Twenty-four middle-aged healthy men were given a low-fat high-carbohydrate (5.5 g fat; L), or a moderately-fatty, (25.7 g fat; M) breakfast of similar energy contents for 28 d. Other meals were under less control. An oral glucose tolerance test (OGTT) was given at 09.00 hours on day 1 before treatment allocation and at 13.30 hours on day 29. There were no significant treatment differences in fasting serum values, either on day 1 or at the termination of treatments on day 29. The following was observed on day 29: (1) the M breakfast led to higher OGTT C-peptide responses and higher areas under the curves (AUC) of OGTT serum glucose and insulin responses compared with the OGTT responses to the L breakfast (P < 0.05); (2) treatment M failed to prevent OGTT glycosuria, eliminated with treatment L; (3) serum non-esterified fatty acid (NEFA) AUC was 59% lower with treatment L than with treatment M, between 09.00 and 13.20 hours (P <0.0001), and lower with treatment L than with treatment M during the OGTT (P = 0.005); (4) serum triacylglycerol (TAG) concentrations were similar for both treatments, especially during the morning, but their origins were different during the afternoon OGTT when the Svedberg flotation unit 20–400 lipid fraction was higher with treatment L than with treatment M (P = 0.016); plasma apolipoprotein B-48 level with treatment M was not significantly greater than that with treatment L (P = 0.086); (5) plasma tissue plasminogen-activator activity increased after breakfast with treatment L (P = 0.0008), but not with treatment M (P = 0.80). Waist:hip circumference was positively correlated with serum insulin and glucose AUC and with fasting LDL-cholesterol. Waist:hip circumference and serum TAG and insulin AUC were correlated with factors of thrombus formation; and the OGTT NEFA and glucose AUC were correlated. A small difference in fat intake at breakfast has a large influence on circulating diurnal NEFA concentration, which it is concluded influences adversely glucose tolerance up to 6 h later.

This paper reviews the evidence for the claims of health benefits derived from the use of probiotics. A brief history of probiotics and the types of probiotics currently used and the criteria for the selection of probiotics is discussed. The ability of probiotics to enhance the nutritional content and bioavailability of nutrients and the scientific evidence for the usefulness of probiotics in alleviating the symptoms of lactose intolerance and in enhancing growth development is examined. The remainder of the review focuses on studies of a specific probiotic, Lactobacillus GG which has been extensively investigated for its health benefits in humans and animals. These studies serve as a model for the potential benefits of probiotics. The ability of Lactobacillus GG to treat or prevent diarrhoeal disease, to serve as an adjuvant for vaccines, to prevent rotavirus-induced diarrhoea, to prevent milk-based allergic reactions, alcohol-induced liver disease and colon cancer are presented. The review concludes with a discussion of the data supporting the safety of probiotics.

Effects of progressive endurance training on energy expenditure (EE) were studied in thirteen elderly sedentary subjects (62.8 (sd 2.3) years) after 7 and 14 weeks of training. Daily EE (DEE) and energy cost of the various usual activities were measured over 48 h by whole-body indirect calorimetry. Free-living DEE (DEEFLC) was calculated from 7 d activity recordings and the energy costs of activities were measured in the calorimeters using the factorial method. DEEFLC did not vary significantly throughout the training period despite the additional energy cost of training sessions (0·60 (sd 0·15) MJ/d), because energy expended during free-living activities (EEACT) decreased by 4·8 (sd 7·1) % (P < 0·05) and 7·7 (sd 8·6) % (P < 0·01) after 7 and 14 weeks of training respectively. Measurements in the calorimeters showed that sleeping metabolic rate transiently increased by 4·6 (sd 3·2) % after 7 weeks of training (P < 0·001) and returned to its initial level after 14 weeks of training. BMR was 7·6 (sd 7·0)%(P < 0·01) and 4·1 (sd 6·1)% (P = NS) higher after 7 and 14 weeks of training respectively, than before training. Likewise, diet-induced thermogenesis increased from 3·7 (sd 2·5) to 7·2 (sd 2·8) % energy intake after 7 weeks of training (P < 0·05), and returned to its initial level after 14 weeks of training (4·2 (sd 2·6) % energy intake). Despite these changes, energy expended during activities and the corresponding DEE did not vary throughout the training period. It was concluded that: (1) DEEFLC remained constant throughout the training period due to a compensatory decrease in free-living EEACT; (2) progressive endurance training induced a transient increase in sleeping metabolic rate, BMR and diet-induced thermogenesis after 7 weeks which was not reflected in the energy expended during activities and DEE.

Resistant starch (RS) has been defined as the sum of starch and starch-degradation products that reach the human large intestine (Champ, 1994), and it is now regarded as a sub-fraction of starch with a positive impact on colonic welfare and lipid metabolism. An early estimate of the RS intake in Europe gave an average value of approximately 4 g/d (Dyssler & Hoffem, 1994a). However, since no information is available for Italy, the aim of the present study was to estimate the intake of RS in the Italian diet by direct analysis of RS in a range of typical foods representing the main sources of starch intake in the country. The selection of representative foods and of food consumption data were based on published results of the National Food Consumption Study conducted during the 1980s by the National Institute of Nutrition on 10 000 households, using weighed-food records plus inventory methodologies (Saba et al. 1990; Turrini et al. 1991). Three main groups of foods were considered: cereals (pasta, rice, bread and bread products, and pastries), potatoes, legumes. Different commercial brands for each sample were purchased, according to the known presence on the market. Samples were prepared ‘as eaten’ and submitted to simulated chewing, followed by total and resistant starch determination using the enzymic procedure published as a result of the EC Concerted Action EURESTA (Champ, 1992). From these results, the estimated average intake of RS in Italy was found to be 8.5 g/d, with regional differences (from 7.2 g/d in the north-west to 9.2 g/d in the south) mainly due to the different consumption of some typical Italian starchy food (bread, pasta, legumes).

The aim of the present work was to investigate the influence of the intestinal microflora on the changes in hepatic cytochrome P450 apoproteins induced by dietary glucosinolates. Ten rats harbouring a conventional digestive microflora were offered either a diet containing 390g myrosinase-free rapeseed meal/kg (n 5) or a control diet devoid of glucosinolates (n 5). A similar trial was performed using germ-free rats. After 4 weeks of exposure to the dietary regimens, animals were slaughtered and their livers removed for preparation of microsomes and analysis of cytochrome P450 (EC 1.14.14.1). The glucosinolate-rich diet decreased the concentration of total cytochrome P450 in conventional rats only (-34%). The bacterial status did not modify the concentration of apoproteins CYP1A2 and CYP2B1/B2, but greatly decreased the concentration of the male constitutive isoform CYP2C11 (-53 and -45% respectively in conventional and germ-free rats). Germ-free rats fed on the glucosinolate-rich diet had a greater concentration of CYP3A (+139%) and a lower concentration of CYP2E1 (-32%) than their counterparts fed on the control diet. However, these differences were absent in conventional animals. On the whole, the influence of the intestinal microflora on the changes in hepatic cytochrome P450 due to the consumption of cruciferous vegetables is very complex and obviously involves different mechanisms according to the apoprotein.

Chemical and physical transformations of solid food begin in the mouth, but the oral phase of digestion has rarely been studied. In the present study, twelve healthy volunteers masticated mouthfuls of either bread or spaghetti for a physiologically-determined time, and the levels of particle degradation and starch digestion before swallowing were compared for each food. The amounts of saliva moistening bread and spaghetti before swallowing were, respectively, 220 (SEM 12) v. 39 (SEM 6) g/kg fresh matter. Particle size reduction also differed since bread particles were highly degraded, showing a loss of structure, whereas spaghetti retained its physical structure, with rough and incomplete reduction of particle size. Starch hydrolysis was twice as high for bread as for spaghetti, mainly because of the release of high-molecular-mass α-glucans. The production of oligosaccharides was similar after mastication of the two foods, respectively 125 (SEM 8) and 92 (SEM 7) g/kg total starch. Starch hydrolysis, which clearly began in the mouth, depended on the initial structure of the food, as in the breakdown of solid food. These significant physical and chemical degradations of solid foods during oral digestion may influence the entire digestive process.

To assess the response of low consumers of fruit and vegetables to a nutrition education intervention programme, data were collected from 104 adults on attitudinal variables related to ‘eating more fruit, vegetables and vegetable dishes’. Questionnaires (based on the theory of planned behaviour) assessing perceived barriers to increasing fruit and vegetable consumption were administered before an action-orientated intervention programme and at the end of the intervention period (8 weeks). Questionnaire scores for belief-evaluations in the intervention groups pre- and post-study indicated that support of family and friends, food costs, time constraints and shopping practicalities (in order to increase intake of fruit, vegetable and vegetable dishes) were barriers to greater consumption of these foodstuffs. Perceived situational barriers to increasing intakes of fruits and vegetables were: limited availability of vegetables, salads and fruit at work canteens, take-aways, friends' houses and at work generally. Following the intervention the number of visits to the shops was perceived as a greater barrier for increasing intakes of fruit and vegetables. Perceived practical opportunities for increasing intakes highlighted drinking fruit juice, taking fruit as a dessert, having fruit as a between-meal snack and eating two portions of vegetables with a meal. About two-thirds of intervention subjects achieved the recommended fruit and vegetable target, but it is concluded that practical issues and situational barriers need to be addressed for the success of future public health campaigns.

There are physical, mental, social and environmental changes which take place with ageing; for example, decreased physical activity, increase in body fat, decrease in lean body mass and consequently decreased energy intake may be associated with physiological functions that affect metabolism, nutrient intake, physical activity and risk of disease. There are now many studies which have found that undernutrition is prevalent and often unrecognized in patients admitted to hospitals and institutions. There is also evidence which links protein-energy undernutrition or its markers with clinical outcomes in acute and non-acute hospital settings and that nutritional supplements can improve outcomes in some of these settings. However, most clinically-available nutrition screening instruments lack sensitivity and specificity, and abnormal nutritional indicators may simply reflect effects of age, functional disability, or severe underlying disease. Thus, causal relationship cannot be assumed without a sufficiently powerful intervention study which adequately adjusts for the effects of non-nutritional factors, such as the number and severity of co-morbid conditions on clinical outcome.

The human colonic flora has both beneficial and pathogenic potentials with respect to host health. There is now much interest in manipulation of the microbiota composition in order to improve the potentially beneficial aspects. The prebiotic approach dictates that non-viable food components are specifically fermented in the colon by indigenous bacteria thought to be of positive value, e.g. bifidobacteria, lactobacilli. Any food ingredient that enters the large intestine is a candidate prebiotic. However, to be effective, selectivity of the fermentation is essential. Most current attention and success has been derived using non-digestible oligosaccharides. Types primarily being looked at include those which contain fructose, xylose, soya, galactose, glucose and mannose. In particular, fructose-containing oligosaccharides, which occur naturally in a variety of plants such as onion, asparagus, chicory, banana and artichoke, fulfil the prebiotic criteria. Various data have shown that fructo-oligosaccharides (FOS) are specifically fermented by bifidobacteria. During controlled feeding studies, ingestion of these prebiotics causes bifidobacteria to become numerically dominant in faeces. Recent studies have indicated that a FOS dose of 4g/d is prebiotic. To exploit this concept more fully, there is a need for assessments of (a) improved determination of the gut microbiota composition and activity; (b) the use of molecular methodologies to assess accurately prebiotic identities and develop efficient bacterial probing strategies; (c) the prebiotic potential of raw and processed foods; and (d) the health consequences of dietary modulation.

Few other aspects of food supply and metabolism are of greater biological importance than the feeding of mothers during pregnancy and lactation, and of their infants and young children. Nutritional factors during early development not only have short-term effects on growth, body composition and body functions but also exert long-term effects on health, disease and mortality risks in adulthood, as well as development of neural functions and behaviour, a phenomenon called ‘metabolic programming’. The interaction of nutrients and gene expression may form the basis of many of these programming effects and needs to be investigated in more detail. The relation between availability of food ingredients and cell and tissue differentiation and its possible uses for promoting health and development requires further exploration. The course of pregnancy, childbirth and lactation as well as human milk composition and the short- and long-term outcome of the child are influenced by the intake of foods and particularly micronutrients, e.g. polyunsaturated fatty acids, Fe, Zn and I. Folic acid supplementation from before conception through the first weeks of pregnancy can markedly reduce the occurrence of severe embryonic malformations; other potential benefits of modulating nutrient supply on maternal and child health should be further evaluated. The evaluation of dietary effects on child growth requires epidemiological and field studies as well as evaluation of specific cell and tissue growth. Novel substrates, growth factors and conditionally essential nutrients (e.g. growth factors, amino acids, polyunsaturated fatty acids) may be potentially useful as ingredients in functional foods and need to be assessed carefully. Intestinal growth, maturation, and adaptation as well as long-term function may be influenced by food ingredients such as oligosaccharides, gangliosides, high-molecular-mass glycoproteins, bile salt-activated lipase, pre- and probiotics. There are indications for some beneficial effects of functional foods on the developing immune response, for example induced by antioxidant vitamins, trace elements, fatty acids, arginine, nucleotides, and altered antigen contents in infant foods. Peak bone mass at the end of adolescence can be increased by dietary means, which is expected to be of long-term importance for the prevention of osteoporosis at older ages. Future studies should be directed to the combined effects of Ca and other constituents of growing bone, such as P, Mg and Zn, as well as vitamins D and K, and the trace elements F and B. Pregnancy and the first postnatal months are critical time periods for the growth and development of the human nervous system, processes for which adequate substrate supplies are essential. Early diet seems to have long-term effects on sensory and cognitive abilities as well as behaviour. The potential beneficial effects of a balanced supply of nutrients such as I, Fe, Zn and polyunsaturated fatty acids should be further evaluated. Possible long-term effects of early exposure to tastes and flavours on later food choice preferences may have a major impact on public health and need to be further elucidated. The use of biotechnology and recombinant techniques may offer the opportunity to include various bioactive substances in special dietary products, such as human milk proteins, peptides, growth factors, which may have beneficial physiological effects, particularly in infancy and early childhood.

To verify the influence of food consistency on satiety mechanisms we evaluated the effects of the same meal in solid–liquid (SM) and homogenized (HM) form on satiety sensation, gastric emptying rate and plasma cholecystokinin (CCK) concentration. Eight healthy men, aged 21-28 (mean 24·5) years were given two meals (cooked vegetables 250 g, cheese 35 g, croutons 50 g and olive oil 25 g, total energy 2573 kJ, with water 300 ml) differing only in physical state: SM and HM. The subjects consumed the meals in randomized order on non-consecutive days. The sensations of fullness, satiety and desire to eat were evaluated by means of a questionnaire, gastric emptying was assessed by ultrasonographic measurement of antral area, and plasma CCK concentration was measured by radioimmunoassay. The vegetable-rich meal was significantly more satiating (P < 0·05) when in the HM form than when eaten in a SM state. Furthermore, the overall gastric emptying time was significantly slowed (255 (sem 11) min after HM v. 214 (sem 12) min after SM; P < 0·05) and CCK peak occurred later (94 (sem 12) min after HM v. 62 (sem 11) min after SM; NS) when the food was consumed in the HM form. Independently of the type of meal, antral area was significantly related to fullness sensations (r2 0·46, P = 0·004). These results demonstrate that meal consistency is an important physical food characteristic which influences both gastric emptying rate and satiety sensation. Moreover, the relationship observed between antral area and fullness sensation confirms that antral distension plays a part in the regulation of eating behaviour.

The present review addresses the role of food constituents in the aetiology of metabolic conditions and chronic diseases, mostly related to energy metabolism and substrate regulation, such as obesity and non-insulin-dependent diabetes mellitus. Second, attention is paid to malnutrition, a major cause of mortality and morbidity in developing countries, which may be a cause of concern in Europe because of the increasing number of elderly people in the population. Finally, the role of diet during exercise, a condition of enormous substrate demands, is evaluated. Based on a critical evaluation of the existing knowledge in the literature, implications for future research in relation to functional foods are discussed.

Human milk was collected from women in their 10th–14th weeks of lactation, and was analysed for amino acids. Corrections were made for losses of amino acids which were presumed to occur during acid hydrolysis, using a non-linear mathematical model that describes the simultaneous processes of amino acid yield and decay. The mean amino acid composition of the human milk was found to be similar to previously reported estimates, although the cysteine content of the human milk in the present study was 20% higher than the average literature estimate. True (corrected for endogenous amino acid excretions) ileal amino acid digestibility of human milk was determined using the 3-week-old piglet as a model animal for the human infant. The piglets were given either human milk (n 6) or a protein-free diet (n 6) for a 6 d experimental period. Cr2O3 was added as an indigestible marker, to both the human milk and protein-free diet. At the end of the experimental period the piglets were anaesthetized and samples of digesta removed from the terminal ileum of each piglet. After sampling the piglets were killed. Endogenous ileal excretions of amino acids were determined in piglets fed on the protein-free diet. The true digestibilities of total N and amino acid N were 88% and 95% respectively. The true ileal digestibility of the non-amino acid N fraction in human milk, when calculated by difference was only 50%. The true digestibility of the amino acids in human milk ranged from 81–101% with threonine (86%) being the least digestible essential amino acid. When the true ileal digestibility values were used to correct the amino acid composition of human milk, the pattern of digestible amino acids in human milk was different compared with the currently recommended pattern of amino acid requirements for the infant.

The influence of Ca supplementation of the usual diet for 14d on biochemical markers of bone turnover was investigated in healthy young adults aged 21–26 years. In a crossover study, eighteen subjects (five male and thirteen female) were randomly assigned to their self-selected diet (about 22 mmol Ca/d) or their self-selected diet with a 20 mmol/d Ca supplement (about 40 mmol Ca/d) for 14d followed by crossover to the alternative diet for a further 14d. During each dietary period fasting morning first void urine samples (last 3d) and fasting blood serum samples (morning of twelfth day) were collected. Ca supplementation reduced urinary excretion of pyridinoline (14%) and deoxypyridinoline (16%) (biochemical markers of bone resorption) but had no effect on biochemical markers of bone formation (serum osteocalcin and bone-specific alkaline phosphatase; EC 3.1.3.1). It is concluded that Ca supplementation of the usual diet in young adults suppresses bone resorption over a 2-week period. If sustained, this could result in suppression of the bone remodelling rate and an increase in bone mass over time. The findings of this short-term study with a relatively small number of young adults highlight the need for a longer-term intervention study of the effect of increased Ca intake on bone mass in this age group.

Compensatory changes in energy intake (EI) and macronutrient metabolism in response to modest covert underfeeding were tested by whole-body calorimetry in eight lean men. Each was studied on three occasions comprising a controlled stabilization day followed by manipulation and outcome days in a whole-body calorimeter. On the manipulation day EI was fixed, and calculated to maintain energy balance (CONTROL) or to provide 85% of CONTROL by removing energy as carbohydrate (CHOred) or as fat (FATred). On the outcome day, ad libitum EI was allowed at fixed mealtimes. CHOred and FATred manipulations generated significantly different energy balances (-1.10 (SE 0.13) MJ, P=0.000; -1.10 (SE 0.12) MJ, P=0.000) and fat balances (-0.61(SE 0.23) MJ, P=0.03; -1.09 (SE 0.20) MJ, P=0.000), but not carbohydrate balances (-0.39 (SE 0.22) MJ, NS; 0.11 (SE 0.23) MJ, NS) by the end of the manipulation day compared with CONTROL. On the outcome day, EI was significantly higher than CONTROL after CHOred (+1.58 (SE 0.33) MJ, P=0.004) and FATred (+1.21 (SE 0.49) MJ, P=0.022) with no differences between treatments. Overall 48 h energy balances averaged close to zero at -0.14, +0.34, +0.04 MJ on CONTROL, CHOred and FATred respectively. Total 48 h energy intakes on CHOred and FATred averaged 101 (SE 1.7)% and 99 (SE 2.5)% of CONTROL, thus demonstrating accurate detection of a mild energy deficit and efficient next-day compensation. Despite significant differences in macronutrient oxidation rates, the energy homeostatic mechanism appeared to be independent of specific macronutrient deficits.

Previous epidemiological studies have suggested an association between low levels of physical activity, fitness and the metabolic cardiovascular syndrome. However, many studies have used subjective non-quantitative questionnaire-based methods for assessing physical activity which do not distinguish between the different dimensions of this complex exposure, and in which measurement error in the exposure has not been estimated. These deficiencies in the measurement of this exposure complicate the interpretation of the results of epidemiological studies, and consequently make it difficult to design appropriate interventions and to estimate the expected benefit which would result from intervention. In particular, it is unclear whether public health advice should be to increase total energy expenditure, or to attempt to raise fitness by recommending periods of vigorous activity. To separate the effects of fitness and total energy expenditure in the aetiology of the metabolic cardiovascular syndrome, we measured the physical activity level (PAL), defined as total energy expenditure: BMR, and fitness (maximum O2 consumption (Vo2max per kg), measured in a sub-maximal test) in a cross-sectional population-based study of 162 adults aged 30–40 years. Heart-rate monitoring with individual calibration was used to measure total energy expenditure using the HRFlex method (Ceesay et al. 1989) which has been validated previously against doubly-labelled water and whole-body calorimetry. The relationship between a single measure of PAL, Vo2max per kg and the usual or habitual level for each exposure was measured in a sub-study of twenty-two subjects who undertook four repeated measures over the course of 1 year. This study design allows the reliability coefficient to be computed, which is used to adjust the observed associations for measurement error in the exposure. Twelve men (16.4%) and sixteen women (18.0%) were defined as having one or more features of the metabolic cardiovascular syndrome. The univariate odds ratio for each increasing quartile for PAL was 0.64 (95 % CI 0.43–0.94) and was 0.49 (95 % CI 0.32–0.74) for Vo2max per kg, suggesting that the association with the metabolic cardiovascular syndrome was stronger for fitness than for PAL. However, after adjustment for obesity and sex, and correction for exposure measurement error, the odds ratio per quartile for PAL was 0.32 (95 % CI 0.13–0.83) and 0.44 (95 % CI 0.24–0.78) for Vo2max per kg. Thus, although univariate analysis would suggest that fitness has a stronger association with the metabolic cardiovascular syndrome than PAL, this conclusion is reversed once confounding and the differences in measurement error are considered. We conclude from the present study that the metabolic cardiovascular syndrome is strongly associated with reduced habitual energy expenditure. The method employed to assess the exposure in the present study demonstrates the utility of assessing a known dimension of physical activity using a physiologically-based and objective measure with repeated estimation to adjust for measurement error. Such quantitative epidemiological data provide the basis for planning and evaluating the expected benefit of population-level interventions.

Curiosity about probiotic bacteria is high. This is not surprising given the rapid growth of the natural products market in the USA, which was 22% in 1995 (New Hope Natural Media, Boulder, CO). Although probiotic cultures are only one component of that market, this trend shows that consumers in the USA are increasingly taking a pro-active stance towards their health and are purchasing products not only to eliminate what is perceived as dietary negatives but to increase the levels of dietary positives (Wrick, 1995). This shift in consumer attitude bodes well for the development of the American probiotic market. This article describes the composition, promotion and labelling of probiotic-containing products, discusses attributes of commercial probiotic bacteria, and estimates the size of the probiotic market in the USA. This market information was obtained from several published (Anon, 1996a,b)and unpublished but commercially available (SPINS Information Services, San Francisco, CA and New Hope Natural Media, Boulder, CO) sources.

The energy value of NSP has been expressed as their metabolizable energy (ME) content. The aim of the present study was to determine whether differences in ME and net energy (NE) contents were similar for insoluble and soluble NSP. Nine healthy young men were offered three diets according to a Latin-square design (3 × 3) with three repetitions: diet C (control), diet B (control + 50 g sugarbeet fibre/d) and diet I (control + 50 g commercial inulin/d). After a 16 d adaptation period to NSP isolate, food intake was controlled (duplicate meal method) and faeces and urine were collected for 8 d. A period of 60 h was devoted to measurement of energy expenditure (EE) by whole-body indirect calorimetry. NSP-isolate ingestion induced significant increases in the number of defecations and stool weight resulting from increases in water, DM and microbial mass excretion. After deduction of microbial N, differences in faecal N excretion between diets were not significantly different. Urinary N excretion was slightly decreased by sugarbeet fibre or commercial inulin ingestion but the N balances for the diets were not significantly different. Diet energy, N and lipid apparent digestibilities decreased by only 1–2 %. Commercial inulin was entirely fermented and fermentability of sugarbeet fibre averaged 0.886 (sd 0.117). Sugarbeet fibre and commercial inulin ME values averaged 10.7 (sd 1.2) and 13.0(sd 2.3) kJ/g DM respectively. NSP-isolate ingestion caused significant (sugarbeet) and nonsignificant (inulin) increases in daily EE. The maintenance NE contents of sugarbeet fibre and inulin averaged 5.0 (sd 5.0) and 11.9 (sd 1.3)kJ/g DM respectively. Differences in maintenance NE contents of NSP isolates were much greater than differences in ME values.