Human Gut Research Articles

Enhancing the stability of a novel D-allulose 3-epimerase from Ruminococcus sp. CAG55 by interface interaction engineering and terminally attached a self-assembling peptide

D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.

BN-BacArena: Bayesian network extension of BacArena for the dynamic simulation of microbial communities.

Simulating gut microbial dynamics is extremely challenging. Several computational tools, notably the widely used BacArena, enable modeling of dynamic changes in the microbial environment. These methods, however, do not comprehensively account for microbe-microbe stimulant or inhibitory effects or for nutrient-microbe inhibitory effects, typically observed in different compounds present in the daily diet. Here, we present BN-BacArena, an extension of BacArenaconsisting on the incorporation within the native computational framework of a Bayesian network model that accounts for microbe-microbe and nutrient-microbe interactions. Using in vitro experiments, 16S rRNA gene sequencing data and nutritional composition of 55 foods, the output Bayesian network showed 23 significant nutrient-bacteria interactions, suggesting the importance of compounds such as polyols, ascorbic acid, polyphenols and other phytochemicals, and 40 bacteria-bacteria significant relationships. With test data, BN-BacArena demonstrates a statistically significant improvement over BacArena to predict the time-dependent relative abundance of bacterial species involved in the gut microbiota upon different nutritional interventions. As a result, BN-BacArena opens new avenues for the dynamic modeling and simulation of the human gut microbiota metabolism. MATLAB and R code are available in https://github.com/PlanesLab/BN-BacArena.

The macroecology of butyrate-producing bacteria via metagenomic assessment of butyrate production capacity.

Butyrate-producing bacteria are found in many outdoor ecosystems and host organisms, including humans, and are vital to ecosystem functionality and human health. These bacteria ferment organic matter, producing the short-chain fatty acid butyrate. However, the macroecological influences on their biogeographical distribution remain poorly resolved. Here we aimed to characterise their global distribution together with key explanatory climatic, geographical and physicochemical variables. We developed new normalised butyrate production capacity (BPC) indices derived from global metagenomic (n = 13,078) and Australia-wide soil 16S rRNA (n = 1331) data, using Geographic Information System (GIS) and modelling techniques to detail their ecological and biogeographical associations. The highest median BPC scores were found in anoxic and fermentative environments, including the human (BPC = 2.99) and non-human animal gut (BPC = 2.91), and in some plant-soil systems (BPC = 2.33). Within plant-soil systems, roots (BPC = 2.50) and rhizospheres (BPC = 2.34) had the highest median BPC scores. Among soil samples, geographical and climatic variables had the strongest overall effects on BPC scores (variable importance score range = 0.30-0.03), with human population density also making a notable contribution (variable importance score = 0.20). Higher BPC scores were in soils from seasonally productive sandy rangelands, temperate rural residential areas and sites with moderate-to-high soil iron concentrations. Abundances of butyrate-producing bacteria in outdoor soils followed complex ecological patterns influenced by geography, climate, soil chemistry and hydrological fluctuations. These new macroecological insights further our understanding of the ecological patterns of outdoor butyrate-producing bacteria, with implications for emerging microbially focused ecological and human health policies.

Association between monosodium glutamate consumption with changes in gut microbiota and related metabolic dysbiosis—A systematic review

AbstractMonosodium glutamate (MSG) is used as a common food additive in some foods. However, based on our search and knowledge, no comprehensive study discussed the effect of MSG on the human gut microbiome. In this study, the effects of MSG on the gut microbiome, liver, and kidney were performed. Data were collected from databases including PubMed, Scopus, Web of Science, and ScienceDirect using the search strategy and keywords. Finally, 14 eligible studies were selected for systematic review. This study provides a new perspective on the effects of MSG on the gut flora, shedding light on the potential relationship between MSG intake and human health.

Nanopore and Illumina sequencing reveal different viral populations from human gut samples.

The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments, including the human intestinal microbiome. However, bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics. Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples. Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina is preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples.

The Role of Prebiotics in Modulating Gut Microbiota: Implications for Human Health.

The human gut microbiota, an intricate ecosystem within the gastrointestinal tract, plays a pivotal role in health and disease. Prebiotics, non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of beneficial microorganisms, have emerged as a key modulator of this complex microbial community. This review article explores the evolution of the prebiotic concept, delineates various types of prebiotics, including fructans, galactooligosaccharides, xylooligosaccharides, chitooligosaccharides, lactulose, resistant starch, and polyphenols, and elucidates their impact on the gut microbiota composition. We delve into the mechanisms through which prebiotics exert their effects, particularly focusing on producing short-chain fatty acids and modulating the gut microbiota towards a health-promoting composition. The implications of prebiotics on human health are extensively reviewed, focusing on conditions such as obesity, inflammatory bowel disease, immune function, and mental health. The review further discusses the emerging concept of synbiotics-combinations of prebiotics and probiotics that synergistically enhance gut health-and highlights the market potential of prebiotics in response to a growing demand for functional foods. By consolidating current knowledge and identifying areas for future research, this review aims to enhance understanding of prebiotics' role in health and disease, underscoring their importance in maintaining a healthy gut microbiome and overall well-being.

Flavonoids Biotransformation by Human Gut Bacterium Dorea sp. MRG-IFC3 Cell-Free Extract.

Human gut bacterium Dorea sp. MRG-IFC3 is unique in that it only metabolizes puerarin, an isoflavone C-glycoside, whereas it shows broad substrate glycosidase activity for the various flavonoid Oglycosides. To address the question on the substrate specificity, as well as biochemical characteristics, cell-free biotransformation of flavonoid glycosides was performed under various conditions. The results showed that there are two different enzyme systems responsible for the metabolism of flavonoid C-glycosides and O-glycosides in the MRG-IFC3 strain. The system responsible for the puerarin conversion was inducible and comprised of two enzymes; one oxidizes puerarin to 3"-oxopuerarin and the other converts 3"-oxo-puearin to daidzein. The second enzyme was only active toward 3"-oxo-puerarin. The activity of puerarin conversion to daidzein was enhanced in the presence of Mn+2 and NAD+ . It was concluded that puerarin C-deglycosylation by Dorea sp. MRG-IFC3 possibly adopts the same biochemical mechanism as the strain PUE, a species of Dorea longicatena.

Analysis of the fermentation kinetics and gut microbiota modulatory effect of dried chicory root reveals the impact of the plant-cell matrix rationalizing its conversion in the distal colon

Aim: The cell matrix of plant foods has received little attention in prebiotic fiber research. We aimed to understand the impact of the plant cell matrix in dried chicory root on its breakdown in the human gut to explain its reported beneficial effects on gut and metabolic health. Methods: We applied in vitro digestion and fermentation models together with an ex vivo gut barrier integrity model. Plant cell matrix intactness in the upper gastrointestinal tract was investigated by scanning electron microscopy. Colonic breakdown of inulin, and chicory root cubes and powder was assessed by gut microbiota analysis using 16S rRNA gene amplicon sequencing and determining the kinetics of changes in pH, gas, and short-chain fatty acid production (SCFA). Finally, effects on gut barrier integrity were explored by exposing colonic biopsies to fermentation supernatants in an Ussing chamber model. Results: The plant cell matrix of dried chicory root cubes remained intact throughout upper gastrointestinal transit. Dried chicory root fermentation resulted in higher final relative abundances of pectin-degrading Monoglobus and butyrate-producing Roseburia spp. compared to inulin and a seven-fold increase in Bifidobacterium spp. in donors where these species were present. Dried chicory root cubes yielded similar total SCFAs but higher final butyrate levels than chicory root powder or isolated inulin with less gas produced. No uniform but donor-specific effects of fermentation supernatants on the maintenance of gut barrier integrity were detected. Conclusion: The intact plant cell matrix of dried chicory root affected its colonic breakdown kinetics and microbiota, underpinning its beneficial effect in vivo .

Exploring the prebiotic characteristics of crude polysaccharides from coconut testa flour: A comparative analysis of local cultivar

Crude polysaccharides (CPs) of coconut testa flour (CTF) of five local cultivars; Commercial hybrid (COM), Gon Thembili (GT), Ran Thembili (RT), San Ramon (SR), and Tall × Tall (TT) were isolated in this study to examine to evaluate potential prebiotic properties and structural characteristics. The isolated CPs were subjected to FTIR analysis to identify functional groups of biomolecules. Digestibility tests were performed on CPs of different coconut cultivars using artificial human gastric juice. CPs were applied to Lactobacillus sp. and Escherichia coli to evaluate their proliferation in vitro. FTIR results showed that the occurrence of peaks between 1200 cm−1 – 1000 cm−1 was indicative of the presence of C–O side groups and C–O–C glycosidic bonds. CPs of all cultivars displayed strong resistance to hydrolysis (94–97 %) by human gastric juice when compared to inulin and fructooligosaccharides (FOS). The higher proliferation of Lactobacillus sp. was detected on all five CPs when compared to that of glucose. The SR cultivar (1.09) showed a higher prebiotic activity score compared to fructooligosaccharides (0.92) and inulin (0.81). These findings suggest that CPs isolated from CTF of local coconut cultivars possess potential prebiotic characteristics that can be used in the preparation of functional foods. Further studies on this aspect are granted to enable the effective use of CTF in promoting human gut health.

New Insights in Prebiotic Utilization: A Systematic Review

The hectic pace of modern life often leads to quick solutions, both in lifestyle and the choice of food we consume. The importance of the gut microbiome and its balance is being increasingly researched, with the prebiotic concept itself becoming a topic of scientific investigation. The aim of this paper is to analyze scientific studies on the understanding of prebiotics conducted between 2019 and 2024 in order to see what new knowledge, new sources, new ways of use, and newly established effects on certain disease states have been discovered during this period. The question that the authors are trying to answer is how specific prebiotics affect the growth and activity of selected probiotic strains in the human gut (have impact on gut microbiome) and what the implications of these interactions are. Four databases were searched: Pubmed/MEDLINE, Springerlink, Google Scholar, and Scopus. The keywords used were prebiotics, functional food, probiotics, gut microbiome, and trends. A systematic review of 30 scientific studies on the topic of prebiotics revealed significant advances in understanding and application. Research particularly indicates how prebiotics stimulate the growth of beneficial probiotic strains, such as Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, and Bifidobacterium. In addition, innovative approaches in food production, including pasta rich in prebiotic fibers, chocolate with inulin and stevia, and the use of fruit by-products, show promising results in creating “healthier” food options. Although the papers had differing objectives and research methodologies, certain similarities were found. All papers emphasized the importance of using prebiotics, although it depended on the type they come from and their impact on the gut microbiome, i.e., the stimulation of probiotic action within the gut microbiome, which consequently has benefits on health. This review serves as a springboard for further research in this exciting field, with the ultimate goal of harnessing the power of prebiotics to improve health outcomes.

CARE: Large Precision Matrix Estimation for Compositional Data

High-dimensional compositional data are prevalent in many applications. The simplex constraint poses intrinsic challenges to inferring the conditional dependence relationships among the components forming a composition, as encoded by a large precision matrix. We introduce a precise specification of the compositional precision matrix and relate it to its basis counterpart, which is shown to be asymptotically identifiable under suitable sparsity assumptions. By exploiting this connection, we propose a composition adaptive regularized estimation (CARE) method for estimating the sparse basis precision matrix. We derive rates of convergence for the estimator and provide theoretical guarantees on support recovery and data-driven parameter tuning. Our theory reveals an intriguing tradeoff between identification and estimation, thereby highlighting the blessing of dimensionality in compositional data analysis. In particular, in sufficiently high dimensions, the CARE estimator achieves minimax optimality and performs as well as if the basis were observed. We further discuss how our framework can be extended to handle data containing zeros, including sampling zeros and structural zeros. The advantages of CARE over existing methods are illustrated by simulation studies and an application to inferring microbial ecological networks in the human gut. Supplementary materials for this article are available online, including a standardized description of the materials available for reproducing the work.

Identification of plastic-degrading bacteria in the human gut

Environmental pollution caused by the excessive use of plastics has resulted in the inflow of microplastics into the human body. However, the effects of microplastics on the human gut microbiota still need to be better understood. To determine whether plastic-degrading bacteria exist in the human gut, we collected the feces of six human individuals, did enrichment cultures and screened for bacterial species with a low-density polyethylene (LDPE) or polypropylene (PP)-degrading activity using a micro-spray method. We successfully isolated four bacterial species with an LDPE-degrading activity and three with a PP-degrading activity. Notably, all bacterial species identified with an LDPE or PP-degrading activity were opportunistic pathogens. We analyzed the microbial degradation of the LDPE or PP surface using scanning electron microscopy and confirmed that each bacterial species caused the physical changes. Chemical structural changes were further investigated using X-ray photoelectron spectroscopy and Fourier-transform-infrared spectroscopy, confirming the oxidation of the LDPE or PP surface with the formation of carbonyl groups (C=O), ester groups (CO), and hydroxyl groups (-OH) by each bacterial species. Finally, high temperature gel permeation chromatography (HT-GPC) analysis showed that these bacterial species performed to a limited extent depolymerization. These results indicate that, as a single species, these opportunistic pathogens in the human gut have a complete set of enzymes and other components required to initiate the oxidation of the carbon chains of LDPE or PP and to degrade them. Furthermore, these findings suggest that these bacterial species can potentially biodegrade and metabolize microplastics in the human gut.

Scoping review on soil microbiome and gut health—Are soil microorganisms missing from the planetary health plate?

Abstract Evidence suggests that soil microorganisms, to which humans have been exposed throughout our evolutionary history, were essential for the evolution of the human gut microbiome and immunological resilience. In recent decades, gut microbial diversity has decreased along with urbanization and global loss of biodiversity. At the same time, the prevalence of chronic, non‐communicable inflammatory diseases has been increasing in urbanized societies. Here, we investigate whether the existing literature supports the hypothesis that ingesting soil microbiota provides immunological resilience. We focus on identifying research gaps and challenges that must be overcome to understand the effects of eating soil on immunological resilience. A scoping review of the peer‐reviewed empirical literature was carried out. The search focused on studies that exposed mammals to direct soil contact and measured the gut microbiota and host response; 12 articles, including four human, six mouse and two other mammal studies, were identified for inclusion in the review. In addition, seven articles related to human health risks associated with soil consumption were included in the review. Studies indicate that ingesting soil‐bound microorganisms supports immunological resilience. There is a lack of intervention studies that include human study subjects that test orally the effect of soil microorganisms on the gut microbiota and immunological resilience. Challenges, particularly soil heterogeneity and risks related to soil biological and chemical properties, have prevented progress to fully understand the effect of oral intake of soil‐bound microorganisms on human gut microbiota and health. The results encourage the development of technologies that allow daily exposure to soil microbiota. Future research should be aimed to precisely test the importance of oral intake of soil‐bound microorganisms in gut microbial homeostasis and immunological resilience. Read the free Plain Language Summary for this article on the Journal blog.

Microencapsulation of ginger oleoresin through the complex coacervates of gum Arabic and soy protein isolate as the wall materials

In this investigation, the extracted active ingredients of the ginger plant so-called oleoresin have been encapsulated using the complex coacervation method to preserve them from the oxidation reaction during storage at 25 ᵒC for 100 days. The coacervation was carried out by soybean protein isolate and gum Arabic, and the system was compared to the inclusion of β-cyclodextrin (β-CD) and a non-encapsulated oleoresin in terms of the protection from the total phenolic content (TPC) and antioxidant properties against oxidants such as DPPH and H2O2. The soybean protein isolate as a plant derivative is cheaper than the animal proteins such as whey protein and gelatin which were extensively used in the encapsulation of ginger oleoresin and does not create sensitivity in human guts. The encapsulation by the complex coacervation showed advantages in higher oleoresin loading efficiency and encapsulation yield during the preparation and also gained higher protection efficiencies in preserving TPC and antioxidant activities of oleoresin against oxidation during the storage in comparison with the β-CD inclusion. The complex coacervates can preserve up to 95.4% of TPC, and about 97.7% and 98.9% of the original antioxidant activity against DPPH and H2O2 after this period, respectively. At the same storage conditions, the preservation efficiency for TPC and antioxidant activities against DPPH and H2O2 were respectively 88.5, 82.8, and 85.9% for the β-CD inclusion.

Bile Salt Hydrolase Activity-Based Probes for Monitoring Gut Microbial Bile Acid Metabolism.

Bile acids are bioactive metabolites that are biotransformed into secondary bile acids by the gut microbiota, a vast consortium of microbes that inhabit the intestines. The first step in intestinal secondary bile acid metabolism is carried out by a critical enzyme, bile salt hydrolase (BSH), that catalyzes the gateway reaction that precedes all subsequent microbial metabolism of these important metabolites. As gut microbial metabolic activity is difficult to probe due to the complex nature of the gut microbiome, approaches are needed to profile gut microbiota-associated enzymes such as BSH. Here, we develop a panel of BSH activity-based probes (ABPs) to determine how changes in diurnal rhythmicity of gut microbiota-associated metabolism affects BSH activity and substrate preference. This panel of covalent probes enables determination of BSH activity and substrate specificity from multiple gut anerobic bacteria derived from the human and mouse gut microbiome. We found that both gut microbiota-associated BSH activity and substrate preference is rhythmic, likely due to feeding patterns of the mice. These results indicate that this ABP-based approach can be used to profile changes in BSH activity in physiological and disease states that are regulated by circadian rhythms.

A Noninvasive Quantitative Method for Evaluating Intestinal Exposure to Microplastics Based on the Excretion and Metabolism Patterns of Microplastics and Their Additives.

Microplastics (MPs) pose potential health risks to the intestinal tract and gut microbiota, a topic that has garnered significant attention. However, the absence of quantitative assessment methods for human gut MP exposure impedes related health risk assessments. Here, we performed long-term continuous exposure experiments on mice using MPs that mimic actual human exposure characteristics. The daily excretion of fecal MPs and the concentrations of phthalates (PAEs) and their metabolites (mPAEs) in serum and urine were detected. The cumulative excretion rate of fecal MPs remains stable at about 93%. A significant linear correlation was observed between MP exposure and concentration of mPAEs in urine for both low MP (LMP; 150 μg of MPs/d) (R2 = 0.90) and high MP (HMP; 360 μg of MPs/d) groups (R2 = 0.97). Moreover, a strong correlation was found between daily PAEs exposure and total MP-associated PAEs exposure in both LMP (R2 = 0.77) and HMP (R2 = 0.88) groups. Based on these findings, we established a noninvasive model and evaluated multiple MP exposure parameters in the human gut across 6 continents, 30 countries, and 133 individuals. This study offers novel insights for the quantitative assessment of in vivo MP exposure and provides technical support for assessing the health risks of MPs.

Isolation and Characterisation of Streptococcus spp. with Human Milk Oligosaccharides Utilization Capacity from Human Milk.

Human milk oligosaccharides (HMO) that promote the growth of beneficial gut microbes in infants are abundant in human milk. Streptococcus, one of the dominant genera in human milk microbiota, is also highly prevalent in the infant gut microbiota, possibly due to its adeptness at utilizing HMOs. While previous studies have mainly focused on HMO interactions with gut bacteria like Bifidobacterium and Bacteroides spp., the interaction with Streptococcus spp. has not been fully explored. In this study, Streptococcus spp. was isolated from human milk and identified to exhibit extensive capabilities in utilizing HMOs. Their consumption rates of 2'-fucosyllactose (2'-FL), 6'-sialyllactose (6'-SL), and lacto-N-tetraose (LNT) closely matched those of Bifidobacterium longum subsp. infantis ATCC 15697. Furthermore, we assessed the safety-related genes in the genomes of the Streptococcus species capable of utilizing HMOs, revealing potential virulence and resistance genes. In addition, no haemolytic activity was observed. These findings expand the knowledge of metabolic interactions and networks within the microbiota of human milk and the early life human gut.

Consistent signatures in the human gut microbiome of old- and young-onset colorectal cancer

The incidence of young-onset colorectal cancer (yCRC) has been increasing in recent decades, but little is known about the gut microbiome of these patients. Most studies have focused on old-onset CRC (oCRC), and it remains unclear whether CRC signatures derived from old patients are valid in young patients. To address this, we assembled the largest yCRC gut metagenomes to date from two independent cohorts and found that the CRC microbiome had limited association with age across adulthood. Differential analysis revealed that well-known CRC-associated taxa, such as Clostridium symbiosum, Peptostreptococcus stomatis, Parvimonas micra and Hungatella hathewayi were significantly enriched (false discovery rate <0.05) in both old- and young-onset patients. Similar strain-level patterns of Fusobacterium nucleatum, Bacteroides fragilis and Escherichia coli were observed for oCRC and yCRC. Almost all oCRC-associated metagenomic pathways had directionally concordant changes in young patients. Importantly, CRC-associated virulence factors (fadA, bft) were enriched in both oCRC and yCRC compared to their respective controls. Moreover, the microbiome-based classification model had similar predication accuracy for CRC status in old- and young-onset patients, underscoring the consistency of microbial signatures across different age groups.

HMOs Impact the Gut Microbiome of Children and Adults Starting from Low Predicted Daily Doses.

Recent studies suggest that the dietary intake of human milk oligosaccharides (HMOs) provides health benefits from infancy up to adulthood. Thus far, beneficial changes in the adult gut microbiome have been observed at oral doses of 5-20 g/day of HMOs. Efficacy of lower doses has rarely been tested. We assessed four HMO molecular species-2'Fucosyllactose (2'FL), Lacto-N-neotetraose (LNnT), 3'Sialyllactose (3'SL), and 6'Sialyllactose (6'SL)-at predicted doses from 0.3 to 5 g/day for 6-year-old children and adults (n = 6 each), using ex vivo SIFR® technology (Cryptobiotix, Ghent, Belgium). This technology employing bioreactor fermentation on fecal samples enables us to investigate microbial fermentation products that are intractable in vivo given their rapid absorption/consumption in the human gut. We found that HMOs significantly increased short-chain fatty acids (SCFAs), acetate, propionate (in children/adults), and butyrate (in adults) from predicted doses of 0.3-0.5 g/day onwards, with stronger effects as dosing increased. The fermentation of 6'SL had the greatest effect on propionate, LNnT most strongly increased butyrate, and 2'FL and 3'SL most strongly increased acetate. An untargeted metabolomic analysis revealed that HMOs enhanced immune-related metabolites beyond SCFAs, such as aromatic lactic acids (indole-3-lactic acid/3-phenyllactic acid) and 2-hydroxyisocaproic acid, as well as gut-brain-axis-related metabolites (γ-aminobutyric acid/3-hydroxybutyric acid/acetylcholine) and vitamins. The effects of low doses of HMOs potentially originate from the highly specific stimulation of keystone species belonging to, for example, the Bifidobacteriaceae family, which had already significantly increased at doses of only 0.5 g/day LNnT (adults) and 1 g/day 2'FL (children/adults).

CLI: A new protocol for the isolation of Lactic Acid Bacteria from complex plant samples

Lactic Acid Bacteria (LAB) are predominantly probiotic microorganisms and the most are Generally Recognized As Safe (GRAS). LAB inhabit in the human gut ecosystem and are largely found in fermented foods and silage. In the last decades, LAB have also has been found in plant microbiota as a new class of microbes with probiotic activity to plants. For this reason, today the scientific interest in the study and isolation of LAB for agronomic application has increased. However, isolation protocols from complex samples such as plant tissues are scarce and inefficient.In this study, we developed a new protocol (CLI, Complex samples LAB Isolation) which yields purified LAB from plants. The sensitivity of CLI protocol was sufficient to isolate representative microorganisms of LAB genera (i.e. Leuconostoc, Lactococcus and Enterococcus).CLI protocol consists on five steps: i) sample preparation and pre-incubation in 1% sterile peptone at 30 °C for 24–48 h; ii) Sample homogenization in vortex by 10 min; iii) sample serial dilution in quarter-strength Ringer solution, iv) incubation in MRS agar plates with 0.2% of sorbic acid, with 1% of CaCO3, O2 < 15%, at pH 5.8 and 37 °C for 48 h.; v) Selection of single colonies with LAB morphology and CaCO3-solubilization halo. Our scientific contribution is that CLI protocol could be used for several complex samples and represents a useful method for further studies involving native LAB.