博士 / 國立臺灣大學 / 微生物與生化學研究所 / 94 / Lactic acid bacteria are extensively applied in many food manufacturing processes, such as cheese and yogurt of fermented dairy products, wine, and pickles, etc. Criteria for selecting lactic acid bacteria were set up to meet the abilities of withstanding environmental conditions similar to the digestive tract as well as specific biological activities. Following an initial screening of over 201 strains selected from the collections, two of the survived strains were identified as Lactobacillus plantarum 7-40 (NTU 102) and Lactobacillus paracasei ssp. paracasei 1-12’. Antibiotics studied revealed that these two strains were resistant to vancomycin, polymyxin and metronidazole. Survival tests at low pH and tolerance against bile salt were also studied. Results showed that survival percentages of these two strains were more than 80% after 3 hr exposure to pH 3.0 and bile concentration of 1.0% (w/v). Growth inhibition of Gram-negative bacteria, such as Pseudomonas aeruginosa, in plague assay was attributed to the production of lactic acid in supernatant by L. plantarum NTU 102 and L. paracasei. ssp. paracasei 1-12’.
Literatures point out that probiotics can reduce serum cholesterol level and improve intestinal microflora in the host animals. Therefore, L. plantarum NTU 102 was evaluated in vivo and in vitro its effects on improving cholesterol and cecum microflora. In the in vivo study, when hamsters fed with L. plantarum NTU 102, the cecum Bifidobacilli increased and Clostridium perfringens decreased. These results showed that L. plantarum NTU 102 improved cecum microflora. In bile salts tolerance test, L. plantarum NTU 102 was found resistant to bile salts. Serum HDL-cholesterol was significantly increased while LDL-cholesterol decreased when hamsters were fed with L. plantarum NTU 102.
Biosurfactant produced by lactic acid bacteria in human digestion tracks was reported to possess the function of altering intestinal microflora. In this research, four strains identified and tentatively named as L. plantarum NTU 102, Lactobacillus plantarum 7-41, Lactobacillus plantarum M9 and Lactobacillus casei M15, were found higher in E24, which were 77.4%, 64.5%, 74.2% and 77.4%, respectively. These 4 strains were inoculated in MRS broth, and cultivated at 37 oC for 48 hours. Broths and cells were harvested, cracked down by autoclaving, followed by precipitation, centrifugation, and finally by freeze drying to obtain dried crude biosurfactant. Emulsification index of these biosurfactants against kerosene were found ranging from 78% to 83%; surface tensions of water were reduced from 72 ± 3 to 45 ± 3 mN/m; critical micelle concentration (CMC) ranged from 0.2 to 0.35%. These biosurfactants were also tested for their tolerances against pH, temperature and salinity. Results showed that surfactant produced by strain L. plantarum NTU 102 was found most stable under 100 oC, pH range 2 ~ 12 and salinity 0.5 ~ 5.5%. In the inhibition tests against pathogenic microorganisms using filter paper disc diffusion method, both living cells and the secreted biosurfactant showed capabilities of inhibiting the growth of Pseudomonas aeruginosa.
In this research, a pilot scale production of L. plantarum NTU 102 in a 130-L fermentor was conducted using Taguchi method for determining optimal cultivation conditions. Orthogonal array was applied for experimental design. Signal noise ratio was used for data analysis, and also to determine influences of each factor to the process. Taguchi method with L9(34) (4-factor-3-level) was used for its control factors and experimental levels, which were listed as follows: cultivation media (0.01%, 0.05%, 0.1% MRS), agitation speed (0, 25, 50 rpm), temperatures (37, 41, 45℃), and cultivation time (12, 18, 24 hrs). Activated stock broth was first directed into a 5-L fermentor for a preliminary study of small scale production, the optimal cultivation conditions were determined as: inoculums 10%, agitation speed 0 rpm, aeration rate 0 vvm, and cultivation time 24 hrs. Then the broth was transferred to the 130-L fermentor for pilot scale production. Optimal conditions were determined for increasing cell mass and reducing production cost, and used as the basis for industrial production. Results showed the optimal conditions based on Taguchi data analysis were 0.1% molasses in 0.55% MRS for medium, 50 rpm for agitation speed, temperature 41℃, and 12 hrs for cultivation time. This condition was verified by the cell mass production of 2.56 g/L, which was the highest amongst the others, and was suggested as the optimal conditions for mass production of L. plantarum NTU 102.
Preservation tests were conducted by comparing activities of frozen cells, which were prepared by freeze drying a mixture of 8 to 1 ratio of cell broth to a medium of 20% skimmed milk. The frozen cell powders were stored at ambient temperature, 5℃, 25℃, and 40℃, respectively. Results showed that frozen cell powders stored at ambient temperature, 5℃, and 25℃, the cell numbers only decreased 1 log cycle from their initial values of 6.05×1011 ~ 7.09×1012 CFU/g after a 60-day storage. However, it was reduced by 6 log cycles when stored at 40℃ for the same period. This also suggested that the frozen cell powder showed stable activity when stored at ambient temperature.
Probiotic culture systems (PCS) were developed for a variety of domestic animals and aquaculture for years. In this research, L. plantarum NTU 102 was evaluated its effects on improving the immunological activities of Litopenaeus vannamei; including total haemocyte count, phenoloxidase activity, respiratory burst, superoxide dismutase activity, phagocytic activity and clearance efficiency to Vibrio alginolyticus. It was susceptible to V. alginolyticus when the shrimps were fed diets containing L. plantarum NTU 102 at 0 (control), 107 and 1010 CFU/kg diet after 48 and 168 hrs. The immune parameters, including phenoloxidase activity, respiratory burst, superoxide dismutase activity and clearance efficiency to V. alginolyticus, and survival rate in challenge trials with V. alginolyticus were significantly increased, but the total haemocyte counts was significantly decreased for the shrimps fed with a feed containing L. plantarum NTU 102 at 1010 CFU/kg diet for 168 hrs. However, no significant difference was found in phagocytosis. It was therefore concluded that administration of L. plantarum NTU 102 in the diet at 1010 CFU/kg diet enhanced the immune ability of L. vannamei and increased its resistance to V. alginolyticus infection.
| Identifer | oai:union.ndltd.org:TW/094NTU05381052 |
| Date | January 2006 |
| Creators | Chiu-Hsia Chiu, 邱秋霞 |
| Contributors | 潘子明 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 193 |
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