Báo cáo khoa học: Human milk provides peptides highly stimulating the growth of bi®dobacteria pptx

Human milk provides peptides highly stimulating the growth of bi®dobacteria Cornelia Liepke, Knut Adermann, Manfred Raida, Hans-JuÈ rgen MaÈ gert, Wolf-Georg Forssmann and Hans-Dieter Zucht IPF PharmaCeuticals GmbH, Hannover, Germany The large intestine of breast-fed infants is c olonized predominantly by bi®dobacteria, which have a protective eect against acute diarrhea. In t his study we report for the ®rst time the identi®cation of human milk peptides that selectively stimulate the growth of bi®dobacteria. Several bi®dogenic peptides were puri®ed chromato graphically from pepsin-treated human milk and identi®ed as proteolytically generated fragments from the s ecretory component of the soluble polyimmunoglobulin receptor and lactoferrin; both of these proteins exhibit antimicrobial eects. Hydrolysis of the identi®ed peptides with the gastrointestinal proteases pepsin, trypsin and chymotrypsin did not lead to the loss o f bi®dogenic activity, indicating their p otential function in vivo. Sequential comparison revealed a similar structural motif within the identi®ed peptides. A correspondingly designed small peptide (prebiotic lactoferrin-derived peptide-I, PRELP-I) was f ound to stimulate the growth of bi®dobacteria as eectively as the native peptides. The combination of antimicrobial and bi®dobacterial growth stimulatory activity in human milk proteins leads to highly speci®c compounds capable o f regulating t he microbial composition of infants' large intestine. Keywords: bi®dobacteria; human milk; intestinal micro¯ora; lactoferrin; peptides. The colonization of the i ntestine with nonpathogenic microorganisms is essential for the h ealth and wellbeing of the newborn. Breast-feeding creates an intraintestin al envi- ronment that favours a simple ¯ora of bi®dobacteria and few other microorganisms [1,2]. Several epidemiological studies have shown that the incidence of gastrointestinal infections is lower in infants who are exclusively breast-fed than in infants who are fed on formula [3±5]. The occurrence of bi®dobacteria in the large bowel is bene®cial for the infant because it prevents the proliferation of pathogens that cause diarrhea, such as salmonella or rotaviruses [6±8]. It has been shown that breast milk contains speci®c bi®dus growth factors, such as certain carbohydrates, supporting the implantation of these bacteria (reviewed in [9]). It h as been assumed also that signi®cant bi®dogenic activity is associated with milk protein [10±14], either by direct growth stimulation o r b y a ntimicrobial effects. However, the molecules and mechanisms underlying the bi®dogenic activity of human milk protein, especially the effect o f peptides resulting from the digestion of these proteins, have hitherto not been well understood. In the present work, we report that the bi®dogenic activity of human milk protein is due to small proteolytic fragments of major milk proteins. These peptides obtained from the digestion of milk protein with the gastric protease pepsin were identi®ed as effective growth factors for bi®dobacteria. MATERIALS AND METHODS Puri®cation of peptides Human milk and c olostrum (collected 2±3 months and 2 d ays after birth, respectively) were obtained after informed consent from healthy voluntary donors. Pepsin (20 mgág )1 milk protein) was a dded to human and bovine milk after acidi®cation to pH 3.5 with HCl, and this mixture was incubated for 2 h at 37 °C. The proteolysis was stopped by boiling for 5 min. After centrifugation ( 20 min, 6000 g, 4 °C), the fat was skimmed off and the supernatant was collected. Tri¯uoroacetic acid was added to the supernatant to a ®nal concentration of 0.1%. Solid particles were removed by centrifugation (20 min, 2500 g,4°C) and ®ltration. For peptide puri®cation, three subsequent HPLC steps were carried out, tracking the maximum growth stimulatory activity o f the resulting fractions on Bi®dobac- terium bi®dum DSM 2 0082. First HPLC: separation o f 20 mL pepsin-treated human milk using a C18-reversed- phase column (Parcosil, 10 mm ´ 125 mm, 5 lm, 100 A Ê , Biotek, O È stringen, Germany); eluent A, 0.1% tri¯uoroacetic acid; eluent B, 0.1% tri¯uoroacetic acid in acetonitrile; ¯ow rate, 2 mLámin )1 ; gradient, 0±6 0% B in 4 5 min; U V detection, 280 nm (Fig. 1A). Second HPLC: rechromato- graphy of bi®dogenic fractions with the same conditions as above using a shallower gradient, 20±50% B in 45 min. Third and ®nal puri®cation step: rechromatography on a cation exchange column (Parcosil Pepkat, 4 ´ 50 mm, 5 lm, 300 A Ê ,Biotek);eluentA,10m M phosphate buffer (pH 4 .5); eluent B, 10 m M phosphate buffer ( pH 4.5) with 1 M NaCl; ¯ow rate, 0.75 mLámin )1 ; gradient, 0±60% B in Correspondence to C. Liepke, IPF PharmaCeuticals GmbH, Feodor- Lynen-Strasse 31, D-30625 Hannover, Germany. Fax: + 49 511 5466 132, Tel.: + 49 511 5466 201, E-mail: c.liepke@ipf-pharmaceuticals.de Abbreviations: CZE, capillary zone electrophoresis; hLACFR, human lactoferrin; hPIGR, human polyimmunoglobulin receptor; PRELP-I, prebiotic lactoferrin-derived peptide-I. (Received 6 September 2001, revised 26 November 2001, accepted 29 November 2001) Eur. J. Biochem. 269, 712±718 (2002) Ó FEBS 2002 60 min; UV detection, 214 nm. The resulting HPLC fractions were desalted prior to testing for bi®dogenic activity by use of a n analytical C 18 column (Vydac, 4.6 ´ 250 mm, 5 lm, 300 A Ê , The Separations Group, Hesperia, CA); eluent A, 0.1% tri¯uoroacetic acid; eluent B, 0.1% tri¯uoroacetic acid in acetonitrile; ¯ow rate, 0.75 mL ámin )1 ; gradient, 20±40% B in 20 min; UV detec- tion, 214 nm. Peptide analysis Peptides were analysed by electrospray MS using a Sciex API III (Perkin Elmer) mass spectrometer. Alternatively, MALDI MS was performed with a LaserTec RBT I I (PerSeptive Biosystems, Framingham, MA). A mino-acid sequencing was carried out with a gas phase sequencer (model 473 A, Applied Biosystems). The purity of peptides was analysed by capillary zone electrophoresis (CZE) with a P/ACE2100CZEsystem(Beckman). Peptide synthesis Peptides were synthesized using Fmoc solid-phase chemistry on a preloaded TentaGel S Trt resin (Rapp Polymere, Tu È bingen, Germany). Crude peptides were puri®ed by reversed-phase HPLC (Vydac C18, 10 lm, 300 A Ê ;gradient, 10±70% B in 30 min; eluent A, 0.07% tri¯uoroacetic acid/water; eluent B, 0.05% tri¯uoroacetic acid in acetonit- rile/water 4 : 1; ¯ow rate, 0.8 mLámin )1 ; UV detection, 215/230 nm). The purity and identity of synthesized peptides were chec ked by analytical HPLC, MS and sequence analysis. Bacterial growth assays All bi®dobacteria strains (Table 1) were purchased from the Deutsche Sammlung fu È r Zellku lturen und Mikroorganis- men (Braunschweig, Germany). All o ther microorganisms (Escherichia coli ATCC 11229, Enterococcus faecalis ATCC 29212, Clostridium dicile ATCC 9684 and Candida albicans ATCC 10231) were tested at the Hygiene Institute of the University o f H eidelberg, Germany. For growth assays, peptides were added to 200 lLof29gáL )1 Elliker broth ( Difco), inoculated with 25 lLofbacterialculture (A 50 of 0.05) 1 . The samples were incubated a t 37 °C under anaerobic conditions for 16±48 h (according to the strain). Bacterial growth was monitored by photometric absorbance measurements at 550 nm and by microscopy. Pepsin-treated Fig. 1. Puri®cation of bi®dogenic peptides from human milk by consecutive chromatographic steps. (A) Reversed-phase HPLC separation of 20 mL pepsin-treated human milk. The black bars indicate the increased cell density of B. bi®dum DSM 20082 cultures supplemented with the peptides from HPLC fractions corresponding to a 0.5 mL aliquot of milk. The fraction exhibiting highest bi®dogenic activity, indicated by an arrow, was selected for f urther puri®cation. (B) Reversed-phase chromatography of t he selected HPLC fraction from (A). The f raction stimulating most eectively the growth of B. bi®dum is indicated by a black arrow and was selected for the next separation step. (C) Cation-exchange chromato- graphy of the s elected HPLC fraction from ( B) resulted in the puri®c ation to homogeneity of t he two bi®dogenic peptides hLACFR-Ib and hLACFR-Ic. (D) Purity of the isolated peptides was proved by CZE. As an example the electropherogram of the peptide hLACFR-Ic is shown. (E) MALDI-MS analysis revealed a molecular mass o f 5801 Da for th e peptide hLACFR-Ic, concurring with the calculated molecular mass of 5798 Da. The variation of 3 Da is within the accuracy of mass measurement (0.5%). Ó FEBS 2002 Human milk peptides as bi®dus factors (Eur. J. Biochem. 269) 713 human milk (10%, v/v) and 100 l M N-acetylglucosamine, both of which stimulate t he growth of bi®dobacteria, were used as positive controls. RESULTS Identi®cation of human milk peptides that stimulate the growth of B. bi®dum Peptides capable of stimulating the growth of bi®dobacteria were puri®ed from pepsin-treated human milk and colo- strum by m eans of reversed-phase and cation-exchange HPLC. A typical reversed-phase separation of pepsin- treated human milk is shown in Fig. 1A. For detection of growth promoting activity within HPLC fractions B. bi®- dum DSM 20082 was used as a test strain. The HPLC fraction exhibiting highest growth stimulatory activity was selected for further puri®cation. In a second reversed-phase separation step bi®dogenic peptides were enriched in one major peak (Fig. 1B). Use of cation-exchange chromato- graphy resulted in the effective separation of two bi®dogenic peptides from inactive components (Fig. 1C). Purity of the active peptides was proved by CZE (Fig. 1D). MALDI MS analysis revealed a m olecular mass o f 5801 Da for one of the puri®ed peptides (Fig. 1E) and 5584 D a for the other (data not shown). Edman degradation determination of the primary str ucture of t he isolated p eptides (Fig. 2A) and subsequent database analysis (Swis sProt) led to the identi®cation of two lactoferrin fragments, termed hLACFR-Ib and hLACFR-Ic. Using this puri®cation procedure, several peptides that exhibited bi®dobacterial g rowth stimulatory activity were isolated from mature human milk and from colostrum. From mature human milk the highly bi®dogenic peptides Table 1. Susceptibility of dierent bi®dobacteria towards bi®dogenic compounds. The spectru m of growth stimulation is dierent for the bi®dogenic compounds t ested, which were p epsin -treated human milk (10% , v/v), 100 l M N-acetylglucosamine, and 1 l M of th e p eptides hLACFR-Ia, hLACFR-Ic, hLACFR-IIa, and PRELP-I. A Ô+Õ indicates that the application of a compound led to an increased cell density of at least + 5 0% after 16±48 h incubation in 29 gáL )1 Elliker broth. Microorganism Bi®dogenic compounds Human milk N-acetylglusamine hLACFR-Ia hLACFR-Ic hLACFR-IIa PRELP-I B. adolescentis DSM 20083T ± ± ± ± ± ± B. breve DSM 20213T + ± + + + + B. longum DSM 20219T + ± + + ± ± B. bi®dum DSM 20215 + + + + + + B. bi®dum DSM 20456 + ± ± ± ± ± B. bi®dum DSM 20082 + + + + + + B. bi®dum DSM 20088 + ± + + + + Fig. 2. Primary s tructure of isolated human b i®dogenic p eptides. ( A) The human b i®dogenic peptides a re derived from the precursor proteins lactoferrin (hLACFR) a nd th e secretory co mpone nt of t he po lyimmunoglobulin re ce ptor (hP IGR). hL ACFR-Ia, -Ib, an d - Ic are from t he N-terminus and hLACFR-IIa and -IIb are from equivalent positions of the C-terminus of lactoferrin. hLACFR-Ia corresponds to the generally known lactoferrin sequence. hLACFR-Ib is obtained from a lactoferrin variant with an arginine deletion at position 3 and hLACFR-Ic is derived from a lactoferrin variant with two amino-acid residues exchanged. (B) Partial sequence alignment of the bi®dogen ic peptides hPIGR-I, hLACFR- Ia, and hLACFR-IIa. Identical residues are hatched with dark grey. The residues probably related to bi®dogenic activity and used for the design of PRELP-I are marked with arrows. 714 C. Liepke et al. (Eur. J. Biochem. 269) Ó FEBS 2002 hLACFR-Ia, hLACFR-Ib, and hLACFR-Ic (Fig. 2A) were puri®ed. These peptides are fragments derived from the N-terminus of three different lactoferrin variants. In the milk from one donor, the peptides hLACFR-Ib and hLACFR-Ic were found, and from a second donor, the peptides hLACFR-Ia and hLACFR-Ic were isolated. Further puri®cation led to t he identi®cation of the lactofer- rin fragments hLACFR-IIa and hLACFR-IIb, wh ich are derived from the lactoferrin C-terminus (Fig. 2 A). From pepsin-digested colostrum, the bi®dogenic peptides hPIGR-Ia and hPIGR-Ib were puri®ed; these represent fragments of t he soluble polyimmunoglobulin receptor (hPIGR) (Fig. 2A). In mature human milk hPIGR-Ia and hPIGR-Ib were not detectable corresponding to the tenfold lower amount of hPIGR in mature milk. Pepsin-treated bovine m ilk also exhibits bi®dogenic activity at about half the level of that of mature human milk (data not shown). However, no bi®dogenic peptides were identi®ed in bovine mild by reversed-phase HPLC of pepsin-treated bovine milk. Bi®dogenic activity of human milk peptides In dose±response studies using the pure human peptides hPIGR-Ib, hLACFR-Ia, hLACFR-Ib, hLACFR-Ic, and hLACFR-IIa, a dose-dependent growth stimulatory activity on B. bi®dum DSM 20082 was con®rmed. Each of the puri®ed peptides stimulates B. bi®dum by 50±250% at a concentration of 0.5±3 l M (Fig. 3 ). The puri®ed milk peptides promote the growth of several strains o f b i®dobacteria that usually colonize infants' intestines (Table 1). Th e peptides hLACFR-Ia and hLACFR-Ic stimulated ®ve out of the seven bi®dobacterial strains tested signi®cantly and the peptide hLACFR-IIa stimulated four out of the seven. In c ontrast, the known bi®dogenic carbohydrate N -acetylglucosamine exhibits growth promoting activity only towards strains of B. bi®- dum. The growth stimulatory activity of N-acetylglucos- amine was detected at a minimum concentration of 50 l M , which i s 100-fold higher than t he minimum effective concentration of the puri®ed bi®dogenic peptides. Structural characteristics of bi®dogenic peptides All bi®dogenic peptides identi®ed share a similar primary structure containing a disul®de bond, and a small hydro- phobic domain located C-terminally next to the cysteines (Fig. 2 B). Based on these s tructural characteristics, a small peptide n amed prebiotic lactoferrin-derived peptide-I (PRELP-I, Fig. 2A) was designed and chemically synthe- sized. PRELP-I exhibits a g rowth stimulatory effect on B. b i®dum DSM 20082, B. bi®dum DSM 20215, B. infantis and B. breve in the same order of magnitude as the peptides isolated previously (Fig. 3). In contrast, the growth of other microorganisms capable of colonizing the human intestine, such as C. dicile, E. coli, E. faecalis and C. albicans,was not affected signi®cantly by the synthetic peptide using the same anaerobic growth conditions used for bi®dobacteria (Fig. 4 ). To con®rm t hat the growth stimulatory activity of bi®dogenic peptides is related to their peptidic character, control experiments were performed. No bi®dogenic activity was observed when applying amino-acid mixtures representing the a mino-acid composition of t he puri®ed peptides, simple disul®de-containing compounds or bi®do- genic peptides after chemical reduction of disul®de bonds. Hydrolysis with the nonspeci®c protease subtilisin led to inactivation of hLACFR-Ia, hLACFR-IIa, and PRELP-I (data not shown). In contrast, treatment of hLACFR-Ia, hLACFR-IIa, and PRELP-I with pepsin, trypsin or chymotrypsin did not lead to the lo ss of bioactivity, indicating the stability of the peptides towards gastroin- testinal proteases. DISCUSSION Previous reports have shown that proteolytic milk hydro- lysate is an effective stimulant for the growth of bi®dobac- teria [10,14]. However, the molecular structures of the substances responsible for this effect have been hitherto unknown. In the present work, we show that proteolytic fragments of major human milk proteins are effective growth factors for bi®dobacteria. Furthermore, it is shown that the bi®dogenic effect of human milk is not based entirely on their carbohydrate content, as the components isolated contain no glycosyl moieties. The isolated fragments of the polymeric immunoglobulin receptor secretory component (hPIGR) and of human lactoferrin (hLACFR) stimulate the bi®dobacterial growth even in the p resence of an e xtraordinarily rich growth medium. These peptides were shown t o be 100 times more effective on a molar b asis than the c arbohydrate N-acetylglusosamine, a typical currently known bi®dogenic carbohydrate [9] (data not shown). M oreover, they stimu- late a larger set of different bi®dobacterial strains (Table 1). All of these results together show that the peptides described in this study act very effectively as prebiotic growth factors. Interestingly, no other bacterial species was found to be stimulated in a similar manner, which in dicates that the action of these peptides is highly speci®c to bi®dobacteria. It can be assumed t hat the bi®dogenic activity of breast milk Fig. 3. Dose±response of bi®dogenic peptides. The selected bi®dogenic peptides exhibit a dose-dependent growth stimulatory eect on B. bi®dum. The plots show the relative cell density of B. bi®dum DSM 20082 cultivated for 16 h in 29 gáL )1 Elliker broth supplemented with: (A) hLACFR-Ia, -Ib, -Ic; (B) hPIGR-Ib; (C) PRELP-I; (D) hLACFR-IIa. Ó FEBS 2002 Human milk peptides as bi®dus factors (Eur. J. Biochem. 269) 715 based on p eptides exceeds the relevance o f the bi®dogenic activity of milk carbohydrates. There are several lines of evidenc e that the ch aracterized peptidic bi®dus factors are important for the establish- ment of the b i®dobacterial ¯ora in the large bowel of breast-fed infants. One ®nding is that peptide bi®dus factors selectively stimulate bi®dobacterial strains usually found in infants' intestine. Furthermore, the time course of the synthesis of the precursor molecules of peptide bi®dus factors is coupled t o the bi®dogenic activity o f human milk; the bi®dogenic activity of colostrum is higher than that of mature milk [15], which coincides with the 10-fold increased excretion rate of the precursor proteins of peptidic bi®dus factors immediately after birth ( rev- iewed in [16]). As a result, the bi®dogenic activity is increased during the critical initial microbial colonization process of the infants' large bowel. In cow's milk, a signi®cantly lower bi®dogenic activity is present because it lacks peptidic and carbohydrate bi®dus factors [16]. As a result, f eeding with unsup plemented c ow's milk-based formulas does not induce the typical bi®d ¯ora observed in breast-fed infants [17]. Sequence comparison of the bi®dogenic peptides identi- ®ed a common structural motif (Fig. 2B) that contains a pair of cysteine residues forming a disul®de bond and two small hydrophobic domains located C-terminally to the two cysteines. From this observation, a simple peptide with the arti®cial sequence CAVGGGCIAL was designed and tested. This peptide exhibits bi®dogenic activity comparable to those of the isolated peptides. The activity is related to the peptidic characte r, as the nonspeci®c protease subtilisin degraded bi®dogenic activity and amino-acid mixtures had no profound activity. I n addition, presence of the d isul®de bridge is essential as its chemical reduction leads to the complete loss of activity. The importance of t he integrity of the disul®de bond fo r bi®dogenic acitivity is i n accordance with the ®ndings of Poch and Bezkorovainy [18] and Ibrahim and Bezkoro vainy [19]. Poch and Bezkorovainy showed that bovine milk j-casein and hog gastric mucin lost their bi®dobacterial growth-promoting activities following reduction-alkylation or oxidation of their disul®de bonds. Consistent with our results they also showed that simple disul®de-containing compounds exhibit no bi®dobacterial growth stimulating activity. This adds to the ®ndings of Ibrahim and Bezkorovainy who suggest that the diverse bi®dobacterial growth-promoting factors reported have a common component, namely s ulfur containing peptides. In contrast with these ®ndings Etoh et al. [20] identi®ed a decapeptide with g rowth p romoting activity towards B. bi®dum , which contains neither cystine nor cysteine. Therefore, they suggest that sulfhydryl groups are not indispensable to growth-stimulating activity. However, they do not indicate in which concentration the decapeptide is active and no experiments were perf ormed evaluating its speci®city for bi®dobacteria or the importance of the peptidic structure for the bi®dogenic activity. Therefore, it is possible that the decapeptide is responsible for bacterial growth stimulation as a nutritional source, in contrast with the bi®dus factors reported previously [18,19] and the bi®dogenic milk peptides presented in this study. The precursor proteins of peptide bi®dus factors are known to have a protective value for the suckling infant. The p olymeric immunoglobulin receptor precursor consists of the secretory component that is usually bound to the immunoglobulins present in milk [21]. Immunoglobulins are highly protective due to their capability to i nactivate intraintestinal pathogens. Our ®nding, that a portion of the secretory immunoglobulins is a prebiotic factor for bene®cial bacteria, reveals a novel function for this complex. As a result, immunoglobulins act protectively by regulating the infants' i ntestinal m icro¯ora. A further precursor protein y ielding p eptidic b i®dus growth factors i s l actofer- rin. Interestingly, the potential protective role of lactoferrin regulating the intestinal micro¯ora h as already been discussed extensively according t o its antimicrobial activity [22±24]. There are antimicrobial lactoferrin fragments called Fig. 4. Growth in¯uencing activity of the synthetic peptide PRELP-I towards intestinal bacteria. PRELP-I stimulates the growth o f bi®dob acteria bu t does not aect the growth of other microorganism s that colonize the human in testine. The plot show s the bacterial growth of B. infantis, Clostridium dicile, Escherichia coli , Enterococcus faecalis,andCandida albicans cultivated for 20 h (72 h Clostridium dicile) under anaerobic conditions in 29 gáL )1 Elliker broth supplemented with PRELP-I at dierent concentrations. 716 C. Liepke et al. (Eur. J. Biochem. 269) Ó FEBS 2002 lactoferricines [ 25,26], w hich are structurally related t o t he bi®dogenic peptides hLACFR-Ia, -Ib, and -Ic. The anti- microbial activity of lactoferrin described previously and our results provide strong evidence that this protein is also important fo r the determination of the intraintestinal bacterial ¯ora of the newborn by combining the same two activities observed for the secretory immunoglobulins; antibiosis and prebiosis. All of the isolated lactoferrin fragments stem from two equivalent positions within the lactoferrin two-lobe struc- ture, which is based on an ancient sequence duplication within lactoferrin and, obviously, yields two domains with similar f unctions: iron b inding and i n¯uencing bacterial growth. Our ®ndings reveal that human milk contains three slightly different variants of lactoferrin, each corre- sponding to one of the isolated peptides hLACFR-Ia, hLACFR-Ib, and hLACFR-Ic. No description of the proteins corresponding to hLACFR-Ib and hLACFR-Ic has yet been published, although the cDNAs of these variants have already been cloned (GenBank accession numbers M73700, M 93150). The difference in these lactoferrin variants may be of ph ysiological relevance because previous reports show that N-terminal variations of lactoferrin in¯uence its antimicrobial properties, lipopolysaccharide b inding and modulate cellular i mmuni- ty reactions [27]. In this work, however, a signi®cant differences in the bi®dogenic activity of the variant peptides was not observed. Several lines of evidence may answer the important question o f whether the bi®dogenic peptides identi®ed can reach the colon of the suckling infant. The precursor proteins of peptide bi®dus factors are major components of human milk. Therefore, high amounts of bioactive peptide fragments can be liberated during digestion. Furthermore, there is evidence t hat a signi®cant amount of bioactive peptides can successfully passage through the small intes- tine, as the structure required for bioactivity is not degraded by the gastrointestinal proteases pepsin, trypsin or chym- otrypsin. This is also supported by the observation that the entire lactoferrin molecule and larger fragments are present in the stools of infants [28]. Our results demonstrate a novel function of a m ajor human milk protein; a d irect and selective g rowth stimulation of bi®dobacteria coupled to antimicrobial properties of the proteins. Bovine milk, a major part of formula diets, has a s igni®cantly lower bi®dobacteria growth-stimulatory a ctivity. H ence, an additional expla- nation as to why breast-feeding i s bene®cial for the suckling infant is provided, re¯ecting the different gastro- intestinal microbiology and physiology of humans and ruminants. The composition of formula diets should re¯ect these requirements. Simple bi®dogenic peptides, such as the peptide PRELP-I might be useful as an additive to infant formulas to improve their value by supporting the implantation of bi®dobacteria, which are bene®cial to t he suckling infant b y competing with pathogenic microorganisms. ACKNOWLEDGEMENTS The authors thank Prof. D r Geiss (Hygiene Institute, University of Heidelberg, Germany) for technical support. This work was supported by the Deutsche Forschungsgemeinschaft, DFG (ZU 98/2±2). REFERENCES 1. Kleessen,B.,Bunke,H.,Tovar,K.,Noack,J.&Sawatzki,G. (1995) In¯uence of two infant formulas and human milk on the development of the faecal ¯ora in newborn infants. Acta Paediatr. 84, 1347±1356. 2. Stark, P.L. & Lee, A. (1982) The microbial ecology of the large bowel of breast-fed and formula-fed infants during the ®rst year of life. J. Med. Microbiol. 15, 189±203. 3. France, G.L., Marmer, D.J. & Steele, R.W. (1980) Breast-feeding and Salmonella infection. Am. J. Dis. Child. 134, 147±152. 4. Larsen, S.A. & Homer, D.R. (1978) Relation of breast versus bottle f eeding to hospitalization f or gastroenteritis in a middle- class U.S. population. J. Pediatr. 92, 417±418. 5. Golding, J., Emmet, P.M. & Rogers, I.S. (1997) Gastroenteritis, diarrhoea and breast feeding. Early Hum. Dev. 49, S83±S103. 6. Gibson, G.R. & Wang, X. (1994) R egulatory eects of b i®do- bacteria on the growth of other colonic bacteria. J. Appl. Bacteriol. 77, 412±420. 7. Bielecka, M., Biedrzycka, E., Biedrzyc ka, E., Smoragiewicz , W. & Smieszek, M. (1998) Interaction o f Bi®dobacterium and Salmonella during associated growth. Int. J. Food Microbio l. 45, 151±155. 8. Saavedra, J.M., Bauman, N.A., Oung, I., Perman, J.A. & Yolken, R.H. (1994) Feeding of Bi®dobacterium bi®dum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus. Lancet 344, 1046±1049. 9. Beerens, H., Romond, C. & Neut, C. (1980) In¯uence of breast- feeding on the bi®d ¯ora of the newborn intestine. Am.J.Clin.Nutr.33, 2434±2439. 10. Kehagias, C., Jao, Y.C., Mikolajcik, E.M. & Hansen, P.M.T. (1977) G rowth response of Bi®dobacterium bi®dum to a h ydro- lytic produc t i solate d f rom bovine c asein. J. Food Sci. 42 , 146±150. 11. Bezkorovainy, A. & Topouzian, N. (1981) Bi®dobacterium bi®dus var. pennsylvanicus growth promoting activity of human milk casein and its derivatives. Int. J. Biochem. 13, 585±590. 12. Balmer, S.E., Scott, P.H. & Wharton, B.A. (1989) Diet and faecal ¯ora in the newborn: lactoferrin. Arch.Dis.Child.64, 1678±1684. 13. Petschow, B.W. & Talbott, R.D. (1990) Growth promotion of Bi®dobacterium species by whey and casein fractions from human and bovine milk. J. Clin. Microbiol. 28, 287±292. 14. Raynaud, M. & Bizzini, B. (1971) P uri®cation et proprie  te  sdu facteurbi®dus2.Ann. Nutr. Alimen. 25, 209±223. 15. Gyorgy, P. (1953) A hitherto unrecognized dierence between human milk and cow's milk. Pediatrics 11,98. 16. Blanc, B. (1981) Biochemical aspects of human milk ) comparison with bovine milk. World Rev. Nutr. Diet 36, 1±89. 17. Yoshioka, H., Iseki, K. & Fujita, K. (1983) Development and dierences of intestinal ¯o ra in the neonatal period in breast-fed and bottle-fed infants. Pediatrics 72, 317±321. 18. Poch, M. & Bezkorovainy, A. (1991) Bovine milk j-casein trypsin digest is a growth enhancer for the genus Bi®dobacterium. J. Agric. Food Chem. 39, 73±77. 19. Ibrahim, S.A. & Bezkorovainy, A. (1994) Growth-promoting factors for Bi®dobacterium longum. J. Food Sci. 59, 189±191. 20. Etoh, S., Asamura, K., Obu, A., Sonomoto, K. & Ishizaki, A. (2000) Puri®cation and identi®cation of a growth-stimulating peptide for Bi®dobacterium bi®dum from natu ral rubber ser um powder. Biosci. Biotechnol. Biochem. 64, 2083±2088. 21. Kaetzel, C.S., Blanch, V.J., Hempen, P.M., Philips, K.M., Piskurich, J.F. & Youngman, K.R. (1997) The polymeric immu- noglobulin receptor: structure and synthesis. Biochem. Soc. Trans. 25, 475±480. 22. Wharton, B.A., Balmer, S.E. & Scott, P.H. (1994) Faecal ¯ora in the newborn. Eect of lactofe rrin and related nut rients. In Lac- toferrin: Structure and Function (Hutchens, T.W., Rumball, S .V. & Lo È nnerdal, B., eds), pp. 91±98. Plenum Press, New York, London. Ó FEBS 2002 Human milk peptides as bi®dus factors (Eur. J. Biochem. 269) 717 23. Heine, W. ( 1996) Ein¯uûfaktoren verschiedener Nahrungsfor- men auf die intestinale M ikro¯ora. Monat. Kinderhkd. 144 , 169±173. 24. Sanchez, L., Calvo, M. & Br ock, J.H. (1992) Biological r ole of lactoferrin. Arch.Dis.Child.67 , 657±661. 25. Bellamy, W ., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K. & Tomita, M. (1992) Identi®cation of the bactericidal domain of lactoferrin. Biochim. Biophys. Acta 1121, 130±136. 26. Tomita, M., Takase, M., Wakabayashi, H. & Bellamy, W. (1994) Antimicrobial peptides of lactoferrin. In La ctoferrin: Structure and Function (Hutchens, T.W., Rumball, S.V. & Lo È nnerdal, B., eds), pp. 209±218. Plenum Press, New York, London. 27. Berkel, V.P.H.C., Geerts, M.E.J., Veen, V.H.A., Mericskay, M., Boer, d. H.A. & Nuijens, J.H. (1997) N-terminal stretch Arg 2 , Arg 3 ,Arg 4 and Arg 5 of human lactoferrin is essential for binding to heparin, bacterial l ipopolysac charide, human lysozyme and DNA. Biochem. J. 328, 145±151. 28. Goldman, A.S., Garza, C., Schanler, R.J. & Goldblum, R.M. (1990) Molecular forms of lactoferrin in stool and urine from infants fed human milk. Pediatr. Res. 27, 252±255. 718 C. Liepke et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . controls. RESULTS Identi®cation of human milk peptides that stimulate the growth of B. bi®dum Peptides capable of stimulating the growth of bi®dobacteria were puri®ed. report for the ®rst time the identi®cation of human milk peptides that selectively stimulate the growth of bi®dobacteria. Several bi®dogenic peptides were