An in vitro batch culture method to assess potential fermentability of feed ingredients for monogastric diets

Animal Feed Science and Technology 123–124 (2005) 445–462

An in vitro batch culture method to assess potential fermentability of feed ingredients for monogastric diets Barbara A. Williams ∗ , Marlou W. Bosch, Huug Boer, Martin W.A. Verstegen, Seerp Tamminga Animal Nutrition Group, Wageningen Institute of Animal Sciences (WIAS), Wageningen University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands

Abstract Interest in fermentation within the monogastric digestive tract is growing, particularly relative to animal health. This is of particular importance in relation to the forthcoming European ban on inclusion of anti-microbial growth promotors in animal diets. Fermentable carbohydrates are recognized as having an important role in fermentation in the monogastric digestive tract, and are often added to diets without having been examined for their actual fermentability, particularly in relation to the target animal. We describe an in vitro method to assess feed ingredients, as potential components of monogastric diets, which stimulate a positive fermentation (i.e., ones which will be well fermented and produce more short-chain fatty acids (SCFA) and less ammonia). This technique requires use of a batch culture containing the test substrate and an inoculum of appropriate origin. During fermentation, cumulative gas production is measured at regular intervals, as an indicator of kinetics of the reaction. When fermentation is complete, organic matter losses and end-products such as SCFA and ammonia, are measured. This paper illustrates use of the technique with 45 carbohydrate-based ingredients using faeces from unweaned piglets as inoculum. By assessing potential fermentability of a large number of ingredients, it is possible to make an informed choice as to which substrates are most suited for inclusion in a diet. By combining results with information about transit time, diets can be designed which should stimulate desirable fermentation along the entire digestive tract. In vitro fermentability

Abbreviations: AAE, acetic acid equivalents; ADF, acid detergent fibre; ADL, acid detergent lignin; BCR, branched chain ratio; CP, crude protein; DM, dry matter; GIT, gastro intestinal tract; NDF, neutral detergent fibre; OM, organic matter; SCFA, short chain fatty acids ∗ Corresponding author. Fax: +31 317 484260. E-mail address: [email protected] (B.A. Williams). 0377-8401/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2005.04.031

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is a potentially valuable characteristic in diet design, in order to stimulate microbial activity in the digestive tract. © 2005 Elsevier B.V. All rights reserved. Keywords: Cumulative gas production; Prebiotic; Fermentation kinetics

1. Introduction Fermentation in the hindgut is recognized as having an important role in terms of gastrointestinal tract (GIT) health, and of the animal itself (Williams et al., 2001). It is known that this fermentation can be steered in a “positive” direction by use of dietary components such as fermentable carbohydrates, or additives such as herbs. The principle is that an appropriate ingredient in the diet will stimulate one or more supposedly appropriate bacterial species. The word “prebiotic” was defined by Gibson and Roberfroid (1995) as: “. . . non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon . . .”. However, given that there is a much larger microbial population within the small intestine of pigs, versus humans, it could be argued that, at least for pigs, this definition could be extended to include microbial activity in the small intestine. Gibson and Roberfroid (1995) also specified lactobacilli and bifidobacteria as potentially beneficial bacteria. Of the easily cultivable species, these two are considered important for human health. However, there may be other beneficial species, which are as yet uncultivated, but detectable using molecular methods (Zoetendaal et al., 1998). In fact, it was shown by Konstantinov et al. (2004) that no bifidobacteria could be detected in the small intestine of unweaned piglets, suggesting that this group may be less important in pigs compared to humans, at least in the age group examined. In contrast, the method described here is a functional approach whereby all species present in the inoculum can respond to a specific feed ingredient in its entirety. While it would be possible to use this method using single bacterial strains, and sterile technique, the research described here uses only inocula of mixed microbial communities. It also examines fermentability of the whole substrate. A method (Bauer et al., 2003a), whereby fermentability of substrates was compared using this technique before and after a preliminary enzymatic treatment showed that there was very little change in fermentability as a result of enzyme treatment, although there were likely to have been losses due to the actual enzymatic treatment. Given that soluble components in some substrates may be fermentable, it was decided to use the whole materials without preliminary treatment. Fermentation can have both positive and negative consequences in the GIT and, to a large extent, this depends on whether fermentation is of carbohydrate or proteinaceous substances. For example, it is known that fermentation of carbohydrates leads to production of mainly straight-chained short chain fatty acids (SCFA) resulting in uptake of ammonia as a source of N for microbial growth (Stewart et al., 1993). However, fermentation of protein results in more branched-chain fatty acids (Macfarlane et al., 1992), release of ammonia and, often, release of other potentially toxic compounds such as amines, short-chain phenols and indoles (Yokoyama et al., 1982; Russell et al., 1983; Macfarlane et al., 1992). Also, it has been shown that several potential pathogens are protein-fermenters, and are therefore more

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likely to grow in conditions which favour protein fermentation (Macfarlane and Macfarlane, 1995). Ideally, therefore, it is of interest to stimulate fermentation of carbohydrates, while minimizing that of proteins, along the entire GIT. Too often, ingredients are added to diets on the assumption that because they are “fibre”, or “soluble”, they will also be fermentable and therefore have positive properties for gut health. However, this is not necessarily the case. Given the interest in use of fermentable components of human and animal diets, it is important to have a method which can evaluate a wide range of potential ingredients to determine their potential fermentability, particularly in response to an appropriate microbial population (i.e., inoculum). Ideally, such an initial evaluation should be completed in vitro, but give some prediction of what could occur in vivo. So theoretically, a rapidly fermentable ingredient would most likely be fermented proximally, and a more slowly fermentable ingredient more distally in the GIT. A very slowly fermentable ingredient may not be substantively fermented at all in vivo, and be expelled in faeces. Methods to measure cumulative gas production have been used for a number of years to assess nutritive value of forages for ruminants (Beuvink and Spoelstra, 1992; Pell and Schofield, 1993; Theodorou et al., 1994; Cone et al., 1996). However, until recently, these methods have not been reported as measures of fermentation kinetics of monogastric feed ingredients. Therefore, a new procedure was developed by the Wageningen University Animal Nutrition Group in the late 1990s, now used routinely, both with manual (Theodorou et al., 1994) and automated systems (Davies et al., 2000). A detailed account of the method is provided here, with an example of how it has been used to characterize feed ingredients for potential inclusion in a weaning piglet diet.

2. Materials and methods 2.1. Media A semi-defined medium, modified from that of Lowe et al. (1985) was used for most studies, because it was thought to contain most of the micro-nutrients which culturable bacterial species require for growth. The medium contains N in several forms, and is therefore suitable for substrates which are mainly a source of energy to the microbial population. For this research, the medium was prepared by mixing (per 100 ml serum bottle) 76 ml basal solution, 1 ml of vitamin/phosphate buffer solution, 4 ml of bicarbonate buffer, and 1 ml of reducing agent. A stream of O2 -free CO2 flowed into the bottles at all times prior to sealing with butyl rubber stoppers and aluminium crimp seals. All solutions were prepared in advance. Basal and bicarbonate solutions were stored at room temperature, while the reducing agent and vitamins were stored at 4 ◦ C. 2.1.1. The basal solution This solution contained 0.6 g KCl, 0.6 g NaCl, 0.2 g CaCl2 ·2H2 O, 0.5 g MgSO4 ·7H2 O, 1.5 g Pipes buffer, 0.54 g NH4 Cl, 1.0 g trypticase, 1 ml resazurin solution (0.2 g resazurin per 200 ml distilled water), 10 ml ‘Trace Mineral Solution’, 10 ml Haemin Solution (0.1 g Haemin dissolved in small amount 0.05 M NaOH, and made up to 1 l with boiled distilled

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water with CO2 bubbling through it), and 10 ml ‘Fatty Acid Solution’, all made up to 1 l of medium with distilled water. The ‘Trace Mineral Solution’ contained 0.025 g MnCl2 ·4H2 O, 0.020 g FeSO4 ·7H2 O, 0.025 g ZnCl2 , 0.025 g CuCl·2H2 O, 0.050 g CoCl2 ·6H2 O, 0.050 g SeO2 , 0.250 g NiCl2 ·6H2 O, 0.250 g Na2 MoO4 ·2H2 O, 0.0314 g NaVO3 , and 0.250 H3 BO3 , all dissolved in this order into a 0.02 molar solution of HCl and made up to 1 l. The ‘Fatty Acid Solution’ for the basal solution contained 6.85 ml acetic acid, 3.00 ml propionic, 1.84 ml butyric, 0.47 ml iso-butyric, 0.55 ml 2-methyl-butyric, 0.55 ml valeric and 0.55 ml iso-valeric acids, per litre of 0.2 M NaOH. 2.1.2. The reducing agent This contained 20.5 g Na2 S·9H2 O and 20.5 cysteine HCl dissolved in 1 l of boiled distilled water with nitrogen gas bubbling through it. This procedure was completed in a fume cupboard due to the danger of inhalation of toxic fumes. 2.1.3. The bicarbonate solution This solution contained 82 g Na2 CO3 (sodium carbonate anhydrous) per boiled distilled water, with CO2 bubbling through it. Prior to use, and after autoclaving, the solution was bubbled through again with CO2 for about 20 min, before being added to the basal solution. These first three solutions were all autoclaved after preparation. 2.1.4. The vitamin/phosphate solution This solution contained 0.0204 g biotin, 0.0205 g folic acid, 0.1640 g calcium dpantothenate, 0.1640 g nicotinamide, 0.1640 g riboflavin, 0.1640 g thiamin HCl, 0.1640 g pyridoxine HCl, 0.0204 g para-amino benzoic acid, 0.0205 g cyanocobalamin (vitamin B12), dissolved in a 1 l of solution containing 54.7 g KH2 PO4 . This solution was then filter-sterilized into sterile bottles. 2.1.5. N-free solution The solutions described above were modified to be essentially N-free, for studies involving fermentation of proteins, as follows: basal solution – no trypticase, Pipes buffer or NH4 Cl. In addition, 1.46 g KH2 PO4 and 3.55 g Na2 HPO4 were added per litre of solution; reducing agent – no cysteine HCl. All other solutions were the same, with the exception that extra solutions containing known amounts of NH4 Cl, or trypticase, were sometimes added as control amounts of N, present as ammonia or peptides/amino acids if required. The ammonia solution contained 5.4 g NH4 Cl per 100 ml of boiled distilled water, and the trypticase solution 10 g of trypticase per 100 ml. The amount added varied depending upon the amount required. Use of such a medium was to avoid selection of species based on absence of a specific micro-nutrient in it, rather than as a result of its ability to use the test substrate. 2.2. Inoculum The inoculum most commonly used was faeces, since interest was focused on hindgut fermentation in monogastric animals. However, it has been shown that fermentation can also occur earlier in the GIT of pigs (Williams et al., 1997). Therefore, small intestinal

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samples can also be collected from fistulated or slaughtered animals, preferably on known diets. For this kind of assessment, it was important to use a diet which was unlikely to influence GIT microflora to favour one test substrate versus another. Choice of inoculum was based on the purpose of the evaluation. Given that microbial populations vary between areas of the GIT, from a theoretical point of view it would be best to choose a source of microorganisms which come both from the GIT area under investigation, and the appropriate animal. Unfortunately, this means that if the samples came from the small intestine, the animals would have to be either fistulated or slaughtered, and this is not always practicable. The advantage of faecal samples is that samples can be collected from the same animals, fed a fixed diet, thereby decreasing variability among runs. In this case, inoculum was from unweaned piglets at three weeks of age, since the ingredients were to be evaluated for potential inclusion in diets of newly weaned piglets. From whichever source, inoculum was always prepared in the same way. Ideally, material was collected under anaerobic conditions directly from the animal, and immediately placed in a pre-warmed CO2 -filled container for transfer to the laboratory. The material was used when it as fresh as possible (i.e., <1 h between rectal collection and inoculation). In the laboratory, the material was weighed per individual pig, and an amount of pre-warmed (39 ◦ C), anaerobic, sterile, saline (9 g/l NaCl) was added in relation to the combined weight, usually in a ratio of 1:5 but sometimes more dilute depending upon the amount of material available, or its fluidity (i.e., sufficiently liquid to allow injection of inoculum using a needle and syringe into the fermentation vessels). The diluted material was then homogenized using a hand-mixer for 60 s and strained through a double layer of cheesecloth with 16 threads per cm in both directions. Based on rumen studies, this should ensure that the resulting inoculum contained both unattached and attached microorganisms. From the moment that the container was opened in the laboratory, the fluid was continually flushed with CO2 and stirred. The inoculum was then dispensed into the pre-warmed bottles containing substrate and medium – usually 5 ml for a bottle containing 82 ml of total medium. For the research described here, faeces was collected from unweaned piglets, which had no access to creep feed, and had not been exposed to antibiotics. Therefore, it was assumed that they had only ingested milk from their mothers. Piglets were housed with their sows in pens with slatted concrete floors, and kept well separated from the sow diet. They suckled freely from their mothers, and faeces was collected per rectum early in the morning. 2.3. Substrates Substrates were chosen to represent a wide range of feed ingredients and additives which might be considered for addition to a weaning piglet’s diet. The abbreviations for the feed ingredients, and their source, are in Table 1 . In the case of chicory, these are commercially available, and what is known of their processing is in the “other” column of the table. If no specific source is mentioned, then materials were obtained from a commercial supplier in the Netherlands. All substrates had been ground through a 1 mm sieve, unless already present in powder form, or otherwise indicated in the table. A partial chemical analysis (i.e.,

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Table 1 Abbreviations and suppliers of substrates tested Abbreviation

Substrate

Supplier

Grinding (mm)

Cosun Food Technology Centre, Roosendaal, NL Cosun Food Technology Centre, Roosendaal, NL Cosun Food Technology Centre, Roosendaal, NL Cosun Food Technology Centre, Roosendaal, NL

–

CHICI4

Chicory inulin-Frutanimal ND® Chicory inulin-Frutafit® HD Chicory inulin-chicory pulp Chicory inulin-EXL

CHICI5

Chicory inulin-CDF-2-A1

CHICI6

Chicory inulin-CDF-2-A2

CHICI7

Chicory inulin-CDF-2-P

Cosun Food Technology Centre, Roosendaal, NL Cosun Food Technology Centre, Roosendaal, NL Cosun Food Technology Centre, Roosendaal, NL Farm in Dalfsen, NL Orffa, Belgium

Inulins CHICI1 CHICI2 CHICI3

JERAR Jerusalem artichoke FOS Fructo-oligosaccharide Insoluble fibres POTFI Potato fibre WHEBR Wheat bran OATPE Oats, peeled CAROB Carob (St. John’s bread fine powder) PEAHU Starches HACS POTST WHEST Pectins SBPUL1 SBPUL2 SBPEC LCPEC HCPEC

–

Completely soluble

1

1

No melasse, contains ∼8% inulin Not completely soluble, partly crystalline structure Chicory fibre-dried at 105 ◦ C-2000 harvest Chicory fibre-dried at 105 ◦ C-2001 harvest Powdered chicory fibre

1

Freeze-dried

–

1 1

Euroduna Rohstoffe GmbH, Barmstedt, Germany

Pea hulls High amylose corn starch Potato starch Wheat starch

Cerestar

Sugarbeet pulp-pure

Cosun Food Technology Centre, Roosendaal, NL Cosun Food Technology Centre, Roosendaal, NL

Sugarbeet pulp-without melasse Sugarbeet pectin Low methylated citrus pectin High methylated citrus pectin Pectin

PECOR Miscellaneous LACTU Lactulose GALAC1 Galactomannanlocustbean gum (Carob) GALAC2 Galactomannan-Guar gum

Orffa, Belgium

Wageningen Centre for Food Sciences, NL Wageningen Centre for Food Sciences, NL

Other

1

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Table 1 (Continued ) Abbreviation GALAC3 DMOLI

FOS XYLAN ISOMA

Substrate

Supplier

Galactomannan-Cassia gum Dried mannan oligosaccharides (cell wall) Fructo-oligosaccharide Xylan-oat spelts Isomalt

Wageningen Centre for Food Sciences, NL

Grinding (mm)

Other

Orffa, Belgium Ferdiwo, Oudewater, NL

Unless otherwise indicated, all substrates originated from normal commercial suppliers within the Netherlands. Grinding occurred, only for those substrates indicated.

dry matter (DM), ash, Kjehldahl N, and crude fat (EE)) was completed for the more complex ingredients. The neutral detergent fibre (NDF) analysis used the method described by Van Soest et al. (1991), and acid detergent fibre (ADF) and acid detergent lignin (ADL) were determined according to Van Soest (1973). Starch analysis was determined as described by Goelema et al. (1998). In brief, total starch (‘A’) was analyzed by extracting soluble sugars with a 400 ml/l ethanol solution, followed by autoclaving for 3 h at 130 ◦ C, and enzymatic degradation to glucose (1 h at 60 ◦ C, pH 5) using an enzyme cocktail containing amyloglucosidase, ␣-amylase and pullulanase. Glucose was measured by spectrophotometry at 340 nm using hexokinase and glucose-5-phosphate (G6P) dehydrogenase. Total starch plus lower molecular weight sugars (‘B’), were determined in the same way, but without ethanol extraction. Gelatinized starch plus lower sugars (‘C’) was determined by hydrolysis with amyloglucosidase. The degree of gelatinization was calculated as a proportion of total starch after correcting for lower sugars as: degree of gelatinization = (C − (B − A)) × 100/A

(1)

These analyses were not completed on substrates which were considered to contain negligible amounts of the components. 2.4. Measurement of cumulative gas production Kinetics of fermentation were assessed by measuring cumulative gas production over time. To do this, the apparatus of Theodorou et al. (1994), which uses a pressure transducer and syringe to manually measure and remove gas from fermentation vessels at regular time intervals, was used. This was modified in Wageningen so that the transducer was connected to a computer to allow direct input of data, from both pressure and volume, into a spreadsheet, similar to Mauricio et al. (1999) although gas volume was measured by hand using a plastic syringe and recorded in a separate column in the spreadsheet for every bottle at every time interval. In this way, pressure and volume from every bottle was regressed to provide a corrected volume at each time per bottle. This method was later automated (Davies et al., 2000), whereby pressure sensors detected a fixed pressure, after which a computer software program allowed opening of a valve to release gas, and the time at which this occurred was recorded. Both methods were used, depending upon the number of samples to be evaluated.

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The automated system can only accommodate 48 vessels, while the upper limit for the manual system is about 200. In the research described here, the manual method was used due to the large number of substrates. Approximately 0.5 g of dry material (or equivalent) was directly weighed into the 100 ml serum bottle fermentation vessels, and the weight of each substrate recorded for four replicate bottles. The DM and ash were also measured for all substrates. For the research described here, following inoculation, bottles were placed in a pre-warmed incubator at 39 ◦ C, and kept there for 144 h, except for pea hulls (168 h), and potato starch and GALAC2 (192 h). The time for this fermentation was usually either 48 or 72 h, but in this case the purpose was to determine maximum fermentability of each substrate and so it was continued until gas production was very low, so that the asymptote had been reached. Following fermentation, supernatants were removed from residual substrate, by vacuum filtration through pre-weighed sintered glass filter crucibles (Schott Duran, porosity #2, Mainz, Germany) containing clean sand. Following thorough rinsing of the bottle, residue in the crucibles was flushed at least thrice with hot water, and dried to constant weight at 103 ◦ C (ISO 6496, 1999). Once the weight was recorded, crucibles were placed in a muffle furnace at 550 ◦ C for determination of remaining ash (ISO 5984, 1978). 2.5. Post-fermentation analyses Normally, fluid samples were collected following fermentation for analysis of SCFA and ammonia, as reported here. Lactic acid was often measured when digesta from the small intestine was used as inoculum, or if there is a specific interest in lactic-acid producing bacteria. It is also possible to incubate many more than the usual four replicates, and remove bottles at regular time intervals, to allow production of end-products to be described in time, which can be correlated with gas production. For the research reported here, DM was determined by drying to a constant weight at 103 ◦ C (ISO 6496, 1999), and ash by combustion at 550 ◦ C (ISO 5984, 1978). The SCFA in fermentation liquids were analyzed by gas chromatography (Fisons HRGC Mega 2, CE Instruments, Milan, Italy), using a glass column fitted with Chromosorb 101, as carrier gas N2 saturated with methanoic acid, 190 ◦ C and using iso-caproic acid as internal standard. SCFA are reported as mmol/g organic matter (OM) weighed into bottles, and total values also in the form of mg AAE (acetic acid equivalents). This latter unit, allows all individual acids to be compared according to their C content (Henry, 1981). The branched chain ratio (BCR) was calculated, by dividing branched-chain acids and including valeric in mg AAE, by the straight chain acids, to indicate the amount of SCFA, which was more likely related to protein fermentation. Ammonia was determined by the method used of Houdijk (1998), by which supernatant was deproteinized using 10 g/l trichloroacetic acid. Ammonia and phenol were oxidized by sodium hypochlorite in the presence of sodium nitroprusside to form a blue complex, measured colorimetrically at 623 nm. 2.6. Curve fitting and statistics For both manual and automated systems, the method resulted in cumulative gas profiles for each bottle. It is important that these “raw” gas results were corrected to gas as ml/g

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DM or OM of substrate. In this case, gas generated from blank bottles was not subtracted from the other bottles, but reported separately (Table 3), because microbial activity in blank bottles, in the absence of an energy source, will be quite different to fermentation in the presence of an energy source. Ruminant gas production studies have been reported frequently, and it is clear that laboratories use different media, and different proportions of inocula. Thus, including a value for blanks allows the reader to make comparisons among studies more readily. There is no reason to believe that this situation would differ for studies of monogastric fermentation. Profiles of cumulative gas production (i.e., ml of gas produced per g OM over time) were fitted to the monophasic model of Groot et al. (1996) as: G = A/(1 + (C/t)B )

(2)

where G is the total gas produced, A the asymptotic gas production, B the switching characteristic of the curve, C the time at which half of the asymptote has been reached (T1/2 ), and t the time (h). The maximum rate of gas production (Rmax ) and the time at which it occurs (Tmax ), were calculated according to equations of Bauer et al. (2001) as: 2

(−B−1) (−B) Rmax = (A(CB )B(Tmax ))/(1 + (CB )(Tmax ))

(3)

Tmax = C(((B − 1)/(B + 1))1/B )

(4)

In this case, differences between substrates within category were tested for significance using a two-way ANOVA using LS Means by SAS (1990), as: Y = µ + SUBi + εi

(5)

where Y is the parameter to be tested, µ the mean, SUBi the effect of the substrate i, and εi the error term. The effect of replicate bottles was examined separately, and was not statistically significant for any parameters, and so was included in the error term. All statistical analyses used the general linear means (GLM) procedure of SAS (1990).

3. Results A wide range of carbohydrate-based substrates was examined. For statistical purposes, they were separated into categories according to their most prominent fraction, being fructans, starches, cell wall products, pectins, and miscellaneous. The DM, OM, N, crude protein (CP), EE, NDF, ADF and ADL values for the substrates are in Table 2. Not all analyses were conducted for all substrates, as noted earlier, although for the substrates within the starch category, a complete starch analysis was completed (Table 3). The choice of substrates rich in carbohydrates was confirmed by their generally low EE and CP. Even within feed category, there were significant differences between all fermentation kinetic parameters (Table 4), indicating that some substrates were much more rapidly fermented than others. For example, wheat bran and carob were least fermentable in terms of both gas production and OM loss, but other substrates also varied.

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Table 2 Dry matter (DM; g/kg), organic matter (OM; g/kg DM), N (g/kg OM), crude fat (EE; g/kg OM), neutral detergent fibre (NDF; g/kg OM), acid detergent fibre (ADF; g/kg OM) and acid detergent lignin (ADL; g/kg OM) of the substrates Substrate Fructans CHICI1 CHICI2 CHICI3 CHICI4 CHICI5 CHICI6 CHICI7 JERAR FOS Starches HACS POTST WHEST Insoluble fibres POTFI WHEBR OATPE CAROB PEAHU Pectins SBPUL1 SBPUL2 SBPEC LCPEC HCPEC PECOR Miscellaneous LACTU GALAC1 GALAC2 GALAC3 DMOLI XYLAN ISOMA

DM

OM

N

EE

NDF

ADF

ADL

954 969 908 944 942 959 926 971 980

999 999 850 999 960 941 947 949 999

0.3 0.1 17.7 0.2 10.8 10.5 11.6 19.5 0.2

1.1 0.8 15.7 1.0 3.1 4.1 3.8 4.7 0.5

368.7

407.9

39.0

67.2 69.9 77.5 65.3

67.4 73.0 86.3 65.1

8.6 10.3 13.9 9.3

922 806 852

996 993 998

0.7 0.2 0.4

0.8 1.2 1.1

855 876 872 940 886

942 929 978 963 971

13.6 30.8 26.1 7.7 12.8

2.5 22.8 61.0 3.1 4.4

317.7 562.8 152.8 302.2 691.6

306.0 163.6 18.1 270.6 623.40

51.3 48.8 8.9 169.9 4.2

941 900 838 890 833 934

930 900 895 976 890 971

20.6 15.5 9.3 2.4 3.7 13.8

10.1 4.2 0.8 0.8 1.1 34.5

331.9 505.4

206.7 256.5

25.0 14.6

997 903 939 913 937 862 985

999 992 993 988 962 917 100

0.5 0.4 7.7 10.7 53.1 1.6 0.0

2.0 0.8 4.7 11.6 14.3 3.5 0.3

24.7

21.7

1.9

There was wide variation between substrates containing large amounts of carbohydrate, both in total SCFA, and in acid proportions, though variation was less within ingredient categories, particularly in the case of fructans, where there was very little variation (Table 5). For total SCFA production, not including those in the “Miscellaneous” category, values are generally highest (and not very different) for those ingredients of greatest structural complexity (i.e., starch, insoluble fibres and pectins), which were well fermented. The biggest variation between ingredients seemed to occur in the pattern of SCFA production which, in some cases, can probably be related to other parameters measured. For example, in the case of high amylase corn starch, the higher concentration of propionic acid

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Table 3 Amounts of total starch (g/kg OM), lower sugars (g/kg OM), gelatinized starch (g/kg OM) and the degree of gelatinization of total starch of some substrates Substrate

Total starch

Lower sugars

Gelatinized starch

Degree of gelatinization

HACS POTST WHEST POTFI WHEBR OATPE

932.1 975.9 956.3 255.3 116.5 616.0

13.4 2.1 NDa 19.2 15.0 1.9

865.2 8.7 262.3 182.0 34.3 350.4

0.93 0.01 0.27 0.71 0.29 0.57

a

Not detected (ND).

may be related to the higher degree of gelatinization versus the other two starches. This has been noted previously, in the case of maize and pea starch, which had been exposed to different pre-treatments (Bosch et al., 2002).

4. Discussion There were differences between substrates both between and within categories, in fermentation kinetics, DM losses, SCFA and ammonia production, indicating that there is large variation between ingredients, and that assumptions should not be made concerning substrate fermentability without prior evaluation. Also, solubility seemed to have little influence on fermentability characteristics of substrates. Production of gas is of little direct use to the host, although for those microbial communities which include methane-producers, it provides a hydrogen-sink for further microbial metabolism. For the host however, a sudden and excessive production of gas could be detrimental. However, the advantage of measuring cumulative gas production is that fermentation kinetics of an ingredient may provide an indication of where the product is likely to be fermented in the GIT. This information can be used to design diets which encourage fermentation in specific areas of the GIT. If a feed product is very rapidly fermentable it is more likely that, in animals with a significant microbial population in the small intestine (such as pigs), it will be fermented earlier in the tract. More slowly fermentable feeds are likely to be fermented later in the tract, such as in the large intestine. If feeds are very slowly fermentable, it is likely that they will be voided in faeces before fermentation is complete. Houdijk (1998), in studies with young ideally-fistulated pigs fed fructo-oligosaccharides and transgalacto-oligosaccharides, demonstrated that fructo-oligosaccharides were not detectable at the terminal ileum, and so had been fermented earlier in the tract. In vitro, fructooligosaccharides were also more rapidly fermentable than transgalacto-oligosaccharides. Given that bicarbonate is one of the buffers present within the GIT, and that it is essential for activity of some bacteria, it was also included in the medium. However, this buffer is also a source of gas produced in vitro. To what extent this matches what is produced in vivo is still unknown. It is well known that both CO2 and hydrogen can be absorbed into blood, and are excreted via the lungs. In fact, this phenomenon has been exploited by use of the hydrogen breath test to detect carbohydrate mal-absorption (Levitt and Donaldson, 1970)

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Table 4 In vitro fermentation kinetics of substrates using inocula from unweaned piglets Substrates

Fructans CHICI1 CHICI2 CHICI3 CHICI4 CHICI5 CHICI6 CHICI7 JERAR FOS Probability MSD Starches HACS POTST WHEST Probability MSD Insoluble fibres POTFI WHEBR OATPE CAROB PEAHU Probability MSD Pectins SBPUL1 SBPUL2 SBPEC LCPEC HCPEC PECOR Probability MSD Miscellaneous LACTU GALAC1 GALAC2 GALAC3 DMOLI XYLAN ISOMA Probability MSD

In vitro fermentation kineticsa OMCV

t1/2

Rmax

tmax

OMLoss

352.50 ab 368.03 a 359.85 ab 367.53 ab 361.10 ab 353.61 ab 343.37 ab 339.94 b 343.06 ab 0.0108 28.04

12.51 d 11.45 de 23.05 ab 17.88 c 9.71 ef 9.18 ef 9.36 ef 8.55 f 28.63 a 0.0001 2.32

19.78 c 23.04 b 13.55 d 13.36 d 25.00 ab 26.29 a 23.98 ab 25.92 a 8.48 e 0.0001 2.33

8.66 b 8.12 b 18.76 a 9.72 b 6.18 c 5.99 c 5.58 c 5.08 c 18.29 a 0.0001 1.88

0.962 a 0.959 ab 0.796 d 0.966 a 0.940 bc 0.940 bc 0.934 c 0.954 abc 0.955 abc 0.0001 0.022

370.28 b 403.12 a 361.05 b 0.0102 29.91

21.55 c 48.78 a 35.56 b 0.0001 4.95

12.40 a 8.68 b 8.24 b 0.0017 2.22

15.10 c 43.21 a 27.89 b 0.0001 3.72

0.970 a 0.929 a 0.924 a 0.1034 0.058

328.31 b 212.80 c 309.17 b 216.89 c 375.62 a 0.0001 35.64

22.09 bc 19.32 bc 24.23 b 16.81 c 47.61 a 0.0001 5.28

11.34 a 7.40 b 10.73 a 8.47 b 5.48 c 0.0001 1.67

15.14 c 11.54 d 19.48 b 10.39 d 22.95 a 0.0001 3.04

0.869 b 0.586 e 0.802 c 0.713 d 0.925 a 0.0001 0.047

332.80 b 365.23 ab 402.54 a 384.59 a 400.99 a 260.93 c 0.0001 37.51

20.73 bc 23.96 b 10.66 e 14.48 d 19.75 c 28.15 a 0.0001 3.50

11.49 c 12.10 c 25.38 a 18.01 b 14.01 c 6.26 d 0.0001 3.27

13.64 b 18.17 a 7.30 c 9.23 c 13.43 b 13.56 b 0.0001 2.30

0.816 d 0.854 cd 0.924 ab 0.964 a 0.895 bc 0.755 e 0.0001 0.045

355.25 a 348.53 ab 324.09 bc 301.24 c 140.64 e 348.21 ab 313.69 c 0.0001 27.74

12.44 e 32.95 bc 51.81 a 36.22 b 25.57 cd 26.16 c 17.05 de 0.0001 8.64

21.60 a 8.60 c 4.58 d 6.13 d 4.59 d 9.08 c 17.46 b 0.0001 2.22

9.30 e 25.14 b 30.18 a 24.63 b 20.70 c 14.92 d 14.59 d 0.0001 2.18

0.967 ab 0.952 abc 0.945 cd 0.956 abc 0.925 d 0.948 bc 0.973 a 0.0001 0.024

Means with different letters within one column differ significantly (Tukey, P<0.05). a MSD: minimum significant difference; OMCV: cumulative gas production (ml/g OM weighed in); t : half 1/2 time of asymptotic gas production (h); Rmax : maximal rate of gas production (ml/h); ttmax : time of occurrence of Rmax (h); OMLoss: organic matter loss.

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Table 5 End-products of in vitro fermentation of substrates using inocula of unweaned piglets Substrate

End-productsa SCFA

Fructans CHICI1 11.05 a CHICI2 10.91 a CHICI3 10.81 a CHICI4 10.47 a CHICI5 10.89 a CHICI6 10.65 a CHICI7 10.28 a JERAR 10.49 a FOS 8.66 b Probability 0.0001 MSD 1.13 Starches HACS 10.73 a POTST 10.05 a WHEST 10.26 a Probability 0.3997 MSD 1.36 Insoluble Fibres POTFI 10.03 a WHEBR 8.17 b OATPE 9.16 ab CAROB 5.79 c PEAHU 10.00 a Probability 0.0001 MSD 1.32 Pectins SBPUL1 10.16 a SBPUL2 10.91 a SBPEC 10.81 a LCPEC 10.67 a HCPEC 8.99 a PECOR 6.80 b Probability 0.0001 MSD 1.98 Miscellaneous LACTU 10.89 a GALAC1 8.19 b GALAC2 7.48 b GALAC3 7.50 b DMOLI 4.43 c XYLAN 10.35 a ISOMA 10.57 a Probability 0.0001 MSD 1.78

Ac

Pr

Bu

Val

AAE

BCR

pH

NH3

5.53 bc 5.38 bcd 7.13 a 4.92 cd 5.84 b 5.79 b 5.67 c 5.56 bc 4.80 d 0.0001 0.67

3.83 a 3.78 ab 1.98 e 3.44 bc 3.49 abc 3.40 c 3.15 c 3.33 c 2.41 d 0.0001 0.35

0.89 bc 0.88 c 0.94 bc 1.09 a 0.92 bc 0.89 bc 0.91 bc 0.93 bc 1.01 ab 0.0001 0.12

0.57 b 0.54 b 0.28 cd 0.77 a 0.37 c 0.32 cd 0.30 cd 0.36 cd 0.27 d 0.0001 0.097

897 a 876 a 827 a 883 a 866 a 841 a 811 a 839 a 689 b 0.0001 88.46

0.148 bc 0.142 bc 0.150 b 0.204 a 0.120 de 0.108 de 0.109 de 0.128 cd 0.103 e 0.0001 0.021

6.30 d 6.28 d 6.58 a 6.35 cd 6.44 bc 6.41 bc 6.41 bc 6.44 bc 6.46 b 0.0001 0.10

53.8 d 50.4 d 91.7 a 53.6 d 69.7 c 69.3 c 70.2 c 81.8 b 6.4 e 0.0001 3.96

4.97 a 4.27 a 4.54 a 0.0765 0.72

4.17 a 2.47 b 3.54 a 0.0007 0.69

0.95 c 2.63 a 1.39 b 0.0001 0.23

0.36 ab 0.24 b 0.44 a 0.0123 0.148

879 a 890 a 870 a 0.862 104

0.120 b 0.123 ab 0.152 a 0.0257 0.030

6.27 c 6.58 a 6.38 b 0.0001 0.06

58.25 c 76.68 a 70.20 b 0.0001 4.37

5.39 b 4.20 c 4.09 c 3.94 c 6.47 a 0.0001 0.83

2.97 a 1.99 c 2.50 b 1.14 d 2.09 c 0.0001 0.40

1.01 b 1.07 b 1.78 a 0.49 d 0.72 c 0.0001 0.13

0.27 b 0.37 a 0.31 b 0.10 c 0.36 a 0.0001 0.050

806 a 690 b 796 a 428 c 765 ab 0.0001 99

0.135 c 0.234 a 0.167 b 0.078 d 0.158 b 0.0001 0.022

6.50 b 6.71 a 6.53 b 6.61 ab 6.49 b 0.0030 0.15

79.65 c 110.42 a 86.90 b 8.35 d 80.21 c 0.0001 3.42

6.17 a 6.95 a 7.47 a 7.21 a 6.78 a 4.47 b 0.0001 1.30

2.26 a 2.29 a 2.23 a 2.14 a 1.37 b 1.60 b 0.0001 0.39

1.05 a 0.93 ab 0.62 c 0.81 b 0.55 c 0.51 c 0.0001 0.17

0.29 a 0.29 a 0.22 a 0.27 a 0.09 b 0.11 b 0.0001 0.088

796 a 839 a 793 a 796 a 638 b 504 b 0.0001 152

0.141 a 0.143 a 0.098 b 0.103 b 0.069 c 0.068 c 0.0001 0.026

6.54 a 6.53 a 6.50 a 6.60 a 6.61 a 6.60 a 0.8499 0.35

90.28 a 87.89 a 83.80 a 69.56 a 30.40 b 9.82 b 0.0001 32.81

5.60 a 4.14 b 4.11 b 3.42 bc 2.39 d 5.73 a 6.15 a 0.0001 0.98

3.70 a 2.45 cd 2.07 d 2.65 bcd 1.19 e 2.72 bc 3.11 b 0.0001 0.58

0.92 cd 1.26 ab 1.06 bc 1.15 abc 0.63 de 1.43 a 0.60 e 0.0001 0.30

0.46 a 0.20 b 0.12 cd 0.15 bc 0.06 d 0.19 bc 0.44 a 0.0001 0.074

875 a 667 cd 592 d 620 d 356 e 826 ab 823 b 0.0001 142

0.126 b 0.078 cd 0.059 d 0.068 d 0.089 c 0.089 c 0.145 a 0.0001 0.019

6.24 c 6.52 b 6.52 b 6.52 b 6.71 a 6.53 b 6.30 c 0.0001 0.09

55.58 b 7.05 f 6.00 f 11.02 e 24.86 c 69.06 a 56.67 b 0.0001 3.27

Means with different letters within one column differ significantly (Tukey; P<0.05). a MSD: minimum significant difference; SCFA: total short chain fatty acids (mmol/g OM weighed in); Ac: acetic acid. Pr: propionic acid. Bu: butyric acid. Val: valeric acid (mmol/g OM weighed in); AAE: acetic acid equivalents produced (mg/g OM weighed in); BCR: branched chain ratio; NH3 (mg/g OM weighed).

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in human studies. It is therefore unlikely that gas produced in vitro will be matched by gas production in vivo, at least not in terms of gas remaining within the GIT. In vitro, gas can best be regarded as a measure of kinetics, rather than a measure of the amount of gas which may be produced in vivo, at least until more is known quantitatively about gas production and its escape from the GIT. Further selection of ingredients for inclusion in a diet must be according to the pattern of end-products, such as the SCFA profile. For example, it is generally considered that production of SCFA can be considered to be favourable, while production of ammonia indicates a shift to protein fermentation, and this is generally considered to have negative effects on health (Macfarlane and Macfarlane, 1995). Therefore, ingredients should be chosen which result in higher production of SCFA, particularly of butyric acid, for those ingredients which are more likely to be fermented in the large intestine, as they are known to be an important energy source for the colonocytes (Roediger, 1989; Roediger and Moore, 1981). Selection should also be made for a lower BCR as an indicator of less branched-chain fatty acid production, and a lower ammonia concentration, both of which suggest reduced protein fermentation. A selection of several ingredients with different kinetic and endproduct characteristics can be made to design diets to stimulate carbohydrate fermentation along the entire tract. Results obtained in vitro may indicate which end-products could potentially be expected from the ingredients (e.g., higher butyrate values from starch – Mason and Just, 1976), though in vivo evaluations are necessary for confirmation. It is often assumed that ingredients which are soluble will be rapidly fermentable and that non-soluble ones will be more slowly fermentable. This is, of course, not necessarily correct, and is a misunderstanding which has developed from use of the rumen in situ technique for ruminant animals, where all material lost from the in sacco bags is often assumed to be fermented. Another frequent misconception is that a “fibrous” material is fermentable but, as is evident from results presented here, even if a substrate is fermentable, there can still be a lot of variation in terms of kinetics and end-products. By using the technique described here, it is possible to make a more realistic assessment of fermentability, although it remains an in vitro technique and cannot, and should not, be expected to replicate what occurs in a living digestive tract. One important source of variation is the microbial population present within the GIT, which is used as inoculum. This varies both between species of animals as well as between individuals, making it virtually impossible (at least in the case of animals) to evaluate ingredients in relation to every microbial population (i.e., of every individual) before their inclusion in a diet. An interesting possibility for use of this method in the future is to use it in combination with molecular techniques, such as polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis, to detect stimulation of specific bacterial species in vitro in response to a specific ingredient. This research has already started (Zhu et al., 2003), but needs to be combined with in vivo research to determine whether similar stimulation can also be detected in vivo. If validated, this would make the combination of techniques an ideal test for prebiotic activity, and would take a lot of the unknowns out of current attempts to design prebiotics for inclusion in human and animal diets. Gas production measurements are now used routinely by our group to evaluate potential feed ingredients for inclusion in pig (Bauer et al., 2001; Bosch et al., 2002; Awati et al., 2003) and poultry (Guo et al., 2003) diets. It has also been used to evaluate specific

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chemical compounds, in combination with specific standard carbohydrates, to determine how fermentation of those carbohydrates can be altered by the presence of that compound. For example, the effect of olive oil polyphenols was evaluated, using starch and glucose as the standard carbohydrates fermented, with a human faecal microflora (Tassiopoulou et al., 2003). Certain carbohydrate additives, as well as a growth-promoting antibiotic, were combined with porcine chyme as substrate and inoculated with pig faeces (Bauer et al., 2003b). Guo et al. (2003) reported use of mixed chicken caecal contents to compare fermentability of mushrooms, a herb, and their polysaccharide fractions for addition to poultry diets. The relation of results obtained using this in vitro method are being examined for their relevance in vivo. There are, of course, complications. For example, in this in vitro test, it is possible to evaluate specific ingredients, which is not possible in vivo since an animal requires many more nutrients than those in any single ingredient. Also, much larger quantities are required to feed animals than are required in vitro, which is not easy if ingredients are only available in small quantities or are expensive. Even so, a start has been made to relate results from in vitro data to various animal parameters. In a large in vivo experiment, comparing a control diet with a test diet containing added lactulose, inulin, sugarbeet pulp and wheat starch (based on fermentability results reported here), digesta SCFA, lactic acid and ammonia were measured, along with determinations of the microbial community and tissue cytokine concentrations. Konstantinov et al. (2003), reported changes in faecal microbial communities using molecular techniques, and showed more diversity and numbers of bacterial species in faeces of piglets fed the test, versus the control, diet. Closer examination was also completed of the microbial community of the small intestine digesta of the same piglets, and it was found that lactic acid concentrations of the small intestine increased (Awati et al., 2002), and that growth of several Lactobacillus species had been stimulated in the presence of the test diet containing fermentable carbohydrates (Konstantinov et al., 2004). Immunological studies were also completed to compare the same animals fed the same diets, and some changes in cytokine concentrations, in the small and large intestines, occurred (Pi´e et al., personal communication). It is clear that there is a link between diet, microbial community and immunological development and, while an in vitro method such as that reported here cannot give an indication of what those links might be, they may suggest which ingredients to include in the diet, so that fermentation will be encouraged in a positive direction, and investigations of those links can occur. On a more basic level, much more research is required to untangle the direct relations between in vitro and in vivo data. However, while there is unlikely to be a quantitative relationship between the two in terms of SCFA, or lactic acid, it could be that if a particular ingredient seems to stimulate those bacterial species in the inoculum which will produce, for example, butyric acid, there is a strong likelihood that those bacteria may also be stimulated somewhere within the GIT of the living animal. Therefore, a qualitative link seems likely.

5. Conclusions This is a detailed description of an in vitro method to assess fermentability of potential food ingredients for monogastrics, both in terms of their kinetics and end-products. It has been shown to be quite flexible in terms of being able to match the test substrates with

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the inoculum of interest. Results from such evaluations allow an informed choice in terms of adding ingredients to animal or human diets for the purpose of stimulating activity of potentially “beneficial” (in the widest sense), bacteria in the GIT.

Acknowledgments The authors thank Lotje Bos who completed the manual run of the 45 substrates as part of her Masters Thesis in our Group. Dick Bongers, Jane-Martine Muijlaaert, and Yang Hongjian, were of invaluable assistance with the gas readings and analyses. Dr. Liesbeth Bolhuis of the Animal Ethology Group kindly allowed us to collect faeces from animals involved in her work. The following people kindly donated the following substrates for this work: Dr. Henk Schools of the Wageningen Centre for Food Sciences – Galacto-mannans; Mr. Aart Mul of “Cosun” – inulin products; Drs. Waterval from Dalfsen – fresh Jerusalem artichoke; Mr. Nils Dubbeldam of “Euroduna” – carob product; Mr. Sjur Tveite of Biotec ASA – MacroGard; Mr. Marlo van Bergen of Ferdiwo – Isomalt. This work was supported by funding from the EU project “Healthypigut” (QLK5-LT2000-00522), which is gratefully acknowledged by the authors. However, the authors are solely responsible for the text of this publication which does not represent the opinion of the EU. The EU is not responsible for the information presented in this text.

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