European Journal of Clinical Nutrition (2006) 60, 847–852
& 2006 Nature Publishing Group All rights reserved 0954-3007/06 $30.00
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ORIGINAL ARTICLE
15
N-excretion of heat-killed Lactobacillus casei in
humans
KD Wutzke and V Sattinger
Children’s Hospital, Research Laboratory, University of Rostock, Rostock, Germany
Objective: In the present study, Lactobacillus casei (L. casei) labelled with 15N was used to follow the metabolic fate of orally
administered heat-killed 15N-labelled L. casei (15N-L.casei) in humans.
Design: Experimental study.
Setting: University of Rostock, Children’s Hospital, Research Laboratory.
Subjects: Twelve healthy adults aged 23–32 years.
Intervention: The subjects received 36 mg/kg body weight heat-killed 15N-L.casei and 500 mg Lactose-[13C]ureide together
with breakfast. Expired air samples were taken over 14 h, whereas urine and faeces were collected over 2 days. A blood sample
was taken after 2 h. 13C- and 15N-enrichments were measured by isotope ratio mass spectrometry (SerCon, UK).
Results: The orocaecal transit time (OCTT) was reached after 4.1 h. The urinary 15N-excretion was 9.3% of the ingested dose,
whereas the faecal excretion was 65.1% of the ingested dose. After 2 h, 15N-enrichment of supernatant, fibrinogen, and plasma
protein precipitate amounted to 254, 11, and 2 p.p.m. excess, respectively.
Conclusions: In comparison to the OCTT of 4.1 h, 15N-enrichment in urinary ammonia and urinary total nitrogen already began
to rise 30 min after 15N-L.casei ingestion, indicating that 15N-L.casei is rapidly digested in the small bowel. This is confirmed
by 15N-enrichments of blood plasma fractions. The ingestion of heat-killed 15N-L.casei led to a total excretion of 74.4% of the
ingested 15N-dose.
European Journal of Clinical Nutrition (2006) 60, 847–852. doi:10.1038/sj.ejcn.1602389; published online 1 February 2006
Keywords:
15
N-labelled Lactobacillus casei;
15
N-excretion; lactose-[13C]ureide; orocaecal transit time
Introduction
Lactobacillus casei (L. casei) is a Gram-positive, facultative
anaerobic member of the commercially important lactic acid
bacteria and possesses a strictly fermentative metabolism
with lactic acid as the major metabolic end product (Kandler
and Weiss, 1986; Axelsson, 1998). L. casei is commonly
found in probiotic dairy foods such as viable yoghurts.
L. casei also complements the growth of Lactobacillus
johnsonii (La1). For development of potential health benefits
to the consumer, the consumed yoghurt bacteria need to be
Correspondence: Professor KD Wutzke, Children’s Hospital, Research
Laboratory, University of Rostock, Rembrandtstrasse 16/17, D-18055
Rostock, Germany.
E-mail: [email protected]
Guarantor: KD Wutzke.
Contributors: VS was principal investigator and doctoral candidate of KDW.
Received 26 June 2005; revised 11 November 2005; accepted 2 December
2005; published online 1 February 2006
viable. Nevertheless, comparisons have been made between
viable and dead probiotic bacteria reported in various studies
assessing the clinical efficacy of heat-killed lactobacilli,
for example, in the treatment of diarrhoea (Xiao et al.,
2003), in the decrease of Helicobacter pylori colonisation
(Cruchet et al., 2003) or in the oral rehydration therapy
(Simakachorn et al., 2000). However, the metabolic fate of
dead lactobacilli remained unknown. In a recently published study, we investigated the metabolic fate of viable La1
doubly labelled with 13C and 15N after oral ingestion in
humans. Our findings showed that 50% of both stable
isotopes were excreted in breath, urine, and faeces. In
continuation of these investigations it was of interest to
evaluate the effect of heat killing of Lactobacilli when used
for oral ingestion.
The aim of the present study was to follow the metabolic fate of orally administered heat-killed 15N-labelled
L. casei (15N-L.casei) during the passage through the
human intestine in correlation to orocaecal transit time
(OCTT).
15
N-excretion of Lactobacillus casei
KD Wutzke and V Sattinger
848
Methods
Preparation of 15N-yeast extract
The preparation of 15N-labelled Saccharomyces cerevisiae
yeast from 15NH4Cl (99.0 atm%, Campro Scientific, Berlin)
has been previously described in detail (Wutzke et al.,
1983, 1992). A total of 125 g (wet weight) of 15N-labelled
yeast cells suspended in 1 l distilled water were killed and
hydrolysed by heating at 1001C for 45 min. The remaining
cells were removed by centrifugation. The supernatant
autoclaved at 1211C for 15 min was used as 15N-yeast extract
(Table 1).
Culture of lactobacilli
L. casei lactobacilli were obtained from ‘Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH’, Braunschweig, Germany. The organisms were inoculated into 50 ml
PY-bouillon (peptone yeast extract medium) and incubated
at 371C for 2 days. All ingredients except vitamins and
ascorbic acid were autoclaved at 1211C for 15 min. Solutions
of vitamins and ascorbic acid were sterilised separately
by filtration. After mixture of the filtrated and autoclaved
solutions, the medium was adjusted to pH 6.8 with 10%
NaOH (Wutzke and Oetjens, 2005).
After incubation, 10 ml of L. casei containing PY-bouillon
were centrifuged at 4000 r.p.m. The resulting sediment
suspended in 5 ml 0.9% NaCl solution was used as an
inoculum (8 109 colony forming units (cfu)) for 1 l of
15
N-L.casei broth medium (Table 1). After incubation at 371C
for 72 h, 15N-L.casei was separated from the culture medium
by centrifugation at 4000 r.p.m. and was purified by washing
4 times with 0.9% NaCl solution. The viable microorganisms
were centrifuged at 14 000 r.p.m. Thereafter, the cell sediment was heated for 15 min at 1001C in an autoclave. The
N-content in the aqueous phase was measured by using an
elemental analyser. The N-extraction caused by the heatkilling procedure was found to be merely 0.7%. The
microscopic examination of the gram stain preparation
showed approximately 95% intact cell walls. Therefore, they
Table 1 Composition of
15
N-yeast extract
Glucose
Ascorbic acid
K2HPO4 3 H2O
MgSO4 7 H2O
MnSO4 2 H2O
NaCl
FeSO4 7 H2O
Ca-pantothenate
Biotin
Tween-80
B vitamin mixture
Distilled water ad
L. casei
European Journal of Clinical Nutrition
15
N-labelled Lactobacillus casei broth medium
1.0 l
50.0 g
50.0 g
12.5 g
0.5 g
50.0 mg
50.0 mg
25.0 mg
1.0 mg
0.25 mg
5.0 ml
0.5 ml
5.0 l
o1.0 mg
were finally stored without centrifugation at 201C in a
freezer. Reactivating tests of the heat-killed 15N-L.casei cells
failed on both agar plates and PY-bouillon.
Subjects
Twelve healthy adults (five female, seven male, mean age:
26.2 years, mean body weight: 67.5 kg, mean body mass
index: 26.2 kg/m2), volunteered for this study. None of the
subjects were receiving any medication or had a history of
gastrointestinal diseases. All subjects were in good health
throughout the study and none of the subjects complained
about any gastrointestinal problems.
The testing protocol was approved by the Committee on
Ethics of the Faculty of Medicine of the University of
Rostock.
Study protocols
Subjects were studied after an overnight fast. All volunteers
received a continental breakfast made up of two wheat
flour rolls, honey, and coffee beginning at 0745 hours.
All volunteers received an individually standardised regular
lunch made up of two wheat flour rolls, tomato soup, and
two small sausages at noon. No other food was allowed
until 4 h after breakfast. Baseline breath, blood, urine, and
faeces samples were collected at 0730 hours, 30 min before
the ingestion of both 15N-L.casei and lactose-[13C]ureide
(13C-LU) to determine the baseline abundance of 15N
and 13C.
The subjects received a predosage of 3 200 mg unlabelled
lactose ureide at 1200, 1600 and 2000 hours on the day
before the ingestion of 13C-LU (Wutzke et al., 1997; Gohr,
2004; Wutzke and Glasenapp, 2004). During breakfast,
15
N-L.casei and 13C-LU were administered simultaneously
as a single oral pulse labelling at 0800 hours in a dosage of
35.8 mg 15N-L.casei per kg body weight and 500 mg 13C-LU
per subject stirred in 125 g L. casei yoghurt (Danone).
The unlabelled commercial yoghurt was heated for 15 min
at 1001C before ingestion. Two expired breath samples
obtained by direct exhalation into Exetainerst were collected in 30 and 60 min-intervals over a period of 14 h for
measuring 13CO2-enrichment (Wutzke et al., 1997).
Total urine volume was collected at 0.5, 1, 2, 4, 12 and 24h intervals over a period of 48 h after 15N-L.casei administration. The urine fractions were pooled between the time
intervals. Faeces scraped from polyvinyl film were also
collected quantitatively over 2 days.
Sample preparation before isotope ratio mass spectrometry
analysis
The sample preparation before isotope ratio mass spectrometry (IRMS) analysis has been recently explained in detail
(Wutzke and Oetjens, 2005). Urine volumes and faecal
masses were homogenised, recorded and stored at 201C
until analysis (Wutzke et al., 1992). Ammonia was separated
15
N-excretion of Lactobacillus casei
KD Wutzke and V Sattinger
849
in vessels by micro diffusion, following alkalisation of the
urine with NaOH and trapped in boric acid. (Faust et al.,
1981; Wutzke et al., 1992).
A blood sample was taken 2 h after 15N-L.casei administration at 1000 hours. After centrifugation of the citrated
blood, the plasma was used for fibrinogen extraction
with CaCl2 solution (Faust et al., 1981). For plasma protein
precipitation, the remaining fibrinogen-free plasma was
treated with Na2WO4. The soluble supernatant was removed
by centrifugation at 4000 r.p.m. (Faust et al., 1981). Fibrinogen, plasma protein precipitation, and supernatant were
stored at 201C until analysis (Wutzke and Oetjens, 2005).
Analytical techniques
Isotope ratio mass spectrometry. The solid (faeces) and liquid
samples were burned in an elemental analyser (SL) at 9001C
to CO2, H2O and total-15N2 before the 15N-measurement.
The 15N-enrichments in 15N-L.casei, fibrinogen, plasma
protein precipitate, supernatant, urine, faeces, as well as
the 13C-enrichment in breath were measured by isotope ratio
mass spectrometry (Tracer mass 20–20, SerCon, Crewe, UK)
as detailed previously (Barrie et al., 1989; Wigger and
Wutzke, 2004; Wutzke and Glasenapp, 2004; Wutzke and
Oetjens, 2005). The data were expressed either as enrichment
d 13C and d 15N over baseline (DOB), as atom percent excess
(APE), as parts per million excess (p.p.m. exc) (Slater et al.,
2001), or as percentage of cumulative urinary and faecal
15
N-excretion (PCE).
Percentage cumulative urinary and faecal 15N-excretion
The percentage cumulative 15N-excretion (PCE) is the
product of the urinary or faecal 15N-excretion (E) in mmol,
respectively, times 100 divided by the 15N-excess dose (D) in
mmol, respectively.
Results
All results are quoted as mean7s.d.
Yield of 15N-L.casei
The yield of bacterial wet mass was 1 g/l broth medium
(3 1011 cells). The 15N-enrichment of 15N-L.casei amounted
to 82.2 atom %, respectively.
Breath
CO2-signals were observed in breath beginning 30 min
after 13C-LU ingestion. The maximum 13CO2-enrichment of
10.4 DOB was reached after 9 h and was still detectable after
14 h (Figure 1). According to our definition, the measured
OCTT was 4.171.7 h (Figure 1).
13
Urine and faeces
Figure 2 shows the 15N-enrichment in urinary total-nitrogen
(N) and ammonia. The mean maximum 15N-enrichments in
total-N (0.019 APE) and ammonia (0.033 APE) were reached
after 3 h. The urinary PCE shows that 9.372.1% of ingested
dose was excreted 48 h after 15N-L.casei administration.
Figure 3 shows that 1.0% (according to 10.9% of the
total-15N recovery after 48 h) is renally excreted before
reaching the OCTT, whereas 8.3% (according to 89.1%) are
excreted after 4.1 h. The faecal 15N-excretion amounted to
65.1% of ingested dose, 2 days after 15N-L.casei ingestion.
Thus, 74.4% of the ingested 15N-dose was excreted in urine
and faeces. The numbers of L. casei cfu/g in wet faeces were
not counted.
Blood plasma fractions
The measured 15N-excess-enrichments amounted to 254791
in supernatant, 11732 in fibrinogen, and 275 p.p.m. exc
PCE ¼ E100=D:
D ¼ 0.745 mmol 15N/g 15N-L.casei (wet weight) ¼ 0.0267
mmol 15N/kg body weight.
OCTT
For measuring the OCTT, we used the lactose-[13C]ureide
13
CO2-breath test by predosing with unlabelled lactose
ureide (Heine et al., 1995; Wutzke et al., 1997; Wutzke
and Glasenapp, 2004). In 2004 our Rostock group applied a
new, simplified and cheaper predosing protocol. It could
be demonstrated that the predosing with 3 200 mg unlabelled lactose ureide is high enough to induce the
enzyme activity and to give reliable results (Gohr, 2004).
The OCTT was calculated from the time interval between
13
C-LU intake and the detection of a significant and
sustained 13CO2 rise in breath of two DOB or more (Wutzke
et al., 1997).
Figure 1 Mean 13CO2-enrichment in breath and the resulting
orocaecal transit time after lactose-[13C]ureide ingestion.
European Journal of Clinical Nutrition
15
N-excretion of Lactobacillus casei
KD Wutzke and V Sattinger
850
Figure 2 Mean
urinary ammonia.
15
N-enrichment in urinary total nitrogen, and
Figure 3 Mean percentage cumulative renal
correlation to the orocaecal transit time.
15
N-excretion in
in plasma protein precipitates, respectively. The fibrinogen and plasma protein enrichments did not differ
significantly.
Discussion
The human gastrointestinal tract (GIT) possesses a very
complex population of microorganisms, known as gut
microbiota, that interact with each other and with the host.
Estimates assess the count of different types of microorganisms in the gut at 400 and the total number of bacterial
cells at 1014; a dimension that far exceeds the total number
of human beings on the earth.
Probiotics are defined as viable microorganisms, including
Lactobacillus species, such as L. johnsonii and L. casei that may
beneficially affect the host upon ingestion by improving the
balance of the intestinal microflora (Fuller, 1989; Lee and
European Journal of Clinical Nutrition
Salminen, 1995; Pfeiffer and Rosat, 1999; Oozeer et al., 2002).
L. casei is an adaptive species, and can be isolated from raw
and fermented dairy products as well as from the intestinal
tract of humans (Kandler and Weiss, 1986). L. casei has
an industrial application as a human probiotic and as
an acid-producing starter culture for milk fermentation
(Fox et al., 1998).
During the passage through the GIT, viable and dead
yoghurt bacteria are exposed to the acidic conditions of the
stomach, followed by the hydrolytic enzymes and bile salts
of the small intestine (Meydani and Ha, 2000). Mainville
et al. (2005) investigated the in vitro digestion of Bifidobacterium infantis, La1, Lactobacillus rhamnosus and other strains by
using a reactor model simulating stomach (pH 2, presence of
pepsin) and duodenum (pancreatin or bile salts) conditions.
They observed that a few strains were not able to survive in
both reactors. Recently published findings revealed that
approximately equal amounts of 13C and 15N were excreted
in faeces (39.9 and 37.6%, respectively) after ingestion of
doubly stable isotope labelled viable La1 lactobacilli in
healthy adults, whereas the urinary excretion amounted to
1.3 and 12.4%, respectively (Wutzke and Oetjens, 2005).
Thus, total excretion of both isotopes was approximately
50% of the ingested dose. However, the metabolic fate of
orally ingested dead lactobacilli remains open. In vivo tests in
humans aimed at the investigation of the digestion of heatkilled lactobacilli are of great interest with respect to some
important findings of these probiotic bacteria (Simakachorn
et al., 2000; Cruchet et al., 2003; Xiao et al., 2003). Therefore,
in the present study we investigated the metabolic fate of
heat-killed 15N-L.casei after oral administration in correlation to the OCTT. Owing to the relatively high costs for
[U-13C6]glucose we refrained from the doubly labelling of
L. casei.
The 15N-L.casei cells and 13C-LU were given simultaneously during breakfast to represent the transit time of
the meal. Furthermore, the 15N-L.casei was ingested together
with heat-killed unlabelled L. casei yoghurt to obtain a better
distribution in the meal. When considering the results
obtained after 15N-L.casei and 13C-LU ingestion, the time
lag between the 15N-increase in urinary total nitrogen (N)
and urinary ammonia and the rise of 13CO2 in breath,
respectively, became obvious: in comparison to the prompt
15
N-enrichment in urinary-N and ammonia 0.5 h after
15
N-L.casei administration, the 13CO2-onset appears approximately 3.6 h later, clearly indicating that 15N-L.casei is
rapidly digested and absorbed in the small bowel and in
the colon (Figures 1, 2 and 3). The corresponding prompt
15
N-enrichments of the blood plasma fractions evidenced
that 15N was incorporated in the metabolic pool 2 h
after 15N-L.casei ingestion. In comparison to the plasma
protein precipitates, higher 15N-enrichments were measured
in fibrinogen, a protein with a high turnover. However, the
highest 15N-enrichment was observed in the 15N-urea, free
15
N-amino acids and other soluble 15N-substrates containing
supernatant, indicating the release of 15N-amino acids after
15
N-excretion of Lactobacillus casei
KD Wutzke and V Sattinger
851
15
N-L.casei digestion and absorption of its degradation
products. Therefore, it can be assumed that a substantial
portion of ingested 15N-L.casei is rapidly digested in the
small intestine and that the metabolites are incorporated
into human tissue.
When comparing the data obtained after ingestion of
doubly labelled viable La1, the differences to the present
study with heat-killed 15N-L.casei become obvious: the faecal
15
N-excretion of heat-killed 15N-L.casei was clearly higher in
comparison to the data derived from viable La1 (65.1 vs
37.6% of ingested dose, respectively) whereas the urinary
15
N-excretion was in the same order of magnitude 9.3 and
12.4% of ingested dose, respectively. Figure 3 shows that
the major part of 15N is renally excreted after reaching the
caecum. Nevertheless, due to the delayed renal 15N-excretion
it is not possible to differentiate between small intestinal and
colonic recovery from the data. Two days after 15N-L.casei
ingestion, the 15N-enrichment in urinary-N had not reached
the baseline level, indicating that additional 15N is renally
excreted over time. It can be assumed that a part of the 15N is
incorporated and recycled in the large protein pool of the
human body. Therefore, the delayed 15N-excretion leads to
an underestimation of the excreted 15N label and to an
overestimation of the incorporated 15N label, respectively.
In comparison to the data of the present study, the lower
faecal 15N-excretion observed in our previous study with
viable 13C-,15N-labelled La1 reflected the colonisation effect
of the probiotic cells in the gut. The higher the part of viable
cells is able to adhere to the intestinal mucosa the lower
their faecal excretion. The delayed degradation of the
viable cells adhering to the mucosa could lead to a prolonged
15
N-recycling, resulting in a similar or higher urinary
15
N-recovery from viable lactobacilli in comparison to heatkilled cells. Nevertheless, it is difficult to compare the
15
N-excretion data of both studies since two different strains
have been used. Owing to the total combustion of
15
N-L.casei to 15N2 in the SL, the origin of the stable isotope
15
N excreted in faeces remains unknown. It may be excreted
as intact dead cells or in form of metabolic degradation
products of 15N-L.casei such as indigestible 15N-protein or
15
N-nucleic acids.
The OCTT of 4.1 h measured by using 13C-LU was in
agreement with the data of 3.7 h calculated by the raffinose
H2 breath test (Wutzke and Oetjens, 2005). After oral
ingestion of 15N-labelled bifidobacteria in infants similar
results were observed. The renal and faecal 15N-excretion
ranged between 9.2–17.4 and 10.8–19.6% of ingested dose,
respectively (Heine et al., 1992).
Our combination of measuring the urinary and faecal
excretion of 15N-labelled metabolic degradation products of
heat-killed 15N-L.casei in correlation to the OCTT is a
novelty. Further studies with heat-killed doubly labelled
La1 and viable 15N-L.casei are in preparation.
Our findings will contribute to the understanding of the
digestion and elimination of viable and dead lactobacilli
when consumed in humans.
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