Investigation of lead and nickel contaminated natural biofilms

Total reflection X-ray fluorescence spectrometric method was developed for elemental analysis of lead and nickel contaminated natural biofilms grown on polycarbonate substrates. The biofilms were grown in the Lake of Velence for 4 weeks and then in the laboratory in contaminated lake water for other 2 weeks. It was established that the accumulation of these bivalent cations were practically the same and in their presence the accumulation of zinc decreased by approximately 20% while the other elements (Ca, K, Fe, Mn, Sr, Ti, Rb) showed smaller changes within the statistical interval. From biological point of view, these two bivalent cations reduced the abundance of the algae, to a similar extent; however, the total biomass production was significantly hampered only by nickel (14%).     

Effect of Vitamin C Source on Its Stability during Storage and the Properties of Milk Fermented by Lactobacillus rhamnosus

The enrichment of commonly consumed foods with bioactive components might be helpful in promoting health and reducing the risk of disease, so the enrichment of probiotic fermented milk with vitamin C can be considered appropriate. The effect of vitamin C addition depends on the source of origin (rosehip, acerola and ascorbic acid in powder form) on the growth and survival of Lactobacillus rhamnosus and the quality of fermented milk on the 1st and 21st day of storage was analyzed. The pH, total acidity, vitamin C, syneresis, color, texture profile and numbers of bacterial cells in fermented milk were determined. The organoleptic evaluation was also performed. The degradation of vitamin C in milk was shown to depend on its source. The lowest reduction of vitamin C was determined in milk with rosehip. The least stable was vitamin C naturally found in control milk. The addition of rosehip and acerola decreased syneresis and lightness of milk color, increasing the yellow and red color proportion. In contrast, milk with ascorbic acid was the lightest during the whole experimental period and was characterized by a very soft gel. The growth of Lactobacillus rhamnosus during fermentation was most positively affected by the addition of rosehip. However, the best survival of Lactobacillus rhamnosus was demonstrated in milk with acerola. On the 21st day of storage, the number of L. rhamnosus cells in the control milk and the milk with vitamin C was >8 log cfu g⁻¹, so these milks met the criterion of therapeutic minimum. According to the assessors, the taste and odor contributed by the addition of rosehip was the most intense of all the vitamin C sources used in the study.     

富硒活性乳酸菌发酵剂的工艺研究

通过L9(33)正交试验确定了富硒活性乳酸菌发酵剂的适宜发酵条件,即发酵温度39℃,加硒量5μg/mL,接种量7%。结果表明,研制的发酵剂硒转化率达50.38%,冷藏5 d后发酵活力达0.48,冷藏7 d后活菌数达7.2×108cfu/mL,符合发酵剂的各项质量要求,可作为生产富硒活性乳酸菌功能性乳制品的工作发酵剂。     

Antibacterial Effect of Cell-Free Supernatant from Lactobacillus pentosus L-36 against Staphylococcus aureus from Bovine Mastitis

This study sought to analyze the main antibacterial active components of Lactobacillus pentosus (L. pentosus) L-36 cell-free culture supernatants (CFCS) in inhibiting the growth of Staphylococcus aureus (S. aureus), to explore its physicochemical properties and anti-bacterial mechanism. Firstly, the main antibacterial active substance in L-36 CFCS was peptides, which inferred by adjusting pH and enzyme treatment methods. Secondly, the physicochemical properties of the antibacterial active substances in L-36 CFCS were studied from heat, pH, and metal ions, respectively. It demonstrated good antibacterial activity when heated at 65 °C, 85 °C and 100 °C for 10 and 30 min, indicating that it had strong thermal stability. L-36 CFCS had antibacterial activity when the pH value was 2–6, and the antibacterial active substances became stable with the decrease in pH value. After 10 kinds of metal ions were treated, the antibacterial activity did not change significantly, indicating that it was insensitive to metal ions. Finally, scanning electron microscopy, transmission electron microscopy and fluorescence probe were used to reveal the antibacterial mechanism of S. aureus from the aspects of cell morphology and subcellular structure. The results demonstrated that L-36 CFCS could form 1.4–2.3 nm pores in the cell membrane of S. aureus, which increased the permeability of the bacterial cell membrane, resulting in the depolarization of cell membrane potential and leakage of nucleic acid protein and other cell contents. Meanwhile, a large number of ROS are produced and accumulated in the cells, causing damage to DNA, and with the increase in L-36 CFCS concentration, the effect is enhanced, and finally leads to the death of S. aureus. Our study suggests that the main antibacterial active substances of L-36 CFCS are peptides. L-36 CFCS are thermostable, active under acidic conditions, insensitive to metal ions, and exhibit antibacterial effects by damaging cell membranes, DNA and increasing ROS. Using lactic acid bacteria to inhibit S. aureus provides a theoretical basis for the discovery of new antibacterial substances, and will have great significance in the development of antibiotic substitutes, reducing bacterial resistance and ensuring animal food safety.     

Lactobacillus plantarum amylase acting on crude starch granules

The microheterogeneous native amylolytic complex secreted by the isolate A6 of Lactobacillus plantarum revealed a selective enzyme specificity loss when submitted to a limited proteolysis under a suboptimum pH condition. A clear electrophoretic profile change toward just one shorter, more acidic, and equally active polypeptide fragment resulted from the pronase E pretreatment. Although the whole enzyme activity remained apparently unaffected for soluble starch, the native parallel activity on intact and nongelatinized starch granules either from cereals or tubers was dramatically reduced. This phenomenon was more clearly documented by scanning electron microscopy using the easiest accessible native substrate: wheat starch granules. The anion-exchange-purified native enzymes from L. plantarum displayed a different optimum pH curve when compared with the thermotolerant α-amylase from Bacillus licheniformis. The α-amylases from the lactic-acid-producing A6 isolate presented an electrophoretic profile easily distinguishable from those from B. liqueniformis and B. subtilis species.     

Potentially Bioaccessible Phenolic and Antioxidant Potential of Fresh and Stored Lentil Sprouts—Effect of Lactobacillus plantarum 299v Enrichment

The phenolic and antioxidant potential of potentially bioaccessible fractions of lentil sprouts was studied. Sprouts were cocultivated with a probiotic to obtain a new functional product and further stored in cool conditions. The fraction obtained after buffer extraction and gastric digestion had higher content of phenolics compared to the control (by 20% and 46%, respectively); however, a 9% decrease was observed in samples obtained after gastrointestinal digestion. After gastrointestinal digestion, the highest content of phenolics (278 µg/g d.w.) was determined in the fresh control sprouts. Compounds neutralizing ABTS and hydroxyl radicals, chelating metal ions, and exhibiting strong reducing power were effectively released after gastrointestinal digestion (e.g., the values of the gastrointestinal digestibility index for chelating power and ability to quench hydroxyl radicals significantly exceeded 1 in all studied samples). It was proved that the enrichment of sprouts with a probiotic and further storage significantly improved the antioxidant potential; compared to the fresh control sprouts, an increase by 45% and 10% was determined after the gastric and gastrointestinal digestion, respectively. Lentil sprouts enriched with L. plantarum 299v may be a new functional product characterized by the high antioxidant capacity of the potentially bioaccessible fraction.     

Purification and Characterization of an Intracellular β-Glucosidase from Lactobacillus casei ATCC 393

The lactic acid bacterium,Lactobacillus casei, produces an intracellular β-glucosidase when grown on Man-Rogosa-Sharpe (MRS) medium with cellobiose as carbon source. The β-glucosidase activity is produced intracellulary, and no extracellulary activity was detected. The enzyme was purified by ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase as estimated by gel filtration was 480 kDa, consisting of six probably identical subunits. The enzyme exhibited optimum activity at 35°C and pH 6.3 with citrate-phosphate buffer. The enzyme was active against soluble glycosides with (1→4)-β configuration and from Lineweaver Burk plots, K_m value of 16 mmol/L was found for β-pNPG. The β-glucosidase was competitively inhibited by glucose, and no glycosyl transferase activity was observed in the presence of ethanol.     

高效液相色谱法测定唾液乳杆菌脱基因毒性作用

采用高效液相色谱法(HPLC)测定唾液乳杆菌对4-硝基喹啉-N-氧化物(4-NQO)基因毒性的抑制作用。Lact.salivariusFDB89与4-NQO(终浓度为20mg/L)共培养后,利用SOS-显色反应法检测唾液乳杆菌的脱基因毒性作用,同时采用高效液相色谱分析共培养前后的4-NQO含量变化。色谱分析条件为:C18反相色谱柱(kromasil100-5C18),以乙腈、水为流动相进行梯度洗脱,流速1mL/min;检测波长365nm。结果表明,唾液乳杆菌转化4-NQO,降低了4-NQO的基因毒性;HPLC测定的4-NQO峰面积与SOS-显色反应测定的基因毒性值呈正相关,相关系数r2为0.991。HPLC方法测定脱基因毒性检出限为1.2ng,菌体浓度在106~109cfu/mL范围内回收率为85%~98%。HPLC法测定唾液乳杆菌的脱基因毒性操作简单,其重复性和精确度均优于SOS-显色反应,可以采用HPLC代替SOS-显色反应。本研究在应用仪器分析方法代替生化分析方法测定乳杆菌脱基因毒性方面提供了一条新思路。     

Effects of Fructose and Oligofructose Addition on Milk Fermentation Using Novel Lactobacillus Cultures to Obtain High-Quality Yogurt-like Products

The incorporation of prebiotics in fermented milk products is one of the best ways to promote health benefits while improving their sensory characteristics at the same time. The aim of this study was to evaluate the effects of the addition of fructose and oligofructose (1% and 2%) on the physicochemical, rheological, sensory, and microbiological quality attributes of fermented milk products inoculated with indigenous probiotic starter cultures of Lactobacillus isolated from Polish traditional fermented foods. The samples were evaluated during 35 days of refrigerated storage. The oligofructose and fructose caused increases in the populations of bacteria in comparison to the control fermented milk products without the addition of saccharides. The degrees of acidification in different fermented milk samples, as well as their viscosity, firmness, syneresis, and color attributes, changed during storage. The highest overall sensory quality levels were observed for the samples supplemented with L. brevis B1 and oligofructose. This study is the first attempt to compare the influences of different sugar sources on the physicochemical, rheological, sensory, and microbiological quality attributes of fermented milk products.     

Nature of the antimicrobial activity of Lactobacillus caseiBifidobacterium bifidum and Bifidobacterium animalis against foodborne pathogenic and spoilage microorganisms

The antimicrobial activities as well as the nature of the inhibitory compounds obtained from Lactobacillus casei, Bifidobacterium bifidum and Bifidobacterium animalis strains were assayed on foodborne pathogenic – Staphyloccoccus aureus subsp. aureus (CCUG ATCC® 25926?) and Escherichia coli (ATCC® 25922?) – and spoilage microorganisms – Pseudomonas aeruginosa (ATCC® 27853?). Test producer strains showed inhibitory effect on all indicator microorganisms in diffusion of cell-free concentrated supernatant by agar in well methods (10.0–22.5 mm) in periods of 24, 48 and 72 h. Inhibitory compounds showed no sensitivity to the action of proteolytic enzyme trypsin and were completely inactivated by adjusting the pH of the cell-free 20 × concentrated supernatant to 7.0. The results demonstrated that antimicrobial substances do not have proteinaceous nature and are caused by the action of organic acids with decreasing medium pH.     

Interstrain Variability of Human Vaginal Lactobacillus crispatus for Metabolism of Biogenic Amines and Antimicrobial Activity against Urogenital Pathogens

Lactobacillus crispatus is the dominant species in the vagina of many women. With the potential for strains of this species to be used as a probiotic to help prevent and treat dysbiosis, we investigated isolates from vaginal swabs with Lactobacillus-dominated and a dysbiotic microbiota. A comparative genome analysis led to the identification of metabolic pathways for synthesis and degradation of three major biogenic amines in most strains. However, targeted metabolomic analysis of the production and degradation of biogenic amines showed that certain strains have either the ability to produce or to degrade these compounds. Notably, six strains produced cadaverine, one produced putrescine, and two produced tyramine. These biogenic amines are known to raise vaginal pH, cause malodour, and make the environment more favourable to vaginal pathogens. In vitro experiments confirmed that strains isolated from women with a dysbiotic vaginal microbiota have higher antimicrobial effects against the common urogenital pathogens Escherichia coli and Enterococcus faecium. The results indicate that not all L. crispatus vaginal strains appear suitable for probiotic application and the basis for selection should not be only the overall composition of the vaginal microbiota of the host from which they came, but specific biochemical and genetic traits.     

Antiinfluenza virus activity of a bacteriocin produced by Lactobacillus delbrueckii

A novel antibacterial substance produced by Lactobacillus delbrueckii has been isolated and characterized (1). The inhibitory agent corresponded to the criteria for bacteriocins. It was active against lactic acid bacteria (LAB) species and several food-borne pathogens. The cell-free supernatant was purified by HPLC gel-filtration. Three preparations at different purification steps were tested for activity on the reproduction of influenza virus A/chicken/Germany, strain Weybridge (H7N7) and strain Rostock (H7N1) in cell cultures of chicken embryo fibroblasts (CEF). The inhibitory effect was shown to be highly selective and specific. Expression of viral glycoproteins hemagglutinin, neuraminidase, and nucleoprotein on the surface of infected cells, virus-induced cytopathic effect, infectious virus yield, and hemagglutinin production were all reduced at nontoxic concentrations of the crude preparation (B1). B1 did not protect cells from infection, did not affect adsorption, and slightly inhibited viral penetration into infected cells. The purification did not enhance the cellular toxicity and increased about 870-fold the virus-inhibitory activity. No inactivating effect on extracellular virus was found.     

Lactobacillus paracasei HY7015 and Lycopus lucidus Turcz. Extract Promotes Human Dermal Papilla Cell Cytoprotective Effect and Hair Regrowth Rate in C57BL/6 Mice

Hair loss is a disease that requires accurate diagnosis and type-specific medical treatment. Many hair loss treatments have some side effects, such as hormone-related effects, so there is a need for safe and effective hair loss treatment. In this study, we investigated the effects of Lactobacillus paracasei HY7015 (HY7015) and Lycopus lucidus Turcz. (LT) extract on hair regrowth and protection. In vitro experiments were conducted to assess the effects of HY7015 and/or LT extract on human follicle dermal papilla cells (HFDPC) of cytoprotective functions such as proliferations, antioxidants, anti-inflammatory, and growth factor expressions. In animal experiments, we investigated hair regrowth rate, hair follicle formation and secretion of growth factors in telogenic C57BL/6 mice. We confirmed the cytoprotective effects of HY7015 and LT through regulations of proliferation, SOD and IL-1β in HFDPC. In mouse experiments, oral administration of HY7015 and LT promoted hair regrowth as well as hair follicle maturation in the dermal skin of C57BL/6 mice, and upregulated VEGF and IGF-1 growth factor levels in mouse serum. In summary, our data demonstrate that ingestions of HY7015 and LT can promote hair regrowth by enhancing cytoprotective effects and expressions of growth factors.     

Exploring the Bile Stress Response of Lactobacillus mucosae LM1 through Exoproteome Analysis

Lactobacillus sp. have long been studied for their great potential in probiotic applications. Recently, proteomics analysis has become a useful tool for studies on potential lactobacilli probiotics. Specifically, proteomics has helped determine and describe the physiological changes that lactic acid bacteria undergo in specific conditions, especially in the host gut. In particular, the extracellular proteome, or exoproteome, of lactobacilli contains proteins specific to host– or environment–microbe interactions. Using gel-free, label-free ultra-high performance liquid chromatography tandem mass spectrometry, we explored the exoproteome of the probiotic candidate Lactobacillus mucosae LM1 subjected to bile treatment, to determine the proteins it may use against bile stress in the gut. Bile stress increased the size of the LM1 exoproteome, secreting ribosomal proteins (50S ribosomal protein L27 and L16) and metabolic proteins (lactate dehydrogenase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenases, among others) that might have moonlighting functions in the LM1 bile stress response. Interestingly, membrane-associated proteins (transporters, peptidase, ligase and cell division protein ftsH) were among the key proteins whose secretion were induced by the LM1 bile stress response. These specific proteins from LM1 exoproteome will be useful in observing the proposed bile response mechanisms via in vitro experiments. Our data also reveal the possible beneficial effects of LM1 to the host gut.     

Preparation,Characterization, and Mechanism of Antifreeze Peptides from Defatted Antarctic Krill (Euphausia superba) on Lactobacillus rhamnosus

Defatted Antarctic krill powder is the main by-product in the manufacturing of krill oil. Exploring a high value-added approach for utilizing this protein-rich material has received much attention in research and industry. Given this, the preparation and primary characterization of antifreeze peptides from defatted Antarctic krill (AKAPs) were carried out in this study. The cryoprotective effect of AKAPs on Lactobacillus rhamnosus ATCC7469 was also investigated. The results showed that Protamex was the optimum protease for AKAP preparation from defatted Antarctic krill. AKAPs were found to be rich in short peptides, with the MW ranging from 600 to 2000 Da (69.2%). An amino acid composition analysis showed that AKAPs were rich in glutamic acid (18.71%), aspartic acid (12.19%), leucine (7.87%), and lysine (7.61%). After freezing, the relative survival rate of Lactobacillus rhamnosus in the 1.0 mg/mL AKAP-treated group (96.83%) was significantly higher than in the saline group (24.12%) (p < 0.05). AKAPs also retarded the loss of acidifying activity of L. rhamnosus after freezing. AKAPs showed even better cryoprotective activity than three commercial cryoprotectants (sucrose, skim milk, and glycerol). In addition, AKAPs significantly alleviated the decrease in β-galactosidase and lactic dehydrogenase activities of L. rhamnosus (p < 0.05). Furthermore, AKAPs effectively protected the integrity of L. rhamnosus cell membranes from freezing damage and alleviated the leakage of intracellular substances. These findings demonstrate that AKAPs can be a potential cryoprotectant for preserving L. rhamnosus, providing a new way to use defatted Antarctic krill.     

Synthesis of a Pentasaccharide Repeating Unit of the Extracellular Polysaccharide Produced by Lactobacillus Delbrueckii Subsp. Bulgaricus 291

A pentasaccharide methyl glycoside has been synthesized efficiently using a modified glycosylation strategy. This pentasaccharide is a repeating unit of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus 291.     

Effect of strain rate on the mechanical properties of Staphylococcus epidermidis biofilms

The quantification of biofilm mechanical properties can serve as a basis for understanding biofilm resilience and for developing biofilm control strategies. One aspect of tensile testing that is likely to be important for a viscoelastic material such as bacterial biofilm, but unfortunately is often overlooked (i.e., not controlled or reported), is the strain rate used during testing. Thus, we performed tensile testing on intact S. epidermidis biofilms using the microcantilever method at 12 strain rate values ranging over approximately 3 orders of magnitude (0.013-9.07 s(-1)). Ultimate or cohesive strength, elastic modulus, and toughness increased with increasing strain rate and approached a plateau at approximately 1.3 s(-1). Failure strain, on the other hand, did not exhibit any trend with strain rate. Given that the mean values of some parameters increased by as much as 1 order of magnitude over the strain rate range used in this work, we suggest that the strain rate during tensile testing should be carefully controlled and reported to facilitate comparisons among different studies. Furthermore, the quantitative expressions developed in this work that relate mechanical property values with strain rate may be useful for modeling the deformation of bacterial biofilms under applied loads.     

Fermentation of Jamaican Cherries Juice Using Lactobacillus plantarum Elevates Antioxidant Potential and Inhibitory Activity against Type II Diabetes-Related Enzymes

Jamaican cherry (Muntinga calabura Linn.) is tropical tree that is known to produce edible fruit with high nutritional and antioxidant properties. However, its use as functional food is still limited. Previous studies suggest that fermentation with probiotic bacteria could enhance the functional properties of non-dairy products, such as juices. In this study, we analyze the metabolite composition and activity of Jamaican cherry juice following fermentation with Lactobacillus plantarum FNCC 0027 in various substrate compositions. The metabolite profile after fermentation was analyzed using UPLC-HRMS-MS and several bioactive compounds were detected in the substrate following fermentation, including gallic acid, dihydrokaempferol, and 5,7-dihydroxyflavone. We also found that total phenolic content, antioxidant activities, and inhibition of diabetic-related enzymes were enhanced after fermentation using L. plantarum. The significance of its elevation depends on the substrate composition. Overall, our findings suggest that fermentation with L. plantarum FNCC 0027 can improve the functional activities of Jamaican cherry juice.     

Combined cup loading, bis(2-hydroxyethyl) disulfide, and protein precipitation protocols to improve the alkaline proteome of Lactobacillus hilgardii

Despite the large number of papers dealing with bacterial proteomes, very few include information about proteins with alkaline pI's, because of the limits inherent in 2-DE technology. Nonetheless, analyses of in silico proteomes of many prokaryotes show a bimodal distribution of their proteins based on their pI's; the most crowded areas lying between pI 4-7 and 9-11. The aim of the present research was to set up a general, simple, and standardizable 2-DE protocol suitable for studying the alkaline proteome of Lactobacillus hilgardii, a Gram-positive bacillus isolated from wine. The method has also been tested on a Gram-negative bacterium able to degrade aromatic pollutants, Acinetobacter radioresistens S13. Optimization of the method was mainly focused on improving protein extraction and IEF (pI 6-11) separation protocols. Concerning IEF, different methods for sample loading (in-gel rehydration and cup loading), and different reducing agents (DTT and bis(2-hydroxyethyl) disulfide (HED)) were tested and compared. The proposed protocol was found to resolve efficiently alkaline proteins from both of our Lactobacillus and Acinetobacter strains, in spite of their different external layers, thus, enabling a more comprehensive study of their proteomes.     

Physical Properties and Prebiotic Activities (Lactobacillus spp.) of Gelatine-Based Gels Formulated with Agave Fructans and Agave Syrups as Sucrose and Glucose Substitutes

This research developed model foods of gelatine-based gels, where carbohydrates from Agave tequilana Weber var. Azul (agave syrups or/and agave fructans) were incorporated into gel formulations as healthy sucrose and glucose substitutes. The sugars (sucrose and glucose) were substituted by agave carbohydrates (agave syrups and agave fructans), obtaining the subsequent gel formulations: 100% agave syrup (F2 gel), 100% agave fructan (F3 gel), and 50% agave syrup–50% agave fructan (F4 gel). The unsubstituted gel formulation was used as a control (F1 gel). The prebiotic activities, physical properties, thermal stability (HP-TLC), and texture of gelatine-based gels were evaluated. The gel formulations showed translucent appearances with approximately 36 g/100 g of water and water activities values between 0.823 and 0.929. The HP-TLC analysis validated that agave fructans did not hydrolyse during the thermal process of gels production. Gels produced with agave syrup and agave fructan (F2-F4 gels) provided higher hardness, gumminess, and springiness values (p < 0.05) than those produced with glucose and sucrose (F1 gel). Gelatine-based gel formulations displayed prebiotic activities correlated to the ability of Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus rhamnosus to use agave carbohydrates as carbon sources. Based on the prebiotic effect and physical and textural properties, the F2 and F4 gel formulations displayed the best techno-functional properties to produce gel soft candies.