Lentini, Giulia (2025) Development of hydrogel-based minibioreactors for the formulation of functional foods. [Tesi di dottorato]

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Abstract

Nutraceuticals and functional foods that prevent diseases by providing beneficial effects on human health are increasingly reaching the interest of consumers and industries that are nowadays more attentive to food quality and health issues. These ingredients include probiotics, prebiotics, and bioactive compounds capable of providing specific benefits. Fermentation is among the most popular and practical techniques for obtaining functionalized ingredients that, following a step of inactivation of microorganisms, give rise to postbiotics. Researchers are looking for innovative strategies to get increasingly efficient processes and products with a boosted effect; microbial immobilization through encapsulation is an effective method for achieving this goal. This research, in collaboration with Kraft Heinz Company, aims to develop an innovative and efficient fermentation process using minibioreactors also obtaining a dry postbiotic with the potential to revolutionize multiple fields of application. The Company supplied and patented the probiotic microorganism a Lacticaseibacillus paracasei CBAL74 and a suspension of hydrolyzed oats at 15% oat flour was chosen as both the encapsulating material and the fermentative substrate, thanks to its natural composition. Three different encapsulation techniques were tested: freeze-drying, emulsion, and extrusion, but following a microscopic study extrusion was chosen as the technique, which allowed obtaining capsules of dimensions (millimetric) capable of ensuring bacterial growth. Once the technique was established, a preliminary study of formulation and fermentation of minibioreactors made by capsules appropriately suspended in a 1:3 ratio in an external liquid composed by the same suspension of hydrolyzed oats, was conducted. The requirements investigated for the capsules were twofold: confinement of bacteria inside them, separating the bacteria from the metabolites naturally and understanding whether they could act as efficient fermenters. The preliminary fermentation study found that impermeability, when achieved, is not associated with microbial growth during fermentation. Therefore, the capsules did not act as minibioreactors; for this reason, the 1% alginate - 1 M CaCl2 formulation was chosen as the best formulation, which allowed to obtain the maximum bacterial growth (capsules: 5.50 x 109 CFU/mL, external liquid: 7.40 x 106 CFU/mL) and lactic acid production (capsules: 15.90 mg/mL; external liquid: 10.00 mg/mL). Then, a suitable fermentation protocol at laboratory scale (1L) of the innovative minibioreactor fermentation system was developed and compared with the traditional free-cell fermentation technique. Before proceeding with this phase of the study, the possibility of using frozen capsules to reduce the processing time related to production was investigated. Frozen capsules, stored and thawed before use, were found suitable and performed, further implementing the process. The process scale-up (1L) showed that fermentation with this innovative minibioreactor system improved bacterial growth and doubled the lactic acid production compared to free-cell fermentation (capsules: 3.3 x 109 CFU/mL and 20.5 mg/mL; free-cell system: 4.7 x 108 CFU/mL and 9.0 mg/mL). The glucose consumption of the entire innovative minibioreactor process was found to be lower, allowing significantly improved yields in terms of cell yield and lactic acid yield compared to free-cell processes (minibioreactor system: 0.40 mg cells/mg glucose and 3.18 mg lactic acid/ mg glucose; free-cell system: 0.076 mg cells/mg glucose, and 1.40 mg lactic acid/ mg glucose). The study of kinetic parameters also confirmed that the minibioreactors, compared to the free-cell process, showed reduced doubling times (td) and a higher number of generations (n), constant growth rate (k), and constant specific growth rate (µ), suggesting promoted bacterial growth. After conducting the study at a lab scale (1L), it was scaled up to 30 L for the minibioreactor's innovative system. Notably, no variation in bacterial growth, lactic acid content, glucose consumption, and process yields in terms of cell and lactic acid during the scale-up was observed, confirming the potential applicability of the process at an industrial scale. The innovative system, consisting of capsules and the external liquid, has been deeply characterized by different perspectives, revealing significant insights into probiotic encapsulation. The morphology of the capsules was investigated by confocal microscopy using oat autofluorescence, which highlighted a compact but non-homogeneous structure given by the interaction between the hydrolyzed oat suspension and the alginate. Confocal microscopy was also used to study the physiological state of the bacteria during fermentation in the capsules and in the external liquid by staining them with appropriate fluorochromes. The results were in line with those obtained by the spread plate method. This highlights that after 24 hours of fermentation, clusters of dead bacteria were found in the capsules' core and the external liquid. Based on the diffusive phenomena found during the fermentation process from the capsules to the external liquid, an approximate estimate of the diffusion coefficient showed that D was equal to 7.2 x 10-11 m2/s. A study validating the capsules as a protector of probiotics during the digestive process was investigated, confirming that the capsules had almost maximum viability at the end of digestion, compared to a 5-log reduction observed for non-encapsulated bacteria. The focus was not only on the fermentation process but on obtaining a dry enhanced postbiotic product, in this regard, once fermented, both the capsules and the external liquid were subjected to a microbial inactivation process and subsequent drying process. The heat treatment to inactivate the bacterial load in the oatmeal suspension (85°C - 1 min) was not sufficient to kill the encapsulated microorganisms. The capsules showed lower thermal conductivity and more rigorous and innovative treatments were needed that did not affect the product's chemical properties. A microwave process was conducted by setting the microwave power at 800 W (transferred to the samples 238 W) for a total treatment duration of 190 sec composed of 40 s microwave treatment and a simulated holding phase of 10. The capsules and the external suspensions were separated for the drying step: the capsules were dried at 37°C for 4 h. It was necessary to optimize the spray drying process parameters and the formulation for the external liquid postbiotic. A recovery yield of 57.40 ± 0.57% was obtained, with a residual moisture of 3.90 ± 0.14%, using the following conditions: sample dilution equal to 1:4; maltodextrin addition 1:1 concerning the initial solid content; Qfeed = 5 [mL/min]; Qair-IN = 50 [L/min]; Patomization = 3.2 [bar]; Tair-IN = 200 °C. Once the postbiotics were obtained, a functional characterization was performed to understand the potential application field of the products. An evaluation of the antioxidant properties revealed that the postbiotic capsules and the external liquid had the same characteristics as the samples corresponding to time zero of the fermentation, demonstrating that the probiotic bacterium used was not able to increase the antioxidant compounds; the comparison was also performed with the postbiotic free cells used as a control confirming that the process did not bring any improvement. Finally, the inflammatory capacity of the postbiotic capsules, the external liquid, and the free cells used as a control, both digested and undigested, was tested to understand the effect of the digestive process on the anti-inflammatory characteristics. All the tested samples have anti-inflammatory capacity, except for the undigested postbiotic free cells; digestion did not negatively affect the regulation of the biomarker level, and, indeed, for Myd88 and NF-kB, the potential of some samples is amplified.

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