Chlamydomonas reinhardtii is a nonpathogenic, nontoxigenic green algae used as a sustainable source of protein in foods. In order to mimic meat-like qualities, a strain rich in protoporphyrin IX (PPIX), an endogenous heme/chlorophyll precursor, was developed using an evolution and selection strategy, and investigations were carried out to evaluate the safety of the novel strain, C. reinhardtii (red), strain TAI114 (TAI114). Digestibility and proteomic evaluations were conducted to determine whether any potentially allergenic or toxic proteins occurred as the result of the mutation process. The genotoxic potential of pure PPIX was evaluated using a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test. Finally, the novel TAI114 biomass was evaluated for general toxicity and identification of target organs in a 90-day repeateddose oral toxicity study in rats. All proteins were rapidly degraded in pepsin at pH 2.0 suggesting low allergenic potential. The proteomic evaluation indicated that TAI114 biomass contains typical C. reinhardtii proteins. PPIX was unequivocally negative for genotoxic potential and no target organs or adverse effects were observed in rats up to the maximum feasible dose of 4000 mg/kg bw/day TAI114 biomass, which was determined to be the no-observed-adverse-effect-level (NOAEL). These results support the further development and risk characterization of TAI114 biomass as a novel ingredient for use in the meat analogue category of food.

There is an economic interest, both for food security and for the non‐meat‐eating population, in the development of novel, sustainable sources of high‐quality protein. The green algae Chlamydomonas reinhardtii has already been developed for this purpose, and the closely related species, Chlamydomonas debaryana, is a complementary source that also presents some additional advantages, such as reduced production cost. To determine whether C. debaryana may have a similar safety profile to that of C. reinhardtii, a wild type strain was obtained, designated TS04 after confirmation of its identity, and subjected to a battery of preclinical studies. Genetic toxicity was evaluated using a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test in a mouse model. No genotoxic potential (e.g., mutagenicity and clastogenicity) was observed in these tests under the employed conditions up to maximum recommended concentrations or doses. To assess general toxicity, a 90‐day repeated‐dose oral toxicity study was conducted in rats. No mortality or adverse effects were observed, and no target organs were identified up to the maximum feasible dose, due to solubility, of 4,000 mg/kg bw/day. The no‐observed‐adverse‐effect level was determined as the highest dose tested. A digestibility study in simulated gastric fluid was conducted and determined that TS04 has low allergenic potential, exhibiting rapid digestion of proteins. Due to the negative results of our evaluation, it is reasonable to proceed with further development and additional investigations to contribute towards a safety assessment of the proposed use in food for human consumption.

Breast feeding has critical effects on the newborns and either mother's health. Some of such healthimproving effects of the mother's milk is associated with the beneficial microbes, lactic acid bacteria (LAB), which are normally present in the mother's milk. Thus, human milk-associated lactobacilli were isolated in this study and some of their probiotic properties was investigated. Afterwards, Lactobacillus strains were screened for low pH and bile acids tolerance. Molecular identification was carried out using 16SrDNA and polymerase chain reaction (PCR). Antibiotic resistance was evaluated with disk diffusion assay and the inhibitory effect of isolates on pathogenic bacteria was examined with well assay and zone inhibition. Isolation experiments resulted in 122 human milk-associated lactobacilli belonging to 12 species. The most dominant species was Lactobacillus casei followed by Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus gasser, respectively. Screening for probiotic properties showed that 19 isolates, belonging to, Lactobacillus, have interesting probiotic characteristics. The most prevalent antibiotic resistance was observed in case of vancomycin (63.15%) and no drug resistance was detected for chloramphenicol, penicillin, rifampin (0%). Three Lactobacillus strains, designated as L4, L14 and L16, were found as potential probiotic strains since they have indicated promising inhibitory effects against the studied pathogenic bacterial strains. Our results shed light on the considerable diversity of lactobacilli in human breast milk. Furthermore, the candidate probiotic strains detected in this research might be used as potential probiotic strains.