Optimization of the Medium Composition of Lactobacillus rhamnous NCU239

Tao Xiong, Yin Huang, Mingyong Xie

State Key Laboratory of Food Science & Technology

College of Life Science & Food Engineering

Nanchang University, Nanchang, P.R.China

xt-ncu@hotmail.com Corresponding author. Tel:+86-791-8160069 Mobile: +86-13697084048

E-mail address:xt-ncu@hotmail.com

Keywords ：：：： Lactobacillus rhamnosus; MRS enrichment medium; Plackett-Burman design;

Steepest ascent path test; Central Composite Design

Abstract.MRS culture medium was used as the basic medium for optimizing the fermented

condition of Lactobacillus. rhamnosus. The four significant factors (beef extract, tween-80, peptone,

diammonium citrate) were screened from these components by using a Plackeet-Burman design.

Steepest ascent path was adopted to approach the optimal region, and then the Central Composite

Design was used to determine the optimal levels of the main factors. Considering the fit model, cost

and operation, the optimal conditions were determined as follows: beef extract 2.6%, tween-80

0.3%, peptone 2%, diammonium citrate 0.4%, with other components remaining the same, 3%

inoculation, 18h culture at 37℃.Under such conditions, the experimental value of cell concentration

were 3.54×109cfu/ml and 2.19-fold live bacteria counts in MRS-based culture medium, showing no

difference with predicted value 3.68×109cfu/ml.

Introduction

Some reports confirmed that LAB(lactic acid bacteria) are beneficial probiotics on the human

body[1,2,3]

. With deep research on LAB's unique features, making the microbial products and lactic

acid bacteria fermented product application and development have been widespread concerned in

food industry.

Lactobacillus rhamnosus belongs to Lactobacillus, rhamnose bacillus species, Gram-positive

bacteria[4]

. It was isolated from the intestine and named by Gorbach and Goldlin 80 years in the 20th

century[5,6]

. Studies have shown Lactobacillus rhamnosus many characteristcs as follows: regulation

of intestinal microecological balance; relieving diarrhea[7]

; resistant to gastric acid and bile salt;

colonized in the intestine, inhibiting invasion and growth of pathogenic bacteria[8,9]

; activating the

immune system and enhancing human immunity[10]

; reducing toxins[11,12]

; regulation of blood

pressure[13]

; anti-oxidation[14]

and other excellent physiological functions.

In this work, using Plackett-Burman design, the path of steepest ascent design and central

composite design, we ultimately got the Lactobacillus rhamnosus optimized MRS medium. It will be

important for the further high cell density culture research.

Materials and Methods

A.Bacteria strain and media

The Lactobacillus rhamnosus NCU 239 Strain uesd in our experiments was stocked by state key

laboratory of food science and technology, Nanchang university.The medium for inoculum

preparation and fermentation contained the following(in g.L

-1): 20 glucose, 10 peptone, 10 beef

extract, 5 yeast extract, 5 sodium acetate, 2 diammonium hydrogen cirate, 2 KH2PO4, 0.25

MnSO4._7H2O, 0.58 MgSO4._7H2O, 1ml tween 80.

Advanced Materials Research Vol. 365 (2012) pp 312-319Online available since 2011/Oct/24 at www.scientific.net© (2012) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/AMR.365.312

All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP,www.ttp.net. (ID: 132.174.255.3, University of Southern California, Los Angeles, United States of America-09/09/13,03:19:54)

B.Inoculum preparation and counting method

To get the precultures, we scraped aseptically with inoculating loop from slant tube, inoculating

into a 250ml conical flask containing 100ml MRS medium and placing in the 37◦C incubator . When

reaching logarithmic growth phase, 3%(v/v) of the culture was used to inoculate fermentation flasks.

The numbers of lactic acid bacteria were counting by Methylene Blue direct count method[15]

.

C.Determination of Lactobacillus rhamnosus growth curve

In order to master the growth pattern of the strain and confirm the measured time of the follow-up

tests, we have identified the growth curve, using the above method. Bacterial counts have been tested

every 3 hours.

D.Experimental designs and optimization

Based on the MRS medium, through the Plackett-Burman design, we screened significant

influnce factors from 10 medium components. According to PB test results, we applied steepest

ascent path design to get a better fit model and approach the optimal response value. Finally, we used

CCD to determine the optimal concentrations of those significant factors.

Results and discussion

A. The growth curve of Lactobacillus rhamnosus

Figure 1. The growth curve of Lactobacillus rhamnosus.

The results were shown in Fig.1. During the 0h~6h, the bacteria was in the lag phase, with low

growth rate. From 6h to 12h, the number of the cell counts were increasing drastically. 15h~21h was

the stable phase of the bacteria. 21h later, the cell growth was coming into the decline phase.

Therefore, 18h was selected as the measured time of the bacteria counts in subsequent trials.

B.The results of the Plackett-Burman design

Based on MRS medium, we get the results of TableI,II as follow.

TABLE I. PLACKETT-BURMAN TEST DESIGN AND RESULTS

Run

Factors Bacteria

counts

108 cfu/ml X

1

X

2

X

3

X

4

X

5

X

6

X

7

X

8

X

9

X

1

0

1 2

3

4 5

6

7 8

9

10 11

12

1 -1

-1

1 -1

-1

-1 1

1

1 -1

1

1 1

-1

-1 1

-1

-1 -1

1

1 1

-1

1 -1

1

-1 -1

1

-1 -1

-1

1 1

1

1 1

-1

1 -1

-1

1 -1

-1

-1 1

1

1 1

1

-1 1

-1

-1 1

-1

-1 -1

1

1 1

1

1 -1

1

-1 -1

1

-1 -1

-1

1 -1

1

1 1

-1

1 -1

-1

1 -1

-1

1 -1

-1

1 1

1

-1 1

-1

-1 1

-1

1 -1

-1

-1 1

1

1 -1

1

-1 -1

1

1 1

-1

-1 -1

1

1 1

-1

1 -1

-1

22.5 15.45

15.2

12.55 12.2

18.45

15.7 12.15

11.15

23.2 18.2

12.25

Advanced Materials Research Vol. 365 313

TABLE II. DIFFERENT FACTORS AND LEVELS IN THE PLAKETT-BURMAN DESIGN AND SIGNIFICANCE OF THE REGRESSION COEFFICIENT

Factors symbol Levels Regression

coefficient P>t

-1 +1

glucose X1 1 5 -0.1167 0.3305

peptone X2 0.5 1.5 1.3667 0.0310

beef extract X3 0.5 1.5 2.5500 0.0166 yeast extract X4 0.25 1 0.3583 0.1171

sodium acetate X5 0.25 1 -0.7917 0.0535

KH2PO4 X6 0.1 0.3 0.1333 0.2952 diammonium

hydrogen cirate X7 0.1 0.3 1.1417 0.0371

MnSO4._7H2O X8 0.02 0.03 0.2583 0.1608

MgSO4._7H2O X9 0.05 0.07 -0.3750 0.1120

tween 80 X10 0.05 0.15 2.158 0.0197

The calculations were performed using JMP 7.0. The fit model (p=0.0419) has statically

significant. Through the t-test, the first four factors which affect the bacteria counts were: beef

extract, tween 80, peptone, diammonium hydrogen citrate.

Beef extract is the main nitrogen source, which contains water-soluble substances such as creatine,

creatinine, amino acids, nucleotides, organic acids, minerals and vitamins, vital for cell’s

growth.Tween80 is a condensate of sorbitol fatty acid ester and ethylene oxide. As a detergent, it can

disperse bacterial cells. On the one hand, it helps bacteria to absorb nutrients,on the other hand, it can

alleviate inhibition of the metabolites around the bacteria (mainly lactic acid), which harmful to cell

growth. Peptone provides carbon,nitrogen, other nutrients for microbial. Diammonium hydrogen

citrate can be a buffer salts, providing a certain amount of inorganic nitrogen at the same time.

Therefore, in the follow experiment-the steepest ascent path, levels of the four factors were

investigated.

C.The results of the steepest ascent path

According to the results in TableII, we had done the below design in Table III. and got the results

as follow:

TABLE III. EXPERIMENTAL DESIGN OF STEEPEST ASCENT AND RESULTS

Run Beef

Extract Tween 80 Peptone

Diammonium

Hydrogen

Cirate

Bacteria

Counts

108cfu/ml

① 1 0.1 1 0.3 20.35

② 1.5 0.15 1.5 0.35 28

③ 2 0.2 2 0.4 29.5

④ 2.5 0.25 2.5 0.45 31.35

⑤ 3 0.3 3 0.5 30.25

Figure 2. Experimental design of steepest ascent.

From Fig.2, the results shows that the cell counts could reach 3.1×109cfu/ml in group 4, as the

highest value of these 5 experiments. Therefor, we could determine the center of the CCD

experiment based on the above results.

314 Future Materials Engineering and Industry Application

D.Optimization using CCD design

Through the above 2 experimental design, we find these four factors and do the CCD design

experiment as Table IV and Table V.

TABLE IV. FACTORS AND LEVELS OF RESPONSE SURFACE DESIGN

TABLE V. CENTRAL COMPOSITE DSESIGN MATRIX AND EXPERIMENTAL RESULTS

Run A B C D Bacteria number

(109cfu/ml））））

1 -1 -1 -1 -1 3.15

2 1 -1 -1 -1 3.45

3 -1 1 -1 -1 3.47

4 1 1 -1 -1 3.59

5 -1 -1 1 -1 3.12

6 1 -1 1 -1 3.7

7 -1 1 1 -1 3.37

8 1 1 1 -1 3.55

9 -1 -1 -1 1 3.11

10 1 -1 -1 1 3.32

11 -1 1 -1 1 3.49

12 1 1 -1 1 3.42

13 -1 -1 1 1 3.27

14 1 -1 1 1 3.36

15 -1 1 1 1 3.57

16 1 1 1 1 3.25

17 -2 0 0 0 3.01

18 2 0 0 0 3.12

19 0 -2 0 0 3.21

20 0 2 0 0 3.6

21 0 0 -2 0 3.56

22 0 0 2 0 3.43

23 0 0 0 -2 3.71

24 0 0 0 2 3.42

25 0 0 0 0 3.57

26 0 0 0 0 3.47

27 0 0 0 0 3.55

28 0 0 0 0 3.63

29 0 0 0 0 3.62

30 0 0 0 0 3.47

31 0 0 0 0 3.45

Factors Symbol Coded level

-2 -1 0 1 2

beef extract(%) A 1.5 2.0 2.5 3.0 3.5

tween 80(%) B 0.15 0.2. 0.25 0.30 0.35

peptone(%) C 1.5 2.0 2.5 3.0 3.5

diammonium hydrogen

cirate(%)

D 0.35 0.4 0.45 0.5 0.55

Advanced Materials Research Vol. 365 315

TABLE VI. ANALYSIS OF VARIANCE

Source Sum of

Squares DF

Mean

Square

F

Value Prob > F

Model 0.95 14 0.068 11.43 < 0.0001 A 0.07 1 0.07 11.9 0.0033

B 0.17 1 0.17 28.16 < 0.0001

C 1.50E-04 1 1.50E-04 0.025 0.8755 D 0.06 1 0.06 10.14 0.0058

A2 0.4 1 0.4 67.18 < 0.0001

B2 0.031 1 0.031 5.23 0.0361 C2 3.10E-03 1 3.10E-03 0.52 0.4795

D2 1.44E-03 1 1.44E-03 0.24 0.6292

AB 0.099 1 0.099 16.76 0.0008 AC 1.00E-04 1 1.00E-04 0.017 0.8982

AD 0.099 1 0.099 16.76 0.0008

BC 0.027 1 0.027 4.6 0.0477 BD 9.00E-04 1 9.00E-04 0.15 0.7017

CD 2.50E-05 1 2.50E-05 4.22E-03 0.949

Residual 0.095 16 5.92E-03 Lack of

Fit 0.068 10 6.75E-03 1.49 0.3241

Pure Error

0.027 6 4.53E-03

Cor

Total 1.04 30

Analysing by Design-Expert, the model F-value(Table 6) of 11.43 implies the model is significant.

The analysis of variance indicated that the model terms of A, B,D,A2,AB,AD(p<0.01) were very

significant; B2, BC(p<0.05) were significant. The lack of fit(p=0.3241>0.05) was not significant and

the correlation coefficient R2=0.9091. These shows that fit model is well and the test error is low.

We can utilize this model to predict the bacteria counts, achieveing the optimized Lactobacillus

rhamnous MRS medium.

The second-order polynomial equation (1) obtained by Design-Expert was as follow:

Y=3.54+0.054A+0.083B-0.00250C-0.050D-0.12A2-0.033B2-0.010C2+0.007083D2-0.079AB-

0.0025AC-0.079AD-0.041BC+0.0075BD+0.00125CD (1)

E. Analysis of the interaction of factors

Figure 3. Responsive surfaces and contour ploy of Y=f(A,B).

316 Future Materials Engineering and Industry Application

Figure 4. Responsive surfaces and contour ploy of Y=f(B,C).

Figure 5. Responsive surfaces and contour ploy of Y=f(A,D).

In Fig.3, when the amount of beef extract is 2%~2.5%, the bacterial counts raise with the

increasing of tween80, reaching maximum when beef extract 2.5%, tween80 3%. While the amount

of beef extract is 2.5%~3%, the increasing of tween80 can't apparently raise bacterial counts. In

Fig.4, fixing the amount of diammonium hydrogen citrate, the amount of cell increased first and then

decreased, reaching peak value when diammonium hydrogen citrate 0.4%, beef extract 2.75%. In

Fig.5, fixing the amount of peptone, the counts raise with the increase of tween80, reaching peak

when peptone 2%, tween80 3%.

F.Determination of medium components

CCD design results showed that beef extract, tween80, diammonium hydrogen citrate were

significant factors, which consistent with the result of PB experiment, while only peptone was not

significant. It may due to peptone's climbing concentration was over in the steepest ascent path

experiment, deviating from the center point.

Depending on the software analysis, considering costs and operation, optimized MRS

components for Lactobacillus rhamnosus are: 2.6% beef extract, 0.3% tween80, 2% peptone, 0.4%

diammonium hydrogen citrate. Under such components, the bacterial counts reach to 3.68×109cfu/ml.

G.Validity experiment of the model

Culturing bacteria under optimal components, 3 replicate experiments were done. The amount of

bacterium reached to 3.54×109cfu/ml, 3.8% lower than the model prediction. Therefore, the model is

feasible, which describe the influence of 4 factors on cell counts. The low value maybe the one

reason that the bacteria's vitality picked from the slant tube in the verification test was weaker than

that of the response surface experiment. Because of decline of vitality, rejuvenation and transfer of

the bacterium are critical for the experiment. In the control group, the amount could reach

1.61×109cfu/ml. The amount of the bacterium cultured in optimized medium is 2.19 times to that

cultured in the MRS basic medium.

Advanced Materials Research Vol. 365 317

Conclusion

In this study, Lactobacillus rhamnosus NCU239 is the test strain. Using Plackett-Burman design,

four significant factors were selected from the MRS medium. Approaching the maximum response

area by steepest ascent path experiment and using response surface method, four quadratic model

was established for Lactobacillus rhamnosus specific MRS medium. Significant test was done for the

model, the factors' levels were optimized.Considering factors such as model,costs and operations, the

optimal levels are as follows : 2.6% beef extract, 0.3% Tween80, 0.2%, peptone and 0.4%

diammonium hydrogen citrate, other components' concentration unchanged as MRS basic medium.

Under the above components, cultured in 37℃, 18h fermentation , 3% inoculum ,the amount of

viable cell reach to 3.54×109cfu/ml ， which coincide with model prediction value

3.68×109cfu/ml,raised to 2.19 times of the amount in MRS basic medium.

Acknowledgment

We are grateful to the Research Program of State Key Laboratory of Food Science and Technology,

Nanchang University (Project No. SKLF-TS-200920) and Ministry of Education Venture Fund for

returned overseas students for financial support.

References

[1]Brashears, M.M., jaroni, D., Trimble, J., 2003. Isolation, selection, and characterization of lactic

acid bacteria for a competitive exclusion product to reduce shedding of Escherichia coli

O157:H7 in cattle. J. Food Prot, 355-363.

[2]Lee, Y .K., Salminen, S., 1995. The coming of age of probiotics. Trends Food Sci. Technol. 6,

241-245.

[3]Tannock, G. W., 1997. Probiotic properties of lactic acid bacteria: plenty of scope for

fundamental R and D. Trends Biotechnol. 6, 270-274.

[4]RE Buchanan, NE Gibbons. Berger Bacterial Identification Manual [M]. Eighth Edition. Beijing:

Science Press. 1995: 797-810.

[5] Qian Cheng, HUO Gui-cheng, Ma Wei. Functional properties and application prospects of

Lactobacillus rhamnosus (LGG) [J]. Food Science and Technology ,2005(9): 94-98.

[6]Ma Pengfei, Chen You-liang,JinNiao-jun. Research progress in functional properties of

Lactobacillus rhamnosus [J]. Science and Technology, 2009, 25(2):82-96.

[7]Sazawal S, Hiremath G, Dhingra U, et al. Efficacy of pro-biotics in prevention of acute diarrhea:

a meta-analysis of masked, randomized, placebo-controlled trials[J]. The Lancet Infectious

Diseases, 2006, 6(6): 374-382.

[8]Alander M, Satokari R, Korpela R, et al. Persistence of colonization of human colonic mucosa

by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption[J]. Applied and

Environment Microbiology, 1999, 65: 351-354.

[9]Collado M C,Meriluoto J, Salminen S. In vitro analysis of probiotic strain combinations to

inhibit pathogen adhesion to human intestinal mucus [J] Food Research International, 2007,

40(5): 629-636.

[10]Miettinen M, Vuopio-Varkia J, Varkia K. Production of human tumor necrosis factor alpha,

interleukin-6, and interleukin-10 is induced by lactic acid bacteria[J]. Infect Immun, 1996, 64:

5403-5407.

318 Future Materials Engineering and Industry Application

[11]Kankaanpa P, Tuomola E, EL-Nezami H, et al. Binding of aflatoxin B1 alters the adhesion

properties of Lactobacillus rhamnosus strain GG in a Caco-2 model[J]. Food Protect, 2000,

63(3); 412-414.

[12]EL-Nezamai H S, Chrevatidis A, Auriola S, et al. Removal of common Fusarium toxins in

vitro. by strains of Lacobacillus and Propionibacterium[J]. Food Additives and Contaminants,

2002, 19(7); 680-686.

[13]Sgouras D, Maragkoudakis P, Petraki K. In vitro and in vivo inhibition of Helicobacter pylori

by Lactobacillus casei strain Shirota. Applied and environmental microbiology, 2004, 1: 518-

526.

[14]Saide Jao, Gilliland Se. Antioxidative activity of Lactobacilli measured by oxygen radical

absorbance capacity. Journal of dairy science, 2005, 4: 1352-1357.

[15]Xiong Tao, Wang Yun, ZengZhe-ling, et al. An improved method for rapid determination of the

number of viable lactic acid bacteria [J]. Food and Fermentation Industries, 2009, 35(10): 132-

134.

Advanced Materials Research Vol. 365 319

Future Materials Engineering and Industry Application 10.4028/www.scientific.net/AMR.365 Optimization of the Medium Composition of Lactobacillus Rhamnous NCU239 10.4028/www.scientific.net/AMR.365.312

DOI References

[7] Sazawal S, Hiremath G, Dhingra U, et al. Efficacy of pro-biotics in prevention of acute diarrhea: a meta-

analysis of masked, randomized, placebo-controlled trials[J]. The Lancet Infectious Diseases, 2006, 6(6):

374-382.

http://dx.doi.org/10.1016/S1473-3099(06)70495-9