Biphasic water-oil systems for functional augmentation of probiotic Lactobacillus acidophilus nanoencapsulated in luteolin-Fe3+ shells.

Single-cell nanoencapsulation (SCNE) has great potential in the enhancement of therapeutic effects of probiotic microbes. However, the material scope has been limited to water-soluble compounds to avoid non-biocompatible organic solvents that are harmful to living cells. In this work, the SCNE of probiotic Lactobacillus acidophilus with water-insoluble luteolin and Fe3+ ions is achieved by the vortex-assisted, biphasic water-oil system. The process creates L. acidophilus nanoencapsulated in the luteolin-Fe3+ shells that empower the cells with extrinsic properties, such as resistance to lysozyme attack, anti-ROS ability, and α-amylase-inhibition activity, as well as sustaining viability under acidic conditions. The proposed protocol, embracing water-insoluble flavonoids as shell components in SCNE, will be an advanced add-on to the chemical toolbox for the manipulation of living cells at the single-cell level.

Effects of selenium nanoparticles produced by Lactobacillus acidophilus HN23 on lipid deposition in WRL68 cells.

Selenium is an essential trace element for most organisms, protecting cells from oxidative damage caused by free radicals and serving as an adjunctive treatment for non-alcoholic fatty liver disease (NAFLD). In this study, We used the lactic acid bacterium Lactobacillus acidophilus HN23 to reduce tetra-valent sodium selenite into particulate matter, and analyzed it through inductively coupled plasma mass spectrometry (ICP-MS), scanning electron microscopy (SEM), X-ray diffraction energy dispersive spectrometry (EDS), and Fourier transform infrared spectroscopy (FTIR). We found that it consisted of selenium nanoparticles (SeNPs) with a mass composition of 65.8 % zero-valent selenium and some polysaccharide and polypeptide compounds, with particle sizes ranging from 60 to 300 nm. We also detected that SeNPs were much less toxic to cells than selenite. We further used free fatty acids (FFA)-induced WRL68 fatty liver cell model to study the therapeutic effect of SeNPs on NAFLD. The results show that SeNPs are more effective than selenite in reducing lipid deposition, increasing mitochondrial membrane potential (MMP) and antioxidant capacity of WRL68 cells, which is attributed to the chemical valence state of selenium and organic composition in SeNPs. In conclusion, SeNPs produced by probiotics L. acidophilus had the potential to alleviate NAFLD by reducing hepatocyte lipid deposition and oxidative damage. This study may open a new avenue for SeNPs drug development to treat NAFLD.

Influence of lactic acid fermentation on the phenolic profile, antioxidant activities, and volatile compounds of black chokeberry (Aronia melanocarpa) juice.

Lactic acid fermentation is an effective method for improving the quality of black chokeberry. This study aimed to investigate the influence of lactic acid bacteria on the phenolic profile, antioxidant activities, and volatiles of black chokeberry juice. Initially, 107  cfu/mL of Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Lacticaseibacillus rhamnosus were inoculated into pasteurized black chokeberry juice and fermented for 48 h at 37°C. All these strains enhanced the total phenolic and total flavonoid contents, with La. acidophilus showing the highest total phenolic (1683.64 mg/L) and total flavonoid (659.27 mg/L) contents. Phenolic acids, flavonoids, and anthocyanins were identified using ultrahigh-performance liquid chromatography-tandem mass spectrometry. The prevalent phenolic acid, flavonoid, and anthocyanin in the lactic-acid-fermented black chokeberry juice were cinnamic acid, rutin, and cyanidin-3-O-rutinoside, respectively. Furthermore, following fermentation, the DPPH and ABTS scavenging capacity, as well as the reducing power capacity, increased from 59.98% to 92.70%, 83.06% to 94.95%, and 1.24 to 1.82, respectively. Pearson's correlation analysis revealed that the transformation of phenolic acids, flavonoids, and anthocyanins probably contributed to enhancing antioxidant activities and color conversation in black chokeberry juice. A total of 40 volatiles were detected in the fermented black chokeberry juice by gas chromatography-ion mobility spectrometry. The off-flavor odors, such as 1-penten-3-one and propanal in the black chokeberry juice, were weakened after fermentation. The content of 2-pentanone significantly increased in all fermented juice, imparting an ethereal flavor. Hence, lactic acid fermentation can effectively enhance black chokeberry products' flavor and prebiotic value, offering valuable insights into their production. PRACTICAL APPLICATION: The application of lactic acid bacteria in black chokeberry juice not only enhances its flavor but also improves its health benefits. This study has expanded the range of black chokeberry products and offers a new perspective for the development of the black chokeberry industry.

Optimal combination of cow and quinoa milk for manufacturing of functional fermented milk with high levels of antioxidant, essential amino acids and probiotics.

The aim of this research was to produce Rayeb milk, a bio-fermented milk product that has important benefits for health and nutrition. The Rayeb milk was divided into five different treatments: T1 from cow milk, T2 from quinoa milk, T3 from a mixture of cow and quinoa milk (50%:50%), T4 from a mixture of cow and quinoa milk (75%:25%), and T5 from a mixture of cow and quinoa milk (25%:75%). As a starting culture, ABT-5 culture was used. The results demonstrated that blending quinoa milk with cow milk increased the total solids, fat, total protein, pH, acetaldehyde, and diacetyl values of the resulting Rayeb milk. Additionally, the total phenolic content, antioxidant activity, minerals, and amino acids-particularly important amino acids-in Rayeb milk with quinoa milk were higher. In Rayeb milk prepared from a cow and quinoa milk mixture, Lactobacillus acidophilus and Bifidobacterium bifidum were highly stimulated. All Rayeb milk samples, particularly those that contained quinoa milk, possessed more bifidobacteria than the recommended count of 106 cfu g-1 for use as a probiotic. Based on the sensory evaluation results, it is possible to manufacture a bio-Rayeb milk acceptable to the consumer and has a high nutritional and health values using a mixture of cow milk and quinoa milk (75%:25% or 50%:50%) and ABT-5 culture.

Improvement and enhancement of oligosaccharide production from Lactobacillus acidophilus using statistical experimental designs and its inhibitory effect on colon cancer.

Colorectal cancer (CRC) is the third cause of death by cancers worldwide and is one of the most common cancer types reported in both Egypt and the United States. The use of probiotics as a dietary therapy is increasing either as a prevention or as a treatment for many diseases, particularly, in the case of CRC. The increasing acceptance of lactic acid bacterial (LAB) oligosaccharides as bioactive agents has led to an increase in the demand for the large-scale production of LAB-oligosaccharides using fermentation technology. Therefore, in the current study, we are using the Plackett- Burman design (PBD) approach, where sixteen experimental trials were applied to optimize the production of the target oligosaccharide LA-EPS-20079 from Lactobacillus acidophilus. Glucose, yeast extract and sodium acetate trihydrate were the top three significant variables influencing LA-EPS production. The maximum concentration of LA-EPS-20079 achieved by L. acidophilus was 526.79 µg/ml. Furthermore, Box-Behnken design (BBD) as response surface methodology (RSM) was used to complete the optimization procedure. The optimal levels of the chosen variables which were 30.0 g/l, glucose; 5 g/l, yeast extract and 10.0 g/l sodium acetate trihydrate with the predicted LA-EPS-20079 concentration of 794.82 µg/ml. Model validity reached 99.93% when the results were verified. Both optimized trials showed great cytotoxic effects against colon cancer line (CaCo-2) with inhibition percentages ranging from 64.6 to 81.9%. Moreover, downregulation in the expression level of BCL2 and Survivin genes was found with a fold change of 3.377 and 21.38, respectively. Finally, we concluded that the optimized LA-EPS-20079 has maintained its anticancer effect against the CaCo-2 cell line that was previously reported by our research group.

Effect of Lactobacillus (L. acidophilus NCIB1899, L. casei CRL 431, L. paracasei LP33) fermentation on free and bound polyphenolic, antioxidant activities in three Chenopodium quinoa cultivars.

The application of lactic acid bacteria (LAB) fermentation to the production of probiotic beverages is a common method for modifying the health-related functional characteristics and phytochemical content of such beverages. This study evaluated the effect of fermentation with Lactobacillus acidophilus NCIB1899, Lactobacillus casei CRL 431, and Lactobacillus paracasei LP33 on the total phenolic contents (PCs), flavonoid contents (FCs), phenolic profiles, and antioxidant capacities of the solvent-extractable (free) and cell-wall-bound (bound) fractions in quinoa varying in bran color. Compared with unfermented beverages, LAB fermentation significantly increased the free PCs and free FCs by 15.7%-79.4% and 7.6%-84.3%, respectively. The bound PCs increased, whereas bound FCs decreased in fermented black and red quinoa juice. The increments of procyanidin B2 , protocatechuic acid, p-hydroxybenzaldehyde, rutin, and kaempferol through 30 h fermentation exceeded 189%-622%, 13.8%-191%, 55.6%-100%, 48.5%-129%, and 120%-325%, respectively. However, the contents of catechin, procyanidin B1 , and ferulic acid decreased with fermentation. Overall, L. acidophilus NCIB1899, L. casei CRL431, and L. paracasei LP33 strains may be suitable for producing fermented quinoa probiotic beverages. L. acidophilus NCIB1899 was superior for fermentation to L. casei CRL431 and L. paracasei LP33. Red and black quinoa had significantly higher total (sum of free and bound) PC and FC concentrations and antioxidant capacities than white quinoa (p < 0.05) because of their higher concentrations of proanthocyanins and polyphenol, respectively. PRACTICAL APPLICATION: In this study, different LAB (L. acidophilus NCIB1899, L. casei CRL431, and L. paracasei LP33) were singly inoculated on aqueous solutions from quinoa to ferment probiotic beverage and to compare the metabolic capacity of LAB strains on nonnutritive phytochemicals (phenolic compounds). We observed that LAB fermentation greatly enhanced the phenolic and antioxidant activity of quinoa. The comparison indicated that the L. acidophilus NCIB1899 strain has the highest fermentation metabolic capacity.

Development of soy protein isolate/sodium carboxymethyl cellulose synbiotic microgels by double crosslinking with transglutaminase and aluminum chloride for delivery system of Lactobacillus acidophilus.

This study was carried out to develop soy protein isolate (SPI)/sodium carboxymethyl cellulose (NaCMC) synbiotic microgels by applying a double-crosslinking technique using transglutaminase and different concentrations of AlCl3 (0 %, 6 %, 7 %, 8 %) and also by adding Lactobacillus acidophilus (L. acidophilus) and pectic oligosaccharide. Synbiotic microgels crosslinked using 8 % AlCl3 (SPI/NaCMC-Al3+8 microgels) showed the highest encapsulation efficiency (92 %). The double-crosslinked microgels exhibited a smooth surface as proved by SEM. FT-IR, XRD, and DSC analyses showed the possible interaction within matrices and demonstrated the higher thermal stability of synbiotic microgels prepared using a higher concentration of AlCl3. All in all, after exposure to simulated digestion fluid, heat treatment (72 °C, 15 s), and refrigerated storage, more cells in double-crosslinked microgels survived compared to single-crosslinked microgels. In particular, probiotic viability was highest in SPI/NaCMC-Al3+8 microgels. These results indicate that the SPI/NaCMC-Al3+8 microgels developed in this study can effectively protect L. acidophilus against the external environment.

Effect of Probiotics Lactobacillus acidophilus and Lacticaseibacillus rhamnosus on Antibacterial Response Gene Transcription of Human Peripheral Monocytes.

Periodontitis and related systemic inflammatory diseases are characterized by imbalanced ratio between pro- and anti-inflammatory factors. Probiotics may control inflammation by altering the inflammatory phenotype of defense cells. We aimed to evaluate the gene transcription of the antibacterial response of monocytes to exposure to probiotic lactobacilli. CD14 + monocytes were obtained by positive selection from peripheral blood mononuclear cells from healthy donors (5 × 104 CD14 + /mL) and cultured with probiotic strains of Lacticaseibacillus rhamnosus (LR-32) and Lactobacillus acidophilus (LA-5) at a 1:10 multiplicity of infection in 24-well plates for 12 h. The gene expression analysis was performed by RT-qPCR using the Kit RT2 human antibacterial response, and in the supernatant, the cytokines were determined by ELISA. Tukey's post hoc test following an ANOVA with a p value of 5% was used for statistical analysis. Both probiotic strains increased the levels of cytokines TNF-α and CXCL-8 in the supernatant compared to the control of non-challenged cells (p < 0.05), but for IL-1Β and IL-6, this effect was observed only for LA-5 (p < 0.05). The fold-regulation values for the following genes for LA-5 and LR-32 were, respectively, IL-12B (431.94 and 33.30), IL-1Β (76.73 and 17.14), TNF-α (94.63 and 2.49), CXCL-8 (89.59 and 4.18), and TLR-2 (49.68 and 3.40). Likewise, most of the other genes evaluated showed greater expression for LA-5 compared to LR-32 (p < 0.05). The positive regulation of inflammatory factors such as IL-1ß promoted by L. acidophilus LA-5 may increase the antibacterial activity of innate defense in periodontal tissues. However, this property may be deleterious by increasing inflammatory response.

Effect of Lactobacillus acidophilus fermentation on the composition of chlorogenic acids and anti-hyperuricemia activity of Artemisia selengensis Turcz.

Background: Lactobacillus fermentation has become a typical food processing method with the development of the modern food industry. Aims: This study aimed to assess the effects of Lactobacillus acidophilus NCUF202.2 fermentation on Artemisia selengensis Turcz (AST) in relieving hyperuricemia (HUA) and the mechanisms involved. Results: The fermented Artemisia selengensis Turcz extracts (FASTE) could significantly increase the content of free polyphenol and enhance the inhibitory effect on xanthine oxidase (XOD) in vitro. The result of ultrafiltration coupled with UPLC-MS/MS screened out the components that might be combined with XOD in the caffeoylquinic acids (CQAs) of Artemisia selengensis Turcz extracts (ASTE) as 5-CQA, 3-CQA, 4-CQA, 1-CQA, 3,4-diCQA, 3,5-diCQA, 1,5-diCQA and 4,5-diCQA as well as caffeic acid (CA) of FASTE except the above. Our results also showed that ASTE and FASTE could alleviate HUA in rats. In contrast, FASTE had a better ability to reduce serum adenosine deaminase (ADA) activity than ASTE. Furthermore, FASTE could restore catalase (CAT) in HUA model rats in vivo, and superoxide dismutase (SOD) activity to a better degree, thereby inhibiting the production of excess malondialdehyde (MDA). Under the intervention of ASTE and FASTE, the levels of serum pro-inflammatory and anti-inflammatory factors in rats tended to be normal. However, FASTE could increase the expression of the uric acid secretion protein OAT1 and decrease the expression of reabsorption proteins URAT1 and GLUT9 in model rats, thereby reducing the serum uric acid (SUA) level in model rats. ASTE and FASTE can increase the abundance of beneficial bacteria, such as Bifidobacterium and Akkermansiaceae. Conclusion: Overall, part of diCQAs and CQAs was decomposed after fermentation. FASTE had a more substantial HUA-relieving effect than ASTE. It is expected to be applied to functional foods and contribute to the research and development of uric acid lowering functional foods.

Novel targets in rectal cancer by considering lncRNA-miRNA-mRNA network in response to Lactobacillus acidophilus consumption: a randomized clinical trial.

We aimed to explore the lncRNA-miR-mRNA network in response to Lactobacillus acidophilus (L. acidophilus) consumption in rectal cancer patients. The candidate miRs were first taken from the GEO and TCGA databases. We constructed the lncRNA-miR-mRNA network using the high-throughput sequencing data. At last, we created a heatmap based on the experimental data to show the possible correlation of the selected targets. The expression levels of selected targets were measured in the samples of 107 rectal cancer patients undergoing placebo and probiotic consumption and 10 noncancerous subjects using Real-Time PCR. Our analysis revealed a group of differentially expressed 12 miRs and 11 lncRNAs, and 12 genes in rectal cancer patients. A significant expression increase of the selected tumor suppressor miRs, lncRNAs, and genes and a substantial expression decrease of the selected oncomiRs, onco-lncRNAs, and oncogenes were obtained after the probiotic consumption compared to the placebo group. There is a strong correlation between some network components, including miR-133b and IGF1 gene, miR-548ac and MSH2 gene, and miR-21 and SMAD4 gene. In rectal cancer patients, L. acidophilus consumption was associated with improved expression of the lncRNA-miR-mRNA network, which may provide novel monitoring and therapeutic approaches.

NOD2 signaling in CD11c + cells is critical for humoral immune responses during oral vaccination and maintaining the gut microbiome.

Nucleotide-binding oligomerization domain containing 2 (NOD2) is a critical regulator of immune responses within the gastrointestinal tract. This innate immune receptor is expressed by several cell types, including both hematopoietic and nonhematopoietic cells within the gastrointestinal tract. Vaccination targeting the gastrointestinal mucosal immune system is especially difficult due to both physical and mechanistic barriers to reaching inductive sites. The use of lactic acid bacteria is appealing due to their ability to persist within harsh conditions, expression of selected adjuvants, and manufacturing advantages. Recombinant Lactobacillus acidophilus (rLA) has shown great promise in activating the mucosal immune response with minimal impacts on the resident microbiome. To better classify the kinetics of mucosal vaccination with rLA, we utilized mice harboring knockouts of NOD2 expression specifically within CD11c + cells. The results presented here show that NOD2 signaling in CD11c + cells is necessary for mounting a humoral immune response against exogenous antigens expressed by rLA. Additionally, disruption of NOD2 signaling in these cells results in an altered bacterial microbiome profile in both control mice and mice receiving L. acidophilus strain NCK1895 and vaccine strain LaOVA.

Desenvolvimento de bebidas lácteas probióticas adicionadas de polpa de morango e suplementadas com L-triptofano elaboradas a partir de leite bubalino e bovino / Development of probiotic dairy beverages with strawberry pulp and supplemented with L-tryptophan from buffalo and bovine milk

O objetivo deste estudo foi desenvolver uma formulação de bebida láctea bubalina probiótica adicionada de polpa de morango, comparando os efeitos do uso do leite de búfala e de vaca na elaboração dos produtos e verificando a possibilidade de suplementação com triptofano nos produtos lácteos probióticos. Como primeira etapa do trabalho, bebidas lácteas probióticas foram elaboradas a partir de leite bubalino e bovino, fermentadas com Streptococcus thermophilus TA040, Lactobacillus bulgaricus LB340 e Lactobacillus acidophilus La5, e formuladas com 0, 25 e 50% de soro em sua formulação. As bebidas foram avaliadas quanto à cinética de fermentação das culturas láticas utilizadas, ao teor de proteína, gordura e sólidos totais não gordurosos, pós-acidificação, viabilidade das culturas fermentadoras e sua capacidade de sobrevivência ao estresse gastrointestinal in vitro. As bebidas lácteas bubalinas apresentaram resultados superiores as bebidas bovinas. O uso do leite de búfala na elaboração das bebidas lácteas promoveu benefícios quanto as culturas láticas presentes nos produtos, exercendo efeito protetivo e influindo na preservação da viabilidade das bactérias ao longo do armazenamento refrigerado e durante a simulação do estresse gastrointestinal in vitro. As bebidas lácteas elaboradas com 25% apresentaram os resultados mais próximos aos obtidos pelos produtos controle, sem adição de soro, sendo selecionadas para a segunda parte do estudo. Nesta etapa, as formulações de bebida láctea com 25% de soro, foram acrescidas de um preparado com polpa de morango e bebidas sem adição da fruta, utilizadas como controle. As bebidas lácteas bubalinas frutadas, apresentaram menor teor de gordura e melhores características reológicas, com maior viscosidade e consistência do que os produtos controle, sem afetar a pós-acidificação, o perfil de ácido graxo, assim como, a viabilidade e a resistência às condições de estresse gastrointestinal in vitro das culturas fermentadoras. A avaliação da possibilidade de suplementar lácteos probióticos com triptofano foi realizada em conjunto com a Universidade de Milão. Para isso, iogurtes probióticos receberam adição de triptofano antes ou após a fermentação, sendo avaliados com relação ao perfil de pós-acidificação, quantidade de triptofano nos produtos, número de células viáveis por plaqueamento e citometria de fluxo ao longo do armazenamento a 25° e 4°C. Complementarmente, a influência da presença do triptofano no crescimento e produção de compostos antimicrobianos pelas culturas láticas, também foi avaliada. A adição de triptofano após a fermentação dos iogurtes, que foram armazenados sob refrigeração (4°C), além de não afetar a pós-acidificação dos produtos, apresentou benefícios quanto a viabilidade L. acidophilus, redução do dano e aumento do número de células vivas, promovendo teor maior do aminoácido nos iogurtes. A presença do triptofano nos meios de cultivo, também influenciou de forma positiva o crescimento de S. thermophilus e L. acidophilus, melhorando o desenvolvimento das bactérias durante a fermentação e influindo em uma maior atividade antilistérica por parte do S. thermophilus. Diante da influência positiva da aplicação do leite de búfala na elaboração das bebidas lácteas, assim como, a adição do triptofano em iogurtes probióticos, a suplementação do aminoácido em bebidas lácteas bubalinas frutadas permitiria a obtenção de um produto funcional, onde seus benefícios estariam relacionados tanto ao consumo do probiótico presente no produto quanto a complementação de triptofano na dieta do consumidor

The aim of this study was to develop a formulation of probiotic buffalo dairy beverage added with strawberry pulp, comparing the effects of using buffalo and cow's milk in the preparation of products and verifying the possibility of tryptophan supplementation in probiotic dairy products. As a first stage of the work, probiotic dairy beverages were made from buffalo and bovine milk, fermented with Streptococcus thermophiles TA040, Lactobacillus bulgaricus LB340 and Lactobacillus acidophilus La5, and formulated with 0, 25 and 50% whey in their formulation. The beverages were evaluated for the fermentation kinetics of the used lactic cultures, the levels of protein, fat and total no fat solids, post-acidification, fermenting cultures viability and their ability to survive gastrointestinal stress in vitro. Buffalo milk use in dairy beverages production promoted benefits regarding the lactic cultures present in the products, exerting a protective effect and influencing the viability preservation of bacteria during the cold storage and simulation of gastrointestinal stress in vitro. Dairy beverages made with 25% whey addition showed results similar to those obtained by the control products, without whey addition, being selected for the second part of the study. In this part, the dairy beverages formulations with 25% whey, were added with a preparation were added with a strawberry pulp preparation and dairy beverages without added fruit, used as a control. Fruity bubaline dairy beverages had lower fat content and better rheological characteristics, with higher viscosity and consistency than control products, without affecting post-acidification, fatty acid profile, as well as viability and resistance to in vitro gastrointestinal condition of fermented cultures. The possibility of supplementing probiotic dairy products with tryptophan was evaluated in partnership with the University of Milan. For this, probiotic yogurts received the addition of tryptophan before or after fermentation, being evaluated in relation to the post-acidification profile, tryptophan amount in the products, viable cell number per plating and flow cytometry during storage at 25°C and 4°C. In addition, the influence of the tryptophan presence on the growth and production of antimicrobial compounds by lactic cultures was also evaluated. The addition of tryptophan after the yogurt fermentation, which were stored under refrigeration (4°C), in addition to not affecting the post-acidification of the products, showed benefits to the viability of L. acidophilus, reduced the damage and increased the number of cells promoting higher amino acid content in yogurts. Tryptophan presence in the culture media also positively influenced the growth of S. thermophiles and L. acidophilus, improving the development of bacteria during fermentation and influencing better antilisteric activity in the part of S. thermophiles. In view of the buffalo milk positive influence observed after the application in dairy beverage preparation, as well as the addition of tryptophan in probiotic yoghurts, amino acid supplementation in fruity buffalo dairy beverages would allow to obtain a functional product, where its benefits would be related both to the consumption of the probiotic present in the product as to the supplementation of tryptophan in the consumer's diet

Effect of exopolysaccharides from cariogenic bacteria on human gingival fibroblasts.

Bacterial biofilm (dental plaque) plays a key role in caries etiopathogenesis and chronic periodontitis in humans. Dental plaque formation is determined by exopolysaccharides (EPSs) produced by cariogenic and periopathogenic bacteria. The most frequent cariogenic bacteria include oral streptococci (in particular S. mutans) and lactobacilli (most frequently L. acidophilus). In turn, the dominant periopathogen in periodontitis is Porphyromonas gingivalis. Development of dental caries is often accompanied with gingivitis constituting the mildest form of periodontal disease. Basic cellular components of the gingiva tissue are fibroblasts the damage of which determines the progression of chronic periodontitis. Due to insufficient knowledge of the direct effect of dental plaque on metabolic activity of the fibroblasts, this work analyses the effect of EPSs produced by S. mutans and L. acidophilus strains (H2O2-producing and H2O2-not producing) on ATP levels in human gingival fibroblasts (HGF-1) and their viability. EPSs produced in 48-hours bacterial cultures were isolated by precipitation method and quantitatively determined by phenol - sulphuric acid assay. ATP levels in HGF-1 were evaluated using a luminescence test, and cell viability was estimated using fluorescence test. The tests have proven that EPS from S. mutans did not affect the levels of ATP in HGF-1. Whereas EPS derived from L. acidophilus strains, irrespective of the tested strain, significantly increased ATP levels in HGF-1. The analysed EPSs did not affect the viability of cells. The tests presented in this work show that EPSs from cariogenic bacteria have no cytotoxic effect on HGF-1. At the same time, the results provide new data indicating that EPSs from selected oral lactobacilli may have stimulating effect on the synthesis of ATP in gingival fibroblasts which increases their energetic potential and takes a protective effect.

Heat-killed Lactobacillus acidophilus mediates Fusobacterium nucleatum induced pro-inflammatory responses in epithelial cells.

Probiotics is widespreadly used nowadays. However, the safety issue with the use of live probiotics is still a matter of contention. In recent years, an expanding body of evidence supports the beneficial role of heat-killed probiotics in the maintenance of systemic health, whereas the role of these heat-killed bacteria on periodontal health remains unclear. This study aimed to evaluate the effects of heat-killed probiotics on periodontal pathogen virulence and associated mechanisms. We demonstrated that heat-killed Lactobacillus acidophilus was able to coaggregate with Fusobacterium nucleatum, the bridging bacteria of oral biofilm, and inhibit the adhesion and invasion of F. nucleatum, leading to a subsequent elimination of pro-inflammatory cytokine production in oral epithelial cells. This coaggregation further caused a suppression of the virulence gene fap2 expression in F. nucleatum. Therefore, heat-killed L. acidophilus might downregulate the pro-inflammatory cytokine expression in epithelial cells via coaggregation with F. nucleatum and suppression of F. nucleatum fap2 expression, which was the first demonstration that heat-killed probiotics modulate periodontal disease pathogenesis via coaggregation. Collectively, this finding provides new evidence that heat-killed probiotics might exert beneficial effects to periodontal health by coaggregating with periodontal pathogens and modulating their virulence.

Inhibition of Human Cervical Cancer Hela Cell Line by Meat-Derived Lactic Acid Bacteria of Lactobacillus plantarum IIA-1A5 and Lactobacillus acidophilus IIA-2B4.

<b>Background and Objective:</b> Two Indonesian lactic acid bacteria of<i> L. plantarum </i>I IA-1A5 and <i>L. acidophilus </i>IIA-2B4 were previously isolated from beef with some functional probiotic properties. Nevertheless, the possibility of these strains to have anticancer activity remains unknown. Current study aimed to evaluate the inhibitory properties of intra-and extracellular protein extracts of these two strains against cervical cancer HeLa cells. <b>Materials and Methods:</b> The intracellular and extracellular proteins extract from <i>L. plantarum </i>IIA-1A5 and <i>L. acidophilus </i>IIA-2B4 were collected and designated as IP-LP, IP-LA, EP-LP and EP-LA, respectively. The effect of these extracts on the viability and morphology of HeLa cells were observed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and confocal microscopy, respectively. <b>Results:</b> Both IP-LP and IP-LA inhibited HeLa cells in a concentration-dependent manner, with IC₅₀ values of 352.62 and 120.97 µg mL¹, respectively. Meanwhile, the inhibition activity was also observed for EP-LP and EP-LA, <i>albeit</i> very low. The inhibition effect was also confirmed by morphological analysis under confocal electron microscopy which showed the changes in the cell shapes and numbers. <b>Conclusion:</b> Altogether, for the first time this study proposed that the probiotic isolated from Indonesian beef are promising to inhibit cancer cell lines.

Proteolysis and Process-Induced Modifications in Synbiotic Yogurt Investigated by Peptidomics and Phosphopeptidomics.

Probiotic and synbiotic yogurt preparations were manufactured at the semi-industrial pilot scale with Lactobacillus acidophilus and Bifidobacteria strains without inulin or fortified with 1 and 3% (w/w) inulin. The pathway of casein breakdown was determined in probiotic, synbiotic, conventional yogurt, and nonstarted milk base using HPLC-ESI-MS/MS-based peptidomics and phosphopeptidomics; in the latter case, casein phosphorylated peptides (CPPs) were previously enriched by hydroxyapatite chromatography. Compared with traditional yogurt, casein proteolysis increased in probiotic and even more in synbiotic yogurt with 1% inulin. Fortification with 3% inulin greatly modified the proteolytic pattern, indicating a characteristic contribution of probiotics to proteolysis. The enhanced proteolysis in synbiotic yogurt exposed the neo-formed peptides to progressively increase enzymatic or chemical modifications, such as dephosphorylation of CPPs, methionine oxidation, and formation of N-terminal pyroglutamic acids. These modifications might constitute molecular signature descriptors of metabolic processes mediated by complex bacterial communities, with technological, nutritional, and sensorial significance.

Lysates of Lactobacillus acidophilus combined with CTLA-4-blocking antibodies enhance antitumor immunity in a mouse colon cancer model.

Previous reports have suggested that many gut microbiomes were associated with the development of colorectal cancer (CRC), and could modulate response to numerous forms of cancer therapy, including checkpoint blockade immunotherapy. Here we evaluated the protective efficacy of Lactobacillus acidophilus (L. acidophilus) cell lysates combined with an anti-CTL antigen-4 blocking antibody (CTLA-4 mAb) in syngeneic BALB/c mice CRC models induce by a single intraperitoneal injection of 10 mg/kg azoxymethane (AOM), followed by three cycles of 2% dextran sulfate sodium (DSS) in drinking water. In contrast to CTLA-4 mAb monotherapy, L. acidophilus lysates could attenuate the loss of body weight and the combined administration significantly protected mice against CRC development, which suggested that the lysates enhanced antitumor activity of CTLA-4 mAb in model mice. The enhanced efficacy was associated with the increased CD8 + T cell, increased effector memory T cells (CD44 + CD8 + CD62L+), decreased Treg (CD4 + CD25 + Foxp3+) and M2 macrophages (F4/80 + CD206+) in the tumor microenvironment. In addition, our results revealed that L. acidophilus lysates had an immunomodulatory effect through inhibition the M2 polarization and the IL-10 expressed levels of LPS-activated Raw264.7 macrophages. Finally, the 16S rRNA gene sequencing of fecal microbiota demonstrated that the combined administration significantly inhibited the abnormal increase in the relative abundance of proteobacteria and partly counterbalance CRC-induced dysbiosis in model mice. Overall, these data support promising clinical possibilities of L. acidophilus lysates with CTLA-4 mAb in cancer patients and the hypothesis that probiotics help shape the anticancer immune response.

A surface-layer protein from Lactobacillus acidophilus NCFM induces autophagic death in HCT116 cells requiring ROS-mediated modulation of mTOR and JNK signaling pathways.

A surface-layer protein (Slp) derived from Lactobacillus acidophilus NCFM has been reported to possess multiple biological properties, including anti-inflammatory, inhibition of apoptosis in pathogen-invaded HT-29 cells and oxidative stress relief. However, its anti-tumor ability and underlying molecular mechanism are unknown. Here, we report that Slp suppresses cell proliferation and induces autophagic cell death in HCT116 cells. Accumulation of Beclin-1 and microtubule-associated protein 1 light chain 3 from II (LC3-II), and the degradation of p62 were observed when cells were treated with various concentrations of Slp (25, 50, 100 µg mL-1) for 24 h. We also found that the mammalian targets of rapamycin (mTOR) and c-Jun N-terminal kinase (JNK) signaling pathways were crucial mediators regulating Slp-induced autophagic cell death. Additionally, treatment with Slp resulted in the obvious formation of reactive oxygen species (ROS). SP600125, a JNK inhibitor, and N-acetylcysteine (NAC), a ROS inhibitor, attenuated Slp-induced autophagic cell death in HCT116 cells. Furthermore, NAC was found to prevent Slp-induced p70 and JNK phosphorylation. Taken together, our results suggest a novel mechanism of action of Slp induced autophagy, acting simultaneously through the ROS-mediated mTOR and JNK signaling pathways in HCT116 colon cancer cells.

Crystal structure of bacterial cyclopropane-fatty-acyl-phospholipid synthase with phospholipid.

The lipids containing cyclopropane-fatty-acid (CFA) protect bacteria from adverse conditions such as acidity, freeze-drying desiccation and exposure to pollutants. CFA is synthesized when cyclopropane-fatty-acyl-phospholipid synthase (CFA synthase, CFAS) transfers a methylene group from S-adenosylmethionine (SAM) across the cis double bonds of unsaturated fatty acyl chains. Here, we reported a 2.7-Å crystal structure of CFAS from Lactobacillus acidophilus. The enzyme is composed of N- and C-terminal domain, which belong to the sterol carrier protein and methyltransferase superfamily, respectively. A phospholipid in the substrate binding site and a bicarbonate ion (BCI) acting as a general base in the active site were discovered. To elucidate the mechanism, a docking experiment using CFAS from L. acidophilus and SAM was carried out. The analysis of this structure demonstrated that three groups, the carbons from the substrate, the BCI and the methyl of S(CHn)3 group, were close enough to form a cyclopropane ring with the help of amino acids in the active site. Therefore, the structure supports the hypothesis that CFAS from L. acidophilus catalyzes methyl transfer via a carbocation mechanism. These findings provide a structural basis to more deeply understand enzymatic cyclopropanation.

Probiotic and Synbiotic Sorbets Produced with Jussara (Euterpe edulis) Pulp: Evaluation Throughout the Storage Period and Effect of the Matrix on Probiotics Exposed to Simulated Gastrointestinal Fluids.

The aims of the present study were to develop and evaluate different formulations of probiotic and synbiotic sorbets produced with jussara (Euterpe edulis) pulp, polydextrose, Lactobacillus acidophilus LA3, and Lactobacillus paracasei BGP1. The pasteurized jussara pulp presented high content of phenolic compounds, especially anthocyanins, which were not inhibitory to the probiotics used in this study. The levels of polyphenols and anthocyanins present in the sorbets were also high and kept stable for 120 days, as well as the populations of both probiotics. On the other hand, probiotic populations reduced ca. 4 log CFU/g when exposed to simulated gastrointestinal fluids. Altogether, the sorbets produced in this study showed interesting results, indicating the viability on producing functional foods with probiotics, prebiotics, and other components that are rich in polyphenols, such as jussara pulp. The combination of these elements can improve the health beneficial effects of these compounds and provide important advantages to the intestinal microbiota of consumers.