Microbiological characteristics of kumis, a traditional fermented Colombian milk, with particular emphasis on enterococci population

Kumis is a traditional fermented cow milk produced and consumed in South West Colombia. The main objective of this research was to studied the enterococcal population, present in 13 kumis samples traditionally manufactured, for their role as beneficial organisms or opportunistic pathogens. The molecular identification of 72 isolates evidenced that Enterococcus faecalis and E. faecium were the dominant species. The genes gelE, esp, asa1, cyl and hyl, all associated with virulence factors in enterococci, were detected in 30 isolates, while 42 were free of virulence determinants. Skim milk media were fermented by all the different isolates and further tested for proteolysis (free NH₃ groups), Angiotensin-I Converting Enzyme (ACE) inhibitory activity and biogenic amines production. Nine E. faecalis and two E. faecium strains produced fermented milk with ACE-inhibitory activity values ranging from 39.7% to 84.35% .The digestion of fermented milk samples by pepsin and pancreatin evidenced an increase in ACE inhibitory activity, with E. faecalis KE09 as the best producer (IC50 = 14.25 μg ml⁻¹). Moreover, the strains showed a very low tyrosine decarboxylase activity and did not produce histamine during 48 h fermentation in milk. This study underlines the that Colombian kumis is a good source of not virulent enterococci able to produce fermented milks with ACE-inhibitory activity.     

产ACE抑制肽乳酸菌的筛选、益生特性评定及应用

目的:筛选出富产血管紧张素转化酶(Angiotensin converting enzyme,ACE)抑制肽的乳酸菌并评定其益生特性.方法:依据蛋白水解度及ACE抑制率对乳酸菌进行初筛,然后依据模拟胃肠消化后的ACE抑制率最终筛出2株乳酸菌ZJUIDS09和ZJUIDS11,进一步评价其耐酸、耐胆盐、抗生素耐药性和抑菌...     

Angiotensin I-converting enzyme inhibitory activity in milk fermented by wild-type and peptidase-deletion derivatives of Lactobacillus helveticus CNRZ32

Development of molecular methods has enabled detailed studies of the proteolytic system of Lactobacillus helveticus, which has a central role in the release of bioactive peptides during the fermentation of milk. The impact of general aminopeptidase (PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) activity of L. helveticus on the level of angiotensin I-converting enzyme (ACE) inhibitory activity in fermented milk was elucidated by taking advantage of peptidase-negative derivatives of L. helveticus CNRZ32. According to the results, increased level of ACE-inhibitory activity was attained in milk fermented by the peptidase-deficient mutants. The ACE-inhibitory activity determined with the PepN-deficient strain decreased towards the end of fermentation, suggesting that PepN gives rise to a provisional accumulation of ACE-inhibitory peptides before their hydrolysis to shorter peptides or free amino acids. The ACE-inhibitory activity determined with the PepX-deficient strain increased throughout the fermentation, indicating specific blocking of the further hydrolysis of ACE-inhibitory peptides.     

Antihypertensive activity of casein-enriched milk fermented by Lactobacillus helveticus

Milk proteins contain peptidic angiotensin I-converting enzyme (ACE) inhibitors, which can be released by proteolysis during milk fermentation by some strains of Lactobacillus helveticus. Reconstituted milk media containing skim milk powder (12%), skim milk powder (10%) with added sodium caseinate (5%) or whey protein isolate (5%) were fermented by L. helveticus strains R211 and R389, and further tested for bacterial growth, proteolysis (free NH₃ groups) and ACE-inhibitory activity. The antihypertensive activity in spontaneously hypertensive rats (SHR) was also investigated with caseinate-enriched milk unfermented (UFM) and fermented by the two strains of L. helveticus. Caseinate-enriched milk fermented by both strains showed higher proteolysis and ACE-inhibitory activity, indicating that ACE-inhibitory peptides are probably released from caseins during milk fermentation. Significant decreases in mean arterial blood pressure in SHR rats were measured following oral administration of UFM milk at doses of 1.0 and 2.5 g kg⁻¹ of body weight, and milk fermented by R211 or R389 strains at doses of 0.5, 1.0 and 2.5 g kg⁻¹ of body weight. The antihypertensive activity of UFM could be explained by the release of ACE-inhibitory peptides from caseins during the digestion process.     

不同益生菌发酵乳ACEI活性比较

测定了6株德氏乳杆菌保加利亚亚种、6株嗜热链球菌、6株鼠李糖乳杆菌、4株双歧杆菌和天然发酵剂Kefir发酵产物的ACEI活性。结果显示,发酵产物的ACEI活性呈现种属和菌株差异性,发酵和贮藏条件对ACEI活性具有显著影响,菌株发酵结束后经冷藏,ACEI活性均有不同程度的下降。德氏乳杆菌保加利亚亚种Lb9菌株在发酵6h时的ACEI活性为60%,发酵产物在冷藏24h和48h后,ACEI活性分别为55.72%和49.64%。嗜热链球菌Stnh16菌株发酵6h时的ACEI活性显著高于其他嗜热链球菌菌株,为52.73%,冷藏24h和48h后,ACEI活性分别为48.82%和42.58%。鼠李糖乳杆菌在发酵18h时的ACEI活性最高,其中F菌株为83.19%,R26为72.72%,LV108菌株为69.44%,但是冷藏24h和48h后,ACEI活性LV108菌株显著高于其他菌株,ACEI活性分别为66.19%和62.91%,显示其适宜用作生产降血压益生菌发酵乳的发酵剂。双歧杆菌不同菌株中,H4在发酵24h时ACEI活性最高,为46.55%。Kefir在发酵24h时,其ACEI活性最弱,仅为15.23%。该研究为筛选降血压益生菌发酵乳的发酵剂及发酵乳生产提供了参考。     

发酵调味品中血管紧张素转化酶抑制肽的研究进展

发酵调味品是采用微生物发酵的方法,生产具有食品调味和佐餐功能的一类食品。因微生物发酵原料和菌种的不同,所产生的调味品功能有所差异,蛋白类原料在微生物酶的作用下,能够产生相对较多的血管紧张素转化酶（ACE）抑制肽。ACE抑制肽是一类能抑制血管紧张素调节酶活性的多肽物质,可以起到预防或治疗高血压的作用。该研究通过对目前发酵调味食品ACE抑制肽的来源、制备方法、分离纯化方法等进行了介绍,并分析了发酵调味品ACE抑制肽今后的研究发展方向和产业发展趋势。     

Optimization of sour milk fermentation for the production of ACE-inhibitory peptides and purification of a novel peptide from whey protein hydrolysate

The effect of fermentation conditions on the production of angiotensin-I converting enzyme (ACE) inhibitory peptide in sour milk fermented by Lactobacillus helveticus LB10 was investigated using response-surface methodology. Optimal conditions to produce the maximum production of ACE-inhibitory peptides were found to be 4% (v/w) inoculum, 7.5 initial pH of medium and 39.0 °C. The fermented milk resulted in 75.46% inhibition in ACE activity. The cell-envelope proteinase, assisted by X-prolyldipeptidyl aminopeptidase of Lb. helveticus LB10 produced the ACE-inhibitory peptides. A novel ACE-inhibitory peptide from whey protein hydrolysate produced by crude proteinases of Lb. helveticus LB10 was purified. The separations were performed with Sephadex^® G-75 and Sephadex G-15 gel filtration chromatography and reversed-phase, high-performance liquid chromatography. The peptide with the RLSFNP sequence was isolated from β-lactoglobulin hydrolysate and its IC₅₀ while inhibiting ACE activity was 177.39 μm.     

Peptide profiles and angiotensin-I-converting enzyme inhibitory activity of fermented milk products: Effect of bacterial strain,fermentation pH,and storage time

Milk was fermented to defined pH values with 13 strains of lactic acid bacteria. The products were evaluated after 1 and 7 days of cold storage, and major peptides in selected products were identified. The Streptococcus thermophilus and Lactobacillus acidophilus strains used did not give rise to products with significant angiotensin-1-converting enzyme (ACE)-inhibition. The four Lactococcus lactis strains behaved similarly in fermentation, proteolysis and ACE-inhibition. The products made with the seven Lactobacillus helveticus strains varied. The highest ACE-inhibitory activity was obtained with two highly proteolytic strains of Lb. helveticus and with the Lactococcus strains. Fermentation from pH 4.6 to 4.3 with these strains slightly increased the ACE-inhibitory activity, whilst fermentation to pH 3.5 with Lb. helveticus reduced the ACE-inhibitory activity. Cold storage dramatically increased the ACE-inhibitory activity of some products. A non-linear correlation was found between peptide amount and ACE-inhibitory activity, and peptides contributing to the ACE-inhibitory activity were identified.     

Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension

Fermented milk is a potential source of various biologically active peptides with specific health benefits. Angiotensin converting enzyme inhibitory (ACE-I) peptides are one of the most studied bioactive peptides produced during milk fermentation. The presence of these peptides is reported in various fermented milk products such as, yoghurt, cheese, sour milk, etc., which are also available as commercial products. Many of the ACE-I peptides formed during milk fermentation are resistant to gastrointestinal digestion and inhibit angiotensin converting enzyme (ACE) in the rennin angiotension system (RAS). There are various factors, which affect the formation ACE-I peptides and their ability to reach the target tissue in active form, which includes type of starters (lactic acid bacteria (LAB), yeast, etc.), substrate composition (casein type, whey protein, etc.), composition of ACE-I peptide, pre and post-fermentation treatments, and its stability during gastrointestinal digestion. The antihypertensive effect of fermented milk products has also been proved by various in vitro and in vivo (animal and human trials) experiments. This paper reviews the literature on fermented milk products as a source of ACE-I peptides and various factors affecting the production and activity of ACE-I peptides.     

Characterization of the angiotensin-converting enzyme inhibitory activity of fermented milks produced with Lactobacillus casei

Our study assayed angiotensin-converting enzyme (ACE) inhibitory activity and fermentation characteristics of 41 food-originated Lactobacillus casei strains in fermented milk production. Twenty-two of the tested strains produced fermented milks with a high ACE inhibitory activity of over 60%. Two strains (IMAU10408 and IMAU20411) expressing the highest ACE inhibitory activity were selected for further characterization. The heat stability (pasteurization at 63°C for 30 min, 75°C for 25 s, and 85°C for 20 s) and resistance to gastrointestinal proteases (pepsin, trypsinase, and sequential pepsin/trypsinase treatments) of the ACE inhibitory activity in the fermented milks produced with IMAU10408 and IMAU20411 were determined. Interestingly, such activity increased significantly after the heat or protease treatment. Because of the shorter milk coagulation time of L. casei IMAU20411 (vs. IMAU10408), it was selected for optimization experiments for ACE inhibitory activity production. Our results show that fermentation temperature of 37°C, inoculum density of 1 × 10⁶ cfu/g, and fermentation time of 12 h were optimal for maximizing ACE inhibitory activity. Finally, the metabolite profiles of L. casei IMAU20411 after 2 and 42 h of milk fermentation were analyzed by ultra-HPLC electron spray ionization coupled with time-of-flight mass spectrometry. Nine differential abundant metabolites were identified, and 2 of them showed a strong and positive correlation with fermented milk ACE inhibitory activity. To conclude, we have identified a novel ACE inhibitory L. casei strain, which has potential for use as a probiotic in fermented milk production.     

Angiotensin-converting enzyme inhibitory activity of Lactobacillus helveticus strains from traditional fermented dairy foods and antihypertensive effect of fermented milk of strain H9

Hypertension is a major global health issue which elevates the risk of a large world population to chronic life-threatening diseases. The inhibition of angiotensin-converting enzyme (ACE) is an effective target to manage essential hypertension. In this study, the fermentation properties (titratable acidity, free amino nitrogen, and fermentation time) and ACE-inhibitory (ACEI) activity of fermented milks produced by 259 Lactobacillus helveticus strains previously isolated from traditional Chinese and Mongolian fermented foods were determined. Among them, 37 strains had an ACEI activity of over 50%. The concentrations of the antihypertensive peptides, Ile-Pro-Pro and Val-Pro-Pro, were further determined by ultra performance liquid chromatography with quadrupole-time-of-flight mass spectrometry. The change of ACEI activity of the fermented milks of 3 strains exhibiting the highest ACEI activity upon gastrointestinal protease treatment was assayed. Fermented milks produced by strain H9 (IMAU60208) had the highest in vitro ACEI activity (86.4 ± 1.5%), relatively short fermentation time (7.5 h), and detectable Val-Pro-Pro (2.409 ± 0.229 µM) and Ile-Pro-Pro (1.612 ± 0.114 µM) concentrations. Compared with the control, a single oral dose of H9-fermented milk significantly attenuated the systolic, diastolic, and mean blood pressure of spontaneously hypertensive rats (SHR) by 15 to 18 mmHg during the 6 to 12 h after treatment. The long-term daily H9-fermented milk intake over 7 wk exerted significant antihypertensive effect to SHR, but not normotensive rats, and the systolic and diastolic blood pressure were significantly lower, by 12 and 10 mmHg, respectively, compared with the control receiving saline. The feeding of H9-fermented milk to SHR resulted in a significantly higher weight gain at wk 7 compared with groups receiving saline, commercial yogurt, and captopril. Our study identified a novel probiotic L. helveticus strain originated from kurut sampled from Tibet (China), which is a valuable resource for future development of functional foods for hypertension management.     

Effects of ultra-high pressure treatment on angiotensin-converting enzyme (ACE) inhibitory activity,antioxidant activity,and physicochemical properties of milk fermented with Lactobacillus delbrueckii QS306

The present study investigated the influence of ultra-high pressure (UHP) treatment on angiotensin-converting enzyme inhibitory (ACEI) activity and quality of milk fermented with Lactobacillus delbrueckii QS306 after storage. By varying treatment pressure, duration of pressure treatment, and duration of fermentation, optimal process parameters for the UHP treatment of milk fermented with QS306 to enhance ACEI activity were found to be 400 MPa, 10 min, and 48 h, respectively. The degree of ACE inhibition of the fermented milk was 91.63 ± 0.02%. After UHP treatment, ACEI activity, apparent viscosity, concentrations of polypeptides and volatile aromatic substances, umami, and richness had increased significantly, bitterness and astringency were significantly reduced, and antioxidant properties were maintained. In addition, UHP fermented milk maintained a high level of ACEI activity and good quality during storage. Thus, the data represent a valuable reference for improving the storage quality of fermented milk and research for the future development of dairy products with high ACEI activity.     

发酵乳中血管紧张素转换酶抑制剂的分离与鉴定

用瑞士乳杆菌生产具有抑制血管紧张素转换酶(ACE)活性的发酵乳并从其中分离血管紧张素转换酶抑制剂(ACEI)。脱脂乳经灭菌后,接种4%(V/V)的发酵剂,37℃培养24h,获得抑制ACE活性较高的发酵乳。对此发酵乳的乳清进行超滤、上海732阳离子树脂交换、SephadexG-15凝胶过滤和HPLC分步纯化,最终得到两种起主要作用的ACEI肽。经氨基酸分析和液相-质谱分析,这两种肽的氨基酸序列分别为:Phe-Pro(IC50:12.05μmol/L)和Ala-Glu-Glu(IC50:12.42μmol/L)。     

微生物发酵法改良大豆肽风味的研究

该文研究了乳酸菌和酵母菌二次发酵法改良大豆肽的风味。实验结果表明,乳酸菌在发酵过程中,大豆肽溶液pH下降,总酸度上升,产生大量有机酸物质,乳酸菌发酵8h,大豆肽的风味最佳。酵母菌发酵过程中,大豆肽溶液中乙醇含量逐渐增加,产生大量醇类物质,酵母菌发酵2h,大豆肽溶液的风味最佳。经过乳酸菌和酵母菌发酵后,大豆肽的苦味和腥味消失,口感变成具有酸爽的杀口感,气味具有酒香味。因此,经过乳酸菌和酵母菌二次发酵工艺后,大豆肽的风味得到明显改善。     

具有血管紧张素转换酶抑制活性的乳酸菌筛选及特性研究

血管紧张素转换酶（Angiotensin Converting Enzyme，ACE）是抗高血压药物的筛选靶位酶。以体外ACE抑制率为筛选指标，从传统发酵食品中筛选出12株具有ACE抑制活性的乳酸菌，研究了12株乳酸菌的ACE抑制活性和蛋白水解活性，以及模拟胃肠消化对乳酸菌发酵乳ACE抑制活性的影响和乳酸菌耐酸耐胆汁的特性。结果表明，CZ2112具有良好ACE抑制活性，经API鉴定系统鉴定为植物乳杆菌，ACE抑制类型为非竞争性抑制，24h发酵产物ACE抑制率可以达到83％，经模拟胃肠消化后抑制率为70％，邻苯二甲醛（OPA）法检测其蛋白水解活性为OPA指数0．43，对胃酸、胆汁具有较好的耐受性。     

干酪乳杆菌LC-15发酵乳血管紧张素转换酶体外抑制活性的研究

血管紧张素转换酶抑制剂筛选模型是一个用于抗高血压药物和功能性食品研究的有效模型。通过考察乳酸菌蛋白质水解能力与ACE抑制活性的关系，以一株具有较高ACE抑制活性的干酪乳杆菌LC-15为研究对象，研究了发酵时间、添加酪蛋白、酶水解处理和模拟胃肠消化对LC-15发酵乳的ACE抑制活性的影响。结果表明，乳酸菌对牛乳蛋白质的水解能力与ACE抑制活性呈现一定的正相关关系；干酪乳杆菌LC-15在复原脱脂乳中发酵至42h时，发酵乳的ACE抑制活性达到最高，此时ACE抑制率为66％；通过在复原脱脂乳中添加4％酪蛋白可以使LC-15发酵乳的ACE抑制活性提高到81％；控制复原脱脂乳水解度为10％，然后再利用LC-15进行发酵得到的发酵乳的ACE抑制活性达到85％；在模拟胃肠环境消化条件下LC-15发酵乳的ACE抑制活力从最初的85％下降到78％。     

Modelling relationship between angiotensin-(I)-converting enzyme inhibition and the bitter taste of peptides

Relationships between angiotensin-(I)-converting enzyme inhibition and the bitter taste of peptides were studied. In cases where ACE inhibition or bitter taste had not been experimentally determined, their activity was estimated using several different peptide quantitative structure–activity relationship (QSAR) models. Significant correlations between increased ACE inhibition and bitterness were found for dipeptides using both observed and QSAR-predicted values. The relationship between ACE inhibition and bitter taste was attributed to the importance of hydrophobicity for both properties. Limited structural variations for dipeptides could make it difficult to have features that limit the effect of C-terminal hydrophobicity, necessary for ACE inhibition, on bitter taste. A similar modelling approach was also done on data from observed bitter oligopeptides derived from milk proteins. The relationship between QSAR-predicted ACE inhibition and observed bitter taste was not as strong as that found for dipeptides. Larger structural variation possibilities for oligopeptides than for dipeptides may thus make it, more feasible to find a highly efficient ACE inhibitory oligopeptide with a negligible bitter taste than a dipeptide.     

乳酸菌发酵酸豆奶中不良风味物质研究进展

随着植物基蛋白饮料在国内外的迅速发展, 酸豆奶必将进入新时代。酸豆奶是指以大豆为主要原料经过乳酸菌发酵后所得的产品, 在植物基蛋白饮料中占有重要地位, 良好的气味特征和口感特征对酸豆奶产品来说至关重要, 而豆腥味、苦涩味等不良风味的存在限制了酸豆奶的发展。酸豆奶风味物质种类繁多, 挥发性风味物质、非挥发性风味物质的成分和含量极大程度上决定了酸豆奶的感官品质。本研究就酸豆奶的发酵菌种、不良风味物质的来源、风味物质的分类和检测方法进行综述, 从发酵菌种与酸豆奶中不良风味物质的角度出发, 为解决酸豆奶在工业生产中不良风味问题提供思路。     

Antihypertensive and antithrombotic activities of a commercial fermented milk product made with Lactobacillus casei Shirota and Streptococcus thermophilus

Peptides which inhibit the human angiotensin‐converting enzyme (ACE) may be released during milk fermentation, and the micro‐organisms or fermentation conditions influence the specific peptides produced. The aim of this study was to evaluate the ACE inhibitory and antithrombotic activities of a fermented milk product commercially available in Mexico. Viable cell numbers, protein hydrolysis and the pH remained constant during refrigerated storage. The IC₅₀ of ACE inhibitory activity was 31.38 mg/mL. Eight peptide fractions exhibited ACE inhibitory activity and six showed antithrombotic activity. Two fractions showed both. This is the first time that both activities have been reported in a commercial probiotic product.     

Antihypertensive activity of milk fermented by Enterococcus faecalis strains isolated from raw milk

A total of 231 microorganisms were isolated from raw cow milk samples and the angiotensin-converting enzyme-inhibitory (ACEI) activity of the resultant fermented milk produced with the isolated microorganisms was assayed. Forty-six of these microorganisms were selected on the basis of high ACEI activity. Four Enterococcus faecalis strains stood out as producers of fermented milk with potent ACEI activity (IC₅₀ (the protein concentration that inhibits 50% of ACE activity): 34–59 μg mL⁻¹). Single doses (5 mL kg⁻¹) of the whey fraction obtained from these fermented milk samples were administered to spontaneously hypertensive rats (SHR) and to normotensive Wistar-Kyoto (WKY) rats in order to investigate their possible antihypertensive activity. Highly significant decreases in the systolic blood pressure (SBP) and in the diastolic blood pressure (DBP) were observed when the fermented milk was administered to SHR. Nevertheless, the fermented milk did not modify the SBP and the DBP of the WKY rats. Raw cow milk is an excellent source of wild lactic acid bacteria able to produce fermented milk with antihypertensive activity and antihypertensive activity of milk fermented by Enterococcus faecalis strains was associated with peptides different from Ile-Pro-Pro and Val-Pro-Pro.