A Survey of the Peptide Profile in Prato Cheese as Measured by MALDI‐MS and Capillary Electrophoresis

In this study, we describe the characterization of the peptide profile in commercial Prato cheese by matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) and capillary electrophoresis (CE). Ten commercial Prato cheese brands were characterized via their physicochemical composition and subjected to fractionation according to solubility at pH 4.6. The pH 4.6 insoluble fraction was evaluated by CE, whereas MALDI‐MS was applied to the fraction soluble at pH 4.6 and in 70% ethanol. CE revealed a characteristic pattern of hydrolysis, with formation of para‐κ‐casein, hydrolysis of α_s1‐casein at the Phe₂₃ ‐ Phe₂₄ bond, and hydrolysis of β‐casein. For the MALDI‐MS data, a complex peptide profile was observed, with the identification of 44 peptides previously reported (24 peptides from α_s1‐casein, 14 from β‐casein, 3 from κ‐casein, and 3 from α_s2‐casein). It was also observed that cheeses with salt‐in‐moisture content greater than 5% showed an accumulation of a bitter‐tasting peptide (m/z 1536, α_s1‐CN f1‐13), suggesting a relationship between the higher salt concentration and the abundance of this peptide. In conclusion, the results showed that even commercial cheeses produced with different raw material and processing conditions showed very similar peptide profiles when assessed at the molecular level, and only 9 peptides were responsible for discrimination of cheeses.     

Characteristics of Miniature Cheddar‐Type Cheese Made by Microbial Rennet from Bacillus amyloliquefaciens: A Comparison with Commercial Calf Rennet

Miniature Cheddar‐type cheeses were produced using microbial rennet from Bacillus amyloliquefaciens (milk‐clotting enzyme [MCE]) or calf rennet (CAR). With the exception of pH, there were no significant differences in gross composition between MCE‐cheese (MCE‐C) and CAR‐cheese (CAR‐C). The pH value of CAR‐C was significantly higher than that of MCE‐C at 40 and 60 d of ripening. The total nitrogen content of the pH 4.6‐soluble fraction obtained from MCE‐C was higher than that obtained from CAR‐C. However, nitrogen content of the 12% TCA‐soluble fraction was similar between CAR‐C and MCE‐C. The extent of α_s1‐casein and β‐casein hydrolysis, measured by urea‐PAGE, was similar in both cheese samples. The hydrolysis of β‐casein was lower than that of α_s1‐casein. Different reverse phase‐high‐performance liquid chromatography peptide profiles of ethanol‐soluble and ethanol‐insoluble fractions were obtained from CAR‐C and MCE‐C. The peptide content in the 2 cheese samples increased throughout ripening; the ratio of hydrophobic to hydrophilic peptides was lower in MCE‐C than in CAR‐C. Compared with CAR‐C, MCE‐C was softer as a result of higher protein hydrolysis. Microbial rennet from B. amyloliquefaciens contributed to higher proteolytic rates, which reduced ripening time.     

High‐Throughput Isolation of Bacteriocin‐Producing Lactic Acid Bacteria,with Potential Application in the Brewing Industry

Lactic acid bacteria (LAB) were isolated from malted cereals by means of a high‐throughput screening approach and investigated for antimicrobial activity against a range of beer‐spoiling bacteria. Putative bacteriocin‐producing strains were identified by 16S rRNA analysis and the inhibitory compounds were partially characterized. Following determination of the inhibitory spectra of the strains, an unspeciated Lactobacillus sp. UCC128, with inhibitory activity against a range of beer‐spoiling strains was subjected to further characterization. A bacteriocin was purified from this strain and analyzed by mass spectrometry to determine the weight of the protein. The result indicated that the bacteriocin was highly similar to pediocin AcH/PA‐1 from Pediococcus acidilactici. The bacteriocin‐producers identified in this study have the potential to be used in the brewing industry to enhance the microbiological stability of beer in conjunction with hurdles already in place in the brewing process.     

Development of high‐performance liquid chromatography and non‐aqueous capillary electrophoresis methods for the determination of fenoxycarb residues in wheat samples

BACKGROUND: Practical methods for the analysis of fenoxycarb residues in wheat samples were developed using high‐performance liquid chromatography (HPLC) and non‐aqueous capillary electrophoresis (NACE). RESULTS: Fenoxycarb residues in wheat were extracted with acetone by ultrasonication, followed by a clean‐up procedure with liquid–liquid extraction with 5% NaCl/dichloromethane. The HPLC was developed using C18 as column, MeOH/water (6:4, v/v) as the mobile phase and 199 nm as the detection wavelength. The optimal NACE condition was established with the running buffer of 20.0 mmol L^?1 NH₄Ac in 95% MeOH (pH* 9.0), and the applied voltage of 30 kV over a capillary of 50 µm i.d. × 48.5 cm × 40 cm effective length. Both methods gave the relatively lower limits of detection (0.008 mg kg^?1 for HPLC and 0.024 mg kg^?1 for NACE) and the higher recoveries (>85.0%). They were successfully applied to the determination of fenoxycarb in wheat samples. CONCLUSION: The results showed that the fenoxycarb residue gradually reduced to trace amounts after about 3 years, which implied that the pharmacological actions of fenoxycarb could last for about 3 years. Meanwhile, more effort should be made to control and reduce fenoxycarb residues because of its potential health risks to consumers. Copyright © 2007 Society of Chemical Industry     

Salt Reduction in a Model High‐Salt Akawi Cheese: Effects on Bacterial Activity,pH, Moisture,Potential Bioactive Peptides,Amino Acids,and Growth of Human Colon Cells

This study evaluated the effects of sodium chloride reduction and its substitution with potassium chloride on Akawi cheese during storage for 30 d at 4 °C. Survival of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium longum) and starter bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), angiotensin‐converting enzyme‐inhibitory and antioxidant activities, and concentrations of standard amino acids as affected by storage in different brine solutions (10% NaCl, 7.5% NaCl, 7.5% NaCl+KCl [1:1], 5% NaCl, and 5% NaCl+KCl [1:1]) were investigated. Furthermore, viability of human colon cells and human colon cancer cells as affected by the extract showing improved peptide profiles, highest release of amino acids and antioxidant activity (that is, from cheese brined in 7.5% NaCl+KCl) was evaluated. Significant increase was observed in survival of probiotic bacteria in cheeses with low salt after 30 d. Calcium content decreased slightly during storage in all cheeses brined in various solutions. Further, no significant changes were observed in ACE‐inhibitory activity and antioxidant activity of cheeses during storage. Interestingly, concentrations of 4 essential amino acids (phenylalanine, tryptophan, valine, and leucine) increased significantly during storage in brine solutions containing 7.5% total salt. Low concentration of cheese extract (100 μg/mL) significantly improved the growth of normal human colon cells, and reduced the growth of human colon cancer cells. Overall, the study revealed that cheese extracts from reduced‐NaCl brine improved the growth of human colon cells, and the release of essential amino acids, but did not affect the activities of potential bioactive peptides.     

Application of polymerase chain reaction–restriction fragment length polymorphism analysis and lab‐on‐a‐chip capillary electrophoresis for the specific identification of game and domestic meats

BACKGROUND: The objective of this study was to adapt and improve previously published polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods aimed at the identification of game and domestic meats, by replacing the gel electrophoretic steps for DNA fragment analysis by a chip‐based capillary electrophoresis system. RESULTS: PCR amplification of a mitochondrial 12S rRNA gene fragment and subsequent digestion of the amplicons with either MseI or a combination of MboII, BslI, and ApoI endonucleases generated characteristic PCR‐RFLP profiles that allowed discrimination among ten relevant game and domestic meat species. The Agilent 2100 Bioanalyzer utilises capillary electrophoresis on a microchip device that is capable of rapidly sizing DNA fragments, offering a valuable recent development for the analysis of complex DNA banding patterns. CONCLUSION: Results obtained in this work indicated that banding resolution on the system was sensitive, with detection of some small DNA fragments that were not observed with the published conventional PCR‐RFLP gel‐based method. Therefore, the new faster and easy handling procedure provides an additional powerful tool that can be employed for meat speciation. Copyright © 2009 Society of Chemical Industry     

High‐pressure homogenization: a non‐thermal process applied for inactivation of spoilage microorganisms in beer

The inactivation of spoilage microorganisms in beer using high‐pressure homogenization (HPH) was studied with the aim of evaluating the possibility of changing the conventional pasteurization process using this particular process. The homogenization pressure required for the inactivation of lactic acid bacteria, acetic bacteria and yeasts was investigated. For the most resistant microorganisms, the pressure inactivation kinetics and the effects of multiple process passes, initial temperature of the beer and the CO₂ concentration were studied. The results indicated that Lactobacillus delbrueckii was the most resistant microorganism tested, requiring 250 MPa to reach a six decimal reduction. Additionally, results showed that L. delbrueckii inactivation followed a second‐order kinetic process. A multi‐pass process and the use of a high initial beer temperature increased inactivation by HPH with L. delbrueckii, allowing the use of 150 MPa to achieve a five log cycle of inactivation. In contrast, a high CO₂ concentration reduced the efficacy of the HPH process. The results that were obtained are useful for high‐pressure homogenization applications in breweries and help to elucidate the effect of this new technology in a beverage that is both alcoholic and carbonated. Copyright © 2013 The Institute of Brewing & Distilling     

Micellar electrokinetic capillary chromatography of methylxanthines‐containing beverages: discussion of the molecular species involved

Micellar electrokinetic capillary chromatography (MECC) experimental conditions were applied to 12 samples of methylxanthine‐containing infusions of different commercial brands of yerba mate, coffee, tea and cocoa as well as two cola drinks. The best resolution in this mode of automated high‐performance capillary electrophoresis (HPCE) was achieved here when using 15 kV voltage in an uncoated fused‐silica capillary of 45 cm length (40 cm effective length), 50 mM sodium dodecylsulfate, 90 mM pH 8.5 borate buffer and UV detection. Theobromine, caffeine and theophylline were separated, and the peak splitting due to tautomeric species was observed. Experimental conditions were controlled, keeping constant the size of the elution window in each analysis. The limit of detection was less than 1 mg l^?1, the limit of quantitation was 2.5 mg l^?1 and the work range was 2.5–300 mg l^?1. This HPCE–MECC system has proved suitable for the analysis/quality control of xanthines in beverages for consumption. Roles of various parameters as well as distinctly charged species of each xanthine and the origin of peak splitting in this MECC system are discussed. Copyright © 2004 Society of Chemical Industry     

Enzymatic extract from flowers of Algerian spontaneous Cynara cardunculus: Milk‐clotting properties and use in the manufacture of a Camembert‐type cheese

The aim of this study was to assess the coagulant properties of crude extract from cardoon flowers (CECF) as a replacement for the imported coagulants used in the local Algerian cheese factories. Optimal coagulation activity of the extract was observed at pH 5 and 60 °C with a CaCl₂ concentration of 0.02 M. The electrophoretic pattern revealed that the crude extract contains mainly cardosins A and B. The study of interactions involved in the formation of milk gel coagulated by CECF showed that hydrogen forces, hydrophobic interactions and calcium bridges contributed highly to the formation of milk gel. Camembert‐type cheese obtained by CECF was judged by trained panellists and was found to be in agreement with standard specifications. The present study highlights the possibility of using Algerian CECF as a promising plant coagulant in cheesemaking for the dairy industry.     

Understanding the role of natural cheese calcium and phosphorous content,residual lactose and salt‐in‐moisture content on block‐type processed cheese functional properties: Cheese hardness and flowability/meltability

Four treatments of natural Cheddar cheese with two levels (high and low) of calcium (Ca) and phosphorus (P), and two levels (high and low) of residual lactose were manufactured. Each treatment was subsequently split prior to the salting step of cheese manufacturing processed and salted at two levels (high and low) for a total of eight treatments. The eight treatments included: high Ca and P, high lactose, high salt‐in‐moisture (S/M) content (HHH); high Ca and P, high lactose, low S/M (HHL); high Ca and P, low lactose, high S/M (HLH); high Ca and P, low lactose, low S/M (HLL); low Ca and P, high lactose, high S/M (LHH); low Ca and P, high lactose, low S/M (LHL); low Ca and P, low lactose, high S/M (LLH); and low Ca and P, low lactose, low S/M (LLL). After 2 months of ripening, each treatment of natural Cheddar cheese was used to manufacture processed cheese using a twin‐screw Blentech processed cheese cooker. All of the processed cheese food formulations were balanced for moisture, fat and salt. Texture and melt‐flow characteristics of the processed cheese were evaluated with different techniques, including texture profile analysis (TPA) for hardness and melt profile analysis. There was a considerable increase in cheese hardness for the processed cheeses prepared from high Ca and P content, and high S/M natural cheeses compared with low Ca and P content and low S/M natural cheeses. Moreover, definite decrease in flow rate and extent of flow was observed for processed cheeses manufactured from high Ca and P content, and high S/M natural cheeses than that of low Ca and P content and low S/M natural cheeses. No considerable trend was observed in hardness and melt‐flow characteristics for the processed cheeses manufactured from high and low residual lactose content natural Cheddar cheeses. This study strongly demonstrates that the characteristics of natural cheese (calcium and phosphorus content, lactose content and salt‐in‐moisture content) used in processed cheese manufacture have a significant impact on processed cheese functionality.     

High‐fat Diet‐induced Intestinal Hyperpermeability is Associated with Increased Bile Acids in the Large Intestine of Mice

Metabolic syndrome is characterized by low‐grade chronic systemic inflammation, which is associated with intestinal hyperpermeability. This study examined the effects of 3 high‐fat diets (HFDs) composed of different fat sources (soybean oil and lard) on the intestinal permeability, tight junction (TJ) protein expression, and cecal bile acid (BA) concentrations in mice, and then analyzed their interrelations. C57/BL6 mice were fed the control diet, HFD (soybean oil), HFD (lard), and HFD (mix; containing equal concentrations of soybean oil and lard) for 8 wk. Glucose tolerance, intestinal permeability, TJ protein expression, and cecal BA concentration were evaluated. Feeding with the 3 HDFs similarly increased body weight, liver weight, and fat pad weight, and induced glucose intolerance and intestinal hyperpermeability. The expression of TJ proteins, zonula occludens‐2 and junctional adhesion molecule‐A, were lower in the colons of the 3 HFD groups than in the control group (P < 0.05), and these changes appeared to be related to intestinal hyperpermeability. Feeding with HFDs increased total secondary BA (SBA) and total BA concentrations along with increases in some individual BAs in the cecum. Significant positive correlations between intestinal permeability and the concentrations of most SBAs, such as deoxycholic acid and ω‐muricholic acids, were detected (P < 0.05). These results suggest that the HFD‐induced intestinal hyperpermeability is associated with increased BA secretion. The abundance of SBAs in the large intestine may be responsible for the hyperpermeability.     

High‐molecular‐weight glutenin subunit composition of Pakistani hard white spring wheats grown at three locations for 2 years and its relationship with end‐use quality characteristics

Eleven Pakistani hard white spring wheat cultivars, along with one durum wheat and two hard white American‐grown wheat cultivars, were evaluated for their high‐molecular‐weight (HMW) glutenin subunit composition via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). The relationships among different quality characteristics and between these characteristics and HMW glutenin subunits were computed. Three to six HMW glutenin subunits were observed in Pakistani bread wheat cultivars. The presence of HMW glutenin subunits was not affected by growth locations or crop years. However, variations in intensities were observed. Correlations were noticed between certain HMW glutenin subunits and some quality attributes, such as protein, farinograph dough development time, farinograph water absorption, loaf volume and mixograph peak height. The presence of HMW glutenin subunit 20 in the older wheat cultivars C591 and C273, known for excellent chapati quality, indicated a possible relationship between this band and chapati quality. This observation will need to be confirmed by testing a larger number of wheat samples known to have characteristics for both good and poor chapati quality. © 2000 Society of Chemical Industry     

Observation of Injured E. coli Population Resulting from the Application of High‐Pressure Throttling Treatments

This study used flow cytometry (FC), epifluorescent microscopy (EM), and conventional culture media (PC) to evaluate the potential for high‐pressure throttling (HPT) to produce injury in E. coli. E. coli cells suspended at a concentration of approximately 8 log (CFU/mL) in Butterfield's phosphate buffer and UHT skimmed milk, were treated with HPT at pressures ranging from 35 to 283 MPa. Cells were stained with SYTO 9 and propidium iodide (Live/Dead Baclight kit) to assess their membrane integrity. MacConkey and Tryptone Soy agars and a modification of the thin agar layer method were used to determine injured and non‐injured cells. PC results indicated a reduction in E. coli counts as pressure increased but no significant injured population was detected in either matrix. However, FC and EM observations indicated that the membrane integrity of a portion of the bacterial population was affected by HPT, producing different degrees of cell injury that could be sublethal. The percentage of this heterogeneous population increased with applied pressure. These results reassert the importance of understanding the potential of new processing treatments to produce sublethally‐injured bacteria, and developing appropriate detection techniques.     

Enzymatic hydrolysis combined with high‐pressure homogenisation for the preparation of polysaccharide‐based nanoparticles from the by‐product of Flammulina velutipes

In this study, protease, α‐amylase and 5‰ β‐glucanase enzymolysis in combination with high‐pressure homogenisation were used for the preparation of polysaccharide‐based nanoparticles from Flammulina velutipes stipes, respectively, named FNP‐1, FNP‐2 and FNP‐3, and the nanoparticles were subsequently characterised. The FNP size distribution ranged from 50 nm to 300 nm, among which FNP‐2 and FNP‐3 were smaller than FNP‐1, based on the SEM images. GC‐MS results showed that these particles were mainly composed of glucose and glucosamine. The FNP dispersions at 1 wt% behaved as non‐Newtonian, shear‐thinning fluids, and the FNP‐3 dispersion presented superior viscoelasticity. With an increasing degree of enzymolysis, the thermal stability of the FNPs decreased. In addition, these particles presented various cation‐exchange properties. Therefore, the Flammulina velutipes polysaccharide‐based nanoparticles obtained from this study can be potentially used as a promising functional food ingredient in the food industry.     

Production of a high‐protein meal and fermentable sugars from defatted soybean meal,a co‐product of the soybean oil industry

The goal of this research was to produce a high‐protein meal by treating defatted soybean meal, a by‐product of soybean oil production, with dilute acid. Treatments were a mild hydrolysis at 80 °C with sulphuric acid at concentrations ranging from 0.5% to 2.0% (w/v) and times varying from 1 to 16 h that were arranged according to a central composite rotatable experimental design. The end products were an enhanced‐protein meal and a carbohydrate concentrate of fermentable and nonfermentable sugars. The highest protein content rise, from 48% to 58%, was for treatments with concentrations of acid ranging between 1.2% and 1.7% and times between 1.0 and 2.6 h. The maximum yield of fermentable sugar was 21.0% d.b. at 2.0% H₂SO₄ and treatments of at least 6 h. The conditions that provide a highest protein and sugar contents were the treatments with concentrations of sulphuric acid ranging from 0.9 to 1.9% H₂SO₄ for 1–4 h.