Increasing monascus pigment production

Non-protein amino acids increase pigment production by Monascus species in the presence of protein amino acids.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention is related to a method of increasing Monascus pigment 
production. 
2. Description of the Prior Art 
It is known that Monascus microorganisms produce pigments, including 
yellow, oranges and red-purples, that can be used to enrich or compensate 
for the loss of color in food processing. Chemical modifications have been 
attempted to decrease the problems which have prohibited the development 
of these pigments for practical use in the food industry. Various native 
materials and simple chemicals have been used and protein amino acids have 
commonly been found to be useful to improve the quality of the Monascus 
pigments. 
However, it is desirable to not only improve the quality of pigment 
production but also to increase the amount of pigment produced. 
SUMMARY OF THE INVENTION 
The present invention is directed to a method of increasing pigment 
production by Monascus species in the presence of a free protein amino 
acid which method comprises treating the Monascus species in the presence 
of a free protein amino acid with a sufficient amount of a non-protein 
amino acid to increase pigment production by the Monascus species. 
Any Monascus species which will produce pigment in the presence of a free 
protein amino acid can be used. Non-limiting representative Monascus 
species include M. purpureus, M. sp. 1356 and M. sp. 1361 (both obtained 
from the University of California, Davis, Calif.), M. major, M. 
rubigenosus and the like. In one embodiment of the invention the species 
is M. sp. 1356 or M. sp. 1361. 
By free protein amino acid is meant any non-polymeric, essentially free 
natural amino acid commonly found in natural protein, which include 
alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, 
glutamic acid, glycine, histidine, isoleucine, leucine, lysine, 
methionine, phenylalanine, proline, serine, threonine, tryptophan, 
tyrosine and valine and the natural hydroxylated derivatives thereof, 
including hydroxyproline and the like. The free protein amino acid can be 
a single free protein amino acid or functional derivative thereof which 
provides a useable source of free acid in the process, a mixture thereof 
or a composition comprising one or more of such free protein amino acids 
which provides a useable source of free acid to the process, including 
digested proteins, polypeptides and the like, such as digested gelatin, 
yeast, yeast extract, tomato juice and the like. For example, in one 
embodiment, the free protein amino acid is lysine, histidine, arginine, 
asparagine, threonine, glutamic acid, proline, glycine, alanine, valine, 
tyrosine, phenylalanine and the like. In another embodiment the source of 
free protein amino acid is glycine, glutamic acid, digested gelatin, 
yeast, yeast extract, tomato juice and the like. Preferably, the source of 
the free protein amino acid is yeast or yeast extract. 
By free non-protein amino acid is meant a wide variety of 
conventionally-known non-polymeric materials containing at least one amino 
group and one carboxylic acid group or functional derivative thereof which 
provides useable source of non-polymeric, essentially free acid in the 
process, which materials are not commonly found as a natural amino acid 
component of natural protein. More than one amino or carboxylic group can 
be present and not necessarily in equal proportions. In one embodiment of 
the invention, the non-protein amino acid is an aliphatic, aryl, 
heterocyclic or aromatic heterocyclic compound containing at least one 
carboxyl group for every amino group and from 1 to 20 carbon atoms, or a 
functional derivative thereof, such as methanoproline, 
azetidine-2-carboxylic acid, p-aminobenzoic acid, aminoisobutyric acid, 
b-al anine and the like. Preferably, the source of free non-protein amino 
acid comprises one carboxylic group for each amino group and from 1 to 10 
carbon atoms or a functional derivative thereof. 
In another embodiment of the invention, the non-protein amino acid is a 
non-protein heterocyclic compound comprising one (amino-)nitrogen ring 
atom in an otherwise carboxylic ring containing 3 to 7 ring-carbon atoms, 
and at least one carboxylic acid group on the ring carbon atoms including 
those disclosed in U.S. Pat. Nos. 4,047,930, 4,555,260 and 4,560,401. The 
ring may be monocyclic or a fused bicyclic ring, and saturated or 
unsaturated. Preferably, the non-protein heterocyclic compound is one 
which comprises one (amino-)nitrogen ring-atom in an otherwise carboxylic 
saturated ring containing 3 to 5 ring carbon atoms and one carboxylic 
group on one of the ring carbon atoms. In one embodiment of the invention, 
the non-protein amino acid is methanoproline or, an azetidinecarboxylic 
acid, preferably, azetidine-3-carboxylic acid. 
Functional derivative sources of either protein or non-protein amino acids 
include microbiologically acceptable salts, such as hydrohalide, alkali or 
alkaline-earth metal salts and the like or the readily decomposable ester, 
amide or hydrazide of such acids, including those comprising or 
substituted by an alkyl, alkenyl or aralkyl group of up to 10 carbon 
atoms. 
The process of the invention is useful for the production and recovery of 
Monascus pigments under conditions and with growth media conventionally 
known and used in the art. 
The amount of free protein amino acid present is that conventionally known 
and used in the art. Monascus sp. are usually cultured in aqueous growth 
media containing as little as about 0.1% wt yeast extract based on the 
total growth media as the source of free protein amino acid but amounts up 
to 1 or even 5 to 10% wt based on the total growth media in otherwise 
conventional kinds of growth media are within the present invention. 
The amount of non-protein amino acid used to treat the Monascus species is 
an amount sufficient to increase pigment production by Monascus in the 
presence of a protein amino acid and is readily determined by simple 
growth test. Generally, the range of non-protein amino acid used is the 
same or less than the range of free protein amino acid which can be used. 
In one embodiment of the invention, the non-protein amino acid used in the 
range of about 0.001 to about 10% wt based on the total growth media. In 
one embodiment of the invention, the non-protein amino acid is present in 
the range of about 1 to 5% wt based on the total growth media. 
The invention also includes a composition for producing enhanced pigment 
production which comprises 
(a) a source of Monascus species, which will produce pigment in the 
presence of a free protein amino acid; 
(b) a free protein amino acid; and 
(c) a free non-protein amino acid. The source of Monascus is conveniently a 
conventional culture medium containing the species. The composition 
conveniently comprises major amount of said culture media and the 
previously specified amount of each acid. 
The pigments are recovered by conventional procedures known in the art to 
obtain the pigments usually in concentrated or freeze-dried form for 
convenient use as colorants, especially for food.

ILLUSTRATIVE EMBODIMENTS 
The following embodiments are presented to illustrate the invention and 
should not be regarded as limiting it in any way. 
EMBODIMENT 1 
Monascus sp. 1356 was subcultured onto media slants of 5 ml containing 4% 
glucose. 0.3% KH.sub.2 PO.sub.4, 1% yeast extract (Difco), 1.5% agar 
(Difco). After 7 days at room temperature, disks were cut from the slants 
with a cork borer. One disc was added to 50 ml of the above medium 
containing 1% yeast extract into which 0.02% amount of non-protein amino 
acids were dissolved. There were three flasks (replicates) per treatment. 
Prior to autoclaving, the pH of the medium was taken and was in the range 
5.8-6.1. After innoculation, the flasks were placed on a shaker and left 
under 24 h illumination. 
After 8 days incubation, the media was decanted off and, the pH was again 
measured. Final pH was in the range 7.1-7.6. The optical density (OD) of 
the media was read at 290 nm, 390 nm and 500 nm. Some samples had to be 
diluted to one sixth the original concentration with water. When this was 
necessary the OD was multiplied by the dilution factor. The mycelial mats 
were removed from the flasks and immediately frozen in liquid nitrogen. 
The mats were then wrapped in aluminum foil, freeze dried and weighed. The 
mats were then crumbled and a sample (50 mg) from each mat was extracted 
in 2 ml methanol, centrifuged and the OD determined against a methanol 
blank. Dilutions, up to six-fold were made when necessary. 
The results of the above tests are set forth in Table I below. 
TABLE I 
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(A) Pigment Production by Monascus Treatment 
OD of Culture Medium into which 
Pigment was Secreted 
290 nm 390 nm 500 nm 
______________________________________ 
Minus Yeast Extract 
Control 0.63 0.26 0.03 
+ Azetidine-3-carboxylic acid 
0.80 0.17 0.03 
+ Methanoproline 0.48 0.18 0.02 
+ Proline 0.84 0.34 0.04 
Plus Yeast Extract 
Control 2.70 0.76 0.21 
+ Azetidine-3-carboxylic acid 
6.43 2.00 0.55 
+ Methanoproline 5.71 2.06 0.46 
+ Proline 2.93 0.79 0.17 
______________________________________ 
(B) OD of the Extract of the Fungal Mat 
290 nm 390 nm 500 nm 
______________________________________ 
Minus Yeast Extract 
Control 2.01 1.94 0.09 
+ Azetidine-3-carboxylic acid 
1.93 0.88 0.16 
+ Methanoproline 1.58 1.66 0.06 
+ Proline 2.19 3.15 0.19 
Plus Yeast Extract 
Control 2.89 0.37 0.26 
+ Azetidine-3-carboxylic acid 
7.93 6.30 2.78 
+ Methanoproline 7.18 6.30 2.05 
+ Proline 2.58 0.71 0.47 
______________________________________ 
EMBODIMENT 2 
Experimental details were similar to Embodiment 1 but incubation was of 
shorter duration. Concentration of the non-protein amino acids were 0.02%. 
Concentration of the yeast extract was 1%. The pH of the media at the 
beginning of the incubation ranged from 5.16 to 5.52. At the end of the 
incubation period the pH ranged from 4.6 to 7.0. 
The results of the test are in Table 2. 
TABLE 2 
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Pigment Production by Monascus 
O.D. of medium into which 
Pigment was Secreted 
Treatment 290 nm 390 nm 500 nm 
______________________________________ 
Control 0.19 0.19 0.03 
+ Azetidine-3-carboxylic acid 
0.70 0.31 0.07 
+ Aminoisobutyric acid 
0.20 0.30 0.06 
+ Beta-alanine 1.04 0.37 0.08 
______________________________________ 
EMBODIMENT 3 
Colonies of Monascus sp. 1361 were innoculated on to solid aqueous media 
containing 50% tomato juice (V-8 brand name). 2% Bakers yeast, 2% agar, pH 
=5.7. The media also contained either 0.001%, 0.01% or 0.1% 
azetidine-3-carboxylic acid. The plates were then scored visually for red 
pigment production. Results are in Table 3. 
TABLE 3 
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Visual Scoring of Pigment Production by Monascus 
Treatment Score # 
______________________________________ 
Control 0 
+0.001% Azetidine-3-carboxylic acid 
2 
+0.01% Azetidine-3-carboxylic acid 
5 
+0.1% Azetidine-3-carboxylic acid 
9 
______________________________________ 
#0 = White: 1 = pink tinges to culture: 5 = 50% culture red 
10 = 100% culture red. 
Results of these experiments demonstrate that non-protein amino-acids, such 
as azetidine-3-carboxylic acid and methanoproline, significantly increase 
(red and orange) pigment production by Monascus species in the presence of 
free protein amino acids. The degree of stimulation is dependent upon dose 
of the non-protein amino acid. Related free protein amino acids, e.g. 
proline, do not increase pigment production to the same extent as 
non-protein amino acids.