Full Paper Bioscience Microflora Vol. 16 (2), 73-77, 1997

Effect of Containing 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 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 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 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 . 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 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 products, fermented milk, that the number and relative percentage of Bifidobac- milk, bifidogenic 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 , 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 shown in Fig 1. The mean defecation fre- rectly measured by inserting a glass electrode in the quency for all volunteers changed notably. It was 0.56 •} EFFECT OF YOGURT CONTAINING BIFIDOBACTERIUM LONGUM BB536 75

Table 1. Influence of Bifidobacterium longum BB536 yogult administration on the fecal microflora.

Volunteers (n = 1.1) consumed 100 g of B. longum BB536 yogult (test period) or standard yogurt (control period)

per day. a Bacterial counts expressed as mean •} SD of log10 per g of wet feces (n = 11) . b Frequency of occurrence .

p < 0.05 (compared with ccontrol period). d p < 0 .05 (compared with interval period). e Not detected .

Table 2. Influence of Bifidobacterium longum BB536 yogult administration on the relative percentages of fecal bacterial groups.

Volunteers (n = 11) consumed 100 g of standard yogurt (control period) or B. longum BB536 yogurt (test period) per day. a Data shown are the mean of two values and SD (n = 11) . b p < 0.05 (compared with control period). p < 0.05 (compared with interval period).

0.18 in the interval period. It increased significantly to between the experimental period was statistically sig- 0.64 •} 0.20 (p < 0.01) and 0.76 •} 0.27 (p < 0.001) af- nificant. The change in the high-frequency group was ter consuming standard yogurt and Bifidus yogurt, re- notable. In this group, the defecation frequency in- spectively. The difference in mean defecation frequency creased after consuming Bifidus yogurt. However, the between the control and test periods was also signifi- increase after consuming standard yogurt was very cant (p < 0.001). For the individual volunteers, the def- small. The consistency of the feces changed to soft and ecation frequency in the interval period ranged from the color changed to yellow after consuming Bifidus 0.25 to 0.92 times per day. The volunteers were grouped yogurt (Table 4). into a low-frequency group (n = 18) having a defeca- DISCUSSION tion frequency lower than 0.5 and a high-frequency group (n = 21) having a defecation frequency higher Yogurt has been well-known for a long period of than 0.5. The change in the low-frequency group was human history as a health food which has many physi- larger than that in the total group, and the difference ological effects (25, 26). These effects are considered 76 T. YAESHIMA,et al.

Table 3. Effect of Bifidobacterium longum BB536 yogurt administration on fecal properties.

Volunteers (n = 11) consumed 100 g of standard yogurt (control period) or B. longum BB536 yogurt

(test period) per day. a Mean •} SD (n = 11) . No statistical differences among periods in eath test item.

Table 4. Influence of Bifidobacterium longum BB536 yogurt administration on fecal characteristics.

Volunteers (n = 39) consumed 100 g of standard yogurt (control period) or B. longum

BB536 yogurt (test period) per day. Data shown are the mean •} SD (n = 39). (1) Fecal consistency was scored by comparison with a standard consistency sheet .

(Hard) 1-2-3-4-5-6 (Watery) (2) Fecal color was scored by comparison with a standard color sheet .

(Yellow) 7-8-9-10-11-12 (Dark brown).

p < 0.05 (compared with interval period).

to be owing to the lactic cultures, metabolites in the yogurt and many other factors which are yet to be iden- tified. Bifidobacterium has long been applied in the pro- duction of yogurt. However, it is difficult to manufac- ture yogurt using only Bifidobacterium because these bacteria require certain growth-promoting nutrients to grow in milk (23). Furthermore, the taste of yogurt pro- duced using only Bifidobacterium is not suitable as a commodity product. The Bifidus yogurt used in this study was produced by fermenting milk with B. longum BB536 and two bacterial species commonly employed in the manufacture of yogurt. There have been many Fig. 1. Effect of Bifidobacterium longum BB536 yogurt ad- reports concerning the physiological effects of yogurt ministration on the defecation frequency. or fermented milk produced by various types of cul- Volunteers consumed 100 g of standard yogurt (control pe- tures (6, 9, 14, 20). However, previous studies have riod) or B. longum BB536 yogurt (test period) per day. shown only the effects of yogurt administration. The (1)Low group: volunteers having low (< 0 .05) frequency in effects of yogurt include the effects of milk compo- the interval period. (2)High group: volunteers having high nents, bacterial cells, bacterial metabolites, and others. (> 0.5) frequency in the interval period. In this study, the authors tried to ascertain the effect of a p < 0.001 (compared with control period), b p < 0.01 (com- BB536 itself on the intestinal environment, fecal char- pared with control period), cp < 0.05 (compared with control period), dp < 0.001 (compared with interval period), cp < 0.01 acteristics and defecation frequency. The effects of (compared with interval period), f p < 0.05 (compared with Bifidus yogurt were compared with those of standard interval period). yogurt produced without BB536. EFFECT OF YOGURT CONTAINING BIFIDOBACTERIUM LONGUM BB536 77

The administration of BB536 was effective to in- (11) Mitsuoka T. 1978. Intestinal Bacteria and Health. Harcourt crease the number and relative percentage of Bifido- Brace Jovanovich Japan, Tokyo. bacterium. The tendency of decrease in ammonia (12) Mitsuoka T. 1984. and ecology of bifidobac- concentration and increase in organic acid content teria. Bifidobacteria Microflora 3: 11-28. after the consumption of Bifidus yogurt is considered (13) Mitsuoka T, Sega T, Yamamoto S. 1965. Eine Vergesserte Methodik der qualitativen und quantitativen Analyse der to be owing to changes in the microflora. Benno and Darmflora vor Menschen und Tieren. Zblt Bakt I Orig 195: Mitsuoka (2) showed change of some bacteria popula- 455-465. tion, decrease of ammonia cotent and low fecal pH af- (14) Morisaki N, Saito Y, Terada A, Hara H, Osabe K, Muraishi ter administering B. longum. Also, Bifidus yogurt was K, Iwana H, Kaneko T, Mitsuoka T. 1993. Effects of yo- effective to increase the defecation frequency in the gurt-administration on fecal flora and putrefactive metabo- volunteers suffering from slight constipation. Standard lites of senile volunteers. Bifidus 6: 161-168. (15) Reddy BS, Rivenson A. 1993. Inhibitory effect of Bifido- yogurt showed some effects as reported in many previ- bacterium longum on colon, mammary and liver carcino- ous papers. However, this study demonstrated that genesis induced by 2-amino-3-methylimidazo[4,5-f] Bifidus yogurt was more effective than the standard quinone, a food mutagen. Cancer Res 53: 3914-3918. yogurt. Our findings clearly show that the administra- (16) Schuler R, Ruppert A, Muller F. 1968. The microorgan- tion of BB536 was effective to improve the intestinal isms of the bifidus group (Lactobacillus bifidus). I. His- environment, fecal characteristics and defecation fre- torical review, nutritional, physiological and therapeutic aspects, morphology, cutural properties and taxonomy. quency. Milchwissenschaft 23: 356-360. REFERENCES (17) Schuler R, Ruppert A, Muller F. 1968. The microorgan- isms of the bifidus group (Lactobacillus bifidus). II. Tech- (1) BallongueJ. 1993.Bifidobacteria and probioticaction. In nology of bifidus culture in the dairy plant. Milchwissen- Bacteria,Salminen S, von Write A (eds), pp. schaft 23: 554-558. 357-428, MarcelDekker Inc, New York. (18) Schuler R, Ruppert A, Muller F. 1968. The microorgan- (2) BennoY, MitsuokaT. 1992.Impact of B. longumon hu- isms of the bifidus group (Lactobacillus bifidus). III. De- man fecal flora.Microbiol Immunol 36: 683-694. tection of L. bifidus in milk products. Milchwissenschaft (3) Challa A, Rao DR. 1997. Bifidobacteriumlongum and 23: 614-618. lactulosesuppress azoxymethane-induced aberrant crypt (19) Seki M, Igarashi M, Fukuda Y, Shimamura S, Kawashima foci in rats. Carcinogenesis18: 517-521. T, Ogasa K. 1978. The effect of Bifidobacterium cultured (4) ColombelJF, CortotA, Neut C, RomondC. 1987.Yogurt milk on the "regularlity" among an aged group. J Jpn Soc with Bifidobacterium longum reduces erythromycin- Nutr Food Sci 34: 379-387. inducedgastrointestinal effects. Lancet 2: 8549. (20) Takiguchi R, Ohe M, Miyamoto M, Toyoda S, Nakajima (5) EndouK, KumemuraM, NakamuraK, FujisawaT, Suzuki I, Benno Y. 1994. Effects of the fermented-milk adminis- K, BennoY, MitsuokaT. 1991. Effectof high cholesterol tration on stool frequency and fecal microflora on consti- diet and polydextrosesupplementation on the microflora, pated female adults. Bifidus 8: 15-20. bacterialenzyme activity, putrefactive products, volatile (21) Tomoda T, Nakano Y, Kageyama T. 1990. Effect of ad- fatty acid (VFA) profile,weight, and pH of the feces in ministration of yogurt containing Bifidobacterium in healthyvolunteers. Bifidobacteria Microflora 10: 53-64. healthy persons. Bifidus 4: 21-24. (6) Hosoda M, He F, Hiramatsu M, Hashimoto H, Benno (22) Ueda K. 1986. Immunity provided colonized enteric bac- Y.1994.Effects of LactobacillusGG strainintake on fecal teria. Bifidobacteria Microflora 5: 67-72. microfloraand defecationin healthyvolunteers. Bifidus 8: (23) Yaeshima T. 1986. Microbiology of Bifidobacterium and 21-28. its application in food products. Bull Jpn Dairy Tech Assoc (7) Ishibashi N, ShimamuraS. 1993.Bifidobacteria research 35: 10-22. and developmentin Japan.Food TechnolJune 126-136. (24) Yamazaki S, Machii S, Tsuyuki S, Kawashima T, Ueda K. (8) KurmannJA, RasicJ. 1991.The healthpotential of prod- 1985. Immunological responses to monoassociated ucts containingbifidobacteria. In TherapeuticProperties Bifidobacterium longum and their relation to prevention of of Fermented Milk, Robinson RK (ed), pp. 117-157, invasion. Immunology 56: 43-50. ElsevierScience Publisher Co. Inc., New York. (25) Yuguchi H, Goto T, Okonogi S. 1992. The nutritional and (9) Link-Amster H, Rochat F, Saudan KY, Mignot O, physiological value of fermented and lactic milk AeschlimannJM. 1994.Modulation of a specifichumoral drinks. In Functions of Fermented Milk, Hosono A (ed), immuneresponse and changesin intestinalflora mediated pp. 217-245, Elsevier Applied Science, New York. through fermented milk intake. FEMS Immunol Med (26) Yuguchi H, Kudo T, Okonogi S. 1992. Chapter 11, Fer- Microbiol10: 55-64. mented milks, lactic drinks and intestinal microflora. In (10) Mayer JB. 1948.Lactic acid-bifidusmilk. Z Kinderheilk Functions of Fermented Milk, Hosono A (ed), pp. 247- 65: 319-345. 273, Elsevier Applied Science, New York.