201029657 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種抑制沙門氏菌活性的益生菌醱酵組合 物，特別係指一種利用具有耐胃酸、耐膽鹽及良好腸道吸附能 力的複合益生菌群進行醱酵所得的食品組合物。 【先前技術】 由於社會結構的改變，導致人們的飲食習慣在不知不覺中 ❺也開始產生轉變，快速、失衡又過度烹調的飲食方式使得很多 文明病也隨之而生，例如免疫調節功能下降、腸胃功能不良等， 這些身體機能的異常都容易導致人體的衰老現象急劇的發生。 人體腸道的菌相受飲食習慣、年齡、環境等因素影響而會 有所改變’目前研究歸納出益生菌具有增強免疫力、減少過敏、 改善東方人乳糖不耐症、減低女性陰填及泌尿道感染、降低膽 固醇堆積、幫助消化、改善幽門螺旋桿菌感染及預防骨質疏鬆 症等作用’因此各種含益生菌的相關製品不斷推陳出新，最普 ©遍常見的便是添加有益生菌之飲料製品或食品組合物。 然該等腸道保健性的食品，大多僅著重在腸胃道菌相維護 或是減低過敏症狀的功能上，但是有許多人在出國旅遊時常 會面臨因衛生條件不佳而引發細菌感染、食物中毒等現象若 能從平日飲食在腸胃道保健之餘同時強化身體對於沙門氏菌等 食物中毒病原菌之抵抗能力，將可以降低外出時因為水土不 服、腸胃不適而降低旅遊興致的可能性。 201029657 【發明内容】 本發明的主要目的在於裎 生 工 有 仕於k供一種抑制沙門氏菌活性的益 菌撥酵組合物，該益生菌齡故4入& β 王圉醱酵組合物係利用寄存於新竹食品 業研究所寄存的複合益生菌雜ρ^βΓ>〇〇ΛΛΐΛ 现土囷砰BCRC980010以蛋白質、糖類、 機酸、膠質為醱酵基質進行醱酵而得。 該複合益生菌群係至少包括有嘻酸乳酸桿菌植物乳酸桿 菌、鼠李糖乳酸桿菌、長形雙又桿菌和蜜蜂糞腸球菌之益生菌201029657 VI. Description of the Invention: [Technical Field] The present invention relates to a probiotic fermenting composition for inhibiting the activity of Salmonella, in particular to a compound probiotic having the ability to resist gastric acid, bile salts and good intestinal absorption. The food composition obtained by fermenting the flora. [Prior Art] Due to changes in social structure, people's eating habits have begun to change without knowing it. The fast, unbalanced and over-cooked diet has led to many civilized diseases, such as decreased immune regulation. , gastrointestinal dysfunction, etc., these abnormalities in bodily functions are likely to cause the aging phenomenon of the human body to occur sharply. The bacterial phase of the human intestines will be affected by dietary habits, age, environment and other factors. The current study concludes that probiotics have enhanced immunity, reduced allergies, improved lactose intolerance in the East, and reduced female vaginal filling and urinary tract. Path infections, lowering cholesterol accumulation, helping digestion, improving Helicobacter pylori infection and preventing osteoporosis. Therefore, various related products containing probiotics are constantly being updated. The most common ones are the addition of beneficial beverage products or Food composition. However, most of these enteral health foods only focus on the maintenance of gastrointestinal bacteria or the function of reducing allergic symptoms, but many people often face bacterial infections and food poisoning due to poor sanitary conditions when traveling abroad. If the phenomenon can strengthen the body's resistance to food poisoning pathogens such as Salmonella from the daily diet in the gastrointestinal health care, it will reduce the possibility of tourism loss due to soil and water discomfort and gastrointestinal discomfort. 201029657 SUMMARY OF THE INVENTION The main object of the present invention is to provide a beneficial bacteria-producing composition for inhibiting the activity of Salmonella, which is used by the probiotics 4 & The compound probiotic ρ^βΓ deposited in the Hsinchu Food Industry Research Institute is now fermented with protein, sugar, organic acid and colloid as a fermentation substrate. The composite probiotic group includes at least probiotic bacteria such as Lactobacillus acidophilus plant Lactobacillus, Lactobacillus rhamnosus, Bifidobacterium longum and Enterococcus faecalis
群該益生菌群白具有耐酸性、耐膽鹽及可吸附於腸胃道之特 色0 進行撥酵前，將該酸酵基質進行充分混合後除去雜菌，將The probiotic group white has acid resistance, bile salt resistance and special characteristics that can be adsorbed to the gastrointestinal tract. 0 Before the fermentation, the acid fermentation substrate is thoroughly mixed to remove the bacteria.
該複合益生菌群BCRC 98_接種於該酸酵基質上，以WO C之咖度進仃醱酵2〇小時以上以獲得一醱酵產物，該產物經由 均勻授掉、脫水、冷;東乾燥後，研磨成粉末即為本發明之益生 菌醱酵組合物。 本發明之益生菌醱酵組合物除了可直接食用外亦可添加 ®於食〇D中’藉由日常服用或是外出攜帶使用，故在應用上十分 的方便。 本發明之益生菌醱酵組合物除了可以抑制人體被沙 的感染外，同時兼具提升人體免疫力、改善腸胃道菌相之功能。 【實施方式】 本發明抑制沙門氏菌活性的益生菌醱酵組合物係經由筛選 出具有耐胃酸、耐膽鹽及良好腸道吸附能力的複合益生菌群， 利用蛋白暂、ϋ , M糖類、有機酸、膠質為醱酵基質進行醱酵所得的 201029657 食品組合物，該益生菌醱酵組合物亦有助於改善腸内菌叢分# 及提升免疫力。 1.菌種篩選： 用於製造本發明益生菌醱酵組合物之菌株係購自國、内外 菌種中心所寄存或是由乳製品、醃潰物、醱酵製品中分離純化 之菌株，在進行產酸能力、耐酸性、耐膽鹽及腸道吸附能力特 性之分析後，選取在酸鹼值為2的條件下作用2小時仍具有70 @ %以上存活率、在膽鹽環境下作用6小時仍具有80%以上存活 率、對腸道黏膜細胞CaCO-2及胃壁黏膜細胞TSGH細胞具有 良好吸附能力的菌株進行不同的複合益生菌群進行配方試驗。 本發明所中所使用的複合益生菌群NBM-Mix包括有嗜酸 乳酸桿菌NBM-01-07-002、植物乳 酸桿菌（Zβcίc>Z^βcz·//w)s^p/α«ίβrM/w)NBM-01-07-003、鼠李糖乳酸 桿菌r/2am«〇iWi)NBM-01-07-001、長形雙叉桿菌 {Bifidobacterium longum) ΝΒΜ-01 -07-004 和糞.腸球菌 ⑩（五《ierococcM·? /aeca/b) ΝΒΜ-01-05-001，係以冷;東甘油管之形 態寄存於新竹食品工業研究所之生物資源保存中心，其寄存編 號為BCRC 980010，上述菌株的耐酸性、耐膽鹽及腸道吸附能力 特性如表1所示。· 表1、益生菌株的耐酸性、耐膽鹽及腸道吸附能力特性 細胞吸附能力測試 菌株名稱 腸道黏膜 胃壁黏膜 耐酸性 耐膽鹽性The composite probiotic group BCRC 98_ is inoculated on the acid fermentation substrate, and is fermented by WO C for more than 2 hours to obtain a fermentation product, which is uniformly distributed, dehydrated, and cooled; Thereafter, grinding into a powder is the probiotic fermentation composition of the present invention. The probiotic fermentation broth of the present invention can be added to the sputum D in addition to being directly eaten, which is very convenient for use by taking it daily or by carrying it out. The probiotic fermented composition of the present invention not only inhibits the infection of the human body by sand, but also has the functions of improving human immunity and improving gastrointestinal bacteria. [Embodiment] The probiotic fermenting composition of the present invention for inhibiting the activity of Salmonella is selected from a group of probiotics having gastric acid tolerance, bile-tolerant salt resistance and good intestinal adsorption capacity, and utilizing protein temporary, hydrazine, M sugar, organic acid. The 201029657 food composition obtained by fermenting the fermentation substrate, the probiotic fermentation composition also helps to improve the intestinal flora and improve immunity. 1. Screening of strains: The strains used to manufacture the probiotic fermented composition of the present invention are purchased from strains of domestic or foreign strains or isolated and purified from dairy products, salted foods, and fermented products. After analyzing the characteristics of acid production, acid resistance, bile salt resistance and intestinal adsorption capacity, it is selected to have a survival rate of 70 @ % or more in the bile salt environment for 2 hours under the condition of pH 2; At the same time, the strains with more than 80% survival rate, good adhesion to intestinal mucosal cells CaCO-2 and gastric wall mucosal cells TSGH cells were tested for different probiotic groups. The composite probiotic group NBM-Mix used in the present invention includes Lactobacillus acidophilus NBM-01-07-002, Lactobacillus plantarum (Zβcίc>Z^βcz·//w)s^p/α«ίβrM/ w)NBM-01-07-003, Lactobacillus rhamnosus r/2am«〇iWi)NBM-01-07-001, Bifidobacterium longum ΝΒΜ-01 -07-004 and feces. Cocci 10 (five "ierococcM·? /aeca/b" ΝΒΜ-01-05-001, is stored in the form of cold; east glycerol tube deposited in the Biological Resources Conservation Center of Hsinchu Food Industry Research Institute, its registration number is BCRC 980010, The acid resistance, bile salt resistance and intestinal absorption capacity characteristics of the above strains are shown in Table 1. · Table 1. Acid resistance, bile salt resistance and intestinal adsorption capacity of probiotic strains Cell adsorption capacity test Name of strain Enteric mucosa Gastric mucosa Acid resistance Bile salt resistance
CaCO-2細胞 TSGH細胞 201029657 _(CFU/cell) (CFU/cell) 嗜酸乳酸桿菌 ΝΒΜ-01-07-002 198±21 214±11 81% 84% 植物乳酸桿菌 NBM-01-07-003 211±11 316±13 74% 84% 鼠李糖乳酸桿菌 NBM-01-07-001 42±23 62±16 79% 87% 長形雙叉桿菌 NBM-01-07-004 106±6 111±10 83% 87% 糞腸球菌 NBM-01-05-001 321±13 386±27 72% - 83% 2.益生菌醱酵組合物製備： 將蛋白質、糖類、乳化劑、膠質進行充分混合以形成一醱 酵基質，本發明之醱酵基質所使用之蛋白質可以選自生乳、奶 粉、乳清蛋白或前述物質的混合物之群組，糖類可選自葡萄糖、 海藻糖、菊糖、寡糖、多酷體或前述物質的混合物之群組。 〇 在此一實施例中所使用的醱酵基質之組成與比例如表2所 示，將該醱酵基質在70-80°C之中溫均勻混合後進行殺菌以去 除環境中雜菌，待該殺菌後的醱酵基質降溫至30-40°C時，將 前述之複合益生菌群BCRC 980010與該醱酵基質以重量比為 1: 19的比例接種至該殺菌後的醱酵基質，並於3 7±1°C下醱酵 20小時以產生一醱酵產物，將該醱酵產物高速攪拌並脫水、進 行冷凍乾燥、研磨以形成本發明之益生菌醱酵組合物。 201029657 表2、 醱酵基質之配方 成分 含量（份） 葡萄糖 3 酵母粉 0.2 海藻糖 3 菊糖 3 生乳 100 乳清蛋白 2.5 脫脂奶粉 4.5 乳化劑 0.15 動物膠 0.25 大豆多醣體 0.35 3.益生菌撥酵組合物對沙門氏菌活性之抑制能力： (1)試管試驗Wiro): 利用五.co/ί BCRC .10675 —、《Sa/zwowe/ZaBCRC 10747及Wap/j/ococcM^y⑽广⑽及BCRC10908為指標菌株，針對 複合益生菌群中的各株益生菌及益生菌醱酵組合物進行抑菌能 ®力的測試，其結果如表3所示，其中可以看出，由複合益生菌 群進行醱酵所得的益生菌醱酵組合物對於三株指標菌株都有最 佳的抑制能力。 201029657 表3、益生菌及益生菌醱酵組合物的抑菌能力 指標菌株 抑菌圈大小 E. coli BCRC 10675 S. typhimurium BCRC 10747 St. aureus BCRC 10908 嗜酸乳酸桿菌 16mm 1 5mm 16mm ΝΒΜ-01-07-002 植物乳酸桿菌 15mm 14mm 1 6mm NBM-01-07-003 鼠李糖乳酸桿菌 12mm 12mm 12mm NBM-01-07-001 長形雙叉桿菌 14mm 16mm 1 5mm NBM-01-07-004 蜜蜂糞腸球菌 15mm 17mm 16mm NBM-01-05-001 益生菌 18mm 17mm 1 8mm 醱酵組合物 (2)動物試驗（i«Wvo): 將本發明之益生菌醱酵組合物溶於水中餵食BALB/c公鼠 (0.03克/小鼠），連續服用7天後，以口服方式灌食2x105 株沙門氏菌進行攻毒，攻毒六天後檢測小鼠脾臟及肝臟中沙門 氏菌之總量，其結果係以總菌數的對數值表示，其中控制組係 以清水取代本發明之益生菌醱酵組合物進行餵食，由表中可看 出，本發明之益生菌醱酵組合物可以有效避免小鼠感染沙門氏 菌的機率。 201029657 控制組 (n=10) 脾臟 肝臟 • 50±0.02 5.41±0.12 實驗組 (n=10) 2.40±0.14 2.42 土 0.08 4·益生菌醱酵組合物對腸道内菌相之影響： ΟCaCO-2 cell TSGH cells 201029657 _(CFU/cell) (CFU/cell) Lactobacillus acidophilus ΝΒΜ-01-07-002 198±21 214±11 81% 84% Lactobacillus plantum NBM-01-07-003 211 ±11 316±13 74% 84% Lactobacillus rhamnosus NBM-01-07-001 42±23 62±16 79% 87% Bifidobacterium longum NBM-01-07-004 106±6 111±10 83 % 87% Enterococcus faecalis NBM-01-05-001 321±13 386±27 72% - 83% 2. Preparation of probiotic fermented composition: Mix well with protein, sugar, emulsifier and colloid to form a 酦The fermentation substrate, the protein used in the fermentation substrate of the present invention may be selected from the group consisting of raw milk, milk powder, whey protein or a mixture of the foregoing, and the sugar may be selected from the group consisting of glucose, trehalose, inulin, oligosaccharide, and multi-cool body. Or a group of mixtures of the foregoing. The composition and ratio of the fermentation substrate used in this embodiment are shown in Table 2. The fermentation substrate is uniformly mixed at a temperature of 70-80 ° C and then sterilized to remove bacteria in the environment. When the sterilized fermentation substrate is cooled to 30-40 ° C, the above-mentioned composite probiotic group BCRC 980010 and the fermentation substrate are inoculated to the sterilized fermentation substrate at a weight ratio of 1:19, and The fermentation was carried out at 37 ± 1 ° C for 20 hours to produce a fermentation product, which was stirred and dehydrated at high speed, freeze-dried, and ground to form the probiotic fermentation composition of the present invention. 201029657 Table 2. Formulation content of yeast substrate (parts) Glucose 3 Yeast powder 0.2 Trehalose 3 Inulin 3 Raw milk 100 Whey protein 2.5 Skim milk powder 4.5 Emulsifier 0.15 Animal glue 0.25 Soy polysaccharide 0.35 3. Probiotics Inhibition of Salmonella activity by the composition: (1) Test tube test (Wiro): Using five co., Ltd., "Sa/zwowe/ZaBCRC 10747 and Wap/j/ococcM^y (10) wide (10) and BCRC10908 as indicator strains For the probiotics and probiotic fermentation compositions of the probiotics in the compound probiotics, the bacteriostatic energy test was carried out. The results are shown in Table 3. It can be seen that the probiotics are fermented by the probiotics. The probiotic fermentation composition has the best inhibitory ability for the three indicator strains. 201029657 Table 3. Antibacterial ability index of probiotic and probiotic fermentation compositions Strain inhibition zone size E. coli BCRC 10675 S. typhimurium BCRC 10747 St. aureus BCRC 10908 Lactobacillus acidophilus 16mm 1 5mm 16mm ΝΒΜ-01- 07-002 Lactobacillus plantarum 15mm 14mm 1 6mm NBM-01-07-003 Lactobacillus rhamnosus 12mm 12mm 12mm NBM-01-07-001 Bifidobacterium longum 14mm 16mm 1 5mm NBM-01-07-004 Bee feces Enterococcus 15mm 17mm 16mm NBM-01-05-001 Probiotics 18mm 17mm 1 8mm Fermented composition (2) Animal test (i«Wvo): Dissolve the probiotic fermented composition of the invention in water to feed BALB/c Male rats (0.03 g/mouse), after 7 days of continuous administration, 2x105 strains of Salmonella were orally administered for challenge. After 6 days of challenge, the total amount of Salmonella in the spleen and liver of the mice was measured. The logarithmic value of the number of bacteria is indicated, wherein the control group is fed with fresh water instead of the probiotic bacterium fermentation composition of the present invention. As can be seen from the table, the probiotic bacterium fermentation composition of the present invention can effectively prevent the infection of the mouse with Salmonella. Probability. 201029657 Control group (n=10) Spleen Liver • 50±0.02 5.41±0.12 Experimental group (n=10) 2.40±0.14 2.42 Soil 0.08 4. Effect of probiotics fermentation composition on intestinal flora: Ο
將本發明之益生菌醱酵組合物溶於水中餵食小鼠(〇们克， 小鼠）丨期採集小鼠糞便，並依據衛生署公佈之「健康食品 之腸胃道功能改善評估方法」★測小鼠糞便中雙歧桿菌 (5的而6flcien’wwspp )與乳酸桿菌)之 菌數變化，其檢測結果如表5所示，其中控制組係以清水取代 本發明之益生菌酸酵組合物進行餵食’其結果係以總菌數的對 數值表示，由表中可看出，經過兩週的飯食後，小鼠體内的雙 歧桿菌與乳酸桿菌菌數即開始有增加的現象，持續的餵食亦有 助於歧桿菌與乳酸桿菌菌數·平穩的成長° 表5、小鼠體内菌相變化 控制組 實驗組 雙歧桿菌 第0週 6.50±0.00 6.65土0.15 第2週 8.51土0.04 8· 1 8±0· 15 第4週 8.53±0.15 9.25±1.03 201029657 控制組 實驗組 乳酸桿菌 第0週 6.45±0.05 6.41±0.00 第2週 8.76±0.55 8'85±0.15 第4週 8.96±1.00 9.26±0.96 5.益生菌睹酵組合物對提升免疫力之影響： 將本發明之益生菌酸酵組合物溶於水中银食小鼠（〇〇3克/ ®小鼠），定期採集小鼠血液，檢測其中IgA和的含量，其 檢測結果如表ό所示’其中控制組係以清水取代本發明之益生 菌翰酵組合物進行銀食’由表中可看出，經過4週的银食後， 小鼠體内IgA和IgG的含量分別提升了 32%與57%，即本發 明之益生菌醱酵組合物對於免疫力的提升有明顯的助益。 ___参6、小鼠血液中IgA和lgG的+量變化__ __JgA 含量（pg/ml) IgG 含量（pg/ml) •---箜制組_實驗組__控制組_實驗組 第 2 週 112.64±1_21 158.82±1.66 100.63±0.36 119.59±12.13 ——159.77±0.63 211.53±2.48 128.91±1.56 201.76±5,41 本發明之益生菌醱酵組合物可與維生素、香精、有機酸、 礦物質、糖類或益生寡糖粉等食品添加物加以混合，做為日常 保健用途進行服用，表7係為利用本發明之益生菌醱酵組合物 製備食品時之一種實施態樣，其係為粉末狀態，可直接呑食或 疋搭配飲品服用，此外本發明之益生菌醱酵組合物亦可直接做 10 201029657 為食品添加物’添加於飲料、食品中，以藉由日常的食物攝取 達到強化消化道及提升抵抗力之效果。 表7、利用本發明之益生菌醱酵組合物製成^^紙成 成分 ， 含量（份） 益生菌群BCRC980010 10 乾燥菌粉 發酵優酷粉 15 葡萄糖粉 29.25 麥芽糊精 25 乳糖 11 朝鮮菊募糖粉 15 (Inulin99%) 蘋果酸 1.5 酵母萃取粉 1.0 維生素B群 0.25 填酸三約 0.5 天然優酪乳香精 1.5 ❹ 综上所述’本發明之益生菌醱酵板合物確實具有抑制沙門 氏菌活性之功能’且同時具備現有產品之整腸健胃及提升免疫 力之功效’因此，不論是添加於食品或是單獨服用，藉由平曰 常態性服用或是外出旅遊攜帶使用’都有助於提升腸胃道功 能，維持身體健康之機能。 201029657 【圖式簡單說明】 無。 【主要元件符號說明】 無0 參 12The probiotics fermented composition of the present invention is dissolved in water to feed mice (〇克, mice), and the feces of the mice are collected in the flood period, and according to the "evaluation method for gastrointestinal function improvement of healthy foods" published by the Department of Health The number of bacteria in Bifidobacteria (5 and 6 flcien'wwspp) and Lactobacillus in mouse feces was changed as shown in Table 5, wherein the control group was replaced with fresh water to replace the probiotic saccharide composition of the present invention. Feeding' results are expressed as the logarithm of the total number of bacteria. It can be seen from the table that after two weeks of meals, the number of Bifidobacteria and Lactobacillus bacteria in the mice began to increase, continuing. Feeding also contributes to the number of Bacillus and Lactobacillus strains. Table 5: Changes in bacterial phase in mice Control group Bifidobacteria in the experimental group 0th week 6.50±0.00 6.65 soil 0.15 Week 2 8.51 soil 0.04 8 · 1 8±0· 15 Week 4 8.53±0.15 9.25±1.03 201029657 Control group experimental group Lactobacillus week 0 6.45±0.05 6.41±0.00 Week 2 8.76±0.55 8'85±0.15 Week 4 8.96±1.00 9.26 ±0.96 5. Probiotics fermentation composition for lifting The effect of the epidemic power: The probiotic lactic acid composition of the present invention is dissolved in silver-fed mice (〇〇3 g/® mice), and the blood of the mice is collected regularly to detect the content of IgA and the detection results thereof. As shown in the table, 'the control group replaces the probiotic yeast composition of the present invention with clear water for silver food'. It can be seen from the table that after 4 weeks of silver food, the contents of IgA and IgG in the mice are increased, respectively. 32% and 57%, that is, the probiotic fermented composition of the present invention has a significant benefit for the improvement of immunity. ___参6, the amount of IgA and lgG in the blood of mice __ __JgA content (pg/ml) IgG content (pg/ml) •---Tanning group _ experimental group __ control group _ experimental group 2 weeks 112.64±1_21 158.82±1.66 100.63±0.36 119.59±12.13 ——159.77±0.63 211.53±2.48 128.91±1.56 201.76±5,41 The probiotic fermented composition of the present invention can be combined with vitamins, flavors, organic acids and minerals. A food additive such as a saccharide or a probiotic oligosaccharide powder is mixed and used for daily health care, and Table 7 is an embodiment of the preparation of the food using the probiotic bacterium fermentation composition of the present invention, which is in a powder state. It can be directly foraged or taken with a drink, and the probiotics fermentation composition of the present invention can also be directly used as a food additive to be added to beverages and foods to enhance the digestive tract by daily food intake. Improve the effect of resistance. Table 7. Preparation of the composition of the probiotic bacteria fermentation composition of the present invention, content (parts) Probiotics group BCRC980010 10 Dry powder powder fermentation Youku powder 15 Glucose powder 29.25 Maltodextrin 25 Lactose 11 Korean chrysanthemum Sugar powder 15 (Inulin 99%) Malic acid 1.5 Yeast extract powder 1.0 Vitamin B group 0.25 Acid-filled tri-0.5 Natural yogurt-flavored flavor 1.5 综 In summary, the probiotic gluten extract of the present invention does inhibit Salmonella activity. The function 'has the efficacy of the whole product in the stomach and boosting immunity of the existing products'. Therefore, whether it is added to food or taken alone, it can be used by taking normal or taking a trip. Improve the function of the gastrointestinal tract and maintain the function of good health. 201029657 [Simple description of the map] None. [Main component symbol description] No 0 Reference 12