Sensitivity of bifidobacteria to oxygen and redox potential in non-fermented pasteurized milk

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International Dairy Journal 16 (2006) 1038–1048 www.elsevier.com/locate/idairyj

Sensitivity of bifidobacteria to oxygen and redox potential in non-fermented pasteurized milk Marie-Pierre Bolduca, Yves Raymondb, Patrick Fustierb, Claude P. Champagneb, Jean-Christophe Vuillemarda, a

Institut des Nutraceutiques et des Aliments Fonctionnels (INAF) and Centre de Recherche en Sciences et Technologie du Lait (STELA), Faculte´ des Sciences de l’Agriculture et de l’Alimentation, Universite´ Laval,Que´bec, QC, Canada G1 K 7P4 b Food Research and Development Centre, Agriculture and Agrifood Canada, 3600, Casavant Ouest,Saint-Hyacinthe, QC, Canada J2S 8E3 Received 1 May 2005; accepted 17 October 2005

Abstract The effect of deaeration, cysteine addition and electroreduction of milk on the viability of various Bifidobacterium strains in pasteurized milk during refrigerated storage at +7 1C for 4 weeks was assessed. Preliminary assays in deaerated milks showed considerable variability to oxygen sensitivity among the eight strains examined during refrigerated storage. Three strains with different sensitivity were selected for subsequent assays. Assays on the effect of oxygen during growth at 37 1C were included in the screening and results suggest that the growth inhibition observed could serve as an indicator of the negative effect of oxygen on stability of bifidobacteria during refrigerated storage in similar oxygen conditions. The electroreduction treatment had a positive impact on the survival of two of the three cultures tested during the 4 week storage period at 7 1C. Addition of cysteine and deaeration alone had similar effects on cell viability during storage, suggesting that the benefit of electroreduction was mainly linked to its action of lowering the level of dissolved oxygen in the samples. Data from this study suggest that electrochemical reduction of milk, as well as deaeration or addition of reducing agents could be applied to enhance the survival of bifidobacteria during extended storage at 7 1C in milk. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bifidobacteria; Oxygen; Redox potential; Viability

1. Introduction Promoting health and healthy habits is a major concern to consumers. As a result, probiotic-containing products gain in popularity and acceptance. Probiotics are defined as live microorganisms that, administered in adequate amounts, confer a beneficial physiological effect on the host (Araya et al., 2002). Most commonly, there are two genera that have gained recognition as probiotic bacteria and they are Lactobacillus spp., especially L. acidophilus, L. casei, L. johnsonii, L. gasseri and L. rhamnosus; and Bifidobacterium spp., especially B. bifidum, B. animalis Corresponding author. Tel.: +1 418 656 5968; fax: +1 418 656 3353.

E-mail address: [email protected] (J.-C. Vuillemard). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.10.030

subsp. lactis, B. infantis, B. adolescentis and B. longum (Ouwehand et al., 2003; Talwalkar & Kailasapathy, 2004a). The main therapeutic and health benefits that have been associated with consumption of probiotic products include prevention of intestinal infections, enhancement of the immune system, prevention of diarrhoeal diseases, anticarcinogenic activity, prevention of hypercholesterolemia, improvement in lactose utilization, prevention of gastrointestinal tract diseases and stabilization of the gut mucosal barrier (Kailasapathy & Chin, 2000; Ouwehand et al., 2003). Dairy products, such as fresh milk, fermented milk, yoghurt and cheese, have been targeted as good carrier foods for probiotic microorganisms mainly because their consumption is widespread making them a good vector to reach many consumers (Lourens-Hattingh & Viljoen,

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2001). It has been suggested that the daily intake should be between 108 and 109 colony-forming units (cfu) (Kurmann & Rasic, 1991), but some authors recommend higher numbers (Charteris, Kelly, Morelli, & Collins, 1998). However, surveys conducted on several commercially available products have shown low population levels at the time of consumption (Donnet–Hughes, Rochat, Serrant, Aeschlimann, & Schiffrin, 1999; Fasoli et al., 2003; Shah, 2000). Several factors can be responsible for this loss of viability during storage and oxygen sensitivity is considered an important problem in the manufacture and storage of probiotic products (Collins & Hall, 1984; de Vries & Stouthamer, 1969; Klaver, Kingma, & Weerkamp, 1993; Talwalkar & Kailasapathy, 2004a). Many strategies have been studied to protect bacteria from the deleterious effects of oxygen toxicity such as the use of special high oxygen consuming strains (Lourens-Hattingh & Viljoen, 2001), the use of ascorbic acid as an oxygen scavenger in yoghurts (Dave & Shah, 1997a), microencapsulation (Talwalkar & Kailasapathy, 2003a), the use of packaging material less permeable to oxygen (Dave & Shah, 1997d) and oxidative stress adaptation (Talwalkar & Kailasapathy, 2004b). Cysteine is a strong reducing agent and it is known for lowering redox potential of solutions. Although the addition of cysteine to probiotic-containing products could affect their flavour, its addition to growth medium or to fermented dairy products has demonstrated that low redox potential values are favourable for the viability of bifidobacteria during storage (Dave & Shah, 1997b, 1998). It is unknown, however, if the addition of cysteine to unfermented milks can also be beneficial. Electrolysis is a process that uses electrical energy to force reactions to occur on the surface of an electrode in contact with the liquid to be treated. In the two compartment system used, the fluid in contact with the cathode is submitted to reduction reactions (electroreduction) (Tallec, 1985). This process already has some applications in the food industry (He´kal, 1983; Inoue & Kato, 2003; Koseki, Nakagawa, Tanaka, Noguchi, & Omochi, 2003; Mondal & Lalvani, 2003) but has never been used to influence microbial stability in milk, or to enhance the stability of bifidobacteria. The objective of this study was to verify if lowering redox potential of milk to negative values by an electroreduction treatment would be effective in improving the viability of three strains of bifidobacteria during refrigerated storage for 4 weeks. The survival of the selected strains of bifidobacteria in electroreduced milk was also compared to their survival in deaerated milk, in milk supplemented with cysteine and in control milk. 2. Materials and methods 2.1. Microorganisms and culture conditions Eight different strains representing five species of Bifidobacterium were used in this study: B. bifidum KYO,

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B. longum Rosell-R023, B. longum 411 BBL, B. breve ATCC 15700, B. bifidum ATCC 29521, B. infantis ATCC 15697, B. longum ATCC 15708, B. animalis subsp. lactis DSM 10140. All strains were obtained from the bifidobacteria collection of the Food Research and Development Centre. Lyophilized cultures were rehydrated in 1 mL of a sterilized rehydration medium (1.5% peptone, 1.0% tryptone and 0.5% meat extract) with subsequent 10% (v/v) inoculation into lactobacilli MRS broth (Difco, Detroit, MI, USA) and incubation for 24 h at 371C in an anaerobic chamber (85% N2, 5% CO2 and 10% H2). One subculture at 1% (v/v) inoculation was carried out in identical incubation conditions. Stock cultures were prepared by mixing the fresh subculture with 20% glycerol and sterile 20% (w/v) reconstituted skim milk in a 2:5:5 ratio, with subsequent storage at
80 1C in portions of 1 mL placed in 2 mL-vials (Wheaton Scientific Products, Millville, NJ, USA).

2.2. Strain selection as a function of oxygen sensitivity Because of equipment limitations, it was not possible to conduct the evaluation of the effect of milk electroreduction on the stability of all eight strains. Therefore, a screening procedure was carried out in order to select cultures that would have different sensitivities to oxygen during storage in milk. Before each independent assay, fresh cultures were prepared by adding one thawed stock culture to 99 mL of lactobacilli MRS broth, and incubating between 16 and 24 h at 37 1C in the anaerobic chamber. The incubation period was variable so that each culture was collected once it had attained the beginning of the stationary growth phase. Microfiltered and pasteurized milk having 2% fat (2 L, Purfiltre, Lactantia-Parmalat, Montreal, Canada) was purchased at a local supermarket and transferred into two 1 L sterilized bottles (Nalgene Labware, Rochester, NY, USA). One of the samples was deaerated by bubbling nitrogen gas until an oxygen concentration of less than 0.1 g L
1 was reached. This operation was performed in a laminar flux hood to minimize contamination of the milk. Immediately after deaeration, the bulk sample was covered with a sterile aluminium paper and immediately transported into the anaerobic chamber for subsequent manipulations, in order to minimize oxygen re-integration. The non-deaerated 1 L-sample was kept in the sterile laminar hood. Both 1 L-bulk samples were transferred in portions of 100 mL into 100-mL sterilized glass bottles (Nalgene) and placed into an ice/water bath until the milk temperature reached 4 1C. Fresh cultures were then used to inoculate the milk samples at a concentration of approximately 107 cfu mL
1. Two uninoculated bottles of each milk (deaerated and non-deaerated) were kept as controls for subsequent measurements.

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2.4. Bifidobacteria survival in electroreduced milk during refrigerated storage

The non-deaerated milks were incubated at 7 1C for 2 weeks in aerobic conditions and deaerated milks were stored at the same temperature in anaerobic jars (3 L, GasPak Systems, BD and Company, Franklin Lakes, NJ, USA) containing three Anaerogen envelopes (Oxoid, Hampshire, UK). Six independent assays were carried out.

The cell viability studies during refrigerated storage were only carried out on three strains based on the results obtained in assays described in Sections 2.2 and 2.3, and they were B. bifidum KYO, B. longum ATCC 15708 and B. lactis subsp. animalis DSM 10140. One millilitre of thawed stock cultures was added to 99 mL MRS medium, which was subsequently incubated anaerobically at 37 1C (16 h for B. bifidum KYO and B. longum ATCC 15708 and 24 h for B. lactis subsp. animalis DSM 10140). Fresh cultures were centrifuged (8000  g, 20 min, 4 1C) and resuspended in 10 mL of pasteurized skim milk (Purfiltre). This suspension was used for inoculation. Four treatments were carried out, with five independent repetitions each. The samples for each treatment were prepared using microfiltered and pasteurized skim milk (Purfiltre). Treatments were applied as follows: For the preparation of the electroreduced pasteurized milk, a 250 mL portion of milk was transferred into an Hshaped membrane electrolysis cell (Fig. 1) previously sterilized by autoclaving (121 1C, 10 min). Electroreduction was performed on the sample by applying a voltage of
1.55 V between the working stainless steel cathode and the Ag/AgCl reference electrode. Milk was agitated using a magnetic stirrer to minimize fouling on the cationic membrane (Neoseptas CMX-SB, Tokuyama, Tokyo, Japan). Milk was treated for 40 min to reach negative redox potential values below
300 mV as well as dissolved

2.3. Cell growth study Growth curves for each of the eight strains were obtained by reading optical density (OD) using the Thermo Bioscreen C (Labsystems, Helsinki, Finland) automated spectrophotometer in order to study the growth behaviour of the strains in different conditions: (a) in a normal lactobacilli MRS medium; (b) in MRS supplemented with cysteine–HCl (0.5%) to study growth in a reduced medium; (c) in MRS supplemented with ascorbic acid (1%) to study growth in a medium with low oxygen level. For each of the three independent assays of this experiment, an additional plate was prepared by adding 50 mL of paraffin oil to each well to study the same media under anaerobic conditions. On each plate, two wells were filled with each medium (200 mL) to obtain a duplicate reading for each strain and inoculated with a 20 mL portion of a fresh culture prepared by thawing a 1 mL frozen vial of each strain and adding it to 9 mL of the appropriate medium. The plates were incubated for 24 h at 37 1C. OD measurements (600 nm) were taken every 15 min, with the Bioscreen C unit plate agitation set at ‘‘moderate shaking’’ for 30 s before each OD reading.

Potentiostat

Reference electrode

Cationic membrane

H+ A-

anode H2O • ½O2 + 2H+ + 2é

cathode ½O2 + 2H+ + 2é → H2O 2H2O + 2é → 2OH- + H2

2é

electrolyte ↑O2

2é

milk ↓O2 ↑H2

Fig. 1. Simplified diagram of an electrolysis cell.

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oxygen concentrations between 2 and 3 mg L
1. The electrolyte used on the anode side was a solution of 0.1 M sulphuric acid. During treatments, redox potential (mV) and dissolved oxygen (mg L
1) were followed. After the treatment, the processed milk, along with the electrolytic cell, was immediately transported to an anaerobic chamber to be transferred into a sterilized 500 mL-glass bottle to cool down in an ice/water bath to 7 1C prior to the inoculation. The deaerated pasteurized milk was prepared by applying vacuum to 500 mL of milk which had been transferred into a sterile vacuum 2 L-Erlenmeyer flask. The vacuum was applied for 30 min with agitation by a stirring rod. After deaeration, the milk was immediately transferred into two sterile 250 mL glass bottles under a nitrogen flow and transported into the anaerobic chamber to minimize oxygen re-entrance where it was allowed to cool to 7 1C prior to inoculation. For the preparation of the cysteine-supplemented milk, 250 mL of deaerated milk was cooled to 7 1C and 2.5 mL of a (10%) cysteine–HCl solution was added. This cysteine solution was prepared in phosphate buffer (pH 7.0) to minimize the impact on the pH of milk upon its addition. Three different types of control samples were used in this study. (i) The first type consisted of inoculated pasteurized milk to which no further treatment was performed. This milk was kept in the same type of vials as the other treated milks; it served as a mean of comparison for growth/ survival of Bifidobacterium strains. (ii) The second type of control consisted of inoculated pasteurized milk, as in (i) above, stored into a larger bottle with a foam cap to allow exchange of gases between milk and the environment. (iii) The third type of control consisted of vials of uninoculated milk for the purpose of following the evolution of psychrotrophic bacteria over the storage period, as well as the evolution of physical properties. Milk inoculation was done in order to obtain an initial count of approximately 107 cfu mL
1. In order to prevent changes in oxygen and redox during sampling, the experiment was not carried out by taking samples at various incubation times from a single flask. Rather, following inoculation, the milk was fractioned into 20 mL-GC vials closed using Teflon caps (one vial/sample). Except for controls (ii) the bottles were completely filled with the milk samples and care was taken to minimize the head space. All samples were incubated at 7 1C for 4 weeks. Deaerated or electroreduced milk samples were stored in anaerobic jars to assure that oxygen could not re-enter the milk. At each given incubation time, the required number of vials were opened for analyses. Five independent repetitions were carried out. 2.5. Analyses Viable counts of bifidobacteria were obtained by spreadplating (0.1 mL) the appropriate peptone–water dilution on MRS-cysteine agar (MRS supplemented with 0.5 g L
1

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cysteine–HCl) and incubating the plate at 37 1C for 48 h in the anaerobic chamber. Psychrotrophic counts (from the control bottles) were done on 3MTM PetrifilmTM aerobic count plates (3M, St-Paul, MN, USA) after incubation at 7 1C for 10 days. Redox level was determined by a platinum electrode Metrohm 6.0402.100 MC (]2092170-6, Brinkmann Instruments, Westbury, NY, USA) connected to an Oakton pH meter (Oakton Instruments, Vernon Hills, IL, USA) and calibrated using a 250 mV oxidoreduction buffer (]2009612-8, Brinkmann). Dissolved oxygen was measured using a VWR Symphony electrode (VWR Scientific Products, West Chester, PA, USA) mounted with the specified membrane and filled with the supplied DO electrolyte solution (cat. no. 14002-830). The electrode was connected to a VWR Symphony SP50D portable DO meter. The electrode was calibrated every 2 h as described in the supplier’s manual. The pH was determined by an Oakton electrode connected to an Oakton pH meter and calibrated using pH 4 and pH 7 buffer solutions. The electrodes were sterilized with a 70% ethanol solution. For strain selection as a function of oxygen sensitivity, the differences between initial and final viable counts (delta) for each strain of bifidobacteria in aerobic and anaerobic conditions were subjected to a T-test using the SAS Software (Version 8.02.02 MOP020601, Raleigh, NC, USA). Analyses of variance for the cell growth studies were done according to a split-plot model with oil effect in the whole plots and the medium effect in the split plot units using the SAS Software. Bifidobacterium population data and parameters measured in Section 2.4 were analysed by the Mixed Procedure coupled to Bonferroni adjustements of the P-values with the same software. 3. Results and discussion 3.1. Screening assays in deaerated milk Table 1 presents the results of the effect of deaeration on the survival throughout 2 weeks of refrigerated storage of eight different strains of Bifidobacterium spp. in pasteurized milk. Although inoculation levels were close to 107 cfu mL
1, there were some small variations between the assays, and data were analysed as the difference between the final and the initial populations (delta). These results clearly demonstrate that oxygen sensitivity varies from one strain to another, which is in agreement with other studies (de Vries & Stouthamer, 1969; Shimamura et al., 1992; Talwalkar & Kailasapathy, 2003b; Talwalkar, Kailasapathy, Peiris, & Arumugaswamy, 2001), and which confirmed the usefulness of the screening step. In this case, B. bifidum KYO (P ¼ 0:9232) was the most tolerant to the presence of oxygen in milk whereas B. infantis ATCC 15697 (P ¼ 0:0098) was the least tolerant. Five strains showed slight viable count increases in milk at +7 1C in both anaerobic and aerobic conditions. Dechter and

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Table 1 Viability of Bifidobacterium spp. and final parameters measured after 2 weeks of refrigerated storage in non-fermented milk Strain

B. B. B. B. B. B. B. B.

bifidum KYO longum Rosell-R023 longum 411 BBL breve ATCC 15700d bifidum ATCC 29521 infantis ATCC 15697d longum ATCC 15708d animalis subsp. lactis DSM 10140

Non-deaerated milk

Deaerated milk

Delta (log10 cfu mL
1)

pHa

Redoxb (mV)

DOc (ppm)

Delta (log10 cfu mL
1)

pHa

Redoxb (mV)

DOc (ppm)

0.53 0.22 0.11 0.53 1.80
2.86
2.54
0.18

6.50 6.52 6.52 5.94 6.41 6.68 6.58 6.62

+255.03 +255.07 +253.03 +241.83 +258.07 +251.77 +254.37 +247.07

5.23 5.23 6.10 4.13 5.20 6.27 6.50 6.30

0.58 0.56 0.09 1.66 1.98
0.16
0.60
0.54

5.96 5.94 6.43 5.31 5.98 6.56 6.52 6.58


428.00
450.67
459.53
422.67
440.13
466.00
486.00
458.00

0.30 0.43 0.37 0.70 0.53 0.23 0.20 0.13

a

Average initial pH of milk was 6.66 in aerobic conditions and 6.62 in anaerobic conditions. Average initial redox potential of milk was 170 mV in aerobic conditions and
70 mV in anaerobic conditions. c Average initial dissolved oxygen was 6.7 ppm in aerobic conditions and 0.3 ppm in anaerobic conditions. d indicates that there is a statistical difference between the aerobic and anaerobic delta according to t-test. b

Hoover (1998) observed the same apparent growth capability of some bifidobacteria at +4 1C. Among the strains that showed increases in viable counts during the 2 week-storage period, better growth was generally observed in the absence of oxygen. Similarly, lower mortality was observed in the absence of oxygen except for B. animalis subsp. lactis DSM 10140. Even though bifidobacteria are categorized as anaerobes, it has been shown that some strains possess an enzymatic mechanism to overcome the deleterious effects of oxygen toxicity (Ahn, Hwang, & Park, 2001; Shimamura et al., 1992; Talwalkar & Kailasapathy, 2003b). Statistical analysis by T-test demonstrated significant differences between the survival in aerobic and anaerobic conditions only for B. breve ATCC 15700, B. infantis ATCC 15697 and B. longum ATCC 15708 strains (P ¼ 0:0279, 0.0098 and 0.0387, respectively). Psychrotrophic microorganisms were absent in all samples at the beginning of the experiment and also after 2 weeks of refrigerated storage under both conditions (results not shown). Cell growth was associated with a decrease in pH (Table 1). Indeed, regression analyses showed that there was a certain relationship (R2 ¼ 0:70) between final pH and delta log values in the samples which were deaerated. This is presumably due to the formation of acetic and lactic acid from lactose fermentation (Sgorbati, Biavati, & Palenzona, 1995). The lowest pH value reached was 5.31 and occurred in anaerobic conditions by the strain B. breve ATCC 15700 (Table 1). There are variations amongst strains of bifidobacteria with respect to growth and viability during storage in an acid environment (Dave & Shah, 1997c, d; Martin & Chou, 1992; Talwalkar & Kailasapathy, 2003a). Although the pH values observed in this study were not as low as that in yoghurt, acidification had not influenced cell multiplication and survival since positive delta values occurred at least until pH 5.3 and 5.9 in dearated and non-dearated milk, respectively.

Milk deaeration caused the initial value of milk redox potential to decrease under zero (
70 mV) (Table 1). In the presence of oxygen, redox potential values of milk after 2 weeks of refrigeration remained positive within the range of +241.83 to +258.01 mV, which is higher than the initial redox potential value of milk (Table 1). On the other hand, redox potential fell to negative values in milk incubated anaerobically. The relationship between oxygen and redox potential remains difficult to explain. However, it is suggested that the relationship between them depends on the chemical composition of the culture medium, which changes progressively due to metabolic activities of the microorganisms (Morris, 2000). This phenomenon suggests that bifidobacteria were exposed to different media properties in each condition (aerobically and anaerobically). 3.2. Growth in MRS media The assays conducted at 7 1C were designed to examine the effect of oxygen on stability of the cultures during storage rather than its effect on growth. Thus, a series of assays were carried out at 37 1C to specifically examine the effect of redox on growth of the eight bifidobacteria. Growth curves obtained using automated spectrophotometry confirmed the data obtained in assays on oxygen sensitivity. Most strains that showed stability to storage in non-deaerated milk (Table 1) also showed growth in unsupplemented MRS under aerobic conditions (Table 2). Supplementation with either ascorbic acid or cysteine did not have statistically significant effects on B. bifidum KYO, B. breve ATCC 15700 and B. bifidum ATCC 29521 (P ¼ 0:1975, 0.1975 and 0.0637). On the other hand, ascorbic acid supplementation led to a significant difference on growth of B. infantis ATCC 15697, B. longum ATCC 15708 and B. animalis subsp. lactis DSM 10140 (P ¼ 0:0028, 0.0001, 0.0043) as compared with their growth in MRS medium. Cysteine supplementation had a

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Table 2 Maximum optical density reached during growth of each strain in different media Strains

B. B. B. B. B. B. B. B.

bifidum KYO longum Rosell-R023a,b longum 411BBLa,b breve ATCC 15700 bifidum ATCC 29521 infantis ATCC 15697a longum ATCC 15708a animalis subsp. lactis DSM 10140a a

MRS

MRS+ascorbic acid

MRS+cysteine

Aerobic

Anaerobic

Aerobic

Anaerobic

Aerobic

Anaerobic

1.7 1.7 0.7 1.7 0.0 0.0 0.0 0.0

2.0 2.0 1.3 1.9 0.0 0.0 0.0 0.0

1.7 1.8 0.7 1.6 0.2 0.3 1.1 0.2

2.0 2.0 1.8 1.8 0.5 0.8 1.3 0.3

1.8 1.8 0.7 1.7 0.1 0.0 0.0 0.0

2.0 2.0 1.8 1.7 0.1 0.0 0.0 0.1

Strains for which the medium effect is statistically different according to a split-plot analysis of variance. Strains for which the oil effect is statistically different according to a Split-plot analysis of variance.

b

significant effect only in the case of B. longum Rosell-R023 (P ¼ 0:0255). Adding a layer of oil had less effect than the supplements, but B. longum Rosell-R023 and B. longum 411 BBL strains were statistically affected by its presence (P ¼ 0:0351; 0:0091). An examination of Tables 1 and 2 shows that the strains most sensitive to oxygen during a 2 week storage period in milk were the ones for which ascorbic acid supplementation facilitated growth. On the contrary, strains that survived well in aerobic or anaerobic milk were generally not affected by either ascorbic acid or cysteine supplementation. Therefore, data suggest that stability at 7 1C in milk and growth at 37 1C in MRS are similarly affected by oxygen and redox level. 3.3. Effect of electroreduction Three strains having different behaviour towards oxygen were chosen for the viability study which incorporated the electroreduction treatment: B. bifidum KYO was selected for its ability to survive as well as to grow in the presence and in the absence of oxygen in the medium, B. longum ATCC 15708 for its high sensitivity to oxygen, as evidenced by high mortality during storage in milk (Table 1) as well as the absence of growth without an oxygen scavenger such as ascorbic acid in the growth medium (Table 2), while B. animalis subsp. lactis DSM 10140 demonstrated a moderate sensitivity to oxygen. 3.3.1. Viability during storage Psychrotrophic counts remained below 100 cfu mL
1 in all samples. Therefore, growth of spoilage microorganisms did not influence bifidobacteria counts of the different milks. Marked differences in the behaviour of the chosen strains during storage were observed (Fig. 2). B. bifidum KYO cells maintained their initial viable population throughout the 4 week-storage period in all milk samples (Fig. 2A). There was no significant difference in microbial counts between the three treatments and the control

samples after each week and even after 4 weeks of refrigerated storage (P ¼ 0:9099). These results are in accordance with the results obtained in the screening assays at 7 1C and indicate that B. bifidum KYO was neither sensitive to the presence of oxygen nor to the variation in the redox state of the milk species during refrigerated storage. On the contrary, B. bifidum ATCC 15708 cells sustained important viability drops in all milk samples (Fig. 2B). However, the viability loss observed in all treated milk samples was minimized to approximately 2 logs in cysteine-supplemented deaerated milk, in deaerated milk and in electroreduced milk, as compared with controls in which approximately 4 log were lost at the end of a 4-week storage period. B. bifidum ATCC 15708 strain showed higher mortality in aerobic conditions than in anaerobic conditions. These results are in agreement with the ones obtained previously (Table 1). Starting on day 14, there was a significant difference (P ¼ 0:0005) between the population of the controls as compared to the population of the treated milks until the end of the storage period (P ¼ 0:0253). After 4 weeks of storage, the same statistical difference can be found but only according to P-values without the Bonferroni adjustments. Although B. animalis subsp. lactis DSM 10140 viability was only slightly affected by storage at 7 1C (Fig. 2C), the electroreduction, deaeration and deaeration with cysteine supplementation treatments had a positive impact on its survival. At the end of the storage period, a statistical difference can be observed between the controls and the treated milks (according to P-values without Bonferroni adjustments). In the deaerated samples, results from these assays are in agreement with those noted in the screening assays (Table 1). However, in the screening assays, it was observed that B. animalis subsp. lactis DSM 10140 had the tendency to survive better in the presence of oxygen whereas in this case, it showed a slight mortality upon exposure to oxygen. This discrepancy can only be explained by the methodologies used and point to the effect of milk sample preparation techniques in ascertaining the

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8 7 log10 (cfu mL-1)

log10 (cfu mL-1)

7.5

7.0

6 5 4

6.5 3 6.0

2 0

(A)

5

10 15 Time (days)

20

25

30

0

5

(B)

10 15 Time (days)

20

25

30

8.0

log (cfu mL−1)

7.5

7.0

6.5

6.0 0

(C)

5

10 15 Time (days)

20

25

30

Fig. 2. Viability of (A) B. bifidum KYO, (B) B. longum ATCC 15708, (C) B. animalis subsp. lactis DSM 10140 in pasteurized milk during 4 weeks of refrigerated in (K) electroreduced milk, (J) deaerated milk, (.) deaerated milk supplemented with cysteine, (,) closed control milk and (’) open control milk.

stability of probiotics during storage assays. Nevertheless, when comparing with B. bifidum ATCC 15708 it could be concluded that B. animalis subsp. lactis DSM 10140 strain exhibits oxygen tolerance. This explains its incorporation in several commercial probiotic formulations (Masco, Ventura, Zink, Huys, & Swings, 2004; Meile et al., 1997). 3.3.2. Redox potential The average initial redox potential of the pasteurized milk used in this experiment was around +220 mV and this value remained constant in the control milk samples and in the uninoculated samples of each strain throughout the 4 week-storage period (Fig. 3). Although the redox potential values for pasteurized milk were not found in literature, it is known that raw milk has a redox potential between +200 and +300 mV under aerobic conditions (Morris, 2000; Sherbon, 1999). There was no statistical difference in redox potential measurements between control samples and uninoculated samples at any time during the storage period. Similar results were observed in the screening assays (Table 1). In this experiment, vacuum was used instead of nitrogen gas bubbling into milk for the deaeration treatments, and this influenced redox and dissolved oxygen data. Deaeration by nitrogen gas was found to generate a greater decrease in redox potential values and in dissolved oxygen levels. Therefore, in this experiment, a softer method was used for deaeration which

resulted in a non-significant decrease of redox potential as compared to the control milk. In this experiment, cysteine was added at a concentration of 0.1% which allowed the redox potential to decrease to an average value of
100 mV (Fig. 3). Cysteine supplementation to pasteurized milk created reducing conditions throughout the 4 week-storage period. However, the redox potential tended to increase towards the end of the storage period. This phenomenon was also observed by Dave and Shah (1997b) during storage of cysteine supplemented yoghurt samples. In the case of B. bifidum KYO strain (Fig. 3A), redox potential measurements of cysteine supplemented milk were statistically different from the redox measurements in all other milk samples except the electroreduced milk on days 21 and 28 (P ¼ 0:4187 and 1.000). In B. longum ATCC 15708 samples, there were significant differences in redox potential measurements between cysteine supplemented samples and all other samples except the electroreduced milk on day 0 (P ¼ 0:2275) (Fig. 3B). During storage of milk samples containing B. animalis subsp. lactis DSM 10140 cells, redox potential of cysteine supplemented milk remained statistically different from all other milk samples at all time (Fig. 3C). Electroreduction was used to lower the value of milk redox potential without the use of chemical additives. This process decreased the redox potential to values well below
200 mV. However, these reducing conditions were

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200

200

100

100

Redox Potential (mV)

Redox potential (mV)

M.-P. Bolduc et al. / International Dairy Journal 16 (2006) 1038–1048

0 -100 -200

0 -100 -200 -300

-300

-400

-400 0

(A)

1045

5

10 15 Time (days)

20

25

30

0

5

10 15 Time (days)

(B)

20

25

30

300

Redox potential (mV)

200 100 0 -100 -200 -300 -400 0

5

(C)

10 15 Time (days)

20

25

30

Fig. 3. Changes in redox potential of (A) B. bifidum KYO, (B) B. longum ATCC 15708, (C) B. animalis subsp. lactis DSM 10140 in pasteurized milk during 4 weeks of refrigerated in (K) electroreduced milk, (J) deaerated milk, (.)deaerated milk supplemented with cysteine, (,) closed control milk, (’) open control milk and (&) uninoculated milk.

temporary since on day 7, all electroreduced milks showed positive redox potential values. This instability of the redox potential in electroreduced milk and the strong increases after 3–4 days of incubation at 7 1C were also observed in previous studies (Bolduc, 2005). In this experiment, statistical differences between the redox potential measurements of electroreduced and control milks were observed only on day 0 and 28. As seen in Fig. 3, redox potential of electroreduced milks had a tendency to decrease towards the end of the storage period, which could be attributed to metabolic activities of the cells (Morris, 2000). 3.3.3. Dissolved oxygen Control and uninoculated samples had an initial oxygen concentration around 6.7 mg L
1 (Fig. 4). After 4 weeks of refrigerated storage, a drop of 1–2 mg L
1 was noted in the uninoculated milk samples. This could be attributed to activity of NADH oxidases and NADH peroxidases of bifidobacteria triggered by the presence of oxygen (Talwalkar & Kailasapathy, 2003b). Deaerated milk, cysteinesupplemented deaerated milk and electroreduced milk all started with a similar oxygen concentration within the range of 1–2.4 mg L
1 (Fig. 4). These values underwent a slight decrease at the beginning of the storage period and remained constant and close to 0.1 mg L
1 until the end of the storage period. The decrease observed during the first week might be due to the enzymatic activities previously mentioned. For each strain, differences between the

measurements of the three controls and those of the three treated milk samples were statistically significant. These data show that there is only a limited correlation between dissolved oxygen and redox level in milk. Thus, the strong effect of vacuum-deaeration on dissolved oxygen (Fig. 4) was not matched by a strong decrease in redox values (Fig. 3), while a strong increase in redox values noted during storage of the electroreduced milk was not matched by an equivalent increase on dissolved oxygen. 3.3.4. pH The evolution of pH showed similar patterns with the three strains, and only the amplitude of the changes differed (Fig. 5). A noteworthy observation is the pHlowering effect of cysteine supplementation to milk. The milk solutions made with cysteine–HCl had an acidic pH in spite of the use of a phosphate buffer (pH 7.0) to prepare the cysteine concentrate. Its addition lowered the initial milk pH by 0.3 unit. For all the strains, initial milk pH was around 6.6–6.7 (except for the one supplemented with cysteine), but at the end of the storage period, there was a significant difference between the pH of control and uninoculated milk and the other treated milk samples. The pH of the control samples and the uninoculated samples remained constant throughout the studied period. On the other hand, the pH of deaerated milk, cysteinesupplemented deaerated milk and electroreduced milk underwent a decrease between 0.2 and 1.0 pH units as a

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8

6

6

Dissolved oxygen (mg L-1)

Dissolved oxygen (mg L-1)

1046

4

2

0

0

5

(A)

10 15 Time (days)

20

25

4

2

0

0

30

5

10 15 Time (days)

(B)

20

25

30

Dissolved oxygen (mg L-1)

8

6

4

2

0

0

5

10 15 Time (days)

(C)

20

25

30

Fig. 4. Changes in dissolved oxygen of (A) B. bifidum KYO, (B) B. longum ATCC 15708, (C) B. animalis subsp. lactis DSM 10140 in pasteurized milk during 4 weeks of refrigerated in (K) electroreduced milk, (J) deaerated milk, (.) deaerated milk supplemented with cysteine, (,) closed control milk, (’) open control milk and (&) uninoculated milk.

6.8

7.0 6.8

6.7

6.6 6.6 6.5

6.2 pH

pH

6.4

6.0

6.4

5.8

6.3

5.6 6.2

5.4

6.1

5.2 0

(A)

5

10 15 Time (days)

20

25

30

0

5

20

25

(B)

10 15 Time (days)

20

25

30

6.9 6.8 6.7

pH

6.6 6.5 6.4 6.3 6.2 6.1 0

(C)

5

10 15 Time (days)

30

Fig. 5. Changes in pH of (A) B. bifidum KYO, (B) B. longum ATCC 15708, (C) B. animalis subsp. lactis DSM 10140 in pasteurized milk during 4 weeks of refrigerated in (K) electroreduced milk, (J) deaerated milk, (.)deaerated milk supplemented with cysteine, (,) closed control milk, (’) open control milk and (&) uninoculated milk.

ARTICLE IN PRESS M.-P. Bolduc et al. / International Dairy Journal 16 (2006) 1038–1048

function of the strain. Even though the B. bifidum KYO population did not significantly change, it was able to acidify the treated milk samples. This ability of bifidobacteria to produce acids without cell division has been demonstrated by Desjardins, Roy, and Toupin (1990). As was observed in the screening assays (Table 1), B. bifidum KYO strain had a more active acidifying activity in treated milks as compared with the controls. Since higher viabilities and greater acidification were noted in the treated samples, it could be hypothesized that the different pH patterns could at least be partially linked to viable populations. B. bifidum KYO and B. animalis subsp. lactis DSM 10140 both maintained high viabilities in the treated milks (Fig. 2), but the greater acidifying activity of B. bifidum KYO (Fig. 5) would be undesirable in unfermented milk, and B. animalis subsp. lactis DSM 10140 would thus probably constitute a better choice from an industrial standpoint. 4. Conclusion This study adds to the literature with respect to variations between bifidobacteria in reference to sensitivity to oxygen. In this regard, data from this study suggest that cells actively growing at 37 1C in MRS media show similar reactions to oxygen as those stored at 7 1C in milk. Strains which showed the highest viability losses during storage at 7 1C in presence of oxygen also had the lowest growth levels at 37 1C when exposed to oxygen. Thus, it appears that the effect of oxygen/redox on growth inhibition of bifidobacteria at 37 1C can serve as an indicator of its negative effect on stability of the strains during refrigerated storage in similar oxygen/redox conditions. In the past, the development of a probiotic-containing food relied on a strain selection process, such as that carried out in our screening assays. However, recent data on health aspects of probiotics in foods point to the importance of the strain itself in the beneficial effects. Therefore, in the development of a probiotic-containing unfermented milk product, a food manufacturer may not wish to carry out a selection process on the basis of stability to redox conditions, but rather add a specific strain having proven clinical effects. If this strain shows high sensitivity to oxygen, then technological adaptations are required. Data from this study suggest that three approaches could be taken in order to enhance the survival of the cultures during storage: deaeration, addition of reducing agents and electrochemical reduction of milk. However, even if a culture becomes stable in a treated product as shown in this study, its acidifying capacity at 41C could modify the milk characteristics. This problem could be overcome by an appropriate strain selection. It must be kept in mind that data from this study were obtained with storage under anaerobic conditions following the deaeration or electrochemical processes. Data by Dave and Shah (1997a) show that oxygen may re-enter the product during storage in plastic cups. Therefore, the use

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of deaeration or electroreduction on milk might require subsequent packaging that would prevent oxygen re-entry. More data need to be obtained on this aspect. Acknowledgements Authors wish to thank Michel Britten, Laurent Bazinet and Nancy Gardner for technical assistance and helpful discussions and Claude Laberge for support in statistical analysis. This work was supported by a research grant from FQRNT-Novalait-MAPAQ. References Ahn, J. B., Hwang, H. J., & Park, J. H. (2001). Physiological responses of oxygen-tolerant anaerobic Bifidobacterium longum under oxygen. Microbiology and Biotechnology, 11(3), 443–451. Araya, M., Morelli, L., Reid, G., Sanders, M.-E., Stanton, C., Pineiro, M., et al. (2002). Guidelines for the evaluation of probiotics in food. Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food (pp.1–11). London ON, Canada, April 30 and May 1. Bolduc, M.-P. (2005). Effet de la modulation e´lectrochimique du lait sur la flore psychrotrophe native et sur la viabilite´ de cultures probiotiques ajoute´es pendant l’entreposage sous re´frige´ration. M.Sc. Thesis, Universite´ Laval, Que´bec city, Canada. Charteris, W. P., Kelly, P. M., Morelli, L., & Collins, J. K. (1998). Ingredient selection criteria for probiotic microorganisms in functional dairy foods. International Journal of Dairy Technology, 51(4), 123–136. Collins, E. B., & Hall, B. J. (1984). Growth of bifidobacteria in milk and preparation of Bifidobacterium infantis of a dietary adjunct. Journal of Dairy Science, 67, 1376–1380. Dave, R. I., & Shah, N. P. (1997a). Effectiveness of ascorbic acid as an oxygen scavenger in improving viability of probiotic bacteria in yoghurts made with commercial starter cultures. International Dairy Journal, 7, 435–443. Dave, R. I., & Shah, N. P. (1997b). Effect of cysteine on the viability of yoghurt and probiotic bacteria in yoghurts made with commercial starter cultures. International Dairy Journal, 7, 537–545. Dave, R. I., & Shah, N. P. (1997c). Improving viability of Lactobacillus acidophilus and Bifidobacterium spp. in yoghurt. International Dairy Journal, 7, 349–356. Dave, R. I., & Shah, N. P. (1997d). Viability of yoghurt and probiotic bacteria in yoghurts made from commercial starter cultures. International Dairy Journal, 7, 31–41. Dave, R. I., & Shah, N. P. (1998). Ingredient supplementation effects on viability of probiotic bacteria in yogurt. Journal of Dairy Science, 81, 2804–2816. Dechter, T. H., & Hoover, D. G. (1998). Survivability and b-galactosidase activity of bifidobacteria stored at low temperature. Food Biotechnology, 12(1/2), 73–89. Desjardins, M.-L., Roy, D., & Toupin, C. (1990). Uncoupling of growth and acids production in Bifidobacterium ssp. Journal of Dairy Science, 73, 1478–1484. de Vries, W., & Stouthamer, A. H. (1969). Factors determining the degree of anaerobiosis of Bifidobacterium strains. Archives Mikrobiologie, 65, 275–287. Donnet-Hughes, A., Rochat, F., Serrant, J. M., Aeschlimann, J. M., & Schiffrin, E. J. (1999). Modulation of non-specific mechanisms of defense by lactic acid bacteria: Effective dose. Journal of Dairy Science, 82, 863–869. Fasoli, S., Marzotto, M., Rizzotti, L., Rossi, F., Dellaglio, F., & Torriani, S. (2003). Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. International Journal of Food Microbiolology, 82, 59–70.

ARTICLE IN PRESS 1048

M.-P. Bolduc et al. / International Dairy Journal 16 (2006) 1038–1048

He´kal, I. M. (1983). Process for the preservation of color and flavour in liquid containing comestibles. United States Patent no. 4 374 714. Inoue,T. T., & Kato, W. K. (2003). Powdery drinks and process for producing the same. International Patent no. WO03053153. Kailasapathy, K., & Chin, J. (2000). Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp. Immunology and Cell Biology, 78, 80–88. Klaver, F. A. M., Kingma, F., & Weerkamp, A. H. (1993). Growth and survival of bifidobacteria in milk. The Netherlands Milk and Dairy Journal, 47, 151–164. Koseki, M., Nakagawa, A., Tanaka, Y., Noguchi, H., & Omochi, T. (2003). Sensory evaluation of taste of alkali-ion water and bottled mineral waters. Journal of Food Science, 68, 354–358. Kurmann, J. A., & Rasic, J. L. (1991). The health potential of products containing bifidobacteria. In R. K. Robinson (Ed.), Therapeutic Properties of Fermented Milks (pp. 117–157). London, UK: Elsevier Science Publishers Ltd. Lourens-Hattingh, A., & Viljoen, B. C. (2001). Yogourt as probiotic carrier food. International Dairy Journal, 11, 1–17. Martin, J. H., & Chou, K. M. (1992). Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: 1- tolerance to pH of yogurt. Cultured Dairy Products Journal, 27(4), 21–26. Masco, L., Ventura, M., Zink, R., Huys, G., & Swings, J. (2004). Polyphasic taxonomic analysis of Bifidobacterium animalis and Bifidobacterium lactis reveals relatedness at the subspecies level: reclassification of Bifidobacterium animalis as Bifidobacterium animalis subsp. animalis subsp. nov and Bifidobacterium lactis as Bifidobacterium animalis subsp. lactis subsp. nov. International Journal of Systematic and Evolutionary Microbiology, 54, 1137–1143. Meile, L., Ludwig, W., Rueger, U., Gut, C., Kaufmann, P., Dasen, G., et al. (1997). Bifidobacterium lactis sp. nov., a moderately oxygen tolerant species isolated from fermented milk. Systematic and Applied Microbiology, 20(1), 57–64. Mondal, K., & Lalvani, S. B. (2003). Electrochemical hydrogenation of canola oil using a hydrogen transfer agent. Journal of the American Oil Chemistry Society, 80, 1135–1141. Morris, J. G. (2000). The effect of redox potential. In B. M. Lund, T. C. Baird-Parker, & G. W. Gould (Eds.), The microbiological safety and

quality of foods, Vol. 1 (pp. 235–250). Gaithersburg, MD, USA: Aspen Publisher Inc. Ouwehand, A. C., Salvadori, B. B., Fonden, R., Mogensen, G., Salminen, S., & Sellars, R. (2003). Health effects of probiotics and culturecontaining dairy products in humans. Bulletin of the IDF 380 (pp. 4–19). Brussels, Belgium: International Dairy Federation. Sgorbati, B., Biavati, B., & Palenzona, D. (1995). The genus Bifidobacterium. In B. J. B. Wood, & W. H. Holzapfel (Eds.), The lactic acid bacteria, Vol. 2: The genera of lactic acid bacteria (pp. 279–305). London, England: Blackie Academic and Professional. Shah, N. J. (2000). Probiotic bacteria: Selective enumeration and survival in dairy foods. Journal of Dairy Science, 83, 894–907. Sherbon, J. W. (1999). Physical properties of milk. In N. P. Wong (Ed.), Fundamentals of dairy chemistry (third ed., pp. 414–419). Gaithersburg, MD, USA: Aspen Publishers Inc. Shimamura, S., Abe, F., Ishibashi, N., Miyakawa, H., Yaeshima, T., Araya, T., et al. (1992). Relationship between oxygen sensitivity and oxygen metabolism of Bifidobacterium species. Journal of Dairy Science, 75, 3296–3306. Tallec, A. (1985). Ge´ne´ralite´s sur l’e´lectrochimie organique et les facteurs expe´rimentaux. In Masson (Ed.), E´lectrochimie organique: Synthe`se et me´canisme (pp. 10–25). Paris, France: Armand Colin Scientifique. Talwalkar, A., & Kailasapathy, K. (2003a). Effect of microencapsulation on oxygen toxicity in probiotic bacteria. Australian Journal of Dairy Technology, 58(1), 36–39. Talwalkar, A., & Kailasapathy, K. (2003b). Metabolic and biochemical responses of probiotic bacteria to oxygen. Journal of Dairy Science, 86, 2537–2546. Talwalkar, A., & Kailasapathy, K. (2004a). A review of oxygen toxicity in probiotic yogurts: Influence on the survival of probiotic bacteria and protective techniques. Comprehensive Reviews in Food Science and Food Safety, 3, 117–124. Talwalkar, A., & Kailasapathy, K. (2004b). Oxidative stress adaptation of probiotic bacteria. Milchwissenschaft, 59(3/4), 140–143. Talwalkar, A., Kailasapathy, K., Peiris, P., & Arumugaswamy, A. (2001). Application of RBGR–a simple way for screening of oxygen tolerance in probiotic bacteria. International Journal of Food Microbiology, 71, 245–248.