chemical engineering research and design 9 2 ( 2 0 1 4 ) 784–791

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Chemical Engineering Research and Design

journal homepage: www.elsevier.com/locate/cherd

Evaluation of a novel low-cost culture medium containing

exclusively milk, potato and glycerol for microbial
transglutaminase production by Streptomyces mobaraensis

Esther Guerra-Rodríguez, Manuel Vázquez ∗

Food Technology Area, Department of Analytical Chemistry, Faculty of Veterinary Science, University of Santiago de Compostela (Campus
Lugo), 27002 Lugo, Spain

a b s t r a c t

The aim of this work was to evaluate the production of microbial transglutaminase (MTG) by Streptomyces mobaraensis
in a reduced nutritional medium based on non-commercial potatoes. Initially, the production was studied com-
paring 3 culture media based on potato: (i) media based on acid hydrolysed potato, (ii) media based on gelified
non-hydrolysed potato and (iii) media based on gelified non-hydrolysed potato supplemented with glucose (20 g/L).
All media were supplemented with nutrients and inductors of MTG production (yeast extract, peptone, sodium
caseinate, glycerol and mineral salts such as sodium phosphate, potassium phosphate, magnesium sulphate). The
best medium was gelified non-hydrolysed potato allowed to obtain up to 2.72 U/mL at 96 h. In a second study, it was
proposed the substitution of all the nutrients and inductors by exclusively skim milk and glycerol. An experimental
design was performed to optimize the composition of milk, potato and glycerol. The economical yield of the process
was also evaluated. Fermentations were carried out and samples were taken at 72 h and 96 h. Results showed that the
maximum MTG activity was obtained at 72 h. A significant effect of potato and milk on MTG activity was observed,
including an effect of interaction between both compounds. Optimal conditions selected were: 600 g/L of skim milk,
40 g/L of potato and 5 g/L of glycerol, yielding a maximum activity of 3.2 U/mL and an economic yield of 8.11 D of
MTG/D of nutrients. The results showed that the medium milk–potato–glycerol was feasible for the production of
transglutaminase, obtaining a high MTG activity in a simple natural medium.
© 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Potato; Milk; Transglutaminase; Streptomyces mobaraensis; Solanum tuberosum

1. Introduction et al., 2006; Castro-Briones et al., 2009; Ramirez et al., 2002;

Transglutaminase has been found in animals, plants, and
microorganisms. The physiological functions of transglu-
taminase from microorganisms remain unclear (Beninati
and Piacentini, 2004). Transglutaminase (protein–glutamine
gamma-glutamyltransferase, EC 2.3.2.13) is a family of
enzymes that have been evolved for the cross-linking of pro-
teins (Sommer et al., 2011).
Microbial transglutaminase (MTG) is used in Food Tech-
nology to restructure protein foods such as meat, dairy
and fish products. Furthermore, stability, water binding,
mechanical and textural properties can be enhanced (Bonato
Tellez-Luis et al., 2002; Uresti et al., 2004). The increas-
ing demand for vegetarian food, the utilization of novel
proteins as functional ingredients from soy, peas, sesame
and sunflower open new horizons for transglutaminase
applications.
Thinking in the industrial production, the enzyme can be
obtained by Streptomyces mobaraensis (Nagy and Szakacs, 2008).
The composition of the media used to produce MTG from
Streptomyces species have been almost the same in all works
published (Ando et al., 1989). It contains yeast extract, pep-
tone, sodium phosphate, potassium phosphate, magnesium
sulphate and a carbon source.

∗
Corresponding author. Tel.: +34 982 822420; fax: +34 982 241835.
E-mail address: manuel.vazquezm@usc.es (M. Vázquez).
Received 30 April 2013; Received in revised form 21 June 2013; Accepted 24 June 2013
0263-8762/$ – see front matter © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cherd.2013.06.027
 chemical engineering research and design 9 2 ( 2 0 1 4 ) 784–791
The carbon source commonly used is glucose. Among oth-
ers, sucrose, starch, solubilized starch and sucrose, starch and
dextrins have also been used (Guerra-Rodríguez and Vazquez,
2013). However, to be economically interesting, industrial
production requires culture media based on cheaper raw
materials. Additionally, the formulation of the culture media
is of critical importance because the composition affects prod-
uct concentration, yield and volumetric productivity (Bahrim
et al., 2010; Portilla Rivera et al., 2009; Tellez Luis et al., 2004b).
Fermentation media can represent almost 30% of the cost
for a microbial production (Portilla Rivera et al., 2009). General
media employed for the growth of Streptomyces are not eco-
nomically attractive. This contains expensive components as
peptone and yeast extract, and many mineral salts.
Cheaper media based on wastes from the agro-industry
have been studied for the production of MTG. For example, the
MTG production on acid hydrolysates of sorghum straw (Tellez
Luis et al., 2004a), acid hydrolysates of wheat straw (Guerra-
Rodríguez et al., 2012b), sugar cane molasses (Portilla Rivera
et al., 2009) and enzymatic or acid hydrolysates of potato
(Guerra-Rodríguez and Vazquez, 2013; Guerra-Rodríguez et al.,
2012b, a) were proposed. The main drawback of acid hydroly-
sis is the generation of degradation products, such as furfural,
5-(hydroxymethyl)-2-furaldehyde and acetic acid that remain
in the glucose solutions. These by-products are microbial
growth inhibitors and must be controlled under lethal concen-
trations to allow posterior fermentations (Guerra-Rodríguez
et al., 2012b; Wang et al., 2012; Zeni et al., 2011).
Enzyme hydrolysis of potato has the advantage that gen-
erates glucose solutions that can be fermented without
further treatments. No microbial growth inhibitors exist in the
enzyme hydrolysis (Delgado et al., 2009; Vazquez et al., 2009).
However, the process has still steps of hydrolysis that are
time consuming and enzyme consuming. Besides the potato
hydrolysates are a carbon source for the microbial growth but
it needs the supplementation with other nutrients that pro-
vide nitrogen and micronutrients. Yeast extract or peptone is
frequently used but their costs are high (Bustos et al., 2004;
Tellez-Luis et al., 2003).
Glycerol (1,2,3-propanetriol or glycerine) was also studied
as carbon source for the production of MTG (Tellez Luis et al.,
2004b). But the other nutrients of the general medium were
also needed.
It is of interesting to find alternative raw materials of
low cost for preparing fermentation media. Non-commercial
potatoes (Solanum tuberosum) have a cost lower than 0.2 D kg
(Montouto-Grana et al., 2012; Vazquez et al., 2009). Non-
commercial potatoes are those that cannot be sold due to
defects, such as small size, fissures, cuts, tuber deformity.
These are agricultural wastes that can be used as media for
the microbial production of transglutaminase. Caseinate and
starch has been reported as inductor of the production of MTG
(Junqua et al., 1997; Tellez Luis et al., 2004a). Glycerol can be
use as carbon source and potato contains mainly starch.
The first aim of this study was to evaluate the status of
potato more feasible for MTG production. Medium based on
acid hydrolysed potato (H), gelified non-hydrolysed potato
(P) and gelified non-hydrolysed potato supplemented with
glucose (20 g/L) (PG) were evaluated. All media were supple-
mented with the usual nutrients (see Section 2).
Considering that milk is a food that contains mostly water,
casein, minerals, vitamins and lactose. The second aim was
to evaluate skim milk, potato and glycerol exclusively as a
novel reduced culture medium for the production of microbial
785
transglutaminase, without acid hydrolysis stages or neu-
tralization of the acid obtained, without addition of other
nutrients such as sodium caseinate, yeast extract, peptone or
magnesium salts and phosphates.
The low-cost medium based on milk (M), potato (P) and
glycerol (G) was optimized for the production of transglutam-
inase by S. mobaraensis. The effect of these compounds were
determined and modelled, including an economical evalua-
tion based on economic yield of nutrients.
2. Materials and methods
2.1. Materials
Potatoes supplied by local farmers (Dozón, Spain) were
washed, sliced, dried and ground to be used in this work.
The water extracted from potatoes was 80.35% of the fresh
weight. The dried potatoes contained residual 8.13% moisture.
Commercial skim milk (Feiraco, Spain) was obtained in local
markets.
The microorganism employed in this work was S. mobaraen-
sis CECT 3230 obtained by the Spanish Collection of Type
Cultures (http://www.cect.org/). The fermentation experi-
ments were carried out in orbital shakers at 28 ◦ C, 250 RPM
allowing a fermentation time of 72 or 96 h.
Experiments were carried out in 250 mL Erlenmeyer flasks
with a final volume of 100 mL and in 5 L fermentor (Minifors,
Infors AG, Bottmingen/Basel, Switzerland) with a working vol-
ume of 3 L.
The culture media based on potato were supplemented
with the following nutrients: Sodium dibasic phosphate (5 g/L),
sodium monobasic phosphate (2 g/L), magnesium sulphate
(0.5 g/L), yeast extract (5 g/L), sodium caseinate (15 g/L) and
glycerol (10 g/L).
The composition of culture media based on skim milk,
potato and glycerol is shown in the Section 3.
2.2. Analytical methods
Samples were centrifuged for 15 min at 10,000 × g at room tem-
perature. The supernatant was also used for glucose, glycerol
and transglutaminase activity determination.
Glucose and glycerol was measured by HPLC using a Rezex
RHM (Phenomenex, Torrance, California, USA) column with
isocratic elution (flow rate of 0.400 mL/min and mobile phase
of 0.025 M H2 SO4 ), a column oven set at 45 ◦ C and a refractive
index detector (LC 2000 plus, Jasco, Tokio, Japan).
Transglutaminase activity was determined with colori-
metric procedure described by Grossowicz et al., 1950.
N-␣-CBZ-gln-gly (Sigma) was used as substrate. A calibration
curve was made using l-glutamic acid ␥-monohydroxamate
(Sigma). One unit of activity (U) was defined as the amount
that causes the formation of 1 ␮mol of hydroxamate in 1 min
at 37 ◦ C.
2.3. Modelling and statistical methods
The set of experiments followed a second-order, incomplete,
factorial design. Skim milk, potato and glycerol were consid-
ered as operational variables (denoted M, P and G, respectively)
and their effects on the select dependent variable (trans-
glutaminase activity for 72 and 96 h) were calculated. For
786 chemical engineering research and design 9 2 ( 2 0 1 4 ) 784–791

Table 1 – Variables involved in the experiments design (Box–Behnken) for the production of transglutaminase.
Nomenclature Units Variation levels

(a) Fixed variables

Agitation A rpm 200
◦
Temperature T C 28
(b) Independent variables (factors)
Milk M g/L (300, 450, 600)
Potato P g/L (20, 30, 40)
Glycerol G g/L (5, 10, 15)
(c) Dimensionless, normalized independent variables
Milk x1 g/L (−1, 0, 1)
Potato x2 g/L (−1, 0, 1)
Glycerol x3 g/L (−1, 0, 1)
(d) Dependent variable (response)
Transglutaminase activity for 72 h TG72 U/mL
Transglutaminase activity for 96 h TG96 U/mL

computation purposes, the normalized, dimensionless vari-
ables x1 , x2 , x3 were defined as:
M − 450
X1 = (1)
150
P − 30
X2 = (2)
10
G − 10
X3 = (3)
5 obtained. Using the non-hydrolysed potato with glucose (PG),
The set of experiments followed the Box–Behnken design (Box
and Behnken, 1960). This design is formed by combining two-
level factorial designs with incomplete block designs. This
procedure creates designs with desirable statistical proper-
ties but with only a fraction of the experiments required for a
three-level factorial design.
Table 1 summarizes the variables involved in the exper-
imental design for the production of transglutaminase. The
values of fixed variables (agitation and temperature) were
selected on the basis of previous studies (Delgado et al., 2009;
Portilla Rivera et al., 2009; Vazquez et al., 2009).
The experiment design was statistically analyzed using the
Design Expert® 7.1.1 software (Stat-Ease, Inc., Minneapolis,
MN, USA). The model was validated through ANOVA test. All
experiments were performed in triplicate.
3. Results
3.1. Study of the best potato processing for MTG
production
The production of MTG by S. mobaraensis was studied com-
paring 3 culture media based on potato: medium based
on acid hydrolysed potato (H), medium based on gelified
non-hydrolysed potato (P) and medium based on gelified non-
hydrolysed potato supplemented with glucose (20 g/L) (PG).
The results of these fermentations are shown in Fig. 1.
Glycerol was depleted quickly. In the medium based on geli-
fied non-hydrolysed potato, the initial concentration of 9.7 g/L
was depleted at 72 h. The microorganism did not have other
accessible carbon source as glucose, and then it use glycerol
as carbon source. In medium PG, 1.9 g/L of glycerol remained
in the medium at 72 h and glycerol was totally depleted at
96 h. In the acid hydrolysed potato medium, the intake rate for
glycerol was slower. From an initial 10.9 g/L of glycerol, 6.2 g/L
remained at 72 h and at the end of the fermentation at 96 h,
3.6 g/L remained in the culture medium.
Glucose is not in the culture medium based on gelatinized
potato since the starch is not hydrolysed. In the medium of
gelatinized potato supplemented with glucose (16 g/L), it was
consumed until at residual value of 5 g/L at 96 h. In the acid
hydrolysed potato medium, the initial glucose concentration
was 20 g/L and 14 g/L remained at 96 h.
The production of MTG was different in function of the
composition of the culture media. Using the media (H) con-
taining acid hydrolysed potato, up to 0.898 ± 0.063 UA/mL was
the MTG activity obtained was 0.990 ± 0.060 UA/mL at 96 h. In
both fermentations the activity decreased at 120 h. The best
result was obtained using the non-hydrolysed potato with-
out glucose (P). A MTG activity of 2.713 ± 0.049 UA/mL was
obtained at 96 h. The activity decreased to 2.643 UA/mL at
120 h.
On the basis of these results, it was proposed the use of a
medium containing non-hydrolysed potato without glucose.
As commented above, this medium also contains many nutri-
ents such as mineral salts and casein. Considering that milk is
a source of casein and that milk also contains mineral salts, in
the next sections it was studied the substitution of all nutri-
ents and inductors just for milk and glycerol. The proposed
medium contains just potato, glycerol and milk.
3.2. Novel process proposed
Briefly, potatoes were washed and sliced. Then they were
dried at 60 ◦ C. The residual moisture was 8.13%. The dried
potatoes were ground and mixed with water, skim milk and
glycerol. This medium was sterilized and inoculated with S.
mobaraensis. The fermentations were stopped at 72 h or 96 h
and the biomass was separated by centrifugation. The liquid
was the enzymatic crude extract containing microbial trans-
glutaminase. Experiments were performed to optimize the
composition of nutrients that allow the highest transglutam-
inase activity in the crude extracts.
3.3. Mathematical modelling
The nutrient composition used (in terms of dimensional and
dimensionless operational variables) as well as the experi-
mental results obtained at 72 h and 96 h of fermentation are
shown in Table 2.
The interrelationship between nutrient composition vari-
ables can be established through an equation including linear,
interaction and second-order terms. The mathematical model
 chemical engineering research and design 9 2 ( 2 0 1 4 ) 784–791
Fig. 1 – Glucose and glycerol uptake and transglutaminase
production by S. mobaraensis in media based on hydrolysed
potato (H), media based on gelified non-hydrolysed potato
and (P) media based on gelified non-hydrolysed potato
supplemented with glucose (PG).
used as first approach was a polynomial model of quadratic
order as shown in Eq. (4):
  
TGh = a0 + ai xi + bi xi2 + cij xi xj (4)
i i i j
where TGh is transglutaminase activity obtained after h hours
of fermentation; xi are the normalized dimensionless variables
(x1 , x2 , x3 ) and a0 , ai , bi , cij are regression coefficients calculated
fitting the experimental data by multiple regression. Partial
models of the quadratic model were also fitted.
The results of transglutaminase activity at 96 h (TG96) were
in the range 0.014–1.198 U/mL meanwhile the results at 72 h
(TG72) were in the range 0.035–2.994 U/mL. As at 72 h the
787
0.75
Cook's Distance
0.50
0.25
0.00
1 3 5 7 9 11 13 15
Run Number
Fig. 2 – Cook’s distance for the case of the model.
activity obtained is higher than at 96 h, only the model for
the fermentation at 72 h is presented.
Table 3 shows the sum of squares for the partial and com-
plete models. Data fitted well to a 2-factor interaction model
(2FI) due to the lowest value (0.0009) for the p-value calcu-
lated. Therefore, the mathematical model 2FI was selected and
fitted. The F-value of 22.68 implies that the model was sig-
nificant. There is only a 0.01% chance that this large “Model
F-value” could occur due to noise. The value of “p-value proba-
bility > F” was lower than 0.0001. Values of “prob. > F” less than
0.0500 indicate that the model terms are significant.
The value of r2 was 0.9445. This statistical parameter is
usually utilized to determine the correlation of a model. How-
ever, it is better to compare the values of “adjusted r2 ” and
“predicted r2 ”. Parameter “adjusted r2 ” is a measure of the
amount of variation around the mean explained by the model,
adjusted for the number of terms in the model. The “adjusted
r2 ” decreases as the number of terms in the model increases if
those additional terms do not add value to the model. The
“predicted r2 ” is a measure of the amount of variation in
new data explained by the model. The “predicted r2 ” obtained
(0.7272) is in reasonable agreement with the “adjusted r2 ”
obtained (0.9028). The predicted r2 and the adjusted r2 should
be within 0.20 of each other. Otherwise, there may be a
problem with either the data or the model. Other statistical
parameter used to evaluate the significances of a model is
“Adequate precision”. This measures the signal/noise ratio.
A ratio greater than 4 is desirable. The ratio obtained (17.52)
indicates an adequate signal. All these statistical parameters
confirm that the empirical model obtained can be used to
model the production of transglutaminase in the medium of
skim milk, potato and glycerol studied.
However, to obtain a robust model, the Cook’s distance can
be determined. This is a measure of how much the regression
changes if a sample is deleted; relatively large values are asso-
ciated with cases with high leverage and large studentized
residuals. Large values should be investigated because they
could be caused by recording errors, an incorrect model, or a
design point far from the remaining cases. “Large” is defined
788 chemical engineering research and design 9 2 ( 2 0 1 4 ) 784–791

Table 2 – Design of experiments and results of transglutaminase activity obtained at 72 and 96 h of fermentation.
Experiments Independent variables Dependent variable Dependent variable

Dimensional (g/L) Dimensionless TG72 h (U/mL) TG96 h (U/mL)

M P G x1 x2 x3

1 600 20 10 1 −1 0 1.166 0.466

2 300 20 10 −1 −1 0 0.449 0.180
3 450 20 5 0 −1 −1 0.672 0.269
4 600 30 5 1 0 −1 2.131 0.852
5 450 30 10 0 0 0 1.158 0.463
6 450 20 15 0 −1 1 0.685 0.274
7 300 40 10 −1 1 0 0.438 0.175
8 450 30 10 0 0 0 0.970 0.388
9 600 30 15 1 0 1 2.051 0.820
10 300 30 15 −1 0 1 0.035 0.014
11 450 40 15 0 1 1 1.328 0.398
12 450 40 5 0 1 −1 1.352 0.541
13 450 30 10 0 0 0 1.129 0.452
14 600 40 10 1 1 0 2.994 1.198
15 300 30 5 −1 0 −1 1.044 0.418

Table 3 – Summary of the sequential model of squares.

Source Sum of squares df Mean square F-value p-value prob. > F

Mean vs. total 18.8616071 1 18.8616071

Linear vs. mean 6.611995 3 2.20399833 16.6558744 0.0003
2FI vs. linear 1.18107775 3 0.39369258 19.3830693 0.0009
Quadratic vs. 2FI 0.09676094 3 0.03225365 2.84065685 0.1695
Cubic vs. quadratic 0.0249285 2 0.01246425 1.21669704 0.4511
Residual 0.02048867 2 0.01024433
Total 26.796858 14 1.91406129

Table 4 – Analysis of variance (ANOVA) deleting experiment 12.

Source Sum of squares df Mean square F-value p-value prob. > F

Model 7.79 6 1.30 63.95 <0.0001

x1 (M) 1.11 1 1.11 54.61 0.0002
x2 (P) 1.37 1 1.37 67.36 <0.0001
x3 (G) 0.01 1 5.33E-003 0.26 0.6243
x1 ·x2 0.84 1 0.85 41.63 0.0003
x1 ·x3 0.22 1 0.22 10.62 0.0139
x2 ·x3 0.12 1 0.12 5.90 0.0455
Residual 0.14 7 0.02
Lack of fit 0.12 5 0.02 2.38 0.3223
Pure error 0.02 2 0.01
Cor total 7.94 13

as a point that is 2–3 times larger than the other points. Fig. 2
shows Cook’s Distance for the model. It can be seen that exper-
iment 12 have a great influence on the model, it suggests an
experimental error and it is necessary to delete this observed
value to improve the fitting.
A new regression model was obtained deleting experiment
12. Table 4 showed the coefficients recalculated by ANOVA.
The F-value increased up to 63.95. The value of “p-value prob-
ability > F” was also lower than 0.0001. The “lack of fit F-value”
of 0.322 implies that the lack of fit is not significant relative to
the pure error (non-significant lack of fit is good).
The value of r2 was 0.9820. The “predicted r2 ” obtained
(0.9327) is in reasonable agreement with the “adjusted r2 ”
obtained (0.9667). The “adequate precision” was also increased
up to 30.00.
All these statistical parameters confirm that the empiri-
cal Eq. (5) obtained can be used to model the production of
transglutaminase in milk–potato–glycerol medium.
TG72h = 2.0585 − 0.0069 M − 0.0448 P − 0.0572 G + 0.0003 M
·P + 0.0003 M · G − 0.0043 P · G (5)
where M, P and G are the uncoded variables in g/L (Table 1).
The evaluation of the coefficients obtained shows that MTG
production is mainly affected by the concentration of potato,
followed by the concentration of skim milk. The linear effect of
glycerol was not significant. However the interaction of glyc-
erol with milk was significant.
The response surface of the model is shown in Fig. 3.
It is shown the effect of the concentrations of milk and
potato on MTG activity obtained at 72 h of fermentation.
The model predicts a maximum activity of 3.21 U/mL using
600 g/L of skim milk, 40 g/L of potato and 5 g/L of glycerol.
 chemical engineering research and design 9 2 ( 2 0 1 4 ) 784–791
Fig. 3 – Prediction of the model for transglutaminase
activity (U/mL) obtained in the fermentation at 72 h on milk,
potato and glycerol medium.
A set of four confirmation experiments was performed to
verify the optimum obtained. The results gave an average of
2.95 ± 0.30 U/mL.
The optimal medium was tested in laboratory fermenter
with a working volume of 3 L. The results showed that the
MTG production was slower. It was obtained up to 1.65 U/mL
at 120 h of fermentation. This is a 56% of the activity obtained
in shaker fermentation.
Fig. 4 shows the monitoring of dissolved oxygen (DO) and
pH during the fermentation process in 3 L fermenter. DO con-
centration is one of the most important environmental factors
affecting cell growth and product formation. It can be seem a
clear high demand of oxygen by S. mobarensis. Following a two-
stage agitation speed control strategy, other authors obtained
up to DO level of 35% in the first 24 h and 25% in the rest of
fermentation (Yan et al., 2005). In our study, DO levels of 35%,
32% and 30% were obtained at 31, 38 and 72 h of fermenta-
tion, respectively. These peaks of DO level could be associated
with changes in the metabolism of the S. mobarensis where the
oxygen demand decreases. This premise is supported by the
pH increase observed after each peak of DO level. Note MTG
is an enzyme that catalyses reactions that release ammonia,
increasing the pH. The maximum level of pH can be associated
100
pO2
pH
80
Dissolved oxygen, %
60
40
20
0 6.2
0 20 40 60 80 100
Time, h
Fig. 4 – Kinetics of pH and dissolved oxygen level in 4 L
fermentation using the milk, potato and glycerol medium.
789
with the maximum MTG activity in the media. In this case, at
107 h should be obtained the maximum MTG activity.
Although MTG activity was lower than that obtained in
shaker fermentation, modifications in the vessel, blades and
operational conditions in the fermenter allowed to obtain sim-
ilar results that in shaker fermentation. These data are not
shown because they are protected under industrial secret. A
patent of this process was obtained and licensed for industrial
exploitation (Vázquez and Guerra-Rodriguez, 2012).
4. Discussion
These results are very good comparing with those obtained
using other agricultural wastes as fermentation media. Using
hydrolysates of sorghum straw, up to 0.348 UA/mL were
obtained (Tellez Luis et al., 2004a). Using sugar cane molasses
with glycerol, it was obtained up to 0.460 UA/mL (Portilla Rivera
et al., 2009). Using glycerol alone as carbon source, up to
0.725 U/mL was obtained (Tellez Luis et al., 2004b). Using enzy-
matic hydrolysates of potato supplemented with yeast extract,
corn steep liquor and casein, up to 1.128 U/mL was obtained
(Guerra-Rodríguez and Vazquez, 2013).
The result obtained in our work, 2.95 ± 0.30 U/mL, was more
than 300% of the obtained using the cited agricultural wastes
as fermentation media.
Moreover the cited results are at 96 h of fermentation. In
our optimized medium the fermentation is speed up and in
72 h around 3 U/mL is obtained.
Many of the formulations based on agricultural wastes are
supplemented with casein. For example, using sugar cane
molasses with glycerol, 38.4 g/L of casein was needed (Portilla
Rivera et al., 2009), using glycerol as carbon source, 20 g/L of
casein was determined as optimum (Tellez Luis et al., 2004b)
and using enzymatic hydrolysates of potato, 13.7 g/L was used
(Guerra-Rodríguez and Vazquez, 2013).
Our medium contains milk, the natural source of casein.
Therefore it is not needed to add casein. Milk is also a source
of mineral salts. Then it is not needed the addition of diba-
sic sodium phosphate, monobasic potassium phosphate and
magnesium sulphate that are used in the other media cited.
The formulations based on agricultural wastes usually
include also peptone and yeast extract that are expensive
nutrients for the industrial production. Our medium is com-
posed exclusively by milk, potato and glycerol, being clearly
cheaper.
The results obtained, around 3 U/mL, compare very well
with the results obtained using mutant strains of S. mobaraen-
sis grew on general media containing mineral salts, peptone,
7.4
yeast extract and others (Yan et al., 2005).
7.2 From a technical point of view, the conditions to allow
the highest transglutaminase activity could be considered
7 the target of the optimization process. However, economic
aspects should be also considered. In order to provide a better
pH
6.8 optimization of the process, the economic yield of trans-
glutaminase (EY) can be defined as monetary units (D ) of
6.6
transglutaminase produced per monetary unit (D ) spent in
nutrients (Bustos et al., 2004). This is an important parame-
6.4
ter for evaluating the economical performance of the process.
From material balances, EY depends on transglutaminase
120 activity obtained according to the following equation:
Y
EY =  I (6)
(C · N)
790 chemical engineering research and design 9 2 ( 2 0 1 4 ) 784–791
Fig. 5 – Prediction of the model for economic yield EY (D /D )
obtained in the fermentation at 72 h on milk, potato and
glycerol medium.
where C is the cost of each nutrient (D /g), I is the sell-
ing price of transglutaminase (D /U) and N is each nutrient
concentration (g/L) and Y is the transglutaminase activity
(in U/L). The dependence of economic yield on the medium
composition can be predicted from the empirical model and
equation above. For calculation purposes, the cost of each
nutrient was fixed in values obtained in local markets: skim
milk, 0.40 D /kg; potato, 0.20 D /kg; glycerol, 0.74 D /kg. The
selling price of transglutaminase (0.00065 D /U) was inferred
from the market value showed in the website of a pro-
ducer https://www.e-shoponline.es/epages/eb4607.sf/es ES/?
ViewObjectID=247497 where 65.61 D /kg is the price of a prod-
uct containing 100 U/g.
The values obtained of EY of the experiments of Table 2 at
72 h showed that the cost of the nutrients is lower than the
income obtained from the transglutaminase generated. The
values of EY are in the range 0.174–7.99 D /D .
The model obtained for the transglutaminase activity can
be optimized to obtain the maximum economic yield. The
composition of medium for the maximum economic yield was
similar than for the maximum transglutaminase activity as
can be seen in Fig. 5.
Therefore, taking into account these considerations, the
technical and economical optimization gave as optimal con-
ditions: using 600 g/L of skim milk, 40 g/L of potato and 5 g/L
of glycerol. It was obtained a maximum economic yield of
8.11 D (obtained selling transglutaminase) per each D spent
in nutrients.
The advantages of the novel medium proposed compared
with those cited can be resumed as following: (a) decreasing
the number of usual medium components; (b) simplifying the
process; (c) decreasing of medium cost; (d) decreasing of fer-
mentation time; (e) increasing of transglutaminase activity; (f)
increasing of economic yield.
5. Conclusions
A novel medium composed exclusively by skim milk, potato
and glycerol was developed. The mathematical models
obtained for the effect of the medium composition on the
obtained transglutaminase activity shows a significant effect
of milk and potato on the transglutaminase activity, being the
effect of glycerol non-significant. This implies that glycerol
can be needed, but in the range of study the differences in
transglutaminase activity are not due to the effect of the level
of glycerol.
The optimization gave as optimal conditions: 600 g/L of
skim milk, 40 g/L of potato and 5 g/L of glycerol. Using this
reduced medium, a maximum activity of 2.95 ± 0.30 U/mL at
72 h was obtained.
As final conclusion, skim milk, potato and glycerol are a
culture medium feasible for the production of transglutami-
nase by S. mobaraensis. This gives an added value to milk and
non-commercial potatoes.
Acknowledgement
The authors are gratefull to Ministerio de Educación y Ciencia
(Spain) for the financial support of this work (Project: AGL2006-
08250/ALI) and the FEDER founds of the European Union.
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