1. Introduction
Protein in the diet is an essential nutrient for animal growth and plays a vital role in gut health. At present, the structure of feed formulation in China is dominated by corn–soybean meal; however, the excessive dependence on imported soybean meal is detrimental to the economic efficiency of farming. In this context, unconventional protein feed is not yet fully utilized, and there seems to be great potential in developing a new type of protein source as soon as possible to alleviate the shortage of protein sources. Scientists have found that yeast feed offers an attractive alternative source to replace other protein sources; yeast feed is not only easily digested and absorbed but also has a balanced amino acid profile that provides animals with a rich source of nutrients [
1].
Some studies have found that, when yeast feed replaces a portion of fishmeal or soybean meal, it can help in maintaining animal growth performance [
2,
3]. Piglets often face challenges after weaning including diarrhea, low feed intake, and weight loss, which can cause serious damage to gut health and function. Fortunately, the addition of yeast feed can alleviate piglets’ post-weaning stress [
4,
5]. The potential for the use of yeast feed to supplement deficient nutrients has become increasingly recognized, since yeast has a positive impact on animal health and nutrition, regulating the intestinal flora and stimulating the immune system to prevent the growth of pathogenic bacteria [
6]. Currently, the strains used to produce yeast feed are
Saccharomyces cerevisiae,
Candida utilis,
Pichia, and so on, which have functions similar to probiotics and are not only beneficial to human beings but may also play an important role in the nutrition and health of animals.
Candida utilis is rich in protein and amino acids and its cell-wall-derived mannan-oligosaccharides and glucans are beneficial to piglet growth and intestinal health as feed additives [
7]. Furthermore, another advantage of adopting
Candida utilis is that industrial and agricultural wastes may be utilized as substrates to ferment edible proteins for application in livestock farming [
8,
9,
10,
11]. In this way, different feeds fermented with
Candida utilis and yeast-containing products may provide different nutritional functions to animals as nutritional sources or probiotics [
12,
13]. However, in order to produce feeds with high protein contents, fermentation parameters, such as optimization of medium composition, culture conditions, need to be continuously optimized to achieve the desired purpose. The response surface methodology is widely used in process optimization studies since it can allow researchers to evaluate the interactions between factors, quickly identify the relationships between factors, and response values and conclude the best optimization parameters [
14,
15]. Based on the properties of
Candida utilis, we selected industrial waste, glucose master liquor, and a corn byproduct, corn steep liquor, as fermentation substrates to produce forage protein feed; this approach not only turns waste into a valuable product but is also a step in the right direction in tackling the problem of environmental pollution.
Overall, this study aimed to analyze the effect of Candida utilis feed on Dongliao black piglets. We first optimized the medium components of Candida utilis feed to achieve the maximum content of yeast cells in the feed, as achieved through a response surface methodology. Then, we added the optimized Candida utilis feed to the diets to observe its effects on the growth performance, intestinal flora, and metabolic level of weaned piglets, as determined using 16S rDNA combined with non-targeted metabolomics. In this way, we sought to determine whether it can be used as a novel protein source, thereby providing a theoretical basis for its future application in the aquaculture industry.
2. Materials and Methods
Glucose master liquor and corn steep liquor were obtained from Jilin Ruisheng Technology Co. (Songyuan, China). The yeast strain Candida utilis CICC1801 was purchased from China Center of Industrial Culture Collection (Beijing, China). Glucose, agar, yeast paste, peptone, yeast extract, ammonium sulfate (NH4)2SO4, magnesium sulfate (MgSO4), and potassium dihydrogen phosphate (KH2PO4) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The entire feeding experiment was conducted at the Agricultural Experimental Base of Jilin University (Changchun, China), and all animals were treated in accordance with the Ethics Committee of Jilin University.
2.1. Culture Medium
The activated culture medium used was a Yeast Extract Peptone Dextrose Medium (YPD); it contained 20 g of glucose, 20 g of peptone, 10 g of yeast paste, and 20 g of agar in 1 L of deionized water. The initial seed medium contained 50 g of glucose, 20 g of yeast extract, 2 g of potassium dihydrogen phosphate, 1 g of magnesium sulfate, and 1 g of ammonium sulfate in 1 L of deionized water, pH 5.5. In addition to these, the slant medium contained 15 g of agar. All culture media were sterilized at 121 °C for 21 min.
2.2. Methods for the Determination of Strain Indicators
Strain activation: The Candida utilis strain preserved on the slant was taken out and inoculated with the YPD medium using the dilution coating method and incubated in an incubator at 30 °C for 48 h for activation. Then, to observe the colony growth status, a single colony with good growth was taken, inoculated in the slant medium, incubated in the incubator at 30 °C for 48 h and shaken in a bottle to make the seed culture after it grew into moss.
Preparation of seed liquids: A ring of Candida utilis was selected from the slant moss and inoculated in a 500 mL triangular flask containing 100 mL of seed medium, incubated for 20 h at 30 °C and shaken at 160 rpm/min, and stored at 4 °C for the determination of each index in the subsequent test.
Determination of the growth curve: Samples were taken every 2 h during seed cultivation, and the absorbance value was measured at 600 nm using a spectrophotometer after diluting 20 times with distilled water. A growth curve of Candida utilis was plotted with time as the horizontal coordinate and the absorbance value as the vertical coordinate.
The optical density (OD) value was determined using a spectroscopic method (λ = 600 nm) [
16], and the number of yeast cells was determined using a counting chamber [
17].
2.3. Single-Factor Experiment Design of Culture Medium Components
Carbon source concentration: Using the seed medium as a reference, five groups of glucose master liquor concentrations of 2%, 4%, 6%, 8%, and 10% were set as the carbon source; other components were unchanged; the pH was adjusted to 5.5; the strain inoculum was 5%; and fermentation was carried out in a shaking bed at 160 rpm for 20 h at 30 °C. The OD value and number of yeast cells of the fermentation liquid were used as evaluation indexes to initially select the appropriate amount of glucose master liquor to be added.
Nitrogen source concentration: The seed medium was used as a reference; the glucose master liquor concentration was 8%; five groups of corn steep liquor concentrations of 1%, 2%, 3%, 4%, and 5% were set as the nitrogen source; other components were unchanged; the pH was adjusted to 5.5, and the culture conditions remained unchanged. The OD value and number of yeast cells of fermentation liquid were used as evaluation indexes to initially select the appropriate amount of corn steep liquor to be added.
Inorganic salt concentration: Using the seed medium as a reference, the glucose master liquor concentration was 8%; the corn steep liquor concentration was 1%; five groups of KH2PO4 concentrations of 0.05%, 0.1%, 0.15%, 0.2%, and 0.25% were set; other components were unchanged; the pH was adjusted to 5.5, and the culture conditions remained unchanged. The OD value and number of yeast cells of fermentation liquid were used as evaluation indexes to initially select the appropriate amount of KH2PO4 to be added.
2.4. Response Surface Methodology
The Box–Behnken design and the response surface methodology (RSM) were used to further optimize the medium components. The variables selected using the single-factor experiment were designed as three levels (low, middle, and high) and coded as −1, 0, and 1, respectively. According to the coding design, Design-Expert software generated a 3-factor, 3-level, 17-run experimental design scheme, using the OD value and number of yeast cells as response values to optimize the medium components.
2.5. Animals, Diets, and Experimental Design
A total of 120 Dongliao black pigs, acquired from the Agricultural Experimental Base of Jilin University, were weaned at 28 days. Using the optimized medium components for actual fermentation production, the fermentation liquid was enzymatically digested by adding alkaline protease. The enzymatic liquid was filtered to obtain the concentrated broth, and then spray-dried, resulting in the finished product of the
Candida utilis feed. One group was fed the basic diet (the CON group) supplemented with
Saccharomyces cerevisiae, and the experimental group was supplemented with 2.5%
Candida utilis feed. The piglets had free access to feed and drinking water at all times. On the last day of the experiment, each pig’s body weight (BW) and feed consumption were recorded, and these data were used to calculate the growth performance indexes ADG, ADFI, and FCR. All diets were fortified to meet or exceed the NRC (2012) nutritional requirements for piglets (
Table 1).
2.6. Sample Collection
On days 19–21 of the trial, collected feces were immediately stored in liquid nitrogen or at −80 °C for subsequent analysis. Blood samples were collected via a 10 mL jugular vein puncture and promptly centrifuged at 3500× g for 10 min to collect serum. These samples were used for serum biochemical assays and a subsequent histologic analysis.
2.7. Serum Biochemical Indicators Analysis
The blood samples were incubated in a 37 °C water bath for 10 min, centrifuged at 3000 r/min for 15 min to collect serum, and stored at −80 °C. Serum biochemical indicators included triglycerides (TGs), total cholesterol (TC), total bilirubin (T-BIL), direct bilirubin (D-BIL), γ-glutamyltransferase (GGT), high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), which were measured using corresponding commercial kits (Medicalsystem Biotechnology Co., Ltd., Ningbo, China) and an automatic biochemical analyzer (MS-880B, Medicalsystem Biotechnology Co., Ltd., Ningbo, China).
2.8. DNA Extraction and Purification and 16S rDNA Amplification Data Analysis
After the fecal samples collected from each pig were completely dissolved, according to the manufacturer’s instructions, DNA from the different samples was extracted using an E.Z.N.A. ®Stool DNA Kit (D4015, Omega, Inc., Norwalk, CT, USA). Nuclease-free water was used as a blank control. The DNA quality was tested on the machine to see whether it qualified, and the V3-V4 region (468 bp) of 16S rDNA was selected for primer design (forward primer: 5′-CCTACGGGNGGCWGCAG-3′; reverse primer: 5′-GACTACHVGGGTATCTAATCC-3′), followed by PCR amplification of the DNA. The amplified products needed to be further verified via 2% agarose gel electrophoresis, and a DNA library was constructed. Qualified DNA libraries were sequenced using an Illumina sequencer and obtained for a bioinformatics analysis.
After the samples were sequenced, raw data were obtained, paired-end sequences were assigned for overlapping splicing, and then the raw data were quality filtered to obtain high-quality data and remove redundant chimeras. Dereplication was carried out using DADA2 to obtain feature tables and feature sequences, and then a diversity analysis and species classification annotation were carried out. Alpha diversity and beta diversity were calculated via normalization to the same sequences randomly. Most bacteria were identified and analyzed based on the sequencing results.
2.9. Serum-Related Metabolites Extraction and Non-Targeted Metabolomics Analysis
The collected serum samples were thawed on ice, and metabolites were extracted with 50% methanol buffer. Briefly, 20 μL of sample was extracted with 120 μL of precooled 50% methanol, vortexed for 1 min, and incubated at room temperature for 10 min; the extraction mixture was then stored overnight at −20 °C. After centrifugation at 4000× g for 20 min, the supernatants were transferred into new 96-well plates. The samples were stored at −80 °C prior to an LC-MS analysis. In addition, pooled QC samples were also prepared by combining 10 μL of each extraction mixture.
All samples were acquired by the LC-MS system by following the machine’s orders. Firstly, all chromatographic separations were performed using a Vanquish Flex UHPLC system (Thermo Fisher Scientific, Bremen, Germany). An ACQUITY UPLC T3 column (100 mm × 2.1 mm, 1.8 µm, Waters, Milford, CT, USA) was used for the reversed-phase separation. The column oven was maintained at 35 °C. The flow rate was 0.4 mL/min, and the mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid). Gradient elution conditions were set as follows: 0~0.5 min, 5% B; 0.5~7 min, 5% to 100% B; 7~8 min, 100% B; 8~8.1 min, 100% to 5% B; 8.1~10 min, 5% B.
A high-resolution tandem mass spectrometer, Q-Exactive (Thermo Scientific, Waltham, MA, USA), was used to detect metabolites eluted from the column. The Q-Exactive was operated in both positive and negative ion modes. Precursor spectra (70–1050 m/z) were collected at 70,000 resolution to hit an AGC target of 3 × 106. The maximum injection time was set to 100 ms. The top 3 configurations to acquire data were set in the DDA mode. Fragment spectra were collected at 17,500 resolution to hit an AGC target of 1 × 105 with a maximum injection time of 80 ms. In order to evaluate the stability of the LC-MS during the whole acquisition, a quality control sample (pool of all samples) was acquired after every 10 samples.
2.10. Integrated Microbiome–Metabolome Analysis
A Spearman correlation analysis was performed to determine the relationships between the differential flora, between the differential metabolites, and between the differential metabolites and differential flora. This was achieved by performing a Spearman correlation analysis on the differential secondary metabolites obtained from the metabolomics screening and on the flora with significantly differential genus levels obtained from the 16S rDNA sequencing analysis. Based on the calculation results, suitable screening conditions were selected to determine the final correlations between the differential flora and differential metabolites and network diagrams, etc.
2.11. Statistical Analysis
All data were initially sorted in Excel 2021, and data from the fermentation trial were analyzed using one-way ANOVA through Design-expert12.0. Subsequent data were analyzed using SPSS (version 20.0), and differences between the control and experimental groups were analyzed using the t-test. The results are expressed as the mean ± standard deviation (SD), and p < 0.05 was considered to indicate a statistically significant difference.
4. Discussion
Yeast can be added to animal feed as an inexpensive, safe, and novel protein source, as it contains a high protein content and well-balanced amino acids.
Candida utilis feed is produced from different agricultural, forestry, and industrial wastes, so its use contributes to eliminating pollutants and the recycling of materials from waste [
18,
19,
20]. The glucose master liquor used in this experiment is the liquid left over after the production of crystalline glucose, which can become a source of pollution if not fully utilized. Corn steep liquor is a byproduct of corn starch production using the wet process. Concentrated corn steep liquor has a mushy aromatic odor, high nutritional value, and low price; promotes microbial growth and metabolism; and is a high-quality nitrogen source in process production. Although the current application of these two raw materials as the main components of fermented protein feeds is relatively limited, they are expected to be widely used based on the application results.
During fermentation, the medium component has a great influence on the growth and reproduction of yeast cells. We firstly optimized the medium component and the single factor results showed that 8% glucose master liquor, 1% corn steep liquor, and 0.15% KH
2PO
4 could meet the growth demand of the organism, and more yeast cells increased the synthesis rate of fermentation products. Although the concentration of the carbon source needs to be sufficient to supply the growth and reproduction of
Candida utilis, a too-high concentration will inhibit the uptake of other growth factors by
Candida utilis and slow down the metabolism rate of the bacterium. Zhang et al. [
21] used glucose and other substances in combination with
Candida utilis and
Saccharomyces cerevisiae to produce a single-cell protein with a protein content of 43.59% and rich in various amino acids. Zhao et al. [
22] used waste capsicum powder as substrate and 3% corn steep liquor as a nitrogen source to ferment feed using
Candida utilis, and this improved the growth rate of the yeast and increased the protein content. Athar et al. [
23] also applied
Candida utilis as a fermenting bacterium to produce feed additives for animal breeding with beet pulp as a substrate and 1% corn steep liquor as a nitrogen source. When yeast cells lack the supply of phosphate and metal elements, the reaction of nutrient uptake stops, leading to the inhibition of fermentation [
24]. If the concentration of inorganic salt KH
2PO
4 is continuously increased, then it may affect the cellular metabolic function of the bacterium and its internal and external osmotic pressures may become too high, resulting in a decrease in the OD value and number of yeast cells [
25]. Favorable results were obtained by optimizing the medium components through response surface methodology, which could be applied in actual production, proving that these raw materials are beneficial to the growth and reproduction of yeast and achieve a considerable protein content in the final fermentation products.
At present,
Candida utilis feed is popularly used in the aquaculture industry, and there are few studies in livestock and poultry farming. Moreover, feeding with
Candida utilis feed could improve the growth performance and nutrient digestibility of aquatic animals, and it could also alleviate the intestinal inflammation caused by soybean meal [
26,
27,
28]. Cruz et al. [
5] studied the effects of
Candida utilis added to the diets of weaned piglets and found that the addition of yeast to diets did not affect growth performance compared with the control. In this study, compared with the CON group, the addition of 2.5%
Candida utilis protein feed to the basic diet caused no significant differences in the average daily gain (ADG), average daily feed intake (ADFI), or F/G of black piglets. Similar results have been reported in cattle [
29]. However, other studies proved that
Candida utilis not only improves the growth performance of weaned piglets but also reduces diarrhea [
30,
31]. These different results may be due to factors such as yeast composition, addition amount, animal category, and feeding environment, but what can be demonstrated is that the use of
Candida utilis as a source of protein has no adverse effects on growth performance in livestock farming and that growth requirements can be maintained.
Serum biochemical indicators refer to the levels of various chemical components in the plasma, including the levels of many metabolites of the body. Serum is similar to the interstitial fluid of the body’s tissues, and it is more accurate in indicating the physiological situation of the body and more sensitive in indicating pathological changes. For example, ALT and AST are the most widely used biochemical indicators of hepatocellular injury in clinical practice. A previous study showed that the addition of yeast extract significantly reduced the AST and ALT levels in the blood of pikeperch without harming their liver, intestinal, or kidney functions [
32]. In this experiment, the addition of 2.5% CU resulted in a decreasing in AST and ALT in the weaned piglets, indicating that the addition of CU did not damage their liver function, but rather maintained their healthy liver function, improved their immunity and facilitated their growth. As TC, TG, LDL-C, and HDL-C are important indicators in blood lipid examinations, they are often used to assess atherosclerosis, or cardiovascular disease, etc. Azizi et al. [
33] found that addition of yeast reduced the TG content in broiler plasma. In this experiment, the addition of
Candida utilis had a tendency to reduce this indicator compared with the control group. This suggests that
Candida utilis, as a novel protein source, has the effect of improving blood lipids, and it has been reported that the addition of probiotics to the diet can inhibit lipid biosynthesis and increase fatty acid catabolism [
34].
The intestinal flora of mammals is diverse and increases with the stage of growth, and, during the weaning of piglets, physiological stress can lead to changes in fecal flora [
35]. Therefore, we assessed microbiota diversity via 16S rDNA sequencing of fecal flora. The results of this experiment showed that
Firmicutes,
Bacteroidota,
Actinobacteriota, and
Proteobacteria were the most dominant phyla in the piglets. This is consistent with the results of [
36]. In addition, the relative abundance of
Actinobacteriota increased significantly compared with the control after the addition of yeast at the phylum level.
Actinobacteriota is the dominant flora in the animal intestinal tract, and its metabolism produces bioactive substances with anticancer and antibacterial effects [
37]. The increase in the abundance of
Actinobacteriota with the addition of yeast protein feed may alleviate weaning stress and improve the intestinal immunity of piglets, but it is likely that the whole experimental period was too short and did not fully reflect the better growth performance. At the genus level, the current results revealed a high relative abundance of
Clostridium_sensu_stricto_1,
Lactobacillus, and
Muribaculaceae_unclassified in the 2.5% CU group compared with the control group. Yu et al. [
38] added
Bacillus licheniformis to piglet diets, which also increased the relative abundance of
Clostridium_sensu_stricto_1 and
Lactobacillus.
Clostridium_sensu_stricto_1 and
Lactobacillus are members of
Firmicutes, most of which are beneficial intestinal bacteria.
Clostridium_sensu_stricto_1 was reported to promote the adhesion of the intestinal mucus barrier to pathogens, and an increasing in its relative abundance in cecum content adversely correlated with levels of inflammatory factors [
39,
40]. The isolates of
Lactobacillus have been found to be able to inhibit pathogen invasion of intestinal epithelial cells and reduce the adhesion of some pathogenic bacteria to the intestinal mucosa [
41].
Muribaculaceae used undigested food to produce short-chain fatty acids, such as propionate and butyrate. They play an important role in regulating gut health in animals [
42]. We also observed an increase in
Collinsella abundance, which was positively correlated with blood T-cell responses and may be associated with enhancing serum immunity in weaned piglets [
43]. In addition, the relative abundance of
Prevotella 9 showed a decreasing trend in the 2.5% CU group, which is consistent with Iakhno et al. [
44]. High levels of
Prevotella not only exacerbate inflammation but are also associated with obesity and high blood pressure [
45]. However, some other different results were found, such as an increasing in the abundance of
Faecalibacterium; thus, further research is needed to investigate the reasons for this. However, it has been confirmed that
Candida utilis feed can regulate the intestinal flora of weaned piglets, thereby increasing the content of beneficial bacterial genera and protecting the intestinal barrier by inhibiting the invasion of pathogenic bacteria and inflammation; this is conducive to reducing the weaning stress of piglets, thus guaranteeing their healthy growth. Furthermore, the abundance of
Clostridium_sensu_stricto_1 in finishing pigs decreased significantly with decreasing protein levels [
46]. Thus, these results could indicate that it is feasible to apply protein feed fermented by
Candida utilis as a novel protein source by virtue of its rich protein content.
In order to explore the effect of
Candida utilis feed on the metabolic levels of the weaned piglets, metabolomics was used to provide a more comprehensive view of the changes in their plasma metabolites. In this experiment, as determined from our metabolomics results, the metabolic pathways enriched by the 12 differential metabolites were tryptophan metabolism, primary bile acid metabolism, histidine metabolism, taurine and hypotaurine metabolism, and caffeine metabolism. Histidine is a semi-essential amino acid, and it is metabolized mainly by gut microbes. Li et al. [
47] found that feeding a probiotic complex to pigs may affect the metabolism of histidine by producing histidine, which was beneficial in improving the digestive function of the pig intestine. Another study revealed the important role of histidine in the treatment of inflammatory responses, oxidative stress, and metabolic disorders [
48]. Tryptophan is an essential amino acid that plays an important role in regulating appetite and the stress response, maintaining the immune system and protecting the health of the body [
49]. Primary bile acid metabolism is involved in glycolipid metabolism, facilitates lipid absorption, regulates intestinal flora, reduces inflammation, and maintains the integrity of the intestinal epithelial barrier [
50]. Hence, it can be concluded that, in weaned piglets, the addition of yeast protein feed can regulate metabolism levels and will lead to a higher content of metabolites related to digestion and immune function. This has a positive regulatory effect on the regulation of intestinal flora, maintenance of intestinal barriers, reduction in the occurrence of diseases, and improvement of the immune system, maintaining the normal growth and development of piglets. In this study, we performed a combined analysis of fecal microbiota 16S rDNA sequencing and untargeted metabolomics and found close associations between the gut flora and in vivo metabolism of weaned piglets fed the
Candia utilis protein feed. A total of 10 plasma metabolites were correlated with 27 genera of bacteria as revealed by the results of the combined multi-omics analysis. Metabolites such as Cyclo-prolylglycine, Telbivudineand Theobromine 6Hydroxycoumarin showed highly significant positive correlations with
Lactobacillus,
Anaerofustis, and
Atopostipes, which are beneficial in
Firmicutes. A study found that Cyclo-prolylglycine exhibited neuroprotective potential in rats with glutamate neurotoxicity [
51]. Telbivudine was used in clinical applications to treat chronic hepatitis B and showed no adverse effects and Theobromine could reduce diet-induced obesity through fat modulation [
52,
53]. 6-Hydroxycoumarin is a natural compound with an aromatic odor widely distributed in Umbelliferae, and it has high clinical value as an anticancer, antifungal, and anti-inflammatory agent [
54]. Therefore, we concluded that the addition of
Candida utilis protein feed can regulate the intestinal flora of piglets, act as an anti-inflammatory, and maintain intestinal homeostasis through the metabolic pathways affected by these metabolites, such as primary bile acid metabolism, histidine metabolism, and tryptophan metabolism.