Isolation and selection of thermotholerant lactic acid bacteria from nem chua

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY ISOLATION AND SELECTION OF THERMOTHOLERANT LACTIC ACID BACTERIA FROM NEM CHUA SUPERVISOR STUDENT Dr NGO THI PHUONG DUNG HUYNH THI YEN PHI Student code: 3064601 Session: 32 (2006-2010) Can Tho, 2010 APPROVAL SUPERVISOR Dr NGO THI PHUONG DUNG STUDENT HUYNH THI YEN PHI Can Tho, November…, 2010 PRESIDENT OF EXAMINATION COMMITTEE Dr NGUYEN VAN THANH ABSTRACT Nem chua is one of the popular traditional fermented products in Vietnam; however, the uncontrolled manufacture of this product in practice presently still has the challenges particularly in the microbiology aspects In fact, “Nem chua” fermentation is unstable and the Vietnamese producers often face problems of quality control due to the proliferation of undesirable microorganisms, depending on microorganisms found in nature and in meat, depending on condition of slaughter, meat quality, , especially temperature which affecting the growth rates of microorganisms It is more meaningful when they are used in industry Therefore, the thesis "Isolation and selection of thermotolerant lactic acid bacteria from Nem chua” was initially brought to find out the lactic acid bacteria which were thermotolerance and high saccharose fermentation results Nine strains were purely obtained These nice strains were VS1, VS2, VS3, VS4, CR1, CR2, CH1, CH2 and CH3 Of which, the CR1 gave the highest concentration of lactic acid fermentation generated when compared to other strains (0.336 g/l) Strains VS3 and VS1 following the CR1, which were at 0.276 g/l, 0.264 g/l respectively The strains of VS1, VS3 and CR1 were be able to grow at high temperature at 43oC Keywords: Isolation, Lactic acid bacteria, Nem chua, sacchorose CONTENTS Abstract Content Introduction Materials and methods Results and discussion 3.1 Isolation lactic acid bacteria 3.2 Identification of the isolates 3.3 Study of the ability of lactic acid bacteria on saccharose fermentation 3.4 Study of the tolerant temperature of lactic acid bacteria Conclusions and Suggestions i ii 8 11 13 18 20 INTRODUCTION The classification of lactic acid bacteria (LAB) was initiated in 1919 by Orla-Jensen and was until recently primary based on morphological, metabolic and physiological criteria The boundaries of the group have been subject to some controversy, but historically the genera Lactobacillus, Leuconostoc, Pediococcus and Streptococcus form the core of the group The general description of the bacteria included are Gram positive, non spore forming, non motile, rod or coccus shaped, catalase-lacking organisms It can grow under anaerobic condition, producing lactic acid as the major end product during the fermentation of carbohydrate (Axelsson, 2004) LAB was divided into two groups, based on products produced from the fermentation of glucose, namely homofermentative lactic acid bacteria and heterofermentative organism LAB is generally associated with habitats rich in nutrients such as milk, cheese, meat, beverages and vegetables It can be grown and enumerated on agar plates, as long as the agar plates are incubated in an oxygen poor environment Most LAB grow on MRS agar and form grayish white colonies on these media (Theron, 1999) Fermentation of various food stuffs by (LAB) is one of the oldest forms of bio-preservation practiced by mankind LAB are widely used as starter cultures in the manufacture of fermented products and milk products such as yoghurt, cheese, meat products, bakery products wine and vegetable (Axelsson, 2004) In the fermentation, raw materials are converted by microorganisms (bacteria, yeast and molds) to products that have acceptable qualities of food In common fermented products such as yogurt, pickles, lactic acid is produced by the starter culture bacteria to prevent the growth of undesirable microorganisms (Ray and Daeschel, 1992) Food fermentations have a great economic value and it has been accepted that these products contribute in improving human health Nem chua is a very popular traditional fermented meat product in Vietnam It is renowned by expatriate communities and international tourists who have taken an interest in Vietnamese cuisine in recent years With this raw consumption, the consumers can be provided with health benefits due to the live lactic acid bacteria (LAB) in the product (H.T.H.Nguyen et al., 2010) However, Nem chua fermentation is unstable and the Vietnamese producers often face problems of quality control due to the proliferation of undesirable microorganisms Objectives: This thesis was carried out with the aims of isolation and selection of thermotolerant LAB from Nem chua The experiments included: * To isolate and identify LAB from Nem chua * To test the ability of saccharose fermentation by LAB * To study the tolerant temperature of LAB 2 MATERIALS AND METHODS +Samples: different types of Nem chua +Mediums: MRS agar (De Man, Rogosa and SharpeMerck, Germany), MRS broth (Merck, Germany),… +Chemicals: ethanol, H2O2, NaOH, saccharose,… +Devices: pH meter (Sartorius, PB-20, Germany) Autoclave sterilization (Breukelen, Netherlands) Microscope (Olympus U-CMAD3, Japan) Water bath & Shaker (Gesellschaft, Germany) 2.1 Isolation LAB from Nem chua samples + Objective: To obtain pure isolates of LAB + Method: - Samples: Three types of commercial Nem Chua labeled as Van Son, Cai Rang and Chua Hue, obtained from the Maximax supermarket-Ho Chi Minh city, Cai Rang market-Can Tho and Co.opMart supermarket-Can Tho city - One gram of Nem chua samples was grinded well - Then put into the test-tube contains ml sterile water and serially diluted (102 to 107) - Take one ml of each diluted sample to spread on selective MRS agar media (MRSA) - The plates were incubated anaerobically at 32°C for 24 hours - Streaked onto MRSA until purity of culture was obtained 2.2 Identification of the isolates + Objective: to partially identify these isolates belong to LAB group Gram staining: is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gramnegative) based on the chemical and physical properties of their cell walls + Method: - Place a little colony bacterial on a slide - Stain the colony with crystal violet solution for - Pour off the stain - Flood slide with Iodine solution for - Pour off the Iodine solution - Destain by washing the slide briefly with alcohol- acetone (2-3 seconds) - Wash the slide completely with water to remove the acetone - Flood the slide with Fuchsin solution for minute - Wash with water + Result: - Gram positive: coloring all Gram-positive bacteria with a purple color - Gram-negative: coloring all Gram-negative bacteria with a red or pink color Catalase reaction: Each of the isolates was tested for catalase by placing a drop of 3% hydrogen peroxide solution on the cells Immediate formation of bubbles indicated the presence of catalase in the cells + Method: Catalase test was carried out by transferring a little colony of the isolates on MRSA onto a clean slide, flooded with a drop of H2O2 and observed for production of effervescence + Result: If bubbles formed, the organism is catalasepositive If not, the organism is catalase-negative Oxydase reaction: Oxidases catalyse electron transport between substrates acting as electron donors in the bacterium and test by Tetramethyl - p - phenylendiamin dihydrochlorid + Method: Transferring a little colony of the isolates from MRSA onto a clean slide Put a filter-paper above Then flooded with a drop of Tetramethyl - p - phenylendiamin dihydrochlorid and observed for discolorment of filter-paper + Result: If oxidase enzymes are presented, the dye is reduced to a deep violet-blue color 2.3 Study of the ability of saccharose fermentation by lactic acid bacteria + Objective: study of saccharose fermentation by the isolates and analysis + Method: - The process of saccharose fermenting was shown in Figure 5 Gr Saccharose + Gr NaCl 186 ml Sterile H2O Stir well until substrates dissolved completely Pasteurize (95°C in 30 min) Autoclave sterilization Inoculate LAB (The density of bacteria’s 107 cells/milliliter) Recap the bottles Cool to 40-45°C Pour into jars Warm to 32oC Analyze the total acid as lactic acid Days: 0, 2, 4, 6, 8,… Figure Process for Saccharose fermentation of LAB (Nguyen Van Chuong, 2008) - Observational criterion: the total acid as lactic acid in days: 0, 2, 4, 6, 8… - The experiment was repeated three times - Data processed by the Statgraphics program (version 3.0) 2.4 Study the tolerant temperature of lactic acid bacteria + Objective: study of the temperature, at which the lactic acid bacteria can survive + Method: - Incubate the isolates with different temperature: 37, 40, 43, 46 and 49oC - Observe the development of the isolates - The experiment was repeated three times RESULTS AND DISCUSSION 3.1 Isolation of LAB A total of of LAB were selected from agar plates of MRS (Table 2) Table 2: Origin of the isolates Ordinal Name of Location the isolate VS1 Van Son sample-Maximax Supermarket-Ho Chi Minh city VS2 Van Son sample-Maximax Supermarket-Ho Chi Minh city VS3 Van Son sample-Maximax Supermarket-Ho Chi Minh city VS4 Van Son sample-Maximax Supermarket-Ho Chi Minh city CR1 Cai Rang sample-CaiRang makertCan Tho city CR2 Cai Rang sample-CaiRang makertCan Tho city CH1 Chua Hue sample- CoopMart Supermarket-Can Tho city CH2 Chua Hue sample- CoopMart Supermarket-Can Tho city CH3 Chua Hue sample- CoopMart Supermarket-Can Tho city Colonies of the isolates were showed from Figure to Figure 14 and the characteristics of the isolates were shown in Table Figure Colony of VS1 Figure Colony of VS4 Figure 12 Colony of CH1 Figure Colony of VS2 Figure Colony of VS3 Figure 10 Colony of CR1 Figure 11 Colony of CR2 Figure 13 Colony of CH2 Figure 14 Colony of CH3 Table 3: Characteristics of the isolates were observed under microscope at X100 Sample Macro Morphology The isolates Shape Color Micro morphology Spore— forming Motile Coccus Rod VS1 Round Grayish white - - ٧ VS2 Round Grayish white - - ٧ VS3 Round Grayish white - - ٧ VS4 Round, Small Grayish white - - ٧ CR1 Round Grayish white - - ٧ CR2 Round Grayish white - - ٧ CH1 Round, small Grayish white - - ٧ CH2 Round Grayish white - - ٧ CH3 Round, small Grayish white - - Note: (-): non spore-forming, (-): non motile 10 ٧ Most of the colonies of the isolate in MRSA were appeared in grayish white color and round Colony of strain VS4, CH1 and CH3 were smaller than colony of VS1, VS2, VS3, CR1, CR2 and CH2 Observing under microscope at X100, all of the isolates were non -motile and non spore-forming 3.2 Identification of the isolates - Gram staining: all of the isolates were reduced with purpose color, meaning that all the isolates were Gram positive Most of the strains had rod shape (VS1, VS2, VS3, VS4, CR2 and CH3) The strain CR1 had long-rod shape, and only trains had cocci shape (CH1 and CH2) Figures of Gram staining were showed from Figure 15 to Figure 23 - Calatase and oxidase reaction: Catalase test was carried out using 0.3% (v/v) H2O2 and oxidase test with Tetramethyl - p phenylendiamin dihydrochlorid The results were showing that these strains didn’t have catalase and oxidase enzymes in cells 11 Figure 15 The isolate of VS1 Figure 18 The isolate of VS4 Figure 21 The isolate of CH1 Figure 16 The isolate of VS2 Figure 17 The isolate of VS3 Figure 19 The isolate of CR1 Figure 20 The isolate of CR2 Figure 22 The isolate of CH2 Figure 23 The isolate of CH3 12 After testing, it can be concluded that all strains were identified as LAB, since they showed fundamental characteristics of the LAB groups, including: - Gram positive - Catalase and oxidase negative - Rods and Cocci shape - The development of these strains on MRSA 3.3 Study of the ability of LABs on saccharose fermentation: - A drop of 1ml cell suspension (approx 10^7 cells/ml) was added into 200 ml 4% (w/v) saccharose medium in Erlenmeyer flasks to test saccharose fermentation of the isolates - Those Erlenmeyer flasks were incubated at 32oC - The total acid as lactic acid generated by time - The results of total acid as lactic acid are shown in Table and Figure 24 13 Table 4: The results of Saccharose fermenting ability of LAB Treatment Total acid as lactic acid by days bc 0,084 cd 0,168 abc 0,240 10 a 0,264 12 b 0,156bcd 0,060 0,048c 0,072d 0,120cde 0,180bc 0,204c 0,168bcd 0,072bc 0,120bc 0,156abcd 0,216ab 0,276b 0,192ab 0,048c 0,084cd 0,096e 0,084d 0,072e 0,060e 0,084abc 0,096bcd 0,132bcde 0,252a 0,336a 0,228a 0,096ab 0,108bcd 0,108de 0,132cd 0,096e 0,084e 0,120a 0,180a 0,192a 0,228ab 0,156d 0,144cd 0,096ab 0,108bcd 0,120cde 0,132cd 0,192cd 0,180bc 0,12a 0,132b 0,18ab 0,252a 0,192cd 0,132d The statistical is significant only in column The same letters show no significant difference statistically in 95% 14 1=LAB strain isolated from Van Son sample (VS1) 2=LAB strain isolated from Van Son sample (VS2) 3=LAB strain isolated from Van Son sample (VS3) 4=LAB strain isolated from Van Son sample (VS4) 5=LAB strain isolated from Cai Rang sample (CR1) 6=LAB strain isolated from Cai Rang sample (CR2) 7=LAB strain isolated from Chua Hue sample (CH1) 8=LAB strain isolated from Chua Hue sample (CH2) 9=LAB strain isolated from Chua Hue sample (CH3) ` In the first days, the total acid as lactic acid was negligible, so the graph started on the second day of fermentation T h e t o t a l a c id a s la c t ic a c id ( g /l) 0.4 0.35 VS1 0.3 VS2 VS3 0.25 VS4 0.2 CR1 0.15 CR2 0.1 CH1 0.05 CH2 CH3 10 12 Days of fermentation Figure 24 The total acid as lactic acid generated by the time of the isolates 15 The figure 24 shows that all of the isolates were capable of producing lactic acid at different levels The isolate CR1 produced highest amount of lactic acid at tenth day of the fermentation, which was at 0.336g/l (total acid value of strain CR1 was significantly different at 5%, comparing with other treatments), followed by VS1 and VS3, which were at 0.264 g/l and 0.276 g/l, respectively However some other strains such as VS4 and CR2 produced lactic acid at very low level (VS4 was at 0.096 g/l, CR2 was at 0.132 g/l) In addition, total acid value was peaked at 10th day of 12days fermenting period in out of strains (VS1, VS2, VS3, CR1 and CH2 strains), whereas the highest total acid of strain VS4 was observed at 6th day of the survey time and others at 8th day (CH1, CR2 and CH3) The total acid as lactic acid generated is lower than the results of Nguyen Van Chuong (2008) when saccharose fermentation by LAB isolated from “Măng chua” of the highest total was 3.8887 (%w/v) It was caused by the difference of the isolate strains and temperature of fermentation The pH value is shown in Figure 25 16 7.5 VS1 6.5 VS2 pH value VS3 VS4 5.5 CR1 CR2 CH1 4.5 CH2 CH3 3.5 10 12 Days of fermentation Figure 25 pH values of LAB with Saccharose fermentation Figure 25 demonstrates that pH value of culture broth changing along the time of incubation It is clearly that there was a decreasing tendency in pH value in all of the treatments Noticeably, culture broth fermented by strain CR1 had its pH value felt down drastically at 10th day (pH was at 4.04), corresponding to its highest level of lactic acid measured Besides, pH value of culture broth of strain CH3, strain VS3 and strain VS1 were also low, which were at (4.12, 4.13, 4.21), respectively, demonstrating that these strains are very active LAB At 12th day of fermentation, lactic acid produced by all strain was reduced, and pH value was therefore increased The 17 results of acid increased in the first stage and decreased at later stages of fermentation were consistent with the study of Nguyen Van Chuong (2008) in the experiment of sucrose fermentation of lactic acid bacteria and Dang Thi Hong Nhung (2010) in experiment on Cocoa fermentation 3.4 Study of the tolerant temperature of lactic acid bacteria The isolates were cultured in MRSA medium After that, they were incubated in different temperature condition, namely 37, 40, 43, 46 and 49oC (Table 5) Results showed that all the strains were well developed at 38 C and 40oC In 43oC, only three strains were developed, including VS1, VS3 and CR1 However, when the temperature was at 46oC or higher, there was no colony appeared in all of the treatment Due to The Humanure Handbook of Joseph Jenkins (2005), Thermophilic bacteria can live in temperatures above 45oC (113°F), so these strains are Mesophilic bacteria They can live and develop in temperature ranges from 20-45°C (68-113°F) Remarkably, strain VS1, VS3 and CR1 can survive at a higher temperature (43oC) than the others 18 Table 5: Tolerant temperature of LAB Temperature 37 40 43 46 49 VS1 + + + - - VS2 + + - - - VS3 + + + - - VS4 + + - - - CR1 + + + - - CR2 + + - - - CH1 + + - - - CH2 + + - - - CH3 + + - - - o ( C) Strain Note: (+): colonies appeared, (-): colonies didn’t appear 19 CONCLUSIONS AND SUGGESTIONS Conclusions - Nice strains of LAB were isolate from three different samples of Nem chua: Van Son, Cai Rang and chua Hue - Of which isolates, the strain of CR1 is capable of generating highest total acid as lactic acid during saccharose fermentation (0.336 g/l) - Strains VS1, VS2 and CR1 can survive and develop at the highest temperatures (at 43oC) beside the other strains Suggestions - Strain CR1 can produce lactic acid at high level and is thermotolerant LAB, so this can be a very applicable LAB strain need to be further researched - Identification of the species level of nice strains, which have been isolated 20 REFERENCES Vietnamese Đặng Thị Hồng Nhung 2010 Khảo sát q trình lên men ca cao quy mơ nông hộ Luận văn tốt nghiệp Cử nhân ngành Công nghệ Sinh học, Viện NC&PT Công nghệ Sinh học, Trường Đại học Cần Thơ Nguyễn Văn Chương 2008 Phân lập vi khuẩn lactic từ tự nhiên để lên men Măng chua Luận văn Thạc sĩ, Viện NC&PT Công nghệ Sinh học , Trường Đại học Cần Thơ English Axelsson, L 2004 Lactic acid bacteria: Classification and physiology Chapter In Lactic Acid Bacteria, 3rd Ed (S Salminen, A von Wright and A Ouwehand, eds.), Marcel Dekker, New York, NY, pp 1-66 Joseph Jenkins 2005 The Humanure Handbook, 3rd edition Publishing, PO Box 607, Grove City, pp 237-239 H.T.T.Nguyet, F.B Elegado, N.T Librojo-Basilio, R.C Mabesa and E.I Dizon 2010 Isolation and characterisation of selected lactic acid bacteria for improved processing of Nem chua, a traditional fermented meat from Vietnam, Beneficial Microbe (1): 67-74 Orla-Jensen S 1919 The lactic acid bacteria Fred Hostand son,Copenhagen Ray, B and M Daeschel 1992 Food biopreservatives of microbial origin CRC Press, In Boca Raton, Florida, pp 3-11 Theron,J 1999 Laboratory manual in microbiology & plant pathogen University of Pretoria, Pretoria, South Africa, pp 160-161 21 ... Therefore, the thesis "Isolation and selection of thermotolerant lactic acid bacteria from Nem chua? ?? was initially brought to find out the lactic acid bacteria which were thermotolerance and high saccharose... methods Results and discussion 3.1 Isolation lactic acid bacteria 3.2 Identification of the isolates 3.3 Study of the ability of lactic acid bacteria on saccharose fermentation 3.4 Study of the tolerant... CONCLUSIONS AND SUGGESTIONS Conclusions - Nice strains of LAB were isolate from three different samples of Nem chua: Van Son, Cai Rang and chua Hue - Of which isolates, the strain of CR1 is capable of