Nov 28, 1994 - as a dietary supplement for reducing the number of potentially harmful E. coli in the monogastric gut. KEY WORDS: lupin seed; fibre; E. coli; gut.
MICROBIAL ECOLOGY IN HEALTH AND DISEASE
VOL.8: 101-105 (1995)
The Inclusion of Lupin (Lupinus angustrolius) Seed Meal or its Fibre Residue in the Diet Reduces the Levels of Escherichia coli in both Small and Large Intestines of the Rat L. A. RUBIO, R. SPENCER*, G. GRANT and A. PUSZTAI The Rowett Research Institule, Bucksburn, Aberdeen, AB2 9SB, Scotland, UK Received 28 November 1994; revised 8 February 1995
An investigation into the anti-nutritive effect of feeding lupin seeds to male Hooded-Lister rats, showed that lupin containing diets decreased the numbers of Escherichiu coli in the caecum, colon and the small intestine. This potentially beneficial lowering of E. coli populations in the gut may be due to the hindrance by the lupin fibre of the attachment of E. coli to gut receptors, since the effect also occurred in the small intestine where the lupin residue particles have not yet been degraded by bacterial fermentation. Accordingly, it may be possible to use lupin residue as a dietary supplement for reducing the number of potentially harmful E. coli in the monogastric gut. KEY WORDS:
lupin seed; fibre; E. coli; gut.
INTRODUCTION Sweet (low in alkaloids) varieties of lupin (Lupinus angustifolius) which grow well in areas where soybean does not6 have attracted attention in animal production because of their high protein and low anti-nutritional factor contents. However, diets based on lupin seed meal have repeatedly been reported not to support the growth of rats and pigs at the same rate as control diets based on lactalbumin or soybean. 132,10,22 Lupin seeds are particularly rich in non-starch polysaccharides (NSP) and low in starch.’ This differentiates them from most of the other legumes used for animal or human consumption in which starch is the main storage carbohydrate. Vegetable-based diets are known to induce significant changes in the number and metabolism of the microbiota colonising the gut; this is due mainly to the relatively high content of oligo- and polysaccharides in the vegetable^,^ which resist hydrolysis by the host digestive enzymes in the small intestine but provide substrates for fermentative bacteria in the caecum and colon.’ Although the reasons for the lower nutritional value of lupin-based diets are unclear, *Author to whom correspondence should be addressed. CCC 0891-060X/95/030101-05 8 1995 by John Wiley & Sons, Ltd
changes in the intestinal microbiota may affect the normal digestive process, altering the absorption of proteins and inducing changes in general nitrogen metabolism. l4 Furthermore, the production of volatile fatty acids (VFA), which are the main metabolites of hind-gut fermentation and provide up to 30 per cent of the energy required for basal metabolism,’ may also be affected. Accordingly, the primary aims of this study were to investigate the effects on the caecal microbiota of rats fed diets based on lupin seed meal and lupin fibre (insoluble non-starch polysaccharide residue), to correlate these effects with changes in the general nitrogen metabolism of the rats and to find out whether potential changes in microbial numbers were confined to the large intestine or also occurred in the small bowel. MATERIALS AND METHODS Purijication of fractions and chemical analysis Lupin seeds (cv ‘Unicrop’) were obtained from the Grain Pool of Western Australia. Lactalbumin and synthetic free amino acids were from Sigma (Poole, Dorset, UK). Lupin seed meal and lupin residue were extracted and analysed as previously
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L. A. RUB10 ET AL.
Table 1. Composition of the diets ~
Lactalbumin control
Diet"
N
~~~
Lupin meal
Lupin residue
glKg diet
Lactalbumin Corn starch Potato starch Oil (maize) Glucose Glycerol Vitamins + Minerals Silicic acid Lupin meal Lupin residue Digestible E, kJ/g Protein Fibre Fat ~~~~~
120 445.6 100 50 109 I5 100 0.4
~
-
110
305.6
386.6
-
-
50 109 I5
100
-
0.4 360
-
-
15.6 100 50 50
15.6 100 165.6 68.3
50 109 I5 00 0.4 -
I0 15.6 00 52.6 50
~
"For feeding regime and details of the diets, see Materials and methods.
described. l 8 The lupin residue fraction contained most of the NSP and lignin from the meal. Nitrogen in diets and faeces was determined in an N analyser (Foss Electric UK Ltd, York, UK) and in urine samples by a micro-Kjeldhal procedure. The alkaloid content of methanolic extracts of lupin seeds was determined by capillary gas-liquid chromatography (GLC). l 7
Animals, diets and feeding regime Male Hooded-Lister rats (Rowett strain), reared and housed in the breeding and experimental small animal unit of The Rowett Research Institute, were used in the study. They were weaned at 19 d of age, given stock diet (Biosure; Special Diets Services, Manea, Cambridgeshire, UK) for 7 d, and then adapted to experimental conditions by giving them the lactalbumin-based control diet (for composition of diets see Table 1 and ref. no. 18) for 3 d. Four rats (housed individually), matched by weight (82 k 1 g) were used per group. Feed was withdrawn overnight before the start of the experiments. Animals were weighed daily, and urine and faecal samples collected every 2 d. Raw (heatuntreated) whole lupin seed meal or the fibre residue were used in the experiments. Diets were formulated to contain the same amount of digestible energy and protein (Table 1). Crude protein was calculated as N x 6.25 for lactalbumin and
x 5.5 for bean protein.I6 The control diet contained lactalbumin and potato starch; these were replaced by whole lupin seed in the lupin meal diet. The residue diet contained lactalbumin and the fibre residue but no potato starch. Appropriate amounts of synthetic amino acids were added to the legume protein-based diets to equalise them to control (lactalbumin) values. All diets were supplemented with vitamins and minerals to target requirements. Animals were strictly pair-fed at about 12g/d which was the normal intake of control animals under free access to the feed.183'9 Experiment 1 was aimed at studying the overall effect on rat large intestinal microbiota of feeding diets based on lupin meal or its fibre fraction (insoluble residue) in comparison with the whole meal. Three diets were used: lactalbumin control, lupin seed meal diet and a lupin residue diet which was formulated to contain the same amount of insoluble material as the lupin seed diet. Experiment 2 involved similar feeding regimes and diets and was designed to assess the E. coli populations in the small and large intestines. The net protein utilisation (NPU) values were calculated for the rats in each instance.
Sampling procedures and bacterial counts Animals were killed under halothane anaesthesia, exactly 2 h after being given 2.5 g of diet on day 10. In experiment 1 at post mortem, the caecum was removed and weighed, and portions (of tissue and digesta) were immediately transferred to C0,-gassed vials for microbial analysis. A further sample was used for the determination of volatile fatty acids (VFAs) by gas chromatography after extraction in 5 per cent phosphoric acid. In experiment 2, in addition to the caecal sample the last 30cm of the small intestine (considered as ileum) and the previous 30 cm (considered as jejunum), and the colon were taken. Samples were homogenised with a Janke-Kunkel Ultra-Turrax T25 at 20,000 rev/min for 10 s. For experiment 1, anaerobic diluent13 was used for the primary homogenate and the preparation of a dilution series. Viable counts were made usin an adaptation of the measured-drop method. The following media, obtained from Unipath (Basingstoke, UK) unless otherwise stated, were used: WilkinsChalgren agar with 5 per cent defibrinated horse blood for total viable anaerobe count, WilkinsChalgren agar containing 5 per cent defibrinated horse blood and G-N anaerobe selective supplement for the isolation of anaerobic gram-negative
k
103
MANIPULATION OF GUT MICROBIOTA
Table 2. Effect of diet on the bacterial composition of the caecal microbiota
E. coli Other coliforms Enterococci Lactobacilli Bifidobacteria Total Anaerobes Bacteroides ~~~~
Lactalbumin
Lupin meal
Lupin residue
Pooled SD
7.04"* 4.60 6.64 8.8 1 7.71 9.74" 9.40"
4.45' 4.92 5.43 8.46 7.25 8.99b 8.67'
4.60' 4.72 5.58 859 7.41 9.28' 9.08"'
0.76 0.44 0.40 0.45 0.5 1 0.25 0.26
~
Means with different superscripts in each row differ significantly (at least W O . 0 5 ) . *Mean log,,, Nolgm wet wt (four animals per group).
bacilli (Bacteroidaceae), Rogosa agar for the Of lactobacilli, and a selective medium consisting of Columbia agar and 0.5 per cent (v/v) propionic acid (Fisons, UK) for the isolation of bifid~bacteria;~ these cultures were incubated for 48 h at 37°C in an anaerobic cabinet (Don Whitley Scientific, Shipley, UK). MacConkey agar No. 3 and Kanamycin aesculin azide agar, incubated at 37°C for 24 h, were used for the isolation of coliforms and enterococci, respectively. Bacteroidaceae, lactobacilli, bifidobacteria and enterococci were presumptively identified by Gram-stain, their ability to grow on the selective media, colonial morphology, and in some cases catalase reaction and bile tolerance. The identification of E. coli and other coliforms isolated on MacConkey agar No. 3 was confirmed by API 20E (bioMerieux, France). l 2 F o r experiment 2, Maximum Recovery Diluent (Unipath CM733B) was used to prepare the homogenate and dilution series; and viable counts of coliforms on MacConkey agar No. 3 , were assessed.
growth
Statistical analysis The results were subjected to one way analysis of variance using the Minitab Statistical Software Package (Minitab, New York, NY).Differences between means were identified by Student's t test. RESULTS The lupin seeds used in this study contained very low levels of alkaloids (0.1 g/Kg) and only traces could be detected in the residue. Rats fed diets
based on lupin seed meal had gained less weight over the 10 d experimental period (32.0 g vs 42.9 g) and had poorer feed:gain ratios (3.6 vs 2.6) than the lactalbumin controls in both experiments. The animals fed lupin seed diets excreted more nitrogen, particularly as urea in urine. This was reflected in their lower NPU (58.5 per cent) when compared with the control rats (94.2 per cent). In contrast, there were no differences in growth or utilisation of the feed (feed:gain ratio) between the control animals and those fed on diets containing the insoluble lupin residue.
Eflects on bacterial composition of the small and large intestinal contents A comparison of the caecal microbiota of rats fed the lupin and lactalbumin diets (Table 1) revealed a general trend of lower concentrations in the lupin-fed animals of all generalspecies except the non-E. coli coliforms (identified as Proteus mirabilis and Klebsiella spp., the former predominating). The numbers of E. coli in the gut of rats given either lupin meal or its residue were significantly less than those in control animals (P
