Quantitative PCR reveals the frequency and distribution of 3 indigenous yeast species across a range of specialty cheeses.

Indigenous microorganisms are important components of the complex ecosystem of many dairy foods including cheeses, and they are potential contributors to the development of a specific cheese's sensory properties. Among these indigenous microorganisms are the yeasts Cyberlindnera jadinii, Pichia kudriavzevii, and Kazachstania servazzii, which were previously detected using traditional microbiological methods in both raw milk and some artisanal specialty cheeses produced in the province of Québec, Canada. However, their levels across different cheese varieties are unknown. A highly specific and sensitive real-time quantitative PCR assay was developed to quantitate these yeast species in a variety of specialty cheeses (bloomy-rind, washed-rind, and natural-rind cheeses from raw, thermized, and pasteurized milks). The specificity of the quantitative PCR assay was validated, and it showed no cross-amplification with 11 other fungal microorganisms usually found in bloomy-rind and washed-rind cheeses. Cyberlindnera jadinii and P. kudriavzevii were found in the majority of the cheeses analyzed (25 of 29 and 24 of 29 cheeses, respectively) in concentrations up to 104 to 108 gene copies/g in the cheese cores, which are considered oxygen-poor environments, and 101 to 104 gene copies/cm2 in the rind. However, their high abundance was not observed in the same samples. Whereas C. jadinii was present and dominant in all core and rind samples, P. kudriavzevii was mostly present in cheese cores. In contrast, K. servazzii was present in the rinds of only 2 cheeses, in concentrations ranging from 101 to 103 gene copies/cm2, and in 1 cheese core at 105 gene copies/g. Thus, in the ecosystems of specialty cheeses, indigenous yeasts are highly frequent but variable, with certain species selectively present in specific varieties. These results shed light on some indigenous yeasts that establish during the ripening of specialty cheeses.

Smoothing temperature and ratio of casein to whey protein: Two tools to improve nonfat stirred yogurt properties.

Consumers are not always ready to compromise on the loss of texture and increased syneresis that nonfat stirred yogurts display compared with yogurts that contain fat. In this study, we investigated milk protein composition and smoothing temperature as a means to control nonfat yogurt microstructure, textural properties, and syneresis. Yogurts were prepared with different ratios of casein to whey protein (R1.5, R2.8, and R3.9). Yogurts were pumped through a smoothing pilot system comprising a plate heat exchanger set at 15, 20, or 25°C and then stored at 4°C until analysis (d 1, 9, and 23). Yogurt particle size and firmness were measured. Yogurt syneresis and water mobility were determined, respectively, by centrifugation and time domain low-frequency proton nuclear magnetic resonance (1H-LF-NMR). Increasing the smoothing temperature increased gel firmness and microgel (dense protein aggregates) sizes independently of the whey protein content. Also, yogurt microgel sizes changed with storage time, but the evolution pattern depended on protein ratio. Yogurt R1.5 showed the largest particles, and their sizes increased with storage, whereas R2.8 and R3.9 had smaller microgels, and R3.9 did not show any increase in microgel size during storage. Micrographs showed a heterogeneous gel with the empty area occupied by serum for R1.5, whereas R2.8 and R3.9 showed fewer serum zones and a more disrupted gel embedding microgels. Induced syneresis reduced with greater whey protein content and time of storage. This is in agreement with 1H-LF-NMR showing less bulk water mobility with increasing whey protein content during storage. However, 1H-LF-RMN revealed higher values of spontaneous serum separation during storage for R1.5 and R3.9 yogurts, whereas these were lower and stable for R2.8 yogurt. Microgels play an important structural role in yogurt textural attributes, and their characteristics are modulated by whey protein content and smoothing temperature. Optimization of these parameters may help improve nonfat stirred dairy gel.

Studying stirred yogurt microstructure using optical microscopy: How smoothing temperature and storage time affect microgel size related to syneresis.

A grainy texture and high syneresis are 2 defects in low-fat stirred yogurt that are often disliked by consumers. In this study, a rheometer controlling the shear rate and temperature was used to simulate the smoothing step of yogurt manufacture. Identical formulations containing whey protein isolate or whey protein concentrate were compared. After the yogurt milk underwent heat treatment, inoculation, and fermentation at 42°C, the yogurt was smoothed at 42°C (Y42) or 20°C (Y20) or during a cooling ramp from 42°C to 20°C (YR). Induced syneresis (serum expelled by centrifugation) was measured on d 3. Sizes of microgels (dense protein aggregates) were investigated on d 0, 4, and 7 by laser diffraction and by image analysis using optical microscopy. Optical microscopy was also used to characterize the reorganized protein network embedding microgels. The type of whey protein ingredient had only a slight effect on the induced syneresis of YR and Y20 treated yogurts, and the major effect came from the smoothing temperature. The Y42 treatment presented the highest induced syneresis; YR and Y20 had similar low induced syneresis values. Images showed a heterogeneous microstructure (large microgels, reorganized gel) and serum separation for Y42; the YR and Y20 networks were homogeneous. Both the image analyses and laser diffraction showed that the microgel size depended on the smoothing temperature. However, only the image analyses made it possible to identify a time dependency effect on microgel size during storage. The number of microgels >104 µm2 continued to increase over time, whereas the number of microgels <103 µm2 decreased. Microscopic observations were less destructive than laser diffraction and highlighted the presence of microgel aggregation during storage.

Short communication: Effect of stirring operations on changes in physical and rheological properties of nonfat yogurts during storage.

The rheological and physical properties of stirred yogurt depend on several parameters, including the mechanical stress caused by stirring, smoothing, and cooling conditions (duration, intensity, or temperature). However, the literature reports little information about the effects of mechanical stress from all of the stirring operations on changes in yogurt properties during storage. The aim of this study was to determine, by means of a technical scale unit, the combined effects of stirring in the yogurt vat, smoothing, and cooling on changes in the rheological properties of nonfat yogurt during storage at 4°C. The yogurt was standardized to 14% total solids, 0% fat, and 4% protein, and was stirred with a technical scale unit using 2 stirring durations (5 min or 10 min), 2 types of cooling systems (plate or tubular heat exchanger), and 2 smoothing temperatures (38°C for yogurts smoothed before cooling or 20°C for yogurts smoothed after cooling). All yogurts were stored for 22 d at 4°C, and we determined the combined effect of the stirring operations on changes in syneresis, apparent viscosity, firmness, consistency, and flow time. During storage, post-acidification was the same for all stirred yogurts and involved restructuring of the protein network, which resulted in an increase in all properties except syneresis, which decreased. The combined stirring operations did not modify changes in syneresis during yogurt storage but did affect flow time, viscosity, consistency, and firmness. Changes in flow time depended on smoothing temperature, and viscosity and consistency depended on the cooling system used. Firmness was the property most affected by all combined stirring operations during storage. Therefore, the technical scale unit was effective for quantifying the combined effects of stirring, smoothing, and cooling on changes in yogurt properties during storage. This study also confirmed that the restructuring of stirred yogurt depended on the mechanical stress that occurred during the stirring process.

Individual and sequential effects of stirring, smoothing, and cooling on the rheological properties of nonfat yogurts stirred with a technical scale unit.

Few studies have considered the impact of unit operations during stirred yogurt manufacture because their operational sequence is difficult to replicate at the laboratory scale. The aim of this study was to investigate the individual and sequential effects of stirring in the yogurt vat, smoothing, and cooling on the rheological properties of yogurts, using a technical scale unit simulating some industrial conditions. The yogurts were prepared from a milk mixture that was standardized to contain 14% total solids, 0% fat, and 4% protein, and then homogenized, heated (94.5°C, 5 min), and inoculated at 41°C with the same thermophilic lactic starter. The operating parameters under investigation were 2 stirring durations in the yogurt vat (5 or 10 min), 2 cooling systems (plate or tubular heat exchanger), and 2 smoothing temperatures (38°C for smoothing before cooling; 20°C for smoothing after cooling). Sampling valves were installed at critical points on the technical scale unit so that the effect of each operation on the properties of stirred yogurt could be quantified individually. Syneresis, apparent viscosity, firmness, and consistency were analyzed after 1 d of storage at 4°C. In general, as the yogurts moved through the technical scale unit, the properties of the yogurts (evaluated after 1 d) changed: viscosity increased but syneresis, firmness, and consistency decreased. The individual effects of the operations showed that smoothing and cooling, compared with stirring duration, made the greatest contribution in terms of modifying yogurt properties. The stirring parameters (5 or 10 min) had similar effects on the yogurts. The use of a plate heat exchanger promoted a decrease in syneresis, whereas a tubular heat exchanger had a greater effect in terms of increasing firmness and consistency. The type of cooling system had no effect on stirred yogurt viscosity. Smoothing at 38°C had a greater effect on the increase in firmness, whereas smoothing at 20°C contributed more to a decrease in syneresis and increases in viscosity and consistency. This study confirms that each unit operation has a defined effect on the rheological properties of a nonfat stirred yogurt, which also depends on the operation sequence.

Characterization of aromatic properties of old-style cheese starters.

Old-style cheese starters were evaluated to determine their ability to produce cheese aroma compounds. Detailed analyses of the aroma-producing potential of 13 old-style starter cultures were undertaken. The proteolytic profile of the starters was established by an accelerated ripening study using a model cheese slurry and compared with those of a commercial aromatic starter and commercial Cheddar cheeses. To evaluate the aromatic potential of the starter cultures, quantification of free amino acids liberated and volatile compounds after 15 d of ripening at 30 degrees C as well as sensory analysis were carried out. Results showed that proteolysis patterns of all 13 starter cultures in the curd model were comparable to those of commercial Cheddar cheeses. All tested cultures demonstrated the ability to produce high amounts of amino acids recognized as precursors of aroma compounds. Several differences were observed between the starters and commercial Cheddar cheeses regarding some amino acids such as glutamate, leucine, phenylalanine, proline, and ornithine, reflecting the various enzymatic systems present in the starters. Starters Bt (control) and ULAAC-E exhibited various significant differences regarding their free amino acid profiles, as confirmed by sensory analysis. In addition, identification of volatile compounds confirmed the presence of several key molecules related to aroma, such as 3-methylbutanal and diacetyl. Besides the aroma-producing aspect, 2 starters (ULAAC-A and ULAAC-H) seem to possess an important ability to generate large amounts of gamma-aminobutyric acid, which contributed up to 15% of the total amino acids present in the model curd after 15 d ripening. gamma-Aminobutyric acid is an amine well-known for its antihypertensive and calming effects.

Fat-free yogurt made using a galactose-positive exopolysaccharide-producing recombinant strain of Streptococcus thermophilus.

To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS(+) Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS(+) strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS(-)) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.

Production of fresh Cheddar cheese curds with controlled postacidification and enhanced flavor.

Cheddar cheese in curd form is very popular in eastern Canada. It is retailed immediately after cheese manufacturing and can be maintained at room temperature for 24 h to provide better texture and mouthfeel. Subsequently, the cheese curds must be stored at 4 degrees C. The shelf life is generally 3 d. In this study, Cheddar cheese curds were produced by adding a high diacetyl flavor-producing strain (Lactococcus diacetylactis) to a thermophilic-based starter. The objective was to achieve both postacidification stability to increase the shelf life and enhanced flavor. The addition of L. diacetylactis increased processing time but did not affect cheese composition or the evolution of proteolysis and texture. During cheese manufacturing, streptococci became the dominant microflora in all cheeses, whereas populations of Lactococcus cremoris and L. diacetylactis decreased. During cheese storage, viable counts of L. diacetylactis and Streptococcus thermophilus increased but the counts of L. cremoris decreased. During cheese manufacturing and storage, the concentrations of lactic acid and diacetyl increased rapidly in cheeses produced with L. diacetylactis. Citric acid and galactose contents remained high in cheese made without L. diacetylactis. Sensory evaluation indicated that cheeses containing the L. diacetylactis strain were more flavorful and also had less sourness and could be stored at 4 degrees C for up to 7 d.

Short communication: extraction of beta-casein from goat milk.

Peptides derived from milk beta-casein have potential biological activities, such as antihypertensive and immunostimulating properties. These biological properties increase the demand for the production of specific bioactive peptides. beta-Casein can be isolated directly from renneted skim milk, based on the preferential solubilization of beta-casein at low temperature. This study was conducted to compare the recovery and purity of beta-casein extracted from goat and cow milks. Rennet casein was prepared from both milks, heat treated, and dispersed in demineralized water at various temperatures. beta-Casein recovery in the soluble phase increased with decreasing incubation temperature. Concentration of beta-casein was 43% higher in goat milk than in cow milk, which had a direct effect on beta-casein recovery. Furthermore, beta-casein was extracted more efficiently from goat rennet casein. As a result, the extraction yield of beta-casein was 53% higher in goat milk than in cow milk. The purity of beta-casein extracted from both milks reached approximately 90% after incubation at 0 degrees C.

Galactose metabolism and capsule formation in a recombinant strain of Streptococcus thermophilus with a galactose-fermenting phenotype.

The capsule-producing, galactose-negative Streptococcus thermophilus MR-1C strain was first transformed with a low-copy plasmid containing a functional galK gene from Streptococcus salivarius to generate a recombinant galactose-fermenting Strep. thermophilus strain named MR-AAC. Then, we compared the functional properties of Strep. thermophilus MR-AAC with those of the parent MR-1C strain when used as starter for fermented products and cheese. In lactose-supplemented laboratory medium, MR-AAC metabolized galactose, but only when the amount of lactose was less than 0.1% (wt/vol). After 7 h of fermentation, the medium was almost depleted of galactose. The parent strain, MR-1C, showed the same pattern, except that the concentration of galactose decreased by only 25% during the same period. It was found that, during milk fermentation and Mozzarella cheese production, the galactose-fermenting phenotype was not expressed by MR-AAC and this strain expelled galactose into the medium at a level similar to the parent MR-1C strain. In milk and in lactose-supplemented medium, capsular exopolysaccharide production occurred mainly during the late exponential phase and the stationary growth phase with similar kinetics between MR-1C and MR-AAC.

Detection and quantification of capsular exopolysaccharides from Streptococcus thermophilus using lectin probes.

The aim of this work was to use fluorescently labeled lectins to develop a convenient and reliable method to determine the relative abundance of capsular polysaccharides (CPS) at the surface of Streptococcus thermophilus MR-1C cells. Fluorescein isothiocyanate-labeled peanut agglutinin isolated from Arachis hypogaea was found to interact specifically with the CPS of Strep. thermophilus MR-1C. This labeled lectin was then used as an effective probe to detect and quantify CPS. A fluorescence-based lectin-binding assay was successfully applied to follow the accumulation of CPS during the growth of Strep. thermophilus MR-1C in milk and in M17 broth supplemented with lactose. Our results showed that in both media, CPS production by Strep. thermophilus MR-1C began during the exponential phase of growth and continued for several hours after the culture reached the stationary growth phase.

Mechanical and structural properties of milk protein edible films cross-linked by heating and gamma-irradiation.

The mechanical properties of cross-linked edible films based on calcium caseinate and two type of whey proteins (commercial and isolate) were investigated. Cross-linking of the proteins was carried out using thermal and radiative treatments. Size-exclusion chromatography performed on the cross-linked proteins showed that gamma-irradiation increased the molecular weight of calcium caseinate, while it changed little for the whey proteins. However, heating of the whey protein solution induced cross-linking. For both cross-linked proteins, the molecular weight distribution was >/=2 x 10(3) kDa. Combined thermal and radiative treatments were applied to protein formulations with various ratios of calcium caseinate and whey proteins. Whey protein isolate could replace up to 50% of calcium caseinate without decreasing the puncture strength of the films. Films based on commercial whey protein and calcium caseinate were weaker than those containing whey protein isolate. Electron microscopy showed that the mechanical characteristics of these films are closely related to their microstructures.

Physicochemical properties and bacterial resistance of biodegradable milk protein films containing agar and pectin.

Free-standing sterilized edible films based on milk proteins, namely calcium caseinate and whey protein isolate, and polysaccharides, namely pectin and agar, were developed. Cross-linking of the proteins was achieved by the combination of thermal and radiative treatments. Autoclaving pectin and agar prior to their addition to the protein solutions generated films with an improved (P < or = 0.05) puncture strength. The presence of proteins and pectin-agar in the film formulation enhanced (P < or = 0.05) the moisture barrier of the films by 18%. A strain of Streptococcus thermophilus was used to assess the biodegradability behavior of the cross-linked films. Microbiological counts and soluble nitrogen analysis confirmed the biodegradability property of the milk protein films containing autoclaved pectin and agar.

Growth and activities of Lactococcus lactis in milk enriched with low mineral retentate powders.

The growth and activities of three strains of Lactococcus lactis ssp. cremoris (Wg2, E8, and HP) and their proteinase-negative variants were studied in skim milk enriched with three types of retentate powder. The performance of these strains in enriched milks was compared with that determined in reconstituted skim milk. Proteinase-positive strains of L. lactis ssp. cremoris exhibited higher maximum specific growth rates than protease-negative variants. Moreover, maximum specific growth rates of lactococci were lower in skim milk than in enriched milk with a high buffering capacity. The performance of proteinase-positive strains was better than that of proteinase-negative variants. Growth of proteinase-positive lactococci in milk media increased alpha-amino groups as determined by the increase of equivalent glutamic acid concentration. Available alpha-amino groups decreased with proteinase-negative variants. Proteinase-positive strain Wg2 exhibited the most proteolytic activity but showed the least specific overall productivity of lactic acid despite high biomass concentration in milk. Among proteinase-positive lactococci, strain E8 produced more lactic acid than other strains, and, among proteinase-negative variants, strain HP had the best specific overall productivity of lactic acid.