Fermentation at non-conventional conditions in food- and bio-sciences by the application of advanced processing technologies.

The interest in improving the yield and productivity values of relevant microbial fermentations is an increasingly important issue for the scientific community. Therefore, several strategies have been tested for the stimulation of microbial growth and manipulation of their metabolic behavior. One promising approach involves the performance of fermentative processes during non-conventional conditions, which includes high pressure (HP), electric fields (EF) and ultrasound (US). These advanced technologies are usually applied for microbial inactivation in the context of food processing. However, the approach described in this study focuses on the use of these technologies at sub-lethal levels, since the aim is microbial growth and fermentation under these stress conditions. During these sub-lethal conditions, microbial strains develop specific genetic, physiologic and metabolic stress responses, possibly leading to fermentation products and processes with novel characteristics. In some cases, these modifications can represent considerable improvements, such as increased yields, productivities and fermentation rates, lower accumulation of by-products and/or production of different compounds. Although several studies report the successful application of these technologies during the fermentation processes, information on this subject is still scarce and poorly understood. For that reason, the present review paper intends to assemble and discuss the main findings reported in the literature to date, and aims to stimulate interest and encourage further developments in this field.

Effect of ultrasound on lactic acid production by Lactobacillus strains in date (Phoenix dactylifera var. Kabkab) syrup.

Date syrup is rich in fermentable sugars and may be used as a substrate for different microbial fermentations, including lactic acid fermentation processes. The beneficial effects of ultrasounds (US) on bioprocesses have been reported for several microorganisms, due to the enhancement of cell growth, as well as improvements in yields and productivities. Therefore, US treatments (30 kHz, 100 W, 10-30 min) were applied to two lactobacilli (Lactobacillus helveticus PTCC 1332 and Lactobacillus acidophilus PTCC 1643), during fermentation using date syrup as substrate. The effects on lactic acid fermentation were evaluated by analyzing cell growth (dry cell weight and viable cell count), substrate consumption (quantification of glucose and fructose), and product formation (quantification of lactic acid) over time. The effects of US were also evaluated on cell membrane permeability. Both lactobacilli were able to grow well on date syrup without the need for addition of further ingredients. The US effects were highly dependent on treatment duration: treatments of 10- and 20-min stimulated lactobacilli growth, while the treatment extension to 30 min negatively affected cell growth. Similarly, the 10- and 20-min treatments increased sugar consumption and lactic acid production, contrarily to the 30-min treatment. All US treatments increased cell membrane permeability, with a more pronounced effect at more extended treatments. The results of this work showed that application of appropriate US treatments could be a useful tool for stimulation of lactic acid production from date syrup, as well as for other fermentative processes that use date syrup as substrate.

Application of High Pressure with Homogenization, Temperature, Carbon Dioxide, and Cold Plasma for the Inactivation of Bacterial Spores: A Review.

Formation of highly resistant spores is a concern for the safety of low-acid foods as they are a perfect vehicle for food spoilage and/or human infection. For spore inactivation, the strategy usually applied in the food industry is the intensification of traditional preservation methods to sterilization levels, which is often accompanied by decreases of nutritional and sensory properties. In order to overcome these unwanted side effects in food products, novel and emerging sterilization technologies are being developed, such as pressure-assisted thermal sterilization, high-pressure carbon dioxide, high-pressure homogenization, and cold plasma. In this review, the application of these emergent technologies is discussed, in order to understand the effects on bacterial spores and their inactivation and thus ensure food safety of low-acid foods. In general, the application of these novel technologies for inactivating spores is showing promising results. However, it is important to note that each technique has specific features that can be more suitable for a particular type of product. Thus, the most appropriate sterilization method for each product (and target microorganisms) should be assessed and carefully selected.

Enhancing the authenticity of animal by-products: harmonization of DNA extraction methods from novel ingredients.

Introduction: The increasing global pressure to explore alternative protein sources derived from animal by-products has opened-up opportunities, but it has also created the need to assess their compliance with labelling statements, to ensure consumer's trust in the composition of both feed and food products. Assessing the authenticity of highly processed animal by-products, particularly within the rapidly expanding Halal food market, presents a significant challenge due to the lack of robust and standardized methodologies. However, the success of DNA based authenticity system is highly dependent on the extracted DNA quantity, quality, and purity ratios from heterogeneous matrices. Material and methods: In this work, nine DNA extraction methods were tested on selected processed animal by-products with high-value and interest for the feed industry: meals from poultry meat, blood and feather, and hydrolysates from swine meat and bone, fish, and black soldier fly. The proposed DNA extraction methods are developed to specifically target swine-specific mitochondrial region, as a case study. Results and discussion: Both the conventional CTAB method and the commercial kits, specifically Invisorb® Spin Tissue Mini and NucleoSpin™ Food, demonstrated superior extraction efficiency and quality ratios. Nevertheless, commercial kits enabled faster detection in comparison to the conventional methods. The absence of swine DNA was successfully validated and confirmed in all animal meals and hydrolysates that did not contain swine in their composition beforehand, demonstrating their compliance with the Halal market requirements.

The Combined Effect of Pressure and Temperature on Kefir Production-A Case Study of Food Fermentation in Unconventional Conditions.

Food fermentation under pressure has been studied in recent years as a way to produce foods with novel properties. The purpose of this work was to study kefir production under pressure (7-50 MPa) at different temperatures (17-32 °C), as a case study of unconventional food fermentation. The fermentation time to produce kefir was similar at all temperatures (17, 25, and 32 °C) up to 15 MPa, compared to atmospheric pressure. At 50 MPa, the fermentation rate was slower, but the difference was reduced as temperature increased. During fermentation, lactic and acetic acid concentration increased while citric acid decreased. The positive activation volumes (Va) obtained indicate that pressure decreased the fermentation rate, while the temperature rise led to the attenuation of the pressure effect (lower Va). On the other hand, higher activation energies (Ea) were observed with pressure increase, indicating that fermentation became more sensitive to temperature. The condition that resulted in a faster fermentation, higher titratable acidity, and higher concentration of lactic acid was 15 MPa/32 °C. As the authors are aware, this is the second work in the literature to study the combined effect of pressure and temperature on a fermentative process.

Effect of High Pressure on Paracoccus denitrificans Growth and Polyhydroxyalkanoates Production from Glycerol.

The performance of fermentation under non-conventional conditions, such as high pressure (HP), is a strategy currently tested for different fermentation processes. In the present work, the purpose was to apply HP (10-50 MPa) to fermentation by Paracoccus denitrificans, a microorganism able to produce polyhydroxyalkanoates (PHA) from glycerol. In general, cell growth and glycerol consumption were both reduced by HP application, more extensively at higher pressure levels, such as 35 or 50 MPa. PHA production and composition was highly dependent on the pressure applied. HP was found to decrease polymer titers, but increase the PHA content in cell dry mass (%), indicating higher ability to accumulate these polymers in the cells. In addition, some levels of HP affected PHA monomeric composition, with the polymer produced at 10 and 35 MPa showing considerable differences relative to the ones obtained at atmospheric pressure. Therefore, it is possible to foresee that the changes in polymer composition may also affect its physical and mechanical properties. Overall, the results of this study demonstrated that HP technology (at specific levels) can be applied to P. denitrificans fermentations without compromising the ability to produce PHA, with potentially interesting effects on polymer composition.

Combined effect of pressure and temperature for yogurt production.

Fermentation under non-conventional conditions has gained prominence in the last years, due to the possible process improvements. Fermentation under sub-lethal pressures is one of such cases, and may bring novel characteristics and features to fermentative processes and products. In this work, the effect of both pressure (10-100 MPa) and temperature (25-50 °C) on yogurt production fermentation kinetics was studied, as a case-study. Product formation and substrate consumption were evaluated over fermentation time and the profiles were highly dependent on the fermentation conditions used. For instance, the increase of pressure slowed down yogurt fermentation, but fermentative profiles similar to atmospheric pressure (0.1 MPa) were obtained at 10 MPa at almost all temperatures tested. Regarding temperature, higher fermentative rates were achieved at 43 °C for all pressures tested. Moreover, the inhibitory effect of pressure increased when temperature decreased, with complete inhibition of fermentation occurring at 50 MPa for 25-35 °C, contrasting to 43 °C where inhibition occurred only at 100 MPa. Therefore, an antagonistic effect seems to occur, since yogurt fermentation was slowed down by pressure increasing, on one hand, and by temperature decreasing, on the other hand. Additionally, some kinetic parameters were calculated and fermentation at 43 °C presented the best results for yogurt production, with lower fermentation times and higher lactic acid productivities. Interestingly, fermentation at 10 MPa/43 °C presented the optimal conditions, with improved yield and lactic acid production efficiency, when compared to fermentation at 0.1 MPa (efficiency of 75% at 10 MPa, against 40% at 0.1 MPa). As the authors are aware, this work gives the first insights about the simultaneous effect of pressure and temperature variation on a microbial fermentation process, which can be combined to modulate the metabolic activity of microorganisms during fermentation in order to improve the fermentative yields and productivities of the desired product.

Adaptation of Saccharomyces cerevisiae to high pressure (15, 25 and 35 MPa) to enhance the production of bioethanol.

Saccharomyces cerevisiae is a yeast of great importance in many industries and it has been frequently used to produce food products and beverages. More recently, other uses have also been described for this microorganism, such as the production of bioethanol, as a clean, renewable and sustainable alternative fuel. High pressure processing (HPP) is a technology that has attracted a lot of interest and is increasingly being used in the food industry as a non-thermal method of food processing. However, other applications of high pressure (HP) are being studied with this technology in different areas, for example, for fermentation processes, because microbial cells can resist to pressure sub-lethal levels, due to the development of different adaptation mechanisms. The present work intended to study the adaptation of S. cerevisiae to high pressure, using consecutive cycles of fermentation under pressure (at sub-lethal levels), in an attempt to enhance the production of bioethanol. In this context, three pressure levels (15, 25 and 35 MPa) were tested, with each of them showing different effects on S. cerevisiae fermentation behavior. After each cycle at 15 and 25 MPa, both cell growth and ethanol production showed a tendency to increase, suggesting the adaptation of S. cerevisiae to these pressure levels. In fact, at the end of the 4th cycle, the ethanol production was higher under pressure than at atmospheric pressure (0.1 MPa) (8.75 g.L-1 and 10.69 g.L-1 at 15 and 25 MPa, respectively, compared to 8.02 g.L-1 at atmospheric pressure). However, when the pressure was increased to 35 MPa, cell growth and bioethanol production decreased, with minimal production after the 4 consecutive fermentation cycles. In general, the results of this work suggest that consecutive cycles of fermentation under sub-lethal pressure conditions (15 and 25 MPa) can stimulate adaptation to pressure and improve the bioethanol production capacity by S. cerevisiae; hence, this technology can be used to increase rates, yields and productivities of alcoholic fermentation.

Lactobacillus reuteri growth and fermentation under high pressure towards the production of 1,3-propanediol.

Lactobacillus reuteri is a lactic acid bacterium able to produce several relevant bio-based compounds, including 1,3-propanediol (1,3-PDO), a compound used in food industry for a wide range of purposes. The performance of fermentations under high pressure (HP) is a novel strategy for stimulation of microbial growth and possible improvement of fermentation processes. Therefore, the present work intended to evaluate the effects of HP (10-35 MPa) on L. reuteri growth and glycerol/glucose co-fermentation, particularly on 1,3-PDO production. Two different types of samples were used: with or without acetate added in the culture medium. The production of 1,3-PDO was stimulated at 10 MPa, resulting in enhanced final titers, yields and productivities, compared to 0.1 MPa. The highest 1,3-PDO titer (4.21 g L-1) was obtained in the presence of acetate at 10 MPa, representing yield and productivity improvements of ≈ 11 and 12%, respectively, relatively to the same samples at 0.1 MPa. In the absence of acetate, 1,3-PDO titer and productivity were similar to 0.1 MPa, but the yield increased ≈ 26%. High pressure also affected the formation of by-products (lactate, acetate and ethanol) and, as a consequence, higher molar ratios 1,3-PDO:by-products were achieved at 10 MPa, regardless of the presence/absence of acetate. This indicates a metabolic shift, with modification of product selectivity towards production of 1,3-PDO. Overall, this work suggests that HP can be a useful tool to improve of 1,3-PDO production from glycerol by L. reuteri, even if proper process optimization and scale-up are still needed to allow its industrial application. It also opens the possibility of using this technology to stimulate other glycerol fermentations processes that are relevant for food science and biotechnology.

Hyperbaric storage of melon juice at and above room temperature and comparison with storage at atmospheric pressure and refrigeration.

Hyperbaric storage (8h) of melon juice (a highly perishable food) at 25, 30 and 37°C, under pressure at 25-150 MPa was compared with atmospheric pressure storage (0.1 MPa) at the same temperatures and under refrigeration (4°C). Comparatively to the refrigerated condition, hyperbaric storage at 50/75 MPa resulted in similar or lower microbial counts (total aerobic mesophiles, enterobacteriaceae, and yeasts/moulds) while at 100/150 MPa, the counts were lower for all the tested temperatures, indicating in the latter case, in addition to microbial growth inhibition, a microbial inactivation effect. At 25 MPa no microbial inhibition was observed. Physicochemical parameters of all samples stored under pressure (pH, titratable acidity, total soluble solids, browning degree and cloudiness) did not show a clear variation trend with pressure, being the results globally similar to refrigeration storage. These results show the potential of hyperbaric storage, at and above room temperature and with potential energy savings, comparatively to refrigeration.

Microorganisms under high pressure--adaptation, growth and biotechnological potential.

Hydrostatic pressure is a well-known physical parameter which is now considered an important variable of life, since organisms have the ability to adapt to pressure changes, by the development of resistance against this variable. In the past decades a huge interest in high hydrostatic pressure (HHP) technology is increasingly emerging among food and biosciences researchers. Microbial specific stress responses to HHP are currently being investigated, through the evaluation of pressure effects on biomolecules, cell structure, metabolic behavior, growth and viability. The knowledge development in this field allows a better comprehension of pressure resistance mechanisms acquired at sub-lethal pressures. In addition, new applications of HHP could arise from these studies, particularly in what concerns to biotechnology. For instance, the modulation of microbial metabolic pathways, as a response to different pressure conditions, may lead to the production of novel compounds with potential biotechnological and industrial applications. Considering pressure as an extreme life condition, this review intends to present the main findings so far reported in the scientific literature, focusing on microorganisms with the ability to withstand and to grow in high pressure conditions, whether they have innated or acquired resistance, and show the potential of the application of HHP technology for microbial biotechnology.