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.     

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.     

Assessment of the immunomodulatory activity of cheese extracts by a complete and easy to handle <Emphasis Type="Italic">in</Emphasis><Emphasis Type="Italic">vitro</Emphasis> screening methodology

A simple and rapid screening methodology based on the in vitro culture of murine hybridoma and human T-lymphocytes was developed to assess the potential immunomodulatory activity of water-soluble extracts (WSE) from cheese. The two immune cell lines were cultured in microplates with or without cheese WSE. The proliferation and the metabolic activity of cells were monitored at their different growth phases by the BrdU (5-bromo-2′-deoxyuridine) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide] assays, respectively. WSE from Abondance cheese enhanced DNA synthesis and the metabolic activity of the hybridoma cells (1.2-and 1.3-to 1.6-fold of the control, respectively) and also of the T lymphocytes (1.5-fold of the control) for almost all of the dilutions tested. To evaluate the validity of the results obtained following this screening methodology, hybridoma and T-lymphocyte cells were cultivated in 50 ml flasks: the maximal cell density was increased by about 10–16% in presence of cheese WSE for both cell lines and the antibody production by the hybridoma cells was increased by 50%.     

Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria

AIMS: To investigate the combined effect of high-pressure treatments (HPT) and milk inoculation with bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Staphylococcus aureus during ripening of raw milk cheese. METHODS AND RESULTS: Cheeses were manufactured from raw milk artificially contaminated with S. aureus at ca 5 log CFU ml(-1), a commercial starter culture and one of seven strains of BP-LAB, added as adjuncts at 0.1%. HPT of cheeses were performed on days 2 or 50 at 300 MPa (10 degrees C, 10 min) or 500 MPa (10 degrees C, 5 min). On day 3, S. aureus counts were 6.46 log CFU g(-1) in control cheese. Milk inoculation with different BP-LAB lowered S. aureus counts on day 3 when compared with control cheese by up to 0.46 log CFU g(-1), HPT at 300 MPa on day 2 by 0.45 log CFU g(-1) and HPT at 500 MPa on day 2 by 2.43 log CFU g(-1). Combinations of BP-LAB with HPT at 300 and 500 MPa on day 2 lowered S. aureus counts on day 3 by up to 1.02 and 4.00 log CFU g(-1) respectively. CONCLUSIONS: The combined effect of milk inoculation with some of the BP-LAB tested and HPT of cheese on S. aureus inactivation was synergistic. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of HPT at lower pressures with BP-LAB inoculation is a feasible system to improve cheese safety in case of deleterious effects on cheese quality caused by HPT at higher pressures.     

Assessment of the rind microbial diversity in a farmhouse-produced vs a pasteurized industrially produced soft red-smear cheese using both cultivation and rDNA-based methods

AIMS: The diversity of the surface flora of two French red-smear soft cheeses was examined by cultivation-dependent and cultivation-independent methods to assess their composition and to evaluate the accuracy of both approaches. METHODS AND RESULTS: Culture-independent methods used involved 16S ribosomal DNA gene cloning and sequencing and single-strand conformation polymorphism analysis (SSCP). The culture-dependent method used involved direct culture and macroscopic observation, polymerase chain reaction of the 16S rRNA gene from DNA extracted from single colonies followed by complete sequencing of the gene. Only few species were recovered by both approaches either in the pasteurized and the farmer cheese. A large diversity of isolates or 16S rDNA sequences related to marine bacteria was identified at the surface of both cheeses. CONCLUSIONS: The results indicated that all three techniques were informative and complementary to allow a more accurate representativeness of the cheese surface biodiversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Cultivation and molecular methods have to be combined in order to obtain an extended view of the bacterial populations of complex ecosystems.     

PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese

AIMS: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product. METHODS AND RESULTS: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) experiments. The analysis of the DGGE profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter addition, the profile of all the dairy samples was identical to the one shown by the NWC. Simple indexes were calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of specific groups of organisms. LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30 degrees C showed high viable counts and the highest diversity in species indicating their importance in the cheese making, which had not been considered so far. Moreover, the NWC profiles were shown to be the most similar to the curd profile suggesting to be effective in manufacture. CONCLUSIONS: The PCR-DGGE analysis showed that in premium quality manufacture the NWC used as starter had a strong influence on the microflora responsible for process development. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approach appeared to be valid as a tool to control process development, starter effectiveness and product identity as well as to rank cheese quality.     

Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized gut microbiome until the Baroque period

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