Analysis of Protein Fractions and Some Minerals

Present in Chan (Hyptis suaveolens L.) Seeds

Cesar Aguirre, Iovanna Torres, Guillermo Mendoza-Hernández, Teresa Garcia-Gasca, and Alejandro Blanco-Labra

C: Food Chemistry
Abstract: Chan (Hyptis suaveolens L.) seeds have been used as food as well as in traditional medicine in several countries of
America, Asia and Africa. Chan seed protein content was 13.9% on dry weight basis. Analysis of its protein composition
showed 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 those
of other grains including maize, with good levels of branched chain amino acids. In general, except for lysine, it has a
well-balanced amino acid composition, providing a good supply of almost all the essential amino acids for the different
age groups. Magnesium content was high, whereas calcium, potassium, and phosphorous were in the average range when
compared to barley, oat, rice, and wheat. The present results indicate that seeds from the chan plant could be relevant
because of their nutritional properties and they have the potential to be widely used in the production of high-quality
food.
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, considering
their high quality composition, they have the potential for a more extended use in the food industry.

Introduction (Salvia hispanica) and no mechanization has yet been developed

Animal proteins are expensive, particularly in terms of their for their harvesting. This plant has played an important role as
market price and environmental impact. On the other hand, as a source of food as well as in traditional medicine in Africa,
functional ingredients in food formulations, plant proteins repre- Asia, and America where, according to the Mendocino codex
sent a more economic and sustainable alternative (Gonzalez-Perez (Kingsborough 1964), chan was cultivated and highly appreciated
and others 2005). The fractionation of grain proteins, particu- by pre-Hispanic cultures. Their high resistance to insect and fungal
larly from those grains that have shown good food properties, attack was possibly known in pre-Hispanic times since chan plants
has long been a significant area of interest. In this study, we in- were co-cultivated with maize, probably to provide protection
vestigated the proteins present in the seeds of a plant known as against insect pests. We have previously characterized a protein
chan (Hyptis suaveolens) (Martinez 1959), also known as “chia protease inhibitor from these seeds (Aguirre and others 2004) that
gorda” and pignut (Holm and others 1979; Vergara-Santana and is probably involved in the defense mechanism of this plant against
Bravo-Magaña 1992). Chan belongs to a group of seeds gener- insects (Aguirre and others 2009).
ically known as chias. It is a widely distributed bush growing Weber and others (1991) reported the proximal analyses of chan
mainly in Mexico, Central America, South America, Asia and the seeds, indicating a high protein content of 22% expressed as total
Pacific Islands. Its height is between 1 to 2 m, its seeds are small; nitrogen on a dried weight basis. Montúfar-López (2007) reported
approximately 2 to 3 mm long, twice as big as amaranth and chia that no toxic effects have been found. Therefore, to learn more
about the proteins, amino acid and mineral composition, the goal
of this study was to characterize the protein fractions present in
MS 20110428 Submitted 4/4/2011, Accepted 9/30/2011. Author Aguirre is chan seeds and to analyze their amino acid and mineral composi-
with Inst. Tecnológico de Roque, Div. de Estudios de Posgrado e Investigación, tion.
Km 8 Carretera Celaya-Juventino Rosas, C.P. 38110, Celaya, Gto. México. Authors
Torres and Blanco-Labra are with Centro de Investigación y de Estudios Avanzados Materials and Methods
del Inst. Politécnico Nacional, Unidad Irapuato, Dept. de Biotecnologı́a y Bioquı́mica.
Km 9.6 Libramiento Norte Carretera Irapuato-León, C.P. 36821. Irapuato, Gto. Chan seeds (Hyptis suaveolens L.) were kindly provided by Dr.
México. Author Mendoza-Hernández is with Dept. de Bioquı́mica, Facultad de M. Vergara from the Univ. of Colima (Mexico). Bovine pan-
Medicina, Univ. Nacional Autónoma de México, Apartado Postal 70–159, México creas trypsin (type I; EC 3.4.21.4) and Nα-benzoyl-DL-arginine
D.F. 04510, México. Author Garcia-Gasca is with Facultad de Ciencias Nat- p-nitroanilide (BApNA) were from Sigma-Aldrich (St. Louis,
urales. Univ. Autónoma de Querétaro. Av. de las Ciencias s/n. Juriquilla, C.P.
76230. Querétaro, Qro. México. Direct inquiries to author Blanco-Labra (E-mail: Mo., U.S.A.). The western blot kit was from Invitrogen Life
alejandroblancolabra@gmail.com). Technologies (Carlsbad, Calif., U.S.A.). The reagents used for
electrophoresis were from Bio-Rad (Hercules, Calif., U.S.A.). All


C 2011 Institute of Food Technologists
R

doi: 10.1111/j.1750-3841.2011.02480.x Vol. 71, Nr. 1, 2012 r Journal of Food Science C15
Further reproduction without permission is prohibited
 Protein and minerals in chan seeds . . .

chemicals used were analytical grade, and deionized water was used the pure HSTI where 5 μg were loaded. Protein electrotransfer-
throughout the study. Amino acid standards were purchased from ence was performed at 250 mA per hour, using a PVDF mem-
Pierce (Rockford, Ill., U.S.A.). brane with a 25 mM Tris buffer, with 192 mM Glycine 0.1% SDS
and 20% methanol. The detection was performed according to
Protein extraction and fractionation procedure the directions of the Western BreezeR Chromogenic Western-
Seeds were milled into a fine powder and stored at 4 ◦ C until use. Blot Immunodetection Kit from Invitrogen Life Technologies
The seed flour was either directly extracted or first defatted with (Carlsbad, Calif., U.S.A.).
C: Food Chemistry

2 different solvents, hexane or a mixture of chloroform/methanol

(2 : 1, v/v). In both cases, the flour/solvent slurry (1 : 10, w/v) Mineral composition
was stirred for 2 h. After solvent defatting, the sample was dried Chan seeds flour was examined using a scanning electron mi-
at room temperature and stored at 4 ◦ C until use. croscope (JEOL JMS-6480LV) equipped with an energy disper-
Fractionation of proteins was carried out according to the mod- sive X-ray analyzer INCAx-sight Oxford Instruments (Abington,
ified Osborne method (1924). The suspensions of flour/water Oxfordshire, U.K.). The presence of phosphorous, potassium, cal-
(1 : 10, w/v) were stirred for 2 h at room temperature and cium and magnesium was detected.
centrifuged at 39200 × g for 1 h at 4 ◦ C. The supernatant,
referred to as the albumin fraction (A), was stored at −20 ◦ C Amino acid analysis
until use. The pellet was re-suspended in 10 mL of a 50 mM Amino acid content was determined in triplicate using an RP-
Tris-HCl, pH 8 buffer solution, containing 0.1 M NaCl and stirred HPLC with pre-column derivatized with phenylisothiocyanate,
as previously stated. The resulting supernatant was designated as according to published procedures (Bidlingmeyer and others 1984;
the 0.1 globulin fraction (G 0.1). The pellet was extracted with Hendrikson and Meridith 1984). In brief, the dried protein sam-
10 mL of a 50 mM Tris-HCl, pH 8 buffer solution containing ples were hydrolyzed, in triplicate (1 mg each), in constantly
0.3 M NaCl. After centrifugation, the supernatant was separated boiling 6 N HCl and melted crystalline phenol was added for
and it was referred as the 0.3 globulin fraction (G 0.3), and the aromatic amino acid protection. Hydrolysis was performed under
pellet was re-suspended in 10 mL of 70% aqueous ethanol and ex- vacuum in a heating block for 24 h at 110 ◦ C. After cooling at
tracted under constant stirring. The resulting supernatant was the room temperature, the samples and a mixture of amino acid stan-
prolamin fraction (P), and the pellet was re-suspended in 10 mL dards were derivatized by adding 20 μL of a solution containing
of a 0.1 M NaOH solution. After centrifugation, the supernatant ethanol/water/triethylamine/phenylisothiocyanate (7 : 1 : 1 : 1,
was now the glutelin fraction (Gl), and the pellet was the residue. v/v) and incubated at room temperature for 20 min. The samples
As in the case of the soluble proteins reported by Kamizake and were dried in a vacuum centrifuge, dissolved in 0.2 mL of 50 mM
others (2003), the protein content was determined by the Bradford sodium phosphate buffer pH 7.4, filtered through a 0.22 μm filter,
method (Bradford 1976). The total protein content was measured and then the sample was subjected to reverse-phase chromatog-
by the Kjeldahl method, as modified by Axman and others (1990) raphy. The phenylthiocarbamyl derivatives were detected by their
using 6.25 as the nitrogen-to-protein conversion factor. absorbance at 254 nm. After separation, the peaks were integrated
and quantified using a standard curve of peak areas previously
Electrophoresis obtained from known concentrations of the amino acid standard
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis mixtures.
(SDS-PAGE) was carried out according to the method of
Schagger and Von Jagow (1987) with 10% polyacrylamide gels, Results and Discussion
with or without 2-mercaptoethanol. A total of 20 μg of each
protein sample were loaded per lane and broad range molecular Protein extraction
weight markers were used. Chan seed total protein content was 13.9% (on dry weight ba-
sis), as determined by Kjeldahl analysis. Similar results (14.22%)
Trypsin inhibitory activity were reported by Edeoga and others (2006). When the protein
Trypsin inhibitory activity was assayed by monitoring the hy- was extracted, either from the whole flour or from the hexane-
drolysis of BApNA at 405 nm in 0.1 M Tris-HCl, pH 8, after defatted flour with the corresponding solvents of the modified
15 min incubation at 37 ◦ C (Erlanger and others 1961). One Osborne procedure, a low total protein extraction of 4.3% and
unit of proteolytic activity was defined as the increase in 0.01 4.4% (w/w flour), respectively, was obtained by the Bradford as-
absorbance units under the assay conditions described. Inhibitor say. However, when a mixture of chloroform/methanol was used
activity was defined as the difference between the proteolytic activ- to defat the sample, previous to the protein extraction, the ex-
ities measured in the absence and in the presence of the inhibitor. tracted protein yield increased to 7.1%. The difference in protein
Inhibition Units (IU) were calculated as follows: quantification between both methods could be explained because
Kjeldahl method determines protein by quantifying total nitrogen,
Enzyme abs − (Enzyme ± Inhibitor) abs whereas in the extracted protein other N-contained compounds
IU = are excluded.
0.01 × Sample (mL)
In the chloroform/methanol defatted flour, globulins repre-
sented 39% of the total protein, the glutelins were the second
largest fraction with 36%, the albumins with 24%, and the pro-
Trypsin inhibitor Western blot lamins were the lowest protein fraction, with only 1%. Protein
Western-blot analysis was performed after SDS-PAGE accord- fractions were similar to those reported for amaranth, another
ing to Schagger and Von Jagow (1987) with 10% polyacrylamide pseudo cereal (Leyva-Lopez and others 1995), and they were very
gels. Total of 20 μg of each fraction were loaded per lane for different from those of maize, rice, and wheat (Fukushima 1991),
Hyptis suaveolens trypsin inhibitor (HSTI), with the exception of whose albumins were lower that chan and whose prolamines were

C16 Journal of Food Science r Vol. 71, Nr. 1, 2012

Protein and minerals in chan seeds . . .

very high compared to chan. Chloroform/methanol defatted flour
increased total extractable protein by 65% in relation to the non-
defatted flour. This difference could be due to the elimination
of lipids that interfere with the protein extraction (Boatright and
Hettiarachchy 1995), plus the effect that has been reported for
the polyphenols that could be present in the sample, and they
also interfere with the protein extraction (Parpinello and others
laboratory we have reported its presence in chan seeds as well as
its lack of interaction with mammalian chymotrypsin, but it does
inhibited trypsin (Aguirre and others 2004). This inhibitor was
present in these seeds in low concentration. In the albumin frac-
tion (Figure 1C) only the monomer form was present, whereas
in the globulin fractions (Figure 1B and 1C), the inhibitor was
present in the dimer form. These results indicate that the presence

C: Food Chemistry
2004). This method differentially favors the protein extraction of sodium chloride in the extraction solution, which increases
of the globulin fractions, whereas the albumin, prolamin, and the ionic strength, apparently determines the aggregation of this
glutelin were not affected. Subsequent analyses were all done after protein. When the inhibitory activity was measured in the three
chloroform/methanol defatting procedure. different flour preparations, the sample that was defatted with chlo-
roform/methanol had the highest level of inhibitory activity (over
2-fold) when compared to the non-defatted flour or the hexane-
Electrophoretic patterns
defatted flour (Figure 2). In Figure 1C, the Western Blot analysis
All protein fractions were subjected to electrophoresis. In
for the HSTI shows different forms of the HSTI depending on
Figure 1, the protein electrophoretic pattern for the chloro-
the way the extraction was done; the albumin fraction (Figure 1C,
form/methanol defatted sample shows a better resolution than
lane 1) favors the extraction of the monomer form, whereas the
those from the hexane-defatted flour or for the non-defatted flour
globulin fraction (Figure 1C, lanes 2 and 3) favors the extraction
(data not shown). Under non-reducing conditions (Figure 1A), al-
of the dimer form. Prolamin and glutelin fractions (Figure 1C,
bumin (lane 1) showed bands at 34.5, 22.2, 13, and 9 kDa. In the
lane 4 and 5, respectively) contained no HSTI.
globulin fractions the method used showed no difference for the
0.1 and 0.3 NaCl buffer solution extracts (Figure 1A, lane 2 and
3). When the electrophoresis was run under reducing conditions
using 2-mercaptoethanol, the only differences observed were that
the 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 and
34.5 kDa.
No similarity was found with the protein electrophoretic pat-
terns in the albumin fraction of the following legumes: Phaseolus
vulgaris, Ciser arietinum, Lens esculenta, Pisum. sativum, and Lupinus
albus. All these legumes show a higher content of the high molecu-
lar weight proteins; whereas Chan seeds as well as several reported
cereals, (Zea mays, Oryza sativa, Triticum aestivum) have almost no
high molecular weight proteins in that fraction (Hamza and others
1988). On the other hand, all those cereals showed more bands in
the glutelin fraction than chan.

Trypsin inhibitor Figure 2–Trypsin inhibitory activities of all of the protein fractions ex-
Trypsin inhibitor is a protein with anti-nutritional properties tracted from Chan flour. (1) Chloroform/methanol-defatted flour, (2) non-
that has also been related to insect resistance in seed crops. In our defatted flour, and (3) Hexane-defatted flour.

(A) 1 2 3 4 5 6 kDa (B) 1 2 3 4 5 6 kDa (C) 1 2 3 4 5 6

200.0 200.0
110.0
97.4 97.4
66.2 66.2
50.0
42.4 45.0 45.0
34.5 34.3
31.0 31.0

22.2 21.5 23.4

21.5
14.4 16.0 14.4
13.0
9.0
6.5 6.5

Figure 1–Electrophoretic patterns of protein fractions from the chloroform/methanol-defatted chan seed flour: (A) non-reduced conditions; (B) reduced
conditions; (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) glutelin
fraction, and (lane 6) purified chan trypsin inhibitor (HSTI).

Vol. 71, Nr. 1, 2012 r Journal of Food Science C17

 Protein and minerals in chan seeds . . .

This inhibitor was present in low concentrations, in contrast

to other grains such as wheat and soybean, where these types of
inhibitors are more abundant (Barber and others 1986; Kakade and
others 1972; Shewry and others 1984; Wong and others 2004).
Amino acid composition
Table 1 shows chan seeds amino acid composition as per-
C: Food Chemistry

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 show
that chan seeds are a good source of aromatic amino acids mainly
Phe and Tyr when compared to soybean, maize, rice, and wheat.
Also branched chain amino acids contents are high, whereas lysine
is low. Finally, the indispensable amino acid percentage contribu-
tion of chan flour with respect to the requirement patterns based Figure 3–Comparison of indispensable amino acids from chan seeds with
on dietary reference intake (DRI) for all age groups is shown in soybean, rice, maize, and wheat when feeding 100 g of flour of the differ-
Table 2, indicating also that chan represents a good contribution of ent foods. Data are expressed as percentage of the requirement pattern.
essential amino acids to the diet with lower contribution of sulfur Lys, Lysine, AAA, Aromatic amino acids, SAA, Sulfur amino acids, BCAA,
Branched chain amino acids, Thr, Threonine.
amino acids and lysine.

Table 1–Amino acid composition (%) of the protein fractions from chan seeds.
Amino acid Albumin Globulin (0.1) Globulin (0.3) Prolamin Glutelin
a
AsX 7.76 8.69 8.31 7.73 10.92
GlXb 23.8 19.35 21.28 18.87 18.87
Ser 8.5 7.96 8.12 8.15 8.33
Gly 11.5 11.98 11.83 11.81 12.91
Hisc 1.35 1.8 1.54 2.02 1.8
Arg 5.71 6.11 5.25 6.65 5.01
Thrc 3.52 3.62 3.36 3.51 3.5
Ala 6.41 7.97 6.88 6.97 7.34
Pro 4.93 5.44 5.54 5.56 5.53
Valc 3.97 4.93 4.71 4.52 4.66
Met+Cysc 3.46 2.54 2.67 2.38 2.18
Ilec 3.13 3.18 3.05 3.33 3.28
Leuc 6.42 7.32 7.31 7.94 6.64
Phe+Tyrc 4.5 6.08 6.06 5.37 6.0
Lysc 5.50 3.03 4.09 5.18 3.02
Trpc nd nd Nd nd nd
a
Asp + Asn.
b
Glu + Gln.
Essential amino acids. nd = not determined.
c

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

C18 Journal of Food Science r Vol. 71, Nr. 1, 2012

Protein and minerals in chan seeds . . .

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

C: Food Chemistry
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
a
RDAs (Recommended Dietary Allowances) (FAO/WHO/UNU. 2004) are set to meet the needs of 97% to 98% of individuals in a group.
b
The 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 composition
In Table 3 mineral analysis of chan flour indicates the presence of
P, Mg, K, and Ca as well as the contribution of each mineral with
respect to recommended dietary allowances (RDAs) or adequate
intakes (AIs) based on DRI (FAO/WHO/UNU 2004) for all age
groups. This analysis shows that chan seeds are a good source of
Mg, providing over 80% of the RDA for infants and 1 to 3 y old,
whereas at least 60% is covered for all other ages. Phosphorous
content was found in the average range when compared to barley,
oat, rice, and wheat (Gebhardt and Thomas 2002).
Conclusions
Analysis of chan seeds showed that they could be an attractive
source of protein, providing a good quantity of the essential amino
acids required for the different age groups, contain high amount of
aromatic and branched amino acids. Even for infants and children
whose amino acids requirements are higher, these grains could still
be 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, as
well as their protein content and amino acid composition indicates
that chan seeds have the potential to become a useful source for
the food Industry.
Acknowledgments
We would like to acknowledge Dr. J.J. Peña-Cabriales and
M.C.J.A. Vera-Núñez for their assistance in the nitrogen deter-
mination, M.E. Mendiola-Olaya for her technical assistance, and
Dr. Martha Vergara for her kind donation of the chan seeds. Grant
CONCYTEG 09–11-K662–095 and 09–11-K119–044.
References
Aguirre C, Valdés-Rodrı́guez S, Mendoza-Hernández G, Rojo-Domı́nguez A, Blanco-Labra
A. 2004. A novel 8.7 kDa protease inhibitor from Chan seeds (Hyptis suaveolens L.) inhibits
proteases from the larger grain borer Prostephanus truncatus (Coleoptera: Bostrichidae). Comp
Biochem Phys B 138:81–9.
Aguirre C, Castro-Guillén JL, Contreras L, Mendiola-Olaya E, González de la Vara L, Blanco-
Labra A. 2009. Partial characterization of a chymotrypsin-like protease in the larger grain
borer (Prostephanus truncatus (Horn)) in relation to activity of Hyptis suaveolens (L.) trypsin
inhibitor. J Stored Prod Res 45:133–8.
Axman H, Sebastianelli A, Arrillaga JL. 1990. Sample preparation techniques of biological
material for isotope analysis. In Hardarson G, editor. Use of nuclear techniques in studies of
soil-plant relationship. Belgium: Ghent. p 41–53.
Barber D, Sanchez-Monge R, Garcia-Olmedo F, Salcedo G, Mendez E. 1986. Evolutionary
implications 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-column
derivatization. J Chromatogr 336:93–104.
Boatright WL, Hettiarachchy NS. 1995. Effect of lipids on soy protein isolate solubility. J Am
Oil Chem Soc 72:1439–44.
Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities
of 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 Ocimum
gratissimum 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 September
1998.
FAO/WHO/UNU. 2007. Protein and amino acids requirements in human nutrition. WHO
Technical Report Series 935. p 135–83, 247–8.
Fukushima D. 1991. Structure of plant storage proteins and their functions. Food Rev Int
7:353–81.
Gebhardt SE, Thomas RG. 2002. Nutritive value of foods. Beltsville, Md.: U.S. Department of
Agriculture, Agricultural Research Service, Nutrient Data Laboratory. Home and Garden,
Bulletin Nr 72, 95 p.
González-Pérez 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 Food
Chem 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 performance
liquid chromatography: precolumn derivatization with phenylisothiocyanate. Anal Biochem
136:65–74.
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 assessment
of different varieties of soybeans. J Agric Food Chem 20:87–90, 122.
Kamizake KKN, Gonçalves MM, Zaia CTBV, Zaia DAM. 2003. Determination of total pro-
teins in cow milk powder samples: a comparative study between the Kjeldahl method and
spectrophotometric methods. J Food Compos Anal 16:507–16.
Kingsborough EK. 1964. In José Corona Nuñez, editor. Códice Mendocino-Antigüedades de
México. México: Secretaria de Hacienda y Crédito Público. Vol. 1. p 1–149.
Leyva-Lopez NE, Vasco N, Barba de la Rosa AP, Paredes-Lopez O. 1995. Amaranth seed
proteins: effect of defatting on extraction yield and on electrophoretic patherns. Plant Food
Hum Nutr 47:49–53.
Martı́nez M. 1959. Plantas útiles de la flora mexicana. México: Editorial Botas.
Montúfar-López A. 2007. Las chı́as sagradas del templo mayor de Tenochtitlán. Arqueologı́a
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 Biochem
166:368–79.
Shewry PR, Lafiandra D, Salcedo G, Aragoncillo C, Garcia-Olmedo F, Lew EJL, Dietler
MD, Kasarda DD. 1984. N-terminal amino acid sequences of chloroform/methanol-soluble
proteins and albumins from endosperm of wheat, barley and related species. FEBS Lett
175:359–63.
Vergara-Santana MI, Bravo-Magaña F. 1992. Memorias. México: 3era Reunión Nacional Selva
Baja Caducifolia, Universidad de Colima.
Weber CW, Gentry HS, Kohlhepp EA, McCrohan PR. 1991. The nutritional and chemical
evaluation of chia seeds. Ecol Food Nutr 26:119–25.
Wong JH, Cai N, Tanaka CK, Vensel WH, Hurkman WJ, Buchanan BB. 2004. Thioredoxin
reduction alters the solubility of proteins of wheat starchy endosperm: an early event in cereal
germination. Plant Cell Physiol 45:407–15.

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