The Studies on the Purification and Composition Analysis as Well as the Application on Food of Extracellular Adhesive Substance Produced by Marine Biofouling Bacteria (I)

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Project title

Code/計畫編號

NSC89-2313-B019-063

Translated Name/計畫中文名

海洋附著細菌胞外黏性物質之純化和組成分析以及在食品上應用之探討(I)

Project Coordinator/計畫主持人

Chorng-Liang Pan

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Food Science

Website

https://www.grb.gov.tw/search/planDetail?id=552926

Year

2000

Start date/計畫起

01-08-2000

Expected Completion/計畫迄

01-07-2001

Bugetid/研究經費

623千元

ResearchField/研究領域

食品科技(農)

本研究在測試海洋附著細菌產生較多細胞外黏性物質 (Extracellular adhesive substances, EAS) 之最適培養基及培養條件之生產條件，並以上述組合成份與條件培養海洋附著細菌以取得最大量的胞外黏性物質產物，將所得 EAS 經萃取、分離、與純化，並進行一般成分分析。源自海洋附著材料片上之 74 株附著細菌分離株，依所呈現的菌落特徵而分類成：一般性黏液、擴散性黏液、聚集黏液、與勾絲狀黏液，在26oC下於mucus screening basal broth (MSBB) 中以靜置或振盪 (150 rpm) 培養，以及在 Marine Agar (MA, Difco) 上塗抹培養，並分別測試 RV (相對黏度)，依菌落型態與 RV 挑選出：聚集黏液—菌株 SDC07、擴散性黏液— 菌株 DB05B、勾絲狀黏液—菌株 SCA38、與一般性黏液— 菌株 SDC01。菌株 SDC07 與 SCA38 在 MSBB 培養基中，於 26oC 下振盪 (150 rpm) 培養 72 hr 之生長情形，由相對黏度初步篩選並配合光學密度及生菌數，判斷 SDC07 約在培養 20-28 hr 期間有較佳的黏度表現，SCA38 約在培養 40-48 hr 期間有較好黏度表現。菌株 DB05B、SDC01 在 MSBB 培養基中於 26oC 下，靜置培養 72 hr 之生長情形，由相對黏度初步篩選並配合光學密度及生菌數，判斷 DB05B 約在培養 52-60 hr 期間有較好黏度表現，SDC01 產胞外黏性物質的期間約在 8-16 hr。在 MSBB 培養液中置換不同碳源，以 26oC 並用 150 rpm 振盪培養 Micrococcus sp. strain SDC07，結果顯示 M. sp. strain SDC07 所產之聚集胞外黏性物質在含有 5% sucrose 之培養基培養 24 hr 後，培養基中可得到 0.5 g/L 之 EAS 產量。當 M. sp. strain SDC07 使用有機氮源成分如 yeast extract 與 polypeptone/yeast extract 時，EAS 產量及菌液的相對黏度均較以無機氮源時為高。M. sp. strain SDC07 培養在添加 92 mM disodium-â-glycerophosphate (DSGP) 所獲得之 EAS 產量較 46 或 139 mM DSGP、K2HPO4、KH2PO4 (0.5-1.5 mM) 高。M. sp. strain SDC07 培養在添加 1 mg MnCl2．7H2O 之 MSBB 中時，其 EAS 之產率是四組金屬離子測試中最高者，可達到 2.25 g/L，RV 為 8.06。在 MSBB 培養液中以不同碳源取代， 26oC 靜置培養 M. sp. strain DB05B，結果顯示 M. sp. strain DB05B 以 fructose 為碳源可得較高之 EAS 產量 (0.29 g/L)。在培養液中添加 FeCl3 後培養 M. sp. strain DB05B， EAS 的產量較高 (0.15 g/L)，但 RV 是以培養基中添加 MnCl2．7H2O 組較高 (1.96)。在培養條件方面，strain SDC07 於 26oC 下培養，可得到較高的 EAS 產量 (0.95 g/L)。M. sp. strain SDC07 培養在起始 pH 值為 5.2 之 MSBB 培養液時，可產生最高之 RV (1.5) 與 EAS 產量 (1.25 g/L)，其約為培養在 MSBB 起始 pH 值 4.2 時所產生 EAS 的 2 倍。M. sp. strain SDC07，以 150 rpm 振盪培養時，所得 RV 1.70 與 EAS 產量 0.98 g/L，均較以 50 與 100 rpm 之振盪速度培養時為高。M. sp. strain DB05B 培養 2 在起始 pH值為 7.2 之 MSBB 中與培養溫度 26oC 時，呈現比較高的 EAS 產量 (0.08 與 0.15 g/L)。將M. sp. strain SDC07、DB05B、SCA38 所產的 EAS 進行一般成分中，發現 EAS 的一般成分 (以乾物重計) 仍以多醣類 (分別為 77.2%、69.0%、71.0%) 為主，其次為粗蛋白 (11.2%、20.5%、16.0%)。將生產聚集性黏液之菌株 SDC07、擴散性黏液之菌株 DB05B、勾絲狀黏液 SCA38 之菌株，所生產之 EAS 以膠體過濾層析分析，結果得知此四種不同特性之黏性菌株所生產的 EAS 均為一種含醣聚合物與蛋白質之複合物。未來將以本年度計畫研究成果為基礎，持續以發酵槽生產 EAS 條件之探討，並將所得 EAS 萃取物進行主要成分確認以及多項食品科技應用性之測試。The investigation was conducted to examine the optimal compositions of the incubation medium and incubation conditionsto culture the extracellular adhesive substances (EAS) producing marine biofouling bacteria. The maximum yield of EAS produced by each tested strains would be recovered from the culture from the combination of individual optimal factors mentioned above. Then, the harvested EAS was extracted, separated, and primary purified. The proximate composition of these EASs were examined also. Seventyfour EAS producing marine biofouling bacterial isolates were selected for four types mucous colony morphologies on the marine agar plate surface, i.e., general mucous colony, spreading mucous colony, aggregating mucous colony, and ropy colony. And according to their colony morphology and relative viscosity of their cultured broth, strain SDC07 for aggregating mucous colony, strain DB05B for spreading mucous colony, strain SCA38 for ropy colony, and strain SDC01 for general mucous colony. Strains SDC07 and SCA38 were cultured in MSBB broth with shaking incubation (150 rpm) under 26oC. The results showed that strain SDC07 had the better optical density (OD) and relative viscosity (RV) during the incubation period of 20-28 hr. And strain SCA38 was presented higher OD and RV after cultured for 40-48 hr. Strains DB05B and SDC01 were cultured in MSBB broth with shaking incubation (150 rpm) under 26oC. The results showed that strain DB05B had the better optical density (OD) and relative viscosity (RV) during the incubation period of 52-60 hr. And strain SC01 was presented higher OD and RV after cultured for 8-16 hr. To investigate the substitutive effects of carbon or nitrogen sources as well as additive effect of phosphates or metal ions on the EAS yield, sugar content, water content and protein content from the marine biofouling bacteria. Micrococcus sp. Strain SDC07 produced 0.5 g/L EAS while 5% sucrose was used. And as yeast extract or polypeptone/yeast extract were used to take place the original nitrogen source, the EAS yield of strain SDC07 were higher than the control group and the inorganic nitrogen replacement groups. The higher EAS productions of this strain were also observed while 92 mM disodium-b-glycerophosphate (DSGP) or 1.0 mg/L MnCl2．7H2O was employed in the incubation medium. The produced EAS amount of latter was 2.25 g/L and its RV reached 8.06. For strain DB05B, the substitution of fructose apparently give the better EAS production (0.29 g/L). And the addition of FeCl3 into the MSBB obtained 0.15 g/L EAS, but the higher RV (1.96) was observed while MnCl2．7H2O is added. In the study of incubation conditions for the maximum EAS production of strain SDC07, following factors gave the higher yield of EAS: 26oC/0.95 g/L; initial pH at 5.2/1.52 g/L and RV = 1.50; shaking speed at 150 rpm/0.98 g/L and RV = 1.70. As to the 3 strain DB05B, the initial pH at 7.2 and incubation temp. at 26oC were given the higher EAS yields for each tested condition, that are 0.08 g/L and 0.15 g/L. The proximate compositions of strain SDC07, DB05B, and SCA38 are primarily carbohydrate (i.e. polysaccharides), and ite content for these three strains are 77.2%, 69.0%, and 71.0%, respectively in dry mass. The second highest content of these EASs was crude protein, and its content were 11.2%, 20.5%, and 16.0%, respectively in dry mass. The results obtained form gel permeation chromatography indicated that the EAS produced by strains SDC07, DB05B, and SCA38 were the same as to be complex compound with both polysaccharide and protein in their structures, although their colony mucous morphologies were different. The data collected from this year will be the base for the future research work. The study on the fermenter conditions for the EAS production will be conducted in second year, and the application of these EAS on the food technology will be developed in the third year.

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The Studies on the Purification and Composition Analysis as Well as the Application on Food of Extracellular Adhesive Substance Produced by Marine Biofouling Bacteria (I)

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