Fermentation of carrot 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 CARROT JUICE BY LACTIC ACID BACTERIA SUPERVISOR STUDENT MSc. HUYNH XUAN PHONG NGUYEN THI AI XUAN Student code: 3082569 Session: 34 (2008- 2013) Can Tho, 2013 APPROVAL SUPPERVISOR MSc. HUYNH XUAN PHONG Can Tho, May STUDENT NGUYEN THI AI XUAN , 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract This study was carried out in term to contribute for added value of the available, inexpensive and high nutrition raw materials as well as to diversify the production with good characteristics. The study was divided into 2 stages. In the first stage, isolation of the strain for fermentation was done from the nature fermented carrot juice. Four isolates of lactic acid bacteria were compared with other 6 strains from products of digested powder (Luong Phuoc Truong, 2012). A strain of Lactobacillus helveticus gave the best results (1.46% w/v). In the second stage, the fermented carrot juice processing was tested with the selected lactic acid bacteria. Of four testing media, carrot juice with 15% (w/v) of saccharose and 30% dilution ratio was found as the best medium. The favorable conditions for fermented carrot juice were determined based on the results of sensory evaluation and bacterial density determination and they were found as follow: at 5 log cell/mL of the bacterial inoculum, for 24 hours of fermentation time and at 37ºC. The result of storage testing showed that suitable temperature for product storage was 4-6ºC, sensory evaluation (4.21) and bacterial density (7.13 log CFU/mL) of final product maintained after 4 weeks. Key words: carrot juice, lactic acid bacteria, Lactobacillus helveticus, probiotic i CONTENTS 1. INTRODUCTION ............................................................... 1 2. MATERIALS AND METHODS.......................................... 3 2.1. Materials ....................................................................... 3 2.2. Methods ........................................................................ 3 3 RESULTS AND DISCUSSION ............................................ 7 3.1 Isolation of lactic acid bacteria ....................................... 7 3.2 Study on low pH tolerance of isolates ............................. 8 3.3 Study on lactic acid fermentation ability of isolates in carrot juice ..........................................................................10 3.4 Identification of the selected lactic acid bacteria by molecular technique ............................................................11 3.5 Study on the effect of dilution ratio and sugar content....12 3.6 Studying the effect of inoculums density, temperature and time of lactic acid fermentation.....................................14 3.7 Study on temperature and time of storage ......................16 4. CONCLUSIONS AND SUGGESTIONS ............................18 REFERENCES .......................................................................19 ii 1. INTRODUCTION Probiotication is one of the methods used to produce fermented functional foods. Addition of probiotics to food provides several health benefits including reduction in the level of serum cholesterol, improvement of gastrointestinal function, enhancement of immune system and reduction in risk of colon cancer (Yoon et al., 2004). Probiotic bacteria are increasingly used in food and pharmaceutical applications to balance disturbed intestinal microflora and related dysfunction of the human gastrointestinal tract. Presently, the majority of products containing probiotics are dairy-based, which include yogurt and fermented milk beverage. However, due to some drawbacks related to dairy products, there are emerging interests in using non-dairy ingredients as substrates for delivering the physiological benefits of probiotics to wider group of consumers (Mattila-Sandholm et al., 2002). Non-dairy substrates that have been used for lactic acid bacteria (LAB) fermentation include soy protein and cereals. In recent years, several studies have reported the use of fruits and vegetables juices as base medium for LAB fermentation. Juices from these sources are deemed to be advantageous because of their low allergenicity and perceived health benefits (Luckow and Delahunty, 2004). Carrots are good source of carbohydrate, calcium, phosphorous, iron, potassium, magnesium, copper, manganese and sulphur. It is an excellent source of vitamin A, B1, B2, C, E, thiamin, folic acid and riboflavin. The intake of carrot as potent antioxidants, appear to be associated with better health. It is not 1 only preventing vitamin A deficiency but also cancer and other diet related human disease. (Beom et al., 1998). Objectives: To isolate and select lactic acid bacteria and to study the favorable conditions of lactic acid fermented carrot juice with biological values. The content of this study includes: - Isolation of lactic acid bacteria. - Study on low pH tolerance of isolates. - Study on lactic acid fermentation ability of isolates in carrot juice. - Identification of the selected lactic acid bacteria by molecular technique. - Study on the effect of dilution ratio and sugar content. - Studying the effect of inoculums density, temperature and time of lactic acid fermentation. - Study temperature and time of storage. 2 2. MATERIALS AND METHODS 2.1. Materials - Dalat and Chinese carrot were bought from Metro super market. - Six lactic acid bacteria were isolated from five probiotics power and papaya juice fermentation (Luong Phuoc Truong, 2012) - Culture medium: MRS (De Man Rogosa and Shape) agar and broth. - Chemicals: Yeast extract, peptone, glucose, manitol, CaCO3, NaCl, NaOH 1N, H2SO4 1N. - Devices: + Petri dish, pippetteman, eppendorf 1.5 mL + Incubator shaker Innova 4200, New Brunskick Scientific, USA + Optical microscope Olympus DP12 BX41 + Autoclave pbi international 22793 + Refrigerator Sanaky Tokyo, Japan + Eppendorf centrifuge 5417R. 2.2. Methods 2.2.1. Isolation of lactic acid bacteria Carrot juice solution was pasteurized. Then, it was incubated for naturally fermented at 37ºC for 24-48 hours. After that, it was cultured in MRS broth at 37ºC for 24 hours. The bacterial suspension was streaked into MRS agar medium, anaerobically incubated at 37ºC for 48 hours. Then, typical colonies were subcultured several times until the purity of culture was obtained. 3 Pure cultures were defined by observing cell morphology under the optical microscope. All pure bacterial isolates were characterized by Gram staining, catalase, oxydase reaction and disintegration of CaCO3. 2.2.2. Study on low pH tolerance of isolates Low pH tolerant ability of isolates was determined by isolate density in growing media containing different level of pH. The cultures were grown at 30ºC for 24 hours in MRS broth and were used as an inoculum (5 log cell/ mL). Then, isolates were inoculated in MRS broth medium with different pH levels (1,5; 2,5 and 3,5) in 2 hours (sterilized already at 121ºC for 15 minutes). Isolates were measured on plant count method at T0 and T2 (afer 2 hours inoculation). The colonies were counted as viable numbers of isolates in low pH 2.2.3 Study on lactic acid fermentation ability of isolates in carrot juice Carrot juice (pasteurized already with 140 mg/L of NaHSO3 in 20 minutes) was distributed in sterile tubes. The juice was supplied with a inoculum with 5 log cell/mL isolates mixture (each tube contained 36 mL carrot juice + 4 mL of inoculum). Each tube represented a single sample and the experiments were performed in triplication. The lactic acid fermentation was performed in 37ºC for 48 hours. The microorganisms were counted on MRS plates. Samples were taken at 48 hours intervals for chemical and microbiological analysis. pH was measured with a pH meter. Sugar content was measured by Brix handheld meter. Lactic acid content expressed as percent lactic acid was determined by 4 titrating with 0.1N NaOH to pH 8.2. Viable cell counts (CFU/mL) were determined by the standard plate method with MRS medium after 48 hours incubation at 30ºC. 2.2.4 Identification of the selected lactic acid bacteria by molecular technique The bacterial isolate was identified by sequence analysis of partial 16S rRNA gene. A couple of primer including forward primer 8F: (5’-AGA GTT TGA TCC TGG CTC AG-3’) and reverse primer 1492R: (5’-GGC TAC CTT GTT ACG ACT T-3’) were employed to amplify the target gene through PCR technique. DNA sequence of bacterial isolate was determined by DNA machine. Nucleotide sequence was aligned and compared with the data obtained from Gene Bank. 2.2.5 Study on the effect of dilution ratio and sugar content The following variants differing with sugar levels: juice with the addition of 6, 9, 12 and 15% sugar and variants differing with dilution ratio 0, 10, 20 and 30% v/v. Fermentation experiments were conducted in test tubes, each containing 40 mL of pasteurized carrot juice. All samples were inoculated with a 24 hours culture (5 log cell/mL) and incubated at 37ºC for 48 hours. The fermentation dynamics was analyzed on basis of the most important physico-chemical (reducing sugar content, pH, lactic acid content) and microbiological parameters (numerical evolution of the microbial inoculum) at 48 hours. In addition, sensory evaluation of the fermented carrot juice show a statistically significant effect of dilution ratio and sugar content on its total quality 5 2.2.6 Studying the effect of initial inoculum density, temperature and time of fermentation The activity was designed with three factors: LAB density at 3 10 , 105 and 107 cells/mL; fermentation temperatures at: room temperatures, 30ºC and 37ºC; fermentation time at: 24, 36 and 48 hours. Total number of treatments was 3 x 3 x 3 = 27 treatments. The total number of experimental units was 27 treatments x 2 replicates = 54 units. Carrot juice was pasteurized by NaHSO3 140mg/L in 20 minutes and then transferred into tube. Inoculate 0.4 mL of bacterial suspension into tube containing 40 mL of pasteurized carrot juice and incubated room temperatures, 30ºC and 37ºC, respectively. Measure Brix level, pH, acid lactic content and evaluated sensory after fermentation 24, 36, and 48 hours. 2.2.7 Study temperature and time of storage From previous activities, selected the favorable conditions including: isolate strains, inoculum density, sugar content (Brix), dilution ratio, temperature and time of fermentation to produce carrot juice at high quality. After fermentation, fermented carrot juice tubes were stored at the different temperature conditions: at room temperature (between 28ºC and 34ºC), in the cabinet cooler (about 20ºC), in the refrigerator (4-6ºC) about 4 weeks of storage. Found the better storage conditions. 2.2.8 Statistical data analysis The statistical data were analyzed by Microsoft Office Excel 2003 and Statgraphics centurion XV (USA) software. 6 3 RESULTS AND DISCUSSION 3.1 Isolation of lactic acid bacteria All pure isolates were classically defined by observing macro and micro morphology. As the result, 4 isolates of lactic acid bacteria were isolated from 2 kinds of carrot (Dalat and Chinese). Colonies of all isolates were round, smooth, grayish white. Because of the differentiation of collected samples, the cell morphology was various as shown in Table 6’. Moreover, 4 isolates were characterized in Gram positive, lack of calatase and oxydase, produced clear zone around colonies in medium containing CaCO3. The result of Gram staining of 2 representative isolates is described in Figure 14. Figure 14. Gram staining of isolates Two lactic acid bacteria were isolated from Dalat carrot. DL52 were defined as V rod-shaped whereas DLII3 looked like rod-shaped. The isolates TQ13 and TQ25 from Chinese carrot were rod-shaped. Four isolates were characterized of basically characteristics of lactic acid bacteria, they had varieties of both morphology and isolated samples. 7 Table 6’. Cell morphology of isolates The Numbers Sample 1* Antibio probiotic powder A Short rod 2* Probio probiotic powder Pro Short rod 3* Ybio probiotic powder Y Short rod 4* Lactomin plus probiotic powder Lac Cocci in short chains 5* Biosubtyl probiotic powder Bio Long rod 6* Papaya juice DD Pairing rod 7 Carrot juice DLII3 Cocci in pairs 8 Carrot juice DL52 rod in V-shaped 9 Carrot juice TQ13 Short rod 10 Carrot juice TQ25 Short rod isolate Cell morphology Note: The isolates were marked * from Phuoc Luong Truong (2012) 3.2 Study on low pH tolerance of isolates Guarner and Schaafsma (1998), Schrezenmier and de Vrese (2001) reported that a probiotic properties is the ability to survive gastrointestinal transit. Low pH tolerance of isolates was determined by monitoring the density of LAB cells after 2 hours inoculated to MRS broth medium at different pH: 1.5; 2.5; 3.5. The results of counting plate density of isolates cells were showed in Table 7. 8 Table 7. Density of isolates in MRS broth at low pH pH 1,5 2,5 3,5 The isolate A Pro Lac Bio Y DD DLII3 DL52 TQ13 TQ25 A Pro Lac Bio Y DD DLII3 DL52 TQ13 TQ25 A Pro Lac Bio Y DD DLII3 DL52 TQ13 TQ25 LogT0 (log CFU/mL) 1.61gki 1.51lm 1.45mm 1.59jklm 1.69gki 1.53lm 1.59jklm 1.53lm 1.56klm 1.89fgh 2.14de 2.21d 2.01ef 2.0efg 2.14def 1.9fgh 1.85ghi 1.8hi 1.74hij 1.81hi 4.42bc 4.58a 4.47ab 4.53ab 4.55ab 4.57ab 4.53ab 4.51ab 4.42c 4.26c LogT2 (log CFU/mL) 6.82d 6.82cd 6.82cd 6.81d 6.83cd 6.82cd 6.87abcd 6.81d 6.81d 6.85abcd 6.84bcd 6.88abc 6.84bcd 6.83cd 6.84bcd 6.86abcd 6.83bcd 6.85bcd 6.83cd 6.85bcd 6.89ab 6.92a 6.83cd 6.86abcd 6.85bcd 6.85bcd 6.86abcd 6.81d 6.89ab 6.82cd * Note: Value in the table was average value of triplications. Difference value of statistics only had meaning in the column; the average values with the same letter were not significant at 95% probability. The density of isolates was used to evaluate the development and survival of isolate cells. Initial cell density was about 4.219 4.46 log CFU/mL. When supplied into the pH 1.5 and 2.5 medium, these 10 isolates could grow and survive after 2 hours incubation at 37ºC temperature. In pH 3.5 medium, isolates grew well, reaching density of about 6.55 to 6.99 log CFU/mL. In low pH supplied media, density of isolates decreased correlatively with the decrease of pH level. 3.3 Study on lactic acid fermentation ability of isolates in carrot juice Table 8. Following indicators fermented carrot juice The Isolates A Pro Lac Bio Y DD DLII3 DL52 TQ13 TQ25 ºBrix 5.00c 6.83a 5.67bc 6.33ab 6.67a 6.83a 6.83a 6.50a 7.00a 5.67bc pH Acid (% w/v) 3.62b 3.23c 4.08a 3.66b 3.21c 4.18a 3.74b 4.17a 4.18a 3.99a 1.31b 1.46a 0.54e 0.81c 1.39ab 0.46f 0.83c 0.67d 0.51ef 0.71d Log T0 (log CFU/mL) 5.16ab 5.17ab 5.16ab 5.19ab 5.15bb 5.16ab 5.17ab 5.18ab 5.21a 5.18ab Log T48 (log CFU/mL) 8.67bc 8.69a 8.33ef 8.43de 8.85ab 8.02g 8.54cd 8.33ef 8.16fg 8.37de * Note: Value in the table was average value of triplications. Difference value of statistics only had meaning in the column; the average values with the same letter were not significant at 95% probability. The results in Table 8 showed that pH after fermentation was lower than initial, roughly 3.21 to 4.18. During fermentation process, isolates converted glucose into lactic acid, reduced the pH of the fermenting liquid. Similarly, Brix levels also decreased after fermentation. One of the basic requirements demanded from probiotic products is a proper number of probiotic bacteria at the level of at least 6 log CFU/mL. The survival rate of all isolates at the level 10 of about 8.02-8.69 log CFU/mL of fermented carrot juice. Addition, the result of lactic acid concentration measured of different level, ranging 0.46-1.46% w/v. The highest lactic acid concentration was Pro with 1.46% w/v. Therefore, this isolates were chosen for next activities. 3.4 Identification of the selected lactic acid bacteria by molecular technique Isolate Pro was selected to sequence 16S rRNA gene and BLAST on NCBI website (http://www.ncbi.nlm.nih.gov). The result showed that the gene sequences of isolate Pro shared 99% similarity with Lactobacillus helveticus. Result of BLAST on NCBI of isolate Pro Lactobacillus helveticus is an important industrial thermophilic starter that is predominantly employed in the fermentation of milk for the manufacture of several cheeses. In addition to its technological importance, a growing body of scientific evidence shows that strains belonging to the Lb. helveticus species have health-promoting properties. Several in vitro studies showed that Lb. helveticus possesses many common probiotic properties, such as the ability to survive gastrointestinal transit, adhere to epithelial cells, and antagonize pathogens (Valentina and Simone , 2012). 11 3.5 Study on the effect of dilution ratio and sugar content The results of Lb. helveticus density and lactic acid content of carrot juice fermentation were presented in Furgre 17’. Lactic acid content (%w/v) 1.40 6% 1.20 9% 1.00 12% 15% 0.80 0.60 0.40 0.20 0.00 0 10 20 30 Bacterial density (LogCFU/ml) Dilution ratio (% v/v) 9.8 6% 9.6 9% 12% 9.4 15% 9.2 9 8.8 8.6 0 10 20 30 Dilution ratio (%v/v) Furgre 17’. Changes of lactic acid content and Lb. helveticus density In the same dilution ratio carrot juice. Figure 17’ compared the lactic acid generated by Lb. helveticus at 6, 9, 12, and 15% sugar. Lb. helveticus was able to ferment well at 15% glucose (1.17% w/v). However, they did not have the same results at ratio 12 dilution 0%. Perhaps high glucose concentrations caused a certain inhibition on bacterial activities. Lactic acid content after fermentation was statistically significant difference at four dilution ratio. Figure 17’ presented the highest lactic acid content was at 20-30% of dilution ratio. On the other hand, a density of bacteria in all dilution ratios was greater than 6 (log CFU/mL). Table 10. Results sensory evaluation carrot juice fermented by dilution ratio and sugar content Dilution ratio (%v/v) 0 10 20 30 Sugar content(%w/v) 6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15 Sensory scores 2.60cd 2.65cd 2.7bcd 2.75bcd 1.60d 2.65cd 3.08bc 2.77bcd 2.75bcd 3.42abc 3.87abc 4.10ab 2.75bcd 2.75bcd 3.25abc 4.55a * Note: Value in the table was average value of triplications. Difference value of statistics only had meaning in the column; the average values with the same letter were not significant at 95% probability. The results showed that fermented juice samples at initial dilutions were statistically significant different (p<0.05). Table 10 presented the highest sensory evaluation was 4.55 at 15% glucose and 30% of initial dilution. It was significantly different from 010% of initial dilution but not from 20% water, because smell of 13 20% dilution ratio was quite sour. In summary, sugar content 15% and 30% dilution ratio were favorable conditions to lactic acid fermentation 3.6 Studying the effect of inoculums density, temperature and time of lactic acid fermentation This activity was set up with 3 levels (30, 28-34, and 37ºC) for temperature factor, 3 levels (3, 5, and 7 log cells/mL) for inoculum density factor, and 3 levels (24, 36, and 48 hours) for time factor. Figure 19, 20 indicated that lactic acid content in samples of 24 hours incubation was lower than those of 48 hours and 36 hours incubation, with the same inoculum density and temperature. Addition, at the same temperature and fermentation time, inoculum density of 7 log cells/mL gave higher lactic acid Lactic acid content (%w/v) content than inoculum of 3 and 5 log cells/mL. 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 24 hour 36 hour 48 hour 30˚C 28-34˚C 37˚C 3 5 7 3 5 7 3 Inoculum density (log CFU/mL) 5 7 Figure 19. Changes of lactic acid content by inoculums density, temperature and time of lactic acid fermentation 14
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