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    <journal-meta>
      <journal-title-group><journal-title>Journal of Biological and Sustainability Sciences</journal-title></journal-title-group>
      <publisher><publisher-name>BIOSUSS Publishing</publisher-name></publisher>
    </journal-meta>
    <article-meta>
      <article-id pub-id-type="publisher-id">snf3vqlbsvt1b5wc4j8tl0fv</article-id>
      <article-id pub-id-type="doi">10.5281/zenodo.21449521</article-id>
      <article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories>
      <title-group><article-title>Statistical Optimization of Pullulanase Production by Aspergillus flavus under Solid-State Fermentation Using Wheat Bran</article-title></title-group>
      <contrib-group><contrib contrib-type="author"><string-name>Sonia Aziz</string-name></contrib><contrib contrib-type="author"><string-name>Rabbia Shafqat</string-name></contrib><contrib contrib-type="author"><string-name>Fahad Mehmood</string-name></contrib><contrib contrib-type="author"><string-name>Syed Muhammad Hamza Abidi</string-name></contrib></contrib-group>
      <aff id="aff1">¹Institute of Biochemistry and Biotechnology, University of Veterinary &amp; Animal Sciences, Lahore, Pakistan ²Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan ³Department of Physiology and Biochemistry, Cholistan University of Veterinary &amp; Animal Sciences (CUVAS) Bahawalpur, Bahawalpur, Pakistan ⁴Institute of Biological Sciences, Khwaja Fareed University of Engineering and Information Technology, Rahim Yar Khan 64200, Pakistan</aff>
      <author-notes><corresp>Syed Muhammad Hamza Abidi</corresp></author-notes>
      <pub-date date-type="pub" publication-format="electronic" iso-8601-date="2025-08-14"><day>14</day><month>08</month><year>2025</year></pub-date>
      
      <volume>1</volume>
      <issue>1</issue>
      <fpage>1</fpage>
      <lpage>11</lpage>
      <permissions>
        <copyright-statement>Copyright © 2025 the authors</copyright-statement>
        <license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/">
          <license-p>This article is distributed under the Creative Commons Attribution 4.0 International (CC BY 4.0). Authors retain copyright.</license-p>
        </license>
      </permissions>
      <abstract><p>Background: Pullulanase is an industrial starch-debranching enzyme whose production cost may be reduced through fungal solid-state fermentation on agro-industrial residues. This study evaluated initial pH, moisture content, wheat-bran quantity, and inoculum volume for pullulanase production by Aspergillus flavus. Methods: A four-factor, five-level central composite design containing 30 runs was constructed. Pullulanase activity was defined as units per gram of dry substrate, and a second-order polynomial model was used to estimate linear, interaction, and quadratic effects. Results: Measured pullulanase activity ranged from 12.4 to 46.9 U/gds across the 30 design runs. A reduced quadratic model retaining the significant linear and pure-quadratic terms was statistically significant (F8,21 = 11.42, p &lt; 0.0001), with R² = 0.813, adjusted R² = 0.742, and adequate precision of 11.3. The model also showed a significant lack of fit relative to pure replicate error at the six centre points (F = 471.6, p &lt; 0.0001), indicating that the fitted quadratic surface does not fully capture the response. Moisture content produced the strongest positive linear effect, followed by inoculum volume, initial pH, and substrate quantity; all pure-quadratic coefficients were negative. Numerical optimization within the tested region predicted a maximum of 41.5 U/gds at pH 5.85, 69.1% moisture, 8.87 g wheat bran, and 6.09 mL inoculum, but the single highest measured activity (46.9 U/gds) occurred at the highest-level factorial combination tested, suggesting the true optimum may lie at or beyond the upper boundary of the studied ranges. Conclusion: Moisture content, inoculum volume, and initial pH significantly influenced pullulanase production by A. flavus under solid-state fermentation, but the significant lack of fit and the corner-biased response indicate that the present central composite design has not yet bracketed the true process optimum. A follow-up design extending toward higher pH, moisture, substrate, and inoculum levels, together with confirmatory strain identification and aflatoxin-safety screening, is recommended before the process can be considered optimized.</p></abstract>
      <kwd-group kwd-group-type="author-generated"><kwd>central composite design</kwd><kwd>pullulanase</kwd><kwd>response surface methodology</kwd><kwd>solid-state fermentation</kwd><kwd>wheat bran</kwd><kwd>Aspergillus flavus</kwd></kwd-group>
      <custom-meta-group><custom-meta><meta-name>funding-statement</meta-name><meta-value>This research received no external funding.</meta-value></custom-meta><custom-meta><meta-name>conflict-of-interest</meta-name><meta-value>The authors declare no conflict of interest</meta-value></custom-meta><custom-meta><meta-name>ethics-approval</meta-name><meta-value>Not Applicable</meta-value></custom-meta><custom-meta><meta-name>data-availability</meta-name><meta-value>The complete central composite design matrix, measured pullulanase activity values, model-fitted values, and residuals are provided in Table 4. Raw assay data, calibration curves, and replicate measurements are available from the corresponding author upon reasonable request.</meta-value></custom-meta><custom-meta><meta-name>author-contributions</meta-name><meta-value>Sonia Aziz: Conceptualization, Investigation, Data curation, Writing—original draft. Rabbia Shafqat: Methodology, Formal analysis, Visualization, Writing—review and editing. Fahad Mehmood: [CRediT role(s) — to be confirmed by author]. Syed Muhammad Hamza Abidi: Supervision, Project administration, Writing—review and editing, Corresponding author. All authors have read and agreed to the published version of the manuscript.</meta-value></custom-meta></custom-meta-group>
    </article-meta>
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