Bioconversion of Apple Pomace into Microbial Protein Feed Based on Extrusion Pretreatment

Apple pomace (AP) is often used directly as animal feed, while the value of feeding is limited by its low protein content. In this study, extrusion pretreatment was performed for AP, and further fermentation was carried out to improve its nutrition value. Strains suitable for extruded apple pomace (EAP) to produce high-quality microbial protein (MP) feed were screened from 12 different strains. Results showed that Aspergillus niger 3.324 (Asn), Candida utilis1314 (Cau), Geotrichum candidum 1315 (Gec), Bacillus subtilis A308 (Bas1), and Lactic acid bacteria (Lac) were screened as the dominant strains, which exhibited higher feeding value. Strong symbiotic effect was observed in fermentation with mixed strains of Asn, Cau, Gec, and Lac at the ratio of 1:1:1:1. Compared with AP, the pure protein content in the optimized fermented EAP (FEAP) was increased by 138% accompanying with a pleasant flavor and taste. And its pure protein content was increased by 19.20% in comparison to that of the fermented apple pomace. The nutrition value of FEAP was characterized by amino acid profiles; it found that FEAP was comparable to other commercial proteins with higher contents of histidine, phenylalanine, threonine, and valine. Combination of fermentation and extrusion technology significantly enhanced pure protein content and nutritional composition of apple pomace, which was revalorized as a nutritive animal feed rich in microbial protein.


Introduction
Apple pomace (AP) is a waste by-product with high value-added obtained after the juice or cider by means of pressing; the main constituents of apple pomace are skins, pulp, and seeds [42]. AP was often used directly as animal feed, while the feeding value was limited due to its low protein content and digestibility [43]. Reprocessing could be applied to produce high-value compounds from AP instead of using it directly as animal food or discarding as waste [14]. In recent years, varieties of interesting ways were investigated by several researchers such as producing ethanol, phenolic antioxidant, pectin, and enzyme preparation [3,10,35]. However, application of these products was limited due to high production cost [13]. Recently, it had been found that AP could be used as an ideal raw material for fermentation of high-value microbial proteins (MP) to increase the added value of AP [17].
Numerous researches [9,26] demonstrated that AP was a suitable substrate for solid state fermentation (SSF); its protein content was enriched by cultured strains in SSF. Rodríguez et al. [26] found that protein and fat contents of AP were improved after fermented by autochthonous cider yeast strains, which enhanced nutritional and bioactive properties of AP. Devrajan et al. [8] showed that a feed of AP using co-culture of Candida utilis and Kloeckera exhibited better acceptability and digestibility. The fungal protein production was investigated by using AP as a solid substrate for Rhizopus oligosporus growth [4]. Those works focused on single or mixed culture of fungi and yeast, while works on co-culture with fungi, yeast, Bacillus, and Lactobacillus were rarely involved. Meanwhile, it was crucial to find mixed strains suitable for EAP fermentation to produce MP feed.
Several pretreatment techniques were investigated to improve the utilization rate of byproducts including acid/alkaline pretreatment, ultrasound, and microwave-assisted pretreatment [11,28]. However, several fermentation inhibitory compounds such as furfural and hydroxymethyl furfurals were generated during acid and microwave pretreatment [22]. Thus, it was extremely important to choose a more suitable preprocessing method.
Extrusion technology emerged as one of the promising method for processing various materials. It has the advantages of simple process, low energy consumption, and so on [46]. Extrusion technology was widely used for lignocellulosic material treatment. The specific surface areas of cellulose were increased by extrusion process; thereby, the enzyme accessibility to cellulose was enhanced [24,31]. Several researchers [7,16] found that the extrusion process could be used to improve the physicochemical properties of extrudate. Additionally, fermentation inhibitors were not produced during extrusion process [20]. In recent years, extrusion technology had been used in the preparation of raw materials for brewing beer and producing soy sauce [6,45]. Those results showed that extrusion pretreatment contributed to improve the properties of raw materials, fermentation capacity, and product quality. To the best of our knowledge, the application of extrusion technology was rarely reported in the fermentation process of MP production.
The aim of the present work was to screen the optimum mixed strains suitable for EAP fermentation to produce MP feed. The influence of single strain, mixed strains combination, and ratio were investigated for EAP fermentation, and the nutrition value of FEAP was characterized by amino acid contents.

Material Pretreatment
AP was pretreated by using single screw extruder (College of Agricultural Engineering and Food Science, Shandong University of Technology). Extrude apple pomace (EAP) was obtained with the extrusion parameters of screw speed 160 rpm, sleeve temperature 110 °C, and material moisture content 26%.

Screening Dominant Strains for EAP Fermentation
In this work, 10% (v/w) strain suspension was inoculated in each 200-mL Erlenmeyer flask containing 45 g sterilized substrate (EAP and bran in 9:1 ratio, blend and water in 1:2 ratio,

Screening the Optimal Mixed Strains Combination for EAP Fermentation
Based on the results of single strain fermentation, Asn was ensured to be included in each combination, because of its strongest ability to secrete cellulase among the dominant strains, hydrolyzing a large amount of cellulose in the fermentation substrate. Therefore, the double-strain combinations included Asn + Cau, Asn + Gec, Asn + Lac, and Asn + Bas1; the three-strain combinations included Asn + Cau + Gec, Asn + Cau + Lac, Asn + Cau + Bas1, Asn + Gec + Lac, Asn + Gec + Bas1, and Asn + Lac + Bas1; the four-strain combinations included Asn + Cau + Gec + Lac, Asn + Cau + Gec + Bas1, Asn + Cau + Lac + Bas1, and Asn + Gec + Lac + Bas1; and the five-strain combination included Asn + Cau + Gec + Lac + Bas1.

Screening the Optimal Mixed Strains Ratio for EAP Fermentation
Based on the test schedule of orthogonal test L 16 (2 4

Analytical Methods
Pure protein and crude protein content were determined by K9860 automatic Kjeldahl nitrogen meter (Shandong Haineng Scientific Instrument Co., Ltd., China). The amino acids were determined by A300 automatic amino acid analyzer (MembraPure, Germany). Cellulase activity of the fermented EAP (FEAP) was analyzed as follows: FEAP (2 g) and 90 mL water and 10 mL citric acid buffer were added in 200-mL Erlenmeyer flask, extracted at 40 °C for 1 h and filtered for later use. Sodium carboxymethyl cellulose solution (Na-CMC, 2 mL) was added in a test tube, which was preheated in a water bath at 50 °C for 2 to 3 min. Then, diluted enzyme solution (0.5 mL) was added in this tube, placing in a water bath at 50 °C for 30 min. Lastly, 3,5-dinitrosalicylic acid (2.5 mL) was added in the tube and boiled for 5 min, and the absorption value was measured at 530 nm after cooling. According to the following formula (1), the cellulase activity of the sample was calculated, where B is the net glucose content detected from the standard curve (mg), n is the dilution times of enzyme solution, 0.5 is the milliliter number of the diluted enzyme solution absorbed during the determination (mL), t is the response time (min), and M is the molar mass of glucose M(C 6 H 12 O 6 ) = 180.2 g/mol. A unit of enzyme activity (U) was the amount of enzyme required to hydrolyze 1 μmol glucose from 1 mg/mL Na-CMC in 1 min at 50 °C.
Effective viable counts of FEAP were analyzed as follows: FEAP (1 g) was taken by the quartering method and diluted to different multiples. Several 1:

Statistical Analysis
The sample analyses were performed in triplicate. SPSS19.0 was used for the analysis of data, and Origin9.0 was used to plot the data. Means were separated using Duncan's comparison at 95% confidence level (p < 0.05).

Effects of Single Strain on FEAP
As shown in Fig. 1, the pure protein content of Asn (Fig. 1a) was significantly higher (p < 0.05) than other fungi strains, indicating that EAP was a suitable substrate for the growth of Asn to produce MP. There was no significant difference (p > 0.05) in crude protein content between Asn and Aso (Fig. 1b). The crude protein content of Asn and Aso were significantly higher (p < 0.05) than that of other two fungi strains, suggesting that Asn and Aso were more suitable to grow on EAP compared with Trh and Trl. In a similar study, Aruna [5] found that crude protein content during Trichoderma viride fermentation of pineapple peels increased from 4.5 to 14.9% with the addition of (NH 4 ) 2 SO 4 as nitrogen source. The cellulase activity of Asn was significantly higher (p < 0.05) than that of all other strains (Fig. 1c), indicating that a large amount of cellulose in EAP was hydrolyzed by the growth of Asn. Effective viable count of Asn was the highest among fungi strains (Fig. 1d), which was corresponding to its pure protein content. Those results demonstrated that the increase of protein content was caused by the MP produced by the growth of strains.
Cau produced a higher pure protein yield compared with other two yeast strains (Fig. 1a), its crude protein content had no significant difference (p > 0.05) with that of Gec (Fig. 1b). However, the protein contents of Cau and Gec were lower than that of Asn and Aso, indicating that a large amount of cellulose in EAP could not be fully utilized by them due to yeast strains that possessed low hydrolytic enzyme activity [41]. Effective viable count of Cau and Gec was significantly higher (p < 0.05) than that of Sac (Fig. 1d), suggesting that Cau and Gec were more suitable to grow in the EAP substance compared with Sac. In a similar research, Zhu et al. [49] reported that Candida utilis and Geotrichum candidum were the ideal fermentation combination during yellow wine wastes as fermentation substrates. It was worth mentioning that the growth of Cau and Gec led fermentation products to aromatic flavor, which could improve its palatability.
The pure protein content of Bas2 was significantly higher (p < 0.05) than other bacillus strains, while that of Bas was the lowest among all strains (Fig. 1a). Those results indicated that the growth of bacillus strains was not conducive to the accumulation of proteins in fermentation products. The main reason might be that the proteins of EAP were hydrolyzed by the proteases produced during the growth of bacillus [39]. Bas1 and Bas2 exhibited stronger ability to produce cellulase than Bas or Bal (Fig. 1c). The effective viable count of Bas1 was significantly higher (p < 0.05) than that of other strains (Fig. 1d), indicating that Bas1was more suitable for growing on EAP, and its spore content was higher correspondingly. Their spores were not easily killed, which were fed into the digestive system of animals as living bacteria [15].
The pure protein of Lac was only higher than that of Bas or Bas1 (Fig. 1a), and its crude protein was only lower than Asn or Aso (Fig. 1b). It was probably because its main role in fermentation was not to increase protein content. The cellulose of EAP substrate was not adequately utilized due to the use of Lac alone showed low cellulase activity. Therefore, pretreatment or mixed fermentation with other strains was required for Lac fermentation. Abdel-Rahmanet al [1] found that cellulase pretreatment was helpful for lactic acid bacteria to ferment on cellulosic substrates. The effective viable count of Lac was lower than that of Bas1, Cau, and Gec (Fig. 1d). Lactic acid bacteria survived in low pH environments, resisting passage through the small intestine [27]. Furthermore, it showed inhibitory effect on gram-negative bacteria [18]. The fermentation product of Lac led to aromatic smells and good palatability, which was consistent with much of researches [2,25]. Finally, Asn, Cau, Gec, Bas1, and Lac were screened as the dominant strains for fermenting EAP to produce MP feed.

Effects of Mixed Strains on FEAP
The pure protein and crude protein content of mixed strains were significantly higher (p < 0.05) than that of a single strain (Fig. 2), demonstrating that there was a mutually beneficial symbiotic relationship between the mixed strains [49]. The pure protein contents of double-strain combinations were lower in comparison with three-or four-strain combinations (Fig. 2a). The pure protein content of Asn + Cau and Asn + Gec showed higher levels than that of single strain (Fig. 1a). The pure protein content of Asn + Cau + Gec was the highest among the three-strain combination, indicating that the fungi and yeast strains could grow synergistically in the fermentation substrate of EAP. In a similar report, Xiao et al. [44] found that the highest protein content of aquatic macrophytes was obtained from the mixed fermentation of Aspergillus niger and Candida utilis. The four-strain combination of Asn, Cau, Gec, and Lac exhibited a significantly higher (p < 0.05) pure protein content than all other combinations, indicating that the nutrient of EAP substrate could be fully utilized by that combination. Meanwhile, organic acids and bacteriocins were produced by Lac, inhibiting gram-positive bacteria [18]. The fermentation product let to a unique smell, thereby increasing the food intake of the animal. Meanwhile, the crude protein content of the Asn, Cau, Gec, and Lac combination was significantly higher (p < 0.05) than that of other combinations. The crude protein content of Asn + Bas1 and Asn + Lac + Bas1 was lower than that of other combinations (Fig. 2b), showing that Bas1 was not conducive to the accumulation of MP in the fermentation products. On the one hand, the reason seemed to be that the proteins in the EAP substrate were hydrolyzed by proteases produced during the growth of Bas1 [39]. On the other hand, a large number of nutrients in the FEAP were utilized by Bas1, which inhibited the growth of other strains.
The cellulose in EAP substrate is hydrolyzed to monosaccharides by cellulase for microbial growth and utilization. Cellulase activity could directly reflect the growth of Asn and the degree of cellulose hydrolysis in EAP substrate [36]. As shown in Fig. 2c, the cellulase activity of the combination with Bas1 was relatively higher due to Bas1 and Asn possess the ability to produce cellulase. However, FEAP in combinations of Lac showed lower cellulase activity. Romanens [38] found that lactic acid bacteria exhibited an inhibitory effect on filamentous fungi. At the same time, the aroma of FEAP was increased by the addition of Lac, which made the animal more receptive for the MP feed. Some researches demonstrated that the aroma profile of the fermentation product was improved by the co-cultures of yeasts and lactic acid bacteria [12,34]. The effective viable count of Bas1in single strain fermentation was higher than that of mixed strains fermentation (Fig. 1d), indicating that antagonistic effects existed between Bas1 and other strains in the mixed strains fermentation [40]. Thus, Bas1 was not considered for MP production by mixed strains fermentation EAP. In summary, the combination of Asn, Cau, Gec, and Lac was screened as the optimal mixed strains combination for converting EAP into MP feed.

Effects of Mixed Strains Ratio on FEAP
As shown in Fig. 3, group A exhibited the highest pure protein content compared with other groups (Fig. 3a), and its crude protein content was only lower than that of group N (Fig. 3b). Those results indicated that the four kinds of strains could grow cooperatively and accumulate their own MP to increase the pure protein content under the ratio of 1:1:1:1 [29]. The increasing crude protein content in FEAP was mainly due to the addition of inorganic nitrogen source. Moreover, fermentable sugars and inorganic nitrogen in the EAP substrate were converted into strains own MP [5]. When the ratio of Asn:Cau:Gec:Lac was 1:1:1:1, the pure protein content of FEAP was increased by 138% compared with EAP (4.13%). Consequently, the mixed strains at the ratio of 1:1:1:1 was more suitable for converting EAP into high-quality MP feed.
As shown in Fig. 3c, cellulase activity of group I was significantly higher (p < 0.05) than other groups. Aspergillus niger played an important role of the substrate hydrolysis [30]; larger Asn amount in the inoculum resulted in higher cellulase activity. The cellulase activity of group G was only lower than that of group I; the inoculation amount of Cau and Gec was higher than others under this mixed strains ratio. That result indicated that Cau and Gec were beneficial to Asn growth, promoting the production of cellulase when their proportion was higher than certain values. However, the growth of protein-producing yeasts was inhibited by the higher Asn proportion in the inoculum. In agreement with previous studies [48], the growth of yeast was inhibited when the colony number of fungi was higher than certain values. A balance among the co-culture strains possessing different functions was achieved, taking the positive effect for producing MP [47].
As shown in Fig. 3d, the effective viable count of group A was the highest (2.46 × 10 9 CFU/g) among other all groups, indicating that the four strains (Asn:Cau:Gec:Lac) were fermented at the ratio of 1:1:1:1 with excellent synergistic relationship. Effective viable count of group I was only lower than that of group A, suggesting that higher Asn proportion in the inoculums was beneficial to increase the effective viable count in the FEAP. However, high proportion of Asn caused a musty taste in the fermentation product, which was not conducive to animal feeding [37]. The proportion of Lac in groups B, D, F, H, J, and N were relatively higher; their effective viable counts were significantly lower (p < 0.05) than that of group A. Although the effective viable count of fermentation product was increased by Lac in the inoculums (Fig. 2d), a higher proportion of Lac had adverse effect in the fermentation process of mixed strains. In summary, the ratio of Asn:Cau:Gec:Lac at 1:1:1:1 was screened as the optimal mixed strains ratio for producing MP feed by fermentation EAP.

Characterization of Microbial Protein from EAP
As illustrated in Fig. 4, the amino acids except for lysine and histidine in FEAP were higher than those in EAP. The main reason was probably that lysine and methionine in EAP were incorporated by the yeast [21]. The amino acids in FEAP and EAP were higher than those in AP, indicating that the nutrition value was increased by extrusion and fermentation technology.
Composition of amino acid profile of the FEAP and other source proteins was shown in Table 2. All amino acids except for cystine in FEAP exceeded that of ruminants feed. The essential amino acids (EAA) in FEAP were higher than the Food and Agriculture Organization (FAO) standards [23]. The sum of EAA in FEAP was higher compared with the proteins produced by Lactobacillus acidophilus or Aspergillus niger [19], which indicated that the MP feeds produced in this study were superior to that of the single strain fermentation. The content of essential amino acids in FEAP conformed to FAO standard and was also compared to other sources of protein, such as soybean meal and fish meal, which had excellent amino acid components as high-quality protein resources for animal feed application [33]. It suggested that FEAP might be an excellent source of protein for animal feeds, especially for non-ruminant animals. For non-ruminant diets, the main limiting amino acids were lysine, methionine, tryptophan, and threonine. Although FEAP contained low lysine, methionine, and tryptophan, high threonine contents would benefit the use of FEAP as a protein source in non-ruminant feed [32]. Thus, supplementation of low-cost MP into commercial protein could significantly cut down the cost of animal feed.

Conclusions
Co-culture of Asn, Cau, Gec, and Lac at the ratio of 1:1:1:1 on EAP substrate exhibited the optimal microbial protein yield. Pure protein content of 9.81%, crude protein content of 23.52%, cellulase activity of 5.44 U/g, and effective viable count of 2.46 × 10 9 CFU/g in FEAP were achieved using those fermentation conditions. The microbial protein produced by mixed strains showed excellent amino acid profiles also was comparable to other source proteins with higher contents of histidine, phenylalanine, threonine, and valine. The application of extrusion and fermentation technology could effectively improve the feeding value of apple pomace and promote the effective utilization of apple pomace resources.

Availability of Data and Material
The data and material could be available on request.

Declarations
Ethics Approval This article is original and contains unpublished material. The corresponding author confirms that all of the other authors have read and approved the manuscript and no ethical issues involved.

Consent to Participate
All the authors agreed to participate in the scientific work. Table 2 Composition of amino acid profile of the FEAP with that of other proteins FAO Food and Agriculture Organization, EAA essential amino acid, NEAA nonessential amino acid