Recent evidences highlighted the presence of Lactococcus lactis during late cheese ripening. For this reason, the role of this microorganism, well known as dairy starter, should be reconsidered ...throughout cheese manufacturing and ripening. Thus, the main objective of this study was to develop a RT-qPCR protocol for the detection, quantification and determination of the viability of L. lactis in ripened cheese samples by direct analysis of microbial nucleic acids. Standard curves were constructed for the specific quantification of L. lactis in cheese matrices and good results in terms of selectivity, correlation coefficient and efficiency were obtained. Thirty-three ripened cheeses were analyzed and, on the basis of RNA analysis, twelve samples showed 106 to 108 CFU of L. lactis per gram of product, thirteen from 103 to 105 CFU/g, and in eight cheeses, L. lactis was not detected. Traditional plating on M17 medium led to loads ranging from 105 to 109 CFU/g, including the cheese samples where no L. lactis was found by RT-qPCR. From these cheeses, none of the colonies isolated on M17 medium was identified as L. lactis species. These data could be interpreted as a lack of selectivity of M17 medium where colony growth is not always related to lactococcal species. At the same time, the absence or low abundance of L. lactis isolates on M17 medium from cheese where L. lactis was detected by RT-qPCR support the hypothesis that L. lactis starter populations are mainly present in viable but not culturable state during ripening and, for this reason, culture-dependent methods have to be supplemented with direct analysis of cheese.
Fermentation is an essential process step to develop precursor compounds for aroma and flavour characteristics of chocolate, as well as preventing germination of the cocoa bean. Despite the ...importance of the role of microorganisms during the chocolate production, to date, there are some discrepancies of the “cocobiota” community found during fermentation and the impact of starter culture in fermented cocoa beans. This review provides both a detailed overview of the starter cultures used in fermented cocoa beans and the microbial diversity involved during this process, and an in-depth discussion of the methods used to identify these microorganisms. In this review, we included only published articles from 2008 to 2018 in English language. A total of forty-seven studies contributed to the description of the cocobiota from 13 different countries. In detail, we observed that the most common fermentation method used is the wooden box, followed by heap. Interestingly, 37% of the studies cited in this review did not mention the type of cocoa variety studied. Most of the techniques used to identify the microbiota are fingerprinting based (DGGE); however, few studies have been using next-generation technologies to elucidate the possible functions and interactions among microbes. Our results showed a greater diversity of yeasts if compared with bacterial involved in the fermentation. This review will help researchers seeking to design starter cultures to drive cocoa bean fermentation, and thus achieve a homogenous mass of fermented cocoa beans as well as serve as a guide for assessing methodologies for the identification of microorganisms.
•The ecology of fermented cocoa is influenced by intrinsic and extrinsic factors.•Saccharomyces, Lactobacillus and Acetobacter are the main taxa in cocoa fermentation.•Several studies focus on the identification of “cocobiota”.•Needed to correct identification and characterisation of starter cultures•Cocoa fermentation must standardise according regions and variety cultivated.
Lactobacilli have been shown to inhibit or suppress cancer cell growth through the release of strain-specific bioactive metabolites and their inclusion in functional foods could exert a health ...promoting activity on human health. Herein, we examined the antiproliferative activity of the Lactiplantibacillus plantarum strains S2T10D and O2T60C, which have been previously shown to exert different butyrogenic activities. Human HT-29 cells were employed as an in vitro colon cancer model and both bacterial strains were found to inhibit their growth. However, the strain S2T10D showed a greater antiproliferative activity which, interestingly, was correlated to its butyrogenic capability. Noteworthy, for the non-butyrogenic strain O2T60C, the growth inhibitory capability was rather limited. Furthermore, both the butyrate-containing supernatant of S2T10D and glucose-deprived cell culture medium supplemented with the same concentration of butyrate found in S2T10D supernatant, induced a pH-independent cancer cell growth inhibition accompanied by downregulation of cyclin D1 at mRNA level. The downregulation of cyclin D1 gene expression was accompanied by cell cycle arrest in G2/M phase and decrease of cyclin B1 and D1 protein levels. This in vitro study underlines the impact of Lpb. plantarum in the growth inhibition of cancer cells, and proposes butyrate-mediated cell cycle regulation as a potential involved mechanism. Since the production of butyric acid in Lpb. plantarum has been proven strain-dependent and differentially boosted by specific prebiotic compounds, our results open future research paths to determine whether this metabolic activity could be modulated in vivo by enhancing this antiproliferative effects on cancer cells.
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•Two Lactiplantibacillus plantarum strains inhibit cancer cells proliferation in vitro.•Greater inhibition shown by strain S2T10D is related to its butyrogenic capability.•Butyric acid production downregulates cyclin D1 expression regardless from acidity.•Lactiplantibacillus plantarum S2T10D induces cell cycle-arrested on HT29 cells.
The microbiota composition of the offspring of women with gestational diabetes mellitus (GDM), a common pregnancy complication, is still little known. We investigated whether the GDM offspring gut ...microbiota composition is associated with the maternal nutritional habits, metabolic variables or pregnancy outcomes. Furthermore, we compared the GDM offspring microbiota to the microbiota of normoglycemic-mother offspring. Fecal samples of 29 GDM infants were collected during the first week of life and assessed by 16S amplicon-based sequencing. The offspring's microbiota showed significantly lower α-diversity than the corresponding mothers. Earlier maternal nutritional habits were more strongly associated with the offspring microbiota (maternal oligosaccharide positively with infant Ruminococcus, maternal saturated fat intake inversely with infant Rikenellaceae and Ruminococcus) than last-trimester maternal habits. Principal coordinate analysis showed a separation of the infant microbiota according to the type of feeding (breastfeeding vs formula-feeding), displaying in breast-fed infants a higher abundance of Bifidobacterium. A few Bacteroides and Blautia oligotypes were shared by the GDM mothers and their offspring, suggesting a maternal microbial imprinting. Finally, GDM infants showed higher relative abundance of pro-inflammatory taxa than infants from healthy women. In conclusion, many maternal conditions impact on the microbiota composition of GDM offspring whose microbiota showed increased abundance of pro-inflammatory taxa.
Gut health in poultry depends on the balance between the host, intestinal microbiota, intestinal microscopic features and diet. The effects of insect meal (a promising alternative protein source for ...poultry feed) on chicken gut morphology have recently been reported, but no data about intestinal microbiota and mucin composition modulation are available. The present study evaluated the effects of dietary Tenebrio molitor (TM) meal inclusion on gut health of free-range chickens by intestinal microbiota, morphology and mucin composition characterization.
One hundred forty female medium-growing hybrids were divided into 2 dietary treatments (control feed C and 7.5% TM inclusion, with 5 replicate pens/treatment and 14 birds/pen) and slaughtered at 97 days of age (2 birds/pen for a total of 10 chickens/diet). The gut microbiota assessment on cecal content samples by 16S rRNA amplicon based sequencing showed higher alpha (Shannon, P < 0.05) and beta (Adonis and ANOSIM, P < 0.001) diversity in birds fed TM diet than C. In comparison with C group, TM birds displayed significant increase and decrease, respectively, of the relative abundances of Firmicutes and Bacteroidetes phyla, with higher Firmicutes:Bacteroidetes ratios (False Discovery Rate FDR < 0.05). The relative abundance of Clostridium, Oscillospira, Ruminococcus, Coprococcus and Sutterella genera was higher in TM chickens than C (FDR < 0.05). On the contrary, TM birds displayed significant decrease of the relative abundance of Bacteroides genus compared to the C group (FDR < 0.05). Gut morphology evaluation by morphometric analysis on small intestine revealed similar villus height, crypt depth and villus height to crypt depth ratio between C and TM birds. Characterization of gut mucin composition by periodic-acid Schiff, Alcian Blue pH 2.5 and high iron diamine staining on small and large intestine showed unaffected mucin staining intensity in TM chickens when compared to C group.
Dietary TM meal inclusion may positively modulate the gut microbiota of the free-range chickens without influencing the intestinal morphology and mucin composition. Since the rapid growth of chickens directly depends on morphological and functional integrity of the digestive tract, the gut health assessment by a post mortem multidisciplinary approach appears to be fundamental.
Lactococcus lactis, is extensively used as starter culture in dairy products. Nevertheless, it has recently been detected in cheese, as metabolically active cells, in advanced ripening stages. In ...this study, we assessed the viability of L. lactis subsp. lactis in model cheeses up to 180 days of ripening by both culture-dependent and -independent methods. In addition, we studied the expression of metC and als genes involved in the production of aroma compounds detected by Gas Chromatography-Mass Spectrometry (GC-MS). Three L. lactis subsp. lactis commercial starters were inoculated in pasteurized milk and model cheeses were manufactured and ripened for six months. Samples were analysed at manufacturing and ripening steps, in terms of viability of L. lactis by both traditional plating and direct analysis of RNA by reverse transcription quantitative PCR (RT-qPCR), and in terms of aroma profile by GC-MS. Relatively to RT-qPCR analysis, L. lactis was found viable throughout the whole process of cheesemaking and aging, with final average loads of 3–4 Log CFU/g at 180 days. On the contrary, the microorganism was not detected, in ripened samples, by traditional plating on M17 medium, suggesting its entering in a viable but not cultivable (VBNC) state. The aroma profiles of the cheeses highlighted the presence of volatile compounds related to cheese flavor as acetoin, diacetyl, 2,3-butanediol and dimethyl disulfide, whose presence was partially correlated to metC and als genes expression. These results add new insights on the capability of L. lactis to persist during late cheese ripening and suggest a potential contribution of the microorganism to cheese flavor formation.
•L. lactis is able to persist in model cheeses, as metabolically active cells, up to six months of ripening.•L. lactis entering in VBNC state is strain dependent and affected by environmental parameters.•Volatile aroma compounds varied, throughout manufacturing and ripening, according to the starters used in cheesemaking.•Starter metC gene expression was partially related to the production of DMDS.•Als and metC gene expression in L. lactis varied according to the three starters used in cheesemaking.
The relationship between diet and intestinal microbiota and mucin composition appears to be fundamental for poultry gut health. The effects of insect meal (whose role as alternative feed ingredient ...is now well recognized) on gut microbiota and mucin composition have recently been reported in
-fed free-range and broiler chickens, but no data are currently available for
(HI)-fed broilers. The present study evaluated the effects of dietary HI meal inclusion on cecal microbiota and intestinal mucin composition of broiler chickens.
A total of 256 male broiler chickens were allotted to 4 dietary treatments (control diet C and 5%, 10% and 15% HI meal inclusion, with 8 replicate pens/treatment and 8 birds/pen) and slaughtered at 35 d of age (2 animals/pen, 16 birds/diet). The cecal microbiota assessment by 16S rRNA amplicon based sequencing showed lower alpha diversity in HI15 chickens (Shannon,
< 0.05) and higher beta diversity (Adonis and ANOSIM,
< 0.001) in birds fed HI diets than C. Furthermore, HI15 birds displayed significant increase of the relative abundance of Proteobacteria phylum (False Discovery Rate FDR < 0.05) when compared to HI10. L-
(
from Lachnospiraceae family),
,
and
genera were found to be characteristic of HI5 cecal microbiota (FDR < 0.05), while broiler chickens fed HI10 and HI15 diets were characterized (FDR < 0.05) by
and
(HI10) and
,
and
genera (HI15). Periodic-acid Schiff, Alcian Blue pH 2.5 and high iron diamine staining on small and large intestine also demonstrated lower mucin staining intensity in the intestinal villi of HI10 and HI15 birds than C (
< 0.05).
Dietary HI meal utilization at low inclusion levels (i.e., 5%) positively influenced either the cecal microbiota or the gut mucin dynamics in terms of selection of potentially beneficial bacteria and increase in villi mucins. However, high inclusion levels (in particular the 15%) may have a negative influence in terms of partial reduction of microbial complexity, reduction of potentially beneficial bacteria, selection of bacteria with mucolytic activity and decrease in villi mucins.
In this study, the fecal microbiota of 153 healthy volunteers, recruited from four different locations in Italy, has been studied by coupling viable counts, on different microbiological media, with ...ribosomal RNA Denaturing Gradient Gel Electrophoresis (rRNA-DGGE). The volunteers followed three different diets, namely omnivore, ovo-lacto-vegetarian and vegan. The results obtained from culture-dependent and -independent methods have underlined a high level of similarity of the viable fecal microbiota for the three investigated diets. The rRNA DGGE profiles were very complex and comprised a total number of bands that varied from 67 to 64 for the V3 and V9 regions of the 16S rRNA gene, respectively. Only a few bands were specific in/of all three diets, and the presence of common taxa associated with the dietary habits was found. As far as the viable counts are concerned, the high similarity of the fecal microbiota was once again confirmed, with only a few of the investigated groups showing significant differences. Interestingly, the samples grouped differently, according to the recruitment site, thus highlighting a higher impact of the food consumed by the volunteers in the specific geographical locations than that of the type of diet. Lastly, it should be mentioned that the fecal microbiota DGGE profiles obtained from the DNA were clearly separated from those produced using RNA, thus underlining a difference between the total and viable populations in the fecal samples.
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.
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