Scrutinizing a Lactococcus lactis mutant with enhanced capacity for extracellular electron transfer reveals a unique role for a novel type-II NADH dehydrogenase.

Lactococcus lactis, a lactic acid bacterium used in food fermentations and commonly found in the human gut, is known to possess a fermentative metabolism. L. lactis, however, has been demonstrated to transfer metabolically generated electrons to external electron acceptors, a process termed extracellular electron transfer (EET). Here, we investigated an L. lactis mutant with an unusually high capacity for EET that was obtained in an adaptive laboratory evolution (ALE) experiment. First, we investigated how global gene expression had changed, and found that amino acid metabolism and nucleotide metabolism had been affected significantly. One of the most significantly upregulated genes encoded the NADH dehydrogenase NoxB. We found that this upregulation was due to a mutation in the promoter region of NoxB, which abolished carbon catabolite repression. A unique role of NoxB in EET could be attributed and it was directly verified, for the first time, that NoxB could support respiration in L. lactis. NoxB, was shown to be a novel type-II NADH dehydrogenase that is widely distributed among gut microorganisms. This work expands our understanding of EET in Gram-positive electroactive microorganisms and the special significance of a novel type-II NADH dehydrogenase in EET.IMPORTANCEElectroactive microorganisms with extracellular electron transfer (EET) ability play important roles in biotechnology and ecosystems. To date, there have been many investigations aiming at elucidating the mechanisms behind EET, and determining the relevance of EET for microorganisms in different niches. However, how EET can be enhanced and harnessed for biotechnological applications has been less explored. Here, we compare the transcriptomes of an EET-enhanced L. lactis mutant with its parent and elucidate the underlying reason for its superior performance. We find that one of the most significantly upregulated genes is the gene encoding the NADH dehydrogenase NoxB, and that upregulation is due to a mutation in the catabolite-responsive element that abolishes carbon catabolite repression. We demonstrate that NoxB has a special role in EET, and furthermore show that it supports respiration to oxygen, which has never been done previously. In addition, a search reveals that this novel NoxB-type NADH dehydrogenase is widely distributed among gut microorganisms.

Bad to the bone? - Genomic analysis of Enterococcus isolates from diverse environments reveals that most are safe and display potential as food fermentation microorganisms.

Enterococci comprise a group of lactic acid bacteria (LAB) with considerable potential to serve as food fermentation microorganisms. Unfortunately, enterococci have received a lot of negative attention, due to the occurrence of pathogenic and multidrug resistant strains. In this study, we used genomics to select safe candidates among the forty-four studied enterococcal isolates. The genomes of the forty-four strains were fully sequenced and assessed for presence of virulence and antibiotic resistance genes. Nineteen isolates belonging to the species Enterococcus lactis, Enterococcus faecium, Enterococcus durans, and Enterococcus thailandicus, were deemed safe from the genome analysis. The presence of secondary metabolite gene clusters for bacteriocins was assessed, and twelve candidates were found to secrete antimicrobial compounds effective against Listeria monocytogenes isolated from cheese and Staphylococcus aureus. Physiological characterization revealed nineteen industrial potentials; all strains grew well at 42 °C and acidified 1.5 hours faster than their mesophilic counterpart Lactococcus lactis, with which they share metabolism and flavor forming ability. We conclude that a large fraction of the examined enterococci were safe and could serve as excellent food fermentation microorganisms with inherent bioprotective abilities.

Simple & better - Accelerated cheese ripening using a mesophilic starter based on a single strain with superior autolytic properties.

In the manufacture of rennet-coagulated cheese, autolysis is a rate-limiting step for ripening. Previously, a highly autolytic and thermotolerant Lactococcus lactis strain, RD07, was generated, which in preliminary laboratory cheese trials demonstrated great potential as a cheese ripening accelerant. RD07 is proteinase positive (Prt+) and capable of metabolizing citrate (Cit+). In this study, we obtained two derivatives of RD07: EC8 lacking the citrate plasmid, and EC2 lacking the proteinase plasmid. EC2 and EC8 retained the autolytic properties of RD07, and autolyzed 20 times faster than Flora Danica (FD) and SD96, where the latter is the parent of RD07. The three strains EC2, EC8 and RD07 were used in a ratio of 90:8:2, to create a simple starter termed ERC. ERC was less sensitive to cooking when cultured in milk and autolyzed well after entering the stationary phase upon facing sugar starvation. The ERC starter was benchmarked against FD and SD96 in laboratory cheese trials. The free amino acid content in cheese prepared using the ERC culture was 31 % and 34 % higher than in cheese prepared using FD and SD96, respectively. Overall, the ERC culture resulted in a more rapid release of free amino acids. A large-scale (5000 L) Gouda cheese trial at a Danish dairy demonstrated that the single strain ERC starter was comparable in performance to FD + an adjunct Lactobacillus helveticus culture. Furthermore, a large-scale Danbo cheese trial demonstrated that ERC could reduce the ripening period by 50 % for long-term ripened (25 weeks) cheese, resulting in better cheese.

Transforming acid whey into a resource by selective removal of lactic acid and galactose using optimized food-grade microorganisms.

The presence of lactic acid and galactose makes spray drying of acid whey (AW) a significant challenge for the dairy industry. In this study, a novel approach is explored to remove these compounds, utilizing food-grade microorganisms. For removing lactic acid, Corynebacterium glutamicum was selected, which has an inherent ability to metabolize lactic acid but does so slowly. To accelerate lactic acid metabolism, a mutant strain G6006 was isolated through adaptive laboratory evolution, which metabolized all lactic acid from AW two times faster than its parent strain. To eliminate galactose, a lactose-negative mutant of Lactococcus lactis that cannot produce lactate was generated. This strain was then co-cultured with G6006 to maximize the removal of both lactic acid and galactose. The microbially "filtered" AW could readily be spray dried into a stable lactose powder. This study highlights the potential of utilizing food-grade microorganisms to process AW, which currently constitutes a global challenge.

Fermented butter aroma for plant-based applications.

Plant-based dairy alternatives are gaining increasing interest, e.g. alternatives to yoghurt, cheese, and butter. In all these products butter flavor (diacetyl + acetoin) plays an important role. We previously have reported efficient butter flavor formation from low value dairy side streams using a dairy isolate of Lactococcus lactis deficient in lactate dehydrogenase. Here, we have tested the ability of this strain, RD1M5, to form butter flavor in plant milks based on oat and soy. We found that oat milk, with its high sugar content, supported more efficient production of butter aroma, when compared to soy milk. When supplemented with glucose, efficient butter aroma production was achieved in soy milk as well. We also carried out an extended adaptive laboratory evolution of the dairy strain in oat milk. After two months of adaptation, we obtained a strain with enhanced capacity for producing butter aroma. Despite of its high sugar content, RD1M5 and its adapted version only metabolized approximately 10% of the fermentable sugars available in the oat milk, which we found was due to amino acid starvation and partly starvation for vitamins. The study demonstrates that dairy cultures have great potential for use in plant-based fermentations.

Consolidated Bioprocessing in a Dairy Setting─Concurrent Yoghurt Fermentation and Lactose Hydrolysis without Using Lactase Enzymes.

Streptococcus thermophilus is a fast-growing lactic acid bacterium (LAB) used in yoghurt and cheese manufacturing. Recently, we reported how this bacterium could serve as a cell catalyst for hydrolyzing lactose when permeabilized by nisin A. To enhance the lactose hydrolyzing activity of S. thermophilus, we mutated a dairy strain and screened for variants with elevated ß-galactosidase activity. Two isolates, ST30-8 and ST95, had 2.4-fold higher activity. Surprisingly, both strains were able to hydrolyze lactose when used as whole-cell lactase catalysts without permeabilization, and ST30-8 hydrolyzed 30 g/L lactose in 6 h at 50 °C using 0.18 g/L cells. Moreover, both strains hydrolyzed lactose while growing in milk. Genome sequencing revealed a mutation in l-lactate dehydrogenase, which we believe hampers growth and increases the capacity of S. thermophilus to hydrolyze lactose. Our findings will allow production of sweet lactose-reduced yoghurt without the use of costly purified lactase enzymes.

Molecular Characterization of Fermenting Yeast Species from Fermented Teff Dough during Preparation of Injera Using ITS DNA Sequence.

Identification of the yeast responsible for Injera fermentation is important in order to be more consistent and for scale-up of Injera production. In this study, yeast were isolated and identified from fermenting teff dough sample collected from household, hotels, and microenterprises, Addis Ababa. Initially, the yeast obtained from fermenting teff dough of different sources were selected on the basis of their CO2 production potentials. Its DNA sequencing of isolated yeast identified Pichia fermentans, Pichia occidentalis, Candida humilis, Saccharomyces cerevisiae, and Kazachstania bulderi. The association of identified yeast to their sources indicated the presence of Pichia fermentans in fermenting dough samples collected from all sources whereas Kazachstania bulderi, Saccharomyces cerevisiae, and Candida humilis were shown to be present in samples collected from households, hotels, and microenterprises, respectively. The phenotypes and CO2 production potentials of this yeast were also documented. This study has confirmed the presence of different yeast species in the fermentation of teff dough and hinted the complex nature of Injera dough fermentation.

In Vivo anti-trypanosomal activity of dichloromethane and methanol crude leaf extracts of Dovyalis abyssinica (Salicaceae) against Trypanosoma congolense.

BACKGROUND: African trypanosomiasis affects both humans and livestock in sub-Saharan countries including Ethiopia. Due to limitations to current chemotherapy, there is an urgent need for the development of new, safe, cheap and effective drugs. In the present study, the leaf of Dovyalis abyssinica was tested for its in vivo antitrypanosomal activity against Trypanosoma congolense field isolate on mice. METHODS: The leaf of D. abyssinica was macerated using dichloromethane and methanol. The extracts at doses of 250, 200, 150 and 100 mg/kg body weight were administered intraperitonealy daily for 7 days to mice infected with T. congolense. Following administration, parasitemia, packed cell volume, rectal temperature, body weight and survival time were monitored. RESULTS: Administration of dichloromethane and methanol extracts at 250 and 200 mg/kg reduced (p<0.05) parasitemia and rectal temperature, and improved (p<0.05) PCV, mean body weight, and mean survival time compared to dimethylsulfoxide treatment. CONCLUSION: Crude dichloromethane and methanol leaf extracts of D. abyssinica displayed anti-trypanosomal activity that may serve as lead for the development of effective alternative antitrypanosomal drugs.

Prevalence and determinants of hypertension in rural and urban areas of southern Ethiopia.

BACKGROUND: Hypertension is an important public health challenge in both economically developed and developing countries. OBJECTIVE: To asses the magnitude of hypertension in rural and urban southern Ethiopia. PATIENTS AND METHODS: A cross sectional survey was conducted on 979 study participants in Sidama Zone, South Ethiopia from November 1-30, 2008. Data were collected using a structured questionnaire and standardized procedures recommended by the WHO MONICA project for the measurement of the anthopometric variables. Analysis was done using SPSS 15.0 version. RESULTS: Out of 979 participating subjects. 485 were from urban and 494 were from rural. The prevalence of hypertension was 9.9% with 10.1% in urban and 9.7% in rural areas ranging from 4.2% in those below 30 years to 29.4% in those above 60 years. Bivariate analysis showed hypertension was highly occurred more in those above 30 years old, in those with the family history of hypertension, and a BMI > or =25 kg/m2. Hypertension also correlated with, less physical activity, extended family size, personal and family history of diabetes mellitus, measured dysglycemia, excess meat consumption and drinking alcohol. Multivariate analysis showed similar correlation of increased possibility of hypertension with being over 30 years, having a family history of hypertension, a BMI > or =25 kg/m2. and excess meat consumption. Tea drinking was found as a protective factor for hypertension on bivariate and multivariate analysis. CONCLUSIONS: Hypertension has equal public health importance in urban and rural settings of southern Ethiopia. Hypertension is common among those age over 30years. overweight, consume excess meat and have family history of hypertension. Drinking tea may have a protective effect for hypertension.