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Analysis of Protein Fractions and Some MineralsPresent in Chan (Hyptis suaveolens L.) SeedsCesar Aguirre, Iovanna Torres, Guillermo Mendoza-Hernandez, Teresa Garcia-Gasca, and Alejandro Blanco-Labra

Abstract: Chan (Hyptis suaveolens L.) seeds have been used as food as well as in traditional medicine in several countries ofAmerica, Asia and Africa. Chan seed protein content was 13.9% on dry weight basis. Analysis of its protein compositionshowed 39% globulins, 36% glutelins, 24% albumins, and 1% prolamins. By defatting the flour with chloroform/methanol,it increased the extracted proteins and improved the protein band resolution after SDS-PAGE, showing 5 albumin bands,8 globulin bands, and 2 prolamin and glutelin bands. The aromatic amino acid content in chan seeds is higher than thoseof other grains including maize, with good levels of branched chain amino acids. In general, except for lysine, it has awell-balanced amino acid composition, providing a good supply of almost all the essential amino acids for the differentage groups. Magnesium content was high, whereas calcium, potassium, and phosphorous were in the average range whencompared to barley, oat, rice, and wheat. The present results indicate that seeds from the chan plant could be relevantbecause of their nutritional properties and they have the potential to be widely used in the production of high-qualityfood.

Keywords: amino acid composition, Hyptis suaveolens, mineral composition, protease inhibitors, protein quality of grains,pseudo-cereals

Practical Application: Chan seeds are presently used in a very limited manner as a food source; however, consideringtheir high quality composition, they have the potential for a more extended use in the food industry.

IntroductionAnimal proteins are expensive, particularly in terms of their

market price and environmental impact. On the other hand, asfunctional ingredients in food formulations, plant proteins repre-sent a more economic and sustainable alternative (Gonzalez-Perezand others 2005). The fractionation of grain proteins, particu-larly from those grains that have shown good food properties,has long been a significant area of interest. In this study, we in-vestigated the proteins present in the seeds of a plant known aschan (Hyptis suaveolens) (Martinez 1959), also known as “chiagorda” and pignut (Holm and others 1979; Vergara-Santana andBravo-Magana 1992). Chan belongs to a group of seeds gener-ically known as chias. It is a widely distributed bush growingmainly in Mexico, Central America, South America, Asia and thePacific Islands. Its height is between 1 to 2 m, its seeds are small;approximately 2 to 3 mm long, twice as big as amaranth and chia

MS 20110428 Submitted 4/4/2011, Accepted 9/30/2011. Author Aguirre iswith Inst. Tecnologico de Roque, Div. de Estudios de Posgrado e Investigacion,Km 8 Carretera Celaya-Juventino Rosas, C.P. 38110, Celaya, Gto. Mexico. AuthorsTorres and Blanco-Labra are with Centro de Investigacion y de Estudios Avanzadosdel Inst. Politecnico Nacional, Unidad Irapuato, Dept. de Biotecnologıa y Bioquımica.Km 9.6 Libramiento Norte Carretera Irapuato-Leon, C.P. 36821. Irapuato, Gto.Mexico. Author Mendoza-Hernandez is with Dept. de Bioquımica, Facultad deMedicina, Univ. Nacional Autonoma de Mexico, Apartado Postal 70–159, MexicoD.F. 04510, Mexico. Author Garcia-Gasca is with Facultad de Ciencias Nat-urales. Univ. Autonoma de Queretaro. Av. de las Ciencias s/n. Juriquilla, C.P.76230. Queretaro, Qro. Mexico. Direct inquiries to author Blanco-Labra (E-mail:alejandroblancolabra@gmail.com).

(Salvia hispanica) and no mechanization has yet been developedfor their harvesting. This plant has played an important role asa source of food as well as in traditional medicine in Africa,Asia, and America where, according to the Mendocino codex(Kingsborough 1964), chan was cultivated and highly appreciatedby pre-Hispanic cultures. Their high resistance to insect and fungalattack was possibly known in pre-Hispanic times since chan plantswere co-cultivated with maize, probably to provide protectionagainst insect pests. We have previously characterized a proteinprotease inhibitor from these seeds (Aguirre and others 2004) thatis probably involved in the defense mechanism of this plant againstinsects (Aguirre and others 2009).

Weber and others (1991) reported the proximal analyses of chanseeds, indicating a high protein content of 22% expressed as totalnitrogen on a dried weight basis. Montufar-Lopez (2007) reportedthat no toxic effects have been found. Therefore, to learn moreabout the proteins, amino acid and mineral composition, the goalof this study was to characterize the protein fractions present inchan seeds and to analyze their amino acid and mineral composi-tion.

Materials and MethodsChan seeds (Hyptis suaveolens L.) were kindly provided by Dr.

M. Vergara from the Univ. of Colima (Mexico). Bovine pan-creas trypsin (type I; EC 3.4.21.4) and Nα-benzoyl-DL-argininep-nitroanilide (BApNA) were from Sigma-Aldrich (St. Louis,Mo., U.S.A.). The western blot kit was from Invitrogen LifeTechnologies (Carlsbad, Calif., U.S.A.). The reagents used forelectrophoresis were from Bio-Rad (Hercules, Calif., U.S.A.). All

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Protein and minerals in chan seeds . . .

chemicals used were analytical grade, and deionized water was usedthroughout the study. Amino acid standards were purchased fromPierce (Rockford, Ill., U.S.A.).

Protein extraction and fractionation procedureSeeds were milled into a fine powder and stored at 4 ◦C until use.

The seed flour was either directly extracted or first defatted with2 different solvents, hexane or a mixture of chloroform/methanol(2 : 1, v/v). In both cases, the flour/solvent slurry (1 : 10, w/v)was stirred for 2 h. After solvent defatting, the sample was driedat room temperature and stored at 4 ◦C until use.

Fractionation of proteins was carried out according to the mod-ified Osborne method (1924). The suspensions of flour/water(1 : 10, w/v) were stirred for 2 h at room temperature andcentrifuged at 39200 × g for 1 h at 4 ◦C. The supernatant,referred to as the albumin fraction (A), was stored at −20 ◦Cuntil use. The pellet was re-suspended in 10 mL of a 50 mMTris-HCl, pH 8 buffer solution, containing 0.1 M NaCl and stirredas previously stated. The resulting supernatant was designated asthe 0.1 globulin fraction (G 0.1). The pellet was extracted with10 mL of a 50 mM Tris-HCl, pH 8 buffer solution containing0.3 M NaCl. After centrifugation, the supernatant was separatedand it was referred as the 0.3 globulin fraction (G 0.3), and thepellet was re-suspended in 10 mL of 70% aqueous ethanol and ex-tracted under constant stirring. The resulting supernatant was theprolamin fraction (P), and the pellet was re-suspended in 10 mLof a 0.1 M NaOH solution. After centrifugation, the supernatantwas now the glutelin fraction (Gl), and the pellet was the residue.As in the case of the soluble proteins reported by Kamizake andothers (2003), the protein content was determined by the Bradfordmethod (Bradford 1976). The total protein content was measuredby the Kjeldahl method, as modified by Axman and others (1990)using 6.25 as the nitrogen-to-protein conversion factor.

ElectrophoresisSodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) was carried out according to the method ofSchagger and Von Jagow (1987) with 10% polyacrylamide gels,with or without 2-mercaptoethanol. A total of 20 μg of eachprotein sample were loaded per lane and broad range molecularweight markers were used.

Trypsin inhibitory activityTrypsin inhibitory activity was assayed by monitoring the hy-

drolysis of BApNA at 405 nm in 0.1 M Tris-HCl, pH 8, after15 min incubation at 37 ◦C (Erlanger and others 1961). Oneunit of proteolytic activity was defined as the increase in 0.01absorbance units under the assay conditions described. Inhibitoractivity was defined as the difference between the proteolytic activ-ities measured in the absence and in the presence of the inhibitor.Inhibition Units (IU) were calculated as follows:

IU = Enzyme abs − (Enzyme ± Inhibitor) abs0.01 × Sample (mL)

Trypsin inhibitor Western blotWestern-blot analysis was performed after SDS-PAGE accord-

ing to Schagger and Von Jagow (1987) with 10% polyacrylamidegels. Total of 20 μg of each fraction were loaded per lane forHyptis suaveolens trypsin inhibitor (HSTI), with the exception of

the pure HSTI where 5 μg were loaded. Protein electrotransfer-ence was performed at 250 mA per hour, using a PVDF mem-brane with a 25 mM Tris buffer, with 192 mM Glycine 0.1% SDSand 20% methanol. The detection was performed according tothe directions of the Western Breeze R© Chromogenic Western-Blot Immunodetection Kit from Invitrogen Life Technologies(Carlsbad, Calif., U.S.A.).

Mineral compositionChan seeds flour was examined using a scanning electron mi-

croscope (JEOL JMS-6480LV) equipped with an energy disper-sive X-ray analyzer INCAx-sight Oxford Instruments (Abington,Oxfordshire, U.K.). The presence of phosphorous, potassium, cal-cium and magnesium was detected.

Amino acid analysisAmino acid content was determined in triplicate using an RP-

HPLC with pre-column derivatized with phenylisothiocyanate,according to published procedures (Bidlingmeyer and others 1984;Hendrikson and Meridith 1984). In brief, the dried protein sam-ples were hydrolyzed, in triplicate (1 mg each), in constantlyboiling 6 N HCl and melted crystalline phenol was added foraromatic amino acid protection. Hydrolysis was performed undervacuum in a heating block for 24 h at 110 ◦C. After cooling atroom temperature, the samples and a mixture of amino acid stan-dards were derivatized by adding 20 μL of a solution containingethanol/water/triethylamine/phenylisothiocyanate (7 : 1 : 1 : 1,v/v) and incubated at room temperature for 20 min. The sampleswere dried in a vacuum centrifuge, dissolved in 0.2 mL of 50 mMsodium phosphate buffer pH 7.4, filtered through a 0.22 μm filter,and then the sample was subjected to reverse-phase chromatog-raphy. The phenylthiocarbamyl derivatives were detected by theirabsorbance at 254 nm. After separation, the peaks were integratedand quantified using a standard curve of peak areas previouslyobtained from known concentrations of the amino acid standardmixtures.

Results and Discussion

Protein extractionChan seed total protein content was 13.9% (on dry weight ba-

sis), as determined by Kjeldahl analysis. Similar results (14.22%)were reported by Edeoga and others (2006). When the proteinwas extracted, either from the whole flour or from the hexane-defatted flour with the corresponding solvents of the modifiedOsborne procedure, a low total protein extraction of 4.3% and4.4% (w/w flour), respectively, was obtained by the Bradford as-say. However, when a mixture of chloroform/methanol was usedto defat the sample, previous to the protein extraction, the ex-tracted protein yield increased to 7.1%. The difference in proteinquantification between both methods could be explained becauseKjeldahl method determines protein by quantifying total nitrogen,whereas in the extracted protein other N-contained compoundsare excluded.

In the chloroform/methanol defatted flour, globulins repre-sented 39% of the total protein, the glutelins were the secondlargest fraction with 36%, the albumins with 24%, and the pro-lamins were the lowest protein fraction, with only 1%. Proteinfractions were similar to those reported for amaranth, anotherpseudo cereal (Leyva-Lopez and others 1995), and they were verydifferent from those of maize, rice, and wheat (Fukushima 1991),whose albumins were lower that chan and whose prolamines were

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very high compared to chan. Chloroform/methanol defatted flourincreased total extractable protein by 65% in relation to the non-defatted flour. This difference could be due to the eliminationof lipids that interfere with the protein extraction (Boatright andHettiarachchy 1995), plus the effect that has been reported forthe polyphenols that could be present in the sample, and theyalso interfere with the protein extraction (Parpinello and others2004). This method differentially favors the protein extractionof the globulin fractions, whereas the albumin, prolamin, andglutelin were not affected. Subsequent analyses were all done afterchloroform/methanol defatting procedure.

Electrophoretic patternsAll protein fractions were subjected to electrophoresis. In

Figure 1, the protein electrophoretic pattern for the chloro-form/methanol defatted sample shows a better resolution thanthose from the hexane-defatted flour or for the non-defatted flour(data not shown). Under non-reducing conditions (Figure 1A), al-bumin (lane 1) showed bands at 34.5, 22.2, 13, and 9 kDa. In theglobulin fractions the method used showed no difference for the0.1 and 0.3 NaCl buffer solution extracts (Figure 1A, lane 2 and3). When the electrophoresis was run under reducing conditionsusing 2-mercaptoethanol, the only differences observed were thatthe intensity of the 50 kDa and 42.2 bands decreased (Figure 1B,lanes 2 and 3), whereas 34.3 kDa and 23.4 kDa bands increased.The glutelin fraction showed 2 poorly defined bands at 50 and34.5 kDa.

No similarity was found with the protein electrophoretic pat-terns in the albumin fraction of the following legumes: Phaseolusvulgaris, Ciser arietinum, Lens esculenta, Pisum. sativum, and Lupinusalbus. All these legumes show a higher content of the high molecu-lar weight proteins; whereas Chan seeds as well as several reportedcereals, (Zea mays, Oryza sativa, Triticum aestivum) have almost nohigh molecular weight proteins in that fraction (Hamza and others1988). On the other hand, all those cereals showed more bands inthe glutelin fraction than chan.

Trypsin inhibitorTrypsin inhibitor is a protein with anti-nutritional properties

that has also been related to insect resistance in seed crops. In our

laboratory we have reported its presence in chan seeds as well asits lack of interaction with mammalian chymotrypsin, but it doesinhibited trypsin (Aguirre and others 2004). This inhibitor waspresent in these seeds in low concentration. In the albumin frac-tion (Figure 1C) only the monomer form was present, whereasin the globulin fractions (Figure 1B and 1C), the inhibitor waspresent in the dimer form. These results indicate that the presenceof sodium chloride in the extraction solution, which increasesthe ionic strength, apparently determines the aggregation of thisprotein. When the inhibitory activity was measured in the threedifferent flour preparations, the sample that was defatted with chlo-roform/methanol had the highest level of inhibitory activity (over2-fold) when compared to the non-defatted flour or the hexane-defatted flour (Figure 2). In Figure 1C, the Western Blot analysisfor the HSTI shows different forms of the HSTI depending onthe way the extraction was done; the albumin fraction (Figure 1C,lane 1) favors the extraction of the monomer form, whereas theglobulin fraction (Figure 1C, lanes 2 and 3) favors the extractionof the dimer form. Prolamin and glutelin fractions (Figure 1C,lane 4 and 5, respectively) contained no HSTI.

Figure 2–Trypsin inhibitory activities of all of the protein fractions ex-tracted from Chan flour. (1) Chloroform/methanol-defatted flour, (2) non-defatted flour, and (3) Hexane-defatted flour.

(C) (A)200.0

97.4 66.2

45.0

31.0

21.5

14.4

6.5

50.0 42.4

34.5

9.0

110.0

13.0

kDa

22.2

1 2 3 4 5 6 (B)

34.3

23.4

16.0

kDa

200.0

97.4

66.2

45.0

31.0

21.5

14.4

6.5

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

Figure 1–Electrophoretic patterns of protein fractions from the chloroform/methanol-defatted chan seed flour: (A) non-reduced conditions; (B) reducedconditions; (lane 1) albumins, (lane 2) 0.1 globulins, (lane 3) 0.3 globulins, (lane 4) prolamins, (lane 5) glutelins, and (lane 6) molecular weight markers.(C) HSTI Western blot. (Lane 1) albumin fraction, (lane 2) 0.1 globulin fraction, (lane 3) 0.3 globulin fraction, (lane 4) prolamin fraction, (lane 5) glutelinfraction, and (lane 6) purified chan trypsin inhibitor (HSTI).

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This inhibitor was present in low concentrations, in contrastto other grains such as wheat and soybean, where these types ofinhibitors are more abundant (Barber and others 1986; Kakade andothers 1972; Shewry and others 1984; Wong and others 2004).

Amino acid compositionTable 1 shows chan seeds amino acid composition as per-

cents of the different protein fractions. The percentage of indis-pensable amino acids with respect to adult requirement patterns(FAO/WHO/UNU 2007) is shown in Figure 3. Our results showthat chan seeds are a good source of aromatic amino acids mainlyPhe and Tyr when compared to soybean, maize, rice, and wheat.Also branched chain amino acids contents are high, whereas lysineis low. Finally, the indispensable amino acid percentage contribu-tion of chan flour with respect to the requirement patterns basedon dietary reference intake (DRI) for all age groups is shown inTable 2, indicating also that chan represents a good contribution ofessential amino acids to the diet with lower contribution of sulfuramino acids and lysine.

Figure 3–Comparison of indispensable amino acids from chan seeds withsoybean, rice, maize, and wheat when feeding 100 g of flour of the differ-ent foods. Data are expressed as percentage of the requirement pattern.Lys, Lysine, AAA, Aromatic amino acids, SAA, Sulfur amino acids, BCAA,Branched chain amino acids, Thr, Threonine.

Table 1–Amino acid composition (%) of the protein fractions from chan seeds.

Amino acid Albumin Globulin (0.1) Globulin (0.3) Prolamin Glutelin

AsXa 7.76 8.69 8.31 7.73 10.92GlXb 23.8 19.35 21.28 18.87 18.87Ser 8.5 7.96 8.12 8.15 8.33Gly 11.5 11.98 11.83 11.81 12.91Hisc 1.35 1.8 1.54 2.02 1.8Arg 5.71 6.11 5.25 6.65 5.01Thrc 3.52 3.62 3.36 3.51 3.5Ala 6.41 7.97 6.88 6.97 7.34Pro 4.93 5.44 5.54 5.56 5.53Valc 3.97 4.93 4.71 4.52 4.66Met+Cysc 3.46 2.54 2.67 2.38 2.18Ilec 3.13 3.18 3.05 3.33 3.28Leuc 6.42 7.32 7.31 7.94 6.64Phe+Tyrc 4.5 6.08 6.06 5.37 6.0Lysc 5.50 3.03 4.09 5.18 3.02Trpc nd nd Nd nd ndaAsp + Asn.bGlu + Gln.cEssential amino acids. nd = not determined.

Table 2–Amino acid content of whole chan flour and percentage contribution of essential amino acids with respect to the require-ment patterns for different age groups.

Children (years) AdultsInfantsAmino acid content of chan seeds (0.5 to 1 y) 1 to 2 3 to 10 11 to 14 15 to 18 >18

Amino acid mg/100 g flour mg/g protein RPd %RPe RP %RP RP %RP RP %RP RP %RP RP %RP

AsXa 155.58 11.19GlXb 772.88 55.60Ser 794.22 57.14Gly 1115.27 80.24Arg 396.69 28.54Ala 939.04 67.56Pro 2385.03 171.59Hisc 801.94 57.69 20 288 18 320 16 361 16 361 16 361 15 385Thrc 649.37 46.72 31 151 27 173 25 187 25 187 24 195 23 203Valc 1068.69 76.88 43 179 42 183 40 192 40 192 40 192 39 197Met + Cysc 273.17 19.65 28 70 26 76 24 82 23 85 23 85 22 89Ilec 742.55 53.42 32 167 31 172 31 172 30 178 30 178 30 178Leuc 1565.30 112.61 66 171 63 179 61 185 60 188 60 188 59 191Phe + Tyrc 2131.22 153.32 52 295 46 333 41 374 41 374 40 383 38 403Lysc 109.05 7.85 57 14 52 15 48 16 48 16 47 17 45 17aAsp + Asn.bGlu + Gln.cEssential amino acids.dRP = Requirement patterns for the different age groups (milligram amino acid per gram protein).e%RP = Percent of requirement patterns.

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Table 3–Mineral composition of 100 g of chan flour and percentage contribution with respect to RDAs/AIs.

Percentage respect to RDAsa or AIsb

Children Adults

Mineral mg/100 g Infants 14 to 18 19 to 30 31 to 50 Pregnancy/composition flour (7 to 12 mo) 1 to 3 4 to 8 9 to 13 (F/M) (F/M) (F/M) >50 lactation

P 280 102b 61a 56a 22a 22a 40a 40a 40a 22a

Mg 250 333b 313a 192a 104a 69/61a 81/62a 78/60a 60a 63a

Ca 200 74b 40b 25b 15b 15b 20b 20b 17b 15b

K 180 26b 6b 5b 4b 4b 4b 4b 4b 4b

aRDAs (Recommended Dietary Allowances) (FAO/WHO/UNU. 2004) are set to meet the needs of 97% to 98% of individuals in a group.bThe AI (Adequate Intakes) is the mean intake for healthy breastfed infants. The AI for other life stage and gender groups is believed to cover needs of all individuals in the group,but it is not possible to specify with confidence the percentage of individuals covered by this intake.The values represent means of triplicates.

Mineral compositionIn Table 3 mineral analysis of chan flour indicates the presence of

P, Mg, K, and Ca as well as the contribution of each mineral withrespect to recommended dietary allowances (RDAs) or adequateintakes (AIs) based on DRI (FAO/WHO/UNU 2004) for all agegroups. This analysis shows that chan seeds are a good source ofMg, providing over 80% of the RDA for infants and 1 to 3 y old,whereas at least 60% is covered for all other ages. Phosphorouscontent was found in the average range when compared to barley,oat, rice, and wheat (Gebhardt and Thomas 2002).

ConclusionsAnalysis of chan seeds showed that they could be an attractive

source of protein, providing a good quantity of the essential aminoacids required for the different age groups, contain high amount ofaromatic and branched amino acids. Even for infants and childrenwhose amino acids requirements are higher, these grains could stillbe useful since most of the essential amino acids are provided. Nu-tritional studies are still required to validate chan seeds; however,the amount of Mg, P, and Ca, important nutritional elements, aswell as their protein content and amino acid composition indicatesthat chan seeds have the potential to become a useful source forthe food Industry.

AcknowledgmentsWe would like to acknowledge Dr. J.J. Pena-Cabriales and

M.C.J.A. Vera-Nunez for their assistance in the nitrogen deter-mination, M.E. Mendiola-Olaya for her technical assistance, andDr. Martha Vergara for her kind donation of the chan seeds. GrantCONCYTEG 09–11-K662–095 and 09–11-K119–044.

ReferencesAguirre C, Valdes-Rodrıguez S, Mendoza-Hernandez G, Rojo-Domınguez A, Blanco-Labra

A. 2004. A novel 8.7 kDa protease inhibitor from Chan seeds (Hyptis suaveolens L.) inhibitsproteases from the larger grain borer Prostephanus truncatus (Coleoptera: Bostrichidae). CompBiochem Phys B 138:81–9.

Aguirre C, Castro-Guillen JL, Contreras L, Mendiola-Olaya E, Gonzalez de la Vara L, Blanco-Labra A. 2009. Partial characterization of a chymotrypsin-like protease in the larger grainborer (Prostephanus truncatus (Horn)) in relation to activity of Hyptis suaveolens (L.) trypsininhibitor. J Stored Prod Res 45:133–8.

Axman H, Sebastianelli A, Arrillaga JL. 1990. Sample preparation techniques of biologicalmaterial for isotope analysis. In Hardarson G, editor. Use of nuclear techniques in studies ofsoil-plant relationship. Belgium: Ghent. p 41–53.

Barber D, Sanchez-Monge R, Garcia-Olmedo F, Salcedo G, Mendez E. 1986. Evolutionaryimplications of the homologies among members of the trypsin/α-amylase inhibitor family(CM proteins) in wheat and barley. Biochim Biophys Acta 873:147–51.

Bidlingmeyer BA, Cohen SA, Tarvin TL. 1984. Rapid analysis of amino acids using pre-columnderivatization. J Chromatogr 336:93–104.

Boatright WL, Hettiarachchy NS. 1995. Effect of lipids on soy protein isolate solubility. J AmOil Chem Soc 72:1439–44.

Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantitiesof protein utilizing the principle of protein-dye-binding. Anal Biochem 72:248–52.

Edeoga HO, Omosun G, Uche LC 2006. Chemical composition of Hyptis suaveolens and Ocimumgratissimum hybrids from Nigeria. Afr J Biotech 5:892–5.

Erlanger B, Kokowsky N, Come W. 1961. The preparation and propierties of two new chro-mogenic substrates of trypsin. Arch Biochem Biophys 95:271–8.

FAO/WHO/UNU. 2004. Vitamin and mineral requirements in human nutrition. 2nd ed.Bangkok, Thailand: Report of a joint FAO/WHO expert consultation 21–30 September1998.

FAO/WHO/UNU. 2007. Protein and amino acids requirements in human nutrition. WHOTechnical Report Series 935. p 135–83, 247–8.

Fukushima D. 1991. Structure of plant storage proteins and their functions. Food Rev Int7:353–81.

Gebhardt SE, Thomas RG. 2002. Nutritive value of foods. Beltsville, Md.: U.S. Department ofAgriculture, Agricultural Research Service, Nutrient Data Laboratory. Home and Garden,Bulletin Nr 72, 95 p.

Gonzalez-Perez S, Vereijken JM, Van Koningsveld GA, Gruppen H, Voragen AGJ. 2005. For-mation and stability of foams made with sunflower (Helianthus annuus) proteins. J Agric FoodChem 53:6459–76.

Hamza MA, Stegemann H, EI-Tabey Shehata A. 1988. Protein in various legume seeds: elec-trophoretic comparison after optimized extraction. Food Chem 28:117–27.

Hendrikson RL, Meridith SC. 1984. Amino acid analysis by reverse-phase high performanceliquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal Biochem136:65–74.

Holm L, Pancho JV. Herberger JP, Plucknett DL. 1979. A geographical atlas of world weeds.New York: John Wiley & Sons. p 391.

Kakade ML, Simons N, Liener IE, Lambart JW. 1972. Biochemical and nutritional assessmentof different varieties of soybeans. J Agric Food Chem 20:87–90, 122.

Kamizake KKN, Goncalves MM, Zaia CTBV, Zaia DAM. 2003. Determination of total pro-teins in cow milk powder samples: a comparative study between the Kjeldahl method andspectrophotometric methods. J Food Compos Anal 16:507–16.

Kingsborough EK. 1964. In Jose Corona Nunez, editor. Codice Mendocino-Antiguedades deMexico. Mexico: Secretaria de Hacienda y Credito Publico. Vol. 1. p 1–149.

Leyva-Lopez NE, Vasco N, Barba de la Rosa AP, Paredes-Lopez O. 1995. Amaranth seedproteins: effect of defatting on extraction yield and on electrophoretic patherns. Plant FoodHum Nutr 47:49–53.

Martınez M. 1959. Plantas utiles de la flora mexicana. Mexico: Editorial Botas.Montufar-Lopez A. 2007. Las chıas sagradas del templo mayor de Tenochtitlan. Arqueologıa

Mexicana 84:82–5.Osborne TB. 1924. The vegetable proteins. In Monographs in biochemistry. 2nd ed.;

New York: Longmans, Green.Parpinello GP, Versari A, Riponi C 2004.Characterization of sugarbeet (Beta vulgaris, L.) protein.

J Sugar Beet Res 41:39–45.Schagger H, Von Jagow G. 1987. Tricine-sodium dodecyl suphate-polyacrilamide gel elec-

trophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem166:368–79.

Shewry PR, Lafiandra D, Salcedo G, Aragoncillo C, Garcia-Olmedo F, Lew EJL, DietlerMD, Kasarda DD. 1984. N-terminal amino acid sequences of chloroform/methanol-solubleproteins and albumins from endosperm of wheat, barley and related species. FEBS Lett175:359–63.

Vergara-Santana MI, Bravo-Magana F. 1992. Memorias. Mexico: 3era Reunion Nacional SelvaBaja Caducifolia, Universidad de Colima.

Weber CW, Gentry HS, Kohlhepp EA, McCrohan PR. 1991. The nutritional and chemicalevaluation of chia seeds. Ecol Food Nutr 26:119–25.

Wong JH, Cai N, Tanaka CK, Vensel WH, Hurkman WJ, Buchanan BB. 2004. Thioredoxinreduction alters the solubility of proteins of wheat starchy endosperm: an early event in cerealgermination. Plant Cell Physiol 45:407–15.

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