Influence of Various Processing Parameters on the Microbial Community Dynamics, Metabolomic Profiles, and Cup Quality During Wet Coffee Processing.

Post-harvest wet coffee processing is a commonly applied method to transform coffee cherries into green coffee beans through depulping or demucilaging, fermentation, washing, soaking, drying, and dehulling. Multiple processing parameters can be modified and thus influence the coffee quality (green coffee beans and cup quality). The present study aimed to explore the impacts of these parameters, including processing type (depulping or demucilaging), fermentation duration, and application of soaking, on the microbial community dynamics, metabolite compositions of processing waters (fermentation and soaking) and coffee beans, and resulting cup quality through a multiphasic approach. A large-scale wet coffee processing experiment was conducted with Coffea arabica var. Catimor in Yunnan (China) in duplicate. The fermentation steps presented a dynamic interaction between constant nutrient release (mainly from the cherry mucilage) into the surrounding water and active microbial activities led by lactic acid bacteria, especially Leuconostoc and Lactococcus. The microbial communities were affected by both the processing type and fermentation duration. At the same time, the endogenous coffee bean metabolism remained active at different stages along the processing, as could be seen through changes in the concentrations of carbohydrates, organic acids, and free amino acids. Among all the processing variants tested, the fermentation duration had the greatest impact on the green coffee bean compositions and the cup quality. A long fermentation duration resulted in a fruitier and more acidic cup. As an ecological alternative for the depulped processing, the demucilaged processing produced a beverage quality comparable to the depulped one. The application of soaking, however, tempered the positive fermentation effects and standardized the green coffee bean quality, regardless of the preceding processing practices applied. Lastly, the impact strength of each processing parameter would also depend on the coffee variety used and the local geographical conditions. All these findings provide a considerable margin of opportunities for future coffee research.

Following Coffee Production from Cherries to Cup: Microbiological and Metabolomic Analysis of Wet Processing of Coffea arabica.

A cup of coffee is the final product of a complex chain of operations. Wet postharvest processing of coffee is one of these operations, which involves a fermentation that inevitably has to be performed on-farm. During wet coffee processing, the interplay between microbial activities and endogenous bean metabolism results in a specific flavor precursor profile of the green coffee beans. Yet, how specific microbial communities and the changing chemical compositions of the beans determine the flavor of a cup of coffee remains underappreciated. Through a multiphasic approach, the establishment of the microbial communities, as well as their prevalence during wet processing of Coffea arabica, was followed at an experimental farm in Ecuador. Also, the metabolites produced by the microorganisms and those of the coffee bean metabolism were monitored to determine their influence on the green coffee bean metabolite profile over time. The results indicated that lactic acid bacteria were prevalent well before the onset of fermentation and that the fermentation duration entailed shifts in their communities. The fermentation duration also affected the compositions of the beans, so that longer-fermented coffee had more notes that are preferred by consumers. As a consequence, researchers and coffee growers should be aware that the flavor of a cup of coffee is determined before as well as during on-farm processing and that under the right conditions, longer fermentation times can be favorable, although the opposite is often believed.IMPORTANCE Coffee needs to undergo a long chain of events to transform from coffee cherries to a beverage. The coffee postharvest processing is one of the key phases that convert the freshly harvested cherries into green coffee beans before roasting and brewing. Among multiple existing processing methods, the wet processing has been usually applied for Arabica coffee and produces decent quality of both green coffee beans and the cup of coffee. In the present case study, wet processing was followed by a multiphasic approach through both microbiological and metabolomic analyses. The impacts of each processing step, especially the fermentation duration, were studied in detail. Distinct changes in microbial ecosystems, processing waters, coffee beans, and sensory quality of the brews were found. Thus, through fine-tuning of the parameters in each step, the microbial diversity and endogenous bean metabolism can be altered during coffee postharvest processing and hence provide potential to improve coffee quality.

Variability within the dominant microbiota of sliced cooked poultry products at expiration date in the Belgian retail.

Sliced cooked poultry products are susceptible to bacterial spoilage, notwithstanding their storage under modified-atmosphere packaging (MAP) in the cold chain. Although the prevailing bacterial communities are known to be mostly consisting of lactic acid bacteria (LAB), more information is needed about the potential variation in species diversity within national markets. In the present study, a total of 42 different samples of sliced cooked poultry products were collected in the Belgian retail and their bacterial communities were analysed at expiration date. A total of 629 isolates from four different culture media, including plate count agar for the total microbiota and de Man-Rogosa-Sharpe (MRS), modified MRS, and M17 agar as three selective agar media for LAB, were subjected to (GTG)5-PCR fingerprinting and identification by gene sequencing. Overall, Carnobacterium, Lactobacillus, and Leuconostoc were the dominant genera. Within each genus, the most encountered isolates were Carnobacterium divergens, Lactobacillus sakei, and Leuconostoc carnosum. When comparing samples from chicken origin with samples from turkey-derived products, a higher dominance of Carnobacteria spp. was found in the latter group. Also, an association between the dominance of lactobacilli and the presence of added plant material and lactate salts was found.

Fermentation Titer Optimization and Impact on Energy and Water Consumption during Downstream Processing.

A common focus of fermentation process optimization is the product titer. Different strategies to boost fermentation titer target whole-cell biocatalyst selection, process control, and medium composition. Working at higher product concentrations reduces the water that needs to be removed in the case of aqueous systems and, therefore, lowers the cost of downstream separation and purification. Different approaches to achieve higher titer in fermentation are examined. Energy and water consumption data collected from different Cargill fermentation plants, i.e., ethanol, lactic acid, and 2-keto-L-gulonic acid, confirm that improvements in fermentation titer play a decisive role in downstream economics and environmental footprint.

Diversity of the dominant bacterial species on sliced cooked pork products at expiration date in the Belgian retail.

Pork-based cooked products, such as cooked hams, are economically valuable foods that are vulnerable to bacterial spoilage, even when applying cooling and modified atmosphere packaging (MAP). Besides a common presence of Brochothrix thermosphacta, their microbiota are usually dominated by lactic acid bacteria (LAB). Yet, the exact LAB species diversity can differ considerably among products. In this study, 42 sliced cooked pork samples were acquired from three different Belgian supermarkets to map their bacterial heterogeneity. The community compositions of the dominant bacterial species were established by analysing a total of 702 isolates from selective agar media by (GTG)5-PCR fingerprinting followed by gene sequencing. Most of the isolates belonged to the genera Carnobacterium, Lactobacillus, and Leuconostoc, with Leuconostoc carnosum and Leuconostoc gelidum subsp. gelidum being the most dominant members. The diversity of the dominant bacterial species varied when comparing samples from different production facilities and, in some cases, even within the same product types. Although LAB consistently dominated the microbiota of sliced cooked pork products in the Belgian market, results indicated that bacterial diversity needs to be addressed on the level of product composition and batch variation. Dedicated studies will be needed to substantiate potential links between such variability and microbial composition. For instance, the fact that higher levels of lactobacilli were associated with the presence of potassium lactate (E326) may be suggestive of selective pressure but needs to be validated, as this finding referred to a single product only.

Exploring the Impacts of Postharvest Processing on the Microbiota and Metabolite Profiles during Green Coffee Bean Production.

The postharvest treatment and processing of fresh coffee cherries can impact the quality of the unroasted green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through two different wet and dry methods to monitor differences in the microbial community structure and in substrate and metabolite profiles. The changes were followed throughout the postharvest processing chain, from harvest to drying, by implementing up-to-date techniques, encompassing multiple-step metagenomic DNA extraction, high-throughput sequencing, and multiphasic metabolite target analysis. During wet processing, a cohort of lactic acid bacteria (i.e., Leuconostoc, Lactococcus, and Lactobacillus) was the most commonly identified microbial group, along with enterobacteria and yeasts (Pichia and Starmerella). Several of the metabolites associated with lactic acid bacterial metabolism (e.g., lactic acid, acetic acid, and mannitol) produced in the mucilage were also found in the endosperm. During dry processing, acetic acid bacteria (i.e., Acetobacter and Gluconobacter) were most abundant, along with Pichia and non-Pichia (Candida, Starmerella, and Saccharomycopsis) yeasts. Accumulation of associated metabolites (e.g., gluconic acid and sugar alcohols) took place in the drying outer layers of the coffee cherries. Consequently, both wet and dry processing methods significantly influenced the microbial community structures and hence the composition of the final green coffee beans. This systematic approach to dissecting the coffee ecosystem contributes to a deeper understanding of coffee processing and might constitute a state-of-the-art framework for the further analysis and subsequent control of this complex biotechnological process. IMPORTANCE: Coffee production is a long process, starting with the harvest of coffee cherries and the on-farm drying of their beans. In a later stage, the dried green coffee beans are roasted and ground in order to brew a cup of coffee. The on-farm, postharvest processing method applied can impact the quality of the green coffee beans. In the present case study, freshly harvested Arabica coffee cherries were processed through wet and dry processing in four distinct variations. The microorganisms present and the chemical profiles of the coffee beans were analyzed throughout the postharvest processing chain. The up-to-date techniques implemented facilitated the investigation of differences related to the method applied. For instance, different microbial groups were associated with wet and dry processing methods. Additionally, metabolites associated with the respective microorganisms accumulated on the final green coffee beans.

Lactic acid bacteria and their controversial role in fresh meat spoilage.

Lactic acid bacteria (LAB) constitute a heterogeneous group that has been widely associated with fresh meat and cooked meat products. They represent a controversial cohort of microbial species that either contribute to spoilage through generation of offensive metabolites and the subsequent organoleptic downgrading of meat or serve as bioprotective agents with strains of certain species causing unperceivable or no alterations. Therefore, significant distinction among biotypes is substantiated by studies determining spoilage potential as a strain-specific trait corroborating the need to revisit the concept of spoilage.

Processing Environment and Ingredients Are Both Sources of Leuconostoc gelidum, Which Emerges as a Major Spoiler in Ready-To-Eat Meals.

Mesophilic and psychrotrophic organism viable counts, as well as high-throughput 16S rRNA gene-based pyrosequencing, were performed with the aim of elucidating the origin of psychrotrophic lactic acid bacteria (LAB) in a ready-to-eat (RTE) meal manufacturing plant. The microbial counts of the products at the end of the shelf life were greatly underestimated when mesophilic incubation was implemented due to overlooked, psychrotrophic members of the LAB. Pseudomonas spp., Enterobacteriaceae, Streptococcaceae, and Lactobacillus spp. constituted the most widespread operational taxonomic units (OTUs), whereas Leuconostoc gelidum was detected as a minor member of the indigenous microbiota of the food ingredients and microbial community of the processing environment, albeit it colonized samples at almost every sampling point on the premises. However, L. gelidum became the most predominant microbe at the end of the shelf life. The ability of L. gelidum to outgrow notorious, spoilage-related taxa like Pseudomonas, Brochothrix, and Lactobacillus underpins its high growth dynamics and severe spoilage character under refrigeration temperatures. The use of predicted metagenomes was useful for observation of putative gene repertoires in the samples analyzed in this study. The end products grouped in clusters characterized by gene profiles related to carbohydrate depletion presumably associated with a fast energy yield, a finding which is consistent with the fastidious nature of highly competitive LAB that dominated at the end of the shelf life. The present study showcases the detrimental impact of contamination with psychrotrophic LAB on the shelf life of packaged and cold-stored foodstuffs and the long-term quality implications for production batches once resident microbiota are established in the processing environment.

Exploring the strain-specific attachment of Leuconostoc gelidum subsp. gasicomitatum on food contact surfaces.

The psychrotrophic lactic acid bacterium (LAB) Leuconostoc gelidum subsp. gasicomitatum has emerged as one of the most prevalent specific spoilage organisms (SSOs) of packaged, cold-stored food products in Northern Europe. The whole genome sequencing of the type strain L. gelidum subsp. gasicomitatum LMG 18811(T) revealed genes encoding for proteins related to adhesion. In the present study the attachment of six food and environmental isolates was monitored on stainless steel (SS) and glass surfaces incubated (7 °C for 5-9 days) in two food simulating substrates (i.e. sweet bell pepper juice and boiled eggs in brine). The selection encompassed unique genotypes, isolated from different food products or sampling sites as well as slime-forming biotypes. The evaluation of the attached cells was performed with the bead vortexing method and a viability staining assay coupled with epifluorescence microscopy. On SS surfaces the slime-formers showed the lowest attachment (3.3-4.5 logCFU/cm(2)), while strain L. gelidum subsp. gasicomitatum ab2, which was isolated from an acetic acid bath in a vegetable salad company, reached significantly higher populations of attached cells exceeding 7 logCFU/cm(2). Strain ab2 formed dense cell aggregations on SS after 9 days of incubation in sweet bell pepper juice. The attachment ability of L. gelidum subsp. gasicomitatum on surfaces documented in the present study extends our knowledge and understanding of the spoilage potential and intra-subspecies diversity of this microbe.

Psychrotrophic lactic acid bacteria associated with production batch recalls and sporadic cases of early spoilage in Belgium between 2010 and 2014.

Between 2010 and 2014 several spoilage cases in Belgium occurring in retail foodstuffs prior to the end of shelf-life have been reported to our laboratory. Overall, seven cases involved strictly psychrotrophic lactic acid bacteria (LAB) contamination in packaged and chilled-stored food products. The products derived either from recalls of entire production batches or as specimens of sporadic spoilage manifestations. Some of these samples were returned to the manufacturing companies by consumers who observed the alterations after purchasing the products. The products covered a wide range of foodstuffs (i.e. meat, dairy, vegetable, egg products and composite food) and denoted different spoilage defects. However, the microbiota determined by means of 16S rRNA gene high-throughput sequencing analysis underpin few LAB genera (i.e. Leuconostoc, Lactobacillus, Weissella and Lactococcus), which are frequently encountered nowadays as specific spoilage organisms (SSO) albeit overlooked by mesophilic enumeration methods due to their strictly psychrotrophic character. The present study confirms the spreading of psychrotrophic LAB in Belgian food processing environments leading to unexpected spoilage, corroborating their spoilage dynamics and prevalence in all kinds of packaged and refrigerated foodstuffs in Northern Europe.

Monitoring psychrotrophic lactic acid bacteria contamination in a ready-to-eat vegetable salad production environment.

A study monitoring lactic acid bacteria contamination was conducted in a company producing fresh, minimally processed, packaged and ready-to-eat (RTE) vegetable salads (stored at 4°C) in order to investigate the reason for high psychrotrophic LAB levels in the products at the end of shelf-life. Initially, high microbial counts exceeding the established psychrotrophic thresholds (>10(7)-10(8)CFU/g) and spoilage manifestations before the end of the shelf-life (7days) occurred in products containing an assortment of sliced and diced vegetables, but within a one year period these spoilage defects became prevalent in the entire processing plant. Environmental sampling and microbiological analyses of the raw materials and final products throughout the manufacturing process highlighted the presence of high numbers of Leuconostoc spp. in halved and unseeded, fresh sweet bell peppers provided by the supplier. A combination of two DNA fingerprinting techniques facilitated the assessment of the species diversity of LAB present in the processing environment along with the critical point of their introduction in the production facility. Probably through air mediation and surface adhesion, mainly members of the strictly psychrotrophic species Leuconostoc gelidum subsp. gasicomitatum and L. gelidum subsp. gelidum were responsible for the cross-contamination of every vegetable handled within the plant.

Spoilage potential of psychrotrophic lactic acid bacteria (LAB) species: Leuconostoc gelidum subsp. gasicomitatum and Lactococcus piscium, on sweet bell pepper (SBP) simulation medium under different gas compositions.

Sweet bell peppers are a significant constituent of retail, chilled-stored and packaged food products like fresh salads, marinades and ready-to-eat (RTE) meals. Previously, through general screening of the Belgian market and by means of source tracking analysis in a plant manufacturing minimally processed, vegetable salads the susceptibility of fresh-cut sweet bell peppers to lactic acid bacterium (LAB) contamination was substantiated. The determination of the metabolic profiles of Leuconostoc gelidum subsp. gasicomitatum and Lactococcus piscium, two major psychrotrophic, spoilage-related LAB species, on sweet bell pepper (SBP) simulation medium under different packaging conditions - 1.) vacuum: 100% N2, 2.) air: 21% O2, 79% N2, 3.) MAP1: 30% CO2, 70% N2 and 4.) MAP2: 50% O2, 50% CO2 - facilitated a better understanding of the spoilage potential of these microbes as well as the presumptive contribution of O2 in the spectrum of produced volatile organic compounds (VOCs) associated with poor organoleptic properties of food products. Generally, none of the applied gas compositions inhibited the growth of the 4 L. gelidum subsp. gasicomitatum isolates, however the presence of O2 resulted in buttery off-odors by inducing primarily the accumulation of diacetyl and pungent "vinegar" smell due to acetic acid. The 3 tested isolates of L. piscium varied greatly among their growth dynamics and inhibition at MAP2. They exhibited either weak spoilage profile or very offensive metabolism confirming significant intraspecies diversity.

Psychrotrophic members of Leuconostoc gasicomitatum, Leuconostoc gelidum and Lactococcus piscium dominate at the end of shelf-life in packaged and chilled-stored food products in Belgium.

Previously, a considerable underestimation (+0.5-3.2 log CFU/g) on the contamination levels of psychrotrophic lactic acid bacteria (LAB) was observed for 33 retail, packaged food products stored at chilling temperature when the mesophilic enumeration technique was implemented as reference shelf-life parameter. In the present study, the microbial diversity of the dominant psychrotrophic LAB recovered after incubation of plates at 22 °C for 5 days was determined using a polyphasic taxonomic approach. A total of 212 LAB isolates were identified using a combination of rep-PCR fingerprinting, amplified fragment length polymorphism (AFLP) analysis and pheS gene sequencing. Leuconostoc gasicomitatum, Leuconostoc gelidum, Leuconostoc spp., Lactococcus piscium and Lactobacillus algidus proved to be the most competent and predominant species that may go undetected by the widely applied mesophilic enumeration protocols (ISO 4833:2003 and ISO 15214:1998). This study has assessed the interspecific variation among potential spoilage LAB, and highlights the significance of implementing a reference shelf-life parameter based on the enumeration of the total psychrotrophic bacterial load for industrial microbiological routine analyses.

Effect of high oxygen and high carbon dioxide atmosphere packaging on the microbial spoilage and shelf-life of fresh-cut honeydew melon.

This study evaluated the potential of modified atmospheres (MAs) combining high oxygen (O2) and high carbon dioxide (CO2) levels to extend the shelf-life of fresh-cut honeydew melon. Firstly, the effect of MA on the growth and volatile organic metabolite production of Candida sake, Leuconostoc mesenteroides and Leuconostoc gelidum, which had all been previously isolated from spoiled commercial fresh-cut honeydew melon, was evaluated separately on honeydew melon agar at 7 °C. Additionally, the effect of selected MAs on the microbial, physico-chemical and sensory quality of commercial fresh-cut honeydew melon cubes was evaluated at 7 °C. The results showed that MAs with high O2 and high CO2 levels greatly retarded the growth, CO2 and volatile metabolite production (i.e. ethanol, 2-methyl-1-butanol, ethyl acetate, phenylacetic acid, nonanal) of C. sake on honeydew melon agar; especially MAs consisting of 50% O2+50% CO2 and 70% O2+30% CO2. In contrast, the MAs evaluated only had a minor effect on the growth and volatile metabolite production of L. mesenteroides and L. gelidum. Overall, the effect of MAs on colour, juice leakage, juiciness, and firmness of fresh-cut honeydew melon was small during storage. Sensory preference was generally for fresh-cut honeydew melon cubes packaged in MA of 50% O2+50% CO2. These were still acceptable on day five of storage and had appreciably lower populations of yeasts and lactic acid bacteria, lower quantities of volatile organic compounds, but slightly stronger colour oxidation compared to honeydew melon that was packaged in air. Additionally, most of the samples packed in air had blown by day five due to the large quantity of CO2 production during storage. Therefore, 50% O2+50% CO2 is a potential MA solution for extending the shelf-life of fresh-cut honeydew melon.

Total mesophilic counts underestimate in many cases the contamination levels of psychrotrophic lactic acid bacteria (LAB) in chilled-stored food products at the end of their shelf-life.

The major objective of this study was to determine the role of psychrotrophic lactic acid bacteria (LAB) in spoilage-associated phenomena at the end of the shelf-life of 86 various packaged (air, vacuum, modified-atmosphere) chilled-stored retail food products. The current microbiological standards, which are largely based on the total viable mesophilic counts lack discriminatory capacity to detect psychrotrophic LAB. A comparison between the total viable counts on plates incubated at 30 °C (representing the mesophiles) and at 22 °C (indicating the psychrotrophs) for 86 food samples covering a wide range - ready-to-eat vegetable salads, fresh raw meat, cooked meat products and composite food - showed that a consistent underestimation of the microbial load occurs when the total aerobic mesophilic counts are used as a shelf-life parameter. In 38% of the samples, the psychrotrophic counts had significantly higher values (+0.5-3 log CFU/g) than the corresponding total aerobic mesophilic counts. A total of 154 lactic acid bacteria, which were unable to proliferate at 30 °C were isolated. In addition, a further 43 with a poor recovery at this temperature were also isolated. This study highlights the potential fallacy of the total aerobic mesophilic count as a reference shelf-life parameter for chilled food products as it can often underestimate the contamination levels at the end of the shelf-life.