Secondary Microbiota Research Articles

​Identification of Lactic Acid Bactria in Dairy Products using Culture-independent Methods: A Review

The microbiota of dairy products is composed of starter and non-starter lactic acid bacteria, as well as other bacteria, yeasts and filamentous fungi that form secondary microbiota and play an important role in cheese ripening. The use of combined LAB strains based on compatibility and/or coexistence tests plays an important role in food production and preservation, while combinations of strains that exhibit mutually inhibitory properties must be avoided. For centuries, researchers have focused on the exploration of autochthonous LAB biodiversity from various ecological dairy niches in order to address its role in texture and flavour formation and to select strains for use as starter cultures. Recent advanced technological tools like next-generation sequencing machines have played a significant role in the fast and accurate characterization and isolation of microbial communities from dairy products. However, the characterization and isolation of LAB using culture-independent methods is also a reliable and cost-effective method for studying a bacterial community without prior cultivation. These methods include single-strand conformation polymorphism (SSCP-PCR), denaturing gradient gel electrophoresis (DGGE), thermal gradient gel electrophoresis (TGGE), real-time PCR (qPCR), terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), automated ribosomal intergenic spacer analysis (ARISA), and fluorescence in situ hybridization (FISH). Therefore the goal of this review is to compare and describe the most recent culture-independent molecular-based methods for the characterization and isolation of LAB in dairy products.

Lactic Acid Bacteria in Raw-Milk Cheeses: From Starter Cultures to Probiotic Functions.

Traditional cheeses produced from raw milk exhibit a complex microbiota, characterized by a sequence of different microorganisms from milk coagulation and throughout maturation. Lactic acid bacteria (LAB) play an essential role in traditional cheese making, either as starter cultures that cause the rapid acidification of milk or as secondary microbiota that play an important role during cheese ripening. The enzymes produced by such dynamic LAB communities in raw milk are crucial, since they support proteolysis and lipolysis as chief drivers of flavor and texture of cheese. Recently, several LAB species have been characterized and used as probiotics that successfully promote human health. This review highlights the latest trends encompassing LAB acting in traditional raw milk cheeses (from cow, sheep, and goat milk), and their potential as probiotics and producers of bioactive compounds with health-promoting effects.

Comparative analysis of Bacterial Vaginosis microbiota among pregnant and non-pregnant females and isolation of phages against Enterococcus faecalis, Enterococcus faecium, and Shigella flexneri strains

Background and objectivesBacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age. It shifts the paradigms of the vagina from healthy, beneficial microbiota to facultative and strict anaerobes. BV remains one of the most arduous and controversial challenges in modern-day clinical microbiology because of its high prevalence and relapse rates. A lot of research has been carried out on it. Still, its etiology is unknown, which gave this infection global importance. The current study was designed to investigate and compare the microbiota of pregnant and non-pregnant females suffering from BV, and phages were isolated against BV microbiota. Material and methodsThe samples were collected from the vagina by using a speculum, and swabs were streaked on different media to isolate bacteria. The microbiological analysis was performed by microscopy, biochemical testing, and antibiotic susceptibility was determined by using Metronidazole and Clindamycin. Furthermore, the phages were isolated and characterized against BV strains. Results and conclusionThe Gram staining showed high prevalence of Staphylococcus (36% vs. 33%), followed by Streptococcus (31% vs. 14%) and Enterococcus (7% vs. 14%) in non-pregnant and pregnant females' respectively. However, the exception was observed in non-pregnant BV positive females, who had Shigella flexneri in their samples. The antibiotic sensitivity showed Metronidazole was resistant against all BV microbiota, and Clindamycin showed susceptibility against 3 strains. Phages were isolated against three bacterial strains, i.e. E. faecalis, E. faecium, and S. flexneri. Bacterial reduction assay showed bacterial growth decreases in the presence of phage suspension, pH stability showed phages’ maximum lytic activity at pH 7 for E. faecalis and E. faecium and pH 9 for S. flexneri. However, the thermal stability showed phages' highest lytic activity at 55 °C for E. faecalis, 70 °C for E. faecium, and 40 °C for S. flexneri. Phage genome isolation showed that all phages nucleic acid was DNA in nature and between 15 and 20kbp. SEM analysis showed they were circular in shape and might belong to the Podoviridae family. This study provides an understanding of pathogens involved in BV and helps the doctors to treat the patients accordingly. Furthermore, this study showed that Bacterial Vaginosis and BV secondary bacteria have associations. BV secondary microbiota is also involved in the pathogenesis of this infection, whereas bacteriophage therapy has the potential to be used as an alternative treatment to antibiotics.

Dynamic changes of microbiota and texture properties during the ripening of traditionally prepared cheese of China.

The dynamic changes of microbiota assessed by high-throughput sequencing and texture properties of handmade cheese were investigated during ripening time. Streptococcus and Lactococcus were found to be the most predominant genera. The proportion of Streptococcus was decreased from 48 to 32% and the proportion of Lactococcus was increased from 41 to 55% with ripening time from 1 to 120 days. Mould and yeast such as Paecilomyces, Candida, Issatchenkia, Rhodotorula, Cryptococcus and Trichosporon were observed. The regression analysis between composition and textural properties indicated that the hardness was increased along with the rising of soluble nitrogen, while the increased soluble nitrogen could result in lower cohesiveness, and the increased fat in dry matter resulted in lower resilience. The physic-chemical parameters were correlated with secondary microbiota such as Cryptococcus and Candida according to the multivariate association analysis (p < 0.05). These findings could provide a baseline to improve the product quality and preserve the traditional characteristics of handmade cheese.

Antipsychotics and the microbiota.

The gut microbiota has been speculated to underpin metabolic changes associated with chronic antipsychotic use. The change in the gut microbiota can also cause abnormal absorbtion from the gut into the blood stream (leaky gut syndrome) that can lead to inflammatory reactions, and thus, secondary damage to the brain and central nervous system. Our article aims to highlight relevant research on antipsychotic's effect on the microbiota and to point out future directions. Antipsychtoic use can result in specific microbiota changes, and it is important to differentiate this from the innate microbiota of the patient. It is important to treat these microbiota changes, as they are correlated with obesity, which is a negative contributor to the cardiovascular health of those suffering with schizophrenia. Ways to prevent antipsychotic-induced side-effects include antibiotic treatment, histamine 3 receptor blockade and metformin use. Given the dearth of current literature, more research is needed, however, to determine, which comes first in people with schizophrenia--an abnormal gut microbiota that elevates one's risk for schizophrenia or psychopharmacologic treatment of schizophrenia leading to secondary microbiota abnormalities or the negative symptoms of schizophrenia leading to obesity and its associated microbiota changes.

Sugary Kefir: Microbial Identification and Biotechnological Properties

Background: The aim of the present study was to assess the microecosystem composition of three different fruit kefir grains used for the fermentation of apple juice (NAJ), cherry juice (SCN), and a solution of sugary water, enriched with plums (BSS). Methods: Yeast and bacterial populations were enumerated using classical microbiological techniques, clustered by RAPD-PCR genotyping, and identified by sequencing of the D1/D2 region of 26S-rRNA gene and the V1-V3 region of 16S-rRNA gene, respectively. The caseinolytic/lipolytic activities and the production of antimicrobial compounds were assessed by well diffusion assays. The proteolytic and lipolytic capacity were further assessed by SDS-PAGE and titrimetric assay, respectively. Results: Yeast and bacterial populations were enumerated at 6.28, 6.58, and 6.41 log CFU/g and at 4.32, 4.85, and 4.34 log CFU/g, regarding BSS, NAJ, and SCN, respectively. Saccharomyces cerevisiae dominated all three sugary kefir grains; Kluyveromyces marxianus formed a secondary microbiota in BSS and NAJ. Bacillus amyloliquefaciens dominated NAJ and SCN; Lactobacillus rhamnosus dominated BSS. Four bacteria and nine yeast isolates exhibited proteolytic activity. Forty bacteria and 45 yeast isolates possessed lipolytic activity. No antibacterial activity was detected. Conclusions: Prevalence of yeast over bacterial populations was evident in all samples assessed. Several bacterial and yeast strains exhibited proteolytic and lipolytic activities, making them suitable candidates for inclusion in starter cultures for milk and sugary kefir fermentation.

Fungal mycobiota and mycotoxin risk for traditional artisan Italian cave cheese

Cave cheese is a surface mold-ripened variety of cheese produced also in South of Italy, exploiting fungal population naturally occurring on cave walls, as part of secondary microbiota for ripening. In this study, 148 fungal strains were isolated from 22 independent cave cheese samples, collected in 13 Italian geographical locations, mostly in Apulian area.DNA-based identification showed the presence of twenty-four fungal species in the outer part of the cheese ripened in caves. Aspergillus westerdijkiae and Penicillium biforme resulted the most frequently isolated species, followed by Penicillium roqueforti and Penicillium solitum. The 86% of cheese sample presented at least one toxigenic species and the 45% revealed the presence of ochratoxigenic species, A. westerdijkiae and A. steynii, suggesting possible mycotoxin risk during ripening stage in caves, confirmed by the presence of ochratoxin A (OTA) in the rind of 36% of samples.In conclusion, cave cheese is a susceptible product for toxigenic mold growth and in particular OTA contamination, therefore adeguate scientific tools for matching organolectic consumer expectations and complete safety of food should be developed, as well as spontaneously molded and not monitored cheeses should not be consumed to avoid mycotoxin risk.

Temporal microbiota and biochemical profiles during production and ripening of Divle Cave cheese

The aim of this study was to evaluate the proteolysis and lipolysis profiles of a raw ewes' milk cheese during production and ripening. The microbial community of this cheese was quantified, and their roles during proteolysis and lipolysis were studied. We observed an accumulation of substantial amounts of peptides during ripening, which might be attributed to the abundant presence of lactic acid bacteria, as well as to secondary microbiota including fungi, yeasts and actinobacteria. Similarly, high lipolysis levels were obtained, which can be explained by the indigenous lipase in milk and microbial lipases. These results should be of great interest to industrial manufacturers of this cheese.

Microbiota levaduriforme en quesos artesanales de Corrientes, Argentina

The artisanal cheese from Corrientes (from the Spanish acronym QAC-Queso Artesanal de Corrientes/Artisanal Cheese from Corrientes) is a soft cheese elaborated with raw cow milk and an artisanal coagulant agent. Lactic bacteria contitute the main flora of this cheese although yeasts are also present in high quantities as secondary microbiota and might play a relevant role in cheese ripening. The aim of this work was to evaluate yeast occurrence during QAC elaboration and ripening, and the effect of seasonal variation. Yeasts were isolated and purified from raw materials and cheese at different ripening stagesl elaborated during the different seasons. Yeast sample counts were in the order of 103 - 107UFC/ml o UFC/g. Ninety yeast strains were classified: 9 from milk, 28 from the coagulant agent, 10 from curd and 43 from cheese. Candida predominated in milk samples while other yeast genera had low incidence. Candida also predominated in the coagulant agent samples, followed by genera Myxozyma and Debaryomyces. The isolates obtained from cheese belonged to the same genera predominating in the coagulant agent, and showed the same order of prevalence.

Microbial population profile during ripening of Protected Designation of Origin (PDO) Silter cheese, produced with and without autochthonous starter culture

An autochthonous starter culture was used in the production of Protected Designation of Origin (PDO) Silter raw milk cheese, and compared to a control cheese in order to study its influence on microbial population dynamics during ripening. Curd and cheese at different ripening periods (0, 30, 60 and 200 days) from two independent dairies were analysed combining culture-based microbiological analysis, Length Heterogeneity-PCR and 16S microbiome profiling.The autochthonous starter determined higher lactic acid bacteria (LAB) levels for the first 30 days of ripening, without interfering with secondary microbiota that determines the tipicality of this cheese. Only a few genera and species persisted in the cheese despite the microbial richness of the curd. In addition, the high levels of different LAB reduced harmful microorganisms. The various analytic methods used resulted in discrepancies in the proportions of Enterococcus, Lactococcus and Lactobacillus spp., but the pivotal role of Streptococcus and Lactococcus genera was evident. The dominant species included those selected to formulate the starter (St. thermophilus, Lc. lactis, Ln. mesenteroides) as well as Lb. paracasei.The addition of autochthonous starter proved to be effective in controlling the first phases of the cheese-making, without compromising cheese typicality.

Lactic acid bacteria population dynamics during spontaneous fermentation of radish (Raphanus sativus L.) roots in brine.

The aim of the present study was to assess the microecosystem development and the dynamics of the lactic acid bacteria population during spontaneous fermentation of radish (Raphanus sativus L.) roots in brine at 20 and 30 °C. In both temperatures, lactic acid bacteria prevailed the fermentation; as a result, the pH value was reduced to ca. 3.6 and total titrable acidity increased to ca. 0.4% lactic acid. Enterococci population increased and formed a secondary microbiota while pseudomonads, Enterobacteriaceae and yeasts/molds populations were below enumeration limit already before the middle of fermentation. Pediococcus pentosaceus dominated during the first days, followed by Lactobacillus plantarum that prevailed the fermentation until the end. Lactobacillus brevis was also detected during the final days of fermentation. A succession at sub-species level was revealed by the combination of RAPD-PCR and rep-PCR analyses. Glucose and fructose were the main carbohydrates detected in brine and were metabolized into lactic acid, acetic acid and ethanol.

Detection and identification of yeasts in natural whey starter for Parmigiano Reggiano cheese-making

The yeast populations of natural whey starter (NWS) samples from dairies of PDO Parmigiano Reggiano cheese production area were enumerated and characterised. Yeasts were detected with an average value higher than 3 log cfu mL−1 and in a case study we highlighted the correlation between the yeast growth and incorrect management of NWS fermentation. Isolated strains showed the typical Kluyveromyces marxianus PCR-RFLP pattern and D1/D2 sequences obtained displayed similarity values ranging from 99% to 100% for the same species. RAPD-PCR fingerprinting revealed 14 biotypes. The ability of several strains to grow at 52 °C demonstrates that the yeasts may be perfectly integrated into the ecosystem of NWS. This work confirms that yeasts are part of the NWS secondary microbiota, composed by wild strains with relevant biotechnological interest.

Comparative study of spontaneously fermented sourdoughs originating from two regions of Greece: Peloponnesus and Thessaly

A total of 167 yeast and 136 lactic acid bacteria strains were isolated from spontaneously fermented wheat sourdoughs from two regions of Greece, namely Thessaly and Peloponnesus. Identification of the isolates exhibited dominance of Torulaspora delbrueckii with sporadic presence of Saccharomyces cerevisiae and, regarding the lactic acid bacteria, dominance of Lactobacillus sanfranciscensis in the sourdoughs from Thessaly and Lb. plantarum subsp. plantarum in the sourdoughs from Peloponnesus. The latter was accompanied by Pediococcus pentosaceus as secondary microbiota. None of the above mentioned strains exhibited amylolytic, lipolytic or proteolytic activities, and none of the lactic acid bacteria strains produced antimicrobial compounds.

Biodiversity and the survival of autochthonous fermented products.

Many microorganisms isolated and characterized during the past century have received considerable attention from the pharmaceutical and/or food fermentation industry because of their potential biotechnological interest. Lactic acid bacteria (LAB), particularly those belonging to beneficial and nonpathogenic genera (Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, and Streptococcus), have traditionally been used in the food industry. They also play an essential role in the dairy industry due to the tremendous level of human consumption of several important fermented products, mainly cheese and acidified/fermented milks. In the USA, more than 4.6 million tons of cheese and more than 1.3 million tons of yogurt were produced in 2005 [1], whereas in the European Community (EU-25) more than 8 million tons of cheese and 6.9 million tons of acidified/fermented milk were produced in 2003 [3]. These figures correspond to a global sales value that surpasses US$ 28.44 billion, with the global sales value of cheese representing about 30% of total dairy-product sales [2]. Nowadays, more than 500 kinds of cheese are manufactured worldwide, and the many varieties are derived from modifications in cheese-making techniques. However, the fermentation process whereby milk is transformed into cheese has not changed to any great extent since humans discovered the process as a means of preserving food (the fermentation of milk dates back to ~3000 BC). Cheese is a dynamic biochemical product and, unlike many processed food products for which stability is essential, it undergoes significant changes during ripening. The unripe curds of many varieties of cheese have bland and largely similar flavors. However, the sequential actions of cheese microbiota and the interactions among the different microorganisms during the ripening period result in the flavor compounds that characterize each variety. Acidifying lactococci species usually occur as dominant bacteria early in the ripening process. Afterwards their numbers decrease as they give way to lactobacilli, which, due to their higher versatility for fermentation, can survive in cheeses at stages when concentrations of major carbohydrates and free water decrease, whereas the sodium chloride load increases. Apart from the above-mentioned major genera of LAB, other bacteria, including non-starter LAB and non-lactic-acid bacteria, as well as some fungi (yeasts, moulds), staphylococci, micrococci, coryneform bacteria, and propionic acid bacteria, which develop in the product particularly during the maturation phase as secondary microbiota, are often responsible for the intensity of flavor in many dairy products. The production of a wide range of antagonistic primary and secondary metabolites, including organic acids, diacetyl, CO2 and even bacteriocins, affects the complex dynamics of microorganisms in cheese. Thus, a well-founded understanding of the microbial balance in this special living ecosystem is essential to improve older varieties of cheese and to develop new ones. Due to the dairy market’s continuous growth, commercial cultures have conventionally been selected to obtain greater quantities of product, homogeneous quality of the end product, and short production times, with the final goal of satisfying the preferences of consumers. Most varieties of commercially important cheeses are manufactured worldwide by means of commercial starter cultures of generic composition (including acidifying and aromatic diacetyl-producing lactococci strains and sometimes also leuconostocs and lactobacilli strains). In Europe, Asia, Africa, and Latin America, however, there are regions where fermented products are still manufactured traditionally. In global markets, these fermented products are now highly appreciated and are considered to be of premium value because of their flavor characteristics, which are not found in varieties of cheese produced industrially. In Europe, such products are elaborated according to well-established rules governing the production of registered designations of origin. Protected designations of origin (PDOs) apply to foodstuffs that are produced, processed, and prepared in a given geographic area using recognized

Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough

The aim of this study was to assess the interactions between Saccharomyces cerevisiae and lactic acid bacteria that either form a stable consortium in Greek wheat sourdoughs (i.e. Lactobacillus sanfranciscensis and L. brevis) or occasionally constitute the secondary microbiota (i.e. Weissella cibaria, L. paralimentarius, Pediococcus pentosaceus and Enterococcus faecium). For this purpose, wheat dough was prepared by using strains of the above mentioned species either as single starters, or in combination of the yeast with each of the lactic acid bacteria strains. The determination of the metabolic products in sourdough samples was performed by HPLC analysis. Presence of lactic acid bacteria had no effect on S. cerevisiae final cell yield but affected negatively the maximum specific growth rate. Ethanol production was primarily affected negatively while the co-culture had a variable effect on glycerol production. On the other hand, the presence of S. cerevisiae favoured mannitol and acetic acid production, had a species-dependent effect on maximum specific growth rate and had no effect on final cfu/g sourdough and lactic acid production by the lactic acid bacteria and at the same time caused the depletion of glucose, fructose and maltose.

Mussel Periostracum from Deep-Sea Redox Communities as a Microbial Habitat: 3. Secondary Inhabitants

Abstract. Vacated microborings in periostracum of live mussels of the Florida Escarpment redox community (depth of 3266 m) become a habitat for a prolific secondary microbiota consisting of a variety of prokaryotic and a few eukaryotic organisms. Periostracum surface is also colonized by diverse microorganisms, dominated by presumed chemolithotrophic bacteria with stacks of intracellular lamellae. Unlike sheltered microflora within borings, the surface community is heavily grazed upon by numerous archaeogastropods and ciliates.