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 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.

Antimicrobial Susceptibility of Lactic Acid Bacteria Strains of Potential Use as Feed Additives - The Basic Safety and Usefulness Criterion.

The spread of resistance to antibiotics is a major health concern worldwide due to the increasing rate of isolation of multidrug resistant pathogens hampering the treatment of infections. The food chain has been recognized as one of the key routes of antibiotic resistant bacteria transmission between animals and humans. Considering that lactic acid bacteria (LAB) could act as a reservoir of transferable antibiotic resistance genes, LAB strains intended to be used as feed additives should be monitored for their safety. Sixty-five LAB strains which might be potentially used as probiotic feed additives or silage inoculants, were assessed for susceptibility to eight clinically relevant antimicrobials by a minimum inhibitory concentration determination. Among antimicrobial resistant strains, a prevalence of selected genes associated with the acquired resistance was investigated. Nineteen LAB strains displayed phenotypic resistance to one antibiotic, and 15 strains were resistant to more than one of the tested antibiotics. The resistance to aminoglycosides and tetracyclines were the most prevalent and were found in 37 and 26% of the studied strains, respectively. Phenotypic resistance to other antimicrobials was found in single strains. Determinants related to resistance phenotypes were detected in 15 strains as follows, the aph(3″)-IIIa gene in 9 strains, the lnu(A) gene in three strains, the str(A)-str(B), erm(B), msr(C), and tet(M) genes in two strains and the tet(K) gene in one strain. The nucleotide sequences of the detected genes revealed homology to the sequences of the transmissible resistance genes found in lactic acid bacteria as well as pathogenic bacteria. Our study highlights that LAB may be a reservoir of antimicrobial resistance determinants, thus, the first and key step in considering the usefulness of LAB strains as feed additives should be an assessment of their antibiotic resistance. This safety criterion should always precede more complex studies, such as an assessment of adaptability of a strain or its beneficial effect on a host. These results would help in the selection of the best LAB strains for use as feed additives. Importantly, presented data can be useful for revising the current microbiological cut-off values within the genus Lactobacillus and Pediococcus.

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.

Contents of Functionally Bioactive Peptides, Free Amino Acids, and Biogenic Amines in Dutch-Type Cheese Models Produced with Different Lactobacilli.

Cheese ripening involves a number of biochemical processes, mainly of a proteolytic nature, which are initially triggered principally by milk-coagulating enzymes and, afterward, by microorganisms or enzymes of microbial origin. The proteolytic reactions affect, primarily, the synthesis of macro- and medium-molecular peptides from casein. In turn, the advanced proteolysis ends in the formation of short peptides and free amino acids. Further reactions may lead to the formation of nutritionally unfavorable biogenic amines. The present study aimed to determine changes in the contents of bioactive peptides (anserine and L-carnosine), free amino acids, and biogenic amines throughout the ripening of cheese models produced with the addition of Lactobacillus genus bacteria. The contents of amino acids varied considerably in the cheese models, depending on the bacterial strain added and ripening time. After five weeks of ripening, the total content of free amino acids in the cheese models ranged from 611.02 (a cheese model with Lactobacillus casei 2639) to 1596.64 mg kg-1 (a cheese model with Lb. acidophilus 2499). After the same time, the contents of the total biogenic amines in the cheese models with the addition of lactobacilli were lower than in the control cheese model (except for the model with Lb. rhamnosus 489). Anserine was detected in all cheese models (79.29-119.02 mg kg-1), whereas no L-carnosine was found over a five-week ripening period in the cheese models with Lb. delbrueckii 490 and Lb. casei 2639. After a five-week ripening, the highest total content of bioactive peptides was determined in the cheese models containing Lb. acidophilus 2499 (136.11 mg kg-1).

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.

Impact of Nisin-Producing Strains of Lactococcus lactis on the Contents of Bioactive Dipeptides, Free Amino Acids, and Biogenic Amines in Dutch-Type Cheese Models.

The goal of this study was to determine changes in contents of free amino acids, biogenic amines, and bioactive dipeptides (anserine and L-carnosine) in cheese models produced with the addition of nisin-producing strains of Lactococcus lactis over their ripening period. After 5 weeks of ripening, contents of total biogenic amines in the cheese models with the addition of L. lactis strains were lower than in the control cheese model. The cheese models examined differed significantly in contents of free amino acids through the ripening period. Individual free amino acids, such as ornithine, were found in some of the cheese models, which is indicative of their specific microbial activities. Both anserine and L-carnosine were detected in all variants of the cheese models. After 5-week ripening, the highest total content of bioactive dipeptides was determined in the cheese models produced with the nisin-producing culture of L. lactis 11454 (142.15 mg∙kg-1).

Prevalence and toxicity characterization of Bacillus cereus in food products from Poland.

The prevalence of Bacillus cereus in a total of 585 samples of food products (herbs and spices, breakfast cereals, pasta, rice, infant formulas, pasteurized milk, fresh acid and acid/rennet cheeses, mold cheeses and ripening rennet cheeses) marketed in Poland was investigated. The potential of 1022 selected isolates of B. cereus to hydrolyze casein, starch and tributyrin, to ferment lactose, to grow at 7 C/10 days, to produce Nhe and Hbl toxin and to possess the ces gene was verified. B. cereus was found in 38.8% of the analyzed samples, reaching levels from 0.3 to 3.8 log CFU g-1 or mL-1. From the 1022 isolates, 48.8%, 36.0%, 98.9%, 80.0% and 25.0% were capable of fermenting lactose, producing amylase, protease, lipase and growing at 7 C/10 days, respectively, indicating spoilage potentiality. The occurrence of toxigenic B. cereus strains in all tested market products, both of plant (55.8% Hbl(+), 70.7% Nhe(+) and 1.7% ces(+) isolates) and animal origin (84.9% Hbl(+), 82.7% Nhe(+) and 0.9% ces(+) isolates) indicates the possible risk of foodborne infections/intoxications that occur as a result of the possibility of the development of B. cereus in favorable conditions and consumption of these products.

Microbiological quality of selected ready-to-eat leaf vegetables, sprouts and non-pasteurized fresh fruit-vegetable juices including the presence of Cronobacter spp.

Bacteria of the genus Cronobacter are emerging food-borne pathogens. Foods contaminated with Cronobacter spp. may pose a risk to infants or adults with suppressed immunity. This study was aimed at determining the microbiological quality of ready-to-eat (RTE) plant-origin food products available on the Polish market with special emphasis on the prevalence of Cronobacter genus bacteria. Analyses were carried out on 60 samples of commercial RTE type plant-origin food products, including: leaf vegetables (20 samples), sprouts (20 samples) and non-pasteurized vegetable, fruit and fruit-vegetable juices (20 samples). All samples were determined for the total count of aerobic mesophilic bacteria (TAMB) and for the presence of Cronobacter spp. The isolates of Cronobacter spp. were subjected to genetic identification and differentiation by 16S rDNA sequencing, PCR-RFLP analysis and RAPD-PCR and evaluation of antibiotic susceptibility by the disk diffusion assay. The TAMB count in samples of lettuces, sprouts and non-pasteurized fruit, vegetable and fruit-vegetable juices was in the range of 5.6-7.6, 6.7-8.4 and 2.9-7.7 log CFU g-1, respectively. The presence of Cronobacter spp. was detected in 21 (35%) samples of the products, including in 6 (30%) samples of leaf vegetables (rucola, lamb's lettuce, endive escarola and leaf vegetables mix) and in 15 (75%) samples of sprouts (alfalfa, broccoli, small radish, lentil, sunflower, leek and sprout mix). No presence of Cronobacter spp. was detected in the analyzed samples of non-pasteurized fruit, vegetable and fruit-vegetable juices. The 21 strains of Cronobacter spp. isolated from leaf vegetable and sprouts included: 13 strains of C. sakazakii, 4 strains of C. muytjensii, 2 strains of C. turicensis, one strain of C. malonaticus and one strain of C. condimenti. All isolated C. sakazakii, C. muytjensii, C. turicensis and C. malonaticus strains were sensitive to ampicillin, cefepime, chloramphenicol, gentamycin, streptomycin, tetracycline, ciprofloxacin and cotrimoxazol, whereas the C. condimenti isolate showed intermediate resistance to streptomycin and cotrimoxazole.

The effect of selected factors on the survival of Bacillus cereus in the human gastrointestinal tract.

Bacillus cereus is a Gram-positive bacterium widely distributed in soil and vegetation. This bacterial species can also contaminate raw or processed foods. Pathogenic B. cereus strains can cause a range of infections in humans, as well as food poisoning of an emetic (intoxication) or diarrheal type (toxico-infection). Toxico-infections are due to the action of the Hbl toxin, Nhe toxin, and cytotoxin K produced by the microorganism in the gastrointestinal tract. This occurs once the spores or vegetative B. cereus cells survive the pH barrier of the stomach and reach the small intestine where they produce toxins in sufficient amounts. This article discusses the effect of various factors on the survival of B. cereus in the gastrointestinal tract, including low pH and the presence of digestive enzymes in the stomach, bile salts in the small intestine, and indigenous microflora in the lower parts of the gastrointestinal tract. Additional aspects also reported to affect B. cereus survival and virulence in the gastrointestinal tract include the interaction of the spores and vegetative cells with enterocytes. In vitro studies revealed that both vegetative B. cereus and spores can survive in the gastrointestinal tract suggesting that the biological form of the microorganism may have less influence on the occurrence of the symptoms of infection than was once believed. It is most likely the interaction between the pathogen and enterocytes that is necessary for the diarrheal form of B. cereus food poisoning to develop. The adhesion of B. cereus to the intestinal epithelium allows the bacterium to grow and produce enterotoxins in the proximity of the epithelium. Recent studies suggest that the human intestinal microbiota inhibits the growth of vegetative B. cereus cells considerably.

Dipeptidase activity and growth of heat-treated commercial dairy starter culture.

Growing expectations of consumers of fermented dairy products urge the search for novel solutions that would improve their organoleptic properties and in the case of rennet cheeses-that would also accelerate their ripening process. The aim of this study was to determine the peptidolytic activities and growth of heat-treated commercial culture of lactic acid bacteria. The analyzed culture was characterized by a relatively high peptidolytic activity. The growth of bacterial culture subjected to heat treatment at 50-80 °C for 15 s, 10 and 3 min was delayed by a few or 10-20 h compared to the control culture. Based on the results achieved, it may be concluded that in the production of rennet cheeses, the application of additional, fermentation-impaired starter cultures (via heating for ten or so minutes) may serve to accelerate their ripening and to improve their sensory attributes.