Functional foods have attracted significant interest in the food animal industry, with a growing focus on their use as carriers for probiotic cultures. The remarkable advantages offered by probiotics, prebiotics, and synbiotics in intensive farming, particularly in their potential to replace antibiotics, have been extensively documented ^[1][2][133,134].

In addition to functional and safety considerations, technological criteria associated with feed production and processing play a crucial role in the selection of probiotics. The incorporation of live microorganisms into animal feed poses significant challenges due to the exposure of probiotic bacteria to high temperatures during production and their vulnerability to adverse conditions such as low water activity, which can negatively impact bacterial viability.

The preservation of probiotic viability and functionality has been discussed in detail in part 3, focusing on different techniques and protective compounds. However, incorporating probiotics into dry products entails several challenges and requires the careful consideration of various criteria to maintain their functionality. Due to these challenges, many studies have explored the incorporation of probiotics in drinking water or mixed in food (as shown in Table 1), primarily due to the advantages of better concentration control and availability in small-scale investigations ^[3][135].

Used Group	LAB Species	Main Effects	Addition Method	Ref.
Poultry
Broiler chicks	Lpb. plantarum	Improved growth performance, intestinal morphology and immune response in broiler chickens under heat stress.	Sprayed on the feed (postbiotic)	[4][54]
1-day-old chickens	Lgb. salivarius	Improved growth performance (weight and longer shank length), increased relative weights of the immune organs and decreased concentrations of odor-causing compounds.	In diet (107, 108, and 109 CFU/kg of feed)	[5][55]
Broiler chicks	P. acidilactici, Lmb. reuteri, Enterococcus faecium and Lb. acidophilus	Modulates the activation of the innate immune response and inhibits the activation of standard C. perfringens immune responses.	Water (postbiotic)	[6][56]
Broiler chicks	Lgb. salivarius	Improved body weight of broiler under low ambient temperature and a trend in reducing the mortality rate.	Mixed in feed	[7][57]
Broiler	Lb. acidophilus, B. subtilis, S. cerevisiae, A. oryzae	Improved overall weight gain and CP retention.	Mix of probiotics added in basal diet (0–30%)	[8][58]
Swine
Weaned piglets	Lpb. plantarum	Increases diversity and richness in the microbial community, promoting intestinal development.	Liquid probiotic via feed (1.25 × 109 CFU/kg of diet).	[9][59]
Weaned piglets	Lpb. plantarum and P. acidilactici	Reduced impact of enterotoxigenic E. coli, being associated with decreased E. coli detection; modulation of the cytokine response, reduction in intestinal damage and clinical signs, and improved growth performance.	Microencapsulated probiotics suspended in sterile peptone water, given orally via sterile syringe (109 CFU/mL)	[10][60]
Weaned piglets	Lb. Johnsonii Lb. mucosae	Higher (p < 0.05) body weight gain, feed intake, and gain/feed ratio than weaned piglets fed basal diet. Probiotic feeding also increased the numbers of lactobacilli and decreased the numbers of E. coli in the feces of weaned piglets.	Probiotic freeze-dried and mixed into the basal diet	[11][61]
Pig farm	Lpb. plantarum	Improved meat quality and physicochemical characteristics.	Drinking water (2.5 × 107 CFU/mL)	[12][62]
Pigs	Lb. acidophilus, B. subtilis, S. cerevisiae, A. Oryzae	Improved overall performance. The overall gain and apparent total tract digestibility of CP were greater in pigs fed substrate fermentation (SF) diets than in pigs fed a liquid diet (LF).	Basal diets supplemented with 0.30% LF and 0.30% SF multi-microbe probiotic products	[13][63]
Ruminants
Post-weaning lambs	Lpb. plantarum	Promotes the development of rumen papillae, enhances the immune status and gastrointestinal health.	In diet (0.9% v/w, CFS, Postbiotic)	[14][64]
Neonatal calves	Lpb. plantarum	Improves gut health to increase growth performance.	Drinking water (probiotic powder, 1.20 × 109 CFU/g)	[15][65]
Preruminant calves	Lb. acidophilus	Improved gut health. Lower incidence of diarrhea and higher cell-mediated immunity in probiotic fed groups.	Fermented milk, microencapsulated and FD (108 CFU/calf/d) were added in the milk or calf starter, depending on calf’s age.	[16][51]
Others
Rainbow Trout	Ltb. Sakei	Positive effect on growth, immunity, serum enzyme activity, gut microbiome, and resistance to Aeromonas salmonicida	Commercial diet coated in probiotic (1.0 × 107 CFU/g)	[17][66]
Common carp	E. casseliflavus	Improved growth and non-specific immune responses of common carp fingerlings (highest weight gain and specific growth rate at 1012 group, lowest feed conversion ratio at 1012 group)	In diet (1010, 1011, 1012 CFU/kg feed)	[18][67]
Rainbow trout	Lmb. fermentum	The encapsulated L. fermentum plus lactulose improved growth performance and avoided the absorption and accumulation of heavy metals in rainbow trout liver and gills	Encapsulated in diet (107 CFU g−1)	[19][68]
1 month old puppies	Lcb. rhamnosus and Lpb. plantarum	Significantly increased Lactobacillus and Faecalibacterium detection in fecal matter. Increased short-chain fatty acids (acetate, propionate and butyrate) concentration in feces. Prevented gastrointestinal infection.	In diet (109 CFU/day)	[20][69]
Young, training and elderly dogs	Lactobacillus casei, Lpb. plantarum and B. animalis	Promoted the average daily feed intake of elderly dogs. Improved average daily weight gain in all dogs. Enhanced the level of serum IgG, IFN-α, and fecal secretory IgA (sIgA), reducing the TNF-α. Increased beneficial bacteria and decreased potentially harmful bacteria.	In diet, 2 × 109 CFU/g (2 g for young, 4 g for training, 10 g for elderly dogs)	[21][70]
Kittens	E. hirae	Promoted intestinal colonization and fecal shedding of live E. hirae during administration. Ameliorated the effects of atypical enteropathogenic E. coli experimental infection on intestinal function and water loss	Probiotic powder (2.85–4.28 × 108 CFU/day) mixed with 100 μL of sterile water and inoculated into canned cat food	[22][52]
Healthy adult cats	Lb. acidophilus	Improved fecal quality parameters, increased Lactobacillus count and decreased total coliform bacteria counts	In diet (5 × 109 CFU/kg of food)	[23][71]
Adult cats	Lb. acidophilus, Lcb. casei, Lb. lactis, B. bifidum, E. faecium and S. cerevisiae	Probiotics and synbiotics positively modulated (p < 0.05) the fecal microbiota of cats, increasing the lactic acid bacteria counts	Commercial kibbles coated with probiotics, supplemented with freeze-dried probiotics and fructooligosaccharides	[24][72]

The matrices serve as the substrate for probiotic microorganisms, providing essential growth nutrients and acting as delivery vehicles. In the international market, several non-dairy food products intended for human consumption have been commercialized, incorporating probiotic LAB ^[25][26][136,137]. These non-dairy food products have been formulated to support the survival and functionality of probiotic strains, offering a diverse range of options for consumers seeking probiotic benefits ^[25][26][136,137].

Currently, commercial animal supplements are formulated with blends of various LAB species, often accompanied by enzymes or prebiotics ^[27][138]. These supplements are available as liquid additives for mixing into the feeding water or in solid–dry form to be incorporated into animal feed. They are typically administered during times of stress for the animals, such as ration changes, weaning, climate variations, transportation, and post-antibiotic treatment. Although there is limited research on incorporating probiotic LAB in solid–dry form into the food matrix for intensively reared animals, studies on incorporating probiotics into non-dairy matrices for human foods provide valuable insights into current advancements in this field.

Pelleting is a widely used thermal treatment method in the manufacturing of animal feeds ^[28][139]. The pelleting process involves various combinations of conditioning temperature and retention time in commercial feed mills. It should be noted that in some feed mills, conditioner temperatures may reach extreme levels of 90 °C, which can significantly impact cell viability ^[29][140]. As mentioned earlier, the use of protective compounds and encapsulation technologies offers new possibilities for customizing feed additives to withstand specific requirements and improve the survival and functionality of probiotics in animal feed. The successful incorporation of probiotics into such matrices requires the careful consideration of several factors. In addition to the previously mentioned criteria of safety and efficacy during selection, other aspects such as marketing, regulatory compliance, and technological considerations must also be taken into account ^[30][141].

Information regarding the appropriate dosage of probiotics and legislative requirements for the concentration of live probiotics in food at the time of consumption is currently inadequate. The food industry generally considers an amount of 10⁶ CFU/mL or g (as indicated by the FDA, Food and Drug Administration, of the United States of America) for human consumption ^[31][8]. In 2014, the International Scientific Association for Probiotics and Prebiotics reached a consensus that the daily intake of probiotics should range from 10⁸ to 10⁹ viable cells, equivalent to consuming approximately 100 g of probiotic-containing food per day ^[32][5]. For animal feed, the specific dosage is yet to be defined and will depend on the target animal species and growth stage. Probiotics exhibit diverse compositions, dosages, and delivery methods, making it challenging to discuss them comprehensively within a single study. Nonetheless, in recent years, research in the field of probiotics has significantly expanded, providing a growing body of knowledge that allows us to move beyond the uncertainties of empirical use.

Numerous factors can adversely affect the viability of probiotics in the food matrix. These factors include acidic or low pH conditions, hydrogen peroxide production, nutrient availability, dissolved oxygen levels, water activity, processing and storage temperatures, as well as potential interactions with other microbial strains and competitive inhibitors, among others ^[27][138]. To ensure consistency between batches and optimize the viability of probiotic strains in the final product, establishing and controlling the processing line and subsequent storage conditions is essential. Failure to address these factors can result in undesired interactions between bacteria and the food matrix, loss of probiotic viability during food processing and shelf life, and reduced viability of microorganisms as they pass through the gastrointestinal tract ^[30][33][34][141,142,143].

Ensuring the survival of probiotics requires the development of effective formulations and the careful selection of matrices or food vehicles ^[35][36][144,145]. Food matrices exhibit significant compositional variations, some of which may contain molecules that provide protective effects and stimulate the growth of probiotics upon reaching the intestine. Prebiotics, for instance, are non-digestible food ingredients that selectively stimulate a limited number of bacteria in the colon, thereby enhancing host health ^[37][38][146,147]. In animal feed, prebiotics can serve as substrates selectively used by microorganisms, conferring health benefits and contributing to the viability and stability of probiotics within the food matrix ^[39][7]. Complex carbohydrates, such as β-glucans, fructans, arabinoxylans, and starches, can be exploited as functional prebiotic ingredients for animal health applications, offering a rich energy source ^[40][148]. It is important to highlight that the combination of appropriate prebiotics and food matrices has the potential to further enhance the survival of orally delivered probiotics. This should be considered when designing novel functional foods ^[41][149]. For instance, barley-derived β-glucans have been shown to provide tolerance to gastrointestinal transit stress for specific probiotic strains like Lpb. plantarum WCFS1, Lb. acidophilus LA5, and Lb. johnsonii CECT 289, while also reducing intestinal inflammation in in vitro studies ^[41][149].

The composition and diversity of food offered to livestock under intensive farming are highly variable, and each probiotic strain may respond differently. Therefore, it is key to conduct survival trials using different probiotic strains and the specific food matrix intended to contain the probiotic bacteria ^[33][142]. Factors such as storage temperature, the water activity of the food, and the type of container employed significantly influence the survival of probiotics, and should be regarded as essential considerations during the development of probiotic foods ^[31][8].

Whole grains present a promising option as vehicles for probiotics due to their rich content of complex carbohydrates, antioxidants, phytochemicals, and other bioactive compounds ^[42][150]. Incorporating probiotics into whole grain formulations can provide animals with the dual benefits of probiotics and additional bioactive components. The components of grains can serve as substrates for fermentation or act as encapsulation materials in probiotic feed formulations ^[43][151].

Food processing involves various technological steps, many of which can have detrimental effects on the viability of probiotic bacteria. Current research on probiotics has striven to evaluate the strain-specific effects of probiotic species in specific animal species. However, the impact of food matrices remains largely unexplored. This knowledge gap hinders the development of innovative probiotic products, hence this resviearch hw highlights the importance of both probiotic strains (and their processing techniques) and food matrices, which are influenced by production and storage conditions, in determining the overall quality of a probiotic product. Pelleted feed is commonly used in intensive farming and is associated with higher feed efficiency. Incorporating dehydrated probiotics into such feed formulations could potentially reduce the susceptibility of these microorganisms to environmental stresses, including oxygen, pH, water activity, and temperature, during the dehydration process ^[44][152]. The inclusion of probiotic feed additives is expected to be a growing trend in the farming of intensively reared livestock, allowing for the large-scale incorporation of these beneficial microorganisms. Further research is needed to standardize the use of specific probiotic strains in the breeding of specific animals, while preserving their demonstrated properties.