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CHAPTER 5

FORMULATION OF LOW COST FERMENTATION MEDIA FOR ENHANCED

BACTERIOCIN AND VIABLE CELLS PRODUCTION BY PROBIOTICS

STREPTOCOCCUS PHOCAE PI80 AND ENTEROCOCCUS FAECIUM MC13

5.1. Introduction

Lactic acid bacteria (LAB) are among the most important probiotic microorganisms typically

associated with gastrointestinal tract where they exercise beneficial effects. Currently, both in the

shrimp hatchery and farming, probiotic bacteria were used for restraining pathogenic vibrios.

Attempts were being made in food industries for controlling food spoilage microorganisms like

Listeria monocytogenes and other food borne pathogens (Hequet et al., 2007; Urso et al., 2006).

Use of beneficial bacteria (probiotic) to displace pathogens by competitive process is being used

in the shrimp hatchery as a better remedy than administering antibiotics and is now gaining

acceptance for control of pathogens in aquaculture (Havenaar et al., 1992). The probiotic bacteria

were able to produce some antimicrobial metabolites including lactic acid, diacetyl, hydrogen

peroxide and bacteriocin or bacteriocin like compounds (Lindgren and Dobrogosz, 1990; Juven

et al., 1992).

Bacteriocins are a complex heterogeneous peptides or proteins that are ribosomally

synthesized by lactic acid bacteria, which can display broad spectrum of antimicrobial activity

against Gram positive and Gram negative pathogenic bacteria (Klanhammer, 1993). Recently,

the role of bacteriocins are emerging in sea food industry and shrimp hatchery because they were

able to restrain the food borne pathogens such as Listeria monocytogenes, Salmonella typhi , and

shrimp pathogens like Vibrio spp., and Aeromonas spp., (Dominguez et al., 2007; Preetha et al.,

2007b). Also, the bacteriocins were potentially used as a natural biopreservatives to avoid the

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bacterial contamination in food. Bacteriocin production by lactic acid bacteria was related to cell

growth, which can be stimulated by several environmental factors. As LAB are exigent,

nutritionally fastidious microorganisms, growth and bacteriocin production are controlled by

nitrogen sources rather than by carbon sources (Parente and Ricciardi, 1999). Moreover,

bacteriocin synthesis can be affected by the presence of cations, surfactants and inhibitors;

however, the influence of these factors can vary between strains.

Probiotic bacteria may grow in alternative culture media without losing their ability to

produce a variety of antimicrobial metabolites, including bacteriocins (Guerra and Pastrana,

2002). Nutritive complex media such as de Man Rogosa and Sharpe (MRS), M17, CM and

Trypticase Soy Broth Yeast Extract (TSBYE) were used for cultivation of probiotic bacteria and

production of bacteriocin (Li et al., 2002; Mataragas et al., 2004; Trinetta et al., 2008).

Moreover, these media were designed for strains specific (eg. MRS and M17 for Lactobacillus)

and also provide favourable environments for cell growth, but they are cost expensive and

therefore not suited for application in industrial scale fermentation for mass production of

bacteriocin and biomass. Recently, there is interest in the development of low cost media

alternate to MRS media that could be used for large-scale industrial applications (Trinetta et al.,

2008). Most of the earlier research work has focused on statistical based experimental designs to

evaluate the influence of medium composition on batch and fed batch fermentation for

bacteriocins production. Preetha et al. (2007a) and Anthony et al. (2009) applied a full factorial

design with the aim of medium production, optimizing antimicrobial compound production by

Micrococcus MCCB 104 and Bacillus licheniformis AnBa9, using response surface methodology

(RSM). RSM is used for studying the effect of several factors influencing the responses by

varying them simultaneously and carrying out a limited number of experiments and their by it

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can improve product yield and reduce process variability, time, cost etc. This technique has been

used for optimizing composition of microbiological media (He et al., 2004) to improve

fermentation processes (Liong and Shah, 2005) and production of bacteriocins (Anthony et al.,

2008).

The objective of this study is to screen different carbon and nitrogen sources, NaCl and

surfactant by conventional one-variable-at-a time method and further optimize statistically by

full factorial central composite design (CCD) for enhanced bacteriocin and viable cell production

by S. phocae PI80 and E. faecium MC13.

5.2. Material and Methods

5.2.1. Bacterial strains and growth condition

Strains Streptococcus phocae PI80 and Enterococcus faecium MC13 were used in this

study and they were maintained in Lactobacillus MRS agar medium (HiMedia, Mumbai, India)

at 4oC. Listeria monocytogenes was chosen as the indicator strain to demonstrate and measure

the bacteriocin activity. This organism was maintained in BHI agar medium (HiMedia, Mumbai,

India) at 4oC for further work.

5.2.2. Assay of bacteriocin activity

In each trial, S. phocae PI80 and E. faecium MC13 were grown in 250 ml Erlenmeyer

conical flask with 100 ml of medium and incubated at 37oC for 16 h. The pH of the medium was

adjusted to pH 6.5 with 1N HCl or 1N NaOH before culture inoculation. The bacteriocin activity

was estimated by the agar well diffusion method and expressed as arbitrary units (AUml-1).

5.2.3. Optimization of medium composition using response surface methodology

The optimization of culture parameters was carried out by classical, non-statistical and

statistical based technology. Each medium variable was selected by non statistical approach

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(OVAT). Statistical method was used for the modeling and evaluation of problems in which the

response of interest increased by several variables and the objective was to optimize this

response. The nutrients such as carbon, nitrogen sources and salts were screened by one-variable

at a time manners (OVAT) and in order to increase the bacteriocin and viable cells production; it

was statistically optimized by response surface methodology using central composite design

(CCD). The minimum level and maximum limits of the variables were determined and a set of

thirty four experiment trials that included sixteen trials for factorial design, twelve trials for axial

points and six trials for the replication of the central points was programmed employing central

composite design. CCD is designed to evaluate the coefficients of a quadratic model. All

experiments were carried out at 37°C and pH 6.5 for 16 h.

5.2.4. Effect of carbon and nitrogen sources on mass culture of probiotics S. phocae PI80

and E. faecium MC13 in medium 1

To augment the growth of S. phocae PI80 and E. faecium MC13 in medium 1 for mass

culture production, various carbon and nitrogen sources (15 g/L) were tested separately with

medium 1. Both S. phocae PI80 and E. faecium MC13 were inoculated and incubated at 37oC for

48 h. The pH of the medium 1was adjusted to pH 6.5 with 1N HCl or 1N NaOH before culture

inoculation. At every 24 h intervals, samples were collected and the wet weight, dry weight,

optical density (OD) and total viable cells were observed for the period of 48 h. The flasks were

prepared in triplicates for all the experiments. The growth of S. phocae PI80 and E. faecium

MC13 were measured at 600 nm against blank in every day for the period of eight days. Among

the carbon and nitrogen sources tested the one which enhanced the growth of S. phocae PI80 and

E. faecium MC13 were selected for further process. Different concentrations of carbon and

nitrogen sources (10-50 g/L) were added separately with medium 1. Subsequently,

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aforementioned same procedures were carried out for this experiment. Among the different

concentrations tested, the one which enhance the growth of S. phocae PI80 and E. faecium MC13

were selected for fed-batch fermentation. At every six intervals, 20 g/L of carbon source and 30

g/L of nitrogen source were added separately, combinational and the pH of medium was adjusted

to 6.5 for the period of 48 h. The wet weight, dry weight, optical density (OD) and total viable

cells were observed at 24 h and 48 h.

5.3. Results

5.3.1. Optimization of media components for bacteriocin and viable cells production by S.

phocae PI80 and E. faecium MC13

The most popularly used CCD of RSM was employed to formulate low cost fermentation

medium 1 and 2. Six components such as tryptone, peptone, maltose, dextrose, NaCl and

triammonium citrate were expected to have an effect on bacteriocin and viable cell production in

medium 1. The low and high level limits of these variables were showed in Table 14. The central

composition design of six variables in coded along with titer as responses is presented in (Table

15 and 16). The maximum experimental value for bacteriocin activity is 25,600 AUml-1, while

the predicted response based on RSM was estimated to be 25,556.34 and 25,535.21 AUml-1 for

S. phocae PI80 and E. faecium MC13 respectively. Moreover the maximum experimental value

of viable cells for S. phocae PI80 and E. faecium MC13 were 12.94 & 12.32 LogCFUml-1, while

the predicted responses were estimated to be 12.97 and 12. 29 LogCFUml-1. The close

correlation between the experimental and predicted data indicates the appropriateness of the

experimental design.

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Table 14. Experimental range and levels of the independent variables (A, B, C, D, E and F

for both Medium 1 and 2) used in the Central Composite Design (CCD)

Independent variables Low level Low Centre High High level

(g/L) star point level factorial point level factorial star point ( ) ( 1) (0) (+1) (+ )

Medium 1 A

Tryptone 2.1 5.0 10.0 15.0 17.8 B

Peptone 1.7 4.0 8.0 12.0 14.2 C

Maltose 1.3 3.0 6.0 9.0 10.6 D

Dextrose (or) Glucose 1.3 3.0 6.0 9.0 10.6 E

NaCl 2.1 5.0 10.0 15.0 17.8 F

Sodium citrate 1.0 2.5 5.0 7.5 8.9

Medium 2 A

Sodium succinate 2.1 5.0 10.0 15.0 17.8 B

Yeast extract 1.7 4.0 8.0 12.0 14.2 C

Dextrose (or) Glucose 1.3 3.0 6.0 9.0 10.6 D

NaCl 2.1 5.0 10.0 15.0 17.8 E

Tween 80 1.3 3.0 6.0 9.0 10.6 F

K2HPO4 0.4 1.0 5.0 3.0 3.5

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Table 15. Coded experimental design and results of the response surface of maximum

bacteriocin and viable cells production by S. phocae PI80 in medium 1

Run A B C D E F Bacteriocin activity Total viable cells

(AU ml-1) (LogCFUml-1) ______________________________________

Actual Predicted Actual Predicted 1 +1 1 1 1 +1 1 16400 14356.34 11.50 11.51 2 1 1 1 1 1 1 6400 6356.34 8.32 8.33 3 +1 1 +1 +1 1 +1 22500 22456.34 12.73 12.73 4 0 0 0 0 0 0 8600 8774.63 10.62 10.21 5 1 +1 1 +1 +1 +1 14400 14356.34 11.48 11.49 6 +1 +1 1 +1 1 +1 16900 16856.34 12.59 12.06 7 +1 +1 +1 1 +1 1 22500 22456.34 12.94 12.95 8 1 1 1 +1 +1 1 12100 12056.34 11.33 11.34 9 1 1 +1 +1 +1 +1 19600 19556.34 12.50 12.51 10 +1 +1 +1 +1 1 1 25600 25556.34 12.81 12.82 11 +1 1 1 +1 1 1 16900 16856.34 12.36 12.37 12 0 0 0 0 0 0 8100 8774.63 9.63 10.21 13 +1 1 +1 1 +1 +1 12100 12056.34 11.52 11.53 14 1 +1 1 1 1 +1 16900 16856.34 12.15 12.16 15 0 0 0 0 0 0 8600 8774.63 10.37 10.21 16 0 0 0 0 0 0 8600 8774.63 10.33 10.21 17 +1 +1 1 1 +1 +1 16900 19556.34 12.53 12.54 18 1 +1 +1 +1 +1 1 19600 14356.34 12.30 12.31 19 1 +1 +1 1 1 1 12100 12056.34 12.00 12.01 20 1 1 +1 1 1 +1 8600 8556.34 10.63 12.64 21 0 0 0

0 0 8600 8742.58 10.11 10.09 22 0 0 0 +

0 0 12100 12242.58 11.63 11.61 23 0 +

0 0 0 0 10000 10142.58 10.71 10.69 24 0 0 0 0 0 0 8600 7244.51 9.63 9.82 25

0 0 0 0 0 8600 8742.58 10.09 10.07 26 0

0 0 0 0 8600 8742.58 10.00 9.98 27 0 0 0 0 +

0 10000 10142.58 10.35 10.33 28 0 0 0 0 0 0 8100 7244.51 9.72 9.82 29 0 0

0 0 0 8100 8742.58 10.01 9.99 30 0 0 0 0 0 +

6400 6542.58 8.71 8.70 31 +

0 0 0 0 0 12100 12242.58 10.62 10.60 32 0 0 0 0 0

14400 14542.58 12.30 12.28 33 0 0 +

0 0 0 12100 12242.58 11.52 11.50 34 0 0 0 0

0 8600 8742.58 10.13 10.11

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bacteriocin and viable cells production by E. faecium MC13 in medium 1

Run A B C D E F Bacteriocin activity Total viable cells

(AUml-1) (LogCFUml-1)

Actual Predicted Actual Predicted 1 0 0 0 0 0 0 10000 10784.16 9.00 9.58 2 +1 +1 +1 +1 1 1 22500 22435.21 12.22 12.19

3 1 1 1 +1 +1 1 14400 14335.21 11.13 11.10 4 +1 1 1 +1 1 1 16900 16835.21 11.80 11.77 5 +1 +1 1 +1 1 +1 19600 19535.21 11.96 11.94 6 1 1 +1 1 1 +1 8100 8035.21 8.34 8.31 7 1 +1 +1 1 1 1 12100 12035.21 10.86 10.84 8 1 +1 +1 +1 +1 1 22500 22435.21 12.29 12.26 9 +1 1 1 1 +1 1 16900 16835.21 11.59 11.56 10 0 0 0 0 0 0 10000 10784.16 9.23 9.58 11 1 1 1 1 1 1 6400 6335.21 8.08 8.05 12 1 1 +1 1 1 +1 10000 9935.21 9.05 9.03 13 +1 1 +1 +1 1 +1 16900 16835.21 11.80 11.77 14 +1 1 +1 1 +1 +1 22500 22435.21 12.21 12.18 15 0 0 0 0 0 0 12100 10784.16 10.32 9.58 16 1 1 +1 +1 +1 +1 16900 16835.21 11.85 11.82 17 1 +1 1 +1 +1 +1 19600 19535.21 12.01 11.98 18 +1 +1 +1 1 +1 1 25600 25535.21 12.32 12.29 19 0 0 0 0 0 0 10000 10784.16 9.32 9.58 20 +1 +1 1 1 +1 +1 19600 19535.21 12.04 12.01 21 0 0 0 0

0 10000 10211.60 9.05 9.14 22 0 0 0 0 +

0 14400 14611.60 11.21 11.30 23 0 0 0 0 0 10000 10211.60 9.04 9.13 24 0 0

0 0 0 10000 10211.60 8.80 8.89 25 0 0 0 +

0 0 16900 17111.60 11.97 12.06 26

0 0 0 0 0 8600 8311.60 8.10 8.19 27 0 0 0 0 0 0 10000 9780.39 9.47 9.81 28 +

0 0 0 0 0 14000 14611.60 11.59 11.68 29 0 0 +

0 0 0 12100 12311.60 11.09 11.18 30 0

0 0 0 0 12100 12311.60 11.13 11.22 31 0 0 0

0 0 10000 10211.60 9.65 9.74 32 0

0 0 0 0 14400 14611.60 12.05 12.14 33 0 0 0 0 0 + 12100 12311.60 10.85 10.95 34 0 0 0 0 0 0 12100 9780.39 11.21 9.81

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The quality of the model can also be checked using various criteria. The calculated

regression equation for the optimization of medium components assessed the bacteriocin activity

as a function of these variables. By applying multiple regression analysis on the experimental

data, the following coded final equations were found to explain bacteriocin activity and total

viable cells in medium 1.

Bacteriocin activity (PI80) = + 8009.57 + 1118.15A + 447.26B + 1118.15C + 1118.15D +

447.26E

2555.77F + 475.00AB + 625.00AC + 247.26AD

744.35AE

1425.00AF

2943.27BC

1837.50BD

275.00BE

119.35BF + 1487.50CD

225.00CE

5.24CF

1756.85DE + 162.50DF

100.00EF + 1326.02A2

+ 897.36 B2

+ 1326.02C2

+ 1326.02D2

+

897.36E2 + 1346.43F

2 - (1)

Bacteriocin activity (MC13) = + 10282.27 + 2012.67A + 734.78B + 670.89C + 2204.35D +

1405.67E + 670.89F

268.75AB + 331.25AC

1438.08AD + 448.10AE

156.25AF +

927.14BC + 356.25BD + 43.75BE

810.36BF

443.75CD + 643.75CE

1296.47CF

1143.58DE

156.25DF + 156.25EF + 686.35A2

+ 1502.85B2

+ 604.70C2

+ 1584.50D2

+

1074.19E2 + 604.70F

2 - (2)

Total viable cells (PI80) = + 3.08 + 0.083A + 0.656B + 0.850C + 0.197D + 0.237E

0.443F

0.005AB

0.013AC

0.004AD + 0.005AE

0.015AF

0.087BC

0.023BD

0.012BE +

0.012BF

0.001CD

0.002CE

0.035CF

0.028DE

0.009DF

0.022EF + 0.008A2 +

0.008B2 + 0.041C2 + 0.026D2 + 0.018E2 + 0.043F2 - (3)

Total vible cells (MC13) = + 9.69 + 1.12A + 0.29B + 0.73C + 0.74D + 0.69E + 0.58F

0.23AB

0.012AC

0.017AD + 0.078AE + 0.074AF + 0.65BC

0.13BD

0.14BE + 0.47BF

0.11CD

0.027CE

0.078CF + 0.34DE + 0.087DF + 0.16EF + 0.052A2

+ 0.76B2

+ 0.091C2

+ 0.45D2

+ 0.17E2 + 0.09F

2 - (4)

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The quadratic model in Eqs. (1-4) with twenty seven terms contains six linear terms,

fifteen two factorial interactions and six quadratic terms. Adequacy and fitness of bacteriocin

activity were evaluated by standard analysis of variance (ANOVA). The ANOVA of the

quadratic regression models demonstrated that the models were highly significant (P< 0.0001;

0.0051) for bacteriocin activity produced by E. faecium MC13 and S. phocae PI80. The fitness of

the model was examined by determination coefficient (R2 = 0.9971; 0.9856), which implied that

the sample variation more than 99.5% was attributed to the variables and only 0.5% of the total

variance could not be explained by the model. The adjusted determination coefficients (Adj R2 =

0.9814; 0.9077) were also satisfactory to confirm the significance of the model. The models also

showed statiscally insignificant lack of fit. Thus the model was supposed to be adequate for

prediction within the variables. The high F-value and a very low probability indicated that the

present model for both bacteriocin activities showed good agreement between predicted and

experimental results. From the ANOVA analysis, all linear, interaction and quadratic terms

except D2 were statisticaly insignificant at probility level (P>0.05) for bacteriocin activity of S.

phocae PI80 (Table 17). But incase of E. faecium MC13, linear terms B and E, interaction terms

AB, AD, AE, BE, BF, CE, DF, and EF were statisticaly insignificant at probility level (P>0.05).

It is evident from the Table 18, linear terms A, B, D and F, interactiovin terms AC, AF, BC, BD,

CD, CF and DE, quadratic terms A2, B2 C2, D2, E2 and F2 were statisticaly significant at probility

level (P<0.05).

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Table 17. Regression analysis (ANOVA) for bacteriocin activity and viable cells production

by S. phocae PI80 in fermentation medium 1

Source Bacteriocin activity Total viable cells _____________________________ ____________________________ F- Value p- Value F- Value p- Value

(Prob>F) (Prob>F) Model 12.65 0.0051 11.97 0.0058 A 10.33 0.0236 0.33 0.5883 B 1.38 0.2936 20.78 0.0061 C 1.15 0.3331 12.58 0.0164 D 12.39 0.0169 1.20 0.3229 E 5.04 0.0748 0.98 0.3683 F 1.15 0.3331 2.38 0.1839 AB 0.60 0.4731 2.93 0.1475 AC 0.91 0.3831 5.14 0.0727 AD 4.04 0.1008 0.23 0.6502 AE 0.39 0.5588 0.20 0.6720 AF 0.20 0.6710 4.93 0.0770 BC 1.68 0.2518 53.93 0.0008 BD 1.06 0.3511 28.33 0.0031 BE 0.06 0.9045 5.08 0.0740 BF 1.28 0.3090 0.80 0.4127 CD 1.64 0.2566 0.03 0.9863 CE 3.45 0.1224 0.05 0.8227 CF 3.28 0.1299 2.22 0.1963 DE 2.55 0.1710 3.76 0.1100 DF 0.20 0.6710 1.28 0.3096 EF 0.20 0.6710 3.66 0.1141 A2 3.17 0.1353 4.73 0.0818 B2 15.18 0.0115 4.80 0.0800 C2 2.46 0.1777 15.32 0.0112 D2 17.87 0.0093 19.09 0.0072 E2 7.76 0.0387 2.89 0.0501 F2 2.46 0.1777 7.99 0.0368

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by E. faecium MC13 in fermentation medium 1

Source Bacteriocin activity Total viable cells

____________________________ ___________________________ F- Value p- Value F- Value p- Value

(Prob>F) (Prob>F) Model 63.63 <0.0001 3.40 0.0881 A 16.46 0.0098 9.31 0.0284 B 2.63 0.1655 0.65 0.4564 C 16.46 0.0098 4.00 0.1020 D 16.46 0.0098 4.09 0.0991 E 2.63 0.1655 3.57 0.1176 F 86.01 0.0002 2.54 0.1721 AB 9.70 0.0264 1.29 0.3082 AC 16.80 0.0094 0.35 0.5804 AD 0.62 0.4680 0.015 0.9698 AE 5.59 0.0645 0.035 0.8585 AF 87.33 0.0002 0.13 0.7305 BC 87.33 0.0002 2.40 0.1822 BD 145.21 <0.0001 0.45 0.5341 BE 3.25 0.1312 0.46 0.5280 BF 0.14 0.7203 1.25 0.3147 CD 95.16 0.0002 0.28 0.6183 CE 2.18 0.2001 0.018 0.8974 CF 20.19 0.0064 0.034 0.8600 DE 31.12 0.0026 0.68 0.4472 DF 1.14 0.3353 0.19 0.6839 EF 0.43 0.5409 0.64 0.4615 A2 61.05 0.0006 0.053 0.8276 B2 27.96 0.0032 11.54 0.0193 C2 61.05 0.0006 0.16 0.7019 D2 61.05 0.0006 3.91 0.1048 E2 27.96 0.0032 0.56 0.4864 F2 62.95 0.0005 0.18 0.6901

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The interaction between the nutrients and their effects on bacteriocin activity and total

viable cells production by S. phocae PI80 and E. faecium MC13 were plotted in the Figure 12

and 13. Each 2D contour and 3D plot presented the effect of two variables on the production of

bacteriocin activity and total viable cells, while other four variables were held at zero level. The

plots are helpful in identification of the type of interactions between test variables. The plots of

response surfaces suggest that the interaction is negligible between the corresponding variables.

An elliptical or saddle nature of the plots indicates the significance of the interactions between

the corresponding variables. From the model equations derived by differentiation of Eqs. (1-4),

we can obtain the maximum prediction points of the model for S. phocae PI80 and E. faecium

MC13 , which were 15g/L of tryptone, 8.0 g/L of peptone, 3.0 g/L of maltose, 9.0 g/L of glucose,

15 g/L of NaCl and 2.5 g/L of sodium citrate; 10.0 g/L of tryptone, 8.0 g/L of peptone, 3.0 g/L of

maltose, 9.0 g/L of glucose, 15.0 g/L of NaCl and 2.5 g/L of sodium citrate. The model

predicted maximum responses for bacteriocin activity (28,719.6; 24,416.2 AUml-1) and total

viable cells (13.467; 13.887 LogCFUml-1). In order to confirm the predicted results of the model

experiments were performed and maximum bacteriocin activity (25,600 AUml-1) was obtained.

This observation suggested that in the case of bacteriocin activity tryptone, peptone, maltose,

glucose, NaCl and sodium citrate exhibited significant interaction. This indicated that these six

factors played an important role for bacteriocin production. Moreover, the optimum composition

for fermentation medium 1 consists of: tryptone (15.0g/L), peptone (8.0g/L), maltose (3.0g/L),

glucose (9.0g/L), NaCl (15.0g/L) and sodium citrate (2.5g/L); which three fold enhanced

bacteriocin production by S. phocae PI80. But, tryptone (10.0g/L), peptone (6.0g/L), maltose

(3.0g/L), glucose (9.0g/L), NaCl (15.0g/L) and sodium citrate (2.5g/L) are found tobe as

optimum composition for E. faecium MC13.

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Figure 12. 3D response surface plots represent the effect of the significant variables and their

interaction with the response variable. The effects of tryptone, glucose (A); tryptone, maltose

(B); tryptone, glucose (C); peptone, glucose (D) and their mutual interaction on bacteriocin and

viable cell production by S. phocae PI80 were expressed in plots.

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Figure 13. 3D response surface plots represent the effect of the significant variables and their

interaction with the response variable. The effects of peptone, NaCl (A); maltose, NaCl (B);

peptone, NaCl (C); maltose, peptone (D) and their mutual interaction on bacteriocin and viable

cell production by E. faecium MC13 were expressed in plots.

122

For fermentation medium 2, six components such as sodium succinate, yeast extract,

dextrose, NaCl, tween 80 and K2HPO4, were expected to have an effect on bacteriocin

production, were selected by preliminary experiments-one-factor-at-a time experiments. The

experimental design and the results are represented in Table 19 and 20. The bacteriocin activity

varied markedly from 4,100 to 22,500 AUml-1 with the different levels of components in the

medium. The concentration of yeast extract, glucose and K2HPO4 strongly induced the

bacteriocin production by S. phocae PI80 with P-values of <0.0001, 0.0019 and 0.0092

respectively. But in case of E. faecium MC13, yeast extract and NaCl have stimulated the

bacteriocin production with P-values of 0.0006 and 0.0062. While, sodium succinate and tween

80 didn t significantly influence bacteriocin activity. The values of the regression coefficients

were calculated and the response variables of bacteriocin activity and total viable cells could be

written as experimental data:

Bacteriocin activity (PI80) = + 16401.16 + 734.78A + 5143.49B + 2396.04C + 734.78D

47.26E + 1661.25F + 456.25AB + 468.75AC

2378.51AD

996.47AE

581.25AF +

2580.00BC

331.25BD + 1868.75BE + 514.79BF

168.75CD

368.75CE + 1899.74CF

883.97DE

1893.75DF + 306.25EF

893.05A2

403.15B2 + 168.40C2 3.05D2

2505.63E2

+ 637.88F2 - (5)

Bacteriocin activity (MC13) = + 8674.09 + 607.00A + 3354.45B + 670.89C + 2012.67D +

1277.89E + 607.00F + 675.00AB

600.00AC

2172.11AD + 312.67AE

1512.50AF +

1569.50BC

312.50BD

137.50BE

1779.11BF + 962.50CD +612.67CE + 1654.45CF +

469.50DE + 625.00DF

600.00EF + 165.67A2 + 1227.11 B2 + 982.16C2 + 1063.81D2 +

594.33E2 + 165.67F2 - (6)

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Total vible cells (PI80) =

1.026 + 0.864A + 0.499B + 0.037C + 0.553D + 0.815E

1.229F +

0.004AB + 0.002AC

0.020AD

0.040AE

0.043AF + 0.015BC

0.010BD + 0.049BE +

0.049BF

0.21CD

0.006CE + 0.217CF + 0.011DE

0.118DF + 0.074EF

0.035A2

0.035B2 + 0.014C2

0.002D2 + 0.086E2 + 0.207F2 - (7)

Total vible cells (MC13) = + 8.92 + 1.28A + 1.20B

0.081C + 0.53D + 0.35E + 1.13F +

0.23AB

0.23AC

0.13AD + 0.23AE

0.13AF + 1.63BC

0.21BD

0.23BE + 1.08BF +

0.21CD + 0.046CE + 0.50CF + 1.40DE + 0.25DF + 0.14EF + 0.33A2

+ 0.32B2

+ 0.28C2

0.10D2

+ 0.009E2 + 0.38F

2 - (8)

The quadratic model in Eq. (5-8) with twenty seven terms contains six linear terms,

fifteen two factorial interactions and six quadratic terms. Adequacy and fitness of bacteriocin

activity and total viable cells were evaluated by ANOVA. The ANOVA of the quadratic

regression models demonstrated that the models were significant at probability values (0.0004;

0.0010) for bacteriocin activity produced by S. phocae PI80 and E. faecium MC13 (Table 21 and

22). The fitness of the model was examined by determination coefficient (R2 = 0.9952; 0.9925),

which implied that the sample variation more than 99 % was attributed to the variables and only

1% of the total variance could not be explained by the model. The adjusted determination

coefficients (Adj R2 = 0.9690; 0.9523) were also satisfactory to confirm the significance of the

model.

124

Table19. Coded experimental design and results of the response surface of maximum

bacteriocin and viable cells production by S. phocae PI80 in medium 2

Run A B C D E F Bacteriocin activity Total viable cells (AUml-1) (LogCFUml-1)

_______________________________________ Actual Predicted Actual Predicted

1 1 +1 +1 1 1 1 10000 9936.03 9.71 9.70 2 1 +1 1 1 1 +1 8600 8536.03 9.23 9.30

3 +1 1 1 1 +1 1 6400 6336.03 8.09 7.99 4 0 0 0 0 0 0 16900 17155.88 12.04 12.25 5 1 +1 +1 +1 +1 1 22500 22436.03 12.70 12.68 6 +1 1 +1 1 +1 +1 12100 12036.03 10.48 12.46 7 0 0 0 0 0 0 16900 17155.88 11.77 12.25 8 +1 1 +1 +1 1 +1 12100 12036.03 10.30 12.28 9 +1 +1 1 1 +1 +1 19600 19536.03 12.48 12.46 10 1 1 1 1 1 1 3600 3536.03 7.60 7.58 11 +1 1 1 +1 1 1 16900 16836.03 12.35 12.33 12 1 +1 1 +1 +1 +1 10000 9936.03 10.12 10.10 13 0 0 0 0 0 0 16900 17155.03 12.30 12.25 14 +1 +1 +1 +1 1 1 22500 22436.03 12.62 12.60 15 +1 +1 +1 1 +1 1 22500 22436.03 12.79 12.77 16 +1 1 1 +1 1 1 14400 14336.03 12.14 12.13 17 1 1 1 +1 +1 1 12100 12036.03 10.61 10.59 18 1 1 +1 +1 +1 +1 10000 9936.03 10.13 10.11 19 0 0 0 0 0 0 16900 17155.88 12.48 12.25 20 +1 +1 1 +1 1 +1 10000 9936.03 10.10 10.08 21 0 0 0 0 +

0 8600 8808.93 10.00 10.08 22 0 0 0 0

0 10000 10208.93 10.09 10.17 23 0 0

0 0 0 12100 12308.93 10.70 10.78 24 0

0 0 0 0 6400 6608.93 8.31 8.39 25

0 0 0 0 0 12100 12308.93 10.23 10.32 26 0 0 0 0 0 0 16900 15646.44 12.56 12.03 27 0 0 0 0 0 0 16900 15646.44 12.42 12.03 28 0 0 0 +

0 0 14400 14608.93 12.17 12.25 29 0 0 0 0 0 14400 14608.93 12.25 12.33 30 0 0 0 0 0 + 9600 19808.93 12.66 12.75 31 0 0 0

0 0 12100 12308.93 12.01 12.10 32 0 0 +

0 0 0 19600 19808.93 12.55 12.63 33 0 +

0 0 0 0 22500 22708.93 12.83 12.92 34 +

0 0 0 0 0 14400 14608.93 12.00 12.08

125


bacteriocin and viable cells production by E. faecium MC13 in medium 2

Runs A B C D E F Bacteriocin activity Total viable cells

(AU ml-1) (LogCFUml-1) ____________________________________ Actual Predicted Actual Predicted

1 1 1 1 +1 +1 1 12100 12082.25 9.23 9.22 2 +1 1 1 1 +1 1 14400 14382.25 9.13 9.12

3 +1 +1 +1 1 +1 1 22500 22482.25 12.16 12.15 4 1 +1 1 1 1 +1 8100 8082.25 8.34 8.34 5 +1 1 +1 1 +1 +1 10000 9982.25 8.88 8.87 6 +1 1 1 +1 1 1 8100 8082.25 8.17 8.16 7 1 +1 +1 +1 +1 1 22500 22482.25 12.25 12.25 8 1 1 +1 +1 +1 +1 22500 22482.25 12.24 12.23 9 1 +1 +1 1 1 1 10000 9982.25 11.84 11.84 10 0 0 0 0 0 0 8100 8646.00 8.10 8.63 11 +1 +1 1 1 +1 +1 12100 12082.25 9.47 9.46 12 1 +1 1 +1 +1 +1 14400 14382.25 9.32 9.31 13 +1 +1 1 +1 1 +1 10000 9982.25 9.05 9.04 14 +1 +1 +1 +1 1 1 16900 16882.25 11.96 11.96 15 0 0 0 0 0 0 8100 8646.00 8.10 8.63 16 1 1 1 1 1 1 4100 4082.25 8.07 8.07 17 +1 1 +1 +1 1 +1 10000 9982.25 9.00 8.99 18 0 0 0 0 0 0 8100 8646.00 9.04 8.63 19 1 1 +1 1 1 +1 8100 8082.25 8.48 8.47 20 0 0 0 0 0 0 10000 8646.00 9.13 8.63 21 0 0 0 0 0 + 10000 10057.97 11.88 11.91 22 0 0 0 0 0 8100 8157.97 8.34 8.38 23 0 0 0 0

0 8100 8157.97 8.65 8.69 24 0

0 0 0 0 6400 6457.97 8.10 8.13 25 0 0 0 +

0 0 14400 14457.97 9.76 9.79 26 0 +

0 0 0 0 16900 16957.97 11.86 11.89 27 0 0 0 0 0 0 10000 8702.17 8.76 9.22 28

0 0 0 0 0 8100 8157.97 8.00 8.03 29 0 0 +

0 0 0 12100 12157.97 9.74 9.77 30 0 0 0 0 +

0 12100 12157.97 9.76 9.79 31 0 0

0 0 0 10000 10057.97 9.99 10.02 32 0 0 0

0 0 8100 8157.97 8.10 8.13 33 +

0 0 0 0 0 10000 10057.97 12.00 12.03 34 0 0 0 0 0 0 8100 8072.17 10.02 9.22

126


by S. phocae PI80 in fermentation medium 2


________________________ ___________________________ F- Value p- Value F- Value p- Value

(Prob>F) (Prob>F) Model 38.07 0.0004 15.71 0.0030 A 3.31 0.1285 25.6 0.0039 B 162.25 <0.0001 5.47 0.0665 C 35.21 0.0019 0.018 0.8998 D 3.31 0.1285 10.51 0.0229 E 1.23 0.3184 8.21 0.0352 F 16.93 0.0092 2.08 0.2093 AB 4.17 0.0966 0.75 0.4247 AC 4.40 0.0900 0.16 0.7042 AD 26.56 0.0036 5.70 0.0626 AE 4.66 0.0833 8.13 0.0358 AF 6.77 0.0482 4.52 0.0868 BC 31.25 0.0025 0.80 0.4127 BD 2.20 0.1983 4.56 0.0858 BE 69.95 0.0004 33.48 0.0022 BF 1.24 0.3154 0.88 0.3902 CD 0.57 0.4841 9.84 0.0258 CE 2.72 0.1598 0.32 0.5963 CF 16.95 0.0092 9.52 0.0273 DE 3.67 0.1136 0.61 0.4705 DF 71.83 0.0004 33.37 0.0022 EF 1.88 0.2288 4.75 0.0811 A2 12.90 0.0157 8.91 0.0306 B2 2.63 0.1659 24.35 0.0043 C2 0.46 0.5283 1.36 0.2961 D2 12.90 0.0157 0.25 0.6360 E2 101.53 0.0002 46.55 0.0010 F2 6.58 0.0503 3.30 0.1291

127


by E. faecium MC13 in fermentation medium 2


_____________________________ ____________________________ F- Value p- Value F- Value p- Value

(Prob>F) (Prob>F) Model 24.65 0.0010 33.47 0.0022 A 1.87 0.2295 21.54 0.0056 B 57.18 0.0006 19.04 0.0073 C 2.29 0.1908 0.088 0.7792 D 20.59 0.0062 3.72 0.1115 E 8.30 0.0346 1.65 0.2552 F 1.87 0.2295 16.82 0.0093 AB 7.56 0.0403 2.34 0.1869 AC 5.98 0.0583 2.27 0.1925 AD 18.36 0.0078 0.17 0.6959 AE 0.38 0.5644 0.53 0.5000 AF 37.97 0.0016 0.68 0.4467 BC 9.58 0.0270 26.91 0.0035 BD 1.62 0.2590 1.82 0.2347 BE 0.31 0.5995 2.37 0.1846 BF 12.31 0.0171 11.85 0.0184 CD 15.38 0.0112 1.88 0.2289 CE 6.23 0.0548 0.090 0.7766 CF 10.65 0.0224 2.53 0.1724 DE 0.86 0.3969 19.82 0.0067 DF 6.48 0.0515 2.73 0.1592 EF 5.98 0.0583 0.91 0.3849 A2 0.37 0.5707 3.82 0.1081 B2 20.18 0.0064 3.63 0.1150 C2 12.93 0.0156 2.67 0.1630 D2 15.16 0.0115 0.38 0.5632 E2 4.73 0.0816 3.07 0.9580 F2 0.37 0.5707 4.96 0.0765

128

Figure 14 and 15 depicted the response surface plots of the significant interactions

between the nutrients and their effects on bacteriocin activity and viable cells produced by S.

phocae PI80 and E. faecium MC13. The response surface plots of the model equation suggested

that increased level of bacteriocin activity was obtained from the increasing concentration of all

factorial factors. According to the model equations (5-6), we can obtain the maximum prediction

point of the model, which was 10 g/L of sodium succinate, 4.0 g/L of yeast extract, 9.0 g/L of

glucose, 10.0 g/L of NaCl, 6.0 g/L of tween 80 and 1.0 g/L of K2HPO4 for S. phocae PI80; 10

g/L sodium succinate, 8.0 g/L of yeast extract, 9.0 g/L of glucose, 15.0 g/L of NaCl, 6.0 g/L of

tween 80 and 3.0 g/L of K2HPO4 for E. faecium MC13. The model predicted a maximum

response for bacteriocin production by S. phocae PI80 (26,185 AUml-1) and E. faecium MC13

(22,832.3 AUml-1). In order to confirm the predicted results of the model experiments were

performed and maximum bacteriocin activity (22,500 AUml-1) was obtained. These observations

suggested that the optimum composition of fermentation medium 2 consists of: sodium succinate

(10.0 g/L), yeast extract (4.0 g/L) glucose (9.0 g/L), NaCl (10.0 g/L), tween 80 (6.0 g/L) and

K2HPO4 (1.0 g/L) which enhanced the bacteriocin production by two fold in S. phocae PI80.

Similarly E. faecium produced two fold increased bacteriocin, cultured in fermentation medium 2

that consists of sodium succinate (10.0 g/L), yeast extract (12.0 g/L), glucose (9.0g/L), NaCl

(10.0g/L), tween 80 (6.0g/L) and K2HPO4 (1.0g/L).

129

Figure 14. Response surface of bacteriocin and total viable cells production by S. phocae PI80

were estimated as the AUml-1and LogCFUml-1. The effects of sodium succinate, tween 80 (A);

NaCl, tween 80 (B); yeast extract, tween 80 (C); yeast extract, sodium succinate (D) and their

mutual interaction on bacteriocin and total viable cells were expressed in plots.

130

Figure 15. Response surface of bacteriocin and total viable cells production by E. faecium MC13

were estimated as the AUml-1and LogCFUml-1. The effects of sodium succinate, K2HPO4 (A);

sodium succinate, NaCl (B); yeast extract, glucose (C); yeast extract, K2HPO4 (D) and their

mutual interaction on bacteriocin and total viable cells were expressed in plots.

131

5.3.2. Mass culture of S. phocae PI80 and E. faecium MC13 in medium 1

Effect of carbon sources (glucose, lactose, fructose, sucrose, maltose and mannose) on

mass cultures production by S. phocae PI80 and E. faecium MC13 were investigated in medium

1. Among the various carbons sources lactose and sucrose (15 g L-1) were found to be the best

for mass cultures of S. phocae PI80 and E. faecium MC13 (Table 23 and 24). Mass culture

production was also studied at various concentrations of lactose and sucrose and it is found that

maximum biomass production by S. phocae PI80 (Wet weight- 365.3±17.3 mg ml-1; dry weight-

136.3±17.1 mg ml-1; OD- 1.601±0.01; total viable cells- 13.408±0.121 LogCFUml-1) and E.

faecium MC13 (Wet weight- 389.1±15.4 mg ml-1; dry weight- 149.5±12.7 mg ml-1; OD-

1.607±0.05; total viable cells- 13.171±0.112 LogCFUml-1) occurred at 20 g L-1 of lactose and

sucrose in 48 h (Table 25 and 26). Subsequently, fed-batch fermentation was carried out in order

to increase the biomass production by S. phocae PI80 and E. faecium MC13. Mass culture of S.

phocae PI80 and E. faecium MC13 have improved two fold by addition of 20 g L-1 of lactose and

sucrose in fed-batch fermentation (Table 27). Effect of nitrogen sources on mass culture

production by S. phocae and E. faecium MC13 showed that yeast extract was most effective than

other tested nitrogen sources (Table 28 and 29). This may be due to the presence of larger

quantities of free amino acids, short peptides and more growth factors in yeast extract. Among

the various concentrations, yeast extract at 30 and 20 g L-1 showed maximum biomass production

by S. phocae PI80 (Wet weight- 457.0±14.2 mg ml-1; dry weight- 160.7±17.1 mg ml-1; OD-

1.743±0.02; total viable cells- 13.199±0.170 LogCFUml-1) and E. faecium MC13 (Wet weight-

451.2±15.1 mg ml-1; dry weight- 157.7±17.2 mg ml-1; OD- 1.731±0.02; total viable cells-

13.012±0.167 LogCFUml-1) (Table 30 and 31). The mass culture production by S. phocae PI80

132

and E. faecium MC13 were also increased by inclusion of yeast extract at 30 and 20 g L-1 via

fed-batch fermentation (Table 32). Moreover, the combination of lactose (20 g L-1) and yeast

extract (30 g L-1) improved biomass production in S. phocae PI80, which is five times higher

than the biomass obtained from normal medium 1 (Table 33). Similarly the mass culture of E.

faecium MC13 was increased (five times) by the fed batch fermentation through the addition of

sucrose (20 g L-1) and yeast extract (20 g L-1) (Table 34).

Table 23. Effect of carbon sources on biomass production by S. phocae PI80 in statistically

designed fermentation medium 1.

Carbon Wet Weight Dry Weight OD Viability Sources (mg/100ml-1) (mg/100ml-1) (600nm) LogCFUml-1

(15g/L) Glucose 24hr 280.0±17.3 81.6±10.1 1.517±0.04 13.021±0.137 48hr 300.0±17.3 94.0±14.0 1.504±0.03 12.929±0.163 Lactose 24hr 351.6±15.8 131.6±13.0 1.509±0.04 13.187±0.312 48hr 370.3±17.0 142.3±13.6 1.579±0.02 13.120±0.142 Fructose 24hr 251.6±18.7 65.0±8.6 1.395±0.01 12.008±0.169 48hr 271.0±13.5 70.3±4.9 1.381±0.02 11.886±0.151 Sucrose 24hr 298.3±15.8 100.0±17.3 1.496±0.02 12.875±0.164 48hr 301.3±13.2 103.0±11.3 1.484±0.04 12.799±0.161 Maltose 24hr 301.6±10.1 100.0±14.4 1.540±0.01 13.020±0.168 48hr 301.6±13.0 101.0±11.8 1.525±0.02 12.929±0.112 Mannose 24hr 280.0±11.5 81.6±15.8 1.528±0.02 12.130±0.212 48hr 301.3±11.5 90.3±6.6 1.534±0.04 12.795±0.168

133

Table 24. Effect of carbon sources on biomass production by E. faecium MC13 in

statistically designed fermentation medium 1.


(15g/L) Glucose 24hr 256.2±14.7 77.7±12.4 1.387±0.01 12.532±0.121 48hr 267.0±12.4 85.4±15.1 1.395±0.05 12.021±0.127 Lactose 24hr 331.2±13.2 120.4±10.2 1.492±0.07 13.012±0.245 48hr 362.3±16.2 134.1±17.1 1.517±0.01 12.862±0.152 Fructose 24hr 238.2±15.3 59.9±10.4 1.361±0.02 12.001±0.146 48hr 253.7±12.1 64.2±12.1 1.378±0.01 11.767±0.101 Sucrose 24hr 342.1±12.4 129.7±14.2 1.512±0.05 13.122±0.178 48hr 364.5±15.3 140.5±17.4 1.528±0.08 13.072±0.214 Maltose 24hr 322.5±10.1 118.7±10.2 1.467±0.02 12.861±0.211 48hr 334.2±13.0 122.1±13.0 1.479±0.04 12.678±0.107 Mannose 24hr 247.9±12.6 75.1±11.1 1.371±0.01 12.178±0.147 48hr 258.1±10.7 78.3±12.7 1.388±0.04 11.891±0.124

134

Table 25. Effect of lactose on biomass production by S. phocae PI80 in statistically designed

fermentation medium 1.

Lactose Wet Weight Dry Weight OD Viability (g/L) (mg/100ml-1) (mg/100ml-1) (600nm) LogCFUml-1

Lactose 10g/L 24hr 296.5±14.1 90.0±17.3 1.556±0.01 12.423±0.156 48hr 300.0±10.4 102.5±12.5 1.552±0.05 12.161±0.132 Lactose 20g/L 24hr 351.4±12.4 128.4±11.3 1.590±0.03 13.722±0.143 48hr 365.3±17.3 136.3±17.1 1.601±0.01 13.408±0.121 Lactose 30g/L 24hr 281.3±14.2 81.7±10.2 1.542±0.03 13.064±0.143 48hr 301.6±15.2 91.2±8.31 1.513±0.03 12.639±0.110 Lactose 40g/L 24hr 280.0±17.3 80.0±11.4 1.515±0.01 12.715±0.162 48hr 281.0±12.5 81.1.±14.7 1.483±0.03 12.342±0.141 Lactose 50g/L 24hr 237.4±18.1 62.4±17.1 1.353±0.02 12.515±0.245 48hr 253.1±13.7 65.2±11.4 1.378±0.04 12.367±0.114

Table 26. Effect of sucrose on biomass production by E. faecium MC13 in statistically


Sucrose Wet Weight Dry Weight OD Viability (g/L) (mg/100ml-1) (mg/100ml-1) (600nm) LogCFUml-1

Sucrose 10g/L 24hr 279.2±12.5 78.9±10.5 1.421±0.03 12.320±0.127 48hr 287.4±14.2 87.1±8.6 1.478±0.02 12.018±0.101 Sucrose 20g/L 24hr 362.4±17.1 140.7±12.5 1.521±0.02 13.701±0.152 48hr 389.1±15.4 149.5±12.7 1.607±0.05 13.171±0.112 Sucrose 30g/L 24hr 360.7±12.6 137.9±14.1 1.517±0.05 13.708±0.107 48hr 378.5±11.2 141.5±17.3 1.582±0.01 13.012±0.127 Sucrose 40g/L 24hr 301.2±14.5 98.1±12.3 1.491±0.02 12.671±0.124 48hr 312.4±12.3 99.7±12.1 1.507±0.01 12.112±0.138 Sucrose 50g/L 24hr 264.7±14.8 79.1±10.5 1.407±0.07 12.121±0.168 48hr 280.8±12.7 80.2±12.1 1.419±0.05 11.891±0.125

135

Table 27. Effect of lactose and sucrose on mass culture of S. phocae PI80 and E. faecium

MC13 in statistically designed fermentation medium 1.

Sources Wet Weight Dry Weight OD Viability (g/L) (mg/100ml-1) (mg/100ml-1) (600nm) LogCFUml-1

Lactose 20g/L 24hr 484.6±15.1 168.5±12.2 1.761±0.01 14.021±0.221 48hr 687.2±13.4 197.2±14.1 1.821±0.03 14.887±0.142 Sucrose 20g/L 24hr 467.5±17.4 166.7±15.2 1.701±0.02 14.127±0.320 48hr 691.3±15.3 201.2±13.2 1.816±0.04 15.017±0.211

Table 28. Effect of nitrogen sources on mass culture of S. phocae PI80 in statistically



(15g/L) Peptone 24hr 321.3±11.4 110.3±13.1 1.584±0.04 13.146±0.245 48hr 343.2±14.2 117.6±17.3 1.648±0.03 12.779±0.111 Tryptone 24hr 330.6±17.1 111.7±11.7 1.615±0.04 13.161±0.134 48hr 344.4±14.7 120.4±13.1 1.656±0.07 12.809±0.167 Beef extract 24hr 331.3±11.4 114.9±17.1 1.612±0.04 13.017±0.145 48hr 342.8±13.2 121.1±9.8 1.652±0.03 12.698±0.136 Yeast extract 24hr 343.5±17.7 126.6±10.3 1.620±0.01 13.640±0.231 48hr 367.1±11.6 129.7±17.3 1.680±0.05 13.064±0.321 Ammonium nitrate 24hr 267.8±13.1 81.2±13.6 1.374±0.02 12.234±0.116 48hr 283.4±15.2 82.3±13.6 1.307±0.09 12.178±0.137 Sodium nitrate 24hr 261.5±14.1 75.3±10.3 1.368±0.01 12.217±0.114 48hr 273.7±17.3 78.1±12.2 1.389±0.01 12.201±0.234

136

Table 29. Effect of nitrogen sources on mass culture of E. faecium MC13 in statistically



(15g/L) Peptone 24hr 310.1±10.2 101.7±13.6 1.487±0.01 12.876±0.212 48hr 324.3±15.1 107.3±10.5 1.496±0.05 12.201±0.145 Tryptone 24hr 301.2±12.5 97.9±10.1 1.451±0.03 12.512±0.124 48hr 312.5±10.4 98.5±12.4 1.472±0.07 12.017±0.131 Beef extract 24hr 292.7±9.2 87.7±10.4 1.459±0.02 12.116±0.127 48hr 303.5±12.1 98.1±14.2 1.462±0.04 11.672±0.175 Yeast extract 24hr 351.2±13.5 130.7±12.5 1.512±0.07 13.371±0.186 48hr 365.1±17.2 132.1±15.8 1.678±0.04 12.912±0.245 Ammonium nitrate 24hr 243.4±12.4 73.4±10.7 1.352±0.02 12.012±0.214 48hr 251.7±14.4 74.6±15.1 1.391±0.03 11.571±0.104 Sodium nitrate 24hr 257.2±12.1 75.5±10.6 1.387±0.02 12.120±0.122 48hr 262.4±12.7 76.4±10.4 1.412±0.05 11.582±0.230

137

Table 30. Effect of yeast extract on mass culture of S. phocae PI80 in statistically designed

fermentation medium 1.

Yeast extract Wet Weight Dry Weight OD Viability (g/L) (mg/100ml-1) (mg/100ml-1) (600nm) LogCFUml-1

Yeast extract 10g/L 24hr 292.1±10.4 98.52±15.2 1.535±0.03 12.221±0.122 48hr 382.3±17.3 135.2±10.2 1.582±0.02 12.007±0.152 Yeast extract 20g/L 24hr 361.2±10.7 121.5±17.3 1.675±0.01 13.713±0.151 48hr 405.4±12.1 142.1±12.4 1.692±0.04 13.167±0.132 Yeast extract 30g/L 24hr 414.2±17.5 152.1±15.3 1.714±0.03 13.867±0.127 48hr 457.0±14.2 160.7±17.1 1.743±0.02 13.099±0.170 Yeast extract 40g/L 24hr 407.6±9.10 150.2±12.1 1.691±0.04 13.711±0.153 48hr 451.6±13.5 160.7±9.56 1.721±0.01 13.101±0.124 Yeast extract 50g/L 24hr 405.3±17.5 151.0±10.7 1.702±0.03 13.541±0.452 48hr 449.2±10.5 160.2±14.2 1.723±0.01 12.867±0.147

Table 31. Effect of yeast extract on mass culture of E. faecium MC13 in statistically


Yeast extract Wet Weight Dry Weight OD Viability (g/L) (mg/100ml-1) (mg/100ml-1) (600nm) LogCFUml-1

Yeast extract 10g/L 24hr 301.4±8.6 98.2±12.1 1.379±0.05 12.781±0.142 48hr 399.1±10.2 137.8±15.3 1.481±0.03 12.102±0.114 Yeast extract 20g/L 24hr 411.4±13.2 150.1±10.3 1.652±0.03 13.761±0.124 48hr 451.2±15.1 157.7±17.2 1.731±0.02 13.012±0.167 Yeast extract 30g/L 24hr 401.8±10.5 149.5±15.4 1.678±0.07 13.478±0.109 48hr 442.1±12.2 155.3±10.6 1.748±0.02 12.871±0.182 Yeast extract 40g/L 24hr 391.4±12.6 134.3±13.1 1.562±0.09 13.421±0.124 48hr 402.7±12.4 148.5±10.4 1.681±0.05 12.521±0.145 Yeast extract 50g/L 24hr 371.5±13.4 125.1±10.2 1.655±0.01 13.422±0.135 48hr 390.2±11.3 135.3±14.2 1.691±0.04 12.402±0.162

138

Table 32. Effect of yeast extract on mass culture of S. phocae PI80 and E. faecium MC13 in



Yeast extract (30 g) 24hr 501.2±17.1 171.5±17.1 1.784±0.01 14.313±0.151 48hr 904.5±12.7 301.1±12.5 1.889±0.04 15.167±0.132

Yeast extract (20 g) 24hr 512.4±12.5 173.4±10.6 1.772±0.07 14.721±0.254 48hr 891.7±15.2 297.5±13.7 1.821±0.04 15.512±0.164

Table 33. Combination of lactose and yeast extract on mass culture of S. phocae PI80 in



Lactose (20 g) Yeast extract (30 g) 24hr 850.2±12.7 286.5±14.2 1.871±0.03 14.927±0.127

48hr 1202.4±15.1 383.7±12.2 1.901±0.01 15.782±0.322

Table 34. Combination of sucrose and yeast extract on mass culture of E. faecium MC13 in



Sucrose (20 g) Yeast extract (20 g) 24hr 874.7±15.2 287.7±17.1 1.892±0.04 15.127±0.224

48hr 1224.5±13.2 391.1±15.2 1.965±0.03 16.267±0.132

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5.4. Discussion

In general, there is no specific low cost medium manufactured for enhancing bacteriocin

production by probiotic bacteria used in aquaculture, food biopreservation and pharmaceuticals.

As a general rule, every microorganism has its own requirement for increasing bacteriocin

production (Todorov, 2008). For this necessity, the present work was undertaken to optimize

carbon source, nitrogen sources, and NaCl and surfactant tween 80 for bacteriocin production by

S. phocae PI80 and E. faecium MC13. Based on the one variable at-a time (OVAT) findings, two

varieties of fermentation media were designed statistically using response surface methodology,

which has been a popular and effective method to solve multivariate problems and optimize

several responses in many types of experimentation (Anthony et al., 2009). The method of RSM

for improvement of fermentation medium was described in detail by Strobel and Sullivan (1999).

In this study the summary of the results shows that bacteriocin production can be improved in

complex low cost media than in commercial lactobacillus MRS medium, which is expensive

(193 Indian Rupees or 4.1 US$ per liter). Therefore, two types of low cost fermentation media

were formulated with commercial components. Among these two fermentation media, medium

2 is four times (1.1 US$ per liter) and medium 1 three times (1.4 US$ per liter) cost less than

MRS medium. However, higher amount of bacteriocin activity and biomass production was

recorded in medium 1 than medium 2. Moreover, medium 1 has higher bacteriocin activity

(25,600 AUml-1) when compared with commercial MRS medium (14,400 AUml-1). The total

viable cells (TVC) were also counted in medium 1(12.94 LogCFUml-1) and medium 2 (12.83

LogCFUml-1) which are higher than the cells obtained from MRS medium.

Generally in the composition of medium 1, increasing concentration of nitrogen sources

such as tryptone (10-15.0 g/L) and peptone (6-8.0 g/L) have increased the bacteriocin production

140

by S. phocae PI80 and E. faecium MC13. This positive effect may be due to the presences of

amino acids, which were precursors for bacteriocin production. Amino acids have already been

reported for nisin (De Vuyst, 1995). In addition, moderate concentrations of carbon sources such

as maltose and glucose have also increased the bacteriocin activity but higher concentrations

reduced the bacteriocin activity. This was in agreement with the results of Rodriques et al.

(2006) who reported that inclusion of peptone and tryptone to the MRS broth significantly

induce biomass production by S. thermophilus A from 1.811 g/L to 4.250 g/L. Bacteriocin

ST414BZ production by L. plantarum ST414BZ was doubled (from 12800 to 25 600 AUml-1)

with tryptone as sole nitrogen sources (Todorov and Dicks, 2006). Glucose are suitable carbon

source for nisin Z and streptococcin AFF22 production (Matsusaki et al, 1996; John and Ingrid,

1991). Preetha et al. (2007a) obtained maximum bacteriocin activity (24.33 mm) and biomass

(1.83 g/L) by Micrococcus MCCB104 at 24 h of fermentation in the presence of glucose, lactose

and glycerol. On contrary Todorov (2008) found that the increasing concentration of glucose and

maltose have improved bacteriocin production. Bacteriocin ST664BZ production by L.

plantarum ST664BZ was increased two-fold (25,600 AUml-1) when glucose was replaced with

sucrose and maltose (Todorov and Dicks, 2006). Delgado et al. (2007) reported, in general salt

affect the growth and bacteriocin production in several bacterial strains. Moreover, their

mechanistic action in control of bacteriocin production is not well understood and also

bacteriocin production differs from each strain (Kabuki et al., 2007). Also, the inclusion of NaCl

at higher level in specific medium didn t stimulate bacteriocin and biomass production. Whereas

low level of NaCl influenced the bacteriocin activity from 89.0mm2 (zone) to 260.9mm2(zone)

and biomass from 3.32 g/L to 5.71 g/L for the culture L. plantarum 17.2b in MRS broth

(Delgado et al., 2007). Leal

Sanchez et al. (2002) and Anthony et al. (2009) reported that the

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presence of higher concentration of NaCl reduced bacteriocin activity from 3.35 Log10 AUml-1 to

1.30 Log10 AUml-1; 18,065 AUml-1 to 13,200 AUml-1 for L. plantarum LPCO10 and B.

licheniformis AnBa9. Similarly, S. phocae PI80 and E. faecium MC13 produced higher amount

bacteriocin and viable cells in the presence of low concentration of NaCl from 1 to1.5%

(Kanmani et al., 2011a). Similar observations were made by Peykov et al., (2008) in bacteriocin

production by Entreococcus faecium 3587; Kayalvizhi and Gunasekaran (2008) in Bacillus

licheniformis MKU3.

In medium 2, yeast extract is sufficient as a single nitrogen source for increased

bacteriocin production by the strain S. phocae PI80 and E. faecium MC13. Yeast extract provides

not only a relatively larger proportion of free amino acids and short peptides (two to three amino

acids long), but also more growth factors than other protein hydrolysates (De Vuyst, 1995; Aasen

et al., 2000; Anthony et al., 2009). The higher and lower concentration of yeast extract didn t

affect the bacteriocin production. For the reason that the nutritionally fastidious cultures growth

and bacteriocin production have been influenced by organic nitrogen sources (Kim et al., 1997).

Todorov and Dicks (2009) reported that the two-fold higher bacteriocin production (10, 2400 AU

ml-1) by Enterococcus muntii ST4SA was observed in the presence of yeast extract at 20 g L-1.

Low level of bacteriocin activity and biomass produced by Bacillus licheniformis MKU3 in

optimal medium containing yeast extract (2.5g L-1) (Kayalvizhi and Gunasekaran, 2008). Higher

amount of bacteriocin activity was observed when probiotic cells grown in the presence of

sodium succinate with minimum concentration. Biosurfactant tween 80 at a concentration of 6.0

g/L has influenced bacteriocin activity but increasing concentration suppressed biomass

production. This finding is in accordance with the report of bacteriocin warnerin production by

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Staphylococcus warneri (Prema et al., 2006). However several authors observed surfactants have

no influence on antimicrobial activity (Xiraphi et al., 2005; Trinetta et al., 2008)

Higher concentration of K2HPO4 suppressed the bacteriocin activity, where as lower

concentration increased bacteriocin activity in strains S. phocae PI80 and E. faecium MC13. This

result is in agreement with the reported bacteriocin production by Lactobacillus pentosus

ST712BZ and Lactobacillus plantarum AMA-K (Todorov et al., 2007). The response surface

plots are the graphical representation of regression equations and also visualize the interaction of

nutrients and amount of each nutrient required for maximum bacteriocin production. In

conclusion, the designed cost effective media for the animal probiotics S. phocae PI80 and E.

faecium MC13 provided higher amount of bacteriocin production. In addition validation of the

model suggested unequivocally, the reliability of RSM for formulation of media for S. phocae

PI80 and E. faecium MC13.

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