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This article presents new data on Bifidobacterium longum MC-42—a strain that has been actively used for the preparation of commercial dairy products in Russia for almost 40 years. It was demonstrated that this strain possesses high activities of β-galactosidase, α-glucosidase, and leucine arylaminidase; inhibits the growth of pathogens such as Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli; and can efficiently remove cholesterol from the cultural medium. The resistance of B. longum MC-42 determined for 15 commonly used antibiotics was in agreement with those previously reported for Bifidobacterium spp. The absence of frequently transmittable antibiotic resistance genes in the genome and the lack of undesirable activity of β-glucuronidase proved the safe use of B. longum MC-42 as a probiotic and starter culture. Additionally, the impact of two growth-promoting additives—yeast extract or milk protein hydrolysate containing supplementation— on the B. longum MC-42 fermentation profile was assessed. The introduction of these additives increases the maximum attainable viable cell count by orders of magnitude, significantly changed the profile of aminopeptidase activities in extracellular extracts, and influenced the antioxidant and antihypertensive properties of the obtained fermented products. [ABSTRACT FROM AUTHOR]

This article presents new data on Bifidobacterium longum MC-42—a strain that has been actively used for the preparation of commercial dairy products in Russia for almost 40 years. It was demonstrated that this strain possesses high activities of β-galactosidase, α-glucosidase, and leucine arylaminidase; inhibits the growth of pathogens such as Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli; and can efficiently remove cholesterol from the cultural medium. The resistance of B. longum MC-42 determined for 15 commonly used antibiotics was in agreement with those previously reported for Bifidobacterium spp. The absence of frequently transmittable antibiotic resistance genes in the genome and the lack of undesirable activity of β-glucuronidase proved the safe use of B. longum MC-42 as a probiotic and starter culture. Additionally, the impact of two growth-promoting additives—yeast extract or milk protein hydrolysate containing supplementation—on the B. longum MC-42 fermentation profile was assessed. The introduction of these additives increases the maximum attainable viable cell count by orders of magnitude, significantly changed the profile of aminopeptidase activities in extracellular extracts, and influenced the antioxidant and antihypertensive properties of the obtained fermented products.

The present study aimed at selecting non-starter lactic acid bacteria strains, with desirable technological and enzymatic activities, suitable as adjunct culture for the Provola dei Nebrodi cheese production. One hundred and twenty-one lactic acid bacteria, isolated from traditional Provola dei Nebrodi cheese samples, were genetically identified by Rep-PCR genomic fingerprinting, using the (GTG)5-primer, and by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-seven strains, included in the qualified presumption of safety (QPS) list, were tested for technological and proteinase/peptidase activities. Results showed that technological features and flavour formation abilities were strain-dependent. Among the selected strains, Lacticaseibacillus paracasei PN 76 and Limosilactobacillus fermentum PN 101 were used as adjunct culture in pilot-scale cheese-making trials. Data revealed that adjunct cultures positively affected the flavour development of cheese, starting from 30 days of ripening, contributing to the formation of key flavour compounds. The volatile organic compound profiles of experimental cheeses was significantly different from those generated in the controls, suggesting that the selected adjunct strains were able to accelerate the flavour development, contributing to a unique profile of Provola dei Nebrodi cheese.

High-fat diets (HFD) have been widely associated with an increased risk of metabolic disorders and overweight. However, a high intake of sources that are rich in monounsaturated fatty acids has been suggested as a dietary agent that is able to positively influence energy metabolism and vascular function. The main objective of this study was to analyze the role of dietary fats on hepatic peptidases activities and metabolic disorders. Three diets: standard (S), HFD supplemented with virgin olive oil (VOO), and HFD supplemented with butter plus cholesterol (Bch), were administered over six months to male Wistar rats. Plasma and liver samples were collected for clinical biochemistry and aminopeptidase activities (AP) analysis. The expression of inducible nitric oxide synthase (iNOS) was also determined by Western blot in liver samples. The diet supplement with VOO did not induce obesity, in contrast to the Bch group. Though the VOO diet increased the time that was needed to return to the basal levels of plasma glucose, the fasting insulin/glucose ratio and HOMA2-%B index (a homeostasis model index of insulin secretion and valuation of β-cell usefulness (% β-cell secretion)) were improved. An increase of hepatic membrane-bound dipeptidyl-peptidase 4 (DPP4) activity was found only in VOO rats, even if no differences in fasting plasma glucagon-like peptide 1 (GLP-1) were obtained. Both HFDs induced changes in hepatic pyroglutamyl-AP in the soluble fraction, but only the Bch diet increased the soluble tyrosyl-AP. Angiotensinase activities that are implicated in the metabolism of angiotensin II (AngII) to AngIV increased in the VOO diet, which was in agreement with the higher activity of insulin-regulated-AP (IRAP) in this group. Otherwise, the diet that was enriched with butter increased soluble gamma-glutamyl transferase (GGT) and Leucyl-AP, iNOS expression in the liver, and plasma NO. In summary, VOO increased the hepatic activity of AP that were related to glucose metabolism (DPP4, angiotensinases, and IRAP). However, the Bch diet increased activities that are implicated in the control of food intake (Tyrosine-AP), the index of hepatic damage (Leucine-AP and GGT), and the expression of hepatic iNOS and plasma NO. Taken together, these results support that the source of fat in the diet affects several peptidases activities in the liver, which could be related to alterations in feeding behavior and glucose metabolism.

The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, β nerve growth factor (β-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as β-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and β−NGF, they could be used to develop new extenders with less hormone concentration. [ABSTRACT FROM AUTHOR]

The bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases. Thus, the addition of aminopeptidase inhibitors to rabbit semen extenders could be a solution to decrease the hormone degradation. This study was conducted to evaluate the effect of the protease activity inhibition on rabbit semen quality parameters and reproductive performance after artificial insemination. Seminal quality was not affected by the incubation with protease inhibitors, being the values of motility, viability, and acrosome integrity not significantly different between the protease inhibitors and the control group. In addition, seminal plasma aminopeptidase activity was inhibited in a 55.1% by the protease inhibitors. On the other hand, regarding the effect of protease inhibitors on reproductive performance, our results showed that the presence of protease inhibitors affected the prolificacy rate (9.2 ± 0.26 and 9.3 ± 0.23 vs. 8.2 ± 0.22 total born per litter for negative control, positive control, and aminopeptidase inhibitors group, respectively; P < 0.05), having this group one kit less per delivery. We conclude that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate. Therefore, the development of new extenders with specific aminopeptidase inhibitors would be one of the strategies to increase the bioavailability of GnRH analogues without affecting the litter size. [ABSTRACT FROM AUTHOR]

Abstract: Ovulation induction in artificially inseminated rabbits by adding GnRH synthetic analogues in the seminal doses is a welfare-orientated method to induce ovulation in rabbits and could have some advantages in field practice. This study was conducted to determine the effect of male genotype on the aminopeptidase activity in rabbit seminal plasma and the effects of dilution rate of semen on availability and reproductive performance when buserelin acetate is added to the seminal dose. To study the aminopeptidase activity, 12 mature bucks belonging to a paternal line and 12 from a maternal line were used. The bucks from the paternal line were used to study the effect of dilution rate on the availability of buserelin acetate after 2 hours of dilution and on the reproductive performance of the doses after artificial insemination of 389 commercial crossbreed does. Aminopeptidase activity in seminal plasma is dependent on the male genotype. The paternal line resulted 27% more aminopeptidase activity than the maternal line (P < 0.05). On the other hand, semen diluted 1:20 exhibited a marked increase in the availability of buserelin acetate and the fertility in this group was significantly higher than females from dilution rate 1:5 group, which showed similar results to that of the negative control group (does inseminated with semen diluted 1:20 in non–GnRH-supplemented extender). We conclude that the bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases and is consequently affected by the dilution rate used to prepare the artificial insemination doses. [Copyright &y& Elsevier]

Abstract: Samples of raw milk and traditional dairy products were collected from different rural areas in the Delta region. 170 isolates from these products were identified using repetitive genomic element-PCR (Rep-PCR) fingerprinting. The identified isolates were tested for efficiency of biomass production and separation, acidifying activity, autolytic and aminopeptidase properties, antagonistic activities and exopolysaccharide production. The obtained results revealed that the Lactobacillus delbrueckii subsp. lactis, Lactobacillus fermentum, Enterococcus faecium Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum and Lactococcus lactis subsp. lactis were the predominant species in Egyptian dairy products. Two percent of Lactococcus, 10% of Lactobacillus and 1% of Enterococcus isolates showed fast acidifying activity. Aminopeptidase and autolytic properties were generally higher for most Lactobacillus strains when compared to other strains. Among these species, lactobacillus paracasei subsp. paracasei was the highest in Aminopeptidase activity and autolytic properties. Antagonistic activity was detected in 40% of Lactococcus, 70% of Lactobacillus and 50% of Enterococcus isolates. Some isolates produced exopolysaccharides in milk and dairy products. [Copyright &y& Elsevier]

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid 8-naphthylamide (SNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an aminopeptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity aminopeptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity. [ABSTRACT FROM AUTHOR]

In the experiment with water from the hypereutrophic Lake Frederiksborg Slotso (Denmark) sampled during the autumn peak of Microcystis growth, the quantity and production of free-living and cyanobacteria-associated heterotrophic bacteria were determined, as well as the extracellular enzymatic (aminopeptidase) activity. The functional diversities of associated and free-living bacterial communities were additionally compared using BIOLOG GN microplates to reveal the possible export of Microcystis-attached bacteria into ambient water. It has been shown that the cell size, production values, and growth rates of associated bacteria were less than those of free-living bacteria. At the same time, the potential aminopeptidase activity of associated bacteria was always higher than that of free-living bacteria. The experimental results have shown significant compositional differences in the structure of bacterial communities from different habitats. [ABSTRACT FROM AUTHOR]

Abstract: The previously developed p-nitroaniline test for assessing the microbial load of meat surfaces has been now adapted to determine the microbial quality of raw ground meat. A good correlation (r =0.91) between bacterial count determined by the pour plate method and the p-nitroaniline test was obtained. The sensitivity of the new method was of the order of magnitude of 104 cfu/g. This method allows the assay of ground meat in approximately 2.5h, it does not require expensive equipment and the results can be interpreted both spectrophotometrically and visually. Additionally, it has been proven that the method is useful in estimating the microbial quality of raw meat irrespective of the species of Gram-negative psychrotrophic bacteria prevailing in the meat during refrigerated storage. [Copyright &y& Elsevier]

Abstract: A rapid method for the estimation of the microbiological quality of refrigerated raw milk was developed based on the aminopeptidase activity of Gram-negative psychrotrophic bacteria, the dominant microbial population in refrigerated foods. Two versions of the test for measuring the aminopeptidase activity are reported (direct and indirect). A good correlation (r=0.93–0.95) between bacterial counts estimated by conventional methods and the aminopeptidase activity determined in milk was obtained. The sensitivity of the indirect assay was 2×104cfumL-1. This sensitivity limit complies with the level of detection required to satisfy regulations in many countries. The aminopeptidase test allows the assessment of raw milk quality in approximately 2.5h, does not require either high-cost equipment or specialised operators, and results can be interpreted both spectrophotometrically and visually. [Copyright &y& Elsevier]

From the observed pattern of aminopeptidase and alkaline phosphatase activities in the Baltic Sea, the question arose whether there is an interaction between the activities of both enzymes. In experiments with 0.8 μm filtered seawater, the effects of commercial alkaline phosphatase on bacterial aminopeptidase, the effects of commercial peptidase on bacterial alkaline phosphatase activity (APA), and the effects of proteins, carbohydrates and inorganic nutrients on the activities of both enzymes were investigated. Addition of commercial alkaline phosphatase stimulated bacterial aminopeptidase activity and, similarly, the addition of commercial peptidase increased the APA in bacteria. The proteins, albumin and casein, stimulated aminopeptidase activity and APA simultaneously. Experiments using ammonium and glucose suggested that stimulation of APA by peptidase could be mediated by nitrogen and carbon availability. There were also some indications that stimulation of aminopeptidase activity by alkaline phosphatase functioned by catalysing phosphate release from organic phosphorus compounds. [ABSTRACT FROM AUTHOR]

The present study aimed at selecting non-starter lactic acid bacteria strains, with desirable technological and enzymatic activities, suitable as adjunct culture for the Provola dei Nebrodi cheese production. One hundred and twenty-one lactic acid bacteria, isolated from traditional Provola dei Nebrodi cheese samples, were genetically identified by Rep-PCR genomic fingerprinting, using the (GTG)5-primer, and by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-seven strains, included in the qualified presumption of safety (QPS) list, were tested for technological and proteinase/peptidase activities. Results showed that technological features and flavour formation abilities were strain-dependent. Among the selected strains, Lacticaseibacillus paracasei PN 76 and Limosilactobacillus fermentum PN 101 were used as adjunct culture in pilot-scale cheese-making trials. Data revealed that adjunct cultures positively affected the flavour development of cheese, starting from 30 days of ripening, contributing to the formation of key flavour compounds. The volatile organic compound profiles of experimental cheeses was significantly different from those generated in the controls, suggesting that the selected adjunct strains were able to accelerate the flavour development, contributing to a unique profile of Provola dei Nebrodi cheese. [ABSTRACT FROM AUTHOR]

High-fat diets (HFD) have been widely associated with an increased risk of metabolic disorders and overweight. However, a high intake of sources that are rich in monounsaturated fatty acids has been suggested as a dietary agent that is able to positively influence energy metabolism and vascular function. The main objective of this study was to analyze the role of dietary fats on hepatic peptidases activities and metabolic disorders. Three diets: standard (S), HFD supplemented with virgin olive oil (VOO), and HFD supplemented with butter plus cholesterol (Bch), were administered over six months to male Wistar rats. Plasma and liver samples were collected for clinical biochemistry and aminopeptidase activities (AP) analysis. The expression of inducible nitric oxide synthase (iNOS) was also determined by Western blot in liver samples. The diet supplement with VOO did not induce obesity, in contrast to the Bch group. Though the VOO diet increased the time that was needed to return to the basal levels of plasma glucose, the fasting insulin/glucose ratio and HOMA2-%B index (a homeostasis model index of insulin secretion and valuation of β-cell usefulness (% β-cell secretion)) were improved. An increase of hepatic membrane-bound dipeptidyl-peptidase 4 (DPP4) activity was found only in VOO rats, even if no differences in fasting plasma glucagon-like peptide 1 (GLP-1) were obtained. Both HFDs induced changes in hepatic pyroglutamyl-AP in the soluble fraction, but only the Bch diet increased the soluble tyrosyl-AP. Angiotensinase activities that are implicated in the metabolism of angiotensin II (AngII) to AngIV increased in the VOO diet, which was in agreement with the higher activity of insulin-regulated-AP (IRAP) in this group. Otherwise, the diet that was enriched with butter increased soluble gamma-glutamyl transferase (GGT) and Leucyl-AP, iNOS expression in the liver, and plasma NO. In summary, VOO increased the hepatic activity of AP that were related to glucose metabolism (DPP4, angiotensinases, and IRAP). However, the Bch diet increased activities that are implicated in the control of food intake (Tyrosine-AP), the index of hepatic damage (Leucine-AP and GGT), and the expression of hepatic iNOS and plasma NO. Taken together, these results support that the source of fat in the diet affects several peptidases activities in the liver, which could be related to alterations in feeding behavior and glucose metabolism. [ABSTRACT FROM AUTHOR]