Purification and properties of an acid phosphatase from Lactobacillus curvatus DPC2024

An acid phosphatase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-Sephacel, Phenyl Sepharose, chelating Sepharose Fast Flow and MonoQ. The purified enzyme was a tetramer with a subunit molecular mass of 26 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. Its optimum activity was at pH 4.5 and 70°C, with more than 65% of its activity retained after pre-heating for 30 min at 70°C. The enzyme was strongly inhibited by NaF (0.1 m ) and ZnCl₂ (1.0 m ); slightly inhibited by hexametaphosphate, tripolyphosphate or pyrophosphate at 1.0 m concentrations; but unaffected by 10 m ascorbic acid. The acid phosphatase hydrolysed a number of phosphate esters but not bis(p-nitrophenyl)phosphate nor uridine-5′-monophosphate. The N-terminal amino acid sequence of the first 20 residues of this enzyme showed 65% homology with an acid phosphatase from Lactobacillus plantarum DPC2739 and some homology with other phosphatases from mammals, yeasts and Escherichia coli.     

Incorporation of Lactobacillus acidophilus in Minas fresh cheese and its implications for textural and sensorial properties during storage

The effect of Lactobacillus acidophilus on instrumental texture profile and related properties of Minas fresh cheese during storage at 5 °C and on sensory performance was investigated. Four cheese-making trials were prepared, two supplemented with a mesophilic type O culture (T1, T2) and two with lactic acid (T3, T4). L. acidophilus was added in T2 and T3. The viability of L. acidophilus, instrumental texture profile analysis and related properties were monitored during storage for up to 21 days. Probiotic cheeses T3 were firmer by the end of storage, due to higher values of pH and hardness. Differences detected were attributed to the starter, rather than to L. acidophilus. Viability of L. acidophilus during storage ranged from 6.04 to 6.93 for T2 and from 5.46 to 6.53 log cfu g⁻¹ for T3, which performed better in sensory evaluation. Minas fresh cheese is a suitable food system for the delivery of L. acidophilus.     

副干酪乳杆菌胞外多糖抗氧化活性分析

为评价干酪乳杆菌生物合成胞外多糖(exopolysaccharides,EPS)抗氧化活性,本文分离纯化高产胞外多糖的三株副干酪乳杆菌(3号、5号和7号)的胞外多糖,并对其DPPH·、·OH和O₂^-清除率分别进行测定,评价其体外抗氧化活性。通过分析EPS对H₂O₂诱导氧化损伤293T细胞的保护作用,评价其胞内抗氧化活性。结果表明,当EPS浓度为400 μg/mL时,其DPPH·、·OH和O₂^-的清除率最高,分别为8.87%(5号)、88.94%(7号)和34.55%(7号),其中对·OH清除能力高于抗坏血酸(65.87%),且三株菌株之间没有明显差异。3号副干酪乳杆菌所产EPS显著提高超氧化物歧化酶(SOD)活性、总抗氧化能力(T-AOC)活性并抑制了丙二醛(MDA)的生成(P<0.05),且与多糖浓度呈正相关。因此,3株副干酪乳杆菌胞外多糖具有良好的抗氧化活性,作为天然抗氧化剂具有良好的应用潜力。     

副干酪乳杆菌FMLP4菌株在泡菜和酸奶发酵中的应用初探

目的:本文研究副干酪乳杆菌FM-LP-4对发酵泡菜和酸奶品质及体外抗氧化能力的影响。方法:利用副干酪乳杆菌FM-LP-4作为发酵菌株发酵泡菜,保加利亚乳杆菌、嗜热链球菌和干酪乳杆菌FM-LP-4混合发酵酸奶,检测发酵泡菜和酸奶的发酵时间、抗氧化能力等指标的变化,同时对发酵产品进行感官评定。结果:副干酪乳杆菌FM-LP-4能显著缩短泡菜发酵时间(p<0.05),降低硝酸盐含量(p<0.05),并能显著提高发酵泡菜的硬度和体外抗氧化能力(p<0.05),感官评分高于自然发酵泡菜。综合分析,接种量3.0%~5.0%为蔬菜发酵的合适接种量。副干酪乳杆菌FM-LP-4能显著提高发酵酸奶的粘度(p<0.05)、保水率和体外抗氧化能力(p<0.05),同时提高酸奶的感官评分。综合分析,保加利亚乳杆菌、嗜热链球菌和副干酪乳杆菌FM-LP-4的接种比例为1:1:1时为酸奶发酵的合适接种比例。结论:副干酪乳杆菌FM-LP-4在抗氧化功能产品开发中具有较好的应用前景。     

副干酪乳杆菌K56调节肠道菌群及润肠通便功能研究

目的:参考保健食品检验与技术评价规范(2003版)中关于调节肠道菌群及通便功能动物实验评价方法评价副干酪乳杆菌调节肠道菌群及润肠通便功能。方法:分别以0.000867、0.00867、0.0867、0.867、8.67 mg/kg灌胃给予副干酪乳杆菌K56菌株(实验室检测菌株规格为1.5×10¹¹ CFU/g),检测小鼠干预14 d前后双歧杆菌、乳杆菌、肠杆菌、肠球菌、产气荚膜梭菌含量,并测定干预30 d后小肠墨汁推进率、首粒黑便时间、排黑便粒数及重量,评价副干酪乳杆菌对小鼠肠道菌群及排便的影响。结果:干预14 d前后比较,各组小鼠粪便双歧杆菌、乳杆菌、产气荚膜梭菌均显著增加(P<0.05),而肠杆菌和肠球菌无显著变化(P>0.05);与对照组比较,以0.00867、0.0867、0.867 mg/kg剂量的K56干预后,小鼠肠道内双歧杆菌及乳杆菌数量显著增加(P<0.05),0.000867、0.00867、0.867 mg/kg剂量的K56干预后产气荚膜梭菌含量显著降低(P<0.05);与模型组比较,0.00867、0.867、8.67 mg/kg剂量组干预小鼠墨汁推进率显著增高(P<0.05),0.0867、0.867 mg/kg剂量组小鼠排黑便粒数与重量显著增加(P<0.05),但各剂量组对小鼠首粒黑便时间与模型对照组无显著性差异(P>0.05)。结论:副干酪乳杆菌K56菌株具有调节肠道菌群和润肠通便的作用,调节肠道菌群最佳剂量为0.0867 mg/kg(对应人体剂量1.0×10⁸ CFU/d),润肠通便最低有效剂量为0.867 mg/kg(对应人体剂量为1.0×10⁹ CFU/d)。     

益生菌副干酪乳杆菌R8双层包埋工艺研究

目的:以益生菌副干酪乳杆菌R8为研究对象,进行双层包埋关键工艺参数的研究,以提高菌体的稳定性,并实现在肠道定向释放的目的。方法:筛选冻干保护剂、益生元;优化微胶囊包埋材料海藻酸钠和固化液中氯化钙浓度;检测和分析制得的微胶囊在人工胃液和人工肠液中的释放特性。结果:筛选出的最佳冻干保护剂为20%全蛋液;最佳益生元为菊粉;最佳包埋液配方为:5%益生菌、2%菊粉、20%全蛋液,1%海藻酸钠;最佳固化液配方为2.5%氯化钙。所得微囊颗粒可耐受胃液破坏,在模拟肠环境中45 min内释放完毕,人工肠液中活菌数为原微囊活菌数的65.6%。全蛋液在制备工艺过程起到保护作用,并提高在储存过程中的稳定性;将菌体/蛋白质/海藻酸钠微胶囊进一步用壳聚糖材料进行覆膜,可实现在肠道定向释放。     

Potential of anticlostridial Lactobacillus isolated from cheese to prevent blowing defects in semihard cheese

Five anticlostridial Lactobacillus strains isolated from cheese were selected for a mixed adjunct culture. Cheese with the mixed adjunct culture (experimental) and without (control) was made in triplicate and ripened as vacuum‐packed and surface‐ripened cheese. Cheese gross composition was similar. Excessive gas formation occurred only in control cheeses. In contrast to control cheeses, the experimental cheeses were dominated by the added adjunct Lactobacillus strains (repetitive‐PCR). Casein breakdown was not influenced, however, the total amount of amino acids and pH was slightly lower in the experimental cheeses. Anticlostridial nonstarter Lactobacillus strains have potential as protective adjunct cultures against blowing defects in cheese.     

Proteolytic activity of some Lactobacillus paracasei strains in a model ovine-milk curd system: Determination of free amino acids by RP-HPLC

Curd slurries were prepared from ovine milk to study the proteolytic properties of various strains of Lactobacillus paracasei. A commercial industrial starter and the strains to be tested in this study, Lb. paracasei strains Pa1, Pa2, Pa3, were inoculated in separate slurries. Free amino acids were analysed on days 0, 2, 5, 7, and 10. As ripening progressed, total free amino acids increased significantly (P < 0.01); content ranged from 150 mg/100 g dry matter (DM) on day 0 to 600 mg/100 g DM on day 10. Generally speaking, CIT, GLN, LEU, ASN, PRO, and 4-HYPRO were the main free amino acids in all four slurries tested, accounting for 60–82% of the total free amino acids. The slurries made using strains Pa1 and Pa3 were similar to the control slurry, and these three slurries exhibited the highest proteolysis levels. Differences between the strains of the species tested were observed.     

Survival of Lactobacillus rhamnosus strains inoculated in cheese matrix during simulated human digestion

Survival of probiotic bacteria during transit through the gastrointestinal (GI) tract is influenced by a number of environmental variables including stomach acidity, bile salts, digestive enzymes and food matrix. This study assessed survival of seven selected Lactobacillus rhamnosus strains delivered within a model cheese system to the human upper GI tract using a dynamic gastric model (DGM). Good survival rates for all tested strains were recorded during both simulated gastric and duodenal digestion. Strains H12, H25 and N24 demonstrated higher survival capacities during gastric digestion than L. rhamnosus GG strain used as control, with H12 and N24 continuing to grow during duodenal digestion. Strains L. rhamnosus F17, N24 and R61 showed adhesion properties to both HT-29 and Caco-2 cells. The ability to attach to the cheese matrix during digestion was confirmed by scanning electron microscopy, also indicating production of extracellular polysaccharides as a response to acid stress.     

瑞士乳杆菌对契达干酪成熟期间品质的影响

干酪的成熟是形成干酪特有的组织状态、质地和风味的关键工序。将分离自内蒙古传统乳制品中的瑞士乳杆菌SMN2-1作为非发酵性乳酸菌添加到契达干酪的生产中,通过检测其成熟过程中理化指标、气味变化和质构特性等指标,分析了瑞士乳杆菌对契达干酪成熟的影响。结果显示:在90 d的成熟期内,两组干酪的蛋白质、脂肪、水分和盐分含量之间差异不显著(p>0.05),但添加瑞士乳杆菌SMN2-1的干酪成熟第90 d的气味明显改变,内聚性、弹性和咀嚼性等物性指标均高于对照,因此添加瑞士乳杆菌SMN2-1可以有效促进蛋白质分解和干酪的成熟,同时改善干酪的风味和质地。     

Identification of micrococcaceae isolated from goat''s milk and cheese in the Poitou-Charentes region

One hundred and ninety strains of coagulase-negative staphylococci were isolated from goat's milk, whey and cheese at various stages of manufacture.

Sixteen different coagulase negative Staphylococci (CNS) species were recovered, 3 of which were predominant: Staphylococcus simulans, Staphylococcus epidermidis and Staphylococcus xylosus.

The prevalent species were recovered at least at two different stages of cheese manufacturing, suggesting a better adaptation to the environment. After 15 days of ripening, the cheeses showed lower counts of Micrococcaceae.     

副干酪乳杆菌固态发酵对红枣碎活性成分和抑菌性影响

本实验以红枣碎为主料,豆粉为辅料,用副干酪乳杆菌进行固态发酵。以探讨发酵过程对基质中部分活性成分和抑菌特性的影响为目的,分析了发酵过程中的活菌数、γ-氨基丁酸(γ-aminobutyric acid,GABA)、不同有机酸、粗多糖、游离氨基酸和多酚含量变化。结果显示:与发酵前相比,固态发酵24 h后活菌数达到最大值6.5×10⁸ CFU/g,此时基质中GABA、乳酸、粗多糖和游离氨基酸含量分别增加了109.92 μg/g、23.14 mg/g、14.29 mg/g和36.70 μg/g,沙门氏菌、大肠杆菌和金黄色葡萄球菌的抑菌圈直径分别为3.12、2.79、1.24 cm,抑菌能力明显增强。因此固态发酵有利于开发富含活菌和多种活性物质的红枣肠道益生产品,为红枣的开发利用提供新途径。

     

Antimicrobial activity of enterococci strains isolated from white cheese

Twelve strains of enterococci isolated from white cheese samples obtained from different regions of Turkey were screened for their antimicrobial activity against some Gram-positive and Gram-negative food-borne pathogens and contaminating bacteria, also against themselves and some other lactic acid bacteria (LAB), including some species of lactococci, lactobacilli and Leuconostoc . Antimicrobial activity was confirmed for all 12 strains, including six Enterococcus faecium , two Enterococcus faecalis and four Enterococcus durans . Antimicrobial agents produced by these enterococci exhibited a spectrum of activity, which is mainly directed against food-borne pathogens, specially Listeria monocytogenes and Bacillus cereus and some other Bacillus spp. and also some enterococci. Tested LAB was insensitive to inhibitory agents produced by them.     

Survival of Lactobacillus acidophilus, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Bifidobacterium animalis and Propionibacterium in cheese-based dips and the suitability of dips as effective carriers of probiotic bacteria

The suitability of cheese-based dips as a delivery vehicle for probiotic bacteria including Lactobacillus acidophilus, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Bifidobacterium animalis, and Propionibacterium freudenreichii subsp. shermanii was studied by evaluating the survival of these organisms in dips. Effects of organic acids, oils and gums, -cysteine and NaHCO₃ on the survival of probiotics in cheese-based dips were also studied. Eight different combinations and five individual bacteria as controls of these probiotic bacteria were added to 21 batches of French onion dip and selective enumeration of these probiotic bacteria was carried out over a period of 10 weeks of storage. The population of L. acidophilus and B. animalis reduced by 1 log and 2 log per g, respectively. However, when the inoculation level of these bacteria were increased to 8 log per g, they maintained a population of more than 6 log over the shelf life. L. paracasei subsp. paracasei and L. rhamnosus remained at the inoculated level or increased slightly during the storage. A rapid increase in the population of P. freudenreichii subsp. shermanii occurred to attain more than the inoculation level following reduction in their number by 3 log. Except bacterial interaction, no other factors showed significant effect on the survival of individual probiotic bacteria. Each of L. acidophilus, B. animalis, L. paracasei subsp. paracasei, and L. rhamnosus showed varied levels of antogonism, while P. freudenreichii subsp. shermanii showed no effect. Any combination of these bacteria can be used as probiotics in cheese-based French onion dip. However, the inoculation level should be 8 log per g for L. acidophilus and B. animalis and 7 log per g for L. paracasei subsp. paracasei, and/or L. rhamnosus to obtain greater than 6 log of individual bacterial population at the end of shelf life.     

副干酪乳杆菌X12肽聚糖的理化性质及其对HT-29细胞增殖的抑制

本文对副干酪乳杆菌X12肽聚糖的理化性质进行分析,并研究其对结肠癌HT-29细胞的抑制作用。采用SDS-PAGE分析其蛋白分子量,HPLC法检测其氨基酸组成,电镜观察其形态,最后检测其对HT-29细胞增殖的抑制作用。结果显示,副干酪乳杆菌X12肽聚糖蛋白分子量低于14.4 kDa,蛋白氨基酸共有15种,其中丙氨酸、谷氨酸、甘氨酸和赖氨酸含量较高,而电镜结果显示肽聚糖保留了原菌体细胞的骨架结构,并具有抑制HT-29细胞增殖的能力,肽聚糖高剂量组(160 μg/mL)抑制HT-29细胞增殖率可达21.02%。     

Production of -aminobutyric acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods

Lactobacillus strains that accumulated γ-aminobutyric acid (GABA) in culture medium were screened to determine strains with high GABA-producing ability. One strain, NFRI 7415, which was isolated from a Japanese traditional fermented fish (funa-sushi), showed the highest GABA-producing ability among the screened strains. Identification tests (i.e., 16S rDNA sequencing and sugar assimilation ability) indicated that NFRI 7415 belongs to Lb. paracasei. The GABA production was further improved by the addition of pyridoxal phosphate to the culture medium and pH regulation of culture medium at pH 5.0. Under optimal cultivation conditions, strain NFRI 7415 produced GABA at a concentration of 302 mm when the glutamate concentration in the culture medium was 500 mm.     

传统酸面团中植物乳杆菌发酵馒头抗氧化特性及挥发性风味物质特征

为了探究具有不同菌种关系的乳酸菌和酵母菌在泡菜发酵过程中的发酵性能,本实验选取不同食品来源的8株乳酸菌和5株酵母菌,通过代谢产物交叉培养及菌株共培养方法,筛选到一对具有互促关系的乳酸菌和酵母菌,即植物乳杆菌（Lactiplantibacillus plantarum）J05和酿酒酵母（Sacchromyces cerevisiae）Y21。分别从培养方式、接种顺序、接种比例等方面分析该组菌共培养关系,并测定了泡菜发酵过程中pH、总酸、还原糖含量、亚硝酸盐的含量。结果显示,添加酿酒酵母Y21静置培养无菌代谢产物对处于生长期的植物乳杆菌J05具有明显促生长作用,先接种酿酒酵母Y21后接种植物乳杆菌J05可促进两种菌的生长,筛选到两株菌共培养的最佳比例为30:1和40:1。在30:1的接种比例下,泡菜发酵进入稳定期后,与自然发酵组、单菌组、互抑组相比较,互促组pH维持在3.4~3.5之间,显著低于其他各组（P<0.05）;总酸含量最高值为1.25 mg/kg,显著高于其他各组（P<0.01）;还原糖含量较低,维持在0.10 g/100 g左右,亚硝酸盐峰值含量为5.38 mg/kg,显著低于除酿酒酵母Y21组外的其他三组（P<0.01）。本实验筛选所得互促菌组发酵性能较强,可缩短泡菜发酵周期,降低泡菜中亚硝酸盐含量。     

Synbiotic potential of fresh cream cheese supplemented with inulin and Lactobacillus paracasei in co-culture with Streptococcus thermophilus

The influence of the addition of Lactobacillus paracasei and Streptococcus thermophilus on the fructan content at the beginning and at the end of storage at 4 ± 1 °C of a potentially synbiotic fresh cream cheese manufactured with inulin was investigated. Three cheese-making trials were prepared, all supplemented with a lactic culture of S. thermophilus (T1, T2 and T3). L. paracasei subsp. paracasei was added in T1 and T2. Inulin was added in T2 and the fructan content was measured after 1 and 21 days of storage. Samples of T2 possessed similar mean concentrations of fructans after 1 and 21 days of storage, 7.32% and 7.27%, respectively, and no significant difference was observed. These results indicated that the metabolism of starter and probiotic bacteria did not degrade the fructans present in those cheeses. Additionally, synbiotic cheeses possessed a fructan content higher than 7 g per 100 g, sufficient to confer prebiotic potential during the entire storage period of these products.     

Characterization of Listeria monocytogenes strains isolated from Gorgonzola cheese rinds

Listeria monocytogenes is a foodborne pathogen frequently present in ripened soft cheeses. Forty-three strains of L. monocytogenes isolated from the rind of ripened Gorgonzola cheeses produced in 24 different dairy plants were characterized by biotyping, serotyping, and molecular typing. Biotyping was performed by studying two phenotypes closely associated with virulence, such as hemolytic and phospholipase C activities. Traditional typing techniques did not allow a discrimination among the 43 strains studied. All strains showed a good hemolytic activity on blood agar, and only slight differences were observed when titration of hemolytic activity of culture supernatants was performed. Also phospholipase activities were quite similar for all the strains. Concerning serotyping, all strains belonged to serotype 1/2a. The molecular characterization was performed by RAPD-PCR. Combined cluster analysis following PCR amplification experiments allowed to group L. monocytogenes strains into few distinguishable profiles. At a level of similarity of 80%, the 43 strains were grouped into only 5 composite profile groups. Although isolated in 24 different plants, the presence of a few closely related strains demonstrated a possible relationship between these cheese isolates; a special ability of these strains to adapt to Gorgonzola cheese processing environment could be suggested.     

Characterization and selection of Lactobacillus sakei strains isolated from traditional dry sausage for their potential use as starter cultures

A total of 36 Lactobacillus sakei strains isolated from traditional dry sausage, produced without the use of starter cultures, were characterized in respect to their technological properties, i.e. growth and acidification kinetics, ability to grow at different temperatures, pH values or salt concentrations, gas production from glucose, heme-dependent catalase activity, inhibitory activity against pathogens and proteolytic and lipolytic activities.Growth rates were comprised between 0.12 and 0.55 gen/h; while acidification rates varied from 0.01 and 0.07 pH unit/h. All isolates were able to grow at pH 4.2–9.6 and at 15 °C on acetate agar supplemented with 4% NaCl, but were not able to grow at 0 and 45 °C and with 10% NaCl in the same pH conditions. However, 97% of the isolates were able to grow at 4 °C, but only 11%, and 55% were able to grow at pH 3.9 and with 6.5% NaCl, respectively. All isolates were homofermentative from glucose. Heme-dependant catalase activity was found for 97% of the isolates and 3% displayed antibacterial activity against Listeria innocua. Most isolates showed leucine and valine arylamidase and only one exhibited a lipase (C14) activity.Two strains were selected using a multi-variate analysis. Both of them showed a panel of properties highly desirable in sausage fermentation. We propose the use of these strains, in combination with coagulase-negative staphylococci as starter cultures in the studied small-scale facility in order to improve and standardize sausage safety and quality while preserving their typicality.