Production of β-galactosidase for lactose hydrolysis in milk and dairy products using thermophilic lactic acid bacteria

The aim of this work was to establish optimal conditions for the maximum production of β-galactosidase using an industrially suitable medium. Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was cultivated in skim milk, whey and whey permeate basal media, supplemented with whey protein products, yeast extract or De Man–Rogosa–Sharpe (MRS) broth, at pH 5.6 and 43°C. All supplementations of the whey and whey permeate basal media resulted in the enhancement of the specific growth rates, rate of lactic acid production and β-galactosidase activity. However, unsupplemented skim milk gave the greatest rate of lactic acid production (3.50±0.269 mg lactic acid ml⁻¹ media h⁻¹) and the highest β-galactosidase activity (5.491±0.116 U activity ml⁻¹ media); far superior to the best whey-based medium supplemented with MRS (2.71±0.176 mg lactic acid ml⁻¹ media h⁻¹ and 3.091±0.089 U activity ml⁻¹ media, respectively). A technologically feasible approach for the reprocessing of the spent skim milk was tested and a conceptual process scheme is proposed.     

Enhanced β-galactosidase production by supplementing whey with cauliflower waste

Among the five Kluyveromyces marxianus strains tested for β-galactosidase production, K. marxianus NCIM 3465 showed maximum enzyme activity of 1.62 IU mg⁻¹ dry weight. Different levels (5–25%, w/v) of dried cauliflower waste were incorporated into whey to evaluate the effect of its supplementation on enzyme production. Although a marginal increase in enzyme production was seen by incorporating 5% and 10% cauliflower waste in whey, nearly 15% increase in β-galactosidase production was observed when cauliflower waste level was increased to 20% compared with whey alone. Supplementing whey with 20% cauliflower waste also decreased the production time. Lactose concentration in whey, mainly responsible for increasing the biological oxygen demand load of the effluent water, decreased from 4.2% to nearly 0% at 24 h. Thus, this study demonstrated that both these by-products/residues could be effectively used for β-galactosidase production at commercial scale.     

Proteins recovered from acid whey/skim milk mixtures heated at alkaline pH values

Skim milk and mixtures prepared by combining acid whey with skim milk at volume ratios of 2:1, 1:1, 1:2, 1:3 and 1:4 were adjusted to pH 7.5 and heated at 90°C × 15 min. Protein was isolated from these heated samples by precipitation at pH 4.6 and it was found that 65% of the whey protein was recovered in each case. Non-recovered proteins included the proteose peptones and small quantities of β-lactoglobulin, α-lactalbumin and bovine serum albumin. The solubility of these isolates, which contained from 10–25% whey protein, decreased to > 95% when the whey protein exceeded ˜16%. Further characterization of the isolate, prepared from the 1:1 volume ratio of acid whey and skim milk, showed that ˜50% of the whey protein was insoluble, bound to casein and non-functional while the other ˜50% was complexed with casein and was soluble. The addition of a reducing agent suggests that sulphydryl bonding alone is not responsible for complex formation.     

Comparison of Rat Hepatic Cholesterol Biosynthesis During Skim Milk Versus Whey Permeate Ingestion

Whey permeate is an ultrafiltrate of whey that is devoid of protein but contains lactose, salts, and other soluble low molecular weight compounds. These experiments compared cholesterol concentrations of blood plasma, hepatic lipids, and hepatic cholesterol biosynthesis of rats ingesting skim milk powder versus whey permeate powder. Groups of young male rats weighing 90 to 92 g were fed a casein-based diet into which skim milk powder or whey permeate powder was incorporated isocalorically. No effects of skim milk or whey permeate on plasma cholesterol concentrations were observed at any time during 5-wk of feeding. However, 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was increased by either skim milk or whey permeate feeding. Hepatic cholesterol, triglyceride, and phospholipid concentrations at wk 5 were unchanged. Plasma and hepatic cholesterol responses of rats to whey permeate ingestion are similar to those that occur with skim milk consumption, and plasma and hepatic cholesterol concentrations do not reflect necessarily an increase in hepatic cholesterol biosynthesis.     

Impact of the acidification process, hydrocolloids and protein fortifiers on the physical and sensory properties of frozen yogurt

In our study, two acidification procedures, three stabilizers (guar gum, xanthan and carboxymethylcellulose) and two protein-fortifying agents (skim milk powder and whey powder) were evaluated based on their impact on the quality characteristics of frozen yogurts. Indirect acidification (blending of plain acidified milk with ice cream mix) was found to favour texture while direct acidification (fermentation of ice cream mix with starter culture), although it improved viscosity of mixes, did not enhance the sensory acceptance of frozen yogurts. The addition of 0.2% xanthan gum and the partial substitution (at the ratio of 3 : 1) of skim milk powder by whey powder increased overall acceptance and creaminess.     

High-pressure treatment of milk in industrial and pilot-scale equipments: effect of the treatment conditions on the protein distribution in different milk fractions

The effects of two different high-pressure (HP) equipments, operating at industrial- and pilot scales, and of the HP-release rate on the contents of non-sedimentable proteins and denatured whey proteins were investigated after treatments of skim milk—from 250 to 650 MPa. Non-sedimentable caseins and denatured whey proteins significantly increased with the pressure level. The industrial-scale equipment produced lower micellar disintegration than the pilot-scale equipment with similar degrees of whey protein denaturation. Ultracentrifugation supernatants obtained from skim milk at 100,000×g and 20 °C for 1 h were also HP-treated for comparative purposes, showing that, in skim milk, the presence of casein promoted the denaturation of whey proteins, although the extent of whey protein denaturation did not influence the release of casein to the soluble phase. Furthermore, most denatured whey proteins remained soluble after treatment in both equipments. In the pilot-scale equipment, the pressure-release rate influenced casein solubilization and whey protein denaturation.     

Gelation of Mixed Gels Containing κ-Carrageenan and Skim Milk Components

ABSTRACT: The gelation characteristics of mixed gels containing κ-carrageenan and skim milk or milk fractions (skim milk permeate or retentate) obtained by ultrafiltration were examined. Increasing the skim milk solids content of mixtures containing carrageenan increased setting temperatures and gel strength. The milk proteins contributed to gel strength but did not influence the setting temperature of mixtures. The binding of denatured whey proteins to casein micelles affected gel network formation of milk-carrageenan mixtures containing 10% milk solids. Network formation in mixed gels containing carrageenan and milk or milk fractions was initiated by the carrageenan component and dictated primarily by the ionic content of the mixtures.     

Influence of milk pre-heating conditions on casein–whey protein interactions and skim milk concentrate viscosity

The viscosity of concentrates (50–55% total solids) prepared from skim milk heated (5 min at 80 or 90 °C) at pH 6.5 and 6.7 was examined. The extent of heat-induced whey protein denaturation increased with increasing temperature and pH. More denatured whey protein and κ-casein were found in the serum phase of milk heated at higher pH. The viscosity of milk concentrates increased considerably with increasing pH at concentration and increasing heating temperature, whereas the distribution of denatured whey proteins and κ-casein between the serum and micellar phase only marginally influenced concentrate viscosity. Skim milk concentrate viscosity thus appears to be governed primarily by volume fraction and interactions of particles, which are governed primarily by concentration factor, the extent of whey protein denaturation and pH. Control and optimization of these factors can facilitate control over skim milk concentrate viscosity and energy efficiency in spray-drying.     

Effects of beta-glucan addition to a probiotic containing yogurt

ABSTRACT:  This study investigated the effects of addition of β-glucan from 2 different cereal sources (oat and barley) on growth and metabolic activity of Bifidobacterium animalis ssp. lactis (Bb-12™) as determined by plating on a selective medium in yogurt during prolonged cold storage. These yogurt batches were compared to unsupplemented and inulin supplemented controls. All batches were also assessed for syneresis. Oat β-glucan addition resulted in improved probiotic viability and stability comparable to that of inulin. It also enhanced lactic and propionic acid production. The barley β-glucan addition suppressed proteolytic activity more than that from oat. These improvements were hindered by greater syneresis caused likely by thermodynamic incompatibility. Small amplitude oscillatory measurements of acidified model mixture of β-glucan/skim milk solids showed formation of casein gel within the β-glucan network. Binary mixtures of β-glucan and skim milk solids had apparent pseudoplastic and non-Newtonian behavior governed mainly by β-glucan contribution. Above critical concentrations, the mixtures underwent phase separation with the lower phase rich in protein. The phase diagram also showed that the addition of β-glucan may be possible at or below 0.24 w/w%.     

Pilot-scale ceramic membrane filtration of skim milk for the production of a protein base ingredient for use in infant milk formula

The protein composition of bovine skim milk was modified using pilot scale membrane filtration to produce a whey protein-dominant ingredient with a casein profile closer to human milk. Bovine skim milk was processed at low (8.9 °C) or high (50 °C) temperature using ceramic microfiltration (MF) membranes (0.1 μm mean pore diameter). The resulting permeate stream was concentrated using polyethersulfone ultrafiltration (UF) membranes (10 kDa cut-off). The protein profile of MF and UF retentate streams were determined using reversed phase-high performance liquid chromatography and polyacrylamide gel electrophoresis. Permeate from the cold MF process (8.9 °C) had a casein:whey protein ratio of ∼35:65 with no α_S- or κ-casein present, compared with a casein:whey protein ratio of ∼10:90 at 50 °C. This study has demonstrated the application of cold membrane filtration (8.9 °C) at pilot scale to produce a dairy ingredient with a protein profile closer to that of human milk.     

In vitro immunogenicity of various native and thermally processed bovine milk proteins and their mixtures

In vitro immunogenicity of various native and thermally processed (72°C/15 s and 100°C/30 s) bovine milk protein fractions, their mixtures, whey, and skim milk, was studied by analyzing the immune response of T helper (Th) cells in human peripheral blood mononuclear cells. The secretion of Th type cytokines induced by the protein stimulants was quantified while determining the heat-induced protein denaturation. Purified whey proteins, caseins and whey fraction, and skim milk provoked substantial immune responses at various degrees, indicating their potent immunogenicity. The protein mixtures prepared using the fractionated whey proteins with or without caseins appeared less immunogenic in both native and heat-treated forms, implying their potential of producing less immunogenic dairy products. The 100°C/30 s treatment significantly altered the immunogenicity of most of the potent protein stimulants, which mostly coincided with their levels of protein denaturation. The 72°C/15 s treatment caused the least protein denaturation but altered the immunogenicity of several protein stimulants notably, including heat-stable caseins and α-lactalbumin.     

Drying and storage of crude β-galactosidase extracts from Lactobacillus delbrueckii ssp. bulgaricus 11842

The retention of β-galactosidase activity in crude cellular extract (CCE) preparations from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was investigated after spray drying at three different outlet air temperatures (40, 50 or 60 °C), freeze drying, and after 30 days storage. Lactose, skim milk and whey protein preparations in concentrations ranging from 5 to 30% (w/w) were used as drying adjuncts. To further investigate the protective role of sugars in the enzyme activity preservation, cellobiose and sucrose were also employed in 5 and 10% concentrations during spray-drying at 60 °C or freeze-drying. The addition of lactose or skim milk in all examined concentrations resulted in significantly (P<0.05) higher β-galactosidase activity retention in comparison to all other CCE spray dried at 60°C. The effect was less pronounced at lower spray drying temperatures and increased whey protein concentrations, especially during freeze drying, when almost complete recovery of the enzyme activity upon reconstitution was achieved. Cellobiose provided less β-gal protection in comparison to lactose or sucrose. Lactose was more effective than sucrose at 5% concentration, but both sugars were equal at 10%. The β-gal activity retention in dry CCE preparations during storage at 7 °C over 30 day period was related to the initial water activity; higher initial a_w of powders obtained at lower spray drying temperature was correlated with significant (P<0.05) β-gal activity loss. Freeze dried and spray dried (60 °C) preparations were more stable in comparison to all other samples, retaining high β-gal activity during storage up to 30 days.     

Use of acid whey protein concentrate as an ingredient in nonfat cup set-style yogurt

Acid whey resulting from the production of soft cheeses is a disposal problem for the dairy industry. Few uses have been found for acid whey because of its high ash content, low pH, and high organic acid content. The objective of this study was to explore the potential of recovery of whey protein from cottage cheese acid whey for use in yogurt. Cottage cheese acid whey and Cheddar cheese whey were produced from standard cottage cheese and Cheddar cheese-making procedures, respectively. The whey was separated and pasteurized by high temperature, short time pasteurization and stored at 4°C. Food-grade ammonium hydroxide was used to neutralize the acid whey to a pH of 6.4. The whey was heated to 50°C and concentrated using ultrafiltration and diafiltration with 11 polyethersulfone cartridge membrane filters (10,000-kDa cutoff) to 25% total solids and 80% protein. Skim milk was concentrated to 6% total protein. Nonfat, unflavored set-style yogurts (6.0 ± 0.1% protein, 15 ± 1.0% solids) were made from skim milk with added acid whey protein concentrate, skim milk with added sweet whey protein concentrate, or skim milk concentrate. Yogurt mixes were standardized to lactose and fat of 6.50% and 0.10%, respectively. Yogurt was fermented at 43°C to pH 4.6 and stored at 4°C. The experiment was replicated in triplicate. Titratable acidity, pH, whey separation, color, and gel strength were measured weekly in yogurts through 8 wk. Trained panel profiling was conducted on 0, 14, 28, and 56 d. Fat-free yogurts produced with added neutralized fresh liquid acid whey protein concentrate had flavor attributes similar those with added fresh liquid sweet whey protein but had lower gel strength attributes, which translated to differences in trained panel texture attributes and lower consumer liking scores for fat-free yogurt made with added acid whey protein ingredient. Difference in pH was the main contributor to texture differences, as higher pH in acid whey protein yogurts changed gel structure formation and water-holding capacity of the yogurt gel. In a second part of the study, the yogurt mix was reformulated to address texture differences. The reformulated yogurt mix at 2% milkfat and using a lower level of sweet and acid whey ingredient performed at parity with control yogurts in consumer sensory trials. Fresh liquid acid whey protein concentrates from cottage cheese manufacture can be used as a liquid protein ingredient source for manufacture of yogurt in the same factory.     

Hydrolysis of lactose by a thermostable β-galactosidase immobilised on DEAE-cellulose

A method is described for the attachment of whole cells of Bacillus stearothermophilus to an ion-exchange support such as DEAE-cellulose by covalent linkage. This system enables full retention of the β-galactosidase activity of the cells. The enzyme activity immobilised in this way is extremely thermostable with a half-life of approximately 15 days at 60°C and pH 7. The pH optimum appears to be about 6.4 and the immobilised system hydrolyses lactose in dairy products such as whey and skim milk.     

Compositional and functional properties of buttermilk: a comparison between sweet, sour, and whey buttermilk

Buttermilk is a dairy ingredient widely used in the food industry because of its emulsifying capacity and its positive impact on flavor. Commercial buttermilk is sweet buttermilk, a by-product from churning sweet cream into butter. However, other sources of buttermilk exist, including cultured and whey buttermilk obtained from churning of cultured cream and whey cream, respectively. The compositional and functional properties (protein solubility, viscosity, emulsifying and foaming properties) of sweet, sour, and whey buttermilk were determined at different pH levels and compared with those of skim milk and whey. Composition of sweet and cultured buttermilk was similar to skim milk, and composition of whey buttermilk was similar to whey, with the exception of fat content, which was higher in buttermilk than in skim milk or whey (6 to 20% vs. 0.3 to 0.4%). Functional properties of whey buttermilk were independent of pH, whereas sweet and cultured buttermilk exhibited lower protein solubility and emulsifying properties as well as a higher viscosity at low pH (pH ≤ 5). Sweet, sour, and whey buttermilks showed higher emulsifying properties and lower foaming capacity than milk and whey because of the presence of milk fat globule membrane components. Furthermore, among the various buttermilks, whey buttermilk was the one showing the highest emulsifying properties and the lowest foaming capacity. This could be due to a higher ratio of phospholipids to protein in whey buttermilk compared with cultured or sweet buttermilk. Whey buttermilk appears to be a promising and unique ingredient in the formulation of low pH foods.     

Effect of heating on the distribution of transforming growth factor-��2 in bovine milk

The objective of this study was to characterize the impact of heat treatments on the distribution of transforming growth factor-beta (TGF-??2) between cream and skim milk and between the casein and whey fractions of skim milk. Skimming removed 45% and 62% of the TGF-??2 from raw and pasteurized milks and only 8% of the total TGF-??2 in skimmed pasteurized milk was found in whey, compared to 37% in whey from raw skimmed milk. The TGF-??2 content of whey decreased as the heat treatment of the milk increased in intensity (thermization > pasteurization > UHT sterilization). Using milk held for 1 or 2 min at temperatures ranging from 57 to 84 °C, it was shown that TGF-??2 in the whey portion decreases at temperatures above 66 °C and becomes undetectable at temperatures higher than 76 °C. Altogether, these data on the heat-induced changes in TGF-??2 content of cream, skim milk, casein and whey reveal a potentially negative impact of certain heat treatments in developing TGF-??2-enriched fractions from milk.     

Probiotic fermented beverages based on acid whey

Acid whey is a byproduct of cheesemaking that is difficult to use because of its low pH and less-favorable processing properties compared with rennet whey. The aim of this study was to evaluate the qualities of fermented beverages made using acid whey. In manufacturing the beverages, we used probiotic cultures Lactobacillus acidophilus LA-5 or Bifidobacterium animalis ssp. lactis BB-12. The production process included combining pasteurized acid whey with UHT milk, unsweetened condensed milk, or skim milk powder. We introduced milk to enrich casein content and obtain a product with characteristics similar to that of fermented milk drinks. The products were stored under refrigerated conditions (5 ± 1°C) for 21 d. During storage, we assessed the beverages' physicochemical properties and organoleptic characteristics. The properties of the beverages depended on their composition, microbial culture, and storage time. Beverages containing L. acidophilus had higher acidity, which increased during storage; the acidity of samples containing B. animalis was more stable. Beverages made with skim milk powder (La1 and Bb1) had higher acetaldehyde content, but this parameter decreased in all samples during storage. The hardness of the samples did not change during storage and was highest in beverage La3, made from whey, condensed milk, and L. acidophilus. Beverage La2, made from whey, milk, condensed milk, and L. acidophilus, had the best sensory properties. The whey beverages we developed provided a good medium for the probiotic bacteria; bacteria count throughout the storage period exceeded 8 log cfu/mL, distinctly higher than the minimum therapeutic dose.     

Characterization of a new isolate of Lactobacillus fermentum IFO 3956 from Egyptian Ras cheese with proteolytic activity

More than 200 isolates were obtained from 15 Egyptian traditional dairy products (Domiatti cheese, Ras cheese and Rayeb milk) collected from local markets of Alexandria, Tanta and Kafr El-Sheikh. Examination with optical microscope of these dairy samples allowed to classify 92 bacilli, 64 of which were identified as lactobacilli. The proteolytic activity of lactobacilli isolates was tested on skim milk agar. Eight isolates showing a high proteolytic activity were further tested on UHT skim milk. The strain showing the highest proteolytic activity was purified and identified as Lactobacillus fermentum IFO 3656. The specific proteolytic activity of this strain and the factors affecting it (pH, temperature and presence of inhibitors) were studied. The proteolysis targeted mainly caseins (73% of whole casein), especially β-casein (85%). Smaller portions of whey proteins were proteolyzed (20%) essentially β-lactoglobulin. The proteolysis process gave rise to medium-sized peptide populations. The optimum conditions for the proteolysis activity of the studied strains were pH 6.5 and 37 °C. Proteolytic activities were very slightly affected by the increase of the temperature to 42 °C or the pH to 8.2. The protease system of Lactobacillus fermentum IFO 3956 is most probably composed from a high amount of metalloproteases and small amount of cysteine and serine proteases.     

Manufacture of acid gels from skim milk using high-pressure homogenization

The effect of high-pressure homogenization (HPH) alone or in combination with a thermal treatment (TT) was investigated for the manufacture of acid gels from skim milk. Raw skim milk was subjected to HPH (0 to 350 MPa) or a TT (90°C, 5 min), or both, in the following processing combinations: 1) HPH, 2) HPH followed by TT, 3) TT followed by HPH, 4) TT, and 5) raw milk (control). After treatments, L* (lightness) values were measured, and then skim milk was acidified with 3% glucono-δ-lactone and rheological properties (G′ and gelation time), and whey holding capacity was evaluated. Treatments in which HPH and TT were combined showed greater L* values than those in which just HPH was applied. In all treatments, the L* values decreased as the pressure was increased up to 300 MPa with little change afterward. Gelation times were lower when HPH was combined with TT compared with the acid skim milk gels that were just pressure treated. The final G′ in gels obtained from skim milk subjected to the combined process (HPH and TT) was greater and pressure-dependent compared with all other gels. A maximum G′ (∼320 Pa) was observed with skim milk subjected to a combination of thermal processing before or after HPH at 350 MPa. Acid gels obtained from HPH milk at 350 MPa showed a linear decrease in whey holding capacity over time, retaining 20% more whey after centrifugation for 25 min compared with samples treated at lower pressures and all other treatments. Our results suggest that HPH in combination with TT can be used to improve the rheological properties and stability of yogurt, thus decreasing the need for additives.     

Small and large deformation rheology and microstructure of κ-carrageenan gels containing commercial milk protein products

The rheological behaviour of commercial milk protein/κ-carrageenan mixtures in aqueous solutions was studied at neutral pH. Four milk protein ingredients; skim milk powder, milk protein concentrate, sodium caseinate, and whey protein isolate were considered. As seen by confocal laser microscopy, mixtures of κ-carrageenan with skim milk powder, milk protein concentrate, and sodium caseinate showed phase separation, but no phase separation was observed in mixtures containing whey protein isolate. For κ-carrageenan concentrations up to 0.5 wt%, the viscosity of the mixtures at low shear rates increased markedly in the case of skim milk powder and milk protein concentrate addition, but did not change by the addition of sodium caseinate or whey protein isolate. For κ-carrageenan concentrations from 1 to 2.5 wt%, small and large deformation rheological measurements, performed on the milk protein/κ-carrageenan gels, showed that skim milk powder, milk protein concentrate or sodium caseinate markedly improved the strength of the resulting gels, but whey protein isolate had no effect on the gel stength.