The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland.

The present study aimed to determine the genotyping diversity and hemolytic properties of 24 strains of Cronobacter spp. (15 Cronobacter sakazakii, 6 Cronobacter malonaticus, 2 Cronobacter turicensis, and 1 Cronobacter condimenti) isolated from commercial ready-to-eat leaf vegetables, sprouts, nuts, and dried fruits. The multilocus sequence typing (MLST) method was used to determine the sequence types (ST) and clonal complexes (CC) of these strains. The study demonstrated the high genotypic diversity of the Cronobacter genus bacteria isolated from plant-based foods. Five novel sequence types (804, 805, 806, 807, and 808) and the presence of novel alleles in the ppsA, gltB, gyrB, and infB loci were detected. In total, 16 of the 24 strains were assigned to the sequence types ST99, ST258, ST17, ST648, ST21, ST494, and ST98. One C. sakazakii strain (s12) isolated from alfalfa sprouts was assigned to the clonal complex CC4, which encompasses strains often associated with severe infections leading to meningitis in infants. In addition, 87.5% and 16.7% of the Cronobacter spp. strains showed ß-hemolysis of equine and sheep red blood cells, respectively. The presence of the pathogenic species C. sakazakii, C. malonaticus, and C. turicensis in ready-to-eat plant-derived food products shows they are potential sources of infection, especially to those with compromised immunity, which substantiates their further multi-faceted characterization. The significance of this study may prove useful not only in epidemiological investigations, but also in assessing the risk of infections caused by the presence of Cronobacter.

The Impact of White Mulberry, Green Barley, Chia Seeds, and Spirulina on Physicochemical Characteristics, Texture, and Sensory Quality of Processed Cheeses.

Processed cheeses (PC) are products resulting from the mixing and melting of rennet cheese, emulsifying salts, water, and possibly various additional ingredients. They are considered good vehicles for new ingredients, including plant-based ones. In addition to the health-promoting effects of plant-based ingredients, some of them can also affect positively the quality characteristics of PC (e.g., texture, taste, and consistency) and their addition may reduce the amount of emulsifying salts used. The aim of the study was to determine the possibility of the addition of 0.5, 1.0, 2.0, and 3.0% white mulberry (M), chia (Ch), green barley (GB), or spirulina (S) to PC and the effects on selected characteristics of these products (chemical composition, pH, water activity, color parameters, texture, and sensory properties). In all PC variants, a significance decrease in the dry matter content was observed with an increase in the additive level. The use of plant-based additives allowed us to reduce the addition of emulsifying salts by 50% compared to their typical amounts and the share of rennet cheese in the PC recipe by approximately 18%, which had a beneficial effect on the nutritional value of these products. The use of 3% GB, Ch, or M as additives to PC enabled a reduction in its sodium content by 27, 27, and 42%, respectively, compared to the control cheese. Among the tested additives, GB caused the greatest increase in the hardness of PC (even at the amount of 0.5%), indicating that is beneficial and can be used in the production of sliced PC. All the additives either significantly reduced the adhesiveness of PC or had no effect on this parameter. In terms of sensory characteristics, the highest acceptable addition of GB was 0.5%, and that for S and Ch was 1%, while the addition of M, even at 3%, was assessed very positively. The results of this research may be helpful in the development of new recipes for processed cheeses obtained in industrial conditions.

The Presence of Plasmids in Lactococcus lactis IL594 Determines Changes in the Host Phenotype and Expression of Chromosomal Genes.

The L. lactis IL594 strain contains seven plasmids (pIL1 to pIL7) and is the parental strain of the plasmid-free L. lactis IL1403, one of the most studied lactic acid bacteria (LAB) strain. The genetic sequences of pIL1 to pIL7 plasmids have been recently described, however the knowledge of global changes in host phenotype and transcriptome remains poor. In the present study, global phenotypic analyses were combined with transcriptomic studies to evaluate a potential influence of plasmidic genes on overall gene expression in industrially important L. lactis strains. High-throughput screening of phenotypes differences revealed pronounced phenotypic differences in favor of IL594 during the metabolism of some C-sources, including lactose and ß-glucosides. A plasmids-bearing strain presented increased resistance to unfavorable growth conditions, including the presence of heavy metal ions and antimicrobial compounds. Global comparative transcriptomic study of L. lactis strains revealed variation in the expression of over 370 of chromosomal genes caused by plasmids presence. The general trend presented upregulated energy metabolism and biosynthetic genes, differentially expressed regulators, prophages and cell resistance proteins. Our findings suggest that plasmids maintenance leads to significant perturbation in global gene regulation that provides change in central metabolic pathways and adaptive properties of the IL594 cells.

The Impact of the Adjunct Heat-Treated Starter Culture and Lb. helveticus LH-B01 on the Proteolysis and ACE Inhibitory Activity in Dutch-Type Cheese Model during Ripening.

Adjunct cultures are used in cheesemaking to improve the sensory characteristics of the ripened cheeses. In addition, it is known that different adjunct cultures are capable of producing enzymes with the specificity to hydrolyze caseins, leading to the release of various bioactive compounds (bioactive peptides, amino acids, etc.). The objective of this study was to evaluate the effect of adjunct heat-treated starter XT-312 and a cheese culture Lb. helveticus LH-B01 on the proteolytic activity and angiotensin converting enzymes inhibitors (ACE) in cheese models during ripening. Seven different cheese models were evaluated for: proteolytic activity using the spectrophotometric method with ortho-phthaldialdehyde (OPA), soluble nitrogen (SN), trichloroacetic acid-soluble nitrogen (TCA-SN) phosphotungstic acid-soluble nitrogen (PTA-SN), total nitrogen (TN), pH, contents of water, fat, as well as for total bacteria count (TBC), count of Lactococcus genus bacteria, count of Lb. helveticus, and number of non-starter lactic acid bacteria (NSLAB). Presence of adjunct bacterial cultures both in the form of a cheese culture LH-B01 and heat-treated XT-312 starter promoted primary and secondary proteolysis, which resulted in acceleration of the ripening process. ACE inhibitory activity and proteolytic activity was the highest throughout of ripening for cheese model with LH-B01 culture. The cheese models with the adjunct heat-treated starter were characterized by lower TBC, NSLAB and lower count of Lactococcus genus bacteria during ripening, compared to control cheeses.

Microbiological Quality of Nuts, Dried and Candied Fruits, Including the Prevalence of Cronobacter spp.

Cronobacter genus bacteria are food-borne pathogens. Foods contaminated with Cronobacter spp. may pose a risk to infants or immunocompromised adults. The aim of this study was to determine the microbiological quality of nuts, seeds and dried fruits with special emphasis on the occurrence of Cronobacter spp. Analyses were carried out on 64 samples of commercial nuts (20 samples), dried fruits (24), candied fruits (8), seeds (4), and mixes of seeds, dried fruits and nuts (8). The samples were tested for the total plate count of bacteria (TPC), counts of yeasts and molds, and the occurrence of Cronobacter spp. Cronobacter isolates were identified and differentiated by PCR-RFLP (Polymerase Chain Reaction - Restriction Fragments Length Polymorphism) and RAPD-PCR (Random Amplified Polymorphic DNA by PCR) analysis. TPC, and yeasts and molds were not detected in 0.1 g of 23.4%, 89.1%, and 32.8% of the analyzed samples. In the remaining samples, TPC were in the range of 1.2-5.3 log CFU g-1. The presence/absence of Cronobacter species was detected in 12 (18.8%) samples of: nuts (10 samples), and mixes (2 samples). The 12 strains of Cronobacter spp. included: C. sakazakii (3 strains), C. malonaticus (5), and C. turicensis (4). The results of this study contribute to the determination of the presence and species identification of Cronobacter spp. in products of plant origin intended for direct consumption.

Effect of Simulated Gastrointestinal Tract Conditions on Survivability of Probiotic Bacteria Present in Commercial Preparations.

Probiotics are recommended, among others, in the diet of children who are under antibiotic therapy, or that suffer from food allergies or travel diarrhea, etc. In the case of toddlers taking probiotic preparations, it is highly recommended to first remove the special capsule, which normally protects probiotic strains against hard conditions in the gastrointestinal tract. Otherwise, the toddler may choke. This removal can impair probiotic survival and reduce its efficacy in a toddler's organism. The aim of this study was to evaluate the survivability of five strains of lactic acid bacteria from the commercial probiotics available on the Polish market under simulated conditions of the gastrointestinal tract. Five probiotics (each including one of these strains: Bifidobacterium BB-12, Lactobacillus (Lb.) rhamnosus GG, Lb. casei, Lb. acidophilus, Lb. plantarum) were protective capsule deprived, added in a food matrix (chicken-vegetable soup) and subjected under simulated conditions of the gastric and gastrointestinal passage. Strain survivability and possibility to growth were evaluated. Obtained results showed that, among all analyzed commercial probiotic strains, the Lb. plantarum was the most resistant to the applied conditions of the culture medium. They showed a noticeable growth under both in vitro gastric conditions at pH 4.0 and 5.0, as well as in vitro intestinal conditions at all tested concentrations of bile salts.

Antibacterial Activity of Biocellulose with Oregano Essential Oil against Cronobacter Strains.

Biocellulose, named "the biomaterial of the future", is a natural and ecologically friendly polymer, produced by selected acetic acid bacteria strains. Biocellulose impregnated with antimicrobial agents can be used as a novel, safe, and biodegradable food packaging material, helping extend the shelf life of some products and may also have the chance to replace typical plastic packaging, which is a big environmental problem these days. This study aimed to evaluate if cellulose impregned with natural oregano essential oil could show antibacterial activity against Cronobacter strains, which can occur in food, causing diseases and food poisoning. Bacterial cellulose was obtained from two acetic bacteria strains, Gluconacetobacter hansenii ATCC 23769 and Komagataeibacter sp. GH1. Antibacterial activity was studied by the disc-diffusion method against chosen Cronobacter strains, isolated from the plant matrix. Oregano essential oil has been shown to penetrate into the structure of bacterial cellulose, and after applying cellulose to the solid medium, it showed the ability to migrate. Biopolymer from the strain K. sp. GH1 was able to better absorb and retain essential oregano oil (OEO) compared to bacterial cellulose (BC) produced by the G. hansenii ATCC 23769. Bacterial cellulose with oregano essential oil from strain Komagataeibacter GH1 showed generally greater inhibitory properties for the growth of tested strains than its equivalent obtained from G. hansenii. This was probably due to the arrangement of the polymer fibers and its final thickness. The largest zone of inhibition of strain growth was observed in relation to C. condimenti s37 (32.75 mm ± 2.8). At the same time, the control sample using filter paper showed an inhibition zone of 36.0 mm ± 0.7. A similar inhibition zone (28.33 mm ± 2.6) was observed for the C. malonaticus lv31 strain, while the zone in the control sample was 27.1 mm ± 0.7. Based on this study, it was concluded that bacterial cellulose impregnated with oregano essential oil has strong and moderate antimicrobial activity against all presented strains of the genus Cronobacter isolated from plant matrix. Obtained results give a strong impulse to use this biopolymer as ecological food packaging in the near future.

Ultrasound-Assisted Osmotic Dehydration of Apples in Polyols and Dihydroxyacetone (DHA) Solutions.

The aim of this work was to analyse the effect of ultrasound-assisted osmotic dehydration of apples v. Elise on mass transfer parameters, water activity, and colour changes. Ultrasound treatment was performed at a frequency of 21 kHz with a temperature of 40 °C for 30-180 min using four osmotic solutions: 30% concentrated syrups of erythritol, xylitol, maltitol, and dihydroxyacetone (DHA). The efficiency of the used solutes from the polyol groups was compared to reference dehydration in 50% concentrated sucrose solution. Peleg's model was used to fit experimental data. Erythritol, xylitol, and DHA solutions showed similar efficiency to sucrose and good water removal properties in compared values of true water loss. The application of ultrasound by two methods was in most cases unnoticeable and weaker than was expected. On the other hand, sonication by the continuous method allowed for a significant reduction in water activity in apple tissue in all tested solutions.

Modeling of Osmotic Dehydration of Apples in Sugar Alcohols and Dihydroxyacetone (DHA) Solutions.

The purpose of this paper is twofold: on the one hand, we verify effectiveness of alternatives solutes to sucrose solution as osmotic agents, while on the other hand we intend to analyze modeling transfer parameters, using different models. There has also been proposed a new mass transfer parameter-true water loss, which includes actual solid gain during the process. Additional consideration of a new ratio (Cichowska et al. Ratio) can be useful for better interpretation of osmotic dehydration (OD) in terms of practical applications. Apples v. Elise were dipped into 30% concentrated solutions of erythritol, xylitol, maltitol, and dihydroxyacetone (DHA) to remove some water from the tissue. To evaluate the efficiency of these solutes, 50% concentrated sucrose solution was used as a control. All of the tested osmotic agent, except maltitol, were effective in the process as evidenced by high values in the true water loss parameter. Solutions of erythritol and xylitol in 30% concentrate could be an alternative to sucrose in the process of osmotic dehydration. Peleg's, Kelvin⁻Voigt, and Burgers models could fit well with the experimental data. modeling of mass transfer parameters, using Peleg's model can be satisfactorily supplemented by Kelvin⁻Voigt and Burgers model for better prediction of OD within the particular periods of the process.

Study on the Use of Microbial Cellulose as a Biocarrier for 1,3-Dihydroxy-2-Propanone and Its Potential Application in Industry.

Can microbial cellulose (MC) be used as a bio-carrier for 1,3-dihydroxy-2-propanone (DHA)? The aim of this study was to examine the possibility of using MC as a biomaterial for DHA transferring into the stratum corneum and inducing changes in skin color. The MC patches were obtained from Gluconacetobacter xylinus strain and incubated in solutions with various concentrations of DHA (g·L-1: 20; 50; 80; 110) at 22 °C for 24 h. Afterwards; the patches were applied onto the skin for 15, 30, or 60 min. Skin color changes were assessed visually compared to a control patches without DHA. The intensity of skin color was increasing with the increase of DHA concentration and time of patches application. Application of MC patches with DHA (50 g·L-1) for 30 min ensured the color which was considered the closest to the desired natural tan effect. MC patches containing DHA can be biocarriers enabling DHA transport into the stratum corneum and causing skin color changes. Study results indicate a new possibility for industrial applications of MC; e.g., as a biocarrier in masking the symptoms of vitiligo or production of self-tanning agents in the form of masks.

Utilization of a waste glycerol fraction using and reusing immobilized Gluconobacter oxydans ATCC 621 cell extract

Background: Depletion of petroleum resources has enforced the search for alternative sources of renewable energy. Introduction of biofuels into the market was expected to become a solution to this disadvantageous situation. Attempts to cover fuel demand have, however, caused another severe problem­the waste glycerol generated during biodiesel production at a concentration of approximately 10% w/w. This, in turn, prompted a global search for effective methods of valorization of the waste fraction of glycerol. Results: Utilization of the waste fraction at 48 h with an initial glycerol concentration of 30 g·L-1 and proceeding with 62% efficiency enabled the production of 9 g·L-1 dihydroxyacetone at 50% substrate consumption. The re-use of the immobilized biocatalyst resulted in a similar concentration of dihydroxyacetone (8.7 g·L-1) in two-fold shorter time, with an efficiency of 85% and lower substrate consumption (35%). Conclusions: The method proposed in this work is based on the conversion of waste glycerol to dihydroxyacetone in a reaction catalyzed by immobilized Gluconobacter oxydans cell extract with glycerol dehydrogenase activity, and it could be an effective way to convert waste glycerol into a valuable product.

The exopolysaccharides biosynthesis by Candida yeast depends on carbon sources

Background: The exopolysaccharides (EPS) produced by yeast exhibit physico-chemical and rheological properties, which are useful in the production of food and in the cosmetic and pharmaceutical industries as well. The effect was investigated of selected carbon sources on the biosynthesis of EPS by Candida famata and Candida guilliermondii strains originally isolated from kefirs. Results: The biomass yields were dependent on carbon source (sucrose, maltose, lactose, glycerol, sorbitol) and ranged from 4.13 to 7.15 g/L. The highest biomass yield was reported for C. guilliermondii after cultivation on maltose. The maximum specific productivity of EPS during cultivation on maltose was 0.505 and 0.321 for C. guilliermondii and C. famata, respectively. The highest EPS yield was found for C. guilliermondii strain. The EPS produced under these conditions contained 65.4% and 61.5% carbohydrates, respectively. The specific growth rate (µ) of C. famata in medium containing EPS as a sole carbon source was 0.0068 h-1 and 0.0138 h-1 for C. guilliermondii strain. Conclusions: The most preferred carbon source in the synthesis of EPS for both Candida strains was maltose, wherein C. guilliermondii strain showed the higher yield of EPS biosynthesis. The carbon source affected the chemical composition of the resulting EPS and the contribution of carbohydrate in the precipitated preparation of polymers was higher during supplementation of maltose as compared to sucrose. It was also found that the EPS can be a source of carbon for the producing strains.

ClaR--a novel key regulator of cellobiose and lactose metabolism in Lactococcus lactis IL1403.

In a number of previous studies, our group has discovered an alternative pathway for lactose utilization in Lactococcus lactis that, in addition to a sugar-hydrolyzing enzyme with both P-ß-glucosidase and P-ß-galactosidase activity (BglS), engages chromosomally encoded components of cellobiose-specific PTS (PTS(Cel-Lac)), including PtcA, PtcB, and CelB. In this report, we show that this system undergoes regulation via ClaR, a novel activator protein from the RpiR family of transcriptional regulators. Although RpiR proteins are widely distributed among lactic acid bacteria, their roles have yet to be confirmed by functional assays. Here, we show that ClaR activity depends on intracellular cellobiose-6-phosphate availability, while other sugars such as glucose or galactose have no influence on it. We also show that ClaR is crucial for activation of the bglS and celB expression in the presence of cellobiose, with some limited effects on ptcA and ptcB activation. Among 190 of carbon sources tested, the deletion of claR reduces L. lactis growth only in lactose- and/or cellobiose-containing media, suggesting a narrow specificity of this regulator within the context of sugar metabolism.

Exopolysaccharides from yeast: insight into optimal conditions for biosynthesis, chemical composition and functional properties - review.

BACKGROUND: xopolysaccharides (EPS) are not a well-established group of metabolites. An industrial scale    of this EPS production is limited mainly by low yield biosynthesis. Until now, enzymes and biosynthesis pathways, as well as the role of regulatory genes, have not been described. Some of yeast EPS show antitumor, immunostimulatory and antioxidant activity. Others, absorb heavy metals and can function as bioactive components of food. Also, the potential of yeast EPS as thickeners or stabilizers can be found. Optimal conditions for the biosynthesis of yeast exopolysaccharides require strong oxygenation and low temperature of the culture, due to the physiology of the producer strains. The medium should contain sucrose as a carbon source and ammonium sulfate as inorganic nitrogen source, wherein the C:N ratio in the substrate should be 15:1. The cultures are long and the largest accumulation of polymers is observed after 4 or 5 days of culturing. The structure of yeast EPS is complex which affects the strain and culture condition. The EPS from yeast are linear mannans, pullulan, glucooligosaccharides, galactooligosaccharides and other heteropolysaccharides containing α-1,2; α-1,3; α-1,6; ß-1,3; ß-1,4 bonds. Mannose and glucose have the largest participation of carbohydrates for. METHODS: t exopolysaccharides (EPS) are not a well-established group of metabolites. An industrial scale    of this EPS production is limited mainly by low yield biosynthesis. Until now, enzymes and biosynthesis pathways, as well as the role of regulatory genes, have not been described. Some of yeast EPS show antitumor, immunostimulatory and antioxidant activity. Others, absorb heavy metals and can function as bioactive components of food. Also, the potential of yeast EPS as thickeners or stabilizers can be found. Optimal conditions for the biosynthesis of yeast exopolysaccharides require strong oxygenation and low temperature of the culture, due to the physiology of the producer strains. The medium should contain sucrose as a carbon source and ammonium sulfate as inorganic nitrogen source, wherein the C:N ratio in the substrate should be 15:1. The cultures are long and the largest accumulation of polymers is observed after 4 or 5 days of culturing. The structure of yeast EPS is complex which affects the strain and culture condition. The EPS from yeast are linear mannans, pullulan, glucooligosaccharides, galactooligosaccharides and other heteropolysaccharides containing α-1,2; α-1,3; α-1,6; ß-1,3; ß-1,4 bonds. Mannose and glucose have the largest participation of carbohydrates formin. RESULTS: t exopolysaccharides (EPS) are not a well-established group of metabolites. An industrial scale    of this EPS production is limited mainly by low yield biosynthesis. Until now, enzymes and biosynthesis pathways, as well as the role of regulatory genes, have not been described. Some of yeast EPS show antitumor, immunostimulatory and antioxidant activity. Others, absorb heavy metals and can function as bioactive components of food. Also, the potential of yeast EPS as thickeners or stabilizers can be found. Optimal conditions for the biosynthesis of yeast exopolysaccharides require strong oxygenation and low temperature of the culture, due to the physiology of the producer strains. The medium should contain sucrose as a carbon source and ammonium sulfate as inorganic nitrogen source, wherein the C:N ratio in the substrate should be 15:1. The cultures are long and the largest accumulation of polymers is observed after 4 or 5 days of culturing. The structure of yeast EPS is complex which affects the strain and culture condition. The EPS from yeast are linear mannans, pullulan, glucooligosaccharides, galactooligosaccharides and other heteropolysaccharides containing α-1,2; α-1,3; α-1,6; ß-1,3; ß-1,4 bonds. Mannose and glucose have the largest participation of carbohydrates forming EPS.

Evaluation of the efficiency of different disruption methods on yeast cell wall preparation for ß-glucan isolation.

Selected methods for yeast cell disruption were evaluated to establish their suitability for cell wall preparation in the process of ß-glucan isolation. The effect of different disruption methods on contents of total saccharides, ß-glucans and proteins in the produced cell walls preparations was analyzed. The degree of cell wall purification from intracellular components was established on the basis of the ratio of solubilised material. The investigated methods included: cell exposure to hot water (autoclaving), thermally-induced autolysis, homogenization in a bead mill, sonication and their combinations. Experimental systems were prepared in water (pH 5.0 and pH 7.0) and Tris-HCl buffer (pH 8.0). The Saccharomyces cerevisiae yeast cell wall preparations with the highest degree of cytosol component release and purification of ß-glucans were produced by 30 min of cell homogenization with zirconium-glass beads (0.5 mm in diameter). This was confirmed by the highest ratio of solubilised material (approx. 64%-67%). The thus-produced preparations contained ca. 60% of total saccharides, 13%-14% of ß(1,3)/(1,6)-glucans, and approx. 35% of crude proteins. Similar results were obtained after autolysis coupled with bead milling as well as with sonication, but the time required for these processes was more than 24 h. Homogenization in a bead mill could be valuable for general isolation procedures because allows one to eliminate the different autolytic activity of various yeast strains.

Application of immobilized cell preparation obtained from biomass of Gluconacetobacter xylinus bacteria in biotransformation of glycerol to dihydroxyacetone.

INTRODUCTION: Dihydroxyacetone (DHA), being a product of glycerol oxidation by acetic acid bacteria, is an important compound widely applied in the cosmetic, food, and pharmaceutical industry, as well as in medicine. Biotransformation of glycerol to DHA is catalyzed by glycerol dehydrogenase (GlyDH, EC 1.1.1.6) bound with the cytoplasmic membrane of bacteria. An attempt was undertaken in this study to conduct glycerol biotransformation with immobilized fractions of a cell preparation with GlyDH activity. The content of dihydroxyacetone obtained with the cell preparation was compared with its content achieved in the reaction with immobilized viable cells of G. xylinus. MATERIAL AND METHODS: Cell walls of Gluconacetobacter xylinus bacteria were disintegrated enzymatically. The resultant preparation was immobilized on calcium alginate or first separated into two fractions (precipitate and supernatant) by centrifugation and then immobilized. DHA content was determined colorimetrically after the reaction with 3,5-dinitrosalicilic acid. Glycerol content was assayed with the refractometric method. RESULTS: After 20 days of the process, the concentration of DHA obtained with immobilized whole cells reached 25 g/l. In turn, the content of DHA obtained in the same period with immobilized fractions of the cell preparation accounted for 16.9 g/l and 8.95 g/l (depending on the fraction applied). CONCLUSIONS: DHA may be obtained in the process independent of G. xylinus metabolic activity using a preparation which displays the catalytic activity of glycerol dehydrogenase and obtained as a result of disintegration of live bacterial cells. The application of such a preparation may in the future eliminate technological problems posed by the presence of bacterial cells and their metabolites in the culture medium.