1. Introduction
Exogenous enzyme supplementation of pig diets was stimulated recently in the pig industry as a strategy to enhance the growth performance of growing–finishing pigs. Various exogenous enzymes, such as proteases, carbohydrases, and phytases, are commercially supplemented alone or as part of a multi-enzyme combination and have shown positive effects on feed utilization and animal performance in pigs. These substrate-specific enzymes convert the indigestible components of feed ingredients into substrates that the pig can digest. Many studies reported that the supplementation of a multi-enzyme has a more positive effect on pigs due to the synergistic interaction between enzymes [
1]. Non-starch polysaccharide (NSPs)-degrading enzymes such as xylanase, mannanase, and glucanase hydrolyze plant-cell-wall components of the diet and assist in the release of nutritional constituents such as proteins, lipids, starch, and minerals trapped within the cell-wall matrix [
2]. Additionally, protease complements the proteolytic activity by hydrolyzing the proteins resistant to endogenous secretory enzymes, improving the digestibility of amino acids, disrupting the feed structure, liberating fat and starch, and inactivating the anti-nutritional factors present in the diet [
3]. Therefore, adding exogenous dietary enzymes may improve the digestibility and utilization of nutrients, benefiting host performance and health. Hence, the swine industry is looking for solutions to improve the availability of nutrients in pig diets.
Pig intestines are colonized by diverse microbes that contribute to several biological functions and play an important role in the physiology and health of the host. It has significant effects in various aspects, such as suppressing pathogenic infections, synthesizing essential vitamins and amino acids, regulating fat metabolism, and forming immune systems [
4]. Undigested nutrition can eventually become a source of pathogenic bacteria in the large intestines, forming amino-acid-derived metabolites that are toxic to the intestinal epithelium of pigs. Therefore, the hydrolysis of NSPs and proteins is crucial for a balanced intestinal microbiota and for improving the health and productivity of the host. However, studies determining the effect of dietary supplementation of enzymes on pigs’ microbial composition and host–microbiome interactions are limited.
Invertebrates have developed symbiotic interactions with different microorganisms that secrete most of the metabolism-specific digestive enzymes to overcome nutritional limitations [
5]. The study of invertebrate gut-associated symbionts currently represents multidimensional industrial capabilities of biotechnologically active enzymes such as amylase, cellulase, protease, lipase, xylanase, and pectinase. Additionally, arazyme is a 51.5 kDa metalloprotease purified from
Serratia proteamaculans HY-3, a Gram-negative symbiotic bacterium of the spider
Nephila clavata [
6]. Purified arazyme showed high relative proteolytic activities and reduced the viscosity and ammonia concentration of intestinal contents, reflecting a significant feed enzyme effect [
7]. However, the experimental evidence of the effects of exogenous enzymes derived from invertebrate symbionts as feed additives for pigs is limited. Therefore, this study focused on evaluating the effects of a multi-enzyme supplement with arazyme on meat quality, gut microbiota, and host-microbiome interactions in pigs.
4. Discussion
Various gut microbes of invertebrates secrete many hydrolytic enzymes and could be new sources of biotechnological applications [
5]. The metalloprotease arazyme used in this study was produced by
Serratia proteamaculans HY-3, a symbiotic bacterium of the spider
Nephila clavata that can degrade a wide range of proteins and has anti-inflammatory effects in both cell and animal models of atopic dermatitis [
7,
16]. Additionally, mannanase and xylanase were purified from
Cellulosimicrobium sp. HY-13, a gut bacterium of
Eisenia fetida [
8] and
Paenibacillus sp. HY-8, a gut bacterium of
Moechotypa diphysis [
9], respectively. This study used invertebrate symbiotic microbe-derived enzymes with high industrial potential and an enzyme mixture containing arazyme as the main component and xylanase and mannanase as synergistic enzymes as a feed additive to evaluate the growth and meat quality of pigs.
Supplementation of exogenous enzymes in a diet, particularly protease as a part of an enzyme mixture, is widely used with the expectation that it will improve the efficiency of nutrient utilization by neutralizing anti-nutritional factors and increasing nutrient digestibility, thereby, improving growth performance [
17]. In our study, dietary supplementation of arazyme, in combination with xylanase and mannanase, affected the final body weight, average slaughter age, ADG, and gain:feed ratio. However, it showed no significant differences in ADFI compared to that of the control group. Other studies also showed that mixtures of enzymes with protease positively affected nutrient digestibility, bacterial populations in the large intestine, and growth performance of pigs [
1,
18]. Microbial exogenous enzymes hydrolyze dietary components in the small intestine into compounds that can be absorbed. Therefore, improved growth performance due to dietary supplementation of exogenous enzymes may enhance the disruption of the dietary cell walls and increase the exposure of the trapped nutrients, leading to growth-promoting effects. Meat quality is one of the most important economic characteristics of pigs, and it determines the suitability of meat for further processing and storage [
19]. Here, the supplementation of arazyme in combination with xylanase and mannanase influenced meat color, cooking loss, shear force, and antioxidant capacity. In contrast, there was no significant effect on pH, drip loss, and TBARS. An earlier study showed that the supplementation of the multi-enzyme mixture (NSPase: 1,4-
β-xylanase 20,000 U/g;
α-amylase 2000 U/ g; Protease 40,000 U/g) influenced the cooking loss and color lightness of breast meat in broilers fed a low-metabolizable energy diet [
20]. However, the influence of dietary supplementation of multi-enzyme protease as the main component and xylanase and mannanase as synergistic components on meat quality has not been previously reported. Therefore, comparisons could not be made with other studies. In the present study, enzyme supplementation changed the free amino acid profiles. The free amino acid content was 0.476% for the treatment group, which was greater than that of the control group (0.404%). In addition, the treatment group had high levels of lysine, valine, leucine, arginine, and isoleucine. These results infer that the meat quality will be excellent as the treatment group shows improved protein composition compared to the control group. Recent studies have shown that certain amino acids directly participate in taste and could also promote protein synthesis and skeletal muscle growth by activating major signaling pathways [
21,
22]. Therefore, these results suggest that supplementing arazyme in combination with xylanase and mannanase could improve the growth and value of meat in growing–finishing pigs. Additionally, our results showed that C18:1n-9, C16:0, C18:0, and C18:2n-6 were the most abundant fatty acids in the longissimus muscle; thus, the results correlated well with those in earlier studies [
23,
24]. Furthermore, our study revealed higher MUFAs and lower SFAs levels in the treatment group compared to that of the control group. A lower composition of SFAs is considered beneficial from a dietary perspective as SFAs (C14:0 and C16:0) are associated with a cholesterol elevating effect [
25]. However, the experimental evidence of exogenous enzymes derived from invertebrate symbionts and their appropriate proportions for improving host and meat quality is limited. Further studies investigating the correlation between exogenous enzyme additives and growth performance, meat quality, and gut microbiota studies are needed to confirm the results from this study.
In this study, feed additive supplementation increased the family Ruminococcaceae and the genera
Lactobacillus and
Limosilactobacillus (formerly known as
Lactobacillus). This is consistent with earlier reports of changes in the gut microbiome when a single enzyme or similar multi-enzyme was added to feed in livestock [
26,
27,
28]. The diet and its utilization in the intestine play an important role in preserving the diversity of gut microbiota; we conjecture that supplementation of exogenous enzymes promotes nutrient utilization and, thus, more diverse microbiota in the intestine. An earlier study showed that
Lactobacillus reuteri 1, belonging to
Lactobacillus, supplemented into the pig diet improved the meat quality by altering the muscle fiber characteristics and increasing flavor substances such as glutamic acid [
29]. Feed additive consumption also increased
Turicibacter and
Oscillibacter.
Turicibacter is a bacterium that ferments organic compounds and is reported to play a positive role in the microbiome immune interaction and promote the growth performance of pigs [
30]. It was reported that
Oscillibacter was abundant in the feces of pigs with high meat quality compared with those of pigs with low meat quality [
31]. Furthermore, treatment consumption increased the abundance of short-chain fatty acids (SCFAs), producing bacteria such as Ruminococcaceae, Muribaculaceae,
Phascolarctobacterium,
Mediterraneibacter, and
Blautia, but decreased the abundance of potential pathogenic bacteria such as
Porphyromonas,
Aerococcus,
Fusobacterium,
Campylobacter, and
Helicobacter. Short-chain fatty acids are a common product of fiber breakdown and affect the energy supply, gut health, and metabolic homeostasis and sustain the electrolyte balance that plays an important role in animal growth performance and meat quality [
32]. Furthermore, feed additives can increase or decrease the abundance of other members of the OTUs belonging to Lachnospiraceae,
Prevotella, and
Clostridium_
sensu_
stricto taxa. Earlier studies reported that
Prevotella and Lachnospiraceae are SCFAs-producing bacteria [
33]. Ingestion of
Clostridium butyricum, belonging to
Clostridium_
sensu_
stricto, was reported to enhance the growth and meat quality of pigs by enhancing the modulation of the host metabolism and intestinal development [
34].
Clostridium improves meat quality by modulating serum lipid metabolism, amino acid, and fatty acid composition [
35]. Due to the lack of genetic information on these OTUs, additional studies at genus or species level are necessary to investigate their precise roles.
Our data revealed that the feed additive significantly improved the biosynthesis of several predicted amino acids. The feed additive enhanced amino acid production that significantly affected the meat quality and growth performance of pigs [
36]. Furthermore, we conducted association analyses of LEfSe-selected OTUs with ALDEx2-selected predicted metabolic pathways. Results revealed that Otu0010 (Ruminococcaceae), Otu0077 (Prevotellaceae), and Otu0031 (
Prevotella) were increased by the feed additive and had a strong positive correlation with the biosynthesis of most amino acids. It was reported that
Prevotella (belonging to Prevotellaceae) and
Oscillibacter (belonging to Ruminococcaceae) positively correlate with amino acids biosynthesis [
37]. Furthermore, it was reported that the predicted amino acids, which are positively correlated with the increased microorganisms, have a beneficial effect on meat quality and the growth performance of pigs [
38]. It was also reported that lysine, arginine, and glutamic acid increase intramuscular fat, an important factor for meat quality [
39]. Threonine helps improve immunity and feed intake [
40], and tryptophan stimulates serotonin secretion to reduce stress and improves meat quality [
41]. In addition, the biosynthesis of fatty acid also increased by feed additive. Fatty acids can be produced by microorganisms and affect meat quality [
42]. It was reported that the influence on flavor and muscle color could be understood by determining the firmness/oiliness of adipose tissue and oxidative stability of muscles [
43]. Further meta-transcriptomic, metabolomic, and proteomic studies of the intestinal microbes in pigs are necessary to confirm the predicted metabolic pathways identified in this study. The major factors affecting the health of pigs and gut microbial communities, including the type of diets and dose of enzyme and the species and age of the animal, were limited in this study. Further research is required on the application of exogenous enzymes, particularly metalloprotease, from invertebrate symbiotic bacteria for feed supplementation.