Biotechnology Techniques 13: 93–95, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Use of surface response methodology to describe biomass production of Bifidobacterium infantis in complex media A. Azaola∗ , P. Bustamante, S. Huerta, G. Saucedo, R. Gonz´alez, C. Ramos & S. Saval1 Universidad Aut´onoma Metropolitana, Departamento Sistemas Biol´ogicos, Planta Piloto. A.P. 23-161, M´exico ´ 16000. D.F.; 1 Universidad Nacional Aut´onoma de M´exico, Instituto de Ingenieria ∗Author for correspondence (Fax: (525) 724 5267; E-mail:
[email protected]) Received 9 October 1998; Accepted 22 December 1998
Key words: bifidobacteria, complex substrates, factorial design, biomass production, surface response
Abstract An optimized medium containing Trypticasein, phytone, yeast extract and glucose is proposed to improve growth of bifidobacteria with high yeast extract concentration and decreased amounts of Trypticasein and phytone. These new growth media overcome nutritional limitations by the type and amount of amino acids contained in these sources and produced an increase from 1.8 to more than 4 g cell mass l−1 .
Introduction The large intestine of humans is inhabited by a complex collection of microbes, mostly bacteria known as the normal microflora. The predominant species are obligatory anaerobes, among them are members of the genus Bifidobacterium, which are reported to have benefical effects on the host. These species colonize the intestinal tract soon after birth and are present in large populations in breast-fed infants (Benno et al. 1984). However, changes in nutritional habits can diminish the number of these bacteria (Finegold et al. 1974). Bifidobacteria have recently received much attention as inclusions in new dairy products as probiotics. For their production, some authors have described optimal conditions for growth of bifidobacteria, but do not explain the influence of the medium ingredients or their interactions (Tien-Meng & Schaffner 1997). Little attention has been paid to the effect of complex media and their role remains unclear (Bibal et al. 1989). To compensate for this, factorial design and mathematical models have been developed (Watier et al. 1996). The development of statistical models allows the microbiologist to make an accurate prediction of the growth medium composition to obtain
optimal bacterial growth with speed and confidence (Baird-Parker & Kilsby 1987; Saval et al. 1993). This work used factorial designs and response surface models to account for the main growth factors and their interaction during growth of Bifidobacterium infantis.
Material and methods Bifidobacterium infantis ATCC 17930 was grown on basal TPYG medium consisting of (gl−1 ): trypticase peptone, 10; phytone peptone, 5; yeast extract, 2.5; glucose, 5; cysteine · HCl, 0.5; K2 HPO4 , 2; MgCl2 · 6H2 O, 0.5; ZnSO4 · 7H2 O, 0.25; CaCl2 , 0.15; FeCl3 , 0.03; 1 ml Tween 80, and 1 ml 0.25% Resazurin (w/v) was added as anaerobiosis indicator. pH was adjusted at 6.9–7.0. 30 ml of medium were added to 50 ml vials; anaerobiosis was produced by gassing pure CO2 , the vials were hermetically sealed and sterilized at 121 ◦ C for 15 min. Subsequently, they were inoculated with an overnight culture of B. infantis to give 0.15–0.20 mg cells ml−1 . Flasks were incubated at 37 ◦ C and 200 rpm. Samples were collected at intervals and the bacterial biomass was separated by centrifugation at 3000 g for 15 min. The cells were resuspended in 5 ml water and the turbidity read at
94 660 nm. Dry cell weight is determined from a calibration curve between dry cell mass and optical density. Factorial designs (Box et al. 1978, Montgomery 1991) were used and each factor was evaluated at a high (+1) and a low level (−1), and these were referred to a central point (0), which was determined twice. In all cases the response was dry weight obtained after 12 hours of growth. The experimental data were designed and analysed by Statgraphics software 6.0 (Statistical Graphics Co.).
Results and discussion To evaluate the effect of the three nitrogen sources in the culture medium, a 23 factorial design was applied with factors trypticase (x1 ), phytone (x2 ) and yeast extract (x3 ). The levels (−1) and (+1) were defined at 50% lower and higher concentrations refered to the basal TPYG medium concentrations 0,0,0 which are considered as central points. Analysis by ANOVA of cell growth for the factorial design showed significant effects for factors x1 (P = 0.0167), x2 (P = 0.0187) and for the interaction x1 x2 x3 (P = 0.0273) with r 2 of 0.9996 and the growth response curve for this experiment is given by the equation Y = −2.94228 +x1 (0.3407) + x2 (0.8017) + x3 (1.085) + x1 x2 x3 (0.01864). Although variable x3 (P = 0.1246) alone does not intervene significantly, the regression coefficient of variable x3 is considered in the growth equation because it is a hierarchical model. These results indicate the importance of the three nitrogen sources, and the surface response curves (not shown) suggest the necessity of increasing the concentration of factors x1 , x2 , x3 to favor cell biomass production. Considering these results, the following three experiments were programmed and were defined as M-1, M-2 and M-3. Table 1 lists the concentration at the central point for each ingredient and were evaluated at the same levels than past experiment. Table 2 shows the results obtained with medium M-1. In this case, the significant effects were only observed with respect to x1 (P = 0.0010) and x2 (P = 0.0031). This is due to the fact that these two ingredients were increased and the interaction effect of the glucose source ceases to be significant since the two first ingredients are other sources of sugars for growth. The surface response curves corresponding to this medium (not shown) indicate that it is necessary to increase the concentrations of all studied substrates. To test if low glucose concentration is a limiting
Table 1. Concentration of substrates in central points for M-1, M-2 and M-3 media. Substrate
Concentration (g l−1 ) Media M-1 M-2 M-3
Trypticase (x1 ) Phytone (x2 ) Yeast extract (x3 ) Glucose (x4 )
15 10 2.5 5.0
15 5 2.5 15
5 3 10 5
∗ In all cases of the factorial design, the (−1)
and (+1) levels were defined 50% higher and lower concentration.
growth factor with a carbon/nitrogen ratio different from normal (10−2 /10−3 ) (Dunn 1985), medium M-2 was prepared in which the phytone peptone concentration was reduced and glucose was increased (Table 1). Growth obtained after 12 h of culture is shown in Table 2. Among the ingredients studied, only x1 (P = 0.0145) had a significant effect; glucose (x4 ), with a three-fold concentration increase did not show a significant effect, nor did the interaction of glucose with the other studied ingredients. Therefore, glucose does not seem to be a limiting factor, but nitrogen does, specifically because of the type and quantity of amino acids present in each complex. Since yeast extract is a rich amino acid source, medium M-3, (Table 1) contained a 4-fold increase in the concentration of yeast extract (x3 ) while trypticase (x1 ) and phytone (x2 ) were decreased to half their concentration, and glucose (x4 ) was kept constant as in the original TPYG medium. Table 2 shows the results for M-3. ANOVA analysis indicated the significant effects corresponding to factors x1 (P = 0.0050), x2 (P = 0.0460), x3 (P = 0.0010), the interaction x1 x3 (P = 0.0305) and the interaction x1 x3 x4 (P = 0.0334). The values for r 2 show the accuracy of the response predicted by the mathematical models. This value is within the interval considered as good (Taillandier et al. 1996).
Conclusions The analysis of the basal TPYG medium revealed by the triple interaction that the three complex nitrogen ingredients are the limiting factors for B. infantis growth. On the contrary, when a 4-fold increase of glucose concentration was added to medium M-2, no effect on growth was observed. In M-3 in which the
95 Table 2. Factorial design and results obtained with modified M-1, M-2 and M-3 media† . run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Factors† x1 x2
x3
x4
Growth (g l−1 )∗ M-1 M-2 M-3
− − − − − − − − + + + + + + + + 0 0
− − + + − − + + − − + + − − + + 0 0
− + − + − + − + − + − + − + − + 0 0
1.69 1.71 1.81 1.96 2.32 2.37 2.56 2.58 2.54 2.58 2.60 2.67 3.18 3.31 3.58 3.76 2.58 2.50
1.69 1.59 1.74 1.84 2.30 2.10 2.10 2.43 2.84 2.66 2.58 2.23 2.72 2.64 2.90 2.89 2.80 2.49
1.88 2.11 2.84 3.10 2.63 2.42 2.98 3.25 2.08 2.97 4.20 3.45 2.49 2.72 4.27 4.11 3.15 3.12
r2
0.98
0.93
0.99
− − − − + + + + − − − − + + + + 0 0
∗ Dry weight cell after 12 h fermentation. † See Table 1.
major nitrogen source is yeast extract, the biomass is considerably increased at the central point. This is due to the type and amount of aminoacids contained in this source. A comparison between final biomass obtained at the central points for M-1, M-2 and M-3 (Table 2) reveals that the increase in nitrogen source compounds, specifically yeast extract, produces an increase in biomass. Biomass values at the central point for basal medium TPYG were 1.84 g l−1 while for media M-1, M-2 and M-3 an increase of up to 60% was registered. In runs 11, 15 and 16 of the factorial design of M-3 more than 4 g l−1 of biomass were obtained when concentrations of trypticase (x1), phytone (x2 )
and yeast extract (x3 ) were high (+1). The proposed growth medium for optimal biomass yield would be as follows (g l−1 ); trypticase peptone, 7.5; phytone peptone, 4.5; yeast extract, 15; glucose, 5. All other components remain the same as in the basal TPYG medium. Significant triple effects in biological processes studied with mathematical models are difficult to explain, however, as with basal medium TPYG in which a significant triple interaction was observed, we can conclude for M-3 that albeit the presence of glucose, x4 , is not significant per se, its absence would probably result in an important decrease in biomass production, and confirmed by the triple interaction exhibited in medium M-3. This is a case in which the biological criterion should prevail over the mathematical criterion.
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