Malolactic fermentation in lingonberry juice and its use as a preservative.

Lingonberry is a common wild berry that is often sold as jams and beverages. It naturally contains high amounts of the weak acid preservative benzoic acid making it an interesting ingredient for shelf-life extension. Despite this, their use as a raw ingredient is limited by the inherently intense sour taste. This study aimed to improve the taste of lingonberry juice by subjecting it to malolactic fermentation in order to reduce the sourness, and to investigate the benzoic acid in lingonberries as a natural preservative in juice blends by determining the microbial stability. After initial screening of lactic acid bacteria, a Lactiplantibacillus plantarum strain was used as the starter for subsequent investigations. Upon raising the pH, all malic acid was completely converted to lactic acid after seven days. The fermented juice was mixed with blackcurrant juice in different proportions. Challenge tests of the blends showed Listeria monocytogenes could not grow in any juice samples, while Candida albicans only grew in the pure blackcurrant juice. Aspergillus brasiliensis growth was delayed in all samples containing benzoic acid in a concentration-dependent manner. The sourness and astringency were substantially reduced in the juice with added L. plantarum compared to the unfermented juice.

Pearl millet flour and green gram milk based probiotic beverage.

The probiotic beverage was developed using germinated and ungerminated pearl millet flour and green gram milk. The germinated and ungerminated pearl millet flour was added to green gram milk at different concentrations (0.5-2.5 %) along with sugar and cardamom. The mixtures were then inoculated with probiotic bacteria Lactobacillus acidophilus incubated at 37 °C for 6 h. Characterization of probiotic beverages was carried out during storage at (4 ± 1)°C for 21 days. The germinated flour beverage had high acidity as compared to the ungerminated flour beverage. The probiotic count in germinated and ungerminated flour beverages ranged from 8.19 to 8.77 × 107 and 8.04 to 8.52 × 107 log CFU/mL, respectively. Antioxidant activity, polyphenol content increased with an increase in the concentration of flour in the beverage. The LC-MS analysis found the existence of vitexin and isovitexin as the main polyphenolic compounds in the probiotic beverage. Non-dairy probiotic beverage prepared with 0.5 % germinated millet flour gave the best taste, color, texture, and rheological properties.

Inactivation activity and mechanism of pulsed light against Alicyclobacillus acidoterrestris vegetative cells and spores in concentrated apple juice.

Alicyclobacillus acidoterrestris has received much attention due to its unique thermo-acidophilic property and implication in the spoilage of pasteurized juices. The objective of this study was to evaluate the sterilization characteristics and mechanisms of pulsed light (PL) against A. acidoterrestris vegetative cells and spores in apple juice. The results indicated that bacteria cells in apple juice (8-20°Brix) can be completely inactivated within the fluence range of 20.25-47.25 J/cm2, which mainly depended on the soluble solids content (SSC) of juice, and the spores in apple juice (12°Brix) can be completely inactivated by PL with the fluence of 54.00 J/cm2. The PL treatment can significantly increase the leakage of reactive oxygen species (ROS) and proteins from cells and spores. Fluorescence studies of bacterial adenosine triphosphate (ATP) indicated that the loss of ATP was evident. Scanning electron microscopy and confocal laser scanning microscope presented that PL-treated cells or spores had serious morphological damage, which reduced the integrity of cell membrane and led to intracellular electrolyte leakage. In addition, there were no significant negative effects on total sugars, total acids, total phenols, pH value, SSC and soluble sugars, and organic acid content decreased slightly during the PL treatment. The contents of esters and acids in aroma components had a certain loss, while that of alcohols, aldehydes and ketones were increased. These results demonstrated that PL treatment can effectively inactivate the bacteria cells and spores in apple juice with little effect on its quality. This study provides an efficient method for the inactivation of A. acidoterrestris in fruit juice.

Characterization of potential probiotic starter cultures of lactic acid bacteria isolated from Ethiopian fermented cereal beverages, Naaqe, and Cheka.

AIMS: To test the in vitro probiotic potential and starter culture capacity of lactic acid bacteria (LAB) isolated from Naaqe and Cheka, cereal-based Ethiopian traditional fermented beverages. METHODS AND RESULTS: A total of 44 strains were isolated from spontaneously fermented Ethiopian cereal-based beverages, Naaqe and Cheka with 24 putatively identified as LAB and 14 identified up to the species level. The species Limosilactobacillus fermentum (6/12; 50%) and Weissella confusa (5/12, 41.67%) were the predominant species identified from Naaqe, while the two Cheka isolates were L. fermentum and Pediococcus pentosaceus. Six LAB strains inhibited eight of the nine gastrointestinal indicator key pathogens in Ethiopia, including Escherichia coli, Salmonella enterica subsp. enterica var. Typhimurium, Staphylococcus aureus, Shigella flexneri, and Listeria monocytogenes. Three of the LAB isolates exhibited strain-specific immunostimulation in human monocytes. Based on these probiotic properties and growth, six strains were selected for in situ evaluation in a mock fermentation of Naaqe and Cheka. During primary fermentations, L. fermentum 73B, P. pentosaceus 74D, L. fermentum 44B, W. confusa 44D, L. fermentum 82C, and Weissella cibaria 83E and their combinations demonstrated higher pH-lowering properties and colony-forming unit counts compared to the control spontaneous fermentation. The same pattern was also observed in the secondary mock fermentation by the Naaqe LAB isolates. CONCLUSIONS: In this study, we selected six LAB strains with antipathogenic, immunostimulatory, and starter culture potentials that can be used as autochthonous probiotic starters for Naaqe and Cheka fermentations once their health benefit is ascertained in a clinical trial as a next step.

Quantitative microbial spoilage risk assessment caused by fungi in sports drinks through multilevel modelling.

The risk of fungal spoilage of sports drinks produced in the beverage industry was assessed using quantitative microbial spoilage risk assessment (QMSRA). The most relevant pathway was the contamination of the bottles during packaging by mould spores in the air. Mould spores' concentration was estimated by longitudinal sampling for 6 years (936 samples) in different production areas and seasons. This data was analysed using a multilevel model that separates the natural variability in spore concentration (as a function of sampling year, season, and area) and the uncertainty of the sampling method. Then, the expected fungal contamination per bottle was estimated by Monte Carlo simulation, considering their settling velocity and the time and exposure area. The product's shelf life was estimated through the inoculation of bottles with mould spores, following the determination of the probability of visual spoilage as a function of storage time at 20 and 30 °C using logistic regression. The Monte Carlo model estimated low expected spore contamination in the product (1.7 × 10-6 CFU/bottle). Nonetheless, the risk of spoilage is still relevant due to the large production volume and because, as observed experimentally, even a single spore has a high spoilage potential. The applicability of the QMSRA during daily production was made possible through the simplification of the model under the hypothesis that no bottle will be contaminated by more than one spore. This simplification allows the calculation of a two-dimensional performance objective that combines the spore concentration in the air and the exposure time, defining "acceptable combinations" according to an acceptable level of spoilage (ALOS; the proportion of spoiled bottles). The implementation of the model at the operational level was done through the representation of the simplified model as a two-dimensional diagram that defines acceptable and unacceptable areas. The innovative methodology employed here for defining and simplifying QMSRA models can be a blueprint for future studies aiming to quantify the risk of spoilage of other beverages with a similar scope.

Occurrence of Alicyclobacillus acidoterrestris in pasteurized and high hydrostatic pressure-treated fruit juices and isolates' characterization.

In recent years, acidophilic, heat-resistant, and spore-forming spoilage bacteria have been identified in pasteurized or treated by high hydrostatic pressure (HPP) fruit juices. Alicyclobacillus acidoterrestris is the bacteria more frequently linked to the spoilage of this type of product because its spores can survive conventional pasteurization and HPP treatments. Under favourable conditions, such as an acidic pH, its spores can germinate and multiply, with the consequent production of guaiacol. Guaiacol is a compound with an undesirable odour ("medicinal", "smoked" or "antiseptic"). In this context, our objective was to determine the prevalence of A. acidoterrestris in 150 Spanish pasteurized and HPP-treated fruit juices purchased from supermarkets or received from manufacturers. Then, the isolates and the reference strain (CECT 7094 T) were characterized to establish differences in terms of (i) growth capacity at different pH and temperatures, and in (ii) guaiacol production capacity. The results showed a high incidence of A. acidoterrestris (18.0 %) in the analysed juices. The 44.4 % of the isolates came from blends of fruit juices. Within juice blends, 9 juices contained apple juice among their ingredients. This represents a 18.8 % of incidence with respect to the total of blended juices with apple. A high incidence in monovarietal apple juices was also observed (3 out of 14 samples). Regarding the characterization of the isolates, EC1 (isolated from an apple concentrate) showed the highest growth capacity at pH 4.0 at temperatures from 20 to 55 °C. Besides, three strains (R42, EC10, and EZ13, isolated from clementine, plum and white grape juice, respectively) could grow at room temperatures (20 and 25 °C). For pH, only EZ13, isolated from white grape juice, was able to grow significantly at pH 2.5. Finally, the production of guaiacol ranged from 74.1 to 145.6 ppm, being the isolate EC1 the one that produced more guaiacol after 24 h of incubation at 45 °C (145.6 ppm). As we have observed, there is a high incidence of A. acidoterrestris in marketed juices and intermediate products despite the treatments received (pasteurization or HPP). Under favourable conditions for the development of this microorganism, it could produce enough guaiacol to spoil the juices before their consumption. Therefore, in order to improve the quality of fruit juices it is necessary to investigate in more detail the origin of this microorganism and to find strategies to reduce its presence in final products.

Contribution of amino acids to Alicyclobacillus acidoterrestris DSM 3922T resistance towards acid stress.

Spoilage of juice and beverages by a thermo-acidophilic bacterium, Alicyclobacillus acidoterrestris, has been considered to be a major and widespread concern for juice industry. Acid-resistant property of A. acidoterrestris supports its survival and multiplication in acidic juice and challenges the development of corresponding control measures. In this study, intracellular amino acid differences caused by acid stress (pH 3.0, 1 h) were determined by targeted metabolomics. The effect of exogenous amino acids on acid resistance of A. acidoterrestris and the related mechanisms were also investigated. The results showed that acid stress affected the amino acid metabolism of A. acidoterrestris, and the selected glutamate, arginine, and lysine contributed to its survival under acid stress. Exogenous glutamate, arginine, and lysine significantly increased the intracellular pH and ATP level, alleviated cell membrane damage, reduced surface roughness, and suppressed deformation caused by acid stress. Additionally, the up-regulated gadA and speA genes and the enhanced enzymatic activity confirmed that glutamate and arginine decarboxylase systems played a crucial role in maintaining pH homeostasis of A. acidoterrestris under acid stress. Our research reveals an important factor contributing to acid resistance of A. acidoterrestris, which provides an alternative target for effectively controlling this contaminant in fruit juices.

Functional Characterization of Lactobacillus plantarum Isolated from Cow Milk and the Development of Fermented Coconut and Carrot Juice Mixed Beverage.

Screening probiotics are crucial for assessing their safety, security, and further production of functional foods for human health. The present study aimed to isolate and identify bacteria from raw cow's milk samples that exhibit health benefits upon consumption. We characterized the probiotic properties of Lactobacillus plantarum (also called Lactiplantibacillus plantarum) strains CMGC2 and CMJC7 isolated from cow milk by in vitro study. The strains exhibited tolerance to simulated gastric conditions and were further identified by 16S rRNA sequencing as Lactobacillus plantarum (L. plantarum) CMGC2 and CMJC7. Both isolates were evaluated in vitro for their probiotic attributes, viz. hydrophobicity, autoaggregation, co-aggregation, lysozyme tolerance, antibacterial activity, antibiotic susceptibility, hemolytic activity, and phenol tolerance. The isolates CMGC2 and CMJC7 showed excellent probiotic attributes; hence, both strains were selected to produce coconut and carrot juice mixed beverages (CCMB). The CCMB was evaluated for the pH, acid-production rate, and total viable bacterial counts. The results showed that the CCMB was an excellent medium for the growth of CMGC2 and CMJC7 as it supported adequate growth of organisms (8.93 CFU/mL and 8.68 CFU/mL, respectively) even after 48 h of incubation. In conclusion, CMGC2 and CMJC7 can be used to develop different beverages possessing nutritive and probiotic values, and these beverages can be used for producing unique products.

Diversity of yeasts in Indian fermented foods and alcoholic beverages.

Ethnic Indian people have been domesticating beneficial microorganisms (bacteria, yeasts, and moulds) by their wisdom of ethno-microbiological knowledge for production of flavoured and socio-culturally preferred fermented foods and alcoholic beverages for more than 8000 years. The purpose of this review is to collate the available literatures of diversity of Saccharomyces and non-Saccharomyces species associated with Indian fermented foods and alcoholic beverages. A colossal diversity of enzyme- and alcohol-producing yeasts under the phylum Ascomycota has been reported from Indian fermented foods and alcoholic beverages. The distributions of yeast species show 13.5% of Saccharomyces cerevisiae and 86.5% of some non-Saccharomyces spp. in Indian fermented foods and alcoholic beverages, based on the reported literatures available till date. There is a research gap on prospect of yeasts research in India. Hence, we suggest that validation of traditional knowledge of domestication of functional yeasts needs to be studied to develop the functional genomics platforms for Saccharomyces and non-Saccharomyces spp. in Indian fermented foods and alcoholic beverages.

Microbiological and chemical characterization of water kefir: An innovative source of potential probiotics for bee nutrition.

We evaluated the microbial composition of water kefir grains and beverage over the course of one year to determine whether the number and type of microorganisms changed over the time. Bacteria and yeast colonies with different morphologies were isolated from water kefir and their antimicrobial activity was evaluated against Paenibacillus larvae and Ascosphaera apis. A chemical characterization of kefir was also carried out. Our results confirmed that bacteria and yeasts were more numerous in kefir grains compared with those in the beverage. The counts of microorganisms declined, although an important microbial community was still present in kefir after the long storage period. Eleven strains which inhibited bee pathogens were isolated from kefir. Genotypic results demonstrated that these isolates included Lentilactobacillus hilgardii, Lentilactobacillus buchneri and Saccharomyces cerevisiae. Thus, water kefir may be an innovative source of potential probiotic strains for bee nutrition in order to control honeybee diseases.

Mathematical modeling of Escherichia coli O157:H7 growth in carrot juice influenced by Thymbra capitata essential oil, heat treatment, and storage temperature.

The aim of this study was to develop a mathematical model describing the survival of Escherichia coli O157:H7 in carrot juice treated with Thymbra capitata essential oil combined with mild heat treatment and stored at different temperatures. The viable count method was used to investigate the effect of the treatment on bacterial survival, and the response surface methodology was used to develop a statistical model fitting the data. The results showed that the variance of bacterial growth is explained by storage temperature (37 %) and heat treatment (35 %), these are followed by Thymbra capitata essential oil (18 %) and their interaction (9 %). Positive multiplicative interaction was obtained for any pair of the studied treatments and cooperative effect synergy was observed over a large domain of these factors. A mathematical model was successfully developed to describe Escherichia coli O157:H7 response to the selected factors, within the study limits, and to estimate the risk of juice contamination and shelf-life. Based on our results, the use of Thymbra capitata essential oil combined with heat treatment may control Escherichia coli O157:H7 growth in carrot juice stored at low temperature.

Antimicrobial activity and mechanism of preservatives against Alicyclobacillus acidoterrestris and its application in apple juice.

Alicyclobacillus acidoterrestris has great influence on the quality of apple juice products. In this study, the antibacterial activity of five preservatives (ε-polylysine, propylparaben, monocaprin, octyl gallate and heptylparaben) against A. acidoterrestris and its underlying mechanism were investigated. Results showed that these five preservatives all exerted antibacterial activity through a multiple bactericidal mechanism, and monocaprin and octyl gallate had the highest antibacterial activity, with the minimum inhibitory concentration (MIC) values of 22.5 and 6.25 mg/L, respectively. Five preservatives all changed the permeability of the cell membrane and destroyed the complete cell morphology, with the leakages of the intracellular electrolytes. Moreover, the treatment of ε-polylysine, propylparaben and monocaprin increased the leakage of intracellular protein; propylparaben and octyl gallate reduced the levels of cellular adenosine triphosphate. Also, monocaprin and octyl gallate may stimulate bacteria to release a large amount of reactive oxygen species, so that certain oxidative damage can kill the bacteria. Furthermore, monocaprin and octyl gallate could effectively inactivate the contamination of A. acidoterrestris in apple juices, with the slightly decrease of soluble sugars and organic acids, without significant adverse effects on total sugars and titratable acids. This research highlights the great promise of using monocaprin and octyl gallate as the safe multi-functionalized food additives for food preservations.

Kombucha analogues around the world: A review.

Kombucha is a traditional healthy beverage usually made by the fermentation of sweetened tea with a symbiotic culture of bacteria and yeast. The consumption of kombucha is associated with numerous health benefits and therefore the beverage has attracted the attention of consumers worldwide. Non-typical substrates (fruits, vegetables, plants, herbs, dairy, and by-products) are being inoculated with the kombucha consortium in an attempt to develop new products. This review paper reviews the fermentation parameters for different non-tea substrates used to make kombucha, in addition to the findings obtained in terms of physico-chemical analysis, biological activities and sensory evaluation.

[Technological features of fermented beverages production using kombucha].

Kombucha is a beverage made by fermenting sweetened brewed tea (substrate) by symbiotic culture of yeast and bacteria. Numerous researches on optimization of fermentation process, determination of the influence of technological factors on physical and chemical properties, formation of taste and flavor profile of the beverages, prevention of industrial product risks are due to the growing popularity of kombucha in Europe and the USA. Technological features of kombucha production are to optimize conditions for the growth of symbiotic culture and substrate fermentation. The duration of the process depends on the composition of the substrate, the ratio of tea mushroom and substrate, temperature, size and shape of fermentation vessel. The aim of the work was to generalize the results of studying the technological features of the production of fermented kombucha type beverages and to identify the factors that affect the chemical composition and safety of the finished beverages. Material and methods. Analytical research was carried out on the main databases for the keyword "kombucha". The criteria for inclusion of articles in the analysis were research articles with open access and presenting detailed technology of kombucha. Results. The technology of kombucha production is based on fermentation of the substrate and obtaining the base of the beverage with high content of organic acids, mainly acetic acid. In order to ensure microbiological safety the acetic acid concentration in the beverage base must be at least 1.2%. The high organic acid content necessitates the use of only glass or stainless steel fermentation equipment approved for food contact. The fermentation temperature ranges from 18 to 32 °C. The fermentation process is monitored according to basic criteria: temperature, pH value, acidity, acetic acid content, ethyl alcohol content, and residual sugar content. Kombucha production process is connected with microbiological, chemical and physical risks which could appear in case of using low quality raw materials, equipment and consumer packaging made of materials which do not correspond to sanitary norms, violating technological regimes, storage conditions of raw materials and ready production. To prevent hazards affecting the quality and safety of the finished product, it is necessary to control the technological process at all stages of production. Conclusion. Following sanitary-hygienic norms and technological regimes allows producing kombucha with a balanced taste and aroma, which meets the safety requirements for fermented beverages.

Inactivation of Escherichia coli in apple cider using atmospheric cold plasma.

Atmospheric cold plasma (ACP) is a promising non-thermal technology that has the potential to inactivate microorganisms in foods. In this work, the inactivation of E. coli K12, acid-adapted E. coli K12, and E. coli O157:H7 in apple cider by ACP was investigated using feed gases as simulated air (SA) (80 % N2 + 20 % O2) and a mixture of 90 % N2 + 10 % O2 with various processing times (0 to 180 s). We obtained the reduced the populations of both acid-adapted and non-adapted E. coli K12 by 5 log CFU/mL within 120 s, and E. coli O157:H7 within 90 s. Additionally, no significant changes in the °Brix, pH, temperature, or titratable acidity (TA) of apple cider were observed after exposure to ACP. However, processing times longer than 120 s resulted in significant changes in the pH values. The highest concentration of ozone and hydrogen peroxide reached to 0.22 ± 0.1 mg/L for CG in 180 s and 0.07 ± 0.01 mg/L for SA in 150 s, respectively. Both acid-adapted and non-acid adapted E. coli K12 was found to be more resistant to ACP processing than E. coli O157:H7 after the 90 s, so it could serve as a surrogate for E. coli O157:H7. When we compared the effect of the gas type on inactivation, non-selective media, the results showed no significant differences between the gas types, while selective media demonstrated significant differences. In optical absorption spectroscopy measurements of plasma species, primarily ozone peaks were observed. Furthermore, the optical absorption spectroscopy also revealed that the inactivation of the bacteria could be attributed to some plasma species with wavelengths between 190 and 308 nm. The findings provided a perspective on the use of ACP as a method for decontaminating fruit juices as a non-thermal processing.

Storage time and temperature affect microbial dynamics of yeasts and acetic acid bacteria in a kombucha beverage.

Kombucha is a mildly sweet, slightly acidic fermented beverage, commercially available worldwide, that has attracted increasing consumers' interest due to its potential health benefits. Kombucha is commonly prepared using sugared black or green tea, but also other plant substrates are frequently utilised. Kombucha is obtained by fermentation using a symbiotic culture of bacteria and yeasts, whose composition varies depending on inoculum origin, plant substrates and environmental conditions. After fermentation, kombucha drinks are usually refrigerated at 4 °C, in order to maintain their biological and functional properties. There are no reports on the fate of microbial communities of kombucha in relation to long-term storage time and temperature. Here, for the first time, we monitored the diversity and dynamics of the microbial communities of a kombucha beverage fermented with different herbs during storage at 4 °C and at room temperature, for a period of 90 days, utilising culture-dependent and independent approaches. Moreover, cultivable yeasts and acetic acid bacteria (AAB) were isolated from the beverage, inoculated in pure culture, identified by molecular methods, and yeasts assessed for their functional properties. Total yeast counts were not affected by storage temperature and time, although their community composition changed, as Saccharomyces species significantly decreased after 45 days of storage at room temperature, completely disappearing after 90 days. On the other hand, Dekkera anomala (Brettanomyces anomalus), representing 52 % of the yeast isolates, remained viable up to 90 days at both storage temperatures, and was able to produce high levels of organic acids and exopolysaccharides. Data from DGGE (Denaturing Gradient Gel Electrophoresis) band sequencing confirmed that it was the dominant yeast species in all samples across storage. Other yeast isolates were represented by Saccharomyces and Zygosaccharomyces species. Among AAB, Gluconobacter oxydans, Novacetimonas hansenii and Komagataeibacter saccharivorans represented 46, 36 and 18 % of the isolates, whose occurrence remained unchanged across storage at 4 °C and did not vary up to 20 days of storage at room temperature. This work showed that the combination of culture-dependent and independent approaches is important for obtaining a complete picture of the distinctive core microbial community in kombucha beverages during storage, elucidating its diversity and composition, and preliminary characterizing yeast strains with putative functional activities.

Molecular approaches to uncover phage-lactic acid bacteria interactions in a model community simulating fermented beverages.

Food microbial diversity and fluxes during the fermentation processes are well studied whereas phages-bacteria interactions are still poorly described in the literature. This is especially true in fermented beverages, and especially in cider, which is an alcoholic fermented apple beverage. The transcriptomic and proteomic responses of the lactic acid bacterium (LAB) Liquorilactobacillus mali UCMA 16447 to a lytic infection by phage UCMA 21115, both isolated from cider, were investigated, in order to get a better understanding of phages-bacteria interactions in such fermented beverage. During phage infection, 122 and 215 genes were differentially expressed in L. mali UCMA 16447 strain at T15 and T60 respectively, when compared to the uninfected condition. The same trends were confirmed by the proteomic study, with a total of 28 differentially expressed proteins found at T60. Overall, genes encoding cellular functions, such as carbohydrate metabolism, translation, and signal transduction, were downregulated, while genes involved in nucleotide metabolism and in the control of DNA integrity were upregulated in response to phage infection. This work also highlighted that phage infection repressed many genes involved in bacterial cell motility, and affected glycolysis.

Pulsed multifrequency thermosonication induced sonoporation in Alicyclobacillus acidoterrestris spores and vegetative cells.

Alicyclobacillus acidoterrestris (AAT) was proposed as an index of pasteurization design for high-acid fruit products due to its spore resistance and repeated spoilage incidences in fruit juices. This study aimed to determine the effectiveness of pulsed multifrequency ultrasound to minimize AAT spores and vegetative cells in aqueous suspension. For this research, an investigation of the reactive oxygen species and antioxidant activity was performed to examine the effect of temperature and frequency on AAT spore inoculation. Total decreases in AAT bacteria were 5.99, 5.74 Log CFU/mL in vegetative cells for dual-frequency thermosonication (DFTS) and dual-frequency ultrasonication (DFUS), respectively, while 5.90 and 5.38 Log CFU/mL in spores for both DFUS and DFTS, respectively. The loss of the percentage of cells in ultrasound (US) and thermosonication (TS) treatments was inversely associated with the rate of O2-and H2O2 development. The fluorescence microscopy revealed a higher bactericidal efficacy of DFTS compared to the DFUS and control. Scanning Electron Microscopy (SEM) and Transmission electron microscopy (TEM) demonstrated ultra-structural modifications such as the interruption of cell walls by cavitation and pores in the membrane structure of the AAT bacteria induced by sonoporation. Several TS frequencies of 20/40/60, 20/40, and 20 kHz treated spores had a higher electrical conductivity than untreated ones, with an improvement of 7.94, 5.68, and 3.72 %, respectively. Fourier-transform infrared (FTIR) spectroscopy revealed major changes in the spectral region of membrane fatty acids and proteins of AAT. Simultaneously, AAT inactivation specific energy rate was significantly reduced using dual-frequency ultrasound, compared to mono-frequency thermosonication. The significant results of this work recommended pulsed DFUS as an alternative application to mono-frequency US in beverage industries.

Deciphering the antibacterial activity and mechanism of p-coumaric acid against Alicyclobacillus acidoterrestris and its application in apple juice.

Alicyclobacillus acidoterrestris has been regarded as the main hazardous factor causing the spoilage of commercially pasteurized fruit juice due to its unique thermo-acidophilic properties. p-Coumaric acid, primarily isolated from plants and having high biological activity, is supposed to be a viable food additive. Herein, the antibacterial activity and mechanism of p-coumaric acid against A. acidoterrestris and its application in apple juice were investigated. The results showed that p-coumaric acid had an active inhibition against A. acidoterrestris vegetative cells, and both minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) were 0.2 mg/mL. MIC of p-coumaric acid against spores was also 0.2 mg/mL, while MBC was >1.6 mg/mL. Treatment of p-coumaric acid caused significant damage of cell integrity, decrease of intracellular ATP, hyperpolarization of cell membrane, degradation of whole cell protein, and malformation of cell morphology. Agarose gel retardation and fluorescence spectroscopy assays revealed that p-coumaric acid could interact with DNA and intercalate into the groove of DNA double helix to disturb normal cellular functions. Furthermore, the addition of p-coumaric acid at MIC caused 2.07 log drop in A. acidoterrestris vegetative cells and effectively inhibited the germination of spores in apple juice storage at 20 °C for 7 days. The low-temperature storage (4 °C) could inactivate A. acidoterrestris cells in apple juice, but the addition of p-coumaric acid further accelerated degradation of vegetative cells and the inactivation time was shortened from 5 days to 3 days compared with control. Importantly, the addition of p-coumaric acid could increase total phenolic content and ABTS radical scavenging activity and had no remarkable influence on pH, total soluble solids, color and sensory qualities of apple juice, regardless of storage temperature. These results revealed that p-coumaric acid could be expected to serve as an alternative or supplement agent for inhibiting the development of A. acidoterrestris in fruit juice.

Microbial Diversity and Interaction Specificity in Kombucha Tea Fermentations.

Despite the popularity of kombucha tea, the distribution of different microbes across kombucha ferments and how those microbes interact within communities are not well characterized. Using metagenomics, comparative genomics, synthetic community experiments, and metabolomics, we determined the taxonomic, ecological, and functional diversity of 23 distinct kombuchas from across the United States. Shotgun metagenomic sequencing demonstrated that the bacterium Komagataeibacter rhaeticus and the yeast Brettanomyces bruxellensis were the most common microbes in the sampled kombucha communities. To determine the specificity of bacterium-yeast interactions, we experimentally quantified microbial interactions within kombucha biofilms by measuring densities of interacting species and biofilm production. In pairwise combinations of bacteria and yeast, B. bruxellensis and individual strains of Komagataeibacter spp. were sufficient to form kombucha fermentations with robust biofilms, but Zygosaccharomyces bisporus, another yeast found in kombucha, did not stimulate bacteria to produce biofilms. Profiling the spent media of both yeast species using nuclear magnetic resonance spectroscopy suggested that the enhanced ability of B. bruxellensis to ferment and produce key metabolites in sucrose-sweetened tea may explain why it stimulates biofilm formation. Comparative genomics demonstrated that Komagataeibacter spp. with >99% genomic similarity can still have dramatic differences in biofilm production, with strong producers yielding five times more biofilm than the weakest producers. IMPORTANCE Through an integration of metagenomic and experimental approaches, our work reveals the diversity and nature of interactions among key taxa in kombucha microbiomes through the construction of synthetic microbial pairs. Manipulation of these microbes in kombucha has the potential to shape both the fermentation qualities of kombucha and the production of biofilms and is valuable for kombucha beverage producers, biofilm engineers, and synthetic ecologists.