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Article

Free Gossypol Removal and Nutritional Value Enhancement of Cottonseed Meal via Solid-State Fermentation with Rhodotorula mucilaginosa TG529

by
Bifan Liu
1,
Huanyu Liu
1,
Daohe Liu
1,
Miao Zhou
1,
Qian Jiang
1,
Xiaokang Ma
1,
Jing Wang
1,
Bi’e Tan
1,2,* and
Chen Zhang
1,3,*
1
Animal Nutritional Genome and Germplasm Innovation Research Center, College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China
2
Yuelushan Laboratory, Changsha 410128, China
3
Institute of Yunnan Circular Agricultural Industry, Pu’er 665000, China
*
Authors to whom correspondence should be addressed.
Agriculture 2024, 14(9), 1463; https://doi.org/10.3390/agriculture14091463
Submission received: 27 June 2024 / Revised: 1 August 2024 / Accepted: 22 August 2024 / Published: 27 August 2024
(This article belongs to the Section Farm Animal Production)
Browse Figures
Versions Notes

Abstract

:
The presence of free gossypol (FG) in cottonseed meal (CSM) greatly limits the use of CSM as a high-quality protein feed. Microbial fermentation is an effective method to simultaneously reduce FG and improve the nutritional value of CSM. In this study, using potato dextrose agar containing acetic gossypol as a selective medium and humus soil from cotton fields as the source, we isolated six strains of fungi capable of tolerating FG. With an inoculation ratio of 8% (8 mL × 106 CFU/mL cells or spores in 100 g fermented CSM), 50% moisture content, and a temperature of 30 °C, CSM was fermented for 5 days. The results showed that strain F had the highest FG removal rate at 56.43%, which was identified as Rhodotorula mucilaginosa (R. mucilaginosa) and named R. mucilaginosa TG529. Further optimization revealed that when the fermentation time was extended to 11 days, TG529 achieved a maximum FG removal rate of 73.29%. Compared to the original sample, treatment with TG529 significantly increased the contents of crude protein, acid-soluble protein, and 18 amino acids, while significantly reducing the contents of crude fiber, neutral detergent fiber (NDF), and acid detergent fiber in fermented cottonseed meal (FCSM). Using atmospheric and room temperature plasma for mutagenesis of TG529, it was found that the mutated TG529 significantly increased the contents of acid-soluble protein and phenylalanine in FCSM, significantly reduced the NDF content, and enhanced the FG removal rate to 76.50%. In summary, this study screened and mutagenized a strain of FG detoxifying fungus, R. mucilagnosa TG529, which can effectively reduce the FG content and improve the nutritional value of CSM by solid-state fermentation.

1. Introduction

The increasingly affluent population has promoted a demand for livestock and poultry products, which has driven the requirement for large-scale use of agricultural products as feed in feed-intensive production technology [1,2]. Protein feed ingredients play a crucial role in compound feed formulations. In countries such as China, the substantial demand and the scarcity of protein feed lead to a significant dependence on imports, especially for soybean meal, fish meal, and other essential ingredients, partially impeding the progress of animal husbandry [3,4]. Consequently, the exploration of novel plant protein resources has attracted considerable attention in academic circles [5].
Cottonseed meal (CSM), an economical by-product of cotton seed husking and pressing, is notable for its approximately 43~52% crude protein, richness in amino acids, as well as vitamins, indicating its substantial potential as a plant protein feed resource [6,7]. Global production of CSM is estimated to reach around 15 million tons each year [8]. In countries like China, India, Brazil, and Pakistan, CSM serves as a protein-rich ingredient in animal feedstuffs [9,10]. Despite CSM having high nutritional benefits, it contains anti-nutritional factors, primarily gossypol, which will have adverse effects on animal health and reproductive capacity, thereby limiting its application in animal feed and leading to inefficient utilization of protein resources [11,12]. The toxicity associated with gossypol is primarily linked to its free form, while the bound form is relatively non-toxic [13]. Consequently, to improve the application of CSM, numerous approaches were studied, such as physical, chemical, and microbial methods to decrease the content of free gossypol (FG) and enzymatic hydrolysis to enhance protein digestibility.
While conventional physical and chemical techniques can remove FG, they often result in environmental pollution, protein denaturation or degradation, and reduce the nutritional value and palatability of CSM. Conversely, microbial fermentation presents a cost-effective and eco-friendly approach to detoxifying FG and decreasing other anti-nutritional components in CSM, converting it into various compounds. The fermentation process also improves the protein levels and essential amino acid contents, as well as enhances palatability, making it a highly regarded and promising method for detoxifying CSM [14,15]. Previous research has indicated that strains like yeast, Bacillus, and lactic acid bacteria were employed for CSM fermentation [16]. Nonetheless, in an aerobic environment, Saccharomyces cerevisiae tends to produce alcohol exceeding the cell biomass, which limits the animal’s ability to obtain nutrient-rich yeast biomass, including proteins, crucial amino acids, and vitamins [17]. While Bacillus can degrade FG, certain Bacillus strains may decarboxylate or deaminate in amino acid metabolism, producing irritant ammonia, which affects the fermentation flavor [18]. In addition, lactic acid bacteria are capable of reducing anti-nutritional factors; however, the enzyme secretion capacity is relatively weak, resulting in minimal enhancements in the nutritional value of CSM [19]. Therefore, numerous strategies for gossypol removal were being developed and researched, yet achieving an optimal method that effectively degrades gossypol without compromising the nutritional quality of CSM by-products remains a significant challenge.
Factors such as moisture content, strain inoculation ratio, and fermentation time play a role in the efficiency and selectivity of the detoxification process. Optimizing these parameters could enhance the usage of low-gossypol CSM and contribute to its application in protein feedstuffs [8]. In addition, strain mutagenesis technology such as atmospheric and room temperature plasma (ARTP) mutation breeding is also being explored as a potential method for improving detoxification levels. As a result, the purpose of this study was to screen an excellent strain from the cotton field humus soil, optimize fermentation parameters of the strain for improving the nutritional value of CSM, and utilize ARTP mutation technology for improving the strain’s FG removal ration, thereby maximizing the utilization of CSM in animal feeding.

2. Materials and Methods

2.1. Materials

Humus soil samples were collected from cotton fields in the in Aral City, China (40°54′ N, 81°29′ E). Feed-grade CSM was purchased from Zhongcheng Tianli Biotechnology Co., Ltd., Beijing, China. Potato dextrose agar (PDA) and malt extract broth were purchased from Haibo Biotechnology Co., Ltd., Qingdao, China. Gossypol acetate (purity ≥ 98%) was purchased from Lvlai Biotechnology Co., Ltd., Shaanxi, China. Cool the autoclaved PDA to 55 ± 5 °C, then add 2% acetic gossypol-saturated ethanol solution and mix thoroughly to prepare the gossypol-selective medium plates. Analytically pure isopropanol, n-hexane, glacial acetic acid, aniline, 3-amino-1-propanol, and absolute ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.

2.2. Isolation of the Gossypol-Biodegrading Strains

A 1.0 g humus soil sample was dispersed into an Erlenmeyer flask containing 99 mL of sterile malt extract broth and incubated for microbial enrichment at 30 °C and 120 r/min. After 24 h, the microbial enrichment solution was diluted by gradient dilution and coated on gossypol-selective medium plates. Then the plates were incubated inverted at 30 °C until colonies formed. After microbial growth, the strains with different colony morphologies were isolated and purified. The purified strains were then inoculated into PDA and incubated statically at 30 °C for 24 h.
According to the method of Zhang et al., the cells and spores were collected, and the concentration of cells or spores was adjusted to 1 × 106 CFU/mL using sterile water to obtain the fermentation seed solution [20]. Inoculated the fermentation seed solution into the CSM at an inoculation ratio of 8% (mL/g), adjusted the moisture content to 50%, mixed thoroughly in a disinfected plastic container, then packed into polyethylene plastic bags (290 mm × 230 mm; fermented feed bag with a one-way valve; Wenzhou Wangting Packaging Co., Ltd., Wenzhou, China) and vacuum-sealed. Fermented at 30 °C for 5 days. After fermentation, the fermented cottonseed meal (FCSM) was dried at 55 °C, and the FG content was determined using the aniline method (ISO 6866-1985) [21], with unfermented CSM as the control, to calculate the FG removal rate for each treatment group. Set up three parallel groups, and the strain exhibiting the greatest FG removal rate was selected for further experimentation.

2.3. Identification of the Strains

Morphological identification was conducted according to the methods of Zhang et al. [20]. The obtained strains were plated on potato dextrose agar medium and cultured at 30 °C for 3 days. The morphology, size, color, glossiness, texture, and edge characteristics of the colonies were observed.
The genomic DNA of the strains was extracted using a TSINGKE Plant DNA Extraction Kit (Qingke Biotechnology Co., Ltd., Beijing, China). Subsequently, the extracted DNA samples were appropriately diluted and used as PCR templates and its 18S rDNA gene fragment was amplified. PCR amplification was performed using the universal fungal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Following purification, the resultant products were submitted to Qingke Biotechnology Co., Ltd., for sequencing. The obtained sequences were subsequently compared with the sequences of existing-type strains in NCBI via BLAST analysis.

2.4. Determination of Growth Curves

The obtained strain was cultured in malt extract broth at 30 °C and 120 r/min for 26 h. The fermentation broth was sampled every 2 h, with three replicates in each group. The OD600 value of the broth was measured. The culture time was plotted on the horizontal axis and the OD600 on the vertical axis to generate growth curves for the strains.

2.5. Optimization of Solid-State Fermentation Conditions

The optimization of the process was carried out using a single-factor analysis approach. While one parameter was optimized, the other parameters remained at constant levels [22]. Following the fermentation procedure in Section 2.2, the strain was collected in the stationary phase for preparing the seed solution. The seed solution was inoculated into CSM at rates of 2%, 4%, 6%, 8%, 10%, and 12% (mL/g), with a fixed moisture content of 50%, and fermented at 30 °C for 3 days; using unfermented CSM as a control, the FG content in FCSM was measured, and the FG removal rate was calculated to determine the optimal inoculation rate. At the optimal inoculation ratio, the CSM moisture content was adjusted to 35%, 40%, 45%, 50%, 55%, and 60%, respectively, and fermented at 30 °C for 3 days; using unfermented CSM as a control, the FG content in FCSM was measured, and the FG removal rate was calculated to determine the optimal fermentation moisture content. With the optimal inoculation rate and moisture content, CSM was continuously fermented at 30 °C for 13 days; samples were taken on days 3, 5, 7, 9, 11, and 13, respectively; using unfermented CSM as a control, the FG content in FCSM was measured, and the FG removal rate was calculated to determine the optimal fermentation duration. Each treatment was repeated three times.
CSM was fermented under optimized conditions, referred to as the FCSM group, and unfermented CSM was used as a control (CSM group). Test the nutritional components to analyze the impact of fermentation on the nutritional value of CSM.

2.6. ARTP Mutation Breeding of Strains

The fermentation broth of the target strain was collected during the logarithmic phase and centrifuged at 4 °C and 3000 rpm for 5 min. The pellet was collected and washed three times with sterile physiological saline solution, then resuspended in sterile physiological saline to adjust cell concentration between 1 × 106 CFU/mL and 1 × 108 CFU/mL with sterile physiological saline. According to the “Technical specification for mutagenesis breeding of microorganisms” [23], mutagenesis of strains was carried out using an ARTP mutagenesis breeder (Qingtianmu Biotechnology Co., Wuxi, China). The experimental parameters were as follows: irradiation distance of 2 mm, gas flow rate of 10 L/min, power of 100 W, and irradiation time of 60 s, 120 s, and 180 s, respectively. After irradiation, the microbial samples were transferred immediately to sterile centrifuge tubes pre-filled with malt extract broth, mixed well, and incubated at 30 °C, 120 r/min for 30 min. Then, the fungal suspension was diluted by gradient dilution, coated on gossypol selective medium plates, and incubated at 30 °C until colonies formed. The non-irradiated microbial sample (wild strain) was used as a control. The number of colonies was counted in each treatment condition, and the mutagenesis lethality rate was calculated. The groups continued to be cultured until the third day. Then, the largest single colony in each group was picked and inoculated into malt extract broth to prepare the fermentation seed solution of the mutant strains. According to the optimal fermentation conditions determined in Section 2.5, following the fermentation procedure in Section 2.2 to ferment CSM. In the FCSM-ARTP group, inoculate the mutant strain, while in the FCSM group, inoculate the wild strain. After fermentation, the FG removal rates of each group were determined, as were the nutritional components of FCSM in each group.

2.7. Chemical Composition of the FCSM

The detects of crude protein, dry matter, and ether extract were according to AOAC methods [24]. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined by following the principles of Van Soest et al. [25]. Crude fiber was determined by following the method of Henneberg and Stohmann [26]. Gross energy was measured using an isoperibol oxygen bomb calorimeter (SDACM3100, Sundy Science And Technology Co., Ltd., Changsha, China), and acid-soluble protein content was determined according to the “QB/T2653-2004” methods [27]. Amino acids were analyzed using a Hitachi LA-8080 automatic amino acid analyzer (Hitachi Ltd., Tokyo, Japan). For each sample, 0.3 g was weighed and transferred into a sample bottle. Then, we added hydrogen peroxide and formic acid solution and oxidized it for 16 h at 0 °C. After the reaction, sodium metabisulfite was added to decompose the excess peroxyformic acid. Subsequently, a 6 M HCl solution (containing 0.5% phenol) was utilized for hydrolysis over 24 h at 110 °C in a constant-temperature drying oven. After the sample bottle was removed and allowed to cool, the pH was adjusted to 2.20. The sample was then transferred to a 200 mL volumetric flask, where it was mixed, filtered, and prepared for analysis.

2.8. Statistical Analysis

The data from previous strain screening and fermentation parameters Optimization were analyzed using SPSS 26.0 (SPSS, Inc., Chicago, IL, USA) with one-way ANOVA and Duncan’s procedure. An unpaired t-test was conducted to compare the nutritional value of CSM before and after fermentation by wild TG529 and to compare the nutritional value of FCSM fermentation with wild TG529 and mutant TG529. The values are presented as the means ± standard deviation (SD), with p < 0.05 signifying significant differences.

3. Results

3.1. Strain Screening and Identification

As shown in Figure 1, six different fungal strains were isolated from humic soil in cotton fields (Figure 1A–F). Among these, strains A, B, C, and D were filamentous fungi, while strains E and F were yeast-like fungi. Compared to unfermented CSM, the FG content in FCSM treated with all six fungi was significantly reduced (p < 0.05). Notably, the FG content was lowest in FCSM fermented by strain F (named TG529) (p < 0.05), and TG529 exhibited the highest FG removal rate from CSM (p < 0.05), at 56.43% (Figure 1G,H). Therefore, strain F was chosen as the experimental strain for further exploration.

3.2. Identification of the FG Biodegrading Strain

The colony morphology and identification results of TG529 are shown in Figure 2. After 3 days of cultivation on PDA, TG529 formed round, raised colonies with a diameter of 3.0 ± 0.5 mm. The colonies were orange-red, smooth, moist, and had well-defined edges (Figure 2A). Microscopic examination revealed ovoid cells undergoing germination and reproduction, with the absence of pseudohyphae, ascospores, and hurling spores (Figure 2B). The ITS region of the strain was amplified and the electrophoresis map of the amplified products is shown in Figure 2C. The results confirmed that strain TG529 belonged to the species Rhodotorula mucilaginosa (R. mucilaginosa) based on the 100% homologous with R. mucilaginosa CBS 316 (Figure 2D). Therefore, TG529 is named Rhodotorula mucilaginosa TG529 (R. mucilaginosa TG529).

3.3. Growth Study of R. mucilaginosa TG529

The growth curve of R. mucilaginosa TG529 was plotted with time and OD600 as the abscissa and ordinate, respectively, as shown in Figure 3. TG529 exhibited no significant lag phase, with rapid proliferation observed from 0 to 16 h during the exponential phase. Subsequently, from 16 to 26 h, the increase in OD600 slowed down, and the strain entered the stationary phase.

3.4. Optimization of Solid-State Fermentation Condition

The effect of moisture content, TG529 inoculation ratio, and fermentation time on the FG removal in FCSM is shown in Figure 4. As the moisture content increased from 35% to 45%, the content of FG gradually decreased (p < 0.05). When the moisture content ranged from 50% to 60%, the FG content increased with additional water (p > 0.05). At 50% moisture content, the FG content reached the lowest level, which was 514.67 mg/kg. The FG removal rate was 56.24% (Figure 4A,B); subsequently, we investigated the impact of the inoculum ratio on FG removal at a 50% moisture content. The results revealed that as the inoculum ratio increased from 2% to 6%, the FG content decreased (p < 0.05). When the inoculum ratio ranged from 8% to 12%, the FG content remained stable (p > 0.05). Therefore, an inoculum ratio of 8% was selected for the CSM fermentation, with an FG removal rate of 56.24% (Figure 4C,D). Finally, under conditions of 50% moisture and 8% inoculum, the FG content gradually decreased as the fermentation time extended from days 3 to 9 (p < 0.05). At the fermentation times of 11 and 13 days, the FG content stabilizes at 312.33 mg/kg and 311.33 mg/kg, respectively, with FG removal rates of 73.29% and 73.45%, respectively (Figure 4E,F). Thus, the optimum fermentation conditions for CSM were 50% moisture, 8% TG529 inoculation ratio, and 11 days of fermentation time.

3.5. Nutritional Value of R. mucilaginosa-Fermented Cottonseed Meal

Table 1 demonstrates that the fermentation of CSM with R. mucilaginosa TG529 remarkably increased the crude protein content from 46.04% to 51.23% and the acid-soluble protein content from 4.58% to 8.49%, while significantly reducing the crude fiber content from 16.45% to 14.30%, NDF content from 34.55% to 29.92%, and ADF content from 21.02% to 20.38% (p < 0.05). Furthermore, as shown in Table 2, R. mucilaginosa TG529 notably elevated the levels of 18 amino acids in FCSM (p < 0.05).

3.6. ARTP Mutagenesis in R. mucilaginosa TG 529

The TG529 strain was exposed to the ARTP mutagenesis breeder for 60 s, 120 s, and 180 s, resulting in mutated strains labeled as A, B, and C (Figure 5A–C), respectively. The lethality rate of TG529 was positively correlated with the duration of ARTP mutagenesis, with lethality rates of 10.06%, 65.32%, and 94.58% for exposure times of 60 s, 120 s, and 180 s, respectively. After fermenting CSM with the 180s-treated TG529, the FG content was the lowest and significantly lower than that of the 0 s and 60 s treatment groups (p < 0.05). While there were no significant differences in FG removal rates among the treatment groups (p > 0.05), the 180 s treatment group achieved the highest FG removal rate of 76.5%, and the lethality rate for this condition exceeded 90%. Therefore, the TG529 mutant strain (TG529-180s) treated for 180 s was retained for further research.
The effect of ARTP treatment on the improvement of the nutritional quality of fermented CSM by strain TG529 is shown in Table 1 and Table 2. Compared with the wild strain, the ARTP-mutated strain significantly increased the contents of acid-soluble proteins and phenylalanine from 8.49% to 10.47%, and from 29.92% to 25.07%, respectively, and decreased the NDF content from 29.92% to 25.07% in FCSM (p < 0.05).

4. Discussion

Microbial fermentation is a well-established and economical method for detoxifying FG and enhancing protein quality and palatability in CSM [15,28]. Fungi, as ancient eukaryotes, are known as the degraders of various complex hydrocarbons, including polycyclic aromatic hydrocarbons [29] and alkanes [18,30]. A number of fungi were demonstrated to efficiently detoxify CSM, including the ascomycetes Aspergillus niger, Aspergillus oryzae, Pleurotus ostreatus, Candida tropicalis, and Saccharomyces cerevisiae [31,32,33,34]. Building on this, our research isolated a total of six strains from cotton fields by gradually increasing the concentration of gossypol acetate in the medium. Through unautoclaved, pulverized, and no screening CSM with the strains A–F obtained, we observed that the F strain effectively reduced FG levels with a detoxification rate of 56.43%, which was identified and named as R. mucilaginosa TG529. A study demonstrated that Lactobacillus mucosae-XR1 isolated from fresh rumen fluid of sheep showed a 40.65% removal rate of FG for unautoclaved, non-pulverized, and no additional nutrient supplementation CSM [35]. Research indicates that R. mucilaginosa can use polyethylene-derived carbon for cellular incorporation and energy gain, ultimately resulting in the degradation of plastic [36]. As a bioremediation agent, R. mucilaginosa is capable of biosorption, coating, and bioaccumulating Pb(NO3)2 [37]. Therefore, we chose R. mucilaginosa TG529 to further explore the removal rate by optimizing the fermentation conditions. Previous studies have demonstrated that C. tropicalis ZD-3 strains were used as the fermentation autoclaved CSM mixed with corn flour and wheat bran, resulting in significant reductions in FG content (549.06 to 29.80 mg/kg) [32]. The CSM sample was autoclave sterilized at 121 °C for 25 min and fermented with the strain Lactobacillus agilis WWK129, finding the gossypol degradability up to 83% [38]. However, autoclave and heat treatment can transform FG into bound gossypol and significantly negatively affect the quality of CSM [35,39,40]. It is reported that bound gossypol is non-toxic as it cannot be absorbed, while a fraction of it may be released as FG during animal digestion. The bound gossypol should be detected in future studies [41,42].
The appropriate fermentation conditions strongly affect the growth of strains, the production of metabolites, and the nutritional composition of feed [43,44]. The water content in the fermentation medium plays a crucial role in the success of microbial fermentation and significantly affects the activity of microorganisms [31]. In the present study, we found that the FG removal ratio (56.14%) was greatest when the water content was 50%. Inoculum size is one of the most important factors in solid-state fermentation, as it significantly influences the fermentation process [45]. A well-chosen inoculum volume can effectively reduce the growth of harmful microbes, enhancing protection against fermentation [31]. In this study, the inoculum ratio affects the rate of removal of FG. With the increase in the inoculation ratio, the removal rate of FG gradually increased. When the addition ratio was 8–12%, it tended to be stable, and the removal rate was 56.24–56.98%. Therefore, the inoculation ratio of 8% was selected for further study. Fermentation time is also a critical factor in fermentation. As time progresses, microbial biomass accumulates, thus promoting the formation of fermented products [46]. Data indicate that the Panus lecomtei BRM044603 showed considerable efficacy in the removal of 70% FG in CSM after 15 days of cultivation [33]. We found that FG removal significantly increased after 11 days of fermentation, subsequently stabilizing, with a removal rate of 73.29%. The result is consistent with the previous study, which demonstrated that cultivation time has a greater impact on the rate of FG detoxification [47].
Microbial fermentation of CSM was demonstrated to effectively decrease the FG content while simultaneously improving the nutritional value and feed palatability [7,15]. The levels of crude protein, acid-soluble protein, crude fiber, and various amino acids are important indicators for assessing the nutritional value of CSM [48]. This study demonstrated that fermenting CSM with the TGF29 strain resulted in a notable increase in levels of crude protein (46.07% to 51.04%), amino acids such as lysine (1.55 to 2.13%), methionine (0.22% to 0.57%), threonine (1.05 to 1.44%), and tryptophan (0.29 to 0.64%), etc., and acid-soluble protein (4.58% to 8.49%), while also leading to a reduction in crude fiber (16.45% to 14.30%), NDF (34.55 to 29.92%), and ADF content (21.02% to 20.38%). According to previous research, Rhodotorula mucilaginosa PY18 can produce extracellular pectinase enzymes, and Fungi harbor powerful enzymatic machinery comprising, for example, manganese peroxidases, lignin peroxidases, and laccases [49,50]. The findings suggest that the fermentation of R. mucilaginosa TG529 has the potential to boost the nutritional quality of CSM. These results are consistent with the research conducted by Rehemujiang et al. [51] which showed that supplementing with a combination of compound bacteria Bacillus clausii and Saccharomyces cariocanus can improve nutrient composition, reduce anti-nutritional factors, and ultimately achieve detoxification. Eliopoulos et al. [52] discovered that fermentation with Pleurotus ostreatus can reduce free, and total gossypol content, and increase protein content at d 11. Research findings that mixed yeast strains culture of both Candida tropicalis and Saccharomyces cerevisiae used for fermentation increase the crude protein, lysine, and decrease in crude fiber and gossypol profile [34]. A notable portion of the increased proteins can be attributed to yeast proteins and inorganic ammonium salt, as stated in previous research [53]. Furthermore, yeast plays a role in enhancing the amino acid quality by decomposing crude protein using its endogenous enzyme system [54]. Therefore, the fermentation process improves the protein quality of CSM and decreases anti-nutritional factors.
ARTP mutation is an innovative and popular technology used for microbial mutation breeding due to its safety, high mutation rate, and high production efficiency. It has proven to be an efficient means of obtaining high-yield strains in the fermentation industry [55,56]. A study conducted by Liu et al. applied ARTP technology to improve the laccase-producing capabilities of Myrothecium verrucaria. The mutated strains exhibited enhanced enzymatic activity, leading to more efficient degradation of 2-chlorophen [57]. Utilizing ARTP for the mutagenesis of TG529, we observed that the 180s mutated strain significantly enhanced the content of acid-soluble protein and phenylalanine in fermented CSM, while markedly reducing the NDF content. Furthermore, the removal rate of FG was elevated to 76.50%, indicating a substantial improvement over the pre-mutation state. These findings suggest that ARTP mutagenesis of TG529 holds promising potential for enhancing the nutritional and functional properties of fermented CSM. Future research should focus on integrating ARTP with other advanced genetic engineering techniques, such as CRISPR-Cas9, to achieve targeted mutations, screening target genes, and gossy-pol-scavenging enzymes. Moreover, exploring the synergistic effects of ARTP with other environmental factors, such as pH and temperature, could further enhance the detoxifica-tion capabilities of microbial strains.

5. Conclusions

In summary, R. mucilaginosa TG529, isolated from cotton field soil, demonstrates significant efficacy in reducing FG content, a process further enhanced through ARTP technology for detoxification. Through optimization of fermentation conditions, the ideal parameters were established: 8% inoculum concentration, 50% water addition, and an 11-day fermentation period. Under these conditions, the fermentation of CSM resulted in notable improvements in crude protein and acid-soluble protein levels. Concurrently, there were significant reductions in crude fiber, NDF, and ADF content. These findings indicate that fermentation of CSM with R. mucilaginosa TG529 can effectively reduce toxic substances and anti-nutritional factors, enhancing nutritional quality and positioning it as a valuable source of high-quality protein feed.

Author Contributions

Conceptualization, B.L. and C.Z.; methodology, B.L., H.L. and D.L.; software, B.L., M.Z. and C.Z.; validation, B.L., X.M., B.T. and C.Z.; formal analysis, B.L., H.L. and D.L.; investigation, Q.J., J.W., X.M., B.T. and C.Z.; resources, X.M., B.T. and C.Z.; data curation, B.L., C.Z., J.W. and B.T.; writing—original draft preparation, B.L., M.Z. and C.Z.; writing—review and editing, B.L., M.Z., Q.J. and C.Z. visualization, B.L., M.Z. and C.Z. supervision, X.M., J.W., B.T. and C.Z.; project administration, B.L., H.L. and D.L.; funding acquisition, X.M., B.T. and C.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Key R&D Program (2021YFD1301004) and (2021YFD1301005); The Scientific Research Project of Hunan Provincial Education Department (23C0088); The Science and Technology Major Project of Yunnan Province (202202AE090032); Natural Science Foundation of Hunan Province (2024JJ1004).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Hunan Agricultural University Secretary.

Data Availability Statement

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Agriculture 14 01463 g001
Figure 1. FG-removal strains and their effects on FG: (A–F) FG-removal strains; (G) FG content in the CSM or FCSM fermentation by (A–F) strains; (H) the removal rate of FG of (A–F). Different letters indicate significant differences (p < 0.05) among groups.
Figure 1. FG-removal strains and their effects on FG: (A–F) FG-removal strains; (G) FG content in the CSM or FCSM fermentation by (A–F) strains; (H) the removal rate of FG of (A–F). Different letters indicate significant differences (p < 0.05) among groups.
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Figure 2. Identification of the FG biodegrading strain: (A) TG529 growing on PDA plates; (B) microscopic view of TG529 at 40× magnification; (C) PCR amplification electrophoresis results of TG529’s 18S rDNA; (D) phylogenetic tree of TG529.
Figure 2. Identification of the FG biodegrading strain: (A) TG529 growing on PDA plates; (B) microscopic view of TG529 at 40× magnification; (C) PCR amplification electrophoresis results of TG529’s 18S rDNA; (D) phylogenetic tree of TG529.
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Figure 3. Growth curve of R. mucilaginosa TG529.
Figure 3. Growth curve of R. mucilaginosa TG529.
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Figure 4. Optimization of CSM fermentation parameters: (A,B) the effects of different moisture contents on the FG content and removal rate, respectively; (C,D) the effects of different inoculum ratios on the FG content and removal rate, respectively; (E,F) the effects of fermentation time on the FG content and removal rate, respectively. Different letters indicate significant differences (p < 0.05) among groups.
Figure 4. Optimization of CSM fermentation parameters: (A,B) the effects of different moisture contents on the FG content and removal rate, respectively; (C,D) the effects of different inoculum ratios on the FG content and removal rate, respectively; (E,F) the effects of fermentation time on the FG content and removal rate, respectively. Different letters indicate significant differences (p < 0.05) among groups.
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Figure 5. Selection of ARTP-mutagenic strains: (A–C) mutagenic strains after different treatment durations of 60 s, 120 s, and 180 s, respectively; (D) mutagenesis lethality rate; (E,F) the effects of different fermentation times on the FG content and removal rate in FCSM, respectively. TG529-w, TG529 wild strain (0 s treatment). Different letters indicate significant differences (p < 0.05) among groups.
Figure 5. Selection of ARTP-mutagenic strains: (A–C) mutagenic strains after different treatment durations of 60 s, 120 s, and 180 s, respectively; (D) mutagenesis lethality rate; (E,F) the effects of different fermentation times on the FG content and removal rate in FCSM, respectively. TG529-w, TG529 wild strain (0 s treatment). Different letters indicate significant differences (p < 0.05) among groups.
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Table 1. Nutritional value of R. mucilaginosa TG529-fermented CSM.
Table 1. Nutritional value of R. mucilaginosa TG529-fermented CSM.
ItemsCSMFCSMFCSM-ARTP
Gross energy (MJ/kg)19.34 ± 0.0519.30 ± 0.0319.33 ± 0.02
Crude protein content (%)46.04 ± 0.08 b51.23 ± 0.02 a51.67 ± 0.32
Acid-soluble protein contents (%)4.58 ± 0.04 b8.49 ± 0.29 Ba10.47 ± 0.23 Aa
Dry matter (%)95.03 ± 0.2295.01 ± 0.3195.92 ± 0.09
Ether extract (%)1.15 ± 0.051.24 ± 0.051.26 ± 0.02
Crude fiber (%)16.45 ± 0.08 a14.30 ± 0.29 b14.20 ± 0.15
NDF (%)34.55 ± 0.42 a29.92 ± 1.21 Ab25.07 ± 2.32 Ba
ADF (%)21.02 ± 0.23 a20.38 ± 0.29 b21.27 ± 0.81
CSM group, unfermented cottonseed meal; FCSM group, fermented cottonseed meal by wild R. mucilaginosa TG529. FCSM-ARTP group, fermented cottonseed meal by mutagenesis R. mucilaginosa TG529; NDF, neutral detergent fiber; ADF, acid detergent fiber. Different lowercase letters indicate significant differences (p < 0.05) between the CSM group and the FCSM group; Different capital letters indicate significant differences (p < 0.05) between the FCSM group and the FCSM-ARTP group.
Table 2. Amina acids profiles of FCSM by R. mucilaginosa TG529 strain.
Table 2. Amina acids profiles of FCSM by R. mucilaginosa TG529 strain.
ItemsCSMFCSMFCSM-ARTP
Lysine1.55 ± 0.01 b2.13 ± 0.02 a2.10 ± 0.10
Methionine0.22 ± 0.02 b0.57 ± 0.04 a0.59 ± 0.08
Threonine1.05 ± 0.05 b1.44 ± 0.12 a1.56 ± 0.09
Tryptophan0.29 ± 0.02 b0.64 ± 0.05 a0.63 ± 0.07
Arginine3.57 ± 0.04 b5.32 ± 0.12 a5.35 ± 0.12
Histidine0.89 ± 0.06 b1.29 ± 0.03 a1.30 ± 0.08
Isoleucine1.02 ± 0.03 b1.64 ± 0.07 a1.65 ± 0.03
Leucine2.02 ± 0.09 b2.54 ± 0.12 a2.50 ± 0.14
Phenylalanine1.96 ± 0.02 b2.50 ± 0.05 aB2.80 ± 0.09 A
Valine1.50 ± 0.05 b2.42 ± 0.10 a2.33 ± 0.07
Alanine1.33 ± 0.02 b1.94 ± 0.03 a1.93 ± 0.13
Aspartic acid3.10 ± 0.03 b4.48 ± 0.13 a4.49 ± 0.19
Cystine0.34 ± 0.04 b0.69 ± 0.02 a0.64 ± 0.11
Glutamic acid6.52 ± 0.30 b10.57 ± 0.32 a10.50 ± 0.15
Glycine1.36 ± 0.01 b1.81 ± 0.07 a1.76 ± 0.21
Proline1.39 ± 0.03 b2.02 ± 0.07 a2.10 ± 0.20
Serine1.23 ± 0.03 b1.75 ± 0.05 a1.67 ± 0.15
Tyrosine0.89 ± 0.03 b1.22 ± 0.13 a1.24 ± 0.14
CSM group, unfermented cottonseed meal; FCSM group, fermented cottonseed meal by wild R. mucilaginosa TG529. FCSM-ARTP group, fermented cottonseed meal by mutagenesis R. mucilaginosa TG529. Different lowercase letters indicate significant differences (p < 0.05) between the CSM group and the FCSM group; Different capital letters indicate significant differences (p < 0.05) between the FCSM group and the FCSM-ARTP group.
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Liu, B.; Liu, H.; Liu, D.; Zhou, M.; Jiang, Q.; Ma, X.; Wang, J.; Tan, B.; Zhang, C. Free Gossypol Removal and Nutritional Value Enhancement of Cottonseed Meal via Solid-State Fermentation with Rhodotorula mucilaginosa TG529. Agriculture 2024, 14, 1463. https://doi.org/10.3390/agriculture14091463

AMA Style

Liu B, Liu H, Liu D, Zhou M, Jiang Q, Ma X, Wang J, Tan B, Zhang C. Free Gossypol Removal and Nutritional Value Enhancement of Cottonseed Meal via Solid-State Fermentation with Rhodotorula mucilaginosa TG529. Agriculture. 2024; 14(9):1463. https://doi.org/10.3390/agriculture14091463

Chicago/Turabian Style

Liu, Bifan, Huanyu Liu, Daohe Liu, Miao Zhou, Qian Jiang, Xiaokang Ma, Jing Wang, Bi’e Tan, and Chen Zhang. 2024. "Free Gossypol Removal and Nutritional Value Enhancement of Cottonseed Meal via Solid-State Fermentation with Rhodotorula mucilaginosa TG529" Agriculture 14, no. 9: 1463. https://doi.org/10.3390/agriculture14091463

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