Lactulose and Intestinal Microflora in Infant Nutrition

Transcription

1 Lactulose and Intestinal Microflora in Infant Nutrition Bifidobacteria Microflora Vol. 5(1), 27-35, 1986 Isao KIYOSAWA,1* Mitsunori TAKASE, Koji YAMAUCHI, Joji ONO, Tomoko YAESHIMA and Shigeo OKONOGI Central Research Laboratory, Morinaga Milk Industry Co., Ltd., Meguro, Meguro-ku, Tokyo 153 (Received 4 September, 1985) In breast-fed newborn infants, a stable microflora having more than 90% Bifidobacterium is usually developed in the colon and the feces within 5 days after birth. As a result, ph value lowers and putrefactive bacteria decrease in the feces. In bottle feeding with the milk containing a certain quantity of lactulose, it is observed that ph value, the population of Bifidobacterium, and lysozyme activity in the feces approach the levels of breast feeding. The ratio of Bifidobacterium to the total anaerobic bacteria, lysozyme activity, and concentration of the organic acids were increased while ph was lowered in the feces of infants who were fed the follow-up formula food containing lactulose. Composition of the organic acids in the feces varied depending upon the age. The values of molar ratio of acetic acid to lactic acid in feces of suckling infants were 2.7 to 5.3 and they were elevated by 5- to 7-fold in those feces of weaning infants, while lactic acid was not detected in the feces of the adult subjects. In the in vitro experiments, all bifidobacterial species of the present study assimilated lactulose. The activity for lactulose assimilation was, on the contrary, deficient in Clostridium dificile. When C. perfringens or E. coli was cultured simultaneously with the Bifidobacterium in the PYF culture medium containing lactulose, the former two microbials were suppressed remarkably. Infant rats were inoculated with C. perfringens and fed the milk containing lactulose. The occurrence number of the rats in which C. perfringens was detected in the colon also tends to decline during the feeding. Key words : Lactulose; intestinal microflora; Bifidobacterium; infant nutrition Lactulose was first prepared by Montgomery and Hudson (13) making use of the Lobry de Bruyn & van Ekenstein rearrangement of aldoses to ketoses in alkaline solution. After that time, several methods of preparing lactulose by the chemical conversion of lactose in alkaline medium were published. Petuely (15) reported that lactulose was effective in the formation of predominant microflora of Bifidobacterium in the intestinal tract of bottle-fed infants. At that time, there were some problems in the bottle feeding resulting from the composition of infant formula. For instance, the ph value, colour and odor of feces were quite different between breast and bottle feedings. The 1 Present address: Faculty of Agriculture, Tamagawa University, Machida, Tokyo 194. differences were thought to be caused by the conditions of intestinal microflora, which was influenced by the constituents of milk. For the purpose of improving the intestinal microflora of bottle-fed infants lactulose was originally prepared from lactose and added to the infant formula in our company in Since that time, the uses of lactulose have been developed in other foods as the growth factor for Bifidobacterium and in medicine for the treatment of chronic constipation and portal systemic encephalopathy. In this review, the effect of lactulose on the fecal properties, including the intestinal microflora, is discussed along with clinical results of bottle-fed infants. 27

2 28 I. KIYOSAWA et al. 1. Effect of Lactulose on the Intestinal Microflora of Low and Normal Birth Weight Newborn Infants After the report by Petuely, the effects of lactulose on the bottle-feeding of infants were reported by several workers (2, 5, 7, 9). Imura et al. also reported the clinical results on the bottle-feeding low birth weight infant with the formula containing 65 mg of lactulose in 100 ml of the solution compared with the no lactulose formula. The infants were fed with the common commercial formula until attaining the body weight of 2,000 g and then changed to feed with the different formulas for seven weeks (Table 1). The proportions of Bifidobacterium counts to total anaerobic bacterial counts in the feces of infants at different periods are Table 1. Composition of the formulas (S. Imura, S. Mochizuki and M. Takada, 1981 (9)). (S. Imura, S. Mochizuki & M. Takada, 1981) Fig. 1. Percentage of Bifidobacterium to total anaerobic bacteria in the feces of low birth weight infant at different periods of feeding. Table 2. Fecal ph-value, lysozyme activity and organic acids content in low birth weight infants (S. Imura, S. Mochizuki and M. Takada, 1981 (9)).

3 LACTULOSE AND INTESTINAL MICROFLORA 29 Table 3. Proportion of organic acids in the feces of low birth weight infants (S. Imura, S. Mochizuki and M. Takada, 1981 (9)). Table 4. Occurrence of the infant subjects carrying Bifidobacterium in feces, and ph value and lysozyme activity in the feces of infants of the age of five days after birth (K. Morita, 1975 (14)). shown in Fig. 1. At the seventh week of the feeding, the proportions of Bifidobacterium were 36.1 % in L(±) Formula, 28.5% in L( ) Formula A and 16.2% in L( ) Formula B. Organic acids content also increased in the feces of L(+) Formula-fed infants and the fecal ph values were lowest in L( +) Formula among the three formulas. Breast feeding was reported to be characterized by the increase of fecal lysozyme activity (2). The reason is that human milk contains about 4,000 times higher lysozyme activity than cow's milk and intrinsic lysozyme activity is highly accelerated by breast feeding. The formula based on cow's milk usually does not contain lysozyme. Therefore, the lysozyme activity in the feces of infants fed with the formula is significantly lower than that of breast-fed infants. However, the addition of lactulose with lactose to the formula is indicated to increase the fecal activity of lysozyme. In this case, the activity also tends to increase by feeding with the formula containing lactulose (Table 2). The proportions of organic acids in the feces among the feeding of different formulas are shown in Table 3. According to the results, more than 80% of the organic acids are occupied by lactic, acetic and succinic acids. In L(+) Formula, the proportions of lactic and acetic acids to total organic acids are higher, and succinic acid lower than in other formulas. In normal newborn infants fed with these formulas, the ph values are lower and the lysozyme activity is markedly higher than those of low birth weight infants on the average as shown in Table 4 (14). The differences of the ph value and lysozyme activity between L(+) and L(-) Formulas become more distinct compared with the results obtained by the low birth weight infant. It is thought that such differences observed in the fecal properties among the different formulas are caused by the different profile of intestinal microflora.

4 30 I. KIYOSAWA et al. 2. Appearance of Bifidobacterium in the Feces of the Newborn Infant In bottle-fed infants, intestinal microflora are influenced by quality of the milk, especially in neonates. On breast feeding, colonization with Bacteroides and Clostridium, but not Bifidobacterium quickly takes place in the feces during the first days. However, a stable microflora consisting mostly of Bifidobacterium is developed within five days after birth. In contrast, the fecal microflora of bottle-fed infants is variable and Bifidobacterium increases slowly compared with that of the breast-fed infant. The relationship between population of Bifidobacterium and ph value in 105 fecal samples of newborn infants with mix feeding (at fifth day after birth) is shown in Fig. 2. As shown in Fig. 2, Bifidobacterium was not detected in only 4 out of 43 fecal samples of ph lower than 5.5. However the occurrence, of which Bifidobacterium was undetected in the feces, was 23 out of 62 fecal samples of the fecal ph values higher than 5.5. The occurrence of the fecal samples from the infants of the age of 15 days to one month after birth, which displayed the presence of Bifidobacterium, was shown in Table 4. Bifidobacterium was detected in 87.5% of the fecal samples from the breast-fed infants but the occurrence of Bifidobacterium were Fig. 2. Correlation between percentage of Bifidobacterium to total anaerobic bacteria and ph value in the feces of infants at 5 days after birth. 66.7%, 57.1 % and 26.7% in the feces from infants fed L(+) Formula, L(-) Formula A and B respectively. These results suggest that the occurrence of Bifidobacterium is distinctly stimulated in the feces of the lower ph values. The frequency of the occurrence of Bifidobacterium is also higher in the feces of the lower ph values. 3. Effect of Lactulose on the Microflora of the Weaning Infant At the period of weaning, additional foods other than breast and artificial milk are introduced to the infant, and intestinal microflora is influenced by the pattern of weaning (6). Hayasawa et al. observed the changes of microflora of the infant from 9 to 12 months of age fed the follow-up formulas and solid foods. The formulas were alternately given to the infants for 3 months according to the Recommended Program of Weaning in Japan (8). Composition of the formulas are shown in Table 5. L(+) Formula A and B contain 65 mg of lactulose in 100 ml, but L(-) Formula does not contain lactulose. The composition of L( ±) Formula A is similar to that of the infant formula for the young infant. L(+) Formula B and L(-) Formula are prepared for older infants i.e. more than nine months of age as a follow-up formula. The counts of Bifidobacterium are almost the same among the formulas, but the proportion of Bifidobacterium counts to total anaerobic bacterial counts is higher and the ph value lower in L(+) Formulas than in L(-) Formula (Tables 6, 7). Organic acids composition is characterized by the decrease of lactic and succinic acids at the weaning period as compared with the period of breast or bottle feeding as shown in Table 7. Total organic acids content is also lower in L(-) Formula than in L(+) Formula. It is evident that lactulose stimulates the production of organic acids by intestinal microflora resulting in the decrease of ph value in the intestinal tract.

5 LACTULOSE AND INTESTINAL MICROFLORA 31 Table 5. Composition of the formulas (H. Hayasawa, M. Takase, K. Kawase, I. Kiyosawa and S. Okonogi, 1982 (6)). Table 6. Intestinal microflora distribution in the feces of weaning infants at 9 to 12 months old Table 7. Fecal ph-value, lysozyme activity and organic acids content in the feces of infants at 9 to 12 months old (H. Hayasawa, M. Takase, K. Kawase, I. Kiyosawa and S. Okonogi, 1982 (6)). 4. Relationship between Bifidobacterium Count and Organic Acids Content Bifidobacterium is known to produce 1.5 moles acetic acid and 1 mole lactic acid by the utilization of 1 mole glucose (3, 4, 16). These organic acids having the bactericidal activity seem to play an important role in the protection of the host from enteric infection. In Table 8, the molar ratios of acetic/ lactic acid in the feces of infants fed only the milk ranged from 2.7 to 5.3 with the exception of However, at the weaning period during which various solid foods other than milk are introduced. to the infant's diet, the ratio increases about 5 to 7 times. In the adults, there is little or no lactic acid in the feces. Large intakes of foods other than milk appear to reduce lactic acid content and then increase the molar ratio of acetic/lactic acid in the feces. It is

6 32 I. KIYOSAWA et al. Table 8. Bifidobacterium count and organic acids content in the feces of infants and adults Fig. 3. Growth of Bifidobacterium bifidum in PYFlactulose of PYF-glucose medium. postulated that the change in the profile of organic acids in the feces, depending upon the age, is caused from the transition in the intestinal microflora. 5. Suppression of E. coil and C. perfringens in the Culture Medium Containing Lactulose Concerning the different population of intestinal microflora between breast and bottle-fed infant, Benno et al. (1) reported that the numbers of some strains of Clostridium such as C. perfringens and C. difficile are significantly higher in bottle-fed infants than in breast-fed infants. Thus it is required to reduce the cell count of these putrefactive microorganisms in feces of the bottle-fed infant to the level in those of the breast-fed infant. In order to observe the effect of lactulose on the suppression of C. perfringens and Escherichia coli, at first, the growth curve of B. bifidum in the PYF medium with or without lactulose is illustrated in Fig. 3. The growth of B. bifidum at mid-lag phase is delayed by 4.1 hr with lactulose in the medium as compared with the case of glucose medium. The growth delays of other microorganisms are also shown in Table 9. Furthermore, according to the expression of relative growth (R. G.) by Mitsuoka and Hara (12), relative growth (R. G.) of some microorganisms is obtained in Table 9. The growth delay of B. breve is almost similar to that of E. coli and slightly different from that of C. perfringens. However, the growth delay and R.G. of B. longum is quite different from these microorganisms. When E. coli or C. perfringens was cultivated simultaneously with B. breve in the PYF culture medium, the numbers of both microorganisms declined remarkably after 48 hr. However, with B. longum, the declines of both microorganisms were smaller than with

7 LACTULOSE AND INTESTINAL MICROFLORA 33 Table 9. Assimilation of lactulose by intestinal bacteria in vitro Table 10. Suppression of E. coli and C. perfringens by Bifidobacterium in the culture medium containing lactulose B. breve (Table 10). In any case, the Bifidobacterium appears to suppress the growth of E. coli and C. perfringens in the culture medium containing lactulose. Furthermore, the effects of lactulose on the microflora of infant rats were observed. Gastric cannulas were inserted into the stomach of newborn rats; milk was continuously infused by means of a pump with syringes connected to the cannulas according to the method of Messer et al. (10). The control group was fed the milk containing 5.3% protein, 12% fat, 9.1 carbohydrates and a quantity of minerals and vitamins. The experimental group was fed the same milk but containing 0.5% of lactulose in addition. In both the experimental and control groups, infant rats at the fourth day after birth were inoculated with about 107 cells of C. perfringens or equal counts of C. perfringens, Enterobacter cloacae and Lactobacillus salivarius isolated from the caecum of mother rats. The portions of caecum and large intestine were removed and homogenized with sterile precautions at seventh day after birth to analyze the intestinal flora. In the experimental groups inoculated with C. perfringens, this microorganism was not detected in 3 out of 8 infant rats infused with the milk containing lactulose, but was detected in all the infant rats infused with the lactulose-free milk for the control experiment (Table 11). In the mixed inoculation of the above 3 bacterial species, C. perfringens was also undetected in 3 out of 5 infant rats infused with the milk containing lactulose while it was undetected in only one out of 6 subjects when they were infused with the lactulosefree milk for the control experiment. These results suggest that lactulose plays an important role in the suppression of C. perfringens in the intestinal tracts of infant rats.

8 34 I. KIYOSAWA et al.

9 LACTULOSE AND INTESTINAL MICROFLORA Conclusions In breast-fed newborn infants, a stable microflora having more than 90 % Bifidobacterium is usually developed in the colon and the feces within 5 days after birth. As a result, ph value lowers and putrefactive bacteria is reduced in the feces. In the bottle feeding with the milk containing a certain quantity of lactulose, the levels of ph value, the population of Bifidobacterium and lysozyme activity in the feces are almost equivalent to those levels of breast feeding. Furthermore, when C. perfringens or E. coli is coexisted with the Bifidobacterium for culturing in the PYF culture medium containing lactulose, the former two microbials were suppresed remarkably. After the infant rats received the intestinal inoculation of C. perfringens, the occurrence of the microbials was lowered in their intestinal tract by feeding the milk containing lactulose, in contrast to the case of feeding the lactulose-free milk. References (1) Benno, Y., K. Sawada, and T. Mitsuoka Effects of feeding on the fecal flora of infants. Proc. III. IPCR Symp. Intestinal Flora (Tokyo), p (in Japanese). (2) Braun, O.H Faekale Lysozymausscheidung bei Sauglingen und Darmflora. Ernahrungsforsch 10: (3) De Vries, W., and A.H. Stouthamer Pathway of glucose fermentation in relation to the taxonomy of bifidobacteria. J. Bacteriol. 93: (4) De Vries, W., Sj. J. Gerbrandy, and A.H. Stouthamer Carbohydrate metabolism in Bifidobacterium bifidum. Biochem. Biophys. Acta 136: (5) Grutte, F.K., and H. Haenel Laktose nd Laktulose in der Sauglingsernahrung. u Ernahrungsforsch 13: (6) Hayasawa, H., M. Takase, K. Kawase, I. Kiyosawa, and S. Okonogi Effects of lactulose added formula on the intestinal flora of infants from 9 to 12 months of age. Proc. III. IPCR Symp. Intestinal Flora (Tokyo), p (in Japanese). (7) Hoffmann, K., and J. Bircher Veranderungen der bacteriellen Darmbesiedlung nach Laktulosegaben. Schweiz. Med. Wschr. 99: (8) Imamura, E Recommended Program of Weaning. Ishiyaku Pub. Co. Ltd., Tokyo (in Japanese). (9) Imura, S., S. Mochizuki, and T. Takada The fecal properties and the intestinal flora of low birth weight infants fed infant formula. Jap. J. Pediatr. 34: (in Japanese). (10) Messer, M., E.B. Thoman, A.G. Terrasa, and P.R. Dallman Artificial feeding of infant rats by continuous gastric infusion. J. Nutr. 98: (11) Mitsuhashi, S., M. Yoshihama, M. Yahiro, I. Nishikawa, E. Deya, K. Ahiko, and T. Mitsuoka Effects of oligosaccharides on intestinal bacterial flora and fecal characteristics. Proc. III. IPCR Symp. Intestinal Flora (Tokyo), p (in Japanese). (12) Mitsuoka, T., and T. Hara Neosugar and intestinal microflora. Reports of Neosugar Research Group, p (in Japanese). (13) Montgomery, E.M., and C.S. Hudson Relations between rotatory power and structure in the sugar group XXVII. Synthesis of a new disaccharide ketose (lactulose) from lactose. J. Am. Chem. Soc. 52: (14) Morita, K Fecal properties and microflora of newborn infant fed the different formulas. Jap. J. Pediatr. 28: (in Japanese). (15) Petuely, F Bifidusflora bei Fraschenkindern durch Bifidogene Substanzen (Bifidusfactor). Z. Kinderheilkd. 79: (16) Scardovi, V The fructose-6-phosphate shunt as peculiar pattern of hexose degradation in the genus Bifidobacterium. Ann. Microbiologia 15: ) Shimoyama, ( T., M. Satomi, T. Ono, T. Narabayashi, T. Nishigami, S. Nishimura, N. Tanida, S. Hori, M. Sujishi, T. Ikemura, T. Kuroiwa, and H. Nishiyama Morbid physiology of colon diseases. Clinic All-round 26: (in Japanese). 18) Tanaka, R., ( T. Yamashita, A. Iwabuchi, K. Aso, H. Arai, T. Sudo, and R. Higashi Studies on the intestinal microflora of healthy human being. (2) Fecal microflora and organic acids of infant. Ann. Rep. Yakult Cent. Inst. Microbiol. Res. 1: ) Ushijima, M., S. ( Tsuruta, T. Kawashima, R. Kato, C. Ida, S. Okonogi, and H. Kobayashi Profile of microflora, organic acids and ammonia in feces of rheumatic patients. Abstracts Book of the Fourth Conference of the Japan Bifidus Foundation Japanese). (Tokyo), p. 10.(in