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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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Christian Solem reports financial support was provided by Technical University of Denmark. Christian Solem reports a relationship with Technical University of Denmark that includes: employment., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Lactococcus lactis, a lactic acid bacterium with a typical fermentative metabolism, can also use oxygen as an extracellular electron acceptor. Here we demonstrate, for the first time, that L. lactis blocked in NAD + regeneration can use the alternative electron acceptor ferricyanide to support growth. By electrochemical analysis and characterization of strains carrying mutations in the respiratory chain, we pinpoint the essential role of the NADH dehydrogenase and 2-amino-3-carboxy-1,4-naphtoquinone in extracellular electron transfer (EET) and uncover the underlying pathway systematically. Ferricyanide respiration has unexpected effects on L. lactis, e.g., we find that morphology is altered from the normal coccoid to a more rod shaped appearance, and that acid resistance is increased. Using adaptive laboratory evolution (ALE), we successfully enhance the capacity for EET. Whole-genome sequencing reveals the underlying reason for the observed enhanced EET capacity to be a late-stage blocking of menaquinone biosynthesis. The perspectives of the study are numerous, especially within food fermentation and microbiome engineering, where EET can help relieve oxidative stress, promote growth of oxygen sensitive microorganisms and play critical roles in shaping microbial communities., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Nisin has a tendency to associate with the cell wall of the producing strain, which inhibits growth and lowers the ceiling for nisin production. With the premise that resistance to the cationic chlorhexidine could reduce nisin binding, variants with higher tolerance to this compound were isolated. One of the resistant isolates, AT0606, had doubled its resistance to nisin, and produced three times more free nisin, when cultured in shake flasks. Characterization revealed that AT0606 had an overall less negatively charged and thicker cell wall, and these changes appeared to be linked to a defect high-affinity phosphate uptake system, and a mutation inactivating the oleate hydratase. Subsequently, the potential of using AT0606 for cost efficient production of nisin was explored, and it was possible to attain a high titer of 13181 IU/mL using a fermentation substrate based on molasses and a by-product from whey protein hydrolysate production., (Copyright © 2022. Published by Elsevier Ltd.)

Nisin is commonly used as a biopreservative in foods. For industrial production, nisin-producing Lactococcus lactis strains are usually grown to high cell densities to achieve the highest possible nisin titer. However, accumulation of lactic acid eventually halts production, even in pH-controlled fermentations. Here, we describe a nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, with low lactate dehydrogenase activity. The ability of Ge001 to produce nisin was tested using dairy waste as the fermentation substrate. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn 2+ and 100 mM Ca 2+ , respectively. A high titer of 12 084 IU/mL nisin could be reached, which is comparable to the highest titers reported using expensive, rich media. Summing up, we here present a 100% natural, robust, and sustainable approach for producing food-grade nisin and acetoin from readily available dairy waste.

Proteolysis is the most significant primary ripening process in cheese, as it produces amino acids that serve as precursors for various flavor compounds. Therefore, the degree of proteolysis is an important indicator of cheese ripening. Here, the potential of nisin-permeabilized lactic acid bacteria as proteolysis accelerant during cheese ripening was explored. Both nisin-permeabilized Lactococcus lactis and Streptococcus thermophilus cells could hydrolyze peptidase substrates lysyl– p -nitroanilide (Lys– p NA) and glycyl–pro– p NA [Gly–Pro– p NA] efficiently. The overall proteolytic capacity of permeabilized cells depended on nisin concentration, strain used, temperature, and pH. The best candidate, S. thermophilus ST057-1, could hydrolyze 80% Lys– p NA and 94% Gly–Pro– p NA at 30 °C and pH 7 when treated with 5 μg/mL nisin. Permeabilized ST057-1 cells, when used as a starter adjunct, led to a significant increase in free amino acid content in model cheeses, up to 1.5 times that of the reference cheese, within 80 days of cheese ripening. This study highlights the potential of utilizing nisin-permeabilized S. thermophilus cells as an adjunct to accelerate proteolysis during cheese ripening. • Nisin-permeabilized lactic acid bacteria had higher peptidase activity. • Nisin promotes autolysis of L. lactis but has less effect on S. thermophilus. • Permeabilized S. thermophilus accelerated proteolysis when used as an adjunct. [ABSTRACT FROM AUTHOR]

Bacterial smear-ripened cheeses are commonly contaminated with fungi and the potentially dangerous pathogen Listeria monocytogenes , which compromises food safety and quality. Here, we present new approach for accelerating smear development on these cheeses, while reducing growth of unwanted microorganisms. We grew the smear microorganism Brevibacterium linens in acid whey (AW) supplemented with a by-product of whey protein hydrolysate production (FCH). B. linens grew to a high cell density (10.27 log 10 CFU/mL), and alkalized the AW-FCH medium. We found that the fermented AW-FCH, with its high pH, rapidly killed L. monocytogenes and yeasts. When culture medium was smeared on the cheese surface, the surface pH was rapidly neutralized, and in just 7 days, the cheeses developed a dense orange color and growth of spoilage fungi was inhibited. Summing up, by growing B. linens to high cell density on low-value dairy byproducts, and applying dense suspensions onto cheeses, we can reduce the time needed for establishing the smear. Added benefits are increased food safety and less spoilage, as quick smear development inhibits growth of unwanted microorganisms on the rind. Preliminary tests at a dairy show that flavor development is comparable to traditional bacterial surface-ripened cheeses with far more complex surface microflora. • Fermentation as a means to valorize low-value dairy by-products. • Dense B. linens CX3 suspensions for accelerating surface smear development. • Strong antimicrobial effect of acid whey fermented with B. linens CX 3. [ABSTRACT FROM AUTHOR]