Bioprospecting of indigenous yeasts involved in cocoa fermentation using sensory and chemical strategies for selecting a starter inoculum.

Cocoa fermentation is the key and most relevant process in the synthesis of aroma and flavor precursor molecules in dry beans or raw material for producing chocolate. Because this process occurs in an uncontrolled manner, the chemical and sensory quality of beans can vary and be negatively affected. One of the strategies for the standardization and improvement of the sensory quality of chocolate is the introduction of microbial starter cultures. Among these, yeasts involved in fermentation have been studied because of their pectinolytic and metabolic potential in the production of volatile compounds. This study was aimed at isolating and characterizing, both sensory and chemically, yeasts involved in cocoa fermentation that could be used as starter cultures from two agro-ecological regions for the cultivation of cocoa in Colombia. The microbiological analyses identified 22 species represented mostly by Saccharomyces cerevisiae, Wickerhamomyces anomalus and Pichia sp. The preliminary sensory analysis of eight of these species showed that Hanseniaspora thailandica and Pichia kluyveri presented sensory profiles characterized by high intensity levels of fruity notes, which could be ascribed to the production of ethyl acetate, isoamyl acetate, and 2-phenylethyl acetate.

Microbiological analysis of frozen profiteroles and mini chocolate eclairs implicated in a national salmonellosis outbreak.

Between November 2018 and May 2019, Canada experienced a nationwide salmonellosis outbreak linked to the presence of Salmonella enterica ser. Enteritidis in frozen profiteroles. Analysis of the implicated food products revealed low levels of Salmonella ranging from 0.2 to 0.7 MPN/100g. Water activity and pH of the food samples ranged from 0.9479 to 0.9867 and 4.6-6.8 respectively indicating conditions conducive to bacterial growth. Higher levels of the hygiene indicators Enterobacteriaceae and coliforms were associated with Salmonella positive samples compared to Salmonella negative samples. Investigation of the relationship between storage conditions, temperature, and pathogen levels during thawing revealed that the profiteroles reached temperatures permissive to pathogen growth (≥5 °C) much sooner than pathogen growth was observed and that the composition of the food matrix can influence bacterial levels upon thawing. Collectively these data can be used to inform guidance to minimize the risk of infection from the consumption of contaminated cream-filled frozen desserts.

Metagenome-Assembled Genomes Contribute to Unraveling of the Microbiome of Cocoa Fermentation.

Metagenomic studies about cocoa fermentation have mainly reported on the analysis of short reads for determination of operational taxonomic units. However, it is also important to determine metagenome-assembled genomes (MAGs), which are genomes deriving from the assembly of metagenomics. For this research, all the cocoa metagenomes from public databases were downloaded, resulting in five data sets: one from Ghana and four from Brazil. In addition, in silico approaches were used to describe putative phenotypes and the metabolic potential of MAGs. A total of 17 high-quality MAGs were recovered from these microbiomes, as follows: (i) for fungi, Yamadazyma tenuis (n = 1); (ii) lactic acid bacteria, Limosilactobacillus fermentum (n = 5), Liquorilactobacillus cacaonum (n = 1), Liquorilactobacillus nagelli (n = 1), Leuconostoc pseudomesenteroides (n = 1), and Lactiplantibacillus plantarum subsp. plantarum (n = 1); (iii) acetic acid bacteria, Acetobacter senegalensis (n = 2) and Kozakia baliensis (n = 1); and (iv) Bacillus subtilis (n = 1), Brevundimonas sp. (n = 2), and Pseudomonas sp. (n = 1). Medium-quality MAGs were also recovered from cocoa microbiomes, including some that, to our knowledge, were not previously detected in this environment (Liquorilactobacillus vini, Komagataeibacter saccharivorans, and Komagataeibacter maltaceti) and others previously described (Fructobacillus pseudoficulneus and Acetobacter pasteurianus). Taken together, the MAGs were useful for providing an additional description of the microbiome of cocoa fermentation, revealing previously overlooked microorganisms, with prediction of key phenotypes and biochemical pathways. IMPORTANCE The production of chocolate starts with the harvesting of cocoa fruits and the spontaneous fermentation of the seeds in a microbial succession that depends on yeasts, lactic acid bacteria, and acetic acid bacteria in order to eliminate bitter and astringent compounds present in the raw material, which will be further roasted and grinded to originate the cocoa powder that will enter the food processing industry. The microbiota of cocoa fermentation is not completely known, and yet it advanced from culture-based studies to the advent of next-generation DNA sequencing, with the generation of a myriad of data that need bioinformatic approaches to be properly analyzed. Although the majority of metagenomic studies have been based on short reads (operational taxonomic units), it is also important to analyze entire genomes to determine more precisely possible ecological roles of different species. Metagenome-assembled genomes (MAGs) are very useful for this purpose; here, MAGs from cocoa fermentation microbiomes are described, and the possible implications of their phenotypic and metabolic potentials are discussed.

Dissecting industrial fermentations of fine flavour cocoa through metagenomic analysis.

The global demand for fine-flavour cocoa has increased worldwide during the last years. Fine-flavour cocoa offers exceptional quality and unique fruity and floral flavour attributes of high demand by the world's elite chocolatiers. Several studies have highlighted the relevance of cocoa fermentation to produce such attributes. Nevertheless, little is known regarding the microbial interactions and biochemistry that lead to the production of these attributes on farms of industrial relevance, where traditional fermentation methods have been pre-standardized and scaled up. In this study, we have used metagenomic approaches to dissect on-farm industrial fermentations of fine-flavour cocoa. Our results revealed the presence of a shared core of nine dominant microorganisms (i.e. Limosilactobacillus fermentum, Saccharomyces cerevisiae, Pestalotiopsis rhododendri, Acetobacter aceti group, Bacillus subtilis group, Weissella ghanensis group, Lactobacillus_uc, Malassezia restricta and Malassezia globosa) between two farms located at completely different agro-ecological zones. Moreover, a community metabolic model was reconstructed and proposed as a tool to further elucidate the interactions among microorganisms and flavour biochemistry. Our work is the first to reveal a core of microorganisms shared among industrial farms, which is an essential step to process engineering aimed to design starter cultures, reducing fermentation times, and controlling the expression of undesirable phenotypes.

Impact of encapsulating probiotics with cocoa powder on the viability of probiotics during chocolate processing, storage, and in vitro gastrointestinal digestion.

Chocolates can be formulated as a functional food via enrichment with probiotics. However, the added probiotics must overcome the challenges of processing and storage conditions and the harsh gastrointestinal environment. The study aimed to overcome these challenges using two different formulations of cocoa powder as alternative encapsulants along with Na-alginate (A1 ) and Na-alginate and fructooligosaccharides (A2 ). Seven different probiotic strains were encapsulated individually using the new formulations and viabilities of these encapsulated probiotics were assessed prior to and after they were added to chocolates. The highest achieved encapsulation efficiencies were 93.40% for formulation A1 (with Lactobacillus casei) and 95.36% for formulation A2 (with Lactobacillus acidophilus La5). The encapsulated probiotics with the new formulations maintained higher viability than the recommended therapeutic level (107 colony forming unit [CFU]/g) for up to 180 and 120 days of storage at 4 and 25 °C, respectively. The tested encapsulants improved probiotics survival when subjected to thermal stress and maintained about 9.0 Logs CFU/g at 60 °C. Additionally, the viable numbers of probiotics in fortified chocolates showed higher than 7 Logs CFU/g after 90 days of storage at 25 °C. Both formulations exhibited significantly (P < 0.05) high survivability of probiotics (8.0 Logs CFU/g) during the in vitro gastrointestinal digestion. This study demonstrated that cocoa powder along with Na-alginate and FOS has the potential to be used as a probiotic encapsulating material, and chocolates could be an excellent carrier for the development of healthy probiotic chocolate products. PRACTICAL APPLICATION: The introduction of cocoa powder as an effective encapsulating agent to deliver probiotics could help the chocolate industry to develop healthy and attractive functional snacks for health-conscious consumers.

Thermo-adaptive evolution to generate improved Saccharomyces cerevisiae strains for cocoa pulp fermentations.

Cocoa pulp fermentation is a consequence of the succession of indigenous yeasts, lactic acid bacteria and acetic acid bacteria that not only produce a diversity of metabolites, but also cause the production of flavour precursors. However, as such spontaneous fermentations are less reproducible and contribute to produce variability, interest in a microbial starter culture is growing that could be used to inoculate cocoa pulp fermentations. This study aimed to generate robust S. cerevisiae strains by thermo-adaptive evolution that could be used in cocoa fermentation. We evolved a cocoa strain in a sugary defined medium at high temperature to improve both fermentation and growth capacity. Moreover, adaptive evolution at high temperature (40 °C) also enabled us to unveil the molecular basis underlying the improved phenotype by analysing the whole genome sequence of the evolved strain. Adaptation to high-temperature conditions occurred at different genomic levels, and promoted aneuploidies, segmental duplication, and SNVs in the evolved strain. The lipid profile analysis of the evolved strain also evidenced changes in the membrane composition that contribute to maintain an appropriate cell membrane state at high temperature. Our work demonstrates that experimental evolution is an effective approach to generate better-adapted yeast strains at high temperature for industrial processes.

Influence of S. cerevisiae and P. kluyveri as starters on chocolate flavour.

BACKGROUND: Fermented cocoa beans (Theobroma cacao L.) are a pivotal raw material for chocolate production. A cocktail yeast applied in the cocoa fermentation process can promote the formation of pleasant metabolites. Saccharomyces, Pichia and Hanseniaspora have been widely used in fermentation to improve the final product organoleptic profile, highlighting that fermentation is a critical point for chocolate flavour precursor production. This study aims to evaluate the impact of Pichia kluyveri and Saccharomyces cerevisiae strains as starter cultures on the fermentation for two cocoa hybrids, FA13 and CEPEC2002. RESULTS: During fermentation processes, volatile organic compounds (VOCs) and protein profiles were assessed. Chocolates produced were also assessed regarding the presence of VOCs. Eighty VOCs were identified using gas chromatography coupled to mass spectrometry analysis. Mass spectrometry provided the protein profile evolution during fermentation and showed that the profiles changed with inoculation type (spontaneous versus inoculated fermentation). Chocolate obtained from FA13 inoculated with S. cerevisiae strain contained a greater amount of organics acids, being categorised as sourer than chocolate produced by spontaneous fermentation of FA13. CEPEC2002 inoculated with S. cerevisiae strain in co-culture with P. kluyveri strain generated less sour and sweeter chocolate than spontaneous fermentation only. CONCLUSIONS: Chocolates from inoculated assays with starter cultures were more accepted by evaluators, highlighting that P. kluyveri and S. cerevisiae influence the composition of VOCs. Besides, protein profiles also changed throughout fermentation. Further investigation should be conducted to clarify protein degradation dynamics during inoculated fermentations to define which of the microbial cultures positively affect the chocolate sensory characteristics. © 2021 Society of Chemical Industry.

Development of a model for the alcoholic fermentation of cocoa beans by a Saccharomyces cerevisiae strain.

The aromatic quality of chocolate requires the use of cocoa with high aromatic potential, this being acquired during the fermentation of cocoa beans. Traditional fermentation is still often carried out on a small scale with wild strains of yeasts and acetic bacteria and under poorly controlled conditions leading to cocoa quality ranging from best to worst. This study is the first part of a project aiming to control quality of cocoa to produce high aromatic quality chocolate by using a mixed starter of selected strains of yeast and acetic bacteria and by controlling the conditions of fermentation. To achieve this objective, a mathematical model of the alcoholic fermentation of cocoa beans has been developed. The growth, glucose consumption and ethanol production of Saccharomyces cerevisiae LM strain in synthetic broth were modeled for the most important intrinsic (pH, glucose, ethanol, free nitrogen and oxygen levels) and extrinsic (temperature, oxygen level) fermentation parameters. The model was developed by combining the effects of individual conditions in a multiplicative way using the gamma concept. The model was validated in liquid synthetic medium at two different inoculation levels 104 and 106 CFU/mL with an increase in temperature that recorded during spontaneous fermentations. The model clearly shows that the level of inoculation and the speed of the increase in temperature clearly drive yeast growth, while other factors including pH and ethanol, free nitrogen and oxygen levels have no significant impact on yeast growth.

Chemical implications and time reduction of on-farm cocoa fermentation by Saccharomyces cerevisiae and Pichia kudriavzevii.

The use of starters during fermentation has been gaining momentum as it can warrant high-quality chocolate. The objective of this study was to investigate the influence of Saccharomyces cerevisiae (Sc) and Pichia kudriavzevii (Pk) during on-farm fermentation on physico-chemical and microbiological characteristics and levels of methylxanthines and bioactive amines of cocoa. Four treatments were used: ScPk (1:1), only Sc, only Pk, and no starter (control). The starters lead to changes throughout fermentation, but provided fermented cocoa with similar pH, titratable acidity, reducing sugars and phenolic compounds. ScPk shortened fermentation time by 24 h. The ScPk fermented and dried cocoa had higher levels of monomeric phenols, methylxanthines, phenylethylamine and lower levels of the putrefactive amines - putrescine and cadaverine (p < 0.05). The results were confirmed by multivariate analysis. Based on these results, the mixture of both yeasts species is a promising starter for cocoa fermentation decreasing duration time and modulating high-quality components.

Survival of commercial probiotic strains and their effect on dark chocolate synbiotic snack with raspberry content during the storage and after simulated digestion

BACKGROUND: A key challenge for manufacturers of pro-health food containing active probiotic microorganisms is to develop a product with attractive sensory features along with maintenance of declared number of microorganisms during storage and transfer by alimentary tract. RESULTS: The highest concentration of polyphenols was observed in snacks without an additive of probiotics as well as those with an additive of L. rhamnosus and B. animalis bacteria and concentration of these compounds increased by 9.5% during six months of storage. None of the products distinguished itself in the sensorial assessment although each was assessed positively. The number of microorganisms was stable and comparatively high during six months of storage at a room temperature and in cooling conditions (108 cfu/g). In the digestion model, an influence of aggressive digestion conditions was examined in the alimentary tract on the number of microorganisms, which allowed to arrange strains from the most resistant (S. boulardii) to the most sensitive (B. breve). It must be noted that currently on the market there is no available snack containing probiotic yeast as well as there is no literature data on works on such formulation of food. CONCLUSIONS: In the newly developed snack made of chocolate, in which sugar has been replaced with maltitol, a raw material was added in the form of raspberry, prebiotic in the form of inulin and a strain of probiotic bacteria, including the unprecedented so far S. boulardii, which stands a high chance to occupy a good place on the market of functional food.

Inactivation of Salmonella and Listeria monocytogenes on dried fruit, pistachio nuts, cornflakes and chocolate crumb using a peracetic acid-ethanol based sanitizer or Advanced Oxidation Process.

Two decontamination methods were evaluated for inactivating a cocktail of Salmonella or Listeria monocytogenes inoculated onto model low moisture foods (LMFs; dried strawberry, dried apple, raisins, chocolate crumb, cornflakes, shell-on or deshelled pistachio nuts). One treatment was based on a peracetic acid-ethanol (PAA-ethanol) sanitizer combination with the other being an Advanced Oxidation Process (AOP) that simultaneously applied UV-C (254 nm), ozone and hydrogen peroxide. The low moisture food was spray inoculated then dried prior to treatment. With Salmonella it was found that a pre-incubation step in 1% w/v glycerol-tryptic soy broth for 1 h prior to plating, significantly increased recovery of the pathogen compared to TSB alone. However, no increased recovery of L. monocytogenes was observed using the TSB-glycerol pre-incubation step. No Salmonella was detected on cornflakes, chocolate crumb and strawberry using 1.25 parts per thousand (‰) PAA-ethanol. The inactivation of Salmonella on deshelled pistachio was significantly higher using 2.5‰ PAA-ethanol sanitizer compared to the AOP treatments tested. Only negligible reductions of Salmonella (<1 log cfu) were obtained with shell-on pistachio treated with PAA-ethanol sanitizer or AOP. Salmonella could be reduced on dried apple slices by >4 log CFU when 5.0‰ PAA-ethanol was applied. L. monocytogenes was more sensitive to PAA-ethanol compared to Salmonella and could be eliminated on all the LMFs apart from shell-on pistachio. An AOP treatment applied 10% v/v hydrogen peroxide, ozone and 54 mJ/cm2 UV-C could significantly reduce Salmonella on dried apple slices compared to when the individual elements (hydrogen peroxide, ozone or UV-C) were applied. Salmonella was also eliminated by AOP on the other LMFs (apart from shell-on pistachio) although the same level of inactivation was achieved by spraying with 10% v/v hydrogen peroxide alone. L. monocytogenes was sensitive to hydrogen peroxide and AOP being eliminated from all the LMFs. Although this may suggest that hydrogen peroxide spray was equivalent to AOP treatment it was noted that no residual H2O2 or changes in visual appearance was evident on samples treated with the latter process. The study has demonstrated that the two decontamination methods assessed can be applied to reduce Salmonella and L. monocytogenes on LMFs although efficacy is dependent on the pathogen and product type.

Is there an impact of the dairy matrix on the survival of Lactobacillus casei Lc-1 during shelf life and simulated gastrointestinal conditions?

BACKGROUND: The selection of the food matrix to be used as a vehicle for a probiotic culture is important because its chemical composition and physicochemical characteristics can affect probiotic survival during the shelf-life of the product and under simulated gastrointestinal conditions (SGIC). The present study aimed to evaluate the influence of the dairy matrix (chocolate fermented milk beverage, chocolate flan or passion fruit flan) on the survival of Lactobacillus casei Lc-1 during refrigerated storage (4 °C for 21 days) and SGIC. RESULTS: Chocolate fermented milk beverage and chocolate and passion fruit flans could be considered as matrices for the incorporation of L. casei, providing suitable counts (6.38-7.84 log cfu g-1 ) during storage. The type of matrix had an impact on the inicial probiotic counts in the products and on the probiotic resistance to the SGIC. The chocolate fermented milk beverage presented higher initial probiotic counts (7.72 versus 6.65-7.28 log cfu g-1 ). The higher pH (5.3-6.8), solid matrix and increased fat content (65.0-72.9 g 100 g-1 ) contributed to the higher resistance to the SGIC of the chocolate and passion fruit flans, allowing recovery of viability during the enteric phase (increases of 1-1.5 log cycles). CONCLUSION: The type of dairy matrix has an impact on the inicial probiotic counts in the products and on the probiotic resistance to the SGIC. Chocolate and passion fruit flans proved to be more suitable options than chocolate fermented milk beverage for the incorporation of Lactobacillus casei Lc-1. © 2019 Society of Chemical Industry.

Changes in Antioxidants and Sensory Properties of Italian Chocolates and Related Ingredients Under Controlled Conditions During an Eighteen-Month Storage Period.

BACKGROUND: While there has been an increasing interest in the health properties of chocolate, limited research has looked into the changes of antioxidants occurring in the time span from production to the best before date, which was a period of 18 months in this study. METHODS: Humidity, ash, pH, acidity, fiber, carotenoids, retinols, tocopherols, sugars, proteins, theobromine, caffeine, polyphenols, fats, the peroxide value, organic acids, and volatile compounds, along with the sensory profile, were monitored at 18-week intervals for 18 months under conditions simulating a factory warehouse or a point of sale. RESULTS: At the end of the storage period, more polyphenols were lost (64% and 87%) than vitamin E (5% and 14%) in cocoa mass and cocoa powder, respectively. Conversely, a greater loss in vitamin E (34% and 86%) than in polyphenols (19% and 47%) was shown in the hazelnut paste and gianduja chocolate, respectively. The sensory profiling of cocoa mass, cocoa powder, and hazelnut paste revealed increases in grittiness and astringency, as well as decreases in melting, bitterness, and toasted aroma. Moreover, in the hazelnut paste and gianduja chocolate, oiliness increased with a toasted and caramel aroma. Furthermore, dark chocolate was more gritty, acidic, and bitter. Milk chocolate lost its nutty aroma but maintained its sweetness and creaminess. CONCLUSIONS: These results should contribute an important reference for companies and consumers, in order to preserve the antioxidants and understand how antioxidants and sensory properties change from the date of production until the best before date.

Linking cocoa varietals and microbial diversity of Nicaraguan fine cocoa bean fermentations and their impact on final cocoa quality appreciation.

Nicaraguan cocoa bean fermentations of several single local cocoa varieties originating from the same region (North Highlands of Nicaragua, San Jose de Bocay/El Cuá) were compared to fermentations of blended cocoa varietals from other producing regions of the country (Waslala and Nueva Guinea) making use of High Throughput Sequencing techniques, metabolite target analysis and sensory evaluation of cocoa liquor samples. A succession of the important cocoa-related yeasts Hanseniaspora uvarum/opuntiae, Saccharomyces cerevisiae and/or Pichia kudriavzevii was seen for single varietals and Nueva Guinea fermentations, while Kazachstania humilis dominated the mid and end phase of the Waslala cocoa fermentations. Tatumella species (mainly Tatumella terrea and Tatumella punctata) predominated the bacterial community at the onset of all fermentations followed by unusually late (generally 2 days into the fermentations) appearance of Lactobacillus fermentum relative to fermentations in other parts of the World. Acetobacter spp. were the main acetic acid bacteria during all fermentations, but also Gluconobacter spp. were involved in some single-variety fermentations. All fermentations proved complete as determined by metabolite analysis with bean sucrose being fully depleted and pulp sugars exhausted after 48-72 h of fermentation. From an organoleptic point of view, all Nicaraguan cocoas of this study reflected fine fruity (citrus or berry-like) flavours with distinct herbal or caramel notes. Floral notes were associated with the cases where P. kudriavzevii was involved in the later stages of fermentation. Intense citrus/fruity character was related to high pulp and bean citrate concentrations. Off-notes were found in some over-fermented batches where Bacillus spp. was detected. No relation between cut-test results and organoleptic appreciation was seen.

Traceability of Functional Volatile Compounds Generated on Inoculated Cocoa Fermentation and Its Potential Health Benefits.

Microbial communities are responsible for the unique functional properties of chocolate. During microbial growth, several antimicrobial and antioxidant metabolites are produced and can influence human wellbeing. In the last decades, the use of starter cultures in cocoa fermentation has been pushed to improve nutritional value, quality, and the overall product safety. However, it must be noted that unpredictable changes in cocoa flavor have been reported between the different strains from the same species used as a starter, causing a loss of desirable notes and flavors. Thus, the importance of an accurate selection of the starter cultures based on the biogenic effect to complement and optimize chocolate quality has become a major interest for the chocolate industry. This paper aimed to review the microbial communities identified from spontaneous cocoa fermentations and focused on the yeast starter strains used in cocoa beans and their sensorial and flavor profile. The potential compounds that could have health-promoting benefits like limonene, benzaldehyde, 2-phenylethanol, 2-methylbutanal, phenylacetaldehyde, and 2-phenylethyl acetate were also evaluated as their presence remained constant after roasting. Further research is needed to highlight the future perspectives of microbial volatile compounds as biomarkers to warrant food quality and safety.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing.

Salmonella is capable of surviving in a low moisture environment for long periods. Once adapted to the xeric conditions, the thermal resistance of Salmonella is enhanced. Cocoa powder is a low water activity (aw) food (LawF) that is an essential component in a wide variety of desserts and ready-to-eat (RTE) foods and drinks. The aim of this study was to evaluate the desiccation and thermal resistance of Salmonella in cocoa powder, as well as to examine the suitability of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing. Natural unsweetened cocoa powder was inoculated with a 3-strain Salmonella cocktail or E. faecium and was equilibrated to aw 0.30 and 0.45 at room temperature, then subjected to isothermal treatments at 70-80 °C or 12-month storage at RT (room temperature, 22.0 ±â€¯0.5 °C, aw 0.30). At 70 and 80 °C, D-values of both Salmonella and E. faecium increased with decreasing aw. D-values of Salmonella at aw 0.30 cocoa powder were 46.2 ±â€¯4.7, 20.5 ±â€¯1.7, and 11.5 ±â€¯0.9 min at 70, 75 and 80 °C, respectively. Higher heat resistance of E. faecium in aw 0.30 cocoa powder was observed with D-values of 59.9 ±â€¯5.0, 28.9 ±â€¯1.8, and 16.1 ±â€¯1.4 min at 70, 75, and 80 °C, respectively. However, E. faecium demonstrated less heat resistance than Salmonella when aw was increased to 0.45. D-values for Salmonella at aw 0.45 were 31.6-7.0 min at 70-80 °C compared to 25.8-4.7 min for E. faecium. During 12 months of storage at RT, surviving E. faecium population in aw 0.30 cocoa powder was higher than that of the Salmonella cocktail; the population decreased by 1.39 and 3.75 logs, respectively. These findings indicate that the suitability of E. faecium as a surrogate organism for Salmonella is influenced by aw of cocoa powder. The aw correlates with thermal inactivation rates in both Salmonella and E. faecium, and should be considered as a significant contributor to the thermal resistance of Salmonella in cocoa powder.

Enriching novel dark chocolate with Bacillus coagulans as a way to provide beneficial nutrients.

The aim of the present study was to develop a novel type of probiotic chocolate with the additive Bacillus coagulans bacteria and determine the concentration of polyphenols and their bioaccessibility. The manufactured chocolate possessed significantly higher concentrations of polyphenols than the control sample. The sensory profiles of the tested probiotic chocolate were similar to those of the control sample. In future, the probiotic chocolate could be regarded as a functional food product by chocolate producers.

A large outbreak of Salmonella enterica serovar Thompson infections associated with chocolate cake in Busan, Korea.

OBJECTIVES: This study aimed to reveal the epidemiologic characteristics of the outbreak of gastroenteritis caused by Salmonella enterica serovar Thompson in Busan Metropolitan City and to identify points for improvement to prevent of food-borne disease outbreak. METHODS: This was a case-control study. The control group comprised asymptomatic students in the same classes of the cases. The presence or absence of symptoms, ingestion of each food provided by school meal service, and commonly ingested foods in addition to those foods in meal service were investigated. Moreover, specimens collected from rectal swab, preserved foods, and environmental surface were tested. RESULTS: Of the 6,092 subjects, 1,111 (1,083 students, 22 school personnel, and 6 foodservice employees) were included in the case group; this corresponded to an 18.4% attack rate. Symptoms included diarrhea (n=1,051, 94.6%), abdominal pain (n=931, 83.8%), febrile sensation (n=502, 45.2%), and vomiting (n=275, 24.8%). The epidemic curves of each 10 schools were unimodal. Investigation of food intake showed a significantly high odds ratio for chocolate cake in 5 out of the 10 schools. Laboratory test detected Salmonella enterica serovar Thompson both in rectal swab specimens of 9 schools and in collected preserved chocolate cakes of 9 schools. Pulsed-field gel electrophoresis test result showed that Salmonella enterica seorvar Thompson isolated from human and foods were the same. CONCLUSIONS: The source of infection for the Salmonella enterica serovar Thompson outbreak in the 10 schools of Busan Metropolitan City is chocolate cake. Traceback investigation for origin of contaminated food in food-borne disease outbreak and safety control during food production should be more enhanced.

Laxative effect of probiotic chocolate on loperamide-induced constipation in rats.

The fecal morphology, defecation frequency, bowel function, intestinal motility, and fecal bacterial composition were evaluated to investigate the laxative effect of probiotic chocolate containing Streptococcus thermophilus MG510 and Lactobacillus plantarum LRCC5193 (LYC) on loperamide-induced constipated rats. Daily oral administration of LYC in constipated rats for two weeks was shown to significantly increase (n = 14) the defecation frequency, fecal moisture content, and relative abundance of fecal Lactobacillus and Faecalibacterium prausnitzii. Moreover, histological analysis of the distal colon of constipated rats revealed that LYC treatment can also increase the thickness of the colonic mucosa and muscle layers, and crypt of Lieberkühn. LYC also significantly increased (n = 5) the intestinal motility and modulated (n = 9) mRNA expression levels of colonic ZO-1 and Cldn-1 in the constipated rats. Altogether, these results demonstrate that probiotic chocolate has potential as a dietary adjunct for the treatment of constipation.

Prophylactic use of probiotic chocolate modulates intestinal physiological functions in constipated rats.

BACKGROUND: This study investigated the in vivo prophylactic effect of probiotic chocolate on constipation. Rats were administered chocolate containing 2.5 × 1010 CFU g-1 of probiotics daily for 4 weeks and treated with loperamide (5 mg kg-1 ) daily at the fourth week of treatment. RESULTS: Probiotic chocolate treatment significantly (P < 0.05) increased the intestinal motility, colon length, fecal moisture content and number of excreted fecal pellets in constipated rats. Moreover, quantitative real-time polymerase chain reaction data and histological images also revealed that both probiotic chocolate LYC and BB12 treatments were capable of upregulating the mRNA expression levels of colonic ZO-1, occludin and AQP8, leading to the maintenance of the defensive barrier function in the constipated rats compared with the negative controls. Interestingly, these treatments also modulated gut bacterial populations by increasing the abundance levels of Lactobacillus and Bifidobacterium, as well as reducing the abundance level of Enterobacteriaceae. CONCLUSION: The present study demonstrated that probiotic chocolate LYC and BB12 could potentially be used as alternative agents for prophylactic constipation. © 2018 Society of Chemical Industry.