*2.1. Nisin–Biogel Preparation*

Nisin–biogel was prepared as described elsewhere [10,11,13,14]. According to previous studies, a final concentration of 200 μg/mL was selected to be used in the clinical trial [10–13]. After preparation, the nisin–biogel was stored at 4 ◦C until further use.

#### *2.2. Dog Selection*

Animals were selected according to the Veterinary Oral Health Council (VOHC) guidelines for trials testing compounds for PD prevention. The dogs were from an official animal rescue institution ("Casa dos Animais de Lisboa"), and all experimental procedures were approved by the Ethical Committee for Research and Teaching (CEIE) of the Faculty of Veterinary Medicine, University of Lisbon, Portugal (N/Ref 014/2020).

The inclusion criteria were as follows: healthy dogs over 2 years old without severe PD and with no history of antimicrobial therapy in the last month. All animals were submitted to a clinical examination, oral handling, and complete blood analysis (hemogram and measurement of urea, creatinine, alanine aminotransferase, alkaline phosphatase, glucose, albumin, and total blood proteins) to detect any deviations that would prevent their inclusion in the study.

#### *2.3. Clinical Trial*

A total of twenty dogs were selected and submitted to a complete periodontal evaluation, dental plaque sampling (for bacterial total counts), scaling, and dental polishing (timepoint 0). The complete removal of dental plaque and calculus was checked using a disclosure solution (GC Tri Plaque ID Gel®, Tokyo, Japan). All procedures were performed under general anesthesia using acepromazine (0.01 mg/kg, IM), propofol (2 mg/kg, IV), and isoflurane. Intraoperative meloxicam (0.2 mg/Kg, SC) and amoxicillin/clavulanate (8.75 mg/kg, SC) were administered to all animals [15]. After that, each dog was randomly allocated to one of two groups: a treatment group (TG, N = 10) or a control group (CG, N = 10). Animals in the treatment group were submitted to a topical dental application of the nisin–biogel (200 μg/mL) every 48 h, as mentioned in Table 1. The control group was composed of dogs that were not submitted to any treatment. Animals were kept in the trial for 90 days. At the end of the clinical trial (T90), all animals were subjected to a new

complete blood analysis, complete periodontal evaluation, dental plaque sampling, scaling, and dental polishing.

**Table 1.** Posology of the nisin–biogel (200 μg/mL) according to the animal's weight.


In addition, two intermediate dental plaque evaluations of all animals were performed on days 30 and 60. Intermediate evaluations included an awake dental plaque evaluation using a disclosure solution (GC Tri Plaque ID Gel®, Tokyo, Japan) that dyed the dental plaque according to its accumulation. Then, a photographic register of the vestibular margins was performed on all animals. After that, the dental plaque coverage of 4 teeth (2 canines and 2 premolars) of each dog was evaluated using the IMAGEJ® program. The percentage of dental plaque coverage was determined after measuring the total tooth vestibular margin and the area of the dyed dental plaque.

Animals in both groups were fed the same dry food and housed in the same building. All animals were observed daily to detect any general side effects, such as changes in feeding habits and behavior, prostration, vomiting, or diarrhea.
