Lactic fermentation of cooked, comminuted mussel, Perna canaliculus.

The endogenous microflora of mussels, filter feeders, can include pathogens with resulting food safety concerns. The aim was to develop a cook-then-ferment technology to extend shelf life and safety of a ready-to-eat mussels. Only after cooking to destroy the mussel's endogenous microflora could an edible product be made as determined by pH decline after fermentation and the fate of common pathogens. Perna canaliculus was bought live at retail on many dates. Fermentation was with commercial lactic cultures incubated under vacuum at 30 °C for four days. Using one culture containing Pediococcus pentosaceus and Staphylococcus carnosus as a model, pH typically declined to 4.5 to 4.7, and common pathogens, Staphylococcus aureus, Salmonella and Vibrio parahaemolyticus were absent or reduced to acceptable levels. The fate of Listeria monocytogenes was studied with five cultures. These were variably effective at inhibition with one clear success, Chr Hansen's T-SC-150 containing a specific strain of Lactobacillus sakei, and flavour-generating Staphylococcus carnosus. This culture's efficacy was confirmed with sterile extracts of LAB challenging L. monocytogenes in vitro. This culture was also the most rapid fermenter by pH fall. Cook-then-ferment technology may be applied to other novel foods to minimise a disruptive endogenous microflora.

Counteracting the effect of reducing nitrate/nitrite levels on dry fermented sausage aroma by Debaryomyces hansenii inoculation.

The reduction of ingoing amounts of nitrate and nitrite in dry fermented sausages was studied together with the impact of Debaryomyces hansenii inoculation on aroma generation. Three different formulations of sausages were manufactured: control (C), reduced in nitrate and nitrite ingoing amounts (R) and reduced R inoculated with D. hansenii (RY). Changes in physicochemical and microbiological parameters, volatile compounds and aroma were investigated at different drying times. Nitrite/nitrate reduction did not seem to affect microbial growth but affected their metabolic activity. Moreover, nitrite/nitrate reduction decreased lipid oxidation and generation of derived volatile compounds. Yeast inoculation limited lipid oxidation and prevented nitrite oxidation. Sausage aroma profile was positively affected by D. hansenii inoculation which contributed to the generation of potent aroma compounds like ethyl ester compounds and 3-methylbutanal. Long drying time impacted sausage aroma profile as well as yeast metabolism. Yeast inoculation counteracted the negative influence of nitrite/nitrate reduction due to its antioxidant capacity, aroma generation and hindered nitrite oxidation.

An integrated approach for the valorization of mango seed kernel: Efficient extraction solvent selection, phytochemical profiling and antiproliferative activity assessment.

A novel valorization strategy is proposed in this work for the sustainable utilization of a major mango processing waste (i.e. mango seed kernel, MSK), integrating green pressurized-liquid extraction (PLE), bioactive assays and comprehensive HRMS-based phytochemical characterization to obtain bioactive-rich fractions with high antioxidant capacity and antiproliferative activity against human colon cancer cells. Thus, a two steps PLE procedure was proposed to recover first the non-polar fraction (fatty acids and lipids) and second the polar fraction (polyphenols). Efficient selection of the most suitable solvent for the second PLE step (ethanol/ethyl acetate mixture) was based on the Hansen solubility parameters (HSP) approach. A comprehensive GC- and LC-Q-TOF-MS/MS profiling analysis allowed the complete characterization of the lipidic and phenolic fractions obtained under optimal condition (100% EtOH at 150 °C), demonstrating the abundance of oleic and stearic acids, as well as bioactive xanthones, phenolic acids, flavonoids, gallate derivatives and gallotannins. The obtained MSK-extract exhibited higher antiproliferative activity against human colon adenocarcinoma cell line HT-29 compared to traditional extraction procedures described in literature for MSK utilization (e.g. Soxhlet), demonstrating the great potential of the proposed valorization strategy as a valuable opportunity for mango processing industry to deliver a value-added product to the market with health promoting properties.

Inoculation with a terroir selected Debaryomyces hansenii strain changes physico-chemical characteristics of Iberian cured pork loin.

Debaryomyces hansenii Lr1, previously isolated from naturally fermented cured pork loin ("lomo embuchado"), was used to inoculate Iberian pork loin under four different conditions. In all cases, specifically inoculated D. hansenii yeasts grew on the surface of the product and affected its physico-chemical and sensory characteristics. Inoculated pork loin kept higher water activity and pH and possessed lower sodium content than control samples at the end of the 90 days ripening period. Moreover, inoculation with Lr1 yeast did not change the general profile of fatty acids but modified the levels of volatile and aroma compounds by decreasing aldehydes and increasing esters and alcohol compounds. Principal Component Analysis (PCA) of the different physico-chemical parameters that were determined showed a clear separation between the samples indicating that they were different. A tendency in consumers acceptance to prefer cured pork loin specifically inoculated with the higher amounts of yeasts used in this study was observed.

Changes in microbial composition on the crust by different air flow velocities and their effect on sensory properties of dry-aged beef.

Beef rumps (middle gluteal) were dry aged for 28 days using different air flow velocities of 0, 2.5, and 5 m/s (DA0, DA2.5, and DA5, respectively). The microbial composition, physicochemical traits (moisture, pH, and shear force), flavor compounds (inosine 5'-monophosphate, reducing sugar, free amino acid, and free fatty acid), and electronic tongue profile were analyzed at day 0, 14, and 28. No molds or yeasts were detected until day 14. On day 28, Pilaira anomala was found to be the most abundant in DA0, whereas DA2.5 and DA5 showed increased composition of Debaryomyces hansenii. With that, the significant changes in physicochemical traits and flavor compounds occurred. In addition, the pattern of flavor compounds and taste attributes from DA0, which had different mold and yeast compositions, were discriminable from DA2.5 and DA5. Therefore, our results suggest that air flow can affect microbial composition on the crust, possibly resulting in different sensory properties of dry-aged beef.

Microbiota of Iberian dry-cured ham as influenced by chemical composition, high pressure processing and prolonged refrigerated storage.

The effect of high pressure processing (HPP) on the microbiota of ripened Iberian ham of different water activity, salt concentration and intramuscular fat content was investigated before and after a 5-month refrigeration period. At the beginning of the refrigeration period, the only significant effects of chemical composition were those of water activity on psychrotrophs and Micrococcaceae in untreated hams, and of the salt-in-lean ratio on lactic acid bacteria in HPP-treated hams. At the end of the refrigeration period, the only significant effect was that of intramuscular fat content on moulds and yeasts in HPP-treated samples. All microbial groups were significantly affected by HPP, with reductions ranging from 1.7 to 2.0 log cycles after treatment. A significant recovery of all microbial groups took place in HPP-treated hams during the refrigeration period, with increases ranging from 0.5 to 1.1 log cycles. In spite of this recovery, microbial levels in HPP-treated hams remained significantly lower than in untreated hams. Staphylococcus accounted for 93.4% of Iberian ham bacterial isolates, with S. equorum as the most abundant species. Representatives of the Tetragenococcus, Carnobacterium and Streptomyces genera, not previously reported in dry-cured ham, were also isolated. Most of the yeast isolates (75.0%) were identified as Debaryomyces hansenii.

Aroma modulation of Cabernet Gernischt dry red wine by optimal enzyme treatment strategy in winemaking.

Cabernet Gernischt (CG) is a famous Chinese wine grape cultivar, the red wine of which is known for its green trait, especially when produced from grapes cultivated in regions with monsoon climate. To modify CG wine aroma, three enzyme preparations (H. uvarum extracellular enzyme, AR2000, and pectinase) were introduced in different winemaking stages with Saccharomyces cerevisiae. Free and bound aroma compounds in young wines were detected using headspace solid-phase micro-extraction and gas chromatography-mass spectrometry, and aroma characteristics were quantified by trained panelists. Results showed that simultaneous inoculation of enzymes and yeasts improved wine aroma. Partial least-squares regression revealed that the green trait was due mainly to varietal compounds, especially C6 compounds, and could be partly weakened by fermentative compounds. Moreover, H. uvarum enzyme treatments enriched the acid fruit note of CG wine by enhancing the synergistic effect of varietal volatiles and certain fermentative compounds, such as esters and phenylethyls.

Diversity of microbiota found in coffee processing wastewater treatment plant.

Cultivable microbiota presents in a coffee semi-dry processing wastewater treatment plant (WTP) was identified. Thirty-two operational taxonomic units (OTUs) were detected, these being 16 bacteria, 11 yeasts and 4 filamentous fungi. Bacteria dominated the microbial population (11.61 log CFU mL- 1), and presented the highest total diversity index when observed in the WTP aerobic stage (Shannon = 1.94 and Simpson = 0.81). The most frequent bacterial species were Enterobacter asburiae, Sphingobacterium griseoflavum, Chryseobacterium bovis, Serratia marcescens, Corynebacterium flavescens, Acetobacter orientalis and Acetobacter indonesiensis; these showed the largest total bacteria populations in the WTP, with approximately 10 log CFU mL- 1. Yeasts were present at 7 log CFU mL- 1 of viable cells, with Hanseniaspora uvarum, Wickerhamomyces anomalus, Torulaspora delbrueckii, Saturnispora gosingensis, and Kazachstania gamospora being the prevalent species. Filamentous fungi were found at 6 log CFU mL- 1, with Fusarium oxysporum the most populous species. The identified species have the potential to act as a biological treatment in the WTP, and the application of them for this purpose must be better studied.

Multiple microbial cell-free extracts improve the microbiological, biochemical and sensory features of ewes' milk cheese.

This study used cell-free enzyme (CFE) extracts from Lactobacillus casei, Hafnia alvei, Debaryomyces hansenii and Saccharomyces cerevisiae to condition or accelerate Pecorino-type cheese ripening. Compositional, microbiological, and biochemical analyses were performed, and volatile and sensory profiles were obtained. Lactobacilli and cocci increased during ripening, especially in cheeses containing CFE from L. casei, H. alvei and D. hansenii (LHD-C) and L. casei, H. alvei and S. cerevisiae (LHS-C). Compared to control cheese (CC), several enzymatic activities were higher (P < 0.05) in CFE-supplemented cheeses. Compared to the CC (1907 mg kg-1 of cheese), the free amino acid level increased (P < 0.05) in CFE-supplemented cheeses, ranging from approximately 2575 (LHS-C) to 5720 (LHD-C) mg kg-1 of cheese after 60 days of CFE-supplemented ripening. As shown by GC/MS analysis, the levels of several volatile organic compounds were significantly (P < 0.05) lower in CC than in CFE-supplemented cheeses. All cheeses manufactured by adding multiple CFEs exhibited higher scores (P < 0.05) for internal structure, acid taste and juiciness than CC samples. This study shows the possibility of producing ewes' milk cheese with standardized characteristics and improved flavor intensity in a relatively short time.

Microbiota of high-pressure-processed Serrano ham investigated by culture-dependent and culture-independent methods.

The microbiota of Serrano dry-cured ham of different chemical composition, subjected or not to high-pressure processing (HPP), was investigated using culture-dependent and culture-independent methods. Microbial counts were submitted to analysis of variance with physicochemical parameters (aw, NaCl concentration, salt-in-lean ratio and intramuscular fat content) or HPP as main effects. In untreated hams, physicochemical parameters significantly affected counts of aerobic mesophiles, psychrotrophs, and moulds and yeasts. NaCl concentration and fat content influenced the levels of four and three of the five studied microbial groups, respectively, whereas no influence of aw was stated. The HPP treatment had a significant effect on counts of all investigated microbial groups. Culture-independent methods showed the presence of bacteria such as Staphylococcus equorum, Staphylococcus succinus, Bacillus subtilis and Cellulosimicrobium sp., moulds like Penicillium commune, Aspergillus fumigatus, Sclerotinia sclerotiorum, Eurotium athecium and Moniliella mellis, and yeasts like Debaryomyces hansenii and Candida glucosophila. Absence of B. subtilis bands and weaker bands of E. athecium were recorded for HPP-treated hams. The higher microbial levels found in lean ham might result in a quicker deterioration. HPP treatment confirmed its suitability as a procedure to control spoilage microorganisms. DGGE did not seem to be sensitive enough to highlight changes caused by HPP treatment in the microbiota of ham, but contributed to the detection of microbial species not previously found in ham.

Evaluation of a cross contamination model describing transfer of Salmonella spp. and Listeria monocytogenes during grinding of pork and beef.

In a previous study, a model was developed to describe the transfer and survival of Salmonella during grinding of pork (Møller, C.O.A., Nauta, M.J., Christensen, B.B., Dalgaard, P., Hansen, T.B., 2012. Modelling transfer of Salmonella typhimurium DT104 during simulation of grinding of pork. Journal of Applied Microbiology 112 (1), 90-98). The robustness of this model is now evaluated by studying its performance for predicting the transfer and survival of Salmonella spp. and Listeria monocytogenes during grinding of different types of meat (pork and beef), using two different grinders, different sizes and different numbers of pieces of meats to be ground. A total of 19 grinding trials were collected. Acceptable Simulation Zone (ASZ), visual inspection of the data, Quantitative Microbiological Risk Assessment (QMRA), as well as the Total Transfer Potential (TTP) were used as approaches to evaluate model performance and to access the quality of the cross contamination model predictions. Using the ASZ approach and considering that 70% of the observed counts have to be inside a defined acceptable zone of ±0.5 log10CFU per portion, it was found that the cross contamination parameters suggested by Møller et al. (2012) were not able to describe all 19 trials. However, for each of the collected grinding trials, the transfer event was well described when fitted to the model structure proposed by Møller et al. (2012). Parameter estimates obtained by fitting observed trials performed at different conditions, such as size and number of pieces of meat to be ground, may not be applied to describe cross contamination of unlike processing. Nevertheless, the risk estimates, as well as the TTP, revealed that the risk of disease may be reduced when the grinding of meat is performed in a grinder made of stainless steel (for all surfaces in contact with the meat), using a well-sharpened knife and holding at room temperatures lower than 4°C.

Effect of commercial starter cultures on volatile compound profile and sensory characteristics of dry-cured foal sausage.

BACKGROUND: The present work deals with the evaluation of the effect of three different commercial starter cultures (Chr. Hansen, Hørsholm, Denmark) on the volatile compound profile and sensory properties, as well as some important physicochemical parameters, of dry-fermented foal sausages at the end of ripening in order to select the most suitable starter culture for this elaboration. The sausage batches were named as follows: CO (non-inoculated control), FS (Lactobacillus sakei + Staphylococcus carnosus), SM (L. sakei + S. carnosus + Staphylococcus xylosus + Pediococcus pentosaceus + Debaryomyces hansenii) and TR (L. sakei + S. carnosus +S. xylosus). RESULTS: The pH values differed significantly among batches, with the highest values corresponding to CO followed by TR, SM and FS. The highest amounts of volatile compounds were found in FS batch. Hexanal was the most abundant compound, especially in FS and SM batches. These batches also showed higher levels of compounds derived from carbohydrate fermentation and amino acid catabolism. Sensory results showed that acid taste was significantly lower in CO batch than in inoculated batches. CONCLUSION: According to most parameters, batches inoculated with FS and SM starters showed marked acidity compared with TR and CO batches, as expected from the manufacturer's indications. Therefore the most suitable starter culture for use in the manufacture of foal sausages in Mediterranean countries such as Spain with a preference for low-acidity products was found to be TR culture.

Coexistence of Lactic Acid Bacteria and Potential Spoilage Microbiota in a Dairy Processing Environment.

Microbial contamination in food processing plants can play a fundamental role in food quality and safety. In this study, the microbiota in a dairy plant was studied by both 16S rRNA- and 26S rRNA-based culture-independent high-throughput amplicon sequencing. Environmental samples from surfaces and tools were studied along with the different types of cheese produced in the same plant. The microbiota of environmental swabs was very complex, including more than 200 operational taxonomic units with extremely variable relative abundances (0.01 to 99%) depending on the species and sample. A core microbiota shared by 70% of the samples indicated a coexistence of lactic acid bacteria with a remarkable level of Streptococcus thermophilus and possible spoilage-associated bacteria, including Pseudomonas, Acinetobacter, and Psychrobacter, with a relative abundance above 50%. The most abundant yeasts were Kluyveromyces marxianus, Yamadazyma triangularis, Trichosporon faecale, and Debaryomyces hansenii. Beta-diversity analyses showed a clear separation of environmental and cheese samples based on both yeast and bacterial community structure. In addition, predicted metagenomes also indicated differential distribution of metabolic pathways between the two categories of samples. Cooccurrence and coexclusion pattern analyses indicated that the occurrence of potential spoilers was excluded by lactic acid bacteria. In addition, their persistence in the environment can be helpful to counter the development of potential spoilers that may contaminate the cheeses, with possible negative effects on their microbiological quality.

Changes in flavour and microbial diversity during natural fermentation of suan-cai, a traditional food made in Northeast China.

We measured changes in the main physical and chemical properties, flavour compounds and microbial diversity in suan-cai during natural fermentation. The results showed that the pH and concentration of soluble protein initially decreased but were then maintained at a stable level; the concentration of nitrite increased in the initial fermentation stage and after reaching a peak it decreased significantly to a low level by the end of fermentation. Suan-cai was rich in 17 free amino acids. All of the free amino acids increased in concentration to different degrees, except histidine. Total free amino acids reached their highest levels in the mid-fermentation stage. The 17 volatile flavour components identified at the start of fermentation increased to 57 by the mid-fermentation stage; esters and aldehydes were in the greatest diversity and abundance, contributing most to the aroma of suan-cai. Bacteria were more abundant and diverse than fungi in suan-cai; 14 bacterial species were identified from the genera Leuconostoc, Bacillus, Pseudomonas and Lactobacillus. The predominant fungal species identified were Debaryomyces hansenii, Candida tropicalis and Penicillium expansum.

Microbial cell-free extracts as sources of enzyme activities to be used for enhancement flavor development of ewe milk cheese.

Freeze-dried cell-free extracts (CFE) from Lactobacillus casei LC01, Weissella cibaria 1XF5, Hafnia alvei Moller ATCC 51815, and Debaryomyces hansenii LCF-558 were used as sources of enzyme activities for conditioning the ripening of ewe milk cheese. Compared with control cheese (CC), CFE did not affect the gross composition and the growth of the main microbial groups of the cheeses. As shown through urea-PAGE electrophoresis of the pH 4.6-soluble nitrogen fraction and the analysis of free AA, the secondary proteolysis of the cheeses with CFE added was markedly differed from that of the CC. Compared with CC, several enzyme activities were higher in the water-soluble extracts from cheeses made with CFE. In agreement, the levels of 49 volatile compounds significantly differentiated CC from the cheeses made with CFE. The level of some alcohols, ketones, sulfur compounds, and furans were the lowest in the CC, whereas most aldehydes were the highest. Each CFE seemed to affect a specific class of chemical compounds (e.g., the CFE from H. alvei ATCC 51815 mainly influenced the synthesis of sulfur compounds). Apart from the microbial source used, the cheeses with the addition of CFE showed higher score for acceptability than the control cheese. Cheese ripening was accelerated or conditioned using CFE as sources of tailored enzyme activities.

Impact of Debaryomyces hansenii strains inoculation on the quality of slow dry-cured fermented sausages.

Debaryomyces hansenii strains, M4 and P2, isolated from natural fermented sausages were inoculated in slow fermented sausages to study their effect on processing parameters, microbial population, volatile compound and sensory characteristics. The inoculation of D. hansenii strains, M4 and P2, did not affect the ripening process as no differences in pH and Aw were detected. The dominance of the inoculated yeast strains along the process was followed by RAPDs of M13 minisatellite. The inoculated yeasts, P2 and M4, were recovered at the end of the ripening process although P2 appeared in higher counts than M4. The sausages inoculated with P2 resulted in a decrease in lipid oxidation values (TBARS) and a reduction of lipid-oxidation derived aldehydes in addition to a highest acid compound abundance. M4 inoculated sausages resulted in highest sulphur containing compound abundance. However, no differences in consumer acceptance were detected. Moreover, both yeast strains were responsible for the generation of ethyl methyl-branched ester compounds in the dry-cured sausages.

Study of the counts, species and characteristics of the yeast population during the manufacture of dry-cured "lacón". Effect of salt level.

The aim of this work was to study the yeast population during the manufacture of dry-cured "lacón" (a Spanish traditional meat product) and the effect of the salting time. For this study, six batches of "lacón" were manufactured with three different salting times (LS (3 days of salting), MS (4 days of salting) and HS (5 days of salting)). Yeast counts increased significantly (P < 0.001) during the whole process from 2.60 to 6.37 log cfu/g. An increased length of salting time did not affect yeast counts throughout the manufacture of dry-cured "lacón", although the highest yeast counts were obtained from LS batches. A total of 226 isolates were obtained from dry-cured "lacón" during drying-ripening stage, of which 151 were yeasts and were identified at the species level using molecular techniques. The total of 151 identified yeasts belonged to 4 different genera: Debaryomyces, Candida, Cryptococcus and Rhodotorula. Debaryomyces hansenii was the most abundant species isolated throughout the whole process as much in the interior as in the exterior of the pieces of three salt levels of "lacón" studied, while Candida zeylanoides was only isolated from the interior of MS and HS batches and from the exterior of LS and HS groups, but at lesser proportion than D. hansenii.

Changes in sour rotten grape berry microbiota during ripening and wine fermentation.

This study investigated the microbiota of sour rotten wine grapes and its impact on wine fermentations. Yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were enumerated and identified on sound and sour rot grapes during the ripening stage. The alteration of the ecological balance induced by sour rot was particularly evidenced by the unequivocal increase of yeast and AAB counts on rotten grapes, since the beginning of ripening. Yeast and AAB species diversity in rotten grape samples were much higher than those found in sound grapes. LAB populations were low detected from both healthy and sour rotten grapes. The yeast species Issatchenkia occidentalis, Zygoascus hellenicus and Zygosaccharomyces bailii and the AAB species Gluconacetobacter hansenii, Gluconacetobacter intermedius and Acetobacter malorum, were recovered from damaged grapes and resulting grape juices in the winery. Acetobacter orleaniensis and Acetobacter syzygii were only recovered from sour rotten grapes. Dekkera bruxellensis and Oenococcus oeni were only recovered after wine fermentation induced by starter inoculation, irrespective of grape health, probably originating from cellar environment. After malolactic fermentation, racking and sulphur dioxide addition the only remaining species were the yeast Trigonopsis cantarellii and Saccharomyces cerevisiae, independently of the grape health status.

Proteolytic activity of lactic acid bacteria strains and fungal biota for potential use as starter cultures in dry-cured ham.

During the processing of dry-cured meat products, sarcoplasmic and myofibrillar proteins undergo proteolysis, which has a marked effect on product flavor. Microbial proteolytic activity is due to the action of mostly lactic acid bacteria (LAB) and to a lesser extent micrococci. The proteolytic capacity of molds in various meat products is of interest to meat processors in the Mediterranean area. Eleven LAB and mold strains from different commercial origins were tested for proteolytic activity against pork myosin, with a view to possible use of these strains as starter cultures for Iberian dry-cured ham. Proteolytic activity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LAB strains with the highest proteolytic activity were Lactobacillus plantarum (L115), Pediococcus pentosaceus (Saga P TM), and Lactobacillus acidophilus (FARGO 606 TM). The best fungal candidate was Penicillium nalgiovense LEM 50I followed by Penicillium digitatum, Debaryomyces hansenii, and Penicillium chrysogenum.

Improving downstream processes to recover tartaric acid, tartrate and nutrients from vinasses and formulation of inexpensive fermentative broths for xylitol production.

BACKGROUND: Vinasses, the main liquid wastes from the distillation process of grape marc and wine lees, are acidic effluents with high organic content, including acids, carbohydrates, phenols, and unsaturated compounds with high chemical oxygen demand, biological oxygen demand and solid concentrations. These wastes can be revalued to provide additional benefits when they are employed as feedstock of some compounds including tartaric acid, calcium tartrate and economic nutrients for the elaboration of fermentable broths. RESULT: This study attempts to recover tartaric acid and calcium tartrate from vinasses. All the tartaric acid initially solubilised was recovered in both processes. The residual streams can be successfully employed as economic nutrients for the xylose to xylitol bioconversion, achieving higher global volumetric productivities (Q(P, xylitol) = 0.232 g L(-1) h(-1)) and products yields (Y(xylitol/S) = 0.57 g g(-1)) than fermentations carried out using commercial nutrients (Q(P, xylitol) = 0.193 g L(-1) h(-1) and Y(xylitol/S) = 0.55 g g(-1) respectively). CONCLUSION: Tartaric acid can be recovered from vinasses in the form of tartaric acid crystals and calcium tartrate. The residual streams generated in the process can be used as economic nutrients for the production of xylitol by D. hansenii.