A comprehensive approach to determine the probiotic potential of human-derived Lactobacillus for industrial use

Food Microbiology 34 (2013) 19e28

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A comprehensive approach to determine the probiotic potential of human-derived Lactobacillus for industrial use V. Gregoret, M.J. Perezlindo, G. Vinderola, J. Reinheimer, A. Binetti* Instituto de Lactología Industrial (INLAIN, UNL e CONICET), Facultad de Ingeniería Química, Universidad Nacional del Litoral (UNL), Santiago del Estero 2829, 3000 Santa Fe, Argentina

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 April 2012 Received in revised form 18 October 2012 Accepted 10 November 2012 Available online 27 November 2012

Specific strains should only be regarded as probiotics if they fulfill certain safety, technological and functional criteria. The aim of this work was to study, from a comprehensive point of view (in vitro and in vivo tests), three Lactobacillus strains (Lactobacillus paracasei JP1, Lactobacillus rhamnosus 64 and Lactobacillus gasseri 37) isolated from feces of local newborns, determining some parameters of technological, biological and functional relevance. All strains were able to adequately grow in different economic culture media (cheese whey, buttermilk and milk), which were also suitable as cryoprotectants. As selective media, LP-MRS was more effective than B-MRS for the enumeration of all strains. The strains were resistant to different technological (frozen storage, high salt content) and biological (simulated gastrointestinal digestion after refrigerated storage in acidified milk, bile exposure) challenges. L. rhamnosus 64 and L. gasseri 37, in particular, were sensible to chloramphenicol, erythromycin, streptomycin, tetracycline and vancomycin, increased the phagocytic activity of peritoneal macrophage and induced the proliferation of IgA producing cells in small intestine when administered to mice. Even when clinical trails are still needed, both strains fulfilled the main criteria proposed by FAO/WHO to consider them as potential probiotics for the formulation of new foods. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Probiotics Lactobacillus Technological characterization Functional characterization

1. Introduction Probiotics are defined as live microorganisms which, when administered in adequate amounts, confer a benefit on the host (FAO/WHO, 2002). Numerous microorganisms are currently used as human probiotics; among them Lactobacillus and Bifidobacterium constitute the most frequently used genera. Even though there has been a long history of safe consumption of lactobacilli in traditional foods, the probiotic strains can only be utilized if they fulfill certain criteria related to safety, technological and functional aspects (Reid, 2005; Vankerckhoven et al., 2008). Although there are no analytical tools to determine the environmental source of a strain after its primary isolation, it frequently represents the initial factor to be considered. In general, the selection of strains from appropriate sources depends on the targeted population, such as neonates, children, pregnant women or elderly, whose microbiota may differ from that of healthy adults (O’Toole and Claesson, 2010). Isolation from the intestinal tract of healthy individuals is generally recommended for probiotic use in humans

* Corresponding author. Tel.: þ54 342 4530302; fax: þ54 342 4571164x2535. E-mail address: anabinetti@fiq.unl.edu.ar (A. Binetti). 0740-0020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2012.11.004

(FAO/WHO, 2002; Reid, 2005). Following isolation, the next step is the identification at genus and species level using internationally accepted methodologies (in particular, sequencing of DNA encoding 16S rRNA) (Reid, 2005; Vankerckhoven et al., 2008). Even when there is a great number of strains being used as probiotics in different food matrixes, the majority of the studies are based on functional properties (Szajewska et al., 2001; Kirjavainen et al., 2003; Reid et al., 2003; Santosa et al., 2006; Ezendam and van Loveren, 2006) and less information is available concerning their capacity to withstand stresses related to food processing. Among technological criteria, strain viability and maintenance of desirable characteristics during product manufacture and storage is a requirement for probiotic strains to assure a beneficial effect on the consumer (Champagne et al., 2011). In this regard, before reaching the intestinal environment, probiotic strains must overcome several technological and biological barriers (low pH in the stomach, bile salts) (Collins et al., 1998). Safety assessment is also a required step in the selection and evaluation of probiotics. Few probiotic strains have been specifically tested for safety based on the long history of safe consumption of lactic acid bacteria. The FAO/WHO guidelines recommend the determination of antibiotic resistance patterns as one of the most important safety test (FAO/ WHO, 2002).

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V. Gregoret et al. / Food Microbiology 34 (2013) 19e28

In spite of the value of in vitro tests for an initial screening of potential probiotic strains, they are insufficient to assert a strain as probiotic. In vivo trials (at least using an animal model) are strongly suggested by the FAO/WHO to prove that the probiotic confers a significant improvement in health. These assays generally include evaluations of the capacity to stimulate the host immune system, so as to prevent enteric infections or other gut-associated pathologies. Even when a high number of probiotic strains is now available for commercial use around the world, the isolation and characterization of new strains is still desirable, mainly in developing countries. The aim of this work was to characterize, from a comprehensive point of view (by in vitro and in vivo tests), three Lactobacillus strains isolated from local newborns, determining some parameters of technological, biological and functional relevance for their potential addition to new functional products. 2. Material and methods 2.1. Strains and culture conditions The Lactobacillus strains tested in this work were isolated in a previous study (Vinderola et al., 2008) from feces of three newborns from Santa Fe, Argentina, frozen stored (80  C) in MRS broth (Biokar, Beauvais, France) supplemented with 15% (v/v) glycerol, and kept at the INLAIN culture collection. They were preliminarily identified by RAPD-PCR techniques as Lactobacillus paracasei JP1, Lactobacillus rhamnosus 64 and Lactobacillus gasseri 37 and selected, based on preliminary characterization, as potential probiotic strains. For RAPD assays, L. paracasei DN114001 (Danone), L. rhamnosus GG (Valio) and L. gasseri ATCC 33323 were used as reference strains. All strains were cultured in MRS broth (37  C, 16 h, aerobic incubation). 2.1.1. Identification of isolates Total DNA of isolates was obtained from overnight cultures by using the GenElute Bacterial Genomic DNA kit (Sigma, St Louis, MO, USA) according to the manufacturer’s instructions. Purified DNA samples were stored at 20  C until use. To determine the bacterial species, the identity of isolates was analysed by amplifying, sequencing and comparing a 1500 bp fragment within their 16S rRNA gene (Edwards et al., 1989). All PCR reactions were performed using 1 mL of diluted (1:50) DNA as template, 2.5 U Taq DNA polymerase (GE Healthcare, Little Chalfont, United Kingdom), 200 nM dNTPs (GE Healthcare) and 400 nM each primer (SigmaGenosys, The Woodlands, TX, USA) in a final volume of 50 mL. Amplifications were performed in a GeneAmp PCR System (Applied Biosystems, Foster City, CA USA) under the following conditions: 3 min at 94  C, 35 cycles of 1 min at 94  C, 2 min at 51  C and 2 min at 72  C, and a final step of 7 min at 72  C. The PCR products were separated on 0.8% (w/v) agarose gels in TBE (89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.3) buffer, stained with GelRed (Biotium, Hayward, CA, USA) and visualized under UV light (Sambrook and Russell, 2001). Amplicons were purified with MicroSpin Columns (GE Healthcare) and their nucleotide sequences were determined by primer extension at the DNA Sequencing Service of Macrogen (Seoul, Korea). The identity of strains at species level was checked by nucleotideenucleotide BLAST of the NCBI database (www.ncbi.nlm.nhi.gov/blast). 2.1.2. RAPD analysis Genotypic diversity among strains was analysed by RAPD-PCR using two single arbitrary primers, B07 (also named P2, Binetti et al., 2007) and M13 (Huey and Hall, 1989), in independent reactions. Amplifications were carried out under the following conditions: an initial denaturation step of 5 min at 94  C, followed by 30

(B07) or 35 (M13) cycles of 1 min at 94  C, 2 min at 36  C (B07) or 20 s at 45  C (M13), 2 min at 72  C, and a final extension step of 7 min at 72  C. PCR reactions were performed in a total volume of 25 mL with 1 mL template diluted DNA, 500 nM (B07) or 200 nM (M13) primer (Sigma-Genosys), 2.5 U Taq Polymerase (GE Healthcare) and 200 nM of each dNTP (GE Healthcare). A negative control (without template) was included in all reactions. Amplification products were analysed by electrophoresis in 1.0% (w/v) agarose gels in TBE buffer, following standard protocols. 2.1.3. Growth in different culture media The culture media used in this assay were skim milk (10% w/v) (San Regim, Buenos Aires, Argentina), cheese whey (5% w/v) and buttermilk (7.8% w/v), all supplemented with 0.3% (w/v) of yeast extract (Burns et al., 2008a). Dried cheese whey and buttermilk were provided from a local dairy industry and reconstituted to give a final lactose concentration of 4.8% (w/v). MRS broth was included as reference medium. Overnight cultures (MRS broth, 16 h, 37  C) of the strains were centrifuged (8000 g, 15 min, 4  C), washed twice with PBS (137 mM NaCl, 8.2 mM Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl, pH 7.0) solution, resuspended in the same volume of PBS and inoculated (2% v/v) in the corresponding culture media, being finally incubated in a water bath for 24 h at 37  C. Colony counts on MRS agar (37  C, 48 h) were carried out periodically. 2.1.4. Growth in selective/differential culture media Cell counts of the strains under study were performed in selective culture media and compared to counts obtained in MRS agar. Bile MRS agar (B-MRS) and lithium chloride sodium propionate MRS agar (LP-MRS) were used as selective culture media according to previous studies (Vinderola and Reinheimer, 2000). Bovine bile (0.15% w/v, B-MRS) and lithium chloride (0.2% w/v) plus sodium propionate (0.3% w/v, LP-MRS) were suggested as adequate to inhibit the growth of the starter microbial population which were present in fermented dairy products carrying probiotic bacteria. In some commercial products, however, these concentrations are not enough to accomplish this aim, higher amounts of bile salts, lithium chloride and sodium propionate being needed (personal observations at the INLAIN). For this reason, the viability of overnight cultures (MRS broth, 37  C) was determined in B-MRS containing increasing amounts of bovine bile (0.15, 0.2, 0.25 and 0.3% w/v) and LP-MRS with increasing concentrations of the selective agents (0.2, 0.25, 0.3 and 0.4% w/w lithium chloride; 0.3, 0.4, 0.45 and 0.6% w/v sodium propionate). MRS agar was used as control medium. Plates were incubated at 37  C (48 h, aerobic incubation). 2.2. Resistance to technological challenges 2.2.1. Resistance to frozen storage Overnight cultures in MRS broth were centrifuged (8000 g, 15 min, 5  C), washed twice with PBS (pH 7.0) buffer and adjusted to ca. 109 CFU/mL. Cell suspensions were centrifuged (8000 g, 10 min, 5  C) and resuspended in skim milk (10% w/v), buttermilk (10% w/v) and cheese whey (10% w/v). MRS broth supplemented with 15% (v/ v) of glycerol as cryoprotectant was used as control medium. Cell suspensions were then frozen stored at 20 and 70  C for 12 months. At the beginning (day 0) and every 30 days for 12 months, cell counts (MRS Agar, 37  C, 48 h, aerobic incubation) were performed. 2.2.2. Tolerance to salts Overnight cultures were inoculated (2% w/v) in MRS broth added with NaCl and with KCl (both 1 and 2% w/v) according to Reinheimer et al. (1997). MRS broth was used as control medium.

V. Gregoret et al. / Food Microbiology 34 (2013) 19e28

21

Cultures were incubated in a water bath at 37  C for 24 h, the absorbance (l ¼ 560 nm) being measured and the relative growth (as compared to the control medium) calculated.

by the Ethical Committee for Animal Experimentation of the Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral (Esperanza, Santa Fe, Argentina).

2.3. Resistance to biological challenges: cell viability and resistance to simulated gastrointestinal digestion after 30 days refrigerated storage

2.5.2. Feeding procedures Animals were divided in 12 experimental groups: control group 1 (animals sacrificed at the beginning of the experience); control group 2 (animals that received tap water); control group 3 (animals that received 1% w/v skim milk); and three groups for each strain that received a cell suspension (ca. 2  108 CFU/mouse) in 1% w/v skim milk for 2, 5 or 7 consecutive days. The cell suspensions were prepared daily from overnight cultures and washed twice with PBS buffer. All animals were given, simultaneously and ad libitum, tap water and a sterile conventional balanced diet (Cooperación, Buenos Aires, Argentina).

Overnight cultures of the strains were harvested by centrifugation (8000 g, 15 min, 5  C), washed twice with PBS and resuspended (107e108 CFU/mL) in skim milk (10% w/v) previously acidified (pH 4.5) with lactic acid (SigmaeAldrich) to partially simulate the conditions of fermented milks. Non-acidified skim milk was used as control. Cell suspensions were stored at 4  C for 30 days and cell counts (MRS agar, 48 h, 37  C, aerobic incubation) were performed at 0 and 30 days. Before and after the refrigerated storage, the resistance of cells to simulated gastrointestinal digestion was assayed. The cell suspension was mixed (1:1) in 2 “saliva-gastric” resembling solution (SGS: CaCl2 0.22 g/L, NaCl 16.2 g/L, KCl 2.2 g/L, NaHCO3 1.2 g/L and porcine pepsin 0.6% w/v). Samples were incubated at 37  C in a water bath, with agitation and pH dynamic control. Cell suspension was gradually acidified with 1 N HCl from pH 5.0 to pH 2.5 in a 90 min period (Marteau et al., 1997). At 0, 60, 70, 80 and 90 min of incubation, cell counts (MRS agar, 37  C, 48 h, aerobic incubation) were performed. In order to simulate the intestinal digestion, 1 mL of this cell suspension was then centrifuged (9300 g, 10 min, 5  C), and the pellet was suspended in 1 mL of bile-pancreatine solution (0.3% w/v bovine bile, SigmaeAldrich; 0.1% w/v pancreatine, SigmaeAldrich, in PBS buffer pH 8.0), incubated with agitation at 37  C, and a cell count (MRS agar, 37  C, 48 h, aerobic incubation) was performed after 60 min of treatment. 2.4. In vitro safety assessment: antibiotic resistance Overnight cultures were streaked on MRS agar (37  C, 48 h) and, from one isolated colony, a second culture in LSM agar (90% v/v Isosensitest Agar, 10% v/v MRS Agar, Oxoid Ltd., Hamshire, England) was performed and incubated at 37  C for 48 h. Individual colonies were suspended in sterile saline solution up to a density corresponding to McFarland standard 1. This suspension was used to inoculate, by means of a sterile cotton swab and in three different directions, an LSM Agar plate, where the Etest (AB Biodisk, Oxoid Inc., Ontario, Canada) strips were then applied. The antibiotics tested were chloramphenicol, erythromycin, streptomycin, tetracycline and vancomycin. Plates were incubated at 37  C for 48 h and the Minimum Inhibitory Concentration (MIC) was determined from the clear ellipse along the strip with no growth of bacteria, following the manufacturer’s instructions. 2.5. In vivo safety and functionality assessment 2.5.1. Animals Sixty BALB/c mice weighing 19e21 g were obtained from the random bred colony of the Centro de Experimentaciones Biológicas y Bioterio, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral (Esperanza, Santa Fe, Argentina). Animals were maintained for a week at the INLAIN animal facility before starting the experiments. Each experimental group consisted of 5 mice housed together in plastic cages and kept in a controlled environment at a temperature of 21  2  C and a humidity of 55  2%, with a 12 h light/dark cycle. Mice were maintained and treated according to the guidelines of the National Institute of Health (NIH, USA). The experiments with animals were approved

2.5.3. Translocation assay and immunofluorescence test for IgA producing cells enumeration After each feeding period, animals were anesthetized intraperitoneally with a rodent cocktail (9 parts of 100 mg/mL ketamine þ 9 parts of 20 mg/mL xylazine þ 3 parts of 10 mg m/L acepromazine þ 79 parts of sterile saline) and sacrificed by cervical dislocation. The liver was removed and homogenized in 5 mL of sterile PBS buffer. One mL of liver homogenate was pourplated in AVRB agar (Britania, Buenos Aires, Argentina) (translocation assay for enterobacteria). Plates were incubated at 37  C for 24 h in aerobiosis. The small and large intestines were removed for histological preparation and paraffin inclusion. Paraffin-sections (4 mm) were stained with haematoxylin-eosin followed by light microscopy examination (double-blind observations). The number of IgA producing (IgAþ) cells was determined on histological slices of samples from the ileum (excluding Peyer’s patches areas) and from the large intestine (Vintiñi et al., 2000). The immunofluorescence test was performed using achain specific antimouse IgA fluorescein isothiocyanate (FITC) conjugate (SigmaeAldrich). Histological slices were deparaffinized and rehydrated in a series of ethanol at decreasing concentrations (from absolute alcohol to 70 alcohol). Deparaffinized histological samples were treated with a dilution (1/100) of the antibody in PBS buffer and incubated in the dark for 30 min at 37  C. Then, samples were washed twice with PBS buffer and examined using a fluorescent light microscope (Nikon Eclipse with a Hg lamp). The results were expressed as the number of positive cells (fluorescent cells)/10 fields. Positive (fluorescent) cells were counted with a magnification of 400 (double-blind counts). Data were reported as the mean of three counts (each one in a different histological slice) for each animal and for each feeding period. 2.5.4. Functionality: ex vivo phagocytosis assay Before liver removal, peritoneal macrophages were aseptically harvested from the different groups of mice at different periods of feeding (Perdigón et al., 1986) by washing the peritoneal cavity with heparin-BSA buffer (0.1% v/v Heparin 5000 UI/mL, Sigmae Aldrich; 0.3% w/v Bovine Serum Albumin, SigmaeAldrich, in PBS buffer), washed twice with sterile saline solution and adjusted to a concentration of 106 cells/mL using a Neubauer counting chamber. A heat-killed (100  C, 15 min) Candida albicans suspension (107 cells/mL) was opsonized with mouse autologous serum (10%) for 15 min at 37  C. The opsonized yeast suspension (0.1 mL) was mixed with 0.1 mL of each macrophage suspension, the mixture being incubated for 30 min at 37  C. The percentage of phagocytosis was measured as the % of activated macrophages (macrophages that phagocyted at least one yeast cell) after a 100cell count using an optical (400) microscope.

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V. Gregoret et al. / Food Microbiology 34 (2013) 19e28 10

Technological experiments were carried out in at least two independent assays. Within each assay, determinations were carried out in duplicate. Data were analysed using the one-way ANOVA procedure of SPSS software (SPSS Inc., Chicago, IL, USA). Differences between means were detected by Duncan’s Multiple Range Test. Data were considered significantly different when P < 0.05.

Log (CFU/mL)

2.6. Statistical analysis

9

8

3. Results 3.1. Identification of isolates and RAPD analysis

7

0

5

10

15

20

25

Time (h)

The nucleotidic sequences of the 16S rRNA gene fragments confirmed the identification at the species level of the strains isolated from neonates’ feces as: L. paracasei JP1, L. rhamnosus 64 and L. gasseri 37. RAPD analysis with primers B07 and M13 allowed us to evaluate the genetic diversity among them (Fig. 1). Primer M13, in particular, showed higher diversity than primer B07 between autochthonal and reference strains, which allowed discriminating the studied lactobacilli from commercial probiotic strains (L. paracasei DN114001 and L. rhamnosus GG) commonly used in Argentina. 3.2. Growth in different culture media Fig. 2 shows the growth kinetics of L. paracasei JP1, L. rhamnosus 64 and L. gasseri 37 in milk, buttermilk and cheese whey supplemented with 0.3% (w/v) yeast extract. All the strains achieved

Fig. 2. Cell counts during the growth of L. paracasei JP1 (squares), L. rhamnosus 64 (circles) and L. gasseri 37 (triangles) in milk (full symbols, full line), buttermilk (empty symbols, full line) and cheese whey (full symbols, dotted line) added with 0.3% (w/v) yeast extract, at 37  C. Figures are representative of three independent assays.

viable counts ranging from 3  108 to 4.5  109 CFU/mL after 24 h of incubation in all tested media. In the case of L. paracasei JP1 and L. gasseri 37, the growths were slightly faster in buttermilk and cheese whey than in milk up to 10 h of incubation, but then growth kinetics were similar in all conditions. No significant (P > 0.05) differences were observed in the cellular yield among the different culture media for all strains. For L. rhamnosus 64, the differences among the tested media during all the experiences were negligible. None of the culture media without the supplementation of yeast extract was suitable for the growth of the strains (data not shown). 3.3. Growth in selective/differential culture media Table 1 shows the cell counts of the strains under study in BMRS agar and LP-MRS agar, using MRS agar as reference medium. Although all strains grew in both culture media with increasing concentration of the selective agents (lithium chloride and sodium propionate or bovine bile salts), the presence of bovine bile significantly affected (P < 0.05) the growth of L. paracasei JP1 and L. rhamnosus 64 (at all concentrations assessed). In the case of L. gasseri 37, no significant differences (P > 0.05) were detected between B-MRS agar and MRS agar. When LP-MRS agar was evaluated, the cell counts of L. gasseri 37 and L. rhamnosus 64 were similar (P > 0.05) to those obtained in the reference medium. Instead, concentrations of 0.4% (w/v) LiCl and 0.6% (w/v) sodium propionate significantly affected (P < 0.05) the cellular recovery of L. paracasei JP1. 3.4. Resistance to technological challenges

Fig. 1. RAPD profiles of Lactobacillus isolates obtained with primer B07 and M13. M: KiloBase DNA Marker (GE Healthcare); lanes 1e7: primer B07; lanes 8e14: primer M13; lanes 1 and 8: L. paracasei DN114001 (Danone); lanes 2 and 9: L. paracasei JP1; lanes 3 and 10: L. rhamnosus GG (Valio); lanes 4 and 11: L. rhamnosus 64; lanes 5 and 12: L. gasseri ATCC 33323; lanes 6 and 13: L. gasseri 37; lanes 7 and 14: negative control.

3.4.1. Resistance to frozen storage L. paracasei JP1 and L. rhamnosus 64 showed a similar behaviour during the storage at 20 and 80  C (Fig. 3A and B). In both cases, skim milk, buttermilk and cheese whey demonstrated to be adequate as cryoprotectants, since reductions in cell counts lower than 0.7 Log orders were observed after 12 months of storage. Instead, MRS þ glycerol was less effective for these strains, particularly at 20  C, where reductions in cell counts of 4.53 and 3.9 Log cycles were observed for L. paracasei JP1 and L. rhamnosus 64, respectively. The same findings, with smaller reductions in cell counts, were detected at 80  C (1.58 and 2.6 Log orders, respectively). L. gasseri 37 proved less resistant to frozen storage in all the suspension media tested (Fig. 3C). At 20  C, reductions in cell counts between 3.7 and 6.4 Log units were observed in all the

V. Gregoret et al. / Food Microbiology 34 (2013) 19e28

23

Table 1 Growth (log CFU/mL) of studied strains in selective/differential media. Log CFU/mL (mean value  SD)*

Strain

MRS

B-MRS**

8.44  0.19a 8.59  0.26 9.19  0.38a

L. paracasei JP1 L. rhamnosus 64 L. gasseri 37

LP-MRS**

0.15

0.2

0.25

0.30

0.2/0.3

0.25/0.4

0.3/0.45

0.4/0.6

7.57  0.08b 7.84  0.19b 9.18  0.38a

7.68  0.10b 7.56  0.22b 9.15  0.32a

7.27  0.04b 7.79  0.26b 9.19  0.40a

7.29  0.04b 7.57  0.12b 9.12  0.42a

8.34  0.06a 8.19  0.02a 9.18  0.09a

8.42  0.11a 8.13  0.07a 9.29  0.20a

8.42  0.09a 8.35  0.33a 9.26  0.16a

7.45  0.01b 8.35  0.40a 9.07  0.29a

* SD: standard deviation; ** bovine bile (B-MRS) and LiCl/sodium propionate (LP-MRS) concentration (% w/v); significantly different (P > 0.05).

conditions assayed. When this strain was maintained at 80  C for 12 months, cell viability decreased between 0.8 and 2.22 Log. At both temperatures, buttermilk showed a slight protective role (reductions of 3.7 and 0.8 Log orders at 20 and 80  C, 10 9

a,b

: values in the same line with different superscript are

respectively), MRS þ glycerol being again the least adequate medium as cryoprotectant. 3.4.2. Tolerance to salts Table 2 shows the relative growth of strains in MRS supplemented with different concentrations of NaCl and KCl. Salts did not inhibit the growth of any of the strains at the evaluated concentrations (1 and 2% w/v), since no significant (P > 0.05) differences were detected between salt supplemented cultures and control.

Log CFU/mL

8

3.5. Resistance to biological challenges: cell viability and resistance to simulated gastrointestinal digestion after 30 days refrigerated storage

7 6 5

A 4 0

2

4

6

8

10

2

4

6

8

10

2

4

6

8

12

10 9

Log CFU/mL

8 7 6 5

B

4 0

12

10 9

Log CFU/mL

8 7 6 5

C 4 0

10

12

Time (months)

Fig. 3. Survival of L. paracasei JP1 (A), L. rhamnosus 64 (B) and L. gasseri 37 (C) during the frozen storage at 20  C (full lines) and 80  C (dotted lines) in MRS (circles), skim milk (squares), buttermilk (triangles) and cheese whey (stars). Figures are representative of three independent assays.

Fig. 4 shows the cell viability and the resistance to simulated gastric digestion after storage (4  C, 30 days) in milk or in acidified milk. When L. paracasei JP1 was suspended in acidified (pH 4.5) milk and subjected to dynamic gastric digestion, the gastric resistance (evaluated as cell viability after 90 min of simulated gastric digestion) was reduced in 0.4 Log orders (Fig. 4A). After the storage at 4  C for 30 days, no significant differences were observed in the level of viable cells compared to time zero, but after the simulated gastric digestion, cell viability significantly decreased in ca. 1.5 Log orders. When the same strain was suspended in non-acidified milk, the loss of gastric resistance was 1.2 Log orders and, after 30 days at 4  C, a viability loss of ca. 0.5 Log orders was observed, but no changes in the profile of gastric resistance was observed compared to that of time zero. For all the conditions assayed, no viable cells were detected for L. paracasei JP1 after the treatment with bilepancreatine solution, indicating that this strain is not resistant to in vitro simulated intestinal digestion. Fig. 4B shows the viability and gastric resistance of L. rhamnosus 64 after its storage at 4  C. At initial time, cell counts decreased ca. 3 Log orders after the simulated gastric digestion, both in acidified and non-acidified milk. This behaviour was practically the same after 30 days of storage in acidified milk, where ca 0.5 Log order reduction in cell counts were detected. For this condition and after simulated gastric digestion, a total loss in cell viability was observed. No viable cells were observed after the simulated intestinal digestion. When L. gasseri 37 was studied (Fig. 4C), the loss of viability in non-acidified milk was 1.85 and 1.53 Log orders at time zero and after 30 days of storage, respectively. When the cells were suspended in acidified milk, they were less affected by the simulated gastric digestion, reducing their viability in 0.64 and 1 Log orders, at time zero and after 30 days of storage, respectively. In this case, L. gasseri 37 resisted a further simulated intestinal digestion, which reduced its cell viability in 0.34 and 0.65 Log orders, in nonacidified and acidified milk, respectively. For all the strains, milk and acidified milk proved more protective than SGS since, at time zero, reductions in cell counts between 5.66 and 9.22 Log orders were observed when this solution was used as suspension medium (data not shown).

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V. Gregoret et al. / Food Microbiology 34 (2013) 19e28

Table 2 Relative growth (%) of studied strains in MRS broth supplemented with different concentrations of salts (NaCl and KCl) and MIC values against different antibiotics. Strain

L. paracasei JP1 L. rhamnosus 64 L. gasseri 37

Relative growth (mean value  SD)*

MIC (mg/mL) (mean value  SD)

NaCl**

CL

EM

SM

TC

VA

3.5  0.7 3.0  0.0 4.0  0.0

0.064  0.0 0.047  0.0 1.5  0.0

20  5.7 12  0.0 6  0.0

128.0  18.0b 0.44  0.4 3.0  0.0

256  0.0 256  0.0 1.5  0.0

KCl**

1%

2%

1%

2%

95.7  2.4a 95.8  2.9a 96.0  5.7a

91.7  2.2a 88.4  9.2a 95.5  6.3a

91.8  9.9a 99.6  0.4a 96.5  5.0a

95.9  0.6a 81.2  9.6a 97.2  3.8a

* Relative growth ¼ ANaCl/KCl/Acontrol  100; SD: standard deviation. ** Salt concentration (% w/v) in MRS broth. a: Values that are not significantly different (P > 0.05) from the control (MRS). CL: chloramphenicol; EM: erythromycin; SM: streptomycin; TC: tetracycline; VA: vancomycin. b: MIC > breakpoints for Lactobacillus, according to EFSA panel (EFSA, 2008).

3.6. In vitro safety assessment: antibiotic resistance 9

The resistance of the strains to five antibiotics commonly used (chloramphenicol, erythromycin, streptomycin, tetracycline as inhibitors of protein synthesis; and vancomycin, an inhibitor of cellwall synthesis) was determined using the commercial E-testÒ strips, in LSM agar. Table 2 shows the MIC values for all strains. L. paracasei JP1 showed resistance only to tetracycline, since the MIC value turned out to be significantly higher than the breakpoint for this bacterial species (4 mg/mL, EFSA, 2008). For the other antibiotics, MIC values were lower than the reported breakpoints. L. rhamnosus 64 and L. gasseri 37 were sensitive to all the antibiotics assayed.

Log (CFU/mL)

7

5

3

A

3.7. In vivo safety and functionality assessment

1 0

20

40

60

80

100

9

Log (CFU/mL)

7

5

3

B 1 0

20

40

60

80

100

9

Log (CFU/mL)

7

The oral administration of Lactobacillus strains, at the chosen doses, induced no undesiderable side effects since there was no translocation of enterobacteria to the liver (translocation assay was negative). The study of the intestine architecture by haematoxylineosin staining showed no excessive or unusual lymphocyte infiltrates, oedema, mucosal atrophy or symptoms of intestinal inflammation. No significant morphological change in the overall architecture of the small and large intestines were observed compared to control mice (pictures not shown). In relation to the in vivo probiotic properties of the strains, significant (P < 0.05) increases in the percentages (between 75 and 110 %) of phagocytosis were observed when all strains were administered during 5 and 7 days (Fig. 5). Also, the number of IgAproducing cells in the lamina propria of the small intestine significantly (P < 0.05) increased for all the strains tested, when compared with the corresponding control values. In the case of L. paracasei JP1, 22% of increase was detected after 10 days of administration. For L. rhamnosus 64, the differences were observed after 7 and 10 days (60 and 57%, respectively) and, in the case of L. gasseri 37, the number of IgA-producing cells increased 41% after 7 days of administration (Fig. 6). No differences in the number of IgA-producing cells were detected in large intestine, for any of the studied strains (data not shown) compared to control animals.

5

4. Discussion 3

C 1 0

20

40

60

80

100

Time (min)

Fig. 4. Survival of L. paracasei JP1 (A), L. rhamnosus 64 (B) and L. gasseri 37 (C) during the simulated gastric digestion of the strains conserved at 4  C in milk (full lines) and acidified (pH 4.5) milk (dotted lines), at initial time (full symbols) and after 30 days (empty symbols). Figures are representative of three independent assays.

It is well established by now that the human body is inhabited by at least 10 times more bacteria than the number of human cells in the body, and that the majority of those bacteria are found in the gastrointestinal tract (Tannock, 2003). Throughout the history of microbiology, most human studies have focused on the diseasecausing organisms found on or in people; in the last 20 years, however, many reports have centred on the benefits of the resident bacteria and, as a consequence, on probiotics. Selected strains of probiotics have specific effects, though these health benefits cannot be extrapolated from one strain to another, not even of the same

V. Gregoret et al. / Food Microbiology 34 (2013) 19e28 20

*

*

18

* *

Percentage Phagocytosis

16

*

*

14

12 10 8

6 4 Control

L.c. JP1

L.r. 64

L.g. 37

Fig. 5. Effect of the oral administration of L. paracasei JP1 (L.c. JP1), L. gasseri 37 (L.g. 37) and L. rhamnosus 64 (L.r. 64) during 2 ( ), 5 ( ) and 7 (-) consecutive days, on the phagocytic activity (%) of peritoneal macrophages. *: Significantly (P < 0.05) different values from control group (mice received 1% w/v milk).

species. In this regard, the human intestinal tract represents an important source for the isolation of new potential probiotics, in particular, those belonging to Lactobacillus and Bifidobacterium genera, the most frequently included in functional food (Forssten et al., 2011). In a previous work, we isolated 19 strains of Lactobacillus from feces of breast-fed infants, most of them L. rhamnosus and, in minor proportion, L. gasseri (Vinderola et al., 2008). These strains were evaluated by means of a set of simple in vitro tests (phage sensitivity; growth and viability in milk at different values of pH; resistance to salts and flavour compound; bacterial interactions; tolerance to simulated gastric juice and bile; hydrophobicity). From those results, two strains, L. rhamnosus 64 (named LrF64) and L. gasseri 37 (LgF37) were identified as potential candidates for new probiotic dairy foods. In this context, they were selected for this work for a further and comprehensive characterization. Additionally, another strain from the same origin, L. paracasei JP1, was included in this work. All these bacterial

*

300

*

275 Number of IgA+ cells in 10 fields

*

250

* 225

200

175

150 Control

L.c. JP1

L.r. 64

L.g. 37

Fig. 6. Effect of oral administration of L. paracasei JP1 (L.c. JP1), L. gasseri 37 (L.g. 37) and L. rhamnosus 64 (L.g. 64) after 2 ( ), 5 ( ) and 7 (-) consecutive days, on the number of IgAþ cells in lamina propria of small intestine. *: Significantly (P < 0.05) different values from control group (mice received 1% w/v milk).

25

species are common members in the microbiota of breast-fed infants (Xanthopoulus et al., 2000). Although the most important properties of probiotic bacteria are based on their beneficial effects on host health, by definition, probiotics must be viable at the time of consumption, even when non-viable probiotics are able to produce some health effects as well (Ouwehand and Salminen, 1998; Kataria et al., 2009). In consequence, new isolates aimed at integrating the formulation of probiotic foods should be able to grow in economic culture media for biomass production at industrial scale and in selective or differential culture media for their enumeration in the final product (Stanton et al., 2003; Forssten et al., 2011). The three lactobacilli strains included in this work were able to grow in milk, buttermilk and cheese whey (all media supplemented with yeast extract), achieving viable counts higher than 108 CFU/mL after 24 h at 37  C. Moreover, when they were frozen stored (20 and 80  C), these low cost media (mainly buttermilk) proved suitable as cryoprotectants. This fact could be due to the higher buffer capacity of buttermilk, compared to MRS or whey, and could be useful to avoid cell injury due to excessive acidification. Scarce data exist about the appropriate strategy to reduce the costs of culture media, for instance, by using low-cost substrates that could come directly from dairy plants as by-products of cheese- and buttermaking processes. In a previous work, we demonstrated that buttermilk and cheese whey were adequate culture media to produce and freeze biomass of Lactobacillus strains (Burns et al., 2008b). In general, B-MRS and LP-MRS are adequate culture media for the selective and differential enumeration of bifidobacteria, Lactobacillus acidophilus or Lactobacillus casei strains in fermented dairy products (Vinderola and Reinheimer, 2000), being able to inhibit the growth of starter cultures (Streptococcus thermophilus and L. delbrueckii subsp. bulgaricus). In our laboratory, these culture media are frequently used for the routine enumeration of probiotic bacteria in commercial products. During the last years, however, a growing number of commercial starter cultures showed to be resistant to the concentration of selective agents (lithium chloride, sodium propionate and bovine bile) used in those culture media (unpublished data). Taking into account these findings, we tested the growth ability of the three strains at increasing concentrations of bile and sodium propionate/lithium chloride. In our work, the growth of L. paracasei JP1 and L. rhamnosus 64 was significantly affected by bovine bile at all concentrations evaluated. Instead, L. gasseri 37 was able to withstand it. When LP-MRS was assayed, all strains grew in the presence of increasing concentrations of both lithium chloride and sodium propionate, except for L. paracasei JP1, which was partially inhibited. Another important factor for the formulation of probiotic foods is the resistance (viability and functionality) of probiotics to technological stress (salts, acidity, refrigeration, freezing etc.). When the strains were stored at 4  C for 30 days in acidified milk (partially simulating the conditions of commercial yogurts), an adequate survival was observed in all cases. Instead, the resistance of treated cells to simulated gastric digestion was variable. L. paracasei JP1 and L. gasseri 37 showed high resistance to dynamic gastric digestion for 30 days of refrigeration. In both cases, a slight additional protection was observed with acidified (pH 4.5) milk when compared with milk. Moreover, L. gasseri 37 showed resistance to both gastric and intestinal successive barriers, as was previously demonstrated for this strain by means of other methodologies (Vinderola et al., 2008). Contrarily, L. rhamnosus 64 exhibited high sensitivity to simulated gastric digestion (losses of viability of 3 Log orders, approximately, at time zero). In this case, acidified milk also exerted a protective effect during the conservation at 4  C, since no changes in its survival were detected. In milk, however, the viability of this strain was

26

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seriously affected after storage. These data suggest that the exposure to high levels of lactic acid may have induced in Lactobacillus a crossed protection to adapt to the conditions of the simulated gastrointestinal tract, as was suggested for other stress factors that usually induce tolerance to successive exposition to the same or other stress factors (Marteau et al., 1990; Noriega et al., 2004). Usman and Hosono (1999) evaluated the viability of L. gasseri in milk (pH 6.3), reporting a low (<1 Log order) loss of viability after one month of storage at 4  C. Recently, Vinderola et al. (2011) demonstrated the variability of gastric acid resistance during the refrigerated storage of L. casei strains in commercial fermented milks in relation to the flavour and storage conditions. Saarela et al. (2009) reported different survival of L. rhamnosus E800 exposed to simulated gastric digestion, depending on the carrier used for freeze-drying. Therefore, the evaluation of gastric acid resistance of each strain in the particular conditions of the food that will serve as a vehicle represents an important test to be considered during the design of new probiotic products (Forssten et al., 2011). In this regard, salts commonly used in cheeses, for example, could affect the viability and functionality of probiotic strains present in the food matrix (Gómes et al., 1998). Reinheimer et al. (1997) studied the incidence of NaCl and KCl (1 and 2%) on the activity of different thermophilic (L. helveticus and S. thermophilus) starters used in cheese production, showing that salt presence reduced their acidifying and proteolytic activities. More recently, Briggiler-Marcó et al. (2007) reported tolerance of L. casei and L. rhamnosus strains to grow in MRS added with NaCl and KCl. In this work, salts (NaCl or KCl) did not inhibit, at the concentrations tested, the growth of any of the strains assayed, representing an interesting issue, for example, in case they were to be considered for the formulation of probiotic cheeses. In order for probiotics to be considered safe, they should be free of transmissible antibiotic-resistance genes (FAO/WHO, 2002; EFSA, 2008; Vankerckhoven et al., 2008), since antibiotic-resistant foodborne species can therefore act as “reservoirs” of resistance genes and persist in the human host environment in the absence of selective pressure (Ammor et al., 2007; Comunian et al., 2010). On the basis of this precautionary principle and to prevent potential risks to human health, the European Food Safety Authority (EFSA) has proposed a framework for the safety evaluation of microorganisms in the food chain, similar in purpose to the Generally Recognised As Safe (GRAS) approach, taking into account experience of use (Qualified Presumption of Safety, QPS). However, a consensus document does not yet exist at European level and it is therefore unclear which methods should be applied to evaluate the safety of probiotic strains. Based on these EFSA-proposals, microdilution method can be considered as a “tentative reference method” for MIC determination. Furthermore, the E-test used in this work was appreciated as a valuable method, comparable to the reference assay (Vankerckhoven et al., 2008). Most of Lactobacillus species are susceptible to antibiotics able to inhibit the synthesis of proteins (chloramphenicol, erythromycin, clindamycin and tetracycline) and more resistant to aminoglycosides (neomycin, kanamycin, gentamicin and streptomycin) (Ammor et al., 2007). Taking into account the microbiological breakpoint values established by EFSA (2008), all strains tested in the present study were sensitive to all antibiotics tested except L. paracasei JP1, which showed resistance to tetracycline. Although L. paracasei, together with the closely related species L. casei and L. rhamnosus, represents one of the most common bacterial species employed as probiotics in numerous fermented dairy products, only limited information exists about its antibiotic resistance (Cataloluk and Gogebakan, 2004; Devirgiliis et al., 2008; Huys et al., 2008; Comunian et al., 2010). In a recent work, the presence of genetic determinants for

tetracycline resistance [tet(M), tet(W)] was demonstrated in L. paracasei strains isolated from traditional Italian fermented foods, showing that the antibiotic resistance (detected in only a low percentage of the strains) was associated to dairy food produced in areas where more intensive practices were applied in animal husbandry (Comunian et al., 2010). Similar results were found by Cataloluk and Gogebakan (2004) reporting that a high proportion of lactobacilli from human origin (including L. casei, L. rhamnosus and L. gasseri) harboured tetracycline and/or erythromycin resistance genes. Tetracycline resistance is very rarely found in L. paracasei (Ammor et al., 2007; Huys et al., 2008), and our results suggest that acquired rather than constitutive resistance took place in the GIT of neonates or their mothers. The in vivo studies on safety and functionality of new isolates intended for human consumption is a prerequisite for the development of functional foods (FAO/WHO, 2002). A regular probiotic feeding might have the risk of bacterial translocation, so this issue has to be carefully investigated in order to determine at which dose and for how long the product can be safely administered to the host (Pavan et al., 2003). In this study, we determined the possible side-effects of the oral administration of the strains at the dose employed (2  108 CFU/mouse/day). Under all conditions, no bacterial translocation was detected, so that the modulation of the intestinal immune response cannot be attributed to inflammatory responses due to a translocation event. Additionally, no differences were observed in the architecture of small and large intestines after the treatment with all strains, indicating the safety of the selected doses for this animal model. One of the health benefits ascribed to probiotic bacteria or food containing them is their capacity to positively modulate the gut immune response, in a strain specific way (López et al., 2010). The activation of the intestinal immune system may lead to the action of different health effects in a therapeutic or preventive way, such as prevention of certain intestinal infections, inflammatory diseases and colon cancer (Yan and Polk, 2010) or the resolution of different diarrheas (Sartor, 2004). The role of IgA is to exert immune exclusion of pathogens and foreign proteins (Kaila et al., 1992; MacPherson et al., 2001). Vintiñi et al. (2000) demonstrated that certain strains of LAB belonging to the species L. casei, L. rhamnosus, and L. plantarum enhance both systemic and mucosal immunity. Paturi et al. (2007), by using a murine model similar to that of our study, demonstrated that the administration of L. acidophilus and L. paracasei caused an increase in the number of IgA producing cells, IL-10 and IFN-g cytokine producing cells in the small intestine. In the systemic immune response, mice also enhanced the secretion of anti-inflammatory cytokine (IL-10). In a more recent work, Tsai et al. (2010) studied the immune enhancing properties of L. paracasei in BALB/c mice, observing an induction of intestinal IgA production that could prevent infections or other intestinal pathologies. In a clinical study, MartínezCañavate et al. (2009) evaluated the immunological effect of the consumption of fermented milks containing probiotic strains of L. gasseri and L. coryniformis in children suffering from allergy. In this case, the decrease in the level of IgE (the main immunoglobulin implicated in allergies) was accompanied by a significant increase in mucosal IgA that could also play a role in maintaining the children health status. In our study, we observed that the strains, administered for 5 and/or 7 days, were able to significantly increase the number of IgAþ cell in the small intestine mucosa without causing an imbalance and translocation of the resident microbiota. This increase in the number of IgAþ cells was accompanied by an increase in the phagocytic activity of peritoneal macrophages, suggesting then that the strains were also able to modulate the mucosal response at distant sites (Kato et al., 1983).

V. Gregoret et al. / Food Microbiology 34 (2013) 19e28

5. Conclusions Following the main selection criteria proposed by FAO/WHO for probiotics, the three strains isolated from feces of local newborns were able to adequately support different technological (resistance to acidic conditions, frozen storage and salts) and biological (simulated gastrointestinal digestion) challenges. Also, L. rhamnosus 64 and L. gasseri 37, in particular, could be considered as safe and functional strains, since they were sensitive to five tested antibiotics and, from the in vivo trial, they were able to stimulate the immune response by enhancing peritoneal macrophages activity and IgA producing cells in the small intestine. Then, both strains can be regarded as potential probiotics for the formulation of new probiotic foods for the local market. Acknowledgements This work has been supported by the PIP project N 112-20080100645, from the Consejo Nacional de Investigaciones Científicas y Tecnológicas (Argentina), entitled “Funcionalidad de bacterias probióticas para alimentos: factores que la influencian y recursos para garantizarla”. References Ammor, M.S., Flórez, A.B., Mayo, B., 2007. Antibiotic resistance in nonenterococcal lactic acid bacteria and bifidobacteria. Food Microbiology 24, 559e570. Binetti, A.G., Suárez, V.B., Tailliez, P., Reinheimer, J.A., 2007. Characterization of spontaneous phage-resistant variants of Streptococcus thermophilus by RAPD analysis and identification of phage-resistance mechanisms. International Dairy Journal 17, 1117e1122. Briggiler-Marcó, M., Capra, M.L., Quiberoni, A., Vinderola, G., Reinheimer, J.A., Hynes, E., 2007. Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses. Journal Dairy Science 90, 4532e4542. Burns, P., Vinderola, G., Binetti, A., Quiberoni, A., de los Reyes-Gavilán, C.G., Reinheimer, J., 2008a. Bile-resistant derivatives obtained from non-intestinal dairy lactobacilli. International Dairy Journal 18, 377e385. Burns, P., Vinderola, G., Molinari, F., Reinheimer, J., 2008b. Suitability of whey and buttermilk for the growth and frozen storage of probiotic lactobacilli. International Journal of Dairy Technology 61 (2), 156e164. Cataloluk, O., Gogebakan, B., 2004. Presence of drug resistance in intestinal lactobacilli of dairy and human origin in Turkey. FEMS Microbiology Letters 236, 7e12. Champagne, C., Ross, R., Saarela, M., Flemming, K., Charalampopoulos, H., 2011. Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices. International Journal of Food Microbiology 149, 185e193. Collins, J.K., Thornton, G., Sullivan, G.O., 1998. Selection of probiotic strains for human applications. International Dairy Journal 8, 487e490. Comunian, R., Daga, E., Dupre, I., Paba, A., Devirgiliis, C., Piccioni, V., Perozzi, G., Zonenschain, D., Rebecchi, A., Morelli, L., De Lorentiis, A., Giraffa, G., 2010. Susceptibility to tetracycline and erythromycin of Lactobacillus paracasei strains isolated from traditional Italian fermented foods. International Journal of Food Microbiology 138, 151e156. Devirgiliis, C., Caravelli, A., Coppola, D., Barile, S., Perozzi, G., 2008. Antibiotic resistance and microbial composition along the manufacturing process of Mozzarella di Bufala Campana. International Journal of Food Microbiology 128, 378e384. Edwards, U., Rogall, T., Blockerl, H., Emde, M., Bottger, E., 1989. Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Research 17 (19), 7843e7853. Europenan Food Safety Authority (EFSA), 2008. Technical guidance prepared by the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on the update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance. EFSA Journal 732, 1e15. Ezendam, J., van Loveren, H., 2006. Probiotics: immunomodulation and evaluation of safety and efficacy. Nutrition Reviews 64, 1e14. FAO/WHO, 2002. Guidelines for the evaluation of probiotics in food. Food and Agriculture Organization of the United Nations and World Health Organization Working Group Report. Available from: . Forssten, S.D., Sindelar, C.W., Ouwehand, A.C., 2011. Probiotics from an industrial perspective. Anaerobe 17, 410e413. Gómes, A.M., Teixeira, M.G., Malcata, F.X., 1998. Viability of Bifidobacterium lactis and Lactobacillus acidophilus in milk: sodium chloride concentration and storage temperature. Journal of Food Processing and Preservation 22, 221e240.

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