Interspecies interactions in dairy biofilms drive community structure and response against cleaning and disinfection.

Interspecies interactions within a biofilm community influence population dynamics and community structure, which in turn may affect the bacterial stress response to antimicrobials. This study was conducted to assess the impact of interactions between Kocuria salsicia and a three-species biofilm community (comprising Stenotrophomonas rhizophila, Bacillus licheniformis, and Microbacterium lacticum) on biofilm mass, the abundance of individual species, and their survival under a laboratory-scale cleaning and disinfection (C&D) regime. The presence of K. salsicia enhanced the cell numbers of all three species in pairwise interactions. The outcomes derived from summing up pairwise interactions did not accurately predict the bacterial population dynamics within communities of more than two species. In four-species biofilms, we observed the dominance of S. rhizophila and B. licheniformis, alongside a concurrent reduction in the cell counts of K. salsicia and M. lacticum. This pattern suggests that the underlying interactions are not purely non-transitive; instead, a more complex interplay results in the dominance of specific species. We observed that bacterial spatial organization and matrix production in different mixed-species combinations affected survival in response to C&D. Confocal microscopy analysis of spatial organization showed that S. rhizophila localized on the biofilm formed by B. licheniformis and M. lacticum, and S. rhizophila was more susceptible to C&D. Matrix production in B. licheniformis, evidenced by alterations in biofilm mass and by scanning electron microscopy, demonstrated its protective role against C&D, not only for this species itself, but also for neighbouring species. Our findings emphasise that various social interactions within a biofilm community not only affect bacterial population dynamics but also influence the biofilm community's response to C&D stress.

Dynamic social interactions and keystone species shape the diversity and stability of mixed-species biofilms - an example from dairy isolates.

Identifying interspecies interactions in mixed-species biofilms is a key challenge in microbial ecology and is of paramount importance given that interactions govern community functionality and stability. We previously reported a bacterial four-species biofilm model comprising Stenotrophomonas rhizophila, Bacillus licheniformis, Microbacterium lacticum, and Calidifontibacter indicus that were isolated from the surface of a dairy pasteuriser after cleaning and disinfection. These bacteria produced 3.13-fold more biofilm mass compared to the sum of biofilm masses in monoculture. The present study confirms that the observed community synergy results from dynamic social interactions, encompassing commensalism, exploitation, and amensalism. M. lacticum appears to be the keystone species as it increased the growth of all other species that led to the synergy in biofilm mass. Interactions among the other three species (in the absence of M. lacticum) also contributed towards the synergy in biofilm mass. Biofilm inducing effects of bacterial cell-free-supernatants were observed for some combinations, revealing the nature of the observed synergy, and addition of additional species to dual-species combinations confirmed the presence of higher-order interactions within the biofilm community. Our findings provide understanding of bacterial interactions in biofilms which can be used as an interaction-mediated approach for cultivating, engineering, and designing synthetic bacterial communities.

Weight Loss Promotion in Individuals with Obesity through Gut Microbiota Alterations with a Multiphase Modified Ketogenic Diet.

The occurrence of obesity and related metabolic disorders is rising, necessitating effective long-term weight management strategies. With growing interest in the potential role of gut microbes due to their association with responses to different weight loss diets, understanding the mechanisms underlying the interactions between diet, gut microbiota, and weight loss remains a challenge. This study aimed to investigate the potential impact of a multiphase dietary protocol, incorporating an improved ketogenic diet (MDP-i-KD), on weight loss and the gut microbiota. Using metagenomic sequencing, we comprehensively analyzed the taxonomic and functional composition of the gut microbiota in 13 participants before and after a 12-week MDP-i-KD intervention. The results revealed a significant reduction in BMI (9.2% weight loss) among obese participants following the MDP-i-KD intervention. Machine learning analysis identified seven key microbial species highly correlated with MDP-i-KD, with Parabacteroides distasonis exhibiting the highest response. Additionally, the co-occurrence network of the gut microbiota in post-weight-loss participants demonstrated a healthier state. Notably, metabolic pathways related to nucleotide biosynthesis, aromatic amino acid synthesis, and starch degradation were enriched in pre-intervention participants and positively correlated with BMI. Furthermore, species associated with obesity, such as Blautia obeum and Ruminococcus torques, played pivotal roles in regulating these metabolic activities. In conclusion, the MDP-i-KD intervention may assist in weight management by modulating the composition and metabolic functions of the gut microbiota. Parabacteroides distasonis, Blautia obeum, and Ruminococcus torques could be key targets for gut microbiota-based obesity interventions.

Efficacy Of Common Salt Application On The Umbilical Granuloma In Infants.

The objective of this study was to determine the efficacy of the application of common plain salt for the treatment of umbilical granuloma in infants. The study design was descriptive case series and was conducted over a period of 12 months at the paediatric unit, Mardan Medical Complex, Mardan, Pakistan. For this study a total of 50 infants with clinically evident umbilical granuloma were selected. Application of a pinch of common plain salt was advised on the granuloma twice a day for three consecutive days. Outcomes in the shape of complete resolution of the granuloma, adverse effects, and recurrence of granuloma were assessed. The number of patients responding well to the treatment was 45 (90%). The treatment showed good response and there was not any adverse effect or recurrence. It was concluded that the common plain salt is a simple, effective, safe, and cheaper option for the treatment of umbilical granuloma in infants.

Effects of Co-Fermentation of Lactiplantibacillus plantarum and Saccharomyces cerevisiae on Digestive and Quality Properties of Steamed Bread.

The leavening of wheat-based steamed bread is carried out either with a pure yeast culture or with traditional starter cultures containing both lactic acid bacteria and yeast/mold. The use of variable starter cultures significantly affects steamed bread's quality attributes, including nutritional profile. In this paper, differences in physicochemical properties, the type of digested starch, the production of free amino acids, and the specific volume of steamed bread under three fermentation methods (blank, yeast, and LP-GM4-yeast) were compared. The digestion characteristics (protein and starch hydrolysis) of steamed bread produced by using either yeast alone or a combination of Lactiplantibacillus plantrum and yeast (LP-GM4-yeast) were analyzed by an in vitro simulated digestion technique. It was found that the specific volume of steamed bread fermented by LP-GM4-yeast co-culture was increased by about 32%, the proportion of resistant starch was significantly increased (more than double), and soluble protein with molecular weight of 30-40 kDa was significantly increased. The results of this study showed that steamed bread produced by LP-GM4-yeast co-culture is more beneficial to human health than that by single culture.

Modulating the Human Gut Microbiota through Hypocaloric Balanced Diets: An Effective Approach for Managing Obesity.

This study aimed to investigate the effects of a hypocaloric balanced diet (HBD) on anthropometric measures and gut microbiota of 43 people with obesity. Fecal samples were collected from the study subjects at weeks 0 and 12, and a detailed analysis of gut microbiota was performed using 16S rRNA gene sequencing. By comparing anthropometric measures and microbiota changes in subjects before and after the HBD intervention, we revealed the potential effects of HBD on weight loss and gut microbiota. Our results indicated that the HBD resulted in a significant decrease in body mass index (BMI), and most of the physiological indicators were decreased to a greater degree in the effective HBD group (EHBD, weight loss ≥ 5%) than in the ineffective HBD group (IHBD, weight loss < 5%). The HBD intervention also modified the gut microbiota of the subjects with obesity. Specifically, Blautia, Lachnoclostridium, Terrisporobacter, Ruminococcus (R. torques, R. gnavus), and Pseudomonas were significantly reduced. In addition, we employed machine learning models, such as XGBRF and GB models, to rank the importance of various features and identified the top 10 key bacterial genera involved. Gut microbiota co-occurrence networks showed the dominance of healthier microbiota following successful weight loss. These results suggested that the HBD intervention enhanced weight loss, which may be related to diet-induced changes in the gut microbiota.

Tuning Thermostability and Catalytic Efficiency of Aflatoxin-Degrading Enzyme by Error-prone PCR.

In our previous work, a recombinant aflatoxin-degrading enzyme derived from Myxococcus fulvus (MADE) was reported. However, the low thermal stability of the enzyme had limitations for its use in industrial applications. In this study, we obtained an improved variant of recombinant MADE (rMADE) with enhanced thermostability and catalytic activity using error-prone PCR. Firstly, we constructed a mutant library containing over 5000 individual mutants. Three mutants with T50 values higher than the wild-type rMADE by 16.5 °C (rMADE-1124), 6.5 °C (rMADE-1795), and 9.8 °C (rMADE-2848) were screened by a high-throughput screening method. Additionally, the catalytic activity of rMADE-1795 and rMADE-2848 was improved by 81.5% and 67.7%, respectively, compared to the wild-type. Moreover, structural analysis revealed that replacement of acidic amino acids with basic amino acids by a mutation (D114H) in rMADE-2848 increased the polar interactions with surrounding residues and resulted in a threefold increase in the t1/2 value of the enzyme and made it more thermaltolerate. KEY POINTS: • Mutant libraries construction of a new aflatoxins degrading enzyme by error-prone PCR. • D114H/N295D mutant improved enzyme activity and thermostability. • The first reported enhanced thermostability of aflatoxins degrading enzyme better for its application.

Synergistic interactions in multispecies biofilm combinations of bacterial isolates recovered from diverse food processing industries.

Most biofilms within the food industry are formed by multiple bacterial species which co-exist on surfaces as a result of interspecies interactions. These ecological interactions often make these communities tolerant against antimicrobials. Our previous work led to the identification of a large number (327) of highly diverse bacterial species on food contact surfaces of the dairy, meat, and egg industries after routine cleaning and disinfection (C&D) regimes. In the current study, biofilm-forming ability of 92 bacterial strains belonging to 26 genera and 42 species was assessed and synergistic interactions in biofilm formation were investigated by coculturing species in all possible four-species combinations. Out of the total 455 four-species biofilm combinations, greater biofilm mass production, compared to the sum of biofilm masses of individual species in monoculture, was observed in 34 combinations. Around half of the combinations showed synergy in biofilm mass > 1.5-fold and most of the combinations belonged to dairy strains. The highest synergy (3.13-fold) was shown by a combination of dairy strains comprising Stenotrophomonas rhizophila, Bacillus licheniformis, Microbacterium lacticum, and Calidifontibacter indicus. The observed synergy in mixed biofilms turned out to be strain-specific rather than species-dependent. All biofilm combinations showing remarkable synergy appeared to have certain common species in all combinations which shows there are keystone industry-specific bacterial species which stimulate synergy or antagonism and this may have implication for biofilm control in the concerned food industries.

Pear pomace soluble dietary fiber ameliorates the negative effects of high-fat diet in mice by regulating the gut microbiota and associated metabolites.

The gut microbiota and related metabolites are positively regulated by soluble dietary fiber (SDF). In this study, we explored the effects of SDF from pear pomace (PP) on the regulation of gut microbiota and metabolism in high-fat-diet-fed (HFD-fed) C57BL/6J male mice. The results showed that PP-SDF was able to maintain the HFD disrupted gut microbiota diversity with a significant increase in Lachnospiraceae_UCG-006, Akkermansia, and Bifidobacterium spp. The negative effects of high-fat diet were ameliorated by PP-SDF by regulating lipid metabolisms with a significant increase in metabolites like isobutyryl carnitine and dioscoretine. Correlation analysis revealed that gut microbiota, such as Akkermansia and Lachnospiraceae_UCG-006 in the PP-SDF intervention groups had strong positive correlations with isobutyryl carnitine and dioscoretin. These findings demonstrated that PP-SDF interfered with the host's gut microbiota and related metabolites to reduce the negative effects caused by a high-fat diet.

Biofilm-based delivery approaches and specific enrichment strategies of probiotics in the human gut.

The use of probiotics has been one of the effective strategies to restructure perturbed human gut microbiota following a disease or metabolic disorder. One of the biggest challenges associated with the use of probiotic-based gut modulation strategies is to keep the probiotic cells viable and stable during the gastrointestinal transit. Biofilm-based probiotics delivery approaches have emerged as fascinating modes of probiotic delivery in which probiotics show significantly greater tolerance and biotherapeutic potential, and interestingly probiotic biofilms can be developed on food-grade surfaces too, which is ideal for the growth and proliferation of bacterial cells for incorporation into food matrices. In addition, biofilms can be further encapsulated with food-grade materials or with bacterial self-produced biofilms. This review presents a newly emerging and unprecedently discussed techniques for the safe delivery of probiotics based on biofilms and further discusses newly emerging prebiotic materials which target specific gut microbiota groups for growth and proliferation.

Application of stable isotopic and mineral elemental fingerprints in identifying the geographical originof concentrated apple juice in China.

Food traceability is an important component of food safety and quality. Currently, there is no authentic established technique to identify the origin of concentrated apple juice (CAJ) in China. In this study, the isotopes of δ13C, δ18O and the contents of 32 elements in CAJ from five production areas (BHB, NWR, SCH, LP and YRAR) were determined. The δ13C, δ18O and 28 elements were significantly different (P < 0.05: post-hoc Duncan's test) in the five production areas. PCA, PLS-DA and OPLS-DA were employed for regional classification of samples. The results show that ten key variables (Tl, Se, δ18O, B, Mg, Sr, Nd, Mo, As, and Na) are more relevant for discrimination of the samples. These findings contribute to understanding the variations of stable isotopic and element compositions in Chinese CAJ depending on geographic origins and offer valuable insight into the control of fraudulent labeling regarding the geographic origins of CAJ.

Preparation and Antioxidant Properties of Germinated Soybean Protein Hydrolysates.

In this study, soybeans during different germination stages were described and compared with regard to morphology, water content, protein, amino acids, and isoflavones. The optimal conditions for the hydrolysis of proteins obtained from germinated soybeans were determined using the response surface methodology. Gel filtration chromatography was used to separate germinated soybean protein hydrolysates after ultrafiltration, whereas 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ABTS•+, and FRAP assays were used to assess the antioxidant activity of different fractions. Findings of this study revealed that protein and isoflavone contents were high in soybean at 24 h following germination (the bud was about 0.5-1 cm). The proteins from germinated soybeans were hydrolyzed and separated into five fractions (G1-G5) and evaluated in terms of their molecular weight and antioxidant activity. Interestingly, the antioxidant activity was found to be higher in germinated soybean protein hydrolysates than in other soybean protein hydrolysates derived from soybean meal protein. This suggests that germination can effectively improve the utilization rate of soybean proteins. The antioxidant activity of G3 was best among G1-G5. The results obtained in this study demonstrate that germination for 24 h when the bud length is about 0.5-1 cm can be applied as a special pretreatment of plant seeds in the development of germinated foods. These findings can be used to identify the structure of the potential antioxidative hydrolysates for their possible exploitation in functional foods.

Trans-kingdom interactions in mixed biofilm communities.

The microbial world represents a phenomenal diversity of microorganisms from different kingdoms of life, which occupy an impressive set of ecological niches. Most, if not all, microorganisms once colonize a surface develop architecturally complex surface-adhered communities, which we refer to as biofilms. They are embedded in polymeric structural scaffolds and serve as a dynamic milieu for intercellular communication through physical and chemical signalling. Deciphering microbial ecology of biofilms in various natural or engineered settings has revealed coexistence of microorganisms from all domains of life, including Bacteria, Archaea, and Eukarya. The coexistence of these dynamic microbes is not arbitrary, as a highly coordinated architectural setup and physiological complexity show ecological interdependence and myriads of underlying interactions. In this review, we describe how species from different kingdoms interact in biofilms and discuss the functional consequences of such interactions. We highlight metabolic advances of collaboration among species from different kingdoms, and advocate that these interactions are of great importance and need to be addressed in future research. Since trans-kingdom biofilms impact diverse contexts, ranging from complicated infections to efficient growth of plants, future knowledge within this field will be beneficial for medical microbiology, biotechnology, and our general understanding of microbial life in nature.

Transcriptional Changes in Bifidobacterium bifidum Involved in Synergistic Multispecies Biofilms.

Bifidobacterium bifidum is part of the core microbiota of healthy infant guts where it may form biofilms on epithelial cells, mucosa, and food particles in the gut lumen. Little is known about transcriptional changes in B. bifidum engaged in synergistic multispecies biofilms with ecologically relevant species of the human gut. Recently, we reported prevalence of synergism in mixed-species biofilms formed by the human gut microbiota. This study represents a comparative gene expression analysis of B. bifidum when grown in a single-species biofilm and in two multispecies biofilm consortia with Bifidobacterium longum subsp. infantis, Bacteroides ovatus, and Parabacteroides distasonis in order to identify genes involved in this adaptive process in mixed biofilms and the influence on its metabolic and functional traits. Changes up to 58% and 43% in its genome were found when it grew in three- and four-species biofilm consortia, respectively. Upregulation of genes of B. bifidum involved in carbohydrate metabolism (particularly the galE gene), quorum sensing (luxS and pfs), and amino acid metabolism (especially branched chain amino acids) in both multispecies biofilms, compared to single-species biofilms, suggest that they may be contributing factors for the observed synergistic biofilm production when B. bifidum coexists with other species in a biofilm.

Characteristics of surface layer protein from Lactobacillus kefiri HBA20 and the role in mediating interactions with Saccharomyces cerevisiae Y8.

In this study, the surface layer protein (SLP) from Lactobacillus kefiri HBA20 was characterized. The SLP was extracted by 5M LiCl. The molecular mass of the SLP was approximately 64 kDa as analyzed via SDS-PAGE. The surface morphology and the adhesion potential of L. kefiri HBA20 in the absence and presence of SLP were measured by AFM. Moreover, the protein secondary structure was evaluated by using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR), respectively. SLP had high ß-sheet contents and low content of α-helix. Thermal analysis of SLP of Lactobacillus kefiri HBA20 exhibited one transition peak at 129.64 °C. Furthermore, SEM measurements were showed that after the SLP were removed from the cell surface, the coaggregation ability with Saccharomyces cerevisiae Y8 of the strain was significantly reduced. In conclusion, the SLP of Lactobacillus kefiri HBA20 has a stable structure and the ability of adhesion to yeast. Molecular docking study revealed that mannan bind with the hydrophobic residues of SLP. Our results will help further understanding of the new surface layer protein and the interaction between L. kefiri and S. cerevisiae.

New Trends in Photodynamic Inactivation (PDI) Combating Biofilms in the Food Industry-A Review.

Biofilms cause problems in the food industry due to their persistence and incompetent hygiene processing technologies. Interest in photodynamic inactivation (PDI) for combating biofilms has increased in recent years. This technique can induce microbial cell death, reduce cell attachment, ruin biofilm biomolecules and eradicate structured biofilms without inducing microbial resistance. This review addresses microbial challenges posed by biofilms in food environments and highlights the advantages of PDI in preventing and eradicating microbial biofilm communities. Current findings of the antibiofilm efficiencies of this technique are summarized. Additionally, emphasis is given to its potential mechanisms and factors capable of influencing biofilm communities, as well as promising hurdle strategies.

Proteomic Analysis Explores Interactions between Lactiplantibacillus plantarum and Saccharomyces cerevisiae during Sourdough Fermentation.

Sourdough is a fermentation culture which is formed following metabolic activities of a multiple bacterial and fungal species on raw dough. However, little is known about the mechanism of interaction among different species involved in fermentation. In this study, Lactiplantibacillus plantarum Sx3 and Saccharomyces cerevisiae Sq7 were selected. Protein changes in sourdough, fermented with single culture (either Sx3 or Sq7) and mixed culture (both Sx3 and Sq7), were evaluated by proteomics. The results show that carbohydrate metabolism in mixed-culture-based sourdough is the most important metabolic pathway. A greater abundance of L-lactate dehydrogenase and UDP-glucose 4-epimerase that contribute to the quality of sourdough were observed in mixed-culture-based sourdough than those produced by a single culture. Calreticulin, enolase, seryl-tRNA synthetase, ribosomal protein L23, ribosomal protein L16, and ribosomal protein L5 that are needed for the stability of proteins were increased in mixed-culture-based sourdough. The abundance of some compounds which play an important role in enhancing the nutritional characteristics and flavour of sourdough (citrate synthase, aldehyde dehydrogenase, pyruvate decarboxylase, pyruvate dehydrogenase E1 and acetyl-CoA) was decreased. In summary, this approach provided new insights into the interaction between L. plantarum and S. cerevisiae in sourdough, which may serve as a base for further research into the detailed mechanism.

Comprehensive Scanning of Prophages in Lactobacillus: Distribution, Diversity, Antibiotic Resistance Genes, and Linkages with CRISPR-Cas Systems.

Prophage integration, release, and dissemination exert various effects on host bacteria. In the genus Lactobacillus, they may cause bacteriophage contamination during fermentation and even regulate bacterial populations in the gut. However, little is known about their distribution, genetic architecture, and relationships with their hosts. Here, we conducted prophage prediction analysis on 1,472 genomes from 16 different Lactobacillus species and found prophage fragments in almost all lactobacilli (99.8%), with 1,459 predicted intact prophages identified in 64.1% of the strains. We present an uneven prophage distribution among Lactobacillus species; multihabitat species retained more prophages in their genomes than restricted-habitat species. Characterization of the genome features, average nucleotide identity, and landscape visualization presented a high genome diversity of Lactobacillus prophages. We detected antibiotic resistance genes in more than 10% of Lactobacillus prophages and validated that the occurrence of resistance genes conferred by prophage integration was possibly associated with phenotypic resistance in Lactobacillus plantarum. Furthermore, our broad and comprehensive examination of the distribution of CRISPR-Cas systems across the genomes predicted type I and type III systems as potential antagonistic elements of Lactobacillus prophage. IMPORTANCE Lactobacilli are inherent microorganisms in the human gut and are widely used in the food processing industries due to their probiotic properties. Prophages were reportedly hidden in numerous Lactobacillus genomes and can potentially contaminate entire batches of fermentation or modulate the intestinal microecology once they are released. Therefore, a comprehensive scanning of prophages in Lactobacillus is essential for the safety evaluation and application development of probiotic candidates. We show that prophages are widely distributed among lactobacilli; however, intact prophages are more common in multihabitat species and display wide variations in genome feature, integration site, and genomic organization. Our data of the prophage-mediated antibiotic resistance genes (ARGs) and the resistance phenotype of lactobacilli provide evidence for deciphering the putative role of prophages as vectors of the ARGs. Furthermore, understanding the association between prophages and CRISPR-Cas systems is crucial to appreciate the coevolution of phages and Lactobacillus.

Synergistic interactions prevail in multispecies biofilms formed by the human gut microbiota on mucin.

Bacterial species in the human gut predominantly exist in the form of mixed-species biofilms on mucosal surfaces. In this study, the biofilm-forming ability of many human gut bacterial strains (133 strains recovered from human faeces) on mucin-coated and non-coated polystyrene surfaces was determined. A significant variation (P < 0.05) in the biofilm-forming ability of many bacterial species on both surfaces was noticed. Based on some preliminary trials, four bacterial species were selected (Bifidobacterium bifidum, Bifidobacterium longum subsp. infantis, Parabacteroides distasonis and Bacteroides ovatus), which could not form any abundant biofilm individually under the in vitro conditions investigated, but produced abundant biofilms when co-cultured in different combinations of two, three and four species, giving an evidence of synergistic interactions in multispecies biofilm formation. There was a 4.74-fold increase in the biofilm mass when all strains developed a biofilm together. Strain-specific qPCR analysis showed that B. bifidum was the most dominant species (56%) in the four-species biofilm after 24 h, followed by B. longum subsp. infantis (36.2%). Study involving cell free supernatant of the cooperating strains showed that cell viability as well as physical presence of cooperating cells were prerequisites for the observed synergy in biofilms. The molecular mechanism behind these interactions and subsequent effects on the functionality of the strains involved were not determined in our study but merit further work.