Effects of Rosa roxburghii Tratt Must on the Growth, Nutrient Composition, and Antioxidant Activity of Pleurotus ostreatus Mycelia

Rosa roxburghii Tratt, a Rosaceae plant endemic to China, produces fruit with high nutritional and medicinal value. The effects of R. roxburghii must on the growth, nutrient composition, and antioxidant activity of Pleurotus ostreatus mycelia was investigated. We measured the mycelial growth rate, proximate composition, amino acid and crude polysaccharide content, and the antioxidant activity of the crude polysaccharides of P. ostreatus mycelia cultivated under different concentrations of R. roxburghii must (2%, 4%, and 8%, v/v). Low concentrations of R. roxburghii must (2% and 4%) promoted mycelial growth, while a high concentration (8%) inhibited mycelial growth. Low concentrations of R. roxburghii must had no significant effects on the soluble substances, fat, ash, and crude fiber in P. ostreatus mycelia, but significantly increased the crude protein and total amino acid contents (p < 0.05). The addition of R. roxburghii must at low concentrations significantly increased the crude polysaccharide content in mycelia (p < 0.05) but had no impact on the scavenging of hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl (DPPH). Therefore, R. roxburghii must at low concentration can be used as a substrate for P. ostreatus cultivation to increase the protein and polysaccharide contents in mycelia.


Introduction
Pleurotus ostreatus, the oyster mushroom, is an edible fungus of the order Agaricales in the class Agaricomycetes and division Basidiomycota [1]. The fruiting bodies of P. ostreatus are highly favored due to their rich nutrients and pleasant flavor, and P. ostreatus mushrooms are widely consumed. P. ostreatus is the third most consumed edible mushroom in the world following Lentinus edodes and Agaricus bisporus, and the most abundantly and widely cultivated edible mushroom in China [2,3]. With the rapid scaling-up of P. ostreatus cultivation, the shortage of substrates and the increase in production costs have become a bottleneck in the development of the P. ostreatus industry. Therefore, there is a pressing need to identify alternative culture substrates for the production of P. ostreatus.
Rosa roxburghii Tratt is a small deciduous perennial shrub in the Rosaceae family. This species is also known as "Cili" because the fruit is covered with prickles [4,5]. The R. roxburghii endemic to China is mainly distributed in the southwestern regions, such as Guizhou, Yunnan, and Sichuan provinces [6]. Studies have shown that both the pomace and must of R. roxburghii are rich in minerals, organic acids, and amino acids [7]. These nutrients are sufficient for the growth of edible mushrooms, and R. roxburghii fruit pomace and must can be used as excellent cultivation substrates. Yang et al. [8] demonstrated that R. roxburghii fruit pomace as a nitrogen source could support the growth of P. ostreatus by measuring the protein content during the cultivation of P. ostreatus with R. roxburghii pomace. Zhang et al. [9] reported the feasibility of P. ostreatus cultivation with R. roxburghii pomace. Yang et al. [10] further optimized the formulation of R. roxburghii pomace as a substrate for P. ostreatus production. Therefore, it is feasible to use R. roxburghii pomace for the cultivation of P. ostreatus. The use of this substrate not only leads to rapid fruiting body formation and mycelial growth, but also reduces the environmental pollution caused by pomace. Whether the must can also be used as a substrate for P. ostreatus cultivation, and whether using the must would affect the nutrient composition of P. ostreatus mycelia are questions that require further investigation.
In this study, the effects of different levels of R. roxburghii must on the growth rate of P. ostreatus mycelia were determined. Then, the proximate composition and the contents of amino acids and crude polysaccharides in the mycelia of P. ostreatus cultivated with must were examined. Finally, the antioxidant activity of the crude polysaccharides in the mycelia cultivated with fruit juice was analyzed.

Sources of Materials
P. ostreatus of the cultivar P10 was purchased from the China Center of Industrial Culture Collection and maintained in a potato dextrose agar medium (PDA: 200 g/L potato infusion, 20 g/L dextrose, and 15 g/L agar) at 4 • C for later usage.
The R. roxburghii fruit of the cultivar Guinong 5 was obtained from Longli County, Guizhou Province, China ( Figure 1). Fresh and mature R. roxburghii fruits were washed with sterile water, crushed with a juice extractor, and filtered through microporous film (0.22 µm) to acquire the must, which was then added to the medium for the cultivation of the mycelium of P. ostreatus.

Effect of R. roxburghii Must on the Growth of P. ostreatus Mycelia
First, PDA containing R. roxburghii must (RM) with a concentration of 3% (v/v) was used to evaluate whether R. roxburghii must would affect the growth of mycelia. The PDA without R. roxburghii must was set as a control. The mycelium discs (1 cm in diameter) of P. ostreatus were placed in PDA medium plates under aseptic conditions and incubated at 28 • C in darkness for 5 days. The diameter of the mycelium was measured, and the colony morphology was noted [11]. Three parallel replicates were performed for each sample.
Then, three different additive amounts of R. roxburghii must with concentrations of 2%, 4%, and 8% (v/v) were added to the PDA. The diameters of the three groups of mycelia were recorded and compared with the control group using the above method.
Cottonseed hull medium was prepared at a substrate-to-water ratio of 1:1.2 (containing 1% lime) and sterilized in a test tube (20 cm × 200 mm) at 121 • C for 30 min. Identical mycelial plugs of P. ostreatus were made with a punch (1 cm in diameter), inoculated into the test tubes with the cottonseed hull medium, and cultured at 28 • C. The mycelial growth was observed and recorded. Each sample had three replicates.

Effects of R. roxburghii Must on the Nutrient Composition of P. ostreatus Mycelia
The mycelia of P. ostreatus cultured in each group were dried in an oven to a constant weight and ground into powder for determination of the nutrient composition.
Determination of the proximate composition: crude protein was measured using the micro-Kjeldahl method [12]; the fat content was measured by Soxhlet extraction [13]; and the measurement of soluble substances, ash, and crude fiber was performed following the methods described by Wang et al. [14].
Determination of amino acids: dry mycelia were hydrolyzed with hydrochloric acid (6 mol/L), and the amino acid content was determined using an automatic amino acid analyzer (A300, MembraPure, Hennigsdorf, Germany). Cysteine and methionine were measured as cysteic acid and methionine sulfone, respectively, after performing acid oxidation and 20% HCl hydrolysis at 150 • C for 20 h. The tryptophan analysis was conducted on a Ba(OH) 2 hydrolysate.

Effects of R. roxburghii Must on the Crude Polysaccharide Content and Antioxidant Activity of P. ostreatus Mycelia
One hundred grams of dry powder from the P. ostreatus mycelia was prepared and the crude polysaccharides extracted with hot water (65 • C, 2.5 h, 1:30, w/w). The extract was filtered and concentrated at 65 • C in a rotary evaporator under reduced pressure, precipitated with 95% ethanol at a 1:3 ratio (extract:ethanol, v/v), and kept overnight at 4 • C. The extract was then centrifuged (4000× g, 30 min). The obtained precipitate was freeze-dried, and the crude polysaccharide preparation was finished. Finally, the yield and content of crude polysaccharides were calculated.
Analysis of antioxidant activity: the assessment of the scavenging ability of the crude polysaccharides of P. ostreatus on hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was conducted according to the method previously described by Zhang [15].

Statistical Analysis
The results were the mean ± standard deviation of a triplicate analysis. Statistical comparisons were conducted using the Student's t test. Differences were considered statistically significant at p < 0.05.

Physicochemical Parameters of R. roxburghii Must
The cultivar of R. roxburghii used in this study was Guinong 5 (Figure 1), which was developed in Longli County, Guizhou Province, China, with an average fruit weight of 21.26 ± 1.58 g. The total sugar content of the fruit juice was 5.22 ± 0.34 g/100 g FW, the total acid content was 987.13 ± 73.69 mg/100 g FW, and the soluble protein content was 0.34 ± 0.02 g/100 g FW (Table 1).

Effects of R. roxburghii Must on the Growth of P. ostreatus Mycelia
To analyze the effects of R. roxburghii must on the growth of P. ostreatus mycelia, the growth of mycelia on the PDA medium with (3% RM v/v) and without (control) must were compared. The results showed that the mycelia were denser and thicker, and the growth was faster with must ( Figure 2A,B). The mycelial diameter and growth rate with must were significantly improved compared to the control ( Figure 2C,D).
Further analysis focused on whether the effects of must on mycelial growth were concentration-dependent. R. roxburghii must was added to the PDA medium at different concentrations, and the mycelial diameter and growth rate were measured. The results showed that the mycelial diameter and growth rate significantly increased with increasing R. roxburghii must concentrations in the range of 0 to 4%. Mycelial diameter and growth rate reached the maximum at 4% RM but decreased at 8% RM ( Figure 3).  In addition, this study analyzed the growth characteristics of P. ostreatus mycelia on a cottonseed hull medium. The mycelial growth on the cottonseed hull medium with different concentrations of R. roxburghii must was similar to that in the PDA medium. Mycelial growth was significantly accelerated by the addition of 2% and 4% RM but was inhibited by 8% fruit juice (Figure 4). The inhibition of mycelial growth with 8% RM in the cottonseed hull medium was less than that in the PDA medium. The results showed that low concentrations of R. roxburghii must promoted the growth of P. ostreatus mycelia, while high concentrations of must inhibited mycelial growth. The optimum concentration of 4% was used for the subsequent experiments.

Effects of R. roxburghii Must on the Nutrient Composition of P. ostreatus Mycelia
To investigate the effects of R. roxburghii must on the nutrient composition of P. ostreatus mycelia, the differences in the proximate composition, amino acid composition, and vitamin content in mycelia with (4%) and without (control) fruit juice were analyzed. Table 2 shows the proximate composition of P. ostreatus mycelia. There were no significant differences in the soluble substances, fat, ash, or crude fiber content in mycelia with and without R. roxburghii must. However, the content of crude protein was significantly increased with the addition of R. roxburghii must.  Table 3 displays the differences in amino acids in the mycelia of P. ostreatus. The total amino acid content in the mycelia cultured with R. roxburghii must (12.29 ± 0.41%) was significantly higher than that in the control (11.23 ± 0.37%), though there was no significant difference in the total content of essential amino acids (5.02 ± 0.18% with RM vs. 5.42 ± 0.17% without). The content of phenylalanine was significantly reduced by the addition of R. roxburghii must, whereas the levels of eight amino acids (threonine, alanine, isoleucine, lysine, proline, tryptophan, arginine, and glutamic acid) were significantly increased. An analysis of the content of vitamins in the mycelia of P. ostreatus cultured with and without R. roxburghii must was also conducted. As shown in Table 4, no significant differences in thiamine, riboflavin, and niacin were observed. Table 4. Vitamin content of the P. ostreatus mycelium grown on PDA (mg/100 g, dry weight basis).

Effects of R. roxburghii Must on the Polysaccharide Content and Antioxidant Activity of P. ostreatus Mycelia
The results showed that there was no significant difference in the yield of crude polysaccharides between the two groups, but R. roxburghii must significantly increased the crude polysaccharide content in the P. ostreatus mycelia ( Figure 5).
Further analysis was conducted on the antioxidant activity of the crude polysaccharides in the mycelia of P. ostreatus cultivated with and without R. roxburghii must. As shown in Figure 6A, the scavenging of hydroxyl radicals increased with the increase in polysaccharide concentration. There was no significant difference in the scavenging of hydroxyl radicals by crude polysaccharides between the two groups. Figure 6B illustrates the scavenging of DPPH by the mycelial polysaccharides of P. ostreatus. The scavenging of DPPH in both groups increased with increasing crude polysaccharide concentration, and no significant difference was found between the two groups.

Discussion
P. ostreatus is an edible mushroom that is widely cultivated around the world because of its unique flavor and high adaptability to cultivation substrates [16]. With the worldwide grain shortage and rising grain prices, especially due to the impact of the COVID-19 pandemic on agricultural production in the past two years, the cultivation of P. ostreatus has been greatly impacted. Therefore, it is necessary to find alternative substrates for P. ostreatus production. As a by-product of the fruit processing industry, pomace can be used as a substrate for P. ostreatus cultivation and has the advantages of low cost, easy accessibility, and low technical complexity [17]. Apple [18], grape [19], and grapefruit [20] pomaces have been studied and confirmed as high-quality substrates for the cultivation of P. ostreatus.
R. roxburghii is endemic to the southern regions of China and has high nutritional and medicinal value. The fruit has received extensive attention and intensive investigation in recent years [21], and the planting area of R. roxburghii is increasing every year. In Guizhou Province, for example, the planting area reached 140,000 hectares in 2021, and the total industrial output value exceeded USD 500 million, an increase of more than 53% over the same period last year. Therefore, China is rich in R. roxburghii resources. Unfortunately, the fruit contains a high phenol content, giving it a sour, astringent, and unpleasant taste, and is, therefore, often used for deep processing to produce juice and pomace. The pomace has been shown to be useful in the cultivation of P. ostreatus, with the advantages of fast fruiting body formation, a short growth cycle, and high production efficiency [8][9][10]. Whether the must of R. roxburghii can also be used as a substrate for the production of P. ostreatus has remained unknown. The results of this study revealed that low concentrations of R. roxburghii must (≤4% v/v) promoted the growth of P. ostreatus mycelia, while a high concentration (8% v/v) inhibited mycelial growth. Therefore, R. roxburghii must at low concentrations can be used as a substrate for the cultivation of P. ostreatus mycelia.
Studies have shown that the growth of P. ostreatus mycelia can be influenced by multiple factors [22]. For example, a high histidine content in media promotes mycelial growth in P. ostreatus [23], while trace elements can also affect the growth of mycelia. In a previous study, Zn 2+ promoted branching and increased the dry weight of mycelia, while Mn 2+ and Cu 2+ inhibited the elongation of P. ostreatus mycelia [24]. The carbon-to-nitrogen ratio in media can also affect the growth rate of P. ostreatus mycelia [25]. The must of R. roxburghii contains a variety of trace elements such as iron, zinc, and copper, in addition to abundant amino acids and vitamins [26]. These nutrients may have various impacts on the mycelial growth of P. ostreatus. Therefore, this study demonstrated that the growth of P. ostreatus mycelia increased and then decreased with increasing concentrations of R. roxburghii must. The underlying reasons for this finding need to be further studied.
The results also showed that must at low concentration (4% v/v) significantly increased the crude protein and amino acid contents in the mycelia of P. ostreatus. This may be explained by the rich carbon and nitrogen sources in R. roxburghii must.
Polysaccharides have been extracted from the fruiting bodies, mycelia, and mycelial fermentation broth of P. ostreatus, and exhibit reported antioxidant, antitumor, and antiviral activities [27,28]. In the present study, addition R. roxburghii must could increase the content rather than the yield of crude polysaccharides in mycelia. The antioxidant activities of mycelial polysaccharides towards hydroxyl radicals and DPPH were similar under both culture conditions.

Conclusions
R. roxburghii must improved the overall antioxidant performance of polysaccharides in the mycelia of P. ostreatus. R. roxburghii must had dual effects on the growth of P. ostreatus mycelia: low concentrations (not more than 4% v/v) promoted the growth of P. ostreatus mycelia, while a high concentration (8% v/v) inhibited the growth of mycelia. The addition of R. roxburghii must had no effect on the soluble substances, fat, ash, and crude fiber in the mycelia of P. ostreatus, but significantly increased the crude protein and amino acid contents. In addition, R. roxburghii must increased the content of crude polysaccharides in the mycelia and thus improved the total antioxidant activity. Taken together, these findings indicate that R. roxburghii must, at appropriate concentrations, can be used as a natural substrate for the cultivation of P. ostreatus mycelia.