1243022 A7 B7 五、發明說明（1 ) 發明背景 發明範圍 (請先閱讀背面之注意事項再填寫本頁) 本發明提供偵測臭滷水發酵程度的方法。 先前技藝說明 臭豆腐是台灣特殊的傳統發酵食品，其通常係藉鹼性 發酵而得。發酵所使用的微生物應可將蛋白質水解成胺基 酸和胜肽。驗性發酵中使用的主要微生物是捍菌屬，其可 分泌細胞外酶以水解蛋白質成胺基酸及肽，藉此在發酵過 程中釋出氨並增加pH值。（Wang，J· and Fung，D.Y.C·， Alkaline-fermented food · a review with emphasis on pidan fermentation. Crit. Rev. Microbial. 22 · 101,1996) 傳統製作臭豆腐的方法包括用鹽醃潰原料後置於自然 環境中，使原料腐敗而生臭滷水，將豆腐塊浸入臭滷水並 發酵4到6小時；及自滷水中取出該豆腐。（Su，Y. C. Traditional fermented foods in Taiwan, In Proceeding of the Orienal Fermented Foods，p. 15, Food Industry Research and Development Institute, Taiwan，Hsinchu, 1980 ). 經濟部智慧財產局員工消費合作社印製 適合製作臭豆腐之臭滷水之評估一般是由經驗豐富的 臭豆腐製造者進行，彼等可分辨經發酵臭滷水之臭味的差 異。然而，有鑑於感官感受力的不同及臭滷水複雜的氣 味，這種評估無法標準化。因此有必要發展偵測臭滷水發 酵程度的標準化方法，以便於決定適合於製造臭豆腐之經 發酵臭滷水。 -4 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1243022 經濟部智慧財產局員工消費合作社印制衣 A7 五、發明說明（2 本發明的目的是提供偵測臭滷水發酵程度的方法。 本發明的其他目的是提供測定臭滴水發酵之最佳程度 之方法。 要說明 圖」表示以八2菌群接種之滷水組合物中的酸驗值變 化、氨釋放量及細菌總數。 圖2表示以A2菌群接種入之滷水組合物在 培養的氨量改變情形。 圖3表示振盪速率對以Α2菌群接種之滷水組合物中氨 濃度變化的影響。 查詳細說明_ 本發明目的係提供偵測臭滷水發酵程度的方法，其包 括測定臭滷水的氨量，其中較高氨量表示高發酵程度：、 根據本發明，頃發現氨在臭滷水發酵期間會連續產 生。事實上’氨量與臭味係成正比。因此，氨量可用於偵 測臭滷水的發酵程度。經由偵測釋放的氨濃度來控制發酵 私度，我們可標準化製造臭豆腐的程序，藉此可製得衛 生、安全且品質穩定的臭豆腐。 、本發明的其他目的是提供測定臭滷水發酵之最佳程度 的方法，其包括測足滷水的氨量，其中如氨量到達預定值 即得到發酵的最佳程度。 根據本發明方法，預定值是由官能品評決定。在一個 具體實施例中，預定值是2 2 0 0到2 8 0 〇毫克/升，較隹 2 5 2 0亳克/升。 … -----------裝---------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 1243022 經濟部智慧財產局員工消費合作社印製 A7 ____ B7 五、發明說明（4 ) 1 % (重量/體積）鹽。均質原料以得到滷水培養基。添加 8 0 0笔升的滷水培養基至i 〇 〇 〇毫升的瓶中，並接著在1 2 i °C滅菌6 0分鐘。然後把A2菌群接種至滷水組合物中，在 3 0 °C發酵。 實例2 氣之測定 如實例1製備的臭滷水發酵丨〇週，每週取出臭滷水以測 足氨量。在1 20〇〇rpm了離心臭滷水丨〇分鐘，從離心上層 取出2 5晕升並稀釋1 0 〇 〇倍。滴入3滴礦物穩定劑（η a c h ) 和聚乙烯醇分散劑及i亳升的奈斯勒試劑（HACH)反應1分 鐘。用分光光度計（攜帶分光光度計 DR/2〇 1 〇，美國HACH股份有限公司）在us奈米下測量 吸光度。如圖1所示，氨量會隨發酵時間增加而增加。 實例3 滷永的佳發酵 把6 0片豆腐塊（4.5公分X 4 · 5公分X i . 2公分）置於乾 淨的塑膠盒。在室溫下浸潰豆腐於10〇〇亳升的臭滷水中4 小時。取出該豆腐並用清水清洗以得到衛生的生臭豆腐。 將如上述製作的生臭豆腐冷藏於4它下整夜後油炸，起始 油炸溫度是15 Ot：接著進行官能喜好評估。 共有50位官能品評者針對外觀、氣味、質地及臭豆腐 Y配合料）的整體喜好進行品評。樣本是隨機供應和編 號。品評結果（評價分為_級，！是相對於傳統臭豆腐極 不喜歡，9是極喜歡）鍵入電腦以統計分析系統（⑷）分 析。評估結果示於表1。 -7 - 本紙張尺度適用f _家標準（CNS)A4 —-------- -------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 1243022 A7 五、發明說明（6 貫例6 酵的最佳振盪 見彳1所述來製備臭滷水。分別在靜置及振盪於 7/^pm&150rpm下發酵該臭滷水；再如實例2所述測定 乱f。如圖3所示，在靜置狀況下臭滷水的發酵會增加到 2 5 2 0毫克/升。所以在靜置狀況發酵是發酵的較佳條件。 實例7 測定 如實例1所述來製備臭滷水並在蓋緊瓶蓋的瓶子及瓶蓋 微鬆的矾中分別發酵。如實例2所述來測定氨量。如表2所 不’在盖緊瓶蓋的瓶中發酵的氨量未達252〇毫克/升。因 此’蓋緊瓶蓋的瓶子不能達成最佳發酵。所以，在瓶蓋微 鬆的瓶中發酵係為較佳。 (請先閱讀背面之注意事項再填寫本頁} 加蓋法 培養前氨量 培養4週後的氨量 (毫克/升） (毫克/升） 瓶蓋蓋緊 18 0 - - - *---- 45 6 瓶蓋微鬆 18 0 3 4 3 0 訂---------線0. 經濟部智慧財產局員工消費合作社印製 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）1243022 A7 B7 V. Description of the invention (1) Background of the invention (Please read the notes on the back before filling out this page) The present invention provides a method for detecting the degree of fermentation of odorous brine. Previous techniques show that stinky tofu is a special traditional fermented food in Taiwan, which is usually obtained by alkaline fermentation. The microorganisms used in the fermentation should hydrolyze the protein into amino acids and peptides. The main microorganism used in experimental fermentation is genus Bacillus, which secretes extracellular enzymes to hydrolyze proteins into amino acids and peptides, thereby releasing ammonia and increasing pH during the fermentation process. (Wang, J · and Fung, DYC ·, Alkaline-fermented food · a review with emphasis on pidan fermentation. Crit. Rev. Microbial. 22 · 101, 1996) The traditional method of making stinky tofu includes pickling raw materials with salt and placing In the natural environment, the raw materials are spoiled to produce stinky brine, the tofu pieces are immersed in the stinky brine and fermented for 4 to 6 hours; and the tofu is taken out of the brine. (Su, YC Traditional fermented foods in Taiwan, In Proceeding of the Orienal Fermented Foods, p. 15, Food Industry Research and Development Institute, Taiwan, Hsinchu, 1980). The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs prints suitable for making stinky tofu The evaluation of stinky brine is generally performed by experienced stinky tofu manufacturers, who can distinguish the difference in the odor of fermented stinky brine. However, given the difference in sensory sensibility and the complex smell of bitter water, this assessment cannot be standardized. It is therefore necessary to develop a standardized method for detecting the fermentation level of stinky brine in order to determine the fermented stinky brine suitable for the production of stinky tofu. -4-This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1243022 Printed clothing A7 of the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (2 The purpose of the invention is to provide the Method for the degree of fermentation of brine. Another object of the present invention is to provide a method for determining the optimal degree of fermentation of odorous drip water. To illustrate the figure, it is shown that the acid value change, ammonia release amount and The total number of bacteria. Figure 2 shows the change in the amount of ammonia in the brine composition inoculated with A2 flora. Figure 3 shows the effect of the oscillation rate on the change in ammonia concentration in the brine composition inoculated with A2 flora. The purpose of the present invention is to provide a method for detecting the degree of fermentation of odorous brine, which comprises measuring the amount of ammonia in odorous brine, wherein a higher amount of ammonia indicates a high degree of fermentation: According to the present invention, it is found that ammonia is continuously produced during the fermentation of odorous brine. In fact, the amount of ammonia is directly proportional to the odor. Therefore, the amount of ammonia can be used to detect the degree of fermentation of stinky brine. The detection of the ammonia concentration to control the fermentation privacy We can standardize the procedure for making stinky tofu, thereby making stinky tofu that is hygienic, safe, and stable in quality. Another object of the present invention is to provide a method for determining the optimal degree of fermentation of stinky brine, which includes measuring the ammonia content of marinated brine. The optimal level of fermentation is obtained when the amount of ammonia reaches a predetermined value. According to the method of the present invention, the predetermined value is determined by the functional product evaluation. In a specific embodiment, the predetermined value is 2200 to 2800 mg. / Liter, compared with 5 2 5 2 0 亳 g / liter. ... ----------- install --------- order --------- line (please first Read the notes on the back and fill in this page) 1243022 A7 ____ B7 printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (4) 1% (weight / volume) salt. Homogeneous raw materials to obtain brine culture medium. Add 8 0 0 liters of brine culture medium was put into a 1000 ml bottle, and then sterilized at 12 ° C for 60 minutes. Then the A2 flora was inoculated into the brine composition and fermented at 30 ° C. Example 2 The determination of gas is the same as the fermentation of odorous brine prepared in Example 1. Weekly, the odorous brine is taken out every week to measure the ammonia content. The centrifugal odor brine was centrifuged at 200 rpm for 10 minutes, and then removed from the upper centrifugal layer and halved and diluted 1000 times. Three drops of a mineral stabilizer (η ach) and a polyvinyl alcohol dispersant and i 亳 liter were added dropwise. Naissler reagent (HACH) reaction for 1 minute. Measure the absorbance at us nanometer with a spectrophotometer (carrying spectrophotometer DR / 2001, American HACH Co., Ltd.). As shown in Figure 1, the amount of ammonia will be It increases with the increase of fermentation time. Example 3 Lu Yongjia's fermentation put 60 pieces of tofu pieces (4.5 cm X 4 · 5 cm X i. 2 cm) in a clean plastic box. Soak the tofu in 100,000 liters of brine at room temperature for 4 hours. The tofu was taken out and washed with water to obtain a hygienic stinky tofu. The stinky tofu prepared as described above was refrigerated at 4 ° C overnight and then fried, and the initial frying temperature was 15 Ot: Then, functional preference evaluation was performed. A total of 50 functional reviewers evaluated the overall preferences for appearance, odor, texture, and stinky tofu (Y compound). Samples are supplied randomly and numbered. The evaluation result (the evaluation is divided into _ grades! It is very disliked compared to the traditional stinky tofu, 9 is extremely liked) Enter the computer for statistical analysis system (⑷) analysis. The evaluation results are shown in Table 1. -7-This paper size applies to f _ house standard (CNS) A4 —-------- ------------------- Order ------ --- Line (please read the precautions on the back before filling this page) 1243022 A7 V. Description of the invention (6 Example 6 The best vibration of the fermentation is described in 彳 1 to prepare the smelly brine. Stand at rest and shake at Ferment the odorous brine at 7 / ^ pm & 150 rpm; then measure the f as described in Example 2. As shown in Figure 3, the fermentation of odorous brine will increase to 2520 mg / L under static conditions. Fermentation under standing conditions is the preferred condition for fermentation. Example 7 Determination of odorous brine as described in Example 1 and fermentation in a bottle with a tightly closed cap and alum with a slightly loose cap. Ammonia was measured as described in Example 2. As shown in Table 2, the amount of ammonia fermented in a bottle with a tightly capped cap does not reach 252.0 mg / L. Therefore, the bottle with a tightly capped cap cannot achieve optimal fermentation. Therefore, the slightly loose cap The fermentation system in the bottle is better. (Please read the precautions on the back before filling out this page} Ammonia before capping method Ammonia after 4 weeks incubation (mg / L) (mg / L) Cap tightly 18 0---* ---- 45 6 bottles Micro pine 1,803,430 --------- lines set 0.5 Ministry of Economic Affairs Intellectual Property Office employees consumer cooperatives printed in this paper scale applicable Chinese National Standard (CNS) A4 size (210 X 297 mm)