Whey Cheeses Containing Probiotic and Bioprotective Cultures Produced with Ultrafiltrated Cow's Whey.

Bovine whey cheese (WC) is a product from southern European countries that presents some challenges: its production process involves high energy inputs; the yield is low; and WC has a short shelf life. The application of ultrafiltration (UF) to bovine whey before manufacture of WC and the employment of protective cultures can reduce these disadvantages. The objective of this research was the production of whey cheeses using ultrafiltrated bovine cheese whey with added probiotics or probiotics plus protective cultures. Three types of WC were produced: control CW without any addition (C); CW with the addition of the probiotic Lactobacillus acidophilus (LA5); and CW with the addition of Lactobacillus acidophilus plus a protective culture containing Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus (LA5FQ4). The WCs were stored under refrigerated conditions for 28 days. The products with added cultures presented lower pH values and higher titratable acidities when compared to the control. Sample LA5 presented the lowest pH and the highest titratable acidity, while LA5FQ4 presented intermediate values. Slight differences were observed between products regarding color parameters, chiefly resulting from storage time. The samples with added cultures were firmer when compared to the control, with LA5 cheeses showing the highest values at the end of the storage. Lactic acid bacteria (LAB) counts were on the order of log 8-9 CFU/g for the products with added cultures. Lower levels of yeasts and molds were detected on the sample with the protective culture (LA5FQ4), so that by the end of storage they presented counts one log cycle lower than C and LA5. Hence, the beneficial impact of the protective culture on the shelf life of the product is evident. Regarding sensory evaluation, LA5FQ4 cheeses obtained the highest scores for all parameters evaluated. It can be concluded that the use of UF associated with the addition of protective cultures can be very useful to reduce the energy consumption of the manufacturing process, to prolong the shelf life of WC and to improve its sensory properties.

Context matters: environmental microbiota from ice cream processing facilities affected the inhibitory performance of two lactic acid bacteria strains against Listeria monocytogenes.

IMPORTANCE: Antilisterial LAB strains have been proposed as biological control agents for application in food processing environments. However, the effect of resident food processing environment microbiota on the performance on antilisterial LAB strains is poorly understood. Our study shows that the presence of microbiota collected from ice cream processing facilities' environmental surfaces can affect the attachment and inhibitory effect of LAB strains against L. monocytogenes. Further studies are therefore needed to assess whether individual microbial taxa affect antilisterial properties of LAB strains and to characterize the underlying mechanisms.

Protective Cultures in Food Products: From Science to Market.

An ultimate goal in food production is to guarantee food safety and security. Fermented food products benefit from the intrinsic capabilities of the applied starter cultures as they produce organic acids and bactericidal compounds such as hydrogen peroxide that hamper most food pathogens. In addition, highly potent small peptides, bacteriocins, are being expelled to exert antibiotic effects. Based on ongoing scientific efforts, there is a growing market of food products to which protective cultures are added exclusively for food safety and for prolonged shelf life. In this regard, most genera from the order Lactobacillales play a prominent role. Here, we give an overview on protective cultures in food products. We summarize the mode of actions of antibacterial mechanisms. We display the strategies for the isolation and characterization of protective cultures in order to have them market-ready. A survey of the growing market reveals promising perspectives. Finally, a comprehensive chapter discusses the current legislation issues concerning protective cultures, leading to the conclusion that the application of protective cultures is superior to the usage of defined bacteriocins regarding simplicity, economic costs, and thus usage in less-developed countries. We believe that further discovery of bacteria to be implemented in food preservation will significantly contribute to customer's food safety and food security, badly needed to feed world's growing population but also for food waste reduction in order to save substantial amounts of greenhouse gas emissions.

Targeting Enterococci with Antimicrobial Activity against Clostridium perfringens from Poultry.

Necrotic enteritis (NE), caused by Clostridium perfringens, is an emerging issue in poultry farming. New approaches, other than antibiotics, are necessary to prevent NE development and the emergence of multidrug-resistant bacteria. Enterococci are commensal microorganisms that can produce enterocins, antimicrobial peptides with activities against pathogens, and could be excellent candidates for protective cultures. This study aimed to screen and characterize Enterococcus strains of poultry origin for their inhibitory activity against C. perfringens. In total, 251 Enterococcus strains of poultry origin plus five bacteriocin-producing (BP+) E. durans strains of other origins were screened for antimicrobial activity against the indicator C. perfringens X2967 strain using the "spot on the lawn" method. We detected thirty-two BP+ strains (eleven Enterococcus faecium, nine E. gallinarum, eight E. faecalis, three E. durans, and one E. casseliflavus). We further studied the antimicrobial activity of the supernatants of these 32 BP+ strains using agar well diffusion and microtitration against a collection of 20 C. perfringens strains. Twelve BP+ enterococci that were found to exhibit antimicrobial activity against C. perfringens were characterized using whole genome sequencing. Among these, E. faecium X2893 and X2906 were the most promising candidates for further studies as protective cultures for poultry farming. Both strains belong to the sequence type ST722, harbor the genes encoding for enterocin A and enterocin B, do not possess acquired resistance genes, do not carry plasmids, and present the acm gene, which is implicated in host colonization. Further research is needed to determine the utility of these strains as protective cultures.

Evolution of Volatile Compounds during Ripening and Final Sensory Changes of Traditional Raw Ewe's Milk Cheese "Torta del Casar" Maturated with Selected Protective Lactic Acid Bacteria.

In traditional soft ripened cheeses made with raw milk, the use of protective cultures is infrequent. In the present work, the effect of selected (for their activity against Listeria monocytogenes) protective cultures of Lactocaseibacillus casei 116 and Lactococcus garvieae 151 was evaluated, on the evolution of volatile compounds throughout the ripening and on the final sensory characteristics of traditional soft ripened "Torta del Casar" cheese. For this, both strains were separately inoculated in raw cheeses and ripened for 90 days. The selected LAB strains did not affect physicochemical parameters, including texture and color of the ripened cheeses. However, they could have a positive effect on the aroma, for the generation of methyl branched acids and for the reduction in compounds derived from ß-oxidation of fatty acids. Thus, these protective cultures, in addition to contributing to their safety, could improve quality of the ripened cheeses.

Selective Survival of Protective Cultures during High-Pressure Processing by Leveraging Freeze-Drying and Encapsulation.

High-Pressure Processing's (HPP) non-thermal inactivation of cells has been largely incompatible with food products in which the activity of selected cultures is intended (e.g., bio-preservation). This work aims to overcome this limitation using a cocoa butter encapsulation system for freeze-dried cultures that can be integrated with HPP technology with minimal detrimental effects on cell viability or activity capabilities. Using commercially available freeze-dried protective cultures, the desiccated cells survived HPP (600 MPa, 5 °C, 3 min) and subsequently experienced a 0.66-log increase in cell counts during 2 h of incubation. When the same culture was rehydrated prior to HPP, it underwent a >6.07-log decrease. Phosphate-buffered saline or skim milk inoculated with cocoa butter-encapsulated culture up to 24 h before HPP displayed robust cell counts after HPP and subsequent plating (8.37−9.16 CFU/mL). In addition to assessing viability following HPP, the study sought to test the applicability in a product in which post-HPP fermentation is desired While HPP-treated encapsulated cultures initially exhibited significantly delayed fermentative processes compared to the positive controls, by 48 h following inoculation, the HPP samples' pH values bore no significant difference from those of the positive controls (encapsulated samples: pH 3.83 to 3.92; positive controls: pH 3.81 to 3.85). The HPP encapsulated cultures also maintained high cell counts throughout the fermentation (≥8.95 log CFU/mL).

High pressure processing, acidic and osmotic stress increased resistance to aminoglycosides and tetracyclines and the frequency of gene transfer among strains from commercial starter and protective cultures.

This study analyzed the effect of food-related stresses on the expression of antibiotic resistance of starter and protective strains and resistance gene transfer frequency. After exposure to high-pressure processing, acidic and osmotic stress, the expression of genes encoding resistance to aminoglycosides (aac(6')Ie-aph(2″)Ia and aph(3')-IIIa) and/or tetracyclines (tetM) increased. After cold stress, a decrease in the expression level of all tested genes was observed. The results obtained in the gene expression analysis correlated with the results of the phenotype patterns. After acidic and osmotic stresses, a significant increase in the frequency of each gene transfer was observed. To the best of the authors' knowledge, this is the first study focused on changes in antibiotic resistance associated with a stress response among starter and protective strains. The results suggest that the physicochemical factors prevailing during food production and storage may affect the phenotype of antibiotic resistance and the level of expression of antibiotic resistance genes among microorganisms. As a result, they can contribute to the spread of antibiotic resistance. This points to the need to verify strains used in the food industry for their antibiotic resistance to prevent them from becoming a reservoir for antibiotic resistance genes.

Biocontrol of ochratoxigenic Penicillium nordicum in dry-cured fermented sausages by Debaryomyces hansenii and Staphylococcus xylosus.

Penicillium nordicum is the main ochratoxin A (OTA) producing mould in dry-cured meat products. The use of autochthonous microorganisms as protective cultures is a promising strategy to control this hazard. The aim of this work was to evaluate the effect of Debaryomyces hansenii and Staphylococcus xylosus isolated from dry-cured meat products as biocontrol agents (BCAs) against P. nordicum during the ripening of dry-cured fermented sausages. The BCAs were added to the dough, and P. nordicum were inoculated on the surface after stuffing. Then, the sausages were ripened following a traditional processing. The growth of the microorganisms was determined by plate count at the beginning and at the end of ripening. To assess the implantation of BCAs in the sausages, the yeasts and staphylococci isolated from the sausages at the end of processing were identified by sequencing the 16S and 18S rRNA respectively, and they were characterized by pulsed field gel electrophoresis (PFGE) of the chromosomal DNA. OTA was quantified by UHPLC-MS/MS. BCAs were able to colonise and develop throughout the processing. Although none of the BCAs reduced the growth of P. nordicum, a OTA decrease was observed in the sausages inoculated with D. hansenii individually or combined with S. xylosus. The drop of OTA amount was particularly marked in the portions where the casing was damaged allowing the mould to grow inside the sausages. In these areas, OTA was not detected in the inoculated batches. In conclusion, D. hansenii could be proposed as BCA individually or in combination with S. xylosus for the biocontrol of OTA hazard in dry-cured fermented sausages.

Strategies for Biocontrol of Listeria monocytogenes Using Lactic Acid Bacteria and Their Metabolites in Ready-to-Eat Meat- and Dairy-Ripened Products.

Listeria monocytogenes is one of the most important foodborne pathogens. This microorganism is a serious concern in the ready-to-eat (RTE) meat and dairy-ripened products industries. The use of lactic acid bacteria (LAB)-producing anti-L. monocytogenes peptides (bacteriocins) and/or lactic acid and/or other antimicrobial system could be a promising tool to control this pathogen in RTE meat and dairy products. This review provides an up to date about the strategies of use of LAB and their metabolites in RTE meat products and dairy foods by selecting the most appropriate strains, by analysing the mechanism by which they inhibit L. monocytogenes and methods of effective application of LAB, and their metabolites in these kinds of products to control this pathogen throughout the processing and storage. The selection of LAB with anti-L. monocytogenes activity allows to dispose of effective strains in meat and dairy-ripened products, achieving reductions form 2-5 logarithmic cycles of this pathogen throughout the ripening process. The combination of selected LAB strains with antimicrobial compounds, such as acid/sodium lactate and other strategies, as the active packaging could be the next future innovation for eliminating risk of L. monocytogenes in meat and dairy-ripened products.

Microbial biopreservatives for controlling the spoilage of beef and lamb meat: their application and effects on meat quality.

Biopreservation is a recognized natural method for controlling the growth of undesirable bacteria on fresh meat. It offers the potential to inhibit spoilage bacteria and extend meat shelf-life, but this aspect has been much less studied compared to using the approach to target pathogenic bacteria. This review provides comprehensive information on the application of biopreservatives of microbial origin, mainly bacteriocins and protective cultures, in relation to bacterial spoilage of beef and lamb meat. The sensory effect of these biopreservatives, an aspect that often receives less attention in microbiological studies, is also reviewed. Microbial biopreservatives were found to be able to retard the growth of the major meat spoilage bacteria, Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae. Their addition did not have any discernible negative impact on the sensory properties of meat, whether assessed by human sensory panels or instrumental and chemical analyses. Although results are promising, the concept of biopreservation for controlling spoilage bacteria on fresh meat is still in its infancy. Studies in this area are still lacking, especially for lamb. Biopreservatives need more testing under conditions representative of commercial meat production, along with studies of any possible sensory effects, in order to validate their potential for large-scale industrial applications.

Role of Exposure to Lactic Acid Bacteria from Foods of Animal Origin in Human Health.

Animal products, in particular dairy and fermented products, are major natural sources of lactic acid bacteria (LAB). These are known for their antimicrobial properties, as well as for their roles in organoleptic changes, antioxidant activity, nutrient digestibility, the release of peptides and polysaccharides, amino acid decarboxylation, and biogenic amine production and degradation. Due to their antimicrobial properties, LAB are used in humans and in animals, with beneficial effects, as probiotics or in the treatment of a variety of diseases. In livestock production, LAB contribute to animal performance, health, and productivity. In the food industry, LAB are applied as bioprotective and biopreservation agents, contributing to improve food safety and quality. However, some studies have described resistance to relevant antibiotics in LAB, with the concomitant risks associated with the transfer of antibiotic resistance genes to foodborne pathogens and their potential dissemination throughout the food chain and the environment. Here, we summarize the application of LAB in livestock and animal products, as well as the health impact of LAB in animal food products. In general, the beneficial effects of LAB on the human food chain seem to outweigh the potential risks associated with their consumption as part of animal and human diets. However, further studies and continuous monitorization efforts are needed to ensure their safe application in animal products and in the control of pathogenic microorganisms, preventing the possible risks associated with antibiotic resistance and, thus, protecting public health.

Effect of Commercial Protective Cultures and Bacterial Fermentates on Listeria monocytogenes Growth in a Refrigerated High-Moisture Model Cheese.

ABSTRACT: Biopreservatives are clean-label ingredients used to control pathogenic and spoilage microorganisms in ready-to-eat foods, including cheese. In a first set of experiments, the efficacies of six commercial biopreservatives in controlling Listeria monocytogenes growth at 4°C were tested in a high-moisture model cheese (pH 6.00, 56% moisture, and 1.25% salt) made of cream, micellar casein, water, salt, lactose, lactic acid, and a single protective culture (PC-1, PC-2, or PC-3 at 106 CFU/g [target]) or bacterial fermentate (CM-1 or CM-2 [cultured milk] or CSV-1 [cultured sugar-vinegar blend], 0.5 or 1.0% target level). Cheeses were inoculated with 3 log CFU/g L. monocytogenes (5-strain cocktail), after which 25-g samples were vacuum sealed and stored at 4°C for 8 weeks. L. monocytogenes populations from triplicate samples were enumerated weekly on modified Oxford agar in duplicate trials. L. monocytogenes growth (≥1-log increase) was observed in approximately 1 week in control cheese and those formulated with 106 CFU of PC-1 or PC-2 per g. Growth was delayed to 2.5 weeks in model cheeses formulated with 106 CFU of PC-3 per g or 0.5% CM-2 and to 3 weeks with 0.5% CM-1 or CSV-1. Growth was further delayed to 6.5 to 7.5 weeks in model cheeses formulated with 1.0% CM-1 or CM-2, while formulation with 1.0% CSV-1 inhibited L. monocytogenes growth for 8 weeks. In a second set of experiments, the combined effects of pH and 0.5% CSV-1 on L. monocytogenes inhibition were investigated. Incorporation of 0.5% CSV-1 delayed L. monocytogenes growth to 3, 6, and >10 weeks in cheeses of pH 6.00, 5.75, and 5.50, respectively, versus growth observed in 1, 1, and 3.5 weeks in control cheeses. These data suggest that certain fermentates have greater antilisterial activity than protective cultures in directly acidified cheeses with direct biopreservative incorporation and refrigerated storage. Further research is needed to optimize the conditions to prevent listerial growth by utilizing protective cultures in fresh, soft cheeses.

Lactobacillus plantarum Strains Isolated from Polish Regional Cheeses Exhibit Anti-Staphylococcal Activity and Selected Probiotic Properties.

Twenty-nine Lactobacillus plantarum strains isolated from different types of Polish regional cheeses (Oscypek and Korycinski) were assessed for selected probiotic properties and anti-staphylococcal activity. Most of the tested L. plantarum strains were considered safe. Whole bacterial cultures (WBC) and cell-free supernatants (CFSs) of L. plantarum strains inhibited growth of Staphylococcus aureus (average inhibition growth zones were 2.8 mm ± 1.2 and 2.8 mm ± 1.1 respectively). Moreover, almost all neutralized, catalase-treated cell-free supernatants (CFN) of L. plantarum cultures also exhibited slight anti-staphylococcal activity in vitro. The most promising strains Os4 and Kor14 were selected for further study. Both strains were able to survive during digestive gastro-intestinal passage model. Live cells of L. plantarum Os4 and Kor14 caused the strongest inhibition of S. aureus adhesion to Caco-2 cells comparing with CFN and heat-killed bacterial cells. S. aureus and L. plantarum (Os4 or Kor14) co-cultured in skim milk resulted in growth inhibition of S. aureus in both 8 °C and 37 °C incubation temperatures. Observed abilities, demonstrated for L. plantarum Os4 and Kor14, confirms that these strains could be used in the food industry as protective cultures.

Quantitative proteomic profiling of ochratoxin A repression in Penicillium nordicum by protective cultures.

Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products.

Use of Carnobacterium spp protective culture in MAP packed Ricotta fresca cheese to control Pseudomonas spp.

Ricotta fresca is a whey cheese susceptible of secondary contamination, mainly from Pseudomonas spp. The extension of the shelf life of refrigerated ricotta fresca could be obtained using protective cultures inhibiting the growth of this spoilage microorganism. A commercial biopreservative, Lyofast CNBAL, comprising Carnobacterium spp was tested against Pseudomonas spp. The surface of ricotta fresca samples were inoculated either with Pseudomonas spp or Pseudomonas and Carnobacterium spp. Samples were MAP packed, stored at 4 °C and analyzed the day of the inoculum and 7, 14 and 21 days after the contamination. Microbiological analyses included total bacterial count, mesophilic lactic acid bacteria, Enterobacteriaceae, Pseudomonas spp, Listeria monocytogenes, moulds and yeasts. Pseudomonas mean initial contamination level was comparable in blank and artificially inoculated samples, respectively with values of 2.15 ±â€¯0.21 and 2.34 ±â€¯0.26 log cfu g-1. Carnobacterium spp. significantly reduced the growth of Pseudomonas spp respectively of 1.28 log and 0.83 log after 14 and 21 days of refrigerated storage. Intrinsic properties and physico-chemical composition were also investigated. Limited variation of pH was observed in samples inoculated with the protective cultures, indicating low acidification properties of Carnobacterium spp. Instead, no significant differences were observed for aW, moisture, fat and proteins during storage and between inoculated and control samples.

Biocontrol strategies for Mediterranean-style fermented sausages.

Naturally fermented meat sausages have a long tradition in Mediterranean countries and are one of the most important groups of traditional foods consumed throughout Europe. Despite all the advances in food science and technology and increased regulatory requirements and concerns for safety and quality during the last decades, the challenge to control important foodborne pathogens in this type of meat products still persists. Simultaneously, growing consumer interest in safe, high quality and minimal processed products, with less additives/preservatives have driven the food industry and scientists in a crusade for innovative technologies to maintain the safety of these products by natural means. Biological control (biocontrol) fits well within this tendency. This review summarizes the latest achievements on biocontrol strategies applied to Mediterranean-style fermented sausages, namely: (i) bioprotective cultures; (ii) bacteriocins; and, (iii) essential oils (EOs).

Recent advances in cured raw ham manufacture.

Cured raw hams are a valuable and popular group of meat products. The consumption and international trade have increased during the last years, therefore new technologies to accelerate the production process and to increase product quality and safety are needed. In the current review, an overview of European protected cured raw hams is presented. Furthermore, traditional methods for cured raw ham production together with recent advantages in the techniques for pretreatment (trimming, blade tenderization, and freeze-thawing), curing/salting (tumbling, vacuum impregnation, pulsed pressure, ultrasound, pulsed electric fields, simultaneous thawing/salting), drying/ripening (Quick-Dry-Slice-process, oil drop application, high temperature short time process) and postprocessing (vacuum and modified atmosphere packaging, high hydrostatic pressure, high pressure carbon dioxide, high pressure carbon dioxide with ultrasound) are described. Moreover, application techniques and effects of protective cultures and starter cultures, such as molds, yeasts, coagulase-negative staphylococci and lactic acid bacteria, on cured raw ham quality and safety are reviewed.

Testing commercial biopreservative against spoilage microorganisms in MAP packed Ricotta fresca cheese.

Ricotta fresca cheese is susceptible to secondary contamination and is able to support the growth of pathogens or spoilage psychotrophic bacteria during storage. The aim of the present study was to evaluate which among three commercial biopreservatives was suitable to be used to control the growth of spoilage microorganisms in sheep's milk MAP ricotta fresca cheese. 144 Ricotta fresca cheese samples were inoculated either with the bioprotective culture Lyofast FPR 2 (including Enterococcus faecium, Lactobacillus plantarum e Lactobacillus rhamnosus) or Lyofast CNBAL (Carnobacterium spp) or the fermentate MicroGARD 430. Not inoculated control and experimental ricotta were MAP packed (30% CO2 and 70% N2) and stored at 4 °C. Triplicate samples were analyzed after 5 h and 7, 14 and 21 days after inoculation for total bacterial count, mesophilic lactic acid bacteria, Enterobacteriaceae, Pseudomonas spp, Listeria monocytogenes, moulds and yeasts. Among the tested biopreservatives only Carnobacterium spp was able to control Pseudomonas spp and Enterobacteriaceae. The maximum reduction in the concentration of Pseudomonas spp and Enterobacteriaceae was respectively 1.93 and 2.66 log10 cfu/g, observed 14 days after production. Therefore, Carnobacterium spp was selected as the culture of choice to conduct a challenge study against Pseudomonas spp.