Paper Number THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(79) 2014, VOL 1 39 IMMOBILIZING LACTOBACILLUS PLANTARUM BACTERIA ON SODIUM ALGINATE AND ITS APPLICATION TO TOMATO JUICE F[.]
THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(79).2014, VOL 39 IMMOBILIZING LACTOBACILLUS PLANTARUM BACTERIA ON SODIUM ALGINATE AND ITS APPLICATION TO TOMATO JUICE FERMENTATION FOR MAKING PROBIOTIC PRODUCTS NGHIÊN CỨU CỐ ĐỊNH VI KHUẨN LACTOBACILLUS PLANTARUM TRÊN NATRI ALGINATE VÀ ỨNG DỤNG LÊN MEN TẠO NƯỚC CÀ CHUA BỔ SUNG PROBIOTIC Ngo Thi Minh Phuong, Nguyen Thanh Hoi The University of Danang, College of Technology; Email: nthoi@ud.edu.vn Abstract - Immobilizing bacteria on sodium alginate brings about some incomparable advantages such as enhancing cell viability, strengthening spore stability, protesting against a number of physicochemical conditions, assuring simple implementation procedures and low-cost preservation Lactobacillus plantarum, considered to be a probiotic, brings out many benefits This study aims at examining the survivability of Lactobacillus plantarum on sodium alginate and its possible applications in fermenting tomato juice to create probiotic products The research results showed that the capsules prepared with 3% (w/v) sodium alginate and 1,5% (w/v) CaCl2 were most appropriate for cell immobilization, and the density of Lactobacillus plantarum was 1,1.107 cells/ml after it had been stored at room temperature for three months The bacterial motility revealed no significant change before and after the immobilization process Also, the sodium alginate capsules which had been reused three times still maintained high lactic acid production ability, creating new products that benefit our health Tóm tắt - Cố định vi khuẩn chất mang natri alginate có nhiều ưu điểm vượt trội như: khả sống sót tế bào cao, tăng cường ổn định tế bào chống lại số điều kiện hóa lí, phương pháp đơn giản, dễ thực hiện, giá thành bảo quản thấp Vi khuẩn Lactobacillus plantarum có nhiều lợi ích xem probiotic Nghiên cứu nhằm mục đích khảo sát khả sống sót vi khuẩn Lactobacillus plantarum cố định natri alginate ứng dụng lên men nước cà chua tạo sản phẩm probiotic Kết nghiên cứu cho thấy: Hạt gel thử nghiệm với natri alginate 3% CaCl2 1,5% tốt cho việc cố định tế bào, mật độ Lactobacillus plantarum sau ba tháng bảo quản nhiệt độ thường 1,1.107 tế bào/ml, hoạt lực vi khuẩn so với trước cố định thay đổi không đáng kể, hạt natri alginate qua lần tái sử dụng sinh axit lactic tốt, đồng thời tạo sản phẩm tốt cho sức khỏe người tiêu dùng Key words - lactobaccillus plantarum; immobilized; sodium alginate; tomato juice; probiotic Từ khóa - lactobaccillus plantarum; cố định; natri alginate; nước cà chua; probiotic Introduction "Immobilizing Lactobacillus plantarum bacteria on sodium alginate and its application in tomato juice fermentation making probiotic products” Preservation of microorganism strains is particularly important It serves as the basis for research and applications related to many fields such as: biology, medicine, agriculture, food technology, etc There are many methods of preservation microorganisms such as freezing, freeze-drying and so on Microorganisms preserved by freeze-drying can be retained for 10-20 years However, this method requires high cost and when strains are taken out to use, the rest is highly likely to be infected Therefore, we have always found new, innovative methods for optimum preservation Among the various techniques of immobilization, immobilizing on alginate gel has outstanding advantageous features It is simple, and generally nontoxic, and allows easy control of particle characteristics such as permeability or thickness of the gel membrane Besides the fact that it is a light and inexpensive method, the capsules can be re-used many times in the production process We use strains of L plantarum for our research because they have proven their ability to survive gastric transit and colonize the intestinal tract of humans and other animals [6] Various special therapeutic or prophylactic properties have been associated with L plantarum, such as reducing diarrhea infection, stomach pain and constipation associated with irritable bowel syndrome, flatulence and positively affecting the immune system of children infected with HIV, they are considered a probiotic Because of the above reasons we chose the theme: Materials and methods 2.1 Materials ➢ Lactobacillus plantarum is activated on the strains taken from the laboratory of Food Technology – Da Nang College of Technology ➢ Tomato 2.2 Methods 2.2.1 Microbiological method [3] The number of cells was identified by counting the number of colonies growing on agar The number of bacterial cells in ml of the sample is calculated as follows: N (CFU / gorCFU / ml ) = − C (n1vd1 + + ni vdi ) N: Number of cells (colony forming units) of bacteria in 1g or 1ml of sample − C: Total number of colonies counted on one selected petri dish − ni: Number of petri dish implanted at ith dilution rate − di: Corresponding dilution factor − V (ml): Volume of sample solution implanted in each petri dish 2.2.2 Biochemical method [3] The produced lactic acid was quantified by titration 40 Ngo Thi Minh Phuong, Nguyen Thanh Hoi with solution of NaOH of 0.1 N The equation to calculate the total acid content is: 𝑣 𝑘 𝑛 1000 𝑉 v: Volume of NaOH consumed for sample preparation k: Coefficient of acid (for lactic, k = 0.009) 1000: Calculation coefficient (by g/l) n: Dilution factor V: Volume of sample 2.2.3 Immobilizing Lactobacillus plantarum bacteria - Observations were made with Sodium alginate is of different concentrations, i.e 1%; 1.5%; 2%; 2.5%; 3%; 3.5%; 4% and CaCl2 0.5%; %; 1.5%; 2%; 2.5%; 3%; 3.5% - The capsules were then created and Lactobacillus plantarum was immobilized on the capsules gel according to the diagram in Figure Tomato Classification Washing Tomato juice Sterilization (121 C, 20 min) Water v Mixing L.p bacteria Fermentation Sugar Filtration Fermented tomato juice Figure Process of tomato juice fermentation Results and discussions Sodium alginate CaCl2 Cacilum alginate capsules with immobilized bacteria 3.1 The activation of Lactobacillus plantarum strains After the activation process, we obtained separated, circularly-shaped, pure, milky, shiny, and smooth colonies that were stuck on the surface of the activation environment (Figure 3) Observing the cells by microscope, we found that they are rod-shaped and sequencing They were concluded to be L.plantarum bacteria After being activated, the L.plantarum bacteria were preserved as in Figure Figure Diagram of the package granulating 2.2.4 Examining the viability of Lactobacillus plantarum on sodium alginate gel capsules during storage time The steps were carried out in the following order: - Survey the density of Lactobacillus plantarum cells during storage time - Soak five sodium alginate capsules which had just been immobilized with bacteria in tubes containing ml of distilled water for 40 minutes - Dilute the samples decimally and spread them on petri dishes to determine the initial cell by counting the number of the formed colonies This procedure was periodically repeated after days, 14 days, 30 days, and month 2.2.5 Physical method The number of apparent cells of Lactobacillus plantarum was determined by measuring the optical density OD600 on a UV - Vis spectrophotometer 2.2.6 The experiment procedure for the production of tomato juice fermentation making probiotic products Figure L.p colonies Figure Strains preservation in tubes 3.2 Lactic acid produced by Lactobacillus plantarum A solution with bacteria density of 109 CFU.ml-1 was put into 100ml flask of milk to be fermented The lactic acid productivity was measured as described in Section 2.2.2 From the experimental results we can draw the following conclusions: 10 8.8 8.3 g/l 7.5 4.7 time, h 24 48 72 Figure Acidity in every 12 hours 96 THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(79).2014, VOL st nd th On the , , days, acid production increased rapidly; especially, after 48 hours, the content of acid was 7.5 g l-1 However, after the 4th day, it decelerated This happened as the amount of bacteria had been increasing while the amount of substrate remained unchanged When the substrate content gradually decreased, the amount of acid was also reduces It is suggested that the content of lactic acid produced during fermentation is relatively larger than it is mentioned in the literature [5] 3.3 The concentration of Sodium alginate and CaCl2 in capsules production From the results in Table 1, the optimal concentration 41 of sodium alginate is 3% This concentration was selected based on some literature references, especially [1], in which it is advised the harder capsules are, the lower the ability of diffusion of substrate into the capsules via microbial cell wall is The formed capsules, however, are strong, and also, the ability to protect immobilized cells from external effects is good, and vice versa The capsules formed by Sodium alginate 3% are spherical and slightly solid, so they have better capacity for cell protection and substrate diffusion during the fermentation process in comparison with hard gel capsules Table Examined concentrations of Sodium alginate and CaCl2 Characteristics Concentration of sodium alginate (%) 1,5 The ability to Create Create a soft Soft, firm create particles particles less particle and able to shrinkage Shape Structure capsule 2,5 3,5 Create good particles Create good Solid particles Particles are Spherical broken particles, shrinkage Spherical Spherical Spherical of Particles are Soft, firm broken Soft, firm Soft, firm Soft, rather Solid solid Solid Undefined shape Undefined shape Solid Concentration of CaCl2 (%) 0,5 1,5 2,5 3,5 The ability to Particles are Create rather Creating create particles broken good good particles particles Creating good particles Create good Create good Create good particles particles particles Structure capsule Soft, firm Soft, firm of Particles are Create broken gel soft Soft, firm 3.4 Lactobacillus plantarum encapsulation procedure The solutions of Sodium alginate 3%, and of CaCl2 1,5% were prepared At the same time, we prepared a solution of Lactobacillus plantarum cells at the density of 109 cells ml-1 (the density is determined by measuring the optical density and calculating the apparent number of cells afterwards) The apparent number of cells ml-1 can be calculated through the optical density (OD) using the following equation [7]: Solid Solid results in Figure 6, it shows that L.plantarum bacteria are very convenient for later storage and use after being immobilized on sodium alginate capsules − − X: Apparent number of cells ml-1 OD: Optical density values measured at a wavelength of 600nm Next, the bacteria solution was mixed with CaCl2 Then, capsules were created by the drip method The formed capsules were removed to drain then put in an oven for drying at a temperature of 380C until constant mass is reached, and finally stored According to [8], the drying temperature of 380C was chosen because the drying time is ensured without inactivating bacteria From the obtained Figure Capsules with immobilized bacteria 3.5 The viability of Lactobacillus plantarum on sodium alginate capsules during storage time The viability of lactic bacteria on Sodium alginate capsules was examined by sowing the samples in a proper nutrition environment and observing the growth and development of colonies on a petri dish 42 Ngo Thi Minh Phuong, Nguyen Thanh Hoi week month month month Figure Survivability of immobilized bacteria during storage Lactic acid produced, g/l The results in Figure show that the number of viable Lactobacillus plantarum bacteria is large After months being stored at room temperature (300C), their survival rates remained high (1,1.107 cells ml-1) It can be said this method of microorganism preservation allows high productivity, easy implementation and and applications In this method, the survival rate is high because the combination between sodium alginate and Ca2+ helps forms an area of alginate and create gels with “egg-box” model The formation of gel particles requires linking mechanism between two or more alginate chains The chain of alginate molecules has folds and clefts so that Ca2+ can get into, locate and make connections Ca2+ links the alginate molecules into the alginate chains, making the chains remain stable Lactobacillus plantarum blended with CaCl2 solution also gets into the clefts and stays immobilized there Accordingly, the gel particles can hold numerous bacterial cells The outer protection layer protects the cells from external conditions, so the bacteria can be preserved in a long period without being infected or inactivated 3.6 Lactic fermentation ability of the gel capsules during the preservation process As described in section 2.2.2, five immobilized gel capsules preserved for one week were put into 100 ml flask of fresh milk to study their lactic fermentation ability The productivity of the bacteria was checked in every 12 hours, which is presented in Figure From the graph, we can infer that: - Compared to the sample before storage (Figure 5), the productivity of Lactobacillus plantarum decreased slightly - After 24 hours of fermentation, the immobilized cells, which were preserved for months produced less acid (2.7 g l-1) than free cells (4.32 g l-1) However, after 96 hours the difference is not significant (8.3 g l-1 vs 8.8 g l1 ,respectively) It means that the preservation process did affect the motility of bacteria, or in other words, the bacteria were inhibited by storage conditions In the first 24 hours, the bacteria needed some time to re-activate The duration was not enough for the bacteria to adapt to the fermentation solution - After 96 hours, the bacteria stored for months became active and adapted to the medium, so the fermentation abilities of all the samples were the same This suggests that under room-temperature storage condition, the preservation process does not considerably affect Lactobacillus plantarum As a result, we are able to conclude that with this preservation method allows the productivity of immobilized bacteria to remain relatively stable after months of storage It can be explained as follows: after the bacterial cells had been immobilized in the gel capsules, the gel matrix surrounded the cells Such structure created pores, enabling substrates and fermentation products to be easily diffused out of the gel capsules Thus, we can well control the reaction and separate the products from the reaction vessel Moreover, because of the gel layer surrounding bacterial cell, the productivity of the immobilized bacteria is stable during storage 3.7 Producing fermented tomato juice making probiotic products 3.7.1 The content of produced lactic acid during reuse L.plantarum strains preserved for over months were used to ferment tomato juice Afterthat, lactic acid formed in the product was quantified and qualified (Figure 9) Content of acid, g/l CFU/ml Tim e Time, h 24 48 72 After week Sau tuần Sau tháng After month After months Sau tháng Figure Productivity of bacteria 96 Figure Content of acids (g/l) As can be seen in Figure 9, the content of lactic acid tends to increase over time This is because right at the end of the first fermentation, we implemented the second and then the third fermentation In fact, this strain had been activated in the previous fermentation, so it did not require activation for the next one Therefore, the amount of acid produced later was greater than that of the previous fermentation THE UNIVERSITY OF DANANG, JOURNAL OF SCIENCE AND TECHNOLOGY, NO 6(79).2014, VOL Also, reusing strains times did not reduce the acid content generated 3.7.2 Tomato juice fermentation making probiotic products The Lactobacillus plantarum strain stored for months on sodium alginate capsules was used to ferment tomato juice, the result of which is shown in Figure 10 43 Conclusion From what have been discussed, we have successfully immobilized Lactobacillus plantarum on substances containing sodium alginate (Sodium alginate 3% and CaCl2 1.5%).The density of Lactobacillus plantarum was 1,1.107 cells/ml when stored at room temperature after three months, proving this method is much more convenient than others The bacterial productivity has no significant change in comparison with that of non-immobilized bacteria after three months of immobilization After the immobilization process, Lactobacillus plantarum can be consecutively reused three times but still maintains high acid fermentation ability Also, the tomato juice fermented by Lactobacillus plantarum strains ensures good- quality probiotic products REFERENCES Figure 10 Fermented tomato juice The tomato juice was then examined as is revealed in Tables and Table Target indexes of products after 72 hours of fermentation Parameters Content of dry matter Content 15 (%) pH Content of acid 6.85 g/l Density of Lactobacillus plantarum cells 109 cells/ml Table Sensory evaluation of formed products Colour Brick red State Liquid Flavor Savour Typical odor of fermented lactic (Smell of tomatoes no longer exists) [1] Mai Ngọc Dũng, “Thủy phân saccharose invertase cố định hạt calium alginate”, Tạp chí phát triển KH&CN, 10 (4) [2] Nguyễn Đức Lượng, Cơ sở vi sinh vật công nghiệp, Nhà xuất bản Đại Học Quốc Gia Hồ Chí Minh, 2002 [3] Lê Thị Thanh Mai, Các phương pháp phân tích ngành cơng nghệ lên men, Nhà x́t bản Khoa học Kĩ thuật, 2005 [4] Bayrock D & Ingledew W M., “Mechanism of viability loss during fluidized bed drying of baker’s yeast”, Food Research International, 1997, 30(6), p: 417 – 425 [5] Binbin Liu, Maohua Yang, Benkun Qi, Xiangrong Chen, Zhiguo Su, Yinhua Wan, “Optimizing l-(+)-lactic acid production by thermophile Lactobacillus plantarum As.1.3 using alternative nitrogen sources with response surface method”, Biochemical Engineering Journal 2010, 15(2), p:212–219 [6] Miriam Zago and al., “Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses”, Food Microbiology 2011, 28, p: 1033 – 1040 [7] Stephanie Hessea, Tetsuo Kondo, “Behavior of cellulose production of Acetobacter xylinum in 13C-enriched cultivation media including movements on nematic ordered cellulose templates”, Carbohydrate Polymers 2005, 60, p: 457 – 465 [8] Zoe Konsoula, Maria Liakopoulou-Kyriakides, “Thermostable amylase production by Bacillus subtilis entrapped in calcium alginate gel capsules”, Enzyme and Microbial Technology 2006, 39, p: 690–696 Sweet and sour (The Board of Editors received the paper on 28/02/2014, its review was completed on 13/05/2014) ... acid content generated 3.7.2 Tomato juice fermentation making probiotic products The Lactobacillus plantarum strain stored for months on sodium alginate capsules was used to ferment tomato juice, ... acid fermentation ability Also, the tomato juice fermented by Lactobacillus plantarum strains ensures good- quality probiotic products REFERENCES Figure 10 Fermented tomato juice The tomato juice. .. according to the diagram in Figure Tomato Classification Washing Tomato juice Sterilization (121 C, 20 min) Water v Mixing L.p bacteria Fermentation Sugar Filtration Fermented tomato juice Figure