Effect of heat treatments on stability of β-lactams in milk

The presence of residues of antimicrobial substances in milk may have serious toxicological and technical consequences. To date, few studies have been done to evaluate the effect of heat treatments on β-lactam residues in milk. However, the few studies that have been conducted estimate losses of antimicrobial activity under different combinations of temperature and time using microbiological methods. The aims of this study were to calculate the kinetic parameters for the degradation of β-lactam antibiotics in milk and to develop prediction models to estimate the concentration losses of these compounds in conventional dairy heat treatments. To do so, we employed a quantitative HPLC method to calculate losses in concentrations of 10 β-lactam antibiotics in milk with different combinations of temperature and time. Increasing the temperature from 60°C to 100°C decreased the half-life of amoxicillin (372 to 50 min), ampicillin (741 to 26 min), cloxacillin (367 to 46 min), and penicillin G (382 to 43 min). These increases in temperature caused further degradation in cephalosporins, which was accompanied by a decrease in half-life times to reach very low values; for instance, 4, 5, and 6 min for cefoperazone, cephurexime, and cephapirin, respectively. Kinetic equations were applied to different heat treatments used in dairy processing. Heat treatments at high temperatures and long times (e.g., 120°C for 20 min) led to a further degradation of β-lactam antibiotics with percentages close to 100% for cefoperazone and cefuroxime. In contrast, when milk was subjected to heat treatments at lower temperatures and times (e.g., 72°C for 15 s), the degradation of β-lactam in milk did not exceed 1% for the 10 antibiotics tested.     

Enhancement of β-glucosidase activity on the cell-surface of sake yeast by disruption of SED1

We determined the genetic background that would result in a more optimal display of heterologously expressed β-glucosidase (BGL) on the cell surface of yeast Saccharomyces cerevisiae. Amongst a collection of 28 strains carrying deletions in genes for glycosylphosphatidyl inositol (GPI)-anchored proteins, the Δsed1 and Δtos6 strains had significantly higher BGL-activity whilst maintaining wild type growth. Absence of Sed1p, which might facilitate incorporation of anchored BGL on the cell-surface, could also influence the activity of BGL on the cell surface with the heterologous gene being placed under the control of the SED1 promoter. For the evaluation of its industrial applicability we tested this system in heterologous and homogenous SED1-disruptants of sake yeast, a diploid S. cerevisiae strain, in which either the SED1 ORF or the complete gene including the promoter was deleted by use of the high-efficiency loss of heterozygosity method. Evaluation of disruptants displaying BGL showed that deletion of the SED1 ORF enhanced BGL activity on the cell surface, while additional deletion of the SED1 promoter increased further BGL activity on the cell surface. Compared to heterozygous disruption, homozygous disruption resulted generally in a higher BGL activity. Thus, homozygous deletion of both SED1 gene and promoter resulted in the most efficient display of BGL reaching a 1.6-fold increase of BGL-activity compared to wild type.     

Effect of storage conditions on furosine formation in milk–cereal based baby foods

The effect of storage during 9 months at 25, 30 and 37 °C on furosine formation in three milk–cereal based baby foods was studied to evaluate development of the Maillard reaction. Furosine was measured by HPLC-UV. Immediately after the manufacturing process, furosine contents were 310–340 mg/100 g protein and at the 9th storage month were 426–603 mg/100 g protein. Storage time and temperature have a significant increase (p < 0.05) of furosine content during storage. Furosine contents were higher in sample containing honey than in those without honey. Interactions (p < 0.05) between storage time and temperature or type of sample were found. A predictive model equation of the evolution of furosine during storage explaining 80% of the variability in furosine content was obtained. The blockage of lysine through storage calculated using the furosine and total lysine provided values ranged from 9.5% to 18.1% for analysed baby foods.     

Effect of Sicilian pasture feeding management on content of α-tocopherol and β-carotene in cow milk

This study was performed to evaluate α-tocopherol and β-carotene contents of pasture milk under ordinary Sicilian farming conditions. Fourteen dairy farms were allocated into 2 balanced groups on the basis of cultivated (CULT) or spontaneous (SPO) pasture type feeding. Bulk milk per farm was collected 4 times from February through April at 3-wk intervals. Pasture botanical and diet composition, diet nutritional quality, milk yield and composition were estimated each time. Pasture intake levels were calculated based on feed analyses, hay and concentrate amounts fed, and milk yield and chemical composition. According to pasture intake, the farms were split into low pasture intake (LPI; <29.5% of dry matter) and high pasture intake (HPI; >29.5% of dry matter) groups. Milk samples per farm were analyzed for α-tocopherol and β-carotene contents by HPLC. The SPO group had higher levels of α-tocopherol and β-carotene in milk (0.7 and 0.3 mg/L, respectively) and milk fat (19.0 and 7.5 mg/kg fat, respectively) compared with the CULT group in milk (0.5 and 0.2 mg/L, respectively) and milk fat (14.6 and 4.9 mg/kg, respectively). High pasture intake compared with LPI increased α-tocopherol in milk fat (18.0 and 16.0 mg/kg of fat, respectively). However, only in the SPO (not in CULT), HPI compared with LPI increased milk α-tocopherol (0.8 vs. 0.6 mg/L, respectively), milk β-carotene (0.3 vs. 0.2 mg/L, respectively), and milk fat β-carotene (8.4 vs. 6.6 mg/kg, respectively). Results may be related to the different botanical composition of the respective pasture types and pasture intake. Spontaneous pasture compared with CULT contained a higher mass proportion of Asteraceae, Fabaceae, Cruciferae, Euphorbiaceae, and Malvaceae plants. Milk and milk fat α-tocopherol levels were higher on test-days (TD)-1, TD-2, and TD-4 compared with TD-3. For HPI farms, milk fat β-carotene content was higher on the first 2 TD compared with the last 2 TD. These differences could be related to plant biological stage. On Sicilian dairy farms, the highest milk α-tocopherol and β-carotene contents may be obtained feeding high levels of SPO pasture in the spring.     

Effect of therapeutic administration of β-lactam antibiotics on the bacterial community and antibiotic resistance patterns in milk

Dairy cows with mastitis are frequently treated with antibiotics. The potential effect of antibiotics on the milk microbiome is still not clear. Therefore, the objective of this research was to investigate the effect of 2 commonly used cephalosporins on the milk microbiota of dairy cows and the antibiotic resistance genes in the milk. The milk samples were collected from 7 dairy cows at the period before medication (d 0), medication (d 1, 2, 3), withdrawal period (d 4, 6, 8), and the period after withdrawal (d 9, 11, 13, 15). We applied 16S rRNA sequencing to explore the microbiota changes, and antibiotic resistance patterns were investigated by quantitative PCR. The microbiota richness and diversity in each sample were calculated using the Chao 1 (richness), Shannon (diversity), and Simpson (diversity) indices. The cephalosporins treatment lowered the Simpson diversity value at the period of withdrawal. Members of the Enterobacter genera were the most affected bacteria associated with mastitis. Meanwhile, antibiotic resistance genes in the milk were also influenced by antibiotic treatment. The cephalosporins treatment raised the proportion of bla_TEM in milk samples at the period of withdrawal. Therefore, the treatment of cephalosporins led to change in the milk microbiota and increase of β-lactam resistance gene in the milk at the time of withdrawal period.     

Effect of storage conditions on the solubility and viscosity of β-glucan extracted from bread under in vitro conditions

The viscosity and solubility of β-glucan in muffins have been shown to be reduced by certain storage conditions, though the effect of storage on bread fortified with barley β-glucan concentrate has not been investigated. Therefore, this study investigated the effect of storage temperature and time (23 °C for 1, 4, and 7 d, 4 °C for 4, 7, and 14 d, and -20 °C for 1, 2, 4, and 8 wk) on the solubility and viscosity of β-glucan upon incorporation into bread at levels corresponding to 0 or 1.5 g β-glucan/serving, with or without vital gluten addition. The firmness and moisture content of bread following each storage treatment were also evaluated. The highest moisture and lowest firmness values were found in fresh bread, though these parameters were still maintained at appreciable levels upon room temperature storage of the 1.5 g β-glucan/serving bread with added gluten and at either room temperature or frozen storage for the 1.5 g β-glucan/serving bread for 4 d. If it is desirable to store bread for 7 d or more, frozen storage should be utilized in order to best maintain bread moisture and firmness levels. It is recommended that β-glucan-fortified bread be consumed fresh for greatest β-glucan solubility and viscosity, though β-glucan solubility of approximately 40% is still achievable upon frozen storage of the bread for up to 2 wk. It is still unclear, however, as to what extent of reductions in the solubility and viscosity of β-glucan would lower its physiological effectiveness. PRACTICAL APPLICATION: Previous research has demonstrated that solubility and thus viscosity of β-glucan, which is an important property associated with its health benefits can be impacted by different storage conditions applied to some bakery products, like muffins. This study demonstrates the extent of changes in the solubility and viscosity of β-glucan incorporated into bread. Therefore, storage time and temperature should be optimized to minimize changes in β-glucan for maintaining its efficacy for its health benefits.     

Effect of high-pressure processing on bacterial inactivation in açaí juices with varying pH and soluble solids content

This study evaluated microbial inactivation effects of high-pressure processing (HPP) applied to açaí juices formulated with varying pH and soluble solids content (SSC). Açaí juice with pH 4.3 and 2.9°Brix was initially inoculated with cocktails of 5 strains of E. coli O157:H7, Listeria monocytogenes or Salmonella spp. and processed at varying pressures (300, 400 and 600 MPa) and dwelling times (1 and 3 min) at 5 °C. The lethality at 400 MPa for 3 min was >6-log CFU/mL. Further inactivation was observed during storage under refrigeration in the populations of Salmonella spp. and L. monocytogenes. In order to study the influence of pH and SSC on inactivation of Salmonella spp. by HPP, pH of açaí juice samples was adjusted to a range between 4.0 and 5.5 and SSC was adjusted between 2.9 and 14.9°Brix. The ability of HPP to provide a 5-log reduction in the population of Salmonella spp. was reduced with increasing pH and SSC. Immediately after HPP the juices with pH 4.0 and 2.9°Brix presented >6-log reduction while the one with 8.9°Brix resulted in 5-log reduction. In one week of refrigerated storage (7 °C), the juices (pH 4.0–14.9°Brix and pH 4.5–2.9°Brix) also presented >6-log reduction in Salmonella spp. concentration. These results indicated that a less intense process (below commonly recommended commercial conditions - 600 MPa/3 min) could be applied for açaí juice thus assuring required safety, in addition to an extra microbial inactivation verified during refrigerated storage.Industrial relevanceOur results showed that viability of cells of pathogenic strains continues to fall after processing and that this effect can be reversed in higher pH and higher concentration of soluble solids. This observation can help the design of more efficient protocols for validation of HPP processing, leading to milder processing conditions that will allow reduction of maintenance and energy costs associated with HPP. In addition, our results help to clarify the criteria to be adopted in the microbiological validation of juices processed by high hydrostatic pressure.     

Effects of β-κ-casein (CSN2-CSN3) haplotypes and β-lactoglobulin (BLG) genotypes on milk production traits and detailed protein composition of individual milk of Simmental cows

The aim of this study was to investigate the effects of CSN2-CSN3 (β-κ-casein) haplotypes and BLG (β-lactoglobulin) genotypes on milk production traits, content of protein fractions, and detailed protein composition of individual milk of Simmental cows. Content of the major protein fractions was measured by reversed-phase HPLC in individual milk samples of 2,167 cows. Protein composition was measured as percentage of each casein (CN) fraction to total CN and as percentage of β-lactoglobulin (β-LG) to total whey protein. Genotypes at CSN2, CSN3, and BLG were ascertained by reversed-phase HPLC, and CSN2-CSN3 haplotype probabilities were estimated for each cow. Traits were analyzed by using a linear model including the fixed effects of herd-test-day, parity, days in milk, and somatic cell score class, linear regressions on haplotype probabilities, class of BLG genotype, and the random effect of the sire of the cow. Effects of haplotypes and BLG genotypes on yields were weak or trivial. Genotype BB at BLG and haplotypes carrying CSN2 B and CSN3 B were associated with increased CN content and CN number. Haplotypes including CSN3 B were associated with increased κ-CN content and percentage of κ-CN to total CN and with decreased percentages of α_S1- and γ-CN to total CN. Allele CSN2 B had the effect of increasing β-CN content and decreasing content of α_S1-CN. Haplotypes including allele CSN2 A¹ exhibited decreased β-, α_S2-, and γ-CN concentrations and increased α_S1- and κ-CN contents, whereas CSN2 I had positive effects on β-CN concentration and trivial effects on content of other protein fractions. Effects of haplotypes on CN composition were similar to those exerted on content of CN fractions. Allele BLG A was associated with increased β-LG concentration and percentage of β-LG to total whey protein and with decreased content of other milk proteins, namely β-CN and α_S1-CN. Estimated additive genetic variance for investigated traits ranged from 14 to 39% of total variance. Increasing the frequency of specific genotypes or haplotypes by selective breeding might be an effective way to change milk protein composition.     

Effect of water activity on sugar crystallization and β-carotene stability of freeze-dried mango powder

Storage water activity (a_w) affects the stability of freeze-dried food. The sugar crystallization and storage stability of β-carotene in freeze-dried mango powder was investigated following storage under various relative vapor pressures (11.3-80.9%). Sugar crystallization was revealed by the loss of sorbed water in the water sorption experiment. However, the sorption isotherms showed unclear divergence between the experimental and predicted values. X-ray powder diffraction and scanning electron microscopy were used to confirm the crystallization. The results showed that increased a_w resulted in higher sugar crystallization. The loss of β-carotene was monitored by high-performance liquid chromatography with a diode-array detector and fitted to a first-order reaction. The rate constant decreased as a_w increased up to 0.43, due to the collapse of the mango powder. An increase in the rate constant above this value of a_w coincided with pronounced sugar crystallization. Choosing the appropriate value of a_w for storage can prevent sugar crystallization and enhance β-carotene stability in freeze-dried fruit powder.     

Effects of metal ions on growth, β-oxidation system,and thioesterase activity of Lactococcus lactis

The effects of divalent metal ions (Ca²⁺, Mg²⁺, Fe²⁺, and Cu²⁺) on the growth, β-oxidation system, and thioesterase activity of Lactococcus lactis were investigated. Different metal ions significantly influenced the growth of L. lactis: Ca²⁺ and Fe²⁺ accelerated growth, whereas Cu²⁺ inhibited growth. Furthermore, Mg²⁺ inhibited growth of L. lactis at a low concentration but stimulated growth of L. lactis at a high concentration. The divalent metal ions had significant effects on activity of the 4 key enzymes of the β-oxidation system (acyl-CoA dehydrogenase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and thiolase) and thioesterase of L. lactis. The activity of acyl-CoA dehydrogenases increased markedly in the presence of Ca²⁺ and Mg²⁺, whereas it decreased with 1 mmol/L Fe²⁺ or 12 mmol/L Mg²⁺. All the metal ions could induce activity of enoyl-CoA hydratase. In addition, 12 mmol/L Mg²⁺ significantly stimulated activity of L-3-hydroxyacyl-CoA dehydrogenase, and all metal ions could induce activity of thiolase, although thiolase activity decreased significantly when 0.05 mmol/L Cu²⁺ was added into M17 broth. Inhibition of thioesterase activity by all 4 metal ions could be reversed by 2 mmol/L Ca²⁺. These results help us understand the effect of metal ions on the β-oxidation system and thioesterase activity of Lactococcus lactis.     

Impact of β-glucan on debittering,bioaccessibility and storage stability of skim milk fortified with shrimp oil nanoliposomes

Shrimp oil was encapsulated in nanoliposomes and fortified into skim milk. Shrimp oil nanoliposomes (SONL) were thermodynamically stable when added into skim milk at 10 mL 100 mL⁻¹. Mild bitterness in fortified skim milk caused by the SONL was masked by adding β-glucan at various levels (0.05–0.2 g 100 mL⁻¹). With the addition of SONL, fortified skim milk appeared more reddish in colour due to the presence of astaxanthin. Addition of β-glucan resulted in the increase in viscosity of the fortified milk by forming network of junction zones. During the storage of skim milk fortified with SONL and 0.1 g 100 mL⁻¹ β-glucan at 4 °C for 15 days, no major quality changes took place. Simulated in vitro digestion studies revealed that 45.41 g 100 g⁻¹ eicosapentaenoic acid (EPA) and 48.86 g 100 g⁻¹ docosahexaenoic acid (DHA) from shrimp oil were bioaccessible for absorption in the gut after digestion.     

Developmental changes in storage proteins and peptidyl prolyl cis–trans isomerase activity in grains of different wheat cultivars

In the present study, storage proteins from five different wheat cultivars were extracted, fractionated and evaluated for their accumulation at different stages of development. SDS–PAGE analysis revealed that the accumulation of high molecular weight glutenin subunits was cultivar and stage dependent. However, low molecular weight glutenin subunits’ accumulation was not altered significantly after 16 days post anthesis in any of the cultivars. The rheological parameters (storage- and loss-modulus) of dough and gluten showed close association with either gliadins or glutenins. Peptidyl prolyl cis–trans isomerase (PPIase) activity, measured at different stages of grains development, showed variability with both the developmental stage and cultivar, and appeared to be primarily due to cyclophilins. Principal component analysis revealed the association of PPIase activity with either gliadin or total proteins, suggesting their significant role in the deposition of storage proteins in wheat.     

Occurrence of β-casein fragments in cold-stored and curdled river buffalo (Bubalus bubalis L.) milk

The safeguard of river buffalo Mozzarella cheese, a Protected Designation of Origin dairy product, has prompted an analytical study to trace the milk and curd used as raw material in cheesemaking. This is to prevent the illegal use of milk or curd from different geographical areas outside of those indicated in the official production protocol. For this purpose, we studied primary proteolysis occurring in fresh and frozen milk and curd to identify a molecular marker that could indicate the raw material used. Whole casein from frozen river buffalo milk was separated using cation-exchange chromatography and sodium dodecyl sulfate-PAGE, and a protein component with an estimated molecular weight of 15.3 kDa was detected. This protein component was revealed in fresh river buffalo milk as a faint electrophoresis band, which drastically increased in intensity in refrigerated and frozen milk as well as in curd and was found to be associated with β-CN through hydrophobic interaction. By using matrix-assisted laser desorption/ionization-time of flight peptide mass mapping, this component was identified as the C-terminal fragment f(69-209) of β-CN (expected molecular weight of 15,748.8 Da). β-Casein f(69-209), originating from the early hydrolysis of Lys₆₈-Ser₆₉ by plasmin, has no counterpart in bovine milk. The increased rate of hydrolysis by plasmin toward the cleavage site Lys₆₈-Ser₆₉ has to be ascribed to the elevated proline content of the peptide 61-73. The favored production of β-CN f(69-209) has also drawn attention to the complementary proteose peptone β-CN f(1-68) that is presumed to play a physiological role in inducing milk secretion similar to that of β-CN f(1-29). The higher in vivo and in vitro production rate, compared with γ₁-CN formation, indicates that β-CN f(69-209) and its complementary fragment are candidate molecular markers to evaluate milk and curd freshness. We used indirect ELISA analysis based on the determination of remaining nonhydrolyzed β-CN to perform a quantitative evaluation of proteolysis.     

Immobilization of recombinant thermostable β-galactosidase from Bacillus stearothermophilus for lactose hydrolysis in milk

A recombinant thermostable β-galactosidase from Bacillus stearothermophilus was immobilized onto chitosan using Tris(hydroxymethyl)phosphine (THP) and glutaraldehyde, and a packed bed reactor was utilized to hydrolyze lactose in milk. The thermostability and enzyme activity of THP-immobilized β-galactosidase during storage was superior to that of free and glutaraldehyde-immobilized enzymes. The THP-immobilized β-galactosidase showed greater relative activity in the presence of Ca²⁺ than the free enzyme and was stable during the storage at 4°C for 6 wk, whereas the free enzyme lost 31% of the initial activity under the same storage conditions. More than 80% of lactose hydrolysis in milk was achieved after 2 h of operation in the reactor. Therefore, THP-immobilized recombinant thermostable β-galactosidase from Bacillus stearothermophilus has the potential for application in the production of lactose-hydrolyzed milk.     

Effect of γ-irradiation on antioxidant activity of black pepper (Piper nigrum L.)

Antioxidant activity and EPR investigations of irradiated ground black pepper (Piper nigrum L.) were evaluated. The black pepper was exposed to γ-irradiation at doses from 5 to 30 kGy. The effect of irradiation on antioxidant properties of black pepper extracts was investigated by radical-scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, by determination of reducing power and content of thiobarbituric acid-reactive substances. Some significant changes were observed in creation of thiobarbituric acid-reactive substances (TBARS). Difference between non-irradiated and irradiated samples at the legal European limit dose of 10 kGy reached, on average, 23% and, at the Food and Drug Administration (FDA) 30 kGy limit, 33%. Irradiation affected significantly the DPPH radical-scavenging activity and reducing power of ground black pepper extracts. The γ-radiation treatment of ground black pepper samples observed by EPR, resulted in the production of three paramagnetic species (GI–GIII) characterized by different origin, thermal behaviour and stability. The axially symmetric EPR resonances, GI and GII, were assigned to the carbohydrate radical structures. The spin Hamiltonian parameters of GIII possessed the characteristic features of “cellulosic” radical species. The EPR measurements, performed 20 weeks after the radiation process, confirmed that temperature increase from 298 to 353 K, caused significant decrease of integral EPR signal intensity for γ-irradiated samples (∼40%), compared to the reference (non-irradiated) ground black pepper, where only 13% drop was found. Significant correlation between EPR and thiobarbituric acid methods was assessed by study of antioxidant activity changes in relation to irradiation doses and also in the case of spice storage, between EPR and reducing power methods.     

Effect of milk protein composition and amount of β-casein on growth performance,gut hormones,and inflammatory cytokines in an in vivo piglet model

The objective of this work was to better understand the effect of differences in milk protein composition, and specifically, a change in β-casein to total casein in a milk-based matrix, on growth performance and metabolic and inflammatory responses using a piglet model. Three formulas were optimized for piglets, with similar metabolizable energy, total protein content, and other essential nutrients. Only the protein type and ratio varied between the treatments: the protein fraction of the control diet contained only whey proteins, whereas 2 other matrices contained a whey protein to casein ratio of 60:40, and differed in the amount of β-casein (12.5 and 17.1% of total protein). Piglets fed formula containing whey proteins and caseins, regardless of the concentration of β-casein, showed a significantly higher average daily gain, average daily feed intake, and feed efficiency compared with piglets consuming the formula with only whey protein. Consumption of the formula containing only whey protein showed higher levels of plasma glucagon-like peptide-1 and ghrelin compared with the consumption of formula containing casein and whey protein. A positive correlation was observed between postprandial time and glucagon-like peptide-1 response. The intestinal pro-inflammatory cytokine tumor necrosis factor α increased significantly in piglets fed the whey protein/casein diet compared with those fed whey protein formula. All formula-fed piglets showed a lower level of IL-6 cytokine compared with the ad libitum sow-fed piglets, regardless of composition. No significant differences in the anti-inflammatory IL-10 concentration were observed between treatment groups. Milk protein composition contributed to the regulation of piglets‘ metabolic and physiological responses, with whey protein/casein formula promoting growth performance and a different immune regulatory balance compared with a formula containing only whey protein. Results indicated no differences between treatments containing different levels of β-casein.