Effect of the controlled fermented quinoa containing protective starter culture on technological characteristics of wheat bread supplemented with red lentil.

Selected antifungal lactic acid bacteria (LAB) isolated from mature spontaneous quinoa sourdough was used as potential starter culture to produce loaf wheat bread containing controlled fermented quinoa (CFQ) supplemented with red lentil (RL) flour. Phylogenetic evolutionary tree led to the identification of Enterococcus hirae as the selected LAB isolate. Furthermore, there was no significant difference (P > 0.05) between bread containing CFQ and control in terms of hardness. The highest loaf specific volume and overall acceptability were also observed in control sample and wheat bread containing CFQ + RL, respectively. Meanwhile, the rate of surface fungal growth on wheat bread enriched with CFQ was significantly lower than the other samples. In accordance with a non-linear multivariable model, positive and negative correlations were observed between porosity and specific volume (+ 0.79), and also specific volume and crumb hardness (- 0.70), respectively. Accordingly, CFQ can be used as bio-preservative to produce clean-label supplemented wheat bread.

Application of Commercial Biopreservation Starter in Combination with MAP for Shelf-Life Extension of Burrata Cheese.

Burrata is a fresh pasta filata cheese manufactured in Italy. Its demand on the worldwide market is constantly growing, and prolonging its shelf-life is an important challenge for the Italian dairy industry. In the present study, combining a commercial bio-protective starter and modified atmosphere packaging (MAP) was evaluated as a strategy to delay the spoilage of product quality. Three experimental samples of burrata were produced by experimental trials at the industrial level and stored for 28 days under refrigerated conditions. Two samples contained the protective starter but were packaged differently (under MAP and immersed in water), and one did not contain the starter and was packaged under MAP. A sample of burrata without a starter and immersed in water was also prepared and used as a control. The combination of MAP and bio-protective starter delayed the degradation of lactose and citric acid, used as indices of microbial activity. In fact, lower counts of Enterobacteriaceae and Pseudomonas were observed in this sample. In contrast, control burrata had the highest level of total Volatile Organic Compounds (VOC) at the end of the storage period, because of higher microbial activity. Even though all samples were judged to be unacceptable after 28 days from the sensory point of view, the sample with bio-protective starter under MAP had the best score after 21 days, obtaining a shelf-life extension of about 7 days with respect to control. In conclusion, the combination of MAP and protective starter culture could be an easy way to extend the shelf-life of burrata stored under correct refrigerated conditions.

Australian consumers' attitudes to packaged fresh meat products with added microbial bioprotective cultures for shelf-life extension.

The application of food-grade microbial cultures to fresh meat products is a promising natural approach for meat shelf-life extension. However, before its adoption into commercial practice, it is essential to understand consumers' attitudes to this approach and the resulting marketed products. This study investigated Australian consumers' willingness to purchase and consume packaged fresh meat products with added microbial cultures for shelf-life extension. A national online survey of over 800 respondents was conducted. Results indicated that most Australian consumers would be willing to buy and eat such products, with 17.8% of respondents less likely to buy and 11.1% unwilling to eat these products. Respondents' purchasing and consumption decisions were influenced by demographic factors, their food and meat shopping and consumption behaviors, and the value, taste, and type of the meat product. Consumer acceptance may be improved by increasing their awareness of the potential use of microbial cultures as natural antimicrobials for food shelf-life extension.

Antifungal activity of protective cultures against the yogurt drink-specific spoilage yeasts.

Fungal agents emerged as post-pasteurization contamination are responsible for the spoilage in yogurt drink. In this work, the antifungal effects of some lactic acid bacteria (LAB) on the spoilage yeasts isolated from yogurt drink (Doogh) were evaluated. First, the microbial growth in the yogurt drink samples during the storage time was investigated, and the isolated microorganisms were identified using biochemical methods and sequencing of the specific amplicons. Yeasts (3-7 log CFU ml-1 ) were found to be the most abundant microorganisms (specific spoilage organisms) in several samples. Using the amplification technique of rDNA by ITS1 and ITS4 primers, the dominant yeasts were identified as Pichia kudriavzevii, Kluyveromyces marxianus, and Candida parapsilosis. Then, the antimicrobial activity of 37 strains of LAB against the isolated yeasts was studied using broth microdilution. Eventually, the strains of Lacticplantibacillus plantarum (245, 24, P6, and P7), Lactiplantibacillus pentosus (20), and Levilactobacillus brevis (30) exhibited significant antifungal activity. In the most effective impacts, lag times of C. parapsilosis, K. marxianus, and P. kudriavzevii were increased by almost 12-19 h, 12-19 h, and 2-6 h, respectively, while the area under the growth curve for these yeasts was reduced to lower than 40%, near 16%, and approximately 67%, in the order given. Overall, these bacteria showed high potential as the substituents for chemical preservatives in yogurt drinks. PRACTICAL APPLICATION: Spoilage yeasts were isolated from yogurt drink and identified by molecular method. Isolated yeasts belonged to Pichia, Kluyveromyces, and Candida genera. Inhibitory effects of 37 strains were evaluated against the spoilage yeasts. Cell-free supernatant was used against the isolated fungi in microdilution method. Several LAB strains showed a significant antimicrobial activity.

Probiotic potential of commercial dairy-associated protective cultures: In vitro and in vivo protection against Listeria monocytogenes infection.

Protective bacterial cultures (PCs) are commercially available to producers to control undesirable microbes in foods, including foodborne pathogens such as Listeria monocytogenes. They are generally recognized as safe for consumption and many are capable of producing bacteriocins. Yet their potential to act as probiotics and confer a health benefit on the host is not known. This study investigated the ability of three commercial PCs to survive human gastrointestinal conditions and exert anti-infective properties against L. monocytogenes. Counts of two PCs of Lactiplantibacillus plantarum remained unchanged after exposure to simulated gastrointestinal conditions, whereas counts of the PC Lactococcus lactis subsp. lactis were reduced by 5.3 log CFU/mL. Cultures of Lactiplantibacillus plantarum and Lactococcus lactis subsp. lactis adhered to human Caco-2 epithelial cells at âˆ¼ 6 log CFU/mL. This pretreatment reduced subsequent L. monocytogenes adhesion and invasion by 1-1.6 log CFU/mL and 3.8-4.9 log CFU/mL, respectively, compared to control. L. monocytogenes-induced cytotoxicity was also reduced from 29.1% in untreated monolayers to âˆ¼ 8% in those treated with PCs. Pretreatment of Caco-2 monolayers with Lactococcus lactis subsp. lactis and one PC of Lactiplantibacillus plantarum reduced L. monocytogenes translocation by ≥ 1.2 log CFU/mL compared to control (≥ 94.5% inhibition). All PCs significantly reduced DextranFITC permeability through Caco-2 monolayers to approximately half that of control. Pretreatment with PCs also reduced L. monocytogenes-induced mortality in Caenorhabditis elegans. These findings demonstrate the potential for commercially produced PCs to exert probiotic effects in the host through protection against L. monocytogenes infection, thus providing an additional benefit to food safety beyond inhibiting pathogen growth, survival, and virulence in foods.

Evaluation of the potential of protective cultures to extend the microbial shelf-life of chilled lamb meat.

The use of protective cultures to inhibit spoilage bacteria is a promising natural preservation technique to extend the shelf-life of fresh meat. This study evaluated the effectiveness of six food-grade protective cultures (containing different combinations of Lactobacillus sakei, Pediococcus pentosaceus, Staphylococcus xylosus, and Staphylococcus carnosus) on naturally contaminated chill-stored (4 °C) lamb meat in different packaging systems. Only slight reductions of common meat spoilage bacteria Brochothrix thermosphacta, Pseudomonas spp., and Enterobacteriaceae were observed in culture-treated samples stored in modified atmosphere packaging (80% O2:20% CO2). Greater inhibitory effects were found in vacuum-packed lamb, with mixed cultures containing either L. sakei, S. carnosus, and S. xylosus or S. carnosus and L. sakei causing the most significant reductions. Protective cultures did not adversely affect meat color or pH. This study demonstrated the potential of protective cultures comprising lactic acid bacteria and coagulase-negative staphylococci in controlling microbial spoilage of lamb and, by inference, other types of meat as a natural solution for shelf-life extension.

Current Trends of Enterococci in Dairy Products: A Comprehensive Review of Their Multiple Roles.

As a genus that has evolved for resistance against adverse environmental factors and that readily exchanges genetic elements, enterococci are well adapted to the cheese environment and may reach high numbers in artisanal cheeses. Their metabolites impact cheese flavor, texture, and rheological properties, thus contributing to the development of its typical sensorial properties. Due to their antimicrobial activity, enterococci modulate the cheese microbiota, stimulate autolysis of other lactic acid bacteria (LAB), control pathogens and deterioration microorganisms, and may offer beneficial effects to the health of their hosts. They could in principle be employed as adjunct/protective/probiotic cultures; however, due to their propensity to acquire genetic determinants of virulence and antibiotic resistance, together with the opportunistic character of some of its members, this genus does not possess Qualified Presumption of Safety (QPS) status. It is, however, noteworthy that some putative virulence factors described in foodborne enterococci may simply reflect adaptation to the food environment and to the human host as commensal. Further research is needed to help distinguish friend from foe among enterococci, eventually enabling exploitation of the beneficial aspects of specific cheese-associated strains. This review aims at discussing both beneficial and deleterious roles played by enterococci in artisanal cheeses, while highlighting the need for further research on such a remarkably hardy genus.

Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.

In this study, we evaluated the efficacy of 3 commercial protective cultures designated PC1 (Lactobacillus spp.), PC2 (Lactobacillus rhamnosus), and PC3 (Lactobacillus rhamnosus) as biopreservatives in queso fresco (QF) against 9 yeast strains (Candida zeylanoides, Clavispora lusitaniae, Debaryomyces hansenii, Debaryomyces prosopidis, Kluyveromyces marxianus, Meyerozyma guilliermondii, Pichia fermentans, Rhodotorula mucilaginosa, and Torulaspora delbrueckii) and 11 mold strains (Aspergillus cibarius, Aureobasidium pullulans, Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, Penicillium roqueforti, Mucor genevensis, Mucor racemosus, Phoma dimorpha, and Trichoderma amazonicum). All fungal spoilage strains were previously isolated from dairy processing environments. A positive control (C) with no protective culture was included. Fungal spoilage organisms were inoculated on cheese surfaces at an inoculum level of 20 cfu/g, and cheeses were stored at 6 ± 2°C throughout the study. For yeast enumeration, cheeses were sampled on d 0, 7, 14, and 21 postinoculation. Significant inhibition was detected for each yeast strain by comparing yeast counts for each cheese treated with protective culture against the control cheese using one-way ANOVA with Bonferroni correction performed individually at d 7, 14, and 21 postinoculation. Mold growth was visually observed and imaged weekly through 70 d postinoculation. Whereas PC3 inhibited Cl. lusitaniae, Mey. guilliermondii, and Ph. dimorpha, PC2 inhibited the outgrowth of Cl. lusitaniae, D. hansenii, and Ph. dimorpha. Protective culture 1 had the broadest spectrum of efficacy across yeast and molds, delaying spoilage caused by 4 distinct yeast strains (Cl. lusitaniae, D. hansenii, D. prosopidis, and Mey. guilliermondii), and inhibiting visible growth of 2 mold strains (P. chrysogenum and Ph. dimorpha). Results demonstrated that commercial protective cultures vary in performance, as indicated by the breadth of mold and yeast inhibition at both the genus and species level. This study suggests that manufacturers looking into using protective cultures should investigate their efficacy against specific fungal strains of concern.

The effect of protective cultures on Staphylococcus aureus growth and enterotoxin production.

Staphylococcus aureus is the causative agent of staphylococcal food poisoning and is a common contaminant in milk. Despite efforts to control S. aureus, recalls and outbreaks continue to occur, highlighting the need for additional interventions. This study determined the potential for protective cultures (PC) that are commercially available to producers to control S. aureus growth in raw milk and attenuate virulence by impeding staphylococcal enterotoxin (SE) production in raw milk and laboratory medium. Cultures of Hafnia alvei and Lactococcus lactis effectively inhibited S. aureus growth in raw milk to counts ~5 log CFU/mL lower than control when cocultured following a cheesemaking time and temperature profile; two cultures of Lactobacillus plantarum inhibited growth to ~1.5 log CFU/mL less than control. Cocultures of S. aureus with Lc. lactis, H. alvei and Lb. plantarum in raw milk reduced SE levels by 24.9%, 62.4%, and 76%, respectively. Lc. lactis also decreased SE production in raw milk in the absence of PC-mediated growth inhibition. Significant reductions in SE production in the absence of pathogen growth inhibition were also achieved in laboratory medium. Together, these results demonstrate the potential for PCs to inhibit S. aureus growth and impede SE production in the absence of growth inhibition.

Compatibility of Commercially Produced Protective Cultures with Common Cheesemaking Cultures and Their Antagonistic Effect on Foodborne Pathogens.

ABSTRACT: The documented survival of pathogenic bacteria, including Listeria monocytogenes (LM), Shiga toxin-producing Escherichia coli (STEC), and Salmonella during the manufacture and aging of some cheeses highlights the need for additional interventions to enhance food safety. Unfortunately, few interventions are compliant with the Standards of Identity for cheese. Protective bacterial cultures (PCs) represent actionable, natural interventions. However, supportive data for commercially produced PCs regarding their efficacy against pathogens and potential antagonism with each other and cheesemaking cultures are scant, thereby impeding their potential use by the cheese industry. The overall objective of this study was to identify commercially produced PCs that exert antimicrobial activity toward pathogens with minimal impact on beneficial cheese microbes. Direct antagonism and agar well diffusion assays were used to determine the impact of 10 commercially produced PCs on the growth of starter cultures and cultures of ripening bacteria and fungi. Deferred antagonism was used to evaluate the potential for antimicrobial effects against LM, STEC, and Salmonella. PCs and starter cultures were cocultured in ultrahigh-temperature-processed milk to determine the effects of coculture on starter acidification profiles when incubated according to a simulated cheesemaking temperature profile (4 h at 35°C followed by 20 h at 20°C). Compatibility assays suggest that PC antagonism is microbe and strain specific. Only one PC negatively impacted the acidification of the starters tested. PC antagonism of ripening bacteria and fungi growth varied but was consistent within species. All PCs displayed deferred inhibition of LM, STEC, and Salmonella growth, but to varying degrees. These data identify commercial PCs with potential for the control of pathogens and characterize their compatibility with cheesemaking cultures for future use by cheesemakers and investigations of their efficacy in the production of cheese.

Biopreservation approaches to reduce Listeria monocytogenes in fresh vegetables.

Two biopreservation approaches for fresh lettuce, rocket salad, parsley and spinach were studied. The potential of Pediococcus pentosaceus DT016, as a protective culture, to suppress Listeria monocytogenes in vegetables during storage was evaluated. The pathogen numbers in the vegetables inoculated with P. pentosaceus DT016 were significantly (p < 0.01) lower throughout the storage period and, at the last storage day, a minimum difference of 1.4 log CFU/g was reported when compared with the vegetables without the protective culture. Moreover, by using two levels of L. monocytogenes (about 6 and 4 log CFU/g), it was observed that the antagonist effect of P. pentosaceus was higher for the lower pathogen numbers. The second approach evaluated a pediocin DT016 solution to inactivate and control L. monocytogenes proliferation. The pathogen load was studied after washing with: water, chlorine and the pediocin solution and along storage at 4  °C. Comparing the various washing solutions, the vegetables washed with pediocin presented significantly (p < 0.01) lower pathogen numbers throughout storage, by a minimum of 3.2 and 2.7 log CFU/g, than in vegetables washed with water and chlorine, respectively. The proposed methodologies are promising alternatives to maintain the safety of fresh vegetables during extended storage at refrigeration temperature.

Evaluation of biopreservatives in Greek yogurt to inhibit yeast and mold spoilage and development of a yogurt spoilage predictive model.

Dairy products, including cultured dairy products such as cheese and yogurt, are susceptible to fungal spoilage. Traditionally, additives such as potassium sorbate have been used to control fungal spoilage; however, with consumer demand for clean-label products, other strategies to control fungal spoilage (e.g., biopreservatives) are increasingly being used in dairy formulations. In order to help the dairy industry better evaluate biopreservatives for control of fungal spoilage, we developed a challenge study protocol, which was applied to evaluate 2 commercially available protective cultures for their ability to control yeast and mold spoilage of Greek yogurt. Greek yogurt formulated with and without protective cultures was inoculated with a cocktail consisting of 5 yeasts and 1 mold to yield inoculum levels of 101 and 103 cfu/g of yogurt. The inoculated yogurts were stored at 7°C and fungal counts as well as time to visible growth, on the yogurt surface, of mycelium mold colonies or yeast were determined over shelf-life. Whereas fungal concentrations increased to spoilage levels (≥105 cfu/g) in all yogurt formulations at both inoculum levels by d 23 of storage at 7°C, no surface mold was observed over 76 d in any of the products formulated with protective cultures. Control yogurts without biopreservatives all showed surface mold by d 23. In order to allow industry to better evaluate the business effects of improved control of surface mold growth that can be achieved with protective cultures, we developed a Monte Carlo simulation model to estimate consumer exposure to visible mold growth in yogurt formulated without fungal inhibitors. Our model showed that initial mold contamination rate has the largest effect on the model outcome, indicating that accurate data on contamination rates are important for use of these models. When air plates were used, in a proof-of-concept approach, to estimate initial contamination rates in a small yogurt manufacturing operation, our model predicted that 550 ± 25.2 consumers (±standard deviation) would be exposed to visible mold growth for every 1 million cups of yogurt produced. With initial contamination rate data for individual facilities, this model could be used by industry to estimate the number of consumers exposed to visible mold spoilage and could allow industry to better assess the value of mold-control strategies.

A Multibacteriocin Cheese Starter System, Comprising Nisin and Lacticin 3147 in Lactococcus lactis, in Combination with Plantaricin from Lactobacillus plantarum.

Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-bacteriocin-producing starters of Lactococcus lactis capable of producing the class I bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of class I bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the class II bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the class II producer proved to be most effective for reducing bacterial load. As emergence of bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications.IMPORTANCE We generated a suite of single- and double-bacteriocin-producing starter cultures capable of generating the class I bacteriocin lacticin 3147 or nisin or both bacteriocins simultaneously via conjugation. The transconjugants exhibited improved bacteriophage resistance and antimicrobial activity. The single producers proved to be as effective as the double-bacteriocin producer at reducing Listeria numbers in laboratory-scale cheese. However, combining the double producer or the lacticin-producing starter with a class II bacteriocin producer, Lactobacillus plantarum LMG P-26358, proved to be most effective at reducing Listeria numbers and was significantly better than a combination of the three bacteriocin-producing strains, as the double producer is not inhibited by either of the class I bacteriocins. Since the simultaneous use of lacticin and nisin should reduce the emergence of bacteriocin-tolerant derivatives, this study suggests that a protective starter system produced by bacteriocin stacking is a worthwhile multihurdle approach for food safety applications.

Lactobacillus plantarum 29 inhibits Penicillium spp. involved in the spoilage of black truffles (Tuber aestivum).

The effect of an antifungal culture of Lactobacillus plantarum to be used in the storage at refrigeration temperature of fresh black truffles was examined. The strain was selected among 29 lactobacilli isolated from foods and evaluated for their viability and acidification activity at 4 °C, as well as for their inhibitory activity against 11 Penicillium strains isolated from truffles stored at refrigeration temperature. Lb. plantarum 29 showed the ability to hold not only the growth of Penicillium isolated from truffles, but also that of P. digitatum DSM 2750, a green mold involved in the spoilage of truffles. The antifungal activity was observed in vitro and in situ, and the sensory characteristics of truffles were preserved during the cold storage.

Identification and Partial Characterization of Antilisterial Compounds Produced by Dairy Yeasts.

Food-grade yeasts make significant contributions to flavor development in fermented foods. Some yeast species also inhibit undesirable bacteria, yeasts and molds, apparently by producing antimicrobial compounds called mycocins. The aim of this study was to evaluate the ability of wild yeasts, isolated from raw milk and cheese in the Quebec province area, to produce antilisterial compounds. Based on an agar-membrane screening test, 22 of 95 isolates, namely one Candida catenulata, one Candida parapsilosis, five Candida tropicalis, four Debaryomyces hansenii, one Geotrichum candidum, nine Pichia fermentans and one Pichia anomala, exhibited a significant inhibitory effect against Listeria ivanovii HPB28. Four in particular, namely C. tropicalis LMA-693, D. hansenii LMA-916, P. fermentans LMA-256 and P. anomala LMA-827, produced substances extractable from culture supernatant and capable of decreasing 18-h growth of L. ivanovii by, respectively, 97, 92, 84 and 78 %. Heating the extracted material (100 °C for 10 min) decreased these values to 72, 62, 58 and 31 %, respectively, while treatment with trypsin or pronase E decreased them to as little as 27 %. The extracts reduced the numbers of viable Listeria monocytogenes by as much as four log cycles within an hour. Transmission electron microscopy revealed a high proportion of lysis among the cells, apparently due to pore formation. This study clearly shows the potential of these four yeast isolates for use as bio-preservatives in a variety of dairy products.

Investigation into the Potential of Bacteriocinogenic Lactobacillus plantarum BFE 5092 for Biopreservation of Raw Turkey Meat.

The bacteriocin-producing Lactobacillus plantarum BFE 5092 was assessed for its potential as a protective culture in the biopreservation of aerobically stored turkey meat. This strain produces three bacteriocins, i.e. plantaricins EF, JK and N. The absolute expression of Lactobacillus plantarum BFE 5092 16S rRNA housekeeping gene, as well as l-ldh, plnEF and plnG genes as determined by quantitative, real-time-PCR, revealed that these genes were expressed to similar levels when the strain was grown at 8 and 30 °C in MRS broth. On turkey meat, Lactobacillus plantarum BFE 5092 did not grow but survived, as indicated by similar viable cell numbers during a 9-day storage period at 8 °C. When inoculated at 1 × 10(7) CFU/g on the turkey meat and subsequently stored at 10 °C, the culture did again not show good growth. Lactobacillus plantarum BFE 5092 could not inhibit the growth of naturally occurring listeriae or Gram-negative bacteria on the turkey meat at 10 °C, or that of Listeria monocytogenes when it was co-inoculated at a level of 1 × 10(5) CFU/g. Gene expression analyses showed that the bacteriocin genes were expressed on turkey meat stored at 10 °C. Moreover, the investigation into the absolute expression of the three plantaricin genes of Lactobacillus plantarum BFE 5092 in co-culture with Listeria monocytogenes on turkey meat by qRT-PCR showed that the plantaricin genes were indeed expressed during the low-temperature storage condition. The Lactobacillus plantarum BFE 5092 strain overall could not effectively inhibit L. monocytogenes and therefore it would not make a suitable protective culture for biopreservation of turkey meat stored aerobically at low temperature.