The effects of microfluidization on the physical, microbial, chemical, and coagulation properties of milk.

This work examines the use of mild heat treatments in conjunction with 2-pass microfluidization to generate cheese milk for potential use in soft cheeses, such as Queso Fresco. Raw, thermized, and high temperature, short time pasteurized milk samples, standardized to the 3% (wt/wt) fat content used in cheesemaking, were processed at 4 inlet temperature and pressure conditions: 42°C/75 MPa, 42°C/125 MPa, 54°C/125 MPa, and 54°C/170 MPa. Processing-induced changes in the physical, chemical, and microbial properties resulting from the intense pressure, shear, and cavitation that milk experiences as it is microfluidized were compared with nonmicrofluidized controls. A pressure-dependent increase in exit temperature was observed for all microfluidized samples, with inactivation of alkaline phosphatase in raw and thermized samples at 125 and 170 MPa. Microfluidization of all samples under the 4 inlet temperature and processing pressure conditions resulted in a stable emulsion of fat droplets ranging from 0.390 to 0.501 µm, compared with 7.921 (control) and 4.127 (homogenized control) µm. Confocal imaging showed coalescence of scattered fat agglomerates 1 to 3 µm in size during the first 24 h. We found no changes in fat, lactose, ash content or pH, indicating the major components of milk remained unaffected by microfluidization. However, the apparent protein content was reduced from 3.1 to 2.2%, likely a result of near infrared spectroscopy improperly identifying the micellar fragments embedded into the fat droplets. Microbiology results indicated a decrease in mesophilic aerobic and psychrophilic milk microflora with increasing temperature and pressure, suggesting that microfluidization may eliminate bacteria. The viscosities of milk samples were similar but tended to be higher after treatment at 54°C and 125 or 170 MPa. These samples exhibited the longest coagulation times and the weakest gel firmness, indicating that formation of the casein matrix, a critical step in the production of cheese, was affected. Low temperature and pressure (42°C/75 MPa) exhibited similar coagulation properties to controls. The results suggest that microfluidization at lower pressures may be used to manufacture high-moisture cheese with altered texture whereas higher pressures may result in novel dairy ingredients.

Characterization of starter-free Queso Fresco made with sodium-potassium salt blends over 12 weeks of 4°C storage.

Development of reduced-sodium cheese to meet the demands of consumers concerned about sodium levels in their diet is challenging when a high-moisture, higher pH, fresh cheese, such as Queso Fresco (QF), depends on its NaCl salt content to obtain its signature flavor and quality traits. This study evaluated the effects of different Na-K salt blends on the compositional, sensorial, microbial, functional, and rheological properties of QF stored for up to 12 wk at 4°C. Queso Fresco curd from each vat was divided into 6 portions and salted with different blends of NaCl-KCl (Na-K, %): 0.75-0.75, 1.0-0.5, 1.0-1.0, 1.0-1.3, 1.0-1.5, and 2.0-0 (control). Within this narrow salt range (1.5 to 2.5% total salt), the moisture, protein, fat, and lactose levels; water activity; pH; and the textural and rheological properties were not affected by salt treatment or aging. The total salt, sodium, potassium, and ash contents reflected the different Na-K ratios added to the QF. Total aerobic microbial count, overall proteolysis, the release of casein phosphopeptides, and the level of volatile compounds were affected by aging but not by the salt treatment. Only the 1.0-1.3 and 1.0-1.5 Na-K cheeses had sensory saltiness scores similar to that of the 2.0-0 Na-K control QF. Loss of free serum from the cheese matrix increased steadily over the 12 wk, with higher losses found in QF containing 1.5% total salt compared with the higher Na-K blends. In conclusion, KCl substitution is a viable means for reduction of sodium in QF resulting in only minor differences in the quality traits, and levels of 1.0-1.3 and 1.0-1.5 Na-K are recommended to match the saltiness intensity of the 2.0-0 Na-K control. The findings from this study will aid cheese producers in creating reduced-sodium QF for health-conscious consumers.

Bacteriocin production by Streptococcus thermophilus in complex growth media.

OBJECTIVE: To test if the production of bacteriocins by Streptococcus thermophilus is influenced when grown in various complex media commonly used for the culturing of lactic acid bacteria. RESULTS: Forty-one strains of S. thermophilus were screened for the production of bacteriocins in tryptone/yeast extract/lactose (TYL), M17-lactose (M17L), M17-glucose (M17G) and MRS media. Two strains, ST144 and ST145, were identified as novel bacteriocin producers, with constitutive production observed only in M17G. Strains ST110, ST114 and ST134 constitutively produced bacteriocins in all growth media but ST114 required growth in MRS for its antimicrobial activity to persist in a 24 h culture. The addition of a synthetic quorum sensing peptide (BlpC) induced bacteriocin production by ST106 in all media tested; and by ST118 in TYL and M17L. Strain ST109, which constitutively produced a bacteriocin in TYL and M17 broths, required BlpC induction when grown in MRS. Real-time PCR analysis showed that the natural expression of blpC in ST109 was lower when grown in MRS, suggesting that something in medium interfered with the blp quorum sensing system. CONCLUSION: As the choice of growth medium influences both bacteriocin production and peptide stability, several types of production media should be tested when screening for novel bacteriocin-producing strains of S. thermophilus.

Effect of high-pressure processing on reduction of Listeria monocytogenes in packaged Queso Fresco.

The effect of high-hydrostatic-pressure processing (HPP) on the survival of a 5-strain rifampicin-resistant cocktail of Listeria monocytogenes in Queso Fresco (QF) was evaluated as a postpackaging intervention. Queso Fresco was made using pasteurized, homogenized milk, and was starter-free and not pressed. In phase 1, QF slices (12.7 × 7.6 × 1 cm), weighing from 52 to 66 g, were surface inoculated with L. monocytogenes (ca. 5.0 log10 cfu/g) and individually double vacuum packaged. The slices were then warmed to either 20 or 40°C and HPP treated at 200, 400, and 600 MPa for hold times of 5, 10, 15, or 20 min. Treatment at 600 MPa was most effective in reducing L. monocytogenes to below the detection level of 0.91 log10 cfu/g at all hold times and temperatures. High-hydrostatic-pressure processing at 40°C, 400 MPa, and hold time ≥ 15 min was effective but resulted in wheying-off and textural changes. In phase 2, L. monocytogenes was inoculated either on the slices (ca. 5.0 log10 cfu/g; ON) or in the curds (ca. 7.0 log10 cfu/g; IN) before the cheese block was formed and sliced. The slices were treated at 20°C and 600 MPa at hold times of 3, 10, and 20 min, and then stored at 4 and 10°C for 60 d. For both treatments, L. monocytogenes became less resistant to pressure as hold time increased, with greater percentages of injured cells at 3 and 10 min than at 20 min, at which the lethality of the process increased. For the IN treatment, with hold times of 3 and 10 min, growth of L. monocytogenes increased the first week of storage, but was delayed for 1 wk, with a hold time of 20 min. Longer lag times in growth of L. monocytogenes during storage at 4°C were observed for the ON treatment at hold times of 10 and 20 min, indicating that the IN treatment may have provided a more protective environment with less injury to the cells than the ON treatment. Similarly, HPP treatment for 10 min followed by storage at 4°C was the best method for suppressing the growth of the endogenous microflora with bacterial counts remaining below the level of detection for 2 out of the 3 QF samples for up to 84 d. Lag times in growth were not observed during storage of QF at 10°C. Although HPP reduced L. monocytogenes immediately after processing, a second preservation technique is necessary to control growth of L. monocytogenes during cold storage. However, the results also showed that HPP would be effective for slowing the growth of microorganisms that can shorten the shelf life of QF.

Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110.

γ-aminobutyric acid (GABA) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic, and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented dairy foods including cheeses and yogurt. The survey of 42 strains of the yogurt starter culture Streptococcus thermophilus by PCR techniques indicated the presence of a glutamate decarboxylase gene (gadB) in 16 strains. DNA sequencing data indicated that the GAD/GABA antiporter locus (gadB/gadC) in GAD(+) S. thermophilus strains is flanked by transposase elements (5' and 3') and positioned between the luxS (5') and the HD-superfamily hydrolase genes (3'). The PCR amplification product of a ca. 2-kb genomic fragment that included the gadB and its putative promoter region was inserted into a shuttle vector, which was used to transform Escherichia coli DH5α. Subsequently, the recombinant plasmid pMEU5a-1/gadB (7.24 kb) was electrotransformed into the GAD-negative strain S. thermophilus ST128. The ST128 transformants carrying the plasmid-encoded gadB produced functional GAD enzyme as evidenced by the conversion of glutamate to GABA at a rate similar to strains with the gadB/gadC operon located on the chromosome. The results demonstrated the potential to impart to non-GABA-producing strains of S. thermophilus and other lactic acid bacteria the GAD(+) phenotype that improves their appeal in possible applications in the development of health-promoting functional foods.

Short communication: characterization of microflora in Mexican Chihuahua cheese.

This work was performed to identify the bacterial species present in 10 Chihuahua cheeses obtained from commercial producers in Mexico using 16S rRNA gene analysis. As expected, some of the agar media initially used for isolation were not very selective, supporting the growth of several unrelated bacterial species. Sequence analysis identified potential pathogens, including Escherichia coli and Staphylococcus aureus, in all raw milk samples and 2 pasteurized milk samples. Streptococcus thermophilus and Lactococcus lactis ssp. lactis were identified in 9 and 6 samples, respectively, and would serve as acidifying agents during cheese production. Lactobacilli were identified in all cheeses, with the most prevalent being Lactobacillus plantarum identified in 7 raw milk and 1 pasteurized milk cheeses. Leuconostoc mesenteroides and Streptococcus macedonicus were identified in 4 raw milk cheeses and both were present in all pasteurized milk samples, suggesting that they may play a role in the development of traditional Chihuahua cheese attributes.

Production of antilisterial bacteriocins by staphylococci isolated from bovine milk.

A collection of 111 staphylococcal isolates recovered from healthy cows in 41 dairy herds in Brazil was surveyed for the production of bacteriocins. The group included 94 coagulase-positive and 17 coagulase-negative strains of staphylococci. All cultures were grown in tryptic soy broth for 18h at 37°C, and cell-free supernatants were tested for antimicrobial activity against several target organisms by using the agar diffusion method. Filtrates of 57 staphylococci showed strong activity against Listeria monocytogenes Scott A, and 52 isolates also inhibited the growth of Stapylococcus aureus Newbould 305, a major causative agent of bovine mastitis in the United States. The plasmid profiles of staphylococci invariably included an 8-kb plasmid. Staphylococcal isolates were tested for the production of aureocins A70 and A53, 2 bacteriocins of coagulase-positive staphylococci known to be associated with 8-kb and 10.2-kb plasmids, respectively. The presence of the A70 or A53 bacteriocin gene was checked by PCR techniques using primers based on nucleotide sequences flanking the structural gene of each bacteriocin. Agarose gel analysis of amplified PCR products of plasmid templates from all 58 isolates showed only a 525-bp fragment corresponding to the structural gene of the bacteriocin aureocin A70. The results indicated that the apparently widespread association of A70-producing staphylococci with healthy cows in Brazil may be beneficial in controlling undesirable bacteria in dairy herds.

Nisin-induced expression of pediocin in dairy lactic acid bacteria.

AIMS: To test whether a single vector, nisin-controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. METHODS AND RESULTS: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0.8-kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml(-1) nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. CONCLUSIONS: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.

Cloning of milk-derived bioactive peptides in Streptococcus thermophilus.

The enzymatic breakdown of milk proteins releases bioactive peptides. Two such peptides are the 11-residue antimicrobial peptide from bovine lactoferrin (BL-11) and the 12-residue hypotensive peptide from alpha(s1)-casein (C-12). These two peptides have now been cloned in Streptococcus thermophilus to develop strains that enhance the functionality and nutritional value of dairy food products. Nucleic acid sequences encoding the peptides were generated by overlapping PCR and were subsequently cloned into a new expression vector under control of the ST(2201) promoter. S. thermophilus transformants were successfully identified using GFP as a selectable marker. The presence of the synthetic gene constructs in S. thermophilus was confirmed by PCR.