Bacterial contamination in starch-to-ethanol fermentations: Can bacteriocin-producing Saccharomyces cerevisiae offer a solution?

The work is dedicated to increase the efficiency of the processing of Fusarium contaminated barley biomass by using pretreatment of grains with antimicrobial lactic acid bacteria in combination with special selected strains of yeast for alcoholic fermentation. Bacteriocins producing lactic acid bacteria (LAB) have been isolated from Lithuanian spontanious rye sourdoughs. These LAB’s were attributing to Lactobacillus and Pediococcus genotypes and Lactobacillus sakei KTU05-06, Pediococcus acidilactici KTU05-07 and P. pentosaceus KTU05-08 species, PCR detection of LAB structural genes has been carried out. The contamination of barley with Fusarium spp. decrease the amount of ethanol during fermentation processes (R2 = 0.868) and, contrary, increase the concentrations of higher alkohols (R2 = 0.867). The efficiency of fermentation process could be increased by selecting yeast strains: a higher ethanol yield and a lower amount of higher alkohols was observed by using K. marxianus var. Bulgaricus. Other yeast strains – K. Marxianus show the same effect on fermentation processes as S. cerevisae. The treatment of contaminated barley with selected LAB has not a significant influence on bioethanol yield. But by using some LAB strains such as P. acidilactici and P. pentosaceus a higher amount of higher alkohols has been developed. Furthermore in all cases this biotechnological solution has positive influence on dried grains with solubles (DDGS) detoxification by decreasing the amount of DON in important raw material for feed production. The best detoxification effect has been achieved by using P. acidilactici or L. sakei for contaminated barley treatment and S. cerevisiae – for fermentation processes. So, by selecting LAB with antimicrobial activity in combiantion with yeast, the efficiency of the processing of Fusarium cantaminated barley can be significantly increased: biomass could be used for bioethanol production and DDGS – for feed.DOI: http://dx.doi.org/10.5755/j01.ct.60.2.1617

Bacterial contaminations in yeast fermentation tanks are a recurring problem for the bioethanol production industry. Lactic acid bacteria (LAB), particularly of the genus Lactobacillus, are the most common contaminants. Their proliferation can reduce fermentation efficiency or even impose premature shutdown for cleaning. We have previously reported that laboratory yeast strains naturally excrete amino acids via transporters of the Drug: H+ Antiporter-1 (DHA1) family. This excretion allows yeast to cross-feed LAB, which are most often unable to grow without an external amino acid supply. Whether industrial yeast strains used in bioethanol production likewise promote LAB proliferation through cross-feeding has not been investigated. In this study, we first show that the yeast strain Ethanol Red used in ethanol production supports growth of Lactobacillus fermentum in an amino-acid-free synthetic medium. This effect was markedly reduced upon homozygous deletion of the QDR3 gene encoding a DHA1-family amino acid exporter. We further show that cultivation of Ethanol Red in a nonsterile sugarcane-molasses-based medium is associated with an increase in lactic acid due to LAB growth. When Ethanol Red lacked the QDR1, QDR2, and QDR3 genes, this lactic acid production was not observed and ethanol production was not significantly reduced. Our results indicate that Ethanol Red cultivated in synthetic or molasses medium sustains LAB proliferation in a manner that depends on its ability to excrete amino acids via Qdr transporters. They further suggest that using mutant industrial yeast derivatives lacking DHA1-family amino acid exporters may be a way to reduce the risk of bacterial contaminations during fermentation.

Bacterial contamination during industrial yeast fermentation has serious economic consequences for fuel ethanol producers. In addition to deviating carbon away from ethanol formation, bacterial cells and their metabolites often have a detrimental effect on yeast fermentative performance. The bacterial contaminants are commonly lactic acid bacteria (LAB), comprising both homo- and heterofermentative strains. We have studied the effects of these two different types of bacteria upon yeast fermentative performance, particularly in connection with sugarcane-based fuel ethanol fermentation process. Homofermentative Lactobacillus plantarum was found to be more detrimental to an industrial yeast strain (Saccharomyces cerevisiae CAT-1), when compared with heterofermentative Lactobacillus fermentum, in terms of reduced yeast viability and ethanol formation, presumably due to the higher titres of lactic acid in the growth medium. These effects were only noticed when bacteria and yeast were inoculated in equal cell numbers. However, when simulating industrial fuel ethanol conditions, as conducted in Brazil where high yeast cell densities and short fermentation time prevail, the heterofermentative strain was more deleterious than the homofermentative type, causing lower ethanol yield and out competing yeast cells during cell recycle. Yeast overproduction of glycerol was noticed only in the presence of the heterofermentative bacterium. Since the heterofermentative bacterium was shown to be more deleterious to yeast cells than the homofermentative strain, we believe our findings could stimulate the search for more strain-specific antimicrobial agents to treat bacterial contaminations during industrial ethanol fermentation.

Bacteriocins from lactic acid bacteria (LAB) are natural antimicrobial peptides or proteins with interesting potential applications in food preservation and health care. The present study was aimed to isolate bacteriocinogenic LAB from dairy products, fermented food and environmental waste samples. One thirty five colonies of LAB were isolated and screened for bacteriocin production by agar overlay method. Among them, Lactobacillus acidophilus NCIM5426 isolated from home made paneer showed maximum zone of inhibition i.e 22-24mm in diameter against food borne ( Listeria monocytogenes , Staphylococcus aureus ) and human pathogens ( Escherichia coli , Salmonella typhi ). The antibacterial compound from the culture supernatant was found to be proteinaceous in nature and therefore, identified as bacteriocin. The bacteriocin of L. acidophilus NCIM5426 was found to be heat-stable (121°C for 15 min) and active over a wide pH range of 4.0-10.0. It showed stability (60%) for 30 days at room temperature (30-32°C). Addition of surfactants (EDTA, SDS, Hexadecyl trimethylamonium bromide) up to 1% to crude bacteriocin showed increase in antibacterial activity where as metal ions (Calcium Chloride, Zinc Sulphate and Mercuric Chloride) in low concentration (0.5-1mgL -1 ) decreased the activity. The bacteriocin was purified to its homogeneity by ammonium sulfate precipitation followed by gel filtration chromatography and HPLC. Molecular weight of bacteriocin was found to be 5.6 and 5.8 KDa by SDS page and LC/MS respectively. Production and activity of bacteriocin was significant (50%) even at higher salt (NaCl) concentration i.e 3%. Our present study demonstrates the possibility of using L. acidophilus NCIM5426 or its bacteriocin as a biopreservative in dairy industry.

Wine consumers predominantly use visual, sensory and textual descriptors as quality/preference indicators to describe olfactory sensations. In this study, different wines were analysed to generate relevant chemical and sensory characterisation data and attributes. Sequential inoculation of Syrah grape must was performed with a combination of Saccharomyces yeast, non-Saccharomyces yeasts and lactic acid bacteria for the possible improvement of Syrah wine quality. Selected anthocyanins, flavan-3-ols, flavonols and phenolic acids were quantified in Syrah wines using the reversed-phase high-performance liquid chromatography photodiode array detection (RP-HPLC-DAD) technique. Sensory (descriptive evaluation) and physicochemical/oenological parameters (Winescan® and OenoFoss™) results were compared to phenolic compound concentrations. Phenolic compound concentrations increased in Syrah wines made with a combination of a Saccharomyces reference yeast, non-Saccharomyces yeasts and lactic acid bacteria. Syrah wines made with a combination of Metschnikowia pulcherrima + Saccharomyces cerevisiae + Oenococcus oeni, and M. pulcherrima + S. cerevisiae + Lactobacillus plantarum, had higher flavonol concentrations compared to wines made without lactic acid bacteria. Syrah wines made with a combination of Saccharomyces cerevisiae (Sc) + Oenococcus oeni (LAB1) were highest in phenolic acid concentrations. Syrah wines made with a combination of M. pulcherrima + S. cerevisiae + L. plantarum had higher total anthocyanins than wines made without lactic acid bacteria. Syrah wine sensory attributes, viz. mouthfeel and astringency, correlated with a combination of lactic acid bacteria and yeast treatments. Syrah wines made with a combination of yeast and lactic acid bacteria (LAB) scored highest in overall quality. Indications are that the S. cerevisiae reference yeast retained more phenolic compounds during fermentation when compared to wines made with a combination of non-Saccharomyces yeasts and LAB. The improved red colour of Syrah wines may be achieved by sequential inoculation with non-Saccharomyces yeast and LAB. This could be beneficial where winemakers use grape cultivars with low anthocyanin levels in the grape skin to produce wines of improved quality.

Probiotic supplementation can assist with manipulating the rumen microbial ecosystem. Lactic acid bacteria and yeast from fermented fish (Budu) as the indigenous food from West Sumatra, Indonesia, are potential probiotics for livestock. This study aims to select the best candidate lactic acid bacteria and yeast strains from fermented fish as ruminant probiotics and evaluate the effect of their supplementation on the characteristics of rumen fermentation, feed digestion, and total gas production in vitro. This study used nine treatments, performed in triplicate, in a completely randomized design. The substrate ratio comprised of 70% Pennisetum purpureum forage and 30% concentrate. Five lactic acid bacteria and three yeast isolates were used in this study. Treatments were as follows: T0: control (basal diet); T1: T0 + Lactobacillus parabuchneri strain 3347; T2: T0 + Lactobacillus buchneri strain 5296; T3: T0 + Lactobacillus harbinensis JCM 16178; T4: T0 + Schleiferilactobacillus harbinensis strain LH991; T5: T0 + L. parabuchneri strain 6902; T6: T0 + Pichia kudriavzevii strain B-5P; T7: T0 + P. kudriavzevii strain CBS 5147; and T8: T0 + commercial yeast (Saccharomyces cerevisiae). The lactic acid bacteria inoculum contained 1.02 × 1011 colony-forming unit (CFU)/mL, while the yeast inoculum contained 1.5 × 1010 CFU/mL. The results showed that four lactic acid bacteria and three yeast produced a higher total gas yield (104-183.33 mL) compared to the control (103 mL). Supplementation with lactic acid bacteria in the rumen fermentation in vitro showed dry matter digestibility of 63%-70% and organic matter digestibility (OMD) of 64%-71%. We observed that total volatile fatty acid (VFA) production in all treatments was significantly higher (86-121 mM) compared to the control (81 mM). The concentration of NH3 production was higher in all treatments (12.33-16.83 mM) than in the control (12.25 mM). Meanwhile, the probiotic supplementation did not cause a significant change in the rumen pH (6.86-7.12). Supplementation with the lactic acid bacteria S. harbinensis strain LH991 consistently demonstrated the best results from the parameters of dry and OMD (70.29% and 71.16%, respectively), total VFA (121.67 mM), NH3 (16.83 mM), and total gas production (149.17 mL). The best results were observed from the yeast candidate P. kudriavzevii strain B-5P, where the results were dry and OMD (67.64% and 69.55% respectively), total VFA (96.67 mM), NH3 (13.42 mM), and total gas production (183.33 mL). Based on the obtained results, lactic acid bacteria S. harbinensis strain LH991 and yeast P. kudriavzevii strain B-5P are attractive candidates to be utilized as probiotics for ruminants based on their potential to improve rumen fermentation in vitro. This probiotic supplementation can increase the digestibility of feed ingredients, production of total VFA and NH3, and total gas produced.

Microbial contamination has serious consequences for the industries that use fermentation processes. Common contaminants such as faster growing lactic acid bacteria or wild yeast can rapidly outnumber inoculated culture yeast and produce undesirable end products. Our study focuses on a rapid method of identification of such contaminants based on autofluorescence spectroscopy of bacterial and yeast species. Lactic acid bacteria (Lactobacillus casei), and yeast (Saccharomyces cerevisiae) were cultured under controlled conditions and studied for variations in their autofluorescence. We observed spectral differences in the spectral range representative of tryptophan residues of proteins, with excitation at 290 nm and emission scanned in the 300 nm - 440 nm range. Excitation scans between 240 nm and 310 nm were also performed for the emission at 340 nm. Moreover, we observed clearly pronounced differences in the excitation and emission in the visible range, with 410 nm excitation. These results demonstrate that bacterial and yeast species can be differentiated using their intrinsic fluorescence both in UV and in the visible region. The comparative spectroscopic study of selected strains of Saccharomyces yeast showed clear differences between strains. Spectrally-resolved laser scanning microscopy was carried out to link the results obtained using ensembles of cells with spectral properties of individual cells. Strongly fluorescent subpopulation were observed for all yeast strains with excitation at 405 nm. The fluorescence spectra showed variations correlated with cell brightness. The presented results demonstrate that using autofluorescence, it is possible to differentiate between yeast and lactic acid bacteria and between different yeast species.

Yeasts are predominant in several fermented foods prepared from ingredients of plant as well as animal origin. The diversity of foods in which, yeasts predominate ranges from alcoholic beverages such as wines (e.g., fruit, palm and rice wines), cereal based leavened products (e.g., sourdough and idli), milk products (e.g., cheese and dahi) and condiments such as soy sauce and papads. In natural food fermentation, yeasts are either dominant alone or mixed with lactic acid bacteria or mycelial fungi. Many yeast strains have been selected from the natural fermentation and successfully utilised as starter culture for industrial food production. They have a significant impact on food quality by improving the taste, flavour, texture, nutritive values, reduction of anti-nutritional factors and improving the functionality (health promoting properties). This chapter focuses on the beneficial role of yeast in fermented foods with special reference in improving the functionality in fermented food products.

In this study, fermentation process was carried out on orange-fleshed sweet potato cubes to produce sweet potato pickle using a mixed culture of Lactobacillus plantarum and Leuconostoc mesenteroides at 30 °C over 12 days period. Spontaneous fermentation was also performed as a control. Samples were withdrawn at various time intervals for analyses of reducing sugar content, total number of lactic acid and non-lactic acid bacteria, lactic acid concentration, pH, and sensory attributes. The results showed that using a mixed culture of L. plantarum and L. mesenteroides could greatly reduce contamination of non-lactic acid bacteria, retaining low amount of reducing sugar, rapidly producing lactic acid and consequently decreasing pH value of the pickle, as well as giving better sensory score. After 12 d of fermentation, sample of pickle inoculated with mixed culture showed the following characters: total lactic acid content 0.5%, total lactic acid bacteria 8.46 log10 CFU mL-1, total non-lactic acid bacteria 1 log10 CFU mL-1, total reducing sugar 0.84 g L-1, texture 64.92 mm 50 g-1 s-1, and hedonic sensory score for both taste and aroma 4 (like) in a scale of 5. These results indicated the potential ability of the mixed culture of lactic acid bacteria to improve the quality of the pickle fermented spontaneously.

The rapid spread of drug-resistant pathogenic microbial strains has created an urgent need for the development of new anti-infective molecules, having different mechanism of action in comparison to existing drugs. Natural antimicrobial peptides (AMPs) represent a novel class of molecules with a broad spectrum of activity and a low rate in inducing bacterial resistance. In particular, linear alpha-helical cationic antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement of the innate defense against microbes. However, until now, many AMPs have failed in clinical trials because of several drawbacks that strongly limit their applicability such as degradation, cytotoxicity, and high production cost. Thus, to overcome the limitations of native peptides, a rational in silico approach to AMPs design becomes a promising strategy that drastically reduce production costs and the time required for evaluation of activity and toxicity. This chapter focuses on the strategies and methods for de novo design of potentially active AMPs. In particular, statistical-based design strategies and MD methods for modelling AMPs are elucidated.

The ongoing battle against viral pandemics continues, with the possibility of future outbreaks. The search for effective antiviral compounds that can combat a diverse range of viruses continues to be a focal point of research. This study investigated the efficacy of two natural antimicrobial peptides (AMPs) (lactoferricin and LL-37), two synthetic AMPs (melimine and Mel4), and nine AMP mimics (758, 1091, 1096, 1083, 610, NAPL, 3-BIPL, 4-BIPL, and Sau-22) against influenza A virus strains H1N1 and H3N2, human adenovirus 5 (HAdV-5), and murine norovirus 1 (MNV-1). These compounds were tested using virus pre-treatment, cell pre-treatment, or post-cell entry treatment assays, electron microscopy, and circular dichroism (CD), alongside evaluations of cytotoxicity against the host cells. After virus pre-treatment, the AMP mimics 610 and Sau-22 had relatively low IC50 values for influenza strains H1N1 (2.35 and 6.93 µM, respectively) and H3N2 (3.7 and 5.34 µM, respectively). Conversely, natural and synthetic AMPs were not active against these strains. For the non-enveloped viruses, the AMP Mel4 and mimic 1083 had moderate activity against HAdV-5 (Mel4 IC50 = 47.4 µM; 1083 IC50 = 47.2 µM), whereas all AMPs, but none of the mimics, were active against norovirus (LL-37 IC50 = 4.2 µM; lactoferricin IC50 = 23.18 µM; melimine IC50 = 4.8 µM; Mel4 IC50 = 8.6 µM). Transmission electron microscopy demonstrated that the mimics targeted the outer envelope of influenza viruses, while the AMPs targeted the capsid of non-enveloped viruses. CD showed that Mel4 adopted an α-helical structure in a membrane mimetic environment, but mimic 758 remained unstructured. The diverse activity against different virus groups is probably influenced by charge, hydrophobicity, size, and, in the case of natural and synthetic AMPs, their secondary structure. These findings underscore the potential of peptides and mimics as promising candidates for antiviral therapeutics against both enveloped and non-enveloped viruses.

Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation

Aflatoxin M1 absorption by non-viable cells of lactic acid bacteria and Saccharomyces cerevisiae strains in Frescal cheese

Global demand for renewable and sustainable energy is increasing, and one of the most common biofuels is ethanol. Most ethanol is produced by Saccharomyces cerevisiae (yeast) fermentation of either crops rich in sucrose (e.g., sugar cane and sugar beet) or starch-rich crops (e.g., corn and starchy grains). Ethanol produced from these sources is termed a first-generation biofuel. Yeast fermentation can yield a range of additional valuable co-products that accumulate during primary fermentation (e.g., protein concentrates, water soluble metabolites, fusel alcohols, and industrial enzymes). Distillers’ solubles is a liquid co-product that can be used in animal feed or as a resource for recovery of valuable materials. In some processes it is preferred that this fraction is modified by a second fermentation with another fermentation organism (e.g., lactic acid bacteria). Such two stage fermentations can produce valuable compounds, such as 1,3-propanediol, organic acids, and bacteriocins. The use of lactic acid bacteria can also lead to the aggregation of stillage proteins and enable protein aggregation into concentrates. Once concentrated, the protein has utility as a high-protein feed ingredient. After separation of protein concentrates the remaining solution is a potential source of several known small molecules. The purpose of this review is to provide policy makers, bioethanol producers, and researchers insight into additional added-value products that can be recovered from ethanol beers. Novel products may be isolated during or after distillation. The ability to isolate and purify these compounds can provide substantial additional revenue for biofuel manufacturers through the development of marketable co-products.

Maotai-flavor liquor, derived from a multi-stage solid fermentation process, is one of the most popular liquors in China. Its quality and flavor are closely related to diverse lactic acid bacteria (LAB). It is therefore significant to characterize LAB for the manufacturing of Maotai-flavor liquor. In this study, LAB in solid state fermentation stages were analyzed through high-throughput sequencing and cultivation-dependent methods during the fermentation process. In total, 65 LAB species were identified in the fermented matrix, showing a much higher LAB diversity than other types of Chinese liquor. In addition, discrepancies were found to exist in the dominant LAB community structures during different fermentation stages, and strains of the Lactobacillus genus were found to be the most dominant LAB. Furthermore, 33 LAB species were identified from the fermentation matrix through the cultivation and 16 S rRNA analysis of single isolates, thus representing 50.8% coverage of the detected LAB species. The relative abundance of isolated LAB in the total abundance of LAB was 95.9% during the fermentation process. In doing so, Lac2 and Lac13, two potentially new LAB species, were isolated. To the best of our knowledge, this study represents the identification of the highest number of LAB species by cultivation-dependent methods from fermented grains of Chinese liquor. In summary, this study monitored the LAB species composition of solid fermented matrix during the fermentation of Maotai-flavor liquor and has highlighted the potential importance of the higher abundance of LAB and the effect it has on the unique flavor of Maotai liquor.