Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 on the hydrolysis and angiotensin-I-converting enzyme inhibition activity of fermented ultra-high-temperature milk and nonfat dried milk powder.

Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation.

Logistic modeling to predict the minimum inhibitory concentration (MIC) of olive leaf extract (OLE) against Listeria monocytogenes.

Olive leaf extract (OLE) has been increasingly recognized as a natural and effective antimicrobial against a host of foodborne pathogens. This study attempts to predict the minimum inhibitory concentration (MIC) of OLE against Listeria monocytogenes F2365 by utilizing the asymptotic deceleration point (PDA) in a logistic model (LM), namely MIC-PDA. The experimental data obtained from the inhibitory rate (IR) versus OLE concentration against L. monocytogenes were sufficiently fitted (R2 = 0.88957). Five significant critical points were derived by taking the multi-order derivatives of the LM function: the inflection point (PI), the maximum acceleration point (PAM), the maximum deceleration point (PDM), the absolute acceleration point (PAA), and the asymptotic deceleration point (PDA). The PDA ([OLE] = 37.055 mg/mL) was employed to approximate the MIC-PDA. This MIC value was decreased by over 42% compared to the experimental MIC of 64.0 mg/mL, obtained using the conventional 2-fold dilution method (i.e., MIC-2fold). The accuracy of MIC-PDA was evaluated by an in vitro L. monocytogenes growth inhibition assay. Finally, the logistic modeling method was independently validated using our previously published inhibition data of OLE against the growths of Escherichia coli O157:H7 and Salmonella enteritidis. The MIC-PDA (for [OLE]) values were estimated to be 41.083 and 35.313 mg/mL, respectively, compared to the experimental value of 62.5 mg/mL. Taken together, MIC-PDA, as estimated from the logistic modeling, holds the potential to shorten the time and reduce cost when OLE is used as an antimicrobial in the food industry.

Characterization of an exopolysaccharide (EPS-3A) produced by Streptococcus thermophilus ZJUIDS-2-01 isolated from traditional yak yogurt.

Yak yogurt, one of the naturally fermented dairy products prepared by local herdsmen in the Qinghai-Tibet Plateau, contains a diverse array of microorganisms. We isolated and identified a novel Streptococcus thermophilus strain, ZJUIDS-2-01, from the traditional yak yogurt. We further purified and carried out detailed structural, physiochemical, and bioactivity studies of an exopolysaccharide (EPS-3A) produced by S. thermophilus ZJUIDS-2-01. The weight-average molecular weight (Mw) of EPS-3A was estimated to be 1.38 × 106 Da by High-Performance Gel Permeation Chromatography (HPGPC). The monosaccharide analysis established its composition to be glucose, galactose, N-acetyl-D-galactosamine, and rhamnose in a ratio of 5.2:2.5:6.4:1.0. The molecular structure of EPS-3A was determined by the combination of permethylation analysis, FT-IR, and NMR spectroscopic techniques. The ζ-potential measurements indicated that EPS-3A had a pKa value of ~4.40. The DSC yielded a melting point (Tm) of 80.4 °C and enthalpy change (ΔH) of 578 J/g for EPS-3A, comparable to those of the xanthan gum (XG), a commercial EPS. EPS-3A exhibited better O/W emulsion stability and flocculating capacity than XG. Furthermore, it also demonstrated similar antioxidant activity to XG and promising in vitro antibacterial properties. This work evidenced that EPS-3A derived from S. thermophilus ZJUIDS-2-01 holds the potential for food and industrial applications.

Chemical composition as an indicator for evaluating heated whey protein isolate (WPI) and sugar beet pectin (SBP) systems to stabilize O/W emulsions.

We investigated changes in the chemical composition of WPI as a result of heating (60 °C, 72 h) with SBP in solution (pH 6.75). The concentration of WPI was kept at a constant (3%), whereas the level of SBP was varied at 1, 1.5, and 3%. The reaction products were examined using the Ellman's reagent, ninhydrin assay, and gel electrophoresis. The results demonstrated that the losses of the free sulfhydryl (-SH) and primary amine (-NH2) contents in WPI were less severe compared to those occurring in the dry-state at similar conditions (mass ratio, temperature, and reaction duration). The mixtures were used as emulsifiers in an O/W emulsion system at pH 3.20 and 6.75 and showed an improved ability to stabilize the average size of the droplets than WPI alone at acidic pH. The mixtures at higher levels of SBP, ≥ 1.5%, however, adversely affected the emulsion stability at neutral pH.

Natural flagella-templated Au nanowires as a novel adjuvant against Listeria monocytogenes.

A simple method was developed for the extraction and purification of bacterial flagella with a yield of a concentration of 113.22 ± 5.64 mg mL-1. Gold (Au) nanowires were synthesized using the bacterial flagella as the template. Transmission Electron Microscopy (TEM) analysis showed that the nanowires were scarcely clustered as stiff (no tendency to bend or fold) and straight nanorods with homogeneous surface and a uniform aspect ratio over 60. Fourier Transform Infrared (FT-IR) spectroscopic studies revealed the deep involvement of the functional groups located within and on the surface of flagellin, including C-N, N-H, O-H, and C[double bond, length as m-dash]O. The profound transformation observed in the absorption profiles of these groups supported the notion that both chemical (reduction) reaction and physical (electrostatic) binding of Au occurred during the formation of Au nanowires. Verbascoside, oleuropein, and olive leaf extract (OLE) have been shown to inhibit the growth of Listeria monocytogenes completely at their respective Minimal Inhibitory Concentrations (MICs) of 20, 64, and 64 mg mL-1. In contrast, the synthesized Au nanowires demonstrated high electrocatalytic activity and reduced the MICs of the three antibacterial compounds by half. Moreover, results from the AMES assays indicated that the synthesized Au nanowires had no mutagenic activities at the catalytic concentration used, 128 µg mL-1. Therefore, the Au nanowires fabricated in this work have the potential to be used as new antimicrobial food packaging materials to enhance food safety.

Assessment of Antioxidant and Antimicrobial Properties of Lignin from Corn Stover Residue Pretreated with Low-Moisture Anhydrous Ammonia and Enzymatic Hydrolysis Process.

Lignin accounts for 15-35% of dry biomass materials. Therefore, developing value-added co-products from lignin residues is increasingly important to improve the economic viability of biofuel production from biomass resources. The main objective of this work was to study the lignin extracts from corn stover residue obtained from a new and improved process for bioethanol production. Extraction conditions that favored high lignin yield were optimized, and antioxidant and antimicrobial activities of the resulting lignin were investigated. Potential estrogenic toxicity of lignin extracts was also evaluated. The corn stover was pretreated by low-moisture anhydrous ammonia (LMAA) and then subjected to enzymatic hydrolysis using cellulase and hemicellulase. The residues were then added with sodium hydroxide and extracted for different temperatures and times for enhancing lignin yield and the bioactivities. The optimal extraction conditions using 4% (w/v) sodium hydroxide were determined to be 50 °C, 120 min, and 1:8 (w:v), the ratio between corn stover solids and extracting liquid. Under the optimal condition, 33.92 g of lignin yield per 100 g of corn stover residue was obtained. Furthermore, the extracts produced using these conditions showed the highest antioxidant activity by the hydrophilic oxygen radical absorbance capacity (ORAC) assay. The extracts also displayed significant antimicrobial activities against Listeria innocua. Minimal estrogenic impacts were observed for all lignin extracts when tested using the MCF-7 cell proliferation assay. Thus, the lignin extracts could be used for antioxidant and antimicrobial applications, and improve the value of the co-products from the biomass-based biorefinery.

Physical and chemical changes in whey protein concentrate stored at elevated temperature and humidity.

In a case study, we monitored the physical properties of 2 batches of whey protein concentrate (WPC) under adverse storage conditions to provide information on shelf life in hot, humid areas. Whey protein concentrates with 34.9 g of protein/100g (WPC34) and 76.8 g of protein/100g (WPC80) were stored for up to 18 mo under ambient conditions and at elevated temperature and relative humidity. The samples became yellower with storage; those stored at 35 °C were removed from the study by 12 mo because of their unsatisfactory appearance. Decreases in lysine and increases in water activity, volatile compound formation, and powder caking values were observed in many specimens. Levels of aerobic mesophilic bacteria, coliforms, yeast, and mold were <3.85 log10 cfu/g in all samples. Relative humidity was not a factor in most samples. When stored in sealed bags, these samples of WPC34 and WPC80 had a shelf life of 9 mo at 35 °C but at least 18 mo at lower temperatures, which should extend the market for these products.

Effect of homogenization and pasteurization on the structure and stability of whey protein in milk.

The effect of homogenization alone or in combination with high-temperature, short-time (HTST) pasteurization or UHT processing on the whey fraction of milk was investigated using highly sensitive spectroscopic techniques. In pilot plant trials, 1-L quantities of whole milk were homogenized in a 2-stage homogenizer at 35°C (6.9 MPa/10.3 MPa) and, along with skim milk, were subjected to HTST pasteurization (72°C for 15 s) or UHT processing (135°C for 2 s). Other whole milk samples were processed using homogenization followed by either HTST pasteurization or UHT processing. The processed skim and whole milk samples were centrifuged further to remove fat and then acidified to pH 4.6 to isolate the corresponding whey fractions, and centrifuged again. The whey fractions were then purified using dialysis and investigated using the circular dichroism, Fourier transform infrared, and Trp intrinsic fluorescence spectroscopic techniques. Results demonstrated that homogenization combined with UHT processing of milk caused not only changes in protein composition but also significant secondary structural loss, particularly in the amounts of apparent antiparallel ß-sheet and α-helix, as well as diminished tertiary structural contact. In both cases of homogenization alone and followed by HTST treatments, neither caused appreciable chemical changes, nor remarkable secondary structural reduction. But disruption was evident in the tertiary structural environment of the whey proteins due to homogenization of whole milk as shown by both the near-UV circular dichroism and Trp intrinsic fluorescence. In-depth structural stability analyses revealed that even though processing of milk imposed little impairment on the secondary structural stability, the tertiary structural stability of whey protein was altered significantly. The following order was derived based on these studies: raw whole>HTST, homogenized, homogenized and pasteurized>skimmed and pasteurized, and skimmed UHT>homogenized UHT. The methodology demonstrated in this study can be used to gain insight into the behavior of milk proteins when processed and provides a new empirical and comparative approach for analyzing and assessing the effect of processing schemes on the nutrition and quality of milk and dairy product without the need for extended separation and purification, which can be both time-consuming and disruptive to protein structures.

Structural and thermal stability of ß-lactoglobulin as a result of interacting with sugar beet pectin.

Changes in the structural and thermal stability of ß-lactoglobulin (ß-LG) induced by interacting with sugar beet pectin (SBP) have been studied by circular dichroism (CD), Fourier transform infrared, and steady-state as well as time-resolved fluorescence spectroscopic techniques. It has been demonstrated that SBP not only is capable of binding to native ß-LG but also causes a significant loss in antiparallel ß-sheet, ∼10%, accompanied by an increase in either random coil or turn structures. In addition, the interaction also disrupted the environments of all aromatic residues including Trp, Phe, and Tyr of ß-LG as evidenced by near-UV CD and fluorescence. When preheated ß-LG was combined with SBP, the secondary structure of ß-LG was partially recovered, ∼4% gain in antiparallel ß-sheet, and Trp19 fluorescence was recovered slightly. Although forming complexes with SBP did not significantly impact the thermal stability of individual secondary structural elements of ß-LG, the environment of Trp19 was protected considerably.

Investigation of molecular interactions between ß-lactoglobulin and sugar beet pectin by multi-detection HPSEC.

Molecular interactions between ß-lactoglobulin (ß-LG) and sugar beet pectin (SBP) were studied using online multi-detection high performance size exclusion chromatography (HPSEC) at neutral pH and 50mM ionic strength. The hydrodynamic properties of various interacting polymer fractions were characterized in detail and compared with those of ß-LG and SBP. Results showed that ∼6.5% (w/w) of native dimeric ß-LG molecules formed complexes with over 35% SBP molecules of varying sizes, 800, 110 and 75 kDa. Although the ß-LG molecules bind to SBP molecules of all sizes and shapes, they tend to favor the intermediate (110 kDa) and small sized (75 kDa) SBP molecules. All resulting complexes possess altered shapes and hydrodynamic properties when compared to unbound SBP and ß-LG. About half of the interacting ß-LG (∼3.5%) molecules were thought to bind to a small amount of non-covalently bound feruloyl groups, possibly present in SBP. When pre-heat treated ß-LG and SBP were combined, more than 16% of ß-LG formed complexes with at least 45% of SBP molecules of varying sizes, Mw∼750-800, 110, and 55-80 kDa. The complexes formed between ß-LG aggregates and/or oligomers and the large SBP molecules (750-800 kDa) adopt the shape of ß-LG aggregates, random coil. Both groups of complexes formed between ß-LG intermediate (110 kDa) and small sized (55-80 kDa) SBP take on the shape of rigid rod. It was speculated that half of the interacting heat-treated ß-LG molecules (∼8%) are complexed with non-covalently bound feruloyl groups in SBP.

Heavy metal and phenol adsorptive properties of biochars from pyrolyzed switchgrass and woody biomass in correlation with surface properties.

In this work, the surface structures of biochars, derived from three types of biomass, switchgrass (SG), hardwood (HW) and softwood (SW) through either fast pyrolysis (FP) in a fluidized-bed reactor (at 500 °C) or slow pyrolysis (at 500° and 700 °C), were studied in detail, and compared with that of the activated carbons obtained by steam activation of the slow pyrolyzed biochars (at 500 °C). The surface acidic functional groups were determined quantitatively by the Boehm Titration method. The adsorptive properties of heavy metals, Zn(2+) and Cu(2+) onto the biochars and the activated carbons were investigated by the adsorption isotherms and SEM images, and correlated with the surface properties. ATR-FTIR and GC techniques were used to analyze the adsorptive behavior of phenol onto the biochars and activated carbons, and the results demonstrated that phenol adsorption capability is directly proportional to the micropore surface area as well as the combined level of the accessible carboxylic and lactonic groups. The relative adsorption capacity with respect to the biomass precursor follows the order: SW > HW > SG.

Physico-chemical characterization of protein-associated polysaccharides extracted from sugar beet pulp.

We have solubilized and separated polysaccharides from sugar beet pulp (SBP) into three fractions with steam assisted flash extraction (SAFE). For pectin, recovery ranged from 8 to 14%, degree of methy-esterification 66-73%, crude protein 1.3-1.7%, M(w) 262-318 kDa, η(w) 0.22-0.23 dL/g, Rg(z) 36-39 nm and Rh(z) 41-42 nm. For alkaline soluble polysaccharides, (ASP I) recovery ranged from 4.0 to 6.5%, crude protein 1.2-4.8%, weight average molar mass (M(w)) 66-68 kDa, weight average intrinsic viscosity (η(w)) 0.27-0.30 dL/g, z-average radius of gyration (Rg(z)) 25-29 nm and z-average hydrated radius (Rh(z)) 10-11 nm. ASP II recovery ranged from 2.0 to 8.6%, crude protein 1.2-4.8%, M(w) 299-339 kDa, η(w) 0.22-0.33 dL/g, Rg(z) 33-34 nm and Rh(z) 30-34 nm. Recovery of the residue mainly cellulose, ranged from 20.3 to 22.3%. The cellulose in this fraction was converted to carboxymethyl cellulose (CMC). The CMC fraction contained 0.33-0.43 crude protein and had an M(w) ranging from 127 to 263 kDa, η(w) 3.6-8.0 dL/g, Rg(z) 35-45 nm and Rh(z) 27-40 nm.

Reactions between ß-lactoglobulin and genipin: kinetics and characterization of the products.

In this paper, we present the first detailed study of the reaction kinetics and the characterization of the products from the endothermic reactions between ß-lactoglobulin and genipin. The effects of the concentration, temperature, and pH were investigated. In the temperature range studied, the reaction was approximately a pseudo-first-order with respect to genipin and 0.22-order and -0.24-order with respect to ß-lactoglobulin for pH 6.75 and 10.5 with corresponding activation energy (E(a)) estimated to be 66.2 ± 3.8 and 9.40 ± 0.36 kJ/mol, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies, validated by matrix-assisted laser desorption ionization-time of flight mass spectrometry, showed the presence of oligomeric, i.e., di-, tri-, quadri-, and pentameric, forms of cross-linked ß-lactoglobulin by genipin at neutral but not alkaline pH; however, an extensive cross-linked network was not observed, consistent with the atomic force microscopy images. It was demonstrated that the reaction temperature and the concentration of genipin but not that of ß-lactoglobulin positively affected the extent of the cross-linking reactions.

Extrusion texturized dairy proteins: processing and application.

The primary proteins in milk, casein and the whey proteins α-lactalbumin and ß-lactoglobulin, have a number of health benefits and desirable functional properties. In a twin-screw extruder, mechanical shear forces, heat, and pressure cause considerable changes in the molecular structures of the dairy proteins, a process known as texturization. These changes further impart unique functional properties to dairy proteins, resulting in new protein-based food ingredients. The new functional behavior depends on the extent of texturization and the degree of structural change imparted and is controlled by adjusting parameters such as extrusion temperature and moisture level. Such texturized proteins can be used to produce puffed high-protein snacks. Softer gels and expanded structures can be made using supercritical fluid extrusion and cold extrusion, techniques that avoid elevated temperatures, minimizing possible damage to the nutritive components and functionality of the texturized dairy proteins. The uses of the texturized dairy ingredient in food products with improved functionality and enhanced nutritive profiles are presented.

Physical properties, molecular structures, and protein quality of texturized whey protein isolate: effect of extrusion temperature.

Although extrusion technology has contributed much to increasing the effective utilization of whey, the effect of extrusion conditions on the functional properties of the proteins is not well understood. In this work, the impact of extrusion temperature on the physical and chemical properties, molecular structures, and protein quality of texturized whey protein isolate (WPI) was investigated at a constant moisture content and compared with WPI treated with simple heat only. The Bradford assay methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high-performance liquid chromatography techniques were used to determine protein solubility and to analyze compositional changes in the two major whey proteins, α-lactalbumin and ß-lactoglobulin. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic techniques were applied to study the secondary and tertiary structures of the proteins. This study demonstrated that extrusion temperature is a critical but not the sole determining factor in affecting the functional properties of extruded WPI.

Implication of C-terminal deletion on the structure and stability of bovine beta-casein.

Bovine beta-casein (beta-CN) with its C-terminal truncated by chymosin digestion, beta-CN-(f1-192), was examined and characterized using circular dichroism (CD) under various temperature conditions. CONTIN/LL analysis of the CD data revealed significant secondary structure disruption in beta-CN-(f1-192) relative to its parent protein,beta-CN, in the temperature range (5 degrees to 70 degrees C) studied. Near-UV CD spectra indicated significant temperature dependent structural changes. Analytical ultracentrifugation results showed significant reduction but not complete abolishment of self-association in beta-CN-(f1-192) compared to whole beta-casein at 2 degrees -37 degrees C. Furthermore, binding experiments with the common hydrophobic probe - 8-anilino-1- naphthalene sulfonic acid (ANS) illustrated that beta-CN-(f1-192) is nearly incapable of binding to ANS relative to whole beta-CN, suggesting a nearly complete open overall tertiary structure brought about by the C-terminal truncation. It has been demonstrated clearly that the tail peptide beta-CN-(f193-209) is important in maintaining the hydrophobic core of beta-CN but the residual association observed argues for a minor role for other sites as well.

Thermal and alkaline denaturation of bovine beta-casein.

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.

Structural determinants of the rate of protein folding.

To understand the mechanism of protein folding and to assist rational design of fast-folding, non-aggregating and stable artificial enzymes, it is essential to determine the structural parameters which govern the rate constants of folding, kf. It has been found that -logkf is a linear function of the so-called chain topology parameter (CTP) within the range of 10(-1)s(-1)< or = kf < or =10(8)s(-1). The correlation between -logkf and CTP is much improved than using previously published contact order (CO) method. It has been further suggested that short sequence separations may be preferred for the establishment of stable interactions for the design of novel artificial enzymes and the modification of slow-folding proteins with aggregating intermediates.