Full Paper Bifidobacteria Microflora Vol. 10 (2), 123-130, 1991 Effect of and Yogurt Supplemented with Bifidobacteriumand/or Lactulose in Healthy Persons : A Comparative Study

Tsunesuke TOMODA,1 Yasuo NAKANO1 and Takashi KAGEYAMA2

Department of Clinical Pathology, Osaka Medical College,1 Takatsuki, Osaka 569 and 2SecondDepartment of Internal Medicine, Osaka Medical College, Takatsuki, Osaka 569

(Received for publication, October 8, 1990)

Abstract Three types of yogurt supplemented with Bifidobacterium alone, lactulose alone, or both Bifidobacteriumand lactulose were fed to healthy persons, who were tested before and after administration. Plain yogurt was fed during a control period. Yogurt containing both Bifidobacteriumand lactulose resulted in greater improvement in the form and frequency of feces than the other . No differences were noted in blood chemistry test results before and after the admin- istration of each type of yogurt. There were no side effects of yogurt administration. The number of Bifidobacteriumin the feces increased after the administration of yogurt containing Bifidobacteriumand/or lactulose, but there were no differences among the three test yogurts. Not only the administered Bifidobacteriumspecies but also the original Bifidobacteriumspecies was increased by eating Bifidobacterium and lactulose-containing yogurt, but the administered Bifidobacteriumspecies was not increased more than the dominant original species. The ammonia content in the feces decreased more after the administration of Bifidobacterium-and/or lactulose-containing yogurt than after that of plain yogurt but there were no differences among the three test yogurts.

Key words: yogurt; Bifidobacterium; lactulose; feces; ammonia; healthy persons

Many reports have been published on yogurt as a promotor of health (3). The effect of the administration of supplements containing Bifidobacteriumand lactulose (one of the growth factors of Bifidobacterium)has also been reported in infants, aged persons and some patients (3, 12). When yogurt is administered to patients, many factors such as medicines or special diets are involved, so the true effect of Bifidobacteriumcannot be observed. In this study, plain yogurt or yogurt containing Bifidobacteriumand/or lactulose was fed to healthy adults, and some answers to questionnaires, blood chemistry results, number and species of Bifidobacteriumin

123 124 T. TOMODA et al the feces and the ammonia content of the feces were examined before and after the administration of each preparation of yogurt.

MATERIALS AND METHODS

The healthy subjects tested in this study were 5 men and 5 women aged 20 to 60 years. Four kinds of yogurt prepared by Morinaga Industry Co., Ltd. were used in this study: A) plain yogurt, B) lactulose-supplemented yogurt, C) Bifidobacterium-supplemented yogurt, and D) both Bifidobacterium- and lactulose- supplemented yogurt. B) contained 0.5 g lactulose/100 g yogurt, C) contained at least 108 /g, and D) contained both. Plain and supple- mented yogurts also contained Streptococcus salivarius subsp. thermophilus and Lacto- bacillus delbrueckii subsp. bulgaricus for fermentation. Each yogurt was given in amounts of 130 g/day for 3-6 weeks, first A, then B, then C, then D, at intervals of 3 months. Subjective findings, as determined by direct questioning, are listed in Table 1. Serum protein and lipid levels and liver function were tested. Fecal samples were prepared by serially 10-fold step dilution methods (2). The diluted samples were spread onto Mitsuoka's BS medium (8) and incubated at 37°C 3 days anaerobically. After incubation, number and species of Bifidobacterium were confirmed from maxi- mum dilution sample. Identification of species was done by Bergey's classification

(14), with tests of fermentation and Gram's staining. The ammonia content was determined by the Amicheck system (Chugai Pharmaceutical Co., Ltd.) for dry chemicals. Diluted fecal samples were applied to test paper. The ammonia in the feces reacted with a pH indicator in the paper to produce a color which was then measured photometrically. The ammonia level was recorded as

g/g feces. ƒÊ Statistical significance was assessed by Student's t-test.

RESULTS

The answers to questions about subjective evaluation of changes in stools, appetite, intestinal conditions (meteorism or borborygmus) and body weight are shown in Table 1. The quality of the feces became better in those given yogurt containing both lactulose and Bifidobacterium. There was also less meteorism or borborygmus when the subjects ate supplemented yogurt than when they ate plain yogurt. Appetite and body weight were not changed significantly in any of the four groups. No side effects were noted. The blood chemistry findings (protein, lipid and liver function) before and after the administration of each type of yogurt are shown in Table 2. No significant differences were noted. The number of Bifidobacteriain the feces in each group is shown in Table 3. It increased significantly after the administration of yogurt containing Bifidobacterium and/or lactulose, but there was no significant difference among the three groups. YOGURT SUPPLEMENTED WITH BIFIDOBACTERIUM AND/OR LACTULOSE 125

Table 1. Effect of yogurt preparations on subjective findings and body weight in 10 healthy persons

However, the number of Bifidobacteriareturned to the pre-treatment level by the end of the 3-month interval between tests. Fermentation (Streptococcus salivarius subsp. thermophilusand delbrueckiisubsp. bulgaricus) were not found in any of the samples of feces tested after the administration of each type of yogurt. The number and species of Bifidobacteriain the feces of 6 healthy persons given yogurt containing lactulose and Bifidobacteriumlongum are shown in Table 4. Five (Cases 1, 3, 4, 5, 6) had dominantly B. adolescentisbefore treatment, but the admin- istration of Bifidobacteriumlongum resulted not only in large amounts of Bifidobacterium longum in the feces, but also increased the numbers of the original Bifidobacterium species (B. adolescentis). In Case 2, the increase in the amount of administered Bifidobacteria(B. longum)was not clear, because Bifidobacteriumlongum was the domi- nant original Bifidobacteriumspecies. But in all tested feces with dominant original B. adolescentis,their number was higher or equal to that of B. longum. In 10-fold step dilution method, classification of Bifidobacteriumspecies in the preceding tube below one order at maximum diluted sample was difficult for numerous Bifido- bacterium. The given Bifidobacteriumand original Bifidobacteriumcould not be clearly distinguished in this study. The fecal ammonia content in each yogurt group is shown in Table 5. It decreased significantly in each yogurt-plus-supplement group, but there was no significant difference among the three groups. In the plain yogurt group the ammonia content of the feces did not change. 126 T. TOMODA et al

Table 2. Serum lipids and protein and liver function before

NS: no significant difference.

Table 4. Number and species of Bifidobacteria in feces before and after administration of yogurt containing lactulose+Bifidobacterium in six healthy persons

Number is bacterial counts/g feces. YOGURT SUPPLEMENTED WITH BIFIDOBACTERIUM AND/OR LACTULOSE 127 and after administration of each type of yogurt

DISCUSSION

Many reports have described the nutritional value of yogurt or fermented milk (3, 5, 6, 13), especially in infants, aged persons and various patients. In patients, the true merits of yogurt may be lessened by medications to cure the disease, so the good effect of yogurt may not be manifested. There have been few studies of the effect of yogurt in healthy persons. In this study, we observed the results in healthy persons eating 4 preparations of yogurt: one plain, one with Bifidobacterium,one with lactulose, and one with both. The supplemented yogurt used in this study was more effective than plain yogurt in improving several subjective findings. Yogurt containing both Bifido- bacteriumand lactulose gave the best results, especially improved form and frequency of feces. Serum lipid and protein, and liver function tests were unchanged by the yogurt preparations. There were no adverse side effects. The effect of Bifidobacterium administration on hypercholesteremia has been reported (1, 4, 16) . In this study, however, the ingestion of yogurt containing Bifidobacteriumand/or lactulose had no effect on serum cholesterol in healthy persons. The fact that yogurt administration caused no changes in blood chemistry in healthy persons shows its probable safety for patients with many diseases. 128 T. TOMODA et al YOGURT SUPPLEMENTED WITH BIFIDOBACTERIUM AND/OR LACTULOSE 129

The number of Bifidobacteriain the feces was increased by the administration of yogurt containing Bifidobacteriumand/or lactulose. Not only the given species of Bifidobacterium,but also the dominant original species of Bifidobacteriumwas increased in the feces. It is thought that the pH of the intestinal tract or other conditions enhance the growth of all Bifidobacteria species when Bifidobacteriumis ingested. However, the increase of given Bifidobacterium(B. longum) did not exceed that of original Bifidobacterium(B. adolescentis). The adherence of original Bifidobacteriamay be more permanent than that of given Bifidobacteria. The increase of Bifidobacteria caused by supplemented yogurt was reduced during the 3-month interval between administrations. The remarkable increases of the number of Bifidobacteriumafter some kinds of yogurt administration were proved in some persons. Reexamination would have to be studied to trace the causes. The increase of number of Bifido- bacterium cannot be confirmed to depend on any speices when original and given Bifidobacteriumwere the same species. A particular method for distinction of original and given Bifidobacteriummust be also developed. Other fermentation bacteria were not found in the feces, so the effects of these bacteria were excluded in this study. All three yogurt preparations reduced ammonia equally in the feces of healthy persons. Ammonia production in the intestinal tract is derived from protein or amino acid decomposition by E. coli and other gram-negative bacilli. However, Bifidobacteriaproduce almost no ammonia (15), so it seems that a predominance of Bifidobacteriadecreases the ammonia concentration in the intestinal tract by inhibiting the growth of ammonia-producing bacteria. Many actions of Bifidobacteriumhave been reported (7, 9), as well as those of lactulose by Petuely (10, 11). Yogurt containing lactulose also increased the num- ber of Bifidobacteriain the intestinal tract. Therefore, yogurt supplemented with Bifidobacteriumand/or lactulose seems to be more effective than plain yogurt. There were no differences among the three types of yogurt tested in this study in regard to increased numbers of Bifidobacteriaand reduced amounts of ammonia in the feces. The development of many preparations of yogurt can be expected to improve intestinal function in normal persons.

REFERENCES

(1) Gilliland, S.E., C.R. Nelson, and C. Maxwell. 1985. Assimilation of cholesterol by Lactobacillus acidophilus. Appl. Environ. Microbiol. 49: 377-381.

(2) Gorbach, S.L., L. Nahas, P.I. Lerner, and L. Weinstein. 1967. Studies on intestinal Micro- flora. I. Effect of diet, age, and periodic sampling on numbers of fecal microorganisms in man. Gastroenterology 53: 845-855.

(3) Hitchins, A.D., and F.E. McDonough. 1989. Prophylactic and therapeutic aspects of fermented milk. Am. J. Clin. Nutr. 49: 675-684.

(4) Homma, N. 1988. Bifidobacteria as a resistance factor in human beings. Bifidobacteria Micro- flora 7: 35-43.

(5) Kolars, J.C., M.D. Levitt, M. Aouji, and D.A. Savaiano. 1984. Yogurt•\an autodigesting source of lactose. N. Engl. J. Med. 310: 1-3.

(6) McDonough, F.E., A.D. Hitchins, N.P. Wong, P. Wells, and C.E. Bodwell. 1987. Mechanism of improved utilization of yogurt and sweet acidophilus milk by lactose intolerant persons. Am. J. Clin. Nutr. 45: 570-574. 130 T. TOMODA et al

(7) Mitsuoka, T., and C. Kaneuchi. 1977. Ecology of the bijidobacteria. Am. J. Clin. Nutr. 30: 1799-1810. (8) Mitsuoka, T. 1980. A color atlas of anaerobic bacteria, Sobunsha, Tokyo (in Japanese). (9) Mutai, M., and R. Tanaka. 1987. Ecology of Bifidobacteriumin the human intestinal flora. Bifidobacteria Microflora 6: 33-41. (10) Petuely, F. 1957. Bifidusflora bei Frashenkindern durch Bifidogene Substanzen (Bifidusfactor). Z. Kinderheilk. 79: 174-179. (11) Petuely, F. 1986. Uber den Bifidusfaktor Lactulose. Bifidobacteria Microflora 5: 3-11. (12) Rasic, J.L., and J.A. Kurman. 1983. Bifidobacteria and their Role, Birkhauser Verlag, Basel, Boston, Stuttgart. (13) Smith, T.M., J.C. Kolars, D.A. Savaiano, and M.D. Levitt. 1985. Absorption of from milk and yogurt. Am. J. Clin. Nutr. 42: 1197-1200. (14) Sneath, P.H.A., N.S. Mair, M.E. Sharpe, and J.G. Holt. 1986. Bergey's manual of systematic bacteriology, Vol. 2. Williams & Willkins, Baltimore. (15) Suzuki, K., and T. Mitsuoka. 1976. Urease activity of intestinal anerobes. Medicine and Biology 93: 359-363 (in Japanese). (16) Thakur, C.P., and A.N. Jha. 1981. Influence of milk, yogurt and calcium on cholesterol-induced atherosclerosis in rabbits. Atherosclerosis 39: 211-215.