Doxycycline-Dependent Inducible and Reversible RNA Interference Mediated by a Single Lentivirus Vector

Most fermented milk prepared by strains of Lactobacillus helveticus showed significant antihypertensive effect in spontaneously hypertensive rats (SHR) by oral administration. However, milk fermented by other species of lactic acid bacteria did not show significant antihypertensive effects. Most of the whey fractions of the milk fermented by L. helveticus or Lactobacillus delbrueckii subsp. bulgaricus showed higher angiotensin I-converting enzyme (ACE) inhibitory activity than the activity of milk fermented by other species. Proteolytic activity in cell wall and peptide content of the fermented milk were higher in L. helveticus strains than other species.     

Cardiovascular Effects of Fermented Milk Containing Angiotensin-Converting Enzyme Inhibitors Evaluated in Permanently Catheterized, Spontaneously Hypertensive Rats

In this study, two strains of Lactobacillus helveticus were used to produce fermented milk rich in angiotensin-converting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of L. helveticus gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.     

Comparative Evaluation of Oral Administration of Probiotic Lactobacilli-fermented Milks on Macrophage Function

Six strains of lactobacilli belonging to three species (Lactobacillus casei, Lactobacillus acidophilus and Lactobacillus helveticus) were evaluated for probiotic attributes viz. acid tolerance, bile tolerance and cell surface hydrophobicity. All the six strains exhibited probiotic attributes with considerable degree of variation. Three Lactobacillus strains selected on the basis of probiotic attributes were used for preparing three different fermented milks. In order to evaluate the effect of feeding these probiotic fermented milks on macrophage cell function, an in-vivo trial was conducted in mice for a period of 2, 5 and 8?days. The control group of mice was fed with skim milk. The phagocytic activity of macrophages increased significantly (P?<?0.05) on feeding fermented milk prepared using L. acidophilus, L. casei and L. helveticus as compared to milk group (control) on 2nd, 5th and 8th day of feeding, respectively. Likewise, the release of ??-glucuronidase and ??-galactosidase from peritoneal macrophages increased significantly (P?<?0.05) on 2nd, 5th and 8th day of feeding as compared to their respective control group (milk). The results thus depict that feeding of probiotic fermented milk enhances phagocytic activity of the macrophages.     

Characterization of New Milk-derived Inhibitors of Angiotensin Converting Enzyme In Vitro and In Vivo

Inhibition of angiotensin converting enzyme (ACE) has been observed with a variety of different peptides, and peptide fragments with inhibitory capabilities have been identified within many different proteins, including milk proteins. The purpose of this study therefore was to identify new short peptides with inhibitory properties from the primary structure of milk proteins and to characterize them in vitro and in vivo, since no milk derived ACE inhibitors have previously been evaluated for their ability to inhibit ACE in vivo. In vitro, 8 of 9 dipeptides were found to be competitive inhibitors of ACE. The IC₅₀ was significantly lower when an angiotensin I-like substrate was used, than when a bradykinin-like substrate was used. Using three different in vivo models for ACE inhibition, a very moderate effect was observed for three of the new peptides, but only for up to 6 or 12 minutes. Nothing was observed with two reference compounds that are reported to be hypotensive ACE-inhibitors derived from milk proteins. This raises the question whether the mechanism of hypotensive action is straightforward inhibition of ACE in vivo.     

一株高ACE抑制活性乳杆菌的筛选鉴定、培养条件优化及其基因组分析

【背景】乳杆菌是人体肠道益生菌,其发酵乳中可检测到血管紧张素转换酶(Angiotensin converting enzyme,ACE)抑制肽。海洋蕴藏着丰富的微生物种质资源,分布着大量的乳杆菌。【目的】从高通量测序结果中发现渤海沉积物中分布着乳杆菌资源。为了进一步开发具有ACE抑制活性的海洋乳杆菌资源,提高乳杆菌发酵乳的ACE抑制活性,筛选瑞士乳杆菌(Lactobacillus helveticus)并对其特性进行研究。【方法】采用高通量测序技术从渤海沉积物中检测乳杆菌,并对其进行富集分离,对筛选出的乳杆菌进行16S rRNA基因鉴定和全基因组测序分析,测定该菌发酵乳的ACE抑制活性,并采用正交实验优化发酵条件。【结果】渤海沉积物中含有乳杆菌并成功筛选出一株瑞士乳杆菌GY-3,其发酵乳具有较高的ACE抑制活性。该菌在发酵温度37°C,接种量3%,且在脱脂乳培养基中添加1.0%葡萄糖,0.6%大豆蛋白胨,1.0%酵母浸粉,0.04%MnSO_4·4H_2O时,抑制活性最高,可达79.52%。通过对该菌基因组进行测序研究,发现其产ACE抑制肽涉及蛋白酶系统、多肽转运系统和肽酶系统。【结论】为扩大海洋源产ACE抑制肽的乳杆菌种质资源、开发高产ACE抑制活性的发酵菌株奠定了基础,进一步研究了如何提高乳杆菌产ACE抑制肽的水平,并对其基因组进行了研究,为今后生物学特性和ACE抑制活性机理的研究奠定了基础,并对降血压相关产品的开发具有重要意义。     

Anti-hypertensive activity of fermented dairy products containing biogenic peptides

A considerable amount of research has been conducted to study the health benefits of fermented milks. One possible mechanism for the beneficial effects is the involvement of biogenic compounds, which are produced by the action of microorganisms and make the products directly beneficial without the need for live bacteria. Research on the health effects of a pasteurized sour milk, which is fermented by a starter culture containing Lactobacillus helveticus as the predominant microorganism, indicated clear evidence for the involvement of biogenic compounds. In this paper, beneficial effects of sour milk are reviewed with special attention to the effect on anti-hypertension and angiotensin-I converting enzyme inhibitory peptides identified in sour milk.     

An in vitro protocol for direct isolation of potential probiotic lactobacilli from raw bovine milk and traditional fermented milks

A method for isolating potential probiotic lactobacilli directly from traditional milk-based foods was developed. The novel digestion/enrichment protocol was set up taking care to minimize the protective effect of milk proteins and fats and was validated testing three commercial fermented milks containing well-known probiotic Lactobacillus strains. Only probiotic bacteria claimed in the label were isolated from two out of three commercial fermented milks. The application of the new protocol to 15 raw milk samples and 6 traditional fermented milk samples made it feasible to isolate 11 potential probiotic Lactobacillus strains belonging to Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus vaginalis species. Even though further analyses need to ascertain functional properties of these lactobacilli, the novel protocol set-up makes it feasible to isolate quickly potential probiotic strains from traditional milk-based foods reducing the amount of time required by traditional procedures that, in addition, do not allow to isolate microorganisms occurring as sub-dominant populations.     

Cardiovascular effects of fermented milk containing angiotensin-converting enzyme inhibitors evaluated in permanently catheterized,spontaneously hypertensive rats

In this study, two strains of Lactobacillus helveticus were used to produce fermented milk rich in angiotensin-converting enzyme (ACE) inhibitors. In vitro tests revealed that the two milks contained competitive inhibitors of ACE in amounts comparable to what has been obtained in previously reported studies. The two milks were administered by gavage to spontaneously hypertensive rats that had had a permanent aortic catheter inserted through the left arteria carotis, and mean arterial blood pressure and heart rate were monitored from 4 to 8 h after administration. Unfermented milk and milk fermented with a lactococcal strain that does not produce inhibitors were used as controls. Highly significant blood pressure effects were observed; i.e., milk fermented with the two strains of L. helveticus gave a more pronounced drop in blood pressure than the controls. Significant differences in heart rate effects were detected with one of the strains.     

Effects of Lactobacillus helveticus fermented milk on bone cells in vitro

Milk fermented with Lactobacillus helveticus (L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey (Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3-1.4 times with the 1 × 10⁻⁵, 1 × 10⁻⁴ and 1 × 10⁻³ solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.     

Peptidomic Analysis of ACE Inhibitory Peptides Extracted from Fermented Goat Milk

Angiotensin converting enzyme (ACE) is considered as main causative agent in growing hypertension and other cardiovascular disorders. Inhibition of ACE by producing and purifying bioactive peptides of fermented goat milk is aimed in this study. Protein extracted from goat milk was hydrolyzed with proteolytic enzymes of LH (Lactobacillus helveticus-cicc22171). ACE inhibitory peptides were purified from fermented samples of goat milk protein by optimizing incubation time to 8 h (S-8), 16 h (S-16), 24 h (S-24) and 36 h (S-36), via ultrafiltration. S-8 was used as control to compare the ACE inhibition trend. Molecular weight cut-off; 10000 Da (PM-10) and Ultracel 3K membrane was used to perform ultrafiltration. Sample with 24 h incubation time was considered as best hydrolyzed as compared to others, by applying Nin-Hydrin reaction and SDS-PAGE analysis. ACE inhibitory assay validated the authenticity of S-24 in inhibiting ACE, in vitro. Furthermore, Q executive hybrid quadrupole-orbitrap mass spectrometry was used to determine molecular structure and amino acid sequence of ACE inhibitory peptides. Three peptides, VLPVPQKAVPQ, VLPVPQKVVPQ and TQTPVVVPPFLQPEIMGVPKVKE containing functional amino acid structure, has been identified with highest ACE inhibitory activity on the basis of intensity, size and higher concentration of hydrophobic amino acids as shown in figure as graphical abstract. Fermented goat milk containing these novel bioactive peptides, can be used as nutraceuticals to inhibit ACE and control hypertension in future.

Graphical Abstract

     

Diversity of lactic acid bacteria and yeasts in Airag and Tarag,traditional fermented milk products of Mongolia

We used culture- and molecular-biology-based methods to investigate microbial diversity in the traditional Mongolian fermented milks “Airag” (fermented mare’s milk) and “Tarag” (fermented milk of cows, yaks, goats, or camels). By rRNA or functional gene sequencing, we identified 367 lactic acid bacteria (LAB) strains and 152 yeast strains isolated from 22 Airag and 31 Tarag samples. The total concentration of LAB in Airag (10^{7.78 ± 0.50} c.f.u. ml^–1; mean ± SD) was significantly lower (P < 0.01) than in Tarag (10^{8.35 ± 0.62} c.f.u. ml⁻¹), whereas the total concentration of yeasts in Airag (10^{7.41 ± 0.61} c.f.u. ml^-1) was significantly higher (P < 0.01) than in Tarag (10^{5.86 ± 1.29} c.f.u. ml^-1). Lactobacillus helveticus and Lactobacillus kefiranofaciens were isolated from Airag as the predominant LAB strains at levels of about 10⁷ c.f.u. ml⁻¹, whereas Lactobacillus delbrueckii subsp. bulgaricus, L. helveticus, and Streptococcus thermophilus were the predominant isolates from Tarag at about 10⁷ c.f.u. ml⁻¹. The lactose-fermenting Kluyveromyces marxianus was isolated predominantly from Airag as its major alcoholic fermentation component. Non-lactose-fermenting yeasts such as Saccharomyces cerevisiae, Issatchenkia orientalis, and Kazachstania unispora were the predominant isolates from Tarag, at about 10⁵ c.f.u. ml⁻¹. The apparent geographic differences in the L. kefiranofaciens and S. thermophilus contents of Tarag strongly suggested that differences among the animal species from which the milk was sourced, rather than geographic distances, were the most important factors influencing the diversity of the microbial composition of traditional fermented milks in Mongolia.     

Impact of Proteolytic <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> MTCC5463 on Production of Bioactive Peptides Derived from Honey Based Fermented Milk

In this study, Lactobacillus helveticus MTCC5463 was evaluated for its proteolytic activity and production of bioactive peptides during fermentation of honey supplemented milk under specified growth conditions. Generally, lactic acid bacteria have a strong proteolytic system. However, L. helveticus MTCC5463 showed maximum proteolytic activity at 4 % level of honey supplementation compared to 6 % and control. Similarly, water soluble extract derived from fermented honey based milks exhibited different level of bioactive peptides productions during fermentation. L. helveticus MTCC5463 showed maximum peptides production at 4 % level of honey supplementation compared to control during HPLC analysis and LC–MS analysis.     

Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks Started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of β-casein (β-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of β-CN f7-14, f47-52, and f169-175 and κ-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the β-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC₅₀s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC₅₀s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.     

Improving anti-hyperglycemic and anti-hypertensive properties of camu-camu (Myriciaria dubia Mc. Vaugh) using lactic acid bacterial fermentation

Camu-camu (Myriciaria dubia Mc. Vaugh) is a tropical fruit rich in phenolic antioxidants with diverse human health benefits. The aim of this study was to improve phenolic antioxidant–linked functionalities of camu–camu relevant for dietary management of early stages of type 2 diabetes (T2D) and associated hypertension using lactic acid bacterial (LAB) fermentation. Dried camu–camu powder combined with soymilk was fermented using two LAB strains, Lactobacillus plantarum & Lactobacillus helveticus individually and evaluated for total soluble phenolic content, total antioxidant activity, α-amylase, α-glucosidase, and angiotensin-I-converting enzyme (ACE) inhibitory activities using in vitro assay models. Overall, fermentation of camu–camu and soymilk combination with both LAB strains resulted in higher α-amylase, and α-glucosidase inhibitory activities, while total soluble phenolic content and antioxidant activity did not change significantly with fermentation. Improvement of ACE enzyme inhibitory activity was also observed when camu–camu (0.5 & 1%) and soymilk combination was fermented with L. plantarum. Therefore such safe and value added fermentation strategy with LAB can be used to improve human health relevant phenolic antioxidant profile in camu–camu and has relevance for designing innovative probiotic beverage to target improved food designs for dietary support for T2D and associated hypertension management.     

内蒙古呼伦贝尔地区传统发酵乳中乳酸菌的多样性分析

【目的】对内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌资源的生物多样性进行研究。【方法】采用纯培养和16S rRNA基因序列分析法对内蒙古呼伦贝尔地区传统发酵乳中的乳酸菌进行多样性分析。【结果】从8份传统发酵乳制品(6份酸牛奶和2份酸马奶)样品中分离到24株乳酸菌,通过16S rRNA基因序列分析和系统进化关系分析将24株乳酸菌鉴定为2株Lactobacillus kefiranofaciens、2株Lactobacillus kefiri、5株Lactobacillus paracasei、3株Lactobacillus plantarum、1株Lactobacillus rhamnosus、6株Lactococcus lactis subsp.lactis、2株Leuconostoc mesenteroides subsp.dextranicum、2株Streptococcus thermophilus和1株Enterococcus faecium。【结论】Lactococcus lactis subsp.lactis为内蒙古呼伦贝尔地区传统发酵乳制品的优势菌种,占总分离株的25%,其次为Lactobacillus paracasei,占总分离株的20.83%。     

Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of beta-casein (beta-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of beta-CN f7-14, f47-52, and f169-175 and kappa-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the beta-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC(50)s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC(50)s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.     

Characterization and electrotransformation of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Lactobacillus paraplantarum</Emphasis> isolated from fermented vegetables

Aims of the study were to characterize two Lactobacillus plantarum-related strains, Lact. plantarum and Lactobacillus paraplantarum isolated from fermented vegetables and, for their potential use as starter strains, compare their growth in various food matrices. Species-level identification of the strains belonging to the Lact. plantarum group was performed by multiplex-PCR with species-specific primers and generation of distinct genotypic profiles was carried out by PFGE-based DNA-fingerprinting. Growth profiles were determined in various food and feed matrices. Compared to Lact. plantarum, Lact. paraplantarum reached higher cell densities in all plant-based matrices and MRS broth. On the contrary to the good growth in plant-based matrices and MRS, poor growth was observed in unprocessed milk. Supplemented lactose did not improve the growth of either tested strain, while predigestion of milk proteins with Lactobacillus helveticus or addition of casitone proved to be an effective means to enhance growth. To find out the applicability of molecular methods, the strains were transformed with replicative plasmids by electroporation. To our knowledge, this is a first report of the electrotransformation of Lact. paraplantarum with a recombinant plasmid.     

Modulation of the Cell-Surface Proteinase Activity of Thermophilic Lactobacilli by the Peptide Supply

The proteolytic system of thermophilic lactobacilli is considered important for bacterial nutrition as well as for the formation of flavor and texture in fermented products. We investigated the influence of peptide content on the cell surface proteinase and intracellular aminopeptidase activities from seven thermophilic lactobacilli strains. The proteinase activities were remarkably reduced in cells grown in the peptide-rich medium MRS or in a chemically defined medium supplemented with Casitone compared with those found in a synthetic medium. The degree of inhibition observed was strain dependent. When proteinase activities were analyzed by their hydrolytic patterns of α- and β-casein degradation, four types of P_III-caseinolytic cleavage specificity were distinguished. Lactobacillus helveticus strains possessed aminopeptidase activities with broader specificity than those found in L. delbrueckii subsp. lactis strains. However, the aminopeptidase activities were not influenced by the peptide content of the medium. Received: 1 February 2002 / Accepted: 27 February 2002     

Classification of Lactobacillus helveticus Strains by Immunological Differences in Extracellular Proteinases

Polyclonal antibodies against an extracellular proteinase of Lactobacillus helveticus CP53 were raised. The antibodies reacted with a 170-kDa enzyme with activity and a 53-kDa protein that seemed to be a degradation product of the 170-kDa proteinase from results of immunoblotting. The antibodies reacted also with a 45-kDa extracellular proteinase of L. helveticus CP790. However, monoclonal antibodies to the CP790 proteinase did not react with the proteinase of L. helveticus CP53. Seventeen strains of L. helveticus were tested for immunological reactivity with the two kinds of antibodies. The strains all had the same reactivity as either strain CP53 or strain CP790. Eleven strains with the 45-kDa proteinase were identified as L. helveticus biovar jugurti because they did not ferment maltose, four other strains with the 170- and 53-kDa proteins were identified as L. helveticus biovar helveticus because they fermented maltose. The remaining two strains dit not fit this pattern; they had both the 170- and 53-kDa proteins, but classification by their sugar utilization showed them to be L. helveticus biovar jugurti.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.