Appl. Microbiol., Volume 4, Issue 3 (September 2024) – 17 articles

Bio-protection is one of the most popular natural protection methods to control food safety and shelf life. Lactic acid bacteria, especially Lactobacilli strains, are used in the food industry for this purpose due to their probiotic properties and, accordingly, bioprotective properties. We aimed to investigate the role of the bacteriocin-producing lactic acid bacteria Lactobacillus sakei and Pediococcus acidilactici in inducing microbiological, physicochemical, and chemical changes in the Turkish-style fermented sausage sucuk. The effects of protective cultures were compared with those of commercial starter cultures consisting of Pediococcus pentosaceus + Staphylococcus carnosus; a non-cultured group was used as a control. L. sakei inoculation and, to a lower extent, P. acidilactici inoculation resulted in the rapid domination of lactic acid bacteria (LAB) in the environment, whereas commercially used starter cultures and the non-cultured group showed lower counts of LAB. Moreover, L. sakei and P. acidilactici succeeded in inhibiting pathogens including S. aureus, E. coli, and Enterobacteriaceae. The number of enterococci decreased notably in the L. sakei-inoculated sucuk samples; however, an increase was determined in the samples inoculated with P. acidilactici. On the other side, the effect of commercial starter cultures was not sufficient for the inhibition of food-borne pathogens in the sucuk samples. Consequently, the inoculation of protective cultures, particularly of L. sakei, can provide a considerable contribution to improving microbial quality and food safety, retarding lipid oxidation, and increasing proteolytic activities in sucuk without important changes in its sensory properties. Full article

Functional vinegar with high γ-aminobutyric acid (GABA) content was manufactured through a two-stage serial co-fermentation of rice wine lees, a by-product of Korean rice wine, using lactic acid bacteria (LAB) and acetic acid bacteria (AAB). The first LAB fermentation elevated GABA content by utilizing monosodium glutamate (MSG) as a precursor. Lactiplantibacillus plantarum KS2020 converted up to 10% of MSG into GABA and indicated a GABA content of 65.49 mg/g. The concentration of LAB-fermented rice wine lees was then optimized for the second co-fermentation, and Acetobacter aceti was used to produce vinegar. Co-fermentation using 40% first LAB-fermented rice wine lees yielded vinegar with 55.34 mg/g acetic acid and 22.61 mg/g GABA. The temperature-dependent reduction in GABA in GABA-enriched vinegar followed the Arrhenius relationship during storage, with an activation energy of 9.94 kcal/mol (20–35 °C, R² = 0.99). The GABA present in the vinegar showed evidence of a temperature-/time-dependent decrease, decreasing by 40% over five months. This study first proved the higher GABA-enriched vinegar production from rice wine lees using Lb. plantarum KS2020 and A. aceti. Full article

Halal means permissible according to Islamic law. Halal meat is obtained by hand slaughtering an animal that is not stunned and that is blessed by a Muslim individual immediately before slaughter. The purpose of this study was to determine the microbiological quality of raw meat from halal meat markets. A total of 138 beef samples were purchased from three halal (n = 72 samples) and three non-halal markets (n = 66 samples) between November 2016 and October 2017. All samples were analyzed for the presence of indicator organisms—aerobic plate counts (APCs), Enterobacteriaceae counts (ECs), total coliform counts (TCCs), and generic Escherichia coli (ECCs). The levels of APCs, ECs, TCCs, and ECCs (mean log CFU/g) in halal samples were 4.93 (100%), 2.89 (91.7%), 2.87 (94.4%), and 1.09 (18.1%), respectively, and those in non-halal samples were 4.92 (100%), 3.07 (95.5%), 3.02 (89.4%), and 1.15 (16.7%), respectively. The levels of TCCs and ECs were higher in halal samples during the summer compared to the other three seasons, whereas the highest ECCs in halal samples were found during autumn. In non-halal samples, significant differences were observed in the ECCs, TCCs, and ECs across seasons, with the highest level of contamination during autumn. Samples having higher levels of indicator organisms (APCs, ECs, TCCs, and ECCs) were more likely to be positive for pathogenic bacteria. The high levels of indicator organisms in both halal and non-halal retail meat samples suggest that the operation size, and not halal or non-halal meat classification, is associated with the microbiological quality. These findings can inform food safety interventions targeting small meat markets in the United States. Full article

Phosphorus (P) solubilization is one of the major traits for plant growth-promoting rhizobacteria since P is easily rendered insoluble in soil. Phosphate-solubilizing microorganisms can be harnessed as an environment-friendly strategy to enhance the mobilization and acquisition of P by crops. Utilization of such microorganisms as microbial inoculants in agriculture serves as an alternative to chemical fertilizers and an approach for more efficient P fertilization. Hence, this study aims to characterize a phosphate-solubilizing isolate and evaluate its potential as a microbial inoculant. Morphological, biochemical, and genetic characterization of the isolate were performed. Then, the mineral phosphate solubilization ability of the isolate was evaluated. Lastly, this study evaluated the plant growth promotion of the isolate as a single inoculant in rice or as a co-inoculant with rhizobia in peanuts. On the basis of biochemical and 16S rRNA analysis, the isolate was identified as Enterobacter sp. Also, it can solubilize P from tricalcium phosphate or aluminum phosphate. Simultaneous with P solubilization, medium acidification, and gluconic acid secretion were observed. Lastly, the Enterobacter sp. isolate could potentially be developed as a biofertilizer in reducing P resource input or to enhance the performance of a rhizobia inoculant. Full article

Fermented foods made through microbial growth and enzymatic conversions have been integral to human diets for at least 10,000 years. Recent interest in fermented foods has surged due to their functional properties and health benefits. Cruciferous vegetables of the genus Brassica, such as cabbage, broccoli, and cauliflower, are commonly used to produce fermented foods like sauerkraut, kimchi, pao cai, fermented turnips, and others. These foods are rich in lactic acid bacteria (LAB) and bioactive compounds, which contribute to their potential health-promoting properties. We examined 12 clinical trials investigating fermented foods of the genus Brassica. These studies, which mainly assessed the health benefits of kimchi or sauerkraut consumption, found that regular intake can alleviate symptoms of irritable bowel syndrome (IBS), aid weight loss, and enhance metabolic health. Seven observational studies also observed health benefits when consuming fermented foods of the genus Brassica. Six of the seven observational studies on kimchi intake linked kimchi intake to reduced obesity risk and other health benefits. An observational study linked sauerkraut and cabbage consumption to reduced breast cancer risk. Despite these findings, the exact roles of various microorganisms and bioactive compounds within these health effects require further investigation. This review underscores the potential of fermented cruciferous vegetables as functional foods, and advocates for more clinical trials and mechanistic studies to understand and optimize their health benefits. Full article

Comparative metatranscriptomics of the bacterial and yeast communities of two milk kefir beverages (MKAA1 and MKAA2) was carried out. They were obtained by fermentation with two different frozen stocks of the kefir grain CIDCA AGK1, differing in rheological features and production of organic acids. We hypothesised that the differences in their physicochemical and rheological properties might be due to the microbial activity in each product. The dominance of lactic acid bacteria, yeast, and a marginal amount of acetic acid bacteria characterised the microbiome. The bacterial families Lactobacillaceae and Streptococcaceae accounted for almost all of the bacterial gene transcripts, with Lactobacillus helveticus, L. kefiranofaciens, L. gallinarum, and Lactococcus lactis being most frequent in the microbiome of the MKAA1 beverage and L. kefiranofaciens, Lc. Lactis, and Leuconostoc mesenteroides being the most prevalent in MKAA2. Dipodascaceae and Saccharomycetaceae were the leading yeast families, represented by Yarrowia lipolytica, Saccharomyces unisporus, and Kluyveromyces marxianus. MKAA1 and MKAA2 shared >75% KEGG Ortologs (KOs) in their bacteria and yeast libraries. The considerable decreases in total expressed genes (KEGG Ortologs) assigned to Lactobacillus helveticus and L. gallinarum might be related to the variations in the rheological features of the beverages, probably by compromising the interrelations with L. kefiranofaciens, which might explain the variations in the rheological features of the beverages. Full article

According to a report by the World Health Organization (WHO), each year, over 550 million individuals worldwide suffer from and 230,000 die from diarrheal illnesses, which accounts for more than half of the global foodborne disease burden. Among them, children face a heightened vulnerability, with approximately 220 million falling ill and 96,000 succumbing to these diseases annually. This work aimed to study the genomic characterization of selected E. coli strains from catfish (Clarias (C.) gariepinus) caught from the Onitsha North axis of the River Niger in Anambra state, Nigeria. A total of 50 fish were randomly purchased from different fishermen over a period of four months. Samples that comprised six different organs (skin, flesh, gills, gonads, guts, and liver) were screened for E. coli strains using cultural and biochemical methods. Multilocus sequence typing (MLST) and core genome (cg)MLST were performed using Ridom SeqSphere+ software. The aerobic plate count (APC) and coliform count ranged from 0.5 × 10⁴ to 3.7 × 10⁴ cfu/g and 0 to 3.0 × 10⁴ cfu/g, respectively. Whole-genome sequencing (WGS) confirmed the presence of E. coli and Klebsiella quasipneumoniae isolates in our samples. We could identify only two serotypes (O102:H7 and O40:H4) of E. coli. Antimicrobial resistance genes (ARGs) and point mutations that conferred antibiotic resistance were extracted from the genome assemblies. Good hygiene is recommended to avoid the cross-contamination of raw C. gariepinus with ready-to-eat food. Full article

A species-specific multiplex-PCR method and phenotypic tests were combined to evaluate biochemical and genotypic differences between 24 representative Leuconostoc mesenteroides diverse isolates previously found to dominate in spoiled, vacuum-packed Anthotyros whey cheeses stored at 4 °C for 40 days and identified by 16S rRNA gene sequencing. Based on their phenotypic (API 50 CHL) profiles, the 24 isolates comprised 6 multi-strain and 7 single-strain biotypes. Only two single-strain biotypes (L4A and L4B) produced slime (dextran) from sucrose, and only four biotypes (L2A–L2C, L3; 7 isolates) fermented L-arabinose; the remaining 15 isolates (biotypes L1A–L1F) were dextran-negative, oligofermenting Ln. mesenteroides variants, able to ferment D-xylose and grow at 37 °C. Based on their multiplex-PCR (rpoB, araA, dsr, and sorA) gene profiles in comparison with those of the type strains of the four Ln. mesenteroides subsp. cremoris (rpoB), dextranicum (rpoB/dsr), mesenteroides (rpoB/araA/dsr/sorA), and jonggajibkimchii (rpoB/araA/dsr), no isolate was assigned to the first two subspecies and only four isolates (L2A and L2C) to the subsp. mesenteroides. Ten isolates shared the subsp. jonggajibkimchii profile, while the other ten ones have a fifth atypical profile (rpoB/dsr/sorA), seemingly being closer to the subsp. dextranicum. Particularly the atypical biotype L1B representatives of the most prevalent psychrotrophic Ln. mesenteroides subsp. jonggajibkimchii (rpoB/araA/dsr) genotype at Anthotyros whey cheese spoilage deserve further biochemical and molecular characterization studies. Full article

Strawberry cultivation holds significant economic and social promise within Peruvian fruit production. However, conventional management practices have led to the excessive use of agrochemicals in this crop. This study proposes an organic approach to strawberry production, integrating less environmentally harmful technologies. The aim was to assess microbial inoculation by using Trichoderma sp. and Pseudomonas putida and the application of organic amendments on strawberry seedlings of the commercial cultivar “San Andreas”. A field experiment was established with evaluations in the vegetative and productive stages. Results indicate that the co-inoculation of Trichoderma sp. and Pseudomonas putida increased leaf area by 7%, and enhanced the aerial part’s fresh and dry biomass by 13% and 28%, respectively, compared to treatment without microbial inoculation. Concurrently, compost application increased the leaf number and aerial dry biomass by 22% and 19% at the end of the vegetative stage, respectively, compared to treatment without organic amendment. In addition, it reduced the days for flowering, maintaining the fruit’s physicochemical attributes. Regarding yield, the amendments application significantly enhanced fruit weight per plant by 40%, especially when applied together with Trichoderma sp., and co-inoculation increased the number of fruits per meter square by 22%. These findings highlight the potential of technologies such as microbial inoculation and organic amendments to enhance strawberry yields and to gradually reduce the use of synthetic fertilizers. Full article

Because of its swelling capacity, compacted bentonite clay is a suitable buffer material in deep geological repositories for high-level nuclear waste. However, this only applies if the swelling capacity is maintained. Accordingly, bentonites have to be stable to changing temperature, humidity, infiltrating fluids or microbial activity. In batch experiments, we investigated combined microbial and thermo-hydro-geochemical effects on the swelling capacity of uncompacted bentonite MX-80. Bentonite was exposed to fluids of different ionic strength and the bacterium Stenotrophomonas bentonitica. Bacterial growth was monitored by counting colony-forming units while the swelling capacity of bentonite was evaluated using in situ XRD at varied temperatures and humidity. The presence of bentonite prolonged the survival of S. bentonitica. However, electron microscopy, XRD and ICP-OES analyses showed neither an interaction of S. bentonitica with bentonite, nor significant changes in the swelling capacity or element composition. The swelling capacity and diffraction peak intensity were, however, strongly reduced by the ionic strength of the fluid and the exposure time. The study highlights that bentonite is affected by thermo-hydro-geochemical and microbial processes to different degrees and that the complexity of different co-occurring factors in potential nuclear waste repositories is important to consider in safety assessments. Full article

Culture-dependent and culture-independent microbiological methods are two approaches used to study microbial community composition. Culture-dependent methods have been the standard method used for many years but have limited utility with unculturable microorganisms. Culture-independent methods, including molecular techniques, enable direct analysis of microbial DNA without requiring cultivation. Both culture-dependent and -independent methods have roles in advancing our understanding of microbiology, and a combination of these approaches often yields a comprehensive depiction of the microbial diversity within a dynamic system. Bacterial activity reaction tests (BARTs) are a common culture-dependent test used to identify bacteria growing in industrial water samples. In this study, next-generation sequencing (NGS) was used to identify the taxa growing in BARTs and compared with the BART reaction patterns. Additionally, several water samples were analyzed by both BART and NGS analysis to determine whether the bacteria found in the water were also present in the BARTs. The results showed overall agreement between NGS and BARTs, though, in some cases, the most abundant taxa found in the water samples differed from those in the BARTs. This highlights the need for further study into the microbial community dynamics of culture-dependent tests to determine whether they are representative of the original sample. Full article

The Bacillus megaterium LVN01 species native to Colombia has demonstrated the ability to metabolize different coproducts or industrial waste (such as fique juice, cane molasses, and residual glycerol) and accumulate polyhydroxybutyrate (PHB), giving it potential in the bioplastics industry. In this research, the potential of liquid digestate as a carbon source for the production of PHA polymers in fermentation processes with this bacterial strain was evaluated. Favorably, it was found that B. megaterium utilizes the nutrients from this residual substrate to multiply appropriately and efficiently synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Bench-scale aerobic batch fermentation, under the operational conditions of this research [volume: 3 L; temperature: 30.8 °C; agitation: 400 rpm; pH: 7.0 ± 0.2; dissolved oxygen: 100% saturation; antifoam: 10% (v/v)], generated maximum values of dry cell weight (DCW) (0.56 g cell L⁻¹) at 60 h, while the maximum PHBV yield (360 mg PHBV L⁻¹) occurred at 16 h, which is very favorable for sustainable degradable bioplastics production. Additionally, GC–MS and NMR analyses confirmed that the PHBV copolymer synthesized by B. megaterium is made up of the monomers 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Furthermore, the thermal properties determined by TGA (Tonset = 283.1 °C; Tendset = 296.98 °C; Td = 290.114 °C) and DSC (Tm = °C 155.7 °C; ΔHf = 19.80 J g⁻¹; Xcr = 18.17%) indicate that it is a thermally stable biopolymer with low percentages of crystallinity, providing flexibility that facilitates molding, adaptation, and application in various industrial sectors. Full article

This study addresses the challenge of finding novel ways to solubilize phosphorus and zinc for agricultural purposes. The aim was to isolate PSMs (phosphorous-solubilizing microbes) and ZnSMs (zinc-solubilizing microbes) from different environments (e.g., soil amendments, land uses, and crop rotation systems) and evaluate their ability to solubilize different insoluble P sources (e.g., β-tricalcium phosphate (β-TCP), calcium-phytate (CaP), and rock phosphate (RP)) and Zn sources (e.g., zinc carbonate (ZnC), zinc oxide (ZnO), and zinc phosphate (ZnP)). Here, 25 isolates capable of solubilizing either P or Zn sources were isolated and classified by species using 16S rRNA and ITS-region sequencing. Notably, Aspergillus awamori, Fusarium circinatum, Fusarium longifundum, and Mucor circinelloides, isolated from cultivated soils and soil amendments, emerged as the most efficient PSMs and ZnSMs. Mucor circinelloides exhibited the highest solubilization ability for broths containing β-TCP, CaP, RP, ZnO, and ZnP, with log2-fold changes of 3.7, 1.8, 8.9, 7.8, and 2.4, respectively, compared to the control. For ZnC and ZnO, Aspergillus awamori displayed the highest Zn solubilization, with a 2.1 and 3.0 log2-fold change. The study highlights the potential of these strains as biofertilizers and underscores the role of Mucor and Fusarium genera in zinc solubilization. Full article

The production of value-added products from microorganisms, such as single-cell protein (SCP), through the valorization of agricultural byproducts enhances circular economy while offering alternative solutions for waste treatment. In this study, SCP was obtained through the biotechnological treatment and valorization of cheese whey, the main byproduct of the dairy industry, for the development of novel edible films. To the best of the authors’ knowledge, this is the first report examining SCP as a biopolymer for edible film production. Specifically, Kluyveromyces marxianus, which has gained QPS and GRAS status, strain EXF-5288 cultivated in deproteinized cheese whey (DCW) lactose (10.0 g/L) in a 3 L fed-batch bioreactor, resulting in a SCP_max of 2.63 g/L with a protein content of up to 49.1% w/w. The addition of increased glycerol concentrations (30, 40, and 50% w/w of dry cells) as plasticizers was examined to develop SCP-based edible films. Regarding physicochemical characterization, increased glycerol concentration significantly increased moisture content (MC%) and solubility (S%), but there was not a significant difference in other parameters. Regarding wettability, SCP-based films could be described as oleophilic surfaces since the degree of oil contact angle (OCA) ranged between 46.7° ± 1.3 and 54.0° ± 0.5. The proposed holistic approach could contribute to the development of sustainable packaging materials through waste treatment. Full article

Human adenovirus (HAdV) F40/41 is an important pathogen in pediatric acute gastroenteritis cases. However, the diversity of study designs and diagnostic methods often leads to misinterpretations of their impact. Our study explored the genetic diversity of HAdV-F40/41 in Brazil using a specific qPCR assay for HAdV species F, combined with a phylogenetic analysis of the partial hexon and fiber genes. Our results demonstrated that HAdV-F41 strains predominated and exhibited higher diversity than HAdV-F40 strains. Based on the hexon gene, Brazilian HAdV-F41 strains were grouped into two genome type clusters (GTC), further divided into subclusters, with most strains clusteringto GTC2. The partial shaft region of the fiber gene exhibited higher conservation among HAdV-F41. The specific qPCR assay for HAdV species F identified HAdV-F in an additional 31.5% (34/108) of previously uncharacterized HAdV-positive samples detected using a non-specific HAdV qPCR assay. Both assays strongly correlated in detecting HAdV-F, and the specific qPCR assay for enteric types can enhance HAdV surveillance, especially when sequencing is not possible. Our study provides novel insights regarding the genetic diversity of HAdV-F species in Brazil. Full article

Beneficial microbes are crucial for improving crop adaptation and growth under various stresses. They enhance nutrient uptake, improve plant immune responses, and help plants tolerate stresses like drought, salinity, and heat. The yield potential of any crop is significantly influenced by its associated microbiomes and their potential to improve growth under different stressful environments. Therefore, it is crucial and exciting to understand the mechanisms of plant–microbe interactions. Maize (Zea mays L.) is one of the primary staple foods worldwide, in addition to wheat and rice. Maize is also an industrial crop globally, contributing 83% of its production for use in feed, starch, and biofuel industries. Maize requires significant nitrogen fertilization to achieve optimal growth and yield. Maize plants are highly susceptible to heat, salinity, and drought stresses and require innovative methods to mitigate the harmful effects of environmental stresses and reduce the use of chemical fertilizers. This review summarizes our current understanding of the beneficial interactions between maize plants and specific microbes. These beneficial microbes improve plant resilience to stress and increase productivity. For example, they regulate electron transport, downregulate catalase, and upregulate antioxidants. We also review the roles of plant growth-promoting rhizobacteria (PGPR) in enhancing stress tolerance in maize. Additionally, we explore the application of these microbes in maize production and identify major knowledge gaps that need to be addressed to utilize the potential of beneficial microbes fully. Full article

Vitamin B₁₂ is a critical nutrient in vegan and vegetarian lifestyles as plant-based vitamin sources are rare. Traditional fermented foods could be enriched by adding vitamin B₁₂-producing bacteria to offer non-animal vitamin sources. The aim was to isolate a vitamin B₁₂ producer that is capable of producing the human-active vitamin even at low pH values so that it can be used in fruit juice fortification. Therefore, fermented foods (homemade and industrial) and probiotics were screened for vitamin B₁₂ production strains. A modified microbiological vitamin B₁₂ assay based on Lactobacillus delbrueckii subsp. lactis DSM 20355 was used to identify vitamin B₁₂-containing samples and the presence of vitamin B₁₂-producing strains. The screening resulted in isolating several positive strains for vitamin B₁₂ formation derived from sourdough. Mass spectrometry confirmed the biosynthesis of solely the human physiologically active form. Species identification carried out by the German Strain Collection of Microorganisms and Cell Cultures resulted in two species: Acetobacter orientalis and Acetobacter malorum, of which two isolates were further characterised. The potential for cobalamin biosynthesises in food matrixes was demonstrated for A. malorum HFD 3141 and A. orientalis HFD 3031 in apple juice at different pH values (2.85–3.80). The isolates synthesised up to 18.89 µg/L and 7.97 µg/L vitamin B₁₂ at pH 3.80. The results of this study suggest that acetic acid bacteria (AAB) and fermented acetic acid foods are promising resources for vitamin B₁₂ and its producers, which might have been overlooked in the past. Full article