Antimicrobial activity of cellulose casing impregnation with chitosan against LAB

NONG LAM UNIVERSITY - HO CHI MINH CITY FACULTY OF FOOD SCIENCE & TECHNOLOGY Antimicrobial Activity of Cellulose Casing Impregnation with Chitosan against Lactic Acid Bacteria A Thesis submitted in partial fulfilment of the requirements for admission to the degree of Bachelor of Food Science and Technology By Student: Nguyen Duy Phu Supervisor: Dr Chitsiri Rachtanapun Dr Vu Thi Lam An Ho Chi Minh City, 2013 NONG LAM UNIVERSITY - HO CHI MINH CITY FACULTY OF FOOD SCIENCE & TECHNOLOGY Antimicrobial Activity of Cellulose Casing Impregnation with Chitosan against Lactic Acid Bacteria A Thesis submitted in partial fulfilment of the requirements for admission to the degree of Bachelor of Food Science and Technology By Student: Nguyen Duy Phu Supervisor: Dr Chitsiri Rachtanapun Dr Vu Thi Lam An Ho Chi Minh City, 2013 Antimicrobial Activity of Cellulose Casing Impregnation with Chitosan against Lactic Acid Bacteria By NGUYEN DUY PHU A Project Report Submitted to the Faculty of Food Science and Technology, Kasetsart University, Thailand and Faculty of Food Science and Technology, Nong Lam University, Vietnam, in Partial Fulfilment of the Requirements for the Degree of Bachelor of Food Science and Technology at Nong Lam University Ho Chi Minh City, 2013 ACKNOWLEDGMENT … To start with, I am deeply grateful to my parents for all things of my life I sincerely thank the teachers of the faculty of Food Science and Technology, Nong Lam University Ho Chi Minh City who have given me precious knowledge during four years studying I shall never forget the hospitality from Kasetsart University Thailand; Dr Siree Chaiseri, the Dean of Faculty of Agro-Industry, Kasetsart University and Dr Sasitorn Tongchitpakdee, the Deputy Head of Department of Food Science and Technology Especially, a big thank from my heart to Dr Chitsiri Rachtanapun, Ms Fuse Pongkwan, Mr Phuthai Buakham who were directly advisor, guided enthusiastically as well as all of members in microbiology lab All are so kind and friendly From this short-time internship, I learned so many new things in my life such as new knowledge, culture and people etc… Moreover, I would like to thank Dr Vu Thi Lam An, Department of Food Microbiology, Faculty of Food Science and Technology, Nong Lam University who guided enthusiastically and helped me during time executing graduation thesis Last but not least, I thank a lots my classmates in Advanced Education Program course 34 who always help me during studying - Ho Chi Minh City, February 2013Nguyen Duy Phu i ABSTRACT Chitosan, a versatile hydrophilic polysaccharide derived from chitin, has a broad antimicrobial spectrum against gram-negative, gram-positive foodborne pathogenic bacteria Antimicrobial mechanisms of chitosan were presented and briefly discussed This research was aimed to produce the antimicrobial casing by combining between chitosan and cellulose casing using vacuum impregnation technique, and then cellulose casing impregnation with chitosan were experimented Cellulose casing was impregnated with chitosan solution % (v/v) for 20 s After that, it was carried out in two steps in a chamber containing the liquid phase of chitosan solution In the first step, pressure was hold at -680 mmHg for or by vacuum pressure In this step, the internal gas in the pores of cellulose casing was expanded and partially flown out In the second step, atmospheric pressure at 760 mmHg was restored in the chamber for 30 s, residual gas was compressed, and the external liquid was flown into the pores of cellulose casing The impregnated casing was then dried in an oven at 50 oC for 30 After that, antimicrobial casing was cut into small pieces (7 cm x cm) and embedded in aseptic plastic The antimicrobial activity of cellulose casing impregnation with chitosan was investigated against Lactobacillus plantarum TISTR 844 and TISTR 850 which are potential food spoilage bacteria in sausages The impregnated casing was immersed in 36 ml MRS broth with ml of log CFU/ml, and then incubated at 37 ± oC at 24 ± h The totals of lactobacilli were enumerated by using the spread plate method The results showed that impregnated casing could inhibit microbial growth and reduce number of L plantarum TISTR 844 by 1.04 log CFU/ml and L plantarum TISTR 850 by 1.37 log CFU/ml compared with controlled sample The result of this research proved the antimicrobial property of chitosan associated with cellulose casing in vacuum impregnation against foodborne pathogenic bacteria ii TABLE OF CONTENTS ACKNOWLEDGMENT i ABSTRACT ii TABLE OF CONTENTS iii ABBREVIATIONS AND SYMBOLS v LIST OF FIGURES vi LIST OF TABLES vii CHAPTER INTRODUCTION CHAPTER LITERATURE REVIEW 2.1 A brief introduction of chitosan 2.1.1 Definition and composition of chitosan 2.1.2 Antimicrobial mechanism of chitosan 2.1.3 Application of chitosan in food industry 2.2 The role of lactic acid bacteria in fermented sausages 2.3 Recent research of chitosan against pathogenic bacteria CHAPTER MATERIALS AND METHOD 3.1 Material 3.1.1 Chitosan 3.1.2 Cellulose casing impregnation 3.1.3 Lactic acid bacteria 10 3.2 Antimicrobial activity of cellulose casing impregnation with chitosan against lactic acid bacteria 10 3.2.1 Purpose 10 iii 3.2.2 Method 10 CHAPTER RESULTS AND DISCUSSION 12 CONCLUSION 14 RECOMMENDATION 15 APPENDIX 16 REFERENCES 24 iv ABBREVIATIONS AND SYMBOLS % (v/v) : Volume concentration B cereus : Bacillus cereus Co : Company DA : Degree of acetylation E coli : Escherichia coli fig : Figure h : Hour L brevis : Lactobacillus brevis L bulgaris : Lactobacillus bulgaris L monocytogenes : Listeria monocytogenes L plantarum : Lactobacillus plantarum L sakei : Lactobacillus sakei L.curvatus : Lactobacillus curvatus LAB : Lactic acid bacteria : Minute MRS : De Man, Rogosa and Sharpe MW : Molecular weight o C : Degree Celsius s : Second S aureus : Staphylococcus aureus spp : Species Tab : Table TISTR : Strain VI : Vacuum impregnation VP : Vacuum pressure v LIST OF FIGURES Fig 2.1.Formula of chitosan … Fig 2.2.The phylogenetic relationship of L plantarum with LAB…………… ………… Fig 4.1.Change in number of lactic acid bacteria for 24 h incubated with cellulose casing with and without chitosan treatment……………………………………………… 12 Fig 4.2.Colonies number of pathogenic bacteria………………………………………… 14 Fig A.1.Automatic pipet …………………………………………………………………17 Fig A.2.Incubator………………………………………………………………………… 18 Fig A.3.Electric balance……………………………………………………………………19 Fig A.4.Vortex mixer………………………………………………………………………19 Fig A.5.Laminar flow… ……………………………………………………………… 19 vi LIST OF TABLES Tab 3.1 Three samples impregnated cellulose casing… ……………………… Tab 4.1 Log reduction between of L plantarum TISTR 844 and TISTR 850 in Cellulose casing with Chitosan treatment after 24 h………………………………… 13 Tab A.1 Typical analysis of peptone………… …… …………………………… 16 Tab A.2 Approximate formula g/l of MRS broth…… ……… ……………………17 Tab A.3 Log reduction starter culture of L plantarum TISTR 844… … …………22 Tab A.4 Log reduction of L plantarum TISTR 844 in cellulose casing with and without chitosan treatment…………………………………………………………… 22 Tab A.5 Log reduction starter culture of L plantarum TISTR 850…… … ………23 Tab A.6 Log reduction of L plantarum TISTR 850 in cellulose casing with and without chitosan treatment…………………………………………………………… 23 Tab A.7 Statistic analyzed of starter culture L plantarum TISTR 844 and L plantarum TISTR 850……………………………………………… …………………………….24 Tab A.8 Statistic analyzed of L plantarum TISTR 844 in cellulose casing with and without chitosan treatment…………………………………………………………… 24 Tab A.9 Statistic analyzed of L plantarum TISTR 850 in cellulose casing with and without chitosan treatment…………………………………………………………… 24 vii APPENDIX 1.1 Peptone: Peptone form meat peptic digested used as nutritive substrate in fermentation and culture media It is prepared from selected animal tissues of which fat and sinew removed Meat peptone provides nitrogen, vitamins, amino acids and carbon in microbiological culture media Its high sulfur compound content makes meat peptone, peptic suited for the detection of bacteria (Clostridia spp., Salmonella spp.) producing hydrogen sulfide As an ingredient in culture media meat peptone, peptic is usually employed in concentrations of 0.3 to 1.0% It is used for the cultivation of yeast and moulds, enterobacteria, and staphylococci, Merck-Millipore, Germany Tab A.1 Typical analysis of peptone Color granules Light yellow-beige Color in solution Yellow-beige pH (5% in water) 6.5-7.5 Loss on drying (105°C) ≤6.0% Sulfated ash (800°C) ≤17.0% Amino-nitrogen (N α) 4.5-6.5% Total nitrogen (Kjeldahl) 12.0-13.0% Nitrite (NO2) Absent 1.2 Agar: Granulated, purified and free from inhibitors for microbiology For solid culture media which can be inoculated on the surface approximately 12-15 g/l, with pH values below 6.0 up to 20 g/l For semi-solid culture media approximately 3-8 g/l For liquid culture media of low viscosity approximately 0.5-1 g/l Store dry and tightly closed protect from light Store at 15 - 25 oC, Merck-Millipore, Germany 1.3 MRS Agar 2%: It was produced by mixed between MRS broth and agar follow ratio 2% solution It used for plating as a medium for cultivation of lactobacilli 16 1.4 MRS broth: DificoTM lactobacilli MRS Broth base for the cultivation of lactobacilli, Merck-Millipore, Germany Suspend 52.25 g in litter of purified water Mix thoroughly Heat with frequent agitation and boil for minute for completely dissolve the powder Autoclave at 121 oC for 15 Test samples of the finished product for performance using stable, typical control cultures Tab A.2 Approximate formula g/l of MRS broth Proteose Peptone No.3 10 g Beef Extract 8.0 g Yeast Extract 4.0 g Magnesium Sulfate 0.2 g Manganese Sulfate 0.05 g Dipotasium Phosphate 2.0 g Dextrose 20 g Polysorbate 80 1.0 g Ammonium Citrate 2.0 g Sodium Acetate 5.0 g Final pH 6.5 ± 0.2 at 25ºC 2.1 Automatic pipet: It is a laboratory tool used to transfer small volumes of liquid, especially small volumes, accurately and precisely P-20, P-200, and P-1000 models which are recommended for volume transfers of from to 20 µ, 50 to 200 µ, and 100 to 1000 µ , Fisher Biotec Proprietary Limited, Australia Fig A.1.Automatic pipet 17 2.2 Bunsen burner: it is essentially a tube connected to a steady supply of flammable gas that is used in the laboratory to provide an adjustable open flame The flame is used to sterilize inoculating loops and needles 2.3 Auto clave: An autoclave is a device used to sterilize equipment and media by subjecting them to high pressure saturated steam at 121 °C for around 15–20 2.4 Incubator: An incubator is a device used to grow and maintain of course microbiological cultures or cell cultures The incubator maintains optimal temperature, humidity and other conditions such as the carbon dioxide and oxygen content of the atmosphere inside Incubators are essential for a lot of experimental work in microbiology lab The most commonly used temperature for bacteria approximately 37 °C, as these organisms grow well under such conditions Fig A.2.Incubator 2.7 Shaker: It used to mix substances with forceful gyroscope nutation, or horizontal or orbital oscillation 2.8 Vortex mixer: It used to rapidly oscillate liquids, suspend cells or dilute samples in microbiology or biochemical lab, Genius Co, USA 2.6 Electric balance: used to simplify complex laboratory measurements which help reduce errors in manual calculations, Mettler Toledo Co, USA 18 Fig A.3.Electric balance Fig A.4.Vortex mixer 2.5 Laminar flow Fig A.5.Laminar flow The laminar flow provides an aseptic work area while allowing the containment of infectious splashes or aerosols generated by many microbiological procedures It protects the working environment from dust and other air-born contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area 3.1 Streaking a slant agar i Remove the caps: While the wire is cooling, remove the first caps by grasping it with a little finger ii Agar slant tubes: The open tubes should be held in a nearly horizontal position After 19 that, flaming the mouth of the tubes for to s with the Bunsen burner iii Insert the Loop: When the inoculation loop has had time to cool, carefully insert it into the agar slant with the bacterial culture iv Streaking:  Wrap fingers of non-dominant hand around the culture tube containing broth for transfer  Using the pinkie finger of your dominant hand twist the cap from the tube Hold in your pinkie and not place it on the counter  Pass the mouth of the culture tube across the flame  Direct the inoculating needle into the broth  Flame the mouth of your broth culture tube and replace the cap Place it in your rack  Pick up the slant in your non dominant hand  Twist off the cap  Flame the mouth of the slant tube  Direct the inoculating needle into the tube and “stab” the agar in the base (butt)  Withdraw on the entry line and when you reach the surface make a simple streak along the face  Flame the mouth of the tube and replace the cap  Flame your inoculating needle and replace in your rack 3.2 Spread plate technique In natural habitats, bacteria usually grow together in populations containing a number of species In order to adequately study and characterize an individual bacterial species, one 20 needs a pure culture The spread plate technique is an easy, direct way of achieving this result In this technique, a small volume of dilute bacterial mixture containing 100 to 200 cells or less is transferred to the center of an agar plate and is spread evenly over the surface with a sterile, L-shaped glass rod The glass rod is normally sterilized by dipping in alcohol and flamed to burn off the alcohol After incubation, some of the dispersed cells develop into isolated colonies A colony is a large number of bacterial cells on solid medium, which is visible to the naked eye as a discrete entity Procedure: With a wax pencil, label the bottom of the agar medium plates with the name of the bacterium to be inoculated, your name and date Pipette 0.1 ml of the respective bacterial culture onto the center of an agar plate Dip the L-shaped glass rod into a beaker of ethanol and then tap the rod on the side of the beaker to remove any excess ethanol Briefly pass the ethanol-soaked spreader through the flame to burn off the alcohol, and allow it to cool inside the lid of a sterile petri plate Spread the bacterial sample evenly over the agar surface with the sterilized spreader, making sure the entire surface of the plate has been covered Also make sure you not touch the edge of the plate Immerse the spreader in ethanol, tap on the side of the beaker to remove any excess ethanol, and reflame Repeat the procedure to inoculate the remaining two plates Invert the plates and incubate for 24 to 48 h at room temperature or 37 oC After incubation, measure some representative colonies and carefully observe their morphology Finally record your results in the report 21 4.1 L plantarum TISTR 844 Tab A.3 Log reduction starter culture of L plantarum TISTR 844 Starter culture 1st 2nd Number of Dish 61 63 colonies Dish 63 67 volume of sample solution inoculated (ml) 0.1 Concentration 10-6 Log CFU/ml 8.79 8.81 Tab A.4 Log reduction of L plantarum TISTR 844 in cellulose casing with and without chitosan treatment Treatment Control V2N30 V3N30 1st 2nd 1st 2nd 1st 2nd Number of Dish 60 61 37 40 6 colonies Dish 62 64 44 46 8.63 7.65 7.84 Volume of sample solution inoculated (ml) 0.1 Concentration 10-6 Log CFU/ml 8.78 8.79 8.60 22 4.2 L plantarum TISTR 850 Tab A.5 Log reduction starter culture of L plantarum TISTR 850 Starter culture 1st 2nd Number of Dish 118 158 colonies Dish 114 157 Volume of sample 0.1 solution inoculated (ml) 10-6 Concentration Log CFU/ml 9.06 9.2 Tab A.6 Log reduction of L plantarum TISTR 850 in cellulose casing with and without chitosan treatment Treatment Control V2N30 V3N30 1st 2nd 1st 2nd 1st 2nd Number of Dish 123 146 76 75 colonies Dish 126 140 80 83 Volume of sample 0.1 solution inoculated (ml) 10-6 Concentration Log CFU/ml 9.09 9.15 8.89 23 8.89 7.69 7.81 Tab A.7 Statistic analyzed of starter culture L plantarum TISTR 844 and L plantarum TISTR 850 Starter culture TISTR 844 L plantarum TISTR 850 N Valid Mean 8.8000 9.1300 Std Error of Mean Std Deviation 01000 01414 07000 09899 Variance Minimum 000 8.79 010 9.06 Maximum 8.81 9.20 Tab A.8 Statistic analyzed of L plantarum TISTR 844 in cellulose casing with and without chitosan treatment Treatment N Valid Mean Std Error of Mean Std Deviation Variance Minimum Maximum L plantarum TISTR 844 Control V2N30 2 8.7850 8.6150 00500 00707 000 8.78 8.79 01500 02121 000 8.60 8.63 V3N30 7.7450 09500 13435 018 7.65 7.84 Tab A.9 Statistic analyzed of L plantarum TISTR 850 in cellulose casing with and without chitosan treatment L plantarum TISTR 850 Treatment Control V2N30 N Valid 2 Mean 9.1000 8.8900 Std Error of Mean 05000 00000 Std Deviation 07071 00000 Variance 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Antimicrobial activity of cellulose casing impregnation with chitosan against lactic acid bacteria 3.2.1 Purpose This experiment executed to evaluate the antimicrobial activity of cellulose casing impregnated... 3.1.2 Cellulose casing impregnation 3.1.3 Lactic acid bacteria 10 3.2 Antimicrobial activity of cellulose casing impregnation with chitosan against lactic acid