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2024 Volume 3
Article Contents
Abstract
INTRODUCTION
The aim of the study
MATERIALS AND METHODS
Sample collection, morphological studies, and isolation
DNA extraction, PCR amplification, sequencing
Taxon treatments
Phylogenetic analyses
RESULTS
Phylogenetic analysis
Morphological analyses
DISCUSSION
Acknowledgments
Conflict of interest
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References
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Study on fermentation kinetics, antioxidant activity and flavor characteristics of Lactobacillus plantarum CCFM1050 fermented wolfberry pulp

  • 1.

    College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China

  • 2.

    Ningxia Runxinyuan Wolfberry development Co., LTD, Zhongning 755100, China

  • 3.

    Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU), Beijing 100048, China

More Information
  • As a superfruit, wolfberry has extremely high nutritional value, and how to enhance the accessibility of its nutrients is the core of current research. This study focused on exploring the kinetic model of Lactobacillus plantarum CCFM1050 fermentation of wolfberry and the potential alterations of antioxidant activity and volatile flavor compounds induced by lactic acid fermentation. we monitored cell counts, product formation, and substrate changes over a 72-h period of wolfberry fermentation. A kinetic model was developed to illustrate cell growth, substrate consumption, and product accumulation during wolfberry pulp fermentation. Phenolic substance analysis revealed a significant increase in total phenol and flavonoid content in wolfberry pulp during fermentation, reaching 1.16 and 1.15 times, respectively, compared to pre-fermentation levels. The elevated levels of phenolic substances led to a substantial increase in DPPH and ABTS free radical scavenging rates in fermented wolfberry pulp, reaching 67.16% and 32.10%, respectively. Volatile components of samples were analyzed using the HS-GC-IMS method, and fingerprints of wolfberry pulp before and after fermentation were established. A total of 51 compounds were identified, including 12 alcohols, seven aldehydes, two acids, eight esters, and 12 ketones, contributing to an enhanced flavor profile in the fermented wolfberry pulp. This study is helpful for understanding the kinetic changes in the lactic acid fermentation of wolfberry, the changes of antioxidant active substances and VOCs, and provides guidance for the industrial processing of wolfberry.

  • INTRODUCTION

    In coastal ecosystems, salt marsh habitats are common and consist of diverse halophytes, grasses, herbs, and shrubs, as well as microorganisms [ 1, 2] . These species-rich ecosystems are highly productive, and investigating fungal diversity in these habitats is important while many areas are still being explored [ 2, 3] . Researchers are currently studying the taxonomy of fungi in marine and semi-marine environments and increasing the number of known taxa recorded [ 4− 6] . Ascomycota was identified as the dominant group in world salt marsh ecosystems, including the highest diversity in Pleosporales, Dothideomycetes [ 2] . Coniothyriaceae is a pleosporalean family with a large number of terrestrial taxa, while taxa associated with salt marsh vegetation have rarely been reported [ 2] .

    Coniothyriaceae was established by Cooke [ 7] to accommodate Coniothyrium species. The type genus and species of Coniothyriaceae are Coniothyrium Corda and C. palmarum Corda, respectively [ 8− 10] . This family was previously linked to Leptosphaeriaceae [ 11] and this was followed by several authors [ 12− 16] . Subsequently, molecular data analyses for phoma-like asexual morphs were performed by de Gruyter et al. [ 17] based on LSU and ITS sequence data and revealed that C. palmarum is phylogenetically distant from Leptosphaeriaceae and closely related to Coniothyriaceae. de Gruyter et al. [ 17] reinstated Coniothyriaceae as a distinct family in Pleosporales and transferred several Phoma and Pyrenochaeta species into this family. Thus, the morphological variations of Coniothyrium species were expanded by the addition of more characters, such as setose pycnidia and conidiogenesis with elongated conidiophores [ 17] . Several authors later updated the placements of many Coniothyrium species with generic level changes and novel genera placed in different families [ 18, 19] .

    Verkley et al. [ 19] studied morphology and phylogenetic relationships of coniothyrium-like and closely related taxa. These species are coelomycetous and characterized in having pycnidial or stromatic conidiomata and small, subhyaline to pigmented, 1- or 2-celled conidia [ 19] . The phenotypic plasticity of these coelomycetous species has made their taxonomic placements uncertain and, thus the majority of them have been placed in Coniothyrium [ 19− 21] . Both Coniothyrium and coniothyrium-like species were identified as polyphyletic within Pleosporales and recent taxonomic treatments were mainly treated with combined morphology and molecular data analyses [ 12, 16, 17, 19, 21− 32] . Coniothyrium sensu stricto is characterized by 1-septate conidia and grouping in Coniothyriaceae [ 17, 19− 21, 33] . Currently, Coniothyriaceae consists of five genera, such as Coniothyrium, Foliophoma Crous , Neoconiothyrium Crous, Ochrocladosporium Crous & U. Braun and Staurosphaeria Rabenh. (≡ Hazslinszkyomyces Crous & R.K. Schumach.) [ 10, 34] .

    Coniothyriaceae members have been identified as pathogens that cause necrotrophic and leaf spots on leaves, and saprobes on dead branches [ 10, 17] . The sexual morph is characterized in having cucurbitaria-like, black, globose ascomata, short central ostiole, textura angularis peridium cells, branched, septate, cellular pseudoparaphyses, 8-spored, cylindrical, bitunicate asci and muriform, ellipsoidal ascospores that are initially hyaline and brown at maturity [ 10] . Asexual morphs are coelomycetous and sometimes differentiated with phoma-like, camarosporium-like, coniothyrium-like, or cladosporium-like asexual characters. They are characterized in having dark, globose, pycnidial conidiomata, with central, sometimes papillate ostiole, cells of textura angularis or textura globulosa in the conidiomatal wall, hyaline macroconidiogenous and microconidial cells and conidia. Conidial morphology is varied as macroconidia and microconidia. Macroconidia are ellipsoid, red-brown, and septation is from the central transverse septum to muriformly septate, while microconidia are hyaline, globose to ellipsoid and aseptate [ 9, 10, 24, 27, 35, 36] .

    The aim of the study

    In this study, we aim to expand the taxonomy of fungi associated with dead plant hosts in salt marsh ecosystems. We investigate salt marsh habitats in Thailand to collect fungal specimens and isolate them to find out the taxonomic novelties. Morphological illustrations, comprehensive descriptions, and multi-gene phylogenetic analyses are provided to confirm the placement of new findings.

    MATERIALS AND METHODS

    Sample collection, morphological studies, and isolation

    Fungal specimens were collected from salt marsh habitats in Pranburi Province, Thailand, 2021. Samples were preserved in sterile Ziploc bags in the laboratory and incubated at room temperature 25 °C. Rehydrated specimens were observed to identify fungal fruiting bodies and macro-morphology was observed by using a Motic SMZ 168 compound stereomicroscope. Micro-morphologies (e.g., conidiomata, conidiogenous cells, conidia) were examined from hand-sectioned structures using a Nikon ECLIPSE 80i compound stereomicroscope, equipped with a Canon 600D digital camera. The measurements of photomicrographs were obtained using Tarosoft (R) Image Frame Work version 0.9.7. Images were edited with Adobe Photoshop CS6 Extended version 13.0.1 software (Adobe Systems, San Jose, California, USA).

    Single-spore isolation was carried out as described by Senanayake et al. [ 37] . Germinated spores were aseptically transferred into fresh malt extract agar medium (MEA) prepared in 50% or 100% concentrations of sterilized natural seawater [ 38] . Culture plates were incubated at 25 °C for six weeks and inspected every week. Herbarium specimens are preserved at Mae Fah Luang University Herbarium (MFLU) in Chiang Rai, Thailand. All living cultures are deposited at Mae Fah Luang Culture Collection (MFLUCC). Facesoffungi and Index Fungorum numbers for new taxa were obtained [ 39, 40] .

    DNA extraction, PCR amplification, sequencing

    The methodologies for DNA extraction, PCR, gel electrophoresis, and sequencing were followed, as detailed in Dissanayake et al. [ 41] . The genomic DNA was extracted from fresh mycelium using the E.Z.N.A Fungal DNA Mini Kit- D3390-02 (Omega Bio-Tek, USA) following the guidelines of the manufacturer. DNA sequences were obtained for the internal transcribed spacer region (ITS1, 5.8S, ITS2), the small subunit (SSU), and the large subunit (LSU) of the nuclear ribosomal RNA gene. PCR thermal cycle programs for each locus region are presented in Table 1. Purification and sequencing were outsourced to the Bio Genomed Co. LTD laboratory (Biogenomed Co., Thailand). Newly generated sequences were submitted to NCBI GenBank ( www.ncbi.nlm.nih.gov/genbank).

    Table 1.  Gene regions, primers, and PCR thermal cycle programs used in this study, with corresponding reference(s).
    Genes/loci PCR primers (forward/reverse) PCR conditions Reference (s)
    ITS and LSU ITS5/ITS4 and LR0R/LR5 94 °C; 2 min (95 °C; 30 s, 55 °C; 50 s, 72 °C; 90 s) × 35 thermal cycles, 72 °C; 10 min [ 42− 44]
    SSU NS1/NS4 95 °C; 3 min (95 °C; 30 s, 55 °C; 50 s, 72 °C; 30 s) × 35 thermal cycles, 72 °C; 10 min [ 42]
     | Show Table
    DownLoad: CSV

    BioEdit v 7.0.9.0 program [ 45] was used to check the quality of the newly generated sequence chromatograms. For primary identification, contig sequences were checked with BLAST searches in NCBI. Sequences for phylogenetic analyses were downloaded from GenBank ( Table 2) following Hyde et al. [ 25] . Each gene matrix was aligned with MAFFT version 7 [ 46] with default parameters and manually adjusted for improvement where necessary using BioEdit v. 7.2 [ 45] . The trimAl v1.4 software was used for the automated removal of spurious sequences or poorly aligned regions in each single gene alignment, and gappyout was selected as the automated trimming method [ 47] . Two separate phylogenetic analyses were conducted: Maximum Likelihood (ML) and Bayesian Inference (BI). LSU, SSU, and ITS concatenated dataset was analyzed for Coniothyriaceae and selected families in Pleosporales.

    Table 2.  Taxa used in the phylogenetic analyses and their GenBank accession numbers. Sequences of new taxon generated in this study are in blue-bold and type strains are in black-bold.
    Species Strain/voucher number GenBank accession numbers
    ITS LSU SSU
    Amarenographium ammophilae MFLUCC 16–0296 KU848196 KU848197 KU848198
    Ascochyta pisi CBS 126.54 GU237772 EU754137 EU754038
    Bipolaris microstegii CBS 132550 NR_120160 NG_042690 NA
    Bipolaris victoriae CBS 327.64 NR_147489 NG_069233 NA
    Comoclathris arrhenatheri MFLUCC 15–0465 NR_165855 NG_068240 NG_068374
    Coniothyrioides thailandica MFLUCC 22-0193 OQ023276 OQ023277 OQ025050
    Coniothyrium carteri LG1401 MS6E KX359604 KX359604 NA
    Coniothyrium cereale CBS 157.78 MH861123 JX681080 NA
    Coniothyrium chiangmaiense MFLUCC 16–0891 KY568987 KY550384 KY550385
    Coniothyrium dolichi CBS 124140 JF740183 GQ387611 GQ387550
    Coniothyrium glycines CBS 124455 JF740184 GQ387597 GQ387536
    Coniothyrium palmarum CBS 400.71 AY720708 EU754153 AY720712
    Coniothyrium palmarum CBS 758.73 NA JX681085 EU754055
    Coniothyrium sp. B9-10-9 MW764153 NA NA
    Coniothyrium sp. P16-10-4 MW764259 NA NA
    Coniothyrium telephii CBS 188.71 JF740188 GQ387599 GQ387538
    Coniothyrium telephii CBS 856.97 JF740189 GQ387600 GQ387539
    Coniothyrium telephii UTHSC:DI16–189 LT796830 LN907332 NA
    Coniothyrium triseptatum MFLU 19–0758 NR_171948 NG_073674 NA
    Curvularia heteropogonis CBS 284.91 JN192379 JN600990 NA
    Didymella azollae A1 MT514913 MT514910 NA
    Foliophoma camporesii MFLUCC 18–1129 KY929151 KY929181 NA
    Foliophoma fallens CBS 161.78 KY929147 GU238074 GU238215
    Foliophoma fallens CBS 284.70 KY929148 GU238078 GU238218
    Libertasomyces myopori CPC 27354 NR_145200 NG_058241 NA
    Libertasomyces platani CPC 29609 NR_155336 NG_059744 NA
    Libertasomyces quercus CBS 134.97 NR_155337 DQ377883 NA
    Melnikia anthoxanthii MFLUCC 14–1010 NA KU848204 KU848205
    Neoconiothyrium hakeae CPC 27616 KY173397 KY173490 NA
    Neoconiothyrium hakeae CPC 27620 KY173398 KY173491 NA
    Neoconiothyrium multiporum CBS 353.65 NR_111617 JF740268 NA
    Neoconiothyrium multiporum CBS 501.91 JF740186 GU238109 GU238225
    Neoconiothyrium persooniae CBS 143175 NR_156386 NG_058509 NA
    Neoconiothyrium viticola CPC 36397 NR_165929 NG_068326 NA
    Neoplatysporoides aloeicola CPC 24435 NR_154230 NG_058160 NA
    Ochrocladosporium elatum CBS 146.33 EU040233 EU040233 NA
    Ochrocladosporium frigidarii CBS 103.81 NR_156512 NG_064123 NA
    Phaeosphaeria chiangraina MFLUCC 13–0231 KM434270 KM434280 KM434289
    Phaeosphaeria musae MFLUCC 11–0133 KM434267 KM434277 KM434287
    Phaeosphaeria thysanolaenicola MFLUCC 10–0563 NR_155642 NG_069236 NG_063559
    Phaeosphaeria oryzae CBS 110110 NR_156557 NG_069025 NG_061080
    Phaeosphaeriopsis dracaenicola MFLUCC 11–0157 NR_155644 NG_059532 KM434292
    Pleospora herbarum MFLUCC 14-0920 KY659560 KY659563 KY659567
    Querciphoma carteri CBS 101633 JF740180 GQ387593 GQ387532
    Querciphoma carteri CBS 105.91 JF740181 GQ387594 GQ387533
    Querciphoma carteri Gv5 MT819903 NA NA
    Querciphoma carteri UASWS2031 MN833930 NA NA
    Shiraia bambusicola NBRC 30753 AB354987 AB354968 NA
    Shiraia bambusicola NRBC 30771 AB354990 AB354971 NA
    Shiraia bambusicola NRBC 30772 AB354991 AB354972 NA
    Staurosphaeria aloes CBS 136437 KF777142 KF777198 NA
    Staurosphaeria aloes CPC 21572 NR_137821 NG_067283 NA
    Staurosphaeria aptrootii CBS 483.95 NR_155186 NA NA
    Staurosphaeria lycii CPC 30998 KY929150 KY929180 NA
    Staurosphaeria lycii CPC 31014 KY929151 KY929181 NA
    Staurosphaeria rhamnicola MFLUCC 17–0813 MF434200 MF434288 MF434376
    Staurosphaeria rhamnicola MFLUCC 17–0814 NR_154461 MF434289 NG_063659
    Stemphylium vesicarium CBS 191.86 MH861935 MH873624 GU238232
    NA: Sequences not available in GenBank.
     | Show Table
    DownLoad: CSV

    Taxon treatments

    Phylogenetic analyses

    In the phylogenetic analyses, maximum likelihood (ML) was executed using IQ-Tree web server ( http://iqtree.cibiv.univie.ac.at/) with bootstrap support obtained from 1,000 pseudoreplicates [ 48, 49] . Bayesian Inference (BI) analysis was performed on the CIPRES Science Gateway portal under MrBayes on XSEDE (3.2.7a) [ 50] . Six simultaneous Markov chains were run for 1,000,000 generations, and trees were sampled every 1,000 th generation, ending the run automatically when the standard deviation of split frequencies dropped below 0.01. The best nucleotide substitution models for each genetic marker were evaluated using jModelTest2 on XSEDE in the online CIPRES Portal ( www.phylo.org/portal2) [ 51, 52] . The best-fit models under the AIC criterion were revealed to be GTR+I+G for ITS and LSU regions while GTR+I for SSU region. Phylogenetic trees were visualized with FigTree version 1.4.0 [ 53] and edited in Microsoft PowerPoint (2019).

    RESULTS

    Phylogenetic analysis

    The combined LSU, SSU, and ITS alignment was used to construct the final phylogenetic analysis ( Fig. 1) of maximum likelihood (ML) and Bayesian inference (BI).

    Figure 1.  Phylogram generated from maximum likelihood analysis based on combined LSU, SSU, and ITS sequenced data. Fifty-eight strains were included in the combined sequence analyses, which comprised 2251 characters with gaps (LSU = 800, SSU = 948, ITS = 503). Single gene analyses were also performed, and topology and clade stability were compared from the combined gene analyses. Ascochyta pisi Lib. (CBS 126.54) and Didymella azollae E. Shams, F. Dehghanizadeh, A. Pordel & M. Javan-Nikkhah (A1) were used as the outgroup taxa. The final ML optimization likelihood is -10163.644. The matrix included 494 distinct alignment patterns including undetermined characters. Estimated base frequencies were obtained as follows: A = 0.245, C = 0.219, G = 0.274, T= 0.262; substitution rates AC = 2.73290, AG = 3.93954, AT = 2.73290, CG = 1.0, CT = 7.93321, GT = 1.0 and the gamma distribution shape parameter α = 0.439534. Bootstrap support values for ML (first set) equal to or greater than 75% and BYPP equal to or greater than 0.95 are given above or below the nodes. The strains from the current study are in red bold and the type strains are in black bold. The scale bar represents the expected number of nucleotide substitutions per site.
    DownLoad: Full-Size Img PowerPoint

    Morphological analyses

    Coniothyrioides Wijes., M.S. Calabon, E.B.G. Jones & K.D. Hyde, gen. nov.

    Index Fungorum number: 555045; Facesoffungi number: 13901 Fig. 2

    Etymology – Resembling Coniothyrium taxa

    Saprobic on a submerged decaying wood in salt marsh ecosystems. Sexual morph: Undermined. Asexual morph: Coelomycetous. Forming conspicuous, round to irregular, black pycnidia. Conidiomata semi-immersed, erumpent through the host substrate, globose to subglobose, solitary, scattered to aggregated, uni-loculate, ostiolate, covered in setae, rigid when dehydrated, black. Setae originated from the outermost layers of conidiomatal wall, divergent, brown, with hyaline apex, septate, smooth-walled, uniformly wide from base to apex. Conidiomatal wall composed of several layers, from outer to inner layers black, dark brown, pale brown to hyaline cells of textura angularis. Conidiophores reduced to conidiogenous cells. Conidiogenous cells lining the inner cavity, doliiform to subcylindrical, smooth-walled, hyaline, enteroblastic, phialidic conidiogenesis with periclinal thickening at the apex. Conidia solitary, ellipsoidal to obovoid, rounded at the apex, aseptate, initially hyaline, becoming pale to dark brown at maturity, smooth-walled, sometimes finely verruculose, with smaller guttules at young and indistinct at maturity.

    Figure 2.  Coniothyrioides thailandica sp. nov. (MFLU 22-0276, holotype). (a) & (b) Appearance of conidiomata on a submerged decaying woody substrate. (c) Longitudinal section of conidioma. (d) Conidiomatal wall. (e) The appearance of setae. (f) & (g) Conidiogenous cells with developing conidia. (h) Conidia. Scale bars: a = 200 μm, b = 100 μm, c = 50 μm, d = 20 μm, e, h = 10 μm, f, g = 5 μm.
    DownLoad: Full-Size Img PowerPoint

    Type species – Coniothyrioides thailandica

    Note – Coniothyrioides gen. nov. is a monotypic genus associated with decaying woody substrates in salt marsh habitats in central Thailand. This genus is characterized in having pycnidial conidiomata with the cells of textura angularis wall surrounded by distinct setae, doliiform to subcylindrical, hyaline conidiogenous cells, and ellipsoidal to obovoid, aseptate and hyaline to brown conidia. Based on some conidial characteristics such as aseptate, hyaline to brown conidia the genus shares similar morphologies to coniothyrium-like taxa [ 19] , by ellipsoidal to subcylindrical conidia sharing similar characters to Coniothyrium and Neoconiothyrium [ 9, 16, 20, 24] . However, other accepted genera in Coniothyriaceae differ from this genus in conidial morphologies: Foliophoma has only hyaline conidia except for F. camporesii D. Pem & K.D. Hyde; Hazslinszkyomyces has muriformly septate conidia [ 27] ; Ochrocladosporium has cladosporium-like conidia [ 35] . Moreover, phylogenetically Coniothyrioides forms a distinct lineage within Coniothyriaceae ( Fig. 1). Coniothyrium carteri (Gruyter & Boerema) Verkley & Gruyter (LG1401_MS6E) was the closest species based on BLAST result of ITS (94.33% similarity) and C. telephii (Allesch.) Verkley & Gruyter (UTHSC:DI16-189) was the closest species LSU sequence data (99.31% similarity) and sequences are lacking for SSU in the GenBank. The genus is known from its asexual morph and the sexual morphology was not observed.

    In our phylogenetic analyses, Foliophoma species were grouped outside of Coniothyriaceae with closer to Libertasomycetaceae and Pleosporaceae species. Foliophoma was introduced by Crous & Groenewald [ 27] to accommodate F. fallens (Sacc.) Crous, in Coniothyriaceae based on the parsimony analyses of single LSU and ITS sequence data. Foliophoma camporesii was later introduced based on morphology and maximum likelihood analyses of LSU-SSU- ITS sequence data by Hyde et al. [ 25] . Based on morphology, Foliophoma species share similar characteristics to the species of Coniothyriaceae in having dark brown conidiomata, conidial wall with textura angularis cells, phialidic conidiogenesis sometimes with periclinal thickening or percurrent proliferation, and mainly ellipsoidal shaped conidia. However, the type species of the genus, F. fallens differs other Coniothyriaceae taxa in having eustromatic conidiomata. Based on this taxonomic uncertainty, more fresh collections with additional coding genes are required to clarify the accurate placement of Foliophoma.

    Coniothyrioides thailandica Wijes., M.S. Calabon, E.B.G Jones & K.D. Hyde, sp. nov.

    Index Fungorum number: 555050; Facesoffungi number: 13902

    Etymology – The name reflects the county Thailand, from where the species was isolated.

    Saprobic on a submerged and decaying woody substrate. Sexual morph: Undermined. Asexual morph: Coelomycetous. Conidiomata 150–200 μm high, 100–150 μm diam. (x̄ = 160 × 130 µm), pycnidial, semi-immersed, erumpent through the host substrate, globose to subglobose, solitary, scattered to aggregated, uni-loculate, ostiolate, covered by setae, rigid when dehydrated, black. Setae 3–5 µm wide, originating from the outermost layers of conidiomatal wall, divergent, brown, with hyaline apex, septate, smooth-walled, uniformly wide from base to apex. Conidiomatal wall 15–20 µm wide, equally thickened, composed of several layers, outermost layers dark brown to black, towards inside pale brown to hyaline cells of textura angularis, surrounded by setae. Conidiophores reduced to conidiogenous cells. Conidiogenous cells 4–5 μm long × 2.5–3.5 μm wide, lining the inner cavity, doliiform to subcylindrical, smooth-walled, hyaline, enteroblastic, phialidic conidiogenesis with periclinal thickening at the apex. Conidia 3–5 × 2.5–3 μm ( ¯x = 4.5 × 2.7 µm, n = 20), solitary, ellipsoidal to obovoid, rounded at the apex, aseptate, initially hyaline, becoming pale to dark brown at maturity, smooth-walled, sometimes finely verruculose, with smaller guttules at young and indistinct at maturity.

    Culture characteristics – On MEA, colony circular with a filamentous margin, reaching 40–45 mm diam. in 25 d at 25 °C, light gray from above, brown from center becoming light gray in the margin below, surface rough, dry, flat, with dense mycelia, edge filiform.

    Material examined – Thailand, Pranburi Province, on a submerged decaying wood, 23 March 2021, Mark S. Calabon, SPAR26 (MFLU 22-0276, holotype), ex-type living cultures, MFLUCC 22-0193.

    GenBank numbers – ITS = OQ023276, LSU = OQ023277, SSU = OQ025050.

    Notes – Coniothyrioides thailandica sp. nov. shares morphological characters with other representatives in Coniothyriaceae in having pycnidial, globose, uni-locular conidioma with a central ostiole, peridial wall with the cells of textura angularis, and doliiform to subcylindrical conidiogenous cells, phialidic conidiogenesis with a periclinal thickening at the apex. The synopsis of asexual morphological characters for the generic types of the family including their hosts and localities is presented in Table 3. Based on the presence of conidiomatal setae, our species (MFLU 22-0276) resembles Neoconio thyrium [ 24] . In addition, our species resembles Foliophoma camporesii (MFLU 17-1006) in having hyaline to brown and aseptate conidia but differs in having larger conidiomata (150–200 × 100–150 vs 40–47 × 40–69 μm) and the presence of setae on the wall ( Table 3). Phylogenetically, our strain (MFLUCC 22-0193) formed an independent lineage within Coniothyriaceae with 94% ML and 0.99 BI statistical support ( Fig. 1). The base pair differences between our stain and the strains represent type species of other genera in Coniothyriaceae are listed ( Table 4). Thus, the evidence based on both morphology and phylogeny, we establish Coniothyrioides as a new genus in Coniothyriaceae with C. thailandica as the type species.

    Table 3.  Synopsis of asexual morphological characters of related genera of Coniothyriaceae.
    Species Conidiomata (µm) Conidiomata wall (µm) Conidiogenous cells (µm) Conidia (µm) Habitat(s) and host(s) Locality Reference
    Coniothyrioides thailandica (holotype: MFLU 22-0276) 150–200 high, × 100–150 diam., pycnidial, semi-immersed, erumpent, dark brown to black, globose to subglobose,
    uni-locular, ostiolate     		15–20 wide, black, dark brown to hyaline cells of textura angularis,
    Brown, septate setae (3–5 µm wide,) with hyaline apex     		4–5 long × 2.5–3.5 wide, hyaline, doliiform to subcylindrical, enteroblastic, phialidic conidiogenesis with periclinal thickening 3–5 × 2.5–3, ellipsoidal to obovoid, aseptate, rounded at apex, initially hyaline, becoming pale to dark brown at maturity On decaying wood in salt marsh habitat Thailand This study
    Coniothyrium
    palmarum (CBS 400-71)     		Immersed, dark brown, globose, pale to uni-locular brown, thick-walled cells of textura angularis hyaline, phialidic conidiogenesis, doliiform to cylindrical Subcylindrical, spherical, ellipsoid or broadly clavate, 0(–1)-septate, apex obtuse, brown, base truncate, sometimes minute marginal frill On Chamaerops humilis ( Arecaceae) Italy [ 16]PP,
    [ 20]GN
    Foliophoma fallens (holotype: CBS 284.70) 120–250 wide, eustromatic, globose, uni-multi locular,
    1–3 ostiolate     		3–6 layers,
    brown textura angularis     		5–7 × 4–5, hyaline, phialidic conidiogenesis with thickening or proliferation at apex, dolliform to subcylindrical,
    periclinal     		(5–)5.5–6(–7) × (3–)4(–5),

    broadly ellipsoidal, aseptate, hyaline, guttulate or granular, apex obtuse, base truncate to bluntly rounded     		Leaf spot on Nerium oleander ( Apocynaceae) Italy [ 27]
    *Foliophoma camporesii (holotype: MFLU 17-1006) 40–47 × 40–69, pycnidial, immersed to semi-immersed, globose to subglobose, ellipsoidal or irregular, carbonaceous 15–40, 1–2-layered of cells of textura angularis 2–4 × 2–3, hyaline, globose to short cylindrical, phialidic conidiogenesis with periclinal thickening or percurrent proliferation at apex 2–6 × 3–5, ovoid to ellipsoidal, aseptate, hyaline when immature, brown at maturity On dead stems of Maclura pomifera ( Moraceae) Italy [ 25]
    Hazslinszkyomyces aloes (≡ Camarosporium aloes: ex-type - CPS 21572) 250 diam, pycnidial, erumpent, brown, globose, central ostiolate 3–6 layers of brown textura angularis
    		5–10 × 4–5, hyaline,
    ampulliform to doliiform, apex with several inconspicuous percurrent proliferation,     		(9–)11–13(–14) × (4–)6–7(–8), ellipsoid, initially hyaline, aseptate, becoming pale brown, subcylindrical
    to clavate or obovoid with 3 transverse eusepta, constricted at median septum or not, apex obtuse, base bluntly rounded to truncate     		Dead bark of Aloe dichotoma ( Xanthorrhoeaceae) South Africa [ 27]
    Neoconiothyrium persooniae (ex-type CPC 32021 = CBS 143175) 100–200 diam, superficial, ellipsoid to obpyriform, 1–2 papillate ostioles, 10–15 diam, with or without setae 3–6 layers, hyaline textura angularis 5–8 × 4–5, hyaline, doliiform to ampulliform, phialidic, with periclinal thickening or percurrent proliferation (5–)6–7(–8) × 3(–4), ellipsoid to subclavate, aseptate, initially hyaline medium brown, becoming cylindrical and at times 1-septate, apex subobtuse, base bluntly rounded On leaves of Persoonia laurina subsp. laurina ( Proteaceae) Australia [ 24]
    Ochrocladosporium elatum (CBS 146.33) – – Integrated as lateral peg-like loci on hyphal
    cells, or erect, subcylindrical, up to 25 µm long, 2.5–4 µm wide,
    with 1–3 terminal loci, occasionally lateral, 1–1.5 µm wide     		Ramoconidia, 10–40 × 3–5, subcylindrical to ellipsoid, hyaline to pale brown, 0(–1)-septate, giving rise to branched
    chains of conidia that are subcylindrical to ellipsoid, aseptate, (7–)8–10(–14) × (3–)4(–4.5), olivaceous brown     		Wood pulp Sweden [ 35]
    '–' observed morphologies on cultures, therefore conidiomata and wall characters are not recorded. '*' species which is not represent a generic type. GN- based on the generic description. PP- based on the photographic plate provided.
     | Show Table
    DownLoad: CSV
    Table 4.  The base pair comparisons of our strain (MFLUCC 22-0193) with the strains representing type species of other genera in Coniothyriaceae.
    Species Strain LSU SSU ITS
    Coniothyrium palmarum CBS 400-71 14/800 (1.75%) 3/948 (0.3%) 69/487 (14.10%)
    Foliophoma fallens CBS 284.70 8/800 (1%) 2/948 (0.2%) 66/497 (13.27%)
    Hazslinszkyomyces aloes CPC:21572 6/800 (0.75%) – 52/497 (10.46%)
    Neoconiothyrium persooniae CBS:143175 20/800 (2.5%) – 49/497 (9.85%)
    Ochrocladosporium elatum CBS 146.33 13/800 (1.62%) – 53/497 (10.66%)
     | Show Table
    DownLoad: CSV

    Members of Coniothyriaceae have high morphological plasticity and it is not adequate to use only morphology for identification at the genus level. Coniothyrium dolichi (Mohanty) Verkley & Gruyter (≡ Pyrenochaeta dolichi Mohanty, CBS 124140) and C. glycines (R.B. Stewart) Verkley & Gruyter (≡ P. glycines R.B. Stewart, CBS 124455) form a separate clade within Coniothyriaceae ( Fig. 1). Based on the morphology observed from corn meal agar medium (CMA) by Grondona et al. [ 54] , C. dolichi differs to our species by having two types of conidiogenous cells including discrete, ampulliform conidiogenous cells and integrated, cylindrical conidiogenous cells on filiform, septate conidiophores in the same conidioma, while our species has doliiform to subcylindrical conidiogenous cells and conidiophores are reduced to conidiogenous cells [ 54] . The pycnidial conidiomata and the ostiole of C. dolichi covered by dark brown, septate setae resembles our species and conidia are aseptate, ellipsoid, and hyaline with more or fewer guttules while our species has brown conidia at maturity [ 54] . The original description of C. dolichi, mentioned that conidia were greenish-yellow in mass similar to coniothyrium-like conidia, as well as to our species [ 55]. Also, a monodictys-like synanamorph was reported in C. dolichi based on its dark brown to black, dictyosporous conidia by differs from our species [ 17, 54, 56] . Coniothyrium glycines produces monophialidic, ampulliform, conidiogenous cells and aseptate, ellipsoidal conidia (4–8 × 1–3 µm), while our species has doliiform to subcylindrical conidiogenous cells [ 57, 58] . The unique character of C. glycines is well-defined, dark brown to black, melanized sclerotia covered with setae which differs from our species and other Coniothyrium taxa. Based on the multi-gene phylogeny provided by de Gruyter et al. [ 17] and the results of our study, the placements of these two species were confirmed in Coniothyriaceae. Also, Coniothyrium triseptatum Dayar., Thyagaraja & K.D. Hyde (MFLU 19-0758) creates a separate lineage in Coniothyriaceae ( Fig. 1) and only sexual morph was reported for this fungus. Therefore, we could not compare the morphology of C. triseptatum with our species [ 31] .

    DISCUSSION

    In this study, we introduced the novel genus Coniothyrioides in Coniothyriaceae, with C. thailandica as the type species, following the guidelines and major criteria for defining generic and species boundaries in Dothideomycetes by Chethana et al. [ 59] and Pem et al. [ 60] . The coelomycetous asexual morph of Coniothyrioides was associated with the decaying and submerged wood in the salt marsh habitats. Traditionally, morphology is used to delimit coelomycetes by considering the characteristics of conidiomata, conidiophores, conidiogenesis, and conidia including host associations [ 20, 32, 61] . However, accurate taxonomy of most coniothyrium-like species is challenging because of their simplicity, plasticity, and morphological variations [ 19] . In our study, genera in Coniothyriaceae differ in some conidial morphologies. For instance, Coniothyrium is characterized by aseptate to 1-septate, ellipsoidal to clavate or cylindrical, brown conidia [ 9, 17, 62] , Foliophoma with aseptate, ovoid ellipsoidal, only hyaline or hyaline-brown conidia, and Hazslinszkyomyces ellipsoidal to obovoid, transversely and muriformly septate, uniformly brown conidia [ 27] . Neoconiothyrium species have aseptate or 1-septate, ellipsoid to subclavate or subcylindrical, hyaline to medium brown conidia [ 24] while Ochrocladosporium species have cladosporium-like pale brown, aseptate or 1-septate conidia that occurring in branched chains [ 35] . Coniothyrioides thailandica is characterized by aseptate, ellipsoidal to obovoid, hyaline to pale or dark brown conidia. However, the phylogenetic analyses in this study reveal these morphological differences are not strong enough for generic delimitation of the family. Some characteristics of Coniothyrioides overlap with those of other accepted genera in the family, such as the conidiogenous cell morphology of Coniothyrium and Foliophoma which have doliiform to cylindrical or subcylindrical, hyaline, phialidic conidiogenesis with periclinal thickening and conidia show aseptate, ellipsoid-associated shapes, and hyaline to brown pigmentation ( Table 3).

    The number of fungi was estimated at between 2.2 to 3.8 million [ 63] , with about 100,000-150,000 known species and fungus-like taxa [ 64 − 66] . There are 151,834 species listed in Species Fungorum [ 67] . An up-to-date online database ( https://coelomycetes.org/) for coelomycetes is being implemented [ 21, 32] . As coniothyrium-like taxa are frequently collected and morphologically similar, it is likely that they will remain unidentified. Therefore, it is to be expected that if molecular data are incorporated in morpho-taxonomic studies of these groups, will help identify many more novel taxa. This has occurred in other genera, which are plant pathogens and ecologically more important. According to Bhunjun et al. [ 65] Coniothyrium is one of the most speciose genera listed in Species Fungorum in 2021 and studies of coniothyrium-like taxa may yield more novel species. A few records of Coniothyriaceae taxa have been identified in salt marsh ecosystems, such as Coniothyrium obiones Jaap (India and Portugal) and as unidentified Coniothyrium species (USA) [ 2] . However, Wanasinghe et al. [ 29] referred the placement of C. obiones in Neocamarosporiaceae based on multi-gene phylogeny. In marine habitats, C. cerealis E. Müll. was isolated as an alga-derived fungus in the Baltic Sea by Elsebai et al. [ 68, 69] .

    In this study, we discussed the morphology and multi-gene phylogenetic analyses results of our new collection to verify its identity and phylogenetic placement in Coniothyriaceae. Based on ecological and geographical data on salt marsh fungi, we noted lack records of Coniothyriaceae worldwide (see Calabon et al. [ 2] ). Thus, we propose that additional collections be conducted in order to identify other Coniothyriaceae taxa and improve our understanding of fungal diversity in salt marsh ecosystems, a topic that is currently understudied.

    Acknowledgments

    S.N. Wijesinghe would like to thank Mae Fah Luang University on behalf of the Graduate Studies Support Grant (Grant No. Oh 7702(6)/125) for financial support and Mushroom Research Foundation (MRF), Thailand. Further, S.N. Wijesinghe would like to thank Dr. Udeni Jayalal, Dr. Samantha C. Karunarathna, and Dr. Gothamie Weerakoon for their precious advice during this study. M.S. Calabon is grateful to the Department of Science and Technology – Science Education Institute (DOST-SEI). K.D. Hyde would like to thank the National Research Council of Thailand (NRCT) grant 'Total fungal diversity in a given forest area with implications towards species numbers, chemical diversity and biotechnology' (Grant No. N42A650547).

  • Kevin David Hyde is the Editorial Board member of Journal Studies in Fungi. He is blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of Kevin David Hyde and his research groups.

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  • Cite this article

    Liu H, Li N, Wang Y, Cheng T, Yang H, et al. 2024. Study on fermentation kinetics, antioxidant activity and flavor characteristics of Lactobacillus plantarum CCFM1050 fermented wolfberry pulp. Food Innovation and Advances 3(2): 126−134 doi: 10.48130/fia-0024-0012
    Liu H, Li N, Wang Y, Cheng T, Yang H, et al. 2024. Study on fermentation kinetics, antioxidant activity and flavor characteristics of Lactobacillus plantarum CCFM1050 fermented wolfberry pulp. Food Innovation and Advances 3(2): 126−134 doi: 10.48130/fia-0024-0012
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Study on fermentation kinetics, antioxidant activity and flavor characteristics of Lactobacillus plantarum CCFM1050 fermented wolfberry pulp

  • 1.

    College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China

  • 2.

    Ningxia Runxinyuan Wolfberry development Co., LTD, Zhongning 755100, China

  • 3.

    Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU), Beijing 100048, China

  • Received: 20 March 2024
  • Revised: 07 May 2024
  • Accepted: 08 May 2024
  • Published online: 27 May 2024
Food Innovation and Advances  3,  2024, 3(2): 126−134  |  Cite this article

Abstract: As a superfruit, wolfberry has extremely high nutritional value, and how to enhance the accessibility of its nutrients is the core of current research. This study focused on exploring the kinetic model of Lactobacillus plantarum CCFM1050 fermentation of wolfberry and the potential alterations of antioxidant activity and volatile flavor compounds induced by lactic acid fermentation. we monitored cell counts, product formation, and substrate changes over a 72-h period of wolfberry fermentation. A kinetic model was developed to illustrate cell growth, substrate consumption, and product accumulation during wolfberry pulp fermentation. Phenolic substance analysis revealed a significant increase in total phenol and flavonoid content in wolfberry pulp during fermentation, reaching 1.16 and 1.15 times, respectively, compared to pre-fermentation levels. The elevated levels of phenolic substances led to a substantial increase in DPPH and ABTS free radical scavenging rates in fermented wolfberry pulp, reaching 67.16% and 32.10%, respectively. Volatile components of samples were analyzed using the HS-GC-IMS method, and fingerprints of wolfberry pulp before and after fermentation were established. A total of 51 compounds were identified, including 12 alcohols, seven aldehydes, two acids, eight esters, and 12 ketones, contributing to an enhanced flavor profile in the fermented wolfberry pulp. This study is helpful for understanding the kinetic changes in the lactic acid fermentation of wolfberry, the changes of antioxidant active substances and VOCs, and provides guidance for the industrial processing of wolfberry.

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    Introduction

    • Wolfberry (Lycium chinense Miller) is a deciduous shrub in the Solanaceae family, found in China and other parts of Asia. Its fruit, bright orange-red oval berries measuring 1−2 cm in length, is traditionally used as an herb and functional food in Asian countries. Polysaccharides, flavonoids, and polyphenols are recognized as the primary bioactive constituents of wolfberry, showcasing a range of effects including anti-aging[1], neuroprotection, hypoglycemic, and hypolipidemic properties[2], immune modulation, anti-tumor activity[3], and cellular protection[4]. As a result, the consumption of wolfberry is anticipated to mitigate the risks associated with certain diseases and conditions linked to oxidative stress, while simultaneously enhancing antioxidant defense mechanisms. To optimize the absorption efficiency of polyphenols and other beneficial substances in wolfberries within the human body and fully harness their antioxidant functions, the adoption of innovative technologies becomes imperative[5]. Lactic acid fermentation emerges as a widely employed technique in the processing of functional foods, renowned for its capacity to significantly enhance various nutritional components. This method holds promise for enhancing the bioavailability and beneficial effects of wolfberry constituents, thereby potentially maximizing their health-promoting properties.

      Fermentation by lactic acid bacteria (LAB) has the potential to enhance a variety of bioactive compounds within the substrate[6], making it a promising alternative for the processing of wolfberry. The organic acids, esters, and other substances produced during fermentation can boost the antioxidant capacity of wolfberry pulp[5], providing beneficial effects on human health post-consumption. Prebiotic compounds, identified as 'selective fermentation agents facilitating specific changes in the composition and/or functioning of gut microbiota, beneficial for the well-being and health of the host', has been observed in research to stimulate probiotic proliferation in fermentation products, thereby improving fermentation efficiency and overall effectiveness. Numerous studies have highlighted the advantages of employing Lactobacillus plantarum in the biological processing of fruits and vegetables. For instance, in addition to improving sensory perceptions, fermentation can significantly augment aroma components in apple juice[7], kiwi fruit[8], and wolfberry juice[9]. This enhances health benefits, as the hydrolase of L. plantarum releases antioxidant compounds from conjugated phytochemicals in fruits and vegetables[10,11]. To the best of our knowledge, the current research on wolfberry mainly focuses on the fermentation of its plant components or extracts, and the fermentation of fruit pulp has not been reflected in the literature. The fermentation process of wolfberry pulp is complex and influenced by various factors such as the amount of added prebiotics, the inoculum size of lactic acid bacteria, and the fermentation time. Therefore, developing an accurate kinetic model is not only advantageous for comprehending and forecasting the dynamic alterations of several crucial parameters throughout the fermentation process, but it also aids in optimizing fermentation conditions. This, in turn, aims to achieve increased efficiency and energy savings through model optimization.

      Following the above discussion, this study was conducted in three distinct parts. Firstly, the changes of substrates, products and bacterial number parameters in the process of fermentation of wolfberry pulp by Lactobacillus plantarum were recorded, and the kinetic model equation of lactic acid fermentation of wolfberry pulp was emphatically explored. In addition, the changes in phenolic compounds and antioxidant activity in unfermented goji berry pulp and fermented goji berry pulp were analyzed. In the third part, the volatile aroma components of wolfberry were detected by GC-IMS to clarify the effect of fermentation on the aroma change of wolfberry pulp.

    Materials and methods

      Materials

    • Dried wolfberry sourced from Ningxia Runxinyuan Wolfberry Development Co., Ltd. (Zhongning, China). Lactobacillus plantarum CCFM1050 obtained from the Fruit and Vegetable Storage and Processing Laboratory of the School of Food Science and Engineering, Northwest A&F University (Yangling, China). DPPH (D4313), ABTS(IA0010), Caffeic acid (SC8010), Gallic acid (SG8040), Rutin (SR8250), and other phenolic standard products were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

      Preparation of wolfberry pulp

    • Dried wolfberry was thoroughly rinsed with tap water. Spoiled berries were removed, and the remaining berries were soaked in tap water at a ratio of 1:5 (w/v). After pulp beating, the mixture was homogenized under a colloid mill for 10 min. The resulting wolfberry pulp was then sterilized at 100 °C for 5 min cooled and set aside for use.

      Preparation of bacterial cultures

    • Lactobacillus plantarum CCFM1050, stored in a glycerol solution at −80 °C was first thawed at room temperature and then activated in 20 mL MRS broth at 37 °C for 12 h. One hundred μL bacterial culture was activated twice, washed with 20 mL of physiological saline, and utilized as an inoculum for wolfberry pulp.

      Prebiotic screening single factor experiment

    • The activated bacterial culture's viability was determined using the coating plate method. A 1.0 × 108 CFU/100 g concentration served as the 1% inoculum. Firstly, inulin, lactulose, and oligosaccharides were individually added to 50 g of wolfberry pulp at 1%, 3%, 5%, 7%, and 9% concentrations. Then after 24 h of fermentation, measurements were taken for total acid content and viable bacterial count.

      Construction of fermentation dynamics model

    • Leveraging the insights gleaned from prior response surface optimization experiments, Lactobacillus plantarum CCFM1050 was employed as the fermentation strain. Primarily, each 100 g of wolfberry pulp received supplementation of 1 × 108 bacterial solution, coupled with the addition of 0.06 g of oligosaccharides for the fermentation process. Next, sampling intervals of 6 h was maintained throughout the fermentation trajectory to scrutinize the dynamics of viable bacteria, reducing sugars, and total acid content. The model's parameters were methodically computed based on the empirical data.

      Dynamic model of bacterial growth

    • The growth of bacterial cells typically adheres to an 'S' curve, a pattern well-suited for simulation by the logistic equation[12]. This equation effectively captures the changes in bacterial growth and the distinct expression of bacterial growth metabolism rates, as observed in the wolfberry pulp fermentation process.

      The logistic equation is expressed as follows:

      1xdxdt=μm(1−XXm) (1)

      At the commencement of fermentation (t = 0) and with the initial microbial population denoted as X = Xm, the integral deformation equation is substituted, yielding the following expression:

      X=X0eμmt1−X0Xm(1−eμmt) (2)

      In the formula, X0 is the initial number of live lactic acid bacteria during growth / (108 CFU/mL); Xm represents the highest viable count of lactic acid bacteria during the growth period/(108 CFU/mL); μm is the maximum specific growth rate/h−1, t denotes time in hours.

      Upon converting Eqn (2) into a deformed form, simultaneous logarithmic transformation of both sides results in:

      lnXXm−X=μmt−ln(XmX−1) (3)
      Product generation kinetics model

    • In bacterial fermentation, the relationship between cell growth rate and product generation rate can manifest in three ways: coupled with bacterial growth, partially coupled, and non-coupled[13]. The product generation rate does not perfectly align with any single model, introducing a degree of error. Consequently, the Leudeking-Piret equation is employed:

      dPdt=αdXdt+βX (4)

      In the formula, P represents the total acid concentration after fermentation, (g/L); X denotes the number of live bacteria, (108 CFU/mL); T is the fermentation time, h; α is the product synthesis constant associated with bacterial growth; β is the product synthesis constant associated with bacterial volume.

      In the context of lactic acid bacteria fermenting wolfberry pulp to produce acid, the process conforms to a partially coupled type, specifically α ≠ 0 and β ≠ 0. At this stage, the bacterial body is in a stable phase: dX/dt = 0, X = Xm, so that:

      β=dPdt×Xm (5)

      When taking t = 0 and P = 0 as the initial conditions, the integral results in:

      P=αA(t)+βB(t)A(t)=X0[eμmt1−X0Xm(1−eμmt)−1]B(t)=Xmμmln[1−X0Xm(1−eμmt)] (6)
      Substrate consumption dynamics model

    • Throughout the fermentation process of lactic acid bacteria, the sustenance of normal physiological activities in cells necessitates specific substrates. The energy generated through substrate consumption serves both microbial growth and the production of new metabolites. The dynamics of substrate consumption can be effectively described using the Luedeking equation:

      −dSdt=γdXdt+δX (7)

      In the formula, S represents the substrate concentration/(g/100 mL), X denotes the number of live bacteria/(108 CFU/mL), T is time/h, γ and δ are substrate consumption parameters.

      When the bacterial body is in a stable phase with dX/dt = 0 and X = Xm, the following can be derived from Eqn (7):

      δ=−dSdt×Xm (8)

      After integration:

      S=S0−γ(t)−δD(t)C(t)=X0[eμmt1−X0Xm(1−eμmt)−1]D(t)=Xmμmln[1−X0Xm(1−eμmt)] (9)

      Determination of chemical substances and antioxidant activity during fermentation

      Determination of total phenolic and total flavonoids content

    • The estimation of total phenolic content was performed by slightly modifying the Folin-Ciocalteu procedure[14]. To put it briefly, 1 mL of the sample, once diluted, was amalgamated with 2 mL of the Folin-Ciocalteu reagent (1:9, v/v). This was followed by the introduction of 2 mL of Na2CO3 solution (75 g/L). The blend was then left undisturbed in a dark environment at room temperature for 30 min. Post this incubation, the absorbance of the concoction was gauged at 760 nm.

      The total flavonoid content (TFC) was evaluated using the AlCl3 colorimetric method, as described by Wu et al.[15]. Initially, 2 mL of the diluted phenolic extract was incubated with 0.25 mL of NaNO2 solution at a concentration of 50 g/L for 5 min. Then, 0.5 mL of AlCl3 solution, prepared at a concentration of 100 g/L, was added, followed by a further 5 min incubation. Subsequently, 1 mL of NaOH solution, with a concentration of 2 mol/L, was introduced and allowed to react for 10 min. The absorbance of the resulting solution was measured at 510 nm.

      DPPH and ABTS radical scavenging rate

    • The DPPH radical scavenging activity was assessed using a modified protocol based on prior methods[15]. A total of 0.4 mL of the appropriately diluted sample was combined with 9.6 mL of a methanolic DPPH solution (at a concentration of 20 mg/mL). The resulting solution was subsequently incubated at a consistent temperature of 37 °C in a light-restricted environment for exactly 30 min. Following this incubation period, the absorbance of the mixture was carefully measured at a target wavelength of 517 nm.

      The ABTS radical scavenging activity was determined with slight modifications to previously described methods[16]. The process commenced with the incubation of 2.45 mmol/L K2S2O8 and 7 mmol/L ABTS in a 1:1 (v/v) ratio, maintained in a dark environment for 16 h to synthesize the ABTS radical cationic reagents. The subsequent step involved diluting the reaction mixture with 80% ethanol to obtain an absorbance of 0.70 ± 0.02 at a wavelength of 734 nm. Thereafter, a 0.6 mL sample was mixed with 5.4 mL of the ABTS radical cation solution, allowing for a 6 min reaction period. The absorbance of the mixture was then precisely measured at 734 nm. The outcome of this assay was expressed as the percentage of ABTS free radical inhibition.

      Phenolic profiles

    • The samples were extracted three times by ethyl acetate, combined with organic phases, evaporated by a rotary evaporator, and then reconstituted with methanol for testing.

      High-Performance Liquid Chromatography (HPLC) was utilized to determine the phenolic profiles, adhering to the methodology delineated in a prior study[15]. The flow rate was set at 1 mL/min, and the detection was facilitated by a UV–visible spectrophotometer, monitoring at a wavelength of 280 nm. The quantification of the phenolic compounds was achieved by correlating the obtained peak areas with those of established external standards.

      Phenolic profiles were quantified by HPLC according to previously reported method[15]. The mobile phase, consisting of a 1% formic acid solution (referred to as solvent A) and acetonitrile (designated as solvent B), were combined according to a specific gradient elution scheme. This program was as follows: from the start to 5 min, the proportion of solvent B was maintained at 5%; from 5 to 25 min, it increased to 12%; from 25 to 40 min, the concentration rose to 30%; between 40 and 50 min, solvent B constituted 45% of the mixture; and from 50 to 60 min, the program returned to an initial 5% concentration of B. The column oven temperature was set at 30 °C and 10 μL of samples were injected into the sampler. Separation was carried out on a Symmetry C18 column (4.6 mm × 250 mm, 5 μm, Waters). The flow rate was set at 1 mL/min, and the detection was facilitated by a UV–visible spectrophotometer, monitoring at a wavelength of 280 nm. The quantification of the phenolic compounds was achieved by correlating the obtained peak areas with those of established external standards.

      GC-IMS detection

    • The analysis of Volatile Organic Compounds (VOCs) was conducted using the FlavourSpec® analytical system (G.A.S., Dortmund, Germany)[17]. The initial step involved the preparation of wolfberry pulp samples, where 5 g were precisely measured and deposited into a 20 mL headspace vial. This vial was then subjected to a thermal treatment at a temperature of 60 °C for 30 min to facilitate the release of VOCs. Following the incubation period, the samples were injected into the gas chromatography system using an injection needle heated to 85 °C, with a specified air volume of 500 μL at the top. The Gas Chromatography-Ion Mobility Spectrometry (GC-IMS) technique was subsequently employed to resolve the volatile components within the sample. The separation process occurred in a capillary column (MXT5, 15 m × 0.53 mm inner diameter). Nitrogen gas, with a purity level of no less than 99.999%, was chosen as the carrier gas. The flow rate of the carrier gas was programmed to initiate at 2 mL/min, which was sustained for the first 2 min. This rate was then incremented to 10 mL/min over 8 min, further increased to 100 mL/min within the subsequent 10 min, and ultimately raised to 150 mL/min in another 10 min. The ions resulting from the analytical process were directed into a drift tube, which was maintained at a temperature of 45 °C. The drift gas, set at a flow rate of 150 mL/min, facilitated the ion mobility within the drift tube, thereby completing the VOC assessment procedure.

      Statistical analysis

    • Sample preparation and analysis were conducted in triplicate to ensure consistency. Data were displayed as Mean ± SEM and processed using Origin 2022 software (Origin Lab, China). Statistical analysis was carried out via one-way ANOVA, with a p-value less than 0.05 indicating statistical significance. HS-GC-IMS data were extracted through the GC-IMS library retrieval method, and a bi-dimensional cross-qualitative approach was employed for qualitative analysis.

    Results

      Selection of prebiotics

    • Some studies have reported that lactulose and inulin promote the growth of Lactobacillus bulgaricus and Lactobacillus casei[18], to improve the fermentation efficiency of wolfberry pulp, we explored the effects of three prebiotics (inulin, lactulose, and galacto-oligosaccharides) on the fermentation of wolfberry. Figure 1a illustrates the impact of adding three prebiotics on total acidity during fermentation. It is evident from Fig. 1a that among the five prebiotics tested, wolfberry pulp supplemented with galactooligosaccharide produced higher total acidity after fermentation compared to pulp supplemented with inulin or lactulose. Different dosages of lactulose did not significantly influence the change in total acidity during fermentation (p > 0.05). Inulin, at varying dosages, had a certain impact on total acid formation, exhibiting a trend similar to that of galactooligosaccharides. The highest total acid content was observed in wolfberry pulp supplemented with 7% galactooligosaccharide, reaching 33.84 mg/mL. Figure 1b illustrates the quantification of viable bacteria after 48 h of fermentation with the three prebiotics at varying concentrations. The supplementation of prebiotics significantly enhanced the population of viable bacteria in fermented wolfberry pulp. The addition of lactulose maximizes the preservation of lactic acid bacteria activity (1.30 × 108 CFU/mL), aligning with findings from Ricardo's study[19]. As the dosage of prebiotics increases, the count of lactic acid bacteria also rises. When the dosage reaches 5%, the number of viable bacteria tends to stabilize. In the experimental group supplemented with galactooligosaccharides, at a 7% addition rate, the population of live bacteria peaks (1.18 × 108 CFU/mL), and as the addition rate continues to increase to 9%, the count of live bacteria decreases (1.17 × 108 CFU/mL). This decline may be attributed to the excessive addition of galactooligosaccharides hindering the growth of lactic acid bacteria. This observation is in harmony with the observed change in total acid depicted in Fig. 1a. Based on the combined data from Fig. 1a & b, galactooligosaccharides were ultimately selected as the prebiotics for augmenting the fermentation of wolfberry pulp.

      Figure 1. 

      Effects of adding three prebiotics (inulin, lactulose, and oligosaccharides) on (a) total acid and (b) viable bacterial count during fermentation process. The different lowercase letters marking in the figure represent different levels of significance (p < 0.05).

      Establishment of fermentation dynamics model equations

      Dynamic model of bacterial growth

    • Figure 2a showed the overall changes in the number of viable bacteria, total acids, and total sugars over 72 h of fermentation. Throughout the 72-h fermentation period, lactic acid bacteria underwent distinct phases, including an adjustment period, logarithmic period, stable period, and apoptosis period. Notably, the construction of fermentation kinetics models typically focuses on the changes in microbial numbers before entering the apoptosis period.

      Figure 2. 

      (a) Changes in total sugar, total acid, and bacterial count within 72 h of fermentation. (b) Fitting curve of the kinetic equation for bacterial growth. (c) Fitting curve of the kinetic equation for total acid production. (d) Fitting curve of the kinetic equation for substrate consumption.

      In Fig. 2b, it is observed that after 54 h of fermenting wolfberry pulp, plant lactobacilli entered the apoptosis period. Consequently, our study concentrated on developing kinetics models specifically within the initial 54 h of fermentation.

      Figure 2a, combined with Eqn (3), yields a plot of lnXXm−X against t:

      lnXXm−X=0.3393t−4.2829,R2=0.9960

      From this, it can be deduced that μm = 0.3393. Given X0 = 0.1 × 108 CFU/mL and Xm = 11.45 × 108 CFU/mL, substituting into Eqn (3), the kinetic equation for bacterial growth during wolfberry pulp fermentation is determined as:

      X=0.1e0.3393t0.9913+0.0087e0.3393t (10)

      Upon fitting the equation with experimental data points, an R² value of 0.9898 was obtained. This high R² value signifies that the equation effectively represents the changes in bacterial growth over time. The robust fit of the equation to the experimental data highlights its reliability and accuracy in capturing the dynamics of bacterial growth during the fermentation process of wolfberry pulp.

      Product generation kinetics model

    • Based on the total acid production curve in Fig. 2a, it is known that Lactobacillus plantarum is in the stable growth phase after 24 h, where dXdt = 0 and X = Xm. Plotting P against t for total acid concentration, the relationship between the two is determined as: P = 1.9374t + 37.8075, with a correlation coefficient R2 = 0.9333.

      From Eqn (5), it is derived that: β = dPdt × Xm = 1.937411.45 = 0.1692. Substituting the parameters μm, X0, Xm, and β into Eqn (6) to plot P-βB(t) against A(t), the relationship between them is found to be: P − βB(t) = 0.0836A(t), with R2 = 0.8995. Thus, α = 0.0836.

      Substituting μm, X0, Xm, β, and α into Eqns (6), the product generation kinetic model is obtained. After calculation, the product generation kinetics equation for wolfberry pulp fermentation process is:

      X=0.4457×(e0.3393t0.9913+0.0087e0.3393t−1)+2.6223×ln(0.9913+0.0087e0.3393t) (11)

      Following the fitting of the equation with experimental data points, an R² value of 0.9949 was achieved. This high R² value indicates that the equation aptly represents the process of total acid generation during the fermentation of wolfberry pulp. The close alignment between the model and experimental data underscores the accuracy and reliability of the equation in capturing the dynamics of total acid production (Fig. 2c).

      Substrate consumption dynamics model

    • Based on the substrate consumption data from the change in Fig. 2a, taking the data during the stable growth phase of bacterial cells, the relationship between total sugar content S and time t is determined as: S = −0.01789t + 0.6359, with a correlation coefficient R2 = 0.9179. From Eqn (8), it is found that: δ = 0.0194.

      Substituting parameters X0, Xm, and δ into Eqn (9) to plot S0 − S − δD(t) against C(t), the relationship between them is: S0 − S − δD(t) = 0.1524C(t), with a correlation coefficient R2 = 0.9114. Thus, γ is determined to be 0.1524. After calculation, the kinetic equation for substrate consumption is:

      X=3.9126−0.007×(e0.3393t0.9913+0.0087e0.3393t−1)−0.0322×ln(0.9913+0.0087e0.3393t) (12)

      Upon fitting the equation with experimental data points, an R² value of 0.9225 was attained. This R² value indicates that the equation effectively represents the process of total sugar consumption during the fermentation of wolfberry pulp. While slightly lower than perfect correlation, the substantial R² value demonstrates the reliability of the equation in capturing the dynamics of total sugar utilization (Fig. 2d).

      Effects of fermentation on phytochemicals and antioxidant activity of wolfberry pulp

      Total phenolic and total flavonoids content and antioxidant activity

    • Phenolic compounds contribute to antioxidant and anti-inflammatory health benefits. It has been pointed out that lactic acid bacteria can enhance the bioavailability of phenolic compounds by converting them into smaller, more absorbable molecules. They also release conjugated phenolics from fermentable materials which further improve its utilization rate and health benefits to the human body[20]. Figure 3 illustrates the alterations in total phenols and flavonoids before and after fermentation, along with the shifts in DPPH radical and ABTS radical scavenging rates. Upon comparing the total phenolic and flavonoid content in wolfberry pulp pre- and post-fermentation, the findings revealed a significant increase after fermentation (p < 0.05). The post-fermentation content of total phenols and flavonoids was 1.15 and 1.16 times higher than their respective levels before fermentation. Typically, phenolic content undergoes augmentation through enzymatic reactions during fermentation, wherein β-Glucosidase catalyzes the hydrolysis of glycosidic bonds, leading to the release of phenolic glycosides[15]. DPPH radical scavenging rate and ABTS radical scavenging rate serve as common metrics for assessing food antioxidant activity. Following fermentation, the DPPH radical scavenging rate in wolfberry pulp significantly increased, reaching 67.16% ± 6.70% (p < 0.05) after 48 h, and the ABTS radical scavenging rate increased from 26.50% ± 1.20% to 32.10% ± 0.90%. The alteration in free radical scavenging rate demonstrated a positive correlation with the heightened content of total phenols and flavonoids. Phenols are recognized as pivotal contributors that profoundly influence the antioxidant capacity of the food system[21]. Concurrently, the metabolic activity of lactic acid bacteria produces various phenolic compounds, which help improve the overall antioxidant capacity of food[22]. To investigate how the fermentation of Lactobacillus plantarum changes the content of phenolic compounds in wolfberry pulp, HPLC was used to determine the content of monomeric phenols in wolfberry pulp.

      Figure 3. 

      (a) Effect of fermentation on the content of total phenols and flavonoids in wolfberry pulp. (b) Effect of DPPH and ABTS radical scavenging rate. * represents different levels of significance (p < 0.05), while ns represents non significance (p > 0.05).

      Phenolic profiles

    • A blank control group (1) was set up along with four experimental groups, namely: after enzymolysis (2); add probiotics (3); cultivation without added prebiotics (4); adding prebiotic inference (5). The detection of five sets of samples provided a detailed explanation of the changes in composition during the production of fermented wolfberry pulp. Eight phenolic compounds, comprising five phenolic acids and three flavonoids in the wolfberry pulp, were qualitatively and quantitatively analyzed using liquid chromatography (Table 1). Chlorogenic acid, the predominant phenolic acid in wolfberry, significantly increased through fermentation (p < 0.05). In wolfberry pulp fermented with oligosaccharides, the chlorogenic acid content was 1.16 times higher than that in the blank control group, aligning with the changes in total phenols. Other phenolic acids, including gallic acid, caffeic acid, and ferulic acid, exhibited varying increases in wolfberry after enzymatic hydrolysis and fermentation. A distinctive pattern was observed in p-Coumaric acid, which increased to more than four times (23.28 ± 0.81 μg/mL) its original value (5.32 ± 0.46 μg/mL) after enzymatic hydrolysis. However, after fermentation, the p-Coumaric acid content decreased again, matching that of the blank group (5.37 ± 0.13 μg/mL). This might be attributed to the poor stability of coumaric acid in low-pH environments. Three flavonoids, catechin, rutin, and quercetin, all showed significant increases after fermentation (p < 0.05). Rutin, a common dietary flavone, possesses pharmacological properties, including antibacterial, anti-inflammatory, anti-cancer, anti-diabetic effects[23]. Among the eight monomeric phenols detected rutin exhibited the highest content, attributable to the inherent richness of rutin in wolfberry itself. The alterations in monomeric phenolic substances in wolfberry pulp before and after fermentation further indicate that fermentation can stimulate the generation of phenolic substances in wolfberry pulp, thereby enhancing its antioxidant activity.

      Table 1.  Phytochemical profiles in wolfberry pulp before and after LAB fermentation (μg/mL).

      Phytochemicals12345
      Gallic acid3.49 ± 0.74c4.74 ± 0.38b4.87 ± 0.30b7.76 ± 0.02a7.92 ± 0.28a
      Catechin33.79 ± 4.83b37.70 ± 2.76a39.95 ± 3.87a42.36 ± 2.01a42.31 ± 2.78a
      p-Coumaric acid5.32 ± 0.46b23.28 ± 0.81a21.09 ± 1.06a5.27 ± 0.55b5.37 ± 0.13b
      Chlorogenic acid36.63 ± 10.11c109.29 ± 3.87b107.56 ± 3.95b165.56 ± 4.08a159.04 ± 8.58a
      Caffeic acid2.68 ± 0.68ab1.39 ± 0.81b4.30 ± 0.79a4.57 ± 0.99a3.11 ± 0.54ab
      Ferulic acid5.97 ± 0.25c8.12 ± 0.80ab8.37 ± 0.82a7.45 ± 0.41abc6.55 ± 0.26bc
      Rutin208.09 ± 19.27b311.27 ± 41.04ab263.13 ± 16.56ab361.47 ± 23.15a265.70 ± 60.17ab
      Quercetin41.60 ± 1.09c39.93 ± 1.76c38.88 ± 0.38c58.21 ± 1.88a53.29 ± 1.79b
      Values with different superscripts in the same row have significant differences (p < 0.05). Among them: without any processing (control) (1); after enzymatic hydrolysis (2); add prebiotics (3); fermentation without added prebiotics (4); adding prebiotic fermentation (5).

      Identification and analysis of volatile components in wolfberry samples

    • Five sets of samples underwent analysis using HS-GC-IMS (Fig. 4). A total of 51 primary volatile aroma components were identified, comprising 12 alcohols, seven aldehydes, two acids, eight esters, 12 ketones, and 10 other compounds. Two among them lacked matching names in the database, suggesting they might constitute unique aroma components of wolfberry. In comparison to the original pulp, enzymatic hydrolysis resulted in an increase in hexanal, 4-methyl-4-hydroxy-2-pentanone, 2-methyl-butan-1-ol, and other substances. However, during subsequent fermentation, these substances were utilized and consumed by microorganisms as substrates (Fig. 5). Fermentation introduced more flavor components to the wolfberry pulp, such as 2-butanone, butyl formate, and 4-methyl-3-penten-2-one. Ketones can exhibit distinct flavor characteristics at low concentrations[24]. HS-GC-IMS detection results indicated that fermentation could augment the content of various ketones in wolfberry pulp. A trace number of harmful components to human health were detected in the samples before fermentation, such as tetrahydrofuran and 2-ethylfuran, which may be characteristic flavor components of wolfberry. The content of furan compounds also significantly decreased after fermentation. This suggested that fermentation could degrade some residual harmful components while enhancing the presence of beneficial aroma components. Principal component analysis demonstrated a significant distinction between the blank control group and the two groups without fermentation treatment, as well as the two groups with fermentation treatment. PC1 and PC2 contributed 77.94% and 20.24%, respectively, resulting in a total contribution rate of 98.18%.

      Figure 4. 

      (a) Fingerprint of volatile aroma components in five samples of wolfberry pulp. (b) Subtraction plot of different wolfberry pulp samples. Among them: without any processing (control) (1); after enzymatic hydrolysis (2); add prebiotics (3); fermentation without added prebiotics (4); adding prebiotic fermentation (5).

      Figure 5. 

      (a) Cluster heat map of aroma components. (b) Circular clustering diagram of aroma components. (c) Principal component analysis of aroma components.

    Conclusions

    • This study demonstrated the substantial impact of lactic acid bacteria fermentation on the flavor, chemical characteristics, and antioxidant activity of wolfberry pulp. Lab fermentation significantly increased the content of total phenols and flavonoids in wolfberry pulp (p < 0.05), subsequently enhancing its antioxidant activity with a positive correlation. The alterations in volatile aroma components in wolfberry pulp before and after fermentation were scrutinized using HS-GC-IMS. The findings revealed that lab fermentation augmented various aldehydes, ketones, and esters, introducing novel flavors like sourness and fruitiness to the fermented wolfberry pulp. Concerning fermentation efficiency, the addition of galactooligosaccharides expedited the fermentation process of wolfberry pulp, elevating the production of total acids and yielding a more favorable fermentation outcome. In summary, this study unveiled the fermentation kinetic model of L. plantarum CCFM1050 on wolfberry pulp with galactooligosaccharides. It concluded that lactic acid fermentation could enhance the flavor and functional properties of wolfberry pulp, offering insights for its advanced processing and potential guidance for the future development of the wolfberry industry.

    Author contributions

    • The authors confirm contribution to the paper as follows: conceptualization, funding acquisition: Peng Q; investigation: Liu H, Wang Y, Chen T; methodology: Liu H, Li N; data curation, formal analysis, draft manuscript preparation: Liu H; validation, software: Li N; resources, supervision, manuscript revision & editing: Yang H, Peng Q. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

      Acknowledgments

      • This work was supported by Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology & Business University (BTBU); Science and Technology Project of Xining, Grant/Award Number: 2022-Y-12; Open Foundation of the Key Laboratory of Seaweed Fertilizers, Ministry of Agriculture and Rural Affairs, Grant/Award Number: KLSF-2023-010. The authors would like to thank the shared instrument platform of the College of Food Science and Engineering of Northwest A&F University, Mrs. Ma (Instrument shared platform of College of Food Science & Engineering, Northwest A&F University) for the assistance with food grade laboratory, Mrs. Wang (Instrument shared platform of College of Food Science & Engineering, Northwest A&F University) for the assistance with HPLC (LC-2030 PLUS, Shimadzu), Mrs. Cao (Scientific Research Center of College of Horticulture, Northwest A&F University) for the assistance with HS-GC-IMS (FlavourSpec®, G.A.S., Dortmund, Germany).

      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 on behalf of China Agricultural University, Zhejiang University and Shenyang 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 (5)  Table (1) References (24)
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    Cite this article
    Liu H, Li N, Wang Y, Cheng T, Yang H, et al. 2024. Study on fermentation kinetics, antioxidant activity and flavor characteristics of Lactobacillus plantarum CCFM1050 fermented wolfberry pulp. Food Innovation and Advances 3(2): 126−134 doi: 10.48130/fia-0024-0012
    Liu H, Li N, Wang Y, Cheng T, Yang H, et al. 2024. Study on fermentation kinetics, antioxidant activity and flavor characteristics of Lactobacillus plantarum CCFM1050 fermented wolfberry pulp. Food Innovation and Advances 3(2): 126−134 doi: 10.48130/fia-0024-0012
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  • Introduction
  • Materials and methods

  • Materials

  • Preparation of wolfberry pulp

  • Preparation of bacterial cultures

  • Prebiotic screening single factor experiment

  • Construction of fermentation dynamics model

  • Dynamic model of bacterial growth
  • Product generation kinetics model
  • Substrate consumption dynamics model

  • Determination of chemical substances and antioxidant activity during fermentation

  • Determination of total phenolic and total flavonoids content
  • DPPH and ABTS radical scavenging rate
  • Phenolic profiles

  • GC-IMS detection

  • Statistical analysis

  • Results

  • Selection of prebiotics

  • Establishment of fermentation dynamics model equations

  • Dynamic model of bacterial growth
  • Product generation kinetics model
  • Substrate consumption dynamics model

  • Effects of fermentation on phytochemicals and antioxidant activity of wolfberry pulp

  • Total phenolic and total flavonoids content and antioxidant activity
  • Phenolic profiles

  • Identification and analysis of volatile components in wolfberry samples

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