Effects of calcium chloride substitution on the physicochemical properties of Minas Frescal cheese.

The aim in this research paper was to investigate the effect of using calcium monophosphate (MCP) and MCP mixed with commercial phosphates salts, in total or partial replacement of calcium chloride (CaCl2) in the manufacture of Minas Frescal cheese. Initially, model cheeses were made to perform the rheological analysis during the coagulation process. Of these, the five best treatments were chosen to carry out the production of Minas Frescal cheese, used only CaCl2 and MCP, and partial replacements of MCP + polyphosphate, MCP + potassium monophosphate (MKP) and MCP. The cheeses showed no significant difference in physicochemical composition, yield and syneresis, however, the cheese with partial replacement of CaCl2 by MCP + polyphosphate and MCP + MKP showed the highest hardness values, like the control. This demonstrates that it is possible to replace calcium chloride without significant changes in the physicochemical characteristics and yield of Minas Frescal cheese, and it is still possible to modulate the hardness of the cheese produced according to the type of calcium/phosphate source used. This allows the industry to replace the source of calcium in the manufacture of Minas Frescal cheese according to the desired hardness.

Pseudomonas carnis isolated from blue discolored fresh cheese and insights into the phylogeny.

Most Pseudomonas spp. are responsible for spoilage in refrigerated foods such as alteration in flavor, texture and appearance. Samples of Minas Frescal cheese with blue discoloration were analysed and contained a high Pseudomonas concentration (7.72 ± 0.36 log CFU/g). Out of the 26 Pseudomonas isolates that were analyzed in our study, 19 demonstrated the capability of producing a diffusible dark pigment. Thus, a pigment-producing isolate (C020) was selected by rep-PCR fingerprinting and subsequently subjected to whole-genome sequencing. The draft genome assembled comprises 42 contigs totaling 6,366,75 bp with an average G + C content of 59.97%, and the species prediction performed by TYGS server, based on the draft genome sequence, identified the C020 as Pseudomonas carnis. In order to investigate the phylogenetic relationships of this isolate with strains already identified of this species, we performed an analysis based on whole-genomic sequences. First, an analysis of all P. carnis genomes deposited in GenBank to date shows that 11% (4/37) are misidentified, and belong to the Pseudomonas paracarnis species. A comparative analysis based on phylogenomic analysis has showed that there is no evolutionary relationship between P. carnis strains carrying second copies of trp genes related to blue discoloration (trpABCDF). This finding reinforces the assertion that these genes are contained in a mobile genetic element. However, it is worth noting that all strains carrying these secondary gene copies have exclusively been isolated from food sources. This observation provides valuable insights into the potential origins and dispersion dynamics of this genetic trait within the species.

Challenges associated with spray drying of lactic acid bacteria: Understanding cell viability loss.

Lactic acid bacteria (LAB) cultures used in food fermentation are often dried to reduce transportation costs and facilitate handling during use. Dried LAB ferments are generally lyophilized to ensure high cell viability. Spray drying has come to the forefront as a promising technique due to its versatility and lower associated energy costs. Adverse conditions during spray drying, such as mechanical stress, dehydration, heating, and oxygen exposure, can lead to low LAB cell viability. This reduced viability has limited spray drying's industrial applications thus far. This review aims to demonstrate the operations and thermodynamic principles that govern spray drying, then correlate them to the damage suffered by LAB cells during the spray-drying process. The particularities of spray drying that might cause LAB cell death are detailed in this review, and the conclusion may enhance future studies on ways to improve cell viability.

The ability of Lactococcus lactis subsp. lactis bv. diacetylactis strains in producing nisin.

Lactococcus lactis subsp. lactis bv. diacetylactis is a relevant microorganism for the dairy industry because of its role in the production of aromatic compounds. Despite this technological property, the identification of bacteriocinogenic potential of obtained strains can offer the additional positive aspect of biosafety. A panel of 15 L. lactis subsp. lactis bv. diacetylactis strains was characterised for the presence and expression of bacteriocin related genes, and further investigated regarding the nisin operon. Eight strains were positive only for nisA, and one strain (SBR4) presented a full nisin operon, with sequencing that was shown to be similar to nisin Z. Only SBR4 presented inhibitory activity against 16 microbial target strains. The growth curves of selected targets strains confirmed the inhibitory activity of SBR4 and consequently the nisin production. This research has demonstrated the inhibitory potential of L. lactis subsp. lactis bv. diacetylactis strain, SBR4, due to its ability to produce nisin Z. This biopreservative potential, associated to previously characterised technological properties, allow the indication of this strain as a promising candidate to be used by the dairy industry as a starter or adjunct culture.

Modification of polysulfone membrane used in the water filtration process to reduce biofouling.

Polysulfone membranes (PSF) were modified with silver nanoparticles obtained by new synthesis (nAgNS), silver nanoparticles obtained commercially (nAgC), silver sulfadiazine (SP), dodecyltrimethylammonium bromide (DOTAB), benzalkonium chloride (CB) or sodium dodecylbenzene sulfonate (DBSS) to improve the efficiency of the water filtration process by reducing biofouling. All membranes had lower hydrophobicity compared with PSF. The zeta potentials of all membranes were negative at pH 7.0, except for CB 10%. In the agar diffusion test, E. coli was considered to be sensitive to the antimicrobial effect of the nAgNS 1%, 3%, 6%, 10% and DOTAB 10%, whereas S. aureus was sensitive to the nAgNS 1%, 3%, 6%, 10%, DOTAB 10%, CB 0.22%, 2% and 10%. The lowest adhesion of E. coli was found in the nAgNS 6% and 10%. In the evaluation of the loss of flow rate during filtration of the E. coli suspension and pure water, nAgNS showed higher flow rate values when compared with PSF. The nAgNS did not release quantities of silver (0.1 mg/l) above the amount considered safe by the World Health Organization. Membranes nAgNS 6% and 10% showed the best anti-biofouling characteristic.

Development of a selective culture medium for bifidobacteria, Raffinose-Propionate Lithium Mupirocin (RP-MUP) and assessment of its usage with Petrifilm™ Aerobic Count plates.

This study aimed to develop a selective culture media to enumerate bifidobacteria in fermented milk and to assess this medium when used with Petrifilm™ AC plates. For this purpose, Bifidobacterium spp., Lactobacillus spp. and Streptococcus thermophilus strains were tested to verify their fermentation patterns for different carbohydrates. All bifidobacteria strains were able to use raffinose. Based on these characteristic, a selective culture medium was proposed (Raffinose-Propionate Lithium Mupirocin, RP-MUP), used with Petrifilm™ AC plates, and was used to enumerate bifidobacteria in fermented milk. RP-MUP performance was assessed by comparing the results with this medium to reference protocols and culture media for bifidobacteria enumeration. RP-MUP, whether used or not with Petrifilm™ AC, presented similar performance to TOS-MUP (ISO 29981), with no significant differences between the mean bifidobacteria counts (p < 0.05) and with high correlation indices (r = 0.99, p < 0.05). As an advantage, reliable results were obtained after just 48 h of incubation when RP-MUP was used with Petrifilm™ AC, instead of the 72 h described in the ISO 29981 protocol.

Development of an alternative culture medium for the selective enumeration of Lactobacillus casei in fermented milk.

Monitoring the populations of probiotic strains of the species Lactobacillus casei in food is required by food industries in order to assure that a minimum concentration of these organisms will be ingested by consumers. In this context, Petrifilm™ AC plates can be used along with selective culture media to allow the enumeration of specific groups of lactic acid bacteria. The present study aimed to assess chemical substances as selective agents for Lb. casei in order to propose a selective culture medium to be used with Petrifilm™ AC plates as an alternative protocol for the enumeration of probiotic strains of this species in fermented milk. Twenty-six probiotic and starter cultures (including six strains of Lb. casei) were plated on de Man Rogosa and Sharpe (MRS) agar with distinct concentrations of nalidixic acid, bile, lithium chloride, metronidazole, sodium propionate, and vancomycin. Vancomycin at 10 mg/L demonstrated selective activity for Lb. casei. In addition, 2,3,5-triphenyltetrazolium chlorine was identified as a compound that did not inhibit Lb. casei, and Petrifilm™ AC plates used with MRS and vancomycin at 10 mg/L (MRS-V) demonstrated more colonies of this organism when incubated under anaerobic conditions than aerobic conditions. Acidophilus milk and yoghurt were prepared, added to Lb. casei strains, and stored at 4 °C. Lb. casei populations were monitored using MRS-V and MRTLV by conventional plating and associated with Petrifilm™ AC plates. All correlation indices between counts obtained by conventional plating and Petrifilm™ AC were significant (p < 0.05), but the best performance was observed for growth on MRS-V. The obtained data indicate the efficiency of using MRS-V associated with Petrifilm™ AC plates for the enumeration of Lb. casei strains in fermented milk. However, the selective potential of this culture medium must be evaluated considering the specific strains of Lb. casei and the starter cultures inoculated in the fermented milk that requires monitoring.

Influence of different culture media on the antimicrobial activity of Pediococcus pentosaceus ST65ACC against Listeria monocytogenes.

Pediococcus pentosaceus ST65ACC is a bacteriocinogenic lactic acid bacteria (LAB) isolated from Brazilian artisanal cheese that is capable of inhibiting different food pathogens, mainly Listeria monocytogenes. The production of bacteriocins can be influenced by several growth conditions, such as temperature, pH, and medium composition. This study aimed to evaluate the effect of different culture media on the production of bacteriocins and antimicrobial activity of P. pentosaceus ST65ACC on L. monocytogenes Scott A. The strains were inoculated alone and in coculture in four different media: BHI broth, MRS broth, meat broth, and reconstituted skim milk (RSM) 10% (w/v). The culture media were then incubated at 37 °C for 96 h, and count analysis, pH measurement, and bacteriocin production were performed at 0, 24, 48, 72 and 96 h. L. monocytogenes was inhibited to nondetectable levels in coculture with P. pentosaceus ST65ACC in MRS broth within 96 h, consistent with the high production of bacteriocin throughout the analysis period (3,200-12,800 AU/mL). However, lower inhibitory activities of P. pentosaceus ST65ACC on L. monocytogenes Scott A were recorded in BHI, RSM, and meat broth, with low or no production of bacteriocins at the analyzed times. The composition of these culture media may have repressed the production and activity of bacteriocins and, consequently, the antagonist activity of P. pentosaceus ST65ACC on L. monocytogenes Scott A. The results showed that the antimicrobial activity was more effective in MRS broth, presenting greater production of bacteriocins and less variability when compared to the other media analyzed.

Assessment of safety and in situ antibacterial activity of Weissella cibaria strains isolated from dairy farms in Minas Gerais State, Brazil, for their food application.

Weissella cibaria W21, W25, and W42 strains have previously been characterized for their antagonism against a range of foodborne pathogens. However, prior to their use as protective agents, further analyses such as their safety and in situ activity are needed. The safety of W. cibaria W21, W25, and W42 strains was predicted in silico and confirmed experimentally. Analyses of their genomes using appropriate software did not reveal any acquired antimicrobial resistance genes, nor mobile genetic elements (MGEs). The survival of each strain was determined in vitro under conditions mimicking the gastrointestinal tract (GIT). Thus, hemolysis analysis was performed using blood agar and the cytotoxicity assay was determined using a mixture of two cell lines (80% of Caco-2 and 20% of HT-29). We also performed the inflammation and anti-inflammation capabilities of these strains using the promonocytic human cell line U937. The Weissella strains were found to be haemolysis-negative and non-cytotoxic and did not induce any inflammation. Furthermore, these strains adhered tightly to intestinal Caco-2 cell-lines and exerted in situ anti-proliferative activity against methicillin-resistant Staphylococcus aureus (strain MRSA S1) and Escherichia coli 181, a colistin-resistant strain. However, the W. cibaria strains showed low survival rate under simulated GIT conditions in vitro. The unusual LAB-strains W. cibaria strains W21, W25, and W42 are safe and endowed with potent antibacterial activities. These strains are therefore good candidates for industrial applications. The results of this study provide a characterization and insights into Weissella strains, which are considered unusual LAB, but which prompt a growing interest in their bio-functional properties and their potential industrial applications.

Microbiological safety of Minas Frescal Cheese (MFC) and tracking the contamination of Escherichia coli and Staphylococcus aureus in MFC processing.

Minas Frescal cheese (MFC) is a traditional food produced and consumed in Brazil, characterized by its soft texture, low sodium, and high moisture content. This study characterized the microbiological contamination by coliforms, Escherichia coli and Staphylococcus aureus, in 99 MFC samples obtained in retail sale and produced by three distinct industrial procedures. Dairy processors were selected to investigate the key points of E. coli and S. aureus contamination during cheese processing. MFC samples produced by the addition of lactic culture presented higher counts of coliforms and E. coli, when compared to other samples (p<0.05). MFC samples produced by the addition of rennet alone presented higher counts of S. aureus when compared to other samples (p<0.05). Fourteen of 19 MFC samples produced by the addition of lactic culture presented E. coli counts higher than 5 × 10(2) colon-forming units/g. The processing steps after pasteurization were identified as the main sources of E. coli and S. aureus contamination of MFC. Based on the results, MFC was characterized as a potential hazard for consumers due to the high frequency of samples contaminated with E. coli and S. aureus counts in noncompliance with Brazilian standards for sanitary quality and safety.

Selective enumeration of propionibacteria in Emmental-type cheese using Petrifilm™ aerobic count plates added to lithium glycerol broth.

Propionibacteria derived from dairy products are relevant starter cultures for the production of Swiss and Emmental-type cheeses, and the monitoring of which is mandatory for proper quality control. This study aimed to evaluate an alternative procedure to enumerate propionibacteria, in order to develop a reliable and practical methodology to be employed by dairy industries. 2,3,5-triphenyltetrazolium chloride (TTC) inhibitory activity was tested against five reference strains (CIRM 09, 38, 39, 40 and 116); TTC at 0·0025% (w/v) was not inhibitory, with the exception of one strain (CIRM 116). Subsequently, the four TTC-resistant strains, three commercial starter cultures (PS-1, PB-I, and CHOO) and twelve Emmental-type cheese samples were subjected to propionibacteria enumeration using Lithium Glycerol (LG) agar, and Petrifilm™ Aerobic Count (AC) plates added to LG broth (anaerobic incubation at 30 °C for 7 d). Petrifilm™ AC added to LG broth presented high counts than LG agar (P<0·05) for only two reference strains (CIRM 39, and 40) and for all commercial starter cultures. Cheese sample counts obtained by both procedures did not show significant differences (P<0·05). Significant correlation indexes were observed between the counts recorded by both methods (P<0·05). These results demonstrate the reliability of Petrifilm™ AC plates added to LG broth in enumerating select Propionibacterium spp., despite some limitations observed for specific commercial starter cultures.

Genomic Analyses of Pediococcus pentosaceus ST65ACC, a Bacteriocinogenic Strain Isolated from Artisanal Raw-Milk Cheese.

Pediococcus pentosaceus ST65ACC was obtained from a Brazilian artisanal cheese (BAC) and characterized as bacteriocinogenic. This strain presented beneficial properties in previous studies, indicating its potential as a probiotic candidate. In this study, we aimed to carry out a genetic characterization based on whole-genome sequencing (WGS), including taxonomy, biotechnological properties, bacteriocin clusters and safety-related genes. WGS was performed using the Illumina MiSeq platform and the genome was annotated with the Prokaryotic Genome Annotation (Prokka). P. pentosaceus ST65ACC taxonomy was investigated and bacteriocin genes clusters were identified by BAGEL4, metabolic pathways were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and safety-related genes were checked. P. pentosaceus ST65ACC had a total draft genome size of 1,933,194 bp with a GC content of 37.00%, and encoded 1950 protein coding sequences (CDSs), 6 rRNA, 55 tRNA, 1 tmRNA and no plasmids were detected. The analysis revealed absence of a CRISPR/Cas system, bacteriocin gene clusters for pediocin PA-1/AcH and penocin-A were identified. Genes related to beneficial properties, such as stress adaptation genes and adhesion genes, were identified. Furthermore, genes related to biogenic amines and virulence-related genes were not detected. Genes related to antibiotic resistance were identified, but not in prophage regions. Based on the obtained results, the beneficial potential of P. pentosaceus ST65ACC was confirmed, allowing its characterization as a potential probiotic candidate.

Phylogenetic characterization and biodiversity of spore-forming bacteria isolated from Brazilian UHT milk.

Among the milk contaminating microorganisms, those which are able to form heat-resistant spores are concerning, especially for dairy companies that use ultra-high temperature (UHT) technology. These spores, throughout storage, can germinate and produce hydrolytic enzymes that compromise the quality of the final product. This study evaluated 184 UHT milk samples from different batches collected from seven Brazilian dairy companies with a possible microbial contamination problem. The bacteria were isolated, phenotypically characterized, clustered by REP-PCR, and identified through 16S rDNA sequencing. The presence of Bacillus sporothermodurans was verified using biochemical tests (Gram staining, catalase and oxidase test, glucose fermentation, esculin hydrolysis, nitrate reduction, and urease test). According to these tests, none of the isolates presented typical characteristics of B. sporothermodurans. In sequence, the isolates, that presented rod-shapes, were submitted to molecular analyses in order to determine the microbial biodiversity existing among them. The isolates obtained were grouped into 16 clusters, four of which were composed of only one individual. A phylogenetic tree was constructed using the sequences obtained from the 16S rDNA sequencing and some reference strains of species close to those found using BLAST search in the NCBI nucleotide database. Through this tree, it was possible to verify the division of the isolates into two large groups, the Bacillus subtilis and the Bacillus cereus groups. Furthermore, most isolates are phylogenetically closely related, which makes it even more difficult to identify them at the species level. In conclusion, it was possible to assess, in general, the groups of sporulated contaminants in Brazilian UHT milk produced in the regions evaluated. In addition, it was also possible to determine the biodiversity of spore-forming bacteria found in UHT milk samples, thus opening up a range of possible research topics regarding the effects of the presence of these microorganisms on milk quality.

Technological Characterization of Lactic Acid Bacteria Strains for Potential Use in Cheese Manufacture.

A total of 26 lactic acid bacteria isolates from both Italian and Brazilian cheeses were tested for their use in cheesemaking. Isolates were screened for salt tolerance, exopolysaccharide and diacetyl production, lipolytic, acidifying, and proteolytic activities. In addition, the aminopeptidase (Pep N and Pep X) activities, were evaluated. Most of the strains demonstrated salt tolerance to 6% of NaCl, while only two L. delbruekii (P14, P38), one L. rhamnosus (P50) and one L. plantarum (Q3C4) were able to grow in the presence of 10% (w/v) of NaCl. Except for 2 L. plantarum (Q1C6 and Q3C4), all strains showed low or medium acidifying activity and good proteolytic features. Furthermore, lipolytic activity was revealed in none of the strains, while the production of EPS and diacetyl was widespread and variable among the tested strains. Finally, regarding aminopeptidase activities, 1 L. delbrueckii (P10), 1 L. rhamnosus (P50), and 1 L. lactis (Q5C6) were considered as the better performing, showing high values of both Pep N and Pep X. Based on data presented here, the aforementioned strains could be suggested as promising adjunct cultures in cheesemaking.

High-intensity ultrasound treatment on casein: Pea mixed systems: Effect on gelling properties.

This study aimed to investigate the suitability of the application of high-intensity ultrasounds (HIUS) to improve the acid induced gelation of mixed protein systems formed by casein micelles (CMs) and pea. The protein suspensions were prepared in different protein ratios CMs: pea (100:0, 80:20, 50:50, 20:80, 0:100) at 8% (w/w) total protein concentration. In the suspensions, the ultrasound treatment produced an increase in solubility, surface hydrophobicity, and a decrease in the samples' viscosity, with more remarkable differences in protein blends in which pea protein was the major component. However, the replacement of 20% of CMs for pea proteins highly affected the gel elasticity. Hence, the creation of smaller and more hydrophobic building blocks before acidification due to the HIUS treatment increased the elasticity of the gels up to 10 times. Therefore, high-intensity ultrasounds are a suitable green technique to increase the gelling properties of CMs: pea systems.

Strategies for the Development of Bioprotective Cultures in Food Preservation.

Consumers worldwide are increasingly demanding food with fewer ingredients, preferably without chemical additives. The trend called "Clean Label" has stimulated the development and commercialization of new types of bioprotective bacterial cultures. These bacteria are not considered new, and several cultures have been available on the market. Additionally, new bioprotective bacteria are being identified to service the clean label trend, extend the shelf life, and, mainly, improve the food safety of food. In this context, the lactic acid bacteria (LAB) have been extensively prospected as a bioprotective culture, as they have a long history in food production and their antimicrobial activity against spoilage and pathogenic microorganisms is well established. However, to make LAB cultures available in the market is not that easy, the strains should be characterized phenotypically and genotypically, and studies of safety and technological application are necessary to validate their bioprotection performance. Thus, this review presents information on the bioprotection mechanisms developed by LAB in foods and describes the main strategies used to identify and characterize bioprotective LAB with potential application in the food industry.

Further culture-independent characterization of the lactic microbiota of Serro artisanal cheese.

This study aimed to provide a further characterization of the lactic microbiota present in Minas artisanal cheese (MAC) from the Serro region by using culture-independent methods, as a complementary analysis of a previous study. The total DNA extracted from MAC samples (n = 55) was subjected to repetitive extragenic palindromic-PCR (rep-PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Rep-PCR analysis showed that core microbiota of Serro MAC was closely related, independent of the production town, farm size, or time of production. The sequencing of PCR-DGGE bands identified the prevalence of Lactococcus lactis in all samples, and Streptococcus salivarius was also identified. Thus, we conclude that when more accurate methods are unavailable, rep-PCR can be used as a culture-independent method to demonstrate if the microbiota is closely related or not among the samples. PCR-DGGE results also matched to the main findings of high-throughput sequencing, previously presented, confirming its confidence to detect the main microbial groups present in the raw milk cheeses.

Genomic Analyses of Weissella cibaria W25, a Potential Bacteriocin-Producing Strain Isolated from Pasture in Campos das Vertentes, Minas Gerais, Brazil.

Weissella is a genus containing Gram-positive, heterofermentative bacteria belonging to the lactic acid bacteria (LAB) group. These bacteria are endowed with promising technological and antimicrobial attributes. Weissella cibaria W25 was isolated from a dairy environment where raw milk cheeses are produced. Therefore, we sequenced and assembled the W25 draft genome sequence, which consists of 41 contigs totaling ~2.4 Mbp, with a G + C content of 45.04%. Then we carried out a comprehensive comparative genomic analysis with W. cibaria 110, known to produce the weissellicin 110 bacteriocin, and four other non-bacteriocin-producing W. cibaria strains.

Colloidal and Acid Gelling Properties of Mixed Milk and Pea Protein Suspensions.

The present study aims to describe colloidal and acid gelling properties of mixed suspensions of pea and milk proteins. Mixed protein suspensions were prepared by adding pea protein isolate to rehydrated skimmed milk (3% w/w protein) to generate four mixed samples at 5, 7, 9, and 11% w/w total protein. Skimmed milk powder was also used to prepare four pure milk samples at the same protein concentrations. The samples were analyzed in regard to their pH, viscosity, color, percentage of sedimentable material, heat and ethanol stabilities, and acid gelling properties. Mixed suspensions were darker and presented higher pH, viscosity, and percentage of sedimentable material than milk samples. Heat and ethanol stabilities were similar for both systems and were reduced as a function of total protein concentration. Small oscillation rheology and induced syneresis data showed that the presence of pea proteins accelerated acid gel formation but weakened the final structure of the gels. In this context, the results found in the present work contributed to a better understanding of mixed dairy/plant protein functionalities and the development of new food products.

Gelling properties of black soldier fly (Hermetia illucens) larvae protein after ultrasound treatment.

H. illucens, black soldier fly larvae (BSFL) is one of the sustainable sources of protein. However, the research on the functionality of BSFL proteins is limited and need to be explored to increase consumer acceptance. The aim of this study is to create a gel system from BSFL protein and evaluate the impact of ultrasound treatment at different exposure time (5, 15, 30 min) on the physicochemical properties of BSFL protein. The highest values for surface hydrophobicity, size, ζ-potential were obtained after 15 min of ultrasound treatment and the same was found for the elastic modulus. Finally, confocal laser scanning microscopy (CLSM) along with image analysis revealed the lowest pore size after 15 min of treatment. The high protein content of BSFL protein extract and its promising gel system herein created, are important features to be considered for further development of insect-based food.