Enhancement in VitaminB
12
Production By
Mutant Strains of Propionibacterium
freudenreichii
Mir Naiman Ali
Asst. Professor. Department of Microbiology, Mumtaz Degree & P.G College, Malakpet, Hyderabad-500036, Andhra Pradesh (India)
naimanali@yahoo.co.in
+91-9348048044
Mazharuddin Khan Mohd.
Prof. & Head Dept. of Microbiology & Biotechnology Mumtaz Degree & P.G College,
Malakpet, Hyderabad-500036, Andhra Pradesh (India)
dr_mazhar_khan@yahoo.co.in +91-9885218310
ABSTRACT
In the present study vitaminB12 production was carried out with parental and mutant strains of
Propionibcterium freudenreichii. Physical mutagenesis was performed by three different doses of U.V. irradiation- 200, 300 and 400erg/mm2and five different irradiation time periods were selected- 30sec, 60sec, 90sec, 120 sec and 150sec. Obtained 15 mutant strains were used to produce vitaminB12 by anaerobic and aerobic fermentation techniques at 300C, 320C and 340C. 200 erg/mm2 U.V. dose and short duration exposure haven’t developed any desirable character in organism but mutants which were exposed to 400 erg/mm2 for 60 seconds developed desirable (resistant to propionic acid) character. High yield of vitmainB12 was produced at 300C by mutants of P. freudenreichii.
Key words: VitaminB12, physical mutagenesis, U.V. radiation, propionic acid, bioassay.
INTRODUCTION:
Vitamins are organic compounds which are necessary for good health & vitality. Vitamins are defined as essential micronutrients that are required in trace quantity and cannot be synthesized by mammals. They are nutrients required in minute quantities to sustain life and their deficiency results in structural and functional disorders of various organs in the body. Among the vitamins the most interesting molecules in the world of science and medicine is vitamin B12 (cobalamin), which was originally discovered as the anti-pernicious anemia
factor in the early 1920s, when two American physicians, Minot and Murphy, demonstrated it to cure pernicious anemia, a disorder first described in 1835, with a diet that included raw liver. In humans, the vitamin is required in trace amounts (approximately 1 mg/day) to assist the action of two enzymes, methionine synthase and (R)-methylmalonyl-CoA mutase (1).
Vitamin B12 is obtained exclusively by fermentation process. The chemical synthesis of vitaminB12 is not feasible as it requires 70 reaction steps which makes it too difficult and expensive, subsequent purification steps unsafe to the operators and the process is not eco-friendly. Since the discovery by Rickes et al (1948) that microorganisms may synthesize vitaminB12, several fermentation processes have been reported for the production of this vitamin on a commercial scale. Merck began production of vitamin B12 by Pseudomonas
denitrificans in 1952 and have improved the efficiency of culture more than 30-fold relative to the performance of the original soil isolates by genetic manipulations and microbial screening (2). It is produced by a number of pharmaceutical companies to meet annual demands worldwide.
VitaminB12 is produced industrially by microbial fermentation, using almost exclusively Pseudomonas
denitrificans and Propionibacterium species. The other fermentative organisms used are species of
Streptomyces, Bacillus, Methanobacterium, Pseudomonas, Klebsiella,Propionibacterium and Flavobacterium.
Regarded As Safe) status from the US FDA (Food and Drug Administration) and produce neither endotoxin nor exotoxins (4) and produce highest yield. But interestingly Propionibacteria produce vitaminB12 intracellularly and excrete mainly propionic acid and acetic acid extracellularly. This is the major problem in vitaminB12 production using Propionibacterium, as the cell growth inhibits due to the accumulation of inhibitory metabolites such as propionic acid and acetic acid which are produced extracellularly. Furthermore it is crucial to neutralize the accumulated propionic acid during the whole fermentation process in order to maintain the culture. To overcome this situation an effort was made in present study through random physical mutagenesis (U.V. irradiation) to develop desirable resistant characters in Propionibacterium freudenreichii organism against the propionic acid leading to continuous production of vitaminB12 in fermentation process.
MATERIALS AND METHODS:
Microorganisms and culture:
Propionibacterium freudenreichii MTCC* 1950 and Lactobacillus delbrueckii Subsp. Lactis type MTCC 911 were procured from MTCC (Microbial Type Culture Collection Centre and Gene Bank) Chandigarh, India, for production of vitaminB12 and for bioassay respectively. The cultures were grown and maintained on nutrient agar and Yeast-Extract Lactate agar plates respectively at 300C. After optimum growth the cultures were stored at 40C till further use.
Physical Mutagenesis:
In the present study physical mutagenesis was selected in order to develop mutants of
Propionibacterium freudenreichii. For physical mutagenesis actively growing culture of
Propionibacterium freudenreichii was inoculated on fifteen culture plates by spread plate technique and these plates were exposed to U.V light (irradiated) with three different doses i.e. 200 erg/mm2, 300erg/mm2 and 400erg/mm2for 30sec, 60sec, 90sec, 120sec and 150 sec respectively as shown in Table-1 and incubated at 300C. The survivors of the first mutagenesis dose were again subjected to a second dose of mutagenesis. Finally the survivors of second dose were used for production of vitaminB12. In this way a total of fifteen (15) physical mutants, five mutants for each dose of U.V. irradiation were selected for the experiments.
Table 1. Details of dose and time of U.V. exposure for physical mutagenesis.
S.No DOSE OF
PHYSICAL MUTAGEN (U.V.LIGHT)
TIME OF EXPOSURE (sec)
STRAIN
1. 200 erg/mm2
30 Set-I A
60 Set-I B
90 Set-I C
120 Set-I D
150 Set-I E
2. 300 erg/mm2
30 Set-II A
60 Set-II B
90 Set-II C
120 Set-II D
150 Set-II E
3. 400 erg/mm2
30 Set-III A
60 Set-III B
90 Set-III C
120 Set-III D
150 Set-III E
Preparation of inoculum:
The inoculum was prepared by inoculating pure culture from above plates into a 200ml inoculum media in 500ml Erlenmeyer flasks and incubated at 300C for 24 hours in Hi-Media Mc. Intosh Jars Anaerobic-system Mark-III with gaspak.
Preparation of fermentation medium:
The production medium used for vitaminB12 production consists of following components:
Table-2. Composition of fermentation medium
COMPONENT PERCENTAGE
L-glutamic acid 1.0
Sucrose 6.0
(NH4)2HO4 0.3
KCL 0.08
MgSO4.7 H2O 0.05
MnSO4.4H2O 0.03
FeSO4.7H2O 0.003
ZnSO4.7H2O 0.002
Co(No3)2.6H2O 0.004
Betaine 1.0
Delta-amino levulinic acid hydrochloride 0.05
5,6-Dimethyl benziminazole 0.1
PH 7.0-7.2
Betaine acts as a source of methyl groups as it aids in the synthesis of methionine by acting as a methyl donor. The medium was prepared by combining all of the ingredients except the delta-aminolevulinic acid hydrochloride and sterilized at 15 lbs steam pressure (1210C) for 15 minutes. Delta-aminolevulinic acid is inactivated by heat and hence it was sterilized by a membrane filtration technique. The concentration of cobalt in the production medium is critical as excess of cobalt suppresses the formation of cobinamide. Delta aminolevulinic acid act as a precursor in the synthesis of vitaminB12 and the production is facilitated by the addition of it in the production medium.
Set up of fermentation process:
VitaminB12 production was carried out with obtained mutants of P. freudenreichii. Three (3) sets of mutant culture flasks (15 flasks in a set) were prepared and vitaminB12 production was carried out at three different temperatures i.e. 300, 320 and 340C with:
Set No. I incubated at 300C, Set No. II incubated at 320C and Set No. III incubated at 340C.
At each temperature fifteen (15) mutants in 15 flasks were incubated with a total of 45 conical flasks. About 5% of mutant inoculum was inoculated in each flask containing fermentation media (100 ml) and incubated for 7 days. The vitaminB12 fermentation is carried out by anaerobic and aerobic stages as mentioned below.
(ii) Anaerobic fermentation stage: The anaerobic fermentation phase lasts for 84hours, during which formation of cobinamide occurred. During the anaerobic phase pH fall was recorded from 7.2-6.5. The production media was supplemented with 5,6-Dimethyl benziminazole as. P freudenreichii cannot readily synthesize this base.
(ii) Aerobic fermentation stage: After 84 hours the flasks with production media were shifted from Mc.Intosh jars to orbital shaking incubator for aerobic phase. The aerobic phase of fermentation also lasted for another 84 hours during which the nucleotide was formed. This nucleotide under aerobic conditions links with previously formed cobinamide to give cobalamin.
Assay of vitaminB12
The amount of vitaminB12 produced was estimated by a microbiological bioassay method using Lactobacillus
milk medium and transferred biweekly. The culture was incubated at 37oC for 18-24 hours and then refrigerated until further use.
A standard stock solution of vitaminB12 (Hi Media) was prepared by diluting stock solution to a series of tubes to give 0.025, 0.05, 0.075, 0.10, 0.25, 0.5 and 0.75 mµg (milli microgram) of Vitamin B12 per tube. Distilled water and double strength medium were added to bring the final volume to 10 ml. The tubes were covered with metal cover lined with cotton and were autoclaved for 5 min at 121oC. The tubes were cooled and 1 drop of inoculum was added to each tube except the blank. The assay was read turbidometrically at 600nm on spectrophotometer after 15-18 hrs of incubation in a constant temperature water bath at 37oC.
RESULTS AND DISCUSSION:
VitaminB12 has been produced in the laboratory at 300, 320C and 340C temperature by mutant and parental strains of Propionibacterium freudenreichii through anaerobic and aerobic fermentation techniques as described in materials and methods, all experiments were performed in triplicates and mean values were considered. It is well established that during production of vitaminB12, production of propionic acid also takes place at anaerobic fermentation stage, which inhibits the vitaminB12 production activity of organism. This results in gradual downfall in vitaminB12 production and ultimately arrests the production of vitaminB12.
A clear effect of temperature, dose and time of U.V. radiation was observed on ability of vitaminB12 production in parental and mutant strains (Table-3).
Table-3. Production of VitaminB12 at different temperatures by Mutant Strains of P.freudenreichii
S.No DOSE OF
PHYSICAL MUTAGEN (U.V.LIGHT)
TIME OF EXPOSURE
(sec)
STRAIN YIELD OF VITAMINB12 (mg/lit)
AT
300C 320C 340C
1. Control --- Parental 19 18 17
2. 200 erg/mm2
30 Set-I A 19 18 18
60 Set-I B 19 18 18
90 Set-I C 19 18 18
120 Set-I D 19 18 18
150 Set-I E 20 19 18
3. 300 erg/mm2
30 Set-II A 19 18 17
60 Set-II B 20 18 17
90 Set-II C 21 18 18
120 Set-II D 22 20 18
150 Set-II E 22 21 19
4. 400 erg/mm2
30 Set-III A 23 22 20
60 Set-III B 24 22 21
90 Set-III C 19 18 18
120 Set-III D 19 18 18
150 Set-III E 19 18 18
It was noticed that at 300C temperature when vitaminB12 reached 19mg in production system, the further production of vitaminB12 ceased in parental as well as in mutant strain flasks, except Set-E (flask). The results are indicating that the short duration exposure to U.V. radiation (200 erg/mm2) did not develop any resistant characters in mutant strains towards the propionic acid which is produced extracellularly along with production of vitaminB12 in production system. Such type of inhibition in vitaminB12 production by Propioonibacterium
vitaminB12 among all the strains used in production of vitminB12 i.e. (23 and 24mg/lit), but the production of vitaminB12 drastically decreased in strains which were exposed for longer time i.e. 90, 120 and 150 sec (Table-3) and Figure-1.
In general the production of vitaminB12 decreased in all mutant strains and parental strains as temperature increases from 300C to 340C, as the lowest yield was recorded at 340C by respective mutants. Based on the results obtained it can be realized that the higher temperature is not suitable for production of vitaminB12 in fact, it is decreasing the yield of vitaminB12. Perhaps at higher temperature even after the mutagenesis mutant strains were unable to show required resistance towards the propionic acid which is produced along with vitaminB12.
Figure-1: VitaminB12 production by different mutant strains of Propionibacterium freudenreichii
High activity of the organism i.e. production of vitaminB12 was recorded at 30oC temperature and low yield was recorded at 32oC and 340C temperature. From Table- 3 and Fighure-1 it is clear that the growth inhibition which is caused by higher concentration of propionic acid under anaerobic conditions can be minimized when temperature was decreased. The above results infer the ability of the mutant strains in the production of vitaminB12. It was observed from studies that the selected UV radiation (dose, duration and fermentation
temperature), were improving the resistance in the organisms against the propionic acid. Further the fermentation parameter temperature also influenced the yield, as high yield was observed at 30oC. Based on the results obtained it is shown that there is high possibility to increase vitaminB12 production in the organism by physical mutagenesis. Modification of temperature in fermentation process may also be beneficial. Hence this technique can be used in strain improvement program and also in the production of vitaminB12.
REFERENCES:
[1] Shrikant A. Survase., Ishwar B. Bajaj., and Rekha S. Singhal., (2006). Biotechnological production of vitamins. Food Technol. Biotechnol 44 (3) 381–396.
[2] Marwaha, S.S., Sethi, R.P., Kennedy, J.F., and Rakesh, K. (1983). Simulation of fermentation conditions for vitamin B12 biosynthesis
from whey. Biotechnol. Adv. 11 481–493.
