Clair-Yves Boquien

The apparent breastfeeding paradox in very preterm infants: relationship between breast feeding, early weight gain and neurodevelopment based on results from two cohorts, EPIPAGE and LIFT

Context Supplementation of breast milk is difficult once infants suckle the breast and is often discontinued at end of hospitalisation and after discharge. Thus, breastfed preterm infants are exposed to an increased risk of nutritional deficit with a possible consequence on neurodevelopmental outcome. Objective To assess the relationship between breast feeding at time of discharge, weight gain during hospitalisation and neurodevelopmental outcome. Design Observational cohort study. Setting Two large, independent population-based cohorts of very preterm infants: the Loire Infant Follow-up Team (LIFT) and the EPIPAGE cohorts. Patients 2925 very preterm infants alive at discharge. Main outcome measure Suboptimal neurodevelopmental outcome, defined as a score in the lower tercile, using Age and Stages Questionnaire at 2 years in LIFT and Kaufman Assessment Battery for Children Test at 5 years in EPIPAGE. Two propensity scores for breast feeding at discharge, one for each cohort, were used to reduce bias. Results Breast feeding at time of discharge concerned only 278/1733 (16%) infants in LIFT and 409/2163 (19%) infants in EPIPAGE cohort. Breast feeding is significantly associated with an increased risk of losing one weight Z-score during hospitalisation (LIFT: n=1463, adjusted odd ratio (aOR)=2.51 (95% CI 1.87 to 3.36); EPIPAGE: n=1417, aOR=1.55 (95% CI 1.14 to 2.12)) and with a decreased risk for a suboptimal neurodevelopmental assessment (LIFT: n=1463, aOR=0.63 (95% CI 0.45 to 0.87); EPIPAGE: n=1441, aOR=0.65 (95% CI 0.47 to 0.89) and an increased chance of having a head circumference Z-score higher than 0.5 at 2 years in LIFT cohort (n=1276, aOR=1.43 (95% CI 1.02 to 2.02)) and at 5 years in EPIPAGE cohort (n=1412, aOR=1.47 (95% CI 1.10 to 1.95)). Conclusions The observed better neurodevelopment in spite of suboptimal initial weight gain could be termed the ‘apparent breastfeeding paradox’ in very preterm infants. Regardless of the mechanisms involved, the current data provide encouragement for the use of breast feeding in preterm infants.

Development of an analytical strategy based on liquid chromatography–high resolution mass spectrometry for measuring perfluorinated compounds in human breast milk: Application to the generation of preliminary data regarding perinatal exposure in France

Perfluorinated compounds (PFCs) are man-made chemicals for which endocrine disrupting properties and related possible side effects on human health have been reported, particularly in the case of an exposure during the early stages of development, (notably the perinatal period). Existing analytical methods dedicated to PFCs monitoring in food and/or human fluids are currently based on liquid chromatography coupled to tandem mass spectrometry, and were recently demonstrated to present some limitations in terms of sensitivity and/or specificity. An alternative strategy dedicated to the analysis of fourteen PFCs in human breast milk was proposed, based on an effective sample preparation followed by a liquid chromatography coupled to high resolution mass spectrometry measurement (LC-HRMS). This methodology confirmed the high interest for HRMS after negative ionization for such halogenated substances, and finally permitted to reach detection limits around the pg mL(-1) range with an outstanding signal specificity compared to LC-MS/MS. The proposed method was applied to a first set of 30 breast milk samples from French women. The main PFCs detected in all these samples were PFOS and PFOA with respective median values of 74 (range from 24 to 171) and 57 (range from 18 to 102) pg mL(-1), respectively. These exposure data appeared in the same range as other reported values for European countries.

Enzymatic methods for determining populations of Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091 in pure and mixed cultures

SummaryStreptococcus cremoris AM2 is characterized by an aminopeptidase and Leuconostoc lactis CNRZ 1091 by an α-galactosidase and a citrate lyase. These strains were grown in pure or mixed cultures, in presence or absence of citrate (15 mM) and at controlled or uncontrolled pH. Cell populations and the activities of the enzymes were measured during microbial growth. Linear correlations were established between the population of S. cremoris AM2 and aminopeptidase activity, and between that of L. lactis CNRZ 1091 and the activities of α-galactosidase and citrate lyase. These correlations held regardless of whether the culture was pure or mixed and if the pH was controlled or not. The presence of citrate did not change citrate lyase and aminopeptidase activities, but inhibited the synthesis of the α-galactosidase and not its activity. The linear relationships permit the determination of bacterial populations in less than 2 h without counting but by measuring enzyme activities.

Microbial dynamics of co- and separately entrapped mixed cultures of mesophilic lactic acid bacteria during the continuous prefermentation of milk.

Four strains of mesophilic lactic acid bacteria were separately or coentrapped in kappa-carrageenan/locust bean gum gel beads and used for continuous prefermentation of UHT skim milk in a stirred-tank bioreactor. Lactic acid and cell productivities of the immobilized cell bioreactor were particularly high and remarkably stable during eight weeks of continuous milk prefermentation (about 18 g h-1 l-1 of lactic acid and 4.9 x 10(12) CFU h-1 l-1, respectively, but important variations of the bacterial populations is prefermented milk and gel beads occurred in any case (co-or separate entrapment). The strain Lactococcus lactis subsp. lactis biovar diacetylactis CDII became dominant, accounting for approx. 90% (released cells) and 78% (immobilized cells) of the total population. Microscopic observations of sections of gels beads showed a progressive destructing of the bead surface with rupture and release of entrapped viable cells from peripheral cavities of the gel. It is believed that these cavities close again after releasing all or part of their cell content, entrapping the different strains of the mixed culture and initiating a new colonization step and a cross-contamination of the beads. On the other hand, experimentations over seven-week periods with pasteurized milk showed the high resistance of the immobilized cell bioreactor to psychrotrophic contamination.

Use of an immobilized cell bioreactor for the continuous inoculation of milk in fresh cheese manufacturing

A system was developed to continuously acidify and inoculate skim milk for the production of fresh cheese. Four strains of mesophilic lactic acid bacteria were entrapped separately in κ -carrageenan/locust bean gum gel beads and used in a stirred bioreactor operated at 26°C with a 25% (v/v) gel load. The pH in the reactor was controlled at 6.0 by adding fresh milk using proportional integrated derived regulation. The bioreactor was operated during 8-h daily cycles for up to 7 weeks with different milks (heat treatment, dry matter content) and differing starting procedures. The heat treatment of the milk was an important factor for process performance: a dilution rate increase of 57% and an inoculation level decrease of 63% were observed with sterilized UHT skim milk (142°C – 7.5 s) compared with pasteurized skim milk (72°C – 15 s). The dry matter content of the milk (8–13% w/w) had no detectable effect on these parameters. A convenient starting procedure of the system was tested; steady-state was reached in less than 40 min following an interruption period of 16–60 h. These results combined with our published data on process performance show the feasibility of using an integrated immobilized cell bioreactor for milk prefermentation in cheese manufacture.

Methods for the determination of aroma compounds in dairy products: a comparative study

Several methods used for sample preparation and for the determination of volatile flavour compounds in dairy products were tested. Steam distillation was an advantageous method for isolating volatile substances. The extraction yields for acetaldehyde, ethanol and diacetyl were >90%, and acetoin was partly separated from diacetyl. After steam distillation of the sample, gas-liquid chromatography was found to be a rapid method for the determination of acetaldehyde, ethanol and diacetyl in a wide range of concentrations corresponding to those found in dairy products. However, the lower limits for reproducible measurements were high (250, 1250 and 65 μM respectively). From the same sample, colorimetry was shown to be an accurate method for the detection of low levels of diacetyl and acetoin (12 and 57 μM respectively). Diacetyl interfered in the colorimetrie determination of acetoin. However, the interference was

Influence of dilution rate and cell immobilization on plasmid stability during continuous cultures of recombinant strains of Lactococcus lactis subsp. lactis.

The influence of dilution rate and cell immobilization on plasmid stability in recombinant strains of Lactococcus lactis subsp. lactis was investigated during continuous cultures. The studied strains, L. lactis IL2682 and IL2683, contained plasmids pIL9 (Lac+), pIL205 (CmR) and plasmids pIL252 (low copy number) and pIL253 (high copy number), respectively, that conferred resistance to erythromycin. Plasmid pIL205 was remarkably stable. Dilution rate did not affect the rate of loss of plasmids pIL252 and pIL253 significantly. Nevertheless, the loss of plasmid pIL253 was apparent after a further 21 generations when the dilution rate was decreased from 0.70 h-1 to 0.55 h-1. Cell immobilization in beads of kappa-carrageenan/locust bean gum improved plasmid stability by factors of 4.5 for pIL253 and 6.5 for pIL252. Thus, 10% of cells containing plasmids pIL252 or pIL253 were still present after 370 or 540 generations, respectively, compared with 50 or 210 generations in free cell cultures.

Effect of temperature on diacetyl and acetoin production by Lactococcus lactis subsp. lactis biovar diacetilactis CNRZ 483

We have investigated the effect of culture temperature on the growth of Lactococcus lactis subsp. lactis biovar diacetilactis , on its acidifying power, on diacetyl and acetoin production, and also on the activities of the principal enzymes involved in the synthesis of these two compounds. The rates of growth and lactic acid production decreased by a factor of ∼ 2 when the temperature decreased from 30 to 18°C. At 18°C, the maximal concentration of diacetyl (0·3mM) was 1·7 times that at 30°C, while that of acetoin was unchanged (5·2 mM). These results are explained by the behaviour of the principal enzymes involved in pyruvate metabolism. The activities of lactic dehydrogenase and acetolactate synthase varied little for culture temperatures between 18 and 30·C. However, the activity of NADH oxidase for a culture temperature of 18°C was 3·7 times that for 30°C, while that of diacetyl reductase for 30°C was 2·7 times that for 18°C. The net effect of temperature on these two activities was an increase in diacetyl production at lower temperature.

Perinatal protein restriction affects milk free amino acid and fatty acid profile in lactating rats: potential role on pup growth and metabolic status

Perinatal undernutrition affects not only fetal and neonatal growth but also adult health outcome, as suggested by the metabolic imprinting concept. Although maternal milk is the only channel through which nutrients are transferred from mother to offspring during the postnatal period, the impact of maternal undernutrition on milk composition is poorly understood. The present study investigates, in a rat model of nutritional programming, the effects of feeding an isocaloric, low-protein diet throughout gestation and lactation on milk composition and its possible consequences on offsprings growth and metabolic status. We used an integrated methodological approach that combined targeted analyses of macronutrients, free amino acid and fatty acid content throughout lactation, with an untargeted mass-spectrometric-based metabolomic phenotyping. Whereas perinatal dietary protein restriction failed to alter milk protein content, it dramatically decreased the concentration of most free amino acids at the end of lactation. Interestingly, a decrease of several amino acids involved in insulin secretion or gluconeogenesis was observed, suggesting that maternal protein restriction during the perinatal period may impact the insulinotrophic effect of milk, which may, in turn, account for the slower growth of the suckled male offspring. Besides, the decrease in sulfur amino acids may alter redox status in the offspring. Maternal undernutrition was also associated with an increase in milk total fatty acid content, with modifications in their pattern. Altogether, our results show that milk composition is clearly influenced by maternal diet and suggest that alterations in milk composition may play a role in offspring growth and metabolic programming.

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

SummaryThe effect of plasmid introduction into Lactococcus lactis subsp. lactis IL2661 on the growth of this strain and on plasmid stability was studied in pure batch cultures. The plasmids used (coding for erythromycin or chloramphenicol resistance) were the following: pIL205 (42 kb), pIL252 (4.6 kb, 6–9 copies), pIL253 (4.8 kb, 45–85 copies) and pE194 (inserted in the chromosome). Growth and acidification of L. lactis subsp. lactis IL2661 were similar to those of the derived recombinant lactococci. The maximal population at the end of the fermentation (9 h) was about 1.1±0.3 × 1010 cfu/ml, and maximal growth rate 0.92±0.07 h−1. Growth yield and lactic acid concentrations were 3.9±0.8 × 1011 cfu/g lactose consumed and 25.6±2.3 g/l, respectively. Different levels of plasmid stability were detected. Plasmid pE194, and plasmids pIL252 and pIL253 in the absence of pIL205, were stable after 10 h of culture. A slight loss (1–2%) of pIL205 was observed in all strains. In the presence of pIL205, plasmids pIL252 and pIL253 were maintained in only 56–95% of the cells. This result suggested an incompatibility between pIL205 and pIL252 or pIL253.