Semi-moist pet foods belong to the category of minimally processed diets and are characterized by their high moisture content of 15 to 40% and water activity of 0.60 to 0.85 (Rolinec et al., 2016; Kiprotich et al., 2021; Deliephan, 2022). Semi-moist pet foods have increased in popularity globally due to their quality and sensory attributes, which are similar to those of fresh diets. They are edible without hydration and have a prolonged shelf life (Qiu et al., 2019). Semi-moist pet foods are a diverse category of diets that are mostly classified according to the macro ingredients used to manufacture the base rations, such as cereals, meats, or dairy products. These foods are often given to companion animals as treats for training and reinforcement of positive behavior.

Commercially, semi-moist pet foods such as chewy dog treats are produced through partial drying or osmotic drying using humectants. Diets are also formulated with humectants to enhance shelf life (Deliephan, 2022). The desired water activity in semi-moist or intermediate-moisture pet food is achieved through the application of salt, sugar, glycerol, or propylene glycol, which immobilize and bind free water, which is necessary for the formation of these diets (Fellows, 2009). Upon formulation, the ingredients are assembled, and the food is manufactured through baking, cooking, extrusion, or thermal dehydration (Fellows, 2009).

Semi-moist pet foods are attractive to many pet owners because they are ready to eat (RTE), highly nutritious, palatable, and microbially safe (Severini et al., 2008). However, semi-moist pet foods are susceptible to mold growth and proliferation due to their high moisture content and water activity, which are favorable to the survival of spoilage and toxigenic fungi (Deliephan et al., 2023). The proliferation of fungi in semi-moist pet foods causes spoilage, which leads to economic losses for pet food manufacturers who must carefully dispose of contaminated products. Moreover, some fungal species in genera like Aspergillus, Penicillium, Fusarium, and Alternaria biosynthesize toxic secondary metabolites, mycotoxins (Deliephan, 2022). Examples of mycotoxins include, but are not limited to, ochratoxins, deoxynivalenol, zearalenone, with aflatoxins being the most important toxins in the semi-moist pet food segment (Pitt and Hocking, 2009). Ingestion of mycotoxins by humans and animals causes mycotoxicosis, which might present as acute or chronic cytotoxicity, hepatotoxicity, mutagenicity, or carcinogenicity (Janik et al., 2020). Restuccia et al. (2019) reported that pets experiencing aflatoxin poisoning exhibited symptoms such as sluggishness, loss of appetite, vomiting, jaundice, or diarrhea. Therefore, it is crucial that the microbial safety of semi-moist pet food is adequate, and the ensuing products are safe for animal consumption.

Thus, to enhance the microbial safety of semi-moist pet foods, moisture and water activity must be strictly controlled using humectants like propylene glycol, glycerol, or sugar (Zicker, 2008). This is because the final moisture content in these foods’ ranges from 25 to 35%, which provides sufficient “active water” required for mold proliferation (Zicker, 2008; Kiprotich et al., 2021). To control mold proliferation, antimycotic agents such as potassium sorbate, calcium propionate, and organic acids (e.g., butyric acids, parabens, etc.) are formulated into semi-moist pet foods during the manufacturing process (Kiprotich et al., 2021). However, there is an increasing demand for pet foods considered “natural” and free of synthetic food additives or preservatives, which are less desirable to pet owners who prefer their animals consume clean-label foods (Kiprotich et al., 2021).

Whey is a liquid by-product of cheese manufacturing obtained after coagulation of casein proteins (Brandelli et al., 2015). It is a cheap carbon source for lactic acid bacteria (LAB), which require nutrients for fermentation. Whey is rich in lactose, soluble proteins, lipids, and minerals essential for the growth and multiplication of LAB (Panesar et al., 2007; Koutinas et al., 2009; Prazeres et al., 2012). The fermentation of whey using LAB leads to the production of important biomolecules such as bacteriocins. These are small heat-stable peptides of bacterial origin with antimicrobial and immune functions (Cotter et al., 2005). The fermentation process also produces several metabolites with antifungal properties, such as organic acids (acetic, lactic, formic, and propionic acids), benzoates, polyphenols, diacetyl, and short-chain fatty acids (Gerez et al., 2013; Cortés-Zavaleta et al., 2014; Miao et al., 2014; Gamba et al., 2015; Luz et al., 2020; Izzo et al., 2020a,b).

Given the increasing demand for pet foods formulated without synthetic preservatives, there is a need to explore natural and sustainable antifungal food additives to control mold in semi-moist products. Recent studies by Luz et al. (2020) show that whey fermented with Lactobacillus plantarum was able to extend the shelf life of pita bread when used as a substitute for calcium propionate. Preliminary studies by Kiprotich et al. (2021) reported an extension in shelf life and inactivation of Aspergillus flavus inoculated in semi-moist pet food treated with whey permeate fermentate (WPF) as a substitute for potassium sorbate. However, preliminary studies have demonstrated that higher doses of WPF were required to completely inactivate the A. flavus, but this caused significant negative changes in product quality and palatability. Thus, there was a need to potentiate the antifungal properties of WPF.

Citrus extracts (CEX) demonstrated limited antifungal activity when used alone to control A. flavus in semi-moist pet food, but preliminary studies showed some potentiation effect when combined with WPF. The hypothesis of this study was that WPF when used in combination with CEX would result in an additive or potentiation effect and thus would reduce the dose of WPF required to inhibit mold proliferation. To the authors’ knowledge, the antifungal properties of WPF and CEX have not been demonstrated alone, or in combination at smaller doses. Therefore, the objective of this study was to investigate the antifungal efficacy of WPF alone or in combination with CEX when challenged against Aspergillus flavus artificially inoculated in semi-moist pet food. The secondary objective was to determine the decimal reduction times (D-Values) and the effect of these antimycotic additives on the shelf life of semi-moist pet food for dogs.

The proliferation of fungi is the most limiting factor in determining the shelf life of semi-moist pet food, as they thrive in conditions of relatively high moisture and water activity. The inclusion of WPF in semi-moist pet food was able to inhibit the proliferation of Aspergillus flavus, a challenging toxigenic mold that produces carcinogenic mycotoxins such as aflatoxin B1, as the preliminary study reported by Kiprotich et al. (2021) showed.

The process of fermenting whey using LAB produces bioactive compounds such as peptides with antifungal properties. For instance, Luz et al. (2020) reported that phenolic compounds which possess antifungal properties had been isolated in a cell-free supernatant that had been manufactured by fermenting whey using a LAB strain, Lactobacillus plantarum. The phenolic compounds produced by the fermentation of whey using L. plantarum were hydroxybenzoic acid, phenyllactic acid, and lactic acid that impeded the growth of Mucor spp., Penicillium digitatum, and Bacillus subtilis (Barman et al., 2017). Dal Bello et al. (2007) demonstrated that fermentation of whey using LAB produced cyclic dipeptides (L-Leu-L-Pro and L-Phe-L-Pro) in culture media that were used to extend the shelf life of sourdough bread.

In the current study, spray dried WPF was added to the semi-moist pet food at different inclusion levels and mixed with water before thermal processing. The hypothesis was that the hydration effect of water added to the batter activated the antifungal compounds present in WPF which impeded the proliferation of A. flavus. However, the maximum amount of WPF that could be added to the formula without compromising the organoleptic properties of the semi-moist pet food was 1.0% (w/w). This was evident as inclusion levels above 1.0% exhibited superior antifungal effects but resulted in a poorly formed product. Increasing the inclusion levels of WPF from 0.25 to 1.0% resulted in increased log reduction but resulted in increased costs of production upon product formulation. Currently, there is limited data that show safety or toxicity effects of WPF or CEX on animal health, however, increasing these compounds in semi-moist pet food had adverse effects on the sensory and palatability of the product.

Citrus extracts obtained from citrus fruit peels or leaves have demonstrated strong antifungal properties against fungal strains such as P. digitatum and P. italicum (Trabelsi et al., 2016). Analysis of citrus extract obtained from peels showed that these extracts contained a wide range of flavonoids in high quantities, coumarins and volatile compounds such as phytoalexins that inhibit proliferation of pathogenic fungi (Chen et al., 2019). However, preliminary studies by Kiprotich et al. (2021), reported that CEX exhibited low antifungal activity when used as a mold inhibitor in semi-moist pet treats. Moreover, the product manufacturer reported that the maximum daily allowable daily intake of CEX is 0.15%, which failed to exhibit any antimycotic effect in semi-moist pet food. Preliminary studies also demonstrated evidence of potentiation as CEX alone exhibited limited to no antimycotic activity when used alone as a mold inhibitor but resulted in increased log reductions when it was combined with WPF, compared to when WPF was applied alone. Nonetheless, additional studies are warranted to investigate the type and quantity of antimicrobial compounds present in the WPF. The composition of CEX that was used in this study also needs to be investigated so that the synergistic antifungal compounds present in this extract responsible for potentiating WPF are understood such that the mechanisms of mycotic inhibition of these two compounds can be investigated.

D-values refer to the time (days) required for an antifungal (potassium sorbate, WPF alone, or WPF in combination with CEX) to reduce the initial population of A. flavus by 90% or 1.0 log CFU/piece. The D-values obtained in this study provide insights into the fungicidal and/or fungistatic nature of the treatments, as they estimate the amount of time required to achieve a 90% kill of the A. flavus inoculated in the semi-moist pet food.

The treatments that contained WPF at 1.0% alone or WPF at 0.5% combined with CEX at 0.15% had the most fungicidal effect, evidenced by the fewer number of days required to achieve a 1.0 log kill of the pathogenic mold. Treatments that contained 0.5% WPF alone or in combination with CEX at 0.015% were more fungistatic compared to the treatments that contained WPF at 0.25% alone or in combination with CEX at 0.015% or 0.15%. The WPF at 0.25% alone or in combination with CEX at 0.015% or 0.15% were completely overwhelmed by opportunistic spoilage mold by day 28, even though the log CFU counts of the A. flavus had reduced from the initial concentration. The PC (0.1% potassium sorbate) had a more fungistatic effect, even though the D-values observed from this treatment were high and comparable to treatments at 0.25% WPF alone or in combination with CEX at 0.015 and 0.15%. The PC was fungistatic because it did not show any signs of visible mold spoilage until day 85, compared to the latter treatments. The D-values obtained from this study followed a similar trend to that of the log reductions. For instance, the NC had the highest D-value compared to the PC and the rest of the treatments. The treatments containing 0.5% WPF when combined with 0.15 and 0.015% of CEX yielded numerically lower (p > 0.05) D-values as compared to other combination treatments as well as 0.25 and 0.5% WPF individually. This finding could be indicative of potentiation between the two treatments in inhibiting mold in semi-moist pet food. However, the D-values for NC, 0.25% WPF alone or in combination with CEX at 0.015% or 0.15%, might be misleading as these semi-moist pet foods that had been treated with these treatments were overwhelmed by opportunistic spoilage mold that may have outcompeted A. flavus. Thus, these values are not evidence of mold inhibition by these treatments. However, additional studies are warranted to study the antifungal (fungicidal and fungistatic) effects of WPF and CEX alone or in combination using microdilution to evaluate fungal conidial germination or the disc diffusion assay, which were not possible in this study because of the powdery nature of WPF that formed colloidal suspensions and the partial solubility of the CEX in water, which complicated these assays.

The shelf life experiments investigated the mold inhibitory effects of the treatments. Semi-moist pet foods are high in moisture and have a water activity that favors the growth of toxigenic and spoilage fungi. Fermentation of whey proteins produce phenolic compounds that inhibit the growth of fungi, for instance, Izzo et al. (2020a) replaced water with whey fermented using L. plantarum during baking and that significantly increased the shelf life of pita bread. Luz et al. (2018) evaluated phenolic compounds that were present in freeze-dried whey fermented with L. plantarum strains 220 and 221 for 72 h. Their analysis revealed a significant number of benzoates (hydroxybenzoic acid, benzoic acid, 1,2-dihydroxibenzene) which possess significant antifungal activity as evidenced by their industrial application in foods for mold inhibition. Luz et al. (2018) also reported a significant number of polyphenols with demonstrated antifungal properties such as sinapic acid, DL-3-phenillactic acid, caffeic acid, p-coumaric acid, salicilic acid, and hydrocinnamic acid. Collectively, these benzoates and polyphenols can be hypothesized as inflicting stressors on fungi, creating multiple hurdles thus eventually inhibiting their growth or inactivating them. In this present study, it took the NC treatment only 10 days for signs of visible mold growth to be observed. The WFP treatments at 0.25% alone or in combination with CEX at 0.015 and 0.15% exhibited visible signs of mold growth in 15, 22 and 38 days, respectively. The PC (0.1% potassium sorbate) treatment seemed to be moderately effective at preventing mold growth. This is because visible signs of mold on the PC samples were observed on day 85. The rest of the treatments with WPF at inclusion levels of >0.5% (w/w) alone or in combination with CEX did not show any signs of visible mold growth even after 365 days of storage in ambient conditions. Our findings from the shelf life study suggest that WPF has significantly superior fungistatic effects compared to potassium sorbate, albeit its combination with CEX potentiates this effect, ultimately increasing the shelf life of semi-moist pet food. Thus, the rationale of pairing WPF with CEX is to create a potentiation effect which in turn reduces the dose of WPF required to control mold which lowers production costs in the pet food industry. Additional studies are warranted to identify the type and concentration of the phenolic and benzoic acids present in WPF to better explain the mechanism of fungal inhibition.

The contamination and proliferation of spoilage and toxigenic fungi in semi-moist pet foods is a perennial challenge for manufacturers, exacerbated by the ever-increasing demand for products that are considered “natural” and have “clean labels” as healthier alternatives for feeding companion animals. The utilization of whey fermented with lactic acid bacteria strains such as L. plantarum can be used to produce value-added and “natural” antifungal compounds that can be used as alternative substitutes to synthetic preservatives such as potassium sorbate to control mold in semi-moist pet foods and baked goods intended for human consumption. However, because larger doses of fermented whey are required to achieve the desired level of microbial safety in semi-moist pet foods, additional research into polyphenols with potentiating or synergistic antimycotic effects is warranted to boost the applicability of WPF. This current study creates a scientific framework for the application of fermented whey protein into semi-moist pet food as this ingredient gains in popularity among pet food brands that sell “natural” and clean label products. In conclusion, WPF at 0.5–1.0% (w/w) inclusion levels can be used alone or in combination with CEX as natural antifungal treatments to control spoilage and toxigenic fungi in semi-moist pet foods for dogs.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

GA, JD, and CR conceived the idea, designed the study, helped with editing, and interpretation of results. SK performed all the experiments, collected data, data analysis, and wrote the primary manuscript. All authors contributed to the article and approved the submitted version.