Gilles Robitaille

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.

Effect of pepsin-treated bovine and goat caseinomacropeptide on Escherichia coli and Lactobacillus rhamnosus in acidic conditions.

Caseinomacropeptide (CMP) is a 7-kDa phosphoglycopolypeptide released from κ-casein during milk digestion and in the cheesemaking process. The objective of the study was to analyze the effect of pepsin-treated CMP from cow and goat milk on the resistance of Escherichia coli and Lactobacillus rhamnosus during acid stress. Bacterial cells in the exponential growth phase were suspended in acidified phosphate buffered saline with or without pepsin-treated CMP. Viability was determined during a 90-min incubation period. Pepsin-treated CMP exhibited bactericidal activity at pH 3.5 when added in a dose-dependent manner to E. coli, decreasing survival by more than 90% within 15 min at 0.25 mg/mL. At pH >4.5, the bactericidal activity disappeared, indicating that pepsin-treated CMP was efficient at low pH only. The effectiveness of pepsin-treated CMP at pH 3.5 was not affected by the presence of glycoconjugates linked to CMP or by the bovine or caprine origin of milk. In contrast, L. rhamnosus, a probiotic, was more resistant to acid stress when pepsin-treated bovine or caprine CMP was added to the media. Viability reached 50% after 60 min of incubation at pH 3 compared with 5% survival in the media without added pepsin-treated CMP. Neither glycosylation extent nor sequence variations between CMP from bovine milk and caprine milk affected the protective activity of hydrolyzed CMP toward L. rhamnosus. This suggests that encrypted bioactive peptides released by the pepsin treatment of CMP had an antibacterial effect on E. coli in acidic media, but improved the resistance of L. rhamnosus to acid stress. The peptide fragment accountable for bactericidal activity is the N-terminal region κ-casein f(106-124).

Proteomic analysis and immunodetection of the bovine milk osteopontin isoforms

The aim of this study was to characterize the osteopontin (OPN) secreted in bovine milk and to determine whether the different forms are the product of spliced variants. Spliced variants of the human gene and secreted osteopontin isoforms have been reported in human tumor tissue. In bovine milk, we identified 2 major forms: one corresponding to the full-length coding transcript and a truncated version of this form. No alternative spliced transcripts were detected in the lactating mammary gland tissue, in milk somatic cells, or in peripheral blood immune cells. The 60-kDa bovine osteopontin (bOPN) and a truncated 40-kDa protein isoform were confirmed by mass spectrometry and further characterized by immunoblotting using a panel of 6 antibodies targeting different domains of the protein. Of the 3 human anti-OPN antibodies targeting the N-terminal segment of the protein, only one detected all forms on sodium dodecyl sulfate-PAGE; one human anti-OPN antibody failed to detect bOPN, whereas the other detected only the 60-kDa protein, albeit barely in its phosphorylated form. Detection was generally more sensitive when the 60-kDa protein was dephosphorylated. Two polyclonal antibodies raised against bOPN were tested: one targeting the milk-purified bOPN (bOPN-121) and one targeting a bovine epitope (synthetic peptide) corresponding to a carboxy-terminal domain of the protein (bOPN-117). The bOPN-121 antibody detected all forms irrespective of the phosphorylation status of bOPN. The bOPN-117 and the mouse anti-human OPN (hOPN-4) antibodies, which recognized different domains of the carboxy-terminal segment of the protein, also preferentially detected the dephosphorylated 60-kDa protein. Whereas phosphorylation had a major effect on detection for several antibodies, deglycosylation slightly decreased immunodetection for the tested antibodies. In particular, phosphorylation is the major posttranslational modification that influenced the weak detection capacity of several antibodies. This fact needs to be taken into account for immunodetection of milk content. In conclusion, the OPN forms secreted in bovine milk are not the product of alternative splicing. The 40-kDa protein appears to be a truncated hypophosphorylated variant of the full-length 60-kDa form, which is highly phosphorylated. Together, the proteomic and immunoblotting analyses used to characterize bovine milk OPN revealed the complex nature of the bovine milk OPN forms.

Growth-promoting effects of caseinomacropeptide from cow and goat milk on probiotics.

Caseinomacropeptide (CMP), a 7-kDa phosphoglycopolypeptide fragment released from κ-casein during milk renneting, is heterogeneous with respect to post-translational glycosylation. Several studies have reported that CMP has growth-promoting activity on lactic acid bacteria belonging to the genera Bifidobacterium. The aim of this study was to evaluate the effect of glycosylation and sequence variations between bovine and caprine CMP on the growth of two probiotics: Lactobacillus rhamnosus RW-9595-M and Bifidobacterium thermophilum RBL67. The growth-promoting activities of CMP (mixture of glycosylated (gCMP) and non-glycosylated (aCMP) fractions), aCMP and gCMP were measured in a basal minimal culture medium using turbidimetric microplate assay at 37 °C. Supplementation of the culture media at 2 mg/ml significantly improved maximum growth by 1.5 to 1.8 times depending on the strain, the additive (CMP, aCMP, gCMP), and the bovine or caprine origin (P < 0.05). CMP preparations also decreased the time needed to reach the inflexion point of the growth curve and increase the cell density at that time (P < 0.05). The effects of CMP preparations were dose dependent and significantly superior to the effect of bovine β-lactoglobulin added to the culture media. As gCMP and aCMP were as efficient as bovine and caprine CMP (P > 0.1), it was concluded that the presence of oligosaccharides linked to CMP was not essential for growth-promoting activity of CMP.

Growth-promoting effects of pepsin- and trypsin-treated caseinomacropeptide from bovine milk on probiotics

Probiotic Lactobacillus and Bifidobacterium species are generally fastidious bacteria and require rich media for propagation. In milk-based media, they grow poorly, and nitrogen supplementation is required to produce high bacterial biomass levels. It has been reported that caseinomacropeptide (CMP), a 7-kDa peptide released from κ-casein during renneting or gastric digestion, exhibits some growth-promoting activity for lactobacilli and bifidobacteria. During the digestive process, peptides derived from CMP are detected in the intestinal lumen The aim of this study was to evaluate the effects of peptic and tryptic digests of CMP on probiotic lactic acid bacteria growth in de Man, Rogosa and Sharpe broth (MRS) and in milk during fermentation at 37 °C under anaerobic conditions. The study showed that pepsin-treated CMP used as supplements at 0.5 g/l can promote the growth of probiotics even in peptone-rich environments such as MRS. The effect was strain-dependent and evident for the strains that grow poorly in MRS, with an improvement of >1.5 times (P<0.05) by addition of pepsin-treated CMP. Trypsin-treated CMP was much less efficient as growth promoter. Moreover, pepsin-treated CMP was effective in promoting the growth in milk of all probiotic lactic acid bacteria tested, with biomass levels being improved significantly, by 1.7 to 2.6 times (P<0.05), depending on the strain. Thus, supplementation of MRS and of milk with pepsin-treated CMP would be advantageous for the production of high biomass levels for Bifidobacteria and Lactobacilli.

Differential allele-specific accumulation of bovine kappa-casein mRNA throughout lactation.

A differential allele-specific accumulation of kappa-casein mRNA that is not linked to the kappa-casein protein variants is described in Holstein cows. Actually, cows genotyped kappa-casein AB were a mixed population. For the first group of kappa-casein AB cows, allele A-specific kappa-casein mRNA contents within mammary epithelial cells were lower than the allele B-specific ones (cows LH), suggesting that the allele A-specific kappa-casein gene was expressed with lower efficiency in mRNA. For the other group of kappa-casein AB cows, allele A- and B-specific kappa-casein mRNA accumulated to a similar level within mammary epithelial cells (cows HH). The objective of this study was to determine whether the accumulation of allele-specific kappa-casein mRNA remained constant throughout lactation for the two groups of cows. Quantitative RT-PCR was used to monitor Holstein cows kappa-casein AB genotyped HH and LH throughout lactation for the proportion of allele B-specific mRNA accumulation relative to the total kappa-casein encoded mRNA within mammary epithelial cells: RNA was extracted from milk somatic cells known to contain a small proportion of mammary epithelial cells. Mean values of allele B-specific mRNA content were 50.6+/-0.5 and 54.0+/-0.9%, for cows HH and cows LH, respectively, and did not vary during lactation (P> 0.10). This suggests that the phenotypic expression of the genetic mutation that causes the differential allele-specific accumulation of kappa-casein mRNA was not affected by physiological and environmental factors, which tend to vary considerably throughout lactation.

Genetic polymorphism of plasminogen in dairy cattle

Plasmin (PLM; E.C. 3.4.4.14) is the major proteolytic enzyme normally present in bovine milk. From a technological point of view, PLM activity in milk is detrimental as it increases the proteolysis of casein to proteose peptones, and this results in reduced storage time, taste defects, loss of cheese yield and quality, and changes in the physicochemical properties of milk (for review, see Fox, 1992). Therefore a reduction of PLM activity in milk would be desirable. Plasminogen (PLG), the zymogen of plasmin, and PLM content are affected by several physiological and environmental factors, and by genetic factors such as breed (Richardson, 1983; Schaar, 1985; Politis et al . 1989; Benslimane et al . 1990). We have addressed the question of PLG polymorphism in dairy cattle.