The Use of Viable and Heat-shocked Lactobacillus helveticus DPC 4571 in Enzyme-Modified Cheese Production

The use of viable or attenuated Lactobacillus helveticus DPC 4571 for use in enzyme-modified cheese production was assessed. Optimal heat shocking conditions for attenuation of DPC 4571 were found to be 69°C for 25 sec. Enzyme-modified cheese was produced from an emulsion of pre-hydrolysed rennet curd, water, and butter fat. This substrate was heat-treated and inoculated with either an equivalent level of viable or attenuated cells of DPC 4571 and further incubated under controlled conditions. The heat-treated products produced using attenuated DPC 4571 had a preferred sensory character with strong cheesy savory notes, enhanced secondary proteolysis, and more key volatile flavor compounds than those produced with viable DPC 4571. However, prolonged incubation (>16 h) resulted in growth of advantageous enterococci, which adversely influenced the sensory profile.     

Isolation and characterisation of a Lactobacillus helveticus ITG LH1 peptidase-rich sub-proteome

Lactobacillus helveticus strains, one of the most nutritionally fastidious lactic acid bacteria, have a potent proteolytic system that makes them very interesting for different uses in the dairy industry. Its applications concern from cheese ripening to the preparation of fermented milk products with biologically active peptides. The cell-free extract (CFE) of Lactobacillus helveticus strain ITG LH1 was analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), using IPG immobiline dry strips (pH 4-7). With the aim to study the proteolytic enzymes expressed by Lactobacillus helveticus ITG LH1 grown in milk medium, a two step-chromatography methodology, based on ion exchange and affinity chromatography, was developed for the preparation of a peptidase-rich sub-proteome from the CFE of stationary growing cells. Several affinity chromatography columns were tested and among them a HiTrap Chelating column was selected as it provided the best performance for the enrichment in peptidases. Peptidase activities were studied using different beta-Naphtylamide (beta-NA) derivatives and specific activities were increased 50- to 100-fold by this chromatographic procedure. Sub-proteome characterisation was performed by 2D-PAGE, pH 4-7, followed by protein digestion with trypsin, analysis by MALDI-TOF mass spectrometry and subsequent database searches using peptide mass fingerprints. Among the most abundant proteins seven peptidases were present, namely the two general aminopeptidases (PepN, PepC), three dipeptidases (PepDA, PepV, PepQ) and two endopeptidases (PepO, PepO3), all of them corresponding to the catalytic classes of metallo- or cysteine-peptidases. Several stress proteins (such as heat shock proteins DnaK and GroEL) and other enzymes implied in bacterial metabolism, namely in the carbohydrate pathways (such as LDH), were also identified in the peptidase-rich sub-proteome.     

Ribotyping and biotyping of Lactobacillus helveticus from the koumiss

The lactic acid bacteria are very important components involved in the milk products processing. Lactobacillus helveticus, a homofermentative thermophilic lactobacillus commonly occurring in cheeses, has been isolated as the prevailing species in natural koumiss in this work. The species identification of six studied L. helveticus strains was based on rep-PCR fingerprinting with (GTG)₅ primer. Biotyping (API 50CH kit, conventional tests) showed phenotypic heterogeneity among isolates and enabled the identification to the genus Lactobacillus only. Ribotyping with restriction enzyme EcoRI yielded a strain-specific restriction patterns and allowed good strain differentiation. Obtained ribotypes of the isolates originating from the koumiss gave unique band patterns with low similarity to the selected reference cultures of L. helveticus. Despite low number of strains analyzed, the ribotype data showed certain heterogeneity that seemed to be not only strain dependent, but also related to the source of isolates. We concluded that analyzed isolates from koumiss represent a new ecovar, L. helveticus ecovar Koumiss.     

瑞士乳杆菌的益生特性

研究了瑞士乳杆菌作为发酵肉制品发酵剂的发酵特性;在模拟胃肠道环境中对酸和胆盐的耐受性;另以Caco-2细胞为体外肠道上皮细胞模型,研究了瑞士乳杆菌对小肠的黏附性和影响黏附的因素。实验结果表明,瑞士乳杆菌不产粘液,不产气,不产H2S,不产氨,不产H2O2,不具有氨基酸脱羧酶活性,不具有硝酸还原酶能力,具有抑制S.aureus和E.coli能力,能耐受一定量的NaCl和NaNO2,发酵葡萄糖产酸。瑞士乳杆菌同时还具有较好的耐酸和耐胆盐性能。通过瑞士乳杆菌的小肠黏附实验发现,生长稳定期的瑞士乳杆菌与Caco-2细胞有很好的黏附效果,当瑞士乳杆菌与Caco-2细胞共培养时间为2h时,黏附趋于饱和,且黏附环境pH值为7.0时黏附性最强。结果显示瑞士乳杆菌具备益生活性,满足发酵肉制品中益生菌制剂的筛选标准,可作为发酵菌种用于发酵肉制品的生产。     

Effects of nitrates and nitrites on Lactobacillus helveticus and Lactobacillus casei dairy cultures

 The aim of this work was to observe the effects of nitrites and nitrates on the titratable acidity of milk after addition of Lactobacillus helveticus (TX 121) and L. casei (CAD 154) dairy cultures and on the levels of nitrate and nitrite. After adding L. helveticus and nitrites the increasing concentrations of the latter brought about a marked decrease in titratable acidity. In milk containing 100 mg·kg^–1 nitrite the resulting concentration was 39.33 mg·kg^–1 NaNO₂. Nitrates caused a less obvious decrease in titratable acidity, giving 13.27 g·l^–1 lactic acid. In milk containing 100 mg·kg^–1 NaNO₃ the resulting concentration was 24.99 mg·kg^–1 NaNO₃. Experiments with L. casei and a nitrite additive revealed a decrease in titratable acidity to 8.89 g·l^–1 lactic acid. After incubation, nitrite levels were reduced from 100 mg·kg^–1 NaNO₂ to 37.81 mg·kg^–1 NaNO₂. Nitrates were also stated to inhibit the titratable acidity of the sample, which decreased to 11.42 g·l^–1 lactic acid. Nitrates were reduced to 46.99 mg·kg^–1 NaNO₃. The present study shows that nitrites, more than nitrates decrease the titratable acidity of milk after addition of L. helveticus and L. casei dairy cultures. A reduction of nitrates and nitrites in milk samples by Lactobacillus was also found. The results of this study can be used in the dairy industry in the production of several types of hard cheese as well as fermented milk products that use L. helveticus and L. casei dairy cultures. Received: 31 March 1999 / Revised version: 6 September 1999     

Detection and enumeration of Lactobacillus helveticus in dairy products

Lactobacillus helveticus, a lactic acid bacterium, is an important species in food fermentation, e.g., cheesemaking, and is considered beneficial to human health. We developed a quantitative real-time polymerase chain reaction (qPCR) method for the detection and quantification of L. helveticus in dairy products. The method uses a set of target-specific PCR primers and a fluorogenic probe and amplifies a part of the pheS gene that encodes the alpha subunit of the phenyalanine-tRNA synthetase. All 24 L. helveticus strains tested were qPCR positive; no signal was observed for 23 strains belonging to closely related species. The limit of detection was ten copies per reaction and the assay covered a linear dynamic range of eight logs. The method was used to detect and enumerate L. helveticus in milk and cheese during ripening; therefore it can be used to study the temporal and spatial distribution of L. helveticus during cheese manufacturing and ripening.     

瑞士乳杆菌特性及应用研究进展

瑞士乳杆菌是乳酸菌的一种,是对人体健康有益的菌种。具有较强的蛋白水解能力,调节肠道菌群能力,增强免疫力及抗高血压的功效,还具有产生细菌素或生物活性肽的潜能,并且可与发酵乳制品中的益生元结合产生合生素,因此被广泛用于食品发酵中。该文综述了瑞士乳杆菌基因组特征,蛋白水解系统,抗高血压,产生的胞外多糖所具有的免疫调节的益生特性,以及其在食品和菌体生物表面活性剂中的应用。     

瑞士乳杆菌增菌培养基的筛选

以冻干后菌体的存活率为依据,首先筛选出存活率最高的瑞士乳杆菌基础培养基为10%的脱脂乳培养基;然后分别采用单因素实验和正交实验,优化培养基成分及用量;最后确定瑞士乳杆菌的增菌培养基配方为:10%脱脂乳+0.1%乳糖+1.0%酵母膏+10%麦芽汁+1.0g/100mL柠檬酸三钠.在此优化培养基中,瑞士乳杆菌培养至10h左右,其活菌数可达8.16×109cfu/mL.     

Immunomodulating effects of peptidic fractions issued from milk fermented with Lactobacillus helveticus

The effect of peptides released during the fermentation of milk on the humoral immune system and on fibrosarcoma growth was studied. Lactobacillus helveticus was able to release peptidic compounds during milk fermentation due to its high proteolytic activity, as was shown by the degree of proteolysis and size-exclusion HPLC elution profiles. Three fractions of these compounds were separated and fed to mice during different periods (2, 5, and 7 d). The humoral immune response was assessed by following the number of IgA-secreting cells, and the antitumor activity was monitored by studying the regression of subcutaneously implanted fibrosarcomas. Feeding during 2 and 7 d with the medium-sized fraction (Fraction II) significantly increased the IgA-producing cells in the intestines, whereas feeding with the large compound fraction (Fraction I) during 5 d and the small compound fraction (Fraction III) during all three feeding periods provided similar increases. A double dose of Fraction II showed the highest IgA-producing cell count. The increase by Fraction III was shown to be caused by the presence of L-Tryptophan. Fraction II significantly decreased the size of fibrosarcoma when previously fed during 7 d, and feeding with Fraction I during 5 d decreased significantly its size after 35 d of growth. Although the mechanisms by which lactic acid bacteria enhance the immune system are not clear, this study clearly shows that bioactive compounds released in fermented milks contribute to the immunoenhancing and antitumor properties of these products. The release of bioactive peptides by lactic acid bacteria can have important implications on the modulation of the cellular immune response.     

切达干酪促熟附属发酵剂的筛选

切达干酪成熟时间较长,成熟期间费用较高,导致生产成本增加。因此干酪的促熟受到人们的普遍关注。试验从中国传统发酵食品中分离出12株具有较高肽酶活力的菌株,经进一步筛选,得到了两株具有较好促进干酪成熟能力的菌株,有望作为附属发酵剂应用于切达干酪生产。     

Detection and identification of Lactobacillus helveticus bacteriophages by PCR

A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Phi-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages.     

瑞士乳杆菌与干酪乳杆菌在不同原料中的发酵特性研究

研究了瑞士乳杆菌和干酪乳杆菌单独与组合,以及与嗜热链球菌和保加利亚乳杆菌组合在纯牛乳、纯豆乳和混合乳中发酵12h后pH值和酸度值变化,以此来说明它们在3种原料中的生长情况.结果表明,瑞士乳杆菌在纯牛乳和混合乳中产酸能力很强,在纯豆乳中产酸能力不够强,生长不够好;干酪乳杆菌在纯豆乳中生长产酸能力比瑞士乳杆菌强;研究还得出瑞士乳杆菌和干酪乳杆菌组合,以及他们分别与嗜热链球菌和保加利亚乳杆菌组合在纯豆乳生长产酸能力相当.     

瑞士乳杆菌发酵乳中生物活性小肽的免疫功能特性研究

利用MTT、中性红吞噬、一氧化氮诱生分泌等方法从已获得的瑞士乳杆菌发酵乳来源小肽中筛选出对小鼠腹腔巨噬细胞具有免疫活性的小肽GLPN,并通过ELISA方法检测TNF-Ω,IL-1β,IL-6的细胞因子水平,以对具有的免疫促进作用效果进行评估.结果表明,GLPN在0.1 g/L质量浓度下所有指标上都能表现出显著的促进作用,而在LPS作用下,GLPN质量浓度达到0.5 g/L也未引起炎症因子的过量表达.     

瑞士乳杆菌冻干保护剂的选择

研究几种高、低分子化合物对冻干瑞士乳杆菌的保护作用.以冻干后的活菌存活率和凝乳时间为考察指标,先进行单因素试验筛选出保护效果最好的4种物质甘油、葡萄糖、组氨酸和牛血清白蛋白,用这4种物质复配,进行复合保护剂正交试验.结果表明:将增菌培养基发酵液(含10%脱脂乳)与冻干保护剂溶液混合冻干,最佳保护剂配方为0.5%甘油+15.0%葡萄糖+150 mg/L组氨酸+1.5%牛血清白蛋白.加入冻干保护剂后,瑞士乳杆菌冻干后存活率可达89%.     

宫廷奶酪中优良乳酸菌菌株的分离与鉴定

利用梯度稀释法和划线纯化法对4种宫廷奶酪中的乳酸菌进行初筛,再通过产酸实验和过氧化氢酶实验进行复筛,得到4株乳酸菌。通过形态学特征观察和16SrDNA序列分子鉴定,确定2株为屎肠球菌(MRSA1、MRSB1),2株为乳明串珠菌(MRSA2、MRSD3)。抑菌实验表明乳明串珠菌MRSA2对大肠杆菌、志贺氏菌、沙门氏菌具有良好的抑菌特性;4株菌均有抗氧化活性,菌悬液的DPPH自由基清除能力和羟自由基清除能力均高于无细胞提取物,其中菌株MRSA2菌悬液DPPH自由基和羟自由基清除能力分别高达60.67%和31.86%。研究筛选出的具有良好抑菌特性和抗氧化活性的乳明串珠菌MRSA2在功能性食品研发中具有潜在的应用价值。     

The inhibitory effects of Lactobacillus casei and Lactobacillus helveticus on Bacillus species isolated from raw milk in various salt concentrations

Nineteen strains of Bacillus were isolated and identified from 111 samples of raw milk. The inhibitory effects of Lactobacillus casei and Lactobacillus helveticus on strains of Bacillus were studied. The inhibitory effects were evaluated (using the well diffusion method) on nutrient agar, with NaCl added at concentrations of 40, 45, 50, 55, 60 and 65 g/L. Lactobacillus casei inhibited 16 strains of Bacillus on nutrient agar without salt, and 18 strains in the presence of salt. When the salt concentration was 6.5%, the inhibitory effect decreased, and L. casei inhibited only eight strains of Bacillus ( Bacillus cereus , Bacillus sphaericus , Bacillus licheniformis , Bacillus macerans , Bacillus firmus , Bacillus coagulans , Bacillus polymyxa and Bacillus stearothermophilus ). Lactobacillus helveticus formed the inhibitory zones against five strains of Bacillus on nutrient agar without salt, but with 4% NaCl concentration, it showed an inhibitory effect on 13 strains; a further two strains were inhibited in the presence of 4.5 and 5.0% salt. In 5.5% NaCl, the same 15 strains were inhibited by L. helveticus, whilst in 6 and 6.5% salt, the inhibitory effect decreased and only six and seven strains were affected, respectively. The maximum inhibitory effect with both L. casei and L. helveticus was observed in the presence of 4.5–5.5% salt.     

瑞士乳杆菌对小鼠肠道微生物区系的影响

本实验探讨瑞士乳杆菌对小鼠肠道微生物区系的影响,分析小鼠灌喂益生菌后肠道内各种微生物变化规律。研究将小鼠分成4组,日食对照组、生理盐水对照组、瑞士乳杆菌灌喂组和大肠杆菌灌喂组,连续灌喂15d,采集小鼠粪便利用选择性培养基对不同组别的小鼠肠道菌群作平板计数分析。研究结果表明,瑞士乳杆菌灌喂组与日食组和生理盐水组相比其肠道中乳酸菌类、双歧杆菌类、肠杆菌类、大肠杆菌类差异极显著（p〈O．01）,肠球菌类含量具有显著性差异（p〈0．05）;大肠杆菌灌喂组其肠道中乳酸菌类、双歧杆菌类、肠杆菌类、大肠杆菌类含量与对照组相比具有显著性差异（p〈0．05）,而肠球菌类的分析结果显示变化差异不显著。研究证实了瑞士乳杆菌能够促进小鼠肠道内有益菌的显著增殖,而对肠道内过路菌和可疑致病菌均有一定的抑制作用,表明灌喂瑞士乳杆菌对小鼠肠道微生物区系具有一定的调节作用。     

Lactobacillus helveticus 9制备酪蛋白源活性肽工艺的研究

基于酪蛋白水解度和多肽质量浓度数据,研究了接种量、培养时间、培养温度及底物浓度对瑞士乳杆茵茵株L.helveticus 9水解酪蛋白的影响.结果表明.L.helveticus 9对酪蛋白的水解作用的最佳条件为接种量为1×108 mL-1,底物质量浓度为50g/L,培养时间为48 h,培养温度为37℃;在此优化条件下,酪蛋白的水解度可迭17.3%,多肽质量浓度达到0.62 g/L.显然,具有较高蛋白水解活力的菌株L.helveticus 9可以用于从酪蛋白中来制备潜在的生物活性肽.     

Inactivation of Lactobacillus helveticus bacteriophages by thermal and chemical treatments.

The effect of several biocides and thermal treatments on the viability of four Lactobacillus helveticus phages was investigated. Times to achieve 99% inactivation of phages at 63 degrees C and 72 degrees C in three suspension media were calculated. The three suspension media were tris magnesium gelatin buffer (10 mM Tris-HCl, 10 mM MgSO4, and 0.1% wt/vol gelatin), reconstituted skim milk sterile reconstituted commercial nonfat dry skim milk, and Man Rogosa Sharpe broth. The thermal resistance depended on the phage considered, but a treatment of 5 min at 90 degrees C produced a total inactivation of high titer suspensions of all phages studied. The results obtained for the three tested media did not allow us to establish a clear difference among them, since some phages were more heat resistant in Man Rogosa Sharpe broth and others in tris magnesium gelatin buffer. From the investigation on biocides, we established that sodium hypochlorite at a concentration of 100 ppm was very effective in inactivating phages. The suitability of ethanol 75%, commonly used to disinfect utensils and laboratory equipment, was confirmed. Isopropanol turned out to be, in general, less effective than ethanol at the assayed concentrations. In contrast, peracetic acid (0.15%) was found to be an effective biocide for the complete inactivation of all phages studied after 5 min of exposure. The results allowed us to establish a basis for adopting the most effective thermal and chemical treatments for inactivating phages in dairy plant and laboratory environments.