fermentation of pineapple juice by lactic acid bacteria

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY FERMENTATION OF PINEAPPLE JUICE BY LACTIC ACID BACTERIA SUPERVISOR STUDENT Dr. NGO THI PHUONG DUNG MACH TU UYEN Student code: 3082566 Session: 34 (2008-2013) Can Tho, May 2013 APPROVAL SUPERVISOR STUDENT Dr. NGO THI PHUONG DUNG MACH TU UYEN Can Tho, May 2013 PRESIDENT OF EXAMINATION COMMITTEE i Abstract Lactic acid bacteria are used popularly for fruit juice fermentation because they are good source of probiotics for human. In this study, lactic acid bacteria were isolated from pineapples and probiotic products. The isolated bacterial strains and some strains from the previous research were screened for application on pineapple fermentation process. There were 4 strains of lactic acid bacteria isolated from pineapple juice. All of them could grow in pH 1.5 medium and reached high density (over 6 log CFU/mL). Moreover, Lactobacillus acidophilus strain produced highest concentration of lactic acid, so it was the most favorable strain for pineapple fermentation. The different diluted ratios of pineapple juice (0%, 10%, 20% and 30% w/v) and different sucrose supplements (0%, 3%, 6%, and 9% w/v) were used for testing fermenting capacity of Lactobacillus acidophilus strain. The pure pineapple juice with 9% of sucrose supplement was found to be suitable for fermentation. Based on the results of sensory evaluation and bacterial density determination, the favorable conditions for pineapple fermentation were determined as follow: temperature at 37°C, fermentation time for 36 hours and initial bacterial level at 5 log cells/mL. The results of storage testing showed that the suitable temperature for product storage was 4 – 6°C, bacterial density (8.057 log CFU/mL) of final product maintained after 3 weeks. Key words: fermentation, lactic acid bacteria, Lactobacillus acidophilus, pineapple, probiotic. iii Contents Abstract ........................................................................................ iii Contents........................................................................................ iv 1. Introduction .............................................................................. 1 Objectives ................................................................................. 3 2. Materials and methods .............................................................. 4 2.1 Material............................................................................... 4 2.2 Methods .............................................................................. 4 2.2.1 Isolation and identification of LAB isolates at genus level...................................................................................... 4 2.2.2 Study on acidity tolerant ability of LAB isolates........ 5 2.2.3 Study on application in producing of fermented pineapple juice by lactic acid bacteria ................................. 5 2.2.4 Study on dilution ratio of pineapple juice and sucrose supplement ratio................................................................... 6 2.2.5 Study on inoculum density, temperature and fermentation time ................................................................. 7 2.2.6 Study on storage temperature and storage time .......... 7 3. Results and discussion.............................................................. 9 3.1 Results of isolation and identification of LAB isolates at genus level ................................................................................ 9 3.2 Acidity tolerant ability of lactic acid bacterium isolates .. 12 3.3 Application in producing of fermented pineapple juice by lactic acid bacteria .................................................................. 14 3.4 Dilution ratio of pineapple juice and sucrose supplements ratio ......................................................................................... 15 3.5 Study on inoculum density, temperature and fermentation time ......................................................................................... 18 3.6 Storage temperature and storage time .............................. 21 4. Conclusions and suggestions ................................................... 24 4.1 Conclusions ...................................................................... 24 4.2 Suggestions ....................................................................... 24 References ................................................................................... 25 iv 1. Introduction Nowadays, scientists as well as nutritional experts always research to find bio-products which are good for health and nutritional. These products are called probiotic products. Probiotics are bacteria that help maintain the natural balance of organisms. A number of probiotic strains have been introduced in the market in dietary and pharmaceutical forms. Lactic acid bacteria (e.g. Lactobacillus) and Bifidobacterium constitute the main group of probiotics commercialized for human consumption. The treatment of gastrointestinal infections continues to be complicated due to the expansion of antibiotic resistances. Of the benefits of probiotics, those related to their preventive and therapeutic uses against gastrointestinal infections have an outstanding position, as reflected in a large number of patents. The mechanisms of action of probiotics against gastrointestinal pathogens addressed in diverse patent applications include: modification of the environmental conditions, competition for nutrients and adhesion sites, production of antimicrobial metabolites and modulation of the immune and nonimmune defense mechanisms of the host (Sanz et al., 2007). Currently, the probiotic product is commonly used, which is type of fermented milk and yogurt. However, many consumers are intolerant of lactose as well as cholesterol in the milk product. Thus, the fruit juice is recommended as a good medium to produce fermented probiotic products (Mattila-Sandholm et al., 2002). Lactic acid bacteria (LAB) are part of the macrobiotic on mucous membranes, such as the intestines, mouth, skin, urinary and 1 genital organs of both humans and animals, and may have a beneficial influence on these ecosystems. They are used widely in probiotic products such as yogurt, fermented meat, fermented vegetables… LAB strains are supported the immunomodulatory role attributed. These products are not only used as food but also used for treating intestinal and stomach diseases because lactic acid bacteria can produce antibiotics to prevent and skill pathogenic bacteria. Fruit juices are good media for bacteria’s growth and probiotic products. Fruits and vegetables are good for health as they contain antioxidants, vitamins, fibers and minerals. There is no allergen in their components. Pineapple is one of the most popular fruits in tropical countries especially in Vietnam. Pineapple, Ananas comosus, belongs to the Bromeliaceae family, from which one of its most important health-promoting compounds, the enzyme bromelain, was named. The Spanish name for pineapple, pina, and the root of its English name, reflects the fruit's visual similarity to the pinecone.There are many vitamins (A, B1, B2, C et al.), enzyme bromelain and minerals (Fe, Mg, K, Zn, Ca et al.) in pineapple. So many health benefits of using pineapple such as potential antiinflammatory, digestive benefits, antioxidant protection, immune support and protection against macular degeneration (Ensminger et al., 1986; Cho et al., 2004; Maurer, 2001). Besides, pineapple juice is a suitable medium for lactic acid bacteria to produce new probiotic products. Therefore, the research “Fermentation of pineapple juice by lactic acid bacteria” was carried out. 2 Objectives To isolate lactic acid bacterium isolates from pineapple, and to study on conditions for fermented pineapple juice by lactic acid bacteria. The content of this study include: - Isolation and identification of LAB isolates at genus level - Study on acidity tolerant ability of LAB isolates - Study on application in producing of fermented pineapple juice by lactic acid bacteria - Study on dilution ratio of pineapple juice and sucrose supplement ratio - Study on inoculum density, temperature and fermentation time - Study on storage temperature and storage time 3 2. Materials and methods 2.1 Material - Pineapple bought from Hung Loi market, Ninh Kieu District, Can Tho City. - Six lactic acid bacterium isolate was isolated from five probiotics powder and fermented papaya juice ( Luong Phuoc Truong, 2012) - Sucrose - Medium  MRS broth (De Man, Rogosa and Sharpe-Merck, Germany): Peptone from casein 10 g/L, meat extract 8 g/l, yeast extract 4 g/L, D (+) glucose 20 g/L, dipotassium hydrogen phosphate 2 g/L, tween 80 1 mL, di-ammonium hydrogen citrate 2 g/L, sodium acetate 5 g/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.04 g/L.  MRS agar: MRS broth + agar 14 g/L. - Chemical: NaOH 0.1N, HCl 0.1N, H2O2 3%, NaHSO3, the Gram stain (Crystal violet, Iod, Acetone, Ethanol, Fuchsin). - Equipment in Food Biotechnology laboratory. 2.2 Methods 2.2.1 Isolation and identification of LAB isolates at genus level - Pineapple juice solution was contained in a glass, which was sterilized. Then, incubated for naturally fermented at 37ºC for 24 – 48 hours. Transferred 1 mL of fermentation solution above into a tube containing MRS broth medium. After 24 hours, transferred bacteria from MRS broth medium to MRS agar medium. 4 - Observation and photograph the bacteria under the light microscope with objectives 100X. - Genus identification by some tests: Gram stain and catalase, oxidase, dissolution of CaCO3. 2.2.2 Study on acidity tolerant ability of LAB isolates Inoculated 1 mL of LAB isolates (6 log cells/mL) into 9 mL MRS broth medium with different pH levels (1.5; 2.5 and 3.5). There were two factors (bacterium strains and pH) and triplication in this experiment. Then, bacterial density of each treatment was determined at initial inoculating time (at T0) and 2 hours after inoculating (at T2) by plate counting method on MRS agar. - Evaluation criterion: cell density of the bacterial strains. - Results: Chose bacterial strains tolerant ability of lowest - Using Statgraphics Centurion XV software to analysis pH. data. 2.2.3 Study on application in producing of fermented pineapple juice by lactic acid bacteria - Pineapple was pressed and put into the tubes with 50 mL volume. - Pasteurized by 140 mg/L of NaHSO3 in 20 minutes, then inoculated the strains of LAB into tubes, each tube contained 36 mL of pineapple juice and 4 mL of LAB. Inoculum density of all samples were 5 log cells/mL. - There were one factor and triplication in this experiment. Incubated at 37ºC for 48 hours and then analyzed the assessment criterion. 5  The evaluation criterion - pH: Measure by pH meter. - Soluble matter content (ºBrix): Measured by Brix handheld meter. - Lactic acid content: Based into total acid index. - Density of LAB: Plate counting method. - Sensory evaluation. 2.2.4 Study on dilution ratio of pineapple juice and sucrose supplement ratio Experiments were conducted randomly with two factors, at 4 levels and triplication. Total number of treatments: 4x4x3 = 48 X factor (%): Percentage of dilution ratio X1 = 0% X2 = 10% X3 = 20% X4 = 30% Y factor (%): Percentage of sucrose ratio Y1 = 0% Y2 = 3% Y3 = 6% Y4 = 9%  Processing: Pineapple juice was diluted at four levels (0%, 10% 20%, 30% w/v). Pasteurized by NaHSO3 (140 mg/L) for 20 minutes. Then, distributed into 48 tubes, each tube contained 40 mL. Each dilution rate was mixed with four sucrose levels. Then they were measured pH and ºBrix. Inoculated 1% bacteria with 7 log cells/mL into each tube (0.4 mL/tube). Then, all of tubes were incubated at 37ºC for 48 hours.  The evaluation criterion - pH - Soluble matter content (ºBrix) - Lactic acid content - Density of LAB 6 2.2.5 Sensory evaluation Study on inoculum density, temperature and fermentation time The experiment was conducted in three factors and duplication. Density of LAB (log cells/mL): X1 = 3 X2 = 5 X3 = 7 Incubating temperature factor (ºC): Y1 = 30 Y2 = 37 Y3 = 28 – 32 (room temperature) Fermentation time factor (hour): Z1 = 24  Z2 = 36 Z3 = 48 Processing: Selected the most appropriate treatments in experiments in section 2.2.4, fixed 2 treatments and continued to do this experiment. Performed the same experiment above but density of LAB are diluted with saline for 5, 7, 9 log cells/mL. Then inoculated into the tube 1% of bacteria (initial concentration the bacteria corresponding 3, 5, 7 log cells/mL). Divided each group and incubated conducted at different time and different temperatures.  The evaluation criterion: - pH - Soluble matter content (ºBrix) - Lactic acid content - Density of LAB - Sensory evaluation 2.2.6 Study on storage temperature and storage time The experiment included two factors and triplication. 7 Factor 1: Storage temperature: the refrigerator (4 – 6ºC), cool room (20 – 25ºC) and room temperature (28 – 32ºC). Factor 2: Storage time: 1, 2 and 3 weeks. The total number of experimental units: 3 x 3 x 3 = 27.  The evaluation criterion: - pH - Soluble matter content (ºBrix) - Lactic acid content - Density of LAB - Sensory evaluation 8 3. Results and discussion 3.1 Results of isolation and identification of LAB isolates at genus level From naturally fermented pineapple juice four bacterial strains were isolated. Besides, this project used six lactic acid bacteria, taken from thesis of Luong Phuoc Truong (2012). The aim of isolation was to compare the usability of pineapple juice by four isolated strains and known strains. Determination of whether these isolated strains in experiment 1 depended on lactic acid bacteria was tested by some basically physical and biochemistry properties. Gram staining Results of gram staining gave information that bacterial species remained purple color after staining crystal violet, thus they were gram positive bacteria. Figure 10. Gram-positive bacteria (X100) Dissolution of CaCO3 Lactic acid from bacteria was dissolved CaCO3 and formed clear zones around colonies on the medium (MRS agar with 1.5% CaCO3). 9 Figure 11. Reduction of CaCO3 by lactic acid Catalase test The results showed that the formation of bubbles did not occur in them, indicated negative results. Figure 12. Catalase-negative Oxidase test Because color changes of filter paper strips were absent in surface of agar plate (Figure 13). Therefore, these bacteria did cytodrome not contain oxydase in Figure 13. Oxidase-negative metabolism. Morphology characteristics of bacteria were briefly described by Table 2. 10 Table 2. Morphology characteristics of bacteria LAB Numbers Sources Colony Cell shape isolates Antibio 1* 2* probiotic White color, circular A shape, convex elevation, power smooth texture. Probio White color, circular probiotic P shape convex elevation, power Short rod Short rod smooth texture White color, circular Ybio probiotic 3* Y shape, convex elevation, Short rod power smooth texture Lactomin plus 4* 5* probiotic White color, circular L shape, convex elevation, power smooth texture Biosubtyl White color, irregular probiotic Bio shape, convex elevation, power Cocci Long rod smooth texture White color, circular 6* Papaya juice DD shape, convex elevation, Rod in pair smooth texture White color, circular Pineapple 7 K1 shape, convex elevation, Short rod juice smooth texture White color, circular Pineapple 8 K2 shape, convex elevation, juice smooth texture 11 Long rod LAB Numbers Sources Colony Cell shape isolates White color, circular Pineapple 9 K3 shape, convex elevation, Short rod juice smooth texture White color, circular Pineapple 10 K4 shape, convex elevation, Short rod juice smooth texture Due to the above mentioned characteristics, four isolated bacteria were lactic acid bacteria with following characteristics such as: gram-positive bacteria, catalase-negative, reducing lactic acid to dissolution of CaCO3, cocci or rod shape and growth in MRS agar. 3.2 Acidity tolerant ability of lactic acid bacterium isolates The results were shown in Table 3. Table 3. The evaluation criterion of LAB density pH 1.5 LAB density at T0 LAB density at T2 (log CFU/mL) (log CFU/mL) Strains ij 6.85efg A 1.545 P 1.53jk 6.87cde Y 1.555i 6.82ghi L 1.52k 6.81 ij Bio 1.53jk 6.93a DD 1.515k 6.86def K1 1.55ij 6.825hi K2 1.56i 6.81 ij K3 1.53jk 6.86abcd K4 1.51k 6.8j 12 pH 2.5 3,5 CV% LAB density at T0 LAB density at T2 (log CFU/mL) (log CFU/mL) A 1.925gh 6.89bc P 1.96e 6.88bcd Y 1.91h 6.89bc L 1.93fgh 6.85efg Bio 1.91h 6.86def DD 1.955e 6.8j K1 1.915h 6.83ghi K2 1.95ef 6.87cde K3 1.92gh 6.82hij K4 1.94efg 6.825hi A 4.28a 6.9b P 4.235d 6.95a Y 4.24cd 6.93a L 4.25bcd 6.935a Bio 4.26abc 6.84fgh DD 4.28a 6.81 ij K1 4.23d 6.88bcd K2 4.26abc 6.87cde K3 4.26abc 6.83ghi K4 4.27ab 6.81ij 47.038% 0.6403% Strains *Note: Value in the table was average value of triplication; the average values with the same letter were not significantly different at the 95% confidence level. After incubation at 37ºC for 2 hours with different pH levels, all strains increased densities. 13 At pH 1.5 and 2.5, the bacterial densities reduced significant at initial time point (T0) with value (1.51 – 1.96 log CFU/mL), but they recovered, increased and achieved value (6.8 – 6.93 log CFU/ml) after incubation at 37ºC for 2 hours. Each bacterial species were specialized to live in basic pH. If they moved stress pH conditions, metabolic imbalances of bacteria and environment occurred, that led to the death of bacteria. At pH 3.5, most of bacteria resistanted and reduced slightly at initial time point (T0) with value (4.23 – 4.28 log CFU/mL), as well as, increased and achieved value (6.8 – 6.95 log CFU/mL) after incubation at 37ºC for 2 hours. 3.3 Application in producing of fermented pineapple juice by lactic acid bacteria The result was shown in Table 4. Table 4. The evaluation criterion after fermentation of pineapple juice LAB strains ºBrix after fermentation pH after fermentation Lactic acid (% w/v) A 8a 3.417bc 0.786cd 4.247b 7.207de P 8a 3.513a 0.768cd 4.223bc 7.28bc Y 8a 3.39bc 1.203a 4.353a 7.38a L 8a 3.407bc 0.882b 4.263b 7.163e Bio 8a 3.383bc 0.882b 4.247b 7.233cd DD 8a 3.417bc 0.732d 4.247b 7.31b K1 7,333c 3.35c 0.84bc 4.247b 7.257cd K2 7,833ab 3.523a 0.81bcd 4.237bc 7.21de K3 7,667b 3.26d 0.773cd 4.067bc 7.283bc K4 7,833ab 3.437b 0.81bcd 4.18c 7.247cd 14 LAB density at LAB density at T0 T48 (log CFU/mL) (log CFU/mL) CV% 3.310% 2.370% 16.003% 1.274% 0.885% *Note: Value in the table was average value of triplication; the average values with the same letter were not significantly different at the 95% confidence level. Over view, ºBrix and pH after fermentation were reduced after inoculating for 48 hours because of converting sucrose to lactic acid by LAB. L. acidophillus from Ybio probiotic powder had a preponderance of lactic acid content. LAB density at initial inoculum was close to inoculum density. The Y strain included highest LAB density (4.353 log CFU/mL) and the difference was significant with other strain. After 48 hours incubated, LAB density was increased above 6 log CFU/mL. That satisfied the requirement of probiotic product. There for, all of these LAB strains could use to apply in producing of fermented pineapple juice. Besides, acid lactic content of L. acidophillus from Ybio probiotic powder was highest (1.203% w/v) than others. So this bacterium was chose to apply in producing of fermented pineapple juice. 3.4 Dilution ratio of pineapple juice and sucrose supplements ratio The result indicated that L. acidophilus isolated from Ybio probiotic powder could be applied in fermentation of pineapple juice. At the same dilution of juice, the lowest bacterial density was measured in the treatment of 0% of sugar. The reason is that nutrient-depleted environment after converting all sugar into lactic acid inhibited the bacterial growth and development. Besides, lactic acid itself could inhibit the development of bacteria (Nguyen Thi Hien, 2006). In contrast, the treatment of 15 9% of sugar content exhibited the highest bacterial density, which means this was optimal for fermentation of pineapple juice. Moreover, bacterial density at all dilution ratios was above 6 log CFU/mL, this will be probably considered for further research of probiotic production. Figure 15. Changes of LAB density by different dilution ratio and sugar content Choosing suitable dilution ratios and sugar content as well as appropriate sensory evaluation determine fermented juice quality. The evaluation was carried out with 5 parameters by 5 undergraduates of Biotechnology (Table 6). The results showed that fermented juice samples at initial dilutions were statistically significant different (p[...]... pH data 2.2.3 Study on application in producing of fermented pineapple juice by lactic acid bacteria - Pineapple was pressed and put into the tubes with 50 mL volume - Pasteurized by 140 mg/L of NaHSO3 in 20 minutes, then inoculated the strains of LAB into tubes, each tube contained 36 mL of pineapple juice and 4 mL of LAB Inoculum density of all samples were 5 log cells/mL - There were one factor and... (ºBrix) - Lactic acid content - Density of LAB - Sensory evaluation 8 3 Results and discussion 3.1 Results of isolation and identification of LAB isolates at genus level From naturally fermented pineapple juice four bacterial strains were isolated Besides, this project used six lactic acid bacteria, taken from thesis of Luong Phuoc Truong (2012) The aim of isolation was to compare the usability of pineapple. .. (6.8 – 6.95 log CFU/mL) after incubation at 37ºC for 2 hours 3.3 Application in producing of fermented pineapple juice by lactic acid bacteria The result was shown in Table 4 Table 4 The evaluation criterion after fermentation of pineapple juice LAB strains ºBrix after fermentation pH after fermentation Lactic acid (% w/v) A 8a 3.417bc 0.786cd 4.247b 7.207de P 8a 3.513a 0.768cd 4.223bc 7.28bc Y 8a 3.39bc... That satisfied the requirement of probiotic product There for, all of these LAB strains could use to apply in producing of fermented pineapple juice Besides, acid lactic content of L acidophillus from Ybio probiotic powder was highest (1.203% w/v) than others So this bacterium was chose to apply in producing of fermented pineapple juice 3.4 Dilution ratio of pineapple juice and sucrose supplements ratio... result indicated that L acidophilus isolated from Ybio probiotic powder could be applied in fermentation of pineapple juice At the same dilution of juice, the lowest bacterial density was measured in the treatment of 0% of sugar The reason is that nutrient-depleted environment after converting all sugar into lactic acid inhibited the bacterial growth and development Besides, lactic acid itself could inhibit... color, circular Pineapple 10 K4 shape, convex elevation, Short rod juice smooth texture Due to the above mentioned characteristics, four isolated bacteria were lactic acid bacteria with following characteristics such as: gram-positive bacteria, catalase-negative, reducing lactic acid to dissolution of CaCO3, cocci or rod shape and growth in MRS agar 3.2 Acidity tolerant ability of lactic acid bacterium... development of bacteria (Nguyen Thi Hien, 2006) In contrast, the treatment of 15 9% of sugar content exhibited the highest bacterial density, which means this was optimal for fermentation of pineapple juice Moreover, bacterial density at all dilution ratios was above 6 log CFU/mL, this will be probably considered for further research of probiotic production Figure 15 Changes of LAB density by different... development of bacteria in pineapple juice Figure 16 Changes of lactic acid content by different inoculum density and fermentation time In general, after 48-hour incubation, lactic acid content in sample-7 log cells/mL-37ºC was higher than that in sample-5 log cells/mL-37ºC after 36-hour incubation, 1.89% w/v and 1.65% w/v, respectively In addition, with the same inoculum and incubation time, lactic acid. .. temperature 18 for the growth and development of bacteria This could lead to high lactic acid was converted At the same temperature and fermentation time, inoculum density of 7 log cells/mL gave higher lactic acid content than inoculum of 3 and 5 log cells/mL This might result from the fact that the higher the inoculum density, the higher lactic acid content Similarly, bacterial density was highest at sample... Gram-positive bacteria (X100) Dissolution of CaCO3 Lactic acid from bacteria was dissolved CaCO3 and formed clear zones around colonies on the medium (MRS agar with 1.5% CaCO3) 9 Figure 11 Reduction of CaCO3 by lactic acid Catalase test The results showed that the formation of bubbles did not occur in them, indicated negative results Figure 12 Catalase-negative Oxidase test Because color changes of filter ... 2001) Besides, pineapple juice is a suitable medium for lactic acid bacteria to produce new probiotic products Therefore, the research Fermentation of pineapple juice by lactic acid bacteria was... producing of fermented pineapple juice by lactic acid bacteria The result was shown in Table Table The evaluation criterion after fermentation of pineapple juice LAB strains ºBrix after fermentation. .. development of bacteria in pineapple juice Figure 16 Changes of lactic acid content by different inoculum density and fermentation time In general, after 48-hour incubation, lactic acid content