Bifidobacterium from Fermented Milks: Survival During Gastric Transit NABIL BERRADA Service d‘Hepato-Gastroenteroiogb Hdpital Charles-Nicolle 1 me de Germont, 76031 Rouen Cedex, France JEAN-FRANCOIS LEMELAND and GILLES LAROCHE Labomtoire de Bact6riologie-Parasitobgie Hatel-Dieu 51 rue de Lecat, 76031 Rouen Cedex, France PIERRE THOUVENOT SeMce de MBdecine Nudeaire Centre Hospitalier Universitaire 54500 Vandoewe ies Nancy, France M A R I N E PlAlA B.S.N. 7 rue de Teheran, 75381 Paris Cedex 08, France ABSTRACT

Two Bij-idobacterium strains contained in two different fermented milks behave very differently when exposed to an in vitro simulated gastric environment. One strain survives very well during at least 90 min (> 107/g), but the second strain studied is much less resistant. These in vitro results, with slight differences, were confirmed by an in vivo study in humans. The assessment of the gastric emptying rate of these products allows an estimation of the amount of Bifidobacterium that may pass into the small intestine. (Key words: Bi@dobacterium, fermented milks, gastric transit) Abbreviation key: TPY = trypticase phytone yeast. INTRODUCTION

The recent growth in the development of socalled ‘%ifidus” fermented milks follows several reports (1, 2, 5 , 6, 7) on the health properties of Bifidobacterium. A prerequisite for any effect of ingested bacteria, including the traditional yogurt cultures, Streptococcus ther-

Received March 2, 1990. Accepted July 23, 1990. 1991 J Dairy Sci 74409-413

mophilus and Lactobacillus delbrueckii ssp. bulgaricus, on either the endogenous flora or the whole human organism is that these bacteria remain viable during gastric transit and that approximately I@ bacteria (e.g., 100 g of a fermented milk containing lo7 bacteridg) may pass into the intestine. Kolars et al. (3) and Savaiano et al. (8) reported that S. thermophilus and L. delbrueckii ssp. bulgaricus are resistant to gastric acidity and consequently are alive, numerous, and active in the human intestine although not natural inhabitants of it. Resistance to gastric acidity has not been demonstrated for Bifidobacterium (6). The objectives of this study were 1) to demonstrate the ability of two Bijidobacterium strains to resist gastric transit in vitro by the means of a simulation and then in vivo and 2) to assess the amount of Bifidobacterium that passes into the intestine by taking into account the gastric emptying rate of the tested fermented milks. MATERIALS AND METHODS Bacterial Strains

We used two different brands of commercial bifidus fermented milks, products number 1 and number 2. The origin of the Bifidobacterium strains used by the manufacturers is Unhown to us.

409

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BERRADA ET AL.

Culture Medla and Reagents

first sample was taken using a gastric tube inserted just before ingestion. Other samples were taken at 30, 60, and 90 min. Each 1-ml sample was immediately neutralized, and dilutions and plate counts were carried out as before. Product number 1 was tested 14 and 24 d after manufactwe; we were only able to test product number 2 14 d after manufacture because the level of Bifidobacterium in product number 2 at 24 d was less than 103/g. Fermented milks were kept at 6'C until ingestion. All 10 subjects tested the two products during three different sessions in a randomized order. Individual values at each time point were analyzed for variance, and the mean differences were subjected to a t test.

Trypticase phytone yeast 0 was used as basic medium (9). The final pH value, after autoclaving at 121'C for 25 min, was 6.5. An inhibitory solution was also used to allow specific growth of Bifidobacterium (30 g propionic acid, 6 g lithium chloride, .4 g neomycin B sulfate, and 100 ml water). The inhibitory solution was sterilized by .45-pfiltration. An antioxygen solution was prepared (3 g L-cysteine hydrochloride and 100 ml water) and s t e d k d by .45 p filtration. The culture medium was obtained by mixing 100 ml melted TPY medium, 5 ml inhibitory solutian (122 pg neomycin/ L in final solution) and 1 ml anti-oxygen solution. The diluent used was a cysteinated Ringer's solution (.3 g/LGcysteine hydrochloride). The final pH value after autoclaving at Gastric Emptying Rate 121'C for 25 min was 6.5. The medium and The 12 healthy young subjects, 6 males and diluent were regenerated for 10 min at 1 W C 6 females (whose characteristics are reported in before use. Table 1) had no digestive pathology, no sign of lactose intolerance, and were taking no medicaIn Vitro Survival Study tion; they ingested 250 ml of product number l The fermented milks were purchased in and 250 ml of product number 2 in two sepastores and used about 8 d after manufacture. rate sessions and in randomized order. FerSome of the samples were used to determine mented milks were served in opaque cups at initial viable counts. Remaining samples were ambient temperature. acidified by adding 1.25N HCl with gentle The ingestion of products began at 1900 h. stirring in order to decrease the pH to 3. The The subjects had been fasting since lunch and quantiw of HCl rqujred for 125 g of product had neither smoked nor drunk since 1600 h; number 1 (initial pH = 4.4) was 8.5 ml, it was their physical activity was normal. A solution 7.8 ml for 125 g of product number 2 (initial of rhenium sulfur colloids, nonabsofbable by pH = 4.5). Samples were then kept at 37'C in the digestive tract, labeled by 50 pCi of darkness. Their pH value after 3 h had not Varied.

Samples were taken after 5 and 90 min at 37'C and quickly neutralizd by mixing 1 ml of sample with .5 ml .111N NaOH and 8.5 ml cysteinated Ringer's solution, resulting in a final dilution of .l. Subsequent .1 dilutions were then carried out using cysteinated Ringer's solution; l ml of the last dilution was plated in Petri dishes on TPY medium. Bacterial counts were performed after a 6-d aaaembic (10% C& and 90% Hz) incubation at 3792. Each test was carried out in duplicate.

In Vivo Survival Study After fasting overnight, 10 healthy young adults ingested 250 g each of product number 1 or number 2. Immediately after ingestion, the J o d of Dahy Science Vol. 74, No. 2, 1991

TABLE 1. QlaracteriStics of the subjects in the gastric

emptying study. Number 1 2 3 4 5 6 7

8 9 10 11

12

Age

Weight

Height

(vr)

(kn)

(can)

45 35 41 20 40

80 80 73 64 69 61 61 53 50 50 52 40

180 179 170 171 170 169 169 163 163 163 160 158

22 33 35 43 40

26 24

BIFIDOBACKWUUM SURVIVAL DURING GASTRIC TRANSIT

411

I

(1

TIME (MINUTES)

Figure 2. In vivo study (10 subjects); product number 1, 14 d 0;product number 1,24 d (+); product number 2, 14 d (0).

99m-technetium was added to the fermented milks. The subjects were informed of the use of radioactive isotopes, and all consented to take part in the study. This study was approved by the Committee of Medical Ethics of the University of Nancy, France. The beginning of ingestion was considered as time zero (t = 0). Scintigraphies 1 min in duration were obtained every 10 min over 3 h in left oblic anterior view and in erect position with a gamma camera equipped with a low energy collimator (Grammatome Ik Sopha Medical, Buc, France) and interfaced to a computer (Matek 1502, Informatec, Paris, France). The subjects were permitted to sit or move around at will in between imaging. Scintigraphies were analyzed by defining the region of interest corresponding to the stomach and d e tennine the radioactivity in this area. The percentage of gastric retention was calculated for each 10-min interval as (decay corrected count at time t)/(count at t = 0) x 100. RESULTS

in Vitro Study

The curves obtained (Figure 1) are the mean value of the two tests performed expressed in decimal logarithms. Product number 1 showed a higher initial BzjUobacterium population than product number 2 (6 x 107/g vs. 6 x ld/g). The Bifiabbacteriwn strain in product number 1 was resistant to simulated gastric conditions. More than lo7 Bifidobacteriumlg sur-

vived after 90 min, indicating that the population decreased by less than one logarithm unit. The Bz$abbacterium strain in product number 2 was much less resistant as indicated by the Clogarithm unit decrease in the population in the first 5 min. After 90 min, only 102 Bifidobacteriumlg remained in the sample from product number 2. In Vivo Survival Study

Results ( F i p 2) are the mean values of the results obtained with the 1 0 subjects, expressed as decimal logarithms. Product number 1 contained, as in the in vitro study, a high initial Bifidobacteriwn population (at least 107/g), at 14 d as well as at 24 d after manufacture. Product number 2 contained a lower initial Bifiabbacteriwn population than product number 1 (l@/g), but this population was higher than the one observed in the in vitro study. During gastric transit, the Bifiabbacterium population io product number 1 decreased by less than 2 logarithm units in 90 min. The survival was the same for product number 1 ingested 24 or 14 d after manufacture, as shown by the parallel decrease curves. The 1Od difference in product age did not alter the resistance of the Bifidobacterium strain in product number 1. The Bifidobacterium strain in product number 2 was less resistant to gastric transit. The population decreased by 4 logarithm units in 90 min (the decrease observed in the in vitro study was much greater). Journal of Dairy Science Vol. 74, No. 2, 1991

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BERRADA ET AL.

the paired Student's c test. No significant difference exists between the two products, as shown by Figure 3 (mean of experimental curves). With product number 1, the stomach was half emptied within about 45 min vs. 41 min with product number 2. Over 80% of the stomach content was emptied after 90 min in both cases. DISCUSSION

The in vitro behavior of the Bifidobacterium strain in product number 2 is similar to that described by Rasic and Kurmann (6): the population of this strain decreases from an initial TIME (MINUTES) 106/g to less than l d / g during storage for 24 d Figure 3. Gastric emptyiog rate; product number 1 (0); at 6'C (product pH = 4.5). This strain is also product number 2 (0). sensitive to gastric acidity. The Bijkbbacreriurn strain in product number 1 exhibits a different behavior. The initial There was no statistical difference between population, slightly higher than that of the subjects. Differences between product number product number 2 strain, remains unchanged 1 and product number 2 were significant during storage for 24 d at 6'C (product pH = (Pc.OOO1).For product number 1, there was no 4.4). This strain is not sensitive to gastric acididifference between results obtained at 14 and ty. There is no difference in resistance to gas24 d, except for initial bacterial counts (R.01). tric acidity between Bifidobucreriu in 14- and W - o l d products. The Bz9hbucterium strain in product numGastric Ernptylng Rate ber 2 does not resist acidic pH, as demonstrated Results, expressed as the time necessary for by its inability to survive at pH 3 in vitro. For 10, 50, and 80% of the gastric content to pass this strain, simulated in vitro conditions were into the intestine (Table 2) were analyzed with more severe than real gastric conditions. The

TABLE 2. ~ a s t r i cemptying rates1 for products

1 and number 2.

Number 1 Subject number 1 2 3 4

5 6 7 8 9 10 11 12

10%2

50%

80%

10%

50%

80%

15 14 7 12 1 18 10 6

51 29 26 55 18 36 35 56 52

107 53 55 88 45 49 74 94 73 63 81

13 7 23 7

61 39 43

97 62 58 51 59 87 65 116 76 55 123 79 77.333 24.035 2.003

25

Mean SD t test

Number 2

46

23 11 16

38 47

13.167 6.913 576

40.750 12.322 1.027

(minutes) necessary for emptyio%. 'Percentage of gastric contents emptied.

Journal of Dairy Science Vol. 74, No. 2, 1991

25

97

8 5 21 19 14 15 16

25 53 29 75 41 30 77 41

72250 20.552 1.713

12.661 6.429 .536

44.917 18.083 1.507

4

BIFIDOBACTERIUM SURVIVAL. DURING GASTRIC TRANSIT

Bifiabbacterium strain in product number 1, however, resists acidity; thus, there is only a slight difference between in vitro and in vivo results, most likely due to the dilution by gastric juices in vivo.

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to the human intestine enough living Bijiabbacterium to ensure a health benefit, as has been demonstrated for yogurt and lactose intolerance (3, 8, 10). Thus, choosing a Bifidobacferium strain resistant to acidity is important for the manufacture of bifidus fermented milks.

CONCLUSIONS

We conclude that only one of the two fermented milks studied contains a Bifidobacferium strain resistant to either the gastric acidity during the 90-min gastric transit or the product acidity during its shelf life (24 d at pH 4.4 and at 6°C). We also conclude that in vitro and in vivo results are consistent, despite slight differences, especially in the case of Bifidobacterium strain in product number 2. These differences may be due to the following: gastric contents are progressively diluted by the secretion of gastric juices, which contribute to lower bacterial concentrations even if bacteria survive; in the stomach, pH just after product ingestion is the product pH, and then it decreases progressively, whereas in the in vitro simulation pH was 3 in the beginning and then remained unchanged. Results obtained on gastric emptying of product number 1 and product number 2 are consistent with previous results for yogurt (4); in this study, the half-emptying of the stomach occurred after about 42 min in lactose-tolerant subjects. We may deduce from these data that in both cases the same amount of product (80%) passes into the intestine after 90 min, which in the case of 125 g (1 c) of product number 1 corresponds to at least 100 g x lo7 Bifidubacteriumlg or lo9 total Bifidobacterium. This is not the case for product number 2 for which the Bifidobacterium population is consistently inferior to that of product number 1 and decreases more rapidly both in vitro and in vivo. In general, we have demonstrated that not all commercial bifidus fermented milks may bring

ACKNOWLEDGMENTS

This work was supported by Gervais-Danone France. The authors are grateful to H. Tonetti and B. Libaud for technical assistance and advice on microbiological methodology. REFERENCES 1 Hotta, M.,Y. Sato, S. Iwata, N. Yamashita, K. Sunakawa, T. Oikawa, R. Tanaka, K. Watanabe, and M. Takayam. 1987. Clinical effects of Bifidobacterium preparations on pediatric intractable diarrhea. Keio J. Med. 36(3):298. 2 Kim, H.S. 1988. Characterization of Laftobacilli and Bifidobacteria as applied to dietary adjuncts. Cult. Dairy Prod. J. 23:6. 3 K o h , J. C., M. D. Levitt, M. Aouji, and D. A. Savaiano. 1984. Yogun, an autodigesting source of lactose. New England J. Med. 3101. 4Mourot, J., P. Thowenot, J. M.Antoine, and G. Debry. 1985. Comparison of gastric emptying rates of yogurt and mitk in man 1st Int. Congr. for Food and Health, Parma, Italy. 5Rasic. J. L. 1983. The role of dairy foods containing bifid* and acidophilus bacteria in nutrition and health. N. Ear. Dairy J. 43:2. 6Rasic, 1. L.. and J. A. Kurmann. 1983. Bifidobacteria and their role. Experientia Suppl. 39, ed. Birkhiiuser Verlag, Basel, Switzerland; Boston, MA; Stuttgart, Germany. 7Romond, C. 1986. Les Bifidobocterium et la sang. Cah. Nutr. Diet. 26215. 8Savaiano. D. A.. A. Abouelanowr, D. E. Smith, and M. D. L-evitt. 1984. Lactose malabsorption from y e gurt, pasteurkd yogurt, meet acidophilus milk. and cultured milk in lactase-deficient individuals. Am. J. Clin. Nutr. 40:1219. 9 SCardOVi, V. 1986. gena^ BifidobadrriWn Orla-Jtnsen 1924, 472AL. Page 1423 in Bmgey’s manual of systematic bacteri010g~.Vol. 2. Williams & W W , Baltimore, m. 10 Wytock, D. H.,and J. A. DiPalma. 1988. All yogurts are not created equal. Am. J. Clin. Nutr. 47454.

Journal of Dairy Science Vol. 74. No. 2, 1991