Lactobacillus plantarum NTU 102生理特性及其在保健食品與水產養殖應用之探討

Abstract

乳酸菌在醱酵食品製造中應用廣泛，舉凡乳酪、優酪乳等各種醱酵乳製品、酒類、醃漬品之製造。本研究從各種乳酸菌製品與自製韓式泡菜樣品中分離出在低pH及膽鹽(bile salt)的環境中可生長之201株菌為革蘭式陽性、缺乏催化酶的菌株。比較這些篩選菌株在pH 2.0、含0.5%膽鹽之MRS培養基中生長菌落數及各菌株在8% skim milk凝乳試驗之速率，再次進行大量篩選工作，符合上述要求者計有45株菌。重覆在pH 2.5及0.5% 膽鹽的環境中篩得2株可滴定酸量較高之乳酸菌，進行初步鑑定分別為Lactobacillus plantarum 7-40 (NTU 102) 及 Lactobacillus paracasei ssp. paracasei 1-12’，此兩株菌對 vancomycin、 polymyxin 及metronidazole具有抗性，另外 L. plantarum NTU 102、L. paracasei ssp. paracasei 1-12’在pH 3.0及1%膽鹽培養基經過3小時之後兩者均有80%以上之存活率。 L. plantarum NTU 102 及 L. paracasei ssp. paracasei 1-12’菌株具有抑制Pseudomonas aeruginosa之效果。 研究報告指出乳酸菌具有降低膽固醇之能力，因此將分離株L. plantarum NTU 102進行體外及動物降低膽固醇試驗。在體外試驗L. plantarum NTU 102具有較佳的膽鹽耐受性。由動物實驗得知，餵食L. plantarum NTU 102菌株之倉鼠其雙歧桿菌數量增加，且產氣莢膜梭菌數量減少，顯示具有改善腸道菌相之功能。餵食L. plantarum NTU 102 乳酸飲料可以提高血清中HDL膽固醇，並且減少LDL膽固醇含量。 有關乳酸菌屬（Lactobacilli）在腸道平衡微生物菌相及抑制病原菌機制，可能與乳酸菌分泌生物表面活性劑有關。因此本研究針對分離菌株 Lactobacillus plantarum M9、Lactobacillus casei M15、L. plantarum NTU 102及Lactobacillus plantarum 7-41是否會分泌生物表面活性劑進行研究。臨界微胞濃度（critical micelle concentration）簡稱為CMC值是評估表面活性劑形成乳化微胞之指標。篩選菌株M9、M15、NTU 102及7-41產生之粗生物表面活性劑，分別配製不同濃度之粗生物表面活性劑溶液，測定其表面張力及乳化指標，其對煤油之乳化指標在78%至83%之間；對水表面張力從72 ± 3 mN/m 可降至 45 ± 3 mN/m左右，四株菌並無顯著性差異；而其CMC值分別為0.35%、0.3%、0.2%及0.35%。針對四株菌之理化特性在不同氯化鈉濃度（0.5~5.5%）、pH (2~12)及溫度 (60，100，120℃)作用30分鐘，只有NTU 102維持較佳之穩定度。 L. plantrum NTU 102 菌株之量產試驗主要以田口方法（Taguchi method）探討之。應用直交表設計實驗，採用信號雜訊比之數據分析方法，探討各個因子對製程之影響。以田口式L9(34) 四因子三階方法來進行此次實驗。由製程設計出控制因子及實驗水準：培養基 (0.01%、0.05%、0.1% MRS)，轉速 (0、25、50 rpm)，溫度 (37、41、45℃) 及培養時間 (12、18、24小時)。首先將活化過後之菌液先以五公升小型液態醱酵槽方式進行研究，得到其最佳培養條件 (接菌量10%、轉速0 rpm、通氣量0 vvm及時間24小時)，之後再移入130公升大型醱酵槽內培養進行實驗，以因應工業上的量產，找出最適培養方法、節省培養成本以及能夠獲得菌體最高產量。所得之結果經由田口式數據分析得到最佳條件組合為：培養基0.1%糖蜜＋0.55%MRS、轉速50 rpm、溫度41℃及培養時間12小時。經由確認實驗所得到菌體之產量為2.56 g/L，較所有條件組合之菌體量高，證實此組合為實驗生產L. plantarum NTU 102產量的最適條件。另外，在凍乾菌體活性貯存試驗以8:1（培養液：20%脫脂牛奶）比例，所得凍乾菌粉分別貯存於常溫、5℃、25℃及40℃，其結果在常溫、5℃及25℃初菌數自6.05×1011~7.09×1012 CFU/g經60天試驗發現只下降1個對數值，但在40℃卻下降6個對數值。顯示此菌粉貯存在常溫其菌體活性尚趨穩定。 在國外將益生微生物製劑應用於水產養殖已行之多年，因此本研究以L. plantarum NTU 102 餵食南美白蝦 (Litopenaeus vannamei)，探討是否會提高其免疫能力。由其結果得知，以餵食含1010 CFU/kg diet 之L. plantarum NTU 102 飼料，在24、48及168小時具有較高之超氧歧化酵素，且與控制組比較亦有顯著性差異。總血球數的變異與控制組比較，經7天餵食 NTU 102菌量在107和1010 CFU/kg diet之南美白蝦有較低之總血球數，而餵食L. plantarum NTU 102組間並沒有明顯的差異性 (p> 0.05)。在酚氧化酵素(PO)方面，發現持續餵食不同濃度L. plantarum NTU 102至7天之兩組，其PO活性增加，且與控制組比較有顯著性差異 (p＜ 0.05)。以V. alginolytics攻擊南美白蝦之試驗中發現，餵食不含L. plantarum NTU 102飼料之南美白蝦，在攻擊實驗後其死亡率會隨著時間增加而增加，且為各試驗組中最高。

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.