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Journal of the Science of Food and Agriculture J Sci Food Agric 87:1717–1725 (2007)
Study on the effects of laminarin,
a polysaccharide from seaweed, on gut
characteristics
Christelle Devill ´
e,∗Myriam Gharbi, Guy Dandrifosse and Olivier Peulen
University of Liege, diGESD (Study Group of Digestive System), Centre of Immunology, B36, 4000 Liege, Belgium
Abstract: This study investigates whether laminarin (β1-3,β1-6-glucan), a polysaccharide from seaweed, exhibits
beneficial properties for human health by analysing its effects on intestinal parameters. Anaerobic batch culture
fermenters were used for the screening of the in vitro utilization of laminarin by the human gut microflora through
the monitoring of biochemical and microbiological parameters. Additionally, the influence of laminarin ingestion
on the composition of intestinal mucus (neutral mucins, sialomucins and sulphomucins) was studied in rats.
Laminarin was almost totally (more than 90% used) fermented after 24 h of incubation with human intestinal
bacteria. It was not selectively used by bifidobacteria and lactobacilli, but increased the production of propionate
and butyrate. Variations of mucus composition were observed in jejunum, ileum, caecum and colon, both in
lumen content and in intestinal wall, of rats after ingestion of this polysaccharide. Due to its effects on mucus
composition, laminarin could influence the adherence and the translocation of bacteria across the epithelial wall.
In conclusion, laminarin seems to be a modulator of the intestinal metabolism by its effects on mucus composition,
intestinal pH and short chain fatty acid (SCFA) production, especially butyrate.
2007 Society of Chemical Industry
Keywords: laminarin; polysaccharides; fermentation; prebiotic; short chain fatty acids (SCFA); mucus
INTRODUCTION
A substantial amount of the research into human
and animal nutrition concerns non-digestible carbohy-
drates exhibiting biological properties which may ben-
efit the host’s health. Several options have been inves-
tigated in attempts to improve bacterial metabolism
in a beneficial way.1–2 One strategy is to supple-
ment the diet with prebiotics. The term ‘prebiotic’
was introduced by Gibson and Roberfroid.3Prebi-
otics are defined as ‘non-digestible food ingredients
that beneficially affect the host by selectively stimulat-
ing the growth and/or the activity of one or a limited
number of bacteria in the colon, and thus improve
host health’.4–8 In general, the prebiotics promote the
growth of Lactobacillus and Bifidobacterium,whichare
viewed as being positive for host health and welfare,
because they inhibit gut colonization by pathogens and
are thought to exert protective effects against acute and
chronic gut disorders.1,9Moreover, the fermentation
of prebiotics leads to the production of lactic acid,
short-chain fatty acids (SCFA) and gases.10 Among
these metabolites, butyrate is known to be the main
energy source for colonic cells as it stimulates epithelial
cell growth.11
Brown seaweeds are a potential source of such bioac-
tive ingredients. Indeed, they contain large amounts
(about 40% of the dry matter) of polysaccharides
resistant to hydrolysis by human endogenous enzymes
and thus are considered as dietary fibres.12 These
carbohydrates have chemical, physicochemical and
fermentation characteristics which are not found
among higher plant carbohydrates. Moreover, the
approval of some seaweeds for human consump-
tion led to a renewal of interest in these sources of
dietary fibres and their properties.13 – 16 In these sea-
weeds, soluble fibre consists of laminarin (β1–3,β1-6-
glucan), fucans and alginates, whereas insoluble fibre
is essentially cellulose.17,18 Research was carried out
to investigate the fermentation and prebiotic effect
of laminarin and oligo-laminarin by human bacteria,
in vitro and in vivo, with gnotobiotic rats.13,14,18 The
results of these studies were relatively different and
need to be clarified. Moreover, few data are avail-
able compared to the interest in algal oligosaccharides
in human nutrition and as putative modulators of
intestinal function.
In this study, we investigated whether laminarin
exhibits biological properties that may benefit the
host’s health to determine its value as a functional food
ingredient. Its effects on different intestinal parameters
were evaluated: (1) the utilization of laminarin by
the human gut microbiota through biochemical
(pH, production of SCFA) and microbiological
parameters (total bacteria growth, selective growth
of bifidobacteria and lactobacillus), and (2) the
effects of laminarin ingestion on the distribution of
intestinal mucins (neutral mucins, sialomucins and
sulphomucins) in rats.
∗Correspondence to: Dr Christelle Devill ´
e, diGESD, University of Liege, B36 (+4), B-4000, Liege, Belgium
E-mail: cdeville@ulg.ac.be
Contract/grant sponsor: FNRS-Spadel Grant, Leon-Fredericq Foundation of the Liege University; contract/grant number: (n◦A 50/5 – VM – GR2)
(Received 30 March 2006; revised version received 4 September 2006; accepted 22 November 2006)
Published online 8 May 2007;DOI: 10.1002/jsfa.2901
2007 Society of Chemical Industry. J Sci Food Agric 0022– 5142/2007/$30.00
C Devill´
eet al.
EXPERIMENTAL
General reagents
All chemicals were purchased from Sigma-Aldrich
(Boornem, Belgium) and from VWR International
(Leuven, Belgium). Microbiological culture media
were obtained from BioTrading (Keerbergen, Bel-
gium).
Test substrates
Two types of laminarin were used in this study:
laboratory laminarin, extracted according to the
technique described by Devill´
eet al.15 and commercial
laminarin (Sigma L-9634) extracted from Laminaria
digitata. Laminarin is a polysaccharide of glucose
(from 26 to 31 glucose units), primarily poly(β-
glucose-1–3) with some β-(1–6) interstrand linkages
and branch points. Glucose was used as a non-
prebiotic control and fructo-oligosaccharides (FOS)
for its well-known prebiotic properties. FOS were
obtained from CoSucra (Fibrulose F97, Warcoing,
Belgium). FOS are extracted from chicory roots and
have a degree of polymerization from 2 to 30, with an
average of 5.
In vitro fermentation of laminarin by human
faecal bacteria
The inoculum was made from fresh faeces anaerobi-
cally collected from three healthy human volunteers,
aged between 25 and 35, who had not taken antibi-
otics for at least three months prior to the sampling
date and had no recent history of gastrointestinal com-
plaints. Details of the process are given by Wang and
Gibson.19 Faecal samples were mixed and homoge-
nized in 100 ml of sterile pre-reduced phosphate buffer
(Na2HPO4/KH2PO4,17.8gL
−1/13.6gL
−1,pH7)
to give a final concentration of 10% (w:v). Sterile
batch culture fermenters (100 mL) were filled with
90 mL of a pre-reduced basal culture medium, con-
taining the carbohydrate sources (1 g) and inoculated
with 10 mL of the faecal suspension (in duplicate for
each carbohydrate tested). The basal culture medium
consisted of peptone water (2.0gL
−1), yeast extract
(2.0gL
−1), NaCl (0.1gL
−1), K2HPO4(0.04 g L−1),
KH2PO4(0.04 g L−1), MgSO4.7H2O(0.01 g L−1),
CaCl2.2H2O(0.01 g L−1), NaHCO3(2.0gL
−1), cys-
teine HCl (0.5gL
−1), bile salts (0.5gL
−1). Tween
80 (2.0 ml L−1), phylloquinone (0.01 ml L−1)and
haemin solution (1.0 mL L−1) were added to the
medium and its pH was adjusted to 7 using HCl
(1 mol L−1). Fermenters were incubated at 37 ◦C.
Samples (5 mL) were removed initially (0 h) and after
6h, 24h and 48h for analysis. All experiments were
carried out in an anaerobic cabinet (Anaerobic Cab-
inet, Ruskinns Technology) containing H2–CO2–N2
(10:10:80, v:v:v). At the end of fermentation or at
the respective time intervals, fermentation fluid was
collected from the fermenters to analyse the pH value,
the turbidimetry (660 nm), the fructose-6-phosphate
phosphoketolase activity (F6PPK, EC 4.1.2.22) of
bifidobacteria, the laminarin and bacterial contents.
All tests were carried out in triplicate.
Turbidimetry
Turbidimetry was determined by absorbance mea-
surement at 660 nm with a spectrophotometer after
homogenization of each sample. The growth of bacte-
ria was evaluated by subtraction of the initial optical
density of the fermentation fluid.
F6PPK assay
F6PPK is specific to the genus Bifidobacterium and
is a key enzyme in the glycolytic fermentation of
this bacteria.20 This enzyme is commonly used for
the detection and identification of bifidobacteria. It
was measured as described by Scardovi,21 except
that hexadecyltrimethylammonium bromide (CTAB)
was used for cell disruption.22 In brief, bacteria
were washed twice with phosphate buffer [KH2PO4
(0.05 mol L−1)/cysteine HCl (500 mg L−1), pH 6.5]
and resuspended in this buffer. This suspension was
incubated with CTAB (450 µgmL
−1) for 5 min at
room temperature. Then, 0.25 mL solution containing
sodium fluoride (3 mg mL−1) and potassium iodoac-
etate (5 mg mL−1) and 0.25 mL of sodium fructose-
6-phosphate (80 mg mL−1) was added to the samples
and incubated at 37 ◦C for 30 min. Following this incu-
bation, 1.5 mL of hydroxylamine HCl (13 g L−1)was
added and the solution was incubated at room temper-
ature for 10 min. Finally, 1 mL of TCA ((150 gL−1),
v:v), 1 mL of HCl (4 mol L−1) and 1 mL of ferric chlo-
ride ((50 gL−1), w:v in 0.1 mol L−1HCl) were added
and the absorbance was measured at 505 nm.
Laminarin assay
Laminarin was estimated by measurement of glucose
concentration produced by the enzymatic hydrolysis
of laminarin.15 In brief, samples were incubated with
laminarinase of Trichoderma spp. (0.0025 U) for 1 h at
37 ◦C. The amount of glucose released was measured
according to the technique described by Hugget and
Nixon23 by using TGO reagent [tris-HCl (0.5 mol
L−1, pH 7), glucose oxidase (20 mg L−1), peroxidase
(900 U L−1), reduced O-dianisidine dihydrochloride
(0.16 mmol L−1), Triton X-100 (0.25%)]. Finally,
the samples were analysed by spectrophotometry at
420 nm. The initial concentration of laminarin was
considered as 100%.
Detection and enumeration of bacteria on selective agars
Bacterial contents were studied by culture on
selective agar. Samples (500 µl) of batch cultures
were serially diluted with pre-reduced peptone water
solution (5 g L−1) enriched with NaCl (2.5gL
−1)and
cysteine HCl (0.5gL
−1). Portions (20 µl) of each
dilution were then inoculated onto a range of agars
designed to be selective for predominant colonic
bacteria. The following growth media were used:
Plate Count Agar (PCA) for total aerobes, Reinforced
Clostridia Agar (RCA) for total anaerobes, Schaedler
1718 J Sci Food Agric 87:1717–1725 (2007)
DOI: 10.1002/jsfa
Laminarin and intestine biochemistry
agar with kanamycin (100 mg L−1) and vancomycin
(7.5 mg L−1)forBacteroides,24,25 Schaedler agar
with paromomycin (7 mg L−1)forBifidobacterium,26
de Man, Rogosa and Sharpe Agar (MRS) for
Lactobacillus.27 Agar plates were placed into an
anaerobic cabinet at least 16 h before inoculation to
allow them to pre-reduce. After incubation, bacterial
colonies were counted and morphology noted.
Analysis of the agar specificity by PCR
To analyse the specificity of selective agars, colonies
were sub-cultured and characterized to genus level
on the basis of PCR results after genomic DNA
extraction. Genomic DNA was isolated using a
WizardGenomic DNA Purification Kit (Promega,
Leiden, The Netherlands). DNA integrity was
checked by electrophoresis in agarose gel (1%)
containing ethidium bromide (0.5µgmL
−1).
PCR was performed on this genomic DNA in a
final volume of 50 µl. Three sets of primers obtained
from Eurogentec (Seraing, Belgium) were used in
separate PCR reactions to check the specificity of the
agars: Bac 1/2 specific for Bacteroides,28 Pbi F1/R2
specific for Bifidobacterium29 and Lac 1/2 specific
for Lactobacillus30 (Table 1). The PCR mixture was
composed of Mg2+free buffer, MgCl2(1.5 mmol L−1
for Bac 1/2, 3 mmol L−1for Pbi F1/R2 and 2.5 mmol
L−1for Lac 1/2), dNTP (200 µmol L−1), primers
(0.8µmol L−1each), DMSO (3% for Bac 1/2) and
Taq polymerase (Promega; 1 U). The PCR was carried
out with a thermal cycler (Thermolyne, Amplitron
II). The program began with an initial 95 ◦Cstepfor
5 min, followed by 35 or 40 cycles consisting of 95 ◦C
for 1 min, annealing temperature for 1 min and 72 ◦C
for 1 min. Final elongation was performed for 5 min at
72 ◦C. The PCR products (10 µL) were separated by
electrophoresis in agarose gel.
Analysis of fermentation end-products
Culture samples (1 mL) were centrifuged at 16 100 ×g
for 3 min to remove bacteria. Analysis of SCFA was
carried out by gas–liquid chromatography (GLC).
The GLC system consisted of a gas chromatograph
(DANI 8521) equipped with a flame ionization
detector. The chromatographic column used was
WCOT fused silica (25 mm ×0.32 mm) coated with
FFAP-CBdf 0.3. The column was used in an isotherm
mode at 140 ◦C and the injector and detector
temperature were set at 270 ◦C. The injection type
was split with a ratio of 1:50. Helium was used
as carrier gas, with a head pressure of 80 000 pa
bars. One millilitre of HCl with heptanoic acid
(internal standard, 10 mmol L−1) was added to
1 mL of samples to form free fatty acids. SCFA
were extracted with 2 mL of diethylether. A sample
of the diethylether extract (1 µl) was injected for
measurement. Acetic, propionic, butyric, isobutyric,
valeric, isovaleric, caproic and isocaproic acids were
assayed. Concentrations of SCFA were expressed as
millimoles per litre.31
Effects of laminarin ingestion on gut parameters
in rats
Animals
Wistar rats (Rattus norvegicus,n=20), initially aged
6 weeks, were used to study the effects of laminarin
ingestion on mucus composition. They were obtained
from the animal house of the Medicine Faculty of
the University of Liege. They were housed in an
environmentally controlled room at 23 ◦Cwitha
12 h light– dark cycle. They were fed with standard
laboratory diet (Carfill, Bruxelles, Belgium) and had
water ad libitum. Day of birth was reported as day
0. In our experiments, there was no body weight
difference between males and females. No distinction
between genders was made. The animal experiments
were approved by the Animal Welfare Committee of
the University of Liege. Throughout the study, the rats
were located in metabolism cages to avoid coprophagy.
Experimental design
Commercial laminarin (10 g kg−1of body weight)
dissolved in water was administrated orally with a
micropipette. Control animals were treated in the same
way, but received only the vehicle (water). Laminarin
was given for 14 days once daily at 9:00 pm. The body
weight of each rat was measured daily. The rats were
killed by cervical dislocation.
Organ preparation
The small intestine, caecum and colon were imme-
diately removed after sacrifice. The duodenum was
removed from the intestine up to the Treitz liga-
ment. The small intestine was divided in two pieces
of equal length called jejunum and ileum. Each organ
was flushed with cold saline (NaCl, 9 g L−1). The
intestinal contents were collected and homogenized
(Ultra-turrax, IKA T8). Organs were homogenized
Table 1. List of PCR primers used for the determination of the specificity of the selective agar selected for the enumeration of the different bacterial
genera
Primer sets Sequences 5–3Target Size (bp) Annealing temperature (◦C) References
Bac 1 TCC ACC TGG GGA GTA CGC CG Bacteroides 220 60 28
Bac 2 TAT GGC ACT TAA GCC GAC ACC
Pbi F1 CCG GAA TAG CTC C Bifidobacterium 898 50 29
Pbi R2 GAC CAT GCA CCA CCT GTG AA
Lac 1 CTC AAA ACT AAA CAA AGT TTC Lactobacillus 250 55 30
Lac 2 CTT GTA CAC ACC GCC CGT CA
J Sci Food Agric 87:1717 – 1725 (2007) 1719
DOI: 10.1002/jsfa
C Devill´
eet al.
in distilled water (1 g wet weight in 4.15 ml) using
the same homogenizer. All samples were immediately
placed at −20 ◦C.
Mucin assay
Neutral, acidic (sialomucins and sulphomucins)
and strictly sulphated (sulphomucins) mucins were
characterized by transposing the tests used in
histochemistry to mucins of the intestinal goblet cells,
as previously described.32,33 Mucins were assayed in
luminal contents and intestinal walls using mucins
from porcine stomach (Sigma) as mucin standard.
Linear range was 0 to 0.5 mg mL−1for each mucin
types. For solubilizing the mucins, the samples
were homogenized with an ultra turrax followed by
centrifugation at 1000 ×gfor 10 min. Histochemical
reagents (periodic acid-Schiff (PAS) Mantle and
Allen’s method,32 alcian blue (AB) pH 2.5 or AB
pH 0.5) were added to the diluted samples. Acidic
mucins were precipitated in the presence of modified
Carnoy fixative (ethanol–formaldehyde–acetic acid,
6:3:1). Centrifugation was carried out at 9500 ×g
for 10 min. The pellet was dissolved in water in the
presence of Triton X-100 and sonicated for 1 min.
Absorbance was read at 600 nm for the AB and at
570 nm for the PAS reaction. Each assay was carried
out in triplicate and the results were expressed as mg
of mucins per g of organ or content (fresh weight).
Statistical analysis
Results are reported as mean ±standard deviation
(SD). Statistical analysis was performed using Stu-
dent’s ttest when the experimental design included
two groups and with one-way analysis of variance
(ANOVA) when the experimental design included
more of two groups. A P-value below 0.05 was con-
sidered to be statistically significant.
RESULTS
In vitro fermentation of laminarin by human
faecal bacteria
The initial pH of the different experimental batch
cultures was 7. Table 2 shows the effects of the
substrates used on the pH value at different incubation
periods – pH decreased, with each substrate tested,
with increasing time of incubation. After 6 h of
incubation, the pH was higher in laminarin containing
medium [commercial (P<0.05) or extracted in
the laboratory (P<0.01)] than with glucose or
FOS. After 24 h and 48 h of incubation, the four
sources of carbohydrates gave similar decreases in
pH values. Table 3 shows the effect of the different
substrates on the turbidimetry at different incubation
periods. The absorbance increased with time and
with all carbohydrates tested. After a 6 h incubation,
turbidimetry was lower and thus bacterial growth faster
(P<0.001) with laminarin (commercial or extracted
in the laboratory) than with glucose or FOS, but
after 24 or 48 h incubation, no significant difference
was observed between glucose, FOS and commercial
laminarin. Bacterial growth in the batch system
incubated with laminarin extracted in the laboratory
was approximately half that obtained with the other
carbohydrates. Table 4 shows the content of laminarin
at the different incubation periods. No significant
decrease was recorded after 6 h of incubation. After
24 h, the main part of the laminarin was used by
the human faecal bacteria (92.24% for commercial
laminarin and 93.35% for laminarin extracted in
the laboratory). F6PPK activity increased when the
bacteria were cultured in the medium containing
glucose or FOS during the first 6 h of incubation. Then
F6PPK activity decreased gradually with time (results
not shown). This activity was constant and lower
for batch systems containing laminarin (commercial
or extracted in the laboratory) compared to control
batches.
Table 2. Effects of different carbohydrate substrates on the pH value at different incubation periods
Substrate 0 h 6 h 24 h 48 h
Glucose 7.00 ±0.07 5.75 ±0.07 4.70 ±0.14 4.20 ±0.14
FOS 7.00 ±0.07 5.95 ±0.14 4.55 ±0.14 3.90 ±0.14
Commercial laminarin 6.90 ±0.21 6.30 ±0.07* 4.60 ±0.07 4.50 ±0.14
Laminarin extracted 7.00 ±0.07 6.35 ±0.07** 4.75 ±0.07 4.65 ±0.21
∗P<0.05 compared with glucose, used as a non prebiotic control, at the same time of incubation.
∗∗ P<0.01 compared with glucose, used as a non prebiotic control, at the same time of incubation.
Table 3. Effects of different carbohydrate substrates on the turbidimetry at different incubation periodsa
Substrate 0 h 6 h 24 h 48 h
Glucose 1.198 ±0.008 1.788 ±0.004 2.119 ±0.003 2.179 ±0.009
FOS 1.230 ±0.013 1.780 ±0.002 2.154 ±0.002 1.305 ±0.004
Commercial laminarin 1.500 ±0.015 1.826 ±0.005* 2.391 ±0.003 2.450 ±0.006
Laminarin extracted 1.930 ±0.014 2.035 ±0.015* 2.351 ±0.007* 2.399 ±0.015*
aValues are mean ±SD of the optical density at 660 nm.
∗P<0.001 compared with glucose, used as a non prebiotic control, at the same time of incubation.
1720 J Sci Food Agric 87:1717–1725 (2007)
DOI: 10.1002/jsfa
Laminarin and intestine biochemistry
Table 4. Laminarin content in batch culture systems at different incubation periodsa
Laminarin 0 h 6 h 24 h 48 h
Commercial laminarin 100.00 ±1.33 100.00 ±0.58 7.76 ±1.53 1.27 ±0.09
Laminarin extracted 100.00 ±0.08 99.39 ±0.09 6.65 ±0.84 2.30 ±0.24
aLaminarin content is expressed as % of initial content (0 h).
Figure 1. Compared effects of glucose and commercial laminarin on
proportion of individual SCFA after 24 h fermentation in vitro with
human faecal bacteria.
Generally, high concentrations of total aerobes,
total anaerobes, Bacteroides, Bifidobacterium and Lac-
tobacillus were maintained in the four batch systems.
Significant differences were only observed with FOS,
which caused a significant increase (P<0.05) of lac-
tobacilli and a decrease of bacteroides compared to
glucose. No other difference was observed among the
carbohydrates (results not shown). SCFA production
was only analysed in the culture media containing
glucose or commercial laminarin. Significant differ-
ences were observed for SCFA production (Fig. 1).
Acetic, propionic and butyric acids represented the
major part of the SCFA produced (99.07% with glu-
cose and 98.03% with laminarin). The total amount
of SCFA produced (data not shown) was greater
(P<0.001) in the culture medium containing lam-
inarin (67.60 ±1.43 mmol L−1) than in the medium
containing glucose (51.56 ±1.48 mmol L−1). Lami-
narin led to a high proportion of acid acetic (63.96%)
and relatively high proportions of propionic (25.91%)
and butyric (8.16%) acids, whereas SCFA production
from glucose was characterized by high acetic acid
production (90.40%) and low propionic (5.88%) and
butyric (2.79%) acid proportions (P<0.001).
Study of the laminarin action on intestinal
characteristics in rats
There were no significant differences in the mean
body weight and the mean weight of intestinal organs
(lumen content and intestinal wall of jejunum, ileum,
caecum and colon) in the control group and in the
laminarin-treated group (data not shown).
The influence of laminarin ingestion on the quantity
and the distribution of mucins in luminal contents
and in homogenates of intestinal wall (jejunum,
ileum, caecum and colon) is shown in Figs 2 and 3,
respectively.
Mucin distribution in luminal contents
Acidic mucins were detected in ileum, caecum and
colon contents, but not in jejunum content, but no
significant difference was observed between the two
groups of rats (control and laminarin). Neutral mucins
were present in the whole intestine. The quantity of
neutral mucins was significantly lower (P<0.05) in
the group ingesting laminarin in the case of jejunum,
ileum and caecum contents and significantly higher
(P<0.05) in the case of the colon content. Strictly
sulphated mucins were not detected in the hindgut
lumen of the intestine of the two groups of rats.
Mucin distribution in intestinal walls
Acidic (sialomucins and sulphomucins), strictly sul-
phated and neutral mucins were detected in the whole
intestine. No significant differences in mucin distri-
bution (neutral, acidic and strictly sulphated) were
observed between the two groups of rats (control and
laminarin) except for a lower quantity (P<0.05) of
Figure 2. Quantity of acidic (top) and neutral (bottom) mucins
expressed in mg g−1of the hindgut content of jejunum, ileum,
caecum and colon of initially 6-week-old rats in control group ()and
in rats ingesting laminarin [143 mg kg−1body weight per day, for
14 days ()]. Gastric mucin preparation was used as standard. The
data are mean values for 10 rats per group, with their SD represented
by vertical bars. ND =not detected; ∗,∗∗ mean value was significantly
different from that for the control group (∗P<0.05, ∗∗ P<0.005).
J Sci Food Agric 87:1717 – 1725 (2007) 1721
DOI: 10.1002/jsfa
C Devill´
eet al.
Figure 3. Quantity of acidic (top), sulphated (middle) and neutral
(bottom) mucins expressed in mg g−1of organ (jejunum, ileum,
caecum and colon) of initially 6-week-old rats in control group ()and
in rats ingesting laminarin [143 mg kg−1body weight per day, for
14 days ()]. Gastric mucin preparation was used as standard. The
data are mean values for 10 rats per group, with their SD represented
by vertical bars. ∗Mean value was significantly different from that for
the control group (P<0·05).
acidic mucins in the caecum wall of rats ingesting
laminarin.
DISCUSSION AND CONCLUSIONS
The metabolism and composition of human intesti-
nal microflora, especially the production of SCFA
(butyrate) and the levels of Bifidobacterium and Lacto-
bacillus, play a role in maintaining intestinal health.10,11
Consequently, many attempts have been made to
increase their proportion in the intestinal tract by
probiotic or prebiotic ingestion. Laminarin is a stor-
age polysaccharide of seaweed which is not hydrolysed
by the human endogenous digestive enzymes.15 The
aim of the present work was to investigate whether
laminarin could exhibit biological properties that may
benefit the host’s health. As some dietary fibres have
been shown to qualitatively or quantitatively (or both)
modify the intestinal metabolism, the composition of
intestinal microflora4,34 and mucus,35 – 37 we studied
the effects of laminarin on these gut parameters. There
are a number of methods currently in use to determine
the fermentative and prebiotic properties of a putative
substrate – pure culture studies with a large range
of intestinal bacteria, fermentation vessels containing
mixed cultures, animal models such as human flora-
associated animals and human trials.2,38,39 In vitro
fermentation has been used in the present study.
Three bacterial genera were studied in this work, cho-
sen to represent interesting bacteria present in the
human microflora – Bacteroides, Bifidobacterium and
Lactobacillus. Bifidobacteria and lactobacilli because
they are known to be beneficial bacteria of the human
microflora, and bacteroides because they represent the
predominant genus in the human intestine.40 Lami-
narin used in our study allowed the growth of human
faecal bacteria that involved a decrease of the pH. After
24 h of incubation, there was no difference between
glucose, FOS and commercial laminarin. At the same
concentration, the growth observed with laminarin
extracted in the laboratory was lower. Two hypothe-
ses are advanced to explain this observation. First,
the laboratory-extracted laminarin could contain some
molecules able to slow down or partially inhibit bacte-
rial growth. Second, this observation could be due to
a difference in the structure of the laminarin extracted
at our laboratory.
Our results suggested that laminarin was used by
human faecal bacteria only after 6 h of incubation
and was almost depleted by 24 h. A delay seems
necessary for the use of laminarin as a source of
carbohydrate by the human faecal bacteria. Such a
lag phase has already been reported and has been
attributed to the time required for the induction
of the enzymatic machinery necessary to hydrolyse
and use this polysaccharide.18 The F6PPK assay
and the results of bacterial composition showed
that laminarin was not selectively fermented by
bifidobacteria and lactobacilli. Laminarin did not
exhibit prebiotic effect like FOS, but its fermentation
led to high proportions of propionate and butyrate
compared with glucose, which mainly produced
acetate. Our results were therefore in agreement with
those of Michel and his collaborators.14 They showed
that laminarin is used by human intestinal bacteria
with the production of propionate and butyrate. In
their study, this polysaccharide did not promote the
growth of lactobacilli and bifidobacteria, contrary to
the observations of Lahaye et al.13 Lahaye and his
collaborators showed that laminarin has a prebiotic
effect and promotes the growth of bifidobacteria. One
of the hypothesis to explain these differences between
their results is the involvement of β1 – 6 linkages in
the polysaccharide to have a prebiotic effect. This
hypothesis was not confirmed in our study since the
laminarin used contained β1–6 links.
1722 J Sci Food Agric 87:1717–1725 (2007)
DOI: 10.1002/jsfa
Laminarin and intestine biochemistry
The acidic pH resulting from laminarin fermen-
tation is probably caused by the high level of total
SCFA production. Both acidification and produc-
tion of SCFA are considered as beneficial for human
health.41 – 47 Indeed, a lower colonic pH is usually
considered as beneficial for inhibition of pathogenic
bacteria growth, mineral biodisponibility in the colon
and cancer prevention. A decrease in colonic pH might
reduce the risk of developing colonic cancer because an
inverse correlation between stool pH and colon cancer
risk has been reported.3,41,42 Fermentation of lam-
inarin produced SCFA, particularly propionate and
butyrate, that are considered to be beneficial.45– 47
In general, the fermentation of carbohydrates, par-
ticularly by bifidobacteria and lactobacilli, produces
SCFA, like propionate and butyrate.10 In our case,
the number of these bacteria was not influenced by
laminarin. Then either laminarin increased metabolic
activity of these bacteria without increasing their num-
ber or it increased the growth of other bacteria pro-
ducing these SCFA. More investigations concerning
butyrate and propionate production by laminarin fer-
mentation are needed. For example, are populations
of butyrate-producing bacteria,48,49 like Clostridium,
Faecalibacterium, Fusobacterium or Roseburia modified
by laminarin?
Butyrate currently attracts more attention than other
SCFA as a desirable metabolite of intestinal bacteria,
through its interactions with colonocytes.11 Moreover,
anti-neoplastic properties have also been attributed to
butyrate.11,45 In developed countries, the colon is the
second most common site of cancer that is a major
cause of sickness and mortality.50,51 The possible
biological properties of butyrate are thus of relevance
and it is therefore desirable to promote its production
in the colon, for example by laminarin ingestion.
A further aim of the study was to determine the
effects of laminarin ingestion on the composition of
intestinal mucus in vivo in rats. Mucus composition of
intestinal contents resulted from mucin secretion and
potentially mucolytic activity of the flora. Variations
in mucus composition may occur through diet, but
also through fermentation. Sharma and Schumacher35
postulated that the quantity and composition of
mucus produced by the epithelial cells are mostly
influenced by the diet in the small intestine and by
the intestinal flora in the large intestine. In our study,
laminarin ingestion seemed to induce some variations
of mucus composition both in the intestinal wall and
in the lumen content of jejunum, ileum, caecum and
colon of rats. The variations of mucus composition
in luminal contents could be explained by some
modifications of the composition of the microflora
after laminarin ingestion, as observed in vitro. Indeed,
research has shown that ingestion of dietary fibres
could induce activity of glycolytic enzymes, which
could alter mucin degradation.36,52 – 54 Moreover, the
fermentation of mucins by different bacteria could
induce a loss of some oligosaccharidic residues. The
methods used to measure the mucins are based
on histological identification methods in which the
staining is dependant on the presence of specific
oligosaccharides. So, the difference observed could
also be caused by a bacterial degradation of the
mucins. Modifications in the mucus composition
could be also correlated to the end-products of
fermentation (SCFA, lactic acid). Indeed, these acids
may induce a decrease in pH resulting in mucus
secretion. So, variations in the specific mucins present
in the mucosa, brought about by diet composition,
could be mediated by the fermentative bacterial
metabolites produced.10,11 Mucus contains different
types of mucins divided in two groups on basis of
their histological staining – neutral mucins and acidic
mucins (sulphomucins and sialomucins). As a result
of the decrease of neutral mucins, the acidic mucins
became predominant and could acidify the mucus.
Indeed, the physico-chemical characteristics of the
sialomucins and the sulphomucins are different from
those of neutral mucins – they make the mucus more
acid and viscous, increase the resistance of the mucus
to bacterial enzymes and are involved in protection
against bacterial translocation.55 Thus, laminarin
seems to modify mucin secretion or metabolism
(or both) and, by these effects, could influence the
adherence and translocation of bacteria across the
epithelial wall and protect against some infections.
However, more investigations are needed concerning
quantitative mucin variations. For example, assaying
the mucins after fractionation of the samples and
analysing the protein and carbohydrate composition
of mucus to highlight changes in protein–carbohydrate
ratio. Moreover, the thickness of the mucus layer could
also be studied to analyse secretion variations.
In conclusion, this study suggests that laminarin
is a potential modulator of intestinal metabolism and
would be promising as a functional food ingredient, by
its effects (direct or indirect) on mucus composition
and on the production of SCFA, essentially butyrate.
However, other investigations are needed and these
results have to be confirmed in humans.
ACKNOWLEDGMENTS
This research was supported by FNRS-Spadel
Grant (n◦A 50/5-VM – GR2) and Leon-Fredericq
Foundation of Liege University. The authors wish
to thank Dr Delcenserie (University of Liege) and Dr
Jonkers (University of Maastricht) for their help.
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