Advances in Kombucha Tea Fermentation: A Review

Kombucha is a carbonated, slightly acidic beverage traditionally produced by the fermentation of sweetened tea by a symbiotic culture of bacteria and yeast (SCOBY). The microbial community of kombucha is a complex one, whose dynamics are still not fully understood; however, the emergence of culture-independent techniques has allowed a more comprehensive insight into kombucha microbiota. In recent times, advancements have been made towards the optimisation of the fermentation process, including the use of alternative substrates, defined starter cultures and the modification of fermentation parameters, with the aim of producing an innovative beverage that is improved in terms of its physiochemical, sensory and bioactive properties. The global kombucha market is rapidly increasing, with the rising popularity of the tea attributed in part to its purported health benefits, despite the lack of research in human subjects to substantiate such claims. Accordingly, the incidence of kombucha home-brewing has increased, meaning there is a requirement for individuals to recognise the potential hazards associated with fermentation and the relevant preventative measures to be undertaken to ensure the safe preparation of kombucha. The aim of this review is to provide an update regarding the current knowledge of kombucha production, microbiology, safety and marketing.

DHA-Rich Aurantiochytrium Biomass, a Novel Dietary Supplement, Resists Degradation by Rumen Microbiota without Disrupting Microbial Activity

We first sought to evaluate the effect of dietary supplementation with the docosahexaenoic acid (DHA)-rich microalgae, Aurantiochytrium limacinum (AURA), on rumen fermentation and the resistance of DHA to degradation and biohydrogenation by rumen microbes through ex vivo fermentation experiments. Subsequently, we sought to quantify the diet-derived DHA content of milk and the impact of AURA on microbial composition and metabolism in a pilot feeding trial with rumen-cannulated dairy cows. To achieve our aims, rumen fluid from cannulated cows was used as inoculum, and the effect of AURA inclusion on fermentation ex vivo was examined. At doses corresponding to the amount of AURA recommended for commercial production animals, only ~10% of DHA was degraded or biohydrogenated by rumen microorganisms. The results show that feeding with AURA had no effect on either total bacterial density or short-chain fatty acid production. Real-time quantitative PCR analysis of the rumen fluid samples collected during a seven-week in vivo trial revealed that microbes related to lactic acid metabolism and methanogenesis were significantly suppressed by the AURA-supplemented diet. The DHA concentration in milk increased over 25-fold with the AURA-supplemented diet and dropped by 30–40% within one week of washout. The addition of A. limacinum biomass to dairy cow diets resulted in positive effects on rumen microbial composition with no adverse effect on fermentation activity. AURA-derived DHA was stable, with only modest degradation in the rumen, and was successfully deposited in milk. This is the first study to investigate the effect of supplementing the diet of dairy cows with a protist-based biomass, namely, on important rumen fermentation parameters and on DHA deposition in milk, using a combination of ex vivo and in vivo approaches.

Nourishing the Human Holobiont to Reduce the Risk of Non-Communicable Diseases: A Cow’s Milk Evidence Map Example

The microbiome revolution brought the realization that diet, health, and safety for humans in reality means diet, health, and safety for the human holobiont/superorganism. Eating healthier means much more than just feeding human cells. Our diet must also nourish the combination of our microbiome and our connected physiological systems (e.g., the microimmunosome). For this reason, there has been an interest in returning to ancestral “complete” unprocessed foods enriched in microbes, including raw milks. To contribute to this inevitable “nourishing the holobiont” trend, we introduce a systematic risk–benefit analysis tool (evidence mapping), which facilitates transdisciplinary state-of-the-science decisions that transcend single scientific disciplines. Our prior paper developed an evidence map (a type of risk–benefit mind map) for raw vs. processed/pasteurized human breast milk. In the present paper, we follow with a comprehensive evidence map and narrative for raw/natural vs. processed/pasteurized cow’s milk. Importantly, the evidence maps incorporate clinical data for both infectious and non-communicable diseases and allow the impact of modern agricultural, food management, and medical and veterinary monitoring outcomes to be captured. Additionally, we focus on the impact of raw milks (as “complete” foods) on the microimmunosome, the microbiome-systems biology unit that significantly determines risk of the world’s number one cause of human death, non-communicable diseases.

Application of a Bacteriophage–Sanitizer Combination in Post-Harvest Control of E. coli O157:H7 Contamination on Spinach Leaves in the Presence or Absence of a High Organic Load Produce Wash

Foodborne illness due to the consumption of contaminated products continues to be a serious public health issue. Bacteriophages might provide a natural and effective way to control and reduce the pathogenic bacterial population on food products. Researchers have conducted various experiments to prove their effectiveness against different pathogens and their ability to act as a natural intervention to control pathogen populations, especially in the food industry. In this study, a cocktail of bacteriophages (phages) was added to wash water in the presence of a high organic load along with commercially used sanitizers (chlorine or Sanidate 5.0) to study the efficacy of the phage–sanitizer combination in the challenge water. It was determined that in the absence of organic loads, the sanitizer by itself or the combination with phages significantly (p < 0.001) reduced the contamination by 3.00–5.00 log CFU/mL. In the presence of organic loads, the sanitizer by itself did not contribute to a significant reduction (p > 0.05) compared to the control. However, the sanitizer–phage combination led to a 3.00-log and 6.00-log reduction (p < 0.001) of the pathogen at the end of 3 and 6 h, respectively, in the presence of high organic loads. Therefore, utilizing a combination treatment (phage–sanitizer) might be one solution to reduce pathogen contamination in the food industry, especially the fresh produce industry, thus providing safe food for consumption.

Effect of Enterocins A and B on the Viability and Virulence Gene Expression of Listeria monocytogenes in Sliced Dry-Cured Ham

Dry-cured ham can be contaminated with Listeria monocytogenes during its industrial processing. The use of bacteriocins could ensure the safety of such meat products, but their effect on pathogen physiology is unknown. Therefore, the impact of enterocins A and B on the L. monocytogenes population, and the expression patterns of five genes (inlA, inlB, clpC, fbpA and prfA) related to adhesion/invasion and virulence regulation have been monitored in sliced dry-cured ham during 30 d of storage in refrigeration (4 °C) and temperature-abuse conditions (20 °C). L. monocytogenes strains S2 (serotype 1/2a) and S7-2 (serotype 4b) counts were reduced by 0.5 and 0.6 log units immediately after the application of enterocins A and B, a decrease lower than previously reported. Differences in gene expression were found between the two strains. For strain S2, expression tended to increase for almost all genes up to day seven of storage, whereas this increase was observed immediately after application for strain S7-2; however, overall gene expression was repressed from day one onwards, mainly under temperature-abuse conditions. L. monocytogenes strains investigated in the present work exhibited a mild sensitivity to enterocins A and B in sliced dry-cured ham. Bacteriocins caused changes in the expression patterns of virulence genes associated with adhesion and invasion, although the potential virulence of surviving cells was not enhanced.

Role of the Mn-Catalase in Aerobic Growth of Lactobacillus plantarum ATCC 14431

Lactobacilli are Gram-positive aerotolerant organisms that comprise the largest genus of Lactic Acid Bacteria (LAB). Most lactobacilli are devoid of the antioxidant enzymes, superoxide dismutases, and catalases, required for protection against superoxide radicals and hydrogen peroxide (H2O2), respectively. However, some lactobacilli can accumulate millimolar concentrations of intracellular manganese and spare the need for superoxide dismutase, while others possess non-heme catalases. L. plantarum is associated with plant materials and plays an important role in fermented foods and gut microbiomes. Therefore, understanding the effects of the environment on the growth and survival of this organism is essential for its success in relevant industrial applications. In this report, we investigated the physiological role of Mn-catalase (MnKat) in Lactobacillus plantarum ATCC 14431. To this end, we compared the physiological and morphological properties of a ΔMnkat mutant strain and its isogenic parental strain L. plantarum ATCC 14431. Our data showed that the MnKat is critical for the growth of L. plantarum ATCC 14431 in the presence of oxygen and resistance to H2O2. The aerobic growth of the mutant in presence or absence of H2O2 was improved in the Mn-rich medium (APT) as compared to the growth in MRS medium. Furthermore, under aerobic conditions the mutant strain possessed atypical cellular morphology (i.e., shorter, and fatter). In conclusion, the MnKat of L. plantarum ATCC 14431 is important for aerobic growth, protection against H2O2, and maintenance of the rod-shaped cell morphology under aerobic conditions.

Slightly Acidic Electrolyzed Water to Remove Methylobacterium mesophilicum, Rhodotorula mucilaginosa and Cladosporium cladosporioides in Households

(1) Background: Slightly acidic electrolyzed water (SAEW) is an effective and safe sterilizing solution. Its active component is hypochlorous acid (HOCl) which has been proved to exhibit a strong disinfectant activity. In this research we evaluated the effectiveness of SAEW in the removal of Methylobacterium mesophilicum, Rhodotorula mucilaginosa and Cladosporium cladosporioides, responsible for pink-colored biofilm and black mold in households. (2) Methods: Two concentrations of SAEW, 20 mg/L and 40 mg/L, were tested against M. mesophilicum, R. mucilaginosa and C. cladosporioides. In vitro experiments and mesh experiments were conducted to test the effectiveness of SAEW. (3) Results: The test results showed that 40 mg/L SAEW was effective in removing R. mucilaginosa and C. cladosporioides, with the population decreasing by approximately two orders of magnitude. For M. mesophilicum, resistance towards SAEW was observed; to obtain a 1.3 order of magnitude decrease in bacterial population, washing 5 times with 40 mg/L SAEW was necessary. Mesh experiments showed that SAEW can remove black mold; (4) Conclusions: Overall results indicated that SAEW was particularly effective for R. mucilaginosa and C. cladosporioides species commonly found in Japanese households.

Isolation, Identification and Characterization of Bioflocculant-Producing Bacteria from Activated Sludge of Vulindlela Wastewater Treatment Plant

The low microbial flocculant yields and efficiencies limit their industrial applications. There is a need to identify bacteria with high bioflocculant production. The aim of this study was to isolate and identify a bioflocculant-producing bacterium from activated sludge wastewater and characterise its bioflocculant activity. The identification of the isolated bacterium was performed by 16S rRNA gene sequencing analysis. The optimal medium composition (carbon and nitrogen sources, cations and inoculum size) and culture conditions (temperature, pH, shaking speed and time) were evaluated by the one-factor-at-a-time method. The morphology, functional groups, crystallinity and pyrolysis profile of the bioflocculant were analysed using scanning electron microscope (SEM), Fourier transform infrared (FTIR) and thermogravimetric (TGA) analysis. The bacterium was identified as Proteus mirabilis AB 932526.1. Its optimal medium and culture conditions were: sucrose (20 g/L), yeast extract (1.2 g/L), MnCl2 (1 g/L), pH 6, 30 °C, inoculation volume (3%), shaking speed (120 rpm) for 72 h of cultivation. SEM micrograph revealed the bioflocculant to be amorphous. FTIR analysis indicated the presence of hydroxyl, carboxyl and amino groups. The bioflocculant was completely pyrolyzed at temperatures above 800 °C. The bacterium has potential to produce bioflocculant of industrial importance.

Differences at Species Level and in Repertoires of Secondary Metabolite Biosynthetic Gene Clusters among Streptomyces coelicolor A3(2) and Type Strains of S. coelicolor and Its Taxonomic Neighbors

Streptomyces coelicolor A3(2) is used worldwide for genetic studies, and its complete genome sequence was published in 2002. However, as the whole genome of the type strain of S. coelicolor has not been analyzed, the relationship between S. coelicolor A3(2) and the type strain is not yet well known. To clarify differences in their biosynthetic potential, as well as their taxonomic positions, we sequenced whole genomes of S. coelicolor NBRC 12854T and type strains of its closely related species—such as Streptomyces daghestanicus, Streptomyces hydrogenans, and Streptomyces violascens—via PacBio. Biosynthetic gene clusters for polyketides and non-ribosomal peptides were surveyed by antiSMASH, followed by bioinformatic analyses. Type strains of Streptomyces albidoflavus, S. coelicolor, S. daghestanicus, S. hydrogenans, and S. violascens shared the same 16S rDNA sequence, but S. coelicolor A3(2) did not. S. coelicolor A3(2) and S. coelicolor NBRC 12854T can be classified as Streptomycesanthocyanicus and S. albidoflavus, respectively. In contrast, S. daghestanicus, S. hydrogenans, and S. violascens are independent species, despite their identical 16S rDNA sequences. S. coelicolor A3(2), S. coelicolor NBRC 12854T, S. daghestanicus NBRC 12762T, S. hydrogenans NBRC 13475T, and S. violascens NBRC 12920T each harbor specific polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) gene clusters in their genomes, whereas PKS and NRPS gene clusters are well conserved between S. coelicolor A3(2) and S. anthocyanicus JCM 5058T, and between S. coelicolor NBRC 12854T and S. albidoflavus DSM 40455T, belonging to the same species. These results support our hypothesis that the repertoires of PKS and NRPS gene clusters are different between different species.

Biospeckle Analysis and Biofilm Electrostatic Tests, Two Useful Methods in Microbiology

The development of more sensitive methodologies, capable of quickly detecting and monitoring a microbial population present in a specific biological matrix, as well as performing to allow for the study of all its metabolic changes (e.g., during the formation of biofilm) to occur, is an essential requirement for both well-being and the food industry. Two techniques, in particular, have gained the attention of scientists: The first is “biospeckle”, an optical technique representing an innovative tool for applications in food quality, food safety, and nutraceuticals. With this technique, we can quickly evaluate and monitor the presence of bacteria (or their proliferation) in a solid or liquid biological matrix. In addition, the technique is helpful in quantifying and optimizing the correct storage time of the pro-biotics, if they are entrapped in matrices such as alginate and follow their survival rate in simulated gastro-intestinal conditions. A second technique with great chances is the “biofilm electrostatic test” (BET). BET undoubtedly represents a fast, simple, and highly reproducible tool suitable for admitting the evaluation of the in vitro bacterial capacity in order to adhere through an electrostatic interaction with a pyro-electrified carrier after only 2 h of incubation. BET could represent the way for a quick and standardized evaluation of bacterial resistance among biofilm-producing microorganisms through a fast evaluation of the potential presence of the biofilm.