Full Paper Bioscience Microflora Vol. 16 (2), 73-77, 1997 Effect of Yogurt Containing Bifidobacterium longum BB536 on the Intestinal Environment, Fecal Characteristics and Defeca- tion Frequenc A Comparisony: with Standard Yogurt Tomoko YAESHIMA,1Sachiko TAKAHASHI,1Nobuko MATSUMOTO,1 Norio ISHIBASHI,1Hirotoshi HAYASAWA1and Hisakazu IINO2 1Nutritional Science Laboratory, Morinaga Milk Industry Co.Ltd., 5-1-83, Higashihara, Zama, Kanagawa 228, Japan, 2Showa Women's University, 1-7, Taishido, Setagaya-ku, Tokyo 154, Japan Received May 26, 1997; Accepted for publication, August 25, 1997 Yogurt containing Bifidobacterium longum BB536 (designated as Bifidus yogurt) was administered to adult volunteers and its effects on the intestinal environment with reference to fecal microflora, ammonia levels , fecal characteristics (color, consistency) and defecation frequency were examined. Bifidus yogurt was manufactured by fermenting milk with B. longum BB536, Streptococcus thermophilus STH-450 and Lactobacillus delbrueckii subsp . bulgaricus LBU-108. Standard yogurt manufactured using only S. thermophilus STH-450 and L. delbrueckii subsp. bulgaricus LBU-108 was used as the control diet. Eleven women volunteers were assigned as subjects to test the effects of Bifidus yogurt on the intestinal environment. Thirty-nine women volunteers were assigned as subjects to test the effects on fecal character- istics and defecation frequency. The volunteers were each administered 100 g of standard yogurt per day for two weeks. After a two-week interval period, each subject was administered 100 g of Bifidus yogurt per day for the subsequent test period. The period of administration of Bifidus yogurt was 2 weeks for testing effects on the intestinal environment and 3 weeks for testing effects on fecal characteristics and defecation frequency. The administration of Bifidus yogurt was effective to increase the number and relative percentage of fecal bifidobacteria significantly. The fecal ammonia concentration tended to decrease and fecal organic acid content tended to increase. The defecation frequency was significantly increased by Bifidus yogurt. The color of the feces changed to yellow and the consistency changed to soft. The administration of Bifidus yogurt was effective to improve the intestinal environment, fecal characteristics and defecation frequency. Key words: bifidobacteria; yogurt; fecal flora; intestinal environment; defecation frequency Bifidobacteria have been recognized as a dominant yogurt, Bifidobacterium species and lactic cultures are component of the fecal microflora of humans and some used for the fermentation process, because Bifido- kinds of animals (12). It is said that bifidobacteria bacterium displays only limited growth in milk (23). contribute to the health of the hosts by suppressing B. longum is the most common species of bifidobac- unfavorable bacteria and by stimulating host immune teria used for yogurt because it shows comparatively functions (1, 8, 11). Due to the ecological and physi- better growth in milk than other species of this genus. ological importance of bifidobacteria, the application B. longum BB536 (BB536) was initially isolated from of viable cells of these bacteria in pharmaceutical prod- a healthy female baby in 1968 (24). It has been used in ucts and foods has been studied (7). Studies on food many dairy products including yogurt. Also, many re- application have focused mainly on dairy products, es- ports concerning the physiological effects of this strain pecially yogurt. The first work concerning the applica- have been published (3, 4, 15, 19, 21, 22). tion to yogurt was reported by Mayer (10). Twenty years In this report, the authors describe the process em- later, Schuler (16-18) reported the application of ployed in manufacturing yogurt containing BB536. This bifidobacteria to many dairy products. In Japan, the first yogurt was administered to healthy adult volunteers to fermented milk products containing bifidobacteria were test its effects on the intestinal environment (including introduced in 1971 (7). Among the many foods now the fecal microflora), fecal characteristics and defeca- available containing bifidobacteria, yogurt (or tion frequency. In order to evaluate the effects of fermented milk) is the most common product, and com- BB536, standard yogurt was administered as the con- pared to other such foods, probably has the largest vol- trol diet. This study was performed under the guide- ume sales in the world. For the manufacture of this lines of the Helsinki Declaration. 73 74 T. YAESHIMA,et al. feces. Samples for the analysis of ammonia concentra- MATERIALS AND METHODS tion and organic acid content were frozen in 2% per- Yogurt. For the manufacture of Bifidus yogurt, three chloric acid and analyzed after thawing. Ammonia strains were used as starters for the fermentation of milk. concentration was measured using an ammonia test kit To prepare the starter cultures, BB536 was cultured in (Wako Pure Chemicals, Tokyo). Organic acids were skim milk containing yeast extract, whereas Strepto- measured by HPLC. The analytical results obtained in coccus thermophilus STH-450 and Lactobacillus each of the experimental periods were averaged and delbrueckii subsp. bulgaricus LBU-108 were cultured the means were compared. in skim milk. Sterilized milk was inoculated with each Fecal characteristics and defecation frequency. The of the three starters, poured into cartons and incubated. volunteers recorded their defecation frequency and The number of viable cells of BB536 was more than assessed the fecal characteristics throughout the exami- 2 •~ 107/ml after two weeks of shelf storage at below nation period. The fecal characteristics (color and con- 10•Ž. Standard yogurt used as the control diet was sistency) were determined and scored by comparison manufactured by fermenting milk with starter cultures with a standard consistency sheet and a standard color of S. thermophilus STH-450 and L. delbrueckii subsp. sheet. The consistency was scored between 1 (Hard) bulgaricus LBU-108. The texture, acidity and taste of and 6 (Soft like watery) at 0.5 unit intervals. The color the two yogurt preparations were almost the same. was scored between 7 (Yellow) and 12 (Dark brown) Experimental schedule. Eleven women volunteers at 0.5 unit intervals. were assigned as subjects for testing the effects of Statistical analysis. Statistical significance was as- Bifidus yogurt on the intestinal environment, which was sessed by Student's t-test with p < 0.05 the determin- examined by fecal analysis. Thirty-nine women volun- ing level. teers were assigned as subjects for testing the effects of RESULTS Bifidus yogurt on fecal characteristics and defecation frequency. The volunteers were each administered 100 The changes in fecal microflora are shown in Table g of standard yogurt per day for two weeks. After a 1. The changes in the relative percentages of dominant two-week interval period, each subject was adminis- bacterial groups in the fecal microflora are shown in tered 1.00 g of Bifidus yogurt per day for the subse- Table 2. The number of Bifidobacterium in the control quent test period. The period of administration of period was 9.7 •} 0.3 in log number. It was significantly Bifidus yogurt was 2 weeks for testing effects on the increased in the test period (p < 0.05). The relative per- intestinal environment and 3 weeks for testing effects centage of Bifidobacterium present in the test period on the fecal characteristics and defecation frequency. was 34.4 •} 19.2%. This value was significantly higher The volunteers were instructed to control their food than the values observed in other periods (p < 0.01). intake throughout the experimental period. That is, they Examination of the data of individual subjects indicated were to avoid eating probiotic products, fermented milk, that the number and relative percentage of Bifidobac- milk, bifidogenic oligosaccharide products and natto terium were highest in the test period during which (soy bean fermented with Bacillus subtilus). Bifidus yogurt was consumed in subjects 7 and 9 among Fecal analysis. Whole freshly voided feces were the 11 volunteers, respectively (data not shown). The collected from the volunteers at the end of each week number and relative percentage of bcteroidaceae were of the experimental period. Samples were kept under higher in the interval period than other periods. anaerobic conditions and refrigerated. Analysis of the Fecal ammonia concentration, pH, water content, and fecal microflora, pH and water contents were performed organic acids are shown in Table 3. The ammonia con- within 24 hours after excretion. The method employed centration tended to decrease in the test period, whereas for bacteriological analysis of the fecal microflora was the values in the control and interval periods were al- basically identical to that reported by Mitsuoka et al. most the same. The fecal pH and water content did not (13). For the enumeration of Clostridium, CC-medium show any notable changes. The levels of total organic (5) was used. For the enumeration of Bifidobacterium, acids and volatile fatty acids were higher in the control BS and RCB media were used. RCB medium was pre- and test periods. pared by modifying reinforced clostridial agar (RCA: The effects of Bifidus yogurt on fecal characteristics BBL) to include 0.5 mg/l metronidazole, 0.5 mg/l are shown in Table 4, and the effect on defecation fre- cephalothin and 50 ml/l horse blood. Fecal pH was di- quency is