Fd Chem. Toxic. Vol. 22. no. 6, pp. 415-418, 1984 0278-6915/84 $3.00 +0.00 Printed in Great Britain Pergamon Press Ltd

H Y D R O C O L L O I D FOOD ADDITIVES A N D RAT CAECAL MICROBIAL E N Z Y M E ACTIVITIES

A. K. MALLETT*, A. WISEr + and I. R. ROWLAND* *British Industrial Biological Research Association and

tMedical Research Council Laboratories, Carshalton, Surrey, England

(Received 4 October 1983)

Abstract--Agar, carboxymethylcellulose, carrageenan, guar gum, gum acacia, locust-beam gum or pectin (50 g/kg diet), given to weanling rats for 4 wk, increased the weight of the caecal wall and the caecal contents. Feeding carboxymethylcellulose, guar gum or pectin significantly increased, and feeding carrageenan decreased, the total bacterial population of the caecum. Feeding carboxymethylcellulose significantly increased in vitro activity of bacterial azoreductase, fl-glucosidase, fl-glucuronidase, nitrate reductase, nitroreductase and urease. Guar gum, gum acacia and locust-bean gum each increased at least three of these activities, in contrast, feeding carrageenan greatly decreased all microbial enzyme activities, while agar decreased fi-glucosidase, /~-glucuronidase and nitroreductase activities.

INTRODUCTION

A number of plant-derived hydrocolloids are incor- porated into processed foods, including confection- ery, dairy products and qow-calorie' diet prepara- tions (Adrian, 1976; Glicksman, 1969). Their use as stabilizing and emulsifying agents results from gel formation due to hydrogen bonding or cross-linking with ionic species in an aqueous environment (Glicks- man, 1969). Materials with a wide range of physico- chemical properties are available to the food industry but all are resistant to attack by mammalian digestive enzymes (Adrian, 1976) and hence reach the diverse microbial population of the large intestine. Hydro- colloid additives rarely show toxicity to laboratory animals, although high dietary concentrations may reduce growth rate or decrease the digestibility of some nutrients (Adrian, 1976).

The metabolic activity of the gut microflora is an important factor in the disposition, toxicity and/or carcinogenicity of many compounds (Goldman, 1981; Rowland, 1981; Scheline, 1980; Williams, 1972). The caecal microflora relies on two sources of nutrition, namely endogenous host secretions (Fau- conneau & Michel, 1970; Hoskins, 1978; Salter, 1973) and undigested residues from the diet (Kim, Bene- yenga & Grummer, 1979; Mauron, 1981; Wise & Gilburt, 1980). Plant cell-wall components are in- cluded in this latter category and these may alter microbial metabolism in the rat caecum (Rowland, Wise & Mallett, 1983).

Previous studies have demonstrated that pectin, a plant cell-wall component with hydrocolloid proper- ties, influences bacterial enzyme activity in the cae- cum (Mallett, Rowland & Wise, 1983a) and may predispose the host to toxic sequelae (Wise, Mallett & Rowland,1982). The present paper reports a study of the effect of feeding a number of hydrocolloid materials (agar, carboxymethylcellulose, car-

++Present address: School of Nutritional Science, Robert Gordon's Institute of Technology, Aberdeen AB9 1FR, UK.

rageenan, guar gum, gum acacia, locust-bean gum and pectin) on a range of caecal microbial enzyme activities that are of toxicological importance to the host animal.

EXPERIMENTAL

Forty eight male Sprague Dawley rats (3 wk old), with conventional microflora were purchased from OLAC (1976) Ltd, Bicester, Oxon. The rats were randomized into eight groups and fed ad lib. either a purified diet (Wise, 1982) or a diet containing 50 g/kg of one of the following hydrocolloids-agar-agar (Mapletons Foods Ltd, Liverpool) t-carrageenan, carboxymethylcellulose (sodium salt), pectin (Sigma Chemical Co. Ltd, Poole, Dorset; nos C1138, C5013 and P9135), gum acacia No. l, guar gum LJR31 or locust-bean gum No. l (L. J. Rickard, Beckenham, Kent). These materials were all 'food-grade" prepara- tions and were used without further purification. They were added to the complete basal diet and were assumed not to contribute any energy (Bright-See, Rao, Hi & Tang, 1978); they did not therefore change the ratio of nutrients to the known energy providers in the diet. The control diet was not supplemented with a non-calorific bulking agent (e.g. cellulose) because of possible changes that might have occurred in the metabolic activity of the gut microflora (Mal- lett, Wise & Rowland, 1983b).

After 4wk the rats were killed by cervical dis- location and the caecal contents from each animal were suspended in 25ml 0.1 M-phosphate buffer pH 7 under anaerobic conditions (Wise & Gilburt, 1982) which were maintained throughout the follow- ing enzyme assays. Azoreductase activity was mea- sured over 60min using 1.5 mM-amaranth as the substrate by following the time-dependent decrease in absorbance at 520nm (Wise et al. 1982). Nitro- reductase activity (1.5 mM-p-nitrobenzoic acid) was determined from the production of p-aminobenzoic acid over 30 min (Wise et al. 1982). Nitrate reductase activity (20 mM-sodium nitrate) was determined from the generation of nitrite over 60 min (Wise et al.

415

416 A.K.."VIALLF.TT ¢'1 a/.

Table 1. Body weight and caecal data of rats given diets containing various hydrocolloids

Weight of Weight of Number of Cnncn of Body caccal caecal caecal caccal

Hydrocolloid weight contents wall bacteria ammonia added (g) (g) (rag) { × lOm)'~ (#mol)

None 240 [ 7 297 14 17 (227 2851 (I.4 2.1) 1174 4171 (4.7 28) (2.7 25)

Agar 249 2.8* 394* 0.5 8.2 (212 315) (I.6 3.5i (296 576) (I.0 211 (4.6 /9}

Carboxymethyl 219* 8. I *** 649"** 5(1"* 74"** cellulose (188 246) (5.4 11) (517 960) (23 64) (34 84)

Carrageenan 209 2.9** 4(/3" 0.3"** 4. I * (175 254) (19 4.2) (361 724) (0.02 2.11 (3.3 5.3)

Guar gum 214" 3.3*** 481)** 33** 23 1168 246) (3,1 4.2) (400 ~57) (23 49) {12 35)

Gum acacia 235 3.4*** 477** 18 24* (218 252) (2.4 5.5) 1345 5301 (ll 58) (16 351

Locust-bean gum 225 3.2* 441" 22 22 (193 248) (I.4 4.61 (338 742) (2.8 481 (12 ~1)

Pectin 229 3.9*** 490** 35* 2~* (204 245) 13.0 5.1) (335 660) (13 71) (19 28)

"}'Microscopic count. Values are medians for six rats (range in parcnlhesest. Fhose marked with asterisks differ

significantly from the control: *P < 0.05: **P < (I.01: ***P < 0.001 (by Mann-Whitney I test).

1982). fl-Glucosidase and fl-glucuronidase activities were determined over 10rain using 3 mM-p-nitro- phenol-fl-D-glucoside and phenolphthalein-fl-D- glucuronide, respectively (Rowland el al. 1983). Ure- ase activity was determined by measuring the rate of ammonia production over 20 min in incubations of caecal contents with 10m~-urea (Sigma Chemical Co., 1981). The latter estimation also included a measurement of caecal ammonia prior to incubation with urea. The total caecal bacterial population was determined by direct microscopic clump counts (Holdeman & Moore, 1973).

The data were analysed by the Mann-Whitney U test, and data obtained for urease activity and ammonia concentration were subjected to Spear- man's rank correlation analysis (Siegel, 1956).

RESULTS

Dietary carboxymethylcellulose and guar gum significantly decreased the final body weight of the rats by 9 11~,,; compared to the control group, yet the other indigestible materials were without significant

effect (Table 1). All the hydrocolloids significantly increased the weight of both the caecal wall and caecal contents (Table l). Feeding carboxymethyl- cellulose, guar gum and pectin significantly increased the total bacterial population of the caecum, but carrageenan decreased the number of bacteria to 2'~,i of the control value (Table 1). The mean caecal ammonia concentration was increased fourfold by dietary carboxymethylcellulose, with smaller increases following dietary pectin and gum acacia (Table 1 ). In contrast, carrageenan decreased caecal ammonia by 76!! ,,.

Feeding carboxymethylcellulose significantly in- c r ea sed the total activity of all the bacterial enzymes determined, the greatest effect occurring with urease a n d fl-glucosidase activities, which were elevated five and eightfold, respectively (Table 2). Dietary guar gum and locust-bean gum also significantly increased the total hydrolytic or reductive activity of the gut microflora toward the majority of substrates used. Feeding gum acacia significantly increased azoreduc- tase, nitroreductase and nitrate reductase activities, but was without effect on hydrolytic functions of the

]able 2. Caecal microbial enzyme activities of rats given diets containing various bydrocolloids

Enzyme activity (l~mol product/caecum hr)

Hydrocolloid Azo fi ]~ Nitrutc added reductasc Glucosidase Glucuroniduse Nitroreductase reductase Ureasc

None 4.7 15 14 I.I I I 79 (2.8 7.5) 18.5 291 (10 331 (06 1.7) (4.(~ 17) (35 466)

Agar 1.3 6. I ** 2.6*** (L [ t*** 7.6 29 (0.05 I1) (2.4 15) 16.0 9.3) 10.02 0.3) I I 0 43) IlO 1721

Carboxymethyl I1"* 123"** 41"** [.S** 51"** 422* cellulose 16.5 19) (43 271)) (33 144) (I.5 2.4) (38 128) 1146 591)

Carrageenan 0.19"** 1.0*** (I,36"** 0.(1(|5"** 1.5** 7 1 *** (0 0.71 ((I.2 2,11 (0 1.7) (1) (I.{)4) (I.3 7.9) (2.9 25)

Guar gum 15** 65** 33** 1.5" 29 283 (5.3 33) (28 92) (IS 65) II.I 2.6) (I . t 65) (66 402)

Gum acacia 12"* 21 21 15"* 43*** 164 (5,2 24) (I.0 731 (12 49) (I.I 2.2) 123 65) 151 5111

Locust-bean gum 10" 43** 26 2. I * 33* 22(,* 12.6 32) {16 126) (6.5 91) {0.5 37) 14.9 62) I l l5 S01}

Pectin 8.4 19 20 1.4 88*** 167 {3.4~11) 17./ 45) 16.9 43) (0,6 2.3) 144 156) (99 438)

Values are medians (or six rats Irange m parentheses). Those marked with asterisks differ significanll3 from the control: *P<0.05: **P<0.01; ***P<I).001 {by Mann-Whitney k testL

Hydrocolloids and microbial enzymes 417

caecal bacteria, while pectin significantly increased only nitrate reductase activity. In contrast, car- rageenan decreased the activity of all enzymes, while agar significantly depressed/3-glucuronidase and ni- troreductase activities.

The diet-related changes in microbial urease activ- ity (Table 2) correlated well (r =0.80; P <0.001) with caecal ammonia (Table 1).

DISCUSSION

The hydrocolloid food additives used in this study all increased the amount of caecal contents, the greatest change occurring with carboxy- methylcellulose. Caecal enlargement may have been due to an osmotic phenomenon resulting from un- degraded hydrocolloid or bacterial fermentation products in the hindgut (Leegwater, de Groot & van Kalmthout-Kuyper, 1974). It was associated with diet-related differences in the consistency of the gut contents (data not shown). However, despite the general increase in caecal weight, the size of the bacterial population was significantly increased only after feeding carboxymethylcellulose, guar gum and pectin. The remaining compounds, in particular car- rageenan, decreased the concentration of bacteria. This effect may have resulted from dilution of the gut microflora by undegraded hydrocolloid or from unspecified bactericidal properties of these com- pounds. However, no attempt was made to analyse the species composition of the microflora.

The gut microflora may use certain complex diet- ary carbohydrates as a source of energy for cell division and metabolism (Cummings, Stephen & Branch, 1981). Some of the plant hydrocolloids used in the study reported here may have been utilized in this manner. The presence or absence of enzymes required to cleave the parent polymer may explain some of the variation in microbial biotransformation activities that occurred in response to feeding the various hydrocolloids. The total metabolic activity of the microflora was independent of the amount of caecal content or the total number of organisms present in the hindgut, suggesting that changes in bacterial biotransformations were dependent either upon enzyme induction or repression, or upon redis- tribution of bacterial species within the gut popu- lation. The results are expressed as activity per cae- cum to demonstrate any diet-related changes in the total activity of the caecal microflora for the range of enzymes studied and to enable the toxic sequelae associated with changes in bacterial metabolism to be considered.

Feeding carboxymethylcellulose increased the total activity of all the enzymes studied, suggesting that the /~-l,4-glucose backbone was susceptible to attack by gut bacteria, a finding reported previously (World Health Organization, 1974). The guar-gum, gum- acacia and locust-bean-gum diets increased the activ- ity of many of the enzymes again suggesting that they provided a microbial energy source. Guar gum and locust-bean gum show a similar chemical com- position and comprise a/~-1,4-mannose polymer with galactose side chains, while gum acacia is a complex polysaccharide based on galactose (Glicksman, 1969). Guar gum is known to be almost completely

fermented by rat gut microflora (Nyman & Asp, 1982). Pectin, a polymer of galacturonic acid (Glicks- man, 1969), is fermented by the gut bacteria and the pectin diet increased nitrate reductase activity in accordance with previous reports (Mallett et al. 1983a; Wise et al. 1982) but had only minor effects on other enzyme activities.

In contrast to the other hydrocolloids, feeding agar and t-carrageenan decreased the enzymic activity of the caecal microflora. These two algal poly- saccharides share a similar galactose backbone yet, unlike the other hydrocolloids, they contain an ap- preciable number of sulphate residues (Glicksman, 1969). ~-Carrageenan is sulphated on alternate units of this backbone, as are 2- and •-carrageenan (Kra- gen & Bourdais, 1973). The degree of sulphation of the galactose polymer determines the physico- chemical properties of the resulting gel structure (Adrian, 1976) and may thereby influence the extent of bacterial degradation. In general, carrageenans are resistant to attack by the gut microflora (Hawkins & Yaphe, 1965; Tomarelli, Tucker, Bauman et al. 1974) which may explain the decrease in the number of caecal bacteria observed in this study. Agar, while showing some of the structural and physico-chemical properties of the carrageenans (Glicksman, 1969), decreased the activity of a number of microbial enzymes, yet was without effect on bacterial numbers.

It is difficult to assess from in vitro observations the consequences of changes in caecal microbial enzyme activities that occur in vi~o, but increased bacterial metabolism may be associated with host toxicity. For example, it has been reported that urease activity, and the concomitant production of ammonia, may be linked with tumour promotion in the gut or with systemic toxicity following the absorption of ammo- nia (Visek, 1978), while increased bacterial nitrate reductase activity results in methaemoglobinaemia following oral nitrate administration (Wise et al. 1982). Bacterial hydrolysis of /]-glucosides and /~-glucuronides may also give a number of toxic sequelae (Goldin, Lombardi, Mayhew & Gorbach, 1981: Williams, 1972); it was increased by carboxy- methylcellulose and guar gum treatment, while hy- drolysis of/~-glucosides was increased by locust-bean gum. The aglycones generated by these decon- jugation reactions may interact either locally with the tissues of the gut (Takada, Hirooka, Hiramatsu & Yamamoto, 1982) or systemically following absorp- tion into the circulation (Hill, Backer & Hill, 1980).

This study demonstrates that hydrocolloid food additives influence the metabolic activity of the rat caecal microflora and may increase or decrease the generation of bacterial products of potential toxicological importance to the host animal.

Acknowledgements--This work was funded in part by the UK Ministry of Agriculture, Fisheries and Food, to whom IRR and AKM are grateful. The authors thank M. J. Davies lbr performing the urease assays and D. J. Gilburt and C. Bearnc for technical assistance.

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