In vitro and in vivo activities of lactic acid bacteria from Italian mountain cheese" and their exploitation in dairy production

The use of health-promoting lactic acid bacteria (LAB) strains as starter or adjunct cultures for dairy productions could facilitate the in situ bio-synthesis of bioactive molecules during the fermentation process, increasing the interest towards dairy products as multifunctional foods. Currently, there is much research about genotypic and technological characterization of raw milk cheeses microbiota, which is rich in biodiversity and could be exploited for improving the sensory attributes and add healthy benefits to the cheese. Traditional Mountain (TM) cheese is made from raw cow’s milk and spontaneously fermented in small farms called “Malga” located in the alpine areas of Trentino region. For the first time, the microbial population of TM-cheese has been characterized in order to select cocci and non-starter LAB suitable for developing new starter or adjunct cultures, respectively. Samples (n = 120) of milk, curd and cheese at different ripening times (24 hours, 1 month and 7 months) were enumerated in selective culture media. Mesophilic and thermophilic cocci dominated during the first 24 hours following production, and mesophilic lactobacilli were dominant at the end of ripening. Six hundred and forty colonies were isolated from curd and cheese 24 hours following production, and 95 more colonies were isolated from cheese after 7 months of ripening. All isolates were genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. Cocci clustered in 231 biotypes belonging to 16 different species, and non-starter LAB (NSLAB) clustered in 70 biotypes belonging to 13 different species. Lactococcus lactis, Streptococcus thermophilus and Enterococcus faecalis were dominant in curd and 24h-cheese; Pediococcus pentosaceus and Lactobacillus paracasei were the main species at the end of ripening. The phenotypic, technological and health-promoting activities of all strains were investigated. In particular, lactococci, streptococci and enterococci were tested for their acidification and proteolytic activity, ability to growth at not optimal temperatures, acetoin production, development of olfactory flavour notes, autolysis rate and ability to inhibit the growth of coliforms. Forty percent of enterococci showed the ability to inhibit raw milk resident coliforms in vitro, but they were excluded as possible starters, owing to the presence of associated risk factors. Among lactococci and streptococci, 4 Lc. lactis subsp. lactis and 2 Sc. thermophilus were fast acidifiers, produced pleasant flavours, and were subjected to the freezedrying stability test. Lc. lactis subsp. lactis 68 and Sc. thermophilus 93 showed the best properties and might be appropriate for cheese production. NSLAB strains were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase activity (AP) and biogenic amines production. Concerning the health-promoting properties, the bile salts hydrolysis (BSH) activity was tested qualitatively, the conjugated linoleic acid (CLA) production was measured spectrophotometrically, and the γ-aminobutyric acid (GABA) production was quantified by UHPLC (Ultra High Performance Liquid Cromatography). Lb. paracasei isolates resulted to be well adapted to the Malga environment and showed the highest AP activity and acetoin production. Some strains harbored very interesting health-promoting properties and produced bioactive substances. In particular, Lb. rhamnosus BT68, Lb. paracasei BT18, BT25, BT31, Pc. pentosaceus BT3, BT13, BT51 produced between 70 and 130 mg/mL of total CLA in vitro. Lb. brevis BT66 converted L-glutamate to a high concentration of GABA (129 ± 8.6 mg/L) and showed BSH activity. These first results revealed that TM-cheese is a reservoir of a high microbial diversity, and the resident LAB could be exploited not only for the applicability in dairy production but also for their health-promoting properties. Lc. lactis subsp. lactis 68 and Sc. thermophilus 93, which showed to be the best performing strains, were tested as starter and adjunct cultures, for the production of 9 experimental TM-cheese wheels in a Malga-farm, respectively. Three control (CTRL) cheeses were produced according to the tradition and any starter or adjunct culture was not added; three starter (STR) and three commercial starter (CMS) cheeses were produced inoculating the vat milk with both selected strains and a commercial Sc. thermophilus strain, respectively. After 24 hours, 1 month and 7 months of ripening the microbial content of all experimental cheeses was investigated. Mesophilic cocci and lactobacilli dominated in cheese samples after 24 hours and 1 month of ripening, while cocci dominated in full-ripened cheese. The total genomic DNA was extracted, and a fragment of the V1-V3 region was amplified and pyrosequenced. Lactococci and streptococci were the most abundant species in CTRL and STR cheese, and Lc. lactis subsp. lactis 68 affected the proliferation of the (raw milk) indigenous Lc. lactis subsp. cremoris during the early fermentation. Moreover, the commercial Sc. thermophilus showed to be dominant towards Lc. lactis subsp. lactis and cremoris naturally present in raw milk and to be responsible in decreasing the abundance of Lactobacillus subp. and Enterococcus sp. The survival of TM-cheese microbiota in vitro was investigated under simulated human gastro-intestinal (GI) conditions. The 9 full ripened experimental TM-cheeses were subjected to a model system that simulates digestive processes in the mouth, stomach and small intestine, comprising sequential incubation in human gastric and duodenal juices. Bacterial counts were performed before and after the simulation: total bacterial count and thermophilic cocci significantly decreased after the simulated digestion. Thirty-six lactobacilli were isolated from cheese after digestion: among them 1 Lb. paracasei, 1 Lb. parabuchneri and 1 Lb. fermentum were tested for their survival after GI transit. Lc. lactis subsp. lactis 68 and Lb. parabuchneri D34 strains were used to ferment whole milk and digested. The load of Lb. parabuchneri D34 decreased by about one log more when grown as pure culture than fermented milk after simulated digestion, suggesting that Lb. parabuchneri D34 had in itself the ability to survive digestion, but the fat content and the cheese structure might protect LAB during the GI transit. Furthermore, our interest towards the GABA producing strains lead us to test the ability of Lb. brevis BT66 to produce GABA in situ during cheese production, through the decarboxylation of glutamate. Twenty experimental micro-cheeses were produced using a commercial starter strain (107 CFU/mL) and Lb. brevis BT66 as adjunct culture. Four different concentrations (102 , 103, 104, 105 CFU/mL) of Lb. brevis BT66 were tested in quadruplicate. In order to follow the microbial evolution, samples of milk, curd and cheese after 20 days of ripening were enumerated in selective media. The control and experimental samples showed a similar trend, suggesting that both milk-resident and starter strains grew during ripening. However, the load of mesophilic lactobacilli in all experimental curd samples was higher than the control ones. The concentration of GABA and glutamic acid in cheese samples after 20 days of ripening was quantified by UHPLC-HQOMS. The amino acidic profiles showed that while the concentration of Lb. brevis BT66 in milk increased, the amount of both glutamic acid (from 324 ± 37 to 202 ± 32 mg/kg) and GABA (from 154 ± 31 to 91 ± 20 mg/kg) significantly decreased in cheese. These results suggested that the experimental strain converted glutamic acid to GABA, but that GABA may have subsequently been converted to succinate by GABA transaminases. The non-protein amino acid GABA has been reported to impact on brain function through the gut:brain axis system, to harbor an anti-obesity and antidiabetogenic effect, to regulate the immune system, the inflammation process and the energy metabolism in mammals including induction of hypotension, diuretic and tranquilizer effects, stimulation of immune cells. Owing to its ability to produce high concentrations of GABA and its BSH activity in vitro, Lb. brevis BT66 was selected to be tested in vivo in mice suffering obesity-associated type-2-diabetes. Another Lb. brevis (strain DPC6108), isolated from the human GI tract and harboring the same properties, was simultaneously investigated. The corresponding rifampicin resistant mutants (rif) were generated; their genotypic profile was obtained by RAPD-PCR and PFGE (Pulsed-Field Gel Electrophoresis) and was identical to the native strain. The conversion rates of monosodium glutamate to GABA were investigated by nextgeneration amino acid analysis: Lb. brevis BT66rif produced 840.5 ± 266 µg/mL of GABA with about 73% of bioconversion and Lb. brevis DPC6108rif produced 1,218.0 ± 393.2 µg/mL with about 87% of bioconversion. The BSH activity was positive to both qualitative and quantitative assays and the results were similar in both native and mutant strains. The rifampicin resistant strains were freeze-dried and tested for their stability at room temperature, +4 and -20 °C. Both spectrophotometer and plate count methods revealed that freeze-dried strains survived at room temperature during 24 hours after suspending in sterile water. The stability of freezedried strains at +4 and -20 °C was investigated enumerating the viable cells in selective medium during 10 weeks and any significant load reduction was not detected in the first 4 weeks following freeze-drying. Both pharmabiotic-producing Lb. brevis BT66rif and DPC6108rif were resistant to freeze-drying, survived transit through mouse GI tract (as proven by a pilot study), and their therapeutic efficiency is being assessed in vivo to treat metabolic obesity and type-2-diabete

Milk metagenomics and cheese-making properties as affected by indoor farming and summer highland grazing

The study of the complex relationships between milk metagenomics and milk composition and cheese-making efficiency as affected by indoor farming and summer highland grazing was the aim of the present work. The experimental design considered monthly sampling (over 5 mo) of the milk produced by 12 Brown Swiss cows divided into 2 groups: the first remained on a lowland indoor farm from June to October, and the second was moved to highland pastures in July and then returned to the lowland farm in September. The resulting 60 milk samples (2 kg each) were used to analyze milk composition, milk coagulation, curd firming, and syneresis processes, and to make individual model cheeses to measure cheese yields and nutrient recoveries in the cheese. After DNA extraction and Illumina Miseq sequencing, milk microbiota amplicons were also processed by means of an open-source pipeline called Quantitative Insights Into Microbial Ecology (Qiime2, version 2018.2; https://qiime2.org). Out of a total of 44 taxa analyzed, 13 bacterial taxa were considered important for the dairy industry (lactic acid bacteria, LAB, 5 taxa; and spoilage bacteria, 4) and for human (other probiotics, 2) and animal health (pathogenic bacteria, 2). The results revealed the transhumant group of cows transferred to summer highland pastures showed an increase in almost all the LAB taxa, bifidobacteria, and propionibacteria, and a reduction in spoilage taxa. All the metagenomic changes disappeared when the transhumant cows were moved back to the permanent indoor farm. The relationships between 17 microbial traits and 30 compositional and technological milk traits were investigated through analysis of correlation and latent explanatory factor analysis. Eight latent factors were identified, explaining 75.3% of the total variance, 2 of which were mainly based on microbial traits: pro-dairy bacteria (14% of total variance, improving during summer pasturing) and pathogenic bacteria (6.0% of total variance). Some bacterial traits contributed to other compositional-technological latent factors (gelation, udder health, and caseins)

Massive survey on bacterial–bacteriophages biodiversity and quality of natural whey starter cultures in Trentingrana cheese production

9openInternationalItalian coauthor/editorThis study focused on the microbial and bacteriophages identification and characterization in cheese-production facilities that use natural whey starter (NWS) cultures for Trentingrana production. Bacterial and phage screening was carried out on cooked not acidified whey and NWS samples isolated from six dairy factories, for 4 consecutive days in four different months. By means of a combined approach, using plate counts, bacterial isolation, and metataxonomic analysis Lactobacillus helveticus was found occurring as the dominant species in NWS cultures and Levilactobacillus brevis as codominant in the cheese factories where the temperature of NWS production was mainly lower than 40°C, suggesting that the variability in the parameters of the NWS culture preparation could differently modulate the bacterial species in NWS cultures. Using turbidity test approach on 303 bacterial isolates from the NWS cultures, 120 distinct phages were identified. L. helveticus phage contamination of NWS cultures was revealed in most of the analyzed samples, but despite the great recovery of bacteriophage contamination cases, the microbial quality of NWS cultures was high. Our results support the presence of natural bacteriophage resistance mechanisms in L. helveticus. The use of NWS cultures probably creates an ideal environment for the proliferation of different L. helveticus strains balanced with their phages without a clear dominance. It is evident, from this study, that the presence of a high biodiversity of NWS bacterial strains is relevant to avoid phages dominance in NWS cultures and consequently to keep a good acidification ability.openMancini, A.; Cid Rodriguez, M.; Zago, M.; Cologna, M.; Goss, A.; Carafa, I.; Tuohy, K.; Merz, A.; Franciosi, E.Mancini, A.; Cid Rodriguez, M.; Zago, M.; Cologna, M.; Goss, A.; Carafa, I.; Tuohy, K.; Merz, A.; Franciosi, E

Sustainable and Health-Protecting Food Ingredients from Bioprocessed Food by-Products and Wastes

Dietary inadequacy and nutrition-related non-communicable diseases (N-NCDs) represent two main issues for the whole society, urgently requesting solutions from researchers, policy-makers, and other stakeholders involved in the health and food system. Food by-products and wastes (FBPW) represent a global problem of increasing severity, widely recognized as an important unsustainability hotspot, with high socio-economic and environmental costs. Yet, recycling and up-cycling of FBPW to produce functional foods could represent a solution to dietary inadequacy and risk of N-NCDs onset. Bioprocessing of FBPW with selected microorganisms appears to be a relatively cheap strategy to yield molecules (or rather molecules mixtures) that may be used to fortify/enrich food, as well as to formulate dietary supplements. This review, conjugating human health and sustainability in relation to food, describes the state-of-the-art of the use of yeasts, molds, and lactic acid bacteria for producing value-added compounds from FBPW. Challenges related to FBPW bioprocessing prior to their use in food regard will be also discussed: (i) loss of product functionality upon scale-up of recovery process; (ii) finding logistic solutions to the intrinsic perishability of the majority of FBPW; (iii) inserting up-cycling of FBPW in an appropriate legislative framework; (iv) increasing consumer acceptability of food and dietary supplements derived from FBPWThis research was funded by the project SYSTEMIC “an integrated approach to the challenge of sustainable food systems: adaptive and mitigatory strategies to address climate change and malnutrition”, Knowledge hub on Nutrition and Food Security, that has received funding from national research funding parties in Belgium (FWO), France (INRA), Germany (BLE), Italy (MIPAAF), Latvia (IZM), Norway (RCN), Portugal (FCT), and Spain (AEI) in a joint action of JPI HDHL, JPI-OCEANS and FACCE-JPI launched in 2019 under the ERA-NET ERAHDHL (n° 696295). Francisca Rodrigues (CEECIND/01886/2020) is thankful for her contract financed by FCT/MCTES—CEEC Individual 2020 Program Contractinfo:eu-repo/semantics/publishedVersio

Corrigendum to “Effects of therapeutic hypothermia on the gut microbiota and metabolome of infants suffering hypoxic-ischemic encephalopathy at birth” [Int. J. Biochem. Cell Biol. 93 (December) (2017), 110-118]

peer-reviewedCorrigendum Refers to: Watkins, C., Murphy, K., Yen, S., Carafa, I., Dempsey, E., O’Shea, C., Vercoe, E., Ross, R., Stanton, C. and Ryan, C. (2017). Effects of therapeutic hypothermia on the gut microbiota and metabolome of infants suffering hypoxic-ischemic encephalopathy at birth. The International Journal of Biochemistry & Cell Biology, [online] 93, pp.110-118. Available at: https://doi.org/10.1016/j.biocel.2017.08.01

Effects of therapeutic hypothermia on the gut microbiota and metabolome of infants suffering hypoxic-ischemic encephalopathy at birth

Neonatal hypoxic ischemic encephalopathy (HIE) in the perinatal period can lead to significant neurological deficits in later life. Total body cooling (TBC) is a neuroprotective strategy used in the treatment of HIE and has been shown to reduce seizures and improve neurodevelopmental outcomes in treated infants. Little is known, however, about the effects of HIE/TBC on the developing gut microbiota composition and subsequent metabolic profile. Ten term infants with HIE who received TBC at 33.5 degrees C for 72 h were recruited. A control group consisted of nine healthy full term infants. Faecal samples were collected from both groups at 2 years of age and stored at -20 degrees C. 16S rRNA amplicon Illumina sequencing was carried out to determine gut microbiota composition and 1H NMR analysis was performed to determine the metabolic profile of faecal water. The gut microbiota composition of the HIE/TBC infants were found to have significantly lower proportions of Bacteroides compared to the non-cooled healthy control group. Alpha diversity measures detected significantly lower diversity in microbial richness in the HIE/TBC infant group compared to the control infants (Shannon index

Metformin and dipeptidase peptidyl-4 inhibitor differentially modulate the intestinal microbiota and plasma metabolome of metabolically dysfunctional mice

Objectives: Recent evidence indicates that gut microbiota is altered considerably by a variety of commonly prescribed medications. This study assessed the impact of 2 antidiabetic therapeutics on gut microbiota and markers of cardiometabolic disease in metabolically dysfunctional mice. Methods: C57BL/6 mice were fed a high-fat diet for 24 weeks while receiving 1 of 2 antidiabetic therapeutics—metformin or dipeptidase peptidyl-4 (DPP-4) inhibitor, PKF-275-055—for the final 12 weeks. Mice were assessed for weight gain, glucose and cholesterol metabolism, and adiposity. In addition, cecal microbiota was analyzed by 16S compositional sequencing, and plasma metabolome was analyzed by liquid chromatography with tandem mass spectrometry. Results: Both therapeutics had similar metabolic effects, attenuating mesenteric adiposity and improving cholesterol metabolism and insulin sensitivity. However, multivariate analyses of microbiota and metabolomics data revealed clear divergence of the therapeutic groups. Although both metformin and PKF-275-055 mice displayed significantly decreased Firmicutes/Bacteroidetes ratios, only metformin harboured metabolic health-associated Akkermansia, Parabacteroides and Christensenella. Paradoxically, metformin also reduced α diversity, a metric frequently associated with host metabolic fitness. PKF-275-055 mice displayed elevated levels of butyrate-producing Ruminococcus and acetogen Dorea, with reduced levels of certain plasma sphingomyelin, phosphatidylcholine and lysophosphatidylcholine entities. In turn, metformin reduced levels of acylcarnitines, a functional group associated with systemic metabolic dysfunction. Finally, several associations were identified between metabolites and altered taxa. Conclusions: This study represents the first direct comparison of the microbiota-modifying effects of metformin and a DPP-4 inhibitor, and proposes several putative microbial targets both in terms of novel therapeutic development and adverse effect prevention

Critical Analysis and Quality Assessment of Nanomedicines and Nanocarriers in Clinical Trials: Three Years of Activity at the Clinical Trials Office

Investigational medicinal products submitted over the course of 3 years and authorized at the Clinical Trials Office of the Italian Medicines Agency as part of a request for authorization of clinical trials were scrutinized to identify those encompassing nanomedicines. The quality assessment reports performed on the documentation submitted were analyzed, classifying and discussing the most frequently detected issues. The identification of nanomedicines retrieved and the information on their quality profiles are shared to increase the transparency and availability of information, providing feedback that can support sponsors in optimizing the quality part of the documentation and of the information submitted. Results confirm that nanomedicines tested as investigational medicinal products in clinical trials are developed and authorized in agreement with the highest standards of quality, meeting safety profiles according to the strong regulatory requirements in the European Union. Some key points are highlighted and indicate that the regulatory approach to innovation in a clinical trial setting could potentially be renewed to ride the wave of innovation, particularly in the nanotechnology field, capitalizing on lessons learned and still ensuring a strong and effective framework

Metagenomics of milk before, during and after summer transhumance to highland pasture in relation to human health and cheese making properties

Summer transhumance is a traditional form of pastoralism to highland pasture in the dairy system of alpine regions and it is practiced all over the world. Positive influences can be obtained for the environment, society, tourism, animal health and welfare. The aim of this study was to analyze the evolution of the milk microbiota before, during, and after summer transhumance to highland pasture in relation to possible effects on animal / human health and cheese-making properties. The study involved 12 healthy, multiparous, mid-lactation Brown Swiss cows reared in a farm located in the Trento province (Northern East Italy). The cows were monitored from June to October and divided into two groups: six cows were used as a control group and kept in the lowland permanent farm (PF); the other six cows were moved to a temporary alpine farm (ALP) from July to September. From each milk, samples were collected every four weeks for genomic DNA extraction and microbiota characterization using community 16S rRNA amplicon (V3\u2013V4 region) based Illumina Miseq sequencing and QIIME2 (2018.2 version). The relative abundance was analyzed after a log10 transformation and identified species were classified into two unfavorable categories: Spoilage and Pathogenic species, and two favorable categories: Probiotics and Dairy species. For the statistical analysis, we used a linear mixed model, where the combined Month 7 Group (MG) effect was used as fixed and the cow effect as random. The results revealed a different behavior in the ALP group compared to the PF group during the 3 months of summer transhumance. The Spoilage bacteria tended to decrease in the alpine pasture (MG effect: p < .001), with significant differences in terms of Pseudomonas, Alicyclobacillus, and Clostridiales (p < .001, p < .001, p < .05 respectively). Pathogenic did not show large differences between the two groups; instead, the Probiotic category, which includes Propionibacterium and Bifidobacterium, showed a gradual increase in the ALP group (p < .001). Dairy species also had a significant increase in the ALP group (p < .001), especially Lactococcus and Lactobacillus (both p < .001). All the microbiological changes disappeared when cows were moved back from alpine pasture to the permanent indoor farm. Summer transhumance to alpine pasture, therefore, has a favorable effect on the milk microbiota, with positive implications for both the cheese-making attitude of milk produced and its possible effect on human health

Nasaruni Academy for Maasai Girls

Dr. Michelle Cude, Associate Professor of Middle, Secondary, and Mathematics Department, was recently granted a 17-18 Fulbright Scholarship for her work in Narok, Kenya. Fueled by having an international exchange teacher, Alice Sayo, in her Methods of Teaching Social Studies course, Dr. Cude’s work began in 2011 when Alice shared her dream of opening a school in her community. Dr. Cude helped to raise $7,000 to buy 5 acres of land, and she did not stop there. Since its opening in 2012, The Nasaruni Academy for Maasai Girls, has grown from 13 students to 80 students as of January \u2717. Nasaruni, meaning “haven” in the Maasai language, is a place for Maasai girls to learn English, Swahili, math, social studies, geography and sciences in the standard curriculum required by the Kenyan government. Dr. Cude serves as the faculty advisor for JMU’s Future Social Studies Educators, a campus club that often fundraises for the Academy through events like Empty Bowls, which made just over$9,000 on March 31, 2017 for necessary improvements at the school