www.elsevier.com/locate/foodres

Food Research International 38 (2005) 159–165

Pre-treatment effects on the extraction efficiency of xanthophyllsfrom marigold flower (Tagetes erecta) using hexane

Jose Luis Navarrete-Bolanos *, Claudia Leticia Rangel-Cruz, Hugo Jimenez-Islas,Enrique Botello-Alvarez, Ramiro Rico-Martınez

Departamento de Ingenierıa Quımica-Bioquımica, Instituto Tecnologico de Celaya, Av. Tecnologico s/n, C.P. 38010 Celaya, Gto, Mexico

Received 1 June 2004; accepted 26 September 2004

Abstract

The marigold flower (Tagetes erecta) is one of the richest natural sources of xanthophylls, mainly lutein. Its saponified extract is

used as an additive in several food and pharmaceutical industries. However, the efficiency in the xanthophylls extraction from this

natural source depends heavily upon an appropriate prior treatment, given to the flower, to increase wall-cells permeability and

facilitate the diffusive mechanisms of mass exchange between immiscible phases during the lixiviation process. In this work, the effect

of different treatments on marigold flower to increase the xanthophylls extraction efficiency is studied. The results clearly indicate the

interrelation that exists between the treatment and the extraction. It is shown that almost full recovery of the xanthophylls contents

can be achieved when the marigold is treated with hydrolytic enzymes synthesized by microbiota associated to marigold flowers.

These results have significant impact on the cost-efficiency of the process.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Marigold treatments; Xanthophylls extraction; Efficiency

1. Introduction

Xanthophylls (oxygenated carotenoids) are compo-

nents with strong demand in international markets.

They are used as additives for poultry (e.g. chicken),crustaceous (e.g. shrimp) and fish (e.g. salmon) feeds

to provide bright colors in egg yolks, skin, and fatty tis-

sues due to its pigmenting properties (Bernhard, Broz,

Hengartner, Kreienbuhl, & Schiedt, 1997; Bletner, Mitc-

hell, & Tugwell, 1966; Hencken, 1992; Levi, 2001). They

have also been used as human nutritional supplement

(nutraceuticals) based on important biological function

that include cancer prevention (Chew, Wong, & Wong,1996), inhibition of the auto-oxidation of cellular lipids

0963-9969/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodres.2004.09.007

* Corresponding author. Tel.: +52 461 61 1 75 75; fax: +52 461 61 1

79 79.

E-mail address: jlnb@itc.mx (J.L. Navarrete-Bolanos).

(Zhang, Coney, & Bertram, 1991), protection against

oxidant-induced cell damage (Martin, Eailla, & Smith,

1996), and prevention of age-related macular degenera-

tion (Fullmer & Shao, 2001; Seddon et al., 1994). Im-

proved xanthophylls production processes are, as aresult, an important issue for many groups that have ex-

plored several alternatives including chemical synthesis

(Kreienbuhl, Rudin, & Rudolph, 2000), and fermenta-

tion technology (Gierhart, 1994; Hirschberg & Harker,

1999; Jacobson, Jolly, Sedmak, Skatrud, & Wasileski,

2000). Despite the success of these innovative efforts,

none of them reports xanthophylls production in quan-

tity and quality comparable to processes that extract thexanthophylls from their natural source. One of these

substrates is the marigold flowers (Tagetes erecta) con-

sidered as one of the richest natural sources of xantho-

phylls, mainly lutein (Ausich & Sanders, 1997; Levi,

2001), that are extracted by lixiviation with hexane fol-

160 J.L. Navarrete-Bolanos et al. / Food Research International 38 (2005) 159–165

lowed by concentration stage to obtain oleoresin. The

oleoresin is used as raw material for further purification

processes to obtain both a fine crystalline lutein and

xanthophylls mixture free from impurities that can be

suitable for human consumption, either in nutritional

supplements or as a food additive (Ausich & Sanders,1997; Khachik, 2001; Levi, 2001; Madhavi & Kagan,

2002). Other efforts have concentrated on the develop-

ment of methods for isomerization of lutein in the oleo-

resin to zeaxanthin, to improve pigmenting properties

(zeaxanthin has double pigmenting power compared

with lutein), which is desirable when the products are

used as additives on food and foodstuff industries

(Rodriguez, 1997). However, the quality and yield ofthe oleoresin obtained from marigold flower lixiviation

has not been discussed. In this direction, developments

based on supercritical fluid extraction (SFE) have dem-

onstrated the feasibility of obtaining enriched extracts

via selective oleoresin extraction from marigold flower

(Naranjo-Modad, Lopez-Munguıa, Vilarem, Gaset, &

Barzana, 2000). However, such SFE processes are, at

present, commercially impractical primarily due to highcapital costs (Kanel & Marentis, 2000). Other alterna-

tives, associated with the extraction from marigold flow-

ers, to increase xanthophylls concentration in flower

flour have also been reported (Barzana et al., 2002; Del-

gado-Vargas & Paredes-Lopez, 1997; Matoushek, 1974;

Navarrete-Bolanos, Jimenez-Islas, Botello-Alvarez, &

Rico-Martınez, 2003; Navarrete-Bolanos, Jimenez-Islas,

Botello-Alvarez, Rico-Martınez, & Paredes-Lopez,2004a; Navarrete-Bolanos, Jimenez-Islas, Botello-Alva-

rez, Rico-Martınez, & Paredes-Lopez, 2004b). Unfortu-

nately, known marigold extracts fail to meet both

quality and yield criteria exhibiting low-pigmenting

strengths and failing to recover the majority of the xan-

thophylls originally present in the marigold. In this con-

tribution, the effect of the different treatments on

xanthophylls concentrations in marigold flower floursis studied, seeking to characterize its relationship on

the extraction efficiency using hexane, and on the quan-

tity and quality of the oleoresin.

2. Materials and methods

2.1. Natural source

Marigold flowers (T. erecta) from a single batch were

used for all experiments. The batch contained flowers

with a yellow-orange coloration ratio of 10:90 and

30% average weight of receptacles. The marigold flowers

were divided in two sets. One set was conserved fresh

and separated in two subsets. One of these subsets was

processed via solid state fermentation. As for the secondfresh flower subset, the petals were separated from their

receptacles and processed via uncontrolled ensilage. For

the second set, the petals were separated from their

receptacles and processed via enzymatic treatments

using a raw enzymatic extract (non-commercial en-

zymes) or commercial cellulose. In order to verify the ef-

fect of the original marigold flower moisture on the

yield, the marigold was processed in two separatebatches: one using fresh petals and the other using dried

petals. The dried petals were obtained by dehydration

under environmental conditions up to a moisture con-

tent of 10% (±1%). Also, to avoid changes on both fresh

and dried marigold, all sets were stored at 4 �C and

sealed in black polyethylene bags until they were used

during experimental assays.

2.2. Traditional uncontrolled ensilage

In order to evaluate the traditional ensilage, the mar-

igold flowers, including theirs receptacles, were stored

for up to seven weeks in a closed yard under uncon-

trolled conditions. At the end, the product obtained

was dried and processed to obtain marigold flower flour.

2.3. Microorganisms culture

A mixed culture of microorganisms (Flavobacterium

IIb, Acinetobacter anitratus, and Rhizopus nigricans)

were propagated and mixed, in the proportions accord-

ing to Navarrete-Bolanos et al. (2003), to obtain the

starter inoculum used in the solid-state fermentation as-

says. Furthermore, the starter inoculum was filtrated toobtain a raw enzymatic extract that was used in the

enzymatic treatment with non-commercial enzymes.

2.4. Enzymatic treatments with non-commercial enzymes

In order to evaluate the effect of raw enzymatic ex-

tract on the marigold petals, the supernatants obtained

from mixed culture filtrated were blended with driedor fresh petals in 1:12 (w/v) ratio and kept on rotary sha-

ker at 28 �C and 175 rpm (Forma Scientific, model 4520)

for 24 h. The samples treated were filtered to separate

their solid and liquid phases. The solid phases, marigold

petals treated, were processed to obtain marigold flower

flour.

2.5. Enzymatic treatment with commercial cellulase

Commercial enzyme solution at 0.05 (±0.01% (w/v))

concentration and endo-1,4-b-DD-glucanase (EC 3.2.1.4

from Aspergillus niger, Sigma Chemical Co., St. Louis,

MO) activity was used for enzymatic treatment with

commercial cellulase according to Navarrete-Bolanos

et al. (2004a). In the same manner as for the enzymatic

treatments with raw enzymatic extract, the commercialenzyme solutions were blended with dried or fresh petals

in 1:12 (w/v) ratio and kept on rotary shaker at 28 �C,

0

5

10

15

20

25

30

35

0 5 10 15 20 25

Time (h)

Xt (

g/K

g flo

ur)

0 20 40 60 80 100 120

Time (h)

Fig. 1. Effect of enzymatic treatment on flower petals: xanthophylls

concentration in samples treated with raw enzymatic extract on dry

petals (j), and fresh petals (m), axis X bottom. Xanthophylls

concentration in samples treated with commercial enzyme solution

on dry petals (d), and fresh petals (r), axis X top.

J.L. Navarrete-Bolanos et al. / Food Research International 38 (2005) 159–165 161

175 rpm for 120 h, the samples treated were filtered and

the solid phases were processed to obtain marigold

flower flour.

2.6. Solid state fermentation

The mixed culture microorganisms was blended with

fresh marigold flower in 1.5:1 (w/v) ratio on a modular

rotating drum fermentation system under the following

conditions: 73.8% moisture content, intermittent agita-

tion every 14.8 h, and 4.1 l inlet air/min. These condi-

tions maximize the total xanthophylls extraction

(Navarrete-Bolanos et al., 2004b) from the fermented

marigold flower flour obtained.

2.7. Marigold flower flour

The products obtained by all proposed processes,

enzymatic treatment, solid state fermentation and ensi-

lage, were dehydrated in a vacuum oven (Shel Lab.

model 1430) to 10% (±1%) moisture content, and milled

(0.5-mm sieve) using a Brinkmann mill (Brinkmann,Westbury, NY). The flour obtained was analyzed by

AOAC (1984) method 970.64 to determine the total xan-

thophylls concentration and to obtain oleoresin via a

lixiviation process.

2.8. Xanthophylls extraction by hexane

The flours obtained from described treatments wereextracted in a batch process using analytical grade hex-

ane (J.T. Baker, Mallinckrodt Baker Inc., Phillipsburg,

NJ) in 1:6 (w/v) flour to hexane ratio and at constant

temperature of 35 �C during 15 min of extraction time

(Navarrete-Bolanos, Jimenez-Islas, Rico-Martınez,

Domınguez-Domınguez, & Regalado-Gonzalez, 2001).

When the extraction was concluded, the light phase

(hexane + xanthophylls) must be separated of the heavyphase (solid). The light phase was concentrated to ob-

tain the oleoresin extract that was weighed (digital ana-

lytical balance, Ohaus-explorer) to quantify the yield

extraction and determine the xanthophylls concentra-

tion according to AOAC (1984) method 970.64. The

heavy phase (solid) was desolventized, and the retained

solvent volume was determined by difference of weights

(Ohaus-explorer balance). Solvent-free solid was alsoanalyzed to determine the xanthophylls concentration

according to AOAC (1984) method 970.64.

2.9. Single-stage extraction

This may be a batch or a continuous operation

depending of the separation objective. A single extrac-

tion batch was used to characterize the mass exchangebetween phases of the process and to establish the con-

ditions at which the maximum extraction is achieved.

A continuous multistage extraction system, called cross-

current extraction, was performed by contacting the lea-

ched solids with fresh leaching solvent. For this system,

the solution to be withdrawn is in contact with the fres-

hest solid and the fresh solvent is added to the solid from

which most of the solute has already been leached. Thesystem can be operated with any number of stages. Its

purpose is to increase the mass transfer over and above

what is possible with a single stage and obtain higher

concentrations on the final product. Once again, when

the extraction was concluded, the phases were separated

and analyzed to quantify the yield extraction.

3. Results and discussion

3.1. Enzymatic treatments

Previously, the relationship between the effects of dif-

ferent treatments on marigold flowers on xanthophylls

extraction had been studied. For the enzymatic treat-

ment, the xanthophylls concentration in flours obtainedvia raw enzyme extract or commercial cellulose treat-

ments showed an increase clearly correlated to the enzy-

matic activity. However, the flours with highest contents

of xanthophylls (29.3 (±0.1) g of total xanthophylls/kg

of flour) are obtained using the raw enzymatic extract

synthesized from microorganisms associated with mari-

gold flower (Flavobacterium IIb, A. anitratus, and R. nig-

ricans). Clearly, this process has significant advantageswhen compared to the enzymatic treatment using com-

mercial enzymes: (1) the treatment with raw enzymatic

extract is more cost-efficient due in part to the straight-

forward hydrolytic enzyme synthesis, and (2) a substan-

tial reduction in processing time can be achieved while

attaining high xanthophylls content (Fig. 1).

162 J.L. Navarrete-Bolanos et al. / Food Research International 38 (2005) 159–165

3.2. Solid state fermentation and ensilage processes

The experimental assays on the rotating drum fer-

mentation system under optimum condition resulted

on marigold flower flour with higher content of xantho-

phylls (17.81 (±0.2) g of total xanthophylls/kg of flour)compared against the xanthophylls concentration in

flours obtained by the commercial ensilage process

(11.54 (±0.5) g of total xanthophylls/kg of flour). In

addition, the commercial ensilage required a long

processing time, nearly four weeks, to obtain this quality

flour (Fig. 2). The difference of the xanthophylls concen-

tration between these processes is associated to the oxy-

gen requirements of the microorganisms. It appears thatthe oxygen depletion, in the commercial ensilage proc-

ess, limits the synthesis of hydrolytic enzymes with cell-

ulase activity, whereas in the solid-state fermentation a

constant supply of oxygen was kept, allowing a steady

enzyme synthesis.

In general, the experiments described here demon-

strate that enzymatic treatment processes are a viable

alternative for improving the yield of xanthophylls ex-tracted from marigold flower. The yields obtained repre-

sent a 153.9% increase with respect to the average

amount obtained using the commercial ensilage process,

and a 64.5% higher with respect to the average amount

obtained using the solid-state fermentation process.

However, these differences can be partially attributed

to variations in both the xanthophylls content of raw

marigold and the particular characteristics of the sub-strate used in each particular method. In the enzymatic

treatment studies, the substrate was composed solely of

marigold flower petals, which have higher xanthophylls

content than the remaining flower parts; while for the

solid-state fermentation system, and commercial ensi-

lage, the substrate was the whole marigold flower,

including its receptacles. The receptacles comprise about

30% of the samples, thus the overall yields (once cor-

0

5

10

15

20

0 1 2 3 4 5 6 7 8

Time (Days)

Xt(

g/K

gflo

ur)

0 1 2 3 4 5 6 7 8

Time (Weeks)

Fig. 2. Effect of solid-state fermentation and uncontrolled ensilage on

marigold flower flours: xanthophylls concentration in samples

obtained by fermentation process (r, axis X bottom), and xantho-

phylls concentration in ensilaged samples (j, axis X top).

rected for this fact) of the enzymatic treatments and

the solid-state fermentation system are similar.

Independently of the treatment, the enzymatic reac-

tion leads to degradation of the wall-cell components

(mainly cellulose and hemicellulose). As a result, the

wall-cells exhibit an increment in its permeability, lead-ing to improved mass transfer of hydro-soluble cell com-

ponents between the solid (petals) and liquid (enzymatic

extract) phases. Furthermore, the hydro-insoluble com-

ponents (e.g. xanthophylls) increase their concentration

in the remaining solid mass. Additionally, because of

these changes in wall-cell permeability, the xanthophylls

extraction from internal parts of the marigold flower

petals, using hexane, is relatively increased.

3.3. Xanthophylls extraction with hexane

All the dried samples of the treatments, previously

described, were lixiviated with hexane to achieve xan-

thophylls extraction. A single-stage continuous extrac-

tion was performed to evaluate the change on

xanthophylls concentrations in hexane, and also toestablish the approximate number of stages in which

the maximum extraction could be achieved. The results,

showed in Fig. 3, indicate the correlation that exists

among different treatments on marigold flower prior to

the extraction process. In the figure, it is observed that

the material treated with raw enzymatic extract from

saprophyte microorganisms leads, in short time, to the

highest yield; the results show that at 10 min of extrac-tion time, the maximum concentration of xanthophylls

in the solvent phase is achieved. After this time, the con-

centration remains practically constant. However, it

may not be necessary to operate the extraction for

10 min. From the figure, it can be noted that after only

4 min xanthophylls recovery of 96% on average is

achieved. In addition, the result also show that the only

sample that reaches equilibrium, within the horizon ofthe experiment, is the one treated with the raw enzy-

matic extract.

0

20

40

60

80

100

120

140

160

0 2 4 6 8 10 12 14 16Time (Min)

Xt (

g/K

g ol

eore

sin)

0

20

40

60

80

100

120

Fig. 3. Effect of treatments in the xanthophylls extraction using

hexane. Samples treated with raw enzymatic extract (j), untreated

samples (r), samples treated by solid-state fermentation (m), and

samples treated by uncontrolled ensilage (d).

Table 1

Estimated values of volumetric mass transfer coefficients

Treatment kca (h�1) R2

T1 43.1690 0.9988

T2 21.1306 0.9988

T3 28.0622 0.9916

T4 35.5172 0.9893

T1: samples treated with raw enzymatic extract, T2: untreated samples,

T3: samples treated by solid-state fermentation, and T4 samples trea-

ted by uncontrolled ensilage.

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8Stages number

Xt(

g/K

gflo

ur)

0

20

40

60

80

100

120

140

160X

t(g/

Kg

oleo

resi

n)

Fig. 4. Number of stages required for xanthophylls extraction by

lixiviation with hexane. Xanthophylls concentration on samples

treated with raw enzymatic extract: light phase (j, axis Y right), and

solid phase (d, axis Y left). Xanthophylls concentration on untreated

samples: light phase (m, axis Y right), and solid phase (r, axis Y left).

J.L. Navarrete-Bolanos et al. / Food Research International 38 (2005) 159–165 163

The diverse phenomena encountered in the lixiviation

assays make it difficult to apply a single theory to ex-

plain them. In general, the differences between xantho-

phylls obtained from different treatments are due to

the presence of slowly dissolving components in the

oleoresin. Each particular xanthophylls mixture con-tains several different substances, as it is evident from

the differences in properties of the oleoresin obtained

after short and long lixiviation processing times. In

addition, if the wall-cells of the marigold flower petals

remain intact upon exposure to hexane, the lixiviation

will mainly involve Knudsen diffusion of the xantho-

phylls through the cell wall. Such diffusion process is

slow giving, as a result, only partial xanthophylls extrac-tion. However, when the marigold petals are pulverized,

as a consequence of the treatment method, the rates of

diffusion of xanthophylls are increased (both Fickian

diffusion and mass convection are involved). These com-

petitive mechanisms of diffusion prevent a simple inter-

pretation, since the characterization of the structures in

the solid matrix after treatments is required to assess the

partial contribution of each of them. A first attempt todescribe these mass-transfer processes, however, can be

formulated via the introduction of a suitable volumetric

mass-transfer coefficient (kca), using the following

(lumped parameter) equations derived from liquid and

solid mass balances in the batch extraction:

Liquid phase:

dW L

dt¼ kca

1þ skcaW S: ð1Þ

Initial condition.

@t ¼ 0; W L ¼ 0: ð1aÞSolid phase:

dW S

dt¼ �kca W S �

skca1þ skca

W S

� �: ð2Þ

Initial condition.

@t ¼ 0; W S ¼ 1; ð2aÞwhere WL is the dimensionless concentration of xantho-

phylls in hexane, CAL=C0

As; WS the dimensionless con-

centration of xanthophylls in marigold, CAs=C0

As; kca

the volumetric mass transfer, min�1; s the retentiontime, min; t the time, min; CAs

the concentration of xan-

thophylls in marigold, g/kg oleoresin; CALthe concen-

tration of xanthophylls in hexane, g/kg oleoresin; and

C0As

the initial concentration of xanthophylls in mari-

gold, g/kg oleoresin.

Integration using the initial conditions leads to:

W S ¼ e�kcað1þ/Þt; ð3Þ

W L ¼ 1

ð1þ 2skcaÞ½1� e�kcað1þ/Þt�; ð4Þ

where

/ ¼ skca1þ skca

: ð5Þ

Eq. (4) is used for calculating the unknown parameters

kca and skca from experimental data for each treatment,

using nonlinear regression with Levenberg–Marquardt

method (Zhang & Chen, 1997). The results of kca to-

gether with the correlation coefficient are shown in

Table 1. There one can observe that the treatment with

raw enzymatic extract produces the highest volumetricmass transfer, in correspondence to the highest xantho-

phylls yield. The large magnitude of kca indicates a large

degree of disintegration of the cell walls. The differences

in magnitude among the parameter kca, for different

treatments, seem to suggest that for the fastest process

it is reasonable to expect that nearly all the xantho-

phylls, initially contained in the solid phase C0As, migrate

to the liquid phase as oleoresin at the end of the extrac-tion stage.

3.4. Multistage crosscurrent extraction

An efficient extraction can only be achieved using

multiple contact operations. Multiple stages allow to in-

crease the mass transfer rates over and above what is

6.77

7

8.73

1

7.52

4

4.79

95.

496

3.42

8

1.97

1

10.7

4415

110

.9.

980

11.6

97

14.7

50

zeax

anth

in

lute

in

Retention times

mä rz

lute

in

zeax

anth

in

2.21

4

3.87

4

7.92

78.

829

14.4

1213

.617

12.7

83

11.9

0010

.272 12

.342

17.8

8317

.722

Retention times

Fig. 5. Xanthophylls profile comparison: final products obtained from the traditional ensilage process (left) and enzymatic treatments (right).

164 J.L. Navarrete-Bolanos et al. / Food Research International 38 (2005) 159–165

possible with a single stage, obtaining higher concentra-

tions on the final product. The xanthophylls extraction

was analyzed in a multistage crosscurrent extraction as

shown in Fig. 4. The results indicate that the flour ob-

tained from enzymatic treatment on flowers using the

raw enzymatic extract can be depleted of xanthophyllswithin five stages. While for the untreated samples (fresh

marigold petals) xanthophylls recovery of only about

60% on average can be expected for this number of

stages. Similar xanthophylls recovery from the untreated

samples, as that of the samples treated with raw enzy-

matic extract in five stages, is reached only after 18

stages.

4. Conclusions

The results presented here confirm that the success ofa lixiviation process depends upon the prior treatment

which may be given to the solid. In this study, the enzy-

matic treatment appears the best option to increase xan-

thophylls yield extraction; however, the treatment

selection depends on the purpose of the product in the

market. If the product is intended as additive in poultry

feed, the treated substrate should be whole marigold

flowers. In this case, the residual solid, after extraction,is used as a support for the saponified extract in an inte-

gration stage. In such a stage, the extract is added to a

powdery matrix (which can be the residual solid) in con-

centrations around 15% (w/w). Therefore, the solid-state

fermentation appears the best treatment process previ-

ous to the xanthophylls extraction stage for this final

application. On the other hand, if the xanthophylls will

be used as additive in food, pharmaceutical, and cos-metic industries, then the treatment using the raw enzy-

matic extract is the best option for treatment process

previous to the xanthophylls extraction stage, since it

maximizes the extraction yield and it is applied to sub-

strates free of marigold flower receptacles. It is impor-

tant to note that both treatment processes are based

on the action of hydrolytic enzymes synthesized by

microbiota associated to marigold flowers. Such on-site

enzyme production has significant advantages. Besides

the demonstrated increase on concentration in flours

and extraction yield, it significantly reduces the costassociated to the enzyme treatment when compared to

commercial alternatives. In addition, the flours obtained

from these treatments show no alteration on the total

xanthophylls profile when compared to the product ob-

tained from the traditional ensilage process (Fig. 5).

Acknowledgments

The authors gratefully acknowledge financial support

by FOMIX (CONACYT/CONCYTEG; 02-09-B-005)

and COSNET (896.03-P).

References

AOAC. (1984). Official methods of the Association of Official

Analytical Chemists (14th ed.). Washington, DC.

Ausich, R.L., & Sanders, D.J. (1997). Process for the formation,

isolation and purification of comestible xanthophyll crystals from

plants. US Patent No. 5,648,564.

Barzana, E., Rubio, D., Santamarıa, R. I., Garcıa-Correa, O., Garcıa,

F., Ridaura-Sanz, V. E., et al. (2002). Enzyme-mediated solvent

extraction of carotenoids from marigold flower (Tagetes erecta).

Journal of Agricultural and Food Chemistry, 50, 4491–4496.

Bernhard, K., Broz, J., Hengartner, U., Kreienbuhl, P., & Schiedt, K.

(1997). Pigmentation with carotenoides. US Patent No. 5,670,548.

Bletner, J. K., Mitchell, R. P., & Tugwell, R. L. (1966). The effect of

Eimeria maxima on broiler pigmentation. Poultry Science, 45,

689–694.

Chew, B. P., Wong, M. W., & Wong, T. S. (1996). Effects of lutein

from marigold extract on immunity and growth of mammary

tumors in mice. Anticancer Research, 16, 3689–3694.

Delgado-Vargas, F., & Paredes-Lopez, O. (1997). Effects of enzymatic

treatments of marigold flowers on lutein isomeric profiles. Journal

of Agricultural and Food Chemistry, 45, 1097–1102.

J.L. Navarrete-Bolanos et al. / Food Research International 38 (2005) 159–165 165

Fullmer, L. A., & Shao, A. (2001). The role of lutein in eye health and

nutrition. American Association of Cereal Chemistry, 46, 408–413.

Gierhart, D. L. (1994). Production of zeaxanthin and zeaxanthin-

containing compositions. US Patent No. 5,308,759.

Hencken, H. (1992). Chemical and physiological behavior of feed

carotenoids and their effects on pigmentation. Poultry Science, 71,

711–717.

Hirschberg, J., & Harker, M. (1999). Carotenoid-producing bacterial

species and process for production of carotenoids using same. US

Patent No. 5,935,808.

Jacobson, G. K., Jolly, S. O., Sedmak, J. J., Skatrud, T. J., &

Wasileski, J. M. (2000). Astaxanthin over-producing strains of

Phaffia rhodozyma, method for their cultivation and their use in

animal feeds. US Patent No. 6,015,684.

Kanel, J. S., & Marentis, R. T. (2000). Process for fluid/dense gas

extraction under enhanced solubility condition. US Patent No.

6,106,720.

Khachik, F. (2001). Process for extraction and purification of lutein,

zeaxanthin and rare carotenoids from marigold flowers and plants.

U.S. Patent No. 6,262,284.

Kreienbuhl, P., Rudin, P., & Rudolph, W. (2000). Method of making

carotenoids. US Patent No. 6,150,561.

Levi, L. W. (2001). Trans-xanthophyll ester concentrates of

enhanced purity and methods of making same. US Patent No.

6,191,293.

Madhavi, D. L., & Kagan, D. I. (2002). Process for the isolation of

mixed carotenoids from plants. US Patent No. 6,380,442.

Martin, K. R., Eailla, M. L., & Smith, J. C. (1996). b-Carotene and

lutein protect HepG2 human liver cells against oxidant-induced

damage. Journal of Nutrition, 126, 2098–2106.

Matoushek, R. F. (1974). Xanthophylls extraction process. U.S.

Patent No. 3,783,099.

Naranjo-Modad, S., Lopez-Munguıa, A., Vilarem, G., Gaset, A., &

Barzana, E. (2000). Solubility of purified lutein diesters obtained

from Tagetes erecta in supercritical CO2 and the effect of solvent

modifiers. Journal of Agricultural and Food Chemistry, 48,

5640–5642.

Navarrete-Bolanos, J. L., Jimenez-Islas, H., Rico-Martınez, R.,

Domınguez-Domınguez, J., & Regalado-Gonzalez, C. (2001). A

statistically-based strategy for obtaining equilibrium data in

xanthophylls lixiviation. Journal of Food Science, 6, 1143–1146.

Navarrete-Bolanos, J. L., Jimenez-Islas, H., Botello-Alvarez, E., &

Rico-Martınez, R. (2003). Mixed culture optimization for marigold

flower ensilage via experimental design and response surface

methodology. Journal of Agricultural and Food Chemistry, 51,

2206–2211.

Navarrete-Bolanos, J. L., Jimenez-Islas, H., Botello-Alvarez, E., Rico-

Martınez, R., & Paredes-Lopez, O. (2004a). Improving xantho-

phylls extraction from marigold flower using cellulolytic enzymes.

Journal of Agricultural and Food Chemistry, 52, 3394–3398.

Navarrete-Bolanos, J. L., Jimenez-Islas, H., Botello-Alvarez, E., Rico-

Martınez, R., & Paredes-Lopez, O. (2004b). An optimization study

on solid state fermentation: Xanthophylls extraction from mari-

gold flower. Applied Microbiology and Biotechnology (Web Release

Date: 20-May-2004).

Rodriguez, G. (1997). Pigmenting efficiency of a natural xanthophyll

by isomerization. US Patent No. 5,973,211.

Seddon, J. M., Ajani, U. A., Sperduto, R. D., Hiller, R., Blair, N.,

Burton, T. C., et al. (1994). Dietary carotenoids, vitamins A, C and

E, and advanced age-related macular degeneration. A multicenter

study. Journal of American Medical Association, 272, 1413–1420.

Zhang, J. Z., & Chen, L. H. (1997). Non-monotone Levenberg–

Marquardt algorithms and their convergence analysis. Journal of

Optimization Theory and Applications, 92(2), 393–418.

Zhang, L. X., Coney, R. V., & Bertram, J. S. (1991). Carotenoids

enhance gap functional communication and inhibit lipid peroxida-

tion in C3H/10T/2 cells: Relationship to their cancer chemopre-

ventive action. Carcinogenesis, 12, 109–114.