577 . 154 : 616.34-008.87 : 599

BACTERIAL BREAKDOWN OF STRUCTURAL STARCHES AND STARCH PRODUCTS IN THE DIGESTIVE TRACT OF RUMINANT AND NON- RUMINANT MAMIVIALS

FRANK BAKER, HAMED NASR, FRANK MORRICE and JABEZ BRUCE Rowett Research Institute, Bucksburn, Aberdeenshire

(PLATES CXLIX-CLVII)

MANY species of bacteria and protozoa are capable of decomposing starch in vitro (Thaysen and Galloway, 1930 ; Porter, 1946). In the digestive tract of mammals starch broken down by bacteria may have important nutritional consequences for the host (Fridericia, 1926 ; Kon et al., 1938 ; Van der Wath and Myburgh, 1941 ; Baker, 1942). Neither the primary site nor the predominant agents of decomposition, however, are necessarily the same in different hosts, and in the same host the mode of breakdown may be influenced by the type of starch in the diet. In the course of numerous microscopic examinations of the gut contents of mammals (Baker and Nasr, 1947 ; Baker and Harriss, 1947-48) it became increasingly apparent that the morpho- logical appearances of starch disintegration in the gastro-intestinal tract under various circumstances, though diverse, nevertheless presented an intelligible pattern whose presentation would be helpful to a study of the bacteriological, biochemical and nutritional con- sequences of the process. The results of our study of this pattern are dealt with in four sections : (I) observations on the gut contents of animals given diets containing various starches ; (11) bacterial ecology of starch breakdown ; (111) cultural investigation of bacterial breakdown of starch in the pig’s cscum ; and (IV) related observations which assist interpretation of the preceding studies.

I. OBSERVATIONS ON THE OUT CONTENTS OF ANIMALS QIVEN DIETS CONTAINING VARIOUS STARCHES

Material and methods

The breakdown of untreated maize- and potato-starch granules was studied microscopically in the digestive tract of rats, mice, hamsters, guinea-pigs, rabbits, domestic pigs, man, sheep and oxen. Chief attention was given to the cecum of non-ruminants and the rumen of ruminants, and to untreated starches prepared from raw potat,o and raw maize ; but the breakdown in viwo and in &ro of a

J. PATH. BACT.-VOL. LXII 617 2 T 2

618 F. BAKER, H . N A S R , F. MORRICE A N D J . BRUCE

number of other starches and starch products was also investigated. If only small quantities of a starch or starch product were available, rats and mice were employed for the in-vivo experiments.

Microscopic methods. These included methods for the demonstration of the structural and histochemical changes which attend the breakdown of starch granules, and the bacterial agents directly or indirectly concerned.

Breakdown of starch granules was detected by changes in birefringence in polarised light and by staining with iodine and Congo red solutions. Bacteria were demonstrated by means of wet preparations stained in iodine and cotton blue. Full details of these procedures are given by Baker and Nasr. Dried and fixed films were also prepared and stained by one or other of the usual methods, and a few specimens were examined with the phase-contrast microscope.

These were collected either immediately after death or through a fistula in the living animal; samples for microscopic examination were fixed without delay in 10 per cent. formalin. Fistula material was collected from sheep and oxen with rumen fistulae only, and from sheep with fistulae in the rumen, duodenum, ileum and caccum. In man, samples were collected from a patient with a pelvic colostomy.

Diets. Rats, mice and hamsters were given the starch under investigation in a diet whose composition was as follows :-starch under investigation 400 g., casein 230 g., sucrose 330 g., margarine 150 g., McCollum’s salt mixture no. 185 (McCollum et al., 1916) 80 g. and Radiostoleum (B.D.H.) 3 ml. Radiostoleum contains vitamin A, 15,000 international units per grsmme and vitamin D,, 3000 international units per gramme.

Guinea-pigs and rabbits were given the usual greenstuff diet heavily dusted with starches. In some experiments whole raw or boiled potatoes were also given. Some sheep and oxen received the starch through a rumen fistula, the animals being given an ordinary diet of hay or pasture. Pigs were fed twice daily on the usual type of mixed cereal and concentrate ration (National Pig Food) and 1 lb. of the appropriate starch was added to each feed.

Collection of gut contents.

Structural features attending the bacterial breakdown of starch granules

Starch-disintegrating bacteria exercise their action in situ upon the surface of the granules. The structural features attending break- down are remarkably uniform in different animal species and include four main features. (1) Indentation of the margins (fig. 8) and pitting of the surface are regularly observed. (2) Progressive alterations are seen in the colour-reaction with iodine, the initial blue-black giving place in sequence to mauve and red reactions. In the earlier stages of breakdown various parts of the granule are differently coloured. In maize starch, the bacterial attack ordinarily begins at the hilum. In potato starch it often begins simultaneously from the hilum and from a number of peripheral foci. As breakdown continues colourless residues are often observed in which the lamellation of the granule is still visible (figs. 26-30). The normal lamellation of untreated starch granules is readily disclosed by the phase-contrast microscope (fig. 19). (3) Birefringence in polarised light is lost (figs. 1-16). In potato starch this process develops irregularly, granules showing a fragmented appearance, with progressive obliteration of the maltese cross. In

J. PATH. BACT.-VOL. LXIl

INTACT STARCH GRANULES

PLATE CXLIX

FIGS. 1-&--Intact starch granules in xylol-balsam under polarised light, showing birefringence and the cross of polarisation. FIG. 1 .-Canna (tous-les-mois). x 500. FIG. 2.-Tapioca. x 500. FIG. 3.-Potato. x 500. FIG. 4.-Potato. x 650. FIG. &-Waxy maize. x 500. FIG. &-Normal maize. x 500.

BACTERIAL BREAKDOWN OF STARCHES 619

maize starch, examined in ruminants and hamsters, the attack commonly begins at the hilum. (4) The staining reaction in Congo red is increased.

The observation that alteration in the iodine reaction runs parallel to loss of birefringence shows that crystallite disorganisation is accompanied by actual dextrinisation of the starch. Moreover, a diffusion of coloured dextrins into the surrounding field can be directly observed in iodine preparations where a number of granules are heaped together. Since it is well established (Hopkins, 1946) that the smallest trace of amylose gives a blue reaction with iodine, the temporary persistence of non-reacting residues in potato starch indicates that the amylose fraction of the granules is more rapidly dispersed than their amylopectin fraction.

Xite of breakdown of maize and potato starches

The features described above yield criteria for detecting the occurrence and site of breakdown of starches in different animal species. The main points may be briefly stated.

In non-ruminants other than man. In rats, mice, rabbits, guinea-pigs and domestic pigs, maize starch is digested in the small gut and relatively few granules reach the caecum (fig. 14). Conversely, potato-starch granules accumulate in the caecum in large numbers and are there attacked by bacteria. In rats and mice caxal contents of animals given potato starch in concentrate diets are invariably light in colour and of pasty consistency. The caecum is often dilated with gas and if the experiment is prolonged it becomes permanently enlarged. The colour and consistence of the faxes of animals given potato starch are more variable than the colour and consistency of their caecal contents. This variability is probably due to differences in the rate of bacterial breakdown in the caecum, which, in turn, will influence the amount of undigested or partly digested starch accumulating in the rectum. In animals given maize starch the faxes are always dark.

In hamsters both maize and potato starch accumulate in the gastric diverticulum where, though to a rather limited extent, bacterial breakdown of both types of starch granule is observed. Large numbers of potato-starch granules pass on to the caecum where further bacterial breakdown results. In some hamsters maize-starch granules also reach the caecum. The faeces of hamsters given potato starch are commonly lighter in colour than those given maize starch. No point can usefully be made of the colour of the caecal contents in rabbits and guinea-pigs because, with the diets used, the faeces are deeply stained by chlorophyll and other residues. The amounts of starch fed to pigs was insufficient to have a decisive effect on the colour of the faeces.

In man. Through the helpful collaboration of Professor W. C.

620 F. BAKER, H . NASR, F . MORRICE AND J . BRUCE

Wilson of Aberdeen University, untreated maize starch and untreated potato starch were given in different meals to a male patient with a pelvic colostomy. Samples were collected from the colostomy opening one hour before and a t 4, 8, 12, 16 and 28 hours after the meal. Two hundred grammes of potato starch were taken, but the patient could not be persuaded to eat more than 100 g. of maize starch. Microscopic examination of the colostomy samples showed no granules of potato starch before or up to 8 hours after the meal. A few granules were detected at 12 hours and a large number at 16 hours after the meal. Many granules were still present a t 28 hours. The granules a t 16 and 28 hours showed clear signs of microbial breakdown. Colostomy samples taken in the same way after maize starch had been given showed no granules before and up to 12 hours after the meal. At 12, 16 and 28 hours a very few granules were observed. It is im- probable that these differences can be attributed to the smaller quantity of maize starch eaten ; instead it reflects the different digestibility of the two starches by the gut enzymes.

I n ruminants. Through the kindness of Drs M. McNaught, E. C. Owen and J. A. B. Smith of the Hannah Dairy Research Institute, and Dr A. T. Phillipson of this Institute, we received for examination material from oxen and sheep obtained through fistulae into the rumen and other parts of the gut. In oxen and sheep, bacterial breakdown of both maize and potato starch is readily demonstrated in the rumen. Maize starch is more rapidly broken down than potato starch. Thus in each of two oxen with a rumen fistula, disintegration of maize starch could be demonstrated 1 hour after the starch was given and it was conspicuous at 2 hours. At 8-14 hours most of the granules had disintegrated. When the same two animals were given potato starch, samples from one of the oxen showed little structural change in the starch granules 4 hours after administration, and even a t 8 hours it was not pronounced. Little further change took place during the next 8-16 hours. In the other animal scarcely any dis- integration of potato-starch granules was observed over 16 hours. Again, in a sheep with separate fistuh in the rumen, duodenum, ileum and caecum, disintegration of maize starch in the rumen was observed a t 2 hours. Only a few granules could be detected after 11 hours. With potato starch, disintegration could be observed in rumen samples at 2 hours but numerous nearly intact granules still remained a t 6 hours. After 11 hours no maize starch reached the duodenum whereas potato-starch granules were still demonstrable. Little maize starch reached the ileum, whereas potato starch was observed in the ileum and caxum after 4 hours. The partly dis- integrated granules which reached the caecum did not appear to undergo any further change and showed few adherent bacteria. These observations indicated that :-( 1) Potato starch has greater resistance to the action of both digestive secretions and micro-organisms than maize starch in all the animal species investigated. (2) In non-

J. PATH. BACT.-YOL. L S I I

BACTERIAL BREAKDOWN OF STARCHES

PLATE CL

FIG. 'I.-Intact starch granules from maranta (st Vincent arrowroot) in xylol under polarised light. x 500.

~~

FIG. 8.-Potato starch from rumen of ox : early phase showing indentation of margins. x 500.

FIGS. 9 and 10.-Potato starch from rumen of ox : later phases. x 500.

FIGS. 11 and 12.-Potato starch from ciecum of pig : early arid late phases. X 650.

FIGS. 8- 12.-Phases of bacterial breakdown of starch, showing deformation of crosses of polarisation and loss of birefringence. Polarised light : in xvlol - balsam.

PLATE CLI FIG. 13.-Maranta starch from faeces of rat : representative field.

FIQ. 14.-Maize starch from faxes of rat : this field contains a single granule, found

x 500.

only after prolonged search. x 500.

FIG. 15.-Maize starch from gastric diverticulum of hamster. x 500.

FIGS. 13- 15.-Phases of bacterial breakdown of starch, showing deformation Polarised light : in of crosses of polarisation and loss of birefringence.

xylol-balsam.

FIG. 16.-Potato starch ground in ball mill to 31 per cent. solubility in cold watcr, Polarised light : in showing deformation of granule and cross of polarisation.

xylol-balsam. x 500.

field illumination : in air. x 500. FIG. 17.-Intact potato-starch granules, showing optically smooth surfaces. Dark-

FIG. 18.-Potato-starch granules ground to 31 per cent. solubility in cold water, showing Dark-field illumination : in air. deformation of granules and pitting of surface.

X 500.

J. PATH. BACT.-VOL. LSII

B.4CTERIAL BREAKDOWN O F STARCHES

PLATE CLI

.I. P ~ T I I . BACT.-Vnr,. r m r

STARCH GRANULES BY PHASE-CONTRAST ILLUXIXATIOX

PLATE CLII

FIG. I9-Intact potato- starch granules by phase-contrast illumin- ation to show lamella- tion. In water. x 550.

FIG. 20.-Intact potato- starch granules by phase-contrast illumin- ation, to show optically smooth surface. In water. x 5.50.

FIG. 21.-Potato starch from c5ccum of rat by phase- contrast illumination, to show granules in process of breakdown, with adherent masses of bacteria. x 270.

PLATE CLIII FIG. 22.-Portion of surface of potato-starch granule from caecum of pig in process

of breakdown, showing bacteria in enzymatic cavities. Phase-contrast illumination. x 1200.

FIG. 23.-Starch granules with surrounding microbial facies of rods from caecum of mouse. Cf. fig. 34. x 380.

FIG. 24.-Surface of potato-starch granule from gastric diverticulum of hamster in formol-saline to show adherent coccoid forms. Phase-contrast illumination. Note wrinkling of surface layer. See also fig. 37. x 600.

Membrane system of starch granule after irradiation at 25378 and extraction with chloral hydrate to remove unaltered starch. In butanol (R.I. 1.397). Such systems are demonstrable in potato but not in maize starch, and may account for the relative resistance of tuber starches to the digestive secretions (see text).

FIG. 26.-Iodine preparation of starch granules from caecum of rabbit in process of breakdown : early phase.

FIG. 27.-Iodine preparation of starch granules from caecum of rabbit in process of breakdown : late phase.

FIG. 25.-Photo-degradation of starch in ultra-violet light.

x 500.

x 500.

x 500.

FIGS. 26 and 27 are from preparations showing dissolution of the blue-reacting (amylose) component of potato starch, with temporary persistence of a non-reacting (amylopectin P) residue.

BACTERI.41. BREAKDOWK OF STARCHES

PLATE CLIII

BACTERIAL BREAKDOWN OF STARCHES 621

ruminants, including man and excepting the hamster, maize starch is broken down by digestive secretions in the small gut and potato starch by bacterial action in the caecum and large gut. (3) In ruminants both maize starch and potato starch are broken down by bacterial action in the rumen. (4) In the non-ruminant hamster the breakdown of starch presents a picture intermediate between that observed in ruminants and the other non-ruminants examined.

E8ect.s of physical treatment on the structure and site of digestion of potato starch in rats and mice

Boiling. As recommended by Kon et al. boiled starch was prepared by making the starch into a thick paste with water in a glass beaker and immersing the beaker in water kept at 100" C. for 15 minutes. The resulting gelatinous material was subsequently incorporated in the diet of the animal. Swelling of the granules was accompanied by rapid diffusion of their amylose into the surrounding liquid and by decrease and finally loss of their birefringence in polarised light. Examination of the caecal contents of rats given boiled potato starch showed that little iodine-reacting material reached the caxum. The caxal contents were darker than those of rats given unboiled potato starch.

Grinding. Mr W. Godden of this Institute drew our attention to the significant effects of grinding upon starch and suggested their relevance to our work. Lampitt et al. (1947) demonstrated that grinding of untreated potato-starch granules is accompanied by a progressive decrease in their birefringence in polarised light, an increase in their solubility in cold water and a decrease in the average molecular weight of the starch. Through the courtesy of Drs S. Lampitt and E. Hughes, samples of the potato starch used in our experiments were ground in a ball mill at Messrs Lyons's laboratories to 31, 86, 98 and 99 per cent. solubility in cold water. Each sample was examined microscopically in Gun's medium under polarised light and in air without a cover-glass by dark-field illumination. At 31 per cent. solubility, marked distortion of the maltese cross was observed in all granules (fig. 16). At 84 per cent. solubility the crosses had disappeared and the granules were rounded and flattened. Such granules still showed strongly birefringent regions in polarised light. At 98 and 99 per cent. solubility the average size of the granules was much diminished but in many of them regions of birefringence in polarised light could still be observed. In the dark field (fig. 17) and by phase- contrast illumination (fig. 20) the surface of the granules was perfectly smooth in unground material but appeared roughened and pitted in material ground to 31 per cent. solubility (fig. 18). These changes became more conspicuous at higher solubilities. When ground potato starches were given to rats or mice only minimal amounts of iodine- reacting residues reached the cmum even a t 31 per cent. solubility,

622 P. BAKER, H . NASR, P. MORRICE AND J . BRUCE

the lowest value tested. The faxes of these animals were darker and harder than those of control animals given unground potato starch.

These results indicate that a form of physical treatmentgrinding -which disturbs the crystallite organisation of the starch granule sufficiently to cause deformation of the cross of polarisation consider- ably reduces the resistance of the granule to the action of the digestive secretions. In rats and mice given the ground starch the resulting increase in digestibility causes a change in the site of digestion. Instead of being digested by bacteria in the caecum, as with untreated potato starch, most of the ground starch is digested in the small intestine and the contribution made by bacteria to the process of breakdown is correspondingly diminished.

Sites of digestion of other tuber or cereal starches

Since these results disclosed that physical structure could deter- mine whether starch was digested by gut secretions or by bacteria, observations were made with other starches which differed among themselves in their structural organisation, namely canna (tous-les- mois), maranta (St Vincent arrowroot), tapioca and rice starch. Mice and rats were used in the investigations. Granules of canna and maranta starch (figs. 1 and 7) are structurally similar to those of potato starch, canna being the largest and maranta the smallest of the three in average dimensions. Tapioca-starch granules are approxi- mately circular in outline and, on the average, much smaller than the other three tuber starches mentioned (fig. 2). Pairs of rats were given canna, maranta, tapioca, potato, maize and rice starch respectively. The pairs of rats given canna and tapioca were killed after 24 days. During this period the faxes of animals given canna, maranta and potato starch were soft and light in colour, those of rats given rice, maize and tapioca starch were darker and harder. Post-mortem examination of animals fed on canna showed a greatly dilated caecum with pasty light-coloured contents containing vast numbers of starch granules in various stages of breakdown and with numerous adherent bacteria. Rats fed on tapioca starch showed a normal cizcum with darker contents, containing few reacting granules but numerous colourless residues in the penultimate phase of disintegration. The pairs of rats given rice, maize, and potato starch were killed after 24 days. Those fed on maize and potato showed the picture already described. With rice, as with maize, very few granules reached the cscum. The two rats given maranta were not killed until they had been fed on the starch for 80 days. The caecum in both animals was then much dilated and its contents showed large numbers of granules with adherent bacteria. The same starches were given to mice, but the diets were poorly tolerated and in consequence the animals were killed after being given the diets for 3 days. The results

BACTERIAL BREAKDOWN OF STARCHES 6 23

with mice were the same as with rats except that, probably because of the short duration of the experiments, dilatation of the caecum with canna, maranta, or potato was not observed. Among the four tuber starches examined, only tapioca was digested in the small gut and its granules were the smallest of the four. Van der Wath and Myburgh supposed that size of the granule, among other things, may influence digestibility of starch, and this observation supports their view. But size is not the only factor, since granules of potato starch ground to 31 per cent. solubility do not accumulate in the caecum although their average size still greatly exceeds that of tapioca.

Normal and waxy maize (figs. 6 and 5)

The proportion of amylose in maize starches is determined by genetic factors (Hector, 1936). Thus in normal maizes the ratio of amylose to amylopectin is roughly 1 to 3, but in the so-called waxy maizes amylose is almost absent. Consequently, normal maize gives an opaque blue-black and waxy maize a transparent brownish reaction with iodine. Other structural features of these starches were dealt with by Baker and Whelan (1950b).

Four pairs of mice were given diets containing waxy and normal maize starches respectively. The animals were killed at 3 days and it was observed that only small numbers of granules of either starch reached the cscum. The absence of any conspicuous difference in the resistance to microbial breakdown of these starches was at first surprising, since our observations had already shown that in potato starch the amylose was more rapidly attacked than the amylopectin component of the granule. It was in agreement, however, with a later observation that in the breakdown of normal maize starch, whether by bacteria, bacterial amylase or digestive secretions, the formation of amylopectin residues is far less conspicuous than in potato starch.

11. BACTERIAL ECOLOGY OF STARCH BREAKDOWN

General and special facies. If we may use the term “microbial facies ’’ to denote “ a constant association in the same natural habitat of several morphologically distinguishable types of micro-organisms ” (Baker and Nasr, p. 35), we may distinguish in the gut of animals a general facies characterising the microbial population as a whole from a special facies associated with the decomposition of particular dietary products. When the general and special facies of various animal species are compared they are found to exhibit more or less distinctive features (Baker and Nasr, 1947 ; Baker and Harriss, 1947-48). In this paper we are primarily concerned with the special bacterial facies associated with the breakdown of untreated starch granules ; but the special facies almost certainly arises from selective

624 P . BAKER, H. NASR, 3'. MORRICE AND J . BRUCE

increase of particular members of the general facies and accordingly they share some features.

Xynthesis of iodine-reacting substances by members of the special facies. In all the animal species investigated, the special bacterial facies of starch breakdown in the gut includes micro-organisms giving a colour reaction with iodine. Where the chemical composition of iodophil bacteria has been studied (Smith and Baker, 1944 ; Hehre and Hamilton, 1946, 1948 ; Nasr and Baker, 1949), the iodine reaction has been shown to be due to the presence in the bacteria of a hexosan polysaccharide whose chemical properties are intermediate between those of amylopectin and glycogen. Synthesis of iodophil poly- saccharide attends the bacterial decomposition of sugars and of such structural polysacoharides as cellulose and starch. The significance of this synthesis for the host animal has been discussed by Baker and Harriss.

The iodine reaction of the special bacterial facies under considera- tion differs in intensity in the various animal species investigated, and the intensity of the reaction attained in individual animals is more variable in some species than in others. In ruminants, both the intensity and the duration of the reaction are greater than in non- ruminants. Among non-ruminants, the greatest intensity is reached and the least individual variation is observed in the domestic pig. I n mice, rats and rabbits the variation observed is greater and the maximum intensity attained is less. The guinea-pig occupies an intermediate position in these respects among the non-ruminants examined. Sufficient observations were not made to assess accurately the intensity or range of variation of the iodine reaction in either hamsters or man.

Some striking differences in the morphological habit of the special bacterial facies of different animal species were observed. Thus, in the ruminants examined, small, large and giant streptococci (for measurements see Baker, 1943) and giant sarcins were the predominant forms (figs. 43-46). Since Baker (1943) has already shown that these types are members of the normal bacterial population of the rumen, the origin of the special from the general facies in this situation is sufficiently obvious. Spore-forming rods were inconspicuous members of the special facies of ruminants on the diets used.

In non-ruminants the extent to which the facies was consistently distinctive depended on the animal species investigated. In guinea- pigs and domestic pigs a high degree of uniformity was maintained ; guinea-pigs showing a facies of curved iodophil rods (fig. 35) and domestic pigs one of longer or shorter clostridial chains (figs. 38-40). The situation encountered in rats (figs. 21, 32 and 33), rabbits (fig. 36), mice (figs. 23 and 34) and man was more complex. In these animals, as already mentioned, the iodine reaction shows considerable individual variation. Where it is strong, a facies of large coccoid forms is

Morphology of the special facies.

hA!TE CLm FIG. 28.-Breakdown of potato-starch granule by pure culture of GI. butyricum isolated

from caecum of pig. Upper granule, complete removal of amylose ; lower granule partial removal of amylose. x 500.

FIG. BQ.-Breakdown of potato starch by pure culture of GI. butyricunt. Partial, removal of amylose. x 500.

FIG. 30.-Breakdown of potato starch by CZ. butyricum amylase in cell-free filtrate. The majority of the undamaged granules, though broken down by the living micro-organism (see figs. 22, 38,39 and 49), are unaffected by the amylase filtrate. We suppose that those affected may have been mechanically damaged before exposure to the amylase. Note removal of amylose and formation of non-reacting residue. x 500.

FIGS. 28-30 are from preparations showing dissolution of the blue-reacting (amylose) component of potato starch, with temporary persistence of a non-reacting (amylopectin 1 ) residue.

Of the 21 types of untreated starch granules treated with cell-free amylase, smooth pea alone showed breakdown of the majority of the granules.

coccoid forms, Granules in early phase of breakdown. x 600.

FIG. 31.-Breakdown of smoot,h pea starch by CZ. butyricum amylase.

x 500.

FIG. 32.-Potato-starch granule from caecum of refecting rat.

FIG. 33.-As fig. 32. Late phase : granule almost completely disintegrated.

FIG. 34.-Potato-starch granule from caecum of mouse. Disintegrating granule with

Facies of iodophil

x 600.

adherent mixed bacteria. x 600.

J. PATH. BACT.-VOL. LSII

BACTERIAL BRE.4XDOWN OF STARCHES

PLATE CLlV

BACTERIAL BREAKDOWN O F STARCHES 625

observed ; where it is weak, the facies is mainly represented by sporing rods. In human colostomy samples collected over a period of 28 hours after a meal containing potato starch it was observed that the coccoid forms appeared at a later stage than the rods. In the hamster, bacterial digestion of starch in the gastric diverticulum was associated with a facies of large coccoid forms (figs. 24 and 37) giving a brownish reaction with iodine, in the caecum by a facies of rods giving a bluish reaction with iodine.

Pleomorphism. For the interpretation of these results account must be taken of pleomorphism among bacteria. In rats given potato starch and dosed with succinylsulphathiazole (vide infra), the special facies showed every transition between very small coccoid forms and large sporing rods resembling clostridia (figs. 52 and 53). Such transi- tions were so often observed within single chains of micro-organisms that no doubt of the bacterial pleomorphism could arise. This raises a possibility that the coccoid forms and rods observed in the facies of other non-ruminants may be pleomorphic variants of one organism rather than distinct species of bacteria, but this cannot be decided on morphological grounds alone. In ruminants no evidence of pleo- morphism was obtained. Although sporing rods play a far more conspicuous role in the microbial breakdown of starch in the caecum of non-ruminant mammals than in the rumen of ruminants, Masson (1950) has now shown that sporing rods may play a preponderant role in the breakdown of starch in the rumen of sheep given diets containing flaked maize. In these diets Masson (private communication) observed that the starch granules were burst and partly dextrinised.

111. CULTURAL INVESTIGATION OF THE BACTERIAL BREAKDOWN OF STARCH IN THE PIG’S CXCUM

Morphological studies of the special microbial facies and its effects. on the starch granule (figs. 22, 41 and 42) afforded the essential back- ground for cultural investigations. It was decided to begin with the domestic pig, since the relative simplicity of the facies promised well for an attempt t o isolate the responsible organism in pure culture. The investigations were pursued by two methods :-in-vitro incubation of the untreated or heated czecal contents of pigs given starch, and attempts to isolate in pure culture the predominant micro-organism of the special facies.

In-vitro incubation of caxal contents

In-vitro incubation of gut contents with various added carbo- hydrates was extensively employed by Quin (1943) for the sheep, by Smith and Baker (1944) for the ox, and by Baker (1942 and un- published observations) for the goat, rabbit and guinea-pig. Such in-vitro incubations were extended to the caecum of the pig in order

626 F. BAKER, H . NASR, F. MORRICE AND J . BRUCE

to ascertain the capacity of the microbial population as a whole to ferment various carbohydrates, including starches, and the contribution made to this process by the bacteria of the special facies.

Incubation of untreated ccecal contents Two pigs were fed twice daily on a diet of National Pig Food

to which a mixture of equal parts of untreated potato and untreated maize starch was gradually introduced, up to one pound of each starch being given with each meal for three days. Ciecal contents were collected immediately after death, suspended in an equal quantity of a sodium bicarbonate buffer (0.1 per cent.) in sterile distilled water and divided into 20 ml. amounts in test- tubes ; 0.2 g. of each of the various carbohydrates tested was added to 20 ml. of buffered czcal contents. Where potato starch was added it was sterilised by dry heat at 110" C. for 30 minutes. This did not seem to interfere with its structure as judged by the unaltered appearance of the lamellation of the granules and the presence of an iodine reaction. The pH of the mixture before and after incubation at 37" C. was determined by universal indicator and the iodine reaction was tested by the addition of Lugol's solution, frothing being taken as evidence of gas production.

Procedure.

Results of incubation. (1) In 4 hours a t 37" C. acid and large quantities of gas were produced from maltose, sucrose, lactose and glucose. (2) There was less rapid production of acid and gas from soluble starch (Analar), commercial dextrin and the two pentose sugars arabinose and xylose. (3) Dulcitol was only slightly fermented. (4) Structural starches (i.e. starches whose granules were intact structures) were far less rapidly fermented than soluble starches. Fermentation of structural potato starch, for example, became apparent only after 24 hours. (5) Among structural starches maize was more rapidly fermented than potato. (6) Fermentation was suppressed by the addition of toluene, 3 drops to 20 ml. (7) Fermentation was accompanied by synthesis of iodophil polysaccharide by a variety of members of the general facies as well as by the clostridia predominating in the special facies. (8) When 1 per cent. of glucose was added to cscal contents with 2 per cent. of potato st,arch and incubation carried out as before, it was found that the addition of glucose prevented the potato starch from being broken down.

Untreated ccecal contents incubated with selective media

Buffered cscal contents containing 1 per cent. of potato starch were incubated for 20 hours a t the temperatures indicated with equal parts of the following selective media :-( 1) " E.C. " medium of Hajna and Perry (1943) a t 37" C. for coliforms ; (2) " E.C." medium a t 45" C. for Bact. coli ; (3) " S.F." medium (Hajna and Perry) at 45' C. for enterococci ; and (4) Winblad's (1941) acetic-acid medium at 37" C. for lactobacilli and yeasts. Controls of caxal contents containing 1 per cent. of potato starch and made up to the same volume with

PLATE CLV

FIG. 36.-Potato starch from ciecum of guinea-pig. Facies of curved iodophil rods. x 600.

FIG. 36.-Potato starch from cecum of rabbit. Facies of rods. x 800.

FIG. 37.-Potato starch from gastric diverticulum of hamster. Facies of large iodophil ooocoid forms. x 800.

FIG. 38.-Potato starch from cecum of pig. Facies of large iodophil clostridia.

FIG. 39.-As fig. 38 ; another field. x 660.

FIG. 40.-Chain of iodophil clostridia from caecum of pig, showing some of the giant

X 600.

forms frequently encountered. x 800.

x 660. FIG. 41.-Potato-starch granule from caecum of pig. Early phase of breakdown.

FIG. 42.-Potato-starch granule from caecum of pig. Late phase of breakdown. x 660.

J. PATH. BACT.--VOL. I X I I

BACTERIAL B R E A K D O W N OB STARCHES

PLATE crAv

BACTERIAL BREAKDOWN OP STARCHES 627

buffer solution were incubated with each selective medium. Potato starch was broken down in the controls but not in any of the selective media.

Incubation of heated ccecal contents Procedure. Samples of buffered cecal contents were heated at 60°C. for

30 minutes, 80" C. for 10 minutes and 100" C. for 5 minutes. After the addition of 1 per cent. of sterile raw-potato starch to each set of heated samples these were incubated with an unheated control sample for 4 hours a t 37°C. The iodine reaction and p H were determined before and after incubation.

Results. In all samples there was an increase in acidity and in the intensity of the iodine reaction on incubation. These changes were least in the controls and increased progressively with the temperature at which the samples were heated. Microscopic examina- tion showed a corresponding increase in disintegration of the potato- starch granules and in the number of spore-forming bacteria.

Amylase activity of ccecal contents before incubation

A clear bacterium-free filtrate prepared from the caecal contents of a pig given potato starch in its food was suspended in an equal quantity of normal saline. Sterility of the filtrate was confirmed by inoculating it into Robertson's meat broth, which was kept a t 37°C. for 7 days. After incubation for 24 hours a t 37°C. with 0.5 per cent. of soluble starch (Analar) the mixture showed no reaction with iodine, whereas the iodine reaction was unaltered for boiled caecal filtrate incubated with starch in the same way. This showed that cell-free amylase was present in the pig's caecum during life.

Amylase activity of ccecal contents after incubation

For 4 days before they were killed, one of two pigs received with each meal an addition of 1 lb. of untreated maize starch and the other received the same amount of untreated potato starch.

From each pig 100 ml. of caecal contents were collected post mortem and added to 300 ml. of the following sterilised phosphate buffer :- KH,PO, (anhydrous), 1 g. ; Na,HPO, (anhydrous), 2 g. ; distilled water, 1 litre. Four samples were prepared, of which one was heated at 60" C. for 30 minutes, one at 80" C. for 10 minutes, one at 100" C . for 15 minutes, while the fourth sample was not heated. After incubation at 37" C. for 48 hours a cell-free filtrate was prepared from each sample. Sterility of the filtrates was checked and they were incubated overnight at 37" C. with 0-5 per cent. soluble starch (Analar). The iodine reaction was used to test whether the starch was broken down.

The change in iodine reaction was greatest in the sample heated at 100" C. and it diminished progressively in the samples heated a t

628 P . BAKER, H . NASR, P . MORRICE AND J . BRUCE

80’ and 60’ C. Hardly any change was demonstrable in the unheated sample after overnight incubation, but an effect was demonstrable when incubation was prolonged to two days. In boiled controls the iodine reaction did not change. After incubation the sterility of all samples was again checked as before.

From these experiments it became clear that the mixed microbial population of the pig’s cecum is able to ferment starch and a wide variety of soluble carbohydrates. Breakdown of starch and soluble carbohydrates is accompanied by the deposition of iodophil poly- saccharide within many of the bacterial species and by the production of acid and gas. Soluble starches are more rapidly fermented than structural starches and structural maize than structural potato starch. These results are in agreement with those obtained in other animals by the workers already mentioned.

The results suggest that in the caecum of the pig lactobacilli, enterococci, yeasts and coliforms do not play a leading part in the breakdown of starch. On the other hand, sporing rods are predominant in caecal samples which, on incubation, attack sugars as well as starch.

Lactobacilli, enterococci, yeasts and coliforms are also capable of attacking a wide range of soluble carbohydrates ; this means that the number of species in the pig’s cecum capable of metabolising the carbohydrate products of starch decomposition is greater than the number capable of decomposing starch itself. This conclusion receives further support from the demonstration that with in-vitro incubation the sporing rods of the special facies are exclusively responsible for the observed amylase production. The demonstration of free amylase in the fresh cecal contents confirms the finding that soluble products of starch decomposition are actually present in the pig’s caecum in life. The capacity of the special facies to metabolise sugars as well as starch sufficiently explains the influence of glucose in retarding the breakdown of starch. The increase in the iodine reaction, i.e. in the rate of polysaccharide formation in the bacteria, upon heating cecal samples before incubation suggests that in unheated samples the liberated products of starch decomposition are partitioned among organisms other than those immediately concerned in the breakdown of starch. The phenomena which we observed in the pig’s cscum show the influence on the mixed bacteria already present of the arrival of a new substrate-starch. The general population shows an increase in fermentative activity and a special group of spore- forming rods assumes sufficient dominance to become recognisable as a special facies.

Isolation of C1. butyricum from the pig’s cmum

Since the work reported in the previous section has established the predominance of sporing rods in the special microbial facies

PLATE CLVI

FIUS. 43-46.-Maize starch from rumen of ox. Facies of large iodophil sarcixm and Different phases of disintegration :--early (fig. 43), late (fig. 44), streptococci.

final (fig. 46). x 600.

FIU. 46.-Potato starch from rumen of ox. relatively restricted breakdown. x 600.

Same.period in rumen aa fig. 43. Note

FIQ. 47.-Gram’s stain. Beijerinck sucrose-agar. x 800.

FIQ. 48.-Iodhe solution ; wet preparation. Iodophil polysaccharide in older cells. x 660.

FIU. 49.-Beijerinck’s medium with dry-sterilised starch aa carbohydrate. Note breakdown of granule. (See also figs. 28 and 29.) x 660.

FIQS. 47-49.-Growth in pure culture of Cl. butyricurn isolated from cecum of pig given untreated potato starch.

J. PATH. BdC'T.--TOL. LLII

BACTERIAL BREAKDOWN OF STARCHES

PLATE CLVI

J. PATH. BACT.-VOL. LXII

BACTERIAL BREAKDOWN O F STARCHES

PLATE CLVII

FIG. 60.-Film from ciecum of rat on FIG. 51.-Film from ciecum of refect- stock diet. Facies of large and small ing rat on potato-starch diet (Kon rods. Gram’s stain. x 1000. et al., 1938). Facies of coccoid

forms. Gram’s stain. X 1000.

FIG. 62.-Film from ciecum of rat on potato-starch diet and dosed with succinylsulphathiazole until the appearance of beri-beri symptoms. Facies of clostridia. Breakdown of starch continued. Gram’s stain. x 1000.

FIG. 53.-Film from ciecum of rat dosed with succinylsulphathiazole. Chain of clostridia showing exten- sive pleomorphism. Gram’s stain. x 1000.

BACTERIAL BREAKDOWN OF STARCHES 629

associated with starch breakdown in the pig's caecum, the way is opened for an attempt to isolate the predominant organism and to study it in pure culture.

Caecal contents of pigs fed on diets containing potato starch were prepared and heated as before and incubated at 37" C. for 48 hours. From the incubated material, which was rich in sporing rods, inoculations were made into a modified Beijerinck fluid medium (Percival, 1920) of the following composition :-sucrose 10 g., peptone 10 g., precipitated chalk 6 g., sodium phosphate (Na,HPO,) 0.1 g., magnesium sulphate 0.1 g., sodium chloride 0.1 g., water 200 ml. Rapid growth developed on incubation, with abundant production of gas within 24 hours, and the culture had a strong smell of butyric acid. Microscopic examination showed immense numbers of iodophil clostridia. The incubated material was again heated and inoculations were made into fresh fluid medium, which was also incubated. Dilutions of the resulting culture were plated on the modified Beijerinck's medium with 2 per cent. agar and incubated in an anaerobic jar for 48 hours a t 37" C.

Procedure.

Results. Small, moist, raised, circular, whitish colonies appeared on the surface of all the plates. From these, subcultures were made in Beijerinck's and other media. Microscopic examination again showed the presence of iodophil rods, in some of which spores were present (fig. 48). Rods containing spores had a typical clostridial morphology. The organism was motile and young cells were Gram- positive (fig. 47). The organism had the cultural characteristics of an iodophil strain of Clostridium butyricum (Prazmowski), as described by Breed et al. (1948). Professor John Cruickshank and Dr Jean Stuart of Aberdeen University kindly confirmed the cultural characters and identity of the organism, a subculture of which was sent to the National Collection of Type Cultures (culture no. 7423). Isolation of this clostridium opened the way to the study of some of its biochemical activities.

Production of amylase by C1. butyricum Cultures grown in the modified Beijerinck's fluid medium were

inoculated into further modifications of the medium in which sucrose was replaced by soluble starch (0.1 to 5-0 per cent.). The effect of adding yeast to the medium was also studied. At various intervals after incubation at 37' C. the subculture was passed through a Seitz sterilising filter (S.B. pad) and a rough indication of the amylase activity of the cell-free filtrate was obtained by incubating known volumes of filtrate and 0.1 per cent. soluble starch solution and observing the time taken to reach the achromic point with iodine. The action of the enzyme on sodium starch glycollate was employed in its further characterisation.

Results. Amylase activity was demonstrated in a filtrate of a subculture of our strain of Cl. butyricum in modified Beijerinck fluid medium containing 0.1 per cent. starch after 3 hours a t 37" C. Activity was increased by the use of young cultures as inocula, by increasing the amount of starch in the medium inoculated, by the addition of yeast and by lengthening the period of incubation of the subculture

Procedure.

3. PATH. BACT.-VOL. IXII 20.

630 F. BAKER, H. NASR, F. MORRICE AND J . BRUCE

to 3 weeks. The filtrate decomposed sodium starch glycollate, which showed that it contained an a-amylase. The enzyme was isolated in dry form by Dr W. J. Whelan and a detailed study of its kinetics and other characteristics has been reported by Whelan and Nasr (1950).

Action of the cell-free amylase on raw starches and starch products

Five ml. of Cl. butyricum filtrate were incubated at 37" C. for 40 hours .with 0.1 per cent. of each of 21 different samples of starch or starch products, as detailed below. Phenylmercuric nitrate, 1 : 500, was added as an antiseptic. Controls of boiled filtrate incubated with the samples investigated always failed to break down starch or the starch products examined. Breek- down of starch was indicated by loss of the iodine reaction and by the results of microscopic examination in the iodine solution.

Results. Among the 21 samples the following non-structural starch polysaccharides * were broken down by the Cl. butyricum amylase :-( 1) potato-starch amylopectin, (2) potato-starch amylose, (3) pea-starch amylose and (4) sodium starch glycollate. With structural starches there was no breakdown of the granules of the following :- ( 5 ) potato, (6) canna, (7) maranta arrowroot, (8) tannia, (9) tapioca, (10) banana, (11) wrinkled pea (" Laxton " : high amylose content), (12) wrinkled pea (" Early bird " : high amylose content), (13) rice, (14) normal maize (amylose and amylopectin), (15) waxy maize (untreated : high amylopectin content) and (16) waxy maize (defatted). In striking contrast the granules of (17) smooth pea starch showed marked loss of iodine reaction and change of structure (fig. 31). This starch contains both amylose and amylopectin. Among altered structural starches no breakdown was observed in (18) soluble starch (Analar). It is noteworthy that two different commercial samples of soluble starch were structural potato starches whose granules showed an essentially similar iodine reaction, birefringence in polarised light and relative insolubility in cold water to those of untreated potato starch. Breakdown was observed in (19) potato starch ground to 31 per cent. solubility in cold water. The red reaction in iodine of the mechanically damaged portions of the ground potato-starch granules indicated regional dextrinisation. In sample (20), potato starch ground to 98 per cent. solubility in cold water, the granules were dextrinised throughout. Sample (2l) , " Pomme "-a commercially processed potato product-contained " grains " which were found to be dissociated tuber parenchyma with intact cell walls. The starch in the interior of these cells formed a structureless mass giving a blue- black reaction in iodine. After treatment with Cl. butyricum enzyme the reaction became reddish, indicating dextrinisation.

Procedure.

* We are indebted to Professor S. Pest and Dr W. J. Whelan, University College of North Wales, for samples 11, 13, 16 and 17, and to Dr S. K. Kon, National Institute for Research in Dairying, for sample 19. For details of the chemical composition and birefringence of the pea starches see Baker and Whelan (19506).

BACTERIAL BREAKDOWN OF STARCHES 631

These results show that the relative proportions of amylose and amylopectin are neither the only nor the primary factors determining the rate of breakdown of starches by Cl. butyricum amylase. Thus chemically extracted amylose and amylopectin (samples 1-3) were dextrinised, whereas neither the structural amylose of wrinkled pea starches (samples 11 and 12) nor the structural Lmylopectin of waxy maize (samples 15 and 16) were affected. The influence of the structural factor is corroborated by the great increase in the rate of breakdown after grinding (samples 19 and 20). These observations are in complete agreement with those already recorded in this paper on the effects of grinding on the digestibility of potato starch in vivo. Similarly it might be that the facility with which smooth pea starch (sample 17), alone amongst the structural starches investigated, is broken down can be attributed to natural faults or fissuring in this type of granule.

Cl. butyricum cell-free amylase is less effective as an agent of starch breakdown than the digestive secretions of any of the animals investigated. Thus maize starch, which was digested in the small gut of non-ruminants, was unaffected after 60 hours’ incubation with Cl. butyricum amylase in vitro (samples 14-16). This may be due either to intrinsic differences between the enzymes or to the operation in the digestive tract of unknown extrinsic factors.

Cl. butyricum in pure culture is a far more powerful agent of breakdown for untreated starches than the cell-free amylase in culture filtrates. Thus structural potato, the most resistant of the tuber starches, was rapidly disintegrated by the living micro-organism (figs. 28, 29 and 49) but unaffected by the enzyme (sample 5). It may be significant that the bacteria of the special facies exercise their action in situ in close apposition to the surface of the granule, since Petrie (private communication) has shown that in plant tissues the corrosion of starch by diastase depends upon actual contact of the cell mitochondria with the surface of the granule. The available evidence emphasises, therefore, the importance of supermolecular organisation in the relative rates of breakdown of different starches and starch products by micro-organisms, cell-free bacterial enzymes and digestive secretions.

Vitamin requirements and capacity for vitamin synthesis of C1. butyricum

The rate of growth in cultures, intensity of iodine reaction and amylase potency of the isolated strain of Cl. butyricum were equally stimulated by the addition of yeast-2 or 3 drops of yeast extract to 5 ml. of culture-or of small amounts of para-aminobenzoic acid and biotin-of each, 0.001 pg. per ml. Investigations were undertaken to determine in greater detail the capacity for vitamin synthesis and the vitamin requirements of our strain of Cl. butyricurn.

632 F . BAKER, H . NASR, 3'. MORRICE AND J . BRUCE

~~~~~~!~~~~

Procedure. A 4-day culture of Cl. butyricum in modified Beijerinck sucrose fluid medium was centrifuged at 3500 r.p.m. for 20 minutes. The deposit was washed and centrifuged three times in succession with sterile saline. The cells were suspended in saline t o match Brown's opacity tube no. 4. This was used to inoculate tubes of a synthetic basal casein-hydrolysate medium of the usual type employed in microbiological assay. Each tube of basal medium was deficient in one only of the following 8 members of the vitamin-B complex :- aneurin, pyridoxin, para-aminobenzoic acid, riboflavin, pantothenic acid, nicotinic acid, biotin and folic acid. A tube of unsupplemented basal medium was inoculated as a control. After 72 hours at 37" C. growth was observed in all tubes except the unsupplemented medium and those deficient in biotin and para-aminobenzoic acid respectively. From this it appeared that if biotin and para-aminobenzoic acid were provided the isolated strain of Cl. butyricum was able to synthesise the remaining six members of the vitamin-B complex. To check this conclusion, the methods of Roberts and Snell (1946) as modified by Mr G . E. Shaw of the Evans Biological Institute (private communication) were used to measure by microbiological assay the amounts of four members of the vitamin-B complex produced by our strain of GI. butyricum in tubes of synthetic medium deficient only in the member of the vitamin-B complex under examination. In filtrates from such cultures incubated for 72 hours at 37' C. the following amounts of the named vitamins were synthesised :---nicotinic acid, 127.0 pmg. per ml. ; riboflavin, 86.0 pmg. per ml. ; folk acid, 13.3 pmg. per ml. ; and pantothenic acid, 225.0 pmg. per ml.

These results show that CZ. butyricum, the predominant member of the special microbial facies associated with the breakdown of

TABLE

Refection experiment. Growth of pigs (lb. live weight) given a diet deficient in vitamin B with 3 different starch supplements

Deflcient diet plus boiled potato starch

No. of d q s rfter de6cienl Uet was giver as the only

food

- 51.5

49.5 48.5 49.0 47.0 49.0

Very ill diet

changed

2 days before

5 12 19 26 33

40

-- 42.0 46.8

42.5 46.0 39.0 43.8 39.0 44.0 38.5 42.3 37.0 43.0

:Very ill : ... diet

changed

Deflcient diet plus untreated maize starch

46.5

51.3 53.5 56.5 61.0 61.0

65.0

I I

53.0 41.5 47.3

56.0 46.0 51.0 55.0 41.5 46.3 58.5 44.0 50.3 57.5 40.5 49.0 53.5 Died on ...

Died on ... ... 37th day

29th day

Diet

starch

Pig no. 140

43.0

50.5 53.0 54.0 59.0 58.0

64.0

-

- pig no. 144

50.0

52-0 54.0 59.0 63.0 64.0

66.0

-I I I

-I- 1-1-

Adequate mixed diet

- pig no. 138 - 43.0

47.0 53.0 59.0 62.5 70.0

75.0

-

- Pig no. 142

57.0

59.0 64.0 70.0 76-0 81.5

91.5

-

Mean

50.0

53.0 68.6 64.5 69-3 75.8

83.3

Vitamin-B-deficient. diet :-casein (vitamin-free) 20 parts, margarine 2.5 parts, salt mixture (Wintrobe, 1939) 5 parts ; total 27.5 parts. Each pig received 5-0 ml. of cod liver oil daily and 70 parts of the appropriate starch supplement.

untreated potato starch in the cmum of the pig, is capable of synthesising members of the vitamin-B complex. This raised the

BACTERIAL BREAKDOWN OF STARCHES 633

possibility that starch breakdown in the pig's caecum might have nutritional value to the host as a source of extra-dietary vitamins, depending on the amounts of the vitamins actually synthesised in vivo and the extent to which they could be utilised by the animal. Under favourable conditions it might be expected that refection- i .e . growth on a diet free from vitamins of the B complex-could be demonstrated in pigs given potato starch as a supplement to the deficient diet. We have now found that two pigs kept on such a diet showed marked although less than the normal increases in weight and remained free from symptoms of avitaminosis, one for 14 and the other for 22 weeks. Four litter-mates given the same diet, 2 with maize starch and 2 with boiled potato starch, failed to refect. Within 4 weeks their growth ceased and they became gravely ill. The two given boiled potato starch died. After they had become gravely ill, the two that were given maize starch were transferred to a normal mixed diet and gradually recovered. The pigs given the untreated potato starch made good progress until the onset of cold weather. One died suddenly on 22nd October 1949 and the other on 12th December 1949. These deaths coincided with a sudden fall of temperature in the unheated building in which the pigs were housed. The essentials of the experiment are given in the table (p. 632).

IV. RELATED OBSERVATIONS

Refection in rats

The phenomenon of potato-starch refection in rats, i .e. their ability to grow and to survive when given diets lacking in vitamin B but containing potato starch, has been dealt with by Kon et al. In the present investigation the caecal contents of rats on stock diet (fig. 50) and of others undergoing potato-starch refection (fig. 51) were kindly supplied by Dr S. K. Kon, National Institute for Research in Dairying, and we had additional material of the same kind from our own experi- ments. In addition the two rats (p. 622) given a vitamin-B-free diet with maranta starch for 80 days increased in weight and showed no symptoms of avitaminosis.

Among rats given vitamin-B-free diets without a refecting agent, e.g. potato starch or maranta starch, acrodynia was the earliest symptom to appear and this was followed by polyneuritis and death. The pairs of animals given potato starches ground to 31, 86 and 98 per cent. solubility in cold water (p. 621) showed the same symptoms and also died. Starch ground to 99 per cent. solubility was examined for site of digestion but not for refection. These experiments require to be extended, since the number of animals that could be employed was limited by the supplies of ground starches available. As they stand, they suggest that the alteration in supermolecular structure of the granule resulting fi'om mechanical treatment was alone sufficient to destroy the ability of potato starch to procure refection.

J. PATH. BACT.-VOL. LXII 2u2

634 F. BAKER, H. NASR, F. MORRICE A N D J . BRUCE

Starch breakdown in rats given succinylsulphathiazole

Kon et al. have shown that the refection in rats given structural potato starch in vitamin-B-free diets is prevented by dosing with succinylsulphathiazole. Caecal contents of rats fed and dosed in this way, in which symptoms of avitaminosis were already apparent, were made available to us by Dr Kon. Microscopic examination of these showed extensive microbial breakdown of the starch granules accumulated in the cacum (fig. 52). In these rats, therefore, it is possible that the bacteria responsible for the breakdown of starch are not those primarily concerned with the synthesis of the vitamin-B complex.

DISCUSSION The variables of starch digestion in the gut

It is now possible to discuss some of the variables which determine the course and outcome of starch digestion in the gut of animals. The supermolecular organisation of the starch is important, and this is influenced by its botanical origin, by the constitution of the individual plant genotype (Hector), and by the physical or chemical treatment which the starch or starch product may have received in its preparation. The architecture and mechanics of the digestive tract of the animal given the starch in its diet are also important. For example, the topography of the tract settles whether the ingesta are first subjected to the action of micro-organisms, as in ruminants, or to that of digestive secretions, as in non-ruminants. The rate of emptying of the viscus where the starch is broken down is of material significance. Thus Hopkins and Leader (1945-46) showed that refection fails in rats given cellulose roughage, presumably owing to the rapid emptying of the gut which it provokes, as suggested to us by the late Dr Marjory Stephenson (private communication). The characteristics of the bacteria associated with starch breakdown will also determine the nature, amount and nutritional value of the microbial products. The ability of the host to make effective use of these products is influenced by the character and efficiency of its mechanism of absorption and resorption and by whether and to what extent it eats its faces. The palatability of the starch product and the extent to which it is tolerated by the digestive system are obviously significant. More detailed characterisation of these and other possible variables opens up a wide field for investigation. Meantime, a few points may usefully be examined more closely.

The nutritional outcome of microbial breakdown of starch

In the breakdown of starch by the digestive secretions of the animal a sequence of hydrolyses culminates in the formation of soluble carbohydrates which are rapidly taken up by the bloodstream. This process is essentially similar in different animal species and affords

BACTERIAL BREAKDOWN O P STARCHES 635

a direct contribution to the energy metabolism of the host. By contrast, in microbial breakdown, the initial hydrolysis brought about by the action of bacterial amylase leads on to a process of fermentation and is accompanied by multiplication of the micro-organisms con- cerned. Microbial breakdown of starch is commonly incomplete, especially in non-ruminants. Because of differences in the bacteria specifically concerned, the process may exhibit distinctive features in various animal species. Thus the number of variables involved is greater in fermentative than in hydrolytic breakdown. In comparison with the direct action of the digestive secretions, microbial breakdown affords a relatively inefficient mechanism for the rapid mobilisation of energy from starch-containing diets. This remains true even in the rumen, where the bulk of the starch fed is broken down by bacteria. On the other hand, since bacterial decomposition of starch is accompanied by microbial synthesis it may afford important contributions to the growth and welfare of the animal. Such a situation is exemplified in refection, where survival of the host depends upon the establishment of an appropriate microbial facies in the gut. Although such a rigorous condition may not be usual in nature or in normal feeding practice, both the balance of the diet and the system of husbandry employed may have an important influence upon the incidence and nutritional value of the two types of starch breakdown in domestic animals. The starch in potatoes is often a major component of a normal pig diet, and according to individual and local practice the potatoes may be given either steamed or raw. The starch of steamed potatoes provides more energy for the pig since it is broken down by gut enzymes, but the bacterial products arising from the breakdown of raw potato starch in the pig’s caxum may often be useful for growth and survival if the diet available is lacking in vitamins of the B-complex.

The inJEuence of the surface membrane on the digestibility of starches

In this work the over-riding influence of supermolecular organisation on the digestibility of starches has been frequently observed. It is especially conspicuous in non-ruminants, where the relative resistance of tuber starches to the digestive secretions leads to their accumulation and subsequent breakdown by bacteria in the caxum. An attempt to gain some insight into gross or fine structural factors specifically concerned in this supermolecular organisation is therefore necessary. Katz and van Itallie (1930), for instance, have shown that cereal and tuber starches have different X-ray powder diagrams. It is possible that such differences, which Bear and French (1941) sought to characterise in crystallographic terms, may be responsible for the observed differences in digestibility of various starches. An alternative and simpler hypothesis is that the differences in digestibility are due

636 F . BAKER, H . NASR, F. MORRICE AND J . BRUCE

to the presence or degree of development of and resistance to enzyme action of surface membranes. Weichsel(l936-37) reported a separation of surface membranes when potato-starch granules dried over P,O, were brought into contact with water. She found that the rate of hydrolysis by taka-diastase was increased by separation of the membranes. By the same treatment she could not demonstrate membranes on wheat starch, and its rate of hydrolysis was unaffected. Baker and Whelan (1950~) observed the separation of membrane systems when dry potato-starch granules were irradiated in air with ultra-violet light (fig. 25). On immersion in water or chloral hydrate the granules became greatly swollen and finally ruptured by osmotic pressure, leaving the membrane systems as residues. The absence of swelling in the residues themselves distinguished them from the artefacts which, as Alsberg (1938) and Samec (1943) showed, may be produced by the direct action of swelling agents on a wide variety of starches. Baker and Whelan (1950~) were unable to demonstrate the formation of membrane residues or osmotic swelling in the granules of irradiated maize starch. That surface membranes may determine the resistance of potato starch to digestive secretions is also suggested by the observation of Kon et al. that the ability of starch to procure refection in rats is diminished when it is stored in alcohol, a treatment which results in uneven shrinkage and consequent fissuring of the surface of the granule. Attempts to assess the relative importance of the several levels within the supermolecular hierarchy of structures determining the digestibility of starch would be premature. In the meantime it is worth insisting upon the direct bearing of observations such as the present upon the problems of nutrition. In the absence of the information which systematic microscopy alone can yield, purely bacteriological and biochemical inquiries lack both a starting point and an essential control. Without some understanding of the significance of the many complex factors involved in the breakdown of starch within the gut of animals, workers must realise that undefined variables are introduced into all experiments, whether conducted in the field, the laboratory or the hospital, in which starch-containing diets are given to animals or man.

SUMMARY

The sites and agents of breakdown of various structural starches and starch products in the gut of different animal species were investigated microscopically. In non-ruminants, excepting the hamster, untreated cereal starches are broken down in the small gut by the digestive secretions. Tuber starches, excepting tapioca starch, escape the action of the digestive secretions and are attacked by bacteria in the czecum. After grinding, potato starch given to rats is digested in the small gut, and the animals fail to refect, i.e. fail to grow and survive in the absence of vitamin B in their diet.

B A C T E R I A L BREAKDOWN OF STARCHES 637

In the hamster, tuber starches are broken down by bacteria in the gastric diverticulum and in the caecum. In ruminants, starches and starch products are broken down in the rumen by bacterial action. Distinctive bacterial facies are associated with bacterial breakdown of starches. In the pig’s caecum Clostridium butyricum is found to be the primary agent of breakdown and a strain isolated from that source synthesised the members of the vitamin-B complex, other than biotin and para-aminobenzoic acid, required for its own growth. By microbiological assay it is possible to measure the amounts of nicotinic acid, riboflavin, folic acid and pantothenic acid liberated by the organism in fluid cultures of synthetic medium. Pigs given vitamin-B-free diets are able to grow and survive if untreated potato starch is added to the diet, but not if the potato starch is rendered soluble by steaming or if untreated maize starch is substituted.

Our thanks are due to Mr W. Smiles, National Institute for Medical Research, for taking the photomicrographs for figs. 19-21.

REFERENCES

ALSBERO, C. L. . . . . . . 1938. Plant Physiol., xiii, 295. BAKER, F. . . . . . . . 1942. Nature, cl, 479.

9 9 . . . . . . . 1943. Ann. Appl . Biol., xxx, 230. BAKER, F., AND HARRISS, S. T. . BAEER, F., AND NASR, H. . . . 1947. J . Roy. Microscop. Soc., lxvii, 27. BAKER, F., AND WHELAN, w. J. . BEAR, R. S., AND FRENCH, D. . 1941. J . Amer. Chem. SOC., Ixiii, 2298. BREED, R. S., MURRAY, E. G. D.,

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