Adam Waśko

PURIFICATION AND CHARACTERIZATION OF PULLULANASE FROM Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co2+ and completely inhibited by Hg2+. The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.

The first report of the physicochemical structure of chitin isolated from Hermetia illucens.

This is the first report on the physicochemical properties of chitin obtained from larvae and imagoes of black soldier flies (Hermetia illucens). Scanning electron microscopy revealed differences in surface morphologies of the two types of chitin. The crystalline index values of chitins from adult flies and larvae were 24.9% and 35%, respectively. This is a trait that differentiates these biopolymers from chitins extracted from other sources described so far. X-ray diffraction patterns and IR spectroscopy revealed that both types of samples of chitin were in an α crystalline form. Also, the results of elemental analysis, thermal stabilities and FTIR spectroscopy of the chitins from larvae and adults of H. illucens were similar, which points to a general similarity in their physicochemical structure.

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k cat/K m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL−1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca2+ and Mg2+, and inhibited by Cu2+, Zn2+, Cd2+, and Fe2+.

Health-promoting properties exhibited by Lactobacillus helveticus strains.

Many strains belonging to lactobacilli exert a variety of beneficial health effects in humans and some of the bacteria are regarded as probiotic microorganisms. Adherence and capabilities of colonization by Lactobacillus strains of the intestinal tract is a prerequisite for probiotic strains to exhibit desired functional properties. The analysis conducted here aimed at screening strains of Lactobacillus helveticus possessing a health-promoting potential. The molecular analysis performed, revealed the presence of a slpA gene encoding the surface S-layer protein SlpA (contributing to the immunostimulatory activity of L. helveticus M 92 probiotic strain) in all B734, DSM, T80, and T105 strains. The product of gene amplification was also identified in a Bifidobacterium animalis ssp. lactis BB12 probiotic strain. SDS-PAGE of a surface protein extract demonstrated the presence of a protein with a mass of about 50 kDa in all strains, which refers to the mass of the S-layer proteins. These results are confirmed by observations carried with transmission electron microscopy, where a clearly visible S-layer was registered in all the strains analyzed. The in vitro study results obtained indicate that the strongest adhesion capacity to epithelial cells (HT-29) was demonstrated by L. helveticus B734, while coaggregation with pathogens was highly diverse among the tested strains. The percentage degree of coaggregation was increasing with the incubation time. After 5 h of incubation, the strongest ability to coaggregate with Escherichia coli was expressed by T104. The T80 strain demonstrated a significant ability to co-aggregate with Staphylococcus aureus, while DSM with Bacillus subtilis. For B734, the highest values of co-aggregation coefficient was noted in samples with Salmonella. The capability of autoaggregation, antibiotic susceptibility, resistance to increasing salt concentrations, and strain survival in simulated small intestinal juice were also analyzed.

Spirulina enhances the viability of Lactobacillus rhamnosus E/N after freeze‐drying in a protective medium of sucrose and lactulose

Aims:  Response surface methodology (RSM) was used to optimize a protective medium for enhancing the viability of Lactobacillus rhamnosus E/N cells during lyophilization.

The effect of conglutinin on production of reactive oxygen species in bovine granulocytes.

Conglutinin is a high molecular-weight lectin originally detected in bovine serum. It belongs to the family of collectins that bind sugar residues in a Ca2+-dependent manner and are effector molecules in innate immunity. Conglutinin appears to play an important role in immune defense mechanisms, showing antiviral and antibacterial activities when tested in vivo and in vitro. The present study evaluated the effect of conglutinin on the respiratory bursts in bovine peripheral phagocytes. Using nitroblue tetrazolium and hydrogen peroxide assays, we showed that sugar ligand-bound conglutinin stimulated the production of superoxide and H2O2 in granulocytes whereas the non-sugar-bound form of conglutinin inhibited these processes. These results indicate that both forms of conglutinin are able to interact with surface leukocyte receptors but have opposite effects on phagocytic activity. Our findings suggest that conglutinin bound to sugar residues on microbial surfaces can induce oxygen burst in phagocytes, and thereby mediates the elimination of pathogens and prevents the spread of infection.

Hermetia illucens as a new and promising species for use in entomoremediation

This study investigated the use of Hermetia illucens (black soldier fly - BSF) larvae as a new species for use in entomoremediation. The H. illucens larvae effectively reduced the dry mass of polluted corn leaves by an average of 49% after 36days for both Cd and Zn (50mg·kg-1), which is a better result than that, which can be obtained by one of the standard proposed pretreatments for biomass polluted after phytoextraction: composting. The presence of heavy metals did not significantly affect the dry mass utilization efficiency. Based on this, we proposed the use of H. illucens as a new post-harvest management method of phytoextraction-polluted biomass. Cadmium mostly accumulates in the puparium, while Zn accumulates in the adults. The high Cd content in the puparium further creates possibility of its application in the metal recovery process. It is also proposed that the general definition of entomoremediation be expanded.

κ-Casein as a source of short-chain bioactive peptides generated by Lactobacillus helveticus

Abstract This paper explores the ability of Lactobacillus helveticus strains to release sequences of short biologically active peptides (containing 2–10 amino acid residues) from casein. The proteolytic enzymes of the tested strains exhibit different patterns of cleavage of CN fractions. The modification of κ-casein (κ-CN) with pyrrolidone carboxylic acid inhibits the proteolytic activity of strains L. helveticus 141 and the reference strain (DSMZ 20075), while the modification with phosphothreonine inhibits enzymes of all the tested bacteria. The peptide sequencing analysis indicated that the examined strains produced functional peptides very efficiently. κ-CN proved to be the main source of short peptides released by bacterial enzymes, and the hydrolysis of κ-CN yielded eighty-two bioactive peptides. The hydrolysis of αS2-casein, αS1-casein, and β-casein yielded six, two, and one short-chain bioactive peptides, respectively. The isolated bioactive peptides exhibited antioxidative, opioid, stimulating, hypotensive, immunomodulating, antibacterial, and antithrombotic activities. A vast majority of the isolated bioactive peptides caused inhibition of the angiotensin-converting enzyme and dipeptidyl peptidase IV. The role of hydrolysis products as neuropeptides is also pointed out. The highest number of cleavage sites in κ-casein and functional activities of short-chain peptides were obtained in hydrolyzates produced by L. helveticus strain T105.

Strains of Photorhabdus spp. associated with polish Heterorhabditis isolates: their molecular and phenotypic characterization and symbiont exchange

The relationships between six bacterial symbionts of the entomopathogenic nematodes Heterorhabditis bacteriophora and Heterorhabditis megidis from Poland to species and subspecies of the genus Photorhabdus were evaluated. This study was based on phylogenetic analysis of sequence data of five genes: 16S rRNA, gyrB, recA, gltX, and dnaN. The bacteria were also characterized phenotypically by biochemical and physiological tests. Our results have revealed that the Photorhabdus strains isolated from H. megidis belong to P. temperata, subsp. temperata and subsp. cinerea. Isolates from H. bacteriophora represent P. luminescens subs. kayaii and P. temperata subs. cinerea. This study for the first time provides evidence for H. bacteriophora and P. temperata subsp. cinerea symbiotic association. In addition, we tested whether the microsymbionts of the Polish H. bacteriophora and H. megidis isolates support the development of non-native nematode host population and colonization of their infective juveniles. It has been shown that the studied Photorhabdus strains can readily swap their nematode host, both at intra- and interspecies level. It supports the hypothesis of different symbiotic associations in the Heterorhabditis–Photorhabdus lineage.

Antioxidative properties of milk protein preparations fermented by Polish strains of Lactobacillus helveticus

BACKGROUND The increasing significance of food products containing substances with antioxidative activi- ties is currently being observed. This is mainly due to the fact that pathogenic changes underlying some diseases are related to the carcinogenic effects of free radicals. Antioxidative compounds play an important role in supporting and enhancing the body’s defense mechanisms, which is useful in preventing some civili- zation diseases. Unfortunately, it has been already proved that some synthetic antioxidants pose a potential risk in vivo. Therefore, antioxidant compounds derived from a natural source are extremely valuable. Milk is a source of biologically active precursors, which when enclosed in structural protein sequences are inactive. The hydrolysis process, involving bacterial proteolytic enzymes, might release biopeptides that act in various ways, including having antioxidant properties. The objective of this study was to determine the antioxidant properties of milk protein preparations fermented by Polish strains of L. helveticus. The research also focused on evaluating the dynamics of milk acidification by these strains and analyzing the textural properties of the skim milk fermented products obtained. METHODS The research studied Polish strains of L. helveticus: B734, 141, T80 and T105, which have not yet been used industrially. The antioxidant properties of 1% (w/v) solutions of milk protein preparations (skim milk powder, caseinoglycomacropeptide and α-lactoalbumin) fermented by these strains were determined by neutralizing the free radicals with 2,2-diphenyl-1-picrylhydrazyl (DPPH˙). Moreover, solutions of skim milk powder (SMP) fermented by the microorganisms being tested were analyzed on gel electrophoresis (SDS-PAGE). The dynamics of milk acidification by these microorganisms was also analyzed L. helveticus strains were used to prepare fermented regenerated skim milk products that were subjected to texture profile analysis (TPA) performed using a TA-XT2i (Stable Micro Systems, Godalming, UK). RESULTS The results suggest that the antioxidant activity of fermented milk protein preparations depended on the type of milk protein preparation and was also related to the strain that conducted the fermentation process. The process of caseinoglycomacropeptide (CGMP) fermentation by DSMZ 20075, T105 and 141 signifi- cantly (p < 0.05) influenced the increase in the antioxidant activities of the protein preparation, the highest values of parameter were obtained in samples fermented by L. helveticus T105 (64.82 ±0.013%), while in the case of α-lactoalbumin (α-la), the strongest free radical scavenging activity (66.67 ±0.020%) was noted for unfermented samples (control). CONCLUSIONS The greatest increase in DPPH scavenging activity (% of inhibition) was noted for fermented SMP solutions. The highest values of the parameter measured were recorded for SMP fermented by the reference strain (85.98 ±0.009%) and T80 (81.66 ±0.013%). Strain T105 demonstrated the most desirable properties with respect to milk acidifying dynamic and texture properties of fermented skim milk products, while the reference strain (L. helveticus DSMZ 20075) and L. helveticus T80 seem to be more desirable in terms of the possibility of obtaining fermented protein preparations with the best antioxidant properties. The Polish strains analyzed here might find application in dairy products and also in developing functional food products. Furthermore, the preparations of milk protein that were fermented by the strains being tested may be a natural source dietary antioxidants.