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Bornet brouns 2002nutritional aspects short chai fructo oligo S

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DIGEST LIVER MS 2002;34lSUPPl.21:S111-40
Nutritional aspects of short-chain fructooligosaccharides:
natural occurrence, chemistry, physiology and
health implications
F.R. J. Bornet
F. Brounsl
Y. Tashiro2
V. Duvillier3
Fmm
Nutri-Health SA, Rueil-Malmaison, France;
‘Scientific Services Group, Vilvoorde,
Belgium & Nutrition and Toxicology
Research Institufe, Maastricht University
Maastricht, The Netherlands; 3 Meiji Seika
Kaisha Ldt,Kawasaki, Japan; 4 Beghin Meiji,
Neuilly-sur-Seine, France.
lddms fw cowmpndenw
Pmf. F. R. J. Borne& Nutri-Health SA, 3,
avenue Paul Doumer, 92500 Rueil-
Malmaison, France.
Fax: +33-l-47521596.
E-mail: frj. bomet@nucri-health-sa. corn
Short-chain fructooligosaccharides occur in a number of edible plants,
such as chicory, onions, asparagus, wheat.. They are a group of linear
fructose oligomers with a degree of polymerjsatjon ranging from n=l up
to
5 (oligosaccharides). Short-chain fructooligosaccharides, to a large ex-
tent, escape digestion in the human upper intestine and reach the colon
where they are totally fermented most/y to lactate, short chain fatty
acids [acetate, propjonate and butyrate), and gas, like dietary fibres. As
a consequence of their fermentation, their caloric value is approximate-
ly 2 Kcal/g. A faecal bulking effect of fructooligosaccharides has been
observed
in humans. An important property of short-chain fruc-
tooligosaccharides is the stimulation of bifidobacterial growth specifically
while suppressing the growth of potentially harmful species such as, for
example, Clostridium perfringens in
the
colon. It is associated with a de-
crease in faecal pH, an increase in faecal or colonic organic acids, a de-
crease in the production of nitrogenous end products in urine and stools,
a decrease in faecal bacterial enzymatic activities and a modification in
faecal neutral sterols. The short-chain fructooligosaccharides enhance
magnesium absorption in humans and have been shown, in animal mod-
els, to reduce colon turnout- development by enhancing both colon bu-
tyrate
concentratjons and local immune system effecters.
Digest Liver Dis 2002;34~Supp1.21:S111-20
Key words: bacteria; colon; short-chain fatty acid; short-chain fructooligosaccharides
Origin and compositional characteristics
By definition, oligosaccharides have a degree of polymerisation (DP) low-
er than 9. Short-chain fructooligosaccharides (SC-FOSS) are a group of lin-
ear Glucosyl c(( 1*2)(fructosyl),P(2+ 1)fructose polymers with a DP rang-
ing from n=l up to 5 (Fig. 1).
Natural
occurrence of
SC-FOSS
Short-chain fructooligosaccharides occur in a number of plants such as
onions, Jerusalem artichokes, asparagus, wheat, rye, and garlic ‘. Onion has
the highest content of SC-FOSS, ranging from 25-40% (dry matter basis) of
which 97% are SC-FOS (GFn, n<5). Garlic and chicory root have a low FOS
content (Table I). In Western countries, the average daily FOS intake of sc-
FOS from natural sources is about 1 g.
Chemical structure and composition of commercial FOS ingredients
SC-FOSS are produced on a commercial scale by two different processes, ei-
ther from sucrose using a food grade fungal fructosyltransferase, (ACTI-
LIGHT@, Beghin Meiji, Neuilly-sur-Seine, France), or from inulin by par-
tial hydrolysis using endo-inulinase (Orafti, Tienen, Belgium).
In SC-FOS synthesis from sucrose, the sucrose plays the dual role of fructose
Short-chain fractooligosaccharides and health
I
Table I. Natural occurrence and distribution of fructooligosaccharides (GFn, n<91 in edible plants. I
Substance %Fructan 6F2 6Fa 6h
PFS-8 Tetal
DS
i Onion
25-40
~ Wheat (Trit. Aest.1
1-4
Chicory
15-20
Jerusalem artich.
16-20
Garlic
25-35
DS: dry substance; DP: degree of polymarisation.
61%
30%
4%
25% 10%
13% 6%
5% 5%
presence of OP <9
presence of DP <9
Glucosyl a (I 9 2) fructose
(sucrose)
(fructosyl) “P (2 + 1) fructose
Gh
I-kestose rs’ F
‘3%
(SC-FOS : n = 1 w 3) IlyStOSO
GF4
fructosyl-nystose
Fig. 1.
General chemical structures of SC-FOS.
onor and fructose acceptor 2. The first reaction on two cially available FOSS are mainly composed of 1 -kestose
sucrose molecules leads to kestose and glucose. The ac- (GR), nystose (GF3) and fructosylnystose (GF4).
tion of the fructosyltransferase on kestose produces nys- Endo-Inulinase splits the glycosidic linkages of chico-
tose and on nystose, produces fructosylnystose. The re- ry inulin producing a mixture of Glucosyl
action is stopped to optimise the ratio between a( 1+2)(fructosyl),P(2+ 1)fructose with n=l to 6 and
GF2/GF3/GF4 at 37%/53%/10%. A chromatography step Fructosyl p(2+ l)(fructosyl),P(2+ 1) fructose with
ensures the purification of SC-FOS. The composition of
the commercial product is given in Figure 2. Commer- n=2 to 7 (Fig. 3). A chromatography step ensures the
purification of FOSS.
ACTILIGHT@ 950
(fvleioligc40, Nutraflora@)
G+F+GF < 5%
FOS: 95% f 2%
GF2: 37% f 6%
GE;: 53% f 6%
I-kestose
GFd: 10% f 4%
‘3%
nystose
-4
fructosyl-nystose
Fig.
2. Composition of commercial fructooligosacharide ingredients
from a sucrose source.
G+F+GF < 3%
FOS: 95% + 2%
+ GFz +
GFz +
GFd : 9%
GF5
t
GF6 + GF, : 8%
Fz +
Fs +
Fg: 58%
F5 f
F6 f
F,: 20%
GF,, + F. (m7): 2%
Food technological properties
of
se-FOS
The SC-FOS mixture has a taste profile which is simi-
lar to that of sucrose, without any cooling effect, but
with a 30% lower sweetness. The water-retention ca-
pacity is higher than that of sucrose. Being non-reduc-
ing sugars, SC-FOS do not lead to Maillard reactions.
They are stable at pH>3 and temperatures up to 130°C.
According to their technological characteristics, sc-
FOS may be used as ingredients in biscuits and cakes,
breakfast cereals and cereal-filled bars, ice and dessert,
dairy products, yoghurt and milk.
Methods to measure short-chain scFOCs in foodproducts
A recent survey conducted by Lee and Prosky 3 con-
cerning the definition of dietary fibre, supports the
view that oligosaccharides which are resistant to hy-
s112
F.A.J. Bernet et at.
drolysis by the alimentary tract, such as SC-FOS, are to
be considered as fibre.
The AOAC Dietary Fibre analytical method for the de-
termination of total dietary fibre involves three enzy-
matic digestion steps with a-amylase, protease and
amyloglucosidase. These enzymatic treatments do not
modify the SC-FOS structures. However, the AOAC
method does not measure SC-FOS because of their
ethanol solubility. Ouarne et al. 4 have developed a spe-
cific and reliable analytical method to measure SC-FOS
in food products. This method involves an invertase
hydrolysis step and detection of sugars using a Dionex
ion chromatograph. The minimal amount of quantifi-
able SC-FOS is 0.75 g per 100 g of food product.
Other methods for the measurement of SC-FOS are
the AOAC Method 997.08 s and the AOAC Method
999.03 h. Both methods use inulinase enzymes to hy-
drolyse FOSS to fructose and glucose. In the latter
procedure, sucrose is selectively quantified by using
a pure sucrase enzyme.
Nutritional aspects
The SC-FOSS have aroused interest in the past decade,
mostly because of their nutritional properties. Fructans,
to a large extent, escape digestion in the human upper
intestine and reach the colon where they are totally fer-
mented, mostly to lactate, short chain fatty acids (SC-
FAs) (acetate, propionate and butyrate), HZ and CO*.
The most important property of FOS is their ability to
stimulate bifidobacterial growth specifically while sup-
pressing the growth of some other species in the colon,
such as Clostridium pet$-&ens. The demonstration of
the potential health benefits of SC-FOSS constitutes an
active field of research in human nutrition.
Digestive fate of SC-FOSS
Numerous in vitro studies have been conducted in ani-
mal models and in humans, showing the indigestibility
of SC-FOS in the small intestine. Kestose (GR) and nys-
tose (GF3) are not significantly hydrolysed by pancreat-
ic homogenate ‘, purified sucrase-isomaltase complex ‘,
nor by small intestinal mucosa homogenate from ani-
mal or human 9. Long-term ingestion of SC-FOS did
not cause induction or suppression of the hydrolysing
enzymes in the rat small intestine ‘. In addition, SC-FOS
did not influence the transmural potential difference of
everted sacs prepared from the jejunum ‘O. When in-
jected intravenously (iv) in rats, SC-FOS are rapidly ex-
creted in urine without degradation, suggesting that
they are not used as an energy source in the body ‘.
The percentage of ingested SC-FOS reaching the colon
has been measured in six healthy volunteers using the
intubation and the slow marker method g. Of the in-
gested SC-FOS, 90% have been recovered at the end of
the ileum. Moreover, the percentages of the constitu-
tive SC-FOS (GR, GF3 and GF4) remain identical to
those of ingested SC-FOS, showing that unabsorbed sc-
FOS remain intact during small intestinal transit.
The utilisation of SC-FOS was studied in vivo in man by
a radiorespirometric method and in vitro by incubation
with human faeces ‘?. The studies showed that SC-FOS
were fermented by intestinal microorganisms mainly to
SCFAs and CO*. SCFAs are absorbed in the colon,
some are metabolised in the colon, the remainder are
metabolised in the liver and peripheral tissues 13. Com-
pared with other fermentable products, like cellulose,
pectin or lactulose, the fermentation of FOS produces
higher percentages of propionic and butyric acid ‘I.
Caloric value of SC-FOSS
SC-FOSS entering the colon are completely fermented
resulting in SCFAs, lactic acid, gases, bacterial mainte-
nance and growth, and heat dissipation 15. This leads to a
loss of energy for the host, which is estimated to be equal
to 50% of the total energy content of carbohydrate 16-‘*.
Molis et al. 9, using the ileal intubation method in
healthy subjects, estimated that the caloric value of sc-
FOS was 9.5 kJ/g, a value somewhat higher than the
6.3 kJ/g reported by Hosoya et al. I’. Those authors
used a radiorespirometric method in healthy subjects
ingesting lU-‘4C1 SC-FOS and also measured gas and
SCFA production from labelled SC-FOS in anaerobic
incubation with faeces. They postulated that SCFAs
were completely absorbed in the large intestine, but
they used a very low mean energy content for SCFAs
(10.0 kJ/g) to calculate the energy value of SC-FOS.
The heat produced by combustion of acetic, propionic,
and butyric acids is 14.6, 20.7 and 25.0 kJ/g, respec-
tively “. Accordingly, the available energy for the hu-
man body from acetic, propionic and butyric acids is
10.9-12.6, 15.5-17.6, and 18.8-21.3 kJ/g, respectively.
According to these figures, the energy value of FOS
should range between 8.4 and 9.2 kJ/g, which is close
to that found by Molis et al. y.
Eflects of SC-FOSS on glucose and lipid metabolism
The glycaemic, insulinaemic and fructosaemic re-
sponses to SC-FOS consumption have been studied in
healthy and diabetic patients 2”. The postprandial plas-
ma responses were not significantly modified after an
oral load of 25 g SC-FOS.
The effect of SC-FOS on glucose tolerance was as-
sessed by Yamashita et al. 2’. Eighteen non-insulin de-
pendent diabetic subjects received 8 g per day of sc-
FOS for 14 days. Fasting glucose levels were signifi-
cantly reduced by 15 mg/dl during a SC-FOS contain-
ing intervention diet. A recent double blind cross-over
study, in 12 healthy subjects I4 showed that over a pe-
5113
Short-chain fructordigosaccharides and health
riod of one month, daily intake of 20 g SC-FOS signif-
icantly decreased basal hepatic glucose production.
The mechanism by which SC-FOS influences glucose
metabolism is unclear. A possible role of SCFAs has
been put forward I3 22. Among the SCFAs produced,
propionate, in particular, was shown to affect hepatic
glucose metabolism in rats and in healthy subjects. A
recent study carried out on normal rats, fed for 4 weeks
with a diet enriched with sodium propionate (7.8 g/100
g food), showed a significant reduction in fasting blood
glucose 23. Long-term oral propionate in healthy sub-
jects (7.5 g sodium propionate/d equivalent to 78 mmol
propionate/d) decreased fasting serum glucose and
maximum insulin increment during a glucose-tolerance
test, as shown by Venter et al. 22. Todesco et al. 24 also
noted a decline in fasting glycaemia in 6 healthy sub-
jects which were supplemented with propionate (9.9 g
sodium propionate/day) for 7 days. Propionate is a glu-
coneogenerator and has been shown to inhibit gluco-
neogenesis from lactate and to stimulate glycolysis in
isolated hepatocytes 2s. Propionate enters the Krebs cy-
cle at the level of succinyl CoA. The inhibiting effect of
propionate on gluconeogenesis may be related to its
metabolic intermediaries, methylmalonyl CoA and suc-
cinyl CoA which are specific inhibitors of pyruvate car-
boxylase 26. In addition, propionate enhances glycoly-
sis, probably by depleting hepatic citrate 27 which is an
important metabolic inhibitor of phosphofructokinase.
Finally, propionate may also influence hepatic glucose
metabolism indirectly by lowering the plasma fatty acid
concentration, which, in itself, is known to be closely
related to the actual rate of gluconeogenesis ?*.
These results suggested that the effects of dietary libre
on glucose metabolism may be exerted, not only
through their action on the upper digestive tract (re-
duction of gastric emptying, lengthening of digestion
and absorption of digestible carbohydrates) but also
through their fermentation products.
A possible hypolipidaemic action of SC-FOS in hu-
mans was expected to be shown after observations in
animal experiments where propionate inhibits choles-
terol synthesis by inhibiting both 3-hydroxy-3-methyl-
glutaryl-CoA (HMG-CoA) synthase 29 and HMG-CoA
reductase j”. Accordingly, this effect has been studied
in non-insulin dependent diabetics 2’ and hyperlipi-
daemic outpatients 1’ 32 However, no hypolipidaemic
.
effects of SC-FOS (20 g/d) were observed in healthy
subjects . l4 The fact that human intervention studies,
thus far, have been unsuccesful, in contrast to animal
experiments, may be related to species differences.
BiJidogenic efSect of SC-FOSS
Unlike other undigestible sugars, such as lactose or
lactulose which are hydrolysed by a wide variety of gut
bacteria, SC-FOS are only fermented in vitro by a lim-
ited range of micro-organisms that include most
species of bifidobacteria (except B.
bifidum) 33-35.
In-
deed, bifidobacteria have relatively high amounts of p-
fructosidase, which is selective for the b-( 1,2) glyco-
sidic bonds present in SC-FOS. After SC-FOS hydroly-
sis, fructose serves as an efficient growth substrate for
the bifidus pathway of hexose fermentation, which is
almost exclusively carried out by bilidobacteria j6.
Numerous studies in humans showed that SC-FOS inges-
tion led to an increase of faecal bilidobacteria 3’ 33-3s 37-4d.
Recent studies 4’ 44 J5 have been conducted in healthy
subjects using a “control” group and a double or single
blind design. The main characteristics and results of the
studies conducted in humans are summarised in Table II.
The P-fructosidase activity in stools was determined by
Bouhnik et al. 44. The P-fructosidase activity signifi-
cantly increased during the 12-day period of SC-FOS
ingestion, and returned to the baseline level in the post-
ingestion period. A significant correlation between fae-
cal P-fructosidase activity and bifidobacteria counts
was observed in subjects receiving SC-FOS but not in
subjects receiving placebo.
More recently, Bouhnik et al. J5 performed a dose re-
sponse study on the effects of SC-FOS on intestinal flo-
ra modulation. Forty healthy volunteers were random-
ly divided into 5 groups. They ingested their usual di-
et and a supplement of SC-FOS for a period of 7-day (of
which the amount differed for each group: 0 g, 2.5 g, 5
g, 10 g or 20 g/d). Faecal bifidobacteria counts were
collected twice, before and at the end of the 7-day pe-
riod. Faecal bifidobacteria counts did not vary in
groups Go and G2.*. while they increased significantly
during SC-FOS ingestion in the Gs, Glo and G20 groups.
A significant correlation between the dose of SC-FOS
ingested and the faecal bifidobacteria counts was ob-
served at the end of the 7-day period.
The relationship between chain length of fructans and
the capacity to stimulate the growth of bifidobacteria
has recently been studied in healthy subjects. ACTI-
LIGHT@ (SC-FOSS, 100% GFn; n<5) and long-chain
fructans (Raftiline@ HP, content of GFn; n<9 lower
than 2%) were ingested for one week at a daily dose of
10 g and compared to a placebo (10 g/d maltodextrins)
by 3 groups each of 8 healthy volunteers. The growth
of bitidobacteria in stools was significantly stimulated
by ACTILIGHTO (increase observed for 7 out of 8 sub-
jects) but not by Raftiline@ HP (increase observed for
3 out of 8 subjects) nor by the placebo (increase in 1
out of 8 subjects) (Fig. 4). In a second set of clinical as-
says, the effect of 10 g/day ACTILIGHTO and 15 g/day
RaftilineO HP have been compared in 2 groups of 25
healthy volunteers during a two-week period. The p-
galactosidase activities in stools, that reflected the
functional activity of the flora, was measured after 1
and 2 weeks of ingestion of the two different fructans.
5114
F.R.J. Bnrnet et al.
Table II. Main characteristics and results of clinical studies conducted in healthy subjects on the prebiotic effects of short-chain FOS ACTILIGHT’.
hIthy
SllbjW3S
Id
6
23
27
(9x31
38
IO
IO
32
[8x41
Me
Iyeersl
-
73+9
36.8*9
25.2zt3.3
20-40
22-39
29.6
Se-FffS deffy
ingartion
w
6
8
;
5
8
4
12.5
2.5
5
:oo
Dolrtkle
f-1
30
14
14
14
14
14
14
12
i
8
8
Blla ceunt
in st$;D~Mf@h
Were After
9.6 9.8
8.8+1 .I 9.7dJ.5
9.8rtO.6 10.2*0.4
9.9kO.6 10.4*0.4
9.7i0.6 10.3zto.4
5.2io.s 6.2iO.6
8.3i1.8 9.4~t2.3
7.9*0.5 9.1kO.3
8.Oi.1 .I 8.2*1 .I
8.1+0.8 9.1*0.4
8.0&l .3 9.5zko.3
8.2rtO.S 9.5rt0.6
statbticsl
signfffeme
[P)
ns
<0.005
<0.05
<0.05
<O.Ol
<O.Ol
co.05
<O.Ol
Ji.05
co.02
<0.002
First Aether
yea6 ref.
Mitsuoka, 1986 31
Mitsuoka, 1987 34
Tokunaga, 1993 4o
Rochat, 1994 41
Williams, 1994 42
Bouhnik, 1996 @
Bouhnik, 1999 45
MS: not significant
The P-galactosidase activity was significantly in-
creased within the two-week period of ingestion of
ACTILIGHT@ but not with Raftiline@ HP (Fig. 5). The
results suggest that SC-FOSS induce a more potent ef-
fect on enzyme induction and bifidus growth than the
long chain fructans.
From currently available scientific evidence, it can be
concluded that stimulation of bifidobacteria growth is
usually associated with a decrease in faecal pH 33 34 4’
and caecal pH 46, an increase in faecal or colonic SC-
FA production 33 39, a decrease in C. perfringens 33 41
and a decrease in the production of putrefactive sub-
Bifidobacteria and immunopotentiating activity
It has been reported that bifidobacteria exert various
effects on immune system-related function, such as,
mitogenic activity 47, adjuvant activity 48 4y, promotion
of macrophages 47 so, stimulation of antibody produc-
tion 5’ 52 and antitumour effects 48 53. Lee et al. 54 tested
the immunopotentiating activity (i.e., to stimulate the
proliferative response of murine immune cells) of 27
1
i5 10
z
4s 9.5
03
.E F
9
ID1
g 0) 8.5 OD8
i-z8
a: 8
9 7.5
7 Placebo lnulin SC-FOS
mkSEM
n = 25 per group
DO D8 Dl5
Ii& 4. Comparison of the bifidogenic afect of IO g/d short-chain fruc-
;ooligosaccharides fACTILIGHT@ PS50) and IO s/d long chain fructan Fig. 5. Comparison of IO s/d short-chain fru@ooligosaccharides [AC-
TILIGHT@ P9501 and 15 g/d long chain fructan finulinl on functional
Iinuiinl in 3 groups of 8 healthy subjects after 8 days of a daily con- activity of faecal flora after 8 and 15 days of a daily consumption in
;umption. healthy subjects.
stances in urine and stools 33. The stimulation of bifi-
dobacteria growth in healthy subjects is inconsistently
associated with significant modifications of faecal bac-
terial enzymatic activities such as P-glucuronidase,
glycocholic acid hydroxylase, nitroreductase and
azoreductase, or modification of faecal neutral sterols,
and, in particular, cholesterol 43 44.
Short-chain fructooligosaccharides and health
micro-organisms in vitro. They showed that bifidobac-
teria strains have a higher immunopotentiating
activity
than do L. casei or L. acidophilus Bifidobacterium
adolescentis. M 100-4, originally derived from human
intestinal microflora, had the strongest mitogenic ac-
tivity on splenocytes and Peyer’s patch cells. This ac-
tivity was shown to be dose dependent and was in-
creased after disruption of the cells by sonication, in-
dicating the existence of an intra-soluble immunopo-
tentiator. Hosono et al. 55 have isolated this soluble
fraction, which contained galactofuranosyl residues as
characteristic constituents, that have not been previ-
ously detected in any other soluble fraction from
Gram-positive bacteria. Bifidobacterium adolescentis
is well known to be one of the major components of
the microflora in the intestines of healthy humans s6.
Butyrate in the colon
Increased interest in fermentable fibre has occurred in
recent years and research has focused on the colon and
the products of colonic fermentation, especially SCFA.
Colonocytes depend, to a large extent, on the availabil-
ity of SCFA derived from bacterial fermentation. Bu-
tyrate oxidation has been shown to make up for more
than 70% of the oxygen consumption by the human
colonic tissue 57, indicating that butyrate is the prime
energy substrate of the colonocyte. Impaired utilisation
of SCFA has been implicated in ulcerative colitis, sug-
gesting an energy-deficient state 58. Mucosal cells of
ulcerative colitis patients demonstrated an absence of
butyrate oxidation, reflecting a metabolic defect in the
mucosa. Moreover, Harig et al. 59 inferred that diver-
sion colitis represented an inflammatory state resulting
from a nutritional deficiency that may be effectively
treated with enema, containing butyrate.
However, butyrate is not only an energy source for
colonocytes.
Sodium butyrate (NaB) exerts an antiproliferative ac-
tivity on many cell types. It is also known as an induc-
er of differentiation of colon carcinoma cell lines.
There is also abundant evidence, from animal and cell
line studies, on the preventive effects of butyrate on
colon cancer and adenoma development. This has been
reviewed in detail by Smith et al. (j”. Butyrate has been
seen to induce gene expression as well as to influence
the rate of gene expression through its effects on post
translational modifications. Key modifications are nu-
clear histone acetylation and phosphorylation which
affect the cell chromatin structure as well as modifica-
tions in hormone receptor binding. Furthermore, bu-
tyrate has been shown to induce apoptosis and, as such,
to reverse the resistance of colonic cancer cells to pro-
grammed cell death.
Pen-in et al. 61 have characterised the modification of
the phenotype of NaB treated PROb rat colon adeno-
carcinoma cells. They focused their study on surface
oligosaccharides that could play a role in their tu-
mourigenicity. Blood-group H antigens, formed by the
addition of fucose on type 1,3 precursors, were less ex-
pressed on butyrate-treated PROb cells, while a(2+3)
linked sialic acids were enhanced. This phenotype was
maintained after NaB withdrawal, whereas cell growth
inhibition was lost. The decrease of H1,3 antigens
would be related to the lower activity of a(2+3) fuco-
syltransferase(s) and competition between fucoses and
sialic acid precursors, borne by CD44v. When subcu-
taneously grafted, NaB-treated PROb cells induced
significantly small tumours. That could result from a
more efficient host response, attributable to the pheno-
type the cancer cells acquired with transient in vitro
NaB treatment, since the lower level of H type I,3 anti-
gens was maintained in growing tumours.
Butyrate stimulates the immunogenicity of the cancer
cells ‘j*. The phenotype of the weakly immunogenic rat
colon cancer PROb cells was modified with sodium bu-
tyrate. After a 4-day in vitro NaB treatment, the lym-
phokine-activated killer cell sensitivity, the expression
of Major Histocompatibility Complex class I, and the
intercellular adhesion molecule 1 of PROb cells, were
increased in a dose-dependent manner,
Perrin et al. 62
tested the efficiency of interleukin 2 (IL-
2) and NaB, alone or in combination, against experi-
mental widespread carcinomatosis induced in rats by
intraperitoneal injection of 2 x 10h PROb colon carci-
noma cells. IL-2/butyrate combination resulted in cas-
es of complete cure of carcinomatosis with specific
protection against PROb cells. IL-2 secretion probably
leads to activation of non-specific effecters such as
natural killer and/or lymphokine-activated killer cells
resulting in a rapid clearance of otherwise immuno-
genic NaB-treated cells.
The complete regression of tumour masses may be at-
tributed, to a butyrate-induced decrease of tumouri-
genicity and an increase of immunogenicity of the can-
cer cells.
Thus, it may be advantageous to provide indigestible car-
bohydrates as an indirect source of butyrate to the large
bowel. Campbell et al. 63 evaluated, in rats, the effects of
selected indigestible oligosaccharides on caecal and fae-
cal SCFA concentration, pH, total large bowel wet
weight and wall weight, and concentrations of intestinal
microbiota. The duration of the study was 14 days. The
SC-FOS containing diet resulted in higher caecal butyrate
concentrations compared with the control, or with the
cellulose or xylo-oligosaccharide containing diets.
Effects of short-chain on colonic tumours in animal
models
A protective effect of all types of dietary fibre against
colon cancer is still questionable 64.
5116
F.R.J. Bernet et al.
To investigate the effects of different type of libres,
rats 6s and
Min
mice 66 have been used: chemically in-
duced and spontaneous cancer models. The relevance
of animal models, as compared to human colon tu-
mour studies depends on the criteria considered (j’.
Azoxymethane (AOM)-induced tumours are similar to
human tumours in many histological, biochemical,
immunological and cellular aspects; but many of the
tumours do not follow the adenoma to carcinoma pro-
gression, frequently arising de novo from flat mucosa.
This model permits the investigation of early stages of
carcinogenesis, the end-point being a consensual pre-
cancerous marker, the aberrant crypt foci. The mouse
model (Min mice) is a model for both familial adeno-
matous polyposis and sporadic colon cancer. The
Min
mice are heterozygous for a non-sense mutation of the
Apt gene, the murine homologue of APC. The
Min
mouse model adenomas are pertinent by their genetic
origin, but they are more frequent in the small bowel
than in the colon, as opposed to the human situation.
These studies provided
data
on later stages of colon
cancerogenesis, and the end-point was the number of
detectable tumours.
A two-part randomised blinded study in rats, mimick-
ing a prospective study in humans, was performed us-
ing a low fibre control diet (CD) and three high fibre
diets: starch free wheat bran (WB), type III resistant
starch (RS) and SC-FOS .
4h Using a randomised block
design, 96 inbred rats were fed for 16, 30 or 44 days to
determine the period of adaptation to the diet, fermen-
tation profiles, and effects on the colon, including mu-
cosal proliferation on day 44. Subsequently, 36 rats fed
the same diets for 44 days were injected with AOM and
checked for aberrant crypt foci 30 days after. After fer-
mentation had stabilised (44 days), only RS and sc-
FOS produced large amounts of butyrate, with a troph-
ic effect in the large intestine. No difference in the mu-
cosal proliferation between the diets was noted at this
time. In the subsequent experiment, one month later,
fewer aberrant crypt foci were present in rats fed high
butyrate producing diets (RS: p=O.O22, SC-FOS:
p=O.O43). Similar effects on a reduction of the number
of aberrant crypt foci in CFl mice treated with AOM
and fed diets containing SC-FOS and exogenous bili-
dobacteria, were published by Koo and Rao 46. Camp-
bell et al. evaluated, in rats, the effects of selected in-
digestible oligosaccharides on caecal and faecal SCFA
concentration, pH, total large bowel wet weight and
wall weight, and concentrations of intestinal microbio-
ta. The duration of the study was 14 days. The SC-FOS-
containing diet resulted in higher caecal butyrate con-
centrations compared with the control, or with the cel-
lulose or xylo-oligosaccharide containing diets.
The same fibre diets have been tested in
Min
mice aged
6 or 7 weeks (n=40) 63. Each group was fed
ad libidum
for 42 days either the control low-fibre diet (CD) or
one of the three high-fibre diets (WB, RS, SC-FOS).
Gut tumours and small intestine lymphoid nodules
were counted. Neither WB nor RS modified the num-
ber of tumours. However, SC-FOS dramatically re-
duced the incidence of colon tumours and concomi-
tantly developed gut-associated lymphoid tissue
(GALT). Interestingly, the SC-FOS effect was limited to
the colon; they were no significant differences between
diets in the number of tumours found in the small in-
testine suggesting strongly that events specific to the
colon were involved. A bifidogenic effect probably oc-
curred in the experiment because Howard et al. 68,
demonstrated that dietary supplementation with the
same SC-FOS enhanced the population of bifidobacte-
ria in mouse colon as soon as 14 days. To obtain an in-
sight into the GALT response to changes in the colonic
ecosystem, the authors looked at the lymphoid tissue
of the small intestine. Examination of the colon, for
that purpose, was impossible because treatment of this
tissue, to study colon tumours, made it impossible to
accurately evaluate colon lymphoid follicles. A signif-
icantly higher number (~~0.05) of macroscopically de-
tectable lymphoid nodules were noted in the small in-
testine with the SC-FOS diet. This suggests that the im-
mune system may play a role in inhibiting tumour for-
mation by eliminating cells that express antigens if
they are immunogenic enough to allow the expansion
of immune cells specific for these antigens.
To investigate whether T cell status may influence
colon tumour formation in Min mice fed a SC-FOS di-
et, Pierre et al. 69 have chosen to immunodeplete mice
with antibodies against target T cells (CD4+ and
CD8+), rather than natural killer (NK) cells, which do
not affect the incidence of intestinal neoplasia in Min
mice 7”. Min mice depleted of CD4+ and CD8+ lym-
phocytes developed twice as many tumours as
im-
munocompetent mice.
To investigate the response of the tissue to SC-FOS at
the effector molecule level, Bassonga et al. assessed
the expression of cytokines present in the colon. They
chose to study the mRNAs since certain cytokines (e.g.
IL- 15) are frequently not translated or secreted by rest-
ing cells. They used a multiprobe ribonuclease protec-
tion assay to study the expression of selected cytokines
in the colon of C57BL/6 and Min mice fed low fibre
diet (CD) or a SC-FOS enriched diet (SC-FOS).
Five cytokines were consistently detected regardless of
the animals or diet (IL-4, IL-5, IL- 13, IL- 15 and inter-
feron alpha (IFN a)). IL-4, IL-5 and IL-13 were ex-
pressed at low but comparable levels and were not sen-
sitive to the diet. IL-IO, IL-9, IL-6 and IL-2, were not
detected. The IL- 15 mRNA was frequently highly ex-
pressed in both Min groups but at a significantly high-
er level (p=O.Ol) in the Min group fed SC-FOS as
com-
s117
pared to the Min group fed CD. IFNo mRNA, when
detected, showed the same pattern of expression as IL-
15. The fact that IFNo appears to be modulated in the
same way as IL-15 supports the hypothesis that IL-15
could be secreted in an active form, since IFNo, a cy-
tokine produced by activated T-cells and which stimu-
lates cytotoxic activity, is a target of active IL-15.
In this context, the possibility that some indigestible
carbohydrates including probiotic stimulate immune
rejection of nascent tumours offers a new perspective
for the prevention of colon cancer and opportunities
for better understanding of GALT to colon cancer.
SC-FOSS and mineral absorption
Another observation of interest is that SC-FOS also im-
proves magnesium absorption by similar influences of
fermentation as those described for calcium. In con-
trast to the effect on calcium, which needs to be veri-
fied also in longer term human trials; a substantial
number of studies have shown an enhanced magne-
sium absorption in animals as well as in humans after
intake of NDO’s 72.
Increased magnesium absorption has also been shown
to enhance bone magnesium content 71 74 and is consid-
ered to be an additional important factor for the reduc-
tion of the risks of osteoporosis 72. Very recently, a
study conducted by Tahiri et al. 75, showed a significant
improvement of magnesium absorption in post-
menopausal women resulting from five weeks’ intake
of ACTILIGHP. The aim of this study was to investi-
gate the effect of moderate daily doses of SC-FOS (10
g/d), on intestinal absorption and status of Mg in post-
menopausal women without hormone replacement
therapy. Twelve healthy post-menopausal women aged
60+6 years (M&SD) received SC-FOS or sucrose
(placebo) treatments for 5 weeks according to a ran-
domised, double blind, crossover design, separated by
a washout period of at least 3 weeks. Subjects ingested
stable isotope 2sMg together with a faecal marker. Sub-
sequently, faeces were collected for 5 to 7 days. Mg
levels were assessed in plasma, erythrocytes and urine
at the beginning and at the end of each treatment.
These measurements allowed the determination of net
Mg absorption and Mg status. The results showed (Fig.
6) that the daily addition of 10 g SC-FOS to the diet in-
creased net Mg absorption by 12% (p<O.O5), from
30.2f5.0 (placebo treatment) to 33.9f7.2% (SC-FOS
treatment), (MBD, ~~0.05). This increase in Mg ab-
sorption was accompanied by an increase in plasma
Mg level as shown by the 8% increase in 25Mg enrich-
ment (~~0.05) and led to a net Mg retention. Accord-
ingly, the authors concluded that the ingestion of mod-
erate doses of SC-FOS did improve intestinal Mg ab-
sorption and status.
Given the important role of appropriate magnesium
~30.016
30.26.0 33.9i7.0
I-------l---
Placebo SC-FOS
Fi6. 6. Individual changes in intestinal Mg absorption after intake of
sc-FOS or palcebo [modified from Tehiri et al., 2001 751
Short-chain fructnoligosaccharides and health
SIIS
availability to achieve a high peak bone mass in early
life as well as the reduction of the rate of osteoporosis
in later life, it can be concluded that NDO, especially
SC-FOS, is an excellent ingredient for inclusion in
functional foods that target persons with an inadequate
dietary Mg intake.
Conclusion
SC-FOSS have aroused interest in the past decade,
mostly because of their nutritional properties. To a
large extent, SC-FOS escape digestion in the human up-
per intestine and reach the colon where they are total-
ly fermented, mostly to lactate and short chain fatty
acids (acetate, propionate and butyrate). The most im-
portant property of SC-FOS is their ability to specifi-
cally stimulate bifidobacterial growth while suppress-
ing the growth of some other bacterial species in the
colon, such as Clostridium perfringens. The prebiotic
effect and functional activity of fructans is dose-de-
pendent and chain-length related. The demonstration
of the health benefits of SC-FOS is a challenge for the
next decade.
List of abbreviations
AOM: Azoxymethane; CD: control diet; UP: degree of polymerisa-
tion; DS: dry substance; GAL1 gut-associated lymphoid tissue;
HMG-CoA: hydroxy-methyl-gluteryl-CoA; IL: interleukin; IFNa: inter-
pheron alpha; iv: intravenously; NaS: sodium butyrate; NK: natural
killer; RS: resistant starch; SCFA: short chain fatty acid; SC-FOS:
short-chain fructooligosaccharide; WB: wheat bran
fR.J. Eornet et al.
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5120
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The ability for such lymphocyte activation as growth of the spleen and Peyer’s patch (PP) cells from unprimed BALB/c mice, and of lymph node (LN) cells from mice immunized with hen’s egg ovomucoid (OM) was assayed by using sonicated microorganisms. The occurrence of immunopotentiating activity was strikingly dependent on the properties of individual strains, rather than on the species or microbial culture conditions. All the tested Bifidobacterium strains, especially B. adolescentis M101-4, showed strong mitogenic activity in those assays, and also enhanced the production of the anti-OM antibody by LN cells, while they did not induce the growth of thymocytes. Similar results were obtained from experiments using germ-free (GF) mice. These results suggest that the activation with B. adolescentis M101-4 was not due to any secondary stimulation of lymphocytes primed with bacteria in the gut or environment, but to direct or indirect activity toward B cells that was intrinsic to the strain.
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Neosugar, a fructooligosaccharide mixture, was tested for genotoxicity in three assays: (1) microbial reverse mutation assays in Salmonella typhimurium (Ames assay) and Escherichia coli WP2 uvr A, (2) the L5178Y mouse lymphoma TK± mammalian cell mutation assay, and (3) an assay for the induction of unscheduled DNA synthesis (UDS) in human epithelioid cells (HeLa S3). Each assay was conducted at a wide range of dose levels, both with and without metabolic activation. Test results gave no indication that neosugar possessed any genotoxic potential. The carcinogenicity and chronic toxicity of neosugar were examined in Fischer 344 rats. Rats were fed diets containing 0, 8000, 20,000, or 50,000 ppm neosugar for 104 weeks. No dose-related effects on survival, growth, hematology, blood chemistry, organ weights, or nonneoplastic lesions were observed. The incidence of rare and spontaneous tumors was comparable between control and neosugar treatment groups, with the exception of pituitary adenomas in male rats. In light of the background incidence of this tumor and an equivocal dose-response trend, it is unlikely that neosugar treatment is related to the incidence of pituitary adenomas in male rats. The results of this study indicate that neosugar is nonmutagenic and that rats are not adversely affected by chronic neosugar exposure.
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Despite an increasing awareness of the relationships between the intestinal microbiota and health, there are few definitive guidelines about dietary interventions to adventitiously influence species composition of the microbiota. Therefore, standard microbiological methods were used to determine changes in the abundance of selected bacteria in anaerobic faecal samples from ten adult human volunteers who consumed 4 g of neosugar, a mixture of short-chain fructooligosaccharides, daily for 2 wks. The diet was not otherwise controlled. Total anaerobic counts increased or remained relatively stable in nine subjects. The percentage of total bacteria counts represented by aerobes increased over ten-fold, but enterics declined by over 90 per cent (from 2.3 per cent to <0.2 per cent). Although bifidobacteria increased from 1 3 per cent to 6.8 per cent of the total bacteria, there was wide individual variation in responses; bifidobacteria were not detected in two subjects at either date (<104 colony forming units/g faeces). Lactobacilli increased in six of the subjects, but were not a significant component of the microbiota at either date (<00001 per cent). Individual variation in responses to supplemental neosugar are probably caused by differences in diet, initial microbiota, and environmental conditions. The results demonstrate that supplementing the diet with neosugar influences the relative abundances of selected bacteria with some of the changes consistent with those considered advantageous.