Fungal Biostarter Effect on the Quality of Dry-Aged Beef

**Background:** Dry-aging of beef enhances tenderness and flavor but is costly and variable, with risks of pathogenic microbial growth. Fungal biostarters, such as *Mucor flavus*, have been proposed to stabilize the process and improve product quality. This study aimed to evaluate the effect of a *M. flavus* strain (KKP 2092p) on the physicochemical characteristics, sensory quality, and bacterial microbiome of dry-aged beef.

**Methods:** Meat samples (entrecote from *Longissimus* muscle, ~3.5 ± 0.5 kg each) were obtained from 14 crossbred cattle. Seven pieces were inoculated with a *M. flavus* suspension (10 mL/kg; biostarter+ group) and seven with Sabouraud broth only (biostarter− control). All samples were dry-aged in a controlled cabinet (DX 1000 Premium S, DryAger) at 1.5 °C, 80–90% relative humidity, for 28 days. Weight loss was measured at 7, 14, 21, and 28 days. pH, color (CIE L*a*b*), water-holding capacity (Grau–Hamm method), shear force (Warner–Bratzler), malondialdehyde content (TBA method), and myofibrillar protein profiles (SDS-PAGE) were assessed. Sensory evaluation was conducted by 40 consumers using a 9-point hedonic scale for raw aroma, grilled aroma, softness, juiciness, flavor, and overall liking. Bacterial communities were characterized via 16S rDNA amplicon sequencing (V3–V4 region) on an Illumina MiSeq platform. Statistical analysis used Student's t-test and ANOVA (p < 0.05).

**Key Results:** Before aging, the two groups did not differ significantly in any quality trait. Weight loss increased significantly over time (7.7%, 13.8%, 19.4%, and 21.4% at 7, 14, 21, and 28 days, respectively), with no significant difference between groups. After 28 days, the biostarter+ group showed a significantly higher pH (5.84 vs. 5.75 in controls; p < 0.05), with an increase of 0.24 units from baseline compared to 0.14 units in controls. Shear force was numerically lower in the biostarter+ group (65.91 N vs. 75.35 N; not significant). Malondialdehyde content was lower in biostarter+ samples (0.52 mg/kg vs. 0.98 mg/kg; not significant). SDS-PAGE revealed significantly lower amounts of myosin light chain LC2 in biostarter+ samples (p < 0.05), indicating greater proteolysis. Sensory evaluation showed significantly higher scores for grilled aroma, softness, juiciness, and overall acceptability in biostarter+ steaks (p < 0.05). Microbiome analysis (37 ASVs from 410,178 quality-filtered sequences) showed higher bacterial species richness in control samples (Chao1: 14–17 vs. 10; Shannon: 0.93–2.25 vs. 1.21–1.23). PCoA based on Bray–Curtis dissimilarity indicated that biostarter+ samples formed a more homogeneous bacterial community, dominated by *Pseudomonas* sp., while controls were more variable and contained *Brochothrix* sp. No food-borne pathogens (*Bacillus cereus*, *Staphylococcus aureus*, *Listeria monocytogenes*, *Escherichia coli*) were detected in any sample.

**Clinical Implications:** Although this is a food science study rather than a clinical trial, the findings have implications for food safety and quality in the meat industry. The use of *M. flavus* as a biostarter can standardize the dry-aging process, improve sensory attributes (particularly tenderness and juiciness), and promote a stable bacterial community without pathogens. This could reduce product variability and spoilage risk, potentially making high-quality dry-aged beef more accessible. However, the authors note that the data are limited and further investigation is needed before commercial application.