The Use of a Novel Ferulic Acid Esterase from Lactobacillus acidophilus K1 for the Release of Phenolic Acids from Brewer's Spent Grain

In this work, an extracellular ferulic acid esterase was produced in bioreactor cultivations of Lactobacillus acidophilus K1 strain. The enzyme was partially purified using ultrafiltration (10 kDa), dialysis (4–6 kDa) and Fast Protein Liquid Chromatography (Sepharose CM, Sephacryl S‐300). A considerable increment of enzyme activity (31‐fold) in the final preparation was achieved. Two distinct bands (approx. 21.5 kDa and 39 kDa) were obtained after SDS‐PAGE. A high similarity of the purified enzyme (LC‐MS/MS analysis) to tannase and ferulic acid esterase from Burkholderia ambifaria MEX‐5 was obtained. The optimal pH and temperature for the enzyme activity were 6.3 and 37°C, respectively. The enzyme preparation effectively released phenolic acids (mainly ferulic and p‐coumaric acid) from brewer's spent grain. This novel enzyme preparation can be used for the utilisation of a valuable and inexpensive by‐product of the brewing industry.     

糖化过程中应用嗜酸乳杆菌K1胞外阿魏酸酯酶释放酚酸

将来自嗜酸乳杆菌K1的胞外阿魏酸酯酶应用于糖化过程,以释放游离酚酸进入到麦汁中.该酶在生物反应器中制成,并部分纯化从而获得单酶.在52℃时,游离阿魏酸和香草酸释放到麦汁中（糖化醪中酶的用量为4.09-14.60 U/L）,在62℃能检测到阿魏酸（酶的添加量为14.60个单位/L）.在26℃时,酶的任一浓度都能使游离P-羟基安息香酸和丁香酸得到有效释放;在52-74℃,游离的P-羟基安息香酸也能释放（酶使用量为14.60U/L）;在26-52℃时,游离儿茶酸也能被酶制剂（酶用量为8.75 U/L和14.60U/L）有效水解;起源于绿原酸的游离咖啡酸在26-62℃也能有效释放.在糖化过程中,虽有细菌酯酶的活性,但没有P-香豆酸释放出来.而由于其较低的热稳定性,在62℃或74℃时,嗜酸乳杆菌K1的阿魏酸酯酶不能释放酚酸.综上所述,嗜酸乳杆菌K1是一个很有前景的产生阿魏酸酯酶的来源物质,在糖化初期可用于抗氧化酚酸的释放.     

Lactobacillus acidophilus Group Lactic Acid Bacteria Applied to Meat Fermentation

Lactobacillus acidophilus group bacteria (L. acidophilus, L crispatus, L. amylovorus, L. gallinarum, L. gasseri, L. johnsonii) , probiotic lactic acid bacteria, were applied to meat fermentation. Of six strains, L. gasseri (predominant lacto-bacilli in human intestinal tracts) JCM1131^T exhibited greatest fermentation performance in meats. This strain resisted gastric acid and bile, and would thus have no detrimental effects in the intestinal tract. Inoculation of the strain depressed the propagation of S. aureus cells and their enterotoxin production during meat fermentation. Results suggest probiotic lactic acid bacteria could be effectively utilized for meat fermentation to produce healthy meat products.     

两种丝状真菌发酵小麦麸皮中酚酸的释放及与阿魏酸酯酶和木聚糖酶的关系

为了评价两种丝状真菌对小麦麸皮降解能力,以及对酚酸释放能力的差异,本实验在不同时间进行总酚和9种酚酸组分的变化监测并且结合阿魏酸酯酶和木聚糖酶活性,电镜观察小麦麸皮表面结构对黑曲霉和棒曲霉进行综合评估。黑曲霉发酵7 d阿魏酸含量达到416.56μg/g麦麸。次之是没食子酸,从7.71μg/g麦麸增加到105.77μg/g麦麸。棒曲霉阿魏酸含量达到200.81μg/g麦麸。次之是丁香酸,没食子酸,对羟基苯甲酸和香豆酸,从分别从原麦麸中含量14.7μg/g麦麸、7.71μg/g麦麸、8.57μg/g麦麸和5.62μg/g麦麸增长至88.4μg/g麦麸,60.38μg/g麦麸,55.56μg/g麦麸,52.93μg/g麦麸。黑曲霉对麦麸的降解能力及对阿魏酸和没食子酸的释放能力都显著优于棒曲霉,而对丁香酸的释放能力略差于棒曲霉。电镜结果显示,黑曲霉降解能力优于棒曲霉。     

The Release of Ferulic Acid and Feruloylated Oligosaccharides During Wort and Beer Production

Ferulic acid, a very attractive natural antioxidant is present in beer in free form, but the main form is the bound form as feruloylated oligosaccharides. Previous research showed that feruloylated oligosaccharides more effectively inhibited lipid and Low Density Lipoprotein oxidation than free ferulic acid. The aim of the present study was to evaluate free and bound ferulic acid concentrations throughout the brewing process in experimental mashes (worts, beers during fermentation, maturation and storage), and to conduct a comparison in commercial beers. Another aim of the study was to investigate methods to increase levels of bound ferulic acid in beer due to the potential health benefits. Specifically, the influence of commercial enzyme preparations on both forms of ferulic acid contents was studied. Five commercial enzyme preparations during mashing were examined: Celluclast, Shearzyme, Viscozyme, Cereflo and Ultraflo. In all experimental beers, the concentrations of esterified ferulic acid were 4–6 fold higher than the corresponding free ferulic acid contents, depending on the enzyme preparation used. Ferulic acid contents in the ester form in experimental beers were in the range of 748.4 mg/hL to 1244.3 mg/hL, whereas the contents of free ferulic acid were in the range of 134.6 mg/hL to 275.2 mg/hL. Comparison of free and bound ferulic acid contents in experimental beers, produced using enzyme preparations and commercial beers found in a local market, showed that concentrations of bound ferulic acid in experimental beers were significantly higher than in commercial beers, whereas concentrations of free ferulic acid in experimental and commercial beers were comparable.     

Partial Purification of Ferulic Acid Esterase from Malted Barley

A partial purification of ferulic acid esterase, which degrades feruloyl glycerol, has been achieved from barley malt in small yields. Coloured and viscous materials were removed from the malt extract using batch‐elution anion exchange chromatography. Further steps included gradient elution anion exchange chromatography and gel filtration chromatography. Estimations of the molecular weight varied greatly from 22KDa to 158 KDa, possibly because the protein interacted with the matrix of the gel exclusion chromatography column and because multiple forms of the enzyme were present. The partially purified feruloyl esterase had an apparent Km of 0.46% feruloyl glycerol. However more than one enzyme may be present and the substrate contains two isomers and so Michelis‐Menten kinetics may not be appropriate.     

微生物发酵产阿魏酸酯酶及释放阿魏酸研究概述

对微生物发酵法产阿魏酸酯酶以及释放阿魏酸的国内外研究进展进行综述,重点介绍阿魏酸酯酶的微生物来源、理化性质及阿魏酸酯酶分子克隆专利,同时对释放阿魏酸的生产工艺及影响因素进行简单总结,为探讨新产品的生产工艺提供参考信息。     

阿魏酸酯酶与纤维素酶协同水解麦麸释放阿魏酸的研究

研究了重组阿魏酸酯酶和纤维素酶协同作用水解去淀粉麸皮对阿魏酸释放量的影响。以高效液相色谱法检测阿魏酸的提取量,结果显示,底物的超声预处理最佳条件为350 W,20 min,是未经超声处理的底物阿魏酸释放率的1.45倍,单独使用阿魏酸酯酶(re Ao Fae A)30 U时阿魏酸的释放率仅为4.1%,单独使用纤维素酶(r Au Cel12A)并不释放阿魏酸,当两种酶协同作用时,阿魏酸的释放率明显提高,经单因素试验确定双酶协同作用的最佳条件为:re Ao Fae A的最适添加量为30 U,r Au Cel12A的最适添加量为70 U,水解时间为10 h,水解温度为40℃,水解p H为5.0,料液质量体积比为1 g:30 m L,此时阿魏酸的释放率为23.6%。该结果表明,去淀粉麸皮中纤维素的降解,对提高阿魏酸酯酶水解释放阿魏酸效率具有重要作用。     

Antibacterial activity of Lactobacillus acidophilus strains isolated from honey marketed in Malaysia against selected multiple antibiotic resistant (MAR) Gram-positive bacteria

A total of 32 lactic acid bacteria (LAB) were isolated from 13 honey samples commercially marketed in Malaysia, 6 strains identified as Lactobacillus acidophilus by API CHL50. The isolates had antibacterial activities against multiple antibiotic resistant's Staphylococcus aureus (25 to 32 mm), Staphylococcus epidermis (14 to 22 mm) and Bacillus subtilis (12 to 19 mm) in the agar overlay method after 24 h incubation at 30 °C. The crude supernatant was heat stable at 90 °C and 121 °C for 1 h. Treatment with proteinase K and RNase II maintained the antimicrobial activity of all the supernatants except sample H006-A and H010-G. All the supernatants showed antimicrobial activities against target bacteria at pH 3 and pH 5 but not at pH 6 within 72 h incubation at 30 °C. S. aureus was not inhibited by sample H006-A isolated from Libyan honey and sample H008-D isolated from Malaysian honey at pH 5, compared to supernatants from other L. acidophilus isolates. The presence of different strains of L. acidophilus in honey obtained from different sources may contribute to the differences in the antimicrobial properties of honey.     

酶解麦麸制备低聚糖和阿魏酸的研究

本研究以黑曲霉(Aspergillus niger)作菌种,采用液体深层发酵法制备出含有阿魏酸酯酶(FAE)和阿拉伯木聚糖酶(AX)的混合酶制剂,探讨了它们协同作用麦麸制备阿魏酸和低聚糖的工艺条件.结果表明,FAE和AX协同作用的最适反应pH为4.4,最适反应温度为50℃.采用混合酶制剂作用于去淀粉麦麸(DSWB)发现,通过3次降解后,麦麸降解率达55.46%,大大高于文献报道的、采用单一酶作用的结果.扫描电子显微镜(SEM)观察发现,DSWB在酶处理前后的微观结构发生了显著变化.     

嗜酸乳杆菌亚油酸异构酶的酶学性质研究

本实验对嗜酸乳杆菌亚油酸异构酶的酶学性质进行了研究.结果表明:以亚油酸为底物,亚油酸异构酶的最适反应温度为40℃;最适反应pH值为4.0,pH值在3.0～6.0范围内有较高的稳定性;不同的金属离子和有机试剂对亚油酸异构酶的影响程度不同,同一种金属离子和有机试剂在不同浓度下也不相同;以亚油酸为底物的酶促反应米氏常数Km=33.33mol/L,Vmax=15.6mol/L·h.     

双酶法降解玉米麸皮制备低聚糖和阿魏酸的研究

利用阿魏酸酯酶和阿拉伯木聚糖酶的混合酶制剂可以实现高效降解玉米麸皮,制备功能性食品原料阿魏酸和低聚木糖。探讨了利用双酶法降解去淀粉玉米麸皮的工艺条件。确定了酶的最适反应条件:温度40℃、pH值4.4,料液比8%。在最适条件下酶解24 h,可以释放出去淀粉玉米麸皮中23.5%的阿魏酸和18.4%的还原糖。探讨了高温高压预处理底物对酶解的影响,结果表明在160℃(0.52 MPa)的高温下预处理去淀粉玉米麸皮30 m in,酶解24 h后释放阿魏酸的量比未经高温高压预处理时提高1倍,还原糖量提高3倍。     

嗜酸乳杆菌对杂色曲霉素吸附作用的研究

嗜酸乳杆菌是肠道内的一种益生菌,也是奶制品中含有的重要的活性微生物。本文探讨了嗜酸乳杆菌及其菌体总蛋白对杂色曲霉素的去除能力,实验结果显示:嗜酸乳杆菌菌体总蛋白对杂色曲霉素的相对吸附率达75.8%,而面包酵母菌体总蛋白为48.9%、小牛血清白蛋白只有32.5%, 嗜酸乳杆菌菌体总蛋白的吸附作用呈一定的量效关系;胰蛋白酶或胃蛋白酶消化使嗜酸乳杆菌菌体总蛋白对杂色曲霉素的相对吸附率由52.2%下降至34.3%和36.1%,但吸附作用没有消失;菌体总蛋白与杂色曲霉素混合温育1、2、3、4、5h检测,结果嗜酸乳杆菌菌体总蛋白在整个实验的时段中的相对吸附率在57.2%～75.7%的较高水平内波动,而面包酵母菌体总蛋白由开始的70.6%相对吸附率在实验5h结束时的19.8%,另外的BSA的吸附率一直维持在14.6%～34.3%之间;酸和热处理菌体使嗜酸乳杆菌对ST的相对吸附率由60.5%分别增加至71.2%和69.5%。本研究的结果为杂色曲霉素的去毒以及食品加工提供了有用的参考数据。     

嗜酸乳杆菌转化菜籽油生成共轭亚油酸条件的优化

文中对嗜酸乳杆菌静息细胞转化菜籽油生成CLA工艺进行了优化研究,通过单因素试验,确定了最适转化液介质、介质浓度、pH、温度、转化时间和细胞浓度,同时,研究了不同金属离子对亚油酸异构酶活性的影响。在该基础上,利用Box-Benhnken中心组合设计和响应面法对转化时间、pH、温度、细胞浓度进行了工艺优化分析,确定了在菜籽油浓度为7.5 mg/mL、脂肪酶的加入量为2 mg/30 mL时,以0.1 mol/L柠檬酸-柠檬酸钠缓冲液为转化液介质条件下,最优转化工艺条件是时间25 h,pH6.5,温度36℃,细胞浓度40 mg/mL,经优化后CLA的生成量可以达到230.12±7.52μg/mL。     

贵州侗族酸肉中一株抗氧化弯曲乳杆菌LAB26的鉴定及特性

从贵州侗族酸肉中筛选出一株乳酸菌,其DPPH自由基清除率、羟自由基清除率、还原能力、抗脂质过氧化能力分别为(59.67±6.68)%、(153.17±5.50)%、(47.31±4.62)%、(55.00±5.19)%,并且菌株有良好耐受NaCl和NaNO2的性能。     

老年人肠道乳酸菌的分离与鉴定及其来源发酵乳杆菌的遗传进化分析

首先对湖北襄阳和恩施地区的健康老年人肠道乳酸菌进行分离与鉴定,然后从中选择发酵乳杆菌（Lactobacillus fermentum）进行多位点序列分型（multilocus sequence typing,MLST）分析。结果显示,基于可培养方法,湖北襄阳与恩施地区老年人肠道乳酸菌可以鉴定为以乳杆菌属（Lactobacillus）为主的5 个菌属,15 个种。总的来说,发酵乳杆菌和唾液乳杆菌（L. salivarius）占老年人肠道源乳酸菌分离株总量的55%左右,表明人体肠道可以作为发酵乳杆菌的重要分离来源。基于8 个持家基因的MLST分析表明,25 株来源于肠道的发酵乳杆菌可以划分为25 个序列型,具有极高的遗传多样性;基于选择压力分析与phi-test,选用的8 个持家基因均受到纯化选择作用,且其中仅基因rpoB显著经历了基因重组事件;另外,基于Prim’s和goeBURST的进化分析表明,一半左右的肠道源发酵乳杆菌倾向于形成单独的分支,表明它们具有一定的宿主特异性,可能的原因是为适应肠道环境,这些发酵乳杆菌经历了有异于发酵食品生境的进化历程。     

Physicochemical Properties,Fatty Acid Profiles,and Sensory Characteristics of Fermented Beef Sausage by Probiotics Lactobacillus plantarum IIA‐2C12 or Lactobacillus acidophilus IIA‐2B4

Probiotics may be used to enhance the functionality and nutritional values of fermented sausages. This study aims to evaluate the physicochemical and sensory properties of beef sausages fermented by lactic acid bacteria of Lactobacillus plantarum IIA‐2C12 and L. acidophilus IIA‐2B4. These strains were isolated from beef cattle and have shown to display probiotic features. While the nutrient contents were not affected by the probiotics, the pH, texture, and color varied among the sausages. Further analysis on fatty acids showed different profiles of saturated (C14:0, C17:0, and C20:0) and unsaturated (C14:1, C18:1n9c, C18:2n6c, and C22:6n3) fatty acids in sausages with probiotics. Gas chromatography–mass spectrometry further revealed some flavor development compounds including acid, alcohols, aldehydes, aromatic, ketones, sulfur, hydrocarbons and terpenes, varied among the sausages. Hedonic test showed no difference in the preference toward aroma, texture, and color for untrained panelists.     

Phenolic Bioactive Modulation by Lactobacillus acidophilus Mediated Fermentation of Cherry Extracts for Anti-Diabetic Functionality,Helicobacter pylori inhibition and Probiotic Bifidobacterium longum Stimulation

Cherry juice from one cultivar was fermented for 0, 24, 48, and 72 h using Lactobacillus acidophilus, and its effects related to management of hyperglycemia, hypertension, inhibition of Helicobacter pylori, and proliferation of probiotic Bifidobacterium longum were evaluated using in vitro models. Cherry extract was fermented by initially adjusting the pH to 6.0 and at the natural acidic pH. Analysis was carried out by adjusting the pH and at fermented acidic pH at each time point from each of the two samples. Total soluble phenolics decreased over a period of 72 h for initial pH adjusted samples whereas it increased or remained constant for natural acidic pH samples. DPPH linked antioxidant activity and α-glucosidase inhibitory activity decreased for samples where final pH was adjusted whereas it increased for natural acidic pH samples. Fermentation led to a decrease in hypertension-relevant ACE inhibitory activity for all samples. Natural acidic pH samples had Helicobacter pylori inhibitory activity at 24, 48 and 72 h. Based on the rationale that simple phenolics in cherry could behave as proline analogs, the potential recovery of the pathogen from inhibition was evaluated with of addition of 0.5 mM proline in the medium. A proline induced growth recovery was observed indicating that the mechanism of inhibition is related to proline dehydrogenase based oxidative phosphorylation. Overall, no inhibition was observed when samples that had H. pylori inhibitory activity were further evaluated for their effect on probiotic Bifidobacterium longum.     

Comparison of Enrichment Media for Routine Detection of Beer Spoiling Lactic Acid Bacteria and Development of Trouble‐shooting Medium for Lactobacillus backi

The beer spoiling lactic acid bacteria (LAB) are known to have a substantial financial impact in the brewing industry and their rapid detection is essential. Thus more effective media for the cultivation of LAB in both routine quality control and special trouble‐shooting situations are needed. In this study, different media were tested for the routine detection of LAB at a commercial brewery. The results showed that the use of an enzyme controlled glucose delivery system, in combination with beer‐MRS medium, can significantly decrease the total analysis time. For more effective trouble‐shooting in contamination incidents a trouble‐shooting media, including the reducing agents L‐cysteine‐HCl and sodium bicarbonate, was developed. The presented medium was shown to improve the growth of beer spoiling L. backi and L. brevis, and is thereby suggested for faster detection of these strains at the breweries.     

嗜酸乳杆菌胞外蛋白调控MAPK和PI3K-AKT信号途径关键蛋白活化水平

探讨并揭示嗜酸乳杆菌(Lactobacillus acidophilus)CICC6005分泌的相关蛋白质促进肠道健康及分子机制具有重要研究价值。本实验在确定了L.acidophilus CICC6005分泌的胞外蛋白抑制HT-29结肠癌细胞增殖的基础上,进一步探讨67 ku和37 ku的胞外蛋白通过何种途径发挥抑制结肠癌细胞增殖。研究以HT-29细胞作为靶细胞,用丝裂原激活的蛋白激酶(mitogen activated protein kinase,MAPK)和磷脂酰肌醇-3激酶-丝氨酸/苏氨酸蛋白激酶(phosphatidylinositol 3-kinase-protein kinase B,PI3K-AKT)信号通路为考察对象,以Western Blotting为手段,分析两个通路中关键的靶点蛋白p38、c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、磷酸化p38(phosphorylated p38,p-p38)、磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase,p-JNK)、磷酸化的细胞外信号调节蛋白激酶(phosphorylated extracellular signal-regulated kinase,p-ERK)、磷酸化蛋白激酶B(phosphorylated AKT,p-AKT)、PI3K的表达水平,以探讨并阐述源于L.acidophilus CICC6005胞外蛋白调控结肠癌细胞增殖状况的分子机制。结果表明,不同质量浓度的67 ku和37 ku胞外蛋白分别作用HT-29细胞一定时间后,两种胞外蛋白均具有下调两个信号通路途径中p-ERK1/2、p-p38、p-AKT、PI3K蛋白的表达,且存在浓度依赖关系;但对p-JNK、ERK1/2、p38、JNK蛋白的表达没有影响。因此,源于L.acidophilus CICC6005分泌的37 ku和67 ku胞外蛋白表现出显著的抑制HT-29细胞增殖的功能,其机制可能与调控MAPK和PI3K-AKT两个信号通路中几个关键的靶点蛋白的活化水平相关。该研究结果提示,食用嗜酸乳杆菌其分泌的胞外蛋白质将达到维护肠道健康的目标。