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Effect of the addition of beeswax on the gel properties and microstructure of white mushroom powder-based hybrid gelator system for 3D food printing

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  • Using white mushroom powder as a raw material, this study proposes a new strategy to apply it to 3D food printing and improve its printing application. The effects of beeswax-gelatin-carrageenan hybrid gelator on the gel strength, rheological properties, microstructure and thermal stability of white mushroom powder-based inks were investigated. The results showed that the addition of beeswax could significantly increase the gel strength of the ink (p < 0.05), and enable 3D food printed objects (cubes and seals) with a smoother surface and greater self-supporting capacity. The rheological results revealed that the hybrid gelator system was a pseudoplastic fluid with shear-thinning behavior, and beeswax addition could increase its elastic modulus and loss modulus. This rheological property was caused by the formation of new crystal forms of beeswax after mixing with the ink in a water bath. It could be seen from the microstructure that the added beeswax was evenly distributed in the network structure, which affected the printing performance during the printing process. Furthermore, the addition of beeswax could reduce the thermal stability of the ink, but had little effect at room temperature. These results suggest that the addition of beeswax had positive effects on the gel properties of the hybrid gelator system, and this work facilitates the practical application of white mushroom in 3D food printing.
  • The genus Nymphaea, belonging to the family Nymphaeaceae and commonly known as water lily, comprises over 60 species of perennial aquatic plants[1]. This precious aquatic plant is distributed across frigid zones to the tropical regions worldwide. In horticulture, water lilies can be divided into two categories based on their ecological habits: tropical water lilies and hardy water lilies[2]. In addition to their aesthetic appeal, economically, Nymphaea species possess a substantial quantity of phytochemicals and nutrients, making them widely utilized for beverage preparation[3], essential oil extraction[4], and as a valuable source of food, nutrition, and medicinal purposes[5,6]. The incorporation of water lily in scented tea offers potential benefits in the tea beverage industry, combining ornamental value and potential health benefits[7]. Understanding the chemical composition and metabolic profiles of water lilies is important for uncovering their biological functions and applications. Metabolomics, a comprehensive study of small molecule metabolites, provides insights into the diverse range of non-volatile and volatile metabolites in water lilies[8,9].

    Non-volatile metabolites encompass a wide range of compounds, including primary metabolites such as carbohydrates, amino acids, and organic acids, as well as secondary metabolites such as flavonoids, alkaloids, and phenolic compounds[10]. These metabolites are essential for the growth, development, and defense mechanisms of plants[11]. Phytochemical screening of water lilies indicates the presence of several bioactive compounds like phenolic, flavonoids, triterpenes, glycosides, carbohydrates, and other compounds[12]. Phenols and flavonoids are the main phytochemicals found in water lilies and are responsible for their health benefits[5]. In addition, the captivating colors of water lily petals are attributed to their richanthocyanin content. For instance, delphinidin 3′-O-(2″-O-galloyl-6″-O-acetyl-β-galactopyranoside was identified as the primary blue anthocyanin in N. colorata[13]. Most detection methods for water lily metabolites are either targeted, with limited coverage, or non-targeted, with lower sensitivity and accuracy[14]. The combination of ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with widely targeted metabolomics techniques provides fast separation, high sensitivity, and broad coverage[15].

    Volatile metabolites, low molecular weight compounds that evaporate at ambient temperatures, are essential for the aroma, pollinator attraction, communication, and defense in plants. Its compositions vary among different species of water lilies. For example, day-blooming species emitting aromatic alcohols and ethers, while nocturnal species have a higher abundance of aromatic ethers, aliphatic esters, and C5-branched chain esters, which may play a role in attracting potential pollinators through olfactory cues[16]. In addition, previous studies using solid phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) detected 79 and 71 volatile compounds in tropical water lilies and hardy water lilies, respectively, with aromatic substances being their major volatile components[17]. However, with the advancements in analytical techniques, researchers have sought to explore more advanced methods for volatile compound analysis. Currently, comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) has been widely adopted for the analysis of volatile compounds in a variety of foods due to its high resolution, high sensitivity, and peak capacity[18,19].

    In this study, we utilized UPLC-MS/MS and GC×GC-TOFMS techniques to comprehensively identify both non-volatile and volatile constituents of water lilies in five different colors. By comparing metabolite profiles across various Nymphaea species and varieties, we can uncover the discrepancies in chemical compounds that maybe linked to variations in scent, coloration, and potential biological activity. The findings of this study will enhance our understanding of the metabolic variability and chemical constitution of water lilies, thereby improving our knowledge of their distinctive attributes and potential uses.

    Methanol, acetonitrile and ethanol were purchased from Merck (Darmstadt, Germany). Ether (GC) was acquired from Tedia (Fairfield, OH, USA). Anhydrous sodium sulfate (AR) and ethyl decanoate (AR, internal standard) were purchased from Sigma-Aldrich (Shanghai, China). Distilled water was obtained from Wahaha Group Company (Hangzhou, China). The carboxen/divinylbenzene/polydimethylsiloxane (CAR-DVB-PDMS; 50/30 μm) microextraction fiber was obtained from Supelco (Bellefonte, PA, USA). The n-alkanes (C3-C9, C8-C40) were purchased from J&K Scientific (Beijing, China).

    As shown in Fig. 1, five different water lily samples were collected from Hangzhou Aquatic Plant Society, including two species — N. lotus (NL), N. rubra (NR), and three varieties — Nymphaea 'Texas Dawn' (TD), Nymphaea 'Blue Bird' (BB), and Nymphaea 'Detective Erika' (DE). Three fully expanded flowers (on the first day after flowering) were collected in the morning, three samples of each variety, and processed immediately.

    Figure 1.  Five different species and varieties of water lilies selected in this study.

    The petals of water lily samples were ground using a mixer mill (MM 400,) with a zirconia bead for 1.5 min at 30 Hz. One hundred mg of sample powder was dissolved with 0.6 mL 70% aqueous methanol; the dissolved sample was placed in the refrigerator at 4 °C for 12 h, during which the sample was vortexed six times; finally, the sample was centrifuged at 4 °C for 10 min at 12,000 r/min, and the extracts were absorbed and filtrated (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China), and then sealed in the injection vial for subsequent UPLC-MS/MS analysis.

    The analysis was conducted by MetWare (Wuhan, China)[20]. UPLC (Shim-pack UFLC SHIMADZU CBM30A) equipped with tandem mass spectrometry (Applied Biosystems 4500 QTRAP) was used for the wide-targeted metabolomic assays of non-volatiles in water lily samples. The analytical conditions were as follows: Waters ACQUITY UPLC HSS T3 C18 column (2.1 mm × 100.0 mm, 1.8 μm); flow rate of 0.35 mL/min; column temperature of 40 °C; injection volume of 4 μL. The mobile phase A was ultrapure water (dissolved in 0.04% acetic acid), and the mobile phase B was acetonitrile (dissolved in 0.04% acetic acid). The mobile phase elution gradient was as follows: the proportion of phase B was 5% at 0.00 min, the proportion of phase B increased linearly to 95% at 10.00 min and maintained at 95% for 1 min, the proportion of phase B was reduced to 5% at 11.00–11.10 min and equilibrated with the proportion of phase B at 5% for 14 min. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.

    Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 4500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source operation parameters were as follows: ion source, turbo spray; source temperature 550 °C; ion spray voltage (IS) 5,500 V (positive ion mode)/–4,500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 50, 60, and 30 psi, respectively; the collision gas (CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. Declustering potential (DP) and collision energy (CE) for individual MRM transitions was carried out with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.

    For the qualitative analysis, the metabolites were identified by matching the retention time, fragmentation patterns, and accurate m/z values to the standards in the self-constructed metabolite database (MetWare, Wuhan, China). The quantitative analysis was conducted based on the signal intensities of metabolites obtained from characteristic ions. MultiaQuant software was used to integrate and calibrate chromatographic peaks. The peak area of each chromatographic peak represented the relative content of the corresponding substance.

    The sample preparation method underwent minor modifications, as outlined in the previous study[19]. Specifically, simultaneous distillation extraction (SDE) method was used to extract the aroma components of water lily flower. The specific steps were as follows: weigh 10.00 g of the water lily flower sample to be tested, put it in a 500 mL round-bottomed flask and add 300 mL of boiling distilled water, and heat it to a slight boil with an electric heating jacket. Add 30 mL of redistilled anhydrous ether into the extraction flask and distill the extract at 50 °C for 1 h. Remove the water from the obtained material with anhydrous sodium sulfate and concentrate under nitrogen, then put into the injection bottle for sealing and storage at –20 °C for measurement.

    The analysis of aroma components in the water lily samples was conducted using a GC×GC-TOFMS system, which consisted of a gas chromatograph (Agilent 7890B; Santa Clara, CA, USA) coupled with a TOFMS instrument (LECO Pegasus 4D; LECO Corporation, St. Joseph, MI, USA). The first dimension (1st D) column was a non-polar DB-5MS column (30 m × 250 μm × 0.25 μm) and the second dimension (2nd D) column was a moderate polar DB-17HT column (1.9 m × 100 μm × 0.10 μm), and both above columns were purchased from Agilent Technologies (Santa Clara, CA, USA). Injection port temperature: 280 °C; Transfer line temperature: 270 °C; Carrier gas: helium (purity 99.999%); No split injection; Modulation time interval: 4.0 s; Sample injection volume: 1.0 μL. The 1st D column temperature program: hold at 60 °C for 3.0 min, ramp at 4.0 °C/min to 280 °C, hold for 2.5 min; 2nd D column temperature program: hold at 65 °C for 3.0 min, ramp at 4.0 °C/min to 280 °C, hold for 2.5 min; Total analysis time: 60.5 min. MS conditions: Electron ionization source; Ionization energy: –70 eV; Mass scanning range: 33 to 600 m/z; Ion source temperature: 220 °C.

    The GC×GC-TOFMS data were processed using the LECO ChromaTOF software. The calculated retention index (RI) values calculated using C8–C40 n-alkanes, and the reference RI values were obtained from the NIST 2014 semi-standard non-polar (DB-5) column. A compound was considered tentatively identified if the difference between the calculated and reference RI values was less than 20.

    Patrial least squares discriminant analysis (PLS-DA) was performed by MetaboAnalyst 5.0 (www.metaboanalyst.ca). The data was log transformed (log10) and normalization by median before PLS-DA. Significantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute Log2FC (fold change) ≥ 1. To mitigate the risk of overfitting, we conducted a permutation test with 100 permutations.

    The identified non-volatile metabolites were annotated using Kyoto Encyclopedia of Genes and Genomes (KEGG) Compound database (www.kegg.jp/kegg/compound), annotated metabolites were then mapped to KEGG pathway database (www.kegg.jp/kegg/pathway.html). Pathways with significantly regulated metabolites mapped to were then fed into metabolite sets enrichment analysis, their significance was determined using p-values obtained from the hypergeometric test.

    A total of 533 non-volatile metabolites including nine categories were tentatively identified for water lilies by a comparison with tandem mass spectrum information from published databases and standards from the MetWare self-constructed metabolite database (Supplemental Fig. S1). As shown in Fig. 2a, these were 151 flavonoids (including 64 flavonoid and flavonoid carbonoside, 51 flavonols, 11 anthocyanins, 11 flavanols, nine dihydroflavone and dihydroflavonol, five isoflavones), 109 phenolic acids, 68 amino acids and derivatives, 57 lipids, 39 nucleotides and derivatives, 30 organic acids, 27 alkaloids, 23 saccharides and alcohols, and 29 other metabolites. Results showed that flavonoids, phenolic acids, lipids, amino acids and derivatives were the dominant non-volatile metabolites in the five water lilies. To better understand the differences in the content of non-volatile components between water lily samples from different species and varieties, the analysis was calculated by Log2FC (fold change) ≥ 2 or ≤ 0.5. The plots reflect the information on differential metabolite up-regulation and down-regulation (Fig. 2b). The numbers of differential metabolites identified in NL, TD, NR, and DE were 329 (237 up, 92 down), 310 (218 up, 92 down), 314 (213 up, 101 down), and 282 (177 up, 105 down), respectively.

    Figure 2.  Overview of the non-volatile components. (a) Quantitative distribution of chemical classes of volatile compounds. (b) Number of differentiated compounds with fold change ≥ 2 or ≤ 0.5. Note: NL, N. lotus; NR, N. rubra; TD, Nymphaea 'Texas Dawn'; BB, Nymphaea 'Blue Bird'; DE, Nymphaea 'Detective Erika'.

    In the present study, all differential metabolites were investigated based on fold change, and a total of 118 non-volatiles were screened in five water lilies, mainly consisted of flavonoids, phenolic acids, amino acids and derivatives, lipids, and organic acids metabolites (Supplemental Table S1 & Supplemental Fig. S2). Specifically, we screened 10 up-regulated and 10 down-regulated metabolites with the highest fold change values in different water lilies (Fig. 3). Comparing the NL samples to the BB samples, we observed higher levels of chrysoeriol-O-malonylhexoside, 6,7,8-tetrahydroxy-5-methoxyflavone, myricetin-O-glucoside-rhamnoside, and kaempferol-3-O-neohesperidoside. On the other hand, the BB samples contained higher levels of isoquercitrin and luteolin-7-O-β-D-gentiobioside. Moreover, the TD samples exhibited high levels of neochlorogenic acid, chlorogenic acid, 1-O-p-coumaroylquinic acid, and tetragallic acid, while the BB samples exhibited high levels of 2'-hydroxygenistein, phloretin 2'-O-glucoside, and 3,4-dihydroxybenzaldehyde. Comparing the NR samples to the BB samples, we found higher levels of cyanidin-3-rutinoside, cyanidin-3-O-galactoside, and myricetin-O-glucoside-rhamnoside. Conversely, the BB samples exhibited higher levels of 7-methoxycoumarin, myricitrin, and naringenin-7-O-glucoside. In comparison to the BB samples, the DE samples displayed higher levels of myricetin-O-glucoside-rhamnoside, cis-4-hydroxy-D-proline, myricetin-3-O-rhamnoside-7-O-rhamnoside, and neochlorogenic acid. On the other hand, the BB samples contained higher levels of myricitrin, isoquercitrin, and naringenin-7-O-glucoside.

    Figure 3.  The highest fold change values of non-volatile metabolites. (a) Fold change plot of NL vs BB. (b) Fold change plot of TD vs BB. (c) Fold change plot of NR vs BB. (d) Fold change plot of DE vs BB. Note: The horizontal coordinate is the log2FC of the differentially metabolized metabolite, and the vertical coordinate is the differentially metabolized metabolite. Red represents up-regulated differentially expressed metabolites, cyan represents down-regulated differentially expressed metabolites. NL, N. lotus; NR, N. rubra; TD, Nymphaea 'Texas Dawn'; BB, Nymphaea 'Blue Bird'; DE, Nymphaea 'Detective Erika'.

    We further identified the enrichment of differential metabolites in the KEGG mapping. The results of pathway enrichment analysis of the detected differential compounds using the KEGG database are shown in Supplemental Fig. S3. A total of 354, 327, 380, 299 differential compounds from BB vs NL, BB vs TD, BB vs NR, BB vs DE samples could be annotated to the relevant metabolic pathways, which were mainly significantly enriched in the pathways of biosynthesis of secondary metabolites, flavonoid biosynthesis, anthocyanin biosynthesis, flavonoids and flavonols biosynthesis, isoflavonoid biosynthesis. Furthermore, we observed significant enrichment in pathways associated with tryptophan metabolism and phenylpropanoid biosynthesis in the BB vs TD group. Additionally, caffeine metabolism exhibited significant enrichment in the BB vs NR group.

    The fragrance of water lily contains volatile compounds such as terpenes, phenylpropanoids, benzenoids, fatty acid derivatives, and amino acid derivatives. These compounds not only attract pollinators, but also play a crucial role in transmitting signals in plant-plant interactions and providing protection and defense for the plant[17]. In this study, the volatile compounds of water lily samples were analyzed by GC×GC-TOFMS (Supplemental Fig. S4). By comparing the MS of the compounds and comparing the chromatographic peaks and tested RI values with the reported RI values, 166 volatiles were identified, including 46 aromatic compounds, 34 alkynes, 22 ketones, 10 alcohols, 18 esters, 20 aldehydes, three carboxylic acids, five heterocyclics, five sulfur-containing compounds, and three other compounds (Fig. 4a). We identified 128, 141, 142, 135, and 129 volatile compounds in BB, NL, TD, NR, and DE, respectively, and 108 of the 166 metabolites were common to all water lily samples (Fig. 4b). We observed that 20 volatile compounds were exclusively detected in specific water lily samples. For instance, in BB variety, compounds such as amorpha-4,11-diene, (Z)-geranylacetone, α-bisabolol, and (E)-β-ionone were found. In NR variety, pyrocinchonic anhydride and 2-tetradecanone were exclusively detected. NL variety exhibited unique compounds including mequinol, 4-ethylresorcinol, p-xylene, and 2,5-hexanedione. TD variety showed the presence of terpilene, (E,E)-2,6-dimethyl-2,4,6-octatriene, 1-nonanol, β-bisabolene, sabinene, 3-carvomenthenone, (4E,6Z)-2,6-dimethyl-2,4,6-octatriene, ipsdienol, and 2-thujene. Lastly, DE variety exclusively contained 2-pentoxyethyl acetate.

    Figure 4.  Overview of the volatile components. (a) Quantitative distribution of chemical classes of volatile compounds. (b) Venn plot; numbers represent the identified metabolites. (c) Relative abundance of different types of volatile compounds. Note: NL, N. lotus; NR, N. rubra; TD, Nymphaea 'Texas Dawn'; BB, Nymphaea 'Blue Bird'; DE, Nymphaea 'Detective Erika'.

    The relative content of volatiles calculated from the total ion chromatograms varied in the concentration and proportion of each chemical class in different samples. Among them, the highest peak area of volatile components was found in BB, followed by NL (Fig. 4c). Alkenes were found to be the most abundant volatile components in BB, accounting for 69.58% of the total volatile components. Aromatic compounds were found to be the most abundant volatile components in NL, accounting for 64.25% of the total volatile components. In addition, alcohols and alkenes accounted for 28.05% and 26.89% of the total volatile components in DE, respectively.

    We investigated the relative amounts of the main 24 chemical compounds released that were greater than 1% in BB, NL, TD, NR, DE, with total relative contents of 90.92%, 91.45%, 83.19%, 77.29%, and 90.83%, respectively (Table 1 & Fig. 5). The concentrations of the identical chemical compounds varied across distinct samples. Within the provided BB sample, three compounds of the alkene class, namely 1,11-dodecadiene (27.30%), (E)-β-famesene (18.28%), and α-farnesene (14.44%), collectively constitute more than half of the total volatile compounds. Among the NL samples, 2,5-dimethoxytoluene (56.18%) emerged as the most abundant compound. In TD and NR samples, dimethyl sulfide was the predominant volatile compound, comprising 33.61% and 18.75% of the total volatile content, respectively. In addition, in DE sample, the concentration of benzyl alcohol (23.83%) was the most abundant, followed by dimethyl sulfide (16.67%) and α-farnesene (16.41%).

    Table 1.  Comparison of the main volatile compounds in five water lily samples.
    No.CompoundsClassRI (ref)[a]RI (cal)[b]CASIonOdor type[c]Flavor[c]Relative content (%)
    BBNLTDNRDE
    1Benzyl alcoholAlcohols1036 ± 41037100-51-679FloralFloral, rose, balsamic0.114.486.328.1323.83
    2BenzaldehydeAldehydes962 ± 3969100-52-777FruityAlmond, burntsugar, sweet8.102.133.175.223.91
    3HexanalAldehydes800 ± 280166-25-141GreenGreen, fatty, leafy0.150.690.613.840.34
    4BenzeneacetaldehydeAldehydes1045 ± 41049122-78-191GreenGreen, floral, honey0.160.141.030.090.85
    51,11-DodecadieneAlkenes1179 ± 217635876-87-967//27.303.6712.627.0411.45
    6(E)-β-FameseneAlkenes1457 ± 2145318794-84-841WoodyWoody, citrus, herbal18.280.390.840.381.27
    7α-FarneseneAlkenes1508 ± 21506502-61-441WoodyCitrus, herbal, neroli14.440.1710.380.1816.41
    8(E)-α-BergamoteneAlkenes1433 ± 3143713474-59-493WoodyWoody2.020.110.030.031.65
    9β-SesquiphellandreneAlkenes1524 ± 2152820307-83-969HerbalHerbal, fruity, woody3.430.120.150.101.37
    10(E)-β-OcimeneAlkenes1049 ± 210483779-61-193/Herbal, sweet0.010.033.890.040.01
    112,5-DimethoxytolueneAromatic compounds1251 ± 5124924599-58-4137//0.3856.180.4912.180.52
    121,4-DimethoxybenzeneAromatic compounds1168 ± 91166150-78-7123GreenGreen, hay, sweet0.053.620.026.630.02
    13PhenolAromatic compounds980 ± 4981108-95-294PhenolicPhenolic, plastic, rubbery0.170.870.561.460.62
    14Acetic acidCarboxylic acids610 ± 1058164-19-745AcidicPungent, sour3.422.012.180.771.58
    15Benzoic acid, methyl esterEsters1094 ± 3109693-58-3105PhenolicPhenolic, wintergreen, almond0.030.570.071.860.03
    16Ethyl acetateEsters612 ± 5613141-78-643EtherealEthereal fruity sweet0.380.390.140.002.06
    17Acetic acid, phenylmethyl esterEsters1164 ± 21166140-11-4108FloralFloral, fruity, jasmine1.880.010.060.020.45
    186-Methyl-5-heptene-2-oneKetones986 ± 2984110-93-043CitrusCitrus, green, musty1.292.162.153.673.05
    19(E)-3-Penten-2-oneKetones735 ± N/A7443102-33-869//0.211.010.311.580.40
    20(E)-GeranylacetoneKetones1453 ± 214483796-70-143FloralFruity, fresh, rose2.150.760.650.721.18
    212-HeptadecanoneKetones1902 ± 719002922-51-258//1.680.411.180.700.80
    22Dimethyl sulfideSulfur-containing compounds520 ± 755375-18-347SulfurousSulfurous, sweet corn4.378.7633.6118.7516.67
    23Carbon disulfideSulfur-containing compounds549 ± 1356575-15-076/Sweet0.551.171.961.291.39
    24BenzothiazoleSulfur-containing compounds1229 ± 8124095-16-9135SulfurousSulfurous, rubbery, vegetable, cooked0.290.480.561.160.53
    Total relative content (%)90.9291.4583.1977.2990.83
    RI, Retention index, Ion, Qualitative ion; [a] RI (ref): The RI values (median ± deviation) were the reference values for semi-standard non-polar (DB-5) column obtained from NIST 2014; [b] RI (cal): The RI values were calculated from C8-C40 n-alkanes; [c] odor type and flavor were obtained from website (www.thegoodscentscompany.com/search2.html). Note: NL, N. lotus; NR, N. rubra; TD, N. 'Texas Dawn'; BB, N. 'Blue Bird'; DE, N. 'Detective Erika'.
     | Show Table
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    Figure 5.  Molecular formulas of major volatile compounds.

    To gain a comprehensive understanding of the variations in volatile compound content among the five water lily samples, we utilized PLS-DA with the peak areas of 166 volatile compounds as input variables. As shown in Fig. 6a, the five samples were distinctly segregated from the remaining samples along the principal component 1 axis (R2X [1] = 37.6%) and principal component 2 axis (R2X [2] = 27.1%). Cross-validation was performed using the leave-one-out method, with the first two principal components explaining 99.6% of the total variance (R2X). The model exhibited good predictive ability (Q2 = 85.1%) and was not overfitted. The evident segregation and high reproducibility observed among the various sample groups substantiated the presence of significant disparities in the volatile compositions of the five water lily species and varieties (Fig. 6a).

    Figure 6.  The partial least squares discriminant analysis (PLS-DA) of the volatile compounds. (a) Score plot of PLS-DA. (b) The loading plot of PLS-DA. (c) Variable importance in the project (VIP) plot of PLS-DA. Note: NL, N. lotus; NR, N. rubra; TD, Nymphaea 'Texas Dawn'; BB, Nymphaea 'Blue Bird'; DE, Nymphaea 'Detective Erika'.

    The variable importance in projection (VIP) value is a comprehensive metric that quantifies the contribution of a variable in describing the data and indicates the significance of an independent variable for the model[21]. By utilizing the PLS-DA model, we identified 42 key volatile compounds with VIP scores of ≥ 1 across all samples (Supplemental Fig. S5). Subsequently, a one-way analysis of variance (ANOVA) was conducted on these compounds, revealing statistically significant differences among the distinct sample groups (p < 0.05). Notably, 26 metabolites of volatile compounds exhibited VIP values exceeding 1.5 (Fig. 6c), with 2,3-butanedione, octanal, 1-methyl-4-(1-hydroxy-1-methylethyl)benzene, acetic acid, phenylmethyl ester, 2,5-dimethoxytoluene, (E)-β-ocimene being among the top-ranked compounds in descending order of VIP values.

    Although previous studies have been reported on the volatile and non-volatile components of water lilies, the number of compounds reported in these studies is relatively limited[2,22]. By employing widely targeted metabolomics, we overcome the limitations associated with both targeted and non-targeted metabolite detection methods. This approach provided us with a high-throughput analysis, increased sensitivity, and wide coverage, enabling a more comprehensive understanding of the metabolomic profile of water lilies. In the present study, we comprehensively identified 533 non-volatile and 166 volatile components in five different colors of water lilies using UPLC-MS/MS and GC × GC-TOFMS techniques.

    It is widely recognized that flower color is a crucial characteristic of ornamental plants and is influenced by various factors. Specifically, the type and concentration of anthocyanins are generally considered to be the primary determinants[23]. Anthocyanins play an important role in plant physiology, serving as attractions for pollinators and herbivores, acting as deterrents against herbivores and parasites, and also influencing visual signals and mimicry of defensive structures[24]. Among the diverse range of flower colors, blue coloration primarily results from the presence of anthocyanins derived from delphinidin[25]. In the petals of Nymphaea 'King of Siam', four anthocyanins were identified: delphinidin 3-O-β-galactopyranoside, delphinidin 3′-O-(2″-O-galloyl-β-galactopyranoside, delphinidin 3-O-(6″-O-acetyl-β-glucopyranoside, delphinidin 3′-O-(2″-O-galloyl-6″-O-acetyl-β-galactopyranoside[13]. In the present study, a total of 11 anthocyanins were detected in five different-colored water lilies. Among them, delphinidin-derived anthocyanins exhibited relatively higher relative abundances in BB, TD, and DE, while its abundance was low in DE. Notably, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, cyanidin-3-rutinoside, cyanidin-O-acetylhexoside, and cyanidin-O-syringic acid, which are glycosides of the cyanidin type, were found to be the most abundant exclusively in the DE samples. In contrast, these compounds were found at very low levels or were not detected in the other four water lily samples.

    In addition, numerous studies conducted on various plant species have provided evidence that flavonoids (including flavones, flavanols, and isoflavonoids) and their glycosides play a significant role in co-pigmentation[26,27]. According to reports, N. lotus stamen extracts contain a higher concentration of flavonoids compared to perianth extracts[22]. The flavonoids identified in the stamen extracts include kaempferol 3-O-galactoside, quercetin 3′-O-xyloside, quercetin 3-O-rhamnoside, isorhamnetin 7-O-galactoside, and myricetin 3′-O-xyloside. Another study indicated that the content of flavonoids in the petals of the N. alba flower is significantly higher than in the stem and root[28]. In addition, a large number of phenolic acids, for example, caffeic acid, chlorogenic acid, p-coumaric acid have been identified in water lilies[12]. Our present study identified various flavonoid glycosides with glucose, rhamnose, galactose, and arabinose as the major sugar ligands. As a result, the number of detected flavonoids and their glycosides in water lilies has significantly expanded. Among them, quercetin, kaempferol, apigenin, myricetin, and luteolin were identified as the five major flavonols present in water lilies. In addition, many previous studies have reported the antioxidant potential of water lily extracts that are associated with the accumulation of its phytochemicals, especially flavonoids[5,7,29]. Therefore, the variations in the composition of flavonoids among distinct water lily species and varieties, as revealed in this investigation, are expected to provide valuable insights into their diverse antioxidant properties.

    Water lilies not only exhibit a wide variety of colors throughout their flowering period but also emit a captivating fragrance, which has garnered significant attention among researchers in recent studies[2,30]. Among the seven species of N. subg. Hydrocallis (Nymphaeaceae), N. lotus was only found to contain detectable levels of 2,5-dimethoxytoluene, and the content was notably high[16]. This is consistent with the results of our study. Dimethyl sulfide, a group of sulfur-containing compounds, was detected at significant concentrations in all five water lilies. Notably, it has been identified as a crucial aromatic volatile in green tea[31] and mandarin juices[32], and even in trace amounts, it contributes to the development of sulfurous notes. Benzyl alcohol, renowned for its delightful floral and sweet essence, has been extensively documented in various Nymphaea species[2]. Its relative concentration in the DE samples was 23.83% of the total volatiles. This high concentration may be the main reason for the sweet and floral aroma of the DE samples. Additionally, according to the report, 2,3-butanedione is known for its buttery, sweet, and creamy flavor, which imparts a cheese-like taste to soy milk[33]. Our research findings indicate that the BB samples had the highest concentration of 2,3-butanedione, which may be responsible for its sweet and creamy flavor, while it was not detected in the NL sample.

    In addition, previous studies reported that the flower aroma of the cold-resistant water lily is influenced by nerolidol and lilac alcohols, with orange and clove flower aromas, while the aroma of tropical water lilies is influenced by ethyl benzoate, acetic acid phenylmethyl ester, and 2-heptadecanone, which provide a fruity and sweet flower aroma[2]. Moreover, the primary volatile compounds identified in the flowers of Nymphaea hybrida were benzyl alcohol, benzyl acetate, benzaldehyde, (E)-α-bergamotene, and these may constitute its main fragrant components[34]. We observed that the NL sample releases a greater proportion of aromatic compounds than the other samples, which may account for its fruity and sweet flavor. In addition, (E)-α-Bergamotene, (E)-β-famesene and 11 other different volatile compounds were reported in N. colorata flowers, which may serve as olfactory cues for insect pollinators[35]. (E)-β-famesene and α-farnesene possess woody, citrus, and herbal aromatic attributes, was found to be more pronounced in BB samples, accounting for 18.28% and 14.44% of the total volatile composition, respectively. These compounds are likely responsible for the woody fragrance observed in BB samples. Overall, the varying concentrations and relative amounts of volatile compounds contribute to the rich and diverse fragrance qualities of these five water lily species and varieties.

    To sum up, our study utilized advanced widely targeted metabolomics to comprehensively investigate the non-volatile and volatile components in five water lily species and varieties. The results revealed significant differences in the composition and abundance of metabolites among the different colors of water lily samples. Regarding non-volatile components, we found that cyanidin-type anthocyanins were abundant in the purple-colored DE samples, while delphinidin-derived anthocyanins were prominent in BB, which exhibited a blue color. Flavonoids, phenolic acids, amino acids, lipids, and organic acids were found to be the dominant non-volatile metabolites, expanding our knowledge of their metabolic profile. Pathway enrichment analysis of the differential compounds indicated the involvement of various metabolic pathways such as flavonoid biosynthesis and anthocyanin biosynthesis in water lily metabolism. In terms of volatile components, water lilies contain diverse volatile compounds, including aromatic compounds, alkynes, ketones, alcohols, esters, and aldehydes. Among these, the concentration ofkey compounds including 1,11-dodecadiene, benzyl alcohol, benzaldehyde, α-farnesene and dimethyl sulfide, showed significant differences among samples. By investigating both non-volatile and volatile metabolites, we have obtained a comprehensive understanding of the metabolic pathways and interplay within the water lily species and varieties. Nevertheless, the intricate mechanisms underlying the synthesis and release of metabolites in water lilies warrant further investigation.

    The authors confirm contribution to the paper as follows: study conception and design: Chen Y, Lv H; data collection: Wei J, Wu Y; resources: Chen S, Yu C; analysis and interpretation of results: Lin Z, Zhu Y; draft manuscript preparation: Yang G. All authors reviewed the results and approved the final version of the manuscript.

    The datasets generated during and/or analyzed during the current study are not publicly available due to management requests, but are available from the corresponding author on reasonable request.

    This work was supported by the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2014-TRICAAS) for financial support.

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

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    Xiao K, Pan L, Tu K. 2022. Effect of the addition of beeswax on the gel properties and microstructure of white mushroom powder-based hybrid gelator system for 3D food printing. Food Materials Research 2:6 doi: 10.48130/FMR-2022-0006
    Xiao K, Pan L, Tu K. 2022. Effect of the addition of beeswax on the gel properties and microstructure of white mushroom powder-based hybrid gelator system for 3D food printing. Food Materials Research 2:6 doi: 10.48130/FMR-2022-0006

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Effect of the addition of beeswax on the gel properties and microstructure of white mushroom powder-based hybrid gelator system for 3D food printing

Food Materials Research  2 Article number: 6  (2022)  |  Cite this article

Abstract: Using white mushroom powder as a raw material, this study proposes a new strategy to apply it to 3D food printing and improve its printing application. The effects of beeswax-gelatin-carrageenan hybrid gelator on the gel strength, rheological properties, microstructure and thermal stability of white mushroom powder-based inks were investigated. The results showed that the addition of beeswax could significantly increase the gel strength of the ink (p < 0.05), and enable 3D food printed objects (cubes and seals) with a smoother surface and greater self-supporting capacity. The rheological results revealed that the hybrid gelator system was a pseudoplastic fluid with shear-thinning behavior, and beeswax addition could increase its elastic modulus and loss modulus. This rheological property was caused by the formation of new crystal forms of beeswax after mixing with the ink in a water bath. It could be seen from the microstructure that the added beeswax was evenly distributed in the network structure, which affected the printing performance during the printing process. Furthermore, the addition of beeswax could reduce the thermal stability of the ink, but had little effect at room temperature. These results suggest that the addition of beeswax had positive effects on the gel properties of the hybrid gelator system, and this work facilitates the practical application of white mushroom in 3D food printing.

    • 3D printing technology, which falls under the category of additive manufacturing (AM), has developed rapidly in recent years, and has breakthrough applications in many fields such as mechanical engineering, aviation, medical and the food industry[1]. 3D food printing (3DFP) includes extrusion, selective laser sintering, binder jetting, and inkjet printing[2]. Specifically, the extrusion-based technique is the most extensively studied 3DFP approach, and the application of 3DFP can be controlled by computers to complete the layers of raw food materials, gradually complete the transformation from 3D digital objects to products, so as to maximize the utilization of materials and reduce the impact on the environment[3]. On this basis, digital objects have the potential to diversify in precision, personalization and innovation, enabling the development of healthy food products according to different needs[4]. 3DFP offers many advantages over traditional food ingredient processing methods, such as flexible innovation, cost reduction, on-demand production and a sense of engagement for the end user[5]. However, despite the rapid development of 3DFP technology, most of its technologies have not yet achieved large-scale industrial production and are in the research and development stages[6]. It is still a relatively new research field with a small number of research papers[7]. First of all, the nature of the printing material determines the restrictions that may be encountered in the food printing process. For example, some foods are not suitable for 3DFP since they do not match the printer for their form and thermal stability, and cannot complete the extrusion stage in a traditional sense[8]. Secondly, the mixed reaction of different components will bring about unknown shape stability and structural characteristics, which brings challenges to the research and development of new products[9]. Therefore, it is possible to conduct in-depth research on traditional printable food inks and non-traditional printing inks, develop food raw materials as semi-solid paste or gel systems, and use different pretreatment methods for non-traditional printing inks to meet food printing needs[10].

      In recent years, food-based hydrocolloids have been frequently mentioned in the 3DFP literature due to their capacity to thicken and gel water-based solutions, affecting their rheological properties (viscoelasticity) and textural properties, thereby making materials more suitable for 3DFP[11]. Xing et al. added different proportions of κ-carrageenan gum, xanthan gum and arabic gum to black fungus to develop a visually appealing modified material for 3DFP[12]. Liu et al. developed a gel model system composed of carrageenan-xanthan-starch, suitable for extrusion-based 3DFP, which improved the mechanical strength of inks[13]. Gelatin is a collagen derived from animal bones, skin and other connective tissues and is easily obtained, with the ability to improve the gelling properties of products[14]. κ-carrageenan has good water affinity and water holding capacity, which can have good compatibility and synergistic effects with other food hydrocolloids[15]. Despite this, the effect of the addition of hydrocolloids on printing inks cannot fully meet today's needs, due to collapse and poor stability. This puts forward new requirements for the hardness and thermal stability of food printing inks, and organogelators have a potential to solve this problem. Different from normal hydrocolloids, beeswax is a complex organogelator rich in fatty acids and hydrocarbons with certain hardness, plasticity and thermal reversibility, and is being gradually used in the food industry[16]. Due to its hardness and thermal reversibility, beeswax is able to positively influence the 3DFP process. The application of food-grade organogelators in 3DFP is rarely reported, though they have better thermal stability, higher viscosity and higher yield stress.

      Edible mushrooms are considered as an important food source, with the global production market expected to reach 20.84 million tons by 2026[17]. Common edible mushrooms include white mushrooms, Shiitake, Oyster and Enoki (among others). Among them, white mushroom (Agaricus bisporus) has the highest market demand owing to its unique flavor, high nutritional value and therapeutic properties[18]. In general, white mushroom is rich in a variety of trace elements including phosphorus, selenium, and copper, as well as active substances such as ergosterol, which have been proven to have medicinal properties such as detoxification, anti-inflammatory, antioxidant and diabetes prevention[19]. On July 13, 2020, the U.S. Food and Drug Administration (FDA, www.federalregister.gov) issued Document No. 2020-13822, proposing amendments to food additive regulations to approve the use of mushroom powder containing vitamin D2 as a nutritional additive in certain food categories. White mushroom was one of the main sources of this mushroom powder, so it had a very broad market prospect. The processing research on white mushroom mainly focused on the addition of commercially powdered raw materials, including the cooking of soup, fried snacks and baked biscuits, which could be used as a dietary fiber supplement[20]. Due to the high yield and nutritional value, the intensive processing products of white mushroom are gradually being developed, while 3D printing of white mushroom products, combined with personalized nutrition customization and appearance design, still require further study. Therefore, the production of white mushroom powder as the raw material for 3DFP has commercial value. Most of the research on 3DFP printing materials focuses on the effect of a single thickener or gelling agent on the printability of printing inks, and there are few related literature on hybrid gelator systems. It is speculated that the addition of beeswax-gelatin-carrageenan hybrid gelators can further enhance the performance of white mushroom powder-based 3DFP.

      In this study, white mushroom powders were used as raw materials while gelatin, κ-carrageenan and beeswax were added to form a hybrid gelator system. Specifically, the current study controlled the amount of beeswax in the printing inks, and ink with different concentration gradients of beeswax (0, 1%, 3%, 5%, 7%, and 9%, w/w) was used for printing. The hybrid gelator system was heated in a water bath and then stirred at high speed to remove air bubbles. These six hybrid gelator systems were used as printing inks for 3DFP. At the same time, the texture properties, thermal stability, microstructure and printing properties of the inks were compared and analyzed, and the effect of the properties of the hybrid gelator system on the printing behavior was explored, the printed products were then comprehensively evaluated. With consumer demand for dietary nutrition and customized appearance, this study, combined with the use of hybrid gelators, could provide a theoretical basis for the realization of 3DFP for non-traditional printable materials such as white mushrooms.

    • Commercial dried white mushroom powders (sieved using an 80 μm mesh sieve) were purchased from Nanjing Zhiqingtang Food Co., Ltd (Nanjing, China). Gelatin (~100 g bloom), κ-carrageenan (food grade), and decolorized beeswax (AR grade) were purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). Printing ink preparation: hydrocolloids (gelatin and κ-carrageenan, w/w, 1:1) were prepared and dispersed in distilled water and heated in a water bath at 80 °C for 10 min until completely dissolved, then mushroom powder and beeswax were added and stirred. Six groups of inks were prepared for printing, and each group of inks was controlled to 100 g, wherein the consumption of gelatin and carrageenan were both 0.7 g, the consumption of distilled water was 67.6 g, the addition of white mushroom powders was 31, 30, 28, 26, 24, and 22 g, corresponding to the addition amount of beeswax 0, 1, 3, 5, 7, and 9 g respectively. A small amount of ink was taken from each group for freeze drying, and the dried product was cut into pieces before being ground into powder for subsequent determinations of crystal structure, thermal stability and microstructure. The effect of the addition amount of beeswax on the printing characteristics of hybrid gelator systems was investigated, and the related mechanisms were further explored.

    • An extrusion 3D printer (FSE-2, BORIMY, China) was used for printing (Fig. 1a & b), and a cube with a diameter of 20 mm (Fig. 1c) was designed with 3DS Max software (2020, Autodesk, USA) to characterize the printing effect. The specific method was to measure the actual height of the product (the distance from the most serious collapse point to the bottom) after 30 min of printing, and the printing accuracy was calculated according to the following formula:

      Accuracy=h0h1×100%

      In the formula, h0 (mm) represents the actual height of cubes after 30 min, and h1 (mm) represents the model set height (20 mm). In addition, a personalized seal shape (Fig. 1d, 40 mm height) was used to further observe the support properties of different inks after printing. In order to further accurately explore the effect of ink composition on printing, the printing parameters were determined as nozzle diameter 1.2 mm, nozzle height 1.1 mm, first layer height 1.3 mm, wire diameter 9.8 mm, nozzle temperature 25 °C, flow rate 100 %, infilling density 100% based on experiments.

      Figure 1. 

      (a) 3D printer appearance, (b) main parts, (c, d) the virtual model used during printing experiments.

    • The definition of gel strength is the initial force required to break the gel, which has important reference significance for the stability and continuity of the gel[21]. The gel strength of the printed samples was measured by a texture analyzer (TA-XT Plus, Stable Micro Systems, UK), referring to the method of Yang et al. with some modifications[22]. The specific parameters were set as follows: probe model P5, pre-test rate 1 mm/s, test rate 1 mm/s, post-test rate 5 mm/s, trigger force 5 g, puncture distance 5 mm, and the measurement was carried out at room temperature (25 °C).

    • Generally, analyzing the flow behavior of ink raw materials is one of the important methods to judge whether it is suitable for extrusion 3D printing[23]. Hence, the influence of varying levels of beeswax on the rheological behavior of the hybrid gelator system could be analyzed by measuring the apparent viscosity and viscoelastic modulus of the inks. A dynamic rheometer (MCR 301, Anton Paar, Austria) was used to characterize the rheological properties of six groups of inks, and the finished sample was placed between a 50 mm parallel plate and a platform (gap 1 mm). The flattened samples were allowed to equilibrate for 1 min at room temperature, excess ink was carefully wiped from the platform prior to the test[24]. The apparent viscosity of inks was recorded in a shear rate ranging from 0.1 to 10 1/s, and frequency sweep was carried out to obtain the elastic modulus (G') and loss modulus (G'') since they were closely related to the viscoelastic properties of the samples. The sweep was performed in an angular frequency oscillating from 0.1 to 10 rad/s, and the measurements were conducted within a linear viscoelastic region with an amplitude strain of 1%. In addition, an appropriate amount of liquid paraffin was added to the edge of the sample during the measurement to prevent the influence of water evaporation on the experiment.

    • In order to characterize the crystal structure of the inks, the freeze-dried powder was placed and compacted in a hole of 15 mm × 20 mm × 1.5 mm in an aluminum sheet, and then tested using an X-ray diffractometer (XRD-smartlab, Rigaku, Japan). According to the method of Zeng et al.[25] with some modifications, Cu (Kα) radiation (wavelength = 1.5406 nm) was considered as the incident X-ray source, and six groups of inks were detected at 40 mA and 40 kV and 2θ scans were performed from 3° to 50° at the rate of 2°/min. All samples were tested at 25 °C.

    • Thermal analysis was carried out by a simultaneous thermogravimetric analyzer (STA449, NETZSCH, Germany) under a nitrogen atmosphere at 50 ml/min according to the method of Zheng et al.[26], and the curves of thermogravimetric (TG) and derivative thermogravimetric (DTG) curves were recorded simultaneously. The temperature of the six groups of inks ranged from 20 °C to 600 °C, and all heating experiments were conducted at 10 °C/min in the specified temperature range. Ten miligrams of each sample were taken for thermal analysis. The experimental process was strictly regulated by the manufacturer according to the established parameters, and an empty alumina crucible was used as a reference during the measurements.

    • The microstructures of printing objects were observed by SEM (SU8100, HITACHI, Japan) according to the method of Liu et al. with some modifications[27]. Specifically, the freeze-dried inks were fixed on a copper plate with conductive double-sided tape and coated with a thin gold film in a vacuum chamber for cross-section observation. Immediately, the groups were diverted to the SEM chamber and the photomicrographs were obtained at a magnification of 800x with an accelerating voltage of 40 kV.

    • Experimental data were presented as mean and standard deviation (SD) from triplicate measurements. Analysis of variance (ANOVA) was used in this study, and the significance level of p-value < 0.05 was operated with a SPSS 18.0 statistical software (SPSS Inc., Chicago, IL, USA). XRD data were analyzed using MDI Jade 6 software. TG and DTG data were analyzed and processed using TA Universal Analysis 2000 software. Graphs were plotted by Origin pro 8.5 software.

    • The continuity and accuracy of ink-extrusion were observed during the printing process, then the printing accuracy was measured and the surface smoothness was observed to evaluate the printing performance of the hybrid gelator systems with different beeswax concentrations after printing. It can be seen from Table 1 that with the increase of the amount of beeswax added, the printing accuracy showed a trend of first increasing and then decreasing, reaching a maximum of 98.35% when the concentration of beeswax was 3% (w/w). It could be observed that when the concentration of beeswax was 1%, 3%, and 5%, the printing accuracy of the cubes was higher than the non-addition group, indicating that the concentration of beeswax at this time could improve the stability and accuracy of cubes. When the added concentration of beeswax was 7%, the printing accuracy was lower than the non-addition group. By observing the printing process, it could be judged that the addition of beeswax at this time affected how smoothly the ink passed through the nozzle, thereby reducing the printing accuracy. In addition, it was observed that when the addition concentration reached 9%, the phenomenon of wire breakage occurred in the printing process, and it was difficult to complete the fusion and accumulation process, so the printing accuracy was not calculated. The phenomenon of broken bars and blocked nozzles in the 3DFP process is related to the viscosity and mechanical strength of the ink[28]. It could be considered that the addition of beeswax could significantly affect its printing accuracy (p < 0.05) and performance.

      Table 1.  Accuracy of the 3D printed compound products at six beeswax levels (0, 1%, 3%, 5%, 7%, 9%, w/w). The model set height was 20 mm.

      Beeswax
      (%, w/w)
      Actual height after
      30 min (mm)
      Accuracy
      (%)
      017.45 ± 0.32b87.25 ± 2.60b
      119.32 ± 0.11d96.60 ± 1.15d
      319.67 ± 0.09d98.35 ± 1.60d
      518.59 ± 0.26c92.95 ± 3.10c
      716.26 ± 0.47a81.30 ± 4.40a
      9NINI
      Different lowercase letters in the same column indicated significant differences (p < 0.05). NI, no information.

      The gel strength of the six groups of composite materials after 3D printing was measured and analyzed by a texture analyzer, and the value reflected the firmness of the hybrid gelator system. Figure 2 reflected the effect of different beeswax additions on the gel strength of the printed samples. It could be seen that with the increase of beeswax, the gel strength of the printed sample was significantly improved (p < 0.05). Shi et al.[29] reported the preparation of an oleogel in which the interaction force between non-covalent bonds such as hydrogen bonds and van der Waals forces increased with the addition of beeswax, resulting in a tighter network structure, increasing the overall hardness. There is a corresponding relationship between the increasing gel strength and the printing accuracy. With reference to Table 1, it could be seen that when the addition of beeswax was 1%, 3%, and 5%, the printing accuracy was improved, indicating that the gel strength at these levels was helpful for printing. When the concentration of beeswax added was 5%, 7%, and 9%, the printing accuracy was lower than non-addition group, which indicated that the gel strength at these levels was not suitable for printing. In addition, when the concentration of beeswax is 3%, the accuracy of the cube printing product was the highest, and the gel strength of the ink reached nearly twice that of the non-addition group. Therefore, the 3% concentration could be used as a standard to further test the printing effect and support performance.

      Figure 2. 

      Gel strength of 3D printed compound products at six beeswax levels (0, 1%, 3%, 5%, 7%, 9%, w/w). a−f: values with different lowercase letters indicated significant difference (p < 0.05).

      Figure 3 is a comparison chart of the printing effect of the ink without beeswax and the ink with 3% beeswax. It can be seen that the printed surface with no beeswax (Fig. 3a & b) was not smooth enough and had many burrs. While the 3% beeswax level products (Fig. 3c & d) had a smooth surface, clear layers and high precision. Taking the seal as an example (Fig. 3d), when printing a product with such a high (40 mm) three-dimensional structure, the printed layers can be well fused to keep the printed shape stable, and the surface was smoother. Through the above analysis of the printing results, it was considered that 3% beeswax would help the 3DFP by white mushroom, and the gel strength of the printed sample was 14.25 ± 0.72 g.

      Figure 3. 

      Printing quality of 3D printed compound products at two beeswax level (a, b: 0; c, d: 3%, w/w).

    • The apparent viscosity curves of the six groups of inks are shown in Fig. 4a. It can be observed that the apparent viscosity of hybrid gelator at different beeswax concentrations decreased as the shear rate increased, showing the shear-thinning behavior. This indicates that the six groups of inks were all shear-thinning pseudoplastic fluids. In addition, the apparent viscosity increased with increasing beeswax concentration, which was consistent with the findings of Shi et al.[29]. Although six hybrid gelator systems showed shear-thinning effects, the minimum force required to push the ink out of the nozzle would vary, thereby affecting the printing process and final accuracy.

      Figure 4. 

      Rheological behavior (a: apparent viscosity; b: G', c: G'') of inks at six beeswax levels (0, 1%, 3%, 5%, 7%, 9%, w/w).

      It can be seen from Fig. 4b and c that the G' of all materials was much higher than G'', indicating that the hybrid gelator systems were mainly elastic in this frequency scanning range, which belonged to a dense network gel. Both G' and G'' increased with oscillation frequency, resulting in an increase in the internal friction of the ink, indicating that the hybrid gelator systems had shear thinning phenomenon. In addition, within the specified oscillation frequency, both G' and G'' increased with the increase of the concentration of beeswax. It was speculated that the addition of beeswax might improve the interaction of the hybrid gelator system, especially the mutual cross-linking of gelatin-carrageenan and beeswax, which enhanced the stability and mechanical strength of the ink.

    • The crystal structures of the six groups of inks were characterized by XRD. It can be clearly observed from Fig. 5, that the five groups of inks with added beeswax showed a high degree of consistency, and two typical diffraction peaks appeared, located at 2θ = 21.37° and 23.75°, respectively. However, this phenomenon was not observed for ink without beeswax addition, and the observed diffraction peaks were also mentioned in the beeswax XRD data of Gaillard et al.[30], which indicated that the diffraction peaks were related to beeswax. With the increase of beeswax addition, the intensity of diffraction peaks showed an increasing trend. The intensity of the diffraction peak of the same group of samples was proportional to the crystallinity, which indicated that the crystallinity of the hybrid gelator system was also increasing with the addition of beeswax. Tian et al. reported that when the beeswax hybrid gelator system was incubated at 45 °C, the diester would undergo a substitution reaction, and then absorb part of the heat of the system to generate hydrocarbons or monoesters, and the hydrocarbons or monoesters would restack to form orthorhombic structures after cooling[31]. During the preparation of the inks, the hybrid gelator systems underwent a similar process, and after stabilization, orthogonal structures were generated, and the orthorhombic content was proportional to the amount of beeswax added. The orthogonal structure has high hardness, which can improve the deformation resistance of the ink[32]. Therefore, this increased stiffness could originate from an increase in the orthogonal structure, which was consistent with the gel strength measurements of this study (Fig. 2). In addition, since the substitution reaction absorbed heat, the process could affect the thermal stability of the ink system.

      Figure 5. 

      X-ray diffractograms of inks at six beeswax levels (0, 1%, 3%, 5%, 7%, 9%, w/w).

    • Thermo-gravimetric analysis (TGA) is one of the most effective methods for analyzing the thermal stability of organic compounds or polymers[33]. Figure 6 shows the mass loss of six groups of inks as a function of temperature, and the thermogravimetric spectra of the inks with different beeswax additions showing roughly the same trend. It could be seen from the TG curve that when the temperature was between 100−250 °C, the inks have a mass loss of about 10% (w/w), which might be related to the water loss phenomenon of the ink. While between 20−100 °C, the mass loss of all inks was small and almost uniform (around 2%−4%, w/w). Therefore, the ink had good thermal stability in 3DFP. In addition, it could be observed in the TG curve that the mass loss of the six groups of inks was not significantly different below 300 °C. Above 300 °C, with the increase in the beeswax level, the loss rate also increased gradually. The composition of the hybrid gelator system was complex, and it was difficult to attribute the mass loss at a certain temperature to a specific substance, but it could reflect the relationship between the mass and temperature of the ink under the action of heat as a whole. It could be observed that around 290−300 °C, the peak apex in the DTG curve of the inks corresponded to the weightless inflection point of the TG curve, and the inflection point marked the transition boundary between the two obvious water phases (water loss events). This demonstrated that the addition of beeswax could make the ink more easily dehydrated under heating conditions from the perspective of the final residual mass, the mass percentage of residual substances in the crucible was negatively correlated with the beeswax level, which illustrates that adding beeswax would reduce the thermal stability of the inks.

      Figure 6. 

      TGA results of the thermal decomposition and its derivative (DTG) of inks at six beeswax levels (0, 1%, 3%, 5%, 7%, 9%, w/w).

    • SEM was used to observe the microstructure of the hybrid gelator system. Figure 7 shows the typical microscopic characteristics of the inks with different beeswax levels. Compared with the ink without beeswax (Fig. 7a), the inks with beeswax added (Fig. 7bf) exhibit coarse particle distribution status. As the beeswax content increases, the ink microstructure exhibits an increasingly rough morphology. As mentioned above, the XRD results indicated that a new crystal structure was formed after mixing. Since the beeswax was solid at room temperature, it could be observed that the beeswax particles were homogeneously embedded in the whole system. White mushroom contains a variety of substances including polysaccharides, proteins and fibers, which could interact with gelatin-carrageenan and increase the action sites of beeswax particles to form a hybrid gelator system with high viscosity and strong mechanical properties.

      Figure 7. 

      SEM images of inks at 800x magnification with six beeswax levels (a−f: 0, 1%, 3%, 5%, 7%, 9%, w/w)

    • In this paper, the effects of different concentrations of beeswax (0, 1%, 3%, 5%, 7%, 9%, w/w) on the printing properties and internal structure of white mushroom hybrid gelator systems were investigated. The results demonstrated that when the addition of beeswax was 3%, the printing accuracy reached a maximum of 98.35%, and the appearance was smoother, and the gel strength was nearly doubled when compared to the control group. This change could be attributed to the generation of orthogonal structures, which increased hardness and affected the gel properties of inks. Microstructurally, the beeswax-added ink exhibited a rough characteristic surface with particle distribution. The hybrid gelator systems had good thermal stability at room temperature, and the results of this study provide a reference for the development of novel white mushroom composite gels for 3DFP.

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

      • Copyright: © 2022 by the author(s). Published by Maximum Academic Press on behalf of Nanjing Agricultural University. 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 (7)  Table (1) References (33)
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    Xiao K, Pan L, Tu K. 2022. Effect of the addition of beeswax on the gel properties and microstructure of white mushroom powder-based hybrid gelator system for 3D food printing. Food Materials Research 2:6 doi: 10.48130/FMR-2022-0006
    Xiao K, Pan L, Tu K. 2022. Effect of the addition of beeswax on the gel properties and microstructure of white mushroom powder-based hybrid gelator system for 3D food printing. Food Materials Research 2:6 doi: 10.48130/FMR-2022-0006

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