Viability, Sublethal Injury, and Release of Cellular Components From Alicyclobacillus acidoterrestris Spores and Cells After the Application of Physical Treatments, Natural Extracts, or Their Components.

Alicyclobacillus acidoterrestris is a spoiling microorganism regarded as one of the most important causes of spoilage of fruit juices and acidic products. In this paper, four strains of A. acidoterrestris (type strain-DSM 3922; two wild strains isolated from soil-C8 and C24; wild strain isolated from a spoiled pear juice CB1) were treated through natural extracts/active compounds from essential oils (EOs), and physical treatments were used to assess their susceptibility and the presence of sublethal injury. The characterization of damage was also performed. The results suggest that it is possible to control A. acidoterrestris through alternative approaches, although the effect relied upon the age of spores. In addition to the mere antimicrobial effect, some treatments could cause a sublethal injury on spores. Lemon extract was the most effective treatment for both the antimicrobial effect and the sublethal injury, as evidenced by the release of proteins, and calcium dipicolinate [dipicolinic acid (DPA)] by fresh spores and only DPA (with an exception for C8) by old spores. A sublethal injury with protein release was also found for physical treatments [US (ultrasound) or heating]. For the first time, this paper reports on the existence of a sublethal injury for A. acidoterrestris, and this evidence could also be a challenge, because injured microorganisms could restore their metabolism, or an opportunity to design new preserving treatments.

Effect of Physical and Chemical Treatments on Viability, Sub-Lethal Injury, and Release of Cellular Components from Bacillus clausii and Bacillus coagulans Spores and Cells.

Bacterial spores are of concern to the food industry due to their ability to survive processing and their potential to subsequently germinate and grow in food. In this paper, two strains belonging to the genus Bacillus (B. clausii DSM 8716 and B. coagulans DSM 1) were studied under in vitro conditions after the application of essential oils, and physical treatments; cells and spores' susceptibility, the extent of sub-lethal injury and the release of cellular components as a function of treatment and targets (cells, spores, old or activated spores) were studied. The highest antimicrobial effect was found for cells treated through citrus extract, while both essential oils and physical treatments could cause a sub-lethal injury on the surviving cells and spores; in addition, the spores of B. coagulans released dipicolinic acid (DPA) and proteins. Sub-lethal injury should be considered when designing a food processing treatment, because injured microorganisms could either repair the damage or be inactivated with a different effect on microbial stability of foods.

Impact of Gluten-Friendly Bread on the Metabolism and Function of In Vitro Gut Microbiota in Healthy Human and Coeliac Subjects.

The main aim of this paper was to assess the in vitro response of healthy and coeliac human faecal microbiota to gluten-friendly bread (GFB). Thus, GFB and control bread (CB) were fermented with faecal microbiota in pH-controlled batch cultures. The effects on the major groups of microbiota were monitored over 48 h incubations by fluorescence in situ hybridisation. Short-chain fatty acids (SCFAs) were measured by high-performance liquid chromatography (HPLC). Furthermore, the death kinetics of Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, Staphylococcus aureus, and Salmonella Typhimurium in a saline solution supplemented with GFB or CB were also assessed. The experiments in saline solution pinpointed that GFB prolonged the survival of L. acidophilus and exerted an antibacterial effect towards S. aureus and S. Typhimurium. Moreover, GFB modulated the intestinal microbiota in vitro, promoting changes in lactobacilli and bifidobacteria members in coeliac subjects. A final multivariate approach combining both viable counts and metabolites suggested that GFB could beneficially modulate the coeliac gut microbiome; however, human studies are needed to prove its efficacy.

Genotypic and Phenotypic Heterogeneity in Alicyclobacillus acidoterrestris: A Contribution to Species Characterization.

Alicyclobacillus acidoterrestris is the main cause of most spoilage problems in fruit juices and acidic products. Since soil borne species often contaminate fruit juices and do not need strict extreme requirements for survival, it is a great concern to investigate whether and how soil species could evolve from their ecological niches in microbial community to new environments as fruit juices. In this study, 23 isolates of thermo-acidophilic, spore-forming bacteria from soil were characterized by cultural and molecular methods. In addition, 2 strains isolated from a spoilage incident in pear juice were typed. Strains phenotyping showed that they could be grouped into 3 different clusters, and some isolates showed identical or quite similar patterns. Analyzing pH and temperature ranges for growth, the majority of strains were able to grow at values described for many species of Alicyclobacillus. Qualitative utilization of lysine, arginine and indole production from tryptophan revealed, for the first time, deamination of lysine and decarboxylation of arginine. Resistance to 5% NaCl as well as the ability to hydrolyze starch and gelatin, nitrate reduction, catalase and oxidase activities confirmed literature evidences. Examining of 16S rRNA, showed that isolates were divided into three blocks represented by effectively soil species and strains that are moving from soil to other possible growing source characterized by parameters that could strongly influence bacterial survival. RAPD PCR technique evidenced a great variability in banding patterns and, although it was not possible to obtain genotypically well-distinguished groups, it was feasible to appreciate genetic similarity between some strains. In conclusion, the investigation of a microbial community entails a combination of metagenomic and classic culture-dependent approaches to expand our knowledge about Alicyclobacillus and to look for new subspecies.

Alicyclobacillus spp.: New Insights on Ecology and Preserving Food Quality through New Approaches.

Alicyclobacillus spp. includes spore-forming and thermo-acidophilic microorganisms, usually recovered from soil, acidic drinks, orchards and equipment from juice producers. The description of the genus is generally based on the presence of ω-fatty acids in the membrane, although some newly described species do not possess them. The genus includes different species and sub-species, but A. acidoterrestris is generally regarded as the most important spoiler for acidic drinks and juices. The main goal of this review is a focus on the ecology of the genus, mainly on the species A. acidoterrestris, with a special emphasis on the different phenotypic properties and genetic traits, along with the correlation among them and with the primary source of isolation. Finally, the last section of the review reports on some alternative approaches to heat treatments (natural compounds and other chemical treatments) to control and/or reduce the contamination of food by Alicyclobacillus.

Spore inactivation and DPA release in Alicyclobacillus acidoterrestris under different stress conditions.

This paper reports on the inactivation of spores of 5 strains of Alicyclobacillus acidoterrestris under different stress conditions (acidic and alkaline pH, high temperature, addition of lysozyme, hydrogen peroxide and p-coumaric acid). The research was divided into two different steps; first, each stress was studied alone, thus pointing out a partial uncoupling between spore inactivation and DPA release, as H2O2 reduced spore level below the detection but it did not cause the release of DPA. A partial correlation was found only for acidic and alkaline pH. 2nd step was focused on the combination of pH, temperature and H2O2 through a factorial design; experiments were performed on both fresh and 4 month-old spores and pinpointed a different trend for DPA release as a function of spore age.