Screening and selection of acid and bile resistant bifidobacteria

International Journal of Food Microbiology 47 (1999) 25–32

Screening and selection of acid and bile resistant bifidobacteria H.S. Chung

a ,c ,

*, Y.B. Kim a , S.L. Chun b , G.E. Ji c

a

Department of Food Science and Technology, Korea University, Seoul 136 -701, Korea b Central Research Institute, Maeil Dairy Industry Co., Pyungtak 451 -860, Korea c Department of Food Science and Nutrition, Hallym University, Chunchon 200 -702, Korea Received 27 November 1997; received in revised form 22 June 1998; accepted 10 November 1998

Abstract Human fecal samples were used as a source of Bifidobacterium strains which are resistant to both acid and bile. The procedure used for screening was as follows: enrichment of Bifidobacterium strains with Bifidobacterium-selective transgalacto-oligosaccharide-propionate (TP) medium followed by acid (pH 2.0) and bile salt stressing (1.5% bile salt (w / v)). Two selected Bifidobacterium strains, designated HJ 30 and SI 31, showed considerably higher rates of survival when incubated in 50 mM phosphate buffer solution adjusted to pH 2.0 or 3.0 or in 50 mM phosphate buffer (pH 7.0) containing 0.5 or 1.0% (w / v) bile salt. HJ 30 and SI 31 were the only strains to have significant growth in Man Rogosa Sharpe (MRS) medium at 0.15% bile salt. All strains tested had similar growth rates in the absence of bile or at an initial pH value of 5.0 or 7.0 as determined by optical density measurements. For SI 31 the number of viable cell counts remained high (6 3 10 7 cfu / ml) for up to 72 h when grown in the skim milk medium, whereas all other strains examined declined to below 10 5 cfu / ml. These results demonstrate that the screening procedures developed in this study are effective for the selection of acid and bile resistant Bifidobacterium strains.  1999 Elsevier Science B.V. All rights reserved. Keywords: Bifidobacterium; Human; Acid and bile resistance

1. Introduction Bifidobacterium species are Gram-positive anaerobic bacteria that inhabit the intestinal tracts of humans and animals. Bifidobacteria were first discovered in 1899 by Tissier at the Pasteur Institute, Paris, France. These bacteria were found to be a predominant component of the intestinal flora in breast-fed infants (Bezkorovainy and Miller-Catchpole, 1989; Mitsuoka, 1990). They are believed to *Corresponding author. 0168-1605 / 99 / $ – see front matter PII: S0168-1605( 98 )00180-9

play an important and beneficial role in the proper balance of normal intestinal flora (Homma, 1988). Establishment of high numbers of Bifidobacterium is reported to form barriers against the proliferation of exogenous pathogens (Gibson and Wang, 1994; Gilliland, 1990). This may arise as a consequence of impeded colonization by the invader or by the control of intestinal pH, specifically, through the release of acetic and lactic acids (Araya-Kojima et al., 1996; Homma, 1988). In addition, humans suffering from lactose malabsorption would benefit from the intake of b-galactosidase producing

 1999 Elsevier Science B.V. All rights reserved.

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Bifidobacterium cells as well as from the reduced lactose content in Bifidobacterium-fermented milk (Mustahpa et al., 1997; Rasic and Kurmann, 1983). Other beneficial effects of the intake of Bifidobacterium may include the reinforcement of immune functions and improve resistance to cancer (Sekine et al., 1985). Incorporation of bifidobacteria into fermented milk products has as a consequence increased around the world (Salminen et al., 1996). Bifidobacteria may be selected that tolerate low pH’s of the stomach and survive the effects of bile produced by the small intestine of humans (Clark and Martin, 1994; Clark et al., 1993). Large variations in Bifidobacterium ability to resist acid or bile have been reported (Berrada et al., 1991; Clark and Martin, 1994; Clark et al., 1993; Ibrahim and Bezkorovainy, 1993). The purpose of this work was to develop a method for the selection of acid and bile resistant bifidobacteria from human fecal samples which are a large reservoir of naturally occurring bifidobacteria.

2. Materials and methods

2.1. Reagents and media Transgalacto-oligosaccharide (TOS) was obtained from Yakurt Ltd., Tokyo, Japan. Oxgall was purchased from Sigma Chemical Co. (St. Louis, Missouri, USA). Transgalacto-oligosaccharide-propionate (TP) medium (Ji et al., 1994) was used as an enrichment medium for Bifdobacterium. HCl or oxgall (Sigma) was added to this medium to give high acid or high bile concentrations. Man Rogosa Sharpe (MRS) medium was purchased from Difco Co. (Detroit, Michigan, USA) and used to cultivate isolated bifidobacteria. Briggs liver (BL) agar medium (Mitsuoka, 1984) was used to plate appropriately diluted Bifidobacterium cultures for viable cell counting. B. adolescentis ATCC 15703 was obtained from American Type Culture Collection (Rockville, Maryland, USA). Bifidobacterium longum 2 (BL2) and Bifidobacterium infantis 420 (BI420) were obtained from Wiesby GmbH and Co. (Gotteskoogstrabe, Germany) and were maintained by propagation in MRS broth with 1% inoculum and 18 h incubation at 378C in Mail Dairy Company

(Pyeongtak, Korea). Cultures were stored in a refrigerator at 3 to 58C between transfers.

2.2. Isolation and cultivation of the strains Fecal samples were collected from infants and adults. The tubes containing the fecal samples were promptly screened for the isolation of resistant strains, as described below. Fecal samples (0.8 g each) were inoculated into 8 ml of TP medium. After an anaerobic incubation for 12 h at 378C, 0.8 ml of the incubated cultures were transferred into fresh TP medium with pH adjusted to 2.0 and incubated anaerobically for another 12 h at 378C. After the acid exposure, an aliquot (0.8 ml) of the incubation medium was transferred into fresh TP medium supplemented with 1.5% oxgall (Sigma), and the incubation continued for another 2 h at 378C. The resulting incubation medium was serially diluted and plated on TP agar medium to select colonies of the resistant Bifidobacterium strains. To isolate reference strains, serially diluted Bifidobacterium cells grown in the regular TP medium were plated on TP agar medium. The present studies utilized Bifidobacterium strains IN33, JS34 and SH35 (own isolates) as reference strains. B. adolescentis ATCC 157303, Bifidobacterium longum BL2 and Bifidobacterium infantis BI420 described above were also included as reference strains for the convenience of comparison to the resistant strains. Microscopic analysis (1000 3 with immersion oil, MODEL CK, Fisher Scientific, Tokyo, Japan) was routinely performed to confirm Bifidobacterium morphology. Bifidobacterium cells were examined for their biochemical and morphological characteristics according to the Bergey’s Manual of Determinative Bacteriology (Scardovi, 1986). The cultures were grown in MRS medium under anaerobic conditions in a microprocessor-controlled anaerobic chamber (Lab Line Instruments, Inc., Melrose Park, Illinois, USA). Cultures were incubated for 18 h at 378C and stored at 3 to 58C between transfers. For the fermentation test, 0.5 ml of 10% substrate solutions, which were membrane filtered through 0.45 mM Acrodisc filter (Gelman Sciences, Ann Arbor, Michigan, USA), were added to 9.5 ml of Peptone Yeast-extract Fildes (PYF) basal medium (Mitsuoka, 1990). After 2.5 day incubation in anaerobic Jars (BBL, Cockeysville, Maryland, USA), the pH of the growth medium was

H.S. Chung et al. / International Journal of Food Microbiology 47 (1999) 25 – 32

measured. Tubes below pH 5.5 were judged to be fermentation positive. The presence of acetate and lactate in the fermented PYF containing glucose medium was assayed by using gas chromatography (5890 A GC, 530 mm HP-20 M column, Hewlett Packard Co., Avondale, Pennsylvania, USA).

2.3. Enzyme test Fructose-6-phosphate phosphoketolase (F6PPK) test was done after growing the cells in MRS medium. After incubation at 378C for 1 day in anaerobic condition, cells were removed by centrifugation at 6000 3 g for 10 min. The F6PPK test was done according to the method of Scardovi (1986). Briefly, cells harvested from 10 ml of MRS medium were washed three times with 50 mM phosphate buffer (pH 6.5) containing 0.05% cysteine ? HCl (w / v) and resuspended in the same solution. After disruption of the cells by sonication (XL 2020 sonicator, Heat Systems Inc, New York, USA) for 10 min in an ice bath, 0.25 ml each of solution I [6 mg NaF and 10 mg Na-iodoacetate / ml (Sigma)] and 80 mg fructose-6-phosphate / ml (Sigma) were added. After incubation at 378C for 30 min the reaction was stopped with 1.5 ml of 140 mg / ml hydroxylamine ? HCl (Sigma). After 10 min at room temperature, 1.0 ml each of 15% (w / v) trichloroacetic acid and 4 M HCl were added. After 5 min incubation at room temperature 1.0 ml of 0.5% (w / v) FeCl 3 ? 6H 2 O was added. The formation of a reddish-brown color was judged to be F6PPK positive. For the catalase test, colonies grown on MRS

27

medium were picked and mixed with 3% H 2 O 2 solution. For the possession of the various glycolytic enzymes, bifidobacteria were grown in 10 ml MRS medium anaerobically for 18 h at 378C. Harvested cells were washed three times with 0.1 M phosphate buffer (pH 6.8) and resuspended in 0.5 ml phosphate buffer. After disruption of the cells by sonication for 10 min, various substrates of 0.02 ml of 20 mM paranitrophenol (PNP)-glycoside (Sigma Chemical Co.) were added (Table 1). After incubation at 458C for 2 h, the reaction was stopped by adding 0.5 ml of 1 M sodium carbonate solution. The formation of a yellow color was judged to be positive for each enzyme.

2.4. Growth in acid and bile One hundred microliters of culture containing about 10 8 to 10 9 cfu / ml were transferred to fresh MRS medium. pH was adjusted to 4.0, 5.0 and 7.0 using 4 N HCl and incubated anaerobically at 378C. The cell growth was measured spectrophotometrically (Spectronic 20, Milton Roy Co., Ivyland, Pennsylvania, USA) at A620 at several timed intervals (12 h). The effects of bile salt were examined in MRS medium (pH 7.0) by adding oxgall (Sigma) to a concentration of 0, 0.06 and 0.15% (w / v).

2.5. Survival in acid and bile One hundred microliters of culture containing about 10 8 to 10 9 cfu / ml were transferred to 5 ml of autoclaved buffer containing 50 mM dibasic sodium

Table 1 Enzymatic patterns of acid and bile / resistant Bifidobacterium strains HJ 30 and SI 31 Enzymes tested

a-Glucosidase b-Glucosidase a-Galactosidase b-Galactosidase b-Xylosidase a-Arabinofuranosidase a-Fucosidase b-Glucuronidase b-Rhamnosidase N-Acetyl-b-glucosaminidase

Substrates used

PNP a-D-glucopyranoside PNP b-D-glucopyranoside PNP a-D-galactopyranoside PNP b-D-galactopyranoside PNP b-D-xylopyranoside PNP a-D-arabinofuranoside PNP a-D-fucopyranoside PNP b-D-glucuronide PNP a-D-rhamnopyranoside PNPN-acetyl-b-D-glucosaminide

Strains HJ 30

SI 31

B. adolescentis ATCCl 5703

1 1 1 1 1 1 2 2 2

1 1 1 1 1 1 2 2 2 2

1 1 1 1 1 1 2 2 2 2

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phosphate that had been adjusted to a pH of 2, 3 and 7 using 4 N HCl. The number of surviving cells was determined following anaerobic incubation (378C) at timed intervals by plating them on BL agar medium. Plates were incubated under anaerobic conditions at 378C for 2 to 2.5 days. The effect of bile salt on cell growth was examined by adding oxgall to a final concentration of 0, 0.5, and 1.0% (w / v).

2.6. Growth in fermented milk Reconstituted skim milk was prepared by dissolving 10 g of skim milk powder (Seoul Milk Co., Seoul, Korea) and 0.05 g cysteine ? HCl in 90 ml of water which had been previously heated to 1108C for 10 min. Cultures were inoculated at 10 6 to 10 7 cfu / ml and incubated anaerobically at 378C for 72 h. Viable cell counts were measured every 12 h by plating on BL agar medium.

2.7. Statistical analysis Data were analyzed by Student-Newman-Keuls (SNK) test following one way analysis of variance (ANOVA) using Sigmastat Statistical Analysis System (Jandel Scientific, San Rafael, California, USA). A probability of P , 0.05 in the two-tailed test was used as the criterion for statistical significance.

BI 420 were also included for comparison. Some of the identifying characteristics of the isolates, HJ 30 and SI 31, can be found in Tables 1 and 2. HJ 30 and SI 31 isolated from different individual samples showed very similar characteristics to each other with respect to carbohydrate fermentation and various glycolytic enzyme patterns. The reference strains were considerably different with regards to sugar fermentation when compared to HJ 30 and SI 31 (data not shown).

3.2. Growth and survival in the presence of bile salt In Table 3 the growth of the Bifidobacterium strains in the presence of three concentrations of bile salt (0, 0.06, and 0.15%) is shown. Although the degree of cell growth during 48 h was similar at 0% bile salt for all of the test strains, a marked difference in the cell growth was observed at the 0.15% oxgall concentration. In the presence of bile salt, HJ 30 and SI 31 showed considerably higher growth than the other strains tested over the 48 h period of incubation. The survival rate of the isolated Bifidobacterium strains at bile concentrations of 0.5 and 1% for a 12 h exposure were very similar at both 0.5 and 1.0% concentrations of bile salt (results not shown). SI 31 and HJ 30 appeared to have the highest resistance against bile salt (results not shown).

3. Results

3.1. Isolation and identification of acid and bile resistant strains During the screening procedure, the microscopical examination of the morphology of the Bifidobacterium revealed that the relative proportion of the Bifidobacterium increased as each step of incubation proceeded. Colonies which passed the screening procedure as described in Section 2 were tested for the presence of fructose-6-phosphate phosphoketolase (F6PPK) activity and acetate / lactate production by gas chromatographic analysis. Two resistant Bifidobacterium strains, HJ 30 and SI 31 were selected and compared to reference Bifidobacterium strains, IN 33, JS 34 and SH 35. B. adolescentis ATCC 15703 and two commercial strains BL2 and

3.3. Growth and survival at acidic pH The growth of the Bifidobacterium strains in MRS medium at pH values of 7.0, 5.0 and 4.0 was investigated. The degrees of cell growth were comparable at pH 7.0 and 5.0 for the various strains, however no growth was observed at pH 4.0 for any of the strains tested (results not shown). In Fig. 1(a) and (b) the survival rate of the isolated strains at pH values of 3 and 2 for 2 h is shown. The rate of decrease in the number of viable cells was much higher at pH 2.0 than at 3.0. SI 31 and HJ 30 showed greatest survival as they declined only 100fold and 1000-fold after 1 h at pH 2, respectively. At pH 3.0, SI 31 and HJ 30 showed only a 10-fold decline in viable cell number after 2 h of incubation.

H.S. Chung et al. / International Journal of Food Microbiology 47 (1999) 25 – 32

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Table 2 Identity characteristics of acid and bile resistant Bifidobacterium strains HJ 30 and SI 31 Characteristics

Spore formation Gram reaction Morphology Anaerobic growth Gas from glucose F6PPK Catalase Motility Production of lactate and acetate Fermentation L-Arabinose Arbutin Cellobiose Fructose Galactose Glucose Lactose Maltose Mannitol Mannose Melezitose Melibiose Raffinose Rhamnose Ribose Salicin Sorbitol Starch Sucrose Trehalose Xylose

Strains HJ 30

SI 31

B. adolescentis ATCCl 5703

2 1 Rod, bifidus form 2 2 1 2 2 1

2 1 Rod, bifidus form 2 2 1 2 2 1

2 1 Rod, bifidus form 2 2 1 2 2 1

1 2 1 1 1 1 1 1 2 2 2 1 1 2 1 1 2 1 1 2 1

1 2 1 1 1 1 1 1 2 2 2 1 1 2 1 1 2 2 1 2 1

1 2 1 1 1 1 1 1 2 2 2 1 1 2 1 1 2 1 1 2 1

3.4. Application of the selected strains for the fermentation of milk In Fig. 2 the growth curve measured by counting the number of viable cells in the reconstituted skim milk is shown. The degree of growth for the various strains tested was similar up to 24 h. However, the rates of cell death were significantly different. The resistant strain SI 31 showed a marked resistance to cell death even after 72 h incubation. It produced coagulation of the fermented skim milk and pH dropped continuously reaching pH 4.1 over a 72 h incubation period (data not shown).

4. Discussion There is growing interest in the use of probiotic Bifidobacterium strains in the dairy industry. Bifidobacterium strains with high resistance to acids or bile salt concentrations may be advantageous for the colonization in the human gut. There are however few studies on the tolerance of Bifidobacterium to bile or acid (Berrada et al., 1991; Clark and Martin, 1994; Clark et al., 1993; Ibrahim and Bezkorovainy, 1993). Ibrahim and Bezkorovainy (1993) reported that B. infantis had the best survival rates followed by B. bifidum, B. breve and B. longum,

H.S. Chung et al. / International Journal of Food Microbiology 47 (1999) 25 – 32

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Table 3 Growth (A 620 )a of Bifidobacterium strains at various concentrations of bile salt Concentration (% w / v)

Time (h)

HJ 30

SI 31

BL 2

BI420

IN 33

JS 34

SH 35

B. adolescentis ATCC 15703

0.0

12 24 36 48 12 24 36 48 12 24 36 48

1.2560.03 1.4760.05 1.4360.09 1.4760.05 0.8860.12 1.0760.05 1.1260.02 1.2360.05 0.6060.14 0.6860.09 0.6760.03 0.7060.07

1.3760.19 1.4860.08 1.4360.09 1.4360.09 0.7060.06 0.9860.02 0.9760.05 1.0860.06 0.4060.01 0.6060.03 0.5960.08 0.6960.07

1.2760.06 1.4360.08 1.5760.09 1.5060.14 0.1260.06 0.1360.05 0.1460.05 0.1860.05 0.0460.04 0.0860.02 0.0760.05 0.0560.01

1.1960.07 1.4060.06 1.5060.08 1.4860.05 0.0860.04 0.1060.08 0.1260.05 0.1460.06 0.0560.03 0.0760.03 0.0760.04 0.0660.04

1.3360.17 1.5060.07 1.5360.01 1.5060.03 0.0560.02 0.2360.05 0.3260.03 0.4360.04 0.0760.02 0.1060.02 0.0860.04 0.0660.05

1.2560.09 1.4960.07 1.4860.05 1.4560.06 0.1460.05 0.7260.05 0.8460.05 0.7060.05 0.0760.03 0.0560.01 0.0460.05 0.0660.01

0.9560.06 1.3360.05 1.5060.09 1.5060.06 0.0760.06 0.0460.05 0.0760.04 0.1560.06 0.0760.03 0.0860.02 0.1360.02 0.1160.02

1.1360.15 1.2360.05 1.5060.03 1.5060.10 0.0660.04 0.2660.05 0.5760.15 0.7260.19 0.0360.02 0.0860.02 0.1160.03 0.0960.02

0.06

0.15

a

O.D. 620 at 0 h at all of the bile concentrations were below 0.03 for all of the strains.

when exposed to bile salt at concentrations ranging from 0 to 3 g / l. In contrast, Clark and Martin (1994) reported that B. longum exhibited the best tolerance to 2.0 or 4.0% bile followed by B. bifidum and B. infantis, which was almost the exact opposite in order of their tolerance to bile. These contrasting observations may reflect the strain-specific resistance to bile. We employed an approach which screens for Bifidobacterium resistance to both acid and bile using human fecal samples as a source of the bacteria. Our approach circumvents these strain specific or technical problems as we focused directly on bifidobacteria with specific resistance to acid and bile only. Various combinations of pH, bile concentration, incubation time and different selective media were tested. TP medium (Ji et al., 1994) which was used in this experiment does not contain antibiotics and instead uses Bifidobacterium-selective nutrient transgalacto-oligosaccharide (TOS) and propionate. Propionate acts as a bacteriostatic agent for several types of bacteria and acts simultaneously to improve growth of bifidobacteria (Kaneko et al., 1994). TOS and propionate are both stable at acidic pH’s or in the presence of bile. Following rigorous testing, we included this medium for our selection experiments. The final screening procedure included enrichment of Bifidobacterium strains in Bifidobacterium-selective TP medium followed by acid and bile salt stressing. Two stress-resistant strains SI 31 and HJ 30 grew

considerably better in the presence of 0.15% bile salt as compared to the reference strains. The strong resistance of SI 31 and HJ 30 to bile was also confirmed by measuring rates of survival in the buffer solution containing 0.5 or 1.0% bile. Abilities of the stressed strains to grow at an initial pH of 5.0 or 7.0 were generally similar to reference strains. However, the number of viable cells at later stages of growth were greater when compared to the reference strains (data not shown). It would, therefore, appear that the selected strains are more resistant to the acids produced during growth. They also exhibited higher rates of survival in the dibasic phosphate buffered solution adjusted to pH 2.0 or 3.0. Their resistance to acid and bile stressing is maintained after continuous subculturing for a period of 2 years in MRS medium (data not shown). The bile and acid resistant strains were maintained at high cell concentrations in reconstituted skim milk for up to 72 h. Higher survival ratios of the SI 31 strain in milk, especially in the latter periods of fermentation, compared with reference strains, may be related to increased acid resistance. Acetate resistance rather than HCl resistance may be more important for the survival in a milk medium since acetate is the major by-product of Bifidobacterium fermentation. HCl resistance may play a more important role during passage of the bacteria through the stomach (Conway et al., 1987). The usefulness of in vitro models of acid or bile resistant bifidobacteria has been well demonstrated in several studies. Ber-

H.S. Chung et al. / International Journal of Food Microbiology 47 (1999) 25 – 32

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Fig. 2. Change in the viable Bifidobacterium cell counts during growth in fermented milk. Standard bars are not shown. Data are mean6S.D. of triplicates. Marks (*) on resistant strains (HJ 30 and SI 31) indicate a significant difference from the most resistant reference strains (P , 0.05).

Fig. 1. Survival of acid and bile resistant Bifidobacterium strains after exposure to pH 3 (a) and pH 2 (b). Data are mean6S.D. of triplicates. Marks (*) on resistant strains (HJ 30 and SI 31) indicate a significant difference from the most resistant reference strains (P , 0.05).

rada et al. (1991) showed that acid-resistant Bifidobacterium survived better in vivo in ten healthy-young adults. Using a dynamic model of the stomach and the small intestine, Marteau et al. (1997) showed that bile exerted a strong influence on the survival of the Bifidobacterium and stated that the investigation of the sensitivities of the potential probiotic strains to bile as a selection step is very important. The successive effects at stress by gastric acid and intestinal bile was expected to be stronger than either of these parameters alone (Marteau et al., 1997). Gilliland et al. (1984) stated that Lactobacillus strains with high acid and bile resistance showed better growth and colonization character in the small intestine than non-resistant strains. Bifidobacterium sp. SI 31 which showed high resistance to acid and bile salt would therefore be useful for products which require higher numbers of cells with the higher potential of the intestinal colonization. Since fermentation of skim milk by SI 31 produced typical yogurt-type coagulation, SI 31 would be a feasible bacteria for the production of fermented-milk products. The specific reason for the acid and bile

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resistance of our selected strains awaits further research. An understanding of acid and bile resistance mechanisms would be beneficial for the future management of probiotic Bifidobacterium (Benjamin and Datta, 1995; Lee et al., 1994; Park et al., 1995). In conclusion, HJ 30 and SI 31 selected by acid and bile resistant stressing procedures have been improved for growth and survival rates in low pH or high bile concentration environments. These results suggest that the screening procedure developed in this study is effective and practical and the strains selected may be useful for industrial application, effective and practical and the strains selected may be useful for industrial application.

Acknowledgements This work was supported by a research grant of the G7 project (1995) supported by the Ministry of Science and Technology of Korea. I would also deeply appreciate Dr. James R. Clarke, East Lansing, Michigan State University for critical reading of this manuscript.

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