CC BY
123
BIOTECHNOLOGIA ACTA, V. 7, No 5, 2014
UDC 602.1:519.673+602.3:579.864
OPTIMIZATION OF TANNASE POSITIVE PROBIOTIC PRODUCTION BY SURFACE RESPONSE METHODOLOGY
1National Institute of Technology Karnataka, India E-mail: prsnbhat@gmail.com 2National Technical University of Ukraine «Kyiv Polytechnic Institute», Kiyv, Ukraine E-mail: biotech@fbt.ntu-kpi.kiev.ua
Received 23.06.2014
Study in conditions in vitro of eight Lactobacillus strains procured from culture repositories for their probiotic potential and extracellular tannase activity was the aim of the research. Based upon acid, bile salt tolerance and antibiotic resistance L. plantarum MTCC 2621 with high tannase activity was selected for production studies. Optimization of nutrient medium in 3 L bioreactor was optimized by Surface Response Methodology based on the Full Factorial Central Composite Design. A factorial design 23 augmented by 6 axial points (a = 1.68) and six replicates at the center point was implemented in 20 experiments. The optimized conditions were found to be pH 5.69, contain of lactose 128.58 g/l, peptone 8 g/l. A tenfold increase in the biomass production was observed using the optimized nutrient medium in bioreactor as compared to initial MRS medium.
Key words: Lactobacillus, optimization of medium, probiotic, response surface methodology.
S. Yumnam1 B. Prasanna1 L. B. Oriabinska2 L. A. Khrokalo2 O. M. Dugan2
Probiotics, according to conclusion of FAO/WHO joint commission, are «live microorganisms which when administered in adequate amount confer a health benefit on the host» [1]. Commonly as probiotics are used the heterogeneous group of lactic acid bacteria, such as lactobacilli, enterococci and bifidobacteria. Also, selected strains must survive in the extreme low pH of stomach and in the presence of bile salt which is to be encountered in the intestine. Lactobacillus is generally considered to be safe as they are the normal inhabitants in human and animal intestine and has long been used for its health benefits [2]. Some of the beneficial effects of Lactobacillus reported are increase immune response [3, 4], reduce antibiotic associated diarrhea [5, 6] and also decrease in irritable bowel syndrome [7-10].
Catechins (gallotannins and elligitannins) and other simple tannins (tannic acid) present in green, black tea and wine are known to have strong antioxidant properties [11]. Accumulating evidence indicates that they have the ability to suppress carcinogens through detoxification of various oxygen-free radicals generated in the body [12]. Enzyme acylhydrolase (EC. 3.1.1.20) popularly known as tannase, hydrolyses tannic acid completely to gallic acid and glucose [13]. Several reports are available
on the production of tannase which hydrolyses gallotannins and elligitannins [14]. The product of enzymatic tannin hydrolysis, gallic acid has antifungal and antiviral properties. Gallic acid acts as an antioxidant and helps to protect human cells against oxidative damage. Several strains of Lactobacillus among bacteria are known to produce tannase. The presence of Lactobacilli with tannase activity in the human alimentary tract may thus have a significant effect on the medicinal properties of tannins [15]. The aims of recent study were the screening a probiotic Lactobacillus strain with high tannase activity and design a nutrient medium for it to achieve higher biomass production.
Materials and Methods
Bacterial strains and culture conditions. Lactobacillus strains, such as L. acidophilus 2902, L. acidophilus 2903, L. brevis 2436, L. casei 2586, L. helveticus 2733, L. lactis 2368 and L.plantarum 2912 obtained from National Collection of Industrially Important Microorganisms (Pune, India) and L. plantarum 2621 from Microbial Type Culture Collection (Chandigarh, India) were used. Frozen stock cultures were transferred to MRS broth (Man-Rogosa-Sharpe, Himedia) and incubated at 35 °C for
62
Experimental articles
24 h and three successive transfers were carried out. The cultures were maintained in MRS agar plates and slants at 4 °C and were subcultured every 30 days. For routine analysis, Lactobacillus strains were subcultured in MRS agar every 48 h.
Extracellular tannase activity. Overnight grown Lactobacillus culture broth were transferred to MRS broth containing 0.2% gallic acid and incubated at 35 °C in a rotary shaker at 150 rpm for 24 h. 1 ml of the culture broth was centrifuged at 8000 rpm for 15 min. To 0.1 ml of supernatant 1ml of 0.3 mM tannic acid in 0.1 M citrate buffer (pH 3.5) was added. After 30 min incubation at 30 °C the reaction was stopped by adding 0.2 ml HCl (2 N). In the control, 0.2 ml of 2 N HCl was added to 0.1 ml of supernatant. After 30 min incubation 1ml of 0.3 mM tannic acid in 0.1 M citrate buffer was added. In the blank 0.1ml of 0.1 M citrate buffer was added to 1ml of 0.3 mM tannic acid in 0.1 M citrate buffer. After 30 min incubation 0.2 ml of 2 N HCl was added. The quantity of gallic acid released during hydrolysis of tannic acid represents the extracellular tannase activity. Gallic acid was measured with the rho-danine reaction. To 200 ці of standard sample, 300ц1 of rhodanine solution (0.667% in methanol) was added and the mixture is vortexed. After exactly 5 min, 4.5 ml of aqueous KOH (0.5 M) solution was added and after 20 min incubation, the absorbance was read at 520 nm. ДА = Absorbance (Test — Blank) — Absorbance (Control — Blank). One unit (U) of tannase activity is defined as 1 pM of gallic acid released per minute in the assay conditions.
Acid and bile salt tolerance assay. For the acid tolerance, 1ml of overnight grown cultures in sterile MRS broth incubated at 35 °C was inoculated in 9 ml of sterile phosphate-buffered saline (PBS) adjusted to pH 2.5 with 2 N HCl and then incubated at 35 °C for 3 h. Bacterial culture inoculated in PBS adjusted to pH 7 was used as a control. After 0th h and 3rd h cells were serially diluted 7 fold in sterile PBS solution and the number of viable bacteria was determined on an MRS agar plate. The survival rate was calculated as log values of colony forming units per ml (CFU / ml).
For the bile salt tolerance, 1ml of overnight grown cultures incubated at 35 °C were transferred into 9 ml sterile MRS broth containing 1% and 2% bile salt (Himedia, India) and then incubated at 35 °C for 24 h. Bacterial cultures grown in sterile MRS broth without bile salt were used as control. After 24 h incubation cells were serially diluted 7 fold in sterile PBS solution and the number of viable bacteria was
determined on an MRS agar plate. The survival rate was calculated as log values of CFU/ml.
Antibiotic resistance. Antibiotic resistance was examined by disk diffusion method [16]. Lactobаcillus strains were grown in sterile MRS broth at 35 °C for 24 h. The suspensions were swabbed on MRS agar plates. Antibiotic discs (Himedia, India) — ampicillin 25 pg, chloramphenicol 25pg, erythromycin 15 pg, kanamycin 30 pg and tetracycline 30 pg were placed on the MRS agar plates and incubated at 35 °C for 48 h. The inhibition zone around the antibiotic disk was measured.
Optimization of medium for biomass production in bioreactor. The batch fermentations were carried out in situ in a 3 L sterilizable stirred bioreactor (Scigenics, India) equipped by dissolved oxygen- (DO) and pH-probes (Mettler Toledo). The DO- and pH-probes were calibrated using standard procedure before launching every batch. The trials were conducted under the following conditions: medium volume 1.6 L, inoculum volume 10%, temperature 35 °C. Aeration and agitation was controlled to keep dissolved oxygen levels above 20% of saturation all the time. pH was maintained by adding appropriate amount of 2 N HCl and 2 N NaOH and temperature was maintained at 35 °C by circulating hot/cold water through the jacket during the fermentation. Sampling was done aseptically for every 1 h interval for analysis. Polypropyleneglycol in concentration of 0.2% was used as the antifoam. 24 h old inoculum developed in MRS broth in shake-flasks was used for the study.
Response surface methodology using central composite design was applied to model the biomass production. The design was based upon the model provided by software Design-Expert (Stat-Ease Inc, Minneapolis, USA). With either a spherical design or a rotatable one, we need number of replicate centre points (points at the origin) to obtain good prediction variance stability. It has been found out that the variance stability will be high when large number of experiments is conducted at centre point. So, actual factorial design 23 was augmented by 6 axial points with a = 1.68 (a is the distance between the «star points» and the centre of the design space. For a full factorial design (23), the formula is a = (23)1/4 and six replicates at the center point and was implemented in 20 experiments 23 (8 trials) plus 6 trials at axial points plus 6 replicate trials at centre points are equal 20 trials. Five levels of variation were selected for each variable shown in Table 1.
63
BIOTECHNOLOGIA ACTA, V. 7, No 5, 2014
Table 1. Experimental design
Coded values (Z) Actual values
Lactose (g/l) Peptone (g/l) pH
-1.68 52.73 5.27 4.39
-1 80 8 4.8
0 120 12 5.4
1 160 16 6
1.68 187.27 18.73 6.41
Biomass measurement. Biomass was determined by drying the pellet obtained by centrifuging 1ml of fermentation broth at 8000 rpm at 4 °C for 12 min. The pellets were dried at 80 °C to constant weight prior weighing. Dry cell weights (DCW) were correlated with optical density measurements at 600 nm in UV-VIS spectrophotometer (LABOMED Inc.) to obtain a calibration of OD600 vs DCW. OD readings were subsequently used to determine DCW.
Preliminary experiments were carried out in 250 ml shaker flasks to determine the individual effects and proper ranges of independent variables using one-factor-at-a-time approach. Lactose concentration, peptone concentration and pH were found to affect the biomass productivity in shaker flasks (in present paper data not shown). Accordingly, these three variables were chosen for bioreactor experiments. Cultivations were carried out with medium containing 0.2 g/l MgSO4 • 7 H2O, 0.04 g/l MnSO4 • H2O; 5 g/l yeast extract with lactose and peptone g/l being the independent variables.
The independent variables will be coded as Z:
Z = (X - X0)/AX (1),
were X — corresponding natural value; X0 — natural value in the centre of domain; AX -increment of X corresponding to one unit of Z.
Second order model of response surface was used to calculate the predicted response (Y):
Y = P0 + SPi Xi + Spii Xi2 + SpijXiXj (2),
where Y — response variable; p0 — interception coefficient; pi — coefficient of the linear effect; pii — the coefficient of quadratic effect and Py — the coefficient of interaction effect. The responses for each run were subjected to multiple non-linear regressions to obtain the coefficients of the polynomial equation. F-test was employed to evaluate the statistical significance of the quadratic polynomial model and the quality of the fit of the model was expressed by the coefficient of determination of correlation R2. The significance of regression coefficient was tested by t-test. The level of significance was given as values less than 0.05. Three-dimensional surface plots were drawn to illustrate the main and interactive effects of the significant variables on dry cell weight. The global optimum levels of the selected variables were obtained by solving the regression equation and by desirability charts. Experimental validation of the predicted model was also performed.
Results and Discussion
Extracellular tannase activity. After 48 h of incubation in enzyme producing medium, extracellular tannase activity of the Lactobacillus strains was determined by measuring the amount of gallic acid produced. Gallic acid was measured due to the rhodanine reaction and the quantity of gallic acid released during hydrolysis of tannic acid represents the extracellular tannase activity.
All the eight Lactobacillus strains showed extracellular tannase activity (Table 2) but L. plantarum 2621 showing highest level. L. plantarum 2912, L. acidohilus 2903, L. helvi-ticus 2733, L. lactis 2368, L. brevis 2436 and L. casei 2586 showed intermediate activity and L. acidophilus 2902 showed least activity. All the eight strains were used for further experiment.
Table 2. Tannase activity of Lactobacillus strains
Strain Activity (U/ml)
L. plantarum 2912 0.004
L. plantarum 2621 0.009
L. acidophilus 2902 0.0007
L. acidophilus 2903 0.004
L. lactis 2368 0.002
L. brevis 2436 0.001
L. helviticus 2733 0.004
L. casei 2586 0.001
Acid and bile tolerance. Survival of probiotics in the gastrointestinal tract is considered to be one of the most important their properties [1]. Different segment of the gastrointestinal tract have varying levels of acid and bile concentration. Stomach and the segments after stomach have the highest acidity and the pH may fall to pH 2.5 and it takes around 3 h for food to be digested. Thirabunyanon et al. [16], Pennacchia et al. [17], Zoumpopoulou et al. [18] also reported about necessity of probiotic survival at pH 2.5.
64
Experimental articles
According to the guidelines by FAO/WHO the test selection of probiotics Lactobacillus strains were carried out in vitro. For the acid tolerance assay strains were exposed to a pH of 2.5 for 3 h and their viability was determined by colony counting. All investigated strains retained viability in experiment (Table 3).
Table 3. The number of viable cells (lg cfu/ml) of Lactobacillus strains incubated at pH 2.5 in 3 h
Strain Control lg cfu/ ml pH 2.5 lg cfu/ml
0 hour 3 hour
L. plantarum NCIM 2912 9.5 10.1 9.7
L. plantarum MTCC 2621 9.5 10.2 10.1
L. acidophilus NCIM 2903 8 8 8
L. acidophilus NCIM 2902 9.3 9.6 9.4
L. lactis NCIM 2368 9.2 10 9.9
L. brevis NCIM 2436 9.4 8.8 8.6
L. casei NCIM 2586 8.6 9.3 9.3
L. helviticus NCIM 2733 9.3 9.5 9.5
Bile concentrations in the intestine may range between 0.5 to 2.0% during first hour of digestion, the levels may decrease during the second hour. Iyer et al. [19] reported about Streptococcus survival at 0.5-2% bile salt concentration. Survival of Lactobacillus strains at 0.3% bile salt was also reported by Maragkou-dakis et al. [20] and Thirabunyanon et al. [17].
For assay of bile tolerance Lactobacillus strains were subjected to 1 and 2% bile salt containing media. The survival of the bacterial strains was determined by plate counting method (Table 4). L. helviticus 2733, L. acidophilus 2903, L. brevis 2436, L. planta-rum 2621 and 2912, L. brevis 2436, L. helviti-cus 2733 survived in bile salt containing media 1% and 2% as well exposed in 24 h. But L. ca-sei 2586, L. lactis 2368 and L. acidophilus 2902 did not show growth both at 1% and 2% bile concentration after 24 hours of incubation.
Antibiotic resistance. Antibiotic resistance of a probiotic strain can be used for development effective therapeutic measures in controlling intestinal infections [21]. Arici et al. [22] reported that probiotics Lactobacillus were resistant to kanamycin and sensitive to erythromycin, chloramphenicol and tetracycline. Verdenelli et al. [23] also reported that L. rhamnosus and L. paracasei to be sensitive to kanamycin and resistant to ampicillin, tetracycline and erythromycin.
Table 4. The number of viable cells (lg cfu/ml) after 24 h incubation in MRS broth containing bile salt
Strain Control, lg cfu/ ml Bile salt concentration (w/v), lg cfu/ml
1% 2%
L. plantarum NCIM 2912 9.5 8.3 8.1
L. plantarum MTCC 2621 9.5 8.6 8
L. acidophilus NCIM 2903 8 9.6 8.7
L. acidophilus NCIM 2902 9.3 0 0
L. lactis NCIM 2368 9.2 0 0
L. brevis NCIM 2436 9.4 10.2 10.0
L. casei NCIM 2586 8.6 9.1 9.1
L. helviticus NCIM 2733 9.3 0 0
Original results of antibiotic resistance test of the strains are shown in Table 5. All eight strains were found to be resistant to ka-namycin and sensitive to ampicillin, chloramphenicol, erythromycin and tetracycline.
Therefore, our results indicate that Lactobacillus plantarum MTCC 2621 and few other strains showed intrinsic antibiotic resistances which is desirable. At the same time, L. plantarum 2621 and 2912, L. acidophilus 2903, L. brevis 2436 and L. helviticus 2733 were found to have tannase activity. However, among all the strains tested Lactobacillus plan-tarum 2621 had shown highest tannase activity (0.009 U/ml, Table 2). Further, L. plantarum 2621 had exhibited highest acid tolerance (Table 3) and reasonably good bile salt tolerance (Table 4) compared to other strains. Based on these results, L. plantarum 2621 was selected for further study.
Optimization of nutrient medium by Response Surface Methodology. The medium was optimized using central composite design for three variables, such as pH, content of lactose and peptone. The experimental and predicted data for the response variable Y (biomass, g/l) are given in Table 6.
The design matrix and the fitness of the each parameter were analyzed by means of Analysis of variance (ANOVA) and a significant second order regression (P < 0.0001) was observed (Table 7). Second-order polynomial equation in coded units for the biomass production was found to be,
Y = -50.86919 + 18.84911A + 0.091773B + 0.29704C + 9.89583x10-3AB — 0.077083AC+ 9.37500x10-5 BA — 1.71342A2 — 5.78870 x10-4B2 — 2.86522x10-3C2.
65
BIOTECHNOLOGIA ACTA, V. 7, No 5, 2014
Table 5. Antibiotic susceptibility test of the Lactobacillus strains
(S — Sensitive, R — Resistant)
Diameter of inhibition zone (mm)
Straine й •і—і a sp о ;3-b ю и н й д Sp o Й. •a ^ as <i <U й Zj /—4 U Ьй >» Й. S o CO w <U Chloramphenicol (25 pg) й •i—l О /—4 >> Ьй a * cd о й со cd ^ tin
L. plantarum MTCC 2621 28(S) 34(S) 28(S) 30(S) 8(R)
L. plantarum NCIM 2912 30(S) 39(S) 25(S) 29(S) 6(R)
L. brevis NCIM 2436 23(S) 26(S) 21(S) 28(S) 6(R)
L. acidophilus NCIM 2902 30(S) 40(S) 25(S) 32(S) 8(R)
L. acidophilus NCIM 2903 20(S) 25(S) 28(S) 21(S) 6(R)
L. casei NCIM 2586 31(S) 41(S) 26(S) 31(S) 1(R)
L. helviticus NCIM 2733 27(S) 34(S) 22(S) 26(S) 9(R)
L. lactis NCIM 2368 30(S) 32(S) 26(S) 32(S) 6(R)
Table 6. Central Composite Design Matrix for observed and predicted response
Run Order pH A Lactose (g/l) B Peptone (g/l) C Biomass in 24 h exposition (g/l) Residual
Actual Predicted
1 4.8 80 8 6.71 6.86 0.15
2 6 80 8 7.6 7.48 -0.12
3 4.8 160 8 7.2 6.95 -0.25
4 6 160 8 8.19 8.52 0.33
5 4.8 80 16 6.1 5.79 -0.31
6 6 80 16 5.4 5.67 0.27
7 4.8 160 16 5.8 5.93 0.13
8 6 160 16 6.9 6.77 -0.13
9 4.39 120 12 5.8 5.97 0.17
10 6.41 120 12 7.4 7.20 -0.20
11 5.4 52.73 12 5.2 5.22 0.02
12 5.4 187.27 12 6.25 6.21 -0.04
13 5.4 120 5.27 9.45 9.39 -0.06
14 5.4 120 18.73 6.98 7.01 0.03
15 5.4 120 12 8 8.33 0.33
16 5.4 120 12 8.5 8.33 -0.17
17 5.4 120 12 8.2 8.33 0.13
18 5.4 120 12 8.5 8.33 -0.17
19 5.4 120 12 8.2 8.33 0.13
20 5.4 120 12 8.6 8.33 -0.27
66
Experimental articles
The Fisher criteria (F-value of 39.25) is indicated the significance of model. Commonly, the determination of coefficient (R2) is estimated the adequacy of the model. Value R2 was 0.9725 and if value exceed 0.75, so it indicates aptness of the model. Adjusted determination coefficient Adj R2 (adjusted for the number of predictors in the model and commonly lower than the R2) was equal 0. 947. Predicted determination coefficient Pred R2 indicates how well a regression model predicts responses for new observations and was equal 0.83. If the Adj R2 and Pred R2 are less than R2 and are nearer to 1, we can conclude that the response equation (second order polynomial equation) is robust and can effectively predicts response for new observations as well. Probability plot of the residuals roughly follows a straight line indicating normal distribution of residuals. Consequently, all of mentioned considerations indicate a good adequacy of the regression model.
The significance of each parameter was determined by Student’s t-test and p-value (Table 7). In our study, model parameters A, B, C, AB, A2 and B2 were found to be significant. Content of lactose and pH of medium have positive effect on biomass production at low concentration as indicated by the positive coefficient but at higher concentrations inhibit the biomass production. The interactive
coefficient was found not to be different from zero at a significance level of 0.05. Response surface curves and contour plots for biomass production, keeping one factor fixed while varying the two other, are demonstrated at figs 1-3. The convex curves commonly indicating that well defined optimum conditions lays in the design space.
From the contour plot it was observed that a high concentration of lactose and pH with a lower concentration of peptone is required for higher biomass production. The optimum levels of the tested variables were pH 5.69, lactose 128.58 g/l and peptone 8 g/l, and the biomass production of 9.15 g/l. was predicted from response surface model. To confirm these data, experimental rechecking was done by using a medium representing these optimal points in the bioreactor. The biomass production of 8.97 g/l was obtained as against the predicted 9.15 g/l which is quite satisfactory. A difference of mere 0.18 g/l, which amounts to 2% error, verifies the validity of the response model and the existence of an optimal point. Biomass production was increased 10 times with the optimized medium as compared to the biomass content in the initial MRS broth. The probiotic biomass also showed tannase activity of 0.0085 U/ml, confirming that the desired tannase activity trait is still present in the biomass produced.
Table 7. Analysis of Variance for Response Surface Quadratic Model
Source Sum of square Af Mean Square F-value P-value Prob > F
Model 27.06 9 3.01 39.25 < 0.0001 iignif.
A (pH) 1.81 1 1.81 23.62 0.0007
B (lactose) 1.2 1 1.2 15.65 0.0027
C (peptone) 6.82 1 6.82 89.1 < 0.0001
AB 0.45 1 0.45 5.89 0.0356
AC 0.27 1 0.27 3.57 0.088
BA 1.80x10-3 1 1.80x10-3 0.024 0.8812
A2 5.48 1 5.48 71.59 < 0.0001
B2 12.36 1 12.36 161.4 < 0.0001
C2 0.03 1 0.03 0.4 0.5436
Residual 0.77 10 0.077
Lack of Fit 0.49 5 0.099 1.8 0.2668 not signif.
Pure Error 0.27 5 0.055
Cor Total 27.83 19
R2 = 97.25% Adj R2 = 94.7%
67
BIOTECHNOLOGIA ACTA, V. 7, No 5, 2014
Fig. 1. Surface Response as a function of lactose and pH (keeping Peptone constant) on biomass production for L.plantarum MTCC 2621
Fig. 2. Surface Response as a function of peptone and lactose (keeping pH constant) on biomass production for L.plantarum MTCC 2621
In the current study, eight tannase positive Lactobacillus strains were taken and in vitro probiotic properties were determined. Among eight strains, L. plantarum 2621 and 2912, L. acidophilus 2903, L. brevis 2436 and L. hel-viticus 2733 were found to possess significant acid tolerance, bile salt tolerance and desirable antibiotic resistance. As Lactobacillus planta-rum 2621 had shown highest tannase activity and significant acid and bile tolerance, it was chosen for further study.
A nutrient medium was designed comprising lactose, peptone and salts making use of “one-factor-at-a-time” approach in shake flasks. Based upon the results, bioreactor trails were conducted to optimize the medium by Response surface modeling technique. Altogether 20 batch trails were conducted in a 3 L bioreactor as per the central composite design. A polynomial response model was generated using software, Design-Expert (Stat-Ease Inc., Minneapolis, USA). The adequacy of the model equation was tested by ANOVA, and response surface and contour plots were generated. Using point prediction tool, optimum points of the variable were predicted and verified by conducting two more bioreactor trials.
Thus a specific nutrient medium for biomass production for L. plantarum 2621 has been designed successfully using response surface methodology. A tenfold increment in biomass production was achieved in the new optimized medium as compared to initial culture medium. The biomass produced had shown significant tannase activity. As the optimization trails were conducted in 3 L bioreactor, the process is ready for scale-up.
Fig. 3. Surface Response as a function of peptone and pH (keeping lactose constant) on biomass production for L.plantarum MTCC 2621
Acknowledgement
All authors would like to acknowledge the support provided by «Department of Science and Technology», Govt. of India and State Agency on Science, Innovations and Information of Ukraine under India-Ukraine Scientific Bilateral Program.
68
Experimental articles
REFERENCES
1. FAO / WHO Food and Agricultural Organiza-
tion of the United Nations and World Health Organization: Guidelines for the evaluation of probiotics in food. 2002, 11 р. Available at http: //www.fao.org/es/esn/food/foddan-
food_probio.
2. Jacobsen C, Rosenfeldt Nielsen V., Hayford A., Moller P., Michaelsen K., Paerregaard A., Sandstrom B., Tvede M., Jakobsen M. Screening of probiotic activities of forty seven strains of Lactobacillus spp. by in vitro techniques and evalution of the colonization ability of five selected strains in humans. Appl. Environ Microbiol. 1999, 659(11), 4949-4956.
3. Kaila M., Isolauri E., Soppi E., Virtanen E., Laine S., Arvilommi H. Enhancement of the circulating antibody secreting cell response in human diarrhea by a human Lactobacillus strain. Ped. Res. 1992, V. 32, P. 141-144.
4. Nagao F., Nakayama M., Muto T., Okumura K. Effects of a fermented milk drink containing Lactobacillus casei strain Shirota on the immune system in healthy human subjects. Bios-ci. Biotechnol. Biochem. 2000, 64(12), 27062708.
5. Black F., Einarsson K., Lidbeck A., Orrhage K., Nord C. Effect of lactic acid producing bacteria on the human intestinal microflora during ampicillin treatment. Scand. J. Infect. Disease. 1991, V. 2, P. 247-254.
6. Gismondo M. R., Drago L., Lombardi A. Review of probiotics available to modify gastrointestinal flora. Int. J. Antimicrob. Agents. 1999, V. 12, P. 287-292.
7. Gupta P., Andrew H., Kirschner B. S., Guanda-lini S. Is Lactobacillus GG helpful in children with Crohn’s disease? Results of a preliminary, open-label stud. J. Ped. Gastroenterol. Nutr. 2000, V. 31, P. 453-457.
8. Guslandi M., Mezzi G., Sorghi M., Testoni P. A. Saccharomyces boulardii in maintenance treatment of Crohn’s disease. Digestive Dis. Sci. 2000, V. 45, P. 1462-1464.
9. Hamilton-Miller J. A review of clinical trials of probiotics in the management of inflammatory bowel disease. Infect. Dis. Rev. 2001, V. 3, P. 83-87.
10. Mattila-Sandholm T. Blum S., Collins J., Crittenden W. de Vos R., Dunne C., Fond n R., Grenov G., Isolauri E. Probiotics: towards demonstrating efficacy. Trends Food Sci. Technol. 1999, V. 10, P. 393-399.
11. Higdon R., Frei A. Tea catechins and polyphenols: health effects, metabolism and antioxidant functions. Crit. Rev. Food. Sci. Nutr. 2003, V. 43, P. 89-143.
12. Lambart A., Elias V. The anti-oxidant and pro-oxidant activities of green tea polyphenols: a role in cancer prevention. Arch. Biochem. Biophys. 2010, V. 501, P. 65-72.
13. Lekha P. K., Lonsane B. K. Production and application of tannin acyl hydrolase: state of the art. Adv. Appl. Microbiol. 1997, V. 44, P.215-260.
14. Bhat T. K., Singh B., Sharma O. P. Microbial degradation of tannins-A current perspective. Biodegradation. 1998, V. 9, P. 343-357.
15. Osawa R., Kuroiso K., Goto S., Shimizu A. Isolation of tannin-degradation Lactobacilli from humans and fermented foods. Appl. Environ. Microb. 2000, V. 66, P. 3093-3097.
16. Thirabunyanon M., Boonprasom P., Niam-sup P. Probiotic potential of lactic acid bacteria isolated from fermented dairy milks on antiproliferation of colon cancer cells. Bio-technol Lett. 2009, V. 3, P. 571-576.
17. Pennacchia C., Ercolin D., Blaiotta G., Pepe O., Mauriello G., Villani F. Selection of Lactobacillus strains from fermented sausages for their potential use as probiotics. Meat Sci. 2004, V. 67, P. 309-317.
18. Zoumpopoulou G., Foligne B., Christodou-lou K., Grangette C., Pot B., Tsakalidou E. Lactobacillus fermentum ACA-DC 179 displays probiotic potential in vitro and protects against trinitrobenzene sulfonic acid (TNBS)-induced colitis and Salmonella infection in murine models. Int. J. Food Microbiol. 2008, V. 121, P. 18-26.
19. Iyer R., Tomar S. K., Kapila S., Mani J., Singh R. Probiotic properties of follate producing Streptococcus thermophiles strain. Food Research Int. 2010, V. 43, P. 103-110.
20. Maragkoudakis P. A., Zoumpopoulou G., Mia-ris C., Kalantzopoulos G., Pot G., Tsakalidou E. Probiotic potential of Lactobacillus strains isolated from dairy products. Int. Dairy J. 2006, 16(2), 189-199.
21. Ei-Naggar M. Comparative study of probiotic cultures to control the growth of E. coli O 157: H 7 and Salmonella typhimurium. Asian Netw. Sci. Inf. Biotechnol. 2004, 3(2), 173-180.
22. Arici M., Bilgin B., Sagdic O., Ozdemir C. Some characteristics of Lactobacillus isolates from infant faeces. Food Microbiol. 2004, V. 21, P. 19-24.
23. Verdenelli M. C., Ghelfi F., Silvi S., Orpiane-si C., Cecchini C., Cresci A. Probiotic properties of Lactobacillus rhamnosus and L. pa-racasei isolated from human faeces. Eur J. Nutr. 2009, 48(6), 355-363.
69
BIOTECHNOLOGIA ACTA, V. 7, No 5, 2014
ОПТИМІЗАЦІЯ ПРОДУКУВАННЯ ТАНАЗОАКТИВНОГО ПРОБІОТИКА НА ОСНОВІ МЕТОДИКИ ВІДГУКУ ПОВЕРХНІ
С. Юмнам1 Б. Прасанна1 Л. Б. Орябінська2 Л. А. Хрокало2 О. М. Дуган2
Національний технологічний інститут штату Карнатака, Індія E-mail: prsnbhat@gmail.com 2Національний технічний університет України «Київський політехнічний інститут»
E-mail: biotech@fbt.ntu-kpi.kiev.ua
Метою роботи було дослідження за умов in vitro пробіотичних властивостей та оптиміза-ції продукування позаклітинного ензиму тан-нази у восьми штамів Lactobacillus, одержаних із музейних культур. Для вивчення промислової продуктивності обрали штам L. plantarum MTCC 2621, толерантний до кислот та жовчі, стійкий до дії антибіотиків, з підвищеною та-назною активністю. Оптимізацію живильного середовища в 3-літровому біореакторі було проведено з використанням методики відгуку поверхні на основі повного факторного аналізу головних компонент. Факторний аналіз 23, розширений шістьма додатковими точками (а = 1,68) і шістьма повторними навколо центральної точки, відтворено в 20 експериментах. Встановлено оптимальні параметри живильного середовища: рН 5,69, вміст лактози — 128,58 г/л, пептону — 8 г/л. За використання оптимізованого живильного середовища одержано 10-кратне підвищення продукції біомаси порівняно із застосуванням стандартного середовища MRS.
Ключові слова: Lactobacillus, пробіотик, оптимізація складу середовища, методика відгуку поверхні.
ОПТИМИЗАЦИЯ ПРОДУЦИРОВАНИЯ ТАННАЗОАКТИВНОГО ПРОБИОТИКА НА ОСНОВЕ МЕТОДИКИ ОТКЛИКА ПОВЕРХНОСТИ
C. Юмнам1 Б. Прасанна1 Л. Б. Орябинская2 Л. А. Хрокало2 А. М. Дуган2
Национальный технологический институт штата Карнатака, Индия E-mail: prsnbhat@gmail.com 2Национальный технический университет Украины «Киевский политехнический институт»
E-mail: biotech@fbt.ntu-kpi.kiev.ua
Целью работы было исследование в условиях in vitro пробиотических свойств и оптимизации продуцирования внеклеточного энзима танназы у восьми штаммов Lactobacillus, полученных из музейных культур. Для изучения промышленной продуктивности избрали штамм L. plantarum MTCC 2621, толерантный к кислотам и желчи, устойчивый к действию антибиотиков, с повышенной танназной активностью. Оптимизация питательной среды в 3-литровом биореакторе проведена с использованием методики отклика поверхности на основе полного факторного анализа главных компонент. Факторный анализ 23, расширенный шестью дополнительными точками (а = 1,68) и шестью повторными вокруг центральной точки, был реализован в 20 экспериментах. Установлены оптимальные параметры питательной среды: рН 5,69, содержание лактозы — 128,58 г/л, пептона — 8 г/л. При использовании оптимизированной питательной среды получено 10-кратное увеличение продукции биомассы по сравнению с применением стандартной среды MRS.
Ключевые слова: Lactobacillus, пробиотик, оптимизация питательной среды, методика отклика поверхности.
70
