MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENT THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY FERMENTATION OF DRAGON FRUIT JUICE BY LACTIC ACID BACTERIA SUPERVISOR STUDENT MSc. HUYNH XUAN PHONG NGUYEN THUY VI Student code: 3082567 Session: 34 Can Tho, 2013 APPROVAL SUPERVISOR STUDENT MSc. HUYNH XUAN PHONG NGUYEN THUY VI Can Tho, 2013 PRESIDENT OF EXAMINATION COMMITTEE Abstract This study was carried out to isolate and select the isolates of lactic acid bacteria applied for the fermentation of dragon fruit juice. Experiments were done including dilution rates, sugar content, inoculum level, incubation temperature, fermentation time, and storage conditions. Six strains of lactic acid bacteria isolated from dragon fruit juice and three strains of probiotic powder were tested. All 6 tested bacterial strains could grow in low pH conditions (1.5 – 3.5) and could be used for fermentation of dragon fruit juice, with the bacterial level was at 6 log CFU/mL. A strain Lactobacillus acidophilus isolated from Antibio powder gave the best capable of dragon fruit juice fermentation (acid content was 1.38% w/v and bacteria concentration was 8.44 log CFU/mL). The favourable conditions of fermentation were determined as follow: pure dragon fruit juice, 9% of supplemented sugar, 37ºC of fermentation temperature, 48 hours of fermentation, and 6 log CFU/mL of inoculum level. The appropriate temperature and time storage for fermented juice were found at 4 – 6ºC for 3 weeks. Keywords: dragon fruit juice, fermentation, lactic acid bacteria, Lactobacillus acidophilus, probiotic i Contents Abstract ...................................................................................... i Contents ...................................................................................... ii 1. INTRODUCTION .................................................................. 1 2. MATERIALS AND METHODS ............................................ 3 2.1 Materials ............................................................................ 3 2.2 Methods ............................................................................. 3 2.2.1. Isolation and identification of LAB isolates at genus level .................................................................................... 3 2.2.2. Study on the acidity tolerant ability of LAB isolates... 4 2.2.3. Study on application in producing of fermented dragon fruit juice by LAB ................................................... 4 2.2.4. Study on dilution rates of dragon fruit juice and sucrose rates ........................................................................ 5 2.2.5. Study on inoculum levels, incubation temperature and fermentation time ......................................................... 5 2.2.6. Study on storage temperature and storage time ........... 5 3. Results and discussion ............................................................. 6 3.1. Isolation and identification of LAB isolates at genus level . 6 3.2. Acidity tolerant ability of LAB isolates.............................. 7 3.3. Application in producing of fermented dragon fruit juice by LAB .................................................................................... 9 3.4. Dilution rates of dragon fruit juice and sucrose rates .......... 10 3.5. Inoculum levels, incubation temperature and fermentation time ......................................................................................... 15 3.6. Storage temperature and storage time ................................ 18 4. Conclusions and suggestions ................................................... 21 4.1 Conclusions........................................................................ 21 ii 4.2 Suggestions ........................................................................ 21 References ................................................................................... 22 iii 1. INTRODUCTION Probiotics are defined as the non-pathogenic microorganisms capable of survive in the digestive process, having positive impact on the health and physiology of the hosts. There are many probiotic products for human and livestock. Lactic acid bacteria (LAB) are able to produce probiotics (Temmerman et al., 2002). Thus, LAB are used in probiotic products such as yogurt, fermented meat, fermented vegetables… These products are not only used as food but also used for treating intestinal and stomach diseases because LAB can produce antibiotics to prevent and skill pathogenic bacteria and germs. Also, LAB are widely used in food fermentation because of their ability to improve flavour, texture and safety of perishable raw materials (Caplice and Fitzgerald, 1999). Fruit juice is a good environment for the growth of bacteria and probiotic products. Fruits and vegetables are good for health because of antioxidants, vitamins, fibers and minerals in components. “Dragon fruit” are commonly known as “thanh long” as in Vietnamese meaning “green dragon”. Dragon fruit contains vitamins A, C, sugar, organic acids,… Dragon fruit juice is a suitable medium for lactic acid bacteria to produce new probiotic products. Therefore, the study “Fermentation of dragon fruit juice by lactic acid bacteria” was carried out. 1 Objectives: To isolate LAB strains from dragon fruit juice, and to study fermented dragon fruit juice producing process by lactic acid bacteria. Contents made - Isolation and identification of LAB isolates at genus level - Study on the acidity tolerant ability of LAB isolates - Study on application in producing of fermented dragon fruit juice by LAB - Study on dilution rates of dragon fruit juice and sucrose rates - Study on inoculum levels, incubation temperature and fermentation time - Study on storage temperature and storage time 2 2. MATERIALS AND METHODS 2.1 Materials - Dragon fruit bought from Xuan Khanh market (Can Tho City) - Probiotics powders: + Antibio powder: Product of Han Wha Pharma Co., Ltd. (Number 472, Namgog-Ri, Yangji - Myon, Yongin - Si, Kyonggi - Do, Korea). + PROBIO powder: Made in the Corporation Imexpharm (No. 4, 30/4, Cao Lanh Town, Dong Thap). + Lactomin powder plus: RexGene Biotech Co. products., Ltd. (Number 641-2, Ochang - Myun, Cheongwon - Kun, Chungbuk, Korea) - Medium: + MRS broth (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, diammonium 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 - Chemicals: NaOH 0.1N, HCl 0.1N, H2O2 3%, NaHSO3, the Gram stain (Crystal violet, Iodine, Acetone, Ethanol, Fuchsin) - Equipments in Food Biotechnology Laboratory, Biotechnology Research and Development Institute, Can Tho University. 2.2 Methods 2.2.1. Isolation and identification of LAB isolates at genus level 3 - Sterilized dragon fruit juice was incubated at 37ºC for 24 – 48 hours. Fermented juice and probiotic powders were incubated in MRS broth medium for 24 hours. LAB were isolated by culturing the incubated media on MRS broth agar medium until getting the pure bacteria. - LAB isolates were identified at genus level through the preliminary tests: Gram stain, catalase, oxidase test and inspection capabilities resolution of CaCO3. 2.2.2. Study on the acidity tolerant ability of LAB isolates LAB isolates were inoculated in MRS broth medium adjusted to different levels of pH (1.5, 2.5 and 3.5). Bacterial density levels of LAB were determined at incubation time (T 0) and after 2 hours of incubation by the counting living method. Acidity tolerant LAB isolates were chosen. 2.2.3. Study on application in producing of fermented dragon fruit juice by LAB 4 mL of LAB with 5 log cells/mL density were inoculated to 36 mL of pasteurized dragon fruit juice. The inoculated juices were incubated at 37ºC for 48 hours. After 48 hours of incubation, the juices were analyzed the assessment criteria including: - pH: measured by pH meter - Soluble matter content (Brix): measured by Brix handheld meter - Content of generated lactic acid: based ion total acid index - Density of LAB: determined by plate counting method - Sensory evaluation 4 2.2.4. Study on dilution rates of dragon fruit juice and sucrose rates 0.4 mL of LAB was inoculated to 39.6 mL of pasteurized dragon fruit juice adjusted to different dilution rates (0, 20, 30 and 40% w/v) and sucrose rates (6, 9, 12 and 15%). The inoculated juices were incubated at 37ºC for 48 hours. After 48 hours of incubation, the juices were analyzed the assessment criteria including: pH, soluble matter content, content of generated lactic acid, density of LAB and sensory evaluation. 2.2.5. Study on inoculum levels, incubation temperature and fermentation time 0.4 mL of LAB was inoculated to 39.6 mL of pasteurized dragon fruit juice adjusted to different levels of inoculum (4, 5, and 6 log cells/mL). The inoculated juices were incubated at different temperature levels (25, 30, and 37ºC) at 12, 24, and 36 hours. After incubation, the juices were analyzed the assessment criteria including: pH, soluble matter content, content of generated lactic acid, density of LAB and sensory evaluation. 2.2.6. Study on storage temperature and storage time The fermented dragon juice products were stored at different temperature levels including: 4 – 6ºC (refrigerator), 20 – 25ºC (cooler) and 28 – 32ºC (room temperature) in 1, 2, 3, and 4 weeks. After storage time, the juices were analyzed the assessment criteria including: pH, soluble matter content, content of generated lactic acid, density of LAB and sensory evaluation. 5 3. RESULTS AND DISCUSSION 3.1. Isolation and identification of LAB isolates at genus level Isolation results The features of isolated bacteria colonies: round, glossy, smooth, creamy white. The colonies of each bacteria strain are identical. Colonies of bacteria isolated from Antibio and Probio powder were larger than dragon fruit ones. There were six isolated bacteria strains, two from dragon fruit juice and four from probiotic powder. Cell morphology of isolated bacteria was various as shown in Table 3’. Table 3’. Cell morphology of isolated bacteria No 1 2 3 4 5 6 Sources Dragon fruit Dragon fruit Antibio probiotic powder Probio probiotic powder Lactomin plus probiotic powder Biosubtyl probiotic powder The isolates TL1 TL2 A Pro Lac Bio Cell morphology Rod Pairing rod Short rod Short rod streptococci Long rod Characteristics of isolated bacteria strains: - Bacteria strains isolated from Antibio and Probio powder were rod-shaped bacteria. They had the same characteristics of Lactobacillus acidophilus, the bacteria in probiotic powder components. - Bacteria strains isolated from Lactomin were streptococci. - Bacteria strains isolated from dragon fruit juice were rodshaped bacteria. However, the length of these bacteria strains was shorter than the length of rod-shaped bactera strains isolated from Antibio and Probio powder. 6 Genus identification Some simple biochemistry experiments were carried out to determine the isolated bacteria strains at genus level. The results showed that: - Gram staining: Bacteria strains remained purple color after staining crystal violet, thus they were Gram-positive bacteria. - Catalase test: All six bacteria strains could not produce bubbles when adding H2O2 3%, indicated negative results. It can be concluded that these isolated bacteria strains belonged to lactic acid bacteria because they: - Are Gram-positive bacteria - Has no enzyme catalase - Are rod-shaped or streptococcus. - Can grow on MRS medium. 3.2. Acidity tolerant ability of LAB isolates According Guarner and Schaafsma (1998), one of the criteria of probiotic products is survival ability of microorganisms after consuming process. In human digestive system, the stomach pH is very low, about 1 – 2, so majority of microorganisms are difficult to survive in this pH. However, microorganisms existed only 1 – 2 hours in the stomach and then move to the large intestine with a neutral pH. After 2 hours of incubation at low pH, the bacteria increased concentration. Initial inoculum level of LAB was 4 log cells/mL. But when inoculating to MRS medium with pH in the range of 1.5 to 2.5, the bacterial density reduced significantly (only 1.08 to 1.40 log CFU/mL), because of shock of bacteria 7 under low pH condition. But only after 2 hours incubation at 37ºC, they recovered and increased concentration (up to 6.31 to 6.62 log CFU/mL). Almost bacteria could resist to pH 3.5. The decrease of bacterial density was not significantly at T 0 (3.34 – 3.58 log CFU/mL). After 2 hours of incubation, the concentration reached to 6.45 – 6.66 log CFU/mL. At T0, the concentration of 6 bacteria strains in pH 3.5 medium were higher than theirs in pH 2.5 medium. And the lowest concentration of bacteria was in pH 1.5 medium. It was showed that the lower pH affect to the survival ability of LAB. Table 4. Density of bacteria in MRS broth with low pH pH 1.5 2.5 3.5 Strains A Pro TL1 TL2 Bio Lac A Pro TL1 TL2 Bio Lac A Pro TL1 TL2 Bio Lac T0 (Log CFU/mL) 1.26 1.08 1.16 1.19 1.12 1.21 1.40 1.37 1.36 1.35 1.39 1.37 3.44 3.58 3.34 3.37 3.36 3.43 T2 (Log CFU/mL) 6.62b 6.48g 6.56c 6.49g 6.48g 6.44i 6.48g 6.40j 6.55cd 6.31k 6.53def 6.31k 6.53def 6.66a 6.54de 6.46h 6.45hi 6.52f 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. 8 All six LAB strains increased the density after 2 hours of incubation at 3 different pH levels (ranging from 6.31 to 6.66 log CFU/mL). The bacterial densities after fermentation were significantly different at the 95% confidence level (Table 4’). The Pro strain reached the highest value (6.66 log CFU/mL) at pH 3.5, next to A strain at pH 1.5 (6.62 log CFU/mL) and TL1 strain at pH 2.5 (6.55 log CFU/mL). All isolated lactic acid bacteria strains were able to adapt and develop in a low pH environment, so they could survive in the environment of the stomach. Therefore, these strains were able to apply in probiotic products. 3.3. Application in producing of fermented dragon fruit juice by LAB This experiment was carried out to determine whether the lactic acid bacteria strains could be applied in the production of fermented dragon fruit juice to create probiotic products, and to compare individual application abilities of LAB strains together. After 48 hours, all lactic acid bacteria strains increased their concentration; solvent content (ºBrix) decreased; lactic acid was produced to reduce the pH of medium (Table 5). Table 5. Following indicators fermented dragon fruit juice Bacteria strains A Pro TL1 TL2 Bio Lac Brix pH 8.0 7.5 7.1 7.9 8.0 7.8 4.31 3.23 4.01 3.95 4.25 4.09 Acid (% w/v) 1.38ª 1.36b 1.28c 1.24d 1.19e 1.23d Log T0 (log CFU/mL) 3.86ab 3.84c 3.82d 3.85bc 3.87ª 3.84c Log T48 (log CFU/mL) 8.44ª 8.36b 8.35bc 8.34c 8.37b 8.27d 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. 9 Brix concentration of all treatments after fermentation decreased insignificantly (from 8.9ºBrix to about 7.1 – 8.0ºBrix). After 48 hours of fermentation, pH decreased from 4.52 to about 3.23 – 4.31. At this pH, lactic acid bacteria could remain. The pH decreased because due to the production of lactic acid during development of bacteria. Concentration of lactic acid produced from A strain (1.38%) and Pro strain (1.36%) were higher than other strain’s and they were significantly different to others at the 95% confidence level. After incubation, densities of LAB increased significantly (from 3.82 – 3.87 to 8.27 – 8.44 log CFU/mL). As statistical data analysis results, at T 0, densities of Bio and A strains were highest (3.87 and 3.86 log CFU/mL), they were significantly different to others at the 95% confidence level. After 48 hours, densities of LAB were higher than 6 log CFU/mL. The highest concentration (8.44 CFU/mL) belonged to A strain (Lactobacillus acidophillus) and it was significantly different to others at the 95% confidence level. Therefore, the Lactobacillus strain isolated from Antibio powder (notated as A) was used in further experiments. 3.4. Dilution rates of dragon fruit juice and sucrose rates The pH and Brix values in different dilution rates of dragon fruit juice and sucrose rates before and after fermentation were presented in Table 5. 10 Table 6. Effects of dilution rates of dragon fruit juice and sucrose rates pH Brix concentration Sugar content Before After Before After (%) fermention fermention fermention fermention 6 3.73 12.4 12.0 9 3.72 19.1 18.5 Juice 4.35 12 3.80 17.4 16.8 15 3.71 17.5 17.0 6 3.72 11.6 11.0 Juice + 9 3.77 14.9 14.0 20% pure 4.42 12 3.78 17.8 17.0 water 15 3.75 19.4 19.0 6 3.69 11.5 11.0 Juice + 9 4.00 14.1 13.5 40% pure 4.35 12 3.73 15.6 15.0 water 15 3.87 19.7 19.1 6 3.81 10.8 10.1 Juice + 9 3.74 13.6 12.8 60% pure 4.36 12 3.74 16.4 16.0 water 15 3.94 17.6 17.0 Note: Values in the table were average values of triplications. Types of juice After 48 hours of incubation with Lactobacillus acidophilus strain isolated from Antibio powder, pH values were reduced (from 4.35 – 4.42 to 3.69 – 4.0) due to bacteria growth converting sugar to lactic acid. At the same time, solute concentration (Brix) also decreased insignificantly. The relationship between sugar concentration and acid index was indicated in Figure 5. 11 density of acid (%w/v) 2.5 2 sugar 6% 1.5 sugar 9% 1 sugar 12% 0.5 sugar 15% 0 juice juice+20% water juice+40% water juice+ 60 % water Figure 5: The change in concentration of acid in different dilution rates of dragon fruit juice and sucrose rates At the same dilution rate, the acid index dropped down when sugar concentration grew up. Acid concentration reached the highest value at 6 % of sugar concentration, and the lowest value at 15% of sugar concentration due to the inhibition of high concentration of sugar. Besides, at the same glucose concentration level, acid indexes were different. In particular, the pure dragon fruit juice had the highest acid index. It was suitable medium for LAB growth. The change of bacterial density was shown in Figure 6. 12 9.4 9.2 Log CFU/ml 9 sugar 6% 8.8 sugar 9% 8.6 sugar 12% 8.4 sugar 15% 8.2 8 juice juice+20% water juice+40% water juice+ 60 % water Figure 6. The change in the density of bacteria In general, the bacterial density decreased when sugar concentration increased, with 12 – 15% of sugar concentration, bacterial density was lower than 9%. The reason was that high sugar concentration inhibited bacteria growth partly (Nguyen Thi Hien, 2006). Overall, with 40% of dilution rate, bacterial density was quite high, nearly equivalent to pure juice. However, in pure dragon fruit juice with 9% of sugar conceatration, the bacterial density achieved the highest value (9.3 log CFU/mL). Thus pure dragon fruit juice was suitable environment for fermentation. The sensory evaluation assessed according to smell, taste and status criteria were presented in Table 6. Table 6. Description of product evaluation criteria Target Smell Description Fragrance, specific characteristics of fruit, acid and sugar concentrations are not strong Taste Sour harmony, not sourer or sweeter Status Homogeneous status, not washier or too condensed 13 The results of sensory evaluations of group of 5 people were presented in Table 7. Table 7. Sensory evaluation results with different dilution rates and sugar content Types of juice Pure juice Juice + 20% water Juice + 40% water Juice + 60% water Sugar content (%) 6 9 12 15 6 9 12 15 6 9 12 15 6 9 12 15 Score 8.1 8.6 8.4 8.4 5.1 5.3 4.8 4.2 2.1 2.3 2.2 1.8 1.5 1.7 1.2 1.6 Note: Values in the Table were average values of 5 replications. The sensory evaluation of pure dragon fruit juice was higher than others. The lowest cores belonged to the samples with 40% and 60 %of dilution rates (1.2 to 2.3 per 10 points) due to less of specific fragrance of fruit. The pure dragon fruit juice had quite high evaluation (more than 8 per 10 points). In particular, the 9%-concentration-rate sample got the highest score due to harmonious flavor. In summary, from the results of the acid index, the concentration of bacteria and sensory evaluation, it could be concluded that the pure dragon fruit juice with 9% of sugar concentration was appropriate for fermentation. 14 3.5. Inoculum levels, incubation temperature and fermentation time Results of fermentation in different inoculum levels, incubation temperature and incubation time were shown in Table 9. Table 9. Effects of inoculum levels and incubation temperature Inoculum concentratio n (log CFU /mL) 4 5 6 Temperature (oC) pH Brix Acid concentratio n (% w/v) 25 30 37 25 30 37 25 30 37 3.54 3.53 3.52 3.53 3.52 3.48 3.55 3.36 3.20 23.73 23.69 23.66 23.72 23.69 23.67 23.64 23.61 23.54 0.54d 0.52e 0.55d 0.55d 0.56d 0.57c 0.54d 0.64b 0.74ª Bacteria concentratio n (log CFU/mL) 4.21e 4.45e 4.51e 5.16d 5.72c 6.05bc 6.13b 6.36ab 6.71ª Note: Values in the Table were average values of triplications. The initial pH and Brix were 4.52 and 23.9, respectively. After fermentation, pH and Brix tended to decline. Final pH of fermentation ranged from 3.20 to 3.55. Brix reduction was not significant and the values at the end of fermentation were in the range from 23.54 to 23.73ºBrix. Effects of incubation temperature and inoculum levels on acid concentration were presented in Figure 7. 15 0.9 concentration of acid (%w/v) 0.8 0.7 0.6 25oC 0.5 0.4 30oC 0.3 37oC 0.2 0.1 0 4 5 6 strain density (log cell/mL) Figure 7. Effects of incubation temperature and inoculum levels on acid concentration At the same inoculum levels, acid concentration at 25ºC was lower than 30ºC’s and 37ºC’s. The acid concentration reached the highest value at 37ºC since this was the optimal temperature for the growth and development of acid lactic bacteria. When inoculum level was 6 log cells/mL, the acid concentration was higher than others at 37ºC. At this level of inoculum, the acid concentration reached the highet value (0.74% w/v) at 37ºC) and it was significantly different to others, next to 30ºC (0.64% w/v). Effects of incubation temperature and inoculum levels on bacterial concentration were shown in figure 8. 16 8 7 6 Log CFU/ml 5 25oC 4 30oC 37oC 3 2 1 0 4 5 6 density of strains (log cell/ml) Figure 8. Effects of incubation temperature and inoculum levels on bacteria concentration As the results, at the same inoculum level, bacterial concentration increased with the increase of incubation temperature. The lowest and highest bacterial density were at 25ºC and 37ºC, respectively. Suitable temperature for growth of lactic acid bacteria was 37ºC. Similarly, bacterial concentration accompanied with inoculum levels to increase. With the 6 log CFU/mL of inoculum level, the bacterial concentrations reached the highest values at 30 and 37ºC (6.71 and 6.36 log CFU/mL, respectively) and the values were significantly different to others. The requirement of probiotic products was the concentration of bacteria must be more than 6 log cells/mL. In this study, conditions satisfied the requirement of probiotic products were 5 log cells/mL inoculum concentration + 37ºC of incubation temperature or 6 log cells/mL + all 3 types of incubation temperature (25, 30, and 37ºC). 17 The samples satisfied the requirement of probiotic products were sensory evaluated. The results were presented in Table 10. Table 10. Sensory evaluation results with different inoculum levels and incubation temperature Density of strains (log cell/mL) Fermented temperature (oC) Marks 5 37 7.4 25 7.4 30 8.3 37 8.9 6 Note: Values in the Table were average values of 5 replications. Results sensory evaluation showed that sample incubated at 37ºC with inoculum level of 5 log cells/mL and sample incubated at 25ºC with inoculum level of 6 log cells/mL had the lowest score. At 6 log cells/mL inoculum level, 30 and 37ºC of incubation temperatures, the sensory quality of the products was higher (8.3 to 8.9 / 10 points). Thus, conditions applied to dragon fruit juice fermentation by LAB were 6 log cells/mL of inoculum level and 37ºC of incubation temperature. 3.6. Storage temperature and storage time Effects of storage temperature and time were presented in Table 10. 18 Table 10. Effects of storage temperature and storage time Acid concentration (%w/v) pH Time (weeks) 4 – 6ºC 0 20 – 25ºC 28 – 32ºC 4 – 6ºC 3.51 20 – 25ºC 28 – 32ºC Bacteria concentration (log CFU/mL) 4 20 28 – – – 6ºC 25ºC 32ºC 0.634 6.87 1 3.53c 2.62f 2.67d 0.631e 1.54d 1.62b 6.86e 7.66b 7.92ª 2 3.58b 2.61g 2.63ef 0.622e 1.59c 1.62b 6.89d 5.87f 5.71g 3 3.66a 2.60g 2.64e 0.622e 1.59c 1.67ª 7.02c 5.42h 5.32i (Note: Values in the Table were average values of triplications) With the samples stored at cool temperature (20 – 25ºC) and room temperature (28 – 32ºC), the bacterial concentrations had increasing trend after 1 week (from 6.87 log cells/mL to 7.66 – 7.92 log cells/mL) an the acid concentrations increased (from 0.634% w/v to 1.54 – 1.67% w/v). It made products become unpleasant and sour. But from the week 2 onwards, the concentrations of bacteria began to reduce due to the inhibition of acid against bacteria (less than 6.0 log cells/mL). Thus, these temperatures were not suitable for storing products. The samples stored at 4 – 6ºC condition had stable bacterial density (6.86 – 7.02 cells/mL) and acid concentration (0.622 – 0.631% w/v). Low temperatures inhibited microbial activity and the products were kept the better features. The 19 bacterial density had a trend to increase from week 1 to week 3 of storage (from 6.87 to 7.02 log CFU/mL). In summary, with products stored at 4 – 6ºC temperature, the bacterial densities and acid concentrations did not change largely comparing to the time of the end of fermentation. Thus, the proper temperature for storage was 4 – 6ºC and the products were maintained quality after 3 weeks of storage in this study. 20 4. CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions - 6 LAB strains were isolated from fermented dragon fruit juice and 3 kinds of probiotic powder. - All isolated LAB strains were able to achieve densities more than 6 log CFU/mL after incubation at 37ºC in two hours in a pH range of 1.5 to pH 3.5. - Lactobacillus acidophillus A strain isolated from Antibio powder had better characteristics in fermentation (1.38% w/v of acid content and 8.44 log CFU/mL of bacterial concentration after fermentation). - Suitable dilution rate and sucrose rate for dragon fruit juice fermentation were 0% and 9%, respectively. - The suitable conditions for the process of dragon fruit juice fermentation: 37ºC of fermentation temperature, 48 hours of fermentation, 6 log celss/mL of inoculum level. - Products stored 4 – 6ºC in 3 weeks were ensure quality and maintained stable concentration of bacteria. 4.2 Suggestions Study on longer storage time. 21 REFERENCES Vietnamese Nguyễn Thị Hiền. 2006. Công nghệ sản xuất mì chính và các sản phẩm lên men cổ truyền. Nxb Khoa học và Kỹ thuật, trang 209 – 211. English Caplice, E. and G.F. Fitzgerald. 1999. Food fermentation: role of microorganwasms in food production and preservation. Int. J. Food Microbiol. 50:131-149. Guarner, F. and G.J. Schaafsma. 1998. Probiotics. International Journal of Food Microbiology, 39:237-238. Temmerman, R., B. Pot, G. Huys and J. Swings. 2002. Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int. J. Food Microbiol. 81: 1-10. 22 [...]... Suitable dilution rate and sucrose rate for dragon fruit juice fermentation were 0% and 9%, respectively - The suitable conditions for the process of dragon fruit juice fermentation: 37ºC of fermentation temperature, 48 hours of fermentation, 6 log celss/mL of inoculum level - Products stored 4 – 6ºC in 3 weeks were ensure quality and maintained stable concentration of bacteria 4.2 Suggestions Study on longer... concentration and acid index was indicated in Figure 5 11 density of acid (%w/v) 2.5 2 sugar 6% 1.5 sugar 9% 1 sugar 12% 0.5 sugar 15% 0 juice juice+20% water juice+ 40% water juice+ 60 % water Figure 5: The change in concentration of acid in different dilution rates of dragon fruit juice and sucrose rates At the same dilution rate, the acid index dropped down when sugar concentration grew up Acid concentration... production of fermented dragon fruit juice to create probiotic products, and to compare individual application abilities of LAB strains together After 48 hours, all lactic acid bacteria strains increased their concentration; solvent content (ºBrix) decreased; lactic acid was produced to reduce the pH of medium (Table 5) Table 5 Following indicators fermented dragon fruit juice Bacteria strains A Pro... identification of LAB isolates at genus level Isolation results The features of isolated bacteria colonies: round, glossy, smooth, creamy white The colonies of each bacteria strain are identical Colonies of bacteria isolated from Antibio and Probio powder were larger than dragon fruit ones There were six isolated bacteria strains, two from dragon fruit juice and four from probiotic powder Cell morphology of isolated... Bacteria strains isolated from Antibio and Probio powder were rod-shaped bacteria They had the same characteristics of Lactobacillus acidophilus, the bacteria in probiotic powder components - Bacteria strains isolated from Lactomin were streptococci - Bacteria strains isolated from dragon fruit juice were rodshaped bacteria However, the length of these bacteria strains was shorter than the length of. .. isolated lactic acid bacteria strains were able to adapt and develop in a low pH environment, so they could survive in the environment of the stomach Therefore, these strains were able to apply in probiotic products 3.3 Application in producing of fermented dragon fruit juice by LAB This experiment was carried out to determine whether the lactic acid bacteria strains could be applied in the production of. .. results of the acid index, the concentration of bacteria and sensory evaluation, it could be concluded that the pure dragon fruit juice with 9% of sugar concentration was appropriate for fermentation 14 3.5 Inoculum levels, incubation temperature and fermentation time Results of fermentation in different inoculum levels, incubation temperature and incubation time were shown in Table 9 Table 9 Effects of. .. fermentation decreased insignificantly (from 8.9ºBrix to about 7.1 – 8.0ºBrix) After 48 hours of fermentation, pH decreased from 4.52 to about 3.23 – 4.31 At this pH, lactic acid bacteria could remain The pH decreased because due to the production of lactic acid during development of bacteria Concentration of lactic acid produced from A strain (1.38%) and Pro strain (1.36%) were higher than other strain’s... 2.3 2.2 1.8 1.5 1.7 1.2 1.6 Note: Values in the Table were average values of 5 replications The sensory evaluation of pure dragon fruit juice was higher than others The lowest cores belonged to the samples with 40% and 60 %of dilution rates (1.2 to 2.3 per 10 points) due to less of specific fragrance of fruit The pure dragon fruit juice had quite high evaluation (more than 8 per 10 points) In particular,... 8.2 8 juice juice+20% water juice+ 40% water juice+ 60 % water Figure 6 The change in the density of bacteria In general, the bacterial density decreased when sugar concentration increased, with 12 – 15% of sugar concentration, bacterial density was lower than 9% The reason was that high sugar concentration inhibited bacteria growth partly (Nguyen Thi Hien, 2006) Overall, with 40% of dilution rate, bacterial