Remote engineering for a cheese whey biorefinery: an Internet-based application for process design,economic analysis,monitoring, and control of multiple plant sites

The proteolysis of cheese whey with the aid of immobilized enzymes is an attractive alternative for this by-product of the dairy industry. Among some possible applications for whey protein hydrolysates, one may cite their use as protein source for individuals with reduced capacity of digestion, or with genetic metabolic disorders (phenylketonuria patients, for instance). The multipurpose plant that processes whey is named here as a cheese whey biorefinery. This work presents the remote control and monitoring of the whey biorefineries using the Internet. In an integrated environment, the web application also enables simulation and economic analyses of the process. This technology might allow small companies to access a remote “engineering centre”, with know-how on plant design and advanced control techniques. The idea can also be extended to large dairy companies, providing the remote control of geographically spread sites of production.     

Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese

The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.     

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.     

Conversion of Lactococcus lactis cell envelope proteinase specificity by partial allele exchange

AIMS: To determine whether conversion of lactocepin substrate binding regions by gene replacement can alter lactocepin specificity in Lactococcus lactis starter bacteria without affecting other important strain properties. METHODS AND RESULTS: We utilized two-step gene replacement to convert substrate-binding determinants in the L. lactis prtP genes encoding group h (bitter) lactocepin in two industrial strains into the corresponding group b (nonbitter) variant. Analysis of lactocepin activity toward alpha(s1)-casein (f 1-23) by reversed-phase high-pressure liquid chromatography demonstrated enzyme specificity among isogenic derivatives had been altered in a manner that was consistent with predicted amino acid substitutions in substrate binding regions. Milk acidification properties of some mutants were not statistically different (P > 0.05) from wild-type parent strains, and strain propensity for autolysis was also not significantly (P > 0.05) changed. CONCLUSIONS: Conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of L. lactis without significantly affecting other important strain properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Methodology outlined in this study can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect, and prtP derivatives developed by this approach should be suitable for commercial application.     

Comparison of phenotypic and genotypic taxonomic methods for the identification of dairy enterococci

The purpose of the study was to assess the phenotypic and genotypic taxonomic congruence in order to allow species allocation of dairy enterococci. A total of 364 enterococci isolated from ewes'milk and cheese from four Portuguese Registered Designation of Origin areas and 25 type and reference strains of Enterococcus spp. were characterized by a polyphasic taxonomical approach involving 40 physiological and biochemical tests, whole-cell protein profiles, amplification of 16S-23S intergenic spacer regions (ITS-PCR) and subsequent restriction analysis (ARDRA). Ribotyping was also performed with reference strains and a subset of 146 isolates. Numerical hierarchic data analysis showed that single-technique identification levels increase from the physiological and biochemical tests to the protein approach, being lower with ITS/ARDRA and ribotyping. Cross-analysis confirmed a higher unmatching level in all pairwise combinations involving physiological and biochemical data. Whole-cell protein profiles followed by ITS/ARDRA identified 89% of the enterococci. Reliable identification of enterococci from milk and cheese could be obtained by analysis of whole-cell protein profiles. ITS-PCR can be used to confirm E. durans and E. faecium and ARDRA further confirms E. faecalis. Results revealed E. faecalis, E. durans, E. hirae and E. faecium as the prevalent species, although species prevalence showed some degree of variation among the areas.     

Purification and biochemical properties of a galactooligosaccharide producing β-galactosidase from Bullera singularis

A beta-galactosidase, catalyzing lactose hydrolysis and galactooligosaccharide (GalOS) synthesis from lactose, was extracted from the yeast, Bullera singularis KCTC 7534. The crude enzyme had a high transgalactosylation activity resulting in the oligosaccharide conversion of over 34% using pure lactose and cheese whey permeate as substrates. The enzyme was purified by two chromatographic steps giving 96-fold purification with a yield of 16%. The molecular weight of the purified enzyme (specific activity of 56 U mg(-1)) was approx. 53 000 Da. The hydrolytic activity was the highest at pH 5 and 50 degrees C, and was stable to 45 degrees C for 2 h. Enzyme activity was inhibited by 10 mM Ag3+ and 10 mM SDS. The Km for lactose hydrolysis was 0.58 M and the maximum reaction velocity (V(max)) was 4 mM min(-1). GalOS, including tri- and tetra-saccharides were produced with a conversion yield of 50%, corresponding to 90 g GalOS l(-1) from 180 g lactose l(-1) by the purified enzyme.     

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese

AIMS: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese. METHODS AND RESULTS: The Mozzarella cheese was made from unpasteurized milk which was inoculated to contain ca 10(5) cfu ml(-1)E. coli O157:H7. Two different heating temperatures (70 and 80 degrees C), commonly used during curd stretching, were investigated to determine their effects on the viability of E. coli O157:H7 in Mozzarella cheese. Stretching at 80 degrees C for 5 min resulted in the loss of culturability of E. coli O157:H7 strains, whereas stretching at 70 degrees C reduced the number of culturable E. coli O157:H7 by a factor of 10. CONCLUSIONS: The results show that stretching curd at 80 degrees C for 5 min is effective in controlling E. coli O157:H7 during the production of Mozzarella cheese. Brining and storage at 4 degrees C for 12 h was less effective than the stretching. Significance and Impact of the Study: Mozzarella cheese should be free of E. coli O157:H7 only if temperatures higher than or equal to 80 degrees C are used during milk processing.     

A lacticin 481-producing adjunct culture increases starter lysis while inhibiting nonstarter lactic acid bacteria proliferation during Cheddar cheese ripening

AIMS: The main aim of this study was to exploit a lacticin 481 producing strain, Lactococcus lactis CNRZ481, as an adjunct for Cheddar cheese manufacture, to increase starter cell lysis and control nonstarter lactic acid bacteria (NSLAB) proliferation in cheese. METHODS AND RESULTS: Lactococcus lactis CNRZ481 was exploited as an adjunct to L. lactis HP for the manufacture of Cheddar cheese at pilot scale (450 l). In these trials, inclusion of the adjunct strain did not compromise acid production by L. lactis HP and cheese was successfully manufactured within 5 h. Experimental cheese exhibited levels of lactate dehydrogenase (LDH) up to five-fold higher than control cheese and a significant reduction in NSLAB growth was also observed throughout the ripening period. CONCLUSIONS: The aims of the study were accomplished as (i) greater enzyme release was achieved through lacticin 481-induced lysis which was associated with an improved flavoured cheese as assessed by a commercial grader and (ii) NSLAB growth was controlled, thus reducing the risk of off-flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of lacticin 481-producing adjuncts for cheese manufacture may prove beneficial for manufacturers who aim to achieve faster ripening through premature and elevated intracellular enzyme release while minimizing inconsistencies in cheese quality because of NSLAB activity.     

Detection of Listeria monocytogenes in Italian-style soft cheeses

AIMS: A rapid detection system specific for Listeria monocytogenes and based on the polymerase chain reaction (PCR) was developed. METHODS AND RESULTS: Primers annealing to the coding region of the actA gene, critically involved in virulence and capable of discrimination between two different alleles naturally occurring in L. monocytogenes, have been utilized. The procedure was applied to recover L. monocytogenes cells in artificially contaminated fresh Italian soft cheeses (mozzarella, crescenza and ricotta). Low levels of L. monocytogenes were detected in mozzarella and crescenza homogenates (0.04-0.4 and 4 CFU g(-1), respectively) whereas in ricotta the detection limit was higher (40 CFU g(-1)). CONCLUSIONS: This PCR-based assay is highly specific as primers used recognize the DNA from different L. monocytogenes strains of clinical and food origin, while no amplification products result with any other Listeria spp. strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted a low-cost and rapid procedure that can be appropriated for the detection in real time of low L. monocytogenes levels in soft cheese.     

Peptides from several italian cheeses inhibitory to proteolytic enzymes of lactic acid bacteria, pseudomonas fluorescens ATCC 948 and to the angiotensin I-converting enzyme

Water-soluble peptides from Mozzarella, Italico, Crescenza, and Gorgonzola cheeses were fractionated by reverse-phase fast protein liquid chromatography. Peptide fractions with inhibitory activity to amino- and endo-peptidases from Lactobacillus delbrueckii ssp. bulgaricus B397, Streptococcus thermophilus 305, and Lactococcus lactis ssp. cremoris Wg2 were found. Enzymes from Lactobacillus casei ssp. casei 2752 were less sensitive. Endopeptidase from Lactobacillus casei ssp. casei 2752 also had a different response to the effect of some inhibitors. It probably showed limited differences in catalysis and substrate positioning. Most of these inhibitory peptides were also effective in reducing the activity of the Pseudomonas fluorescens ATCC 948 endopeptidase and the angiotensin I-converting enzyme. Inhibitory peptide fractions from Mozzarella, Italico, and Crescenza cheeses had a certain degree of hydrophobicity while the peptide fraction from Gorgonzola cheese eluted in the initial part of the acetonitrile gradient. One of the inhibitory peptides contained in the water-soluble extract of Crescenza cheese was further purified and sequenced. It corresponded to the β-casein fragment 58-72.