Coles, 2005

Animal Feed Science and Technology
123–124 (2005) 421–444
In vitro digestion and fermentation methods,

including gas production techniques, as applied to
nutritive evaluation of foods in the hindgut of
humans and other simple-stomached animals
L.T. Coles a , P.J. Moughan a,∗ , A.J. Darragh b
a Riddet Centre, Massey University, Private Bag 11-222, Palmerston North, New Zealand
b Institute of Food, Nutrition and Human Health,
Massey University, Private Bag 11-222 Palmerston North, New Zealand
Abstract
For the purposes of food evaluation, in vitro digestion/fermentation methods are ethically supe-
rior, faster and less expensive than in vivo techniques, whilst still offering a degree of animal–food
interaction that pure chemical analysis lacks. One such in vitro fermentation method is the in vitro
gas production technique, which utilises the relationship between degradation and fermentative gas
production to evaluate the nutritional parameters of foodstuffs. Several different methodologies have
been proposed for the gas production technique, each varying in its complexity, shortcomings and
benefits. Although the gas production technique has been used almost exclusively with ruminants, it
may also be of value for nutritive evaluation of foods for man and other monogastric animals. The
benefits of the technique include being able to run large batches simultaneously at low cost, the ability
to measure fermentation kinetics of soluble as well as insoluble fractions of food, and the ability to
easily make relative comparisons among different foodstuffs. This contribution reviews the in vitro
gas production technique, and in vitro hindgut digestion assays generally, for their application in
predicting in vitro hindgut digestion and fermentation in humans and monogastric farm animals. It is
concluded that currently available in vitro digestion methods of relevance to human food evaluation
Abbreviations: DF, dietary fibre; DM, dry matter; GI, gastrointestinal; NSP, non-starch polysaccharides; OM,
organic matter; RS, resistant starch; SCFA, short chain fatty acids; VFA, volatile fatty acids
∗ Corresponding author. Tel.: +64 6 350 6100; fax: +64 6 350 5671.
E-mail address: [email protected] (P.J. Moughan).
0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.anifeedsci.2005.04.021
422 L.T. Coles et al. / Animal Feed Science and Technology 123–124 (2005) 421–444
lack standardisation, in vivo validation and justification to support their specific methodology, and
have not been tested with a wide range of fermentative substrates.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Gas production; Fermentation; Digestibility; Monogastric; Hindgut
1. Introduction
Historically, and with regard to evaluating digestibility and availability of dietary en-
ergy in humans, in vitro digestion methods have focused primarily on upper tract digestion,
the hindgut being considered of little nutritional significance (Dobbins and Binder, 1981;
Cummings, 1983; Ramakrishna et al., 1990). However, the need for accurate in vitro meth-
ods to study digestion and fermentation in the hindgut of humans has become increas-
ingly apparent given the recently recognised role of the hindgut in nutrition and gut health
(Cummings, 1996; Williams et al., 2001a).
This review addresses in vitro methods, including the in vitro gas production technique,
for the study of hindgut digestion and fermentation in humans and, where appropriate, as a
means of comparison, in monogastric farm animals. The need for and purpose represented
by in vitro digestion methods is discussed. The design features of batch in vitro fermen-
tation methods are outlined along with a discussion of the criteria that a sound method
should fulfill, and critical assessment of current in vitro hindgut digestion/fermentation
methods.
2. The need for in vitro hindgut digestion/fermentation methods
In vitro hindgut digestion/fermentation methods for humans have been developed pre-
dominantly for pathophysiological studies, and specifically as a tool for greater understand-
ing of colon cancer, its dietary causes, and possible prevention. The role of dietary fibre
(DF) and its fermentation has been of considerable interest.
In addition to its physiological roles in maintenance of health and gut function, the
dietary fibre component of food has a nutritional role. For humans on a typical Western
diet low in dietary fibre, hindgut fermentation contributes around 3–11% of maintenance
energy needs (Cummings, 1983; McNeil, 1984; McBurney et al., 1987; McBurney and
Thompson, 1989; Cummings and Macfarlane, 1991), while for pigs, the value is more
significant and may range from 7 to 40% (Yen et al., 1991; Breves et al., 1993; Freeman et al.,
1993).
As diets rich in dietary fibre become more commonplace, a suitable means of studying
and describing their fate in the digestive system is necessary. It is difficult and expensive
to study nutrient digestion and fermentation in the human hindgut directly and there are
important differences in hindgut function among different species of simple-stomached
animals making the choice of an in vivo model difficult. Thus, there is an important potential
role for in vitro techniques.
L.T. Coles et al. / Animal Feed Science and Technology 123–124 (2005) 421–444 423
3. Application and limitations of in vitro hindgut digestion/fermentation methods
Simulation of the gastrointestinal (GI) tract function of any animal species presents a
great challenge (Moughan, 1999). The complex microbial environment of the large intestine
makes this task especially demanding when attempting to develop a rapid and simple hindgut
in vitro digestion method.
In assessing an assay, it needs to be asked what the purpose of developing such a method
is, especially in terms of the context in which it will be used. Specifically, there is a need to de-
termine whether the primary concern is repeatability, rapid turnaround of results and relative
accuracy (i.e., ranking), or absolute accuracy. Whilst the latter consideration is of consider-
able value to researchers, the former address the primary aim of a routine laboratory in vitro
digestion method for evaluating feedstuffs and foods. The ideal in vitro digestion method
would have the ability to provide highly accurate results in a short time. However, in reality it
must be accepted that, at present, it is assumed that a degree of accuracy is often sacrificed for
rapid results, and emphasis is placed on the assay’s ability to rank digestibility of foods rela-
tive to one another. Any in vitro method is inevitably going to fail to match the accuracy that
may be achieved by actually studying a food in vivo (Fuller, 1991). Specifically, it is not pos-
sible to simulate influx of endogenous compounds to the digestive tract and their subsequent
digestion and absorption, and it is difficult to replicate the effect of anti-nutritional factors
and subtle interactions between the host, the food and the bacteria present in the digestive
tract.
That in vitro methods have limitations and drawbacks does not discount them from
being a valuable research tool. Even in vivo digestibility methods have their own set of
shortcomings and difficulties depending on how outputs are classified. Also, the accuracy
of the in vivo method itself will impact the benchmark for in vitro results. The avail-
ability of accurate in vivo data is crucial for critical evaluation of any potential in vitro
method.
Whilst they may lack the accuracy of more complex in vitro digestion/fermentation
methods, the simplest in vitro methods are generally those suited to routine
laboratory application. This is partly because, by their nature, they are easier to com-
plete in any standard laboratory without the need for specialised equipment or mate-
rials and specially trained staff. Additionally, the more complex a method, the greater
the possibility that errors are introduced into it through multiple steps in the method-
ology, and through operator error and inter-laboratory differences. Thus, repeatabil-
ity both at an intra- and inter-laboratory level becomes important. If a method is to
be used for standard evaluation purposes in different parts of the world where com-
parisons are required, it is essential that both repeatability and reproducibility are as-
sured.
4. The monogastric hindgut
To understand the rationale underlying an in vitro hindgut digestion/fermentation

method, and to identify possible merits and shortcomings, it is of value to briefly discuss
large intestinal physiology and the fermentation process.
4.1. Hindgut physiology
Anatomically, the human large intestine is devoid of the well developed caecum present
in other monogastric animals such as the rat and, to an even greater extent, the pig, which
also exhibits extensive sacculation and elongation of the large intestine. Like the rumen, the
large intestine of simple-stomached animals is essentially a fermentation chamber where
material is degraded by bacteria numbering approximately 1010 –1012 /g gut contents in
humans (Macfarlane and Cummings, 1991). The profile of the large intestinal microflora
is diverse and, although the human tract contains several hundred species, there are some
30–40 anaerobic bacterial species that constitute 99% of the total microbial population, the
rest consisting of other facultative organisms (Drasar and Hill, 1974; Finegold and Sutter,
1978; Finegold et al., 1983).
By the time undigested food reaches the hindgut, it will have been in the human body
for about 2–6 h. This length of time is relatively short when compared to the 20–80 h, on
average, that food will reside in the adult human colon (Burkitt et al., 1972; Cummings et
al., 1992; Williams et al., 2001a).
4.2. Substrates for fermentation
The large intestine is constantly supplied with undigested dietary components and other
materials of endogenous origin, such as the host’s enzymes, mucus and desquamated gut
mucosal cells. A large part of the undigested diet at the end of the ileum will be comprised
of non-starch polysaccharides (NSP), a major component of dietary fibre. Dietary fibre is
a term that has historically been open to interpretation and subject to a raft of definitions.
Cummings (1996) provides valuable insight, and argues that the most appropriate view is
that dietary fibre is simply plant cell wall material, and should be measured as total NSP. It
is not uncommon, however, for the definition of DF to include the term ‘non-digestible’ and
the regulatory food labelling authorities in many countries include not only NSP, but also
lignin, oligosaccharides and starch resistant to host enzymes, termed resistant starch (RS),
to be classified as DF. For purposes of this review, DF is defined as the “non-digestible part
of plants”.
The principal dietary substrates available for hindgut fermentation are carbohydrates,
and it is this group of substrates that limits colonic flora growth (Macfarlane and
Cummings, 1991). Of the carbohydrates, and also overall, the most important contribu-
tors to flow of material entering the human large intestine are NSP and RS. A Western
diet provides 10–25 g/day of NSP (which includes cellulose, hemicellulose, pectins and
gums) to the large intestine, and from 5 to 35 g/day of RS (Cummings, 1996). Other car-
bohydrates include oligosaccharides, sugars, sugar alcohols and synthetic carbohydrates.
Sugars and sugar alcohols collectively contribute 2–5 g/day, while oligosaccharides con-
tribute 2–8 g/day (Cummings, 1996).
While carbohydrates are quantitatively the most important, they are not the only sub-
strates available for colonic fermentation. Some dietary protein (1–12 g/day) also escapes
digestion, as do some lipids. In addition to dietary sources, any gut endogenous proteins,
lipids, bile and mucins that reach the hindgut (4–8 g/day) are also potential substrates for
fermentation therein (Cummings, 1996). In all these cases, rapid fermentation occurs.
Lignin is a small and highly insoluble polymer and, whilst not a carbohydrate, it is present
in cell walls of some plants. Lignin is neither digestible nor fermentable and is thought to
inhibit fermentation of associated proteins and carbohydrates (Cummings, 1996).
4.3. Products of fermentation
Bacteria in the hindgut produce enzymes to digest fermentable substrates entering the
large intestine. Anaerobic fermentation enables bacteria to use products of this process to
supply their own energy needs for maintenance and growth, thus increasing their biomass.
The anaerobic fermentation of carbohydrates, quantitatively the most important process in
the hindgut, can be described by the equation of Ewing and Cole (1994) as:
57.5C6 H12 O6 + 45H2 O

→ 65acetate + 20propionate + 15n-butyrate + 140H2 + 95CO2 + 288ATP
The primary products of fermentation in the hindgut are the short chain fatty acids (SCFA)
n-butyrate, propionate and acetate, and gaseous hydrogen and carbon dioxide. Proportion-
ally, in both humans and pigs, acetate is the predominant SCFA, followed by propionate, and
then butyrate (Breves and Stuck, 1995). However, molar ratios of the different SCFA vary
depending on the substrate, as does rate of fermentation (Salvador et al., 1993). The SCFA
are well absorbed (about 95%) but have different fates. Acetate reaches muscle tissue where
it contributes to energy supply and whilst propionate is processed by the liver, little more
about its fate has been studied in man (Cummings, 1996). Butyrate is utilised directly by
colonic epithelium, which derives 60–70% of its energy from products of bacterial fermen-
tation (Roediger, 1980, 1989). Hydrogen gas is either exhaled from the body via the lungs,
voided as flatus or is metabolised further to methane, sulphide and acetate (Cummings,
1996) by methanogenic, sulphate-reducing and acetogenic microbial species. However, not
all individuals possess methanogenic bacteria in their GI tract and the reasons for this and
the consequences of it to the colonic ecosystem are not fully understood (Macfarlane and
Cummings, 1991).
Proteins, peptides and amino acids are rapidly fermented to produce ammonia, amines,
phenols and branched chain fatty acids, all of which are products unique to protein degrada-
tion, in addition to other products of carbohydrate fermentation (Cummings, 1996). Unused
products from bacterial fermentation of nitrogenous compounds, mainly ammonia, amines,
phenols and branched chain fatty acids, may enter the bloodstream and, with the exception of
the branched chain fatty acids, are also largely excreted in the urine and faeces (Cummings,
1996). There are minimal quantities of free amino acids in human ileal effluent (Chako
and Cummings, 1988), yet a wide range of amino acid fermenting species populate the
hindgut. Some bacterial species in the hindgut are able to utilise peptides and free amino
acids directly (Payne, 1975). However, the amino acids available to the majority of these
amino acid fermenting species must result from hydrolysis of peptides by proteases and pep-
tidases. The hydrolysis reaction releases peptides and amino acids that then are available for
assimilation and further metabolism, including fermentation, by bacteria. Colonic bacteria
are therefore able to release amino acids via hydrolysis, as well as having their own mech-
anisms for amino acid synthesis. Moughan (2003) reviewed the evidence for amino acids
being available to the host as a result of hindgut hydrolysis. It appears that absorption of
intact amino acids or peptides from the large intestine is of little nutritional importance to the
host, but large amounts of ammonia may be absorbed and, under most conditions, these are
of minor nutritional importance, although they may have a toxic effect (Clinton et al., 1988).
Although SCFA are the only energy-yielding products of fermentation available to the
host, only a small quantity become available, with the majority of SCFA being used by the
hindgut bacteria for protein synthesis, resulting in an increase in microbial biomass. The
increased biomass is expelled in the faeces along with the non-absorbed SCFA, and any
carbohydrates in excess of fermentative capacity.
End products of fermentation differ according to each genus of bacteria (Macfarlane
et al., 1995) and the relative proportion and amounts of each of the SCFA produced are
dependent on the substrate passing through the hindgut and substrate transit time. Many of
the health effects of dietary fibre are due to its fermentation in the large intestine (van Loo
et al., 1999), and consequently, there is interest in in vitro models to study the SCFA profile
resulting from bacterial fermentation of different foods.
5. In vitro hindgut digestion and fermentation methods
5.1. Introduction
Continuous, semi-continuous and batch (static) systems are the main types of in vitro
models used for hindgut studies in man. These systems remove fermentation products
continuously, intermittently, or not at all. Karppinen (2003) presented a brief discussion of
batch in vitro fermentation methods proposed for studying the human large intestine, as
have others (Barry et al., 1995; Edwards et al., 1996). Earlier, Rumney and Rowland (1992)
undertook an in-depth review, but as applied to semi-continuous and continuous in vitro
fermentation methods for humans. There are a number of detailed critical reviews of in
vitro digestibility assays as applied to simple-stomached farm animals (Boisen and Eggum,
1991; Fuller, 1991; Moughan, 1999; Boisen, 2000; van Kempen et al., 2004).
Simulation of hindgut digestion, in vitro, requires the presence of hindgut microbial
enzymes to digest the substrate. These enzymes are provided either as purified enzymes
commercially available “off-the-shelf” or directly as the result of enzyme production by
live microbes present in the in vitro system. Purified enzymes usually consist of a selection
of either individual or mixed digestive enzymes that are known to be produced by bacteria
in the hindgut (Boisen and Fernández, 1997). Live microbes are supplied as an inoculum
prepared from freshly voided human faeces or caecal/colonic contents. It is important to
note that although both purified enzymes and live microbes have the potential to simulate
digestion in the hindgut, only live microbes are able to ferment a substrate. This is because
enzymes alone are unable to simulate the fermentation process, which involves bacteria
taking up the simpler molecules resulting from digestive breakdown by bacterial enzymes
and synthesising these into energy yielding and gaseous byproducts. While the use of
purified enzymes has been well explored for in vitro hindgut digestion methods in pigs
and cattle, this is not so for humans. Consequently, in vitro hindgut digestion methods for
human studies have generally used live microbes as the enzyme source.
5.2. Types of in vitro hindgut digestion/fermentation methods
5.2.1. Semi-continuous and continuous systems

In vitro digestion methods best suited to studying microbial ecology of the large intestine,
versus the digestive fates of foods, are semi-continuous and continuous.
A specific group of semi-continuous and continuous in vitro methods are the dynamic
in vitro hindgut models, which use either live microbes, purified enzymes or both. These
models are multi-compartmental, computer-controlled systems that are capable of dynami-
cally interacting with the substrate by adjusting pH, temperature and other assay parameters
as substrate passes through the system. The Dutch TIM-2 (TNO gastro-Intestinal Model-2;
Minekus et al., 1999) uses a series of linked glass chambers with flexible internal walls.
Physiological temperature is maintained and the peristaltic movements and mixing of the
large intestine are simulated by periodically pumping water into the space between the glass
and the flexible wall and by computer-controlled contractions exerted on the wall, respec-
tively. Products of fermentation are removed from the anaerobic system using hollow fibre
membranes and the system is able to simulate digestion in a variety of animals, including
pigs, chickens and humans. In combination with the corresponding upper tract dynamic
in vitro model, TIM-1, the system aims to simulate digestion in the entire digestive tract
(Havenaar and Minekus, 1996).
As with other semi-continuous or continuous in vitro digestion systems, dynamic systems
often have their application focused on large intestinal microbial ecology, rather than hindgut
digestion. Furthermore, semi-continuous and continuous systems do not lend themselves
easily to routine evaluation in standard laboratories and, in many cases, require specialised
equipment and computer systems. Consequently, batch in vitro digestion systems will be
dealt with in more detail.
5.2.2. Batch systems

Batch systems are simpler, considerably easier to run and the method of choice for
routinely studying products of fermentation in the large intestine and for food evaluation
purposes. In contrast to semi-continuous and continuous systems, the typical incubation
time for batch in vitro systems is 24 h.
Batch in vitro hindgut digestion methods typically use a faecal slurry from faecal samples,
a buffer and sometimes a nutritive solution and/or a redox agent. The substrate is inoculated
with the faecal slurry and incubated. The degree of fermentation is usually measured in terms
of NSP degradation, SCFA or gas production, or dry matter (DM)/organic matter (OM)
disappearance. Gas production techniques are a special type of batch in vitro fermentation
system and will be discussed separately in more detail later.
The use of purified enzymes as an alternative to a faecal slurry is an area that warrants
investigation for humans given its success in feed evaluation for pigs. The concept of using
purified enzymes in porcine assays evolved over a long period of time after a progression
away from use of biological inocula. Initially, the seminal research of Tilley and Terry
(1963) with rumen fluid was applied to pigs by using rumen fluid (Vervaeke et al., 1979,
1989) and, more logically, intestinal fluids of pigs (Furuya et al., 1979; Ehle et al., 1982).
Following on from this, Löwgren et al. (1989) compared the effectiveness of inoculum from
pig duodenum, ileum or faeces, and in subsequent studies (Graham et al., 1989) showed
that a 48 h incubation with ileal or faecal inoculum could be used to predict faecal apparent
digestibility of dietary fibre in the pig.
Van der Meer and Perez (1992) developed a total tract in vitro method for predicting OM
digestibility using consecutive incubations with pepsin/pancreatin and cellulase to simu-
late the stomach/small intestine and hindgut, respectively. Whilst a high correlation was
found between pig in vivo/in vitro digestibility results using the method, the single enzyme
preparation to simulate the hindgut would seem to be an oversimplification. Boisen and
Fernández (1997) later refined an earlier assay (Boisen and Fernández, 1991) for predicting
total tract digestibility, which included an enzymatic step to simulate hindgut digestion
after consecutive pepsin and pancreatin incubations. Rather than using cellulase for the
hindgut enzymes, Boisen and Fernández (1997) introduced a complex enzymatic prepara-
tion known as Viscozyme® into their in vitro method to predict total tract DM digestibility.
Viscozyme® is a multi-enzyme complex containing a wide range of carbohydrases includ-
ing arabinase, cellulase, ␤-glucanase, hemicellulase, pectinase and xylanase. The method of
Boisen and Fernández (1997) has been the subject of a number of in vivo/in vitro digestibility
comparisons in several monogastric species. Specifically, rat faecal digestibility using oats
(Pettersson et al., 1996), and pig apparent faecal energy and OM digestibility using hulled
and hulless barleys (Beames et al., 1996), have been found to correlate well with in vitro
results. In a study similar to that conducted by Beames et al. (1996), and involving feeding
barley to pigs, Chen (1997) found that in vitro DM estimates had a low correlation with the
in vivo apparent digestibility of energy, although in vitro/in vivo digestibilities with wheat-
milling byproducts were highly correlated. The method of Boisen and Fernández (1997)
has been shown to be highly repeatable (Chen, 1997), and has subsequently been officially
adopted in Denmark for practical energy evaluation of mixed diets for pigs (Spanghero and
Volpelli, 1999). No such parallel exists for humans and consequently there is a lack of a
standard in vitro hindgut digestion method for evaluation of foods. This is not to say that
there has been an absence of in vitro hindgut digestion methods proposed for humans, as
there are a large number available that use live microbes. Table 1 provides an extensive list
of such batch in vitro models proposed in relatively recent times. Barry et al. (1995) have
provided a reference point for earlier examples.
5.3. Failings of batch in vitro hindgut fermentation methods
In addition to the basic design of all batch in vitro fermentation methods reported in the
literature for human studies being similar, almost without exception, the methods all fail in
one or more of the following ways:
5.3.1. Lack of explanation for the rationale to choose the parameters and conditions
Every method differs to at least some degree, yet few explanations are given as to why
the researchers selected the particular parameters. Such lack of justification adds little
credibility to the methodology.
5.3.2. Lack of inter-laboratory investigation to test or standardise the method

To enable results to be compared, and for any in vitro method to be considered robust
and repeatable, the method needs to prove that it can produce the same results, within
Table 1
A summary of batch in vitro fermentation methods proposed for studying digestion and fermentation in the human
large intestine
Literature reference Fermentative Inoculum source Parameters measured Incubation
substrate (h)
Wang et al. (2004) Animal protein by Pig faeces NSP, SCFA, starch 48
itself or with
addition of: potato
starch, sugar beet
pulp, wheat bran
Goñi et al. (2002) Seaweed Rat caecal contents Protein 24
Langkilde et al. (2002) Banana flour Human faeces SCFA, RS, pH, H2 , 24
CH4
Granito et al. (2001) Haricot bean fibre Human faeces SCFA, pH, gas 24
production
Birkett et al. (2000) Human ileal Human faeces SCFA, pH, NH3 , 48
effluent from high glucose
or low, mixed
starch diet
Fernandes et al. (2000) Lactulose, Human faeces SCFA 24
rhamnose, guar
gum, cornstarch
Karppinen et al. (2000) Brans (rye, wheat, Human faeces SCFA, pH, gas 24
oat), inulin production, neutral
sugars
Monsma et al. (2000) Oat bran, wheat Rat caecal contents SCFA, neutral and 96
bran amino sugars
Christensen et al. Wheat flour, wheat Pig faeces SCFA 72
(1999) bread, oat bran
Goñi and Commercial Rat caecal contents SCFA, DM, gas 24
Martin-Carrón purified dietary production
(1998) fibre supplements
Hoebler et al. (1998) Wheat bran, barley Human faeces SCFA, neutral sugars 24
bran, beet fibre
Lebet et al. (1998a) Pea hulls, apple Human faeces SCFA, pH, H2 , 24
pomace, celery cell neutral sugars, gas
walls, oat bran production
concentrate
Wisker et al. (1998) NSP (mixed fibre), Human faeces NSP, neutral sugars 48
citrus concentrate,
wholemeal bread
Casterline et al. (1997) Brans (wheat, oat, Human faeces SCFA 48
corn), fruit and
vegetable fibres
(pea, apple, pear,
fig), ␤-glucan,
pectin, starch, soy
fibre
Fardet et al. (1997) Beet fibre, barley Human faeces SCFA, neutral and 24
bran acidic sugars
Stevenson et al. (1997) Pectin, ispaghula, Rat caecal contents SCFA, protein 24
cornstarch production
Table 1 (Continued )
Literature reference Fermentative Inoculum source Parameters measured Incubation
substrate (h)
Bourquin et al. (1996) Oat fibre, wheat Human faeces SCFA, OM, potential 24
bran, corn bran, water holding
xanthan gum, gum capacity
karaya, guar gum,
gum arabic, soy
fibre, citrus pectin
Edwards et al. (1996) Pregelatinised potato Human faeces SCFA, pH, RS 24
starch, raw potato
starch, retrograded
amylose, glassy pea
starch
Barry et al. (1995) Cellulose, sugarbeet Human faeces SCFA, NSP, pH 24
fibre, soybean fibre,
maize bran, pectin
Guillon et al. (1995) Pea fibre, apple fibre Human faeces SCFA, pH 24
Monsma and Marlett Canned peas, Rat caecal SCFA 96
(1995) psyllium seed husk contents/faeces
fibre
Silvester et al. (1995) Ileal effluent Human faeces SCFA, NH3 24
containing resistant
starch
Sunvold et al. (1995) Cellulose, beet pulp, Cattle ruminal fluid, SCFA, OM 48
citrus pulp, citrus faeces from: cat, dog,
pectin horse, human, pig
Daly et al. (1993) Xanthan gum Human faeces SCFA, pH, H2 24
McBurney and Sauer Pea fibre Human faeces SCFA 24
(1993)
Salvador et al. (1993) Wheat bran, sugar Human faeces SCFA 24
beet, maize, pea
hulls, cocoa
Bourquin et al. (1992) Oat bran, wheat Human faeces SCFA, DM 48
bran, corn fibre
Guillon et al. (1992) Sugar beet fibre Human faeces SCFA, pH 24
Titgemeyer et al. Oat fibre, corn bran, Human faeces SCFA, neutral 48
(1991) soy fibre, sugarbeet sugars, uronic acid
fibre, pea fibre, gum
arabic, guar gum,
apple pectin, citrus
pectin
Adiotomre et al. (1990) Wheat bran, pectin, Human faeces SCFA, pH 24
guar gum, gum
arabic,
carboxymethylcellu-
lose, gellan,
tragacanth gum,
xanthan gum, gum
karaya
Vince et al. (1990) Lactulose, pectin, Human faeces SCFA, NH3 48
arabinogalactan,
cellulose
DM: dry matter, NSP: non-starch polysaccharides, OM: organic matter, RS: resistant starch, SCFA: short chain
fatty acids.
reasonable limits of statistical variation, at multiple laboratories. If it cannot, there is little

chance that it can be used as a standard method on a global scale. Generally, there is a lack
of inter-laboratory testing.
5.3.3. Lack of validation with in vivo data

Few papers describing the development of in vitro fermentation methods include a dis-
cussion of validation. The method of Barry et al. (1995) is widely used, and has been tested
on an inter-laboratory scale, yet validation tests used rats as the in vivo model for NSP
degradation (Nyman et al., 1986), rather than humans. There is uncertainty as to whether
rats can be used as a suitable predictor for digestion in the human large intestine and, in
addition, several issues regarding the suitability of the study by Nyman et al. (1986), as a
comparative baseline, have been raised (Bach Knudsen et al., 1994).
Other methods to have been subjected to validation are those of Christensen et al. (1999)
and McBurney and Sauer (1993). Here pig ileal digesta was used as the fermentable sub-
strate and pig faecal inocula (Christensen et al., 1999) or human faecal inocula (McBurney
and Sauer, 1993) for provision of fermentation capability. In both cases, validation
was completed using pigs, rather than humans, despite the method’s application to the
latter.
A lack of relevant in vivo comparison and validation is perhaps not surprising given
the expense and time-consuming nature of human balance studies. Notable exceptions are
the large studies undertaken by Daniel et al. (1997) and Wisker et al. (1998) that com-
pared NSP degradation in vivo for mixed diets in man, with results from an in vitro
fermentation method adapted from one proposed by Goering and Van Soest (1970) for
ruminants. Whilst a general relationship existed between the in vitro and in vivo re-
sults, the degree of relationship improved as the fermentability of the substrate increased.
Bourquin et al. (1996) also compared their in vitro results with human in vivo results
and found that substrates which were highly fermentable in vitro, were similar in vivo.
This observation is not entirely unexpected given that highly fermentable substrates are
easily, and almost completely degraded, in vitro and in vivo, making it less likely that
substantive overestimation or underestimation of the fermentability of the substrate will
occur in vitro. Furthermore, the enzymatic preparation of DF residues may cause fer-
mentability of substrates to be overestimated in vitro, since the structural properties of
the cell wall may become compromised in a manner that would not occur in vivo, thus
making the DF more susceptible to in vitro bacterial degradation (Wisker et al., 1998).
The effect of such pre-treatment is likely to be less important for highly fermentable
substrates.
Although these comparisons are encouraging, there is still a need for a comprehensive,
well-controlled and methodologically relevant validation of potential in vitro assays with
data obtained from in vivo human studies.
5.3.4. Lack of testing with a range of substrates

Most in vitro methods have focused on NSP degradation when studying hindgut fer-
mentation in man. It is now well accepted that NSP are not the only substrates available for
fermentation in the human hindgut (Cummings, 1996) and a robust in vitro method should
also aim to predict fermentation of other materials entering the hindgut, including protein,
fats, and other carbohydrates such as resistant starch, sugars (including sugar alcohols) and
oligosaccharides.
6. Design considerations for a batch in vitro digestion/fermentation method
When developing any in vitro digestion method, along with cost and practicality, the
objective is to have an in vitro method that is representative of in vivo events. For this
reason, it is imperative to constantly refer back to the species in question so that simulated
physiological conditions such as pH, temperature, and incubation time are appropriate.
Hindgut in vitro fermentation methods also need to provide a suitable environment for
functioning of hindgut bacterial species present in the inoculum. The components of all
batch in vitro fermentation methods developed to date are essentially the same, but the way
in which they are applied differs. Of all the possible factors that differ, the most important
to consider are the inoculum concentration, the incubation time, the presence and type of
buffer, the fluid surface/volume ratio, the form of the substrate and the output ultimately
measured.
6.1. Substrate
Monogastric species ferment food after upper-tract digestion, which means that substrate
that is available for fermentation is not the food as eaten. Therefore, it is unsuitable to test
the food as eaten directly, since that would expose the inocula to food components not
normally available for fermentation.
Appreciating this, it is common for researchers to pre-digest the food, or only test those
components of specific interest, for example by using dietary fibre isolates or resistant starch
as the substrate. Some have taken this a step further, and used ileal digesta as substrate.
Although use of ileal digesta does increase the complexity of the assay, the substrate in this
case is in the form that it would actually arrive at the ileo-caecal junction in vivo (Monsma
Table 2
Combinations of sources of ileal digesta and inoculum for published in vitro hindgut digestion/fermentation assays
Substrate Inoculum source Literature reference
Human ileal digesta Human faeces McBurney and Thompson (1989), Silvester et
al. (1995), Fernandes et al. (2000), Birkett et
al. (2000), Langkilde et al. (2002)
Pig ileal digesta Human faeces McBurney and Sauer (1993), Fardet et al.
(1997), Hoebler et al. (1998)
Rat caecal contents Monsma et al. (2000)
Pig faeces Christensen et al. (1999), Wang et al. (2004)
Pig caecal contents Fondevilla et al. (2002)
Rat ileal digesta Rat caecal contents/faeces Monsma and Marlett (1995, 1996), Bullock
and Norton (1999)
and Marlett, 1996). While dietary fibres may be resistant to host enzymatic degradation,
they still undergo physical changes in the upper tract (Salvador et al., 1993; Åman et al.,
1994). Combinations of ileal digesta and various inocula that have been investigated are in
Table 2.
6.2. Inoculum
Whilst for species such as the pig, caecal contents for use in an in vitro fermentation
system may be obtained, it is impractical to obtain human colonic contents and so faeces
are routinely used in the inoculum. It is essential to use freshly voided faeces that have been
collected and processed under anaerobic conditions to preserve viability of the microbial
population. A discussion of the validity of using faeces, as opposed to colonic contents, is
presented in Section 7.
Inoculum donors are healthy adult subjects, who have not taken antibiotics for at least 3
months prior, and are consuming their normal Western diet. It has been shown that adapting
donors to the substrate to be tested does not affect in vitro results (Barry et al., 1995; Daniel
et al., 1997), although some have chosen to use adapted donors (Titgemeyer et al., 1991;
Wisker et al., 2000). Most methods use at least three donors to provide faecal material for
the pooled inocula, although five or more are recommended to help reduce the effect of
biological variation among individuals (Edwards et al., 1996). Some researchers also limit
the donors to methane non-producers (Lebet et al., 1998a; Granito et al., 2001) to reduce
variation in the inoculum.
The ratio of inoculum to substrate has been found to be an important factor and propor-
tionally more SCFA are produced after 24 h when less polysaccharide has been added to
the fermentation system (Stevenson et al., 1997). It has been hypothesised (Stevenson et
al., 1997) that the reason for this is related to access to the substrate and that any more than
10 g/l of substrate may overload the system.
6.3. Nutritive medium
The nutritive medium contains an N source and other essential minerals. One advantage
of using a high concentration of faecal inoculum is that such nutritive components are
naturally present and do not need to be added to the incubation vessel (Edwards et al.,
1996).
6.4. Buffer
Without inclusion of a buffer in the in vitro fermentation system, the acidic nature of
SCFA produced during fermentation causes the pH to rapidly drop below the pH 6–7 range
normally observed in the colon (McBurney and Van Soest, 1991). In vivo, SCFA are rapidly
absorbed across the colon wall, thereby preventing colonic contents from becoming too
acidic (McBurney and Van Soest, 1991). In the closed environment of a batch in vitro system,
however, no such removal mechanism operates. A buffer (either carbonate, phosphate, or
both) is usually included in the fermentation vessel. The phosphate-only buffers have the
advantage that inoculum preparation under CO2 is not required to preserve the effectiveness
of the buffer (Edwards et al., 1996), thereby allowing oxygen-free N2 to be used as an

alternative means for ensuring an anaerobic environment.
6.5. Stirring
To mimic the peristaltic action of the colon, stirring is often included in the methodology.
This also prevents substrate from settling to the bottom of the incubation vessel or being
poorly dispersed, thereby becoming less accessible for fermentation (Edwards et al., 1996).
However, it has been found that shaking has no effect on fermentation or the increase in
bacterial mass (Stevenson et al., 1997).
6.6. Fluid surface area/volume ratio
The surface area/volume ratio of fluid in the incubation vessel may influence fermenta-
tion results. A surface area/volume ratio of 1:1 has been shown to allow more fermentation
than a ratio of 1:4 when samples of pectin and starch were fermented in vitro (Stevenson
et al., 1997). This is most likely to be associated with the increased surface area avail-
able for gaseous exchange at the liquid–gas interface to remove the gaseous fermentation
products.
6.7. Measurements
To assess fermentability, there are at least two options, being to measure metabolite
production or substrate disappearance. Measuring SCFA production, which is the primary
indicator of fermentability, falls into the first category. In the second category are methods
that measure losses of DM, OM, gross energy or loss of specific components such as NSP,
RS, protein, carbohydrate or other fermentable substrates. Measurements may be taken at
the end of the incubation period, or at stages during the incubation to study kinetics of
fermentation. The latter necessitates multiple samples, since there are multiple end points
for each substrate tested.
6.8. Incubation time
The majority of methods use 24 h, which is convenient in the laboratory. Whilst 24 h

is shorter than the average transit time through the entire human large intestine, it has
been shown to be comparable to the average transit rate of material through the prox-
imal colon (Metcalfe et al., 1987), which is considered to be the main fermentation
chamber in humans (Edwards et al., 1996). Wisker et al. (1998) found a good rela-
tionship between in vitro and in vivo NSP degradation after 24 h of fermentation, but
not after 48 h. Although Monsma and Marlett (1996) argued that 96 h is necessary to
characterise the fermentation pattern of carbohydrates in vitro, incubations longer than
24 h may be affected by end product inhibition that results in a declining microbial
population and the death of bacteria (Vince et al., 1976; Brøbech Mortensen et al.,
1991).
7. The gas production technique
The in vitro gas production technique is a specific example of a batch in vitro method used
for fermentation studies and, as with other batch in vitro methods, facilitates the necessary
anaerobic environment by preparation of the inoculum under strictly anaerobic conditions
and fermentation in a closed system. The technique is similar in setup to other batch in
vitro methods, with the only differences being that the fermentation vessel may be either a
syringe, flask or bottle, and that a means of recording the pressure in the fermentation vessel
is employed, such as an attached pressure transducer. In the case of the automated versions,
pressure readings are recorded automatically by a computer system. Whilst SCFA and
degraded substrates may be analysed at the conclusion of the assay, in terms of methodology
it is the gas evolved as a result of fermentation that is the primary measurement, rather
than the disappearance of fermentable substrate. Gas may be measured either at a single
endpoint, or alternatively at various stages during fermentation to study kinetics of the
fermentation process. The cumulative gas production technique of Theodorou et al. (1994)
is an example of the latter. Gas that is measured is not only that produced as a result
of microbial fermentation (i.e., hydrogen, methane, carbon dioxide), but also the carbon
dioxide that is liberated indirectly as a result of the SCFA produced reacting with the
bicarbonate buffer that is used in all gas production methods. Although it is not possible
to separately measure gas originating via the different pathways, both are produced either
directly or indirectly as a result of microbial fermentation of substrate and are therefore
indicative of fermentative activity.
The gas production technique is well-established in the area of ruminant feed evaluation,
and a great deal of research using the technique in this context has been undertaken, and
dates back over three decades. Despite this wealth of knowledge, the use of gas production
techniques in monogastric feed evaluation is less common and has only emerged relatively
recently. Like its ruminant counterpart, the method measures the volume of gas that is
produced (or the pressure that this gas exerts) as a result of fermentative degradation of a
substrate.
While some studies have investigated the technique for equine application (Lowman et
al., 1999; Moore-Colyer and Longland, 2001; Murray et al., 2003), and less commonly with
rabbits (Calabró et al., 1999), the majority of studies have been applied to pigs.
Ahrens et al. (1991) modified the method of Menke et al. (1979) for use with pigs,
using caecal contents as the inoculum and centrifuged (freeze dried) caecal contents as the
substrate. The ruminant method was modified by using smaller syringes and corresponding
smaller quantities of buffer and inoculum. The fermentation of pectin and lactitol were
investigated in vitro by direct addition to the caecal substrate and compared with in vivo
sampling of caecal contents following diets containing corresponding amounts of pectin
and lactitol. Good agreement was found for a 24 h in vitro incubation and in vivo measures
for pH, ammonia and total volatile fatty acids (VFA).
It was not long after, that the research group at Wageningen University began applying
the cumulative gas production technique (Theodorou et al., 1994) that they had been using
with ruminants, to monogastric animals. Both poultry (Williams et al., 1997a) and pigs were
initially investigated (Williams et al., 1997b, 1998) and, since then, the group has undertaken
numerous studies using the technique with pigs to investigate differences among feedstuffs
(van Laar et al., 2000, 2002; Williams et al., 2000, 2001b, 2001c, 2003; Bosch et al.,
2002), unweaned and adult animals (Bauer et al., 2001) and also the effect of enzymatic
pre-treatment on fermentation kinetics of feedstuffs (Bauer et al., 2003).
Fondevilla et al. (2002) used the same technique to compare Iberian and Landrace pigs
fed acorns using ileal digesta as the substrate and caecal contents as the inoculum. Becker
et al. (2003) recently investigated potential forages for pregnant sows using an adaptation
of the fully automated version proposed by Cone et al. (1996). An adaptation of the syringe
method of Menke and Steingass (1988) was used by Boudry et al. (2003) to investigate
affects of in vitro pre-digestion on fermentability of feedstuffs for pigs, whilst fermentation
of oligosaccharides in pigs has also been studied using a gas production technique (Smiricky-
Tjardes et al., 2003a, 2003b), but only with a single endpoint using a fluid displacement
version.
Campbell and Fahey (1997) used a fluid displacement version to study fermentation of
psyllium, methylcellulose, pectin and Solka Floc® with human faecal inocula. Aside from
the latter study, there is an absence of literature on the application of the gas production
technique with humans.
For simple-stomached animals it is common, for logistical reasons, to use faecal inocula
for the gas production technique rather than caecal or colonic contents. Even for ruminants,
the use of faeces vs. rumen fluid has the same logistical advantages. It is well established,
however, that rumen fluid gives a different fermentation profile to faeces, and so mathe-
matical models have been developed for ruminants to account for differences, such as a
longer lag time (France et al., 2000). These mathematical models are promising and a case
for developing similar models for monogastrics is supported by studies of Monsma and
Marlett (1995), who found differences between in vitro fermentation profiles for rat cae-
cal and faecal inocula. Specifically a longer lag-phase was observed with faecal inocula
(Monsma and Marlett, 1996), which is somewhat analogous to the lag-phase phenomenon
observed with rumen fluid versus cattle faeces when using a gas production technique
(Mauricio et al., 1997). Monsma and Marlett (1995) argued that the same differences be-
tween use of colonic contents and faeces exist in humans, and so faecal inocula will not
necessarily provide an accurate representation of in vivo fermentation in the colon. In both
pigs and humans, the profile of colonic bacteria varies according to whether samples are
taken from the proximal, transverse or distal colon (Williams et al., 1997b). Despite this,
Williams et al. (1997b) believed that faecal microbial inocula are indicative of colonic
microflora. The approach of Edwards et al. (1996), of using faecal inoculum with a high
concentration of microbes, to prevent the initial lag time that often occurs when using fae-
ces as opposed to caecal contents, may be another means of overcoming differences due to
inoculum source. Consideration needs to be given to such alternatives, as it is difficult to
obtain representative human colonic samples.
8. Merits and shortcomings of in vitro digestion/fermentation methods
In vitro digestion methods that use purified enzymes have the advantage of generally
being more reproducible than those that use live microbes, since there is very little variation
in the standardised enzyme preparations used (Moughan, 1999). It is also easier to handle
purified enzymes than faecal samples, and inoculum donors do not need to be sourced.
Among the disadvantages is that selection of enzymes chosen is not necessarily represen-
tative of the enzymes produced by bacterial species that inhabit the hindgut. Additionally,
lack of microbes per se means that the dynamic interactions occurring between substrate
and microbes are not represented. Such methods determine hindgut hydrolysis (digestion)
rather than fermentation.
Of the methods detailed in the literature that use purified enzymes, that presented by
Boisen and Fernández (1997) for pigs, shows the most promise and has the advantage that
it has been developed specifically in combination with an upper tract method to provide
a more suitable substrate for the hindgut step. A similar total tract in vitro method for
human studies has been developed by Lebet et al. (1998b) which employs an enzymatic in
vitro upper tract method similar to that of Aura et al. (1999). Like the approach of Aura et
al. (1999), dialysis is included, which makes the simulated upper tract component of the
method more complicated than that of Boisen and Fernández (1997). The corresponding
hindgut step of the method proposed by Lebet et al. (1998a), uses live microbes and is
essentially the same as that proposed by Barry et al. (1995).
Of the methods available for humans that use live microbes, that of Barry et al. (1995) has
the advantage that it has been tested on an inter-laboratory scale. Another method that uses
live microbes presented by Edwards et al. (1996) for fermentation of resistant starch, has
received some positive feedback, as well as also having been tested in an inter-laboratory
study. The main advantage of this method is its simplicity, since it requires no nutritive
medium and only employs a simple phosphate buffer, thus negating the need for continuous
bubbling with CO2 during inoculum preparation. The system has been validated however,
only for RS.
Gas production techniques have unique benefits over and above other in vitro methods
that use live microbes. Firstly, the gas production technique allows rapid comparisons be-
tween fermentative substrates to be made relatively easily. This is because the amount of
gas produced can be measured readily, and thus a direct comparison between the amounts
of gas produced allows for a non-quantitative estimate of the fermentability of different
substrates to be made. Gas production curves allow fermentation kinetics of substrates to
be compared, and the relative fermentability of substrates according to the gas produced to
any time can be a useful indicator of the comparative fermentability of different substrates
at different stages of fermentation. The gas production technique allows for less sample
and equipment, if multiple end times of fermentation are required. While other hindgut in
vitro digestion methods necessitate destruction of the sample to determine DM, the gas
production technique preserves the sample. If the amount of gas produced at multiple end
points, such as 6, 12, 24 and 48 h, was desired, only one incubation vessel and one sample
of the substrate would be required for each replicate, rather than the four or more required
with other in vitro methods. The equipment required for the non-automated versions of the
method is relatively uncomplicated and available in most standard laboratories.
Shortcomings of the non-automated version of the gas production technique are that it
is labour-intensive and subject to operator-error. For the automated version, capital invest-
ment is a limitation for many laboratories. The automated versions have the advantage that
personnel do not need to manually record the volume of gas produced if multiple readings
over time are required. The high labour cost and time required for reading measurements
with the manual technique inevitably mean that fewer readings are taken than with the auto-
mated version which, depending on purpose, may compromise the results. By automatically
recording the pressure in the headspace of the incubation vessel, there is the ability to make
many measurements during the incubation time period. Yet the higher cost of the automated
system means that for many laboratories the investment may be difficult to justify.
9. Conclusions
There is agreement, based on a limited number of suitable validation studies, that in vitro
batch fermentation assays for humans are able to rank substrates in a similar manner to in
vivo results. It is yet to be demonstrated empirically, however, that the energy provided to
humans as a result of fermentation in the hindgut can be accurately predicted in vitro by any
of the methods. There is a need for such in vivo validation studies to be completed and a sound
in vitro system to be correspondingly developed and standardised for a wide range of foods.
In the interim, currently available methods such as those described by Barry et al. (1995)
and Edwards et al. (1996) are uncomplicated and have been subjected to some degree of
validation. The porcine digestion method proposed by Boisen and Fernández (1997) has
been subjected to a number of validation studies and stands out as offering considerable
promise for total tract studies. The method, given the practical advantages of using purified
enzymes, warrants adaptation to humans.
Gas production techniques are useful to readily and simply compare feedstuffs/foods,
species, inter-individual variation and fermentation kinetics associated with these factors,
using a minimum amount of sample. However, as with other in vitro methods proposed for
studying hindgut digestion and fermentation in simple-stomached animals and humans, the
technique is currently limited to relative comparisons. An in vitro method is needed which
accurately predicts the absolute amount of energy available to the host from any substrate
in a rapid, standardised and reliable manner.
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Animal Feed Science and Technology

123–124 (2005) 421–444

In vitro digestion and fermentation methods,

Massey University, Private Bag 11-222 Palmerston North, New Zealand

E-mail address: [email protected] (P.J. Moughan).

Keywords: Gas production; Fermentation; Digestibility; Monogastric; Hindgut

2. The need for in vitro hindgut digestion/fermentation methods

3. Application and limitations of in vitro hindgut digestion/fermentation methods

4. The monogastric hindgut

To understand the rationale underlying an in vitro hindgut digestion/fermentation

4.1. Hindgut physiology

4.2. Substrates for fermentation

4.3. Products of fermentation

57.5C6 H12 O6 + 45H2 O

5. In vitro hindgut digestion and fermentation methods

5.2. Types of in vitro hindgut digestion/fermentation methods

5.2.1. Semi-continuous and continuous systems

5.2.2. Batch systems

5.3. Failings of batch in vitro hindgut fermentation methods

5.3.2. Lack of inter-laboratory investigation to test or standardise the method

reasonable limits of statistical variation, at multiple laboratories. If it cannot, there is little

5.3.3. Lack of validation with in vivo data

5.3.4. Lack of testing with a range of substrates

6. Design considerations for a batch in vitro digestion/fermentation method

6.3. Nutritive medium

of the buffer (Edwards et al., 1996), thereby allowing oxygen-free N2 to be used as an

6.6. Fluid surface area/volume ratio

6.8. Incubation time

The majority of methods use 24 h, which is convenient in the laboratory. Whilst 24 h

7. The gas production technique

8. Merits and shortcomings of in vitro digestion/fermentation methods

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