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Open Access |
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| Fatty Acid Methyl Ester Profile from Lupinus in the Identification of
Sweet and Bitter Species from this Gender with Oil of Lupinus Uncinatus Schlecht Seeds |
| Sergio Loredo-Dávila1*, Vicente Espinosa-Hernández1, María Antonieta Goytia-Jiménez2, Luis Diaz-Ballote3, Ramón Marcos Soto-
Hernández1 and Pamela G. Marrone4 |
| 1Department of Soil Science, Montecillos Campus, Carretera México-Texcoco, Montecillo, State of Mexico, C.P. 56230, México |
| 2Training Programme for Young Researchers and Entrepreneurs, Universidad Autónoma Chapingo. Km. 38.5 Carretera Mexico–Texcoco, Chapingo, Mexico, C.P. 56230, Mexico |
| 3Department of Applied Physics, CINVESTAV-IPN, Mérida C.P. 97310, Mexico |
| 4Bio Innovations, Inc. 2121 Second St. Suite 107B Davis, CA 95618, USA |
| *Corresponding author: |
Sergio Loredo-Dávila
Department of Soil Science
Montecillos Campus
Carretera México-Texcoco
Mexico
Tel: +52 5531308514
E-mail: checoloredo@hotmail.com |
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| Received May 27, 2011; Accepted August 18, 2012; Published August 24, 2012 |
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| Citation: Loredo-Dávila S, Espinosa-Hernández V, Goytia-Jiménez MA, Diaz-
Ballote L, Soto-Hernández RM, et al (2012) Fatty Acid Methyl Ester Profile from
Lupinus in the Identification of Sweet and Bitter Species from this Gender with Oil
of Lupinus Uncinatus Schlecht Seeds. J Nutr Food Sci 2:158. doi:10.4172/2155-9600.1000158 |
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| Copyright: © 2012 Loredo-Dávila S, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited. |
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| Abstract |
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| This paper describes the implementation of a strategy to develop identification of Lupinus cultivars with fatty acid
methyl esters relation about alkaloids. The genus Lupinus is abundant in Mexico, Lupinus uncinatus is an endemic
plant of Mexico, and this has not been studied in terms of chemical composition and information about length, width
and weight from seeds. This work is focused on analyzing the content of fatty acids methyl esters from L. uncinatus seeds. Where it was determined percentage oil as well as the concentration of twenty two fatty acid methyl esters
and their corresponding equivalence TUFA (Total Saturated Fatty Acid), MUFA (Monounsaturated Fatty Acids) and
PUFA (Polyunsaturated Fatty Acid). As a result of the analysis of lupine seeds, it was observed that the behavior of
sweet spices versus bitter species has an ongoing relationship, with a higher content of PUFA (bitter) to higher MUFA
(sweet) and measures of seeds. |
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| Keywords |
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| Lupinus sweet; Lupinus bitter; Oil composition; Methyl
palmitate; Trans-9-elaidic methyl ester; Cis-9-oleic methyl ester; Trans-
9,12-octadecadienoic acid methyl ester; Cis-9,12-octadecadienoic |
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| Introduction |
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| Lupine (Lupinus L.) is a universal plant with numerous useful
properties. It may be used both as fodder and for soil fertilization.
As fodder, low-alkaloid lupine species such as yellow fodder lupine
(Lupinus luteus L.) and narrow-leaved forage lupine (Lupinus
angustifolius L.) are used. Of course, lupines produce alkaloids not in
order to supply them to man or animals. Various alkaloids function
in plants as insecticides, herbicides, fungicides or pest protectors [1].
Lupinus seeds are characterized by high protein content. Therefore,
attention is particularly focused on the quantity and quality of protein
(the amino acid profile) due to the potential use of these in the diet of
humans and animals. However, much less attention has been focused
on the content and quality of Lupinus oil. Unlike protein whose level
varies in a wide range (30-50%) depending on a particular variety,
the oil content is considerably lower (5-10%) [2]. On the other hand
Lupinus uncinatus Schlecht grown in Sierra Nevada at Mexico State
over the mountain Tláloc, between 2932-2994 masl (meters above sea
level) and slope of 2-23%. It is an annual, biannual, or perennial plant,
more than 1 m tall, herbaceous, of hollow stems, stipules 7 to 9 mm
long, petiole of 1.5-12.5 cm length, 5 to 8 leaflets 3 to 4 cm long and 6 to
8 mm wide, the fruits are dehiscent pods 4.5 to 5.5 cm long, containing
9 to 49 pods per branch and 1 to 7 mature seed per pod, flowering starts
since March and the first mature seed appear in the middle of May. It
is found in the soil with pH 6.3, 3.32% soil Organic Matter content,
0.13 % total Nitrogen, 14.7 mg kg-1soil Phosphorus and 1.46 cmolg-1
soil Potassium [3]. This study analyses the chemical composition and
variability between the oils from seeds of wild L. uncinatus collected in
Sierra Nevada at Mexico State over the mountain Tláloc. |
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| Materials and Methods |
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| Plant material |
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| Seeds of L. uncinatus Schlecht was collected in May, 2009. The study area is located on the oriental slope of the Sierra Nevada in Tláloc
mountain in Mexico State, Mexico, between 19° 23 “43” y 19º 28 “37”
North and between 98º 42 “51” and 98° 48 “12”. |
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| Image hardware and software |
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| A flatbed scanner (Xerox phaser 6110) and Phaser 6110 MFP
software was used for image acquisition, ADOBE PHOTOSHOP 9.0
software was used to ensure identical orientation of the specimen. The
digital image analysis was carried out by standalone version of IMAGN
J 1.40g, a program developed at the United States National Institute
of Health (freely available at http://rsb.info.nih.gov/ij/ or http://www.
scioncorp.com/pages/download_now.htm). A standard personal
computer was used for image analysis. |
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| Image acquisition and analysis |
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| The seed was placed on the scanner bed with the vertical side down
with about 1 cm space between the specimens to avoid seed to seed
contact. Grey image of seeds was acquired with the scanner software.
The scanner resolution was 300 dpi. Images were stored in tiff format
and exported to the PHOTOSHOP program for possible corrections
regarding the orientation. Subsequently the IMAGEN J program was
used for feature extraction. The test was for 25 seeds [4]. Seeds were weighed to determine the number of seeds per gram by counting out
replicates of 100 seeds and placing them on a balance until 1.0 g of seeds
are obtained. |
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| Analysis oils |
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| Material: HPLC grade hexane and methanol were purchased from
Baker (Deventer, Nertherlands). Potassium hydroxide in methanol was
freshly prepared, dissolving 11.2 g in 100 mL of methanol (2 N). The
following fatty acid methyl esters (FAMEs) were purchased from Sigma
Aldrich (St Louis, MO); Methyl butyrate (C4:0, 99.9% purity), methyl
hexanoate (C6:0, 99.7%), methyl octanoate (C8:0, 99.9%), methyl
decanoate (C10:0, 99.9%), methyl undecanoate (C11:0, 99.5%), methyl
laurate (C12:0, 99.8%), methyl tridecanoate (C13:0, 99.4%), methyl
myristate (C14:0, 99.7%), myristoleic acid methyl ester (C14:1, 99.9%),
methyl pentanodecanoate (C15:0, 99.6%), cis-10-pentadecenoic
acid methyl ester (C15:1, 99.0%), methyl palmitate (C16:0, 99.9%),
methyl palmitoleate (C16:1, 99.7%), methyl heptadecanoate (C17:0,
99.9%), cis-10-heptadecenoic (C17:1, 99.9%), methyl stearate (C18:0,
99.9%), trans-9-elaidic methyl ester (trans-C18:1, 96.9%), cis-9-oleic
methyl ester (cis-C18:1, 99.9%), trans-9,12-octadecadienoic acid
methyl ester (trans-C18:2,99.6%), cis-9,12-octadecadienoic (cis-C18:2,
99.6%), methyl arachidate (C20:0, 99.9%), gamma-linolenic acid
methyl ester (C18:3n6, 99.5%), cis-11-eicosenoic (C20:1, 99.9%),
methyl linoleate (C18:3n3, 99.9%), methyl heneicosanoate (C21:0,
99.5%), cis-11,14-eicosadienoic acid methyl ester (C20:2, 99.9%), cis-
8,11,14-eicosatrienoic acid methyl ester (C20:3n6, 99.1%), methyl
erucate (C22:1n9, 99.7%), cis-11,14,17-eicosatrienoic acid methyl
ester (C20:3n3, 99.2%), cis-5,8,11,14-eicosatetraenoic acid methyl
ester (C20:4n6, 99.3%), methyl behenate (C22:0, 99.8%), cis-5,8,11,14,
17-eicosapentaenoic acid methyl ester (C20:5, 99.9%), methyl
tricosanoate (C23:0, 99.9%), cis-13,16-docosadienoic acid methyl ester
(C22:2, 99.9%), methyl lignocerate (C24:0, 99.8%), methyl nervonate
(C24:1, 99.9%) and cis-4,7, 10, 13, 16, 19-docosahexaenoic acid methyl
ester (C22:6, 99.7%). |
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| Extraction of the crude oil |
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| L. uncinatus samples (24.0 g) were dried for 24 hours at 90°C in
a ventilated oven (Thomas Scientific) and then ground in a blender
(Oster) grinder. Oil was extracted from ground L. unicinatus samples
using the Soxhlet technique in an Avanti 2050 Soxtec from Foss
Tecator (Denmark). Samples were weighed (2.5 g) into Whatman #4
filter paper, which was then folded and placed into cellulose extraction
thimbles. Hexane (60 mL) was placed in aluminum extraction cups and
the cups were placed in the position. Hexane was boiled for 40 min with
the thimble in the hexane. Then the thimble was raised and the hexane
continued to boil for 60 min (called the rinse phase). After rinsing, the
remaining hexane was removed for 30 min. Then the cups with the
oil were drying for 20 min. After the extraction process, the oil was
transferred to amber vials covered with cap and placed in a freezer. |
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| Preparation of fatty acid methyl ester (FAMEs) |
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| FAMEs were prepared by transmethylation using CH3OK in CH3OH according to the international standard ISO 5509 [5]. The
mixture was purchased as a 100 mg neat mixture, containing C4 to
C24 FAMEs (2 to 4% relative concentrations). The whole sample was
diluted in 10 mL of hexane. Weigh 100 mg oil extracted from seeds of L.
uncinatus. The sample was dissolved in 10 mL of hexane and 100 μL of 2
N potassium hydroxide in methanol was added. It was put in the vortex,
and centrifuged. The clear supernatant solution is 10 mg/mL which was
analyzed by CG-FID. |
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| GC-FID analysis |
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| The FAMEs were analyzed with an Agilent 7890A GC equipped
with an FID. Automated split injection was performed using an Agilent
7683B Automatic sampler operated at 250°C. The instrumental and
analytical conditions are based in norm UNE-EN ISO 5508:1996. An
HP-INNOWax column (30 x 0320 mm id x 0.25 μm film thickness)
(19091N-113Agilent) was used. The carrier gas was Helium (constant
pressure approximately 230 kPa at 50°C, 33 cm/s at 50°C), the auxiliary gases were H2 40 mL.min-1, Air 450 mL.min-1 and Helium 30 mL.min-1.
Detector temperature was set at 280°C; the temperature program
was: 2 min at 50°C, from 50°C to 220°C at 30°C/min then 20 min at
220°C, from 220°C to 255°C at 5°C/min then 5 min at 255°C. Analyses
were processed with ChemStation Workstation software (Version
REV. B.04.01 SP1). The analysis was performed at least in triplicate.
Peaks were identified by comparison of retention times with standard
compounds. |
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| Results and Discussion |
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| Sizes and measures of seeds length, width and weight of each
specimen were measured (Table 1) and the average of each seed was
calculated with length 6.13 mm, width 4.74 mm and weight 0.044 g. The
length was plotted versus the width (Figure 1). |
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Table 1: Statistical parameters of Lupinus uncinatus Schlecht (expressed as
Standard Deviation value “SD”). |
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Figure 1: Dimensional seeds length (mm) as a function of the width (mm). |
|
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| The oil content for individual sample combination ranged from
6.97% to 9.64% (mean 7.76 ± 1.11 standard deviation). These values
appear to be in good agreement with literature data. The genus Lupinus
typically contains 5-20% oil [5], Roth-Maier and Kirchgessner [6]
reported that the oil levels in some varieties of white Lupin (Lupinus
albus) are 7.6%. Uzun et al. [7] obtained 10.75% in two varieties of
Lupinus albus, while Wilson et al. [8] reported Lupinus albus contained
9% oil, Boschin et al. [9] states that the seed of a variety of Lupinus albus
L., contains about 9-14% oil. Other studies [10] report about Lupinus
albus samples containing 14.7% oil. Lupinus angustifolius was reported
with 6.4% oil and Lupinus mutabilis with 16-20% oil [8,10] agrees that Lupinus mutabilis contains about 20% comparison with soybean oil
from dry seeds which can vary from 12% to 26%. |
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| The percent composition of the FAMEs in L. uncinatus is presented
in Table 2. On average, FAMEs ranked in the following order of
abundance: cis+trans |
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Table 2: Fatty acid composition (expressed as Standard Deviation value “SD”) of Lupinus uncinatus Schlecht. |
|
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| C18:2>C16:0>cis+transC18:1>C18:3n3>C18:0>C22:6>C24:0>C22
:0+C20:5>C20:0>C17:1>C22:1n9=C22:2>C16:1>C13:0=C14:1>C8:13
=C15:0>C15:1=C20:1>C23:0>C14:0=C17:0. |
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| Concerning TSFA (Total Saturated Fatty Acid), the percentage
is 24%; in respect to MUFA (Monounsaturated Fatty Acids) 17%
and PUFA (Polyunsaturated Fatty Acid) are 58% and 1% coelute
C22:0+C20:5 (Figure 2a, 2b). |
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Figure 2a: Score plot of MUFA, TSFA, PUFA. |
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Figure 2b: Loading plot of MUFA, TSFA, PUFA. |
|
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| Literature data for some species of lupines has been a different
distribution between TSFA, MUFA and PUFA (Table 3). |
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Table 3: Lupinus TSFA, MUFA and TUFA in percentage. |
|
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| On the other hand, data were analyzed for TSFA, MUFA and PUFA
L. unicinatus against those reported in the literature, separating the data
into two groups, bitter and sweet (Figure 3). In sweet group the MUFA
were the highest concentration than TSFA and PUFA. In the case of bitter
group the PUFA were the highest concentration than TSFA and PUFA.
Based on the results of L. uncinatus, it could be predicted that is a species
bitter waiting to checking alkaloid content by quantitative methods. |
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Figure 3: Comparison between bitter and sweet species of Lupinus for TSFA,
MUFA and PUFA. |
|
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| Conclusion |
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| It was concluded that the fatty methyl ester profile is a signal to
selection of the Lupinus bitter or sweet. In addition, it also will be
synergistic effect in the development of plant because the oils are a
primary metabolism, and then changes with the evolution of plant.
With the signal is possible, the comparison in plants of this gender
at different age. Therefore, further experimentation is required to
determine the role of alkaloids with fatty methyl ester profile and in
this way; the investigators can have a new rapid test of sweet or bitter
Lupinus cheaper than test of alkaloids. |
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| Acknowledgements |
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| This work was supported by CONACYT (CVU/Becario): 270356/223250 and
Laboratory Training Program for Young Scientists and Entrepreneurs, Universidad
Autonoma Chapingo. Km. 38.5 Carretera México–Texcoco, Chapingo and specially
guidance of Maria Antonieta Goytia Jiménez, whose help made this research
possible. |
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| References |
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