Fermentation of D-Tagatose by Human Intestinal Bacteria and Dairy ...

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sun, Cl. butyricum, Cl. paraputri cum); Bacteroides species. (8 different isolates—B. fragilis (3), B. thetaiotaomicron, B. uniformis, B. intermedius, B. o×atus and B.
ORIGINAL ARTICLE

Fermentation of D -Tagatose by Human Intestinal Bacteria and Dairy Lactic Acid Bacteria Hans Bertelsen1 , Hans Andersen2 and Michael Tvede3 From 1 Arla Foods Innovation, Nr. Vium., Soenderupvej 26, 6920 Videbaek, Denmark, 2 Arla Foods Innovation, Roerdrumvej 2, 8220 Brabrand, Aarhus, Denmark and 3 Department of Clinical Microbiology, Juliane Mariesvej 22, 2100 Copenhagen , Denmark Correspondence to: Hans Bertelsen, Arla Foods Innovation, Nr. Vium. Soenderupvej 26 6920 Videbaek, Denmark. Fax: »45 99944396; Tel: »45 99944444; E-mail: [email protected] k

Microbial Ecology in Health and Disease 2001; 13: 87 – 95 A number of 174 normal or pathogenic human enteric bacteria and dairy lactic acid bacteria were screened for D -tagatose fermentation by incubation for 48 hours. Selection criteria for fermentation employed included a drop in pH below 5.5 and a distance to controls of more than 0.5. Only a few of the normal occurring enteric human bacteria were able to ferment D -tagatose, among those Enterococcus faecalis, Enterococcus faecium and Lactobacillus strains. D -Tagatose fermentation seems to be common among lactic acid bacteria. Most of the analyzed dairy lactic acid bacteria fermented D -tagatose, and among those Lactobacillus, Leuconostoc and Pediococcus strains fermented most strongly, but also strains of Enterococcus, Streptococcus and Lactococcus fermented D -tagatose. None of the analyzed BiŽ dobacterium strains fermented tagatose. Key words : D -tagatose, fermentation, human enteric bacteria, lactic acid bacteria, tagatose-6phosphate pathway.

INTRODUCTION D -Tagatose

is naturally occurring, sweet (1, 2) ketohexose related to D -galactose and is the C-4 epimer of D-fructose. D -Tagatose occurs in Sterculia setigera gum, a partially acetylated acidic polysaccharide (3) and D-tagatose is also found in low concentrations in heated cows milk, produced from lactose (4). In microbiology tagatose is well known as phosphorylated intermediates of the tagatose-6phosphate pathway, which is an important pathway for lactose and galactose degradation in certain Gram positive bacteria, especially the lactic acid bacteria (5, 6). An economical process to produce D -tagatose from lactose has been developed (7, 8). D -Tagatose is a promising low calorie bulk sweetener, 92% the sweetnes of sucrose, utilising dairy by-products, whey and ultraŽ ltration-permeat, as raw materials. The estimated small intestinal absorption of radiolabelled D -tagatose in rats is approximately 20% (9). Similarly, less than 26% of ingested D -tagatose was absorbed in pigs according to measurements of the disappearance of D -tagatose from the digesta (10). Unabsorbed D -tagatose is fermented by intestinal microorganisms to short-chain fatty acids (SCFA). No D-tagatose was found in faeces of pigs ingesting a 10% D-tagatose diet (10). Similarly no D -tagatose was found in human faeces after a 30 g intake of D-tagatose (11). In adapted rats fed a diet with 10% © Taylor & Francis 2001. ISSN 0891-060 X

about 2% of the ingested dose of 1 4 C-D tagatose was recovered in faeces (9). In unadapted rats the recovery of D-tagatose in faeces was much higher, approximately 25% of the ingested dose. Adaptation of the micro ora in pigs and rats are indicated by the Ž nding of increased numbers of D -tagatose degrading bacteria and disappearance of watery stools after a few days of D tagatose ingestion (9, 10). Adaptation in pigs also resulted in increased in ×itro fermentation capacity of D -tagatose and increased mol% of butyrate (12). Furthermore, concentrations of butyrate in the cecum and colon of pigs were increased in a dose-response manner from ingestion of D -tagatose (13). Similarly, increased mol% of butyrate was observed in in ×itro fermentation of faecal samples from adapted human volunteers and in addition changes in faecal micro ora was observed, reduced counts of E. coli and increased numbers of lactobacilli and lactic acid bacteria (14). Only a limited number of reports exist on the ability of various microorganism to utilise D-tagatose. A study reports that only few enteric microorganisms are able to grow on D -tagatose (15). Other studies report high frequency of D-tagatose fermentation among species of Lactobacillus (16– 18) However, none of the strains of biŽ dobacteria were able to ferment D-tagatose (19). The objective of the present study is to screen pure strains of intestinal bacteria as well as dairy starter cultures for D -tagatose fermentation. D -tagatose,

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MATERIALS AND METHODS Human intestinal bacteria Forty-Ž ve isolates of normal (34 strains) or pathogenic human enteric bacteria (11 strains) were chosen for fermentation of D -tagatose in vitro. The following strains, isolated from healthy persons, were chosen as representative for normal human micro ora: Clostridium species (9 different species — Cl. innocuum, Cl. perfringens, Cl. sordellii, Cl. bifermentans, Cl. tertium, Cl. sporogenes, Cl. ramo sun, Cl. butyricum, Cl. paraputriŽ cum); Bacteroides species (8 different isolates — B. fragilis (3), B. thetaiotaomicron, B. uniformis, B. intermedius, B. o×atus and B. melaninogeni cus ); Fusobacterium species (two strains, F. necrophorum and F. nucleatum ); Enterococcus species, (Enterococcus faecium and Enterococcus faecalis); Staphylococcus aureus (1 strain), Enterobacteriaceae (7 different isolates — Enterobacter cloacae, Citrobacter freundii, Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, Klebsiella pneumoniae, Acinetobacter calcoaceticus); BiŽ dobacterium species (2 different isolates); Lactobacillus species (3 different isolates). Eleven enteric pathogens isolated from Danish patients with diarrhoea were tested: Campylobacter coli, Campy lobacter jejuni, Salmonella typhi (2 strains), Salmonella enteritidis, Yersinia enterocolitica (type 3), Shigella sonnei, Shigella  exneri, Aeromonas hydrophil a, E. coli 0157 and Ž nally Clostridium difŽ cile. Twenty two additional different enteric isolates from normal healthy humans were tested for tagatose fermentation as well: Enterococcus faecalis (4 strains); Enterococcus faecium (5 strains); BiŽ dobacterium (5 strains); Lactobacil lus (8 strains). Dairy type lactic acid bacteria The lactic acid bacterial strains used in this study were from the culture collection held at Arla Foods Innovation Centre. The strains are either isolated from a diversity of products or obtained from commercial companies and culture collections. A number of lactic acid bacteria strains (107), characterized by their ability to synthesize lactic acid and belonging to the genera Leuconostoc (10 strains — species Leuc. dex tranicum, Leuc. lactis, Leuc. cremoris, Leuc. mesenteroides); Enterococcus (11 strains — species E. a×iaum, E. faecium, E. durans, E. casseli a×us); Streptococcus (8 strains — species S. sali×arius, S. thermophilus ); Lactococ cus (11 strains — species Lc. lactis, Lc. cremoris), Pediococ cus (7 strains — species P. pentosaceus, P. dextrinicus, P. par×ulus, P. acidilactici ); Lactobacillus (44 strains — species Lb. acidophilus, Lb. cur×atus, Lb. casei, Lb. rhamnosus, Lb. paracasei, Lb. delbrueckii, Lb. bulgaricus, Lb. fermentum, Lb. hel×eticus, Lb. plantarum, Lb. reuteri ) and BiŽ dobac terium (16 strains — species B. animalis, B. infantis, B. biŽ dum, B. longum, B. adolescentis, B. bre×e, B. boum, B. catennulatum, B. globosum, B. indicum, B. steroides) were used for fermentation of D -tagatose in vitro.

Fermentation Human intestinal bacteria. For the fermentation assay, 1% D -tagatose was added to  uid media containing peptoneyeast-extract. The medium contained peptone, 0.5 g; trypticase 0.5 g; yeast extract 1.0 g; resazurin solution 0.4 ml; 0.9 NaCl solution 4.0 ml; destilled water 100 ml; hemin solution 1.0 ml; vitamin K1 0.02 ml; cystein-HCl, H2 O 0.05 g. The media was prepared at the State Serum Institute, Copenhagen Denmark. All tested strains, were inoculated in pure culture to the media and incubated for 48 hours at 37°C in an anaerobic glove box chamber (Forma ScientiŽ c). All strains were grown in media without added tagatose as control. After incubation the media was checked for any contamination by growth using BHI-agar plates. Fermentation of D -tagatose after incubation for 48 hours at 37°C, was measured by a pH-meter. AcidiŽ cation (fermentation) was regarded positive when pH was B 5.5 and the difference between inoculated and uninoculated (controls) media was \ 0.5. Decrease in pH value \ 0.5 and B 1.0 was judged as weak fermentation and a decrease in pH-value \ 1.0 was judged as strong fermentation (20). Dairy type lactic acid bacteria. The cultures were grown in MRS (Leuconostoc, Pediococcus, Lactobacillus, BiŽ dobac terium) or M17 (Lactococcus, Enterococcus, Streptococcus ). The media contained no sugar but were supplemented with 2% of D -tagatose. The medium M17 contained tryptone 5.0 g; soyapeptone 5.0 g; meat digest 5.0 g; yeast extract 2.5 g; asorbic acid 0.5 ml; magnesium sulphate 0.25 g; di-sodium-glycerophosphate 19.0 g, H2 O add to 850 ml. The medium MRS contained peptone 10.0 g; Lab-lemco powder 8.0 g; yeast extract 4.0 g; tween 80 1.0 ml; K2 HPO4 , 3H2 O 2.62 g; CH3 COONa, 3H2 O 5.0 g; tri-ammonium-citrate 2.0 g; MgSO4 , 7H2 O 0.2 g; MnSO4 , 4H2 O 0.05 g; H2 O add to 800 ml. All cultures were inoculated as pure culture (1% of a culture in the stationary phase about 108 –109 ) to the media and incubated aerobic for 48 hours at 30°C (Lactococcus, Pediococcus, Leuconostoc ) or 37°C (Enterococcus, Streptococcus, Lacto bacillus ). BiŽ dobacterium was incubated in an anaerobic chamber at 37°C. All strains were grown in media without added tagatose as a control. Fermentation was evaluated by pH values measured after 48 hours of incubation. Fermentation after incubation was measured by a pH-meter. Fermentation of D -tagatose was regarded positive when pH was B 5.5 and the difference between inoculated media with and without added tagatose (controls) was \ 0.5. Decrease in pH value between 0.5 and 1.0 was judged as weak fermentation; a decrease in pH value \ 1.0 was judged as strong fermentation. RESULTS & DISCUSSION The present study showed that fermentation of D -tagatose is not very widespread among the different genera of

Fermentation of

human enteric bacteria. Of 34 isolates of normal human enteric bacteria representing normal human micro ora, only Ž ve were positive, two out of three Lactobacillus tested, Enterococcus faecalis and two strains of Clostridium (Table I). None of the 11 strains of pathogenic enteric bacteria tested were positive according to the criteria used, however, a decrease in pH from 7.73 to pH 5.99 by Salmonella Typhi 1 may indicate slight growht on D tagatose (Table II). To evaluate whether fermentation of tagatose was a general habit for Enterococcus faecalis and Lactobacillus species, another fermentation assay was carried out with twenty-two different enteric isolates from normal humans (Table III). Testing of those enteric isolates conŽ rmed the ability of Enterococcus faecalis and Lactobacillus to ferment D -tagatose. All of four Enterococcus faecalis strains and six out of eight Lactobacillus tested

D -tagatose

by human intestinal bacteria

were positive, whereas none of the Ž ve tested strains of BiŽ dobacterium were able to ferment D -tagatose. Only one of Ž ve strains of Enterococcus faecium fermented tagatose (Table III). Most of the tested dairy type lactic acid bacteria fermented D-tagatose very extensively. Strong fermentation was seen in 61 strains of the dairy lactic acid bacteria and in 38 of these strains the fermentation of D -tagatose was very intensive-with a decrease in pH value of more than 2 pH units (Table IV). The most strongly fermentative strains were found among Lactobacillus, Leuconostoc and Pediococcus. Less strongly but still good acid producers were found among Streptococcus and Lactococcus. Enterococcus strains were somewhere between the two groups. Of the 107 tested dairy type lactic acid bacteria, 45 strains were not able to ferment D -tagatose (Table IV). None of

Table I Normal occurence enteric human bacteria tested for fermentation of 1% tagatose No.

Bacterial species

pH in control a

pH in 1% tagatose

Score b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Clostridium innocuum Clostridium perfringens Clostridium sordellii Clostridium bifermentans Clostridium tertium Clostridium sporogene s Clostridium ramosum Clostridium butyricum Clostridium paraputri Ž cum Bacteroides fragilis (23780) Bacteroides fragilis (23040) Bacteroides fragilis (23159) Bacteroides thetaiotaomicron Bacteroides uniformis Bacteroides intermedius Bacteroides o×atus Bacteroides melaninogenicus Fusobacterium necrophorum Fusobacterium nucleatum Enterococcus faecium Enterococcus faecalis Staphylococcus aureus Enterobacter cloacae Citrobacter freundii Escherichia coli 1108 Proteus mirabilis Klebsiella oxytoca Klebsiella pneumonia Acinetobacter calcoaceticus BiŽ dobacterium sp. 23829 BiŽ dobacterium sp. 22070-4 Lactobacillus sp. 19070-2 Lactobacillus sp. 19058-4 Lactobacillus sp. 19020-10

7.50 7.53 7.54 7.44 7.05 – 7.52 6.63 6.96 7.37 7.26 7.30 6.53 7.26 7.02 6.56 6.65 7.00 7.01 7.42 8.83 7.01 8.11 7.40 7.70 8.17 7.59 7.94 8.28 6.95 6.98 6.37 6.40 7.01

5.10 7.32 7.44 7.58 5.51 7.52 5.65 6.07 6.62 6.92 6.96 6.82 6.36 6.83 6.79 6.42 6.62 6.65 6.84 6.49 4.77 7.24 8.09 7.62 7.19 7.60 5.71 8.14 8.05 6.46 6.44 4.17 4.14 6.57

Strong positive Negative Negative Negative Weak positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Strong positive Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Strong positive Strong positive Negative

a

Controls used in this study was 1% tagatose medium with no inoculation. Assays with pH \5.5 is negative. whereas assays with pH B5.5 and where the difference to control is more than 0.5 pH units. were estimated as fermentation. pH decrease between 0.5-1.0 was called weak fermentation and pH decrease beyond 1.0 pH unit were estimated as strong fermentation. b

89

90

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Table II Human enteric pathogenic bacteria tested for fermentation of 1% tagatose No.

Bacterial species

pH in control a

pH in 1% tagatose

Score b

1 2 3 4 5 6 7 8 9 10 11

Campylobacter coli Campylobacter jejuni Salmonella typhi 1 Salmonella typhi 2 Yersinia enteritidis Yersinia entrocolitica Shigella sonnei Shigella  exneri Aeromonas hydrophil a Clostridium difŽ cile Escherichia coli 0157 c

7.37 7.37 7.73 7.86 7.49 6.33 6.44 6.44 7.98 6.86 7.65

7.09 7.15 5.99 7.15 7.69 6.12 6.54 6.38 8.10 6.20 7.80

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

a b c

Controls used in this study was 1% tagatose medium with no inoculation as in Table I. Estimation of fermentation as in Table I. All strains except E. coli 0157 were isolated from patients with gastroenteritis.

the BiŽ dobacterium strains were able to ferment D tagatose. A total of 44 Lactobacillus strains were tested for tagatose degradation, 26 of them were positive. All species of Lactobacillus casei -Lb. rhamnosus -Lb. paracasei were capable of fermenting D -tagatose. Other fermenting strains belonged to the species Lb. fermentum, Lb. hel×eticus, Lb. bulgaricus and Lb. plantarum. All the tested Enterococcus were positive, but this genera contains both intensive and moderate intensive strains. The fast growth of Enterococcus faecalis reported by Schweisfurth et al. (15) is conŽ rmed in this study. The Ž ndings in this study of high frequency of D -tagatose fermentation by human enteric Lactobacillus strains is in accordance with Morelli et al. (18) as all the four tested Lactobacillus, isolated from human faeces, were able to ferment D -tagatose. Similar a study by Lenzner et al. (1968) testing 93 strains of Lactobacillus isolated from faeces, gastric juice and vaginal secretion, showed a high frequency of D -tagatose fermentation as 46 were positive. All the 38 tested Lactobacillus casei (subsp. casei, subsp. rhamnosus and subsp. alactosus ) strain were positive and 7 out of 10 Lactobacillus acidophilus were positive, whereas none of the 15 tested strains of Lactobacillus plantarum, the 13 tested strains of Lactobacillus fermentum and the 10 tested strains of Lactobacillus bre×is were positive. Further testing of 138 strains of Lactobacillus casei and Lactobacil lus plantarum, isolated from various materials including cheese, conŽ rmed that all Lactobacillus casei strains were able to ferment D -tagatose whilst those of Lactobacillus plantarum were negative (21). Another study with bacteria isolated from human intestine conŽ rmed that Lactobacillus plantarum is unable to ferment D -tagatose while Lacto bacillus casei subsp. rhamnosus is positive (22, 23) and also that Lactobacillus coryniformis is not able to ferment D tagatose (23, 24). A study by Pidoux et al. (17) testing Lactobacillus strains, isolated from sugary keŽ r grains,

similarly showed that not all Lactobacillus strains are able to ferment D -tagatose as 3 strains of homofermentative Lactobacillus paracasei subsp. paracasei were positive and 2 strains of heterofermentative Lactobacillus hilgardii were negative. In our study we also found that all strains of Lactobacillus casei subsp. casei, Lactobacillus casei subsp. rhamnosus and Lactobacillus paracasei subsp. paracasei (Lactobacillus casei subsp. plantarum ) were able to ferment D -tagatose. A few Lactobacillus plantarum strains were found positive, which is not in accordance with Bengmark et al. (22) and Lenzner and Lenzner (21). None of the tested commercial Lactobacillus plantarum strains were positive. On the other hand it was found that the commercial Lactobacillus fermentum strains used in this study did ferment D -tagatose. This was not found in the study by Lenzner et al. (17). The Lactobacillus acidophilus strains used in this analysis gave variable results but only a few were regarded as positive. The differences detected among Lactobacillus acidophilus could be explained by the known heterogeneity of this species. All the eight tested dairy Enterococcus faecium were able to ferment D-tagatose, but in the study with the human intestinal Enterococus faecium only one of six strains were able to degrade D -tagatose. The inability of BiŽ dobacterium to ferment D -tagatose reported by Roy and Ward (19) is conŽ rmed in this study. The genus BiŽ dobacterium is often considered to belong to the lactic acid group, but they are phylogenetically unrelated and have a special pathway for sugar fermentation, unique to the genus, which clearly separates them from the lactic acid bacteria group. Differentiation between Lacto bacillus and BiŽ dobacterium on the basis of D -tagatose fermentation would be a very easy Ž rst step, although it is not fully conclusive as not all lactobacilli are able to ferment D -tagatose. D -Tagatose is found in nature only in low concentration in various milk products e.g. heated cows milk, produced

Fermentation of

from lactose (4) and thus it is not surprising, that the ability to ferment D -tagatose is not widespread among micro-organisms. The tested lactic acid bacteria showed a high frequency of D -tagatose fermentability and the ability is found in both homofermentative, heterofermentative and facultative heterofermentative groups of lactic acid bacteria and thus the screening gave no overall hints to the pathway of D -tagatose fermentation. The study by Lenzner et al. (16) indicated, that there was no interdependence between the ability of lactobacilli to ferment D -tagatose and lactose. An obvious entry of D -tagatose into established fermentative pathways would be through the tagatose-6-phosphate pathway found in Staphylococcus (25), in non-pathogenic Mycobacterium (26) and in lactic acid bacteria (5). If D -tagatose is taken up via a sugar PEP-dependent PTS, this action also converts D -tagatose into tagatose-6-phosphate and is thus ready for fermentation via the tagatose-6-phosphate pathway. Recently it was documented, that a mutant Lactobacillus casei defect in PTS uptake no longer was able to ferment D -tagatose (27). Some of the tested lactic acid bacteria, especially in the genus of Streptococcus and Lactobacillus are found in the oral cavity as part of the dental plaque bacteria. However, D -tagatose has been demonstrated to be non-cariogenic in two studies using human volunteers. The pH telemetric evaluation of the cariogenicity of D-tagatose were performed both on acute intake of D -tagatose and after

D -tagatose

by human intestinal bacteria

adaptation to D -tagatose. The results showed no critical decrease in the pH (i.e., below pH of 5.7) of inter dental plaque, either during the rinsing periods or the 30-minute periods following rinsing with D -tagatose. This contrasts with plaque pH after rinsing with sucrose, which always fell below the critical pH-value of 5.7, due to the glycolytic production of bacterial acids (28). The lactobacilli are important inhabitants of the intestinal tract of man and animals with functional beneŽ ts like maintenance of the normal micro ora, pathogen interference, exclusion and antagonism, immunostimulation and immunomodulation, anticarcinogenic and antimutagenic activities, deconjugation of bile acids and lactase presentation in ×i×o (29). As the viability of live bacteria in food products and during transit through the gastrointestinal tract may be variable, an alternative is to stimulate growth of beneŽ cial colonic bacteria by non-digestible food, the prebiotic concept (30). Any foodstuff that reaches the colon is a prebiotic candidate. Pig studies and rat studies have shown D -tagatose only to be partly absorbed in the small intestine with the major part fermented in the colon (9, 10) and human studies have shown increased H2 production upon ingestion of D -tagatose (11). Another condition for a food ingredient to be a prebiotic candidate, is selective fermentation by potentially beneŽ cial bacteria in the colon. The data presented in this article shows, that D -tagatose fermentation is widespread within the lactic acid bacteria, but not in most other common human

Table III Screening of 22 different enteric isolates from normal humans for tagatose No.

Isolates

pH-control

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Enterococcus fecalis-24087 Enterococcus fecalis-00583 Enterococcus fecalis-00561 Enterococcus fecalis-17243 Enterococcus faecium-17575 Enterococcus faecium-17243 Enterococcus faecium-17494 Enterococcus faecium-23266 Enterococcus faecium-23335 BiŽ dobacterium -21776-5 BiŽ dobacterium -18780 BiŽ dobacterium -18791 BiŽ dobacterium -18914-3 BiŽ dobacterium -18913-1 Lactobacillus -19015-6 Lactobacillus -22062-5 Lactobacillus -22864 Lactobacillus -22911 Lactobacillus -18911-2 Lactobacillus -18929-5 Lactobacillus -18826-1 Lactobacillus -18854-A

6.10 6.12 5.92 5.92 6.52 6.61 6.64 6.71 6.68 6.66 6.79 6.83 6.80 5.64 5.82 6.99 5.70 5.73 5.74 5.74 5.40 5.55

a b

91

a

pH-tagatose

Score b

4.24 4.20 4.17 4.18 5.72 5.83 5.78 5.95 5.22 5.90 5.93 6.64 6.01 5.23 3.81 6.77 5.18 4.97 5.35 3.93 3.80 3.94

Strong positive Strong positive Strong positive Strong positive Negative Negative Negative Negative Strong positive Negative Negative Negative Negative Negative Strong positive Negative Weak postitive Weak positive Negative Strong positive Strong positive Strong positive

Controls used in this study was 1% tagatose medium with no inoculation as in Table I. Estimation of fermentation as in Table I.

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H. Bertelsen et al.

Table IV Screening of 107 dairy type lactic acid bacteria for fermentation in 2% tagatose medium No

Genera:species:strains

Origin

1 2 3 4 5 6 7 8 9 10

Leuconostoc Leuconostoc Leuconostoc Leuconostoc Leuconostoc Leuconostoc Leuconostoc Leuconostoc Leuconostoc Leuconostoc

lactis lactis cremoris mesenteroides mesenteroides mesenteroides mesenteroides mesenteroides mesenteroides mesenteroides

MD MD MD MD MD MD MD MD MD MD

11 12 13 14 15 16 17 18 19 20 21

Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus Enterococcus

22 23 24 25 26 27 28 29

Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus Streptococcus

30 31 32 33 34 35 36 37 38 39 40

Lactococcus Lactococcus Lactococcus Lactococcus Lactococcus Lactococcus Lactococcus Lactococcus Lactococcus Lactococcus Lactococcus

lactis lactis lactis lactis lactis lactis lactis lactis lactis lactis lactis

41 42 43 44 45 46 47

Pediococcus Pediococcus Pediococcus Pediococcus Pediococcus Pediococcus Pediococcus

pentosaceus pentosaceus pentosaceus pentosaceus dextrinicus par×ulus acidilactici

48 49 50 51 52 53 54 55 56 57 58

Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus

a

Score b

pH-tagatose

pH-control

1142 1195 1216 1316 1321 1322 1324 1317 1325 1326

3.78 3.93 3.74 3.72 6.59 6.67 6.73 3.75 3.80 4.14

6.63 6.45 6.72 6.63 6.76 6.70 6.68 6.66 6.76 6.51

Strong positive Strong positive Strong positive Strong positive Negative Negative Negative Strong positive Strong positive Strong positive

a×iaum casseli a×us durans faecium faecium faecium faecium faecium faecium faecium faecium

MD strain 190 MD strain 691 MD strain 1387 MD strain 454 MD strain 1342 MD strain 1348 MD strain 1355 MD strain 1386 MD strain 1788 NCIMB 40371 UTI strain c

4.39 4.11 4.53 4.73 4.14 4.30 4.54 4.36 4.34 4.29 4.36

6.57 6.55 6.26 6.59 6.45 6.57 6.47 6.27 6.24 6.29 6.25

Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong Strong

sali×arius subsp. Thermph. sali×arius subsp. Thermph. sali×arius subsp. Thermph. thermophilus thermophilus thermophilus thermophilus thermophilus

LMG 6896 LMG 7952 t2 LMG 7953 t3 Chr. Hansen strain Wisby strain UTI strain UTI strain UTI strain

4.69 4.32 4.55 4.94 6.51 4.37 4.42 5.01

6.48 6.27 6.47 6.25 6.33 6.27 6.26 6.23

Strong positive Strong positive Strong positive Strong positive Negative Strong positive Strong positive Strong positive

DSM 4645 LMG 6897 LMG 6890 LMG 7930 DSM 20481 MD strain 1992 MD strain 1720 MD strain 1281 MD strain 2002 MD strain 2037 Commercial strain

6.40 6.49 4.93 6.72 6.67 6.50 4.86 4.89 4.88 4.90 6.58

6.76 6.63 6.43 6.74 6.70 6.57 6.40 6.45 6.40 6.54 6.68

Negative Negative Strong positive Negative Negative Negative Strong positive Strong positive Strong positive Strong positive Negative

MD MD MD MD MD MD MD

3.71 3.78 3.69 3.71 4.31 4.20 6.61

6.60 6.52 6.51 6.46 6.50 6.39 6.72

Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Negative

6.34 6.49 6.47 6.45 6.46 5.23 6.52 3.87 6.48 6.44 6.56

6.51 6.51 6.51 6.58 6.53 6.73 6.60 6.59 6.55 6.57 6.94

Negative Negative Negative Negative Negative Strong positive Negative Strong positive Negative Negative Negative

cremoris cremoris lactis lactis lactis 134 lactis 2 lactis lactis lactis lactis

acidophilus acidophilus acidophilus acidophilus acidophilus acidophilus acidophilus acidophilus acidophilus acidophilus acidophilus

subsp. subsp. subsp. subsp. subsp. subsp. subsp.

mes. mes. mes. mes. dext. dext. dext.

strain strain strain strain strain strain strain strain strain strain

strain strain strain strain strain strain strain

1261 1262 1340 1327 1328 1330 1338

LMG 7943 T NCFB 1697 NCFB 2745 MD strain 1989 MD strain 1990 NCFB 1693 NCFB 1360 Wisby strain Chr. Hansen strain Wisby strain Commercial strain

positive positive positive positive positive positive positive positive positive positive positive

Fermentation of

D -tagatose

by human intestinal bacteria

93

Table IV (Continued ) a

Score b

No

Genera:species:strains

Origin

pH-tagatose

pH-control

59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91

Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus

Commercial strain CRL 1014 MD strain 1313 MD strain 296 MD strain 1727 MD strain 1732 MD strain 1810 LMG 6901 T MD strain 444 CNRZ 206 NCFB 1750 Scandairy strain LMG 6413 MD strain 1202 Scandairy strain Scandairy strain LMG 7955 T MD strain 1927 MD strain 1397 MD strain 1388 Wisby strain MD strain 1116 CNRZ 211 DSM 20174 MD strain 1104 MD strain 1114 MD strain 1126 MD strain 1176 MD strain 1143 MD strain 1159 MD strain 1165 MD strain 1212 MD strain 4017

6.06 3.67 3.63 4.62 3.47 3.75 3.79 6.44 6.64 4.78 4.02 4.44 3.95 3.57 3.53 3.70 3.80 4.50 3.70 3.46 3.90 3.80 6.62 6.44 6.60 6.90 6.81 6.68 3.91 3.97 4.99 5.50 6.72

6.83 6.42 6.34 6.58 6.31 6.29 6.65 6.48 6.64 6.85 6.51 6.63 6.44 6.05 6.35 6.50 6.50 6.90 6.40 6.25 6.50 6.50 6.62 6.71 6.63 6.58 6.66 6.90 6.45 6.52 6.65 6.76 7.08

Weak positive Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Negative Negative Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Strong positive Negative Negative Negative Negative Negative Negative Strong positive Strong positive Strong positive Strong positive Negative

92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107

BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium BiŽ dobacterium

DSM 20083 UTI strain DSM 20456T Chr. Hansen strain Chr. Hansen strain DSM 20213 T DSM 20432 DSM 20103 T DSM 20092 DSM 20214 Wisby strain DSM 20219 T Scandairy strain Scandairy strain Wisby strain DSM 20089

6.99 6.46 6.48 6.48 6.93 6.91 6.85 6.92 6.81 7.00 6.46 6.97 6.75 6.95 6.91 6.84

7.05 6.48 6.72 6.46 6.95 6.90 6.85 7.01 7.00 7.02 6.46 7.00 6.82 6.68 6.85 6.88

Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

a b c

acidophilus acidophilus casei casei casei cur×atus delbrueckii bulgaricus bulgaricus delbruedkii bulgaricus delbruedkii bulgaricus fermentum fermentum fermentum hel×eticus hel×eticus casei rhamnosus paracasei paracasei paracasei paracasei paracasei paracasei paracasei paracasei paracasei rhamnosus rhamnosus plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum plantarum reuteri adolescentis animalis biŽ dum lactis BB12 biŽ dum BB11 bre×e boum catenulatum globosum indicum infantis 420 longum longum longum longum steroides

Controls used in this study was medium without tagatose but inoculated with bacteria. Estimation of fermentation as in Table I. UTI¾Ukrainian Dairy Technology Institute.

intestinal bacteria. Although it is prerequisite for a prebiotic ingredient to be a substrate for beneŽ cial bacteria, it is important to note that growth in pure culture does not necessarily mean growth of the same bacteria on the same substrate in the highly competitive environment of the colon. However, human ingestion of 3 times 10 g per day of D -tagatose for 13 days, was characterised by changes in

microbial population density, coliform bacteria were reduced, and lactobacilli and lactic acid bacteria were increased (14). In pig studies, D -tagatose altered the composition and population of colonic micro ora, as evidenced by increased numbers of D -tagatose degrading bacteria, increased fermentation capacity of D -tagatose and changes in SCFA proŽ le. In ×itro fermentation of

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colonic samples from pigs adapted to D -tagatose for 17 days showed 46 mol% of butyrate, compared to a 17 mol% from fermentation of colonic samples from pigs fed a sucrose control diet (12). Concentrations of butyrate in the caecum and colon of pigs were increased in a dose-response manner to ingestion of D -tagatose (13). Butyrate is not a major end product of fermentation in lactic acid bacteria, so obviously D -tagatose is a substrate for other enteric bacteria producing butyrate or one may speculate that the lactic acid produced by lactic acid bacteria is the substrate for butyrate producing bacteria. In conclusion, D -tagatose is only fermented by a limited number of the tested enteric bacteria and besides from one Clostridium species the positive strains belong to the lactic acid bacteria group, Lactobacillus and Enterococcus. None of the 11 tested enteric pathogens were able to metabolise D -tagatose. The high frequency of D -tagatose fermentation among enteric Lactobacillus and Enterococcus were conŽ rmed on testing dairy type lactobacilli and Enterococcus species. In addition high frequency of D tagatose fermentation was found in other lactic acid bacteria genera, Leuconostoc, Lactococcus, Streptococcus and Pediococcus, as well. None of the tested enteric or dairy type BiŽ dobacterium were able to ferment D -tagatose. The data presented in this article alone does not support any prebiotic potential for D -tagatose, but helps explaining the observed increased number of Lactobacilli and Lactic acid bacteria after ingestion of D-tagatose in humans (14). ACKNOWLEDGEMENTS We gratefully acknowledge Dr. Carl-Alfred Alpert, INRA, Laboratoire de Recherches sur la Viande, Domaine de Vilvert, France for discussion on lactic acid bacteria metabolism. The skilful technical assistance of Michala Sand is greatly appreciated.

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D -tagatose

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