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Research Background: Increasing awareness of the importance of nutrition in health promotion and disease prevention has driven to the development of foods for special medical purposes (FSMPs). In this study, the probiotic strain Lacticaseibacillus paracasei ssp. paracasei ( Lacticaseibacillus casei 431 ® ) was incorporated into FSMPs to develop an innovative product. The aim was to investigate the influence of the FSMP matrix on the specific probiotic properties of L. casei 431 ® in vitro ., Experimental Approach: A series of in vitro experiments were performed as part of the probiotic approach. After evaluation of antibiotic susceptibility profiles, functional properties such as survival under simulated gastrointestinal tract (GIT) conditions, bile salt deconjugation activities, cholesterol assimilation, antagonistic activity against spoilage bacteria and adhesion to Caco-2 cell line monolayers and extracellular matrix proteins were investigated., Results and Conclusions: The L. casei 431 ® strain, both the lyophilised strain and the strain isolated from the FSMP matrix, effectively survived the simulated adverse gastrointestinal conditions without significant effects of the food matrix. The effect of the FSMP matrix on the deconjugation activity of the bile salts of L. casei 431 ® was minimal; however, cholesterol assimilation was increased by 16.4 %. L. casei 431 ® had antibacterial activity against related lactic acid bacteria regardless of whether it was used in FSMPs or not. Conversely, the probiotic strain isolated from FSMP matrix had significantly higher inhibitory activity against six potential pathogens than the lyophilised culture. The autoaggregation ability of the L. casei 431 ® cells was not affected by the FSMP matrix. The adhesion of L. casei 431 ® bacterial cells to the extracellular matrix proteins was reduced after treatment with proteinase K, with the highest adhesion observed to laminin. The adhesion of L. casei 431 ® reduced the binding of E. coli 3014 by 1.81 log units and the binding of S . Typhimurium FP1 to Caco-2 cell lines by 1.85 log units, suggesting the potential for competitive exclusion of these pathogens., Novelty and Scientific Contribution: The results support the positive effect of the FSMP matrix on the specific probiotic properties of L. casei 431 ® , such as antibacterial activity, bile salt deconjugation and cholesterol assimilation, while the incorporation of this probiotic strain adds functional value to the FSMPs. The synergistic effect achieved by the joint application of L. casei 431 ® and innovative FSMP matrix contributed to the development of the novel formulation of an improved functional food product with added value., Competing Interests: CONFLICT OF INTEREST The authors declare no conflict of interest., (Authors.)

Natural tetracycline (TC) antibiotics were the first major class of therapeutics to earn the distinction of 'broad-spectrum antibiotics' and they have been used since the 1940s against a wide range of both Gram-positive and Gram-negative pathogens, mycoplasmas, intracellular chlamydiae, rickettsiae and protozoan parasites. The second generation of semisynthetic tetracyclines, such as minocycline and doxycycline, with improved antimicrobial potency, were introduced during the 1960s. Despite emerging resistance to TCs erupting during the 1980s, it was not until 2006, more than four decades later, that a third--generation TC, named tigecycline, was launched. In addition, two TC analogues, omadacycline and eravacycline, developed via (semi)synthetic and fully synthetic routes, respectively, are at present under clinical evaluation. Interestingly, despite very productive early work on the isolation of a Streptomyces aureofaciens mutant strain that produced 6-demethyl-7-chlortetracycline, the key intermediate in the production of second- and third-generation TCs, biosynthetic approaches in TC development have not been productive for more than 50 years. Relatively slow and tedious molecular biology approaches for the genetic manipulation of TC-producing actinobacteria, as well as an insufficient understanding of the enzymatic mechanisms involved in TC biosynthesis have significantly contributed to the low success of such biosynthetic engineering efforts. However, new opportunities in TC drug development have arisen thanks to a significant progress in the development of affordable and versatile biosynthetic engineering and synthetic biology approaches, and, importantly, to a much deeper understanding of TC biosynthesis, mostly gained over the last two decades.

Some of the most frequently used antibiotics in apiculture for the treatment of bacterial brood diseases are oxytetracycline, chloramphenicol, sulphathiazole and streptomycin. Therefore, the aim of this research is to determine the degradation kinetics of the residua these antibiotics leave behind in different types of honey stored in dark at 25 °C. The examined honey samples ( N =74) originating from the Croatian market had satisfactory physicochemical properties and pollen characteristics. Quantitative analyses of antibiotic residues were done using HPLC and ELISA methods. No antibiotic residues were found in any of the tested acacia, chestnut, floral, meadow and honeydew honey samples ( N =74). In the next step, each of the tested honey samples was supplemented with one of the following antibiotics (in mg/kg): oxytetracycline 10, chloramphenicol 200, sulphathiazole 200 or streptomycin 0.5. Relatively high mass fractions of antibiotics have been added to the honey because the stability of each antibiotic is highly dependent on the spiked antibiotic mass fraction and also on its chemical structure. During a 6-month storage in the dark at 25 °C, the dynamics of degradation of the studied antibiotics was proven to differ dependent on the type of honey into which the antibiotic was added. The half-life of oxytetracycline in the acacia, floral, meadow and honeydew honey stored in the dark at 25 °C was 15, 16, 17 and 19 days, respectively, while in the chestnut honey the decomposition failed to be seen even after 60 days of storage. In all examined honey samples, the half-life of chloramphenicol and sulphathiazole was proven to be longer than 6 months. The fastest decomposition was seen of oxytetracycline added into the acacia honey, followed by streptomycin and sulphathiazole added into the same, while the longest decomposition delay was seen when chloramphenicol was added into the floral honey. According to the results obtained using a linear model of degradation kinetics, the longest oxytetracycline degradation was expected to occur in chestnut honey (116 days). Chloramphenicol and sulphathiazole are preserved the longest in floral (for 661 and 581 days, respectively) and streptomycin in meadow honey (for 321 days).