Competitive Exclusion Is a Major Bioprotective Mechanism of Lactobacilli against Fungal Spoilage in Fermented Milk Products.

A prominent feature of lactic acid bacteria (LAB) is their ability to inhibit growth of spoilage organisms in food, but hitherto research efforts to establish the mechanisms underlying bioactivity focused on the production of antimicrobial compounds by LAB. We show, in this study, that competitive exclusion, i.e., competition for a limited resource by different organisms, is a major mechanism of fungal growth inhibition by lactobacilli in fermented dairy products. The depletion of the essential trace element manganese by two Lactobacillus species was uncovered as the main mechanism for growth inhibition of dairy spoilage yeast and molds. A manganese transporter (MntH1), representing one of the highest expressed gene products in both lactobacilli, facilitates the exhaustive manganese scavenging. Expression of the mntH1 gene was found to be strain dependent, affected by species coculturing and the growth phase. Further, deletion of the mntH1 gene in one of the strains resulted in a loss of bioactivity, proving this gene to be important for manganese depletion. The presence of an mntH gene displayed a distinct phylogenetic pattern within the Lactobacillus genus. Moreover, assaying the bioprotective ability in fermented milk of selected lactobacilli from 10 major phylogenetic groups identified a correlation between the presence of mntH and bioprotective activity. Thus, manganese scavenging emerges as a common trait within the Lactobacillus genus, but differences in expression result in some strains showing more bioprotective effect than others. In summary, competitive exclusion through ion depletion is herein reported as a novel mechanism in LAB to delay the growth of spoilage contaminants in dairy products.IMPORTANCE In societies that have food choices, conscious consumers demand natural solutions to keep their food healthy and fresh during storage, simultaneously reducing food waste. The use of "good bacteria" to protect food against spoilage organisms has a long, successful history, even though the molecular mechanisms are not fully understood. In this study, we show that the depletion of free manganese is a major bioprotective mechanism of lactobacilli in dairy products. High manganese uptake and intracellular storage provide a link to the distinct, nonenzymatic, manganese-catalyzed oxidative stress defense mechanism, previously described for certain lactobacilli. The evaluation of representative Lactobacillus species in our study identifies multiple relevant species groups for fungal growth inhibition via manganese depletion. Hence, through the natural mechanism of nutrient depletion, the use of dedicated bioprotective lactobacilli constitutes an attractive alternative to artificial preservation.

Rates of pyruvate carboxylase, glutamate and GABA neurotransmitter cycling, and glucose oxidation in multiple brain regions of the awake rat using a combination of [2-13C]/[1-13C]glucose infusion and 1H-[13C]NMR ex vivo.

Anaplerosis occurs predominately in astroglia through the action of pyruvate carboxylase (PC). The rate of PC (Vpc) has been reported for cerebral cortex (or whole brain) of awake humans and anesthetized rodents, but regional brain rates remain largely unknown and, hence, were subjected to investigation in the current study. Awake male rats were infused with either [2-13C]glucose or [1-13C]glucose (n = 27/30) for 8, 15, 30, 60 or 120 min, followed by rapid euthanasia with focused-beam microwave irradiation to the brain. Blood plasma and extracts of cerebellum, hippocampus, striatum, and cerebral cortex were analyzed by 1H-[13C]-NMR to establish 13C-enrichment time courses for glutamate-C4,C3,C2, glutamine-C4,C3, GABA-C2,C3,C4 and aspartate-C2,C3. Metabolic rates were determined by fitting a three-compartment metabolic model (glutamatergic and GABAergic neurons and astroglia) to the eighteen time courses. Vpc varied by 44% across brain regions, being lowest in the cerebellum (0.087 ± 0.004 µmol/g/min) and highest in striatum (0.125 ± 0.009) with intermediate values in cerebral cortex (0.106 ± 0.005) and hippocampus (0.114 ± 0.005). Vpc constituted 13-19% of the oxidative glucose consumption rate. Combination of cerebral cortical data with literature values revealed a positive correlation between Vpc and the rates of glutamate/glutamine-cycling and oxidative glucose consumption, respectively, consistent with earlier observations.

Evaluation of the antidiabetic potential of Psidium guajava L. (Myrtaceae) using assays for α-glucosidase, α-amylase, muscle glucose uptake, liver glucose production, and triglyceride accumulation in adipocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Psidium guajava L. (Myrtaceae) leaves are used as an herbal antidiabetic remedy in several parts of the world. On Madagascar, both the bark and leaves are used for treatment of diabetes. MATERIALS AND METHODS: Dilution series of ethanolic extracts of P. guajava leaves and bark were used for determining inhibitory activities against yeast α-glucosidase and porcine α-amylase. Skeletal muscle glucose uptake was measured using 2-deoxy-D-(1-3H)-glucose in murine C2C12 skeletal muscle cells. Hepatic glucose-6-phosphatase activity in rat hepatoma H4IIE cells and triglyceride accumulation in murine 3T3-L1 adipocyte-like cells were assessed using Wako AutoKit Glucose assays and AdipoRed reagent, respectively. Cells were incubated for 18 h with the maximal non-toxic concentrations of the plant extracts determined by the lactate dehydrogenase cytotoxicity assay. RESULTS: Ethanolic extracts of P. guajava leaf and bark inhibited α-glucosidase with IC50 values of 1.0 ± 0.3 and 0.5 ± 0.01 µg/mL, respectively. In the α-amylase inhibition assay, the ethanolic extract of bark of P. guajava showed an IC50 value of 10.6 ± 0.4 µg/mL. None of the extracts were able to reduce glucose-6-phosphatase activity in rat hepatoma H4IIE cells. In contrast, P. guajava leaf extract significantly increased 2-deoxy-D-[1-3H]-glucose uptake in C2C12 muscle cells (161.4 ± 10.1%, p = 0.0015) in comparison to the dimethyl sulfoxide (DMSO) vehicle control, as did the reference compounds metformin (144.0 ± 7.7%, p = 0.0345) and insulin (141.5 ± 13.8%, p = 0.0495). Furthermore, P. guajava leaf and bark extracts, as well as the reference compound rosiglitazone, significantly enhanced triglyceride accumulation in 3T3-L1 cells (252.6 ± 14.2%, p < 0.0001, 211.1 ± 12.7%, p < 0.0001, and 201.1 ± 9.2%, p < 0.0001, respectively) to levels higher than the DMSO vehicle control. Moreover, P. guajava leaf extract significantly enhanced the triglyceride accumulation in 3T3-L1 cells compared to rosiglitazone. CONCLUSION: The results demonstrated that P. guajava leaf and bark extracts can be used as a natural source of α-glucosidase inhibitors. In addition, the bark extract of P. guajava was an effective α-amylase inhibitor. Moreover, P. guajava leaf extract improved glucose uptake in muscle cells, while both leaf and bark extracts enhanced the triglyceride content in adipocytes in culture. P. guajava leaf and bark extracts may thus hypothetically have future applications in the treatment of type 2 diabetes.