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Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, we studied the roles of microbial interactions in flavour formation in a year-long Cheddar cheese making process, using a commercial starter culture containing Streptococcus thermophilus and Lactococcus strains. By using an experimental strategy whereby certain strains were left out from the starter culture, we show that S. thermophilus has a crucial role in boosting Lactococcus growth and shaping flavour compound profile. Controlled milk fermentations with systematic exclusion of single Lactococcus strains, combined with genomics, genome-scale metabolic modelling, and metatranscriptomics, indicated that S. thermophilus proteolytic activity relieves nitrogen limitation for Lactococcus and boosts de novo nucleotide biosynthesis. While S. thermophilus had large contribution to the flavour profile, Lactococcus cremoris also played a role by limiting diacetyl and acetoin formation, which otherwise results in an off-flavour when in excess. This off-flavour control could be attributed to the metabolic re-routing of citrate by L. cremoris from diacetyl and acetoin towards α -ketoglutarate. Further, closely related Lactococcus lactis strains exhibited different interaction patterns with S. thermophilus , highlighting the significance of strain specificity in cheese making. Our results highlight the crucial roles of competitive and cooperative microbial interactions in shaping cheese flavour profile. Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, the authors identify microbial interactions as a mechanism underlying flavour formation in Cheddar cheese.

Biogenic amines have been widely studied because of their potential toxicity in fermented foods. Several lactic acid bacteria have the potential to decarboxylate the amino acid tyrosine to tyramine. In this work, we identified two strains of Lactobacillus curvatus, Lbc1 and Lbc2, endowed with the ability to produce tyramine, a metabolic feature that limits their application in starter cultures for fermented meat. To overcome this limitation, we set out to eliminate tyramine production from L. curvatus strains by using classical strain improvement. About 4,000 mutant isolates of both strains were screened using a colorimetric method, and then potential tyrosine decarboxylase-negative mutants were selected. Firm identification of loss-of-function mutants was performed by analytical determination of tyrosine and tyramine in cultivation medium. Of the 8,000 mutants screened, only one mutant of Lbc1 and two mutants of Lbc2 had completely lost the potential to produce tyramine. Subsequently, one tyrosine decarboxylase-negative mutant of both Lbc1 and Lbc2 was characterized in more detail. DNA sequencing of the Lbc1 mutant tdcA gene disclosed two missense mutations in the promoter distal part of the coding sequence. These two mutations result in two amino acid changes in the encoded tyrosine decarboxylase, Pro87Thr and Ser130Leu, presumably inactivating the enzyme activity. The DNA sequence of the other characterized mutant, derived from strain Lbc2, showed that insertion of a 6-bp fragment at nucleotide position 1348 in the tdc gene is presumably the factor leading to loss of activity. With the successful elimination of the undesirable tyramine-producing phenotype without the use of recombinant DNA technology, these developed L. curvatus mutant strains can be safely used in the dairy industry or in the manufacture of various food products.

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a β- d -xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli . BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para -nitrophenyl-β- d -xylopyranoside ( p NPX), para -nitrophenyl-α-L-arabinofuranoside ( p NPA), β-(1 → 4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2–4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I − V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for p NPA: p NPX of 1:25. BXA43 was stable below 40°C and at pH 4.0–8.0 and showed maximum activity at pH 5.5 and 50°C. K m and k cat for p NPX were 15.6 ± 4.2 mM and 60.6 ± 10.8 s -1 , respectively, and substrate inhibition became apparent above 18 mM p NPX. Similar kinetic parameters and catalytic efficiency values were reported for β- d -xylosidase (XynB3) from Geobacillus stearothermophilus T‒6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to p NPX. BXA43 showed clearly metal inhibition for Zn 2+ and Ag + , which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg 505 Tyr 506 present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus.

Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, we studied the roles of microbial interactions in flavour formation in a year-long Cheddar cheese making process, using a commercial starter culture containing Streptococcus thermophilus and Lactococcus strains. By using an experimental strategy whereby certain strains were left out from the starter culture, we show that S. thermophilus has a crucial role in boosting Lactococcus growth and shaping flavour compound profile. Controlled milk fermentations with systematic exclusion of single Lactococcus strains, combined with genomics, genome-scale metabolic modelling, and metatranscriptomics, indicated that S. thermophilus proteolytic activity relieves nitrogen limitation for Lactococcus and boosts de novo nucleotide biosynthesis. While S. thermophilus had large contribution to the flavour profile, Lactococcus cremoris also played a role by limiting diacetyl and acetoin formation, which otherwise results in an off-flavour when in excess. This off-flavour control could be attributed to the metabolic re-routing of citrate by L. cremoris from diacetyl and acetoin towards α-ketoglutarate. Further, closely related Lactococcus lactis strains exhibited different interaction patterns with S. thermophilus, highlighting the significance of strain specificity in cheese making. Our results highlight the crucial roles of competitive and cooperative microbial interactions in shaping cheese flavour profile.

Research innovations are constantly occurring in universities, research institutions and industrial research laboratories. These are reported in the scientific literature and presented to the scientific community in various congresses and symposia as well as through direct contacts and collaborations. Conversion of these research results to industrially useful innovations is, however, considerably more complex than generally appreciated. The long and winding road from the research laboratory to industrial applications will be illustrated with two recent examples from Chr. Hansen A/S: the implementation in industrial scale of a new production technology based on respiration by Lactococcus lactis and the introduction to the market of L. lactis strains constructed using recombinant DNA technology.

Abstract Research innovations are constantly occurring in universities, research institutions and industrial research laboratories. These are reported in the scientific literature and presented to the scientific community in various congresses and symposia as well as through direct contacts and collaborations. Conversion of these research results to industrially useful innovations is, however, considerably more complex than generally appreciated. The long and winding road from the research laboratory to industrial applications will be illustrated with two recent examples from Chr. Hansen A/S: the implementation in industrial scale of a new production technology based on respiration by Lactococcus lactis and the introduction to the market of L. lactis strains constructed using recombinant DNA technology.

The Bifidobacterium animalis subsp. lactis BB-12 gene BIF_00092, assigned to encode a [beta]-d-xylosidase (BXA43) of glycoside hydrolase family 43 (GH43), was cloned with a C-terminal His-tag and expressed in Escherichia coli. BXA43 was purified to homogeneity from the cell lysate and found to be a dual-specificity exo-hydrolase active on para-nitrophenyl-[beta]-d-xylopyranoside (pNPX), para-nitrophenyl-[alpha]-L-arabinofuranoside (pNPA), [beta]-(1[arrow right]4)-xylopyranosyl oligomers (XOS) of degree of polymerisation (DP) 2-4, and birchwood xylan. A phylogenetic tree of the 92 characterised GH43 enzymes displayed five distinct groups (I-V) showing specificity differences. BXA43 belonged to group IV and had an activity ratio for pNPA:pNPX of 1:25. BXA43 was stable below 40°C and at pH 4.0-8.0 and showed maximum activity at pH 5.5 and 50°C. K ^sub m^ and k ^sub cat^ for pNPX were 15.6±4.2 mM and 60.6±10.8 s^sup -1^, respectively, and substrate inhibition became apparent above 18 mM pNPX. Similar kinetic parameters and catalytic efficiency values were reported for [beta]-d-xylosidase (XynB3) from Geobacillus stearothermophilus T6 also belonging to group IV. The activity of BXA43 for xylooligosaccharides increased with the size and was 2.3 and 5.6 fold higher, respectively for xylobiose and xylotetraose compared to pNPX. BXA43 showed clearly metal inhibition for Zn^sup 2+^ and Ag^sup +^, which is different to its close homologues. Multiple sequence alignment and homology modelling indicated that Arg^sup 505^Tyr^sup 506^ present in BXA43 are probably important for binding to xylotetraose at subsite +3 and occur only in GH43 from the Bifidobacterium genus.

Small newborns are vulnerable to mortality and lifelong loss of human capital. Measures of vulnerability previously focused on liveborn low-birthweight (LBW) babies, yet LBW reduction targets are off-track. There are two pathways to LBW, preterm birth and fetal growth restriction (FGR), with the FGR pathway resulting in the baby being small for gestational age (SGA). Data on LBW babies are available from 158 (81%) of 194 WHO member states and the occupied Palestinian territory, including east Jerusalem, with 113 (58%) having national administrative data, whereas data on preterm births are available from 103 (53%) of 195 countries and areas, with only 64 (33%) providing national administrative data. National administrative data on SGA are available for only eight countries. Global estimates for 2020 suggest 13·4 million livebirths were preterm, with rates over the past decade remaining static, and 23·4 million were SGA. In this Series paper, we estimated prevalence in 2020 for three mutually exclusive types of small vulnerable newborns (SVNs; preterm non-SGA, term SGA, and preterm SGA) using individual-level data (2010–20) from 23 national datasets (∼110 million livebirths) and 31 studies in 18 countries (∼0·4 million livebirths). We found 11·9 million (50% credible interval [Crl] 9·1–12·2 million; 8·8%, 50% Crl 6·8–9·0%) of global livebirths were preterm non-SGA, 21·9 million (50% Crl 20·1–25·5 million; 16·3%, 14·9–18·9%) were term SGA, and 1·5 million (50% Crl 1·2–4·2 million; 1·1%, 50% Crl 0·9–3·1%) were preterm SGA. Over half (55·3%) of the 2·4 million neonatal deaths worldwide in 2020 were attributed to one of the SVN types, of which 73·4% were preterm and the remainder were term SGA. Analyses from 12 of the 23 countries with national data (0·6 million stillbirths at ≥22 weeks gestation) showed around 74% of stillbirths were preterm, including 16·0% preterm SGA and approximately one-fifth of term stillbirths were SGA. There are an estimated 1·9 million stillbirths per year associated with similar vulnerability pathways; hence integrating stillbirths to burden assessments and relevant indicators is crucial. Data can be improved by counting, weighing, and assessing the gestational age of every newborn, whether liveborn or stillborn, and classifying small newborns by the three vulnerability types. The use of these more specific types could accelerate prevention and help target care for the most vulnerable babies.

Introduction: Danish children consume too much sugar and not enough whole grain, fish, fruit, and vegetables. The Nordic region is rich in such foods with a strong health-promoting potential. We lack randomised controlled trials that investigate the developmental and health impact of serving school meals based on Nordic foods. Aim: This paper describes the rationale, design, study population, and potential implications of the Optimal well-being, development and health for Danish children through a healthy New Nordic Diet (OPUS) School Meal Study. Methods: In a cluster-randomised cross-over design, 1021 children from 3rd and 4th grades (8-11 years old) at nine Danish municipal schools were invited to participate. Classes were assigned to two 3-month periods with free school meals based on the New Nordic Diet (NND) or their usual packed lunch (control). Dietary intake, nutrient status, physical activity, cardiorespiratory fitness, sleep, growth, body composition, early metabolic and cardiovascular risk markers, illness, absence from school, wellbeing, cognitive function, social and cultural features, food acceptance, waste, and cost were assessed. Results: In total, 834 children (82% of those invited) participated. Although their parents were slightly better educated than the background population, children from various socioeconomic backgrounds were included. The proportion of overweight and obese children (14%) resembled that of earlier examinations of Danish school children. Drop out was 8.3%. Conclusions: A high inclusion rate and low drop out rate was achieved. This study will be the first to determine whether school meals based on the NND improve children's diet, health, growth, cognitive performance, and early disease risk markers.