PRODUCCIÓN DE TEOBROMINA CON CEPAS INOCUAS AEROBIAS Y ANAEROBIAS
THEOBROMINE PRODUCTION WITH AEROBIC AND ANAEROBIC SAFETY STRAINS
Escamilla Hurtado, M. L. y Verde Calvo, J. R.*
Departamento de Biotecnología. Div. C.B.S. Universidad Autónoma Metropolitana. Unidad Iztapalapa.
Av. San Rafael Atlixco # 186. Col. Vicentina. Deleg. Iztapalapa Ap. P. 55-535. C.P. 09340. México. D.F.
Tel. (55) 5804-4722 Fax. (55) 5804-4712. e-mail: jrvc@xanum.uam.mx
Abstract
Theobromine is a nutraceutic of interest for the food industry because it is a component of chocolate flavor, and
serves as a stimulant of the central nervous system. Several innocuous microbial strains were evaluated for their
capacity of producing theobromine in submerged axenic cultures. Three strains produced 16.1-21.9 mg L-1 of
theobromine under aerobic conditions, through the mechanism of oxidative demethylation of caffeine. Alternatively,
the mechanism of the methylation of precursors was also studied under anaerobic conditions. The addition of Lmethionine and low levels of Aw of the culture medium, 0.967 for bacteria and 0.93 for yeasts, stimulated the
production of theobromine from xanthine or xanthosine to 330.1-597.04 mg L-1 and 26.81-79.45 mg L-1,
respectively; and Lactobacillus pentosus NRLL-B-227 was the most efficient strain to do so. This anaerobic
metabolism had not been previously observed in microbial cultures; allowing the possibility of using natural sources
of xanthines for the production of theobromine by innocuous strains.
Resumen
La teobromina es un nutracéutico de interés para la industria por ser componente del sabor a chocolate, y porque
estimula el sistema nervioso central. Se evaluaron diversas cepas microbianas inocuas para producir teobromina en
cultivos axénicos sumergidos. Tres cepas formaron 16,1-21,9 mg L-1 de teobromina en aerobiosis por el mecanismo
de demetilación oxidativa de la cafeína. Alternativamente, se estudió el mecanismo de metilación de precursores en
anaerobiosis. La adición de L-metionina y los niveles bajos de Aw en el medio, 0,967 para bacterias y 0,93 para
levaduras, estimularon la producción de teobromina, 330,1-597,04 mg L-1 y 26,81-79,45 mg L-1, a partir de xantina o
xantosina, respectivamente; siendo Lactobacillus pentosus NRRL-B-227 la cepa más eficiente. Este metabolismo
anaerobio no había sido observado previamente en cultivos microbianos y abre la posibilidad de aprovechar fuentes
naturales de xantinas para obtener teobromina con cepas seguras.
Resumo
A teobromina é um nutracêutico de interesse para a indústria pelo fato de ser um dos
componentes do sabor do chocolate, além de estimular o sistema nervoso central. Foram
avaliadas diversas cepas microbianas inócuas na produção de teobromina em cultivos axênicos
imersos. Três cepas formaram 16,1-21,9 mg L-1 de teobromina em anaerobiose pelo mecanismo
da desmetilação oxidativa da cafeína. Alternativamente, foi estudado o mecanismo de metilação
dos precursores em anaerobiose. A adição de L-metionina e os baixos níveis de Aw no meio,
0,967 para bactérias e 0,93 para leveduras, estimularam a produção de teobromina, 330,1-597,04
mg L-1 e 26,81-79,45 mg L-1, a partir de xantina o xantosina, respectivamente; sendo
Lactobacillus pentosus NRRL-B-227 a cepa mais eficiente. Este metabolismo anaeróbio no tinha
sido observado previamente em cultivos microbianos, criando a possibilidade no aproveitamento
de fontes naturais de xantinas na obtenção de teobromina a partir de cepas seguras.
Keywords: Aw, caffeine, Lactobacillus pentosus, theobromine, xanthine, xanthosine.
Palabras clave: Aw, cafeína, Lactobacillus pentosus, teobromina, xantina, xantosina.
INTRODUCTION
Theobromine (3,7-dimethylxanthine, or 2,6-dihydroxy-3,7-dimethylpurine) has the structure of an
alkaloid purine, and is a natural component of cocoa, coffee, tea and ‘mate’. This compound is of current interest
because it is a nutraceutic, aside from being the predominant methylxanthine in chocolate flavor; in low doses it
produces a slight cortical activation in the brain that leads to an increased state of euphoria; and it acts as a diuretic,
and a cardiac, respiratory, digestive, renal stimulant, as well as a stimulant for other physiological functions.
Theobromine can also cross the placenta and reach a developing fetus (Litter, 1988; Xatzung, 1996; Eteng et al.,
1997;
Reginatto
et
al.,
1999;
Nutrition
F.A.Q.,
The
Hershey
Co.,
2007
http://www.hersheys.com/nutrition/theobromine.asp). Theobromine is produced for the pharmaceutical industry
through the purification of cacao and other vegetable extracts (Li y Hartland, 1996; Lin et al., 1998). The
concentration of theobromine in a chocolate bar (43 g) is 23-195 mg (Nutrition F.A.Q., 2007), and 230-280 mg in a
cup of chocolate (250 mL) (Eteng et al., 1997).
The biosynthetical pathways of theobromine and caffeine have been studied in coffee, cocoa, tea and mate
cells (Ogutuga y Northcote, 1970; Kato et al., 1996; Regginato et al., 1999; Zheng et al., 2004). The precursors of
these reactions are xanthine, xanthosine, and N-methyl transferases enzymes, which have been characterized by
Mazzafera et al. (1994), Shulthess and Baumann (1995), and McCarthy and McCarthy (2007). These N-methylation
reactions had never been previously observed in the natural metabolism of microbes, although kinetic parameters
have been determined for the enzymes that transform xanthine to theobromine and caffeine in Escherichia coli
cultures that have been modified with cDNA from coffee (Uefuji et al., 2003). Theobromine has been produced by
aerobic microbial cultures through the action of the theobromin-dimethilase enzyme, which catalyses the oxidative
demethylation reaction in caffeine (Asano et al., 1993, 1994; Bang et al., 1999). Pseudomonas putida 352, isolated
from soil, produced via this mechanism the highest levels of theobromine, 20 g L -1, reported thus far (Asano et al.,
1993). Various pure cultures of filamentous fungi were isolated from coffee waste, such as Penicillium, Aspergillus,
Rhizopus, and Phanerochaete, also degrade caffeine. Thus far, research studies have mainly focused on the
detoxification of coffee waste for the production of microbial biomass (Roussos et al., 1994; Hakil et al., 1999;
Brand et al., 2000, Gutiérrez-Sánchez et al., 2004).
The objective of this work is to determine the growth conditions of various microorganisms, considered to
be inocuous for humans, necessary for them to produce theobromine. The oxidative demethylation pathway for
caffeine will also be evaluated in aerobic cultures; as well as the alkylation by electrophilic aromatic substitution of
xanthine and xanthosine in anaerobic cultures.
MATERIALS AND METHODS
Experimental Design
Three sets of experiments were carried out using innocuos microbial strains in order to produce
theobromine. Each set had two stages: the propagation of the colonies and the production of theobromine. The first
one compared the performance of five pure-culture strains of diverse microbial groups under aerobic conditions; and
caffeine served as a precursor of theobromnine for the cultures during the production stage. During the second set of
experiments, four microbial groups were evaluated, as well as two levels of water activity (Aw) under anaerobic
conditions; and xanthine or xanthosine were used as precursors. The third set compared the performance of three
species of one of the microbial groups, which was selected based on its performance during the second experiment,
under growth conditions that were similar to those of cultures grown with a low level of Aw, but two growth media
were evaluated: with glycerol or at low humidity.
Microbial species
The pure cultures of the following species were used during the first series of experiments:
Saccharomyces cerevisiae INRA-CIRM-CLIB 94 (1-Sc), Lactobacillus pentosus NRRL-B-227 (2-Lpe), Pediococcus
pentosaceus MITJ-10, isolated from cider at the UAM-I (Verde et al., 1995) (3-Pp), Bacillus subtilis (from the
UNAM-CFQ100-B-14 pure culture) (4-Bs) and Rhizopus oligosporus (from the UNAM-CFQ100-F-50 pure culture)
(5-Ro). During the second experimental set, the first three pure cultures and the Kluyveromyces marxianus var.
marxianus NRRL-Y-1195 (6-Km) culture were used. In the third set of experiments, the following pure cultures
were used: Lb. pentosus NRRL-B-227 (2-Lpe), Lb. plantarum NRRL-B-813 (7-Lpa) and Lb. casei subsp. rhamnosus
GG from Culturelle, U.S.A. (8-Lc) capsules.
Growth media
To preserve the pure cultures during the experiments all solid media were prepared in assay tubes that were
sterilized in the autoclave (121°C, 15 min). Pure culture 5-Ro and 4-Bs were grown on PDA agar and nutrient rich
agar, respectively. Lactic bacteria were grown in MRS agar and yeasts in Dextrose-Sabouraud agar. The remaining
three cultures were enriched with 1 mL L-1 of a vitamin solution (8 mg of biotin; 4 mg of folic acid, and 100 mL -1 or
2 capsules of Manibee-C®). All media was from Merck.
The composition of the medium of propagation of the Series 1 was based on that proposed by Asano et al.
(1994), with some modifications (g L-1): D-fructose, 10; casein peptone, 5; monosodium L-glutamate, 1; caffeine,
0.5, K2HPO4, 0.5, FeSO4, 0.04 , and 1 mL of vitamin solution, the pH was adjusted to 7.0. Organic and inorganic
components were sterilized separately (121 ° C, 15 min), in deionized water. To the propagation cultures were
added: 0.5 g L-1 of caffeine (900 µL / tube caffeine solution at 0.278%) and 0.136 g L-1 of ZnCl2 (100 µL / tube
ZnCl2 solution, to 0.68%). These solutions were sterilized by microfiltration.
The propagation media used in the second set of experiments was composed of (g L-1): 20 g L-1 D-fructose,
5 g L-1 casein peptone, 2 g L-1 monosodic glutamate, 1 mL of vitamin solution, 2.5 g L-1 KH2PO4, 2.5 g L-1 Na2HPO4
7·H2O and 0.2 g L-1 MgSO4 7·H2O. The pH of the media was adjusted to 5.6 for the yeasts and to 7 for the bacteria.
The media was sterilized in the autoclave as previously stated. In this experimental series two mediums for
production were prepared with two levels of Aw, for yeast N1 = 0.93 and N2 = 0.99; and for bacterias N1 = 0.967 and
N2 = 0.99. The basal composition of the production medium with the high Aw (N2) was similar to the propagation
medium (with a pH of 7) and the N1 level was prepared substituting part of the water for sterile glycerol at 20 and
30% (p/p) for bacteria and yeast, respectively. The following reagents were added to each tube with 5 mL of sterile
production media: 1 g L-1 of xanthine (900 µL/tube of 0.667% p/p xanthine solution) and 0.5 g L -1 of methionine
(100 μL/tube of 3% p/p L-methionine solution); or 0.066 g L-1 of xanthosine (900 µL/tube of 0.05% p/p xanthosine
solution) and 0.033 g L-1 of methionine (100 μL/tube of 0.2% p/p L-methionine). These solutions were sterilized via
microfiltration.
The medium for the propagation cultures for the third experimental set was similar to that of the second one
with the following changes: 1 g L-1 of peptone and 6 g L-1 of monosodic glutamate. In this experimental set no Aw
(N2) media were prepared for production cultures, but two media with Aw (N1) were used: with glycerol (as already
described) and with low humidity. This last one was prepared by concentrating the indicated components 10 times
for the N2 level of the second set, and it was sterilized by microfiltration. All reagents used were by SIGMA.
Cultures
All cultures were axenic and submerged, and the production cultures were done in triplicates. During the
first experimental set, the cultures were shaken at 80 rpm; and cultures 1-Sc, 3-Pp and 5-Ro were incubated at 28-
30C, and 2-Lpe and 4-Bs at 35-37 ºC. The working volume used for the propagation cultures was 4 mL; and 72h
after, production cultures were started in the same tube by adding the sterile solutions with the additives, which
increased the volume to 5ml. Theobromine production was assessed at 18h and 72 h.
In the second and third experimental sets, all cultures were grown for 48 h. They were static with a
volume of 6 mL and grown in an anaerobic CO2 atmosphere. Yeasts and bacterial cultures, 3-Pp and 7-Lpa, were
incubated at 28-30 ºC, and the 2-Lpp and 8-Lc cultures at 35-37 ºC. When the propagation cultures were finished,
microbial counts were carried out using a Neubauer camera, and aliquots of these cultures were poured onto sterile
production cultures, in order to adjust the initial cell count to 5x107 cells mL -1. The production cultures from the
third set with a low humidity media were started with a microbial concentration 10 times higher (5x10 8 m.o. mL-1).
Chemical assays
The aqueous activity (Aw) of the growth media was measured using the AQUA-LAB CX-2 Decagon
Devices Inc. Equipment. The amount of glycerol that must be added to the growth media with a low Aw (N 1) in the
second and third experimental sets was determined by interpolation of standard curve: Aw vs. 0-30% glycerol
solutions in the growth media (p/p). In the third set, the concentration of the components of the low humidity media
was determined by measuring the value of Aw in the solutions that were concentrated 1-10 times, starting from a Aw
(N2) media.
In the first set, samples of the production media were collected at 0, 18, and 72h; and for series 2 and 3,
sampling was done at 0 and 48h. The samples from the growth broth were centrifuged for 30 min at 4°C and 3000
rpm (2016 g) in a Beckman J2-MI centrifuge with a Beckman JA-20.1 rotor. The supernatant was microfiltered
through 0.44  membranes. Theobromine, caffeine, xanthine and xanthosine concentrations were analyzed with six
replicas of diluted samples, by modifying the HPLC chromatography method of Asano et al. (1993). A Thermo
Separations Products’ HPLC chromatographer with a Chrompak ZORBAX C18 (100ª) 5µ of 250 x 4.6 mm column
was used at 25°C. The mobile phase used was methanol/ 1% acetic acid aqueous solution (80:20 v/v), with a 0.8 mL
min-1 flow rate and a 20 μL injection volume. The detector was of a UV-visible diode array detector that gave
readings of 250 nm for xanthosine, 265 nm for xanthine and theobromine, and a 273 nm for caffeine. The data was
collected with a Chem Station v8.3 Agilent, by interpolating standard concentration curves. All reagents were
SIGMA. The data was then analyzed with the statistical program Statgraphics Plus v4 (ANOVA and Duncan’s
multiple range test) to evaluate the effect of Aw, and the consumption of the initial reagents and production of
theobromine by each culture, for all experimental sets.
RESULTS AND DISCUSSION
This work focused on determining the required conditions of several microbial strains, which are
generally considered to the innocuous for humans, for the production of theobromine. The Unites States and the
European Union have issued official catalogues that consider lactic bacteria, yeasts and fungi for industrial uses to be
generally innocuous for humans. In addition, health protection agencies in several countries recognize species that
comprise the native microbial flora of traditionally fermented foods (GRAS) as being safe (FDA/CFSAN/OFAS.
2007
http://www.cfsan.fda.gov/~dms/opa-micr.html;
Curtis-Stevens
and
O’Brien-Nabors,
2009
http://www.foodadditives.org/cultures/fdtechnology.pdf). The lactic bacteria and yeast species used in the present
work are found on those lists. Rh. Oligosporus and B. Sultilis produce enzymes for the food industry and are endemic
of the traditional fermentative processes for many Asian products like soy, tempeh, natto, soy sauce, etc. (Steinkraus,
1983). The fore mentioned government institutions also established that any microorganisms that have been recently
isolated from other sources must be exhaustively tested to be deemed innocuous and approved.
Cultures with caffeine.
All research on the bionsynthesis of theobromine by non-genetically modified microorganisms that
has been published so far, has studied the oxidative demethylation reaction of caffeine, which is shown in Figure 1a
(Asano et al., 1993, 1994; Eteng et al., 1997; Bang et al., 1998). In experimental set 1, this metabolism was carried
out. At 18 h of growth, the suggested time period by Asano et al. (1993), none of the strains produced theobromine,
and thus they were incubated for 72 h. During this time frame all strains that were evaluated consumed caffeine, but
only the three with metabolic respiration produced theobromine. The results of this experimental set are presented in
Table 1. A low dispersion of the data was obtained in all cases (V.C.< 10%). The pure culture that produced the most
theobromine was the P. Putida 352 strain. The later could be due to the lack of adaptation of the strains to the growth
media, or the degradation of caffeine can follow metabolic pathways that do not use theobromine, such as those
observed in vegetables (Mazzafera et al., 1994; Eteng et al., 1997). The 1-Sc strain consumed the most caffeine, and
was produced the second largest amount of theobromine. All strains in the first experimental set demonstrated their
potential use for diminishing the toxicity of residues from the processing plants on aerobic cultures, due to their
capacity for degrading caffeine and forming a biomass, even though some of them do not produce theobromine.
The effect of anaerobiosis, pH and methionine.
Experimental sets 2 and 3 evaluated various strains of lactic bacteria and yeast, such as innocuous
fermentative metabolic species. Preliminary strains that did not produce theobromine were grown under the
following conditions: using xanthine as a precursor, a low pH growth media, a high Aw (0.99 N2) and without a
methyl donor. In order to reach similar levels of metabolic biosynthesis of theobromine to those observed in plants,
set 2 incorporated anaerobic cultures with methionine, which served as a methyl group donor; and the pH was
adjusted to 7 (Ogutuga y Northcote, 1970; Waldhauser et al., 1997). Under these conditions, the methylation or the
alkylation by aromatic electrophilic substitution of the xanthine and xanthosine precursors were observed in these
microorganisms. These metabolic routes had been previously studied in tea, coffe, cacao and mate; and are
summarized in Figures 1b and 1c, respectively (Ogutuga y Northcote, 1970; Eteng et al., 1997; Uefuji et al., 2003;
McCarthy y McCarthy, 2007).
Results from set 2 are shown in Table 2, and values of V.C.<10% were obtained here as well. All strains
assimilated the precursors; and in almost all cases, xanthine was more efficiently consumed than xanthosine (44.088.5% and 18.64-46.11%, respectively, in relation to 100% of the initial content). This can be explained due to the
availability of xanthosine, which is limited by its low solubility in aqueous media. All cultures from set 2 produced
more theobromine than those of set 1; among them, those of the 1-Sc yeast produced up to 23.27 times more
theobromine under anaerobic conditions with a low Aw (N1) than under aerobic conditions.
The effect of the aqueous activity on the growth media
Kim et al. (2000) observed that methoxilation reactions are stimulated in the presence of organic solvents. A
low Aw environment is established under these conditions that decrease the activation energy of the enzymes;
favoring a transition state and increasing its activity as a result. Similarly in cultures from the second set, the effect of
lowering the value of Aw by the addition of glycerol to the media was evaluated. The levels of Aw were chosen to
be within the tolerance limits for the yeasts and the lactic bacteria.
It was observed in the second experimental set that the level of N1 significantly stimulated xanthine
consumption by the 2-Lpe and 6-Km (P< 0.01) strains, and by the 3-Pp and 1-Sc (P< 0.1) strains. The low level of
Aw also favored the production of theobromine through the set since xanthine served as a precursor (P< 0.01).
When the consumption of xanthine at this Aw level (N1) was compared among the strains, three homogeneous
groups emerged from the lowest to highest consumption (P< 0.01): a) 1-Sc and 2-Lpe strains, b) 3-Pp strain, and c)
6-Km strain. Theobromine production was grouped in a similar manner (from lowest to highest, P< 0.01): a) 6-Km,
1-Sc and 3-Pp strains, and b) 2-Lpe strain. The 2-Lpe strain performed the best with xanthine and Aw (N 1) in the
second sets. Its yield for theobromine was 65.38%, according to the reaction in Figure 1b.
The low level of Aw (N1) also favored the consumption of xanthosine in growth cultures from all strains in
the second set of experiments (P< 0.05), in relation to that observed in the N2 level; although if the consumption of
this precursor is compared within each of the Aw levels, then the strains behaved in a homogeneous manner (P>
0.1). Theobromine production was significantly higher with a low Aw (N 1) for the 2-Lpe, 3-Pp and 6-Km strains (P<
0.05) than with an N2 level. Cultures from each of the four strains with an N1 Aw level produced different amounts
of theobromine (from lowest to highest, and P< 0.05): a) 1-Sc strain, b) 6-Km strain, c) 3-Pp strain, and d) 2-Lpe
strain. According to these results, the 2-Lpe strain was also noteworthy in producing theobromine when xanthosine
served as a precursor.
Effect of the glycerol or low humidity media.
In the third set of experiments, three Lactobacillus species were evaluated, since it was this specie,
which produced the most theobromine in the past experimental set. Also two growth media both with a 0.967 Aw
were compared. The results are shown in Table 2, all had a V.C.< 10%. The strains that were evaluated consumed
more xanthine in the media with glycerol than in the low humidity one, with respect to the initial contents (65.186.4% and 45.7-76.2%, respectively), and the same occurred with xanthosine (64.1-80.6% and 41.0-69.1%,
respectively). Although the low humidity media had initial concentrations of its components that were 10 times
higher, this did not lead to a higher theobromine production; for example, 2-Lpe and 7-Lpa strains accumulated more
theobromine in the media with glycerol and xanthine than in the low humidity media ((P≤ 0.1), and something
similar also happened with the 8-Lc strain (P< 0.05). In the glycerol media, strains were divided into two
homogeneous groups based on their xanthine consumption (from lowest to highest and P< 0.01): a) the 7-Lpa strain,
and b) the 8-Lc and 2-Lp strains. These three strains produced different amounts of theobromine under these growth
conditions (from lowest to highest, P< 0.01): a) the 8-Lc strain, b) the 7-Lpa strain, and c) the 2-Lpe strain. When the
direct/raw/net theobromine production values for 2-Lpe in the glycerol/low humidity media were compared, a 1.01:1
ratio was obtained, in spite of the 1:8.81 ratio of xanthine consumption.
Comparando los valores directos de producción de teobromina por la cepa 2-Lpe en los medios con glicerol/
baja humedad, se obtuvo una relación de 1,04:1, a pesar de que el consumo de la xantina fue de 1:8,81.
In regards to precursor consumption, the glycerol and xanthosine cultures showed two homogenous groups
(from lowest to highest, P< 0.05): a) 2-Lpe and 7-Lpa strains, and b) the 8-Lc strain. There was no significant
difference in theobromine production between any strains when the two growth media were compared; but within the
glycerol group, three strains did produced different amounts (from lowest to highest and P< 0.05): a) the 7-Lpa
strain, b) the 8-Lc strain, and c) the 2-Lpe strain. The highest, 2-Lpe, had a xanthosine consumption of 6.39/1 in low
humidity/glycerol media, and produced 1.02 more theobromine in the glycerol media. Thus, media with a high
concentration of their initial components (low humidity media) are not recommended because theobromine yields are
very low compared to the consumption of their precursors.
The theobromine yields of almost all of the observed microbial cultures with xanthosine exceed the
expectations of the stoichiometric equation in Figure 1c; suggesting that at least one other alternate biosynthetic
pathway must exist. One of them could be the one proposed by Ogutuga and Northcote (1970), who observed the
metabolism of tea cells, and noticed that free purines were methylated de novo when nucleic acids were degraded,
thus forming diverse methylxanthines. Yet, it is still necessary to determine whether the metabolic pathways of
microorganisms under anaerobic conditions correspond to those studied in plant cells. Howerever, our results may be
used to develop future methods for the production of theobromine using innocuous microbial strains by taking
advantage of natural xanthenes sources found in coffee, tea, cacao, mate, and guarana (Litter, 1988; Sotelo y
Álvarez, 1991; Mazzafera et al., 1994; Lin et al., 1998; Vitória y Mazzafera, 1999).
Los rendimientos de teobromina observados en casi todos los cultivos microbianos con xantosina exceden
las expectativas de la ecuación de balance estequiométrico de la Figura 1c. Esto sugiere que existe al menos otra vía
alterna de biosíntesis. Una de éstas podría ser la sugerida por Ogutuga y Northcote (1970), quienes al observar el
metabolismo de las células de té, notaron que ocurrían reacciones de metinación de las purinas liberadas ‘de novo’, al
degradar los ácidos nucléicos, y se formaron diversas metilxantinas. Aún es necesario demostrar si las vías
metabólicas de los microorganismos en cultivos anaerobios corresponden a las estudiadas en las células vegetales;
sin embargo, los presentes hallazgos pueden iniciar los estudios tendientes a desarrollar procesos de producción de
teobromina con cepas microbianas inocuas, aprovechando fuentes naturales de xantinas, que se encuentran en los
desechos de café, tés, cacao, mate, cola y guaraná (Litter, 1988; Sotelo y Álvarez, 1991; Mazzafera et al., 1994; Lin
et al., 1998; Vitória y Mazzafera, 1999).
CONCLUSIONS
All innocuous strains evaluated in the first experimental set consumed caffeine, but only those with
metabolic respiration? produced theobromine and had slow growth rates and low yields. Four strains of lactic
bacteria and two of yeast formed theobromine in cultures with xanthine and xanthosine, under conditions similar to
those of synthesis in plant cells. These strains performed better when a methyl donor was added, a neutral pH was
used, the value of Aw decreased in the media used.
Lb. pentosus NRRL-B-227 was the strain that produced the most theobromine, 597.04 g L -1 starting from
the xanthine precursor, during 48h anaerobic conditions, with methionine, a 0.967 Aw (established with glycerol)
and a pH of 7.
Todas las cepas inocuas evaluadas en la serie 1 consumieron cafeína, pero sólo las de metabolismo
respiratorio produjeron teobromina, con cultivos lentos y rendimientos bajos. Cuatro cepas de bacterias lácticas y dos
de levaduras formaron teobromina en cultivos con xantina y xantosina, en condiciones semejantes a las de la síntesis
que realizan las células vegetales. Éstas mejoraron su desempeño cuando se añadió un donador de metilo, se utilizó
pH neutro y se disminuyó el valor de Aw del medio.
Lb. pentosus NRRL-B-227 fue la cepa que produjo más teobromina, 597,04 g L-1 a partir del precursor
xantina, en cultivos anaerobios de 48 h, con metionina, Aw 0,967 (establecido con glicerol) y pH 7,0.
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