Human Milk MicroRNA and Total RNA Differ Depending on Milk Fractionation

MicroRNA have been recently discovered in human milk signifying potentially important functions for both the lactating breast and the infant. Whilst human milk microRNA have started to be explored, little data exist on the evaluation of sample processing, and analysis to ensure that a full spectrum of microRNA can be obtained. Human milk comprises three main fractions: cells, skim milk, and lipids. Typically, the skim milk fraction has been measured in isolation despite evidence that the lipid fraction may contain more microRNA. This study aimed to standardize isolation of microRNA and total RNA from all three fractions of human milk to determine the most appropriate sampling and analysis procedure for future studies. Three different methods from eight commercially available kits were tested for their efficacy in extracting total RNA and microRNA from the lipid, skim, and cell fractions of human milk. Each fraction yielded different concentrations of RNA and microRNA, with the highest quantities found in the cell and lipid fractions, and the lowest in skim milk. The column‐based phenol‐free method was the most efficient extraction method for all three milk fractions. Two microRNAs were expressed and validated in the three milk fractions by qPCR using the three recommended extraction kits for each fraction. High expression levels were identified in the skim and lipid milk factions for these microRNAs. These results suggest that careful consideration of both the human milk sample preparation and extraction protocols should be made prior to embarking upon research in this area. J. Cell. Biochem. 116: 2397–2407, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Investigating the relationship between raw milk bacterial composition, as described by intergenic transcribed spacer–PCR fingerprinting, and pasture altitude

Aims:  To assess the bacterial biodiversity level in bovine raw milk used to produce Fontina, a Protected Designation of Origin cheese manufactured at high-altitude pastures and in valleys of Valle d'Aosta region (North-western Italian Alps) without any starters. To study the relation between microbial composition and pasture altitude, in order to distinguish high-altitude milk against valley and lowland milk.
Methods and Results:  The microflora from milks sampled at different alpine pasture, valley and lowland farms were fingerprinted by PCR of the 16S–23S intergenic transcribed spacers (ITS-PCR). The resulting band patterns were analysed by generalized multivariate statistical techniques to handle discrete (band presence–absence) and continuous (altitude) information. The fingerprints featured numerous bands and marked variability indicating complex, differentiated bacterial communities. Alpine pasture milks were distinguished from lowland ones by cluster analysis, while this technique less clearly discriminated alpine pasture and valley samples. Generalized principal component analysis and clustering-after-ordination enabled a more effective distinction of alpine pasture, valley and lowland samples.
Conclusions:  Alpine raw milks for Fontina production contain highly diverse bacterial communities, the composition of which is related to the altitude of the pasture where milk was produced.
Significance and Impact of the Study:  This research may provide analytical support to the important issue represented by the authentication of the geographical origin of alpine milk productions.     

Media for hybridoma growth and monoclonal antibody production

For the economical production of monoclonal antibodies (MAbs), the cell-culture medium must be optimized for three different phases: growth of the hybridomas, MAb productivity of the hybridomas, and MAb purification or downstream processing. Medium improvements are necessary to meet these requirements for large-scale MAb production. Information bearing on this issue is being addressed in two research areas, cell biology and biochemical engineering, and is reviewed in this article.     

Potential occurrence of adhering living Bacillus spores in milk product processing lines

AIMS: The hygienic risk associated with microbial soil on surfaces of milk processing lines was evaluated, based on experimental results. METHODS AND RESULTS: From a panel of Bacillus spores isolated from milk products, B. cereus CUETM 98/4, was found to be highly resistant to heat (D100=3.32 min in whole milk) and oxidant disinfectant (70% lethality of adherent spores with Ikalin 2%). From adhesion trials, up to 1.1 x 10(7) spores cm(-2) were found to be adherent to solid surfaces when suspended in saline or in custard (10(5) and 10(7) cfu ml(-1)), and over 10% of these adherent spores would resist the cleaning procedure. CONCLUSION: A highly contaminated milk (10(5) cfu ml(-1)) subjected to a current sterilization process (8 log reduction) led to a residual contamination of less than 1 cfu in the representative processing line after a complete production run. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the fact that under appropriate processing conditions (efficient sterilization and cleaning procedures), even disinfection would be sufficient to eliminate any contamination risk. Conversely, the disinfection procedure becomes an essential step under inappropriate processing conditions.     

Lactation curve model with explicit representation of perturbations as a phenotyping tool for dairy livestock precision farming

In the context of dairy farming, ruminant females often face challenges inducing perturbations that affect their performance and welfare. A key issue is how to assess the effect of perturbations and provide metrics to quantify how animals cope with their environment. Milk production dynamics are good candidates to address this issue: i) they are easily accessible, ii) overall dynamics throughout lactation process are well described and iii) perturbations are visible through milk losses. In this study, a perturbed lactation model (PLM) with explicit representation of perturbations was developed. The model combines two components: i) the unperturbed lactation model that describes a theoretical lactation curve, assumed to reflect female production potential and ii) the perturbation model that describes all the deviations from the unperturbed lactation model with four parameters: starting date, intensity and shape (collapse and recovery). To illustrate the use of the PLM as a phenotyping tool, it was fitted on a data set of 319 complete lactations from 181 individual dairy goats. A total of 2 354 perturbations were detected, with an average of 7.40 perturbations per lactation. Loss of milk production for the whole lactation due to perturbations varied between 2 and 19% of the milk production predicted by the unperturbed lactation model. The number of perturbations was not the major factor explaining the loss of milk production, suggesting that there are different types of animal response to challenges. By incorporating explicit representation of perturbations in a lactation model, it was possible to determine for each female the potential milk production, characteristics of each perturbation and milk losses induced by perturbations. Further, it was possible to compare animals and analyze individual variability. The indicators produced by the PLM are likely to be useful to move from raw data to decision support tools in dairy production.     

Approche morphométrique des dents jugales déciduales d’Equus caballus arcelini (sensu lato, Guadelli 1991) : critères de détermination et estimation de l'âge d'abattage

Morphometrical approach of Equus caballus arcelini (sensu lato, Guadelli 1991) jugal milk teeth: determination criteria and mortality age estimation. This paper presents the main morphometrical results of the study of the last western European Pleistocene horse (Equus caballus arcelini sensu lato, Guadelli 1991) jugal milk teeth. In association with observed morphological characters, morphometrical data helps determination. The main purpose of this discrimination between the different jugal milk teeth is the estimation of the mortality age, according to the crown-height method. In an archaeological context, the use of this method on horses' dental series enables to study the issue of human settlements' seasonality. To cite this article: O. Bignon, C. R. Palevol 5 (2006).     

Determining the source of Bacillus cereus and Bacillus licheniformis isolated from raw milk, pasteurized milk and yoghurt

Aims:  Strain-specific detection of Bacillus cereus and Bacillus licheniformi s in raw and pasteurized milk, and yoghurt during processing.
Methods and Results:  Randomly selected isolates of Bacillus spp. were subjected to PCR analysis, where single primer targeting to the repetitive sequence Box elements was used to fingerprint the species. The isolates were separated into six different fingerprint patterns. The results show that isolates clustered together at about the 57% similarity level with two main groups at the 82% and 83% similarity levels, respectively. Contamination with identical strains both of B. cereus and B . licheniformis in raw and pasteurized milk was found as well as contaminated with different strains (in the case of raw milk and yoghurt/pasteurized milk and yoghurt). Several BOX types traced in processed milk samples were not discovered in the original raw milk.
Conclusions:  BOX-PCR fingerprinting is useful for characterizing Bacillus populations in a dairy environment. It can be used to confirm environmental contamination, eventually clonal transfer of Bacillus strains during the technological processing of milk.
Significance and Impact of the Study:  Despite the limited number of strains analysed, the two Bacillus species yielded adequately detectable banding profiles, permitting differentiation of bacteria at the strain level and showing their diversity throughout dairy processing.     

Questioning current definitions for breastfeeding research

ABSTRACT: BACKGROUND: The aim of this paper is to examine how breastfeeding is defined for research purposes. DISCUSSION: Current breastfeeding definitions focus on the amount of breast milk an infant receives and do not encompass how a baby is fed. Our concerns are that key variables are not measured when mothers are pumping or expressing their milk and bottle feeding. It seems the breastfeeding relationship is not considered in the definition. CONCLUSION: While we appreciate the implications of full versus partial breastfeeding in research studies, we also believe the method of infant feeding to be significant. Researchers should develop new definitions.     

Inclusion of cow records in genomic evaluations and impact on bias due to preferential treatment

Background

Today, genomic evaluations are an essential feature of dairy cattle breeding. Initially, genomic evaluation targeted young bulls but recently, a rapidly increasing number of females (both heifers and cows) are being genotyped. A rising issue is whether and how own performance of genotyped cows should be included in genomic evaluations. The purpose of this study was to assess the impact of including yield deviations, i.e. own performance of cows, in genomic evaluations.

Methods

Two different genomic evaluations were performed: one including only reliable daughter yield deviations of proven bulls based on their non-genotyped daughters, and one including both daughter yield deviations for males and own yield deviations for genotyped females. Milk yield, the trait most prone to preferential treatment, and somatic cell count, for which such a bias is very unlikely, were studied. Data consisted of two groups of animals from the three main dairy breeds in France: 11 884 elite females genotyped by breeding companies and 7032 cows genotyped for a research project (and considered as randomly selected from the commercial population).

Results

For several measures that could be related to preferential treatment bias, the elite group presented a different pattern of estimated breeding values for milk yield compared to the other combinations of trait and group: for instance, for milk yield, the average difference between estimated breeding values with or without own yield deviations was significantly different from 0 for this group. Correlations between estimated breeding values with or without yield deviations were lower for elite females than for randomly selected cows for milk yield but were very similar for somatic cell count.

Conclusions

This study demonstrated that including own milk performance of elite females leads to biased (over-estimated) genomic evaluations. Thus, milk production records of elite cows require specific treatment in genomic evaluation.     

Effect of grain type and processing method on rumen fermentation and milk rumenic acid production

It was hypothesized that differences in starch degradability account for observed differences in rumen vaccenic acid (t11-18:1) and milk rumenic acid (RA) concentrations. To test this hypothesis, starch degradability was varied through grain source and by processing. Eight Holstein cows in mid-lactation were assigned to two 4 × 4 Latin squares with four 21-day periods and four diets: dry rolled barley, ground barley, dry rolled corn and ground corn. Diets contained similar starch content and were supplemented with whole sunflower seed to provide similar total polyunsaturated fatty acid (PUFA) (18:2n-6 + 18:3n-3) contents. Forage/concentrate ratios of all diets were 42 : 58. Rumen, plasma and milk samples were collected in the third week of each period. In situ degradation rates (%/h) for rolled corn, ground corn, rolled barley and ground barley were 5.4, 8.9, 17.0 and 19.4, respectively, for dry matter (DM) and 6.3, 10.8, 25.3 and 43.8, respectively, for starch. DM intakes were greater for corn-based diets (CBD) than for barley-based diets (BBD) with no difference between rolled and ground diets. Daily minimum rumen pH was less (5.2 v. 5.5) and pH duration <5.8 (h/d) was greater (7.4 v. 4.3) for BBD than for CBD. Milk fat content and yield were less for BBD than for CBD with greater values observed for rolling compared with grinding. Variability in milk fat yield was strongly related (R2 = 0.55; P < 0.01) to total starch intake (45%) and milk c9t11-CLA (10%) and none of the t-18:1 isomers or CLA isomers that are typically associated with milk fat depression entered the model. The concentrations (%) of t10-18:1 and t11-18:1 were greater for BBD than for CBD in rumen contents (t10-18:1, 3.5 v. 1.3; t11-18:1, 3.2 v. 1.9), plasma (t10-18:1, 1.2 v. 0.2; t11-18:1, 0.97 v. 0.58) and milk (t10-18:1, 3.8 v. 1.0; t11-18:1, 2.6 v. 1.7) despite greater total PUFA intakes for CBD. Milk RA concentration was greater for BBD than for CBD (1.46 v. 0.89) but was not influenced by the method of grain processing. This study clearly demonstrated that the milk content and profile of t-18:1 and CLA isomers were more strongly influenced by the source of grain starch (barley > corn) than by the method of grain processing indicating that factors inherent in the source of starch were responsible for the observed differences and these factors could not be modified by the processing methods used in this study.     

Rapid differentiation and enumeration of the total, viable vegetative cell and spore content of thermophilic bacilli in milk powders with reference to Anoxybacillus flavithermus

AIMS: The development of a rapid method for the selective detection and enumeration of the total and viable vegetative cell and spore content of thermophilic bacilli in milk powder by PCR. METHODS AND RESULTS: Quantitative PCR and microscopy indicate the presence of up to 2.9 log units more cells in milk powder than accounted for by plate counting due to the majority of cells being killed during milk processing. Two approaches for viable and dead cell differentiation of thermophilic bacilli by quantitative PCR were evaluated, these being the nucleic binding dye ethidium monoazide (EMA) and DNase I digestion. The former agent exposed to a viable culture of Anoxybacillus flavithermus caused considerable cell inactivation. In contrast, DNase I treatment had no effect on cell viability and was utilized to develop DNA extraction methods for the differential enumeration of total, viable vegetative cells and spores in milk powder. Moreover, the methods were further applied and evaluated to 41 factory powder samples taken throughout eight process runs to assess changes in numbers of vegetative cells and spores with time. DNase I treatment reduced vegetative cell numbers enumerated with PCR by up to 2.6 log units. The quantification of spores in the factory milk powders investigated indicates on average the presence of 1.2 log units more spores than determined by plate counting. CONCLUSIONS: The method presented in this study provides the ability to selectively enumerate the total and viable cell and spore content of reconstituted milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides a tool to monitor the extent of thermophilic contamination during milk powder manufacturing 60-90 min after sampling.     

Breast milk expression among formally employed women in urban and rural Malaysia: A qualitative study

ABSTRACT: BACKGROUND: Many women are unable to practice exclusive breastfeeding because they are separated from their infants while working. Expressing their breast milk helps them to continue breastfeeding. This study explores the perception and experiences related to the feasibility, acceptability and safety of breast milk expression among formally employed women in Kelantan, Malaysia. METHODS: A qualitative method using in-depth interviews was conducted from December 2008 to December 2009 among Malay women from urban and rural areas. A snowball sampling method was used to recruit the informants, and the interviews, which were facilitated by an interview guide, were audio-recorded and transcribed verbatim. Thematic analysis was conducted, with construction of codes and themes from each interview. RESULTS: Analysis of the interviews with 20 informants identified three themes related to breast milk expression. The themes were as follows: (i) lack of feasibility of expressing breast milk, (ii) negative feelings about expressing breast milk, and (iii) doubts about the safety and hygiene of expressed breast milk. The informants who did not practice exclusive breastfeeding believed that expressing their breast milk was not feasible, commonly because they felt there were not enough facilities for them. They also had negative feelings such as embarrassment. The safety and hygiene of the expressed breast milk was also their main concern. CONCLUSION: More practical and focused education, as well as provision of facilities, is needed for women to effectively and safely express and store their breast milk. The issue of inadequate milk production should be emphasized, especially by encouraging them to express their breast milk as a way to improve milk production.     

Pavement model in Internet of intelligences and applications in risk analysis online

In this article, I suggest a model to study the risk issue that can be mapped into a query on how to pass a road safely. The model is called the pavement model, which can be used in the Internet of intelligences. The model has three components: object, value of success or failure, and duration of the value. An object is mapped into a section of road, the value is defined by the truth value of success or failure passing the road, and the duration represents the number of parts with the value in the session. The case of choosing milk powder shows that the model can perfectly integrate childcare experiences to be a rank that indicates which milk powder should be the first choice and which milk powder is not recommended to buy. The case of assessing typhoon risk shows that the model can dynamically assess typhoon risks with respect to different months and landing situations.     

Stem Cell Research: A Novel Boulevard towards Improved Bovine Mastitis Management

The dairy industry is a multi-billion dollar industry catering the nutritional needs of all age groups globally through the supply of milk. Clinical mastitis has a severe impact on udder tissue and is also an animal welfare issue. Moreover, it significantly reduces animal value and milk production. Mammary tissue damage reduces the number and activity of epithelial cells and consequently contributes to decreased milk production. The high incidence, low cure rate of this highly economic and sometimes deadly disease is an alarming for dairy sector as well as policy makers. Bovine mammary epithelial cells (MECs) and their stem cells are very important in milk production and bioengineering. The adult mammary epithelium consists of two main cell types; an inner layer of luminal epithelial cells, which produce the milk during lactation, and an outer layer of myoepithelial cells resting on a basement membrane, which are responsible for pushing the milk through the ductal network to the teat cistern. Inner layer of columner/luminal cells of bovine MECs, is characterized by cytokeratin18, 19 (CK18, CK19) and outer layer such as myoepithelial cells which are characterized by CK14, α-smooth muscle actin (α-SMA) and p63. Much work has been done in mouse and human, on mammary gland stem cell research, particularly in cancer therapy, but stem cell research in bovine is still in its infancy. Such stem/progenitor cell discoveries in human and mouse mammary gland bring some hope for application in bovines. These progenitors may be therapeutically adopted to correct the structural/cytological defects in the bovine udder due to mastitis. In the present review we focused on various kinds of stem/progenitor cells which can have therapeutic utility and their possibilities to use as a potential stem cell therapy in the management of bovine post-mastitis damage in orders to restore milk production. The possibilities of bovine mammary stem cell therapy offers significant potential for regeneration of tissues that can potentially replace/repair diseased and damaged tissue through differentiation into epithelial, myoepithelial and/or cuboidal/columnar cells in the udder with minimal risk of rejection and side effects.     

Glycation research in amino acids: a place to call home

This is an introduction to a collection of review articles by leading investigators in the field of protein glycation research, see following articles in this issue. With this we launch a section of this journal now established for presentation of research results, reviews and commentaries on protein glycation and related topics. Glycation is the spontaneous, non-enzymatic reaction of protein with saccharides and saccharide derivatives. Although studied in the modern scientific era for over 100 years, its importance in the biology, medicine, food and nutrition, pharmacology and toxicology, and technological processing remains intriguingly undisclosed. In this section of amino acids, research on glycation is a qualifier for publication. Glycation research now has a place to call home.     

Of mammals and milk: how maternal stress affects nursing offspring

1. A large body of research shows that maternal stress during an offspring’s early life can impact its phenotype in both the short and long term. In the Vertebrata, most research has been focused on maternal stress during the prenatal period. However, the postnatal period is particularly important in mammals because maternal milk provides a conduit by which maternal hormones secreted in response to stressors (glucocorticoids, GCs) can reach offspring. Moreover, lactation outlasts gestation in many species.
2. Though GCs were first detected in milk over 40 years ago, few studies have explored how they affect nursing offspring, and no reviews have been written on how maternal stress affects nursing offspring in the natural world.
3. We discuss the evolution of milk and highlight its importance in each of the three mammalian lineages: monotremes (subclass Monotremata), marsupials (infraclass Marsupialia), and eutherians (infraclass Placentalia). Most research on the effects of milk GCs on offspring has been focused on eutherians, but monotremes and marsupials rely on their mothers’ milk for a proportionally longer period of time, and so research on these taxa may yield more insight.
4. We show that GCs are important for milk production, both during an individual nursing bout and over the entire lactation period, and review evidence of GCs moving from maternal blood to milk, and eventually to nursing offspring. We examine evidence from rodents and primates of associations between GC levels in lactating females (either blood or milk) and offspring behaviour and growth rates. We discuss ways that maternal stress may impact these offspring phenotypes outside of milk GCs, such as changes to: (1) milk output, (2) other milk constituents (e.g. macronutrients, growth factors, cytokines), and (3) maternal care behaviour.
5. Critical to understanding the fitness impacts of elevated maternal GC levels during lactation is to place this within the context of the natural environment. Species-specific traits and natural histories will help us to understand why such maternal stress produces different offspring phenotypes that equip them to cope with and succeed in the environment they are about to enter.

     

双歧异黄酮奶粉对化疗荷瘤小鼠减毒增效作用的研究

目的：研究双歧异黄酮奶粉对化疗后荷瘤小鼠免疫功能的影响。方法：以S180荷瘤C57BL／6小鼠为模型,系统地研究双歧异黄酮奶粉对瘤重和细胞功能的影响。结果：双歧异黄酮奶粉对荷瘤鼠化疗所引起的免疫功能低下具有明显的恢复作用,可显著提高化疗荷瘤鼠T细胞转化能力,明显促进化疗荷瘤鼠白介素—2(IL—2)分泌水平。结论;双歧异黄酮奶粉能够拮抗肿瘤抗原和化疗所引起的免疫抑制。     

用聚丙烯酰胺凝胶电泳和微流体芯片电泳鉴别6种液态奶

目的:研究6种液态奶制品蛋白电泳图谱的区别,建立奶制品的蛋白质学鉴别方法。方法:以5种纯牛奶、羊奶、水牛奶、骆驼奶、牦牛奶、黄豆浆为研究对象,通过SDS-PAGE和Agilent 2100微流体芯片电泳法进行分析比较。结果:骆驼奶、黄豆浆与其他研究对象的图谱有明显区别,而牛奶、羊奶、水牛奶、牦牛奶的差异却不是很大;采用微流体芯片电泳可有效地对豆奶、骆驼奶进行区分,还可在一定程度上鉴别牛奶、羊奶、水牛奶和牦牛奶。结论:Agilent2100系统作为一种新型半自动微流体芯片技术,可以快速、高效、准确地应用于液态奶制品的蛋白成分分析及鉴别。     

Initial insights on the performances and management of dairy cattle herds combining two breeds with contrasting features

Finding ways of increasing animal production with low external inputs and without compromising reproductive performances is a key issue of livestock systems sustainability. One way is to take advantage of the diversity and interactions among components within livestock systems. Among studies that investigate the influence of differences in animals’ individual abilities in a herd, few focus on combinations of cow breeds with contrasting features in dairy cattle herds. This study aimed to analyse the performances and management of such multi-breed dairy cattle herds. These herds were composed of two types of dairy breeds: ‘specialist’ (Holstein) and ‘generalist’ (e.g. Montbeliarde, Simmental, etc.). Based on recorded milk data in southern French region, we performed ANOVA: (i) to compare the performances of dairy herds according to breed-type composition: multi-breed, single specialist breed or single generalist breed and (ii) to test the difference of milk performances of specialist and generalist breed cows (n = 10 682) per multi-breed dairy herd within a sample of 22 farms. The sampled farmers were also interviewed to characterise herd management through multivariate analysis. Multi-breed dairy herds had a better trade-off among milk yield, milk fat and protein contents, herd reproduction and concentrate-conversion efficiency than single-breed herds. Conversely, they did not offer advantages in terms of milk prices and udder health. Compared to specialist dairy herds, they produce less milk with the same concentrate-conversion efficiency but have better reproductive performances. Compared to generalist dairy herds, they produce more milk with better concentrate-conversion efficiency but have worse reproductive performances. Within herds, specialist and generalist breed cows significantly differed in milk performances, showing their complementarity. The former produced more milk for a longer lactation length while the latter produced milk with higher protein and fat contents and had a slightly longer lactation rank. Our results also focus on the farmers’ management of multi-breed dairy herds underlying herd performances. Three strategies of management were identified and structured along two main axes. The first differentiates farmers according to their animal-selection practices in relation with their objectives of production: adapting animal to produce milk with low-feeding inputs v. focussing on milk yield trait to intensify milk production. The second refers to the purpose farmers give to multi-breed dairy herds: milk v. milk/meat production. These initial insights on the performances and management of multi-breed dairy herds contribute to better understanding the functioning of ruminant livestock systems based on individual variability.     

Next issue (April 2007): Milk Processing

Edited by Prof. Knut Heller, Karlsruhe, Germany. Highlight articles: – Bacteriophages in dairy products: Pros and cons – Liberation of bioactive peptides from milk proteins – Lactobacillus delbrueckii subsp. bulgaricus for yoghurt with mild taste – Prebiotic galacto-oligosaccharides production using Lactobacillus reuteri – Lactobacillus kefiri biotransformation of pentoxifylline – Modification of milk proteins by microbial transglutaminase ... and much more: Read the next issue of BTJ!