J Sci Food Agric 1997, 73, 101È105

A Simple In Vitro Fermentation System forPolysaccharides—The Effects of Fermenter FluidSurface Area/Fluid Volume Ratio and Amount ofSubstrateAgnes Stevenson, Callum J Buchanan, Rocio Abia and Martin A Eastwood*

Gastrointestinal Laboratory, Department of Medicine, Western General Hospital, University ofEdinburgh, Crewe Road, Edinburgh, EH4 2XU, UK

(Received 15 February 1996 ; revised version received 2 July 1996 ; accepted 2 July 1996)

Abstract : A simple batch fermentation system for dietary non-starch poly-saccharides using simple in vitro methods has been studied to identify if the posi-tion of the bottle (Ñuid surface area/Ñuid volume ratio) and shaking inÑuencefermentation and bacterial mass. Shaking has no e†ect on the fermentation orbacterial mass increase. The amount of pectin, ispaghula or starch inÑuences theresult as does the surface to volume ratio.

Key words : polysaccharides, methodology, dietary NSP, in vitro fermentation,biomass, surface/volume ratio, pectin, ispaghula, starch

INTRODUCTION

The fermentation of non-starch polysaccharides (NSP)and starch in the colon has an important physiologicalfunction. Humans are able to absorb and metaboliseshort-chain fatty acids (SCFA) formed in the colon fromfermentation (Hoverstad et al 1982). Such fermentationis difficult to study in human and animal experiments.There is therefore, a need for simple in vitro systems forinvestigating the fermentation of isolated NSP. In vitrofermentation systems are useful in identifying the poten-tial e†ects of dietary polysaccharides on colonic fermen-tation before undertaking expensive human or animaltrials. The conditions of such a system have not beenfully explored. There appears to be no agreement on theamount of substrate, amount of medium or the size ofthe vessel used for the fermentation studies (McBurneyand Thompson 1989 ; Titgemeyer et al 1991 ; Bourquinet al 1993 ; Mortensen and Nordgaard-Anderson 1993).

The studies described in this paper investigate theinÑuence of the Ñuid surface area/Ñuid volume ratio inthe fermentation vessel, the concentration of substratefermented and the e†ect of shaking or static conditionsof the fermentation bottle. The production of SCFA,which reÑects the extent of polysaccharide fermentation,

* To whom correspondence should be addressed.

and the amount of protein, which equates with the bac-terial biomass, are measured at three di†erent concen-trations of the polysaccharide substrate.

MATERIALS AND METHODS

Three polysaccharide substrates were used, pectin (HPBulmer Ltd, Hereford, UK) ; ispaghula (mixed husk andseed, MADAUS, Germany) and corn starch (SigmaChemical Company Ltd, Dorset, UK).

In vitro fermentation

Polysaccharide substrate (0É25, 0É5 or 1É0 g of eachpreparation) was added to 80 ml of pre-reduced auto-claved medium (Goering and Van Soest 1979) inmedical Ñat bottles (300 ml volume, 13É5 cm high,6É5 cm width and 3É5 cm deep). A 20 ml sample ofcaecal inoculum (80 g rat caecum contents litre~1 pre-reduced medium) was added. Strict anaerobic condi-tions were maintained by continuous Ñushing withoxygen-free carbon dioxide before sealing with suba-seals (BDH Merck Laboratories, Glasgow, UK). Thebottles were incubated at 37¡C for 24 h. Control fer-mentations contained no additional polysaccharide.

The fermentation bottles were positioned in the incu-bator standing or lying to give a Ñuid surface area/Ñuid

101J Sci Food Agric 0022-5142/97/$09.00 1997 SCI. Printed in Great Britain(

102 A Stevenson et al

volume ratio (SA/V) of 1 : 4 or 1 : 1 or 1 : 1S(S \ shaken). When shaking was being studied, a waterbath at 37¡C was used, with the samples agitated at50 rev min~1. The control samples were incubatedusing the same conditions as the substrate samples. Allexperiments were performed in triplicate.

The Ñuid surface area cm~1 ratio in each positionwas calculated from the internal measurements of thetotal inoculated Ñuid within the bottle (width ] length)lying or standing. The volume (ml) was the total volumeof inoculated Ñuid (100 ml).

A 2É0 ml sample of inoculated Ñuid was removed at 0and 24 h and added to 0É5 ml of 1 M NaOH beforeanalysis for SCFA. An internal standard (4-methyl-valerate, 10 g litre~1) was added to correct for extrac-tion efficiency. Aliquots (800 kl) were extracted with3 ] 3 ml diethyl ether and the ether extracts pooled. A3 kl sample of the extract was assayed by gas chroma-tography using 100 g kg~1 SP/1200/10 g kg~1 H3PO4chromsorb W-AW, 175È147 km mesh. SCFA wereseparated over a temperature range of 80È180¡C(gradient 15¡C min~1) using oxygen-free nitrogen as thecarrier gas and detected using a Ñame ionisation detec-tor (Spiller et al 1980). While removing samples of thefermentation Ñuid for analysis of SCFA, 2 ml wasremoved and added to 0É2 ml of 10 g litre~1 sodiumazide. Samples were stored at [ 20¡C until analysis forprotein content. Protein was assayed using the Bio-Radprotein assay (Bio-Rad Laboratories Ltd, Hemel Hemp-stead, Hertfordshire, UK) and measured in a UVÈVisspectrophotometer (Pye Unicam Ltd, York Street,Cambridge, UK) at optical density 595 nm. A standardcurve was prepared using bovine serum albumin (SigmaChemical Co, Poole, Dorset, UK). The samples wereanalysed at the same time as the standards.

Statistical analysis

The results were compared by one-way analysis ofvariance (one-way ANOVA) to test for signiÐcant di†er-

ences. Means of the groups were compared using theStudentÏs t-test. Di†erences at P\ 0É05 were consideredsigniÐcant (Snedecor and Cochrane 1980). A statisticalpackage (Minitab, release 7.1, 1989, State College, PA,USA) was used.

RESULTS

Pectin fermentation

The average production of total SCFA for the controlsample, with no added polysaccharide substrate was26 mmol litre~1 after 24 h. Agitation of the fermenta-tion Ñask did not a†ect the amount of SCFA producedfor any polysaccharide at any amount. An example ofthis is given in Table 1 for pectin, static and agitated ata SA/V ratio of 1 : 1.

Table 2 shows the net production of SCFA at 24 hwhen various concentrations of pectin are fermentedusing di†erent SA/V ratios in either shaken or unshakenconditions.

No SCFA were detected at 0 h with pectin exceptwhen a SA/V ratio of 1 : 1 was used without shaking.After 24 h fermentation the amounts of SCFA producedby each concentration of pectin under the same fermen-tation conditions were signiÐcantly di†erent (P\ 0É05).The amounts of SCFA produced being reduced as theamount of pectin is reduced (P\ 0É05), with the excep-tion of 5 g and 2É5 g litre~1 at a SA/V ratio of 1 : 4where SCFA net production remained constant. After24 h, increased amounts of SCFA (Table 2) were pro-duced with a SA/V ratio of 1 : 1 as opposed to 1 : 4 with10 g or 5 g litre~1 pectin (P\ 0É05). This was not seenwith the 2É5 g litre~1 samples.

No di†erences were found in the relative amounts ofacetate, butyrate and propionate in the fermentationsystem for pectin, regardless of fermentation conditionsand amount of total SCFA produced.

TABLE 1Production of SCFA (mmol litre~1) at 24 h fermentation using three concen-

trations of pectin, with or without agitation mean (^SD), n \ 6

SA/V ratio Acetic Propionic Butyric T otal

10g litre~1 pectin1 : 1 76É6 (13É9) 10É1 (0É3) 11É7 (0É7) 98É4 (4É7)1 : 1Sa 78É3 (5É3) 10É2 (1É5) 11É0 (1É0) 99É5 (7É2)

5g litre~1 pectin1 : 1 51É5 (4É4) 8É0 (0É8) 8É1 (0É8) 67É4 (3É0)1 : 1S 45É7 (9É4) 7É1 (0É7) 7É2 (0É8) 63É3 (7É3)

2É5g litre~1 pectin1 :1 33É6 (5É6) 6É4 (1É7) 5É8 (1É6) 44É5 (8É1)1 : 1S 28.3 (6É0) 5É2 (0É4) 4É9 (0É5) 38É4 (6É6)

a S, shaken.

A simple in vitro fermentation system for polysaccharides 103

TABLE 2Production of protein and SCFA and SCFA protein ratio at various polysaccharide con-

centrations and fermentation conditions at 24 h (mean ^ SD, n \ 6)

SA/V ratio Protein SCFA SCFA/protein(mg litre~1) (mmol litre~1) mg litre~1 (mmol mg~1 protein)

Pectin g litre~11 : 4

2É5 417 (59) 43É0 (6É6) 0É1035É0 470 (34) 40É6 (5É0) 0É086

10É0 592 (41) 85É5 (8É9) 0É1441 : 1

2É5 388 (72) 44É5 (8É1) 0É1155É0 504 (63) 67É4 (3É0) 0É134

10É0 634 (99) 98É4 (14É7) 0É1551 : 1S

2É5 437 (32) 38É4 (6É6) 0É0885É0 539 (53) 63É3 (7É3) 0É117

10É0 699 (39) 99É5 (7É2) 0É142

Ispaghula g litre~11 : 4

2É5 411 (73) 29É5 (2É7) 0É0725É0 415 (67) 39É6 (7É1) 0É095

10É0 582 (88) 67É3 (10É6) 0É1161 : 1

2É5 300 (43) 32É5 (6É4) 0É1085É0 443 (22) 45É1 (15É7) 0É102

10É0 532 (114) 61É1 (5É9) 0É1151 : 1S

2É5 351 (40) 31É5 (6É8) 0É0965É0 467 (64) 39É8 (9É9) 0É085

10É0 542 (102) 65É7 (13É2) 0É121

Starch g litre~11 : 4

2É5 344 (64) 36É1 (3É6) 0É1055É0 496 (6) 40É8 (3É3) 0É082

10É0 630 (93) 59É2 (11É7) 0É0941 : 1

2É5 441 (45) 39É3 (2É2) 0É0895É0 584 (33) 59É8 (5É0) 0É102

10É0 788 (121) 91É3 (6É9) 0É1161 : 1S

2É5 523 (15) 39É6 (3É6) 0É0765É0 726 (66) 63É9 (11É7) 0É880

10É0 1071 (75) 94É4 (10É4) 0É880

Control 275 (80) 26É0 0É095

Ispaghula fermentation

The net amounts of SCFA produced after 24 hdecreased as the amount of ispaghula was reduced. Theresults were signiÐcantly di†erent (P\ 0É05) betweeneach concentration of ispaghula for each situation.

There was no signiÐcant di†erence (P[ 0É05)between the net amount of SCFA produced for any per-centage of ispaghula used in each fermentation condi-tion (1 : 4 ; 1 : 1 and 1 : 1S) at 24 h.

No di†erences were found in the relative amounts ofacetate, butyrate and propionate in the fermentationsystem for ispaghula regardless of fermentation condi-tions.

Starch fermentation

As the amount of starch in the fermentation bottledecreased, the net amounts of SCFA produced after

104 A Stevenson et al

24 h of fermentation were signiÐcantly reduced(P\ 0É05) under each of the fermentation conditions.After 24 h of fermentation the amounts of SCFA pro-duced by each concentration of starch are signiÐcantlyincreased at 1 : 1 SA/V ratio compared with 1 : 4 with10 g litre~1 starch (P\ 0É05). When 2É5 g litre~1 starchwas used, changes in the SA/V ratio did not produce asigniÐcant di†erence in the amount of SCFA.

No di†erences were found in the relative amounts ofacetate, butyrate and propionate in the fermentationsystem for starch regardless of fermentation conditions.

Protein production during fermentationTable 2 shows the amount of protein produced duringthe fermentation of various amounts of substrate,pectin, ispaghula and starch and SA/V ratio. Proteinconcentrations increase with time. The 10 g litre~1 sub-strate results in a higher protein increase than 5É0 and2É5 g litre~1 (P\ 0É05). Using a smaller fermentersurface area less protein is produced when pectin andstarch are the substrates (P\ 0É05) but not whenispaghula is used. Shaking during fermentation does notappear to inÑuence the amounts of protein, althoughsome di†erences are apparent when starch is a sub-strate.

DISCUSSION

This paper examines the e†ect of fermentation condi-tions in a simple batch system using rat caecal inoculumand deÐned polysaccharide sources, pectin, ispaghulaand starch. Pectin is a rapidly fermented poly-saccharide. Ispaghula is fermented but more slowly(Edwards and Eastwood 1992) and starch is an a-glucanof considerable dietary interest both in the soluble andresistant starch form, the resistant form passing to thecolon to be fermented (Englyst and Kingman 1990). Theuse of a simple Ñask fermenter system would allowrapid identiÐcation of the fermentation properties ofpolysaccharides on events in the colon, eg fermentationand stool weight (Adiotomre et al 1990).

The fermentation of pectin produces more SCFAthan ispaghula and starch. Reducing the SA/V ratio ofthe bottle from 1 : 1 to 1 : 4 is associated with areduction of net SCFA production with pectin andstarch but not ispaghula. As the SA/V ratio is increased,more bacteria have increased access to the expandedsubstrate surface/substrate mass, thereby fermenting thesubstrate more readily and hence producing moreSCFA.

For none of the experimental conditions or substrateconcentrations did shaking have any e†ect on the fer-mentation process.

The amount of Ðbre added to the fermenter systemappears to be important. There is proportionately more

SCFA produced after 24 h when less polysaccharide isadded to the fermentation system. This indicates thatthe rate of fermentation is inversely dependent uponsubstrate mass. Accessibility of the substrate bulk to thebacteria may be a factor. An assumption is that theaccessibility of the substrate is the same at all stages offermentation. If, however, there was a residue of sub-strate less accessible to the bacteria, fermentation wouldbe substrate dependent. Such a hypothesis has yet to betested. There is less total SCFA at lower concentrationsof pectin although the amount of SCFA per mg proteinis greater. The pH in the medium may alter as a resultof the increased fermentation but this was not measuredin these experiments. This would indicate that there isan optimal concentration of pectin and an increaseabove this amount would have little e†ect on net SCFAproduction. Measuring protein in the fermentationsample is a means of assessing bacterial biomass. Anincrease in the concentration of substrate also increasedthe amount of protein produced and by implication thebacterial biomass. The medium used contains an excessof nitrogen (as ammonium salts) and therefore the pro-duction of bacterial protein is a useful indicator of bac-terial biomass and is not nitrogen limited. The simplemethodology described here will enable such interestingÐndings to be explored in detail.

There are no signiÐcant di†erences in the amount ofSCFA produced with shaking with any of the poly-saccharides regardless of the SA/V ratio. Shakingduring the fermentation process was not an importantfactor in determining results.

The SA/V ratio inÑuences fermentation. The fermen-tation of pectin and starch gave di†erent yields of SCFAafter 24 h when SA/V ratios of 1É4 and 1É1 were com-pared. Ispaghula showed no signiÐcant di†erences forSCFA production for the di†erent SA/V ratios. TheSA/V ratio inÑuenced the protein production in thestarch experiments. A SA/V ratio of 1 : 4 produced lessprotein after 24 h compared with other surface area/volume ratios.

The SA/V ratio inÑuences some of the fermentationresults but is substrate dependent. A SA/V ratio of 1 : 1allows more fermentation of the substrate than a 1 : 4ratio. This is presumably by increasing the rate ofgaseous exchange between the liquid and gaseousphases. In the case of ispaghula there will be an increasein the area occupied by insoluble material at the bottomof the Ñask allowing bacteria greater access to theispaghula. Shaking the fermentation system did notinÑuence results and therefore shaking need not be partof the procedure. Some of the fermentations weremade with shaking and showed no net increase inSCFA production. Shaking was carried out with bottlesin the same position as those with a 1 : 4 SA/V ratioand would represent a further increase in the e†ectivesurface area available for gaseous exchange. There is anideal surface area gaseous exchange, in this case the

A simple in vitro fermentation system for polysaccharides 105

removal of fermentation gases and from the(H2 CH4)liquid medium and a further increase in surface area hasno signiÐcant e†ect on fermentation. Using a 10 glitre~1 substrate system may slow fermentation, poss-ibly through overloading the inoculum.

These results indicate that the fermentation pattern ofa polysaccharide may be studied using a single batchsystem. It is advisable to deÐne the type and amount ofnon starch polysaccharide and the Ñuid surface area/Ñuid volume ratio.

ACKNOWLEDGEMENT

The authors would like to thank SmithKline Beechamfor Ðnancial support of this work.

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