Mónica Herrero

Application of Flow Cytometry to Segregated Kinetic Modeling Based on the Physiological States of Microorganisms

ABSTRACT Flow cytometry (FC) has been introduced to characterize and to assess the physiological states of microorganisms in conjunction with the classical plate-counting method. To show the applicability of the technique, in particular for the development of kinetic models, pure culture fermentation experiments were followed over time, using both prokaryotic (Lactobacillus hilgardii) and eukaryotic (Saccharomyces cerevisiae) microorganisms growing in standard culture media (MRS and YPD). The differences observed between the active and viable cells determined by FC and CFU, respectively, allowed us to determine that a large number of cells were in a viable but nonculturable (VBNC) state, which resulted in a subpopulation much larger than the damaged-cell (double-stained) subpopulation. Finally, the determination of the evolution of viable, the VBNC, and the dead cells allowed us to develop a segregated kinetic model to describe the yeast and the bacteria population dynamics and glucose consumption in batch cultures. This model, more complete than that which is traditionally used, based only on viable cell measurements, describes better the behavior and the functionality of the cultures, giving a deeper knowledge in real time about the status and the course of the bioprocesses.

Characterization of ø393-A2, a bacteriophage that infects Lactobacillus casei

Bacteriophage o393-A2, isolated from an artisanal cheese whey sample, is a temperate phage able to generate stable lysogens through integration of its DNA into the bacterial genome. One-step growth kinetics of its lytic development revealed eclipse and latent periods of 100 and 140 min, respectively, with a burst size of about 200 p.f.u. per infected cell. ϕ393-A2 virions have an isometric head and a long, non-contractile tail terminating in a baseplate. The capsid is composed of two major and at least nine minor structural polypeptides. The phage genome consists of a double-stranded DNA molecule of 44 kbp bearing 3′-protruding cohesive ends. A physical map of the phage DNA has been constructed for six restriction enzymes. The whole ϕ393-A2 genome has been cloned in Escherichia coli using plasmid- and phage-derived cloning vectors.

Use of flow cytometry to follow the physiological states of microorganisms in cider fermentation processes.

ABSTRACT The flow cytometry (FC) technique used with certain fluorescent dyes (ChemChrome V6 [CV6], DRAQ5, and PI) has proven useful to label and to detect different physiological states of yeast and malolactic bacterium starters conducting cider fermentation over time (by performing sequential inoculation of microorganisms). First, the technique was tested with pure cultures of both types of microorganisms grown in synthetic media under different induced stress conditions. Metabolically active cells detected by FC and by the standard plate-counting method for both types of microorganisms in fresh overnight pure cultures gave good correlations between the two techniques in samples taken at this stage. Otherwise, combining the results obtained by FC and plating during alcoholic and malolactic fermentation over time in the cider-making process, different subpopulations were detected, showing significant differences between the methods. A small number of studies have applied the FC technique to analyze fermentation processes and mixed cultures over time. The results were used to postulate equations explaining the different physiological states in cell populations taken from fresh, pure overnight cultures under nonstress conditions or cells subjected to stress conditions over time, either under a pure-culture fermentation process (in this work, corresponding to alcoholic fermentation) or under mixed-fermentation conditions (for the malolactic-fermentation phase), that could be useful to improve the control of the processes.

Controlled malolactic fermentation in cider using Oenococcus oeni immobilized in alginate beads and comparison with free cell fermentation.

Cells of Oenococcus oeni (formerly Leuconostoc oenos) immobilized in alginate beads were used as starter culture to conduct malolactic fermentation in cider production. Concentrations of major organic acids and volatile compounds were monitored during the process, and results were compared to those obtained when using free cells in the same conditions. The rates of malic acid consumption were similar but lower ethanoic acid content and higher concentration of alcohols were detected with immobilized cells. These features have beneficial effects on the organoleptic properties of cider. A comparison between the kinetic behavior in immobilized and free cells, based on the data obtained for the malic acid consumption, has been developed solving the homogeneous diffusion model when it is applied to the system with immobilized cells.

Quantitative approach to determining the contribution of viable-but-nonculturable subpopulations to malolactic fermentation processes.

ABSTRACT Different sizes of viable-but-nonculturable cell subpopulations of a lactic acid bacterium strain were induced by adding increasing amounts of SO2. The experimental data obtained here were fitted to a segregated kinetic model developed previously. This procedure allowed us to determine in quantitative terms the contribution of this physiological state to malolactic fermentation.

Simultaneous and sequential fermentations with yeast and lactic acid bacteria in apple juice

A complex substrate, reconstituted concentrated apple juice, was used for testing the principal processes during yeast and malolactic bacteria fermentations. Interactions between microorganisms were studied based on two controlled inoculation procedures, and at different fermentation temperatures. Temperature had a more important effect on yeast growth than the presence of malolactic bacteria in the medium. Acceleration of the death phase of the bacterial population was detected at increased temperatures. In all cases, malic acid degradation was affected by the fermentation temperature. When experiments were carried out with simultaneous inoculation, acidification of the medium took place at both temperatures tested (15°C and 22°C), that was not observed when the malolactic bacteria were inoculated after completion of alcoholic fermentation by yeasts.

Prevalent lactic acid bacteria in cider cellars and efficiency of Oenococcus oeni strains

Malolactic fermentation (MLF) is an important step in cider production in order to allowing for improvement of microbiological stability and organoleptic characteristics of cider. Induction of this fermentation by using starter cultures enables a better control over this bioprocess, but although it is a common practice in winemaking, starters specifically focussed for cider MLF are not yet commercially available. Proper starter cultures need to present the ability to degrade l-malic acid conferring pleasing sensory characteristics while avoiding toxicological risks. In this work, lactic acid bacteria (LAB) were first isolated from MLF industrial cider samples, obtained in a cellar in the main cider-producing region of Spain, Asturias. Isolates, identified by molecular tools, belonged to the Lactobacillus brevis and Oenococcus oeni species. After a phylogenetic analysis, representative strains of both identified species were evaluated in order to determine their fermentation capacity, showing O. oeni the best behaviour in this cider fermentation, as previously demonstrated for wine in the literature. Consequently, and with the aim to test the influence at strain level, selection of O. oeni isolates as starters for cider fermentation has been undergone. In order to check the influence of geography over biodiversity, O. oeni strains from six different industrial cellars representing the distinct producing areas in the region (located in a ratio of 30 km) were analyzed by using a specific RAPD method. In this way, isolates were typed in five distinct groups, mainly corresponding to each producing area. All strains isolated from the same cellar showed the same RAPD profile revealing the significance of geographical origin in the indigenous cider LAB. Molecular tools were applied to reject those isolates exhibiting presence of genes related to organoleptic spoilage (exopolysaccharides and acrolein production) or food safety (biogenic amine production), as key selection criteria. Representative strains of each of the five O. oeni RAPD groups were tested as pure cultures to evaluate their technological utility for cider production. Experimental data of malic acid degradation and cell concentration obtained were fitted to previously selected kinetic models aimed to optimization and prediction of bioprocess performance. Four strains revealed as suitable potential starter cultures for conducting MLF in cider production.

Environmental impact of a traditional cooked dish at four different manufacturing scales: from ready meal industry and catering company to traditional restaurant and homemade

PurposeThe environmental burdens of the same dish (a traditional hot stew with pulses and pieces of pork sausages and ham) cooked at four different production scales was analyzed by life cycle assessment (LCA): (a) canned, industrially manufactured and consumed at home; (b) catering company, serving the product for schools; (c) restaurant, cooked in a traditional way and served; and (d) homemade, cooked, and consumed at household level.MethodsThe LCA methodology was applied following the ISO 14044:2006 guidelines. For the inventory analysis, industrial data were obtained from a ready meals factory. Other primary data were directly obtained from the systems analyzed (catering, restaurant and homemade levels). Databases (Ecoinvent, LCA Food DK, BUWAL250, IDEMAT 2001, ETH-ESU 96) were used together with the SimaPro v7.3.3. For the impact assessment, the Eco-indicator 99 method and the CML 2 baseline method were used. In cases (c) and (d), different scenarios for the origin of raw materials and source of energy for cooking were considered. In level (a), an additional scenario considering a 50% reduction of food wastes was also investigated.Results and discussionThe main contribution was meat ingredients, followed by energy consumption. Despite the higher environmental loads in transportation, the factory showed an environmental performance similar to cooking at home with gas. These results can be explained by the implementation of heat recovery systems at industrial scale. The restaurant showed the worst environmental performance. The main reason was that all the energy consumed in the restaurant (even not directly related to cooking) was attributed to the exclusive purpose of serving the food, since no other activities were carried out in the business. Consumer’s choices such as the preference for eating in a restaurant or the energy used for cooking turned out to be important differentiating factors.Conclusions and recommendationsLCA allowed critical aspects to be identified in order to improve sustainable food production and consumption patterns. Electricity consumption and the amount of wastes sent to landfill turned out to be critical control points. In the case of complex dishes such as stews, the higher scale systems in the study (the factory and catering company), with proper energy and environmental practices, can have lower environmental burdens than small-scale systems, such as homemade cooking using a ceramic-glass cooktop or consumption in traditional restaurants. To reinforce the role of education, specific programs on the need to save food and the environmental impact of dietary choices must be implemented at schools.

Chapter 15 – Life Cycle Assessment Focusing on Food Industry Wastes

Life Cycle Assessment (LCA) and other related approaches, such as Life Cycle Thinking and Financial Life Cycle Cost, are valuable environmental management tools useful for the elucidation of major gaps in knowledge of food wastes and food losses in the supply chain and the industrial sector. Lack of knowledge and awareness regarding the heavy consequences of food discharged as waste can partly explain this enormous problem, affecting mainly industrialized countries, which requires urgent attention. Otherwise, resources and energy may be still consumed in vain, despite basic human needs and environmental concerns. With a Life Cycle Thinking perspective, environmental education and specific employment projects should be viable options to promote even lower impact habits in consumers. In this chapter, a brief overview of LCA methodology will be given. LCA can help to assess the necessity to improve valorization technologies and to face the challenge of innovative scenarios for food wastes. In this chapter, both valorization technologies and treatments for the biodegradable fraction or for nonbiodegradable wastes from the food industry are reviewed from an LCA perspective. Finally, as an example, a case study of LCA applied to waste management in the cider-making sector is provided.

Life Cycle Assessment in Biotechnology

Life cycle assessment (LCA) is a technique for assessing environmental impacts in order to identify the key points of a process/product/service as well as suggesting alternatives to improve its environmental performance. The LCA methodology allows a ‘cradle-to-grave’ perspective, considering that all stages involved in the life cycle of a product or activity have a responsibility on the environmental consequences of it. Related terms such as life cycle engineering, social life cycle, and life cycle thinking are emerging with a wider perspective. In this article, an overview of the main aspects of the LCA methodology is provided. Application of LCA in food biotechnology, pharmaceuticals, biopolymers, biofuels, and waste management of the biodegradable fractions is reviewed. Not only the utility of LCA to biotechnological processes, but also its main limitations are presented. The current tendencies in the development of LCA highlight the need to update the tools applicable to different areas with the increasing demand for more accurate environmental information. The main footprints commonly used as LCA indicators have been combined recently with the final goal of obtaining a single indicator useful for decision-making. The interest in LCA for product design and the interactions of LCA with other environmental tools are also commented on.