RESUMEN
Renneting is an enzymatic process that turns milk into curd which is then transformed into cheese. Rennet-induced coagulation of caseins (CNs) is the critical step during this process and the key is the primary hydrolysis of κ-CN's Phe105-Met106 bond by chymosin. This article comprehensively reviews the existing data on the extent/degree of κ-CN hydrolysis during renneting of bovine milk and critically evaluates its determination methods. The data show that under normal cheese-making conditions, milk gelation occurs at a degree of κ-CN hydrolysis <80%, which varies due to several factors including analytical and estimation approaches. The common approach involves isolating the macropeptides released, by precipitating whey proteins and residual CN in 1%-12% trichloroacetic acid (TCA), then assuming that the maximum amount obtained is 100% κ-CN hydrolysis. The drawback is that the estimated degree of κ-CN hydrolysis may be higher than the actual value as TCA partially precipitates the macropeptide fractions. Moreover, macropeptide isolation seems unnecessary based on current advances in chromatographic and electrophoretic techniques. The present work proposes a simple mass balance-based approach that will provide accurate estimates in future studies. The accuracy of measuring the degree of κ-CN hydrolysis has implications on the precision of the data in relation to its partitioning (% distribution between the curd and whey) which is essential for improving whey quality.
RESUMEN
Prenatal inflammation may predispose to preterm birth and postnatal inflammatory disorders such as necrotizing enterocolitis (NEC). Bioactive milk ingredients may help to support gut maturation in such neonates, but mother's milk is often insufficient after preterm birth. We hypothesized that supplementation with bioactive ingredients from bovine milk [osteopontin (OPN), caseinoglycomacropeptide (CGMP), colostrum (COL)] supports gut, immunity, and NEC resistance in neonates born preterm after gram-negative infection before birth. Using preterm pigs as a model for preterm infants, fetal pigs were given intraamniotic injections of lipopolysaccharide (LPS; 1 mg/fetus) and delivered 3 days later (90% gestation). For 5 days, groups of LPS-exposed pigs were fed formula (FOR), bovine colostrum (COL), or formula enriched with OPN or CGMP. LPS induced intraamniotic inflammation and postnatal systemic inflammation but limited effects on postnatal gut parameters and NEC. Relative to FOR, COL feeding to LPS-exposed pigs showed less diarrhea, NEC severity, reduced gut IL-1ß and IL-8 levels, greater gut goblet cell density and digestive enzyme activities, and blood helper T-cell fraction. CGMP improved neonatal arousal and gut lactase activities and reduced LPS-induced IL-8 secretion in intestinal epithelial cells (IECs) in vitro. Finally, OPN tended to reduce diarrhea and stimulated IEC proliferation in vitro. No effects on villus morphology, circulating cytokines, or colonic microbiota were observed among groups. In conclusion, bioactive milk ingredients exerted only modest effects on gut and systemic immune parameters in preterm pigs exposed to prenatal inflammation. Short-term, prenatal exposure to inflammation may render the gut less sensitive to immune-modulatory milk effects. NEW & NOTEWORTHY Prenatal inflammation is a risk factor for preterm birth and postnatal complications including infections. However, from clinical studies, it is difficult to separate the effects of only prenatal inflammation from preterm birth. Using cesarean-delivered preterm pigs with prenatal inflammation, we documented some beneficial gut effects of bioactive milk diets relative to formula, but prenatal inflammation appeared to decrease the sensitivity of enteral feeding. Special treatments and diets may be required for this neonatal population.
Asunto(s)
Caseínas/administración & dosificación , Corioamnionitis/dietoterapia , Enterocolitis Necrotizante/prevención & control , Alimentos Fortificados , Inmunidad Mucosa , Fórmulas Infantiles , Intestinos/inmunología , Osteopontina/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Nacimiento Prematuro , Animales , Animales Recién Nacidos , Caseínas/inmunología , Línea Celular , Corioamnionitis/inducido químicamente , Corioamnionitis/inmunología , Corioamnionitis/metabolismo , Calostro/inmunología , Modelos Animales de Enfermedad , Enterocolitis Necrotizante/etiología , Enterocolitis Necrotizante/inmunología , Enterocolitis Necrotizante/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Microbioma Gastrointestinal , Edad Gestacional , Humanos , Recién Nacido , Absorción Intestinal , Intestinos/microbiología , Intestinos/patología , Lipopolisacáridos , Valor Nutritivo , Osteopontina/inmunología , Fragmentos de Péptidos/inmunología , Permeabilidad , Embarazo , Sus scrofaRESUMEN
The bovine milk protein osteopontin (OPN) may be an efficient means to prevent bacterial adhesion to dental tissues and control biofilm formation. This study sought to determine to what extent OPN impacts adhesion forces and surface attachment of different bacterial strains involved in dental caries or medical device-related infections. It further investigated if OPN's effect on adhesion is caused by blocking the accessibility of glycoconjugates on bacterial surfaces. Bacterial adhesion was determined in a shear-controlled flow cell system in the presence of different concentrations of OPN, and interaction forces of single bacteria were quantified using single-cell force spectroscopy before and after OPN exposure. Moreover, the study investigated OPN's effect on the accessibility of cell surface glycoconjugates through fluorescence lectin-binding analysis. OPN strongly affected bacterial adhesion in a dose-dependent manner for all investigated species (Actinomyces naeslundii, Actinomyces viscosus, Lactobacillus paracasei subsp. paracasei, Staphylococcus epidermidis, Streptococcus mitis, and Streptococcus oralis). Likewise, adhesion forces decreased after OPN treatment. No effect of OPN on the lectin-accessibility to glycoconjugates was found. OPN reduces the adhesion and adhesion force/energy of a variety of bacteria and has a potential therapeutic use for biofilm control. OPN acts upon bacterial adhesion without blocking cell surface glycoconjugates.
RESUMEN
Chymosin-induced cleavage of κ-casein (κ-CN) occurs during the first enzymatic phase in milk coagulation during cheese manufacturing, where the hydrophilic C-terminal peptide of κ-CN, named caseino-macropeptide (CMP), is released into the whey. The CMP peptide is known for its rather heterogeneous composition with respect to both genetic variation and multiple posttranslational modifications, including phosphorylation and O-linked glycosylation. An approach of liquid chromatography coupled with mass spectrometry was used to investigate (1) the overall protein profile and (2) the release of various forms of CMP after addition of chymosin to individual cow milk samples from 2 breeds, Danish Jersey (DJ) and Danish Holstein-Friesian (DH). The cows were selected to represent distinct homo- and heterozygous types of the κ-CN genetic variants A, B, and E (i.e., genotypes AA, BB, AB, EE, and AE). Initially, investigation of the protein profile showed milk with κ-CN BB exhibited the highest relative content of κ-CN, whereas AE milk exhibited the lowest, and after 40min of renneting >90% of intact κ-CN was hydrolyzed by chymosin in milk representing all κ-CN genotype. By in-depth analysis of the CMP chromatographic profile, multiple CMP isoforms with 1 to 3 O-linked glycans (1-3 G) and 1 to 3 phosphate groups (1-3 P) were identified, as well as nonmodified CMP isoforms. The number of identified CMP isoforms varied to some extent between breeds (21CMP isoforms identified in DJ, 26CMP isoforms in DH) and between κ-CN genetic variants (CMP variant A being the most heterogeneous compared with CMP B and E), as well as between individual samples within each breed. The predominant forms of glycans attached to CMP were found to be the acidic tetrasaccharide {N-acetyl-neuraminic acid α(2-3)galactose ß(1-3)[N-acetyl-neuraminic acid α(2-6)]N-acetyl galactose} or trisaccharides {N-acetyl-neuraminic acid α(2-3)galactose ß(1-3)N-acetyl galactose and galactose ß(1-3)[N-acetyl-neuraminic acid (α2-6)]N-acetyl galactose}. The CMP release was calculated to follow first-order kinetics and was determined by the measurement of CMP content during renneting. The highest rate of release for all CMP isoforms occurred from 0 to 2min after chymosin addition. Concurring results from both breeds showed that CMP variant A with 1-2 P had the highest reaction rate of CMP release, followed by CMP B 1-2 P and then by CMP E 1-2 P (only in DH). All the identified glycosylated CMP isoforms had lower reaction rates of release compared with that of nonglycosylated CMP, thus glycan modifications seemed to negatively influence the reaction rate of chymosin-induced hydrolysis of κ-CN.