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To investigate the association between the microbiota in mothers and gut microbiota in infants from 0 to 6 months, the microbiotas in infant feces, maternal feces, and breast milk were determined by 16S rRNA gene sequencing. The contribution of each maternal microbiome to the infant was assessed using fast expectation-maximization for microbial source tracking calculations. The levels of short-chain fatty acids (SCFAs) and secretory immunoglobulin A (sIgA) in the feces of infants were also determined using gas chromatography and IDK-sIgA ELISA to gain a more comprehensive understanding of the infant gut microbiome. The results of this study showed that in addition to Firmicutes (E1) and Bifidobacterium (E2), the dominant microorganisms of the intestinal microbiota of infants aged 0-6 months include Proteobacteria, which is different from previous findings. Acetic acid, the most abundant SCFA in the infant gut, was positively correlated with Megasphaera (P < 0.01), whereas sIgA was positively correlated with Bacteroides (P < 0.05) and negatively correlated with Klebsiella and Clostridium_XVIII (P < 0.05). The maternal gut microbiota contributed more to the infant gut microbiota (43.58% ± 11.13%) than the breast milk microbiota, and significant differences were observed in the contribution of the maternal microbiota to the infant gut microbiota based on the delivery mode and feeding practices. In summary, we emphasize the key role of maternal gut health in the establishment and succession of infant gut microbiota.IMPORTANCEThis study aims to delineate the microbial connections between mothers and infants, leveraging the fast expectation-maximization for microbial source tracking methodology to quantify the contribution of maternal microbiota to the constitution of the infant's gut microbiome. Concurrently, it examines the correlations between the infant gut microbiota and two distinctive biomolecules, namely short-chain fatty acids (SCFAs) and secretory immunoglobulin A (sIgA). The findings indicate that the maternal gut microbiota exerts a greater influence on the infant's gut microbial composition than does the microbiota present in breast milk. Infants born via vaginal delivery and receiving mixed feeding display gut microbiota profiles more similar to their mothers'. Notably, the SCFA acetate displays positive associations with beneficial bacteria and inverse relationships with potentially harmful ones within the infant's gut. Meanwhile, sIgA positively correlates with Bacteroides species and negatively with potentially pathogenic bacteria. By delving into the transmission dynamics of maternal-infant microbiota, exploring the impacts of metabolic byproducts within the infant's gut, and scrutinizing how contextual factors such as birthing method and feeding practices affect the correlation between maternal and infant microbiota, this research endeavors to establish practical strategies for optimizing early-life gut health management in infants. Such insights promise to inform targeted interventions that foster healthier microbial development during the critical first 6 months of life.
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To investigate the changes in the intestinal flora in the Chinese elderly with cardiovascular disease (CVD) and its correlation with the metabolism of trimethylamine (TMA), the intestinal flora composition of elderly individuals with CVD and healthy elderly individuals was analyzed using 16S rRNA sequencing, the TMA levels in the feces of elderly were detected using headspace-gas chromatography (HS-GC), and four kinds of characterized TMA-producing intestinal bacteria in the elderly were quantified using real-time fluorescence quantitative polymerase chain reaction (qPCR). The results showed that Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, and Verrucomicrobia are the dominant microorganisms of the intestinal flora in the Chinese elderly. And there were significant differences in the intestinal bacteria composition between healthy elderly individuals and those with CVD, accompanied by a notable difference in the TMA content. The richness and diversity of the intestinal flora in the elderly with CVD were higher than those in the healthy elderly. Correlation analysis indicated that certain significantly different intestinal flora were associated with the TMA levels. Our findings showed a significant difference in TMA-producing intestinal flora between healthy elderly individuals and those with CVD. The TMA levels were found to be positively and significantly correlated with Klebsiella pneumoniae, suggesting that this bacterium is closely linked to the production of TMA in the elderly gut. This may have implications for the development and progression of CVD in the elderly population.
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Enfermedades Cardiovasculares , Heces , Microbioma Gastrointestinal , Metilaminas , Humanos , Metilaminas/metabolismo , Anciano , Masculino , Femenino , Enfermedades Cardiovasculares/microbiología , Heces/microbiología , China , ARN Ribosómico 16S/genética , Bacterias/clasificación , Bacterias/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Persona de Mediana Edad , Pueblo Asiatico , Anciano de 80 o más Años , Pueblos del Este de AsiaRESUMEN
Background: Gastric cancer (GC) and type 2 diabetes (T2D) contribute to each other, but the interaction mechanisms remain undiscovered. The goal of this research was to explore shared genes as well as crosstalk mechanisms between GC and T2D. Methods: The Gene Expression Omnibus (GEO) database served as the source of the GC and T2D datasets. The differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) were utilized to identify representative genes. In addition, overlapping genes between the representative genes of the two diseases were used for functional enrichment analysis and protein-protein interaction (PPI) network. Next, hub genes were filtered through two machine learning algorithms. Finally, external validation was undertaken with data from the Cancer Genome Atlas (TCGA) database. Results: A total of 292 and 541 DEGs were obtained from the GC (GSE29272) and T2D (GSE164416) datasets, respectively. In addition, 2,704 and 336 module genes were identified in GC and T2D. Following their intersection, 104 crosstalk genes were identified. Enrichment analysis indicated that "ECM-receptor interaction," "AGE-RAGE signaling pathway in diabetic complications," "aging," and "cellular response to copper ion" were mutual pathways. Through the PPI network, 10 genes were identified as candidate hub genes. Machine learning further selected BGN, VCAN, FN1, FBLN1, COL4A5, COL1A1, and COL6A3 as hub genes. Conclusion: "ECM-receptor interaction," "AGE-RAGE signaling pathway in diabetic complications," "aging," and "cellular response to copper ion" were revealed as possible crosstalk mechanisms. BGN, VCAN, FN1, FBLN1, COL4A5, COL1A1, and COL6A3 were identified as shared genes and potential therapeutic targets for people suffering from GC and T2D.
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Antibacterial nanoagents have been increasingly developed due to their favorable biocompatibility, cost-effective raw materials, and alternative chemical or optical properties. Nevertheless, there is still a pressing need for antibacterial nanoagents that exhibit outstanding bacteria-binding capabilities and high antibacterial efficiency. In this study, we constructed a multifunctional cascade bioreactor (GCDCO) as a novel antibacterial agent. This involved incorporating carbon dots (CDs), cobalt sulfide quantum dots (CoSx QDs), and glucose oxidase (GOx) to enhance bacterial inhibition under sunlight irradiation. The GCDCO demonstrated highly efficient antibacterial capabilities attributed to its favorable photothermal properties, photodynamic activity, as well as the synergistic effects of hyperthermia, glucose-augmented chemodynamic action, and additional photodynamic activity. Within this cascade bioreactor, CDs played the role of a photosensitizer for photodynamic therapy (PDT), capable of generating ËO2- even under solar light irradiation. The CoSx QDs not only functioned as a catalytic component to decompose hydrogen peroxide (H2O2) and generate hydroxyl radicals (ËOH), but they also served as heat generators to enhance the Fenton-like catalysis process. Furthermore, GOx was incorporated into this cascade bioreactor to internally supply H2O2 by consuming glucose for a Fenton-like reaction. As a result, GCDCO could generate a substantial amount of reactive oxygen species (ROS), leading to a significant synergistic effect that greatly induced bacterial death. Furthermore, the in vitro antibacterial experiment revealed that GCDCO displayed notably enhanced antibacterial activity against E. coli (99+ %) when combined with glucose under simulated sunlight, surpassing the efficacy of the individual components. This underscores its remarkable efficiency in combating bacterial growth. Taken together, our GCDCO demonstrates significant potential for use in the routine treatment of skin infections among diabetic patients.
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Antibacterianos , Glucosa Oxidasa , Fotoquimioterapia , Puntos Cuánticos , Puntos Cuánticos/química , Puntos Cuánticos/efectos de la radiación , Glucosa Oxidasa/química , Fotoquimioterapia/métodos , Antibacterianos/farmacología , Antibacterianos/química , Escherichia coli/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , Cobalto/química , Cobalto/farmacología , Luz , Carbono/química , Carbono/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Reactores Biológicos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The ever-growing use of nature-derived materials creates exciting opportunities for novel development in various therapeutic biomedical applications. Living cells, serving as the foundation of nanoarchitectonics, exhibit remarkable capabilities that enable the development of bioinspired and biomimetic systems, which will be explored in this review. To understand the foundation of this development, we first revisited the anatomy of cells to explore the characteristics of the building blocks of life that is relevant. Interestingly, animal cells have amazing capabilities due to the inherent functionalities in each specialized cell type. Notably, the versatility of cell membranes allows red blood cells and neutrophils' membranes to cloak inorganic nanoparticles that would naturally be eliminated by the immune system. This underscores how cell membranes facilitate interactions with the surroundings through recognition, targeting, signalling, exchange, and cargo attachment. The functionality of cell membrane-coated nanoparticles can be tailored and improved by strategically engineering the membrane, selecting from a variety of cell membranes with known distinct inherent properties. On the other hand, plant cells exhibit remarkable capabilities for synthesizing various nanoparticles. They play a role in the synthesis of metal, carbon-based, and polymer nanoparticles, used for applications such as antimicrobials or antioxidants. One of the versatile components in plant cells is found in the photosynthetic system, particularly the thylakoid, and the pigment chlorophyll. While there are challenges in consistently synthesizing these remarkable nanoparticles derived from nature, this exploration begins to unveil the endless possibilities in nanoarchitectonics research.
We have highlighted the Cell-derived Nanomaterials for Biomedical Applications through the lenses of our team who have experiences with working on cell membrane, thylakoids, and studying the impact of nanoparticles on biological phenomenon such as nanomaterialsinduced endothelial leakiness (NanoEL). In this review, we have discussed the progress and the wide potential of nanoarchitectonics in plant systems, animal cells and microorganisms. Due to our unique backgrounds, our take on this topic may be the novelty of the review.
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Background: Lactoferrin (LF) is a multifunctional glycoprotein found in human milk and body fluids, which has been shown to play a vital role in regulating the immunity and supporting the intestinal health of infants. Aim: This study evaluated the association between maternal/parturient factors and LF concentration in the breast milk of Chinese mothers. Methods: 207 breast milk samples were collected from healthy mothers with in the first year of lactation. Maternal and parturient information was collected for these participants through questionnaires. The content of lactoferrin in breast milk was detected by liquid chromatography, and macronutrient concentration in breast milk was measured by human milk analyzer in only 109 samples. Results: Our findings demonstrated that the LF content was much higher within the first month of lactation than it was after that period (p < 0.05). When compared with normal and lean mothers, the LF content of obese mothers was considerably higher (p < 0.05). The parity and LF content showed a favorable correlation. The proportion of LF to total protein tended to decrease as lactation progressed. Protein, fat, dry matter, and energy content were significantly positively correlated with LF content (p < 0.001). Conclusion: Early breast milk tends to have a higher level of LF, and the change of LF concentration in breast milk is associated with the parity and body mass index of the mother.
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Lactoferrina , Leche Humana , Embarazo , Lactante , Femenino , Humanos , Leche Humana/química , Lactoferrina/análisis , Índice de Masa Corporal , Lactancia Materna , Lactancia/fisiología , ParidadRESUMEN
The impact of five human milk oligosaccharides (HMOs)-2'-fucosyllactose (2FL), 3'-sialyllactose (3SL), 6'-sialyllactose (6SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT)-on the gut microbiota and short-chain fatty acid (SCFA) metabolites in infants aged 0-6 months was assessed through in vitro fermentation. Analyses of the influence of different HMOs on the composition and distribution of infant gut microbiota and on SCFA levels were conducted using 16S rRNA sequencing, quantitative real-time PCR (qPCR), and gas chromatography (GC), respectively. The findings indicated the crucial role of the initial microbiota composition in shaping fermentation outcomes. Fermentation maintained the dominant genera species in the intestine but influenced their abundance and distribution. Most of the 10 Bifidobacteria strains effectively utilized HMOs or their degradation products, particularly demonstrating proficiency in utilizing 2FL and sialylated HMOs compared to non-fucosylated neutral HMOs. Moreover, our study using B. infantis-dominant strains and B. breve-dominant strains as inocula revealed varying acetic acid levels produced by Bifidobacteria upon HMO degradation. Specifically, the B. infantis-dominant strain yielded notably higher acetic acid levels than the B. breve-dominant strain (p = 0.000), with minimal propionic and butyric acid production observed at fermentation's conclusion. These findings suggest the potential utilization of HMOs in developing microbiota-targeted foods for infants.
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Space-division multiplexing (SDM) has been expected to support the continuous growth of transmission capacity. However, it suffers from high computation complexity that limits its physical implementations. In this paper, we propose and experimentally demonstrate a low-complexity MIMO equalization method to leverage the sparsity of weights and reduce the complexity by L1&L2-regularization in long-haul space-division multiplexing (SDM) systems. The L1-regularization finds the sparse solution of equalizer filters and substitutes it for optimal solution, reducing the complexity with performance degradation. On the other hand, the L2-regularization tends to produce a smoother estimation than L1 regularization and is therefore more robust to large variance. We conduct a 39.87-GBaud QPSK coherent optical transmission experiment based on a 4-core coupled-core fiber with the transmission distance from 1206-km to 7236-km. Comparisons on the equalization performance and computational complexity show that the sparse equalizer using L1&L2-regularization achieves a 30% reduction in complexity at the similar system performance, compared with the traditional time-domain MIMO.
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Ionic liquids (ILs) are thought to have negative effects on human health. Researchers have explored the effects of ILs on zebrafish development during the early stages, but the intergenerational toxicity of ILs on zebrafish development has rarely been reported. Herein, parental zebrafish were exposed to different concentrations (0, 12.5, 25, and 50 mg/L) of [Cn mim]NO3 (n = 2, 4, 6) for 1 week. Subsequently, the F1 offspring were cultured in clean water for 96 h. [Cn mim]NO3 (n = 2, 4, 6) exposure inhibited spermatogenesis and oogenesis in F0 adults, even causing obvious lacunae in the testis and atretic follicle oocytes in ovary. After parental exposure to [Cn mim]NO3 (n = 2, 4, 6), the body length and locomotor behavior were measured in F1 larvae at 96 hours post-fertilization (hpf). The results showed that the higher the concentration of [Cn mim]NO3 (n = 2, 4, 6), the shorter the body length and swimming distance, and the longer the immobility time. Besides, a longer alkyl chain length of [Cn mim]NO3 had a more negative effect on body length and locomotor behavior. RNA-seq analysis revealed several downregulated differentially expressed genes (DEGs)-grin1b, prss1, gria3a, and gria4a-enriched in neurodevelopment-related pathways, particularly the pathway for neuroactive ligand-receptor interaction. Moreover, several upregulated DEGs, namely col1a1a, col1a1b, and acta2, were mainly associated with skeletal development. Expression of DEGs was tested by RT-qPCR, and the outcomes were consistent with those obtained from RNA-Seq. We provide evidence showing the effects of parental exposure to ILs on the regulation of nervous and skeletal development in F1 offspring, demonstrating intergenerational effects.
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Líquidos Iónicos , Contaminantes Químicos del Agua , Animales , Masculino , Femenino , Humanos , Pez Cebra/metabolismo , Líquidos Iónicos/toxicidad , Testículo , Espermatogénesis , Contaminantes Químicos del Agua/toxicidad , Contaminantes Químicos del Agua/metabolismoRESUMEN
Ionic liquids (ILs) have been developed as alternatives to traditional solvents, and their toxicity may be affected by alkyl chain length. Currently, there is limited evidence for whether parental exposure to different alkyl chain length ILs will induce intergenerational toxicity in zebrafish offspring. To address this knowledge gap, the parental zebrafish (F0) were exposed to 25 mg/L [Cnmim]BF4 (n = 4, 6, 8) for 7 days. Following this, fertilized F1 embryos from the exposed parents were reared in clean water for 120 h. Increased mortality, higher deformity rate, increased pericardial edema rate, and a shorter swimming distance and average speed were detected in the unexposed F1 embryonic larvae from the exposed F0 compared to the F1 generation from the unexposed F0. Parental exposure to [Cnmim]BF4 (n = 4, 6, 8) resulted in cardiac malformations and dysfunction in F1 larvae, including increased pericardial areas, increased yolk sac areas and decreased heart rate. Moreover, the intergenerational toxicity of [Cnmim]BF4 (n = 4, 6, 8) in F1 offspring appeared to be alkyl chain length-dependent. Parental [Cnmim]BF4 (n = 4, 6, 8) exposure led to global transcriptomic changes involved in developmental processes, nervous system process, cardiomyopathy, cardiac muscle contraction, and metabolic signalling pathways such as PI3K-Akt, PPAR and cAMP pathways in unexposed F1 offspring. Overall, the present study provides evidence that the neurotoxicity and cardiotoxicity of ILs in zebrafish can be markedly transmitted to offspring, and the intergenerational developmental toxicity is probably linked to transcriptomic alterations, highlighting the necessity of assessing ILs' environmental safety and human health risks.
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Líquidos Iónicos , Contaminantes Químicos del Agua , Animales , Humanos , Líquidos Iónicos/toxicidad , Pez Cebra/fisiología , Transcriptoma , Fosfatidilinositol 3-Quinasas/metabolismo , Perfilación de la Expresión Génica , Larva/metabolismo , Contaminantes Químicos del Agua/toxicidad , Contaminantes Químicos del Agua/metabolismoRESUMEN
Considering the transmission characteristics of the coronavirus disease 2019 (COVID-19), there are certain time delays in the transition from susceptible individuals to exposed individuals after contact with exposed, symptomatically infected, and asymptomatically infected individuals. A COVID-19 model with time delays and exposed infection is developed and then the global dynamics of this model is investigated by an improved method; moreover, the numerical simulations are carried out. It is shown that the COVID-19-free equilibrium T0 is globally asymptotically stable (GAS) if and only if the control reproduction number Rc≤1, while T0 is unstable and the COVID-19 equilibrium T∗ is GAS if and only if Rc>1. The numerical results reveal that strengthening quarantine measures is helpful to control the COVID-19 epidemic in India. Furthermore, when Rc<1, the numbers of symptomatically infected, asymptomatically infected, and quarantined individuals eventually tend to the zero equilibrium state, and with the increase in the time delay, the three kinds of variables change faster and their peaks become larger; when Rc>1, the three kinds of variables eventually tend to the positive equilibrium state, which are oscillatory and the amplitudes of the oscillation enlarge as the value of time delay increases. The numerical results show that when Rc<1, the smaller the value of time delay, the smaller the final epidemic size. In short, the longer it takes time for susceptible individuals to transform exposed individuals, the harder COVID-19 will be controlled.
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COVID-19 , Epidemias , Humanos , COVID-19/epidemiología , SARS-CoV-2 , Cuarentena/métodos , India/epidemiologíaRESUMEN
Introduction: Obesity is a growing health problem among young people worldwide and is associated with gut conditions. This study aimed to explore the relationship between obesity, intestinal microbiota, fecal short-chain fatty acids (SCFAs), and lipopolysaccharide (LPS) in young college students. Methods: 16S rRNA gene sequences, SCFA and LPS contents, and obesity status were analyzed in 68 young college students (20-25 years old). Results: There were significant differences in intestinal microbial beta diversity among students with different body mass index (BMI). The abundance and proportion of Firmicutes and Bacteroides had no significant correlation with BMI. The contents of butyric acid and valeric acid in the feces of obese students were low, and the content of SCFAs had no significant correlation with BMI and LPS. The content of LPS in the feces of obese people was significantly higher than that in healthy people, and there was a significant positive correlation between LPS content and BMI. Conclusion: In general, there was a correlation between intestinal microbiota, SCFA, LPS, and BMI in young college students. Our results may enrich the understanding of the relationship between intestinal conditions and obesity and contribute to the study of obesity in young college students.
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Introduction: The psychrophilic bacterium Pseudomonas lurida (P. lurida) and its thermostable alkaline proteases can seriously damage raw milk quality. Methods: In this study, specific primers were designed for P. lurida's gyrB and aprX genes, and a real-time loop-mediated isothermal amplification (RealAmp) rapid detection method was developed for the early monitoring of P. lurida and its proteases in raw milk. A phylogenetic tree of the gyrB and aprX genes of P. lurida was constructed to analyze the homology of the design sequence of the RealAmp primer. The DNA of 2 strains of P. lurida and 44 strains of non-P. lurida were detected via RealAmp to analyze the specificity of the primer. Results: It was found that aprX-positive proteases were produced by P. lurida-positive strains only when Pseudomonas fluorescens was negative. The dissociation temperatures of gyrB and aprX in the RealAmp-amplified products were approximately 85.0°C and 90.0°C, respectively. Moreover, DNA was detected through a 10-fold dilution of P. lurida in a pure bacterial solution and artificially contaminated skimmed milk. The limit of detection of P. lurida DNA copy number in the pure bacterial solution was 8.6 copies/µL and that in the 10% skimmed milk was 5.5 copies/µL. Further, 144 raw milk samples throughout the year from three farms in Hebei province were analyzed using RealAmp. The highest detection rate of P. lurida was 56% in the first and third quarters, and that of proteases was 36% in the second quarter. The detection rates of P. lurida and its proteases were the highest in samples collected from pasture 2 (52 and 46%, respectively), and the ability of P. lurida to produce proteases reached 88%. Discussion: In conclusion, RealAmp established an early and rapid method for the detection of P. lurida and its proteases in raw milk samples, allowing the identification and control of contamination sources in a timely manner to ensure the quality of milk and dairy products.
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Milk protein concentrate (MPC) is considered an ideal substitute of cow milk because of its similar protein and nutrition. In this study, MPC was hydrolyzed to peptides by alcalase and neutrase, and the properties of hydrolysate were evaluated. When MPC was hydrolyzed at the ratio of alcalase and neutrase of 1:1 and enzyme to substrate ratio of 6000 U/g MPC at 50°C, pH 8.5 for 3 h, the proportion of peptides with molecular weights <1 kDa was 85.31%, and the antigenicity reduction rates of casein and ß-lactoglobulin were 33.76% and 22.38%, respectively. Moreover, LC-MS/MS peptide identification revealed that the alcalase and neutrase disrupted a total of 65 epitopes of casein and 21 epitopes of whey protein, which further elucidated the mechanism of complex enzyme hydrolysis to reduce milk protein allergenicity.
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Caseínas , Proteínas de la Leche , Animales , Bovinos , Femenino , Proteínas de la Leche/análisis , Hidrólisis , Cromatografía Liquida , Espectrometría de Masas en Tándem , Proteína de Suero de Leche/análisis , Alérgenos , Péptidos/química , Epítopos , Subtilisinas/metabolismoRESUMEN
Raw milk microbiota is complex and influenced by many factors that facilitate the introduction of undesirable microorganisms. Milk microbiota is closely related to the safety and quality of dairy products, and it is therefore critical to characterize the variation in the microbial composition of raw milk. In this cross-sectional study, the variation in raw milk microbiota throughout the year (n = 142) from three farms in China was analyzed using 16S rRNA amplicon sequencing, including α and ß diversity, microbial composition, and the relationship between microbiota and milk quality parameters. This aimed to characterize the contamination risk of raw milk throughout the year and the changes in quality parameters caused by contamination. Collection month had a significant effect on microbial composition; microbial diversity was higher in raw milk collected in May and June, while milk collected in October and December had the lowest microbial diversity. Microbiota composition differed significantly between milk collected in January−June, July−August, and September−December (p < 0.05). Bacterial communities represented in raw milk at the phylum level mainly included Proteobacteria, Firmicutes and Bacteroidota; Pseudomonas, Acinetobacter, Streptococcus and Lactobacillus were the most common genera. Redundancy analysis (RDA) found strong correlations between microbial distribution and titratable acidity (TA), fat, and protein. Many genera were significantly correlated with TA, for example Acinetobacter (R = 0.426), Enhydrobacter (R = 0.309), Chryseobacterium (R = 0.352), Lactobacillus (R = −0.326), norank_o__DTU014 (R = −0.697), norank_f__SC-I-84 (R = −0.678), and Subgroup_10 (R = −0.721). Additionally, norank_f__ Muribaculaceae was moderately negatively correlated with fat (R = −0.476) and protein (R = −0.513). These findings provide new information on the ecology of raw milk microbiota at the farm level and contribute to the understanding of the variation in raw milk microbiota in China.
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An accurate method that rapidly detects the number of viable but nonculturable (VBNC) Cronobacter sakazakii was developed by combining propidium bromide with quantitative LAMP (PMA-QLAMP). The gyrB gene was the target for primers design. The optimal PMA treatment conditions were determined to eliminate the DNA amplification of 108 CFU/mL of dead C. sakazakii without affecting any viable C. sakazakii DNA amplification. Compared with the DNA of 24 strains of common non-C. sakazakii strains found in raw milk and dairy products, the DNA of only six C. sakazakii strains from different sources was amplified using PMA-QLAMP. The ability of PMA-QLAMP to quantitatively detect non-dead C. sakazakii in a 10% powdered infant formula (PIF) solution was limited to 4.3 × 102 CFU/mL and above concentrations. Pasteurizing 106 CFU/mL viable C. sakazakii yielded the maximum ratio of the VBNC C. sakazakii. PMA-QLAMP-based detection indicated that, although approximately 13% of 60 samples were positive for viable C. sakazakii, the C. sakazakii titers in these positive samples were low, and none entered the VBNC state under pasteurization. PMA-QLAMP showed potential as a specific and reliable method for detecting VBNC-C. sakazakii in pasteurized raw milk, thereby providing an early warning system that indicates potential contamination of PIF.
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ABSTRACT: Thermostable alkaline protease (TAP) harbored by Pseudomonas fluorescens decomposes protein in milk and dairy products, leading to milk and dairy product spoilage during storage. Thus, a specific, sensitive, rapid, and simple method is required to detect TAP-harboring P. fluorescens. Two sets of primers targeting the aprX and gyrB genes of P. fluorescens were designed. The detection system and conditions were optimized, and a real-time loop-mediated isothermal amplification (real-time LAMP) method was developed for the simultaneous detection of TAP-harboring P. fluorescens in two separate reaction tubes. The phylogenetic tree targeting aprX showed that P. fluorescens and Pseudomonas lurida clustered on the same branch. The phylogenetic tree targeting gyrB showed that P. fluorescens clustered on the same branch with 95% confidence value, whereas P. lurida clustered on different branches. DNA of 16 strains of P. fluorescens and 34 strains of non-P. fluorescens was detected by real-time LAMP. TAP-harboring P. fluorescens can only be identified when the real-time LAMP detection results of both aprX and gyrB are positive. The dissociation temperatures of aprX and gyrB in the real-time LAMP-amplified products were approximately 90.0 and 88.0°C, respectively. The detection limits of the real-time LAMP targeting aprX and gyrB were 4.9 CFU per reaction in pure culture and 2.2 CFU per reaction in skimmed milk. The coefficient of variation of the repeatability test was less than 2%, indicating that the established real-time LAMP of P. fluorescens targeting gyrB and aprX has good stability and repeatability. Real-time LAMP was used to test 200 raw milk samples for the presence of TAP-harboring P. fluorescens in 3 h, and the coincidence rate of the results with those obtained using the traditional method, which takes at least 5 to 7 days, was 100%. Real-time LAMP will be a practical and effective method for accurate and rapid identification of TAP-harboring P. fluorescens in raw milk.
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Pseudomonas fluorescens , Proteínas Bacterianas , Endopeptidasas , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , Filogenia , Sensibilidad y EspecificidadRESUMEN
To quantify viable probiotic Lacticaseibacillus paracasei (L. paracasei) in fermented milk accurately and quickly, propidium monoazide combined with quantitative loop-mediated isothermal amplification (PMA-qLAMP) was applied. The optimal PMA treatment conditions for treating a L. paracasei suspension were determined using an orthogonal test to eliminate the DNA amplification of 108 CFU/mL of dead L. paracasei. Primers were designed based on the species-specific gyrB gene of L. paracasei. A phylogenetic tree based on the gyrB gene showed that L. paracasei clustered on the same branch with 91% support. Compared with the 16 strains commonly found in fermented milk, three strains of L. paracasei showed positive PMA-qLAMP results, and the melting temperature was approximately 82.4°C. There was a linear relationship (R2 = 0.9983) between the Ct values and the logarithm of the concentration of viable bacteria. The PMA-qLAMP detection limit for the L. paracasei artificially added to fermented milk was 7.3 × 102 CFU/mL. There was no significant difference between the logarithm values of the concentration of viable L. paracasei of 50 fermented milk samples within shelf life using the PMA-qLAMP and plate count methods (P > 0.01). PMA-qLAMP is specific and accurate for obtaining reliable results faster than when using plate counts.
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Azidas , Productos Lácteos Cultivados , Lacticaseibacillus paracasei , Viabilidad Microbiana , Leche , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Propidio/análogos & derivados , Animales , Azidas/metabolismo , Productos Lácteos Cultivados/microbiología , Girasa de ADN/genética , Lacticaseibacillus paracasei/clasificación , Lacticaseibacillus paracasei/genética , Lacticaseibacillus paracasei/aislamiento & purificación , Leche/microbiología , Filogenia , Propidio/metabolismoRESUMEN
In this clinical trial, the safety and effectiveness of Lactobacillus paracasei N1115 (LP N1115) were investigated as a potential probiotic to enhance gut development in young children born by caesarean section. Infants and young children between the ages of 6 months and 3 years were administered with a probiotic consisting of LP N1115 strain (n = 30) or placebo supplement (n = 30) over an 8 weeks intervention. And the stool consistency, bowel habits, salivary cortisol, fecal microbiota, and short-chain fatty acid metabolism were investigated. Efficacy data were obtained from 58 participants who completed the study. Overall, the placebo functioned similarly to LP N1115 group in relation to stool consistency, gastrointestinal symptoms, salivary cortisol, and short-chain fatty acids. However, the scoring data relating to the 6-18 months subgroup receiving LP N1115 remained stable over 8 weeks in comparison to placebo. Analysis of the fecal microbiota using 16S rRNA amplicon sequencing revealed that the phyla Firmicutes represented 62% of the microbial relative abundance in the feces of the subjects during the intervening period. No significant changes in alpha- or beta-diversity were noted between the placebo and LP N1115 groups overtime and at each time point. Differential abundance analysis indicated an increase in Lactobacillus in LP N1115 group at weeks 4 (p < .05) and 8 (p < .05) in comparison to the placebo group. These results suggest that probiotic supplementation with LP N1115 was well tolerated by the young children and subtle changes in the microbiome were noted throughout the intervention period.
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Fermented food products have been consumed for thousands of years in China, so fermented Chinese foods may contain huge lactic acid bacterial resources. Here, we report the draft genome sequence of a Lactobacillus plantarum isolate, JMCC0013, collected from traditional Chinese fermented milk, which provides a precious resource for the genomic analysis of Lactobacillus strains.