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J Sci Food Agric 1992, 60,419-423 Germination Alters the Chemical Composition and Protein Quality of Lupin Seeds Suzanne G Dagnia," David S Pettersoqb* Roma R Bellc and Frank V Flanagan" a Edith Cowan University, Western Australia, Australia Department of Agriculture, Baron Hay Court, South Perth, Western Australia 61 5 1, Australia Curtin University of Technology, Western Australia, Australia (Received 13 August 1991 ; revised version received 29 May 1992; accepted 18 September 1992) Abstract: The chemical composition and protein quality of the kernels from Lupinus angustijolius seeds were compared with that for sprouts after 6 days germination. Germination resulted in an apparent increase in protein content from 395 g kg-' to 435 g kg-' DM. Fat and carbohydrate contents decreased. The oligosaccharide content of the sprouted lupin fell to a negligible level, while the phytate and alkaloid concentrations fell from 4.7 g kg-' to 1.6 g kg-I and from 0.72 g kg-' to 0.16 g kg-'. respectively. The quality of lupin kernel protein was poor with a protein efficiency ratio (PER) of 1.45+0.15. Supplementation of kernel with DL-methionhe (2.0 g kg-') increased the protein quality (PER = 2.87k0.17) to that of casein (PER = 2.86k0.18). Germination reduced protein quality (PER = 0.44-tO.I6), and did not improve apparent protein digestibility (APD kernel = 80 f 4 YO; APD sprout = 77 f5 YO). Supplementation of sprout protein with DL-methionine (2.0 g kg-') increased the protein quality (PER = 2.57 020). The total sulphur-containing amino acid concentration of lupin kernel protein, 1.9 g per 16 g N was low. and decreased further to 1.3 g per 16 g N in the sprout, a drop of 32%. The results showed that germination of lupin seeds reduced the concentration of the anti-nutritive factors; however, it also reduced protein quality. Key words: lupin, germination, protein quality, nutritional quality, artinutrients. el a1 1986). The cell wall material from the lupin kernel has cholesterol lowering properties (Evans el al 1990). Therefore, research into the use of lupin as a human food seems warranted. It is a generally held belief that sprouting increases the nutritive value of legume seeds. Past research has determined that germination improves the nutritional value of Phaseolus radiatus, P angularis, Glycine hispida, G m a s (Lee and Karunanithy 1990), red kidney beans (El-Hag et a l l 9 7 8 ) and L albus (EJ-Habbal eta! 1987) by increasing the proportions of proteins and ascorbic acid, and/or by decreasing the content of anti-nutritive factors. However, the nutritional value of navy and pinto beans (P. uulgaris) was not improved following germination ; protein quality was unchanged, and trypsin inhibitor activity persisted (Chang and Harrold 1988). Although there was no information in the literature on the effect of germination on the nutritive value of L INTRODUCTION Legumes are widely incorporated into human diets. The soya bean has been used for many thousands of years in the Orient as a vegetable, in beverages (especially as a 'milk') and as a base for fermented foods. It is also used in a wide range of processed foods in Western nations (Shurtleff and Aoyagi 1985). Seeds of Lupinus angusti$olius have an appearance and chemical composition similar to the soya bean. The kernels are high in protein, and Low in fat and anti-nutritive factors (Yanez ef al 1983; Lee 1986; Gross 1988; Petterson and Crosbie 1990). Lupin protein is low in the sulphur-containing amino acids. However, when supplemented with DLmethionine (1.0-2.0 g kg-'), protein quality is high (Sgarbieri and Galeazzi 1978; Yanez et al I983 ;Aguilera * To whom correspondence should be addressed. 30 419 J F A 60 S G Dugniu, D S Petterson, R R Bell, F V Flunugun 410 TABLE 1 PER, PER ratio. apparent NPU and A P D scores for lupin kernels and sprouts" Triwtiireiit Divrorj. proii+i grolrp CIJllfC'Il l PER Ratio NPU .4 P D 507 (5.2)* 100.4 (5.8)** 100.0 (6.3)** 15.4 (5.6)*** 89.9 (7.0)**** 0.43 (005)* 0.58 (0.03)** 080 (0.04)* 0.63 (0.04)** 0.44 (0.13)* 0.92 (0.01)** 0.77 (0.05)*** 048 (0.09)* < 0.001 < 0~001 ( N x 6.25) (g k g - ' ) I05 I10 I09 Kcrncls Kerncls" Casciii Sprouts Sprouts" 83 x4 ANOVA' 1.45 ( 0 1 5 ) * 2.87 (0.I7)** 2.86 (0. I8)** 0.44 (016)*** 2.57 (020)**** < 0.001 < 0.001 I' Values arc means with standard deviations shown in brackets for 10 rats per group. Means for the same variable with a different nuinbsr of superscript asterisks are significantly different by Scheffe's test ( P < 0.05). I , Suppleincntcd with m-mcthionine (2 g kg I ) . ' ANOVA is one way analysis of variancc. ringu.sri/dius. i t was thought likely that the concen- trations of anti-nutritive factors would decrease and protein content would increase, thus improving the nutritional quality. This study aimed to determine whether germination would lead to a change in nutritive value of lupins with respect to concentration of anti-nutritional factors and to protein. in terms of both quantity and quality as measured by amino acid profile and digestibility. MATERIALS AND METHODS Germination of seed Lupin seeds ( L ungir.vt~foliuscv Gungurru) were soaked in tap water for 24 h and germinated under natural lighting at 30--25OC.The seeds were rinsed and drained daily over the 6 day germination period. A kernel sample was prepared by manually de-hulling lupin seeds. After germination any seeds that had not already shed their hulls were also de-hulled to provide a comparable sample for analysis. The lupin sprout and kernel samples were freeze-dried and milled prior to analysis. Seven batches of sprouts were prepared and sub-samples of each combined for analysis. Analytical methods Nitrogen. potassium, sodium and phosphorus were determined by digesting the samples with sulphuric acid and hydrogen peroxide, and analysing the diluted digest through continuous flow colorimetry and flame photometry in a Leco four-channel autoanalyser. The fat content was determined using a Tecator Soxtec apparatus. using Shell X4 as the solvent. Phytate extraction and assay was by the rapid colorimetric procedure of Latta and Eskin (1980). All of the above analyses were conducted in duplicate. Amino acids were separated by ion exchange chromatography and measured after reaction with ninhydrin. The method of analysis is based on Millipore-Waters manuals (Waters Associates 1984; Walker 1986). The samples were oxidised with performic acid, to ensure a good recovery of cystine and methionine, prior to hydrolysis at 110°C for 24 h in constant boiling hydrochloric acid under nitrogen. The values obtained for threonine, serine and isoleucine were corrected for incomplete hydrolysis and partial decomposition using results from standard ANRC casein. Tryptophan is not detected by this method. An extra hydrolysis, without performic acid, enabled determination of phenylalanine, tyrosine and histidine. Protein efficiency ratio (PER) followed AOAC methods (AOAC 1990). Apparent net protein utilisation (NPU) and apparent protein digestibility (APD) were calculated from nitrogen balance data obtained from metabolic collections conducted during the third week of the PER study. Experimental diet Milled lupin kernels or spouts (with or without the addition of 2.0 g kg-' DL-methionhe) were incorporated into four diets to provide about 100 g of protein per kg of diet (the exact levels are shown in Table 1). The diets were adjusted to provide equal amounts of fat (80 g kg-'), crude fibre (10 g kg-I), moisture (100 g kg-I), and ash (50 g kg-'), based on proximate analyses of kernels and sprouts. Casein was used as the reference protein in the fifth diet. The diets were supplemented with vitamins and minerals according to AOAC (1990) procedures. Germination and nutritional quality of lupin seed 42 1 TABLE 2 Chemical analysis of lupin and sprouts (g kg-') dry basis)" '(YO) * Analyte sprouts (day @ day adaptation period, experimental diets and water were fed ad libiizcnz. Body weight and feed consumption were recorded every 6 days, and on the 28th day of the study. Faeces and urine were collected for four consecutive days during the third week of the experiment. _ _ ___.____ Protein Fat Ash Carbohydrate' Oligosaccharide Phytate Calcium Sodium Potassi um Phosphorus Alkaloid (total) I .8 <3 <3 - 3.8 2.9 2.2 1.4 2.2 1.8 10.7 395.0 82.0 25.5 385.5 59.0 4.7 1.o 0.6 8.9 2.9 0.72 453.1 41.0 39.8 332.1 3.0 I .6 2.6 2.7 8.4 3.5 0.16 Values are pooled samples analysed in duplicate. CV--co-efficient of variation for assay. ' Carbohydrate by difference. " Data analysis PER values were calculated from average weight gain and nitrogen intake figures using the formula: PER = change in body weight (g) per gram of protein consumed. PER values were calculated by dividing the PER for the test material by the PER for casein. NPU and APD figures were determined from nitrogen (N) intake and excretion during the 4-day metabolic collection, using the following formulae: NPU = (N intake-(faecal N + urinary N))/N intake; and AD = (N intake - faecal N)/N intake. The data obtained were analysed using one-way analysis of variance (ANOVA) and Scheffe's test. RESULTS Experimental design Biochemical evaluation Fifty 21-day-old (Wistar) male laboratory rats were obtained from the Animal Resource Centre (Murdoch University, Australia). They were randomly allocated to one of five groups, and housed in individual metabolic cages in an environmentally controlled room (temperature, 20-22°C; relative humidity, 50%). After a 4 The concentrations of anti-nutritive factors (oligosaccharides, phytates and alkaloids), carbohydrate and fat decreased during germination (Table 2). Ash and mineral contents increased. There was an apparent gain in protein concentration in the sprouted lupin when expressed per kg DM. TABLE 3 Amino acid profile of lupin kernels and sprouts (g per 16 g N)" Atnino acidb Cyscys as cysteic acid (cystine) Methionine (as methionine sulphone) Methionine +cystine Aspartic acid Threonine Serine Glutamic acid Proline GIycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Kernels Sprouts (dav 6 ) Change (Oh) 1.2 0.7 1.9 9.5 3.3 47 22.4 3.9 4.5 3.6 40 4.3 7.4 4.1 4.3 4.8 2.6 11.9 0.7 0.6 1.3 23.8 2.7 4.6 8.4 1.6 3.0 3.6 3.9 3.6 5.7 2.4 3.4 3.5 2.1 5.4 -42 - I4 - 32 + 151 - 18 -2 - 63 - 59 - 33 0 -2 - I6 - 23 -41 -21 - 27 - 19 - 55 " Estimates based on pre-oxidation, 24 h and 72 h acid hydrolyses. Note: tryptophan was not determined. 30-2 S G Dagnia, D S Petterson, R R Bell, F V Flanagan 422 TABLE 4 Body weights. average daily weight gain, food intake, dry matter digestibility and nitrogen excretion values for groups of rats used in PER study of lupins" Treatment group - Kernels Kernels" Casein Sprouts Sprouts" ANOVA' Initial body weight ( g ) Final body weight ( g ) 28 day food intake ( g ) Dry matter digestibility (YO) 354 (53)* 1.75 (0.30)* 565 (64)** 5.75 (0.90)** 516 (55)** 5.19 (0.60)** 034 (014)*** 285 (37)* 3.73 (0.39)**** 536 (52)** c 0.001 < 0001 93.2(1.6)* 93.4 (0.6)* 962 (0.5)** 909 (l.6)*** 91.8 (0.7)*.*** < 0.001 Aijerage daily weight gain (g) _ _ 66.7(7.4) 65.9(7.0) 65.3(8.4) 66.8(7.6) 65.2(7.2) NS" 1 15.6(1 3.6)* 227.0(28.7)** 210.7(21.2)** 76.4 (94)*** 169.6(15.9)**** < 0.001 Urinary N Faecal N k) k) 0.30(0.06)* 0.28 (008)* 0.33 (@03)* 0.15 (0.05)* 0.22(0.03)** 0.09(0.01)*** 0.15 (007)** 0.31 (008)* < 0.001 0.10 (001)*** 0.14 (0.02)* < 0.001 " Values are means with standard deviations shown in brackets. Means for the same variable with a different number of superscript asterisks are significantly different by Scheffe's test ( P < 005). ' Supplemented with DL-methionine (2 g kg-'). ' ANOVA is one-way analysis of variance. " NS is not significant. Germination resulted in a decrease in the content of most amino acids, with the exception of aspartic acid (Table 3). Most notably, the proportions of the total sulphurcontaining amino acids decreased by about one-third. Protein quality The results indicated that the quality of lupin kernel protein was poor; however, supplementation with DLmethionine increased the quality to that of casein (Table 4). The PER study showed a substantial increase in sprout protein quality after supplementation. However, the quality of the supplemented sprout protein was significantly less than the quality of the casein and supplemented kernel proteins. NPU was low in rats fed either kernels or sprouts, but improved when diets were supplemented with DLniethionine (2.0 g kg-') (Table 4). Germination did not improve protein digestibility since apparent digestibility was 77*5% and 80?4% for sprouts and kernels, respectively. DISCUSSION The concentration of oligosaccharides, alkaloids and phytate decreased after 6 days germination. The reduction in the concentrations of anti-nutritive factors in the sprout is of benefit for human nutrition. The oligosaccharide content was reduced to a negligible level therefore. flatulence, and other side-effects associated with the consumption of high concentrations of oligosaccharides. will be greatly reduced when the germinated seeds are consumed. Sprouting lowered the concentrations of the bitter alkaloids. In addition to improving flavour, this would have also reduced the already minimal risk of any untoward effects. The benefit of a low phytate content in the sprout would be an increase in mineral bioavailability, since phytate can bind strongly with divalent ions, inhibiting their absorption. Phytate has also been shown to inhibit the action of some digestive enzymes, by binding calcium ions (necessary for trypsin and a-amylase activity) or interacting with enzyme substrates (Liener 1989). The protein quality of the lupin kernels and sprouts was low. Supplementation of the kernel protein with DLmethionine (2 g kg-') increased the quality to that of casein. Sprout protein quality also improved with supplementation ; although not to the level of the casein or supplemented kernels. The lower protein quality of the sprouts is likely to be due to the reduction of the content of sulphur-containing amino acids during germination. The proportions of other essential amino acids, including threonine, lysine, leucine, isoleucine and phenylalanine, decreased during germination. The fact that the supplemented sprouts had a lower protein quality than the supplemented kernels suggested that an amino acid other than the sulphur amino acids became limiting in the supplemented sprout diet. Further work would be necessary to identify this second limiting amino acid in sprouts. The apparent increase in protein can be attributed to the utilisation of fats and carbohydrates as energy sources for the developing sprouts. The aspartic acid concentration increased three-fold during germination (Table 3). This change can be attributed to the release of amino groups from other amino acids to oxaloacetate in the shift from storage protein to functional protein during the course of germination. Consumption of lupin sprouts is most likely to be as part of a mixed and varied diet, particularly in Western countries where there is a wide range of dietary protein sources. The loss of sulphur amino acids is unlikely to Germination and nutritional quality of lupin seed have an adverse effect on the quality of a mixed diet, and would be outweighed by the benefits offered by the reduction in the anti-nutritional factors, which also means that they cannot have any adverse effect on the rest of the dietary intake. A study of the time course for the losses of sulphur amino acids and anti-nutrients might indicate an optimal time for sprouting. ACKNOWLEDGEMENTS The authors thank the Grain Legumes Research Council of Australia for financial support, Dr R L Davies and J Baker for the amino acid analyses, and Dr D J Harris for his assistance with other assays. REFERENCES Aguilera J F, Prieto C, Fonolla J, Gil F 1986 Protein and energy utilization in rats of diets based on lupin seed (Lupinus albus oar. Multolupa). Animal Feed Sci Techno1 15 3340. AOAC 1990 Oficial Methou3 of Analysis of the Oflcial Analytical Chemists (1 5th edn), ed Hilrich K. Association of Official Analytical Chemists, Arlington, USA, pp 1095-1096. Chang K C, Harrold R L 1988 Changes in selected biochemical components, in oitro protein digestibility and amino acids in two bean cultivars during germination. J Food Sci 53 783-787. El-Habbal M S. Attia N Y 1987 Chemical evaluation of lupin seeds during germination. Ann Agric Sci 32 957-966. 423 El-Hag N, Haard N F, Morse R E 1978 Influence of sprouting on the digestibility coefficient, trypsin inhibitor and globulin proteins of red kidney beans. J Food Sci 43 1874-1875. Evans A J, Cheung P, Cheetham N W H 1990 An evaluation of dietary fibre from Lupinus angustifolius. Abstracts. Sixth International Lupin Conference, Tecumo, Chile, p 52. Gross R 1988 Lupins in human nutrition. In Proc Fifth International Lupin Conference, Poznan, Poland, pp 5 1-63. Latta M, Eskin M 1980 A simple and rapid colorimetric method for phytate determination. J Agric Food Chem 26 I3 13-1 3 15. Lee C H 1986 Lupin seed for human consumption. In Proc Fourth International Lupin Conference, Geraldton, Western Australia, pp 64-75. Lee C K, Karunanithy R 1990 Effects of germination on the chemical composition of Glycine and Phaseolus beans. J Sci Food Agric 51 437445. Liener I E 1989 Antinutritional factors in legume seeds: State of the Art. In Recent Advances of Research in Antinutritional Factors in Legume Seeds, ed Huisman J, van der Poel T F B & Liener I E. Pudoc, Wageningen, The Netherlands, pp 6-13. Petterson D S, Crosbie G B 1990 Potential for lupins as a food for humans. Food Aust 42 266-267. Sgarbieri V C, Galeazzi M A M 1978 Some physiochemical and nutritional properties of a sweet lupin (Lupinus albus var. Multolupa) protein. J Agric Food Chem 26 1438-1442. Shurtleff W, Aoyagi A 1985 The Soyfoods Industry and Market Directory and Databook (5th edn). The Soyfoods Centre, CA, USA. Walker B 1986 Analysis of Amino Acidr using Ion-Exchange Ninhydrin on the M600.Millipore-Waters, Sydney, Australia. Waters Associates 1984 The Watersm Amino Acid Analysis System Operators’ Manual. Waters Associates, MA, USA. Yanez E, Ivanovic D, Owen D F, Ballester D 1983 Chemical and nutritional evaluation of sweet lupins. Annals Nutr Metab 27 513-520.