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MICROBIAL ECOLOGY IN HEALTH AND DISEASE VOL. 7: 247-256 (1994) Effects of Gluconic Acid on Human Faecal Bacteria T. ASANO*t, K . YUASAt, K. KUNUGITAt, T. TERAJIt AND T. MITSUOKAt ?Chemical Products Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 5-2-3, Tokodai, Tsukuba, Ibaraki, 300-26 and $Department o j Food Hygiene, Nippon Veterinary and Animal Science University, 1-7-1, Kyonan-cho, Musashino-shi, Tokyo, 180, Jupan Received 22 April 1994; revised 8 July 1994 Gluconate was fermented selectively by the Bijidohacterium adolescentis group and some species of other genera, including Clostridium clostridiiforme, C. innocuum, Propionibacterium acnes, Megasphaera elsdenii, Enterococcus jaecium and Klebsiella pneumoniae; however it was not utilised by most other bacteria including the Bacteroidaceae. No other organic acid salts were utilised by B. adolescentis. These salts weakly inhibited the growth of C. perjringens in vitro, as did gluconate. The absorption rate of gluconate from the ligated small intestinal loop in rats was 19.9 per cent under conditions when 100 per cent of glucose was absorbed. The effects of ingestion of gluconate on human faecal bacteria was studied in ten healthy adult males. They ingested 9 g/d or 3 g/d of glucono-6-lactone (anhydride of gluconic acid). With the 9 g/d ingestion, the number of bifidobacteria significantly increased (P<0.001), whereas C. perfringens decreased and Enterobacteriaceae remained constant. The concentrations of bifidobacteria also increased ( W 0 . 0 5 ) following 3 g/d ingestion. KEY WORDS-Gluconic acid; Gluconate; Absorption; Faecal flora; Bifidobacteria. INTRODUCTION In the human intestinal microbiota useful bacteria coexist with harmful bacteria. It is assumed that harmful bacteria such as Clostridium perjringens produce putrefactive products or toxins, and may contribute to diarrhoea, constipation, cancer, hypertension and/or ageing. On the other hand, useful bacteria such as bifidobacteria are thought to inhibit the growth of harmful bacteria and stimulate immune function, so they are helpful for maintenance of human health.' Since the possible beneficial role of bifidobacteria became known, they have been used as dietary supplements or as starter cultures for yogurt and other cultured milk products in many areas of the world including Japan, Scandinavia and Europe. In Japan, bifidobacteria growthpromoting substances, such as oligosaccharides, are utilised in various food products as dietary supplements to promote the multiplication of bifidobacteria in the human intestine. Various indigestible oligosaccharides such as fructoolig~saccharides,~ isomaltooligosaccha*Author to whom correspondence should be addressed CCC 0891-060X/94/050247-10 0 1994 by John Wiley & Sons, Ltd. rides,6 soybean oligosaccharides,2 galactooligosaccharides' and xylooligosaccharides" have been developed as food materials which promote the growth of bifidobacteria. There is no report on organic acids which promote the growth of bifidobacteria. We have directed our attention to gluconic acid, and have studied its effect on the growth of bifidobacteria. Gluconic acid is an oxide of glucose, and its anhydride is glucono-8-lactone. Gluconic acid forms gluconate salts with various cations such as sodium, calcium, potassium and zinc. These gluconic acid salts are widely utilised in various food products as acids, coagulant and mineral supplement. Gluconic acid exists naturally in rice, honey, wine (60450 p.p.m.), vinegar, beer (40-60 p.p.m.) and grape juice (60-380 p.p.m.). On an industrial scale, gluconic acid is produced from starch by fermentation. There are few reports on the effect of gluconic acid on the growth of bacteria or its absorption in animals. In the present study, we investigated the effect of gluconate on the growth of various intestinal bacteria in vitro, the absorption of gluconate in rats in situ and the effect of ingestion of glucono-&lactone on human faecal bacteria. 248 MATERIALS AND METHODS Utilisation by intestinal bacteria in vitro A total of 88 strains of intestinal bacteria was used in fermentation tests. The carbohydrates tested were sodium gluconate (Fujisawa Pharmaceutical Co., Ltd), fructooligosaccharides (Wako Pure Chemical Industries Ltd) and glucose (Nacalai Tesque, Inc.). The test medium was PYF broth, PY broth4 containing 4 per cent (vlv) Fildes solution,' with 0.5 per cent of each carbohydrate. The control medium lacked the added test carbohydrate. Tubes containing 3 ml of the test medium were inoculated with 30 pl of the test organisms which were precultured in GAM broth (Nissui Pharmaceutical Co., Ltd). The inoculated tubes were incubated at 37°C for 7 d anaerobically by the Anaero-Pak method (Mitsubishi Gas Chemical Co., Inc.). After incubation, the optical density (OD) at 660 nm and pH of the medium were measured. The efficiency of carbohydrate utilisation by each bacterial strain was evaluated quantitatively by calculating the difference in OD between the test medium and the control medium. The difference in OD was scored in the following manner: - , <0.099; f , 0.100-0.199; +, 0.200-0.399; + +, 0.400-0-599; + + +, B0.600; v, variable (strains may be either + or -). Effect on bacterial growth in vitro The bacterial strains used in this test were B. adoIescentis ATCCl5703 and C. perfringens GKK 16. The organic acids tested were D-gluconic acid (Fujisawa), D-glucuronic acid (Nacalai), citric acid (Wako), DL-tartaric acid (Nacalai), m-malic acid (Wako), maleic acid (Nacalai), fumaric acid (Wako), m-lactic acid (Nacalai) and acetic acid (Nacalai). Fructooligosaccharides (Wako) and isomaltooligosaccharides (Wako) were aslo included as the representative of bifidobacteria growthpromoting substances. The organic acid salt solution was prepared in a concentration of 5 per cent (wtIvo1.) neutralised by the addition of 6 N sodium hydroxide. The basal medium was Semisolid GAM without dextrose (Nissui). The test medium was prepared by the elimination of agar from the basal medium and with an added 10 per cent (vol./vol.) of each organic acid salt solution to 0.5 per cent final concentration. Tubes containing 5ml of the test medium were inoculated with 50 p1 of 100-fold dilutions of the test bacteria which were T. ASANO ET AL. precultured in GAM broth. The inoculated tubes were incubated at 37°C for 12 h anaerobically by the Anaero-Pak method. The OD at 660nm of the cultured test medium was measured relative to uninoculated blank medium containing each organic acid. The effect of each sample on the growth of B. adolescentis or C. perfringens was evaluated quantitatively by the percentage difference of the OD in the test medium and the control medium. Intestinal absorption in rats in situ Twelve 7 wk-old male Wistar rats (Charles River Japan Inc.) weighing approximately 280 g were divided into four groups of three rats each. The test solution was either 100mM of sodium gluconate or 100mM of glucose in saline (9g sodium chlorideI1). The rats were anaesthetised with ether after fasting for 24 h. A central longitudinal incision was made into the abdominal wall and the small intestine was exposed. A small intestinal loop about 10 cm in length was made by ligation with a silk suture in the portion of either upper intestine (jejunum) or lower intestine (ileum). 0.5 ml of the test solution was injected into the ligated loop and the whole small intestine was put back into the abdominal cavity. At 30 min after injection, the rats were killed and the ligated loop (test loop) was cut off. The contents in the loop were flushed out with 20ml of saline. At this time, a control loop was made of the intestinal portion next to the test loop and as soon as 0.5 ml of the test solution was injected into the loop, the contents were flushed out with 20 ml of saline. The residual gluconate or glucose in the ligated loop was measured in the contents. Measurement of gluconate was carried out by Food Analysis (F-Kit) D-Gluconic acidb-Glucono-6-lactone (Boehringer Mannheim), and glucose was by Glucose-CII-Test-Wako (Wako). The absorption rate of the sample from the ligated loop was calculated from the amount of the residual sample in the test loop (T) and that in the control loop (C) according to the following formula: Absorption rate (%)=(1 - TIC) x 100 Efect on human faecal bacteria The test sample used in the human volunteer tests was glucono-6-lactone (Fujisawa) powder which reverted to gluconic acid in solution. Two volunteer studies were carried out independently GLUCONATE AND FAECAL BACTERIA with a dose of 9 g/d and 3 g/d respectively. The subjects were ten healthy male volunteers in each test. The ages of the subjects ranged from 25 to 50 yr in the 9 gld ingestion study, and from 26 to 44 yr in the 3 gld study. They had stopped ingesting medicines and antibacterial agents 1 mth prior to and during the test period. However, they could consume food without restrictions other than foods with abundant live cultures, and were otherwise asked to maintain their normal diet throughout the test. These studies were performed in accordance with the Helsinki Declaration as updated in Tokyo, 1975. The subjects ingested glucono-6-lactone three times daily in one-third portions of the appointed dose after a meal for two consecutive weeks. Fresh faecal specimens were collected once a week six times in total. The control specimens were collected 1 wk before and just before starting ingestion. The specimens during ingestion were collected 1 wk and 2 wk after starting ingestion, and further control specimens were collected 1 wk and 2 wk after stopping ingestion. Faecal specimens collected from each subject were immediately refrigerated, and the faecal bacteria were analysed within 3 h. The method of Mitsuoka et a[.*,’ was used for faecal microbial analysis. The results were expressed as log,, of the number of bacteria per gram wet weight of faecal material. The paired t test and chi-square test were used for statistical analysis of the results. RESULTS Utilisation of gluconate by various intestinal bacteria in vitro The results of fermentation tests in vitro are shown in Table 1. Among members of the genus Bijidobacterium, gluconate was utilised selectively by the B. udolescentis group such as B. adolescentis, B. pseudocutenulaturn and B. catenulatum, but was not utilised by other bifidobacteria including B. longum and B. b$dum. In the genus LactobacilEus, gluconate was weakly utilised by L. casri and L. fermentum. In addition, gluconate was utilised by facultative bacteria including Enterococcus faecium and Klebsiella pneumoniae and anaerobic species including Clostridium clostridiiforme, C. innocuum, Propionibacterium acnes and Megusphaera elsdenii. However, gluconate was not utilised by the major part of intestinal bacteria 249 including Bacteroidaceae which are among the predominant bacteria. Eflects of orgunic acids and carbohydrates on the growth of B. adolescentis and C. perfringens in vitro The results of the bacterial growth tests in vitro are shown in Table 2. The growth of B. adolescen(is was promoted by gluconate. The maximal effects were observed at concentrations of 0.5 per cent and 1 per cent of gluconate, and a similar phenomenon was noted with glucose. Other organic acids either had little effect on, or suppressed the growth of, B. adolescentis, especially maleate which strongly inhibited growth. On the other hand, the growth of C. perfringens was weakly suppressed by gluconate in a concentrationdependent manner. Other organic acids similarly suppressed, and the inhibitory effects of citrate and maleate were particularly strong. Intestinal absorption of gluconate in rats in situ The results of the absorption tests are shown in Table 3. In the upper portion of the small intestine, only 19.9 per cent of injected gluconate was absorbed from the ligated loop under conditions when 100 per cent of glucose was absorbed. Similarly in the lower portion of the intestine, the absorption rate of gluconate was 11.6 per cent compared with 49.3 per cent for glucose. EfSrct of glucono-d-lactone ingestion on human fuecal bacteria Faecal bacteria results for the 9 gld of glucono&-lactoneingestion test are shown in Table 4.The changes in bacterial concentrations of the representative intestinal bacteria in the 9 g/d and 3 gld ingestion tests are shown in Figure 1. Bacteroidaceae, bifidobacteria and eubacteria were selected to represent the predominant bacteria, and C. perfringens and Enterobacteriaceae were selected as representatives of potentially harmful bacteria. In the 9 g/d ingestion test (Figure lA), the concentration of bifidobacteria significantly increased (P<O.OO1) during ingestion, whereas C. perfringens decreased in both levels and frequency of occurrence. The number of Enterobacteriaceae, which utilised gluconate in vitro, did not increase during ingestion. In the 3 g/d ingestion test also (Figure 1 B), the concentration of bifidobacteria significantly increased (P<0-05) during ingestion, 250 T. ASANO ET AL. Table 1. Utilisation of gluconate by various intestinal bacteria ~ Bacterial species Bifidobacterium B. adolescentis B. pseudocatenulatum B. catenulatum B. angulatum B. dentium B. longum B. bijidum B. breve B. infantis B. animalis Lactobacillus L. acidophilus L. salivarius L. gasseri L. casei L. fermentum Eubacterium E. aerofaciens E. limosum E. lentum E. nitritogenes Bacteroides B. fragilis B. distasonis B. vulgatus B. thetaiotaomicron B. ovatus B. uniformis B. melaninogenicus Fusobacterium F. necrophorum l? nucleatum F. varium Clostridium C perfringens C. dificile C. paraputrificum C. butyricum C. clostridioiforme C. ramosum C. innocuum C. sordellii C. sporogenes C. bifementans C. botulinum Propionibacterium P. acnes Peptostreptococcus P. magnus P. anaerobius P. asaccharolyticus ~ ~ ~ ~~~~~~ Strains (no.) Sodium gluconate Fruc tooligosaccharides Glucose 5 ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ +++ +++ +++ + 1 2 2 2 2 1 1 +++ +++ V +++ - +++ +++ +++ +++ - +++ +++ ++ 1 1 - f f + ++ ++S ++ +++ +++ +++ + + + ++ ++ - f + + + + 6 2 - 1 1 1 1 1 1 1 +++ + ++ + 2 1 1 + - +++ ++ +++ +++ ++ ++ +++ ++ + +++ ++ ++t f f - 25 1 GLUCONATE AND FAECAL BACTERIA Table 1. Continued ~ ~ Bacterial species Peptostreptococcus P. prevotii P.micros Veillonella V. parvura V. alcalescens Megasphaera M . elsdenii Enterococcus E. faecaiis E. faecium Streptococcus S. pyogenes S. mutans Staphylococcus S. aureus S. epidermidis Escherichiu E. coli Klebsiella K. pneumoniae Proteus P. mirabilis ~ _ Strains (no.) _ _ _ Sodium gluconate 1 1 ~ ~~ ~ ~ ~ Fructooligosaccharides ~ ~ _ ~ Glucose f - 1 1 - 1 ++ 2 ++ +++ 1 1 1 1 1 - - +++ + ++ 1 + + 1 f 4 Bacterial growth assessed by differences in optical density (OD) at 660 nm between control and test experiments: - , c0.099; f , 0.1004199; +, 0.200-0.399; + +, 0.400 -0.599; + + +, >0.600; v, variable (strains may be either + or - ). but the extent of the increase was smaller than that of the 9 g/d ingestion test. The changes in relative ration of the numbers of some dominant bacteria to the total number of bacteria are shown in Figure 2.The ratios of bifidobacteria in the control periods before starting ingestion and after stopping ingestion ranged from 17-0 to 24.2 per cent in both 9 g/d and 3 g/d ingestion tests. In the 9 gid ingestion test, the ratio of bifidobacteria increased to 45.5 per cent during ingestion, while the ratio in the 3 g/d ingestion test increased to 35.1 per cent. DISCUSSION In vitro fermentation tests showed that gluconate was utilised selectively by B. adolescentis group bacteria including B. adolescentis, B. pseudocatenulatum and B. catenulatum. Other Bijidobacteria species isolated from the intestine of human adults, such as B. longum and B. bijidum, did not utilise gluconate. Yaeshima et al. l 3 identified 56 strains of bifidobacteria isolated from the faeces of human adults, and identified them as 31 strains of B. adolescentis, 17 strains of B. pseudocatenulatum, four strains of B. catenulatum and four strains of B. longum. From among the 56 strains, 52 strains belonged to the B. adolescentis group, and utilised gluconate, with the exception of some strains of B. pseudocatenulatum. In the present study, we isolated 51 strains of bifidobacteria from the faeces of the subjects, and identified them as 47 strains of the B. adolescentis group and four strains of B. longum on the basis of carbohydrate fermentation patterns. These results suggest that the major Bifidobacteria species within the intestines of human adults belong to the B. adolescentis group, which can utilise gluconate. Since gluconate is not utilised by the numerically dominant intestinal bacteria, including Bacteroidaceae which are the most predominant bacteria, it can be efficiently utilised by bifidobacteria. Although Enterobacteriaceae utilised gluconate in vitro, they did not increase in concentration in the human volunteer _ 252 T. ASANO ET AL. Table 2. Effects of organic acid and carbohydrates on the growth of B. adoolescentis and C. perfringens in vitro Organic acid or carbohydrate (Yo) Concentration Control Fructooligosaccharides Isomaltooligosaccharides Glucose 0.5 0.5 0.25 0.5 1 Gluconate Glucuronate Citrate Tartrate Malate Maleate Fumarate Lactate Acetate Data are expressed as ratio 2 0.25 0.5 1 2 0.5 0.5 0.5 B. adolescentis ATCC15703 C. perfringens GKKl6 100 152.4 178.9 172.4 197.2 20 1.2 174.8 142.3 172.8 172.8 100 99.6 135.6 161.0 201.3 201.3 194.9 93.6 86.9 74.6 69.5 95.3 0.2 80.1 144.3 87.8 0.5 77.2 97.2 93.1 0.5 0 0.5 63.0 89.4 91.9 0.5 0.5 70.3 36.7 65.3 71.8 69.9 (W,) of the OD in test medium to the OD in control medium. Table 3. Absorption of gluconate from a rat ligated intestinal loop Absorption rate (YO) Small intestine Gluconate Glucose Upper portion Lower portion 19.9 i 6.0 11.6+ 15.2 100 f 0.0 49.3 7.4 + Values refer to mean f SD of three rats. 0.5 ml portion of sample was injected into the ligated intestinal loop and the amount of residual sample was measured after 30 min. tests. The reason why they do not increase in vivo is not clear, but it is possibly a consequence of the increase in bifidobacteria. Gluconate, like other organic acids, weakly suppressed growth of C. perfringens in vitro, and it also decreased the number of C. perfringens in the volunteer test. However the in vitro effect is so weak that it may not be the cause of the in vivo effect. Some oligosaccharides, which could not suppress the growth of C. perfringens in vitro, also decreased the number of this bacterium in the volunteer tests. 11,12 In these cases, the decrease of C. perfringens may have resulted from the environmental changes in the intestine caused by the increase of bifidobacteria, and the same reason could be applied in the case of gluconic acid. One of the conditions necessary for bdidobacteria growth-promoting factor to be effective is that it reaches the large intestine where it may be utilised by bifidobacteria. Therefore, it is necessary for gluconic acid not to be absorbed from the small intestine. At first we were afraid that ingested gluconic acid would be absorbed from the small intestine, since its structural formula is so similar to glucose. Unexpectedly, only 20 per cent of injected gluconate was absorbed from the ligated intestinal loop of rats under conditions where 100 per cent of glucose was absorbed. This result suggests that most of the ingested gluconate is not absorbed from the small intestine and reaches the large intestine. It is said that some oligosaccharides which are absorbable from the intestine or are unstable in gastric juice, need high ingested doses to gain the effect in vivo. Since gluconic acid does not have these defects, it could be an efficient growth-promoting substance for bifidobacteria. In 253 GLUCONATE A N D FAECAL BACTERIA hhhhhhhhh 0 0 0 0 0 0 0 0 0 2 2 2 = 2 ZcZz 254 T. ASANO ET AL. 8 11.2 v) aJ 1110.8- b Enterobacteriaceae Total A --7 65- 4cd P Bifidoba cteri um 9.6- 8 9.49.2 3- Eubacteri um 00 I I 1 I I 2 I 11.2 I 8 d I I I I I 1 7- 5 I Enterobacteriaceae 6- 10.4 10.2 Bifidob$cterium cd L c1 aJ 9.8 DD 0 E D - Eubacterium 9.6 g-4L717J 9.2 -7 0 7 14 21 28 Figure I. Effects of glucono-F-lactone ingestion with the dose of 9 g/day (A) and 3 &day (B) on human faecal bacteria in ten volunteers. Values are expressed as mean of log,, bacterial concentration from each individual. Frequency of occurrence (YO)is shown in parentheses. Significant difference form the concentration of day 0: *P<0.05,***P<O.OOI fact, gluconod-lactone effectively increased bifidobacteria in the human volunteer tests. T h s shows that the minimum daily dose of gluconic acid is less than 3 g, which is sufficiently comparable to the minimum dose of other oligosaccharide^.^,^ GLUCONATE AND FAECAL BACTERIA h x a- a \ M fm W M n x cd YM m W Q 255 T. ASANO ET AL. From these results, we conclude that gluconic acid is utilised effectively by bifidobacteria and contributes to increasing the number of faecal bifidobacteria. Furthermore this effect is attained with a relatively small daily dose of 3 g. Gluconic acid is already used as a food additive, for example in acids, as a coagulant and in mineral supplements such as calcium salt. The present study demonstrates that gluconic acid can be used like other oligosaccharides as a bifidobacteria growthpromoting substance, and not only as a food additive. REFERENCES 1. Fildes P. (1920). New medium for the growth of B. influenza. British Journal of Experimental PatholOgy 1, 129-130. 2. Hayakawa K, Mizutani J, Wada K, Masai T, Yoshihara I, Mitsuoka T. (1990). Effects of soybean oligosaccharides on human faecal flora. Microbial Ecology in Health and Disease 3, 293303. 3. Hidaka H, Eida T, Takizawa T, Tokunaga T, Tashiro Y. (1986). Effects of fructooligosaccharides on intestinal flora and human health. Bifidobacteria and MicroJora 5, 37-50. 4. Holdeman LV, Cat0 EP, Moore WEC. (1977). Anaerobic laboratory manual, 4th edn. Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg. 5. Ito M, Deguchi Y, Miyamori A, Matsumoto K, Kikuchi H, Matsumoto K, Kobayashi Y, Yajima T, Kan T. (1990). 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