FEMSMicrobiologyEcology101 (1992)81-88
© 1992Federationof European MicrobiologicalSocieties0168-6496/92/S05.00
Publishedby Elsevier
F~'MSEC 00392
Estimation of short-chain fatty acid production from protein
by human intestinal bacteria based on branched-chain fatty
acid measurements
Medical Research Council. Dunn Clinical Nutrition Centre. Cambridge, UK
Received4 December 1991
Revisionreceived 10March 1992
Accepted20 March 1992
Key words: Branched-chain fatty acids; Short-chain fatty acids; Protein breakdown; Fermentation
1. SUMMARY
The impc.'-tance of protein breakdown and
amino acid fermentation in the overall economy
of the large intestine has not been quantitated.
We have therefore measured the production of
branched chain-fatty acids (BCFA) both in vitro
and in vivo in order to estimate the contribution
of protein to fermentation.
In vitro batch-culture studies using human faecal inocula showed that short-chain fatty acids
(SCFA) were the principal end products formed
during the degradation of protein by human
colonic bacteria. Approximately 30% of the protein broken down was converted to SCFA.
Branched-chain fatty acids (BCFA) constituted
16% of the SCFA produced from bovine serum
albumin and 21% of the S C F A generated when
casein was the substrate. B C F A concentrations in
Correspondence to: G.T. Macfarlane,MedicalResearchCouncil, DunnClinicalNutritionCentre, 100TennisCourt Road,
Cambridge,CB2 IQL, UK.
gut contents taken from the human proximal and
distal colons were on average, 4.6 and 6.3 mmol
kg-* respectively, corresponding to 3.4% and
7.5% of the total SCFA. These results suggest
that protein fermentation could pote,tiaily account for about 17% of the SCFA found in the
caecum, and 38% of the SCFA produced in the
sigmoid/rectum. Measurements of B C F A in portal and arterial blood taken from individuals undergoing emergency surgery, indicated that net
production of BCFA by the gut microflora was in
the region of 11.1 mmol day-*, which would
require the breakdown of about 12 g of protein.
These ,Jata highlight the role of protein iti the
colon and may explain why many colonic diseases
affect mainly the distal bowel.
2. INTRODUCTION
A wide range of substrates are potentially
available as sources of carbon and energy for
bacteria growing in the human colon. They are
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G.T. Macfarlane, G.R. Gibson, E. Beatty a n d J.H. C u m m i n g s
82
used as a marker to estimate SCFA production
from protein in the large intestine.
3. MATERIALS AND METHODS
3.1. Production of SCFA from proteins in batchculture
Washed-gut bacteria were obtained from fresh
faeces from 3 individuals as previously described
[13]. Portions (250 ml) of the cell suspension were
added to l-! glass fermenters which contained
250 ml of a medium which had the following
composition (g I-1): K2HPO4, 16.0; KH2PO4,
12.0; NaHCO 3, 10.0; CaC! 2 • 2H20, 0.2; MgSO 4 •
7H20, 0.8; NaCI, 7.0; either casein (BDH) or
bovine serum albumin. 20.0; cysteine, 1.2; haemin,
0.1. The following vitamins were also included
(mg 1-1): menadione, 2.0; biotin, 0.04; p.aminobenzoic acid, 0.1; vitamin B12, 0.1; thiamine, 0.2.
The mineral salts medium had been previously
autoclaved and cooled under high purity nitrogen
gas (Air Products), the filter-sterilised vitamins
were added when the medium had cooled.
Anaerobic conditions were maintained by sparging the cultures with nitrogen (2.4 1 h-1). Growth
temperature was 3"P'C and culture pH (7.0) was
controlled using a Modular Fermenter pH Controller (Gallenkamp). Samples (4 ml) were taken
periodically up to 72 h for chemical analyses.
3.2. Chemical measurements
Cells were removed from the samples by centrifugation (27000 × g, 8 min). Proteins (insoluble
in 10% (w/v) trichloroacetic acid (TCA)) and
peptides (soluble in 10% (w/v) TCAJ were measured by the Lowry method [i4]. SCFA, including
BCFA, were detected by gas chromatography using procedures described by Holdeman et al. [15].
These methods do not discriminate between isovalerate and 2-methylbutyrate.
3.3. Measurements of fatty acids in gut contents
Colonic contents were obtained from eight
sudden death-victims at autopsy, between 2 and 4
hours after death, as described by Cummings et
al. [16]. Samples were taken from the caecum,
ascending colon, transverse colon, descending
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principally substances that have escaped digestion in the small bowel and are both dietary and
endogenous in origin, consisting of resistant
starches Ill, plant cell-wall polysaccharides [2]
and a variety of mucopolysaccharides [3] and proteins [4].
Approximately 12 g of protcinaceous materials
are known to enter the colon each day in small
intestinal digesta [4]. However, it is likely that
colonic mucins and sloughed epithelial cells from
the gut mucosa contribute significantly to the
pool of organic nitrogen containing compounds
available for fermentation by the microflora.
These materials are broken down by gut bacteria,
to short-chain fatty acids (SCFA), branched-chain
fatty acids (BClZA), the gases CO 2 and H 2, and
ammonia, phenols, indolcs, amines and other
non-volatile organic acids [5].
Many of these products of fermentation are
absorbed by the host, and undergo a variety of
metabolic fates within the body. Some, such as
ammonia, phenols, indoles and amines are harmful [6], whereas others are beneficial, including
acetate, which is metabolised in the brain and
muscle [7,~] and butyrate, which is essential as a
source of energy for the colonic epithelial cells
[9].
Qualitatively, much is now known of the products of protein degradation formed by intestinal
bacteria, but little quantitative information is
available concerning the contribution made by
proteins and peptides to SCFA generation in the
colon. One method of approaching this problem
is to measure protein breakdown products, such
as BCFA, which are unique to the process.
Branched-chain fatty acids are formed by the
metabolism of branched-chain amino acids such
as valine, leucine and isoleucine. The corresponding BCFA products of these amino acids are
isobutyrate, isovalerate and 2-methyibutyrate respectively [10l. Considerable amounts of BCFA
can be produced in Sticldand reactions by many
clostridia [11], where the branched-chain amino
acids typically serve as the electron donors and
give rise to BCFA that are one carbon atom
shorter than the parent amino acid [12].
In this paper, we report in vitro and in vivo
studies in which the formation of BCFA has been
83
colon, sigmoid/rectum and immediately frozen
at - 2 f f ' C for later use. Slurries (10% (w/v) in
distilled water) were made with the samples,
which were subsequently centrifuged (27000 x g,
10 rain) to remove particulate matter and bacteria. Fatty acids were then analysed by gas chromatography as before.
3.5. Effect of carbohydrate on BCFA production by
colonic bacteria
Slurries (10% (w/v) in 0.1 M sodium phosphate buffer, pH 6.5) were made with fresh faeces from 2 individuals as described by Macfarlane
3.6. Effect o f carbohydrate on BCFA assimilation
by colonic bacteria
Slurries of faecal bacteria were made as described above, and placed into the 2 fermenters.
Each contained 20 mM NH4CI, 20 mM isobutyrate and 20 mM isovalerate. To test the effect
of carbohydrate on BCFA uptake, glucose was
added to one of the fermenters, to give a concentration of 100 raM. Samples were taken at zero
time, and after 24 h of incubation, for measurements of short chain fatty acids.
3. Z Dry weight measurements
These were made using methods described by
Keith and Herbert [21].
3.8. Chemicals
Unless otherwise indicated, all chemicals and
reagents were obtained from Sigma.
4. RESULTS
4.1. Production of SCFA from casein and bocine
serum albumin
Both casein (56%) and bovine serum albumin
(49%) were utilized by mixed populations of
colonic'bacteria. By weight about 31% of the
protein was converted to SCFA in the casein
fermenters, whereas 29% of bovine serum albumin appeared as fatty acids. The relative amounts
of the principal SCFA are shown in Fig. 1. On a
mmol basis, acetate was the predominant fatty
acid produced from both proteins, accounting for
approximately 50% of the total. BCFA consti-
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3.4. Determination o f BCFA production rates in
t'ivo
Samples of portal and arterial blood were obtained from thirteen individuals during emergency surgery following trauma at Baragwanath
Hospital, Soweto, South Africa. The bloods were
collected in lithium heparin (Diotech) and centrifuged at 2000 × g (5 rain). The plasma fractions
were retained and frozen :.~t - 2 0 " C until use.
After defrosting, 1 hal was freeze-transferred [17]
and then freeze-dried in a dessicator with phosphorus pentoxide (BDH), giving sodium salts of
the BCFA. The samples were acidified with 100
/zi phosphoric acid (1 M) and vorte~ mixed. They
were then immediately injected onto a l y e Unicam Series 204 gas chromatograph containing a
25 m, 0.53-ram i.d. fused silica-bonded phase 21
column (SGE, Australia), fitted with an 0.5-m
guard column containing deactivated fused silica,
to trap non-volatile components. The carrier gas
was helium, with an inlet pressure of 14 psi. The
injection port, colutnn oven and flame ionisation
temperatures were 200°C, 100*C and 250"C respectively. 2-Methylvalerate was used as an internal standard. The results were processed with a
Spectrophysics SP4270 computing integrator. As
with the method of Holdeman et al. [15], this
procedure does not permit separation of isovalerate and 2-metbylbutyrate. Daily absorption rates
of BCFA were estimated by using a portal blood
flow of 1 I rain -1 [18,19]. Thus, BCFA concentrations multiplied by 1440 (the number of minutes
in a day) gives a value for the daily production
rate.
et al. [20]. Aliquots (400 ml) were added to 2
identical glass fermenters (0.5-1 working volume),
containing 50 ml of buffer and 5 g Peptone water
(Oxoid). Fifty ml of buffer was then added to one
fermenter and 50 ml of a solution of gelatinised
soluble starch (BDH) to the other, to give a
concentration of 10 g 1- i The culture dry weights
at zero time were 8.3 mg m l - i ± 0.74 SD in = 4).
The cultures were then incubated for 28 h at
37"C. Culture pH (6.5) and anaerobic conditions
were controlled as before. Samples were taken
periodically for BCFA measurements.
6O
<[
u.
O
(n
m
:
0
200
50
40
5
30
4
~100
0,.
I=
a
¢,J
2
t,-
¢~
113
m
O
4o
~
2o
O ~
O
Proximal
colon
Distal
colon
Fig. 2. Total SCFA (open boxes) and BCFA (closed boxes)
concentrations in human colonic contents. Results are mean
values from 8 (proximal colon) or 6 (distal colon) individuals±
SEM.
0 ~4co/ere
C ~PeO~/on,~ete
4 ~ e e
,
-'*ate
Fig. l. Relative amounts of different SCFA produced by
mixed populations of intestinal bacteria grown on either bovine
serum albumin or casein as sole sources of carbon and nitrogen (1O g I-t). The bacteria were grown anaerobiealb' in
batch culture. Results are from samples taken after 72 h of
incubation and are mean values obtained from 3 individuals.
tuted 16% (8% isobutyrate, 8 % i s o v a l e r a t e / 2 methylbutyrate) and 21% (5% isobutyrate, 16%
i s o v a l e r a t e / 2 - m e t h y l b u t y r a t e ) of total S C F A
formed in bovine serum albumin and casein cultures respectively. O t h e r SCFA detected were
p r o p i o n a t e , b u t y r a t e , valerate, i s o c a p r o a t e ,
caproate a n d caprylate.
4.2. Fatty acid concentrations in different regions
of the colon
M e a s u r e m e n t s of fatty acids in gut contents
obtained from h u m a n sudden-death victims (Fig.
2) showed that total concentrations decreased
from a m e a n of 142 m M in the proximal colon
(caecum a n d ascending colon) to approximately
96 m M in the distal colon (descending colon and
sigrnoid/rectum). In contrast, B C F A concentrations increased from 4.5 to 6.3 raM. Although this
increase in B C F A was small in quantitative terms,
the results, when expressed as a percentage of
total SCFA, showed that relative proportions of
B C F A increased from a b o u t 3.4% of total S C F A
in the proximal colon to a r o u n d 4.9% in the
transverse a n d 7.5% in t h e distal colon (Fig. 3).
4.3. Estimation of SCFA p~duction from protein
in the colon
If It is assumed from the in vitro fermentation
results in Fig. 1 that B C F A comprise approximately one fifth o f all fatty acids g e n e r a t e d from
16
g
e
e
o
E~
a.
o
e
"
|
!
C
A
_
!
i
T
D
I
SIR
Region of e o l o n
Fig. 3. BCFA as a percentage of total SCFA in different
regions of the colon. C = caecum (n = 8), A = ascending colon
(n~8), T=transverse colon (n=7), D=descending colon
(n = 6), S/R = sigmoid rectum (n = 6). Results are mean values ± SEM.
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c
~
85
80
6O
- -
C
-
A
Region
b
¢p
i
i
T
D
of
i ....
S~R
¢.~
colon
Fig. 4. Theoretical percentage of' SCFA produced from protein in different regions of the eolon &sestimated from BCFA
concentrations. See legend to Fig. 3.
v
~.
v ~
Time
protein, it is clear from the values shown in Fig. 3
that the potential contribution m a d e by protein
towards S C F A production in the gut rises progressively from a b o u t 17% in t h e prox/mal colon,
to a r o u n d 3 8 % in the distal colon (Fig. 4).
¢.4. BCFA production by the colon
M e a s u r e m e n t s of B C F A in portal and arterial
bloods d e m o n s t r a t e d n e t production of isobutyrate a n d i s o v a l e r a t e / 2 - m e t h y l b u t y r a t e by the
gastrointestinal tract (Table 1). M e a n net daily
production of B C F A was estimated to be 11.1
mmol (range 25-49).
Table
1
BCFA production by the human gastrointestinal tract as indicated by concentrations in portal and arterial blood
lsobutyrate
Portal blood a
Arterial blood a
A/V difference a
Mean daily production b
21.1 (2.2)
15.5 (2.3)
5.5 (1.0)
7.9
lsovalerate/
2-Methylbutyrate
43.7 (5.8)
41.7 (7.7)
2.2 (3.0)
3.2
Samples were taken during emergency surgery. Results are
means of measurements made with 13 individuals. Values in
parathesis are SEM.
a ~ m o l I-;.
b A / V difference× 1440 (retool).
J
10
20
30
[hi
Fig. 5. BCFA pr(gluction by mixed populations of gut bacteria
grc~,,n in the presence (a) and absence (b) of carbohydrate (10
g I -I starch). P,.j,i~,ne water (10 g I t) was the amino acid
source in both fermenters. Isobutyrate. e, isovalerate. O.
Results are mean values obtained from 2 experiments.
4.5. Influence of carbohydrate on BCFA production
Mixed populations o f colonic bacteria prod u c e d b o t h isobutyrate a n d i s o v a l e r a t e / 2 methylbutyrate from p e p t o n e in stiffed-batch culture (Fig. 5). B C F A formation was more rapid in
t h e presence o f 10 g l-1 starch than in its absence. Total B C F A production differed little aft e r 28 h of incubation, however qualitative differe n c e s were evident in that although i s o v a l e r a t e /
2-methylbutyrate was the principal B C F A formed
in both fermenters, comparatively more isobutyrate was produced in the absence of carbohydrate.
4.6. lnjquence of carbohydrate on the assimilation
of isobufyrate and isocalerate by colonic bacteria
E x p e r i m e n t s to d e t e r m i n e w h e t h e r t h e
metabolism of B C F A by intestinal bacteria was
affected by carbohydrate indicated that in the
absence of glucose, 14% of the isobutyrate a n d
22% of the L~ovalerate was assimilated by the
bacteria. In the glucose fermenters, 14.5% of the
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i:ii
f'6
86
Table 2
Utilization of isobutyrate and isovalerate by mixed populations of human colonic bacteria
Time (h)
Concentration (raM)
Control
0
24
Test
lsobutyrate
lsovalerate
Total SCFA
produced a
lsobL.tyrate
Isovalerate
Total SCFA
produced ~
19.0
16.3
20.3
15.9
5.7
20.0
17. !
19.8
17.3
68.8
isobutyrate and 13% of isovalerate were utilized.
The production of SCFA by the glucose cultures
confirmed that the bacteria had fermented the
carbohydrate (Table 2).
5. DISCUSSION
The proximal colon receives food residues and
other substrates from the small intestine and is
rich in carbohydrate and protein. Fermentation is
most active in this region of the gut, as evidenced
by its higher SCFA concentrations (Fig. 2) and
acidic pH [16]. As digesta move distally through
the colon, carbohydrate becomes depleted, the
activities of saccharolytic bacteria are reduced
and SCFA concentrations decline.
Large amounts of proteins and peptides are
present throughout the colon however [20], and
the large intestine is a strongly proteolytic environment [22,23] which enables these polymers to
be mobilised as substrates for amino acid fermenting species [24]. High cell population densities of amino acid fermenting bacteria are present in the gut, and many species produce BCFA,
including eubacteria, Megasphaera elsdenii, saccharolytic and asaccharolytic bacteroides and a
range of anaerobic Gram-positive cocci [15].
Results in Fig. 1 show that SCFA were major
products of protein breakdown by intestinal bacteria. Different ratios of SCFA were produced
from BSA and casein which probably reflects the
different amino acid composition of the readily
digestible parts of the proteins as well as the
specificities of the proteases and peptidases involved in their hydrolysis. BCFA consituted a
significant proportion (approx. 20%) of total
SCFA produced which together with the in vivo
measurements (Figs. 2 and 3) enabled an estimate of the contribution of protein to overall
SCFA production in the colon to be made.
Because 95% of SCFA found in the large
intestine are absorbed by the host [25], these
estimations assumed that BCFA were assimilated
in a similar way to other SCFA. To confirm this,
BCFA were measured in portal bloods (Table 1).
The importance of taking blood samples during
emergency surgery is that patients would not have
previously been treated with ant~iotics, as would
occur with elective surgery. Arterial/venous differences in BCFA were calculated because these
fatty acids, are produced in muscle and peripheral
tissues, but blood in the portal vein comes directly from the large intestine. Total BCFA levels
were high in arterial blood (57 p.mol ! - 1 ) a n d the
A / V d,fference across the gut (7.7 /traol 1-1)
represents only 13% of total BCFA in portal
blood. This shows that while there is net production by the gut, BCFA, arising largely from muscle, are the major source to the liver in man. A
wide range in BCFA production values by the gut
was seen from - 2 5 to 49 mmol day-~. Net production of isobutyrate by the gut was seen in 12
out of the 13 subjects, but in 5 subjects, net
uptake of isovalerate/2-methylbutyrate was observed. The contrasting behaviour of the gut towards these BCFA may represent either much
greater production through fermentation of va-
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Bacteria were grown in batch cultures, Concentration of each BCFA was 20 mM at zero time. The control fermenters contained 20
mM NH4CI. The test fermenters contained 20 mM NH4CI and 100 mM glucose. Values are means from experiments made with
bacteria obtained from 2 individuals.
a SCFA excreted by the bacteria during the incubation, other than isobutyrate and isovalerate.
87
ing for 17% o f total SCFA in the proximal bowel
but 38% in the distal region. Since many products
o f putrefaction, such as a m m o n i a and phenolic
compounds, are toxic and potentially damaging to
host tissues, the data may help to explain why
many diseases of the large bowel seen in W e s t e r n
populations occur predominantly in the distal
colon.
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line, or a specific r e q u i r e m e n t for i s o v a l e r a t e / 2 methylbutyrate for leucine a n d isoleucine synthesis.
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SCFA formed, it can b e calculated from t h e daily
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whereas slightly less isobutyrate was produced in
the presence of carbohydrate (Fig. 5). Moreover,
the rate at which B C F A were formed was stimulated by carbohydrate. Carbohydrate availability
affects B C F A production by a n a e r o b i c bacteria in
different ways. For example, glucose represses
B C F A formation by d o s t r i d i a [26] and Megasphaera elsdenii [27], b u t stimulates the process in
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B C F A have b e e n r e p o r t e d to be highly stitaulatory to the growth of m a n y r u m e n b a c t e r i a
[28,29]. D a t a in Table 2 indicate t h a t isohutyrate
a n d isovalerate were n o t assimilated to a significant degree by h u m a n colonic bacteria after 24 h
of incubation. T h e addition of glucose, which
might have b e e n expected to increase the req u i r e m e n t for a m m o n i a and c a r b o n skeletons for
a m i n o acid synthesis, did not stimulate uptake o f
B C F A by the bacteria. In view of t h e availability
of pre-formed organic nitrogen containing compounds in the h u m a n colon [20], further studies
are n e e d e d to d e t e r m i n e w h e t h e r B C F A have a
major biosynthetic function in vivo.
In conclusion, we have used t h e occurrence o f
B C F A in colonic contents to estimate the contribution m a d e by protein to S C F A production in
the large intestine. O u r results show t h a t alt h o u g h carbohydrate fermentation p r e d o m i n a t e s
in the large intestine as a whole, the breakdown
of proteins by the microflora becomes quantitatively more i m p o r t a n t in the distal colon account-
88
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