9.44a 9.19a 9.20a 9.30a 9.24a

9.22a 8.91a 8.56a 7.51a 9.17a 7.05a

8.75a 7.07ab 5.85abc 4.11bc 5.33abc 2.52c

7.52a 5.70b 5.15c 3.33e 4.25d 0.50f

6.81a 5.90ab 5.51b 1.17d 3.59c 1.36d

6.55a 5.77a 5.42a 1.71b 4.57a 0.15b

P569 P570 P572 P577 P579

e 5.52b 5.00ab 5.06ab 4.82a 5.08ab

e 5.64a 5.17a 5.27a 4.48a 5.26a

e 5.63a 5.26a 5.23a 4.83a 5.01a

e 5.55a 4.95a 5.20a 4.72a 4.85a

e 4.07a 2.39a 3.80a 3.50a 3.55a

e 3.26a 2.02a 2.10a 3.57a 2.38a

e 3.79a 2.49a 2.99a 3.44a 2.39a

Values with different superscripts indicate statistically significant (P < 0.01) differences for a given organism and time.

203

Table 3 Reuterin concentration (mmol/l) in cheese model made with the commercial lactic culture (CLC) MA 016 and different reuterin-producing L. reuteri. Lactic cultures

1d

5d

10 d

15 d

20 d

25 d

30 d

CLC CLC CLC CLC CLC CLC

0.00 0.13 1.44a 0.68 0.81 1.59a

0.00 0.30 2.46a 1.83a 0.55 2.29a

0.00 0.25 2.14a 5.71a 1.10 5.52a

0.00 0.89a 1.57a 5.16a 1.27 4.18a

0.00 0.49 0.70 4.65a 1.49 4.80a

0.00 0.28 0.11 2.78a 1.21a 3.10a

0.00 0.23 0.08 2.59a 1.15 2.85a

a

þ þ þ þ þ

P569 P570 P572 P577 P579

Antimicrobial activity detected against E. coli K12.

highest antimicrobial activity detected (4 AU/ml) corresponded to cheeses made from milk inoculated with L. reuteri INIA P572 and INIA P579. 3.5. Reuterin production in yogurt The inoculated milk had a mean L. reuteri count of 5.78 log cfu/ ml. In the yogurt models manufactured with commercial lactic culture plus L. reuteri INIA P570, INIA P572, INIA P577 or INIA P579, L. reuteri levels were 5.74e6.25 log cfu/ml after 1 d, and slightly decreased to 5.38e5.88 log cfu/ml on day 28, while in yogurt made with L. reuteri INIA P569, L. reuteri reached 4.95 log cfu/ml on day 1, and declined to 3.02 log cfu/ml on day 28 (Table 4). In control yogurt S. thermophilus counts were 9.18 log cfu/ml on day 1, and increased to 9.76 log cfu/ml at the end of the refrigeration period, whereas counts of L. delbrueckii subsp. bulgaricus reached 4.70 log cfu/ml on day 1 and slightly decreased to 4.47 on day 28. No significant differences (P < 0.01) in commercial starter levels were observed in yogurt models made with reuterin producers with respect to control yogurt without L. reuteri (Table 4). The pH of control yogurt decreased from 4.53 on day 1 to 4.24 on day 28. Differences because of milk inoculation with reuterinproducing L. reuteri were 0.06 units or lower (data not shown). Unpleasant odors in the yogurts manufactured with the different reuterin-producing L. reuteri were not detected after a preliminary sensory evaluation carried out by a number of trained panelists. Yogurts made with L. reuteri INIA P572 or INIA P579 showed reuterin concentrations 0.90 mM from day 6 onwards, reaching maximum values of 1.73 and 1.53 mM, respectively. Yogurts made with L. reuteri INIA P570 achieved concentrations of 1.21 and 1.12 mM on days 3 and 6, respectively, and decreased gradually during storage to 0.34 mM on day 28. Reuterin concentrations detected in yogurts made with L. reuteri INIA P569 or INIA P577 were lower than 0.21 and 0.42 mM, respectively, throughout refrigeration period at 6  C (Table 4). An antimicrobial activity of 2 AU/ml was detected in yogurts made with L. reuteri INIA P570, INIA P572 or INIA P579, which showed reuterin concentrations higher than 1 mM, except for the cheese made with L. reuteri INIA P579, where antimicrobial activity was detected on day 21 (0.90 mM) and was not detected on day 28 (1.05 mM) (Table 5). 4. Discussion The analysis by PFGE of the chromosomal DNA restriction patterns has been widely used in lactobacilli as the most appropriate technique for the genotyping of bacterial isolates (Jacobsen et al., 1999). The PFGE analysis using restriction enzymes ApaI and SmaI allowed us to discriminate the seven L. reuteri isolates into five different pulsotypes. The ability to produce biogenic amines by strains from various dairy starter cultures has been reported (Burdychova & Komprda, 2007). Lactobacilli are considered to be generally safe. However, decarboxylating Lactobacillus strains can be responsible in cases in

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Table 4 Log counts (cfu/ml) of S. thermophilus. L. delbrueckii subsp. bulgaricus and L. reuteri strains in yogurt model made with the commercial lactic culture (CLC) YC-X16 and different reuterin-producing lactobacilli.

S. thermophilus

L. delbrueckii

L. reuteri

Lactic cultures

1d

3d

6d

9d

14 d

21 d

28 d

CLC CLC CLC CLC CLC CLC

þ þ þ þ þ

P569 P570 P572 P577 P579

9.18a 9.20a 9.26a 9.10a 9.18a 9.16a

9.13a 9.13a 9.17a 9.08a 9.14a 9.17a

9.13a 9.16a 9.15a 9.14a 9.07a 9.08a

9.13a 9.06a 9.08a 9.11a 9.14a 9.05a

9.10a 9.03a 8.97a 9.10a 9.00a 8.78a

9.07a 9.06a 8.81a 8.58a 8.91a 8.59a

9.76a 9.40a 9.00a 8.64a 9.36a 8.98a

CLC CLC CLC CLC CLC CLC

þ þ þ þ þ

P569 P570 P572 P577 P579

4.70a 4.80a 4.53a 4.49a 4.49a 4.83a

4.46a 4.82a 4.61a 4.58a 4.53a 4.85a

4.77a 4.80a 4.55a 4.60a 4.59a 4.73a

4.79a 4.82a 4.78a 4.56a 4.77a 4.55a

4.99a 5.11a 5.18a 4.47a 5.12a 3.72a

4.73a 4.66a 4.60a 4.76a 4.81a 4.89a

4.47a 4.60a 4.12a 4.38a 4.46a 4.45a

CLC CLC CLC CLC CLC CLC

þ þ þ þ þ

P569 P570 P572 P577 P579

e 4.95a 5.88ab 6.06b 5.74ab 6.25b

e 4.99a 6.00b 6.30b 5.64ab 6.48b

e 4.06a 5.27ab 5.35ab 4.82ab 6.13b

e 4.94a 5.67a 6.19a 5.27a 5.95a

e 4.92a 5.84a 6.17a 5.24a 6.00a

e 4.51a 5.61ab 6.02ab 5.24ab 6.08b

e 3.02a 5.50b 5.78b 5.38b 5.88b

Values with different superscripts indicate statistically significant (P < 0.01) differences for a given organism and time.

which large amounts of biogenic amines have been formed in cheese (Joosten, 1987). It is therefore significant to consider this safety trait as a condition of strains intended to be used as starter or protective cultures because they may cause toxicological problems (Ten Brink, Damink, Joosten, & Huis In ’t Veld, 1990). Here we demonstrated that the five L. reuteri strains tested are not able to produce the most important biogenic amines. L. reuteri is considered one of a limited number of indigenous Lactobacillus species in the human intestine and some L. reuteri strains are established probiotic agents for more than 20 years, being included in different commercial products. The production by some strains of b-HPA gives L. reuteri a competitive advantage in its ecological niches, such as gastrointestinal tract, where glycerol could be converted into reuterin (Morita et al., 2008). Therefore, in this study, we investigated the ability of five L. reuteri strains to produce reuterin in two dairy model systems when supplemented the milk with an optimized concentration of glycerol. L. reuteri was not able to grow in yogurt during storage at 6  C, and resulted in a slight reduction in the counts of the adjunct culture after 28 d. Similar results has been reported for L. reuteri RC14 in milk at refrigeration temperatures (Hekmat & Reid, 2007). L. reuteri did not multiply in cheese model during storage at 12  C, but showed higher reductions in the counts than in yogurt in all cases, except for the yogurt made with L. reuteri INIA P569. This observation seems to be more related to the storage temperature, which could accelerate the cell death process, than to the pH values or reuterin concentration in dairy products. Inoculation of milk with reuterin-producing L. reuteri reduced the counts of L. lactis from commercial starter in the cheese models

Table 5 Reuterin concentration (mmol/l) in yogurt model made with the commercial lactic culture (CLC) YC-X16 and different reuterin-producing L. reuteri. Lactic cultures

1d

3d

6d

9d

14 d

21 d

28 d

CLC CLC CLC CLC CLC CLC

0.00 0.21 0.74 0.74 0.27 0.55

0.00 0.08 1.21a 0.73 0.25 0.84

0.00 0.06 1.12a 0.90 0.28 0.94

0.00 0.18 0.77 1.23a 0.42 1.16a

0.00 0.13 0.52 1.47a 0.24 1.53a

0.00 0.07 0.41 1.73a 0.20 0.90a

0.00 0.03 0.34 1.35a 0.29 1.05

a

þ þ þ þ þ

P569 P570 P572 P577 P579

Antimicrobial activity detected against E. coli K12.

studied from day 10 onwards compared to control cheese made without L. reuteri strains, but did not affect the counts of S. thermophilus and L. delbrueckii subsp. bulgaricus from commercial starter in the yogurt models. The reductions in the cheese starter culture were especially significant in cheeses made with L. reuteri INIA P572 or P579 (Table 2), which showed the highest concentrations of reuterin, up to 4 mM, between days 10 and 20. On the contrary, slight reductions after 30 d, were observed in cheese made with L. reuteri INIA P569, P570 or P577, with no significant differences in the population of L. lactis with respect to cheese without reuterinproducing culture. Reuterin concentrations in cheeses made with these strains were lower than 2.5 mM during the 30 d of storage. In yogurts made with reuterin-producing lactobacilli, reuterin levels were lower than 1.5 mM throughout the entire storage period. Addition of reuterin-producing L. reuteri strains did not resulted in important differences in the pH values of the dairy products. Our study with two dairy models has considered different factors that could affect reuterin production and constitutes a powerful tool to discriminate between different reuterin-producing strains. Reuterin is considered stable at 4  C in milk, but according to Lüthi-Peng, Schärer, and Puhan (2002) its stability decreases as the temperature increases. A decrease in the reuterin concentration during storage at 10  C has been described in a semisolid dairy product supplemented with reuterin (2 AU/ml) as biopreservative by Arqués et al. (2008b). The addition of L. reuteri ATCC 55730 as a co-culture to meat starter cultures and 250 mM of glycerol in dry fermented sausage lowered the counts of E. coli O157:H7 by 1.3 log units compared to control sausage. However, although reuterin production may be the cause of the reduction of the pathogen, reuterin was not detected in the sausages (Muthukumarasamy & Holley, 2007). Our results demonstrate, for the first time to our knowledge, the continuous in situ production of reuterin by reuterin-producing L. reuteri strains added to milk in dairy products throughout time. The addition of reuterin-producing L. reuteri and glycerol to milk provide a continuous relief of reuterin in the dairy products that achieved potentially inhibitory concentrations against a range of Gram-negative and Gram-positive pathogenic and spoilage bacteria (Stevens, Vollenweider, & Lacroix, 2011). It has been reported (Axelsson et al., 1989; Cleusix et al., 2007) that the concentrations of reuterin required to inhibit the growth of lactic acid bacteria are three to five fold higher than those required

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to inhibit other bacteria like E. coli, Salmonella sp. or Listeria sp. Likewise, reuterin has a higher antimicrobial activity against Gramnegative than against Gram-positive pathogenic bacteria (Arqués, Rodríguez, Nuñez, & Medina, 2011). The antimicrobial activity of the HPA-system depends on chemical and physical properties of a food such as pH, salt concentration and temperature, which should be taken into account to the optimization of practical applications to control pathogens. In the present work, the antimicrobial activity of reuterin was evaluated in neutralized supernatants, and in cheese and yogurt other relevant factors like the combined effect of reuterin and lactic acid and/or sodium chloride could enhance reuterin antimicrobial activity (El-Ziney & Debevere, 1998; El-Ziney,Van den Tempel, Debevere, & Jacobsen, 1999; Rasch et al., 2007). Indeed, a combination of high salt, low pH and reuterin inhibits Listeria innocua more efficiently than reuterin at physiological salt concentrations and neutral pH (Rasch et al., 2007). On the contrary, similar effect on E. coli by reuterin has been reported at different salt concentration and pH (Rasch, 2002). The composition of the HPA-system is concentration- and pHdependent (Sung, Chen, Liang, & Hong, 2003; Vollenweider, Grassi, König, & Puhan, 2003). The HPA-system antimicrobial activity has been widely studied and demonstrated, but there is still some controversy about the antimicrobial activity role of its three isoforms. However, according to some authors (Schaefer et al., 2010; Vollenweider et al., 2010) the aldehyde form reacts with sulfhydryl groups of amino acids, leading to trigger oxidative stress response. In the present work, no abnormal odors in the dairy products made with the different reuterin-producing L. reuteri strains were detected after a preliminary sensory evaluation, which is in agreement with previous studies. Addition of L. reuteri to Fior di latte or reduced-fat Edam cheeses did not produce detectable defects in the sensory attributes of the cheese (Minervini et al., 2012; Tungjaroenchai et al., 2004). It has also been reported that the addition of L. reuteri could generate considerable levels of volatile sulfur compounds with potential to affect the favor and aroma properties of the cheese (Sreekumar et al., 2009). However, the effect on dairy sensory characteristics of the addition of L. reuteri as adjunct culture is considered strain specific and thus, should be individually evaluated in each food product. The application of reuterin-producing L. reuteri INIA P572 or P579 plus 50 mM of glycerol lead to the in situ production of reuterin in concentrations that could control pathogenic contaminants potentially present during the manufacture and storage of dairy products. Work is in progress to evaluate the protective potential of reuterin produced in cheese and the effect of reuterin-producing lactobacilli as adjunct cultures on the chemical and sensory properties of dairy products. Acknowledgments This work was supported by project RTA 2010-00116-00-00 and AGL2010-16600 from the Spanish Ministry of Economy and Competitiveness. References Andreoletti, O., Budka, H., Buncic, S., Colin, P., Collins, J. D., De Koeijer, A., et al. (2008). Scientific opinion of the panel on biological hazards on a request from EFSA on the maintenance of the QPS list of microorganisms intentionally added to food or feed. The EFSA Journal, 923, 1e48. Arqués, J. L., Rodríguez, E., Nuñez, M., & Medina, M. (2008a). Inactivation of gramnegative pathogens in refrigerated milk by reuterin in combination with nisin or the lactoperoxidase system. European Food Research and Technology, 227, 77e82. Arqués, J. L., Rodríguez, E., Nuñez, M., & Medina, M. (2008b). Antimicrobial activity of nisin, reuterin, and the lactoperoxidase system on Listeria monocytogenes and

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