Changes in microbiological parameters, pH and titratable acidity during the refrigerated and room temperature storage of dahi prepared from the milk of Vechur cows: an indigenous cattle breed of Kerala.

Vechur cow is an indigenous cattle breed of Kerala listed as a critical breed by FAO. This research communication is related to the hypothesis that the changes occurring in microbiological quality parameters of Vechur cow milk dahi (VCMD) during storage will be superior to other milk and reflective of the traditional concepts of therapeutic properties attributed to Vechur milk. Microbiological quality of the VCMD stored at room (30 ± 1°C) and refrigerated (4 ± 1°C) temperatures in terms of total viable, coliform, yeast and mold and lactococcal counts is reported in this study, together with titratable acidity and pH. Results are compared with cross-bred cow milk dahi (CCMD) as control. On refrigerated storage, despite the comparable initial microbiological quality, VCMD exhibited significantly lower total viable, lactic acid bacteria, yeast and mold counts than CCMD, from the fifth day onwards for the first two parameters and the tenth day onwards for the last parameter. VCMD exhibited significantly higher pH values than CCMD from the fifth day onwards whereas the titratable acidity was significantly lower from the tenth day onwards. Though this study does not delineate the factors contributing towards the lower microbial population observed in VCMD, it provides an impetus to further researches for scientifically validating its traditionally-reported medicinal properties.

The Tape Measure Protein Is Involved in the Heat Stability of Lactococcus lactis Phages.

Virulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low-quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a few Lactococcus lactis phages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (of the Lactococcus lactis 936 [now Sk1virus] phage group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-type L. lactis phages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of the Sk1virus group.IMPORTANCE Virulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low-quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese by-products. Recently, a few Lactococcus lactis phages, members of the Sk1virus group, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulent Sk1virus phage. Identifying such a genetic determinant for heat stability is a first step in understanding the emergence of this group of thermostable phages.

Biodiversity of bacteriophages infecting Lactococcus lactis starter cultures.

In the current study, we characterized 137 Lactococcus lactis bacteriophages that had been isolated between 1997 and 2012 from whey samples obtained from industrial facilities located in 16 countries. Multiplex PCR grouping of these 137 phage isolates revealed that the majority (61.31%) belonged to the 936 group, with the remainder belonging to the P335 and c2 groups (23.36 and 15.33%, respectively). Restriction profile analysis of phage genomic DNA indicated a high degree of genetic diversity within this phage collection. Furthermore, based on a host-range survey of the phage collection using 113 dairy starter strains, we showed that the c2-group isolates exhibited a broader host range than isolates of the 936 and P335 groups.

Exploring Structural Diversity among Adhesion Devices Encoded by Lactococcal P335 Phages with AlphaFold2.

Bacteriophages, or phages, are the most abundant biological entities on Earth. They possess molecular nanodevices to package and store their genome, as well as to introduce it into the cytoplasm of their bacterial prey. Successful phage infection commences with specific recognition of, and adhesion to, a suitable host cell surface. Adhesion devices of siphophages infecting Gram-positive bacteria are very diverse and remain, for the majority, poorly understood. These assemblies often comprise long, flexible, and multi-domain proteins, which limit their structural analyses by experimental approaches. The protein structure prediction program AlphaFold2 is exquisitely adapted to unveil structural and functional details of such molecular machineries. Here, we present structure predictions of adhesion devices from siphophages belonging to the P335 group infecting Lactococcus spp., one of the most extensively applied lactic acid bacteria in dairy fermentations. The predictions of representative adhesion devices from types I-IV P335 phages illustrate their very diverse topology. Adhesion devices from types III and IV phages share a common topology with that of Skunavirus p2, with a receptor binding protein anchored to the virion by a distal tail protein loop. This suggests that they exhibit an activation mechanism similar to that of phage p2 prior to host binding.

Effects of Protein, Calcium, and pH on Gene Transcription, Cell-Envelope Peptidase Activity of Lactococcus lactis Strains, and the Formation of Bitter Peptides.

Calcium- and protein-rich fermented milk products, such as concentrated yoghurts and fresh cheeses, may contain undesired bitter peptides, which are generated by the proteolytic cleavage of casein. Up to now, it is not clear whether this process is caused by endogenous milk enzymes, such as plasmin and cathepsin D, or whether proteolytic enzymes from applied starter cultures, such as the lactococcal cell-envelope peptidase PrtP, are involved. A sensory analysis of fresh cheese products made from milk concentrates fermented with prtP-negative and -positive Lactococcus lactis strains revealed bitterness in the products fermented with prtP-positive L. lactis strains. Two prtP-positive strains, LTH 7122 and LTH 7123, were selected to investigate the effect of increased calcium concentrations (additional 5 mM and 50 mM CaCl2) at neutral (pH 6.6) and acidic (pH 5.5) pH-values on the transcription of the prtP gene and its corresponding PrtP peptidase activity in milk citrate broth (MCB). For both strains, it was shown that prtP transcription was upregulated only under slightly elevated calcium conditions (5 mM CaCl2) after 5 h of growth. In concordance with these findings, PrtP peptidase activity also increased. When higher concentrations of calcium were used (50 mM), prtP expression of both strains decreased strongly by more than 50%. Moreover, PrtP peptidase activity of strain LTH 7123 decreased by 15%, but enzymatic activity of strain LTH 7122 increased slightly during growth under elevated calcium concentrations (50 mM CaCl2). Fermentations of reconstituted casein medium with 3.4% (w/v) and 8.5% (w/v) protein and different calcium concentrations using strain LTH 7122 revealed no clear relationship between prtP transcription and calcium or protein concentration. However, an increase in PrtP peptidase activity under elevated protein and calcium conditions was observed. The activity increase was accompanied by increased levels of bitter peptides derived from different casein fractions. These findings could be a possible explanation for the bitterness in fermented milk concentrates that was detected by a trained bitter panel.

The Lactococcus lactis Pan-Plasmidome.

Plasmids are autonomous, self-replicating, extrachromosomal genetic elements that are typically not essential for growth of their host. They may encode metabolic capabilities, which promote the maintenance of these genetic elements, and may allow adaption to specific ecological niches and consequently enhance survival. Genome sequencing of 16 Lactococcus lactis strains revealed the presence of 83 plasmids, including two megaplasmids. The limitations of Pacific Biosciences SMRT sequencing in detecting the total plasmid complement of lactococcal strains is examined, while a combined Illumina/SMRT sequencing approach is proposed to combat these issues. Comparative genome analysis of these plasmid sequences combined with other publicly available plasmid sequence data allowed the definition of the lactococcal plasmidome, and facilitated an investigation into (bio) technologically important plasmid-encoded traits such as conjugation, bacteriocin production, exopolysaccharide (EPS) production, and (bacterio) phage resistance.

Molecular Structure of Lactoferrin Influences the Thermal Resistance of Lactococcal Phages.

The protective effect of whey proteins on phages of lactic acid bacteria during heat treatment limits the recycling of whey proteins into cheese. To investigate this protective effect, we used lactoferrin (LF) as a whey protein model as a result of its unique physicochemical properties and its antiviral activity. First, the thermal inactivation of lactococcal thermoresistant virulent phage P1532 was measured in LF at 95 °C and under different pH values. Phage inactivation results revealed a strong protective effect of LF on P1532 phage at pH 5 but none at pH 7. The structural conformational changes of LF were then monitored by Fourier transform infrared and circular dichroism spectroscopies. Spectroscopic analysis showed that LF was unfolded after heating at pH 7, while it preserved its tertiary and secondary structures when heated at pH 5. There is a direct correlation between the thermal stability of LF and its ability to protect P1532 phage from heat treatment.

Investigation of the protective effect of whey proteins on lactococcal phages during heat treatment at various pH.

The incorporation of whey protein concentrates (WPC) into cheese is a risky process due to the potential contamination with thermo-resistant phages of lactic acid bacteria (LAB). Furthermore, whey proteins can protect phages during heat treatment, thereby increasing the above risk. The main objective of this work was to understand this protective effect in order to better control LAB phages and maximize whey recycling in the cheese industry. First, the inactivation of a previously characterized thermo-resistant lactococcal virulent phage (P1532) was investigated at 95 °C in WPC, in individual whey components ß-lactoglobulin, α-lactalbumin, and bovine serum albumin as well as under different heat and pH conditions. The structural changes of the tested proteins were also monitored by transmission FTIR spectroscopy. Phage inactivation results indicated that the protective effect of whey proteins was pH and time dependent at 95 °C and was not restricted to one component. FTIR spectra suggest that the protection is related to protein molecular structures and to the level of protein aggregates, which was more pronounced in acidic conditions. Moreover, the molecular structure of the three proteins tested was differently influenced by pH and the duration of the heat treatment. This work confirms the protective effect of WPC on phages during heat treatment and offers the first hint to explain such phenomenon. Finding the appropriate treatment of WPC to reduce the phage risk is one of the keys to improving the cheese manufacturing process.