Plant-based Media Research Articles

Omics-based analysis of Akkermansia muciniphila cultivation in food-grade media.

Background and Aim: Over the past years, the gut microbiota and its correlation to health and disease has been studied extensively. In terms of beneficial microbes, an increased interest in Akkermansia muciniphila (A. muciniphila) has been observed since its discovery. Direct evidence for the role of A. muciniphila in host health has been provided in both mice and human studies. However, for human interventions with A. muciniphila cells, industrial-scale fermentations are needed, and hence, the used cultivation media should be free of animal-derived components, food-grade, non-allergenic and allow for efficient growth to high densities to provide cost-effective production platforms. In this study, we assessed the growth and performance of A. muciniphila in batch bioreactors using newly developed plant-based media. Methods: The bioreactors were supplemented with varying carbon sources, including different ratios of N-acetylglucosamine (GlcNAc) and glucose. We monitored the growth of A. muciniphila in the plant-based medium using optical density (OD600) measurements and microscopy. In addition, we used a combination of biochemical analysis as well as transcriptional and proteomics analysis to gain detailed insight into the physiology. Results: Comparisons between growth on these media and that on mucin revealed differences at both transcriptome and proteome levels, including differences in the expression of glycosyltransferases, signaling proteins, and stress response. Furthermore, elongated cells and higher OD600 values were observed using the plant-based media as compared to cultivation media containing mucin. Conclusion: These differences do not hamper growth, and therefore, our data suggest that the food-grade medium composition described here could be used to produce A. muciniphila with high yields for therapeutic purposes.

Enhancing food preservation with postbiotic metabolites γ-aminobutyric acid (GABA) and bacteriocin-like inhibitory substances (BLIS) produced by Lactobacillus brevis C23 co-cultures in plant-based medium

Lactic acid bacteria (LAB) can produce γ-aminobutyric acid (GABA) with antioxidant properties and sedative effects when it binds to the GABA receptor in the human brain. LAB can also produce bacteriocin-like inhibitory substances (BLIS) with antimicrobial capabilities during carbohydrate fermentation. GABA and BLIS are natural compounds with potential health benefits and food preservation properties. Lactobacillus brevis C23 was co-cultured with three different LABs as inducers, which produced the highest GABA content and BLIS activity. They were cultured in various plant-based media to obtain an edible and better-tasting final product over commercially available media like MRS broth. A coconut-based medium with additives was optimized using response surface methodology (RSM) to increase GABA and BLIS production. The optimized medium for maximum GABA production (3.22 ± 0.01 mg/mL) and BLIS activity (84.40 ± 0.44%) was a 5.5% coconut medium containing 0.23% glucose, 1.44% Tween 20, 0.48% L-glutamic acid, and 0.02% pyridoxine. Due to the presence of GABA, the cell-free supernatant (CFS) as a postbiotic showed higher antioxidant activity than other food preservatives like nisin and potassium sorbate. Finally, microbiological tests on food samples showed that the postbiotic was more effective than other preservatives at combating the growth of LAB, molds and coliform bacteria, making it a possible food preservative.

Diversity of fungal microbiome obtained from plant rhizoplanes

Microbial communities, and their ecological importance, have been investigated in several habitats. However, so far, most studies could not describe the closest microbial interactions and their functionalities. This study investigates the co-occurring interactions between fungi and bacteria in plant rhizoplanes and their potential functions. The partnerships were obtained using fungal-highway columns with four plant-based media. The fungi and associated microbiomes isolated from the columns were identified by sequencing the ITS (fungi) and 16S rRNA genes (bacteria).Statistical analyses including Exploratory Graph and Network Analysis were used to visualize the presence of underlying clusters in the microbial communities and evaluate the metabolic functions associated with the fungal microbiome (PICRUSt2). Our findings characterize the presence of both unique and complex bacterial communities associated with different fungi. The results showed that Bacillus was associated as exo-bacteria in 80 % of the fungi but occurred as putative endo-bacteria in 15 %. A shared core of putative endo-bacterial genera, potentially involved in the nitrogen cycle was found in 80 % of the isolated fungi. The comparison of potential metabolic functions of the putative endo- and exo-communities highlighted the potential essential factors to establish an endosymbiotic relationship, such as the loss of pathways associated with metabolites obtained from the host while maintaining pathways responsible for bacterial survival within the hypha.

Application of fruit juice for proliferation of Bacillus to control fungal phytopathogens

Plant-based media have recently been of interest as potential natural media for microbial culture. This study was conducted to apply inexpensive and available fruits to culture Bacillus antagonizing three fungal phytopathogens including Phytophthora capsici GTC 2.6.1, Rhizoctonia solani GTC 2.7.1, and Sclerotium rolfsii GTC 2.9.1. The results showed that the juice media of dragon fruit, Cavendish banana, watermelon, pineapple, and MT1 seedless guava could all be used to culture two screened antagonistic Bacillus strains including B08 and B18. Surveys on the media from watermelon juice indicated that juice concentration (based on mass of fruit pulp), initial pH, concentration of traditional medium added to the juice medium had different effects on the growth of two strains of Bacillus sp. B08 and B18. These preliminary results demonstrated the potential application of the juice in biomass production of antagonistic Bacillus strains. This approach is certainly safe for the environment and has the potential to improve fruit consumption and reduce the cost of microbial fertilizer production, so it needs more research attention.

Quantification of diversity sampling bias resulting from rice root bacterial isolation on popular and nitrogen-free culture media using 16S amplicon barcoding.

Culturing bacteria from plant material is well known to be conducive to strong bias compared to the actual diversity in the original samples. This bias is related to the bacterial cultivability, chemical composition of the media and culture conditions. Recovery bias is often observed but has never been quantified on different media using an amplicon barcoding approach whereby plant microbiota DNA extractions are compared to DNA extracted from serial dilutions of the same plant tissues grown on bacterial culture media. In this study, we: i) quantified the bacterial culturing diversity bias using 16S amplicon barcode sequencing by comparing a culture-dependent approach (CDA) focused on rice roots on four commonly used bacterial media (10% and 50% TSA, plant-based medium with rice flour, nitrogen free medium NGN and NFb) versus a culture-independent approach (CIA) assessed with DNA extracted directly from root and rhizosphere samples; ii) assessed enriched and missing taxa detected on the different media; iii) used biostatistics functional predictions to highlight metabolic profiles that could potentially be enriched in the CDA and CIA. A comparative analysis of the two approaches revealed that among the 22 phyla present in microbiota of the studied rice root samples, only five were present in the CDA (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Verrucomicrobia). The Proteobacteria phylum was the most abundant in all CDA samples, showing high gamma-Proteobacteria enrichment. The diversity of the combined culture media represented about a third of the total microbiota diversity, and its genus diversity and frequency was documented. The functional prediction tool (PICRUSt2) detected nitrogenase enzyme enrichment in bacterial taxa sampled from nitrogen-free media, thus validating its predictive capacity. Further functional predictions also showed that the CDA mostly missed anaerobic, methylotrophic, methanotrophic and photosynthetic bacteria compared to the CIA, thereby generating valuable insight that could enable the design of ad-hoc culture media and conditions to increase the rice-associated microbiota cultivability.

The Potential of an Inexpensive Plant-Based Medium for Halal and Vegetarian Starter Culture Preparation

The restrictions for halal and vegetarian fermented products apply not only to the food ingredients, but also to the inoculum media. The utilization of a medium for lactic acid bacteria (LAB) leads to some issues from animal-derived proteins sources that may be doubtful for halal and/or vegetarian use. This study aimed to develop a plant-based medium for culturing and maintaining LAB. The result demonstrated that 10 g/L soybean powder in sweet potato extract was suitable for cultivating Lactiplantibacillus plantarum TISTR 2075 with no significant difference (p < 0.05) from MRS (de Man, Rogosa and Sharpe) in the cell number (9.12 log CFU/mL) and specific growth rate (0.04). The feasibility of a plant-based medium to grow and maintain the LAB strains from different origins was evaluated. Compared to MRS, Lpb. plantarum TISTR 2075, Lpb. plantarum MW3, and Lacticaseibacillus casei TISTR 1463 could grow almost as well in a plant-based medium. This medium was also suitable for maintaining the viability of LAB during storage, especially when subjected to slant agar stock culture. It is practical and costs at least 10 times less than MRS. Thus, this study created a low-cost plant-based medium that could be used in laboratories, especially for applications in halal and vegetarian food products.

Cross Cultivation on Homologous/Heterologous Plant-Based Culture Media Empowers Host-Specific and Real Time In Vitro Signature of Plant Microbiota

Alliances of microbiota with plants are masked by the inability of in vitro cultivation of their bulk. Pure cultures piled in international centers originated from dissimilar environments/hosts. Reporting that plant root/leaf-based culture media support the organ-specific growth of microbiota, it was of interest to further investigate if a plant-based medium prepared from homologous (maize) supports specific/adapted microbiota compared to another prepared from heterologous plants (sunflower). The culture-independent community of maize phyllosphere was compared to communities cross-cultivated on plant broth-based media: CFU counts and taxa prevalence (PCR-DGGE; Illumina MiSeq amplicon sequencing). Similar to total maize phyllospheric microbiota, culture-dependent communities were overwhelmed by Proteobacteria (>94.3–98.3%); followed by Firmicutes (>1.3–3.7%), Bacteroidetes (>0.01–1.58%) and Actinobacteria (>0.06–0.34%). Differential in vitro growth on homologous versus heterologous plant-media enriched/restricted various taxa. In contrast, homologous cultivation over represented members of Proteobacteria (ca. > 98.0%), mainly Pseudomonadaceae and Moraxellaceae; heterologous cultivation and R2A enriched Firmicutes (ca. > 3.0%). The present strategy simulates/fingerprints the chemical composition of host plants to expand the culturomics of plant microbiota, advance real-time in vitro cultivation and lab-keeping of compatible plant microbiota, and identify preferential pairing of plant-microbe partners toward future synthetic community (SynComs) research and use in agriculture.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation

BackgroundThe widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium’s composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host.ResultsCommonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28—which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM—which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)—RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels.ConclusionsThe use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

Optimization of the production of Weissella confusa MBF8-1 lysate in plant-based media by using response surface methodology

Optimization of the production of Weissella confusa MBF8-1 lysate in plant-based media by using response surface methodology

Overproduction of Some Plant Growth Promoters by Rhizospheric Microorganisms on Natural Medium

Considering the nutritional values of Mentha viridis. L and Aloe veraplants, these plants can be utilized for the production of alternative cultivation media. The cost of artificial culture media is very high, and some components may be unavailable. Use of the plant-based culture media would drastically reduce the expense of the synthetic media. Fifteen bacterial isolates were isolated from Aloe vera rhizosphere, nine bacterial and nine actinomycetes isolates were isolated from Mentha viridis rhizosphere both cultivated in Sirs EL- Layan, El-Menoufia governorate, Egypt. In-vitroscreening was done for the production of indole acetic acid (IAA) and phosphorus solubilization. Results revealed that bacterial isolate No. MB4 produced a high amount of IAA(36.51 μg/ml) on the Mentha-based culture medium, No. A6 showed maximum IAA production (16.25μg/ml) on the Aloe vera-based culture medium and isolate No. MA6 was efficient in phosphorus solubilization (867.85μg/ml) that was isolated from the Mentha viridis. L rhizosphere.16s rRNA analysis of these isolates revealed they are (Pseudomonas monteilii strain CIP 104883, Streptomyces rochei strain DW3 and Kosakonia radicincitans strain DSM 16656 respectively.

Effect of plant extracts onPseudomonas syringaepv.actinidiaegene expression, motility and virulence

Pseudomonas syringae pv. actinidiae (Psa), the causative agent of bacterial canker of kiwi, was firstly isolated in 1992, but the second outbreak dating back to 2008 is responsible for the current pandemics that has led to major economic losses worldwide. Despite the high economic impact of Pseudomonas syringae pv. actinidiae (Psa) on kiwifruit industry, and the intense research efforts, the molecular mechanisms involved in pathogenesis are still not fully characterized. In particular, the precise mechanism of recognition/communication between Psa and its host plant has not been elucidated yet. Our findings show that plant crude extracts or xylem juices of Actinidia deliciosa and A. chinensis were able to increase Psa virulence in comparison with artificial growing media such as Luria Broth or Nutrient agar. The study of gene expression in response to plant-based or artificial growing medium was performed by qPCR and showed a differential expression of many virulence-related genes. In addition, genes related to biofilm formation, motility and inter-cell signaling (e.g. quorum sensing in the broad sense) were differentially expressed according to the type of growing media. Interestingly, the expression of many of the studied genes responded differentially both to plant extracts and Psa cell density. The molecular data were corroborated by phenological studies testing the effect of plant-based medium on biofilm formation, motility and symptom development in micro-cuttings of Actinidia spp. The present study demonstrates the existence of a not yet identified, host-specific group of molecules able to activate the pathogenesis machinery of Psa. Moreover, these molecules could lead to different physiological and molecular behavior depending on Psa population density. Finally, it was also demonstrated that these molecules were differentially present in leaves and xylem sap collected by different Actinidia species.

Effect of Rocket (Eruca sativa) Extract on MRSA Growth and Proteome: Metabolic Adjustments in Plant-Based Media.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in food has provoked a great concern about the presence of MRSA in associated foodstuff. Although MRSA is often detected in various retailed meat products, it seems that food handlers are more strongly associated with this type of food contamination. Thus, it can be easily postulated that any food could be contaminated with this pathogen in an industrial environment or in household and cause food poisoning. To this direction, the effect of rocket (Eruca sativa) extract on MRSA growth and proteome was examined in the present study. This goal was achieved with the comparative study of the MRSA strain COL proteome, cultivated in rocket extract versus the standard Luria-Bertani growth medium. The obtained results showed that MRSA was able to grow in rocket extract. In addition, proteome analysis using 2-DE method showed that MRSA strain COL is taking advantage of the sugar-, lipid-, and vitamin-rich substrate in the liquid rocket extract, although its growth was delayed in rocket extract compared to Luria–Bertani medium. This work could initiate further research about bacterial metabolism in plant-based media and defense mechanisms against plant-derived antibacterials.

Plant powder teabags: a novel and practical approach to resolve culturability and diversity of rhizobacteria.

We have developed teabags packed with dehydrated plant powders, without any supplements, for preparation of plant infusions necessary to develop media for culturing rhizobacteria. These bacteria are efficiently cultivated on such plant teabag culture media, with better progressive in situ recoverability compared to standard chemically synthetic culture media. Combining various plant-based culture media and incubation conditions enabled us to resolve unique denaturing gradient gel electrophoresis (DGGE) bands that were not resolved by tested standard culture media. Based on polymerase chain reaction PCR-DGGE of 16S rDNA fingerprints and sequencing, the plant teabag culture media supported higher diversity and significant increases in the richness of endo-rhizobacteria, namely Gammaproteobacteria (Enterobacteriaceae) and predominantly Alphaproteobacteria (Rhizobiaceae). This culminated in greater retrieval of the rhizobacteria taxa associated with the plant roots. We conclude that the plant teabag culture medium by itself, without any nutritional supplements, is sufficient and efficient for recovering and mirroring the complex and diverse communities of rhizobacteria. Our message to fellow microbial ecologists is: simply dehydrate your plant canopy, teabag it and soak it to prepare your culture media, with no need for any additional supplementary nutrients.

Prospects of innovative functional foods based on biologically active dietary supplement (BADS) "VITAZAR"

The development of syn- and metabiotics is one of the most important technological lines of reserch in the production of functional foods. The positive effect of these products is achieved by the addition of enrichment components into the formulations that provide selective stimulation of the of intestinal normal flora's growth or activates it’s metabolic activity. In this regard, scientific and practical interest seem to study the possibility of expanding the range of synbiotic products containing not less than107 CFU / ml of the probiotic microorganisms on the dairy, vegetable-dairy (protein-free milk), or only on the plant-based medium without dairy ingredients. In the research work we have studied the conditions for the accumulation of biomass of probiotic microorganisms consortium, consisting of L. casei subsp. Rhamnosus, L. acidophilus, L. plantarum, L. fermentum, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis on nutrient mediums containing 1-20 % wheat germ flour Vitazar. The quantitative assessment of the accumulation of probiotic microflora biomass and its metabolites in milk-vitazar based, whey- vitazar based, water-vitazar based nutrient mediums has been carried out . The concentration of Lactobacterium and Bifidobacterium in active form in quantity of 109 CFU / ml has been reached. It was found out that Vitazar, being a source of dietary fibers and a wide range of carbohydrates, is capable to support the growth of Lactobacterium and Bifidobacterium, forming the favorable environment. The accumulation of probiotic microflora has been observed by the increasing of titratable acidity due to lactose fermentation by β-galactosidase, produced by microorganisms of the consortium. The main stages of the process of the accumulation microbiomass have been identified in the study of the kinetics of the fermentation process. The derived fermented pastes, containing 20 % and 30 % Vitazar are recommended as a component of formulations of functional products.

Adaptive evolution of the FADS gene cluster within Africa.

Long chain polyunsaturated fatty acids (LC-PUFAs) are essential for brain structure, development, and function, and adequate dietary quantities of LC-PUFAs are thought to have been necessary for both brain expansion and the increase in brain complexity observed during modern human evolution. Previous studies conducted in largely European populations suggest that humans have limited capacity to synthesize brain LC-PUFAs such as docosahexaenoic acid (DHA) from plant-based medium chain (MC) PUFAs due to limited desaturase activity. Population-based differences in LC-PUFA levels and their product-to-substrate ratios can, in part, be explained by polymorphisms in the fatty acid desaturase (FADS) gene cluster, which have been associated with increased conversion of MC-PUFAs to LC-PUFAs. Here, we show evidence that these high efficiency converter alleles in the FADS gene cluster were likely driven to near fixation in African populations by positive selection ∼85 kya. We hypothesize that selection at FADS variants, which increase LC-PUFA synthesis from plant-based MC-PUFAs, played an important role in allowing African populations obligatorily tethered to marine sources for LC-PUFAs in isolated geographic regions, to rapidly expand throughout the African continent 60–80 kya.

DyP-like peroxidases of the jelly fungus Auricularia auricula-judae oxidize nonphenolic lignin model compounds and high-redox potential dyes

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (approximately 300 U l(-1)). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l(-1). Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as "dye-decolorizing peroxidases". Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn(2+)) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic beta-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.

Micropropagation of mature British wild cherry

Shoot tips from accessions of wild cherry (Prunus avium L.) selected from British woodland, and also theP. avium rootstock cvs. F12/1 and Charger, were successfully establishedin vitro, and most were easily micropropagated on Murashige and Skoog (MS)-based media. In one accession, adventitious shoots occasionally developed from the extrafloral nectaries positioned at the base of leaf petioles of the initial explants. Micropropagation of cv. Charger was improved by culture on a Quoirin and Lepoivre and Woody Plant-based medium, and by supplementing the medium with 1-phenyl-3-(1, 2, 3-thiadiazol-5yl)urea (thidiazuron). Poor shoot production by F12/1 on 1, 3, 5-trihydroxybenzene (phloroglucinol)-free media was not improved by up to 18 months of regular subculture. Study of cv. F12/1 showed that shoots produced on a MS-based medium with 1 mM phloroglucinol, 0.49 μM indolebutyric acid, 4.4 μM benzyladenine (BA) and 0.29 μM gibberellic acid (GA3), were easier to root over several subcultures than shoots produced on a similar medium with no GA3 and only 2.2 μM BA, but only in a rooting medium supplemented with 1 mM phloroglucinol.