Journal of the Science of Food and Agriculture J Sci Food Agric 81:1522±1529 online: 2001) DOI: 10.1002/jsfa.965

Nutritional assessment of protein from ( L) processed at different pH values, in growing rats Teresa Nestares, Mercedes Barrionuevo, Gloria Urbano and Magdalena Lo´pez-Frı´as* Department of Physiology, School of Pharmacy and Institute of Nutrition and Food Technology, University of Granada, E-18071 Granada, Spain

Abstract: The effect of the commonly used processing techniques of soaking at different pH values) and on the digestive and metabolic utilisation of protein from common beans Phaseolus vulgaris L) in growing rats was studied. Before the cooking step, beans were soaked in solutions of pH 2.6, 5.3 or 8.4. Analyses of beans soaked at moderately acid pH 5.3) showed a signi®cant increase in essential amino acids, especially cysteine. Food intake and growth indices were also dependent on pH. All indices were better in animals fed with beans soaked in a basic pH solution than when an acid soaking solution was used. In contrast with the ®ndings expected on the basis of the chemical analysis of protein composition of the food, the worst results for food intake and growth were found in rats fed with beans soaked at pH 5.3. There were no signi®cant differences in apparent digestibility coef®cient between different groups of animals. The highest metabolic utilisation of protein balance) was found in animals fed with beans soaked in a basic solution; however, the values were insuf®cient to account for the faster rate of growth in this group. The better growth rate was probably due to the improved utilisation of in beans soaked in the basic pH 8.4) solution. # 2001 Society of Chemical Industry

Keywords: common ; nutritive utilisation; Phaseolus vulgaris; protein; processing techniques; pH

1 INTRODUCTION commonly consumed foods such as ice cream or Recent problems linked to meat consumption have led regionally consumed foods such as miso in Japan2 and to renewed interest in vegetarian diets. This phenom- kishk in eastern Europe.3 Bean protein has also been enon is reinforced by the fact that physicians have used,in combination with rice,in a nutritious weaning pointed out that consumers eat too many animal food.4 products rich in saturated ) and not enough plant Like other pulses,common beans contain several foods. The increasing preference for vegetarian diets, antinutritional factors5±7 that may limit their con- already noticeable in Europe and the USA,may sumption and the nutritive utilisation of their protein. eventually mean an increase in the consumption of These antinutritional factors can be eliminated or ,long considered the `poor man's meat'. Thus reduced by appropriate cooking or with other simple the image of pulses is evolving. technologies,6 although processing will modify the The importance of grain products as suppliers of nutritive utilisation of protein.8±11 protein is often ignored. Legumes are the plant foods The protein content and nutritional value and the that contain the highest levels of protein,and can effect of antinutritional factors in bean seeds have been supply an average of 16±20% of the total protein investigated in detail.6,7,12,13 However,recent work intake. The food industry is already diversifying its has brought to light the importance of the pH of the range of ready-to-eat cooked products in order to soaking or cooking solution,in view of the effect of pH stimulate pulse consumption and is making attempts on other seed components such as antinutritional to incorporate pulses into some ready-to-eat cooked factors,14 starch15 and ®bre.16,17 Modi®cations in products. Common beans Phaseolus vulgaris L) are a these factors can affect the nutritive utilisation of good source of protein and have been used as a protein.9,13 In addition,protein solubility substitute for other sources of plant soy) and animal under different pH conditions varies and may be a milk) protein in the industrial preparation of major cause of the poor digestibility of this

* Correspondence to: Magdalena Lo´pez-Frı´as, Depto de Fisiologı´a, Facultad de Farmacia, Campus de Cartuja, Universidad de Granada, E-18071 Granada, Spain E-mail: [email protected] (Received 10 July 2001; accepted 14 August 2001)

# 2001 Society of Chemical Industry. J Sci Food Agric 0022±5142/2001/$30.00 1522 Nutritional assessment of common bean protein legume.11,18 These pH-dependent changes differ Amino acids widely depending on the technology and processing The amino acid composition of the proteins was used.19,20 determined by high-performance liquid chromatogra- Because of the effects of external conditions phy using the Waters Pico-Tag method with pre- location and agricultural practices),21 processing column derivatisation and phenylisothiocyanate.25 and seed composition on the nutritive utilisation of Protein hydrolysis was performed in 6mol lÀ1 hydro- protein,different cultivars grown in different regions cloric acid in sealed,evacuated tubes at 110 °C for should be investigated individually,22 as should the 24h. Cysteine and methionine were determined as in¯uence of different processing and cooking tech- cysteic acid and methionine sulphone respectively, niques. The present study was designed to evaluate the obtained by oxidation with performic acid before chemical and biological nutritional quality of protein 6mol lÀ1 HCl hydrolysis.26 Tryptophan was not from a common bean cultivar habitually grown in determined. southern Spain,where it is the third most widely consumed legume.23 In addition,we investigated how soaking in basic solution affected the nutritive utilisation of bean protein. We hoped to show Biological methods whether soakingÐa simple and cheap process com- Experimental design and diet monly used in Spain and known to improve the For nutritional evaluation we used a biological nitro- digestibility and nutritional value of legumesÐ gen balance technique,recording food intake and impaired,enhanced or had no effect on protein quality changes in body weight and calculating nitrogen intake in P vulgaris beans. and faecal and urinary nitrogen excretion. Three experiments were done simultaneously in which processed common beans were the only source of food: SA,beans soaked in strongly acid solution and cooked; MA,beans soaked in moderately acid solution EXPERIMENTAL and cooked; B,beans soaked in basic solution and Samples cooked. Each experiment lasted 10 days. The diet and Raw,dried common beans R)  P vulgaris L) were demineralised water Milli-Q,Ultrapure System; grown in Andalusia southern Spain). The seeds were Millipore,Bedford,MA,USA) were available ad subjected to three different treatments that differed in libitum throughout the experimental period. During the pH of the soaking solution: SA,soaking in the ®rst 3 days the rats were allowed to adapt to the strongly acid solution pH 2.6) and cooking; MA, diet and experimental conditions. The main experi- soaking in moderately acid solution pH 5.3) and mental period comprised the next 7 days,during cooking; B,soaking in basic solution pH 8.4) and which body weight and food intake were recorded and cooking. faeces and urine were collected for subsequent analysis. Faeces were dried,weighted and homoge- Processing techniques nised. Urine was collected on 0.5% HCl v/v),®ltered Soaking Whatman Filter Paper No 40; Whatman,Maidstone, Raw seeds were soaked at room temperature for 9h in Kent,UK) and diluted. distilled water pH 5.3),citric acid solution 0.1%,pH 2.6) or sodium bicarbonate solution 0.07%,pH 8.4). Animals The seed-to-solution ratio was 1:3 w/v). The soaking In each experiment we used 10 young albino Wistar liquid was drained off and the seeds were blended and lyophilised. rats ®ve males,®ve females) reared in the University of Granada Laboratory Animal Services. The growing recently weaned) animals,with an initial body weight Cooking of 54.39Æ0.18g,were housed in individual metabolic Soaked common beans were cooked by boiling in cages kept in a thermoregulated room 22Æ1°C) distilled water for 35min at a seed-to-water ratio of with controlled 12h light/dark periods lights on at 1:6.67 w/v). The cooking water was drained off and 09:00). the seeds were crushed and lyophilised.

Analytical techniques Biological indices Moisture content The following indices and parameters were deter- Moisture content of the samples was determined by mined for each group according to the formulae AOAC method 925.10.24 given below: intake expressed as dry weight),body weight,protein ef®ciency ratio PER) eqn 1)),feed Crude protein conversion ef®ciency FCE) eqn 2)),apparent Total nitrogen content was determined according to digestibility coef®cient ADC) for protein eqn 3)), the Kjeldahl method24 and the crude protein content nitrogen retention nitrogen balance) eqn 4)) and was calculated NÂ6.25). percentage nitrogen retention/nitrogen absorption

J Sci Food Agric 81:1522±1529 online: 2001) 1523 TNestares et al

Table 1. Composition of nitrogen of Phaseolus vulgaris beans processed at comparisons using Statgraphic Statistical Graphics 2.1 different pH (g kgÀ1 dry matter basis) System software Statistical Graphics Corp,Rockville, Diet Total nitrogen content Crude protein NÂ6.25) MD,USA) with an IBM Personal System/2 Model 20 computer. Differences were considered signi®cant SA 33.8Æ0.1a 211.5Æ0.8a when P <0.05. MA 33.8Æ0.2a 211.2Æ0.9a B 34.3Æ0.1a 214.2Æ0.9a

SA, beans soaked in strong acid medium and cooked; MA, beans soaked in moderate acid medium and cooked; B, beans soaked in basic medium and RESULTS cooked. Chemical analysis The same letter in the same column indicates no signi®cant differences Nitrogen content in all the processed bean diets was $P <0.05). Values are meansÆSEM from ®ve replications. similar Table 1). The amino acid composition of the raw and pro- cessed bean diets is shown in Table 2. The percentage %R/A) eqn 5)). relative amounts of essential amino acids in dietary weight gained proteins are shown in Table 3,referred to chicken egg PER ˆ 1† albumin.27 The chemical score for bean protein protein intake showed that the main limiting amino acids were diet intake cysteine,methionine and threonine. With soaking at FCE ˆ 2† weight gained moderately acid pH 5.4),less cysteine was lost,and also less threonine but to a lesser degree. I À F ADC ˆ  100 3† I Biological analysis Food intake was signi®cantly greater when beans had Balance ˆ I À F ‡ U† 4† been soaked in the basic pH solution; there were no signi®cant differences between the groups fed the diets I À F ‡ U† %R/A ˆ  100 5† prepared from beans soaked in strongly or moderately I À F acid solution Table 4). Daily weight gain Table 4) Body weight and diet/protein intake were expressed was signi®cantly lower in rats fed with beans soaked in as g per rat per day. strongly acid solution group SA). Soaking in basic medium B) led to signi®cantly greater weight gains in this group than in the others. When weight gain in Statistical analysis grams was expressed per gram of protein ingested The results from all experiments and analyses were PER),the relative results were the same Table 4). tested statistically by analysis of variance for multiple Soaking in the strongly acid solution SA) led to a

Amino acid a RSAMABSA‡CMA‡CB‡C Asp 11.6 14.0 20.4 14.2 14.3 19.5 13.1 Glu 22.1 22.9 35.9 21.9 22.7 31.5 19.2 Ser 9.20 8.50 11.6 9.10 9.50 10.6 8.60 Gly 5.40 6.60 10.2 5.60 5.60 8.90 5.00 His 3.40 3.90 4.10 3.80 4.10 3.30 3.40 Thr 5.60 6.20 9.80 6.00 5.80 7.70 5.40 Ala 6.80 6.80 9.50 7.10 7.00 9.10 6.60 Arg 8.00 7.70 13.6 7.90 7.50 11.6 7.10 Pro 7.70 8.00 11.5 7.70 8.10 9.50 7.90 Val 8.70 10.20 15.3 9.50 10.1 13.6 8.90 Ile 7.10 8.50 11.9 7.00 8.70 11.2 7.20 Leu 14.8 17.1 22.6 14.6 16.9 21.0 13.7 Phe 8.70 9.49 14.0 8.80 9.50 12.6 8.50 Lys 8.90 10.61 14.4 9.40 10.6 14.3 9.30 Met 2.40 2.51 3.30 2.30 2.50 2.80 2.50 Cys 0.90 0.92 3.30 0.90 0.90 2.80 0.80 Total Aas $%) 13.40 14.39 21.13 13.59 14.38 19.00 12.70 Total branched Aas 30.6 35.7 49.9 31.1 35.7 45.8 29.9 Total sulfur. Aas 3.20 3.41 6.60 3.20 3.40 3.40 3.30

R, raw beans; SA, beans soaked in strong acid medium; MA, beans soaked in moderate acid medium; B, beans soaked in basic medium; SA‡C, SA plus cooking; MA‡C, MA plus cooking; B‡C, B plus Table 2. Amino acid (AA) composition of raw and cooking. processed Phaseolus vulgaris beans (g kgÀ1 dry Data shown are means of two replications. a matter) Tyrosine was not detected in our samples.

1524 J Sci Food Agric 81:1522±1529 online: 2001) Nutritional assessment of common bean protein

Table 3. Chemical score in processed Phaseolus Table 6. Metabolic utilisation of protein from processed Phaseolus vulgaris vulgaris beans (%) beans Amino acid SA MA B Total urinarynitrogen Group mg per rat per day) Balance %R/A Phenylalanine 74.47 98.43 65.83 Isoleucine 103.08 130.90 84.50 SA 118.40Æ9.10a 56.36Æ6.27a 32.22Æ4.60a Leucine 113.94 140.44 91.64 MA 136.60Æ5.60a 52.23Æ3.31a 28.66Æ1.81a Lysine 91.29 122.13 79.11 B 127.30Æ6.20a 77.59Æ5.40 37.89Æ2.48a Methionine 33.09 36.80 33.35 Balance=nitrogen intakeÀ$faecal nitrogen‡urinary nitrogen);%R/A= Cysteine 12.69 37.46 10.40 [balance/$nitrogen intakeÀfaecal nitrogen)]Â100; SA, beans soaked in Valine 95.14 127.78 82.91 strong acid medium and cooked; MA, beans soaked in moderate acid Threonine 68.68 90.28 62.68 medium and cooked; B, beans soaked in basic medium and cooked. The same letter in the same column indicates no signi®cant differences SA, beans soaked in strong acid medium and $P <0.05). Values are meansÆSEM of 10 Wistar rats. cooked; MA, beans soaked in moderate acid medium and cooked; B, beans soaked in basic medium and cooked. not signi®cantly modi®ed by processing in comparison to the raw beans 218.8g kgÀ1,unpublished data). lower food conversion ef®ciency than soaking at pH The amino acid composition of raw beans was 5.3 or 8.4 Table 4). similar to that reported by others5,11,31,32 for this The digestive utilisation of protein,calculated as the legume. Total amino acid content in the raw diet, ADC Table 5),was higher in group B,although the expressed as amino acidkgÀ1 diet 134g kgÀ1,un- difference was only signi®cant in comparison to group published data),was lower than protein content SA. 218.8g kgÀ1,unpublished data),as was also found Nitrogen balance was signi®cantly higher in group B by Candela et al 33 for P vulgaris. This value was not in comparison to the two acid solution groups Table signi®cantly modi®ed by soaking in strongly acid 6). 143.8g kgÀ1) or basic 127.0g kgÀ1) solution before The ratio of nitrogen retained to nitrogen absorbed cooking,although soaking in plain water 211.3g %R/A) was highest in group B,although the differ- kgÀ1,unpublished data) and soaking in water followed ences between groups were not signi®cant Table 6). by cooking 190.0g kgÀ1) did increase amino acid content. After soaking at pH 5.3,percentage total amino acid content of the diet was the same as DISCUSSION percentage protein content. This difference in amino Chemical analyses of protein acid content between diets was probably due to the pH The beans tested in these experiments had a mean of the soaking solutions. As noted by Marquez and protein content of 211.5g kgÀ1,which was close to the Lajolo18 and Salunkhe et al,34 the protein fraction of values given by other authors.28±30 Protein content was common bean is solubilised to different degrees by

Table 4. Food intake and weight change in rats fed Phaseolus vulgaris bean diets Bodyweight gain Drymatter intake Protein intake Group g per rat per day) g per rat per day) g per rat per day) PER FCE SA 0.90Æ0.14 6.80Æ0.17a 1.44Æ0.04a 0.63Æ0.09 9.80Æ1.94 MA 1.83Æ0.19 7.16Æ0.17a 1.51Æ0.04a 1.22Æ0.13 4.18Æ0.34a B 2.63Æ0.15 7.71Æ0.21 1.65Æ0.05 1.59Æ0.07 2.99Æ0.14a

PER=weight gained/protein intake; FCE=diet intake/weight gained; SA, beans soaked in strong acid medium and cooked; MA, beans soaked in moderate acid medium and cooked; B, beans soaked in basic medium and cooked. The same letter in the same column indicates no signi®cant differences $P <0.05). Values are meansÆSEM of 10 Wistar rats.

Nitrogen intake Total faecal nitrogen Absorbed nitrogen Group mg per rat per day) mg per rat per day) mg per rat per day) ADC SA 218.00Æ5.17a 59.31Æ2.39a 158.69Æ4.75a 72.75Æ1.03a MA 230.10Æ5.70a 55.31Æ2.40a 174.80Æ5.38a 75.90Æ1.04ab B 264.20Æ7.25 59.33Æ3.55a 204.90Æ5.03 77.65Æ0.94b

ADC, apparent digestibility coef®cient; SA, beans soaked in strong acid medium and cooked; MA, beans soaked Table 5. Digestive utilisation of protein in moderate acid medium and cooked; B, beans soaked in basic medium and cooked. from processed Phaseolus vulgaris The same letter in the same column indicates no signi®cant differences $P <0.05). Values are meansÆSEM of 10 beans Wistar rats.

J Sci Food Agric 81:1522±1529 online: 2001) 1525 TNestares et al soaking in acid or basic solutions,and this would lead The proportion of branched amino acids was lowest in to different changes in amino acid content. Minka et this group Table 2),and appetite in rats reportedly al 35 also found that amino acid composition was decreases when the serum concentration of these affected by the use of a basic pH solution or distilled amino acids increases.42 This effect may have occurred water for cooking. either directly,as a result of increased brain levels of These differences in amino acid composition of the free amino acids,or indirectly,through blockage of the diets were not found for chickpea9 or faba bean36 uptake of neutral amino acids such as tryptophan and tested under the same conditions. The different tyrosine in the brain.43 These two amino acids are behaviours are not surprising: Carbonaro et al 11 precursors for the synthesis of neurotransmitters studied protein solubility in different legumes and involved in appetite control. found that protein was solubilised through different As suggested by Tovar et al,44 the hydrolysis of mechanisms in dry bean compared to faba bean and starch in beans soaked in a basic solution probably chickpea. According to these authors,in dry bean the also contributed to the increased palatability of the B electrostatic interactions involving ionisable amino diet. In addition,soaking in an acid solution probably acids with opposite charge were probably responsible led to a greater proportion of hydrolysis-resistant for the low protein solubility in water pH 6). This starch,which may have had physiological effects would account for the higher amino acid content we similar to those reported for dietary ®bre,with found at a similar pH value 5.3). In any case,no important consequences on the amount of foodstuff general pattern of behaviour can be established for eaten.45 amino acids,as the ®ndings vary depending on the Using a basic pH soaking solution reduced the time speci®c amino acid and legume involved.33 required to reach a more palatable texture in P Our chemical scores for protein in processed beans vulgaris.46 If cooking time had been the same in all showed that the limiting amino acids were cysteine and our experiments,we would have obtained a better methionine. However,the high protein content of texture after soaking in basic solution,which would bean means that this legume supplies enough essential have made the diet more palatable. amino acids to cover the growing rat's nutritional requirements,as speci®ed by the US National Effect on protein digestive utilisation Research Council.37 For this study we calculated The digestive utilisation of cooked bean protein nutritional requirements from food intake Table 4) expressed as the ADC 72±77%) was similar to the and amino acid content Table 2) as previously 77% value reported by Tobin and Carpenter21 as the reported.38 mean percentage of coef®cients found by different authors who tested bean as the only source of protein Biological analyses of protein in diets fed to young rats. The range of values between Effect on food intake studies 70±85%) was ascribed by Tobin and Recordings of food intake expressed with reference to Carpenter to differences in processing methods. body weight during the 7day main period of the Under our experimental conditions we found no experiment showed that this value was signi®cantly signi®cant differences in ADC only when the differ- lower in rats fed with cooked beans than in weanling ence in pH of the soaking solutions was high. This rats fed a diet adjusted to 12 or 20% protein content contrasts with the in vitro ®ndings of Marquez and 36,38 18 11 casein‡ DL-methionine). The lower food intakes Lajolo and Carbonaro et al; however,our observa- for beans than in rats fed with a 12% casein/ tion is consistent with the conclusions of the latter methionine diet may have been due in part to the group of authors regarding protein solubility. Accord- greater protein supply from this legume. In rats,higher ing to these authors,only very high pH values higher supplies of dietary protein increase the serum con- than the 8.4 of our basic soaking solution) are able to centration of branched amino acids,and this effect modify the solubility of protein from common bean would partially account for decreased appetite.9 The and other legumes; this would explain why we found lower intake of the cooked bean diets in comparison no differences in protein solubility between our three with the 20% casein/methionine diet may have been pH groups. due in part to a difference in protein quality,despite the fact that heating was reported to enhance the Effect on protein metabolic utilisation palatability of beans.39 The amino acid imbalance in The metabolic utilisation of bean protein,measured beans may have reduced intake40 by causing large, as nitrogen balance,was better than expected in view unspeci®c changes in plasma and brain amino acid of the results of our chemical analyses,in contrast to pro®les.41 our earlier ®ndings for chickpea9 and in contrast to There was no signi®cant difference in food intake another study in which bean was used as the source between animals fed with beans soaked in the two acid of protein in a supplemented diet.5 The good solutions; however,intake was signi®cantly greater in metabolic utilisation in the present study re¯ects the group fed with beans soaked in the basic solution. the greater digestive utilisation of protein in all We assume that palatability was enhanced by several groups,especially in the group fed with beans soaked factors in beans soaked in the bicarbonate solution. in a basic solution. This ®nding most likely re¯ects

1526 J Sci Food Agric 81:1522±1529 online: 2001) Nutritional assessment of common bean protein the good availability of carbohydrates in bean,which absorption by increasing active transport of carbo- is further improved by soaking in basic solution,as hydrates in the digestive tract. explained below. The good PER for the MA diet,despite the low Our analytical methods did not take into account a digestive and metabolic utilisation of bean protein, number of other factors such as the possible interac- probably re¯ects a better utilisation of starch. An tions between protein and other components of the earlier study by Sathe et al 49 showed that starch diet. The results of in vivo studies suggested that the hydrolysis was maximal at pH 6 close to the pH 5.3 of chemical structure of legume proteins may adversely our less acid soaking solution). affect nitrogen metabolism by reducing the rate of release of certain amino acids.47 Also,Carbonaro et 11 al showed that,after heat processing at different pH CONCLUSIONS values,protein aggregation can result in a slower rate In conclusion,the amino acid composition,chemical of amino acid or peptide) absorption than is required score and crude protein content of processed beans for ef®cient protein synthesis,thus leading to amino showed that the high protein content and amino acid acid imbalances. In addition,amino acids might be pro®le of this legume were adequate to satisfy the trapped in protein aggregates in a biologically unavail- nutritional requirements of growing rats. Soaking at able form.11 These factors may account for part of the basic pH improved the palatability of beans,whereas difference in metabolic utilisation of bean protein after soaking at acid pH led to no signi®cant increase in treatment at different pH values. intake. The improved nutritive utilisation of protein in Protein balance in the MA diet was similar to that beans cooked after soaking in basic solution was not found by Tobin and Carpenter21 for bean cooked in related with increases in body weight; rather,weight different ways and given to rats as the only source of gain was more closely related with modi®cations in food. In the SA diet,protein balance was slightly carbohydrates. Soaking beans with bicarbonate salt better; however,the best nitrogen retention was solution,then cooking in a fresh change of water,can obtained after soaking in basic solution and cooking. thus be recommended to preserve and enhance the This effect was not necessarily associated with nutritional value of common beans. increased body weight; for example,in groups MA and SA,body weight increased much less than we would have expected from the calculated nitrogen ACKNOWLEDGEMENTS retention value. We thank Karen Shashok for translating parts of the In the group of animals fed the basic-soaked diet, original manuscript into English. protein digestible utilisation and nitrogen retention were similar to or slightly higher than the values in rats fed with acid-soaked beans,whereas the PER in the REFERENCES former group was more than twofold as high as in the 1 Kebary KMK and Hussein S,Quality of ice cream as infuenced other two groups. 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