Ras cheese, a type of hard cheese, is widely recognized and holds considerable importance in Egypt and various
other regions. This cheese is often subjected to high-humidity storage conditions, fostering the proliferation of
fungi and yeasts that can impact its quality. This study aims to examine the ability of various Lactobacillus strains
as protective adjunct cultures against fungal growth, and to determine the mycotoxin production and enzymatic
activities of isolated fungi found in Ras cheese. The experimental design comprised several treatments: Control
was made using Lactobacillus bulgaricus and Streptococcus thermophilus; T1, T2 and T3 involved control +
Lactobacillus acidophilus, Lactobacillus helveticus and Lacticaseibacillus casei, respectively. On Czapek dextrose agar
medium, control exhibited the highest fungal count at fresh status (525 ± 46 CFU/g), whereas T3 displayed the
lowest value (277 ± 60 CFU/g). T2 with Lb. helveticus showed the lowest total fungal count (302 ± 79 CFU/g)
and had higher antifungal activity in stored cheese than Lb. acidophilus (344 ± 53 CFU/g) and Lb. casei (323 ±
73 CFU/g) at 90-day ripening. T1 yielded the highest count in fresh samples (363 ± 66 CFU/g), while T3 presented the lowest count after 90-days (243 ± 59 CFU/g) on PDA medium. Moreover, Aspergillus flavus and
Aspergillus niger were the predominant fungi in control after 90-days, with reduced occurrences in probiotic treated cheeses. The absence of mycotoxins was verified in Ras cheese; however, isolates of A. flavus showed
the ability to generate aflatoxin B1 and G1, whereas isolates of A. niger could produce ochratoxin A. Both
A. flavus and A. niger isolates exhibited the potential to produce lipases and proteases. The introduction of
probiotics into Ras cheese may serve as an essential environmentally friendly approach to mitigate fungal
presence and diminish the production of undesirable enzymes that compromise the quality and safety of hard
cheeses.