Flavour by design: food-grade lactic acid bacteria improve the volatile aroma spectrum of oat milk, sunflower seed milk, pea milk, and faba milk towards improved flavour and sensory perception.

BACKGROUND: The global market of plant-based milk alternatives is continually growing. Flavour and taste have a key impact on consumers' selection of plant-based beverages. Unfortunately, natural plant milks have only limited acceptance. Their typically bean-like and grassy notes are perceived as "off-flavours" by consumers, while preferred fruity, buttery, and cheesy notes are missing. In this regard, fermentation of plant milk by lactic acid bacteria (LAB) appears to be an appealing option to improve aroma and taste. RESULTS: In this work, we systematically studied LAB fermentation of plant milk. For this purpose, we evaluated 15 food-approved LAB strains to ferment 4 different plant milks: oat milk (representing cereal-based milk), sunflower seed milk (representing seed-based milk), and pea and faba milk (representing legume-based milk). Using GC‒MS analysis, flavour changes during anaerobic fermentations were studied in detail. These revealed species-related and plant milk-related differences and highlighted several well-performing strains delivered a range of beneficial flavour changes. A developed data model estimated the impact of individual flavour compounds using sensory scores and predicted the overall flavour note of fermented and nonfermented samples. Selected sensory perception tests validated the model and allowed us to bridge compositional changes in the flavour profile with consumer response. CONCLUSION: Specific strain-milk combinations provided quite different flavour notes. This opens further developments towards plant-based products with improved flavour, including cheesy and buttery notes, as well as other innovative products in the future. S. thermophilus emerged as a well-performing strain that delivered preferred buttery notes in all tested plant milks. The GC‒MS-based data model was found to be helpful in predicting sensory perception, and its further refinement and application promise enhanced potential to upgrade fermentation approaches to flavour-by-design strategies.

Co-cultures of Propionibacterium freudenreichii and Bacillus amyloliquefaciens cooperatively upgrade sunflower seed milk to high levels of vitamin B12 and multiple co-benefits.

BACKGROUND: Sunflower seeds (Helianthus annuus) display an attractive source for the rapidly increasing market of plant-based human nutrition. Of particular interest are press cakes of the seeds, cheap residuals from sunflower oil manufacturing that offer attractive sustainability and economic benefits. Admittedly, sunflower seed milk, derived therefrom, suffers from limited nutritional value, undesired flavor, and the presence of indigestible sugars. Of specific relevance is the absence of vitamin B12. This vitamin is required for development and function of the central nervous system, healthy red blood cell formation, and DNA synthesis, and displays the most important micronutrient for vegans to be aware of. Here we evaluated the power of microbes to enrich sunflower seed milk nutritionally as well as in flavor. RESULTS: Propionibacterium freudenreichii NCC 1177 showed highest vitamin B12 production in sunflower seed milk out of a range of food-grade propionibacteria. Its growth and B12 production capacity, however, were limited by a lack of accessible carbon sources and stimulants of B12 biosynthesis in the plant milk. This was overcome by co-cultivation with Bacillus amyloliquefaciens NCC 156, which supplied lactate, amino acids, and vitamin B7 for growth of NCC 1177 plus vitamins B2 and B3, potentially supporting vitamin B12 production by the Propionibacterium. After several rounds of optimization, co-fermentation of ultra-high-temperature pre-treated sunflower seed milk by the two microbes, enabled the production of 17 µg (100 g)-1 vitamin B12 within four days without any further supplementation. The fermented milk further revealed significantly enriched levels of L-lysine, the most limiting essential amino acid, vitamin B3, vitamin B6, improved protein quality and flavor, and largely eliminated indigestible sugars. CONCLUSION: The fermented sunflower seed milk, obtained by using two food-grade microbes without further supplementation, displays an attractive, clean-label product with a high level of vitamin B12 and multiple co-benefits. The secret of the successfully upgraded plant milk lies in the multifunctional cooperation of the two microbes, which were combined, based on their genetic potential and metabolic signatures found in mono-culture fermentations. This design by knowledge approach appears valuable for future development of plant-based milk products.

Genome-based selection and application of food-grade microbes for chickpea milk fermentation towards increased L-lysine content, elimination of indigestible sugars, and improved flavour.

BACKGROUND: Plant-based milk alternatives are more popular than ever, and chickpea-based milks are among the most commercially relevant products. Unfortunately, limited nutritional value because of low levels of the essential amino acid L-lysine, low digestibility and unpleasant taste are challenges that must be addressed to improve product quality and meet consumer expectations. RESULTS: Using in-silico screening and food safety classifications, 31 strains were selected as potential L-lysine producers from approximately 2,500 potential candidates. Beneficially, 30% of the isolates significantly accumulated amino acids (up to 1.4 mM) during chickpea milk fermentation, increasing the natural level by up to 43%. The best-performing strains, B. amyloliquefaciens NCC 156 and L. paracasei subsp. paracasei NCC 2511, were tested further. De novo lysine biosynthesis was demonstrated in both strains by 13C metabolic pathway analysis. Spiking small amounts of citrate into the fermentation significantly activated L-lysine biosynthesis in NCC 156 and stimulated growth. Both microbes revealed additional benefits in eliminating indigestible sugars such as stachyose and raffinose and converting off-flavour aldehydes into the corresponding alcohols and acids with fruity and sweet notes. CONCLUSIONS: B. amyloliquefaciens NCC 156 and L. paracasei subsp. paracasei NCC 2511 emerged as multi-benefit microbes for chickpea milk fermentation with strong potential for industrial processing of the plant material. Given the high number of L-lysine-producing isolates identified in silico, this concept appears promising to support strain selection for food fermentation.

Fermentation of plant-based milk alternatives for improved flavour and nutritional value.

Non-dairy milk alternatives (or milk analogues) are water extracts of plants and have become increasingly popular for human nutrition. Over the years, the global market for these products has become a multi-billion dollar business and will reach a value of approximately 26 billion USD within the next 5 years. Moreover, many consumers demand plant-based milk alternatives for sustainability, health-related, lifestyle and dietary reasons, resulting in an abundance of products based on nuts, seeds or beans. Unfortunately, plant-based milk alternatives are often nutritionally unbalanced, and their flavour profiles limit their acceptance. With the goal of producing more valuable and tasty products, fermentation can help to the improve sensory profiles, nutritional properties, texture and microbial safety of plant-based milk alternatives so that the amendment with additional ingredients, often perceived as artificial, can be avoided. To date, plant-based milk fermentation mainly uses mono-cultures of microbes, such as lactic acid bacteria, bacilli and yeasts, for this purpose. More recently, new concepts have proposed mixed-culture fermentations with two or more microbial species. These approaches promise synergistic effects to enhance the fermentation process and improve the quality of the final products. Here, we review the plant-based milk market, including nutritional, sensory and manufacturing aspects. In addition, we provide an overview of the state-of-the-art fermentation of plant materials using mono- and mixed-cultures. Due to the rapid progress in this field, we can expect well-balanced and naturally fermented plant-based milk alternatives in the coming years.

Systems metabolic engineering of Escherichia coli for production of the antitumor drugs violacein and deoxyviolacein.

Violacein and deoxyviolacein are interesting therapeutics against pathogenic bacteria and viruses as well as tumor cells. In the present work, systems-wide metabolic engineering was applied to target Escherichia coli, a widely accepted recombinant host in pharmaceutical biotechnology, for production of these high-value products. The basic producer, E. coli dVio-1, that expressed the vioABCE cluster from Chromobacterium violaceum under control of the inducible araC system, accumulated 180 mg L(-1) of deoxyviolacein. Targeted intracellular metabolite analysis then identified bottlenecks in tryptophan supporting pathways, the major product building block. This was used for comprehensive engineering of serine, chorismate and tryptophan biosynthesis and the non-oxidative pentose-phosphate pathway. The final strain, E. coli dVio-6, accumulated 320 mg L(-1) deoxyviolacein in shake flask cultures. The created chassis of a high-flux tryptophan pathway was complemented by genomic integration of the vioD gene of Janthinobacterium lividum, which enabled exclusive production of violacein. In a fed-batch process, the resulting producer E. coli Vio-4 accumulated 710 mg L(-1) of the desired product. With straightforward broth extraction and subsequent crystallization, violacein could be obtained with 99.8% purity. This demonstrates the potential of E. coli as a platform for production of tryptophan based therapeutics.

A novel strategy to simultaneously enhance bioaccessible lipids and antioxidants in hetero/mixotrophic Chlorella vulgaris as functional ingredient.

Microalgae are a promising source of polyunsaturated fatty acids as well as bioactive antioxidant compounds such as carotenoids, phenolics and tocopherols. However, the accumulation of these biomolecules is often promoted by conflicting growth conditions. In this study, a phased bioprocessing strategy was developed to simultaneously enhance the lipid and antioxidant amounts by tailoring nitrogen content in the cultivation medium and applying light stress. This approach increased the overall contents of total fatty acids, carotenoids, phenolics, and α-tocopherol in Chlorella vulgaris by 2.2-, 2.2-, 1.5-, and 2.1-fold, respectively. Additionally, the bioaccessibility of the lipids and bioactives from the obtained biomasses improved after pulsed electric field (5 µs, 20 kV cm-1, 31.8 kJ kg-1sus) treatment (up to +12%) and high-pressure homogenization (100 MPa, 5-6 passes) (+41-76%). This work represents a step towards the generation of more efficient algae biorefineries, thus expanding the alternative resources available for essential nutrients.

Biochemical and Morphological Characterization of Heterotrophic Crypthecodinium cohnii and Chlorella vulgaris Cell Walls.

Microalgae are attractive for the food and cosmetic industries because of their nutrient composition. However, the bioaccessibility and extractability of nutrients in microalgae are limited by the rigid and indigestible cell wall. The goal of this study is to explore the cell wall polysaccharides (CWPSs) composition and morphology in heterotrophic Crypthecodinium cohnii and Chlorella vulgaris biomasses during growth. Our results showed that glucose was the major component of CWPSs and exopolysaccharides in C. cohnii. C. vulgaris CWPSs have a similar sugar profile in exponential and stationary phases, essentially composed of rhamnose and galactose. C. vulgaris cell wall thickness increased from 82 nm in the exponential phase to 114 nm in the stationary phase and consisted of two main layers. C. cohnii's cell wall was 133 nm thick and composed of several membranes surrounding thecal plates. Understanding of the microalgae cell wall helps developing a more efficient and targeted biorefinery approach.

Complete genome sequence of the myxobacterium Sorangium cellulosum.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

Towards methionine overproduction in Corynebacterium glutamicum--methanethiol and dimethyldisulfide as reduced sulfur sources.

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur source for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis has predicted a high methionine yield for these reduced compounds provided that they can be utilized. Wild type cells were able to grow on methanethiol and on dimethyldisulfide as sole sulfur source, respectively. Isotope labeling studies with mutant strains exhibiting targeted modification of methionine biosynthesis gave detailed insight into the underlying pathways involved in assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as enzyme catalyzing this reaction. Deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur source. Plasmid based overexpression of metY in the delta metY background restored the capability to grow on methanethiol or dimethyldisulfide as sole sulfur source. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased two-fold in the engineered strain. This positive effect was limited by depletion of the metY substrate O-acetylhomoserine, requesting for further metabolic engineering targets towards competitive production strains.

Biochemical and Nutritional Evaluation of Chlorella and Auxenochlorella Biomasses Relevant for Food Application.

Microalgae are a source of potentially healthy and sustainable nutrients. However, the bioaccessibility of these nutrients remains uncertain. In this study, we analyzed the biomass composition of five commercial Chlorella and Auxenochlorella strains, and Chlorella vulgaris heterotrophically cultivated in our laboratory. Protein accounted for 65 ± 3% (w w-1) dry matter (DM) in all biomasses, except for the lab-grown C. vulgaris that contained 20% (w w-1) DM protein. The fatty acids content was comparable and ranged between 7 and 10% (w w-1) DM. Most of the biomasses had a ω6-polyunsaturated fatty acids (PUFAs)/ω3-PUFAs ratio <4, as recommended by nutritional experts. A recently published harmonized protocol for in vitro digestion was used to evaluate fatty acids and protein bioaccessibilities. Protein bioaccessibility ranged between 60 and 74% for commercial Chlorella and Auxenochlorella biomasses and was 43% for the lab-grown C. vulgaris. Fatty acids bioaccessibility was <7% in commercial biomasses and 19% in the lab-grown C. vulgaris. Taken together, the results show that microalgae are promising sources of bioaccessible protein. The limited fatty acids bioaccessibility indicates the need for alternative upstream and downstream production strategies.

Numerical bias estimation for mass spectrometric mass isotopomer analysis.

Mass spectrometric (MS) isotopomer analysis has become a standard tool for investigating biological systems using stable isotopes. In particular, metabolic flux analysis uses mass isotopomers of metabolic products typically formed from (13)C-labeled substrates to quantitate intracellular pathway fluxes. In the current work, we describe a model-driven method of numerical bias estimation regarding MS isotopomer analysis. Correct bias estimation is crucial for measuring statistical qualities of measurements and obtaining reliable fluxes. The model we developed for bias estimation corrects a priori unknown systematic errors unique for each individual mass isotopomer peak. For validation, we carried out both computational simulations and experimental measurements. From stochastic simulations, it was observed that carbon mass isotopomer distributions and measurement noise can be determined much more precisely only if signals are corrected for possible systematic errors. By removing the estimated background signals, the residuals resulting from experimental measurement and model expectation became consistent with normality, experimental variability was reduced, and data consistency was improved. The method is useful for obtaining systematic error-free data from (13)C tracer experiments and can also be extended to other stable isotopes. As a result, the reliability of metabolic fluxes that are typically computed from mass isotopomer measurements is increased.

Investigation of the central carbon metabolism of Sorangium cellulosum: metabolic network reconstruction and quantification of pathway fluxes.

In the present work, the metabolic network of primary metabolism of the slow-growing myxobacterium Sorangium cellulosum was reconstructed from the annotated genome sequence of the type strain So ce56. During growth on glucose as the carbon source and asparagine as the nitrogen source, So ce56 showed a very low growth rate of 0.23 d-(1), equivalent to a doubling time of 3 days. Based on a complete stoichiometric and isotopomer model of the central metabolism, 13C metabolic flux analysis was carried out for growth with glucose as carbon and asparagine as nitrogen sources. Normalized to the uptake flux for glucose (100%), cells recruited glycolysis (51%) and the pentose phosphate pathway (48%) as major catabolic pathways. The Entner-Doudoroff pathway and glyoxylate shunt were not active. A high flux through the TCA cycle (118%) enabled a strong formation of ATP, but cells revealed a rather low yield for biomass. Inspection of fluxes linked to energy metabolism revealed that S. cellulosum utilized only 10% of the ATP formed for growth, whereas 90% is required for maintenance. This explains the apparent discrepancy between the relatively low biomass yield and the high flux through the energy-delivering TCA cycle. The total flux of NADPH supply (216%) was higher than the demand for anabolism (156%), indicating additional reactions for balancing of NADPH. The cells further exhibited a highly active metabolic cycle, interconverting C3 and C4 metabolites of glycolysis and the TCA cycle. The present work provides the first insight into fluxes of the primary metabolism of myxobacteria, especially for future investigation on the supply of cofactors, building blocks, and energy in myxobacteria, producing natural compounds of biotechnological interest.

Appropriate sampling for intracellular amino acid analysis in five phylogenetically different yeasts.

Methanol quenching and fast filtration, the two most common sampling protocols in microbial metabolome analysis, were validated for intracellular amino acid analysis in phylogenetically different yeast strains comprising Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia pastoris, Schizosaccharomyces pombe and Zygosaccharomyces bailii. With only few exceptions for selected amino acids, all yeasts exhibited negligible metabolite leakage during quenching with 60% cold buffered methanol. Slightly higher leakage was observed with increasing methanol content in the quenching solution. Fast filtration resulted in identical levels for intracellular amino acids in all strains tested. The results clearly demonstrate the validity of both approaches for leakage-free sampling of amino acids in yeast.

Metabolic flux engineering of L-lysine production in Corynebacterium glutamicum--over expression and modification of G6P dehydrogenase.

In the present work, metabolic flux engineering of Corynebacterium glutamicum was carried out to increase lysine production. The strategy focused on engineering of the pentose phosphate pathway (PPP) flux by different genetic modifications. Over expression of the zwf gene, encoding G6P dehydrogenase, in the feedback-deregulated lysine-producing strain C. glutamicum ATCC 13032 lysC(fbr) resulted in increased lysine production on different carbon sources including the two major industrial sugars, glucose and sucrose. The additional introduction of the A243T mutation into the zwf gene and the over expression of fructose 1,6-bisphosphatase resulted in a further successive improvement of lysine production. Hereby the point mutation resulted in higher affinity of G6P dehydrogenase towards NADP and reduced sensitivity against inhibition by ATP, PEP and FBP. Overall, the lysine yield increased up to 70% through the combination of the different genetic modifications. Through strain engineering formation of trehalose was reduced by up to 70% due to reduced availability of its precursor G6P. Metabolic flux analysis revealed a 15% increase of PPP flux in response to over expression of the zwf gene. Overall a strong apparent NADPH excess resulted. Redox balancing indicated that this excess is completely oxidized by malic enzyme.

Physiological response of Corynebacterium glutamicum to oxidative stress induced by deletion of the transcriptional repressor McbR.

In the present work the metabolic response of Corynebacterium glutamicum to deletion of the global transcriptional regulator McbR, which controls, e.g. the expression of enzymes of L-methionine and L-cysteine biosynthesis and sulfur assimilation, was studied. Several oxidative stress proteins were significantly upregulated among about 40 proteins in response to deletion of McbR. Linked to this oxidative stress, the mutant exhibited a 50 % reduced growth rate, a 30 % reduced glucose uptake rate and a 30 % reduced biomass yield. It also showed metabolic flux rerouting in response to the deletion. NADPH metabolism was strongly altered. In contrast to the wild-type, the deletion strain supplied significantly more NADPH than required for anabolism, indicating the activity of additional NADPH-consuming reactions. These involved enzymes of oxidative stress protection. Through redirection of metabolic carbon flux in the central catabolism, including a 40 % increased tricarboxylic acid (TCA) cycle flux, the mutant revealed an enhanced NADPH supply to provide redox power for the antioxidant systems. This, however, was not sufficient to compensate for the oxidative stress, as indicated by the drastically disturbed redox equilibrium. The NADPH/NADP+ ratio in C. glutamicum DeltamcbR was only 0.29, and thus much lower than that of the wild-type (2.35). Similarly, the NADH/NAD+ ratio was substantially reduced from 0.18 in the wild-type to 0.08 in the mutant. Deletion of McbR is regarded as a key step towards biotechnological L-methionine overproduction in C. glutamicum. C. glutamicum DeltamcbR, however, did not overproduce L-methionine; this was very likely linked to the low availability of NADPH. Since oxidative stress is often observed in industrial production processes, engineering of NADPH metabolism could be a general strategy for improvement of production strains. Unlike the wild-type, C. glutamicum DeltamcbR contained large granules with high phosphorus content. The storage of these energy-rich polyphosphates is probably the result of a large excess of formation of ATP, as revealed by estimation of the underlying fluxes linked to energy metabolism.

Sampling for metabolome analysis of microorganisms.

In the present work we investigated the most commonly applied methods used for sampling of microorganisms in the field of metabolomics in order to unravel potential sources of error previously ignored but of utmost importance for accurate metabolome analysis. To broaden the significance of our study, we investigated different Gram-negative and Gram-positive bacteria, i.e., Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Gluconobacter oxydans, Pseudomonas putida, and Zymononas mobilis, and analyzed metabolites from different catabolic and anabolic intracellular pathways. Quenching of cells with cold methanol prior to cell separation and extraction led to drastic loss (>60%) of all metabolites tested due to unspecific leakage. Using fast filtration, Gram-negative bacteria also revealed a significant loss (>80%) when inappropriate washing solutions with low ionic strength were applied. Adapting the ionic strength of the washing solution to that of the cultivation medium could almost completely avoid this problem. Gram-positive strains did not show significant leakage independent of the washing solution. Fast filtration with sampling times of several seconds prior to extraction appears to be a suitable approach for metabolites with relatively high intracellular level and low turnover such as amino acids or TCA cycle intermediates. Comparison of metabolite levels in the culture supernatant and the cell interior revealed that the common assumption of whole broth quenching protocols attributing the metabolites found exclusively to the intracellular pools may not be valid in many cases. In such cases a differential approach correcting for medium-contained metabolites is required.