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2024 Volume 9
Article Contents
Abstract
Introduction
Materials and methods
Identification of genes in the genome of S. miltiorrhiza
Multiple sequence alignment and phylogenetic tree construction
Gene structure analysis and cis-elements of SmDofs promoters
RNA-seq and qRT-PCR detection
Co-expression analysis
Subcellular localization
Dual-luciferase assay
Measuring tanshinones and phenolic acids contents by high-performance liquid chromatography
Statistical analysis
Results
Identification of the SmDof genes
Sequence alignment and phylogenetic traits of SmDof proteins
Structural feature of SmDof genes
Cis-acting elements in the promoter region of SmDof genes
Expression patterns of SmDofs and synthetase genes involved in tanshinones and phenolic acids biosynthesis pathway in various tissues and under ABA treatment
Co-expression relationship of SmDofs with the biosynthetic genes involved in tanshinones and phenolic acids biosynthesis in S. miltiorrhiza
Expression pattern and subcellular localization analysis of SmDof12 and SmDof29 genes
Validation of the target genes of SmDof12 and 29 proteins by Dual-LUC assay
Discussion
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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ARTICLE   Open Access    

Economical substrate formulation for mushroom cultivation and food production of mushroom crackers to reduce postharvest waste

  • 1.

    Faculty of Agro-Based Industry, Universiti Malaysia Kelantan Jeli Campus, 17600 Jeli, Kelantan, Malaysia

  • 2.

    Institute of Food Security and Sustainable Agriculture, Universiti Malaysia Kelantan Jeli Campus, 17600 Jeli, Kelantan, Malaysia

More Information
  • Received: 02 September 2023
    Revised: 02 March 2024
    Accepted: 13 March 2024
    Published online: 29 March 2024
    Studies in Fungi  9,  Article number: e002 (2024)  |  Cite this article
  • Agriculture residues of oil palm waste are a big issue for palm oil producing countries. The residues from oil palm fronds are the most crucial to convert wealth. This study focused on oil palm-related agro biomass for mushroom substrate formulation for grey oyster mushroom cultivation. Mushrooms are a highly perishable vegetable that turn into postharvest waste within 4 to 7 d at normal temperatures. Therefore, in this study, the unsold mushroom was converted as a cracker food product to reduce the postharvest losses, especially for small-scale mushroom growers. The agriculture biomass used for substrate preparation is a combination of oil palm frond (OPF), oil palm empty fruit bunch (EFB), palm pressed fiber (PPF), and sawdust (SD). SD as a commercial substrate was used as a control in this study, and rice bran (RB) and lime (L) were used as supplement ingredients for all the treatments. The treatments were according to mixed formulation with the ratio of T0 (control: 97.2% SD, 0.8% RB + 2% L), T1 as a mixed ratio (60% RS + 22.2% EFB + 15% PPF + 0.8% RB + 2% L) and T2 as a mixed ratio (60% OPF + 22.2% EFB + 15% SD + 0.8% RB + 2% L). The total yield in four cycles showed 1.2 kg in T0 (sawdust), 1.4 kg in T1 (majority of rice straw), and 1.5 kg (majority of oil palm frond) in T3 treated substrate. In this study, the oil palm frond was received free of charge as compared to sawdust and rice straw. Therefore, it showed that using the oil palm frond not only gave a high yield of mushrooms at the same time, it was 100 X lower in cost. Next, the unsold yielded mushrooms were used for cracker preparation. The results obtained from this study indicate that mushroom crackers contain fat (11.34%), protein (2.19%), and carbohydrate (76.55%) while being high in moisture (7.87%) and ash (2.06%) compared to commercial potato crackers. Overall acceptance of sensory evaluation towards mushroom crackers showed a high 'extremely like' percentage, contributing about 66%. Thus, this study found that 66% of participants 'extremely liked' the new innovative mushroom crackers. Overall, the results show that oil palm substrate can be an alternative economical substrate for grey oyster mushroom cultivation and food products from mushrooms will be new items in the snack industry.

  • Introduction

    Salvia miltiorrhiza is one of the most commonly used Chinese medical herbs. As a representative species of the Lamiaceae, it is widely used in the treatment of cardiovascular and cerebrovascular diseases[1]. Its main active components are phenolic acids and tanshinones, of which phenolic acids consist of salvianolic acid A (Sal A), salvianolic acid B (Sal B), caffeic acid (CA), and rosmarinic acid (RA)[2−4], while tanshinones include dihydrotanshinone (DT), cryptotanshinone (CT), tanshinone I (TI), and tanshinone IIA (TIIA). The above active ingredients have various pharmaceutical values including anti-tumor, antioxidant, and anti-inflammatory effects[5−8]. In recent years, several transcription factors have been reported to participate in and regulate the synthesis of secondary metabolites of S. miltiorrhiza[9]. DNA binding with one finger (Dof) family is a typical transcription factor (TF) family with zinc finger proteins domain, which is unique to plants and plays an important role in modulating plant growth and development[10]. The Dof family has two main regional domains, namely the N-terminal conserved DNA binding domain and the C-terminal transcriptional regulation domain[10]. The N-terminal of the Dof protein is usually a highly conserved C2-C2 zinc finger domain consisting of 50−52 amino acids, and it can bind to the AAAG cis-acting element in the promoter region of the target gene[11]. The DNA binding domain is a key region, that is considered to be a bidirectional domain and can interact with other proteins[11−13]. The transcriptional regulatory domain of the C-terminal region may perform a variety of functions as it interacts with different regulatory proteins to activate the expression of target genes[11,13].

    Dof proteins play a vital role in plant carbon and nitrogen metabolism[14,15], abiotic stress[16], hormone regulation[17], flowering control[18], light responses, and others[19]. Dof gene (ZmDof1) was first discovered in Zea mays[20], and it was thought to participate in the process of carbon metabolism by regulating the expression of the C4 photosynthetic phosphoenolpyruvate carboxylase (C4PEPC) gene in Z. mays[21]. JcDof3 interacts with F-box protein to regulate photoperiodic flowering and affect the flowering time[22]. In addition, multiple studies have shown that Dof genes are involved in various environmental changes[23]. OsDof18 is associated with the transport of ammonium salt in rice, thus regulating the utilization efficiency of nitrogen in rice[24] , and it can also restrict the biosynthesis of ethylene and increase prophase primary root elongation[17]. The expression of ThDof1.4 and ThZFP1 of Tamarix ramosissima can increase the content of proline and enhance the scavenging ability of ROS, thus improving the tolerance of Tamarix to salt stress and osmotic stress[25]. In Arabidopsis thaliana, AtCDF3 was highly induced by drought, low temperature, and abscisic acid (ABA), and the overexpression of AtCDF3 in transgenic plants enhanced their tolerance to drought, cold, and osmotic stress[26]. SlDof22 is involved in the production of ascorbic acid and the process of tomato salt stress in tomato[27]. These studies uncovered the importance of Dofs in the life cycle of plants.

    Plant hormones are trace compounds involved in the whole process of plant growth and development, and influence the growth and development of plants[28]. ABA, as an important plant hormone can accelerate the shedding of plant organs, and can impact the synthesis of secondary metabolites by stimulating the corresponding transcription factors in plants[9]. In S. miltiorrhiza, ABA can induce the expression of SmbZIP1 leading to the upregulation of SmC4H1 to promote the accumulation of phenolic acids[9]. ABA can also significantly promote the expression of HMGR, FPS, CYP71AV1, and CPR, thus increasing the content of artemisinin in Artemisia annua[29].

    In recent years, the Dof gene family has been gradually identified in many plants due to the continuous publication of the high quality of plant genomes. There were 36 Dof genes in Arabidopsis[30], 103 Dof genes in Camelina sativa[31], 34 Dof genes in melon[32], and 51 Dof genes in blueberry[33]. However, the Dof family has not been fully explored in the whole genome of S. miltiorrhiza. Due to the importance of the Dof gene in various physiological processes of plants, it is necessary to study its specific role in S. miltiorrhiza. In the present study, genome and transcriptome data of S. miltiorrhiza were used to identify the Dof genes. Then, multiple sequence matching, evolutionary tree analysis, gene structure, and cis-acting element analysis were systematically investigated in the whole genome of S. miltiorrhiza. To predict the function of SmDofs in regulating the biosynthesis of tanshinones and phenolic acids in S. miltiorrhiza, co-expression analysis of the biosynthetic pathway genes related to tanshinones and phenolic acids and the SmDofs was performed based on the transcriptome data induced by ABA, and then the target gene of candidate SmDofs were validated by the dual luciferase (Dual-LUC) assay. This study enlarges the understanding of the SmDof gene family, and reveal the potential molecular mechanism of SmDofs in regulating the biosynthesis of tanshinones and phenolic acids in S. miltiorrhiza.

    Materials and methods

    Identification of genes in the genome of S. miltiorrhiza

    The genome sequences were downloaded from the S. miltiorrhiza database[34]. Based on the Pfam database (http://pfam.xfam.org/), the Hidden Markov Model (HMM) file of the Dof gene family (PF02701.18) was obtained, and the whole genome of S. miltiorrhiza compared using the HMMER search program in HMMER3.0 software package to obtain the gene sequence of the initial screening[35]. SMART (http://smart.embl-heidelberg.de/) and MOTIF Search (www.genome.jp/tools/motif) are employed to predict the structure of the candidate protein domains. ExPASy (http://web.expasy.org/compute_pi/) was used to calculate the sequence length, molecular weight, and isoelectric point[36]. Finally, WoLF PSORT (https://wolfpsort.hgc.jp/) was introduced to predict the subcellular localization of the identified Dof proteins[37].

    Multiple sequence alignment and phylogenetic tree construction

    The conserved domain of SmDofs protein was studied by multiple sequence alignment using the DNAMAN 7.0 software. The AtDof protein sequences of A. thaliana were downloaded from the TAIR database (www.arabidopsis.org)[38]. AtDofs and SmDof proteins were analyzed using MEGA 6.0. The phylogenetic tree was constructed using the neighborhood join method (NJ) with the bootstrap value set to 1,000[39].

    Gene structure analysis and cis-elements of SmDofs promoters

    The organization of exons, introns, and untranslated regions of the SmDof genes were analyzed using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/), and visualized by loading the GFF files of SmDof genes of S. miltiorrhiza to the TBtools (v.2.003) software, which was also used for analyzing and searching for conserved motifs[40]. PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was introduced to study the cis-acting elements in a length of 1,500-bp in the upstream of the initiation codon of the 31 SmDof genes in S. miltiorrhiza. According to the functional annotations of cis-acting elements, the candidate elements were gathered for further research[41].

    RNA-seq and qRT-PCR detection

    Two transcriptome datasets of S. miltiorrhiza, of which one is generated from four tissues including flower, stem, leaf, root, and another is collected from hairy roots induced by ABA, were adopted to analyze the expression level of SmDof genes[42]. TBtools (v.2.003) software was employed to draw a heat map to exhibit the expression level of the SmDof genes derived from transcriptome dataset[40]. To detect the expression profile of candidate SmDof genes, hairy roots of S. miltiorrhiza were treated with 50 μM ABA and collected after treatment for 0-, 0.5-, 1-, 2-, 4-, and 8-h, respectively[7] . The collected samples were quickly placed in liquid nitrogen and stored in the refrigerator at −80 °C for subsequent RNA extraction. Total RNA was extracted from S. miltiorrhiza hairy roots using the Plant Total RNA Extraction Kit (Vazyme Biotech Co., Ltd, China). Meanwhile, the concentration and purity of the extracted RNA was measured by spectrophotometer, and then the RNA integrity was observed by electrophoretic analysis with 1% agarose gel. Reverse transcription was performed with the cDNA Synthesis Kit (Vazyme Biotech Co., Ltd, China), and a total of 100 ng RNA was prepared for cDNA synthesis reaction with a volume of 50 μL[43]. Quantitative primer pairs were designed using the Primer 5.0 software. SuperReal PreMix Plus kit (Vazyme Biotech Co., Ltd, China) was used in ABI Step One Plus real-time PCR System. Quantitative real-time PCR (qPCR) was performed using 10 μL real-time PCR reaction solution, including 1 μL cDNA was used as a template; the upper and downstream primers were 0.2 μL, respectively; 5 μL Taq Pro SYBR qPCR Master Mix and 3.6 μL ddH2O. The PCR reaction conditions were as follows: 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, a total of 40 cycles, each sample was triply repeated. SmActin was used as the internal reference gene to normalize the expression level of Dof genes. The method of 2−ΔΔCᴛ was used to calculate the relative expression level of SmDofs[7].

    Co-expression analysis

    The co-expression relationship between the SmDof genes and the biosynthetic genes involved in tanshinones and phenolic acids biosynthesis was resolved. Pearson correlation coefficient > 0.8 and p-value < 0.05 was set as the cutoff. Then, the co-expression relationship was visualized with the Cytoscape software[44].

    Subcellular localization

    To dissect the subcellular localization profiles of SmDof proteins, the open reading fragment (ORF) cDNA sequences of SmDof12 and SmDof29 are amplified and inserted into the vector of PHB-YFP to generate the fusion recombinant of PHB-SmDof12-YFP and PHB-SmDof29-YFP, and then they are transformed into Agrobacterium tumefaciens GV3101 and injected into N. benthamiana leaves for transient transformation, respectively[3]. pHB-YFP was used as the negative control. The transgenic N. benthamiana leaves were cultivated in the dark for 24 h and then transferred to the light for 24 h. YFP signals from infected N. benthamiana leaves were visualized using a high-resolution microscope observation system. The nuclei of epidermal cells of infected N. benthamiana leaves were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (10 μg/mL) for 2 h before observation.

    Dual-luciferase assay

    To investigate the ability of SmDofs to transcriptionally activate the tanshinones biosynthetic genes, Dual-luciferase (Dual-LUC) assays were performed as previously reported[45]. Each of the recombinant plasmids of PHB-SmDof12-YFP and PHB-SmDof29-YFP was introduced into A. tumefaciens strain GV3101 to be the effector, and PHB-YFP plasmid was used as a negative control. The promoters of PAL and GGPPS were inserted into pGREEN0800 vector as the reporter constructs to drive the expression of the firefly luciferase gene, respectively. The Renilla luciferase gene driven by CaMV 35S promoter was used as an internal control. And then, each of them was co-transformed into A. tumefaciens strain GV3101 with the helper plasmid pSoup19. The reporter strains were mixed with effector strains at a ratio of 1:1 to inject into N. benthamiana leaves. Leaves were collected after 48 h for determination of fluorescence values according to the manufacturer's instructions (Promega, Madison, WI, USA)[9]. Three biological replicates were measured for each sample.

    Measuring tanshinones and phenolic acids contents by high-performance liquid chromatography

    Different tissues including roots, stems, leaves, and flowers of S. miltiorrhiza were collected and dried in an oven. The dried tissues were then ground to powder for compound analysis. Extraction of tanshinones and phenolic acids and high-performance liquid chromatography (HPLC) detection were done as the previous report[6,9]. The total content of tanshinones and phenolic acids were quantified by comparing the standard curves and retention times, with solutions without extracts added as the controls.

    Statistical analysis

    All the detections performed in the present study, including qRT-PCR, HPLC, and Dual-LUC assays, were triply repeated. Gene expression levels, tanshinone contents, and phenolic acid contents were presented as the mean value ± SD. SPSS 16.0 software (SPSS) was employed to analyze statistical significance by single-sample t-test and one-way analysis of variance. p-value < 0.05 was regarded to be statistically significant.

    Results

    Identification of the SmDof genes

    The Hidden Markov model (HMM) of the Dof domain (PF02701.18) was employed to search for Dof genes in S. miltiorrhiza. A total of 31 Dof genes were detected, and the gene was named SmDof1-SmDof31, respectively (Supplementary Table S1). The results of Pfam and SMART analysis showed that all of these proteins contained complete Dof domains[23]. The CDS length, protein molecular weight (MW), isoelectric point (pI), and subcellular location of each SmDof gene in S. miltiorrhiza were further analyzed (Table 1). Of the 31 proteins, SmDof25 and SmDof22 had the lowest number of amino acids, decreasing to 168, while SmDof16 had the highest number of amino acids, reaching to 511. The pI of SmDofs ranges from 6.01 (SmDof5) to 10.55 (SmDof17), and the molecular weight ranges from 18,463.7 (SmDof22) to 55,341.6 (SmDof16). Subcellular localization prediction revealed that 27 SmDofs were located in the nucleus, while four SmDofs including SmDof19, 21, 22, and 25 located in chloroplasts (Table 1).

    Table 1.  Length, molecular weight, isoelectric point, and subcellular localization of 31 SmDof proteins in S. miltiorrhiza.
    Gene ID Name Length (aa) MW (Da) pI Subcellar
    localization
    SMILT016590.1 SmDof1 304 33,168.7 8.66 nucleus
    SMILT016591.1 SmDof2 246 26,202.9 9.8 nucleus
    SMILT016651.1 SmDof3 242 26,184.9 8.96 nucleus
    SMILT021318.1 SmDof4 225 23,560.2 8.6 nucleus
    SMILT032678.1 SmDof5 224 24,689.4 6.01 nucleus
    SMILT003591.1 SmDof6 241 25,256.1 4.66 nucleus
    SMILT009582.1 SmDof7 306 32,436.3 4.69 nucleus
    SMILT017417.1 SmDof8 301 33,248.9 6.7 nucleus
    SMILT020107.1 SmDof9 332 36,827.9 7.94 nucleus
    SMILT023380.1 SmDof10 318 34,176.8 9.72 nucleus
    SMILT025505.1 SmDof11 283 30,690.9 8.48 nucleus
    SMILT025760.1 SmDof12 274 30,006.1 8.47 nucleus
    SMILT028288.1 SmDof13 249 27,303.1 8.99 nucleus
    SMILT030586.1 SmDof14 334 36,582.9 6.92 nucleus
    SMILT031093.1 SmDof15 230 23,444.9 8.49 nucleus
    SMILT000323.1 SmDof16 511 55,341.6 5.23 nucleus
    SMILT000784.1 SmDof17 265 27,611.4 10.55 nucleus
    SMILT000789.1 SmDof18 198 22,350 9.04 nucleus
    SMILT001058.1 SmDof19 190 20,795.1 9.27 chloroplast
    SMILT001687.1 SmDof20 216 24,023.7 9.28 nucleus
    SMILT002891.1 SmDof21 268 29,359.3 4.54 chloroplast
    SMILT004451.1 SmDof22 168 18,463.7 8.83 chloroplast
    SMILT005491.1 SmDof23 266 29,274.4 9.31 nucleus
    SMILT007077.1 SmDof24 251 27,427 9.06 nucleus
    SMILT007580.1 SmDof25 168 18,625.9 9.22 chloroplast
    SMILT009335.1 SmDof26 191 21,529.9 9.5 nucleus
    SMILT010473.1 SmDof27 240 24,863.9 7.82 nucleus
    SMILT012697.1 SmDof28 248 26,508.7 9.28 nucleus
    SMILT019592.1 SmDof29 283 30,740.7 8.39 nucleus
    SMILT023561.1 SmDof30 337 35,816.5 9.51 nucleus
    SMILT024154.1 SmDof31 258 27,841.9 8.06 nucleus
     | Show Table
    DownLoad: CSV

    Sequence alignment and phylogenetic traits of SmDof proteins

    To dissect the characteristics of the domain within SmDof proteins, DNAMAN software was employed to conduct multiple amino acid sequence alignment. The results showed that all the SmDof proteins contained a conserved domain in its core sequence, namely CX2CX21CX2C zinc finger structure (Fig. 1). The conserved domain consists of 50 amino acid residues, of them four cysteine residues are relatively conserved within the zinc finger domain in the N-terminal region of SmDof proteins[11].

    Figure 1.  Multiple sequence alignment of the 31 SmDof proteins. Different colors represent identical and conserved amino acid residues, and the red box shows the conserved zinc-finger domain.
    DownLoad: Full-Size Img PowerPoint

    To further explore the evolutionary relationships among the SmDof genes, a phylogenetic tree of a total of 67 Dof proteins (Supplementary Table S2) in Arabidopsis (36 members) and S. miltiorrhiza (31 members) were constructed. The total number of Dof genes in S. miltiorrhiza and A. thaliana is comparatively secure, and it indicates the conservative features of this gene family. Sixty seven Dof proteins are divided into five groups based on the branch of the tree, Groups I−V (Fig. 2). There are 31 SmDof gene families in S. miltiorrhiza, among them, six SmDofs are distributed in Group I and Group IV, 11 in Group II, and eight in Group III. In Arabidopsis, Groups I to IV contain 7, 0, 0, 10, and 19 Dof genes, respectively. The variable number of the five subgroups is beneficial for us to evaluate the degree of gene expansion or loss during the evolution of the two species.

    Figure 2.  Evolutionary relationship of SmDof proteins in S. miltiorrhiza and Arabidopsis. Varied colors represent different groups. There were five groups, Groups I−V, with the green circles representing the SmDof proteins of Arabidopsis and the orange circles representing the Dof proteins of S. miltiorrhiza.
    DownLoad: Full-Size Img PowerPoint

    Structural feature of SmDof genes

    To further investigate the functional regions of SmDof proteins, the conserved motif was predicted by the MEME program utilizing a two-component finite mixture model. In total, 15 motifs were identified in all the SmDof proteins, and we found that many groups of SmDofs shared a similar conserved motif. As shown in Fig. 3a and b, motif 1 is included in all SmDof proteins. Among all groups, Group I contained the most SmDof members being consisted of motifs 1, 2, 3, 8, and 15. The common motifs among the SmDof proteins are indicative of conserved evolutionary relatedness and similar biological functions.

    Figure 3.  Phylogeny, conserved motifs and gene structure of SmDof proteins in S. miltiorrhiza. (a) SmDof proteins evolutionary tree. (b) Conserved motifs of the 31 SmDof proteins. Different colors represent 15 different motifs, and the bottom line represents the length of the sequence. (c) Exon/intron structures of SmDofs. Green represents UTRs and yellow represents CDS.
    DownLoad: Full-Size Img PowerPoint

    To study the structure of SmDof genes, the full-length cDNA sequences of all SmDof genes with the corresponding genomic DNA were aligned (Fig. 3c). The number of exons in SmDof ranged from 1 to 2. There were no more than two introns in each SmDofs. The variation in the number of exons may indicate that the SmDof genes may have diverse functions related to the medicinal substance biosynthesis, growth, or development in S. miltiorrhiza.

    Cis-acting elements in the promoter region of SmDof genes

    PlantCARE was introduced to analyze the promoter sequence of 31 SmDof genes from the translation initiation site (ATG), and 55 cis-acting elements were identified. Among of them, they were related to plant cell development, plant hormones, environmental stress, and light response, respectively (Fig. 4). The results show that 22 light-responsive elements get the most abundant compared to other elements, and 31 SmDof genes have light-responsive elements like Box 4, MRE, GT1-motif. In addition, 12 cis-acting elements related to plant hormones were identified. In addition, there are five cis-acting elements associated with cell development, like CAAT-box, HD-Zip 1, MBSI, CCAAT-box, and MSA-like. There are four cis-acting elements associated with environmental stress, like TC-rich repeats, AT-rich element, LTR, and MBS (Fig. 4). It is implied that most of the SmDofs may play an important role in response to plant hormones and are light responsive. This is in agreement with the previous studies on Dof gene families in sugarcane, which is thought to be involved in light response, metabolism, and other functions[19].

    Figure 4.  Cis-acting elements of SmDof promoters in S. miltiorrhiza. Dof family cis-acting element of S. miltiorrhiza. Different colors represent different classes of cis-acting elements and motifs. Green represents cis-acting elements associated with light, yellow represents cis-acting elements associated with plant hormones, purple represents cis-acting elements associated with cell development, and blue represents environmental stress.
    DownLoad: Full-Size Img PowerPoint

    Expression patterns of SmDofs and synthetase genes involved in tanshinones and phenolic acids biosynthesis pathway in various tissues and under ABA treatment

    To gain a deeper understanding of SmDof expression patterns, four tissues including root, stem, leaf, and flower were collected to measure the total content of tanshinones and phenolic acid by HPLC, and subjected to transcriptome sequencing to investigate the expression of SmDofs. The results showed that the total phenolic acids and tanshinones content were all highest in root compared to other tissues (Fig. 5a), and a total of five genes (SmDof6, 12, 13, 27, 29) were highly expressed in the root, which is harvested in practice as the medicinal tissue[3] (Fig. 5b & Supplementary Table S3).

    Figure 5.  Expression profiles of SmDof genes and synthetase genes involved in tanshinones and phenolic acids biosynthesis pathway. (a) Contents of tanshinones and phenolic acids in different tissues of S. miltiorrhiza. (b) Expression profiles of SmDof gene in various tissues of S. miltiorrhiza. (c) Expression profile of SmDof genes under the treatment of ABA induction based on the transcriptome dataset. Red and blue boxes indicate high and low expression levels of SmDofs, respectively. (d) Expression profiles of synthetase genes involved in tanshinones and phenolic acids biosynthesis pathway under the induction of exogenous ABA detected by qRT-PCR. Three biological replicates were performed and the mean ± SD was taken, SD was represented by the error line. * indicates significant difference in t-test (*p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    To mine the candidate SmDofs in response to ABA treatment, the expression variation of candidate SmDof gene exhibiting at least a 2-fold increase more than the control was set as the cutoff. In total, 11 SmDof genes including SmDof9, 16, 18, 21, 22, 23, 24, 25, 26, 28, and 29 showed an obvious increase compared to the control, among which three SmDofs (SmDof22, 25, and 26) exhibited the highest increase reaching to a 17-fold increase over the control. Whereas, seven SmDof gens including SmDof4, 6, 12, 14, 15, 18, and 20 downregulated the expression levels more than 2-fold than the control (Fig. 5c &Supplementary Table S4). Moreover, qRT-PCR was employed to examine the expression level of synthetase genes involved in the tanshinones and phenolic acids biosynthesis pathway. As shown in Fig. 5d, several genes were revealed including PAL, C4H, TAT, RAS1, and CYP98A14 in phenolic acid biosynthesis pathway and GGPPS in tanshinone biosynthesis pathway upregulated significantly under the induction of exogenous ABA, in particular, PAL, and GGPPS were the most up-regulated. Therefore, the above results provide a valuable dataset for mining functional SmDof genes in regulating medicinal substance metabolite synthesis in S. miltiorrhiza.

    Co-expression relationship of SmDofs with the biosynthetic genes involved in tanshinones and phenolic acids biosynthesis in S. miltiorrhiza

    As reported by Shi et al., ABA can affect the expression of the biosynthetic genes involved in tanshinones and phenolic acids biosynthesis, thereby promoting the medicinal metabolites accumulation in S. miltiorrhiza hairy roots[42]. Therefore, the co-expression relationship between the 31 SmDofs with the biosynthetic genes related to the biosynthesis of tanshinones and phenolic acids in S. miltiorrhiza was dissected. The results showed that 15 SmDofs (including SmDof4, 5, 8, 10, 12, 13, 14, 15, 17, 19, 20, 28, 29, 30, 31) co-expressed with SmRAS, SmHPPR, SmC4H, Sm4CL, SmCYP98A14, SmPAL, or SmTAT genes, respectively, with the Pearson correlation coefficient > 0.8 and p-value < 0.05. Moreover, 15 SmDofs (including SmDof4, 6, 9, 10, 11, 12, 13, 14, 15, 17, 20, 27, 28, 29, 30) exhibited a co-expression pattern with SmCYP76AH1, SmKSL, SmCPS, SmGGPPS, SmDXR, or SmDXS2 genes, respectively, and the correlation coefficient was greater than 0.8. It is noteworthy that SmDof4, 10, 12, 13, 14, 15, 17, 20, 28, 29, 30 not only co-express with tanshinones biosynthetic genes, but also co-express with phenolic acids biosynthetic genes, implying that the above 11 SmDof genes may play a vital role in promoting the accumulation of the above two types of medicinal substances.

    Expression pattern and subcellular localization analysis of SmDof12 and SmDof29 genes

    Transcriptome dataset and co-expression analysis were integrated to mine the candidate SmDof genes in association with the biosynthesis of tanshinones and phenolic acids in S. miltiorrhiza. By the transcriptome dataset from various tissues, five SmDof genes were found including SmDof6, 12, 13, 27, and 29 all expressed vigorously in the root (Fig. 5b), which is thought to be the main tissue to accumulate the medicinal substances in practice[3]. According to the results of co-expression analysis, SmDof12 had the highest negative correlation coefficient (reaching −0.917) with the SmGGPPS gene related to the biosynthesis of tanshinones. Whereas, SmDof29 got the highest correlation with the SmPAL gene involved in the phenolic acids biosynthetic pathway, with the correlation coefficient of 0.912 (Fig. 6). Those results push us to validate the expression profile of the two SmDof genes. As expected, the expression profiles of SmDof12 and SmDof29 detected by qRT-PCR analysis were consistent with the transcriptomic dataset (Fig. 7a, b & Supplementary Table S5), of which indicated the reliability of the transcriptome dataset derived from four types of tissues and hairy root lines treated with ABA in S. miltiorrhiza.

    Figure 6.  Co-expression analysis of SmDof genes and the genes involved in the biosynthetic pathway of phenolic acids and tanshinones. R > 0.5 indicates a positive correlation; R < –0.5 indicates a negative correlation. The data were obtained from the ABA transcriptome dataset.
    DownLoad: Full-Size Img PowerPoint
    Figure 7.  Functional characterization of SmDof12 and SmDof29. (a) Expression patterns of SmDof12 and SmDof29 in four tissues of S. miltiorrhiza. The fold changes of the relative gene expression level in the other three tissues are all normalized to the expression level in flower. (b) Expression patterns of SmDof12 and SmDof29 in hairy roots of S. miltiorrhiza treated with ABA. The fold changes in the relative gene expression level were all normalized to the control expression without induction at the 0 h time point. (c) Subcellular localization of SmDof12 and SmDof29 in tobacco. 35S-YFP is the control group, yellow is the fluorescence of YFP, and blue is the nucleus. Scale bar = 50 μm. (d) Dual-Luc assay showed that SmDof12 could inhibit the activity of SmGGPPS promoter and SmDof29 could promote the activity of SmPAL promoter. Three biological replicates were performed and the mean ± SD was taken, SD was represented by the error line. * indicates significant difference in t-test (* p < 0.05, ** p < 0.01).
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    And then, the subcellular localization of the SmDof12 and SmDof29 in epidermal cells from 45-day-old N. benthamiana leaves were studied by transient expression analysis of the two genes fused with YFP, respectively. Robust fluorescence was observed only in the nuclei in 35S-SmDof12-YFP and 35S-SmDof29-YFP, while the 35S-YFP control displayed fluorescence throughout the whole cell (Fig. 7c), suggesting that the SmDof12 and SmDof29 proteins are all localized in the nuclei in S. miltiorrhiza.

    Validation of the target genes of SmDof12 and 29 proteins by Dual-LUC assay

    According to the results of co-expression analysis, it pushes the exploration of the underlying mechanism of SmDof12 and SmDof29 in regulating tanshinones and phenolic acids. By Dual-LUC assay (Fig. 7d), it was revealed that SmDof12 could uniquely inhibit the transcription of the SmGGPPS promoter, leading to a 3-fold decrease compared to the 35S-YFP control, whereas, SmDof29 significantly activated the SmPAL promoter up to 1.69-fold compared to the control. Those results indicated that SmDof12 might inhibit the biosynthesis of tanshinones by decreasing the activity of the SmGGPPS promoter, while SmDof29 activated the transcription of SmPAL to increase the production of phenolic acids in S. miltiorrhiza.

    Discussion

    Dof genes widely exist in plants and have been validated to participate in diverse biological functions[14,15]. S. miltiorrhiza is a valuable traditional Chinese herbal plant and has been used widely in clinic treatments[1]. Genome-wide identification of SmDof gene lays a foundation for the subsequent study of its function. In the present study, a total of 31 Dof genes were identified in S. miltiorrhiza, and the number of SmDof genes was comparative to that of A. thaliana (36 members)[30], rice (30 members)[32], and tomato (34 members)[46]. The genome size of the above plants varied greatly, but the number of Dof proteins was not related to the size of the genome thus implying its conserved function in the above plant species.

    Multiple sequence alignment uncovered the conserved domain within the SmDof proteins in S. miltiorrhiza. Phylogenetic tree construction showed that the SmDofs got low homology with Arabidopsis, and only Group I together with Group IV had more than six Dof genes getting high sequence similarity between S. miltiorrhiza and Arabidopsis. Previous studies have confirmed that phylogenetic analysis can provide a valuable theoretical basis for functional prediction of similar genes in different species[46]. Genes clustering in the same subgroup are relatively conserved in gene structure, gene expression patterns, and functional evolution[47]. By phylogenetic tree construction, it was found that AtDof5.4 and SmDof7 had high homology, and they were grouped into the same branch. Previous studies have verified that AtDof5.4 is a negative regulator modulating cell proliferation and expansion in Arabidopsis[48], so it is speculated that SmDof7 may also have the same function as AtDof5.4. Indeed, SmDof7 got the highest expression level in the stem and root, indicating that SmDof7 might regulate the cell proliferation and expansion in stem and root of S. miltiorrhiza.

    The diverse structure and organization of the Dof genes, is associated with the evolution and functional differentiation of this gene family in certain species[49]. Gene structures analysis of all the Dof genes in S. miltiorrhiza exhibited visible variation between different subgroups, while similar structures were observed within the same subgroup (Fig. 3c). In general, the structure of the 31 SmDof genes was relatively simple and contained one or two exons, of which it was similar to the previous studies on melon[15]. However, 11 SmDof genes (including SmDof 11, 16, 17, 18, 19, 22, 23, 24, 26, 29, 31) had only one intron or even no intron (Fig. 3c). As previously reported, the intron-less genes may be associated with the quick stress response[50].

    In previous reports, many Dof genes have been validated to be a key regulatory center involved in secondary metabolic synthesis, abiotic stress response, and hormone regulation pathway[23,51,52]. In grape (Vitis vinifera L.), VyDof8 was validated to be induced by a variety of abiotic stress. Overexpression of VyDof8 in tobacco (Nicotiana tabacum) significantly elevated ABA accumulation and drought tolerance during prolonged droughts compared to the control plants[53]. The expression pattern of candidate genes in a certain tissue or under stress signal is often closely related to the function of these genes[54]. Therefore, in this study, the transcriptome dataset of S. miltiorrhiza was introduced to dissect the expression profiles of SmDof gene families in root, stem, leaf, and flower. Most of the SmDof genes (23 out of 31) are expressed in the root of S. miltiorrhiza. It is speculated that some members of the 23 SmDof genes may be related to the growth and development of S. miltiorrhiza roots. It was also revealed that SmDof3 and 16 are highly expressed in flower (Fig. 5b). As a Dof gene, CDF3 in tomato (Lycopersicon esculentum) getting high gene sequence homology with SmDof3 and 16 were validated to regulate the flowering time through modulating the expression of FT-like genes[55]. Therefore, the research project whether overexpression of SmDof3 or 16 in S. mitiorrhiza has a significant impact on regulating its flowering time or growth is worthy of inquiry. The varied expression profiles of SmDof genes in various tissues provide basic data to explore their functions.

    By investigating cis-acting elements within promoters, it indicates that the SmDof genes are related to light response, hormone-related response, cell development, and environmental stress (Fig. 4). In the present study, several SmDof genes (eg. SmDof12 and SmDof29) were found to contain ABA-responsive elements (ACGTG) in putative promoter regions. Through the transcriptome dataset together with gene expression validation detected by qRT-PCR, it was confirmed that SmDof12 and 29 expressed vigorously in S. miltiorrhiza root (Fig. 5b; Fig. 7a, b). Furthermore, SmDof12 and 29 were verified to co-express with the metabolic pathway genes involved in tanshinones or phenolic acids biosynthesis (Fig. 6). This pushes the exploration of the underlying molecular mechanism of how SmDof12 and 29 to regulate the expression of the downstream target gene to modulate the tanshinones and phenolic acids biosynthesis. Subsequently, SmGGPPS and SmPAL were validated to be the target of SmDof12 and SmDof29 by Dual-LUC assay, respectively (Fig. 7d). Through the same strategy, in Scutellariae baicalensis, it is verified that SbNAC25 reduces the synthesis of flavonoid by downregulating the expression of FNSII-2, OMT2, CHI, and F6H2 genes[56]. The fact that the gene expression profile in special tissues and under certain stress treatment detected by transcriptome sequencing and qRT-PCR validation in combination with gene co-expression analysis is a quite valid strategy to mine the candidate regulatory genes and their downstream target genes involved in the biosynthetic pathway of secondary metabolite in many plants[57,58].

    Conclusions

    In the present study, the SmDof gene families in S. miltiorrhiza were characterized based on the whole genome, transcriptome dataset, and qRT-PCR expression analysis. Two SmDof genes (SmDof12 and SmDof29) were mined by gene co-expression strategy from the identified 31 SmDofs, and their target genes of SmDof12 and SmDof29 were validated by Dual-LUC experiments. This study is the first comprehensive analysis of the SmDof gene families in S. miltiorrhiza, and provides valid data for further exploring the underlying molecular mechanism of SmDofs in response to ABA induction. It may also be beneficial to elucidate the diverse biological functions of Dof genes in other plants.

  • The authors confirm contribution to the paper as follows: study conception and supervision: Zhou W, Kai G, Zhu J; study design: Wang X, Wang Q, Hao S; experiment performance and data analysis: Wang X, Wang Q, Hao S; manuscript suggestions: Wang X, Zhou W; draft manuscript preparation: Wang X, Zhou W. All authors reviewed the results and approved the final version of the manuscript.

  • All data generated or analyzed during this study are included in this published article and its supplementary information files.

    • Acknowledgments

    This work was supported by National Natural Science Foundation of China (82373979), Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Vareties (2021C02074-3), WenZhou Key Scientific and Technological Innovation Project (ZN2022006) and Zhejiang Provincial Natural Science Foundation of China (LZ24H280002). We appreciate the great experimental support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University.

  • The authors declare that they have no conflict of interest.

  • [1]

    Daniel JR, Baars J, Tan Q. 2017. Current overview of mushroom production in the world. In Edible and Medicinal Mushrooms: Technology and Applications, eds. Diego ZC, Giménez PA. New York, United States: John Wiley & Sons. pp. 5−13. https://doi.org/10.1002/9781119149446.ch2
    [2]

    Balan V, Zhu W, Krishnamoorthy H, Benhaddou D, Mowrer J, et al. 2022. Challenges and opportunities in producing high-quality edible mushrooms from lignocellulosic biomass in a small scale. Applied Microbiology and Biotechnology 106:1355−74

    Google Scholar

    [3]

    Poh PE, Wu TY, Lam WH, Poon WC, Lim CS. 2020. Oil palm plantation wastes. In Waste Management in the Palm Oil Industry. Green Energy and Technology. Cham: Springer. pp. 5−20. https://doi.org/10.1007/978-3-030-39550-6_2
    [4]

    Thakur MP. 2018. Advances in post-harvest technology and value additions of edible mushrooms. Indian Phytopathology 71:303−15

    doi: 10.1007/s42360-018-0060-9

    CrossRef   Google Scholar

    [5]

    Dawadi E, Magar PB, Bhandari S, Subedi S, Shrestha S and Shrestha, J. 2022. Nutritional and post-harvest quality preservation of mushrooms: A review. Heliyon 8:e12093

    doi: 10.1016/j.heliyon.2022.e12093

    CrossRef   Google Scholar

    [6]

    Riaz MN. 2006. Extruded snacks. In Handbook of food science, technology, and engineering. Vol.4. pp. 168
    [7]

    Godswill AC. 2019. Proximate composition and functional properties of different grain flour composites for industrial applications. International Journal of Food Sciences 2:43−64

    doi: 10.47604/ijf.1010

    CrossRef   Google Scholar

    [8]

    Naher L, Mazlan NA, Hamzah NAB, Islam S, Ab Rhaman S. 2022. Palm press fibre and rice straw for cultivation grey oyster mushroom (Pleurotus sajor-caju). Sains Malaysiana 51:1305−15

    doi: 10.17576/jsm-2022-5105-03

    CrossRef   Google Scholar

    [9]

    Sulaiman NS. 2019. Physical properties and sensory acceptability of cracker made from pumpkin (Cucurbita moschata) flour during storage. Thesis. Universiti of Malaysia Kelantan, Malaysia. http://discol.umk.edu.my/id/eprint/4713/
    [10]

    Loh SJ. 2020. Comparative studies of physicochemical and texture profile analysis of sweet potatoes (Ipomoea batatas) cookies from different variation of sweet potatoes. Thesis. University of Malaysia Kelantan, Malaysia. http://discol.umk.edu.my/id/eprint/4194/
    [11]

    Khalid SK, Ismail-Fitry MR, Mat Yusoff M, Halim HH, Jaafar AH, et al. 2021. Different maturities and varieties of coconut (Cocos nucifera L.) flesh as fat replacers in reduced-fat meatballs. Sains Malaysiana 50:2219−28

    doi: 10.17576/jsm-2021-5008-07

    CrossRef   Google Scholar

    [12]

    Setaiboma W, Kristanti D. 2021. Quality of physicochemical and sensory of mushroom (Pleurotus ostreatus) chicken nuggets with carrageenan and konjac as hydrocolloids. IOP Conference Series: Materials Science and Engineering 1011:012014

    doi: 10.1088/1757-899X/1011/1/012014

    CrossRef   Google Scholar

    [13]

    Lu X, Brennan MA, Serventi L, Mason S, Brennan CS. 2016. How the inclusion of mushroom powder can affect the physicochemical characteristics of pasta. International Journal of Food Science and Technology 51:2433−39

    doi: 10.1111/ijfs.13246

    CrossRef   Google Scholar

    [14]

    Tunick MH, Onwulata CI, Thomas AE, Phillips JG, Mukhopadhyay S, et al. 2013. Critical evaluation of crispy and crunchy textures: a review. International Journal of Food Properties 16:949−63

    doi: 10.1080/10942912.2011.573116

    CrossRef   Google Scholar

    [15]

    Nicole TZH, Nichelle TS, Elizabeth TE, Yuliarti O. 2021. Formulation of functional crackers enriched with fermented soybean (tempeh) paste: rheological and microstructural properties. Future Foods 4:100050

    doi: 10.1016/j.fufo.2021.100050

    CrossRef   Google Scholar

    [16]

    Haverkort AJ, Linnemann AR, Struik PC, Wiskerke JSC. 2023. On Processing Potato 2. Survey of Products, Processes and Operations in Manufacturing. Potato Research 66:339−83

    doi: 10.1007/s11540-022-09563-y

    CrossRef   Google Scholar

    [17]

    Djafari Petroudy SR, Rahmani N, Rasooly Garmaroody E, Rudi H, Ramezani O. 2019. Comparative study of cellulose and lignocellulose nanopapers prepared from hard wood pulps: Morphological, structural and barrier properties. International Journal of Biological Macromolecules 135:515−20

    doi: 10.1016/j.ijbiomac.2019.05.212

    CrossRef   Google Scholar

    [18]

    Butnariu M, Sarac I. 2019. What is sodium glutamate and what effects it has on health. Journal of Applied Biotechnology and Bioengineering 6:223−26

    doi: 10.15406/jabb.2019.06.00195

    CrossRef   Google Scholar

    [19]

    Bera TK, Kar SK, Yadav PK, Mukherjee P, Yadav S, et al. 2017. Effects of monosodium glutamate on human health: A systematic review. World Journal of Pharmaceutical sciences 5(5):139−44

    Google Scholar

    [20]

    MacLeod M. 2022. Home of Food Label. Reading and Understanding Food Labels and Nutrition Info Beaumont Health. www.beaumont.org/conditions/reading-a-food-label (Retrieved December 31, 2022)
    [21]

    Yahya F, Yusof N, Chen C. 2017. Effect of varying ratios of oyster mushroom powder to tapioca flour on the physicochemical properties and sensory acceptability of fried mushroom crackers. Malaysian Applied Biology 46:57−62

    Google Scholar

    [22]

    Ng SH, Robert SD, Wan Ahmad WA, Wan Ishak WR. 2017. Incorporation of dietary fibre-rich oyster mushroom (Pleurotus sajor-caju) powder improves postprandial glycaemic response by interfering with starch granule structure and starch digestibility of biscuit. Food Chemistry 227:358−68

    doi: 10.1016/j.foodchem.2017.01.108

    CrossRef   Google Scholar

    [23]

    Granite S. 2022. Ash determination: Ash content: Food samples. Precisa. www.precisa.com/blog/ash-content-in-food-analysis (Retrieved December 25, 2022)
    [24]

    Rahmah NL, Sukardi, Wulantiasari W, Wijayanti N. 2020. Physicochemical properties of oyster mushroom flavouring powder. IOP Conference Series: Earth and Environmental Science 443:012009

    doi: 10.1088/1755-1315/443/1/012009

    CrossRef   Google Scholar

    [25]

    Zhou S, Ma F, Zhang X, Zhang J. 2016. Carbohydrate changes during growth and fruiting in Pleurotus ostreatus. Fungal Biology 120:852−21

    doi: 10.1016/j.funbio.2016.03.007

    CrossRef   Google Scholar

    [26]

    Roshita I, Liyana NF, Adzemi MA, Zain SHM. 2015. The growth and post harvest performances of different species of osyter mushroom (Pleurotus sp.) cultivated on sawdust and oil palm frond. Malaysian Applied Biology 44:75−82

    Google Scholar

  • Cite this article

    Naher L, Mustaffa Bakri NA, Muhammad Sukhri SAN, Nik Hassan NR, Mohd Firdaus Ganga H, et al. 2024. Economical substrate formulation for mushroom cultivation and food production of mushroom crackers to reduce postharvest waste. Studies in Fungi 9: e002 doi: 10.48130/sif-2024-0003
    Naher L, Mustaffa Bakri NA, Muhammad Sukhri SAN, Nik Hassan NR, Mohd Firdaus Ganga H, et al. 2024. Economical substrate formulation for mushroom cultivation and food production of mushroom crackers to reduce postharvest waste. Studies in Fungi 9: e002 doi: 10.48130/sif-2024-0003
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Economical substrate formulation for mushroom cultivation and food production of mushroom crackers to reduce postharvest waste

  • 1.

    Faculty of Agro-Based Industry, Universiti Malaysia Kelantan Jeli Campus, 17600 Jeli, Kelantan, Malaysia

  • 2.

    Institute of Food Security and Sustainable Agriculture, Universiti Malaysia Kelantan Jeli Campus, 17600 Jeli, Kelantan, Malaysia

  • Received: 02 September 2023
  • Revised: 02 March 2024
  • Accepted: 13 March 2024
  • Published online: 29 March 2024
Studies in Fungi  9,  Article number: e002  (2024)  |  Cite this article

Abstract: Agriculture residues of oil palm waste are a big issue for palm oil producing countries. The residues from oil palm fronds are the most crucial to convert wealth. This study focused on oil palm-related agro biomass for mushroom substrate formulation for grey oyster mushroom cultivation. Mushrooms are a highly perishable vegetable that turn into postharvest waste within 4 to 7 d at normal temperatures. Therefore, in this study, the unsold mushroom was converted as a cracker food product to reduce the postharvest losses, especially for small-scale mushroom growers. The agriculture biomass used for substrate preparation is a combination of oil palm frond (OPF), oil palm empty fruit bunch (EFB), palm pressed fiber (PPF), and sawdust (SD). SD as a commercial substrate was used as a control in this study, and rice bran (RB) and lime (L) were used as supplement ingredients for all the treatments. The treatments were according to mixed formulation with the ratio of T0 (control: 97.2% SD, 0.8% RB + 2% L), T1 as a mixed ratio (60% RS + 22.2% EFB + 15% PPF + 0.8% RB + 2% L) and T2 as a mixed ratio (60% OPF + 22.2% EFB + 15% SD + 0.8% RB + 2% L). The total yield in four cycles showed 1.2 kg in T0 (sawdust), 1.4 kg in T1 (majority of rice straw), and 1.5 kg (majority of oil palm frond) in T3 treated substrate. In this study, the oil palm frond was received free of charge as compared to sawdust and rice straw. Therefore, it showed that using the oil palm frond not only gave a high yield of mushrooms at the same time, it was 100 X lower in cost. Next, the unsold yielded mushrooms were used for cracker preparation. The results obtained from this study indicate that mushroom crackers contain fat (11.34%), protein (2.19%), and carbohydrate (76.55%) while being high in moisture (7.87%) and ash (2.06%) compared to commercial potato crackers. Overall acceptance of sensory evaluation towards mushroom crackers showed a high 'extremely like' percentage, contributing about 66%. Thus, this study found that 66% of participants 'extremely liked' the new innovative mushroom crackers. Overall, the results show that oil palm substrate can be an alternative economical substrate for grey oyster mushroom cultivation and food products from mushrooms will be new items in the snack industry.

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    Introduction

    • Edible mushrooms are a rich source of nutrition as well as income generation for many poor communities. The mushroom industry is quickly gaining the attention of many entrepreneurs, particularly young ones. Due to concerns about health issues, mushrooms are in high demand among the general public, particularly in developed countries, as a medicinal product as well as a health food worldwide[1]. Grey oyster mushroom (Pleurotus sajor caju) is a popular mushroom in the edible group of mushrooms. The cultivation of grey oyster mushrooms requires high humidity of 95%−100% with temperatures between 28 to 30 °C , no exposure to sunlight and a good substrate material of lignocellulose agro-biomass[2]. Oil palm plants are a good source of lignocellulose plants. On the other hand, palm oil-producing countries including Malaysia, Indonesia, Africa, Papua New Guinea, and America face problems due to the vast amount of the plant residue management, especially for oil palm fronds[3]. Based on the economic conditions of using oil palm ago-residues such as oil palm frond and empty fruit bunch to convert mushroom substrates, will especially benefit the rural farmers. The global mushroom market is showing that the demand is projected to rise from 15.25 million tonnes in 2021 to 24.05 million tonnes in 2028 at a CAGR of 6.74% in the forecast period. Hence, the future economic value of mushrooms is predicted to be very high. However, to fulfil the demand of the market, growers need different types of good quality mushroom substrate rather than depending on a single substrate. In Malaysia, the grey oyster is one of the most popular edible mushrooms, mainly cultivated using rubber sawdust substrate. Therefore, mushroom growers often face problems with material supply due to the high demand for rubber sawdust. Besides mushroom cultivation, sawdust is also used for poultry farming; therefore, using oil palm-based residue substrates can be introduced in two factors as waste management and also for a newly formulated substrate.

      Mushrooms are a highly perishable vegetable crop. Mushrooms structurally do not consist of cuticles, which influences the high moisture content, and only 10% is fiber content. According to Thakur[4], numerous phytochemicals, enzymes, primary metabolites, and secondary mycometabolites cause sudden degradation, short shelf life, and high postharvest losses (30%–35%). In addition, fresh mushrooms have a short shelf life, which is within 1 to 8 d which will reduce their economic value[4]. After harvest, mushrooms undergo a series of quality degradations, including moisture loss, discolouration, texture changes, off-flavour, and nutrient loss[5]. The moisture content of fresh mushrooms ranges from 85% to 95%[6].

      As a result of their high moisture content, mushrooms should be preserved at low temperatures to prevent microbial infection. Mushrooms gradually lose moisture during the postharvest period, which causes ongoing weight reduction. Due to water loss and enzyme activity, postharvest mushrooms' colour shows a browning tendency, impacting customer purchase decisions[5]. Thus, the growers of mushrooms, especially in rural areas, often face postharvest losses. Therefore, our study also focused on easy product conversion from mushrooms.

    Materials and methods

      Raw material collection

    • Empty fruit bunch, palm pressed fiber, and oil palm fronds were freely collected from the palm oil plantation at Felda Kemahang, Tanah Merah, Kelantan, Malaysia. Rice straw was purchased from a paddy farmer in the Tumpat, Kelantan area after the harvesting season, and sawdust was purchased from the soil oil mill at Jeli, Kelantan.

      Preparation of substrates

    • The substrates, such as the empty fruit bunches (EFP), were shredded into small pieces, and the rice straw was cut into small pieces using a cutter machine. The oil palm fronds were cut into an appropriate size due to the hardwood used and ground using a grinder machine at ATP. After cutting, the substrates such as EFB, PPF, and RS were soaked overnight to reduce any excess water. On the next day, all the substrates were rinsed with clean water and put on the newspaper or plastics to dry for a few days under sunlight. All the dried materials were again ground using a grinding machine to obtain a smooth size (1.00 mm) for ease of mycelia penetration during mycelium colonization.

      Preparation of substrate in each polyethylene filling bag

    • All the composition substrates including calcium carbonate, and rice bran as an additional supplement for mycelia growth, were mixed with a mixing machine and took around half an hour to mix well as shown in Table 1. Distilled water was added to retain moisture for mycelia growth, and each treatment was added to least two bottles (9.5 L/bottle). Moisture content should be in the 70 to 75% range. Before filling the substrates into the sawdust bag, the pH reader was checked for each treatment.

      Table 1.  Substrate composition.

      Substrate/
      treatment      		Composition of substrate
      T0 (Control)97.2% SD + 0.8% RB + 2% L
      T1 (Mixed)60% RS + 22.2% EFB + 15% PPF + 0.8% RB + 2% L
      T2 (Mixed)60% OPF + 22.2% EFB + 15% SD + 0.8% RB + 2% L
      OPF, Oil Palm Frond; EFB, Empty Fruit Bunch; PPF, Palm Pressed Fibre; SD, Sawdust; RB, Rice bran; L, Lime.

      The mixed prepared substrates for each treatment were then filled into a polyethylene (PE) bag (9'' × 15''), and the medium was pressed manually by hand to ensure it was as compact as possible, resulting in 500 g/bag. The medium was then closed with a PVC neck set. The blocks were then autoclaved to prevent contamination. After autoclaving, the sterilized substrate bags were put in a mushroom lab to cool. Next, 1-2 g of mushroom spawn were added to the blocks and all the blocks were arranged on the racks by each treatment for incubation in the mushroom lab. After mycelium was fully colonized, all the blocks showed primordia to produce the mushroom fruiting body. The harvested mushroom was recorded for total yield and biological efficiency to determine the best potential substrate mushroom for production. The biological efficiency was conducted based on the following formula:

      Biological efficiency, BE (%)= Weight of harvest  Weight of dry substratte ×100%

      Processing of food crackers from grey oyster mushroom

    • Unsold and leftover fresh mushrooms were cleaned and dried in sunlight for 2 d. The dried mushrooms were then ground into powder using a blender at the Food Laboratory (UMK, Malaysia). For the cracker preparation, the ingredients of PH (not stated due to copyright) were boiled to make a mash. A cup of water was transferred to a pot to boil, PH was then slowly added to the boiled water. In the meantime, further boiled water was added, and the ingredient CFH (not stated due to copyright) was slowly added until a rough dough formed in the pot. After mixing the CFH, the dough was transferred to a large plate. Next, another dry ingredient, such as a teaspoon of pepper powder, was added to the dough. Mushroom powder (approximately 10%) was added according to the final weight of 250 g of the dough. The dough was then divided into four to five parts. After 24 h under refrigeration, each dough piece was cut into thin pieces. Next, the crackers were dried using sunlight for at least two days to ensure the crackers were well dried. Finally, then the dried crackers were analysed for proximate analysis and sensory evaluation of participants.

      Proximate analysis

    • Proximate analysis is a chemical analysis method to identify food substance nutritional content such as protein, carbohydrates, fat, and fibre[7]. The analysis results were presented as grades in units of %. The proximate analysis had benefits as an assessment of the quality of the food ingredients, especially on the standard of food substances they should contain.

      Protein analysis

    • Protein content of the mushroom powder, mushroom cracker and commerical crackers was determined using the Kjeldhal method according to the procedures of AOAC with some modification. This method involves three stages which are digestion, distillation and titration following the procedure of Naher et al.[8]. Crude protein was calculated using the following formulas:

      i) Calculate for N2 content:

      %ofN2=(T−B)×N×1.4007weightofsample(g)

      ii) Crude protein = N2% × 6.25

      Ash, fat and carbohydrate analysis

    • Two grams of each mushroom cracker, mushroom powder and commercial cracker were weighed and transferred onto clean, dry, and pre-weighed crucibles, respectively. The samples and crucible were kept in a muffle furnace at 550 °C for 6 h. Then, the sample was cooled in a desiccator and weighed. The fat content was determined using the Soxhlet extractor method with some modifications following the procedure of Naher et al.[8].

      The ash and fat content for mushroom cracker and powder was calculated using the following formula:

      Percentage(%)ofAsh/Fatcontent=W1−W2W×100

      Where: W1 = weight of final cup, W2 = weight of initial cup, W = weight of sample

      The formula for the percentage of carbohydrates determined the carbohydrate content of mushroom crackers, mushroom powder, and commercial crackers. The equation below was used to calculate the carbohydrate content:

      Totalcarbohydrate%=100−(Moisture+Protein+Ash+Fat)

      Analysis of mushroom cracker attributes

    • The physical attributes of mushroom crackers were analysed based on texture (crispiness, hardness, cohesiveness), moisture, and colour. The moisture analysis of the mushroom crackers was performed using the moisture analyzer A&D Heat-Drying Moisture Meter MX-50. A moisture analyzer weighed the sample of mushroom crackers. Then, the analyzer heated the sample at 180 ℃ until the sample dried. Lastly, the sample was weighed again, and the result recorded.

      The texture analysis of a mushroom cracker was designed to mimic biting person. The Brookfield CT3 Texture Analyzer with TA-MTP fixture was used to run the mushroom cracker test. The chip sample was penetrated using a stainless-steel cylinder probe type TA7 with a trigger load of 5 g and a speed of 5.00 mm/s[9]. The texture analysis parameters were set, and the mushroom cracker and commercial potato cracker (control) were positioned beneath the probe. Texture analyzers concentrate on the mushroom cracker's hardness, breaking strength, and cohesion. The data sample was recorded in triplicate.

      The Hunter Lab Colorimeter's optical sensor was placed on top of the mushroom cracker. A colour metre (Konica Minolta CR-400) was used to determine the product's colour for the instrumental measurement[10]. The mushroom cracker and commercial potato cracker (control) samples were tested. Each sample's colour was measured in the CIE L* a* b* colour space, and the results were reported in terms of lightness (L*), redness or red-green (a*), and yellowness or yellow-blue (b*)[11]. The data samples were recorded in triplicate.

      Sensory evaluation

    • The method of sensory evaluation was applied by evaluation of four sensory attributes, which are the number of crackers, texture or feel, colour, richness and overall acceptability of the cracker samples using a 4-point hedonic scale, in which the lowest value (1) stands for extreme dislike while the highest value (4) represents an extreme like (Table 2)[12]. Fifty participants were selected to take part in the determination of the sensory evaluation of mushroom crackers. Before tasting a mushroom cracker, plain water was served to participants to neutralize their mouthfeel and the tasting was carried out under good lighting to determine the colour of the crackers.

      Table 2.  Survey form for the sensory evaluation of mushroom crackers.

      ItemsExtremely
      like 4      		Like 3Dislike 2Extremely dislike 1
      Number of crackersCracker in every biteCracker in 75% chipsCracker in 50% chips< 50% cracker
      Texture/
      feel      		Consistently crispy and crunchy chewyChewy middle, crispy edgesCrunchy
      only and
      not crispy      		Less crunchy and not crispy
      ColourEven golden brownBrown with pale centreVery brownBurned
      RichnessEdible Less oilyMedium oilyHigh oily

      Statistical analysis

    • All the parameters including total yield of mushrooms, biological efficiency from each treatment, proximate, physical attributes of mushroom crackers as well as sensory assessment information were collected and processed using IBM SPSS version 26 for the statistical analysis. The significance of the differences in the data was determined using an independent t-test, as well as ANOVA analysis. The significant differences in the mean values were determined at the 95% confidence interval level of (p < 0.05).

    Results

      Yield and income performance

    • The species of grey oyster (Pleurotus ostreatus) mushroom was cultivated for two months in the mushroom house, at UMK Jeli campus, Malaysia. A total of 45 blocks were cultivated in three different treated substrates in three replicates. The total yield was recorded until the 4th cycle for two months. The results showed (Fig. 1) that oil palm frond majority substrate (T2) produced a higher yield (1.5 kg), while the 2nd highest (1.4 kg) was rice straw substrate (T1) and the lowest (1.2 kg) was sawdust substrate (T0).

      Figure 1. 

      Total yield performance of grey oyster mushroom on different treated substrates.

      For income performance, the individual block preparation cost, yield in four cycles, selling price and net income of each substrate per block are shown in Table 3. T2 was 100 g/block which was higher than T0 and T1. For net income comparison, we make T0 or control a constant of 100% yield, which was compared with T1 and T2 total yield performance. The result showed T2 yield performance was 115% which was 15% higher than T0 (Table 3). The total cost per block also sowed in oil palm form was lowest in T2 as RM 0.60 as compare to T0 and T1. Therefore, the net income of T2 (RM 0.70) was higher than the control (RM 0.38) and T1 (RM 0.53) in Table 3.

      Table 3.  Income performance of each of the substrate treated mushroom yield.

      ProductsCost (RM)/blockYield/ block /selling priceYield performance/ income/block
      T0 (Sawdust commercial/control)0.7486.7 g/RM 1.12100%/RM 0.38
      T1 (RS + EFB + PPF)0.7593 g/RM 1.2107% (7% >)/RM 0.53
      T2 (OPF + EFB + sawdust)0.60100 g/RM 1.30115% (15% >)/RM 0.70

      Proximate analysis of mushroom cracker and commercial potato cracker

    • Proximate analysis was performed to determine the protein, fat, ash, moisture and carbohydrate content. The result shows that there was a significant difference (p < 0.05) in ash, fat and moisture content between commercial potato and mushroom cracker (Table 4). While, no significant difference (p > 0.05) was found in protein and carbohydrate between commercial potato and mushroom cracker (Table 4).

      Table 4.  Proximate analysis of mushroom cracker and commercial cracker.

      SampleMushroom cracker (1 g)Commercial potato
      cracker (1 g)
      Protein (%)2.19 ± 0.902.22 ± 0.10
      Fat (%)11.335 ± 0.306112.8283 ± 0.2475
      Ash (%)2.0567 ± 0.12011.4667 ± 0.2566
      Moisture (%)7.8733 ± 0.22196.15 ± 0.5
      Carbohydrate (%)76.5467 ± 0.1250377.3350 ± 0.5327

      Colour properties

    • The colour property analysis of mushroom cracker and commercial potato cracker is shown in Table 5. The results found that mushroom crackers had the lowest value (55.89 ± 1.0017) of L* while commercial potato crackers showed higher values (62.8033 ± 0.1721). According to the value, L* indicate lightness. This shows that the colour of the mushroom cracker is darker than the commercial potato cracker because due to dark colour mushroom powder from the grey oyster mushroom. There was a significant difference (p < 0.05) shown in L* between mushroom and commercial potato crackers (Table 4).

      Table 5.  Independent t-test for colour analysis.

      Mushroom crackerCommercial potato crackerSig.
      (2-tailed)
      MeanS.D.MeanS.D.
      L*55.891.001762.80330.17210.006
      a*6.751.719711.72670.52580.029
      b*18.16671.501631.550.75020.001

      Next, a* is an indicator for the colour of the crackers to be red or green. The results obtained show that commercial potato crackers are much higher (11.7267 ± 0.5258) than mushroom crackers (6.75 ± 1.7197). This shows that commercial potato crackers have an orange to red colour while mushroom cracker shows less red colour as mushroom crackers have no artificial colour added. Hence, there is a significant different between mushroom crackers and commercial potato crackers which was (p < 0.05) shown in Table 5. Moreover, b* is indicated for yellow or blue colour. The highest b* value indicating yellowness of the sample was observed for the commercial potato cracker (31.55 ± 0.7502). A lower b* was noted in the mushroom cracker (18.1667 ± 1.5016) due to the incorporation of mushroom powder. Due to the mushroom powder's natural brown colour, the mushroom cracker's yellowness was concealed. Consequently, a decreasing trend in b* values was seen as mushroom inclusion increased[13]. Therefore, there is significant difference (p < 0.05) between mushroom crackers and commercial potato crackers.

      Texture profile analysis

    • Texture profile analysis (TPA) is used in a wide variety of fields to measure mechanical qualities like hardness, cohesiveness and springiness by repeatedly compressing a sample with a probe at a predetermined rate. The test was equipped using probe TA 7 Knife edge 60 mm W. A number of texture properties were chosen to analyse the crackers such as hardness, cohesiveness and springiness. Table 3 shows the texture properties of mushroom crackers and commercial potato crackers. For the hardness properties, the mushroom cracker (1,117.00 ± 126.74 g) shows a lower value than the commercial potato cracker (2,481.00 ± 115.8836 g). The hardness of the crackers can be determined by the sensory and terminology of hardness is opposite to crispiness. The lower the hardness is, the more crunchiness of the cracker. So, it can be seen that mushroom crackers are much crispier compared to the commercial potato crackers. Customers favour crackers with a high crispiness score, and low hardness will be displayed[14]. The hardness of the cracker is often related to the interaction of ingredients used. Both crackers were significantly different (p > 0.05) in hardness towards each other.

      Cohesiveness is a measure of how a cracker withstands deformation. Based on the results in Table 6, mushroom crackers show higher cohesiveness, which indicate about 1.00 ± 0.1473. While, commercial potato crackers indicate 0.1867 ± 0.0851 which is less compared to the mushroom cracker. Low cohesiveness indicates high brittleness or crumbliness of the cracker. Therefore, the higher cohesiveness of the mushroom cracker might be because the proteins in the mushroom cracker formed a three-dimensional cross-linked protein network that could withstand more deformation before breaking[15]. Other than that, the lower the cohesiveness value, the more prone it is to breakage. Therefore, there is no significant difference (p < 0.05) between the cohesiveness of mushroom crackers and commercial potato crackers.

      Table 6.  Independent t-test for texture properties.

      Mushroom crackerCommercial potato crackerSig.
      (2-tailed)
      MeanS.D.MeanS.D.
      Hardness (g)1,117.00126.742481.00115.88360.000
      Cohesiveness1.000.14730.18670.08510.003
      Springiness (mm)6.490.897214.619.51030.215

      In addition, springiness properties refer to how quickly and fully a deforming force is recovered. For springiness, the commercial potato cracker has a higher value (14.61 ± 9.5103 mm) than the mushroom cracker (6.49 ± 0.8972 mm). The higher springiness value is because of the extended storage time as commercial potato crackers have been developed on a large-scale during processing time[16]. So, this would affect the springiness of the cracker. Both crackers showed no significant difference (p > 0.05) in springiness attributes between each other.

      Sensory evaluation of mushroom crackers

    • The survey result of the participants' sensory evaluation of mushroom crackers is shown in Table 7. Sensory evaluation of four sensory attributes: the number of crackers consumed, texture or feel, colour, richness, and overall acceptability of the crackers samples, using a 4-point hedonic scale, in which the lowest value (1) stands for 'extreme dislike'. In contrast, the highest value (4) represents 'extremely like'. The percentage of 50 participants determines this sensory evaluation. It was presented that the highest percentage level acceptance of mushroom crackers of 74% are 'extremely like', which means that the respondent eats 100% of the mushroom crackers, whereas another 22% represent 'like', which means they consume 75% of the mushroom crackers while 'dislike' contributed to 4%. Next, for the texture of the mushroom, the largest scale is 'extremely like', contributing 74%.

      Table 7.  Sensory evaluation of mushroom crackers.

      ScaleSensory attribute
      AcceptableTextureColourRichnessOverall acceptance
      Extremely like74%74%38%78%66%
      Like22%26%62%22%33%
      Dislike4%0001%
      Extremely dislike00000

      In crackers, the colour attribute symbolizes the exterior colour of the crackers. The highest percentage of mushroom crackers were in the 'like' scale which contributed 62%, meaning that the crackers are brown in colour. Therefore, another 38% voted 'extremely like', which means the crackers are golden brown.

      Then for the richness of mushroom crackers, the highest scale percentage was 'extremely like', contributing to 78%, meaning that the mushroom cracker is edible. The participants observed that mushroom crackers could be eaten. Therefore, another 22% vote for the 'like' scale indicates that the mushroom crackers are lower in oil. Finally, the largest scale percentage of overall acceptance is 'extremely like', which contributed up to 66%, while another 33% and 1% are 'like' and 'dislike', respectively.

    Discussion

    • Mushrooms become a high-value food worldwide. Therefore, the demand for mushrooms is increasing day by day. A perfect substrate combination results in good growth of mycelia that helps for profitable production of mushroom fruit bodies in commercial cultivation. This study showed that T2 treatment combinations with 60% OPF (oil palm frond) + 22.2% EFB (empty fruit bunch) + 15% SD (sawdust) + 0.8% RB + 2% L recorded higher (1.5 kg in four cycles) production. While, treatment T1 as 60% RS (rice straw) + 22.2% EFB + 15% PPF + 0.8% RB +2% L) produced 1.3 kg and T0 as 97.2% SD (sawdust) + 0.8% RB + 2% L) produced 1.2 kg mushroom fruit bodies. It is interesting that T2 and T0 material composition is quite similar, as oil palm frond and sawdust mainly contain lignin and cellulose, whereas T1, which is a rice straw substrate, mainly contains cellulose-based material. Physiological attributes for substrate in terms of Carbone (c), Nitrogen (N), minerals, and moisture capacity content play important roles in mycelial development. The material of lignocellulose has less moisture vapour evaporation compared to cellulose-based material[17]. Thus, palm-based substrate maintained moisture in the substrate while with the rice straw substrate, several droplets on the substrate bag can be seen that cause issues for mycelial growth.

      The income performance result was also high in oil palm-based substrate (T2). For potential commercial substrates, there is a need to compare its cost price and net income to determine the maximum utilization of the substrate for mushroom production and worth for income generation. In this study, oil palm-based substrate was received free of charge, while sawdust and rice straw were purchased. Hence, oil palm based substrate can be profitable for mushroom farmers as well as a country's economic revenue.

      As for nutritional content, there was not much difference in protein content between commercial crackers and mushroom crackers, commercial potato cracker content was slightly higher (2.22% ± 0.10 %) compared to the mushroom crackers (2.19% ± 0.90 %). This is may be due to MSG (monosodium glutamate). MSG contains glutamate, rich in protein[18]. Another study by Bera et al.[19] defined that glutamate from MSG is the most abundant amino acid (the main component of protein). In preparation of mushroom crackers or even decoration time, this study did not use seasoning with MSG. This is because although MSG contains amino acids, it also contains artificial salt. Too much dietary sodium can cause an increase in blood pressure or health issues. Currently, cracker consumers are aware of MSG salt-processed crackers. Cracker lovers are looking for healthy quality ingredients with minimal processing and they should not contain excess salt while the taste should be similar to commercial crackers[2]. On the other hand, mushrooms contain umami flavor, which is the 5th state group that contains natural MSG flavor; therefore, for the preparation of mushroom crackers there is no need for artificial salt. In this study, mushroom crackers were made with very few ingredients, such as rice flour and potato. Conversely, commercial potato crackers usually contain several artificial ingredients that enhance the artificial protein amount. So, it can be noted that mushroom cracker contain completely natural protein. Table 3 shows that fat content in commercial potato crackers is slightly higher (12.8283 ± 0.2475) than in mushroom crackers (11.335 ± 0.3061). The literature suggests that the recommended fat content range is between 10% and 30% fat[20]. Therefore, mushroom crackers and commercial potato crackers are still in the recommended range. In addition, the higher protein content contributions increased in mushroom powder and constricted the starch-lipid interaction, causing a reduction in oil absorption during frying[21]. The ash content in the mushroom cracker proceeded to be higher, which is about 2.0567 ± 0.1201, compared to the commercial cracker (1.4667 ± 0.2566). The relatively high content of ash also shows fiber richness in the food[22]. Therefore, mushroom crackers show that the products are rich in fiber compared to commercial crackers. However, some studies have revealed that the ash content in processed food can be more than 10%, but in natural food, it must be less than 5%[23]. The mushroom crackers in this study are referred to as minimally processed, whereas commercial potato crackers are fully processed food. In recent years, cracker lovers require processed or minimally processed foods[2]. However, the moisture content in mushroom crackers was 7.8733%, which is slightly higher than that of commercial crackers at 6.15%. Higher moisture content is a result of the capacity of fibers and polysaccharides to retain water[24]. If the moisture level is too high (more than 10%), the texture and flavour will suffer, and the shelf life will be shortened[4]. Crackers with a low moisture level (5%) are more prone to breakage, which results in waste. As shown, commercial crackers are in the range of 5 to 10%, and the moisture content of mushroom crackers was also in a range of 5 to 10%, so it can be noted that the production of mushroom crackers is the commercial standard level. For carbohydrate content, there was no significant difference found between commercial crackers (77.3350%) and mushroom crackers (76.5%). Carbohydrates mainly originated from flour and sugar. In the mushroom cracker, no sugar or artificial additives were added except for flour and the mushroom itself. In commercial crackers, besides flour, a few more ingredients, such as sugar or additives, are added to enhance self-life. Mushroom carbohydrates contain good qualities, such as trehalose, xylitol, and sorbitol, which can act as natural additives.

      Besides natural sugar, the mushrooms also contain polysaccharides such as glycogen, β-glucan, heteroglycan, and chitin[25]. Among these polysaccharides, β-glucan is one of the dietary fibers that can reduce human blood cholesterol and glucose levels that affect cardiovascular heart disease and diabetes for health[25]. Therefore, it can be noted that mushrooms contain good quality carbohydrates which contribute to the healthy food attribution of mushroom crackers.

      As for colors, L* indicates lightness, a* is an indicator of the color of crackers being red or green, and b* indicates yellow or blue color. In all aspects of colour, commercial potato cracker values were higher than mushroom crackers (Table 3). In preparation of mushroom crackers, no artificial colour was added. Texture comparison between mushroom crackers and commercial potato crackers showed no significant difference (Table 3) which showed mushroom crackers as being a potential for commercial standard in taste.

      Sensory acceptability of mushroom crackers showed a higher rate of 72% which shows that the mushroom crackers were 'extremely accepted'. Richness means the texture with crispiness which was high at 78%. It means that the participant accepted the texture of the mushroom crackers that are constantly crispy and crunchy. Usually, customers favour crackers with a high crispiness, and low hardness[14]. In terms of colour, 62% showed that mushroom crackers looked to be brown. This is due to the colour of mushroom crackers being brown rather than golden because of the dark colour of mushroom powder from grey oyster mushrooms[26]. Hence, the mushroom powder contributes to the darkening after frying[21]. However, the colour did not affect the overall acceptance, which showed that 66% of participants 'extremely like' the mushroom crackers.

    Conclusions

    • Mushroom is a potential agri-food which is referred to as vegetable meat. Since demand for mushroom is increasing dramatically, various types of cultivation substrate are needed to avoid raw material scarcity. The results of this study showed that oil palm plant material-based substrate produced the highest yield of 1.5 kg. The net income performance was 15% highest for oil palm substrate compared to rice straw and sawdust substrate. The food product of mushroom crackers overall acceptance level showed 66% participants accepted mushroom crackers. Therefore it can be concluded that the preparation of oil palm products is cost-effective, and growers can easily adopt them for their income generation, which can influence economic sustainability. The production of mushroom crackers can reduce postharvest losses and open the door for extra income for the growers.

    Author contributions

    • The authors confirm contribution to the paper as follows: study conception and design: Naher L; data collection: Mustaffa Bakri NA, Muhammad Sukhri SAN, Nik Raihan NH, Mohd Firdaus Ganga H; analysis and interpretation of results: Md Zain N, Abdul Rahman N; draft manuscript preparation: Naher L, Ch'ng HY, Mokhtar SI. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • All data generated or analyzed during this study are included in this published article.

      Acknowledgments

      • The authors would like to acknowledge the Ministry of Finance, grant code R/MOF/A0700/01204A/2020/00724 for providing financial support. We also acknowledge the Universiti Malaysia Kelantan, Jeli Campus for all the laboratory facilities.

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Rights and permissions
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (1)  Table (7) References (26)
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    Cite this article
    Naher L, Mustaffa Bakri NA, Muhammad Sukhri SAN, Nik Hassan NR, Mohd Firdaus Ganga H, et al. 2024. Economical substrate formulation for mushroom cultivation and food production of mushroom crackers to reduce postharvest waste. Studies in Fungi 9: e002 doi: 10.48130/sif-2024-0003
    Naher L, Mustaffa Bakri NA, Muhammad Sukhri SAN, Nik Hassan NR, Mohd Firdaus Ganga H, et al. 2024. Economical substrate formulation for mushroom cultivation and food production of mushroom crackers to reduce postharvest waste. Studies in Fungi 9: e002 doi: 10.48130/sif-2024-0003
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  • Introduction
  • Materials and methods

  • Raw material collection

  • Preparation of substrates

  • Preparation of substrate in each polyethylene filling bag

  • Processing of food crackers from grey oyster mushroom

  • Proximate analysis

  • Protein analysis

  • Ash, fat and carbohydrate analysis

  • Analysis of mushroom cracker attributes

  • Sensory evaluation

  • Statistical analysis

  • Results

  • Yield and income performance

  • Proximate analysis of mushroom cracker and commercial potato cracker

  • Colour properties

  • Texture profile analysis

  • Sensory evaluation of mushroom crackers

  • Discussion
  • Conclusions
  • Author contributions
  • Data availability
  • Acknowledgments
  • Conflict of interest
  • Rights and permissions
  • About this article
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