Note
Bioscience Microflora Vol. 24 (4), 119–123, 2005
Comparison of Growth Promoting Effects on Bifidobacterium spp. by Bovine Lactoferrin Hydrolysates Woan-Sub KIM*, Md. Morshedur RAHMAN, Haruto KUMURA and Kei-ichi SHIMAZAKI Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University, Nishi 9, Kita 9, Kita-ku, Sapporo 060-8589, Japan Received June 4, 2005; Accepted for publication, August 15, 2005
The effect of bovine lactoferrin hydrolysates on the growth of four species of bifidobacteria, B. bifidum, B. longum, B. breve and B. infantis was investigated. It was observed that the growth of 3 species of bifidobacteria (B. bifidum, B. breve and B. infantis) was stimulated by bovine lactoferrin hydrolysates. These results suggest the possibility that lactoferrin, digested in the intestine, acts as a bifidogenic factor for the growth of bifidobacteria. Key words: bifidobacteria; lactoferrin; prebiotic; growth promoting factor
controversy in the literature over whether or not lactoferrin is a bifidobacterial growth inhibitor or promoter, which has not been resolved. The purpose of this paper was to investigate the effects of bovine lactoferrin hydrolysates supplementation on the growth of four species of bifidobacteria, B. bifidum, B. longum, B. breve and B. infantis.
INTRODUCTION
Bifidobacteria are generally considered to benefit human health together with lactobacilli in vivo. Therefore, they are widely used in probiotic preparations and foods (17). Bifidobacteria are generally characterized as Gram-positive, non-spore forming, nonmotile and catalase-negative anaerobes (21). They distribute to various ecological niches in the human gastrointestinal tract, the exact ratio of which mainly depends on age and diet. Biological functions of bifidobacteria include synthesis of vitamins, supplementation in digestion and absorption, inhibition of growth of exogenous organisms, and stimulation of the immune system (12). Growth factors for bifidobacteria have been investigated and identified as inorganic salts, vitamins, nitrogen and carbohydrates. Milk is known to be a complex mixture of various kinds of bioactive materials that are indispensable for infant nutrition, and a number of bioactive components have been identified in milk proteins. Some of them reveal their biological activity only after undergoing proteolytic digestion. Lactoferrin is an iron-binding glycoprotein that is present in mammalian exocrine secretions and in specific granules of polymorphonuclear leukocytes (1, 2). Mature bovine milk contains 0.1 to 0.3 mg/ml of lactoferrin whereas colostrum contains 2 to 5 mg/ml (3). In vitro studies have demonstrated that lactoferrin is bacteriostatic and occasionally bactericidal for a large number of organisms especially in the iron-poor state (22), however, it is a growth promoter for other organisms (19, 20). There has been considerable
MATERIALS AND METHODS
Proteins and chemicals Bovine lactoferrin (BLf) was donated by Morinaga Milk Industry Co. Ltd. (Zama, Japan). Lactobacilli DeMan-Rogosa-Sharpe (MRS) broth was purchased from Merck (Darmstadt, Germany), and α-Cyano-4cinamic acid (CHCA), 3, 5-dimethoxy-4-hydroxy cinnamic acid (sinapinic acid), trifluoroacetic acid (TFA), trypsin and pepsin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile was acquired from LAB-SCAN (Labscan, Co., Ireland). All other reagents were of the highest purity available. All solutions were prepared using Milli-Q water. Strains, culture conditions and measurement of growth The bifidobacterial strains, used in this study were B. bifidum ATCC 15696, B. infantis ATCC 15697, B. longum ATCC 15707 and B. breve ATCC 15700. Prior to use, all bifidobacteria were cultured for 24 h anaerobically at 37°C in MRS broth using an anaerobic jar (Becton, Dickinson and Company, USA). Ten microliters of activated culture (3.0 × 107 cfu/ml) were freshly inoculated into 10 ml MRS broth. Fermentations of the test tubes were performed anaerobically by covering with paraffin liquid (Kishida Chemical Co., Japan) at 37°C. Hydrolysates of BLf were filtersterilized (pore size 0.22 µ m) and added to the autoclaved medium to give final concentrations of 0.01,
*Corresponding author. Mailing address: Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University, Nishi 9, Kita 9, Kita-ku, Sapporo 060-8589, Japan. Phone: +81-11-706-3642. Fax: +81-11-706-4135. E-mail:
[email protected]
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0.1 and 1 mg/ml. Control incubations of bifidobacteria in MRS were performed without added hydrolysates of BLf. The growth of bifidobacteria was monitored by measuring the optical density at 660 nm using Mini photo 518 (Taitec, Co., Japan) in defined time intervals. Values for each sample were determined in triplicate. Preparation of lactoferrin hydrolysates Tryptic hydrolysates of BLf were prepared by trypsin digestion at 37°C for 12 h. The reaction was terminated by heating at 80°C for 15 min. Peptic hydrolysates of BLf were prepared according to the method described by Recio and Visser (15), i.e. BLf was adjusted to pH 3.0 with 1M HCl and digested with 3% (wt/wt of substrate) pepsin for 12 h at 37°C. The reaction was terminated by heating at 80°C for 15 min and the pH was adjusted to 7.0 by the addition of 1 M NaOH. Analytical methods The degree of hydrolysis of BLf hydrolysates were determined by matrix-assisted laser desorption ionization times of flight mass spectrometry (MALDI-TOF MS). α-CHCA and sinapinic acid were used as the matrix in these experiments. Before applying the sample solution, treatment with Zip Tip (Millipore, Co., USA) was carried out to remove salt. Then, samples were mixed in a 1:1 ratio with matrix solution. One microliter of the mixed sample was applied to a plate and air-dried. The TOF MS instrument employed in this study was a Voyager DE STR system (Perseptive Biosystems, Inc., Framingham, MA, USA). MALDI conditions were as follows: laser power, 2300-2600 nm; accelerating voltage, 20-25 kV; with guide wire, 0.035-0.05% of accelerating voltage; grid voltage, 93-95% of accelerating voltage; and operating in the positive mode detection. Statistical analysis The results are expressed as means ± SD, the significance of differences being determined by Student’s t test. RESULTS AND DISCUSSION
Tryptic and peptic hydrolysates of BLf were prepared in order to know their effects on the growth of bifidobacteria at various concentrations. The degree of hydrolysis was determined by MALDI-TOF MS. The BLf hydrolysates produced by trypsin digestion for 12 h showed a distribution of low molecular mass peptides (< 3500 Da) (data not shown). These hydrolysates of BLf were used to test the growth promotion activity on Bifidobacterium spp. The growth responses of four
species of bifidobacteria at various concentrations (0.01, 0.1 and 1 mg/ml) of bovine lactoferrin hydrolysates were observed at different time points. The growth was compared with that of a control (absence of BLf hydrolysates). Both tryptic and peptic hydrolysates of BLf promoted the growth of B. bifidum ATCC 15696 (Fig. 1A and B). Compared to the control, tryptic hydrolysates showed the highest growth promotional effect on B. bifidum (Fig. 1A) at a concentration of 1 mg/ml. The growth of this strain was also stimulated at 0.1 mg/ml but no effect was observed at a concentration of 0.01 mg/ml. The growth response at 1 mg/ml was higher than the growth response at 0.1 mg/ml. Peptic hydrolysate also significantly promoted the growth of B. bifidum at both 1 mg/ml and 0.1 mg/ml (Fig. 1B). Figure 1, C and D, shows the growth of B. longum ATCC 15707 in the presence or absence of BLf hydrolysates. The tryptic (Fig. 1C) and peptic (Fig. 1D) hydrolysates of BLf did not stimulate the growth of B. longum. Interestingly, both the tryptic and peptic hydrolysates exhibited an inhibitory effect on its growth at a high concentration (1 mg/ml). The growth patterns of B. infantis ATCC 15697 are shown in Fig. 1E and F. Tryptic hydrolysate (Fig. 1E) and peptic hydrolysate (Fig. 1F) of BLf significantly stimulated the growth of B. infantis at concentrations of 0.01, 0.1 and 1 mg/ml. The growth of B. infantis increased with increasing hydrolysate concentrations. Figure 1, G and H, shows the growth responses of B. breve ATCC 15700. Tryptic hydrolysate of BLf showed significantly higher growth effects at concentrations of 0.01, 0.1 and 1 mg/ml than the control (Fig. 1G). On the other hand, peptic hydrolysates of BLf significantly stimulated the growth of B. breve at only 1 mg/ml (Fig. 1H). Tryptic hydrolysate of BLf showed a higher growth promotion effect than peptic hydrolysate in B. breve. In the present study, we used MRS broth for cultivation of bifidobacteria, but this medium is not an optimal medium for cultivating bifidobacteria. Accordingly, the growths in MRS medium differed with species of bifidobacteria. Especially, lower growth than other bifidobacteria was shown in the case of B. bifidum (Fig. 1A and B). However, we did not consider this a problem, because the purpose of this research was to observe the growth promotion effect of addition of BLf hydrolysate, and the effects of the growth promotion were tested from the lag phase to the exponential phase of bifidobacteria. The present study demonstrated the growth stimulation effects of BLf hydrolysates (generated by trypsin and pepsin) on bifidobacteria. The
BIFIDOBACTERIA AND LACTOFERRIN
Fig. 1. Changes of optical density during the growth of bifidobacteria in MRS broth supplemented with bovine lactoferrin hydrolysates: A and B, Bifidobacterium bifidum ATCC 15696; C and D, Bifidobacterium longum ATCC 15707; E and F, Bifidobacterium infantis ATCC 15697; G and H, Bifidobacterium breve ATCC 15700 (A, C, E, G: bovine lactoferrin hydrolysates produced by trypsin digestion; B, D, F, H: bovine lactoferrin hydrolysates produced by pepsin digestion).
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growth of four species of bifidobacteria, B. bifidum, B. longum, B. breve and B. infantis was compared in the presence or absence of BLf hydrolysates. It was observed that the growth of bifidobacteria was stimulated by BLf hydrolysates (Fig. 1A, B, E–H), however, no growth stimulation effect on B. longum was observed (Fig. 1C and D). Previously, we reported that growth of B. longum was not stimulated by the addition of transferrin family proteins (Lactoferrin, Transferrin or Ovotransferrin) to MRS broth. Furthermore, we also reported that B. longum does not possess a binding protein for transferrin family proteins. Therefore, we concluded that the presence of a transferrin family binding protein on bifidobacteria is responsible for such growth stimulation effect (10, 11). Many studies have shown the antimicrobial activity of lactoferrin and transferrin against Gram-negative bacteria. According to Reiter et al. (16), two strains of E. coli were inhibited by addition of colostral whey dialyzed or diluted in Kolmer saline, or addition of precolostral calf serum or lactoferrin. Bishop et al. (3) also studied the bacteriostatic effects of apo-BLf against strains of coliform bacteria associated with bovine mastitis. They reported that a low concentration of apoBLf (0.02 mg/ml) resulted in marked inhibition of growth for all coliforms. In addition, Dionysius et al. (6) examined in vitro antibacterial effects of various forms of lactoferrin on enterotoxigenic strains of E. coli using a microassay for bacterial growth. Native and apo-BLf exhibited variable activity against 19 strains of E. coli, whereas holo-BLf showed no effect. At a concentration of 0.2 mg/ml of apo-BLf, most strains could be distinguished as either sensitive or resistant to inhibition. Dial et al. (5) examined in vitro and in vivo antimicrobial efficacies of BLf against Helicobacter species. Lactoferrin showed bacteriostatic effects against H. pylori when it was cultured in lactoferrin concentrations above 0.5 mg/ml. The antibacterial action of lactoferrin is most frequently attributed to its ability to chelate iron, which in turn may restrict the availability of this essential nutrient for susceptible organisms. Other biological functions have also been suggested for lactoferrin, such as involvement in various aspects of the immune system and inflammation, and some reports have indicated that lactoferrin stimulates cell growth and acts as a growth factor or iron carrier molecule (4, 7, 13, 18). Earlier studies have found that lactoferrin from cow’s milk promotes the growth of bifidobacteria in vitro (14). We also previously reported the growth-promoting activity of bovine lactoferrin on L. acidophilus and bifidobacteria (10, 11). Few studies have systematically
evaluated the impact of lactoferrin on the composition of intestinal bacteria; the addition of BLf to infant formula (1-2g/L) has been reported to cause an increase in intestinal bifidobacteria counts when fed to adult mice (9). Recent studies have shown that lactoferrin can bind DNA and activate transcription, which might explain the molecular basis of growth regulation (8). In the present study, we demonstrated a growth promotion effect of BLf hydrolysates, which were generated by trypsin and pepsin digestion of BLf. Therefore, lactoferrin may have dual mechanisms for inhibiting enteric bacterial pathogens through direct inhibition of bacterial growth or development of favorable change in the composition and metabolism of the non-pathogenic indigenous intestinal bacteria. The lactoferrin hydrolysate described in the present study has commercial value as a natural preservative agent for use in foods and cosmetics, and as a functional component of new clinical foods for prevention or treatment of gastrointestinal disease. Acknowledgements. The authors would like to express appreciation to S. Teraguchi (Morinaga Milk Industry Co., Ltd. Japan) for his support. REFERENCES (1) Baggiolini M, De Duve C, Masson PL, Heremans JF. 1970. Association of lactoferrin with specific granules in rabbit heterophil leukocytes. J Exp Med 131: 559570. (2) Bellamy WR, Wakabayashi H, Takase M, Kawase K, Shimamura S, Tomita M. 1993. Role of cell-binding in the antibacterial mechanism of lactoferricin B. J Appl Bacteriol 75: 478-484. (3) Bishop JG, Schanbacher FL, Ferguson LC, Smith KL. 1976. In vitro growth inhibition of mastitis-causing coliform bacteria by bovine apo-lactoferrin and reversal of inhibition by citrate and high concentrations of apo-lactoferrin. Infect Immun 14: 911-918. (4) Byatt JC, Schmuke JJ, Comens PG, Johnson DA, Collier RJ. 1990. The effect of bovine lactoferrin on muscle growth in vivo and in vitro. Biochem Biophys Res Commun 173: 548-553. (5) Dial EJ, Hall LR, Serna H, Romero JJ, Fox JG, Lichtenberger LM. 1998. Antibiotic properties of bovine lactoferrin on Helicobacter pylory. Dig Dis Sci 43: 2750-2756. (6) Dionysius DA, Grieve PA, Milne JM. 1993. Forms of lactoferrin: Their antibacterial effect on enterotoxigenic Escherichia coli. J Dairy Sci 76: 25972600. (7) Hagiwara T, Shinoda I, Fukuwatari Y, Shimamura S. 1995. Effects of lactoferrin and its peptides on proliferation of rat intestinal epithelial cell line, IEC-
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