Fermentation of mango juice by lactic acid bacteria

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MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY FERMENTATION OF MANGO JUICE BY LACTIC ACID BACTERIA SUPERVISOR STUDENT MSc. HUYNH XUAN PHONG NGUYEN LE VAN Student code: 3082484 Session: 34 Can Tho, 2013 APPROVAL SUPERVISOR STUDENT MSc. HUYNH XUAN PHONG NGUYEN LE VAN Can Tho, May …, 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract In order to take advantage of available fruit and contribute to the diversification of products from mango, this research was carried out in term of added value of fruit juice fermented by lactic acid bacteria (LAB). Four isolates of lactic acid bacteria from mango were obtained. The results of research showed that all 10 strains of lactic acid bacteria were able to develop in pH from 1.5 to 3.5 after 2 hours of incubation at 37°C with a density from 6.80 to 6.91 log cells/mL. After fermentation, the lactic acid content of 10 bacterial strains was in a range from 0.48 to 0.96% w/v, density after 2 hours of incubation at 37°C from 8.85 to 9.3 log cells/mL. Of which, strains isolated Lactobacillus acidophilus from Ybio yeast powder gave the best results with the following levels of lactic acid after fermentation was 0.96% w/v. The appropriate ratio for fermentation mango juice was 40% for dilution and 9% for blending sugar. The suitable conditions for mango juice fermentation were as follow: at 37°C for 36 hours and at 5 log cells/mL. In these conditions the sensory criteria and the density of bacteria could reach requirements for probiotic products (> 6 log cells/mL). These levels could identically maintain during the storage at temperature 4-6oC for 2 weeks. Keyword: fermentation, L. acidophilus, lactic acid bacteria, mango, probiotic. i CONTENTS Abstract ................................................................................. i Content ................................................................................... ii 1. Introduction ....................................................................... 1 2. Materials and methods ........................................................ 3 2.1 Material ............................................................................ 3 2.2 Methods ............................................................................ 3 2.2.1 Isolation and identification of LAB isolates at genus level. ................................................................................ 3 2.2.2 Study on the tolerance at low pH of LAB isolates .... 3 2.2.3 Study on the ability to produce mango juice fermented by LAB ............................................................ 4 2.2.4 Study on the ratio of dilution and blending sugar ...... 4 2.2.5 Study on the effects of LAB density, fermentation temperature and time ........................................................ 5 2.2.6 Study on temperature and time for storage conditions ......................................................................... 5 3. Results and discussion ........................................................ 6 3.1 Isolation of lactic acid bacteria .......................................... 6 3.2 The ability of LAB growing in low pH condition ............. 7 3.3 Applicability of mango juice fermented by LAB ............... 9 3.4 The ratio of dilution and blending sugar ............................ 11 3.5 Effect of inoculum density, temperature, fermentation time ........................................................................................ 16 3.6 The effect of temperature and time for storage conditions .. 19 4. Conclusions and suggestions ............................................... 22 4.1 Conclusions ................................................................ 22 ii 4.2 Suggestions ................................................................ 22 References........................................................................ 23 iii 1. INTRODUCTION Food is an indispensable necessity to humans. Growing society, human life is more and more enhanced. So, eating is not a main demand. The demand of enjoying and exploring the effect of food on health are top priority. Since, the relationship between food and dietary which have health benefits as well as help the body fight the illness has become the trend research of the nutritionist and scientists to create high-value products in terms of biological products, is known as "probiotics". The probiotic concept has been defined by Fuller (1989) to mean “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”. Salminen et al. (1999) proposed that probiotics are microbial cell preparations or components of microbial cells that have a beneficial effect on the health and well-being of the host. There are many probiotic products use for human, livestock, plants. It is produced and used of liquid or powder form. Lactic acid are Gram-positive bacteria (Fooks et al., 1999), ferment carbohydrates into energy and lactic acid (Jay, 2000). Depending on the organism, metabolic pathways differ when glucose is the main carbon source: homofermentative bacteria, whereas the heterofermentative transform a glucose molecule into lactate, ethanol and carbon dioxide (Caplice and Fitzgerald, 1999; Jay, 2000; Kuipers et al., 2000). In addition, LAB produce small organic compounds that give the aroma and flavor to the fermented product (Caplice and Fitzgerald, 1999). LAB are widely distributed in the nature. They could be isolated from 1 soils, water, plants, silages, waste products and also from the intestinal tract of animals and humans (Axelsson, 1998). LAB are widely used in probiotic products such as yogurt, nem chua, pickle,... These products are not only used for eating but also used to treat intestinal , stomach, due to lactic acid bacteria have the ability to produce antibiotics to prevent and kill bacteria and pathogenic microbes. Juice is a good environment for the growth of bacteria and probiotic products. Fruits and vegetables are foods with health benefits because they contain antioxidants, vitamins, fiber and minerals. Moreover, they do not contain any allergens for consumers. In fruits, mango is a popular fruit and has high nutritional value. Ripe mango has attractive yellow color, sweet and sour, savoury aroma is more preferred. Mangoes contain vitamin A, C, sugar, organic acids, so mango is widely used both unripe and ripe fruit. Mangoes are eaten fresh, making juice, jam, candy. In order to take advantage of available fruit and contribute to the diversification of products from mango, this research was carried out in term for added value of fruit juice fermented by lactic acid bacteria. Objectives: Study on the conditions of mango juice fermentation by lactic acid bacteria. 2 2. MATERIALS AND METHODS 2.1 Materials - Mango: Thanh Ca and Cat Chu. - Six isolates of lactic acid bacteria from Luong Phuoc Truong (2012). - Medium: MRS broth, MRS agar. - Chemicals, equipments in Food Biotechnology laboratory. 2.2 Methods 2.2.1 Isolation and identification of LAB isolates at genus level Mango juice was contained in a flask, incubated at 37oC for 24 - 48 hours. Transfered 1 mL mango juice fermented into a test tube containing MRS broth environment. After 24 hours, transfered bacteria from MRS broth to MRS agar environment. Continue transfer bacteria on MRS agar environment until pure. Observe the bacteria under the microscope objective lens X100. Identification through the preliminary test: Gram stain, catalase, oxidase test and dissolution of CaCO3. 2.2.2 Study on the tolerance at low pH of LAB isolates Transferring LAB isolates from experiment 1 into tubes containing MRS broth environment with 3 different levels of pH (1.5, 2.5 and 3.5). This was the 2 experimental factors (strain and pH) with 3 replications. Count the density of bacteria in individual treatments at incubation time (T0) and after 2 hours of incubation by plate counting method. 3 Monitor the indicators: density of the bacteria strains. Analyze the data with statistics program STATGRAPHICS Centurion XV. 2.2.3 Study on the ability to produce mango juice fermented by LAB Ripe mangoes were pressed (pasteurized with 140 mg/L of NaHSO3 in 20 minutes) and put in the centrifuge tubes. Inoculate each of LAB isolates into each tube. This was the 1 experimental factor (strains) with 3 replications. Incubation at 37°C, analyzed after 48 hours. Count density of lactic acid bacteria: plate counting method. Monitor the indicators: Brix, pH, level of lactic acid after fermentation and the density of lactic acid bacteria. Analyze the data with statistics program STATGRAPHICS Centurion XV. 2.2.4 Study on the ratio of dilution and blending sugar This was the 2 experimental factors with 3 replications. Ratio of dilution (%): 30, 40, 50, 60 Ratio of blending surose (%): 6, 9, 12, 15 After pressing, 480 mL of mango juice was diluted with ratios 30, 40, 50, 60 % and was blended sugar with rate 6, 9, 12, 15 %. After dilution and blending, these samples were pasteurized by NaHSO3 (140 mg/L in 20 minutes). Then the best LAB isolate in experiment 3 was inoculated in the samples and incubated at 37oC for 48 hours. Monitor the indicators: Brix, pH before and after fermentation, lactic acid content after fermentation and the 4 density of LAB, the sensory evaluation. Analyze the data with statistics program STATGRAPHICS Centurion XV. 2.2.5 Study on the effects of LAB density, fermentation temperature and time This was the 3 experimental factors at 3 levels with 2 replications. Density (log cells/mL): 3, 5, 7. Incubation temperature (ºC): 30, 37, 28-32. Time for fermentation (hours): 24, 36, 48. After pressing, mango juice was diluted and blended sugar by the appropriate rate in experiment 4, pasteurized by NaHSO3. Then, LAB isolate was inoculated with density were 3, 5, 7 log cells/mL, respectively. Incubated these samples at 30oC, 37oC, 28-32oC for 24, 36, 48 hours. Monitor the indicators: Brix, pH and the density of LAB, the sensory evaluation. Analyze the data with statistics program STATGRAPHICS Centurion XV. 2.2.6 Study on temperature and time for storage conditions This was the 2 experimental factors with 3 replications. Temperature for storage: 4-6oC, 20-25oC, 28-32oC. Time for storage: 1, 2 weeks. Monitor the indicators: Brix, pH and the density of LAB, the sensory evaluation. Analyze the data with statistics program STATGRAPHICS Centurion XV. 5 3. RESULTS AND DISCUSSION 3.1 Isolation of lactic acid bacteria After several transfers in MRS agar medium and visual observations, 4 isolates of lactic acid were isolated from 2 kinds of mango. Colonies of all isolates were round, smooth, grayish white. The cell morphology was shown in Table 2’ Table 2’. The characteristics of isolates Cell No Sample The isolate morphology 1 Cat Chu C213 Rod in pairs 2 Cat Chu C111 Rod in pairs 3 Thanh Ca T223 Rod in pairs 4 Thanh Ca T133 Cocci in pairs 5* Antibio A Short rod 6* Probio P Short rod 7* Ybio Y Short rod 8* Lactomin plus L Cocci 9* Biosubtyl Bio Long rod 10* Papaya ĐĐ Rod in pairs Note: The strains are marked * which were isolated from Truong (2012). 6 2 isolates from Cat Chu (C213, C111) were rod in pairsshaped, but the colony of C213 was smaller than C111. 2 isolates from Thanh Ca (T223, T133) were rod in pairs-shaped and cocci in pairs-shaped. 4 isolates were characterized in Gram positive, lack of catalase and oxydase, produced clear zone around colonies in medium containing CaCO3. Figure 6: Representative Figure 7: CaCO3 test oxydase test of C213 of T223 isolate isolate From the above characteristics, it could be concluded that 4 isolates belong to lactic acid bacteria group. 3.2 The ability of LAB growing in low pH condition The results in Table 3 showed that all bacterial strains at different pH levels have increased density after 2 hours of incubation at 37°C. At time T0, the density of bacteria in the MRS medium at pH 1.5 and 2.5 decreased significantly (from 5 log CFU/mL to 1.46-2.16 log CFU/mL). The reason was that the pH 1.5 and 2.5 are too low. So, most bacteria should be shocked. But only after 2 hours of incubation at 37°C, they worked to restore and increased the density up to 6.8-6.9 log CFU/mL. 7 At pH 3.5, most of bacteria could tolerate this pH. So, the density of LAB at the time T0 decreased slightly from 4.66 to 4.77 log CFU/mL. After 2 hours of incubation at 37°C, the density of bacteria increased up to 6.81-6.91 log CFU/mL. Density of 10 isolates at pH 3.5 was higher than at pH 2.5 and density of 10 isolates at pH 1.5 was lowest. This showed that the lower the pH affects the ability to survive and activity of LAB. Through this experimental study showed that all isolate were able to adapt and develop in a low pH medium, so they could tolerate the environment in the stomach. Therefore, these strains were able to apply in probiotic products. Table 3. The density of bacteria in MRS Broth environment at low pH. pH 1,5 Isolates logT0 Cv logT2 Cv (log CFU/mL) (%) (log CFU/mL) (%) j A 1.48 P j 1.46 Y j 1.49 L j 0.39 0.39 0.39 1.49 0.39 j Bio 1.49 ĐĐ j 0.67 1.48 0.00 C111 j 1.48 0.00 C213 1.47j 0.39 T223 j 1.46 T133 j 1.46 0.79 A 1.95h 2.56 0.39 8 e 0.15 de 0.31 cde 0.08 de 0.85 e 0.86 de 0.22 abcde 0.84 6.81 6.82 6.83 6.82 6.80 6.82 6.86 6.81e 0.90 abcde 0.29 e 6.81 0.34 6.84bcde 0.58 6.86 2,5 P 1.88i Y f 6.88abcd 0.80 2.45 bcde 0.34 bcde 0.30 L i 1.89 0.53 Bio 2.13f 1.18 ĐĐ g C111 2.06 2.67 g 2.04 C213 g 2.07 T223 1.88i 1.98 6.84 6.84 6.82de 0.29 abcde 0.37 ab 0.65 6.86 6.9 1.21 de 6.82 0.29 0.81 6.82cde 0.37 T133 h 1.93 0.79 bcde 6.84 0.58 A 4.72c 0.53 6.84bcde 0.39 P c 0.24 abc 0.73 abcde 1.27 a 0.88 de 0.15 bcde 0.22 cde 0.08 e 0.96 bcde 0.72 abcde 0.22 Y L Bio 3,5 2.16 0.53 ĐĐ C111 C213 T223 T133 4.73 a 4.77 0.24 bc 4.74 0.24 cd 4.71 0.25 e 4.66 0.33 ab 4.77 0.21 c 4.73 0.32 cd 4.71 0.25 de 4.67 0.12 6.89 6.85 6.91 6.82 6.84 6.82 6.81 6.84 6.85 Note: The data in the table is the average of triplicates. The difference was statistically significant mean only in columns. The same characters show no difference statistically significant at 95%. 3.3 Applicability of mango juice fermented by LAB Nowadays, probiotic products are commonly used as fermented milk and yogurt. Besides, juice is offered as a good 9 environment for the fermented probiotic products (MattilaSandholm et al., 2002). Table 4. Some indicators of mango juice after fermentation Isolates Brix ab pH c Acid log T0 log T48 (% w/v) (log CFU/mL) (log CFU/mL) a a Y 8.0 3.02 0.96 4.88 9.30a A 7.67b 3.14bc 0.73c 4.82abc 9.0a P 7.67b 3.42a 0.81bc 4.76c 8.89a L 8.87a 3.2b 0.48d 4.85abc 9.15a Bio 8.5ab 3.21b 0.90ab 4.78bc 8.85a ĐĐ 8.0ab 3.07bc 0.69c 4.85ab 9.25a C213 8.0ab 3.42a 0.92ab 4.83abc 9.03a C111 8.0ab 3.39a 0.91ab 4.83abc 9.08a T223 8.12ab 3.21b 0.75c 4.80abc 8.91a T133 8.83a 3.11bc 0.75c 4.77bc 8.85a Cv (%) 7.23 4.89 19.24 1.14 3.66 Note: The data in the table is the average of triplicates. The same characters show no difference statistically significant at 95%. The results in Table 4 showed that after 48 hours of mango juice fermentation, all 10 isolates of lactic acid increased density, produced lactic acid, reduced the amount of dissolved substances (oBrix) and pH of the environment. Brix of the treatments after fermentation was lower than the original in a range 8.0-8.87, because LAB used sugar in the mango juice to enrichment and generate lactic acid released to the 10 environment. After 48 hours of fermentation mango juice, pH of the environment of the isolates decreased from 3.83 to 3.02-3.42. As the results presented in the previous experiments, LAB isolates were able to survive and grow at this pH. The cause of the decrease in pH was due to the development of lactic acid bacteria that produced lactic acid. Content of lactic acid produced from 4 isolates from Ybio powder, Biosubtyl powder and cat chu have no significant differences with each other and higher than the other isolates. Density of lactic acid bacteria strains in time T0 were nearly equal to the initial concentration of bacteria strains (4.76-4.88 log CFU/mL) and after incubation all isolates had a significant enrichment activity (8.85-9.30 log CFU/mL). Results showed statistically processed, the density of lactic acid bacteria strains at time T0 had no significant differences with each other and significantly different from 3 strains P, Bio,T133. After 48 hours of fermentation the density of bacterial strains were > 6 log CFU/mL, consistent with standards of probiotic products. Therefore, all strains were capable of application in the production of fermented mango. However, lactic acid content of 4 isolates from Ybio powder, Biosubtyl powder and Cat Chu had no significant differences with each other and higher than those of isolates. Among those strains, the L. acidophilus isolate from Ybio had the best content of lactic acid. So, L. acidophilus isolate was selected for the next experiments. 3.4 The ratio of dilution and blending sugar 11 The results in Table 5’ showed that pH values before and after fermentation was difference. After 48 hours of incubation, pH was reduced from about 4.5-4.61 to 3.63-3.84. It caused by bacterial growth, changed glucose into lactic acid. At the same time, content of solution (oBrix) also decreased but not significantly. Table 5’. Some indicators of mango juice fermentation Blending Sucrose (%w/v) Dilution (%) pH after fermentation Brix after fermentation 6 3.73bcde 15.33h 9 3.75abcde 17.43ef 12 3.77abcd 20.17b 15 3.83ab 21.8a 6 3.72cdef 14i 9 3.66ef 16g 12 3.76abcde 18.33d 15 3.68def 20.5b 6 3.84a 12.33j 9 3.71cdef 15.83g 12 3.79abc 17f 15 3.79abc 19.33c 6 3.74abcde 10.83k 9 3.67def 13.83i 30 40 50 12 3.7cdef 15.67gh 15 3.63f 17.5e Cv (%) 2.09 17.83 60 12 Note: The data in the table is the average of triplicates. The same characters show no difference statistically significant at 95%. At the dilution rate of 30% of mango juice, the content of lactic acid reduced while sugar content increased (Figure 8). The high content of sugar inhibited the growth and development of bacteria, acid content decreased. The lowest acid content when the dilution rate was 60%. Besides, at the same sugar content the samples which had dilution ratio 30%, 40% had high content of lactic acid. Therefore, at the dilution rate 30, 40% bacteria growth was better than those of dilution ratios. In addition, when compared with the other treatments the sample at the rate of 40% diluted and 9% sugar content gave the highest acid content. 13 Figure 8. The change of lactic acid content according to sugar content and dilution rate In general, the density of LAB reduced when dilution rate and the content of sugar increased. Density of LAB was highest in 30% dilution and 9% sugar. On the other hand the density of bacteria at all dilution rates were above 6 (log CFU/mL). It was suitable for the production of probiotic products. Figure 9. The change of bacterial density according to sugar content and dilution rate 14 Statistical results of sensory evaluation in Table 7 showed that the samples with dilution ratio of 50% and 60% got lowest score because these samples had a distinct aroma of mango. The sample with 40% dilution and 9% content sugar had highest score because this sample contained lactic acid, sweet taste and aromatic smell. Table 7. Sensory evaluation results of blending sugar content and dilution rate Dilution ratio of juice (% v/v) 30 40 50 60 Sugar content (% w/v) Score of sensory evaluation 6 9 12 15 6 9 12 15 6 3.6cd 4.0b 3.75c 3.3fg 3.4ef 4.65a 3.65cd 3.5de 2.6i 9 12 15 6 9 12 15 2.8h 2.95h 3.15g 2.2j 2.5i 2.8h 2.9h 15.49 Cv (%) Note: The maximum score is 5. The data were the average of 5 sensory evaluators. In short, from the results of pH, Brix, lactic acid content, the density of bacteria and the results of the sensory evaluation 15
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