Isolation , characterization and comparative study of lactobacillus sp. using biochemial tests and ftir analysis
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The document summarizes a study that aimed to isolate, characterize, and compare Lactobacillus strains from different milk product samples. Various tests were performed on isolated strains including Gram staining, biochemical tests, analysis of growth parameters like absorbance and pH, and FTIR analysis. Results showed that the Lactobacillus ATCC 7469 strain and a strain isolated from Bifilac produced the highest amounts of lactic acid and had growth most similar to the reference strain based on FTIR analysis. The study characterized and compared Lactobacillus isolates from different milk sources.
Isolation , characterization and comparative study of lactobacillus sp. using biochemial tests and ftir analysis
- 1. Project by: Vaibhav Kumar Maurya 1. ISOLATION , CHARACTERIZATION AND COMPARATIVE STUDY OF LACTOBACILLUS SP. USING BIOCHEMIAL TESTS AND FTIR ANALYSIS
- 2. INTRODUCTION: The Lactobacillus is a gram positive, facultative anaerobic or microaerophilic, having low GC content, acid tolerant, non sporulating, rod or cocci . Lactic acid bacteria comprises Lactobacillus, Leuconostoc,Pediococcus, Lactococcus , Streptococcus (at core) and Enterococcus, Vagococcus, Weisella (as peripheral). Members of Lactic acid bacteria ferment carbohydrates into energy and lactic acid . Depending upon strain type metabolic pathways differ : Homofermentative and Heterofermentative. FTIR was applied to charaterize Lactobacillus from different samples. In this Comparitive study, ATCC 7469 was used as reference strain to characterize and analyse the spectra. 2.
- 3. OBJECTIVES: The aim of this project was to isolate, characterize and do a comparative study of Lactobacillus sp., from various sources of milk products. Isolation of Lactobacillus strains from different samples. Characterization by using conventional Gram’s Staining and Biochemical Tests. Determination of best strain by studying the effects of various parameters like absorbance, pH, free lactic acid formation and colony count. FTIR analysis for comparitive study of Lactobacillus strains isolated from different samples with ATCC 7469 sample .
- 4. ISOLATION OF LACTOBACILLUS SP. Conical flask of 100ml MRS broth + 1 conical flask of 100ml MRS agar. Autoclave 1g bifiliac was poured into each flask of MRS broth & flask were kept on shaker overnight. 15-20ml MRS agar was poured into each autoclaved petri plate. 50µl of broth was spread plated with an L-rod on the MRS agar plates. Overnight incubation at 37ºC , Gram’s Staining and Biochemical tests Lactobacillus colonies were subcultured and streaked onto an autoclave plate of MRS agar. 6.
- 5. Kulfi sample Bifiliac sample
- 6. Curd sample Milk sample 10.
- 7. BIOCHEMICAL CHARACTERIZATION BIOCHEMICAL TESTS: 1. Voges-Proskauer Test (VP test) Principal : Acetylmethylcarbinol to Diacetyl production . Procedure : 1. 100ml of Mr VP medium was prepared and autoclaved. 2. 15 ml of medium was poured into 6 test tubes. 3. Each test tubes was inoculated with a single colony of lactobacillus isolated from Milk, Kulfi, Curd, Bifiliac,Lactobacillus ATCC and left overnight. Result : Deep rose colour in culture in 15 minutes. 11.
- 9. 2. Methyl Red Test: Principal : Production and stabilization of high conc.of acid end products. Procedure : • 1.100 ml of MR VP medium was prepared and autoclaved. • 2. 15ml of medium was poured into 6 test tubes. • 3. Each test tube was inoculated with a single colony of Lactobacillus,isolated from Milk,Kulfi, Curd,Bifiliac and Lactobacillus ATCC. • 4. Left for overnight on shaker. • 5. The next day, 1ml methyl red indicator was pippeted and added to each boiling tube and left at room temp for 15-20 mins Result : pH 4 – Red colour ( +ve ). 13.
- 10. 12. Milk Sample Kulfi Sample Curd Sample Bifiliac Sample
- 11. 3. Catalase test: Principle : Degrade H2O2 by producing the enzyme Catalase. Procedure : 1.A single colony was touched with a loop and smear onto a clean slide. 2. 2 drops of hydrogen peroxide was added to smear. 3. Presence or absence of free oxygen gas bubbles was checked . Result : Bubbles of free oxygen gas is produced . 15.
- 12. 4. Citrate Utilization test : Principal : Alkaline medium changes bromothymol blue indicator from green to deep Prussian blue . Procedure : 1. 200ml of Simmons Citrate Agar was prepared and autoclaved . 2. 15ml agar each was poured into 6boiling tubes . 3. Each tube is Inoculated with a colony . 4. The boiling tubes were incubated at 35’C for 48 hours and colour change observed . Result : No microbial growth at the surfaces of the slants and the medium remained green thus indicating a negative result . 16.
- 13. 5. Growth at 15ºC and Growth at 45ºC : Procedure : 1. Single colonies wer touched with a loop and streaked onto two MRS agar plates . 2. One MRS agar plate was incubated at 15’C and another was incubated at 45’C overnight . 3. Plates were checked for growth the next day . Result : No growth of colonies was seen at 15’C. However,growth was seen at 45’C . 18.
- 14. • Growth was Observed at 45ºC • Growth was not observed at 15ºC 19.
- 15. Tests ATCC MILK ICE CREAM LOCAL CURD BIFILIAC VP test positive Positive Positive Positive Positive MR test Positive Positive Positive Positive Positive Citrate utilization test Negative Negative Negative Negative Negative Catalase test Negative Negative Negative Negative Negative Growth at 15ºC Negative Negative Negative Negative Negative Growth at 45ºC Positive Positive Positive positive positive Summary of Biochemical Tests:
- 16. EFFECT OF VARIOUS PARAMETERS ON THE CURDLING OF THE MILK 1. ABSORBANCE: The absorbance of the milk inoculated with Lactobacillus ATCC 7469 and Lactobacillus isolated from Milk, Vellore local curd, Ice cream, Bifilac was checked and growth curve plotted. TIME/SO URCE ATCC MILK ICE CREAM VELLOR E CURD BIFILIAC 1 hour 0.03 0.041 0.034 0.022 0.038 2 hours 0.031 0.042 0.035 0.022 0.038 3 hours 0.039 0.043 0.037 0.023 0.039 4 hours 0.042 0.055 0.043 0.031 0.052 5 hours 0.044 0.067 0.062 0.045 0.069 6 hours 0.052 0.079 0.084 0.069 0.077 7 hours 0.069 0.091 0.099 0.088 0.099 8 hours 0.078 0.109 0.103 0.096 0.12 9 hours 0.084 0.117 0.134 0.108 0.147 10 hours 0.11 0.141 0.171 0.134 0.165 11 hours 0.112 0.141 0.172 0.156 0.165 12 hours 0.112 0.142 0.173 0.156 0.166 Fig. Values of absorbance obtained at 600 nm
- 17. 0 0.05 0.1 0.15 0.2 0 5 10 15 ODat600nm hour ComparativeGraph ATCC MILK ICE CREAM VELLORECURD BIFILIAC
- 18. 2. pH: The pH of the milk inoculated with Lactobacillus ATCC 7469 and Lactobacillus isolated from milk and Vellore local curd. Ice cream. Bifilac was seen to decrease, indicating the production of Lactic Acid. Fig. Values of pH obtained Source/ Time ATCC MILK VELLOR E CURD ICE CREAM BIFILIAC After 1st hour 6.56 6.4 6.48 6.5 6.4 After 2nd hour 5.0 5.15 4.85 5 5.38 After 3rd hour 4.5 4.6 4.63 4.81 4.7 After 4th hour 4.4 4.32 4.37 4.4 4.15 After 5th hour 4.15 4.1 4.3 4.33 4.16
- 20. 3.LACTIC ACID PRODUCTION: Using the ether extraction method, the amount of free lactic acid produced for 100cc of milk was calculated as per the formula below: cc. of 0.2N alkali used __________________ X 105 = cc of 0.2 NaOH required to neutralize the lactic acid in 100 cc. of milk cc of ether decanted Fig. Values of cc. of 0.2N alkali used to neutralize the free lactic acid in ether extract for each sample Source/Tim e Bifiliac Milk Ice cream Vellore curd ATCC 1 hour 0.025 0.05 0.05 0.05 0.1 2 hour 0.075 0.06 0.07 0.07 0.15 3 hour 0.08 0.08 0.08 0.08 0.31 4 hour 0.8 0.15 0.32 0.14 0.64 5 hour 1 0.5 0.72 0.9 1.2
- 21. Fig. Values of cc. of free lactic acid produced per 100cc. of milk Hence, it was seen that the most amount of lactic acid of 34.12cc. for 100cc. of milk after 5 hours, was produced by Lactobacillus ATCC 7469 nearest to it Lactobacillus sp. from Bifilac that produces 26.2 cc for 100 cc of milk. Bifilac Milk Ice cream Vellore local curd ATCC 7469 1 hour 0.66 cc 1.34 cc 1.35 cc 1.34 cc 1.62 cc 2 hours 1.93 cc 1.59 cc 1.89 cc 1.88 cc 3.93 cc 3 hours 2.0 cc 2.17 cc 2.11 cc 2.10 cc 8.17 cc 4 hours 21.04 cc 3.98 cc 8.4 cc 3.64 cc 16.8 cc 5 hours 26.5 cc 13.15 cc 18.9 cc 23.69 cc 34.12 cc
- 22. 4. COLONY COUNT : Samples were collected after every two hours for every source and spread plated on MRS agar and incubated overnight to obtain colonies of the desired strain. After 2 hours After 4 hours After 6 hours After 8 hours After 10 hours After 12 hours Bifiliac 54 137 201 369 422 430 ATCC 61 141 250 400 439 444 Milk 72 169 279 391 470 480 Vellore Curd 65 150 220 319 405 410 Ice Cream 40 120 222 336 400 403 Fig. Colony count
- 23. FTIR ANALYSIS Preparation of samples for FTIR. Lyophilization of samples. Processing of samples for FTIR. Spectral analysis of samples.
- 24. - The process of curdling of milk involves the conversion of lactose to lactic acid, The prominent groups of lactic acid are the carboxylic group (OH-C=O) which lies in the range of wave numbers 1760-1670 cm-1 and the alkyl group (-CH3) which lies in the range of wavenumbers 2960-2850 cm-1 . - The detection and increase of these peaks in the spectra obtained by FTIR indicate the positive production of lactic acid. An increase in peak height is directly proportional to an increase in quantity. SPECTRAL ANALYSIS
- 25. Fig. Comparison of ATCC 7469 sample at 1st hour and 7th hour
- 26. Fig. Comparison of Milk sample at 1st hour and 7th hour
- 27. Fig. Comparison of Bifilac sample at 1st hour and 7th hour
- 28. Fig. Comparison of Ice Cream sample at 1st hour and 7th hour
- 29. Fig. Comparison of Vellore Local Curd sample at 1st hour and 7th hour
- 30. CONCLUSION The aim of this project was to isolate, characterize and do a comparative study of Lactobacillus sp. from various sources of milk products . Characterization was done by using conventional Gram’s Staining and Biochemical Tests which have shown the presence of Lactobacillus to be positive. After the Gram staining and Biochemical Tests to confirm the presence of our desired strains we processed our sample by using FTIR.The importance of this FTIR to characterize the organisms on genus and species level and determine the best strain, as it states the amount of similarities between the organisms. Effect of various parameters like Absorbance ,pH, amount of free lactic acid formation and colony count gave the idea of best strain on comparing with reference ATCC 7469 strain.
- 31. 22.