A multifaceted investigation of lactococcal strain diversity in undefined mesophilic starter cultures.

The dairy fermentation industry relies on the activity of lactic acid bacteria in robust starter cultures to accomplish milk acidification. Maintenance of the composition of these starter cultures, whether defined or undefined, is essential to ensure consistent and high-quality fermentation end products. To date, limited information exists regarding the microbial composition of undefined starter culture systems. Here, we describe a culture-based analysis combined with a metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, we describe a qPCR-based genotype detection assay, which is capable of discerning nine distinct lactococcal genotypes to characterize these undefined starter cultures, and which can be applied to monitor compositional changes in an undefined starter culture during a fermentation. IMPORTANCE: This study reports on the development of a combined culture-based analysis and metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, a novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes (based on lactococcal cell wall polysaccharide biosynthesis gene clusters), was used to monitor compositional changes in an undefined starter culture following phage attack. These analytical approaches facilitate a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important QC aspect due to the increasing demand for consistent and high-quality dairy products.

Investigating the growth kinetics in sourdough microbial associations.

In this study we investigated the effect of the single strain in stabilization of type I sourdough microbial associations by crossing six different Fructilactobacillus sanfranciscensis with five Kazachstania humilis strains. Furthermore, we compared three predictive models, Zwitwering based on Gompertz's equation, Baranyi and Roberts' function and Schiraldi's function to evaluate which one best fitted the experimental data in determining the behaviour of co-cultivated microorganisms. Specific growth rates (µm) and lag time (λ) values for each mixed population were assessed. Results showed that the different F. sanfranciscensis strains significantly steer the growth kinetics within the pair and affect the ratio bacterial/yeast cells, as data analysis confirmed, whereas K. humilis accommodates to the bacterial strain. To compare the growth models, Root Mean Square (RMS) values were calculated for each predicted curve by implementing an algorithm based on an iterative process to minimize the deviation among observed and calculated data. Schiraldi's function performed better than the others, revealing, on average, the smallest RMS values and providing the best fitting for over 70% of co-cultivation experiments. Models prove to be consistent in predicting growth kinetics of microbial consortia too.

The Baseplate of Lactobacillus delbrueckii Bacteriophage Ld17 Harbors a Glycerophosphodiesterase.

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca(2+) ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage.

Selected Probiotic Lactobacilli Have the Capacity To Hydrolyze Gluten Peptides during Simulated Gastrointestinal Digestion.

The aim of this study was to demonstrate the capacity of probiotic lactobacilli to hydrolyze immunogenic gluten peptides. Eighteen commercial strains of probiotic lactobacilli with highly variable peptidase activity (i.e., aminopeptidase N, iminopeptidase, prolyl endopeptidyl peptidase, tripeptidase, prolidase, prolinase, and dipeptidase), including toward Pro-rich peptides, were tested in this study. Ten probiotic strains were selected on the basis of their specific enzyme activity. When pooled, these 10 strains provided the peptidase portfolio that is required to completely degrade the immunogenic gluten peptides involved in celiac disease (CD). The selected probiotic mixture was able to completely hydrolyze well-known immunogenic epitopes, including the gliadin 33-mer peptide, the peptide spanning residues 57 to 68 of the α9-gliadin (α9-gliadin peptide 57-68), A-gliadin peptide 62-75, and γ-gliadin peptide 62-75. During digestion under simulated gastrointestinal conditions, the pool of 10 selected probiotic lactobacilli strongly hydrolyzed the wheat bread gluten (ca. 18,000 ppm) to less than 10 ppm after 360 min of treatment. As determined by multidimensional chromatography (MDLC) coupled to nanoelectrospray ionization (nano-ESI)-tandem mass spectrometry (MS/MS), no known immunogenic peptides were detected in wheat bread that was digested in the presence of the probiotics. Accordingly, the level of cytokines (interleukin 2 [IL-2], IL-10, and interferon gamma [IFN-γ]) produced by duodenal biopsy specimens from CD patients who consumed wheat bread digested by probiotics was similar to the baseline value (negative control). Probiotics that specifically hydrolyze gluten polypeptides could also be used to hydrolyze immunogenic peptides that contaminate gluten-free products. This could provide a new and safe adjunctive therapy alternative to the gluten-free diet (GFD).IMPORTANCE This study confirmed that probiotic Lactobacillus strains have different enzymatic abilities for hydrolyzing polypeptides, including the Pro-rich epitopes involved in the pathology of CD. Ten lactobacilli with complementary peptidase activities that hydrolyze gluten peptides during simulated gastrointestinal digestion were selected and tested. The results collected showed the potential of probiotic formulas as novel dietary treatments for CD patients.

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages.

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations.

Candida milleri species reveals intraspecific genetic and metabolic polymorphisms.

Candida milleri, together with Candida humilis, is the most representative yeast species found in type I sourdough ecosystems. In this work, comparison of the ITS region and the D1/D2 domain of 26S rDNA gene partial sequences, karyotyping, mtDNA-RFLP analysis, Intron Splice Site dispersion (ISS-PCR) and (GTG)5 microsatellite analyses, assimilation test of different carbohydrates, and metabolome assessment by FT-IR analysis, were investigated in seventeen strains isolated from four different companies as well as in type strains CBS6897(T) and CBS5658(T). Most isolates were ascribed to C. milleri, even if a strong relatedness was confirmed with C. humilis as well, particularly for three strains. Genetic characterization showed a high degree of intraspecific polymorphism since 12 different genotypes were discriminated. The number of chromosomes varied from 9 to 13 and their size ranged from less than 0.3 to over 2 Mbp. Phenotypic traits let to recognize 9 different profiles of carbon sources assimilation. FT-IR spectra from yeast cells cultivated in different media and collected at different growth phases revealed a diversity of behaviour among strains in accordance with the results of PCR-based fingerprinting. A clear evidence of the polymorphic status of C. milleri species is provided thus representing an important feature for the development of technological applications in bakery industries.

New Genomic Techniques applied to food cultures: a powerful contribution to innovative, safe, and sustainable food products.

Nontransgenic New Genomic Techniques (NGTs) have emerged as a promising tool for food industries, allowing food cultures to contribute to an innovative, safe, and more sustainable food system. NGTs have the potential to be applied to microorganisms, delivering on challenging performance traits like texture, flavour, and an increase of nutritional value. This paper brings insights on how nontransgenic NGTs applied to food cultures could be beneficial to the sector, enabling food industries to generate innovative, safe, and sustainable products for European consumers. Microorganisms derived from NGTs have the potentials of becoming an important contribution to achieve the ambitious targets set by the European 'Green Deal' and 'Farm to Fork' policies. To encourage the development of NGT-derived microorganisms, the current EU regulatory framework should be adapted. These technologies allow the introduction of a precise, minimal DNA modification in microbial genomes resulting in optimized products carrying features that could also be achieved by spontaneous natural genetic evolution. The possibility to use NGTs as a tool to improve food safety, sustainability, and quality is the bottleneck in food culture developments, as it currently relies on lengthy natural evolution strategies or on untargeted random mutagenesis.

Increasing the heme-dependent respiratory efficiency of Lactococcus lactis by inhibition of lactate dehydrogenase.

The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism.

Exploration of the genomic diversity and core genome of the Bifidobacterium adolescentis phylogenetic group by means of a polyphasic approach.

In the current work, we describe genome diversity and core genome sequences among representatives of three bifidobacterial species, i.e., Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium pseudocatenulatum, by employing a polyphasic approach involving analysis of 16S rRNA gene and 16S-23S internal transcribed spacer (ITS) sequences, pulsed-field gel electrophoresis (PFGE), and comparative genomic hybridization (CGH) assays.

The effects of liquid versus spray-dried Laminaria digitata extract on selected bacterial groups in the piglet gastrointestinal tract (GIT) microbiota.

In this study, the effects of supplementing animal feed with a liquid and spray-dried fucoidan and laminarin extract, derived from the seaweed Laminaria digitata on the porcine gastrointestinal microbiota, specifically the communities of Lactobacillus, Bifidobacterium and enterobacteria were evaluated. Twenty four piglets were fed one of three diets over a 21-day period to determine the effect that each had on the bacterial communities. The dietary treatments were as follows; (1) control diet, (2) control diet plus spray-dried formulation of laminarin fucoidan (L/F-SD) extract, (3) control diet plus a liquid formulation of (L/F-WS) extract. Control diet consisted of wheat, soya bean meal, soya oil and a vitamin and mineral mixture. The L/F-SD and L/F-WS supplemented diets had equal proportion of 500 ppm laminarin and fucoidan. At the end of the 21 day feeding period all animals were sacrificed and samples were collected from the ileum, caecum and colon. Counts were determined for Lactobacillus, Bifidobacterium and enterobacteria. Plate count analysis revealed that the L/F-SD diet caused a statistically significant 1.5 log and 2 log increases in the Lactobacillus and Bifidobacterium counts of ileum samples respectively. A greater difference was observed with the L/F-WS diet in that Lactobacillus and Bifidobacterium increased by 2 log and 3 log respectively. Alterations in the Lactobacillus species composition of the gastrointestinal tract (GIT) were analysed using specific PCR - denaturing gradient gel electrophoresis (PCR-DGGE). The DGGE profiles indicated that Lactobacillus species richness decreased along the gastrointestinal tract i.e. the number of dominant species detected in the colon was less than those detected in the ileum and caecum irrespective of the diet consumed. Consumption of both the L/F-SD and L/F-WS diets resulted in a richer Lactobacillus species composition in the ileum, with the L/F-SD diet being associated the emergence of Lactobacillus agilis in the colon. The study indicated that the L/F-WS extract was superior to the L/F-SD extract in increasing the titre of beneficial bacteria in the gastrointestinal tract (GIT).

Bacteriophage-host interactions as a platform to establish the role of phages in modulating the microbial composition of fermented foods.

Food fermentation relies on the activity of robust starter cultures, which are commonly comprised of lactic acid bacteria such as Lactococcus and Streptococcus thermophilus. While bacteriophage infection represents a persistent threat that may cause slowed or failed fermentations, their beneficial role in fermentations is also being appreciated. In order to develop robust starter cultures, it is important to understand how phages interact with and modulate the compositional landscape of these complex microbial communities. Both culture-dependent and -independent methods have been instrumental in defining individual phage-host interactions of many lactic acid bacteria (LAB). This knowledge needs to be integrated and expanded to obtain a full understanding of the overall complexity of such interactions pertinent to fermented foods through a combination of culturomics, metagenomics, and phageomics. With such knowledge, it is believed that factory-specific detection and monitoring systems may be developed to ensure robust and reliable fermentation practices. In this review, we explore/discuss phage-host interactions of LAB, the role of both virulent and temperate phages on the microbial composition, and the current knowledge of phageomes of fermented foods.

Needle in a Whey-Stack: PhRACS as a Discovery Tool for Unknown Phage-Host Combinations.

The field of metagenomics has rapidly expanded to become the go-to method for complex microbial community analyses. However, there is currently no straightforward route from metagenomics to traditional culture-based methods of strain isolation, particularly in (bacterio)phage biology, leading to an investigative bottleneck. Here, we describe a method that exploits specific phage receptor binding protein (RBP)-host cell surface receptor interaction enabling isolation of phage-host combinations from an environmental sample. The method was successfully applied to two complex sample types-a dairy-derived whey sample and an infant fecal sample, enabling retrieval of specific and culturable phage hosts. IMPORTANCE PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory.

Complete genome sequence of Bifidobacterium animalis subsp. lactis BLC1.

Bifidobacterium animalis subsp. lactis BLC1 is a probiotic bacterium that is widely exploited by food industries as the active ingredient of various functional foods. Here we report the complete genome sequence of B. animalis subsp. lactis BLC1, which is expected to provide insights into the biology of this health-promoting microorganism and improve our understanding of its phylogenetic relatedness with other members of the B. animalis subsp. lactis taxon.

Biodiversity of lactic acid bacteria and yeasts in spontaneously-fermented buckwheat and teff sourdoughs.

In this study, four different laboratory scale gluten-free (GF) sourdoughs were developed from buckwheat or teff flours. The fermentations were initiated by the spontaneous biota of the flours and developed under two technological conditions (A and B). Sourdoughs were propagated by continuous back-slopping until the stability was reached. The composition of the stable biota occurring in each sourdough was assessed using both culture-dependent and -independent techniques. Overall, a broad spectrum of lactic acid bacteria (LAB) and yeasts species, belonging mainly to the genera Lactobacillus, Pediococcus, Leuconostoc, Kazachstania and Candida, were identified in the stable sourdoughs. Buckwheat and teff sourdoughs were dominated mainly by obligate or facultative heterofermentative LAB, which are commonly associated with traditional wheat or rye sourdoughs. However, the spontaneous fermentation of the GF flours resulted also in the selection of species which are not consider endemic to traditional sourdoughs, i.e. Pediococcus pentosaceus, Leuconostoc holzapfelii, Lactobacillus gallinarum, Lactobacillus vaginalis, Lactobacillus sakei, Lactobacillus graminis and Weissella cibaria. In general, the composition of the stable biota was strongly affected by the fermentation conditions, whilst Lactobacillus plantarum dominated in all buckwheat sourdoughs. Lactobacillus pontis is described for the first time as dominant species in teff sourdough. Among yeasts, Saccharomyces cerevisiae and Candida glabrata dominated teff sourdoughs, whereas the solely Kazachstania barnetti was isolated in buckwheat sourdough developed under condition A. This study allowed the identification and isolation of LAB and yeasts species which are highly competitive during fermentation of buckwheat or teff flours. Representatives of these species can be selected as starters for the production of sourdough destined to GF bread production.

Lactic Acid Bacteria Adjunct Cultures Exert a Mitigation Effect against Spoilage Microbiota in Fresh Cheese.

Lactic acid bacteria (LAB) have a strong mitigation potential as adjunct cultures to inhibit undesirable bacteria in fermented foods. In fresh cheese with low salt concentration, spoilage and pathogenic bacteria can affect the shelf life with smear on the surface and packaging blowing. In this work, we studied the spoilage microbiota of an Italian fresh cheese to find tailor-made protective cultures for its shelf life improvement. On 14-tested LAB, three of them, namely Lacticaseibacillus rhamnosus LRH05, Latilactobacillus sakei LSK04, and Carnobacterium maltaromaticum CNB06 were the most effective in inhibiting Gram-negative bacteria. These cultures were assessed by the cultivation-dependent and DNA metabarcoding approach using in vitro experiments and industrial trials. Soft cheese with and without adjunct cultures were prepared and stored at 8 and 14 °C until the end of the shelf life in modified atmosphere packaging. Data demonstrated that the use of adjunct cultures reduce and/or modulate the growth of spoilage microbiota at both temperatures. Particularly, during industrial experiments, C. maltaromaticum CNB06 and Lcb. rhamnosus RH05 lowered psychrotrophic bacteria of almost 3 Log CFU/g in a 5-week stored cheese. On the contrary, Llb. sakei LSK04 was able to colonize the cheese but it was not a good candidate for its inhibition capacity. The combined approach applied in this work allowed to evaluate the protective potential of LAB strains against Gram-negative communities.

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli.

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.

Sourdough in gluten-free bread-making: an ancient technology to solve a novel issue?

The increasing demand for high quality gluten-free (GF) bread, clean labels and natural products is raising the need for new approaches in GF bread-making. Sourdough is the foremost fermentation used for baking purposes and it has been proven to be ideal for improving the texture, palatability, aroma, shelf life and nutritional value of wheat and rye breads. These characteristic features derive from the complex metabolic activities of the sourdough-resident lactic acid bacteria and yeasts, e.g. acidification, production of exopolysaccharides, proteolytic- amylolytic- and phytase activity, and production of antimicrobial substances. These effects have been extensively studied and well described for traditional baking, whereas little is known about the role of sourdough in GF baking. Yet, the microbiological and qualitative characterisation of local GF fermented products indicate an overlap with the microbiota of wheat/rye fermentation and suggest that the positive metabolic activities of the sourdough microbiota are still retained during fermentation of GF crops. Thus, the use of sourdough in GF baking may be the new frontier for improving the quality, safety and acceptability of GF bread.

A 12-Year Cohort Study of Doc-Stoppage in Professional Mixed Martial Arts.

Purposes: To determine actions during bouts that generate serious enough injury to stop the bout; verifying the injury incidence, types, and prevalence of doctor stoppages (doc-stoppage); and identify potential risk factors by analyzing technical-tactical profiles for injury in sanctioned mixed martial arts bouts taking place over a 12-y period. Methods: This research analyzed 440 paired mixed martial arts matches separated by doc-stoppage (n = 220) and no doc-stoppage (n = 220) from 2002 to 2014. Technical knockouts for doc-stoppage were diagnosed and managed by attending ringside doctors, and the time-motion variables were categorized into total combat time separated by low- or high-intensity activities per round, stand-up, or groundwork actions, P ≤ .05. Results: The main cause of injuries in doc-stoppage situations was due to facial injuries (>90%), with 87.1% occurring after striking actions during the second round. Lacerations were the leading type of injury, which occurred with 80% frequency. The results showed differences between doc-stoppage and no doc-stoppage for standing combat with low-intensity actions (130.6 [8.5] s vs 83.3 [6.9] s for first round; 115.7 [10.5] s vs 100.1 [9.6] s for second round, and 121.5 [19.5] s vs 106.3 [11.7] s for third round) and total strike attempts (34.5, 23.0-51.8 vs 25.0, 12.0-40.8); in standing combat, head strike attempts (21, 10-33 vs 11, 4-21) and body strikes (2.5, 1.0-5.8 vs 1.0-2), and in groundwork combat, head strikes landed (0.0-3.0 vs 0.0-5.0). Conclusions: This research showed higher values of strike attempts with 2 main orientations, namely the head (on the ground and in stand-up actions) and body (in stand-up actions), and may provide important information regarding the technical knockout and when it can be called by officials supervising mixed martial arts bouts.

10 Years on Time-Motion and Motor Actions of Paired Mixed Martial Arts Athletes.

This study verified the performance probabilities by mixed martial arts rounds by the same athletes, doing paired comparisons of time-motion and actions before and after 10 y. The sample was composed of 845 Ultimate Fighting Championship rounds of 45 athletes separated into before (M1, age range 34-44 y) and after (M2, age range 44-54 y). Motor-control (takedowns, submissions, chokes, locks, strike actions to the head, and body and leg strikes attempted and landed) and time-motion (high and low intensities and standing and ground times) analyses were done. The main results showed significant differences (P ≤ .05) in total strikes landed (M1 22 [13; 34] > M2 18 [10; 31.7]), total strikes attempted (M1 41 [24.5; 62] > M2 35 [21; 48]), single head strikes attempted (M1 19 [9; 34.5] > M2 16.5 [9; 28]), single body strikes landed (M1 1 [0; 4] > M2 1 [0; 2]), single body strikes attempted (M1 2 [0; 5] > M2 1 [0; 3]), takedowns attempted (M1 1 [0; 2] > M2 1 [0; 2]), standing combat time (M1 2:10.28 [1:38.95] > M2 1:55.56 [1:32.17]), and low-intensity time (M1 2:11.45 [1:38.95] > M2 1:56.26 [1:31.89]). Variables that increased the probability to be associated with over the years were body strikes landed, head strikes landed, total strikes landed, and single strikes attempted, whereas body strikes attempted, head strikes attempted, total strikes attempted, and submission attempted had a negative association with mixed martial arts years of experience. Therefore, M2 athletes should be focused on standing combat time combined with strikes-landed actions, targeting the head-it has the highest potential performance probability and avoid unsuccessful body-strike attempts and submissions-which has the lowest potential performance probability over 10 years.