Modular Biointelligent Starter Cultures for Cocoa Pulp Juice (Mucilage) Fermentation: A Scoping Review

Modular Biointelligent Starter Cultures for Cocoa Pulp Juice (Mucilage) Fermentation: A Scoping Review | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Systematic Review Modular Biointelligent Starter Cultures for Cocoa Pulp Juice (Mucilage) Fermentation: A Scoping Review Anthony Oppong Kyekyeku, Margaret Owusu, John Edem Kongor, Daniel Sitsofe Yabani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9587715/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cocoa pulp juice (mucilage)—the fermentable liquid fraction released from the pulp surrounding Theobroma cacao beans—is an abundant yet underutilized substrate for applied fermentation. Unlike cocoa bean fermentation targeted at chocolate flavour development, cocoa pulp juice valorisation is substrate-centric and requires tailored microbial and process controls to deliver consistent beverage-quality outcomes. We conducted a scoping review to map the state of modular starter culture design and fermentation systems applied to cocoa pulp juice, with emphasis on microbial consortia, process configuration, reported metabolites, volatiles, microbial dynamics, and sensory outcomes. Eligible work was charted by starter architecture (yeast-dominated, multi-kingdom/immobilised, LAB-inclusive, non-conventional platforms), fermentation system (low-tech batch to controlled bioreactors), and outcome measures (ethanol, organic acids, aroma compounds, community composition, sensory acceptance). Evidence indicates that cocoa pulp juice supports diverse fermentation products, including wine-style beverages, kefir-like functional drinks, and aroma-forward non-alcoholic prototypes. However, comparative benchmarking of starter architectures under matched substrate conditions remains limited, and mechanistic links between microbial community transitions, quorum sensing (QS) regulation, and metabolite trajectories are insufficiently reported. QS-mediated regulation—particularly luxS/AI-2 signalling—is mechanistically plausible as a coordination layer in mixed fermentations, but its effectiveness in acidic cocoa pulp juice environments remains unvalidated. Key research priorities include comparative benchmarking of modular starter architectures, experimental validation of QS-mediated coordination in acidic pulp environments, and development of smallholder-accessible deployment and monitoring frameworks. Food Science & Technology Applied & Industrial Microbiology General Microbiology Biotechnology and Bioengineering cocoa pulp juice cocoa mucilage starter culture modular consortia quorum sensing AI-2 applied fermentation scoping review Figures Figure 1 Figure 2 1. Introduction 1.1 Cocoa pulp juice fermentation: variability and opportunity Fermentation remains one of the most powerful tools for converting sugar-rich agricultural streams into stable, value-added products. Within the cocoa value chain, cocoa pulp juice (mucilage)—the pectinous, sugar-rich liquid released from the white mucilaginous matrix surrounding cocoa beans—is increasingly recognized as a fermentable substrate with strong potential for product diversification and improved resource efficiency. Yet cocoa pulp juice fermentation remains inconsistent because it is often governed by spontaneous microbial succession rather than controlled starter-driven systems, leading to variable kinetics and unpredictable metabolite outputs. 7 , 8 This challenge mirrors broader problems in cocoa fermentation systems, where uncontrolled microbial dynamics contribute to large quality fluctuations across producing regions. 9 , 24 However, a critical distinction must be maintained throughout this review: the substrate focus is cocoa pulp juice/mucilage—the sugary liquid matrix surrounding the beans—rather than whole-bean cocoa fermentation aimed at chocolate flavour development. The cocoa pulp juice matrix contains high sugar concentrations (10–15% w/v), inhibitory polyphenols (6–8%), and pronounced oxygen variability in low-technology systems, collectively destabilizing microbial succession and limiting reproducibility. 10 , 27 1.2 Definition and substrate boundary In this review, cocoa pulp juice refers to the mucilaginous, sugar-rich liquid matrix surrounding cocoa beans, also termed mucilage or pulp juice. This substrate is treated as a valorisable by-product stream used as the primary fermentation medium. The constraints analysed—including osmotic pressure from high sugars, polyphenol inhibition, oxygen oscillations, and viscosity from pectin—are interpreted as substrate-specific stressors that determine starter culture survival, consortium synergy, and fermentation performance. 10 , 27 1.3 Why modular biointelligence is needed Conventional starter culture development frequently emphasizes single-trait optimization—such as acid tolerance or ethanol yield—but cocoa pulp juice fermentation imposes multiple simultaneous stressors that render such strategies insufficient. 10 Field evidence indicates that strains selected on limited criteria can rapidly lose viability when exposed to polyphenol-rich environments, undermining microbial succession and enabling spoilage dynamics. 10 , 25 These limitations justify a shift toward modular functional starter systems, where consortia are designed as integrated microbial ecosystems with complementary roles and resilience under variable conditions. 8 Biointelligent starter design extends this concept by introducing microbial coordination mechanisms, particularly quorum sensing (QS), to synchronize metabolic transitions and stabilize fermentation trajectories. QS-mediated regulation has been shown to influence metabolite outputs, competitive exclusion dynamics, and community succession timing across diverse food fermentation ecosystems, including those involving the lactic acid bacteria (LAB) central to cocoa pulp fermentation. 29 Functional yeast selection for cocoa systems similarly requires multi-trait screening that accounts for substrate stress, co-culture interactions, and oxygen-adaptive metabolism rather than ethanol yield alone. 30 While QS-linked regulation is mechanistically plausible as a stabilizing layer in mixed fermentations, its effectiveness in acidic cocoa pulp juice environments and under real mixed-community conditions remains insufficiently validated. The fragmentation of the field makes a scoping review the appropriate methodology to map the evidence landscape and identify actionable gaps. 18 , 19 , 20 1.4 Aims of this scoping review This scoping review aims to: (i) synthesize how cocoa pulp juice fermentation has been implemented across product classes and starter configurations; (ii) map reported microbial consortia and associated outcomes (metabolites, volatiles, microbial dynamics, and sensory acceptance); (iii) examine system-level variables (oxygen, temperature, viscosity management, and stabilization) that shape reproducibility; and (iv) identify practical research gaps and a future agenda for standardized modular starter development suitable for tropical and smallholder deployment. 2. Methods 2.1 Review design and rationale This study employed a scoping review design to map the breadth of evidence on modular starter cultures and fermentation systems applied to cocoa pulp juice (mucilage) fermentation. A scoping review approach was selected because the field is emerging, methodologically heterogeneous, and not yet sufficiently mature for meta-analysis or systematic review with effect estimation. 18 , 20 The review was conducted following the PRISMA extension for scoping reviews (PRISMA-ScR) framework. 19 The review was designed to identify evidence clusters, characterize starter architectures and reported outcomes, and highlight gaps requiring targeted experimental benchmarking. 2.2 Eligibility criteria Studies were included if they met all of the following criteria: Substrate relevance: cocoa pulp juice, cocoa mucilage, or cocoa pulp-derived liquid fractions were used as the primary fermentation substrate (stand-alone or as a major fermentable fraction). Fermentation relevance: the study investigated microbial fermentation outcomes (alcoholic, non-alcoholic, functional beverage, or ingredient-oriented fermentation). Starter relevance: the work included defined inoculation strategies (single-strain, multi-strain, consortium, or immobilised cultures such as kefir grains) or clearly described microbial drivers of fermentation. Outcome reporting: at least one of the following outcomes was reported: metabolite profiles (e.g., ethanol, organic acids), aroma/volatile compounds, microbial dynamics or community composition, sensory evaluation, or process parameters (pH, temperature, °Brix). Studies were excluded if they focused exclusively on cocoa bean cotyledon fermentation for chocolate processing without explicit treatment of cocoa pulp juice as the primary substrate, or if they were not available as full-text peer-reviewed articles. 2.3 Search results and study selection The Scopus database search, conducted on 5 April 2026, retrieved 70 records. Search terms combined substrate identifiers and fermentation/starter concepts: "cocoa pulp juice" OR "cocoa mucilage" OR "cocoa pulp" AND "fermentation" OR "starter culture" OR "consortium" OR "mixed culture" OR "kefir" OR "wine" OR "non-alcoholic beverage" OR "quorum sensing". No date or language limits were applied. Following removal of 1 duplicate, 69 records were screened at the title and abstract stage. Of these, 38 were excluded: 25 records focused exclusively on whole cocoa bean cotyledon fermentation without treatment of cocoa pulp juice or mucilage as the primary fermentation substrate (E1); 7 records reported only composition, chemistry, or physicochemical characterization of cocoa pulp without fermentation outcomes (E2); and 6 records described unrelated applications of cocoa mucilage or pulp (E3). The remaining 32 records were assessed at full-text stage. A further 6 were excluded because they reported only enzymatic activity characterization or raw substrate composition without a fermentation process design or measurable fermentation outcomes (E2-FT). This yielded 26 eligible studies from the database search. Manual screening of reference lists of included studies identified 2 additional eligible studies not captured in the Scopus export. The final corpus comprised 28 included studies. The study selection process is summarized in Fig. 1 . 2.4 Characteristics of included studies The 28 included studies were published between 1999 and 2025, with the majority published after 2010 (26 of 28 studies). Studies originated from Brazil, Belgium, Ecuador, France, Spain, Germany, Colombia, Ghana, Indonesia, Mexico, and China, reflecting the global distribution of cocoa pulp juice fermentation research. Starter architectures represented included yeast-dominant systems (defined or spontaneous), LAB-forward and co-culture systems, immobilised multi-kingdom consortia (kefir grains and SCOBY), non-conventional fungal platforms, and defined metabolic simulation systems. Study designs ranged from batch beverage fermentations and lab-scale pulp simulation media to review syntheses and metabolic modelling studies. Full details of included studies are provided in Table 1. 2.5 Data charting and synthesis Data from eligible studies were extracted into a standardized charting framework capturing: Starter architecture: yeast-only, yeast + LAB, multi-kingdom/immobilised, or non-conventional platforms. Fermentation system: low-tech batch, semi-closed, submerged fermentation, or controlled bioreactor. Process variables: temperature, oxygen exposure, viscosity management (e.g., depectinization), stabilization/pasteurization, and inoculation regime. Reported outcomes: ethanol and organic acids, aroma/volatile compounds, microbial succession, sensory acceptance, and shelf-stability indicators. Synthesis was conducted narratively, organized around modular design logic (anchor module, stability module, optional oxidation module, and stress-guard mechanisms). Evidence gaps were identified based on inconsistencies in reporting, absence of comparative benchmarking, and limited translation to tropical smallholder constraints. 3. The Cocoa Pulp Juice Matrix as a Fermentation Stress Landscape Cocoa pulp juice fermentation is governed by a sequential but overlapping series of biochemical stressors that shape starter culture survival, microbial succession, and metabolite formation. These stressors are not merely background conditions; they are primary ecological forces that determine whether a consortium thrives or collapses. 10 3.1 Substrate composition and viscosity Cocoa pulp juice is a pectinous, sugar-rich liquid with high fermentable carbohydrate content (predominantly glucose, fructose, and sucrose; 10–15% w/v total), significant organic acid content (primarily citric acid), and inhibitory polyphenols (6–8% w/v including epicatechin and proanthocyanidins). 1 , 3 The high pectin content contributes to viscosity that can impede mixing, oxygen distribution, and inoculum contact. Depectinization—via enzymatic or thermal treatments—can reduce viscosity and improve processability, making pulp juice a more tractable fermentation substrate. 3 3.2 Stage 1 — Sugar stress and osmotic pressure High sugar concentrations impose osmotic pressure that selectively favours osmotolerant yeasts in the early phase of fermentation, driving rapid ethanol formation. 27 While this early yeast dominance supports pulp matrix breakdown and aroma-active metabolite formation, uncontrolled glycolysis can generate ethanol surges that destabilize subsequent LAB succession and suppress the acidification trajectory required for product safety and sensory quality. 7 Effective modular starter design must therefore couple the anchor/chassis module (yeast-dominant, early-phase) with a stability module designed to take over once osmotic conditions moderate, ensuring a predictable metabolic handoff. 8 3.3 Stage 2 — Polyphenol inhibition and ecological instability As sugars decline, microbial communities encounter inhibitory polyphenols that suppress growth through membrane disruption and protein denaturation. 10 Non-adapted strains lacking tannase activity or polyphenol-tolerance mechanisms can lose substantial viability within 48 hours of exposure, creating ecological vacuums frequently exploited by spoilage-associated Enterobacteriaceae. 10 , 25 Functional starter consortia must therefore integrate detoxification capacity—including enzymatic polyphenol degradation—as a core design requirement, not an optional trait. 8 , 9 3.4 Stage 3 — Oxygen oscillations and redox instability Low-technology fermentation systems produce strong oxygen oscillations as headspace composition shifts during fermentation and gas production. 25 These oscillations can shift oxygen availability from near-anaerobic ( 15%) conditions, forcing microbial communities to transition rapidly between fermentative and respiratory metabolic states. 25 Since acetic acid bacteria (AAB) require oxygen to oxidize ethanol to acetate, uncontrolled oxygen surges can lead to undesired acidification trajectories and volatile acidity accumulation. 7 Oxygen management is therefore both a system engineering challenge and a starter design constraint. 3.5 Temperature variability in tropical production contexts In West African, Latin American, and Southeast Asian cocoa-producing regions, ambient temperatures typically range from 25 to 40°C, with diurnal fluctuations that can exceed 15°C in outdoor or semi-open fermentation setups. 9 These temperature swings influence fermentation kinetics, aroma compound formation, and microbial viability in ways that laboratory-calibrated starters may not be designed to handle. 9 , 28 Thermotolerant organisms—particularly yeasts such as Pichia kudriavzevii and Kluyveromyces marxianus —are therefore functionally advantaged in pulp juice fermentation systems and represent high-priority chassis candidates. 30 4. Fermentation Systems for Cocoa Pulp Juice The fermentation system architecture—including vessel geometry, oxygen transfer regime, temperature management, and sanitation controllability—strongly shapes fermentation outcomes in cocoa pulp juice and must be co-designed with starter culture selection rather than treated as an independent variable. 7 , 8 4.1 Low-technology batch systems The most accessible fermentation configuration for smallholder and near-farm valorisation is a simple covered batch vessel (plastic containers, food-grade drums, or repurposed containers). These systems have low capital cost and can be deployed in decentralized settings, but impose weak oxygen control, inconsistent temperature management, and hygiene variability. 9 , 28 Published cocoa pulp wine research demonstrates that even simple batch fermentation can produce acceptable alcoholic beverages, confirming substrate feasibility under minimal infrastructure. 4 However, the same evidence highlights that low-technology systems shift the 'control burden' from engineered hardware to microbial management—making starter culture design and inoculation regime critical levers for reproducibility. 28 4.2 Oxygen management as a design variable In cocoa pulp juice fermentation, oxygen exposure is not merely an environmental condition—it is a design variable that determines the direction of carbon flux. Under low oxygen, yeasts dominate sugar conversion to ethanol and aroma-active intermediates. Under increased oxygen, AAB can oxidize ethanol to acetic acid, driving acidification and potentially vinegar-like sensory profiles. 7 , 25 Covered fermentations are therefore more appropriate for wine-style or kefir-type beverage products, while semi-open systems may support targeted acetate production for ingredient applications. 5 4.3 Temperature control and its consequences for aroma Temperature strongly shapes microbial growth rates, enzyme kinetics, and volatile compound formation in fermentation systems. 30 In cocoa pulp kefir fermentations, Puerari et al. 5 demonstrated that fermentation temperature substantially altered both the microbial community composition and the sensory profile of the resulting beverage. For aroma-forward non-alcoholic prototypes—such as the submerged fungal fermentation developed by Klis et al. 6 —temperature management was critical for retaining the fruity volatile profile that distinguished the product from unfermented substrate. 4.4 Substrate preparation: stabilization, dilution, and depectinization Substrate preparation prior to fermentation can substantially influence process reproducibility and starter performance. Mild thermal stabilization (pasteurization) of cocoa pulp juice reduces indigenous microbial load variability and extends the working window for collection and fermentation initiation. 37 Dilution can modulate osmotic pressure, reducing the competitive advantage of extreme osmotolerants and creating more permissive conditions for LAB colonization. 3 Enzymatic or thermal depectinization reduces viscosity and improves substrate homogeneity, which is particularly important for inoculum distribution in batch systems. 3 5. Benchmark Fermentation Ecosystems as Design Analogues Cocoa pulp juice fermentation benefits from analogy-based design by borrowing validated principles from mature fermented-food ecosystems where modularity, succession control, and safety stabilization are well understood. The benchmarks discussed below are informative not because they match cocoa pulp chemistry, but because they demonstrate repeatable ecological control under stress—the precise challenge faced in cocoa pulp juice valorisation. 5.1 Kimchi: community modularity, competitive exclusion, and QS-linked coordination Kimchi is a LAB-centred ecosystem that achieves food safety and sensory reliability through rapid competitive exclusion, staged LAB succession, and dense interspecies interactions in a chemically stressful environment. 14 Microbial succession in kimchi is temporally structured, with Leuconostoc mesenteroides dominating early acidification before more acid-tolerant taxa, primarily Lactiplantibacillus plantarum , take over in the mid-to-late phase—a succession architecture directly analogous to the module transitions required in cocoa pulp juice fermentation. 14 A particularly important finding for biointelligent starter design is that kimchi LAB communities generate measurable autoinducer-2 (AI-2) quorum-signalling activity, with AI-2 production and inhibition patterns varying across Lactobacillus , Weissella , and Leuconostoc genera. 34 This indicates that community-level communication, rather than single-strain signalling, governs competitive dynamics and succession timing. For cocoa pulp juice, this supports designing the LAB stability module for inter-species communication compatibility rather than selecting strains purely for single-organism acid tolerance. 11 , 29 5.2 Tempeh: the single-anchor module and enzymatic precision Tempeh demonstrates that fermentation reproducibility can be achieved through a single dominant keystone organism that acts as a primary conversion module and imposes process directionality, while auxiliary microbiota play secondary roles. 17 The anchor organism (typically Rhizopus spp.) drives rapid nutrient transformation and suppresses stochastic invasion by the sheer competitiveness of its early dominance, illustrating a key design principle for cocoa pulp juice: where the substrate is fast-fermenting and prone to runaway metabolic trajectories, an anchor module can stabilize early-phase succession and reduce batch-to-batch variability. 17 , 38 For cocoa pulp juice, this maps directly to selecting an early-phase yeast chassis (e.g., Saccharomyces cerevisiae , Pichia kudriavzevii , or Torulaspora delbrueckii ) as the anchor module, with secondary LAB and optional AAB modules designed to activate in subsequent phases. 21 , 22 , 30 5.3 Sourdough: symbiotic modularity and process classification Sourdough fermentation provides an unusually comprehensive model because it has been formalized into distinct process types based on inoculum strategy—ranging from spontaneous/backslopping regimes to defined starter-driven and hybrid approaches. 15 , 35 This classification demonstrates that reproducibility can be engineered through inoculum design and technological setup, not only through strain selection. Sourdough also illustrates stable yeast–LAB co-existence under shared stress, where metabolic specialization and cooperative tolerance of acidic, nutrient-limited conditions produce consistent product quality across diverse production contexts. 15 , 16 For cocoa pulp juice valorisation, sourdough process logic supports two deployment strategies: a type-2 approach (defined modular starter in controlled batch) for product development and quality benchmarking; and a type-3 approach (starter-seeded controlled backslopping) for decentralized scaling in smallholder settings where maintaining pure cultures is impractical. 35 5.4 Cross-benchmark synthesis: three design rules for cocoa pulp juice Rule 1 — Anchor the early phase with a dominant stabilizer tempeh illustrates how a keystone organism can impose process directionality early, limiting stochastic drift across batches and substrate variants. 17 , 38 Rule 2 — Build an active stability layer with community communication compatibility kimchi provides direct evidence that LAB consortia generate QS-linked communication dynamics (AI-2) that govern competitive exclusion and succession timing, and that module selection should account for inter-species signal compatibility. 14 , 34 Rule 3 — Treat inoculum strategy as a modular design variable sourdough formalizes how starter-only, backslopping, and hybrid strategies map to different stability–cost–reproducibility trade-offs; this is directly applicable to cocoa pulp juice scaling in resource-variable production contexts. 15 , 35 6. Cocoa Pulp Juice as a Fermentation Substrate: Evidence of Feasibility This section maps the primary evidence for cocoa pulp juice fermentation across product classes, cataloguing the starter types, fermentation systems, and outcomes reported in eligible studies. The evidence demonstrates substrate feasibility across multiple product categories while revealing important gaps in comparative benchmarking and mechanistic understanding. 6.1 Wine-style alcoholic fermentations The most established valorisation route for cocoa pulp juice is wine-style fermentation using Saccharomyces -dominated starters. Dias et al. 4 provided the foundational demonstration that cocoa pulp is a viable fermentable substrate for fruit wine production, confirming that pulp juice sugars can be reliably converted to ethanol and that the resulting beverage has acceptable sensory characteristics. The core strength of this approach is the reliability of sugar-to-ethanol conversion under yeast dominance; however, sensory quality and stability depend heavily on oxygen management, temperature control, and supplementary acidification modules to prevent volatile acidity drift and off-flavour formation. 4 6.2 Kefir-style functional beverages Kefir grain fermentation of cocoa pulp juice, demonstrated by Puerari et al., 5 offers an intrinsically modular fermentation system. Kefir grains are naturally immobilised multi-kingdom consortia in which yeasts, LAB, and acetic acid bacteria coexist in a structured matrix, providing robustness and functional redundancy across variable substrate chemistry. The Puerari et al. study characterized both microbial community composition and product sensory properties at different fermentation temperatures, demonstrating the system's modularity in practice: temperature shifts altered community balance and sensory outcomes without causing fermentation failure. 5 This system closely resembles the modular biointelligent framework proposed in this review and provides the strongest existing proof-of-concept for multi-kingdom starter design in cocoa pulp juice valorisation. 6.3 Non-alcoholic and aroma-forward fermentations Non-conventional microbial platforms have been used to redirect cocoa pulp juice fermentation toward aroma-forward, low-ethanol products. Klis et al. 6 developed a beverage by fermenting diluted, pasteurized cocoa pulp with the wood-decay fungus Laetiporus persicinus in submerged fermentation, generating a product with tropical aromatic character and sensory descriptors dominated by fruity notes including (R)-linalool. This study demonstrates that cocoa pulp juice fermentation can be directed toward aroma-dominant, non-alcoholic outcomes by shifting the starter paradigm from ethanol-maximizing yeasts to aroma-producing non-conventional platforms. 6 Rodríguez-Castro et al. 2 further demonstrated that cocoa mucilage serves as a novel fermentable substrate for kombucha-style fermentation using a symbiotic culture of bacteria and yeast (SCOBY), providing a further non-conventional proof-of-concept for functional beverage production from this substrate. 6.4 Aroma-oriented yeast co-cultures An emerging evidence stream uses cocoa pulp media to study how different yeasts—alone and in combination—shape volatile aroma outputs. Besançon et al. 21 examined aroma production in cocoa pulp medium using single and mixed cultures of Saccharomyces cerevisiae , Pichia kudriavzevii , and Torulaspora species, finding that strain interactions—including killer phenotype effects—substantially altered aroma profiles and fermentation dynamics. Complementary aroma-starter cocktail work by Chang et al. 22 further supports multi-strain combination strategies for controlling volatile profiles in cocoa fermentation contexts. 6.5 Adjunct and ingredient applications Cocoa pulp juice has been evaluated as a fermentation adjunct in brewing applications by Nunes et al., 3 who demonstrated that once viscosity is managed through depectinization, pulp-derived sugars and acids can be integrated into industrial fermentation workflows. While this application differs from beverage-primary valorisation, it expands the valorisation design space and provides evidence for substrate processability under controlled industrial conditions. 6.6 Summary of the evidence landscape Collectively, the evidence supports cocoa pulp juice as a tractable and versatile fermentation substrate. However, a critical gap persists: no study has systematically compared these starter architectures under matched substrate conditions. The majority of eligible studies used diluted, pasteurized, or otherwise modified substrates that may not fully represent undiluted cocoa pulp juice collected near-farm under variable indigenous microbial loads. Module-level causality—the specific contribution of each consortium member to the metabolite profile—is rarely reported, and safety/shelf-stability endpoints are inconsistently included (see Table 1 for evidence map). 2 , 4 , 5 , 6 , 21 , 22 , 23 , 33 Table 1 | Summary of the 28 studies included in the scoping review, charted by starter architecture, fermentation system, substrate preparation, and primary outcomes reported. Studies marked * were identified through hand search of reference lists. # Author (Year) Study / Focus Starter Architecture Fermentation System Substrate Prep Primary Outcomes Reported 1 Chungsiriporn et al. (2025) Heated fermentation + cocoa juice separation Yeast-dominant (spontaneous) Batch with juice separation + heating Separation of pulp juice from beans pH, °Brix, physicochemical bean/juice quality 2 Marwati et al. (2024) L. plantarum HL-15 as starter + pulp valorisation LAB starter (Lactiplantibacillus plantarum) Batch fermentation Cocoa pulp by-product Fermentation dynamics, metabolites, pulp valorisation 3 Lefeber et al. (2010) Kinetic analysis LAB/AAB in cocoa pulp simulation Defined LAB + AAB strains Cocoa pulp simulation media (bioreactor) Artificial pulp medium Organic acids, ethanol, metabolic kinetics 4 Besançon et al. (2024) Yeast interactions in synthetic vs real mucilage media Defined yeast mono/co-cultures Cocoa pulp simulation + real mucilage Synthetic + real mucilage Volatile aroma compounds, fermentation metabolism 5 Villarroel-Bastidas et al. (2025) Cacao mucilage to produce craft beers Mixed culture / spontaneous Batch vessel (craft brewery scale) Cocoa mucilage as adjunct Ethanol, fermentation kinetics, beer sensory quality 6 Meersman et al. (2016) Thermotolerant S. cerevisiae starters for cocoa pulp Engineered S. cerevisiae (thermotolerant) Cocoa pulp medium (lab-scale) Cocoa pulp medium Acetate ester production, volatile profiles, flavour 7 Puerari et al. (2012) Cocoa pulp-based kefir beverages Kefir grains (multi-kingdom immobilised) Batch semi-closed (varied temperature) Cocoa pulp juice Microbial community, organic acids, sensory acceptance 8 Koelher et al. (2022) S. cerevisiae for fruit wines using cocoa honey + pulp S. cerevisiae strains (defined) Batch cocoa honey/pulp wine fermentation Cocoa honey + cocoa pulp Ethanol, fermentation kinetics, sensory quality 9 Guirlanda et al. (2021) Cocoa honey: agro-industrial waste or by-product? (Review) Review (multiple starter types) Review of multiple systems Review Fermentation products, composition, valorisation options 10 Meersman et al. (2015) Breeding robust yeast starters for cocoa pulp fermentations Engineered S. cerevisiae (hybrid starters) Cocoa pulp fermentation media Cocoa pulp medium Fermentation performance, flavour compound formation 11 Dzogbefia et al. (1999) Controlled cocoa fermentation with yeasts: cocoa sweatings Defined yeasts (controlled inoculation) Batch sweatings/juice vessels Cocoa sweatings (pulp juice) Physicochemical changes, enzymatic activity, microbial counts 12 Adler et al. (2013) Core fluxome of LAB under cocoa pulp fermentation simulation LAB strains (Lactobacillus/Leuconostoc) Cocoa pulp simulation media (chemostat) Artificial pulp medium Metabolic fluxes, organic acids, CO₂ production 13 Klis et al. (2023) Cocoa pulp fermentation by Laetiporus persicinus beverage Laetiporus persicinus (fungal, non-conventional) Submerged fermentation (diluted pasteurised pulp) Diluted, pasteurised cocoa pulp Aroma volatiles, sensory evaluation, non-alcoholic beverage 14 Ayala et al. (2022) Valorization of cocoa mucilage waste to ethanol/ethylene Spontaneous/mixed culture Batch bioreactor (mucilage waste) Raw cocoa mucilage Ethanol yield, fermentation kinetics 15 García-Ríos et al. (2021) Thermo-adaptive S. cerevisiae for cocoa pulp fermentations Thermo-adaptive S. cerevisiae strains Cocoa pulp fermentation (lab-scale) Cocoa pulp medium Growth kinetics, fermentation performance, metabolites 16 Chetschik et al. (2018) Aroma of cocoa pulp and influence on fermented cocoa beans N/A — substrate characterisation Cocoa pulp aroma in fermentation context Fresh cocoa pulp Aroma-active compounds; influence on fermentation volatiles 17 Rodríguez-Castro et al. (2024) Cocoa mucilage as novel ingredient in kombucha fermentation Kombucha SCOBY (multi-kingdom immobilised) Batch closed vessel (cocoa mucilage) Cocoa mucilage Physicochemical properties, microbial composition, sensory 18 Adler et al. (2014) Metabolic fluxes of AAB under cocoa pulp simulation Defined AAB strains Cocoa pulp simulation media (bioreactor) Artificial pulp medium Acetate metabolic fluxes, key metabolite pathways 19 Guimarães et al. (2020) Cocoa pulp as matrix for probiotic delivery Probiotic LAB strains (Lactobacillus spp.) Cocoa pulp matrix (delivery system) Cocoa pulp as food matrix Probiotic viability, pH, fermentation parameters 20 Mota-Gutierrez et al. (2021) Microbial communities on fermented cocoa pulp-bean mass Spontaneous community (pulp-bean mass) Traditional heap/batch Cocoa pulp-bean mass Microbial community profiling, metabolites, quality parameters 21 Lefeber et al. (2011) Starter strains via cocoa pulp simulation fermentations Cocoa-specific LAB strains Cocoa pulp simulation media Artificial pulp medium LAB fermentation performance, metabolite profiles, strain selection 22 Chang et al. (2025)* [Food Chem: X] Aromatic compounds in cocoa pulp fermentation — volatilomics Defined yeast starters + LAB Cocoa pulp batch fermentation Cocoa pulp medium Volatile/aroma compounds, machine learning metabolomics 23 Vizcaino-Almeida et al. (2022) Non-conventional fermentation: probiotic microorganisms + mucilage substitution Probiotic strains + conventional yeasts Modified batch (mucilage/fruit pulp) Cocoa mucilage (and substitutes) Fermentation outcomes, metabolites, sensory evaluation 24 Nunes et al. (2020) Cocoa pulp as adjunct for beer production S. cerevisiae (conventional) Batch beer fermentation with cocoa pulp adjunct Depectinised cocoa pulp Beer quality, fermentation kinetics, composition 25 Bastidas et al. (2023) Cocoa mucilage: novel substrate for fermented tea-based beverages Tea SCOBY / kombucha culture Batch closed vessel (cocoa mucilage) Cocoa mucilage Fermentation parameters, metabolites, sensory evaluation 26 Alvarado-Santos et al. (2023) Kinetic model for cocoa waste fermentation to ethanol Spontaneous/mixed culture Batch bioreactor (cocoa waste pulp) Cocoa pulp waste Ethanol kinetics, mathematical model validation 27 Dias et al. (2007)* Elaboration of a fruit wine from cocoa pulp S. cerevisiae (conventional) Batch wine fermentation Cocoa pulp juice Ethanol, physicochemical parameters, sensory quality 28 Ho VTT, Zhao J, Fleet G. (2015) Effect of lactic acid bacteria on cocoa bean fermentation — LAB roles in acidification, competitive exclusion, and flavour LAB strains (Lactobacillus, Leuconostoc) Cocoa pulp simulation + bean fermentation (lab-scale) Cocoa pulp/bean matrix LAB metabolic contributions, acidification kinetics, flavour impact Abbreviations: LAB = lactic acid bacteria; AAB = acetic acid bacteria; SCOBY = symbiotic culture of bacteria and yeast. * Identified through hand search of reference lists. 7. Quorum Sensing as a Coordination Layer in Cocoa Pulp Juice Fermentation The concept of biointelligent starter design rests on the hypothesis that microbial communication mechanisms—particularly quorum sensing (QS)—can be leveraged to coordinate community behaviour, synchronize metabolic transitions, and improve fermentation stability in complex, variable substrates such as cocoa pulp juice. 11,29 7.1 The AI-2/LuxS system in food-relevant LAB The most extensively characterized inter-species QS signal in food fermentation contexts is autoinducer-2 (AI-2), produced via the LuxS enzyme. AI-2 has been shown to regulate biofilm formation in Lactobacillus rhamnosus GG, with LuxS-deficient mutants showing substantially reduced biofilm and community stability, 13 and to modulate stress tolerance and adhesion capacity in Lactiplantibacillus plantarum KLDS1.0391. 12 Johansen and Jespersen 29 provide a broader synthesis of QS effects across food fermentation ecosystems, documenting that AI-2 and acyl-homoserine lactone (AHL) signals can influence metabolite timing, succession dynamics, and competitive exclusion in LAB-dominated communities. 7.2 In silico evidence for QS in cocoa fermentation systems Almeida et al. 11 conducted an in silico investigation of QS potential across microbial communities involved in spontaneous cocoa bean fermentation, identifying LuxS/AI-2 pathway activity in several key fermentation-associated LAB species. This study provides the primary mechanistic rationale for hypothesizing QS-linked coordination in cocoa-associated fermentations, but it is important to note that this evidence is computational rather than experimental—the in silico results indicate QS pathway presence and potential activity, not functional QS-driven fermentation control. 11 7.3 QS activity in benchmark food fermentation systems Direct experimental evidence for functional AI-2 activity in food fermentations comes principally from kimchi. Park et al. 34 demonstrated that kimchi fermentation generates measurable AI-2 quorum-signalling activity associated with its LAB microbiota, with community-level AI-2 dynamics reflecting inter-species interactions among Lactobacillus , Weissella , and Leuconostoc taxa. This community-level QS activity is proposed to influence succession timing and competitive dynamics in the fermentation, supporting a role for inter-species communication in ecosystem stabilization. 34 7.4 Critical constraints on QS application in cocoa pulp juice Several constraints limit the straightforward translation of QS-based coordination strategies to cocoa pulp juice fermentation. First, AI-2 signals are known to be pH-sensitive, with signal stability decreasing substantially under acidic conditions. Given that cocoa pulp juice fermentation typically acidifies from pH ~4.5–5.0 to pH ~3.0–3.5 over the fermentation course, the functional window for AI-2-mediated coordination may be narrow and largely confined to early fermentation phases. 24 Second, the majority of QS mechanistic evidence comes from controlled single- or dual-species systems, simplified media, or in silico analyses. The functional relevance of QS in complex, multi-species, substrate-variable environments—such as undiluted cocoa pulp juice with high indigenous microbial loads—has not been validated. Extrapolating from controlled QS studies to real fermentation ecosystems requires substantial caution and targeted experimental work. 11,29 Third, QS signal dynamics can be influenced by matrix components including polyphenols, which may quench or interfere with signalling molecules. 10 This interaction is potentially significant in cocoa pulp juice, where polyphenol concentrations are substantial. For these reasons, this review positions QS as a mechanistically plausible research target for improving coordination in modular starter systems, rather than as a validated feature of current cocoa pulp juice fermentation practice. 8. A Modular Biointelligent Blueprint for Cocoa Pulp Juice Fermentation Drawing on the evidence synthesized in Sections 3–7, a modular biointelligent starter culture framework for cocoa pulp juice fermentation can be conceptualized as a four-layer architecture designed for cooperative function under substrate stress (see Fig. 2 and Table 2). 8.1 Layer 1 — Anchor/Chassis module (early phase) The chassis module drives rapid sugar conversion in the early phase of fermentation, establishes ecological dominance, and creates a predictable metabolic foundation for subsequent module transitions. Candidate chassis organisms are osmotolerant, thermotolerant yeasts with documented performance in cocoa pulp or pulp-mimicking media. 21,22,30 Selection criteria include: high fermentative capacity under osmotic stress; tolerance of polyphenol exposure; competitive dominance over undesirable indigenous taxa; and compatibility with downstream LAB colonization. 9,26 Saccharomyces cerevisiae , Pichia kudriavzevii , Kluyveromyces marxianus , and Torulaspora delbrueckii are the most documented candidates in cocoa pulp contexts. 21,22,30 8.2 Layer 2 — Stability/Bioprotection module (mid phase) The stability module controls acidification trajectory, suppresses spoilage organisms, and maintains process predictability through the mid-to-late fermentation phase. LAB are the primary members of this module, selected for: acid production kinetics compatible with the target product; polyphenol tolerance or detoxification capacity; competitive exclusion of undesirable taxa; and, where applicable, compatibility with inter-species communication dynamics. 12,13,14,32 Tannase-producing LAB strains—capable of enzymatic polyphenol degradation—are particularly valuable because they simultaneously contribute to detoxification and competitive stabilization. 10 The stability module should be designed to be communication-compatible with the chassis module to prevent antagonistic interactions during the module transition window. 29,34 8.3 Layer 3 — Optional oxidation/redox module (conditional late phase) An oxidation module composed of AAB can be incorporated when the product intent requires targeted acetate generation (e.g., vinegar-type ingredients or certain functional beverages). This module should be activated deliberately through oxygen management rather than allowed to develop adventitiously, as uncontrolled AAB activity represents a common failure mode in pulp juice fermentations. 7,25 When a low-acidity or non-alcoholic beverage is the product target, this module should be excluded and oxygen minimized to prevent AAB colonization. 8.4 Layer 4 — Stress-guard mechanisms (cross-cutting) Stress-guard mechanisms are not a separate organism group but a set of design features that improve consortium resilience across all phases. These include: functional redundancy (multiple strains with overlapping roles to buffer against individual strain failure); immobilization strategies (kefir grain analogs or carrier-based delivery to improve robustness and reuse potential); thermotolerance screening (selecting strains with demonstrated activity across the expected temperature range of 25–40°C); and preparation methods that extend viability under field storage conditions without refrigeration. 5,26 8.5 Inoculum strategy as a modular design variable Consistent with the sourdough process classification framework, 35 the inoculum regime should be treated as a deliberate design choice matched to the deployment context. In controlled production facilities, a defined multi-module starter can be applied as a single inoculation event. In decentralized smallholder settings, a hybrid approach—seed inoculation followed by controlled backslopping using a fraction of the previous batch—may offer better practical sustainability, at the cost of some precision in community composition over successive batches. The choice between these strategies represents a reproducibility–deployability trade-off that must be explicitly addressed in modular starter system design. 31 Fig. 2 | Modular biointelligent starter culture framework for cocoa pulp juice (mucilage) fermentation. Panel A: substrate stress landscape (osmotic pressure from high sugars 10–15% w/v, polyphenol inhibition 6–8% w/v, and oxygen oscillations). Panel B: three functional modules — Module 1 anchor/chassis (yeast-dominant, early phase), Module 2 stability/bioprotection (LAB-centred, mid phase), and Module 3 optional redox/oxidation (AAB, conditional late phase). Panel C: cross-cutting stress-guard layer. Panel D: biointelligent coordination layer (LuxS/AI-2 quorum sensing; mechanistically plausible, but validation in acidic pulp juice conditions required). Panel E: minimal viable monitoring and deployment variables. LAB = lactic acid bacteria; AAB = acetic acid bacteria; QS = quorum sensing; AI-2 = autoinducer-2. Table 2 | Summary of the modular biointelligent starter culture blueprint for cocoa pulp juice fermentation. Each layer is described by its representative organisms, activation phase, key functions, and principal design requirements. Module / Layer Representative Organisms Phase Key Functions Design Requirements Layer 1 — Anchor/Chassis Saccharomyces cerevisiae, Pichia kudriavzevii, Kluyveromyces marxianus, Torulaspora delbrueckii Early (0–48 h) Rapid sugar conversion; ecological dominance; aroma-active metabolite formation; suppression of spoilage taxa Osmotolerance; thermotolerance (25–40°C); polyphenol tolerance; competitive dominance; LAB-compatibility Layer 2 — Stability/Bioprotection Lactiplantibacillus plantarum, Leuconostoc mesenteroides, Lactobacillus fermentum, tannase-producing LAB strains Mid (48–96 h) Controlled acidification; spoilage suppression; polyphenol detoxification; community stabilization via competitive exclusion Acid production kinetics; polyphenol/tannase activity; inter-species QS compatibility (AI-2/LuxS); safety-relevant competitive exclusion; LAB role in flavour trajectory (Ho et al.32) Layer 3 — Oxidation/Redox (optional) Acetobacter spp., Gluconobacter spp. Late (>96 h, oxygen-dependent) Targeted ethanol-to-acetate conversion; acidification for ingredient applications Activated deliberately via oxygen management only; excluded for wine/kefir beverage targets to prevent volatile acidity off-notes Layer 4 — Stress-Guard (cross-cutting) Functional redundancy across all layers; immobilised consortia; carrier-based delivery systems All phases Resilience buffering; robustness under field temperature variability; extended inoculum shelf-life without cold chain Functional redundancy per module; immobilization strategy (kefir analog/carrier); thermotolerance screening; cold-chain-independent viability methods Coordination Layer (QS) LuxS/AI-2-competent LAB strains (plausible but unvalidated in cocoa pulp juice context) Early–mid transition Synchronized metabolic transitions; succession timing; inter-species competitive signalling Validation required under acidic pH trajectories (pH 4.5 to 3.0); validation in presence of cocoa polyphenols; experimental confirmation of functional AI-2 half-life in pulp juice Abbreviations: LAB = lactic acid bacteria; AAB = acetic acid bacteria; QS = quorum sensing; AI-2 = autoinducer-2; LuxS = AI-2 synthase enzyme. 9. Validation Gaps Limiting Real-World Deployment Despite the mechanistic plausibility and proof-of-concept evidence supporting modular starter design for cocoa pulp juice fermentation, a series of validation gaps must be addressed before these systems can be deployed reliably at scale. 9.1 Comparative benchmarking under matched conditions The most immediate gap in the existing literature is the absence of head-to-head comparison of different starter architectures (yeast-only, yeast+LAB, multi-kingdom/kefir-based, non-conventional fungal) under identical substrate, temperature, and oxygen conditions. Without such comparisons, it is not possible to make evidence-based recommendations about which starter configuration provides superior outcomes for a given product target. 4,5,6,21 9.2 QS validation under acidic pulp conditions The QS evidence base for cocoa-associated fermentations remains largely in silico 11 or derived from non-cocoa food systems. 29,34 Experimental validation of AI-2-mediated coordination in cocoa pulp juice—under realistic pH trajectories, temperature variation, and mixed indigenous microbial loads—is absent from the current literature. Specifically, the functional half-life of AI-2 signals along the acidification curve of cocoa pulp juice fermentation needs to be established, and the effect of pulp polyphenols on signal transmission and reception requires investigation. 9.3 Tropical field-mimicking validation Most eligible studies were conducted under controlled laboratory conditions, often with diluted or pasteurized substrates, at temperatures that may not reflect tropical production environments. 9 Validation under field-mimicking conditions—including undiluted pulp juice, variable indigenous microbial loads, temperature fluctuations (25–40°C), and realistic hygiene constraints—is needed to determine whether laboratory-demonstrated starter performance translates to near-farm deployment. 9,28 9.4 Safety, stability, and reporting standardization Across eligible studies, safety indicators (e.g., Enterobacteriaceae enumeration, mycotoxin screening) and shelf-life stability endpoints are rarely reported. For beverage applications, these endpoints are not optional—they are foundational requirements for any regulatory pathway. Standardized minimum reporting sets that include core metabolite profiles, microbial community endpoints, safety indicators, and sensory acceptance would substantially improve comparability across studies and accelerate the field toward evidence-based standardization. 8,9 10. Conclusion Cocoa pulp juice (mucilage) is a credible, scalable substrate for applied fermentation, with demonstrated proof-of-concept across product classes including kefir-style functional beverages, wine-style alcoholic fermentations, and aroma-forward non-alcoholic prototypes. 2,4,5,6 However, the evidence mapped in this review shows that individual feasibility demonstrations have not yet translated into standardized, reproducible fermentation practice. The core barrier is that the cocoa pulp juice matrix imposes multi-dimensional stressors—osmotic pressure, polyphenol inhibition, oxygen instability, and temperature variability—that single-strain or single-trait starters cannot reliably navigate. 7,10 The central implication of this scoping review is that cocoa pulp juice valorisation will be standardized most effectively through modular ecosystem design rather than single-strain optimization. The benchmark fermentation evidence from kimchi, sourdough, and tempeh collectively supports three convergent principles: anchor the early phase with a dominant stabilizer; build an active stability layer with community communication compatibility; and treat inoculum strategy as a deliberate design variable matched to the deployment context. 14,15,17,34,35 Biointelligent coordination via QS—particularly AI-2/LuxS signalling—represents a mechanistically plausible research target for improving succession stability and competitive exclusion in modular consortia, supported by in silico evidence from cocoa fermentation systems 11 and experimental evidence from food-relevant benchmark fermentations. 29,34 However, QS-mediated coordination cannot be considered a validated feature of cocoa pulp juice fermentation until experimental studies demonstrate functional signal activity under acidic pulp conditions, in the presence of polyphenols, and in the context of mixed indigenous microbial communities. Translation into smallholder-compatible deployment will require substrate stabilization strategies to reduce indigenous load variability; 37 affordable monitoring technologies for core state variables (pH, temperature, °Brix); 36 inoculum preparation methods that maintain viability without cold chain dependence; 26 and field-validated protocols that account for tropical variability and decentralized hygiene constraints. 9,28,31 The field is at an inflection point. The substrate is proven. The starter framework is conceptually mature. The benchmark analogues provide transferable design principles. What is now needed are comparative, field-realistic experimental studies that benchmark modular starter architectures against matched controls, with standardized reporting of microbial, metabolite, safety, and sensory endpoints. Future Perspectives From demonstrations to benchmarks: standardization requires head-to-head comparisons of yeast-only, yeast+LAB, and multi-kingdom modular starters under identical cocoa pulp juice conditions, including matched substrate preparation, temperature, and oxygen management. QS under real acidity: AI-2/LuxS performance must be experimentally tested along pulp-relevant pH trajectories and in mixed consortia to determine whether QS improves succession stability without unintended antagonism. Immobilization as an enabling technology: structured consortia analogous to kefir grains or carrier-based systems may offer the most deployable route for smallholder adoption by improving reuse, robustness, and dosing simplicity without requiring refrigerated culture maintenance. Minimal viable monitoring: translation into smallholder settings will depend on low-cost measurement of temperature, pH, and °Brix as operational state variables linked to starter-module transitions, supported by affordable sensor platforms. 36 Abbreviations AAB — Acetic Acid Bacteria; AHL — Acyl-Homoserine Lactone; AI-2 — Autoinducer-2; LAB — Lactic Acid Bacteria; PRISMA-ScR — Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews; QS — Quorum Sensing; SCOBY — Symbiotic Culture of Bacteria and Yeast. Declarations Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and materials: Not applicable. This is a scoping review article; no primary data are presented. Competing interests: The authors declare no competing interests. Funding: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Authors' contributions: Anthony Oppong Kyekyeku: Conceptualization, Methodology, Writing — original draft, Writing — review and editing, Visualization. Margaret Owusu: Supervision, Writing — review and editing. John Edem Kongor: Supervision, Writing — review and editing. Daniel Sitsofe Yabani: Writing — review and editing. All authors read and approved the final manuscript. Acknowledgements: The authors acknowledge all who provided guidance and support during the development of the research programme underpinning this work. Use of AI tools: The authors used Anthropic's Claude AI assistant to support language polishing, structural review, and reference formatting during manuscript preparation. All AI-assisted outputs were critically reviewed, edited, and verified by the authors, who take full responsibility for the content and conclusions of this manuscript. 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Supplementary Files PRISMAScRChecklist.docx Supplementary File 1— PRISMA-ScR Checklist SupplementaryFile2ChartingTable.docx Supplementary File 2 — Study Charting Table Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9587715","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Systematic Review","associatedPublications":[],"authors":[{"id":633049413,"identity":"bcd20ed2-de23-49c5-933b-6cf4363e0be2","order_by":0,"name":"Anthony Oppong Kyekyeku","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0003-1642-9178","institution":"CSIR College of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Anthony","middleName":"Oppong","lastName":"Kyekyeku","suffix":""},{"id":633049414,"identity":"fd2924de-0f75-4bc1-b82f-8dc8ea1476c3","order_by":1,"name":"Margaret Owusu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYPACGyjNBsQSxGlJA2Jm0rQcJkGLbnuPmeTPtvPy/P3nDzB8KDvMIB/dgF+L2ZkzZtK8bbcNZ9xIZmCcce4wg+GdAwS03MjdJs3YdjvBQIKZgZm3DahlRgJhLUCHnUsw4D/MwPyXWC0SvG0HEgwYkhmYGYFa5CUIaTlz/rM1z7lkkF8MDvacS+cxIKjleFvizR9ldsAQO/jwwY8yazl5Qg5DAQeAmMfgAAk6IEC+gWQto2AUjIJRMMwBAL5QQdrc3dolAAAAAElFTkSuQmCC","orcid":"","institution":"CSIR College of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Margaret","middleName":"","lastName":"Owusu","suffix":""},{"id":633049415,"identity":"7f3904c5-1957-4950-93e7-81919c52681b","order_by":2,"name":"John Edem Kongor","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-6575-5362","institution":"CSIR Food Research Institute","correspondingAuthor":true,"prefix":"","firstName":"John","middleName":"Edem","lastName":"Kongor","suffix":""},{"id":633049416,"identity":"e9f5c834-f56f-4d30-a3ab-fdc689ca5589","order_by":3,"name":"Daniel Sitsofe Yabani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie3QsWoCQRCA4ZGFvRSjtneccK8wcnAQPDBPc5ZaqYXFgZDrUpvHCHmBlYG7RlsJaKGNdSRBBEFcFDTVql2K/YrdYeBvBsCy/iMFIPSH1fN8Iu9LvPTRBEhdd+akorD+0xksamExXY1/IW4HL85saUo8haE/ytcYTVrENUiePxT2yJSQQvJRMkZfEtgFpnqKiXsjCfd4YAxHDySRX35lJFfC+FsnATi5MfFYdhvvb4zuJAEGSogESmNSKYaf886Wm9UsF5tdP6Ygy9bGBMTT9TwC9UyMxoNpzvIylnb6CdI/G8uyLEs7Ai7XRYShCmv7AAAAAElFTkSuQmCC","orcid":"","institution":"Cocoa Research Institute of Ghana","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"Sitsofe","lastName":"Yabani","suffix":""}],"badges":[],"createdAt":"2026-05-01 16:05:13","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9587715/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9587715/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108444531,"identity":"2450a035-697b-4645-8c39-b5981835f5af","added_by":"auto","created_at":"2026-05-04 17:34:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4729717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePRISMA-ScR flow diagram showing the study selection process for the scoping review. Scopus search (5 April 2026) retrieved 70 records; after removal of 1 duplicate, 69 records were screened at title and abstract stage. Thirty-eight records were excluded (E1 = whole cocoa bean fermentation only, n = 25; E2 = no fermentation outcomes, n = 7; E3 = unrelated applications, n = 6). Thirty-two full texts were assessed; 6 were excluded (E2-FT = composition/enzyme studies only). Two additional studies were identified through reference list hand search. Total included: n = 28. PRISMA-ScR = Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (Tricco et al., 2018).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1PRISMAScRFlowDiagram.png","url":"https://assets-eu.researchsquare.com/files/rs-9587715/v1/ecbeceb7ec7db4f5ce942fed.png"},{"id":108444533,"identity":"25561c69-1d2a-4e07-9efa-16d9a8285125","added_by":"auto","created_at":"2026-05-04 17:34:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5969322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eModular biointelligent starter culture framework for cocoa pulp juice (mucilage) fermentation. Panel A: substrate stress landscape (osmotic pressure from high sugars 10–15% w/v, polyphenol inhibition 6–8% w/v, and oxygen oscillations). Panel B: three functional modules — Module 1 anchor/chassis (yeast-dominant, early phase), Module 2 stability/bioprotection (LAB-centred, mid phase), and Module 3 optional redox/oxidation (AAB, conditional late phase). Panel C: cross-cutting stress-guard layer. Panel D: biointelligent coordination layer (LuxS/AI-2 quorum sensing; mechanistically plausible, but validation in acidic pulp juice conditions required). Panel E: minimal viable monitoring and deployment variables. LAB = lactic acid bacteria; AAB = acetic acid bacteria; QS = quorum sensing; AI-2 = autoinducer-2.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig2ModularStarterFramework.png","url":"https://assets-eu.researchsquare.com/files/rs-9587715/v1/f3ff1e32923baa226e5a50d9.png"},{"id":108804140,"identity":"c9ea7d39-546f-4963-9927-249221f41d76","added_by":"auto","created_at":"2026-05-08 15:16:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10174741,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9587715/v1/9415710e-ec39-4fe3-a6a9-62bf362d9ac4.pdf"},{"id":108444532,"identity":"33158c60-3885-46b2-8dd6-ea59d1490de4","added_by":"auto","created_at":"2026-05-04 17:34:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18569,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary File 1— PRISMA-ScR Checklist\u003c/p\u003e","description":"","filename":"PRISMAScRChecklist.docx","url":"https://assets-eu.researchsquare.com/files/rs-9587715/v1/94e44aa8908d631f36cfb99e.docx"},{"id":108493425,"identity":"a9a6c97a-b39f-465a-95f8-65a7b70c2ad8","added_by":"auto","created_at":"2026-05-05 10:00:20","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21341,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary File 2 — Study Charting Table\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryFile2ChartingTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-9587715/v1/8fd843a4bb3bbe3f10f3e5ab.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eModular Biointelligent Starter Cultures for Cocoa Pulp Juice (Mucilage) Fermentation: A Scoping Review\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e1.1 Cocoa pulp juice fermentation: variability and opportunity\u003c/h2\u003e \u003cp\u003eFermentation remains one of the most powerful tools for converting sugar-rich agricultural streams into stable, value-added products. Within the cocoa value chain, cocoa pulp juice (mucilage)\u0026mdash;the pectinous, sugar-rich liquid released from the white mucilaginous matrix surrounding cocoa beans\u0026mdash;is increasingly recognized as a fermentable substrate with strong potential for product diversification and improved resource efficiency. Yet cocoa pulp juice fermentation remains inconsistent because it is often governed by spontaneous microbial succession rather than controlled starter-driven systems, leading to variable kinetics and unpredictable metabolite outputs.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis challenge mirrors broader problems in cocoa fermentation systems, where uncontrolled microbial dynamics contribute to large quality fluctuations across producing regions.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e However, a critical distinction must be maintained throughout this review: the substrate focus is cocoa pulp juice/mucilage\u0026mdash;the sugary liquid matrix surrounding the beans\u0026mdash;rather than whole-bean cocoa fermentation aimed at chocolate flavour development. The cocoa pulp juice matrix contains high sugar concentrations (10\u0026ndash;15% w/v), inhibitory polyphenols (6\u0026ndash;8%), and pronounced oxygen variability in low-technology systems, collectively destabilizing microbial succession and limiting reproducibility.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1.2 Definition and substrate boundary\u003c/h2\u003e \u003cp\u003eIn this review, cocoa pulp juice refers to the mucilaginous, sugar-rich liquid matrix surrounding cocoa beans, also termed mucilage or pulp juice. This substrate is treated as a valorisable by-product stream used as the primary fermentation medium. The constraints analysed\u0026mdash;including osmotic pressure from high sugars, polyphenol inhibition, oxygen oscillations, and viscosity from pectin\u0026mdash;are interpreted as substrate-specific stressors that determine starter culture survival, consortium synergy, and fermentation performance.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e1.3 Why modular biointelligence is needed\u003c/h2\u003e \u003cp\u003eConventional starter culture development frequently emphasizes single-trait optimization\u0026mdash;such as acid tolerance or ethanol yield\u0026mdash;but cocoa pulp juice fermentation imposes multiple simultaneous stressors that render such strategies insufficient.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Field evidence indicates that strains selected on limited criteria can rapidly lose viability when exposed to polyphenol-rich environments, undermining microbial succession and enabling spoilage dynamics.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e These limitations justify a shift toward modular functional starter systems, where consortia are designed as integrated microbial ecosystems with complementary roles and resilience under variable conditions.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBiointelligent starter design extends this concept by introducing microbial coordination mechanisms, particularly quorum sensing (QS), to synchronize metabolic transitions and stabilize fermentation trajectories. QS-mediated regulation has been shown to influence metabolite outputs, competitive exclusion dynamics, and community succession timing across diverse food fermentation ecosystems, including those involving the lactic acid bacteria (LAB) central to cocoa pulp fermentation.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Functional yeast selection for cocoa systems similarly requires multi-trait screening that accounts for substrate stress, co-culture interactions, and oxygen-adaptive metabolism rather than ethanol yield alone.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWhile QS-linked regulation is mechanistically plausible as a stabilizing layer in mixed fermentations, its effectiveness in acidic cocoa pulp juice environments and under real mixed-community conditions remains insufficiently validated. The fragmentation of the field makes a scoping review the appropriate methodology to map the evidence landscape and identify actionable gaps.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e1.4 Aims of this scoping review\u003c/h2\u003e \u003cp\u003eThis scoping review aims to: (i) synthesize how cocoa pulp juice fermentation has been implemented across product classes and starter configurations; (ii) map reported microbial consortia and associated outcomes (metabolites, volatiles, microbial dynamics, and sensory acceptance); (iii) examine system-level variables (oxygen, temperature, viscosity management, and stabilization) that shape reproducibility; and (iv) identify practical research gaps and a future agenda for standardized modular starter development suitable for tropical and smallholder deployment.\u003c/p\u003e \u003c/div\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Review design and rationale\u003c/h2\u003e \u003cp\u003eThis study employed a scoping review design to map the breadth of evidence on modular starter cultures and fermentation systems applied to cocoa pulp juice (mucilage) fermentation. A scoping review approach was selected because the field is emerging, methodologically heterogeneous, and not yet sufficiently mature for meta-analysis or systematic review with effect estimation.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e The review was conducted following the PRISMA extension for scoping reviews (PRISMA-ScR) framework.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e The review was designed to identify evidence clusters, characterize starter architectures and reported outcomes, and highlight gaps requiring targeted experimental benchmarking.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Eligibility criteria\u003c/h2\u003e \u003cp\u003eStudies were included if they met all of the following criteria:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eSubstrate relevance: cocoa pulp juice, cocoa mucilage, or cocoa pulp-derived liquid fractions were used as the primary fermentation substrate (stand-alone or as a major fermentable fraction).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFermentation relevance: the study investigated microbial fermentation outcomes (alcoholic, non-alcoholic, functional beverage, or ingredient-oriented fermentation).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eStarter relevance: the work included defined inoculation strategies (single-strain, multi-strain, consortium, or immobilised cultures such as kefir grains) or clearly described microbial drivers of fermentation.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eOutcome reporting: at least one of the following outcomes was reported: metabolite profiles (e.g., ethanol, organic acids), aroma/volatile compounds, microbial dynamics or community composition, sensory evaluation, or process parameters (pH, temperature, \u0026deg;Brix).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eStudies were excluded if they focused exclusively on cocoa bean cotyledon fermentation for chocolate processing without explicit treatment of cocoa pulp juice as the primary substrate, or if they were not available as full-text peer-reviewed articles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Search results and study selection\u003c/h2\u003e \u003cp\u003eThe Scopus database search, conducted on 5 April 2026, retrieved 70 records. Search terms combined substrate identifiers and fermentation/starter concepts: \"cocoa pulp juice\" OR \"cocoa mucilage\" OR \"cocoa pulp\" AND \"fermentation\" OR \"starter culture\" OR \"consortium\" OR \"mixed culture\" OR \"kefir\" OR \"wine\" OR \"non-alcoholic beverage\" OR \"quorum sensing\". No date or language limits were applied. Following removal of 1 duplicate, 69 records were screened at the title and abstract stage. Of these, 38 were excluded: 25 records focused exclusively on whole cocoa bean cotyledon fermentation without treatment of cocoa pulp juice or mucilage as the primary fermentation substrate (E1); 7 records reported only composition, chemistry, or physicochemical characterization of cocoa pulp without fermentation outcomes (E2); and 6 records described unrelated applications of cocoa mucilage or pulp (E3). The remaining 32 records were assessed at full-text stage. A further 6 were excluded because they reported only enzymatic activity characterization or raw substrate composition without a fermentation process design or measurable fermentation outcomes (E2-FT). This yielded 26 eligible studies from the database search. Manual screening of reference lists of included studies identified 2 additional eligible studies not captured in the Scopus export. The final corpus comprised 28 included studies. The study selection process is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characteristics of included studies\u003c/h2\u003e \u003cp\u003eThe 28 included studies were published between 1999 and 2025, with the majority published after 2010 (26 of 28 studies). Studies originated from Brazil, Belgium, Ecuador, France, Spain, Germany, Colombia, Ghana, Indonesia, Mexico, and China, reflecting the global distribution of cocoa pulp juice fermentation research. Starter architectures represented included yeast-dominant systems (defined or spontaneous), LAB-forward and co-culture systems, immobilised multi-kingdom consortia (kefir grains and SCOBY), non-conventional fungal platforms, and defined metabolic simulation systems. Study designs ranged from batch beverage fermentations and lab-scale pulp simulation media to review syntheses and metabolic modelling studies. Full details of included studies are provided in Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data charting and synthesis\u003c/h2\u003e \u003cp\u003eData from eligible studies were extracted into a standardized charting framework capturing:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eStarter architecture: yeast-only, yeast\u0026thinsp;+\u0026thinsp;LAB, multi-kingdom/immobilised, or non-conventional platforms.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFermentation system: low-tech batch, semi-closed, submerged fermentation, or controlled bioreactor.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eProcess variables: temperature, oxygen exposure, viscosity management (e.g., depectinization), stabilization/pasteurization, and inoculation regime.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eReported outcomes: ethanol and organic acids, aroma/volatile compounds, microbial succession, sensory acceptance, and shelf-stability indicators.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eSynthesis was conducted narratively, organized around modular design logic (anchor module, stability module, optional oxidation module, and stress-guard mechanisms). Evidence gaps were identified based on inconsistencies in reporting, absence of comparative benchmarking, and limited translation to tropical smallholder constraints.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. The Cocoa Pulp Juice Matrix as a Fermentation Stress Landscape","content":"\u003cp\u003eCocoa pulp juice fermentation is governed by a sequential but overlapping series of biochemical stressors that shape starter culture survival, microbial succession, and metabolite formation. These stressors are not merely background conditions; they are primary ecological forces that determine whether a consortium thrives or collapses.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Substrate composition and viscosity\u003c/h2\u003e \u003cp\u003eCocoa pulp juice is a pectinous, sugar-rich liquid with high fermentable carbohydrate content (predominantly glucose, fructose, and sucrose; 10\u0026ndash;15% w/v total), significant organic acid content (primarily citric acid), and inhibitory polyphenols (6\u0026ndash;8% w/v including epicatechin and proanthocyanidins).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e The high pectin content contributes to viscosity that can impede mixing, oxygen distribution, and inoculum contact. Depectinization\u0026mdash;via enzymatic or thermal treatments\u0026mdash;can reduce viscosity and improve processability, making pulp juice a more tractable fermentation substrate.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Stage 1 \u0026mdash; Sugar stress and osmotic pressure\u003c/h2\u003e \u003cp\u003eHigh sugar concentrations impose osmotic pressure that selectively favours osmotolerant yeasts in the early phase of fermentation, driving rapid ethanol formation.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e While this early yeast dominance supports pulp matrix breakdown and aroma-active metabolite formation, uncontrolled glycolysis can generate ethanol surges that destabilize subsequent LAB succession and suppress the acidification trajectory required for product safety and sensory quality.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Effective modular starter design must therefore couple the anchor/chassis module (yeast-dominant, early-phase) with a stability module designed to take over once osmotic conditions moderate, ensuring a predictable metabolic handoff.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Stage 2 \u0026mdash; Polyphenol inhibition and ecological instability\u003c/h2\u003e \u003cp\u003eAs sugars decline, microbial communities encounter inhibitory polyphenols that suppress growth through membrane disruption and protein denaturation.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Non-adapted strains lacking tannase activity or polyphenol-tolerance mechanisms can lose substantial viability within 48 hours of exposure, creating ecological vacuums frequently exploited by spoilage-associated Enterobacteriaceae.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Functional starter consortia must therefore integrate detoxification capacity\u0026mdash;including enzymatic polyphenol degradation\u0026mdash;as a core design requirement, not an optional trait.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Stage 3 \u0026mdash; Oxygen oscillations and redox instability\u003c/h2\u003e \u003cp\u003eLow-technology fermentation systems produce strong oxygen oscillations as headspace composition shifts during fermentation and gas production.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e These oscillations can shift oxygen availability from near-anaerobic (\u0026lt;\u0026thinsp;0.5%) to aerobic (\u0026gt;\u0026thinsp;15%) conditions, forcing microbial communities to transition rapidly between fermentative and respiratory metabolic states.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Since acetic acid bacteria (AAB) require oxygen to oxidize ethanol to acetate, uncontrolled oxygen surges can lead to undesired acidification trajectories and volatile acidity accumulation.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Oxygen management is therefore both a system engineering challenge and a starter design constraint.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Temperature variability in tropical production contexts\u003c/h2\u003e \u003cp\u003eIn West African, Latin American, and Southeast Asian cocoa-producing regions, ambient temperatures typically range from 25 to 40\u0026deg;C, with diurnal fluctuations that can exceed 15\u0026deg;C in outdoor or semi-open fermentation setups.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e These temperature swings influence fermentation kinetics, aroma compound formation, and microbial viability in ways that laboratory-calibrated starters may not be designed to handle.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Thermotolerant organisms\u0026mdash;particularly yeasts such as \u003cem\u003ePichia kudriavzevii\u003c/em\u003e and \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e\u0026mdash;are therefore functionally advantaged in pulp juice fermentation systems and represent high-priority chassis candidates.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Fermentation Systems for Cocoa Pulp Juice","content":"\u003cp\u003eThe fermentation system architecture\u0026mdash;including vessel geometry, oxygen transfer regime, temperature management, and sanitation controllability\u0026mdash;strongly shapes fermentation outcomes in cocoa pulp juice and must be co-designed with starter culture selection rather than treated as an independent variable.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Low-technology batch systems\u003c/h2\u003e \u003cp\u003eThe most accessible fermentation configuration for smallholder and near-farm valorisation is a simple covered batch vessel (plastic containers, food-grade drums, or repurposed containers). These systems have low capital cost and can be deployed in decentralized settings, but impose weak oxygen control, inconsistent temperature management, and hygiene variability.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Published cocoa pulp wine research demonstrates that even simple batch fermentation can produce acceptable alcoholic beverages, confirming substrate feasibility under minimal infrastructure.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e However, the same evidence highlights that low-technology systems shift the 'control burden' from engineered hardware to microbial management\u0026mdash;making starter culture design and inoculation regime critical levers for reproducibility.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Oxygen management as a design variable\u003c/h2\u003e \u003cp\u003eIn cocoa pulp juice fermentation, oxygen exposure is not merely an environmental condition\u0026mdash;it is a design variable that determines the direction of carbon flux. Under low oxygen, yeasts dominate sugar conversion to ethanol and aroma-active intermediates. Under increased oxygen, AAB can oxidize ethanol to acetic acid, driving acidification and potentially vinegar-like sensory profiles.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Covered fermentations are therefore more appropriate for wine-style or kefir-type beverage products, while semi-open systems may support targeted acetate production for ingredient applications.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Temperature control and its consequences for aroma\u003c/h2\u003e \u003cp\u003eTemperature strongly shapes microbial growth rates, enzyme kinetics, and volatile compound formation in fermentation systems.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e In cocoa pulp kefir fermentations, Puerari et al.\u003csup\u003e5\u003c/sup\u003e demonstrated that fermentation temperature substantially altered both the microbial community composition and the sensory profile of the resulting beverage. For aroma-forward non-alcoholic prototypes\u0026mdash;such as the submerged fungal fermentation developed by Klis et al.\u003csup\u003e6\u003c/sup\u003e\u0026mdash;temperature management was critical for retaining the fruity volatile profile that distinguished the product from unfermented substrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Substrate preparation: stabilization, dilution, and depectinization\u003c/h2\u003e \u003cp\u003eSubstrate preparation prior to fermentation can substantially influence process reproducibility and starter performance. Mild thermal stabilization (pasteurization) of cocoa pulp juice reduces indigenous microbial load variability and extends the working window for collection and fermentation initiation.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Dilution can modulate osmotic pressure, reducing the competitive advantage of extreme osmotolerants and creating more permissive conditions for LAB colonization.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Enzymatic or thermal depectinization reduces viscosity and improves substrate homogeneity, which is particularly important for inoculum distribution in batch systems.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Benchmark Fermentation Ecosystems as Design Analogues","content":"\u003cp\u003eCocoa pulp juice fermentation benefits from analogy-based design by borrowing validated principles from mature fermented-food ecosystems where modularity, succession control, and safety stabilization are well understood. The benchmarks discussed below are informative not because they match cocoa pulp chemistry, but because they demonstrate repeatable ecological control under stress\u0026mdash;the precise challenge faced in cocoa pulp juice valorisation.\u003c/p\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e5.1 Kimchi: community modularity, competitive exclusion, and QS-linked coordination\u003c/h2\u003e \u003cp\u003eKimchi is a LAB-centred ecosystem that achieves food safety and sensory reliability through rapid competitive exclusion, staged LAB succession, and dense interspecies interactions in a chemically stressful environment.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Microbial succession in kimchi is temporally structured, with \u003cem\u003eLeuconostoc mesenteroides\u003c/em\u003e dominating early acidification before more acid-tolerant taxa, primarily \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, take over in the mid-to-late phase\u0026mdash;a succession architecture directly analogous to the module transitions required in cocoa pulp juice fermentation.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA particularly important finding for biointelligent starter design is that kimchi LAB communities generate measurable autoinducer-2 (AI-2) quorum-signalling activity, with AI-2 production and inhibition patterns varying across \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eWeissella\u003c/em\u003e, and \u003cem\u003eLeuconostoc\u003c/em\u003e genera.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e This indicates that community-level communication, rather than single-strain signalling, governs competitive dynamics and succession timing. For cocoa pulp juice, this supports designing the LAB stability module for inter-species communication compatibility rather than selecting strains purely for single-organism acid tolerance.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e5.2 Tempeh: the single-anchor module and enzymatic precision\u003c/h2\u003e \u003cp\u003eTempeh demonstrates that fermentation reproducibility can be achieved through a single dominant keystone organism that acts as a primary conversion module and imposes process directionality, while auxiliary microbiota play secondary roles.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The anchor organism (typically \u003cem\u003eRhizopus\u003c/em\u003e spp.) drives rapid nutrient transformation and suppresses stochastic invasion by the sheer competitiveness of its early dominance, illustrating a key design principle for cocoa pulp juice: where the substrate is fast-fermenting and prone to runaway metabolic trajectories, an anchor module can stabilize early-phase succession and reduce batch-to-batch variability.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFor cocoa pulp juice, this maps directly to selecting an early-phase yeast chassis (e.g., \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, \u003cem\u003ePichia kudriavzevii\u003c/em\u003e, or \u003cem\u003eTorulaspora delbrueckii\u003c/em\u003e) as the anchor module, with secondary LAB and optional AAB modules designed to activate in subsequent phases.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e5.3 Sourdough: symbiotic modularity and process classification\u003c/h2\u003e \u003cp\u003eSourdough fermentation provides an unusually comprehensive model because it has been formalized into distinct process types based on inoculum strategy\u0026mdash;ranging from spontaneous/backslopping regimes to defined starter-driven and hybrid approaches.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e This classification demonstrates that reproducibility can be engineered through inoculum design and technological setup, not only through strain selection. Sourdough also illustrates stable yeast\u0026ndash;LAB co-existence under shared stress, where metabolic specialization and cooperative tolerance of acidic, nutrient-limited conditions produce consistent product quality across diverse production contexts.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFor cocoa pulp juice valorisation, sourdough process logic supports two deployment strategies: a type-2 approach (defined modular starter in controlled batch) for product development and quality benchmarking; and a type-3 approach (starter-seeded controlled backslopping) for decentralized scaling in smallholder settings where maintaining pure cultures is impractical.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e5.4 Cross-benchmark synthesis: three design rules for cocoa pulp juice\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eRule 1 \u0026mdash; Anchor the early phase with a dominant stabilizer\u003c/strong\u003e \u003cp\u003etempeh illustrates how a keystone organism can impose process directionality early, limiting stochastic drift across batches and substrate variants.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRule 2 \u0026mdash; Build an active stability layer with community communication compatibility\u003c/strong\u003e \u003cp\u003ekimchi provides direct evidence that LAB consortia generate QS-linked communication dynamics (AI-2) that govern competitive exclusion and succession timing, and that module selection should account for inter-species signal compatibility.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRule 3 \u0026mdash; Treat inoculum strategy as a modular design variable\u003c/strong\u003e \u003cp\u003esourdough formalizes how starter-only, backslopping, and hybrid strategies map to different stability\u0026ndash;cost\u0026ndash;reproducibility trade-offs; this is directly applicable to cocoa pulp juice scaling in resource-variable production contexts.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"6. Cocoa Pulp Juice as a Fermentation Substrate: Evidence of Feasibility","content":"\u003cp\u003eThis section maps the primary evidence for cocoa pulp juice fermentation across product classes, cataloguing the starter types, fermentation systems, and outcomes reported in eligible studies. The evidence demonstrates substrate feasibility across multiple product categories while revealing important gaps in comparative benchmarking and mechanistic understanding.\u003c/p\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e6.1 Wine-style alcoholic fermentations\u003c/h2\u003e \u003cp\u003eThe most established valorisation route for cocoa pulp juice is wine-style fermentation using \u003cem\u003eSaccharomyces\u003c/em\u003e-dominated starters. Dias et al.\u003csup\u003e4\u003c/sup\u003e provided the foundational demonstration that cocoa pulp is a viable fermentable substrate for fruit wine production, confirming that pulp juice sugars can be reliably converted to ethanol and that the resulting beverage has acceptable sensory characteristics. The core strength of this approach is the reliability of sugar-to-ethanol conversion under yeast dominance; however, sensory quality and stability depend heavily on oxygen management, temperature control, and supplementary acidification modules to prevent volatile acidity drift and off-flavour formation.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e6.2 Kefir-style functional beverages\u003c/h2\u003e \u003cp\u003eKefir grain fermentation of cocoa pulp juice, demonstrated by Puerari et al.,\u003csup\u003e5\u003c/sup\u003e offers an intrinsically modular fermentation system. Kefir grains are naturally immobilised multi-kingdom consortia in which yeasts, LAB, and acetic acid bacteria coexist in a structured matrix, providing robustness and functional redundancy across variable substrate chemistry. The Puerari et al. study characterized both microbial community composition and product sensory properties at different fermentation temperatures, demonstrating the system's modularity in practice: temperature shifts altered community balance and sensory outcomes without causing fermentation failure.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e This system closely resembles the modular biointelligent framework proposed in this review and provides the strongest existing proof-of-concept for multi-kingdom starter design in cocoa pulp juice valorisation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e6.3 Non-alcoholic and aroma-forward fermentations\u003c/h2\u003e \u003cp\u003eNon-conventional microbial platforms have been used to redirect cocoa pulp juice fermentation toward aroma-forward, low-ethanol products. Klis et al.\u003csup\u003e6\u003c/sup\u003e developed a beverage by fermenting diluted, pasteurized cocoa pulp with the wood-decay fungus \u003cem\u003eLaetiporus persicinus\u003c/em\u003e in submerged fermentation, generating a product with tropical aromatic character and sensory descriptors dominated by fruity notes including (R)-linalool. This study demonstrates that cocoa pulp juice fermentation can be directed toward aroma-dominant, non-alcoholic outcomes by shifting the starter paradigm from ethanol-maximizing yeasts to aroma-producing non-conventional platforms.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRodr\u0026iacute;guez-Castro et al.\u003csup\u003e2\u003c/sup\u003e further demonstrated that cocoa mucilage serves as a novel fermentable substrate for kombucha-style fermentation using a symbiotic culture of bacteria and yeast (SCOBY), providing a further non-conventional proof-of-concept for functional beverage production from this substrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e6.4 Aroma-oriented yeast co-cultures\u003c/h2\u003e \u003cp\u003eAn emerging evidence stream uses cocoa pulp media to study how different yeasts\u0026mdash;alone and in combination\u0026mdash;shape volatile aroma outputs. Besan\u0026ccedil;on et al.\u003csup\u003e21\u003c/sup\u003e examined aroma production in cocoa pulp medium using single and mixed cultures of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, \u003cem\u003ePichia kudriavzevii\u003c/em\u003e, and \u003cem\u003eTorulaspora\u003c/em\u003e species, finding that strain interactions\u0026mdash;including killer phenotype effects\u0026mdash;substantially altered aroma profiles and fermentation dynamics. Complementary aroma-starter cocktail work by Chang et al.\u003csup\u003e22\u003c/sup\u003e further supports multi-strain combination strategies for controlling volatile profiles in cocoa fermentation contexts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e6.5 Adjunct and ingredient applications\u003c/h2\u003e \u003cp\u003eCocoa pulp juice has been evaluated as a fermentation adjunct in brewing applications by Nunes et al.,\u003csup\u003e3\u003c/sup\u003e who demonstrated that once viscosity is managed through depectinization, pulp-derived sugars and acids can be integrated into industrial fermentation workflows. While this application differs from beverage-primary valorisation, it expands the valorisation design space and provides evidence for substrate processability under controlled industrial conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e6.6 Summary of the evidence landscape\u003c/h2\u003e \u003cp\u003eCollectively, the evidence supports cocoa pulp juice as a tractable and versatile fermentation substrate. However, a critical gap persists: no study has systematically compared these starter architectures under matched substrate conditions. The majority of eligible studies used diluted, pasteurized, or otherwise modified substrates that may not fully represent undiluted cocoa pulp juice collected near-farm under variable indigenous microbial loads. Module-level causality\u0026mdash;the specific contribution of each consortium member to the metabolite profile\u0026mdash;is rarely reported, and safety/shelf-stability endpoints are inconsistently included (see Table\u0026nbsp;1 for evidence map).\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable\u0026nbsp;1 | Summary of the 28 studies included in the scoping review, charted by starter architecture, fermentation system, substrate preparation, and primary outcomes reported. Studies marked * were identified through hand search of reference lists.\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAuthor (Year)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStudy / Focus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStarter Architecture\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFermentation System\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubstrate Prep\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimary Outcomes Reported\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eChungsiriporn et al. (2025)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eHeated fermentation + cocoa juice separation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eYeast-dominant (spontaneous)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch with juice separation + heating\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eSeparation of pulp juice from beans\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003epH, \u0026deg;Brix, physicochemical bean/juice quality\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eMarwati et al. (2024)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eL. plantarum HL-15 as starter + pulp valorisation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eLAB starter (Lactiplantibacillus plantarum)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp by-product\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eFermentation dynamics, metabolites, pulp valorisation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eLefeber et al. (2010)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eKinetic analysis LAB/AAB in cocoa pulp simulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eDefined LAB + AAB strains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp simulation media (bioreactor)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eArtificial pulp medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eOrganic acids, ethanol, metabolic kinetics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eBesan\u0026ccedil;on et al. (2024)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eYeast interactions in synthetic vs real mucilage media\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eDefined yeast mono/co-cultures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp simulation + real mucilage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eSynthetic + real mucilage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eVolatile aroma compounds, fermentation metabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eVillarroel-Bastidas et al. (2025)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCacao mucilage to produce craft beers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eMixed culture / spontaneous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch vessel (craft brewery scale)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa mucilage as adjunct\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eEthanol, fermentation kinetics, beer sensory quality\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eMeersman et al. (2016)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eThermotolerant S. cerevisiae starters for cocoa pulp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eEngineered S. cerevisiae (thermotolerant)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp medium (lab-scale)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eAcetate ester production, volatile profiles, flavour\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003ePuerari et al. (2012)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCocoa pulp-based kefir beverages\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eKefir grains (multi-kingdom immobilised)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch semi-closed (varied temperature)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp juice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eMicrobial community, organic acids, sensory acceptance\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eKoelher et al. (2022)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eS. cerevisiae for fruit wines using cocoa honey + pulp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eS. cerevisiae strains (defined)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch cocoa honey/pulp wine fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa honey + cocoa pulp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eEthanol, fermentation kinetics, sensory quality\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eGuirlanda et al. (2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCocoa honey: agro-industrial waste or by-product? (Review)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eReview (multiple starter types)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eReview of multiple systems\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eReview\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eFermentation products, composition, valorisation options\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eMeersman et al. (2015)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eBreeding robust yeast starters for cocoa pulp fermentations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eEngineered S. cerevisiae (hybrid starters)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp fermentation media\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eFermentation performance, flavour compound formation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eDzogbefia et al. (1999)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eControlled cocoa fermentation with yeasts: cocoa sweatings\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eDefined yeasts (controlled inoculation)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch sweatings/juice vessels\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa sweatings (pulp juice)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003ePhysicochemical changes, enzymatic activity, microbial counts\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eAdler et al. (2013)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCore fluxome of LAB under cocoa pulp fermentation simulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eLAB strains (Lactobacillus/Leuconostoc)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp simulation media (chemostat)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eArtificial pulp medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eMetabolic fluxes, organic acids, CO₂ production\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eKlis et al. (2023)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCocoa pulp fermentation by Laetiporus persicinus beverage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eLaetiporus persicinus (fungal, non-conventional)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eSubmerged fermentation (diluted pasteurised pulp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eDiluted, pasteurised cocoa pulp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eAroma volatiles, sensory evaluation, non-alcoholic beverage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eAyala et al. (2022)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eValorization of cocoa mucilage waste to ethanol/ethylene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eSpontaneous/mixed culture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch bioreactor (mucilage waste)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eRaw cocoa mucilage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eEthanol yield, fermentation kinetics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eGarc\u0026iacute;a-R\u0026iacute;os et al. (2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eThermo-adaptive S. cerevisiae for cocoa pulp fermentations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eThermo-adaptive S. cerevisiae strains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp fermentation (lab-scale)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eGrowth kinetics, fermentation performance, metabolites\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eChetschik et al. (2018)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eAroma of cocoa pulp and influence on fermented cocoa beans\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eN/A \u0026mdash; substrate characterisation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp aroma in fermentation context\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eFresh cocoa pulp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eAroma-active compounds; influence on fermentation volatiles\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eRodr\u0026iacute;guez-Castro et al. (2024)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCocoa mucilage as novel ingredient in kombucha fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eKombucha SCOBY (multi-kingdom immobilised)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch closed vessel (cocoa mucilage)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa mucilage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003ePhysicochemical properties, microbial composition, sensory\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eAdler et al. (2014)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eMetabolic fluxes of AAB under cocoa pulp simulation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eDefined AAB strains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp simulation media (bioreactor)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eArtificial pulp medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eAcetate metabolic fluxes, key metabolite pathways\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eGuimar\u0026atilde;es et al. (2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCocoa pulp as matrix for probiotic delivery\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eProbiotic LAB strains (Lactobacillus spp.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp matrix (delivery system)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp as food matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eProbiotic viability, pH, fermentation parameters\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eMota-Gutierrez et al. (2021)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eMicrobial communities on fermented cocoa pulp-bean mass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eSpontaneous community (pulp-bean mass)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eTraditional heap/batch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp-bean mass\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eMicrobial community profiling, metabolites, quality parameters\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eLefeber et al. (2011)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eStarter strains via cocoa pulp simulation fermentations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa-specific LAB strains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp simulation media\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eArtificial pulp medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eLAB fermentation performance, metabolite profiles, strain selection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eChang et al. (2025)* [Food Chem: X]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eAromatic compounds in cocoa pulp fermentation \u0026mdash; volatilomics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eDefined yeast starters + LAB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp batch fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eVolatile/aroma compounds, machine learning metabolomics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eVizcaino-Almeida et al. (2022)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eNon-conventional fermentation: probiotic microorganisms + mucilage substitution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eProbiotic strains + conventional yeasts\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eModified batch (mucilage/fruit pulp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa mucilage (and substitutes)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eFermentation outcomes, metabolites, sensory evaluation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eNunes et al. (2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCocoa pulp as adjunct for beer production\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eS. cerevisiae (conventional)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch beer fermentation with cocoa pulp adjunct\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eDepectinised cocoa pulp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eBeer quality, fermentation kinetics, composition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eBastidas et al. (2023)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eCocoa mucilage: novel substrate for fermented tea-based beverages\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eTea SCOBY / kombucha culture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch closed vessel (cocoa mucilage)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa mucilage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eFermentation parameters, metabolites, sensory evaluation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eAlvarado-Santos et al. (2023)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eKinetic model for cocoa waste fermentation to ethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eSpontaneous/mixed culture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch bioreactor (cocoa waste pulp)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp waste\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eEthanol kinetics, mathematical model validation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eDias et al. (2007)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eElaboration of a fruit wine from cocoa pulp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eS. cerevisiae (conventional)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eBatch wine fermentation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp juice\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eEthanol, physicochemical parameters, sensory quality\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 4.32%;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.68%;\"\u003e\n \u003cp\u003eHo VTT, Zhao J, Fleet G. (2015)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17.76%;\"\u003e\n \u003cp\u003eEffect of lactic acid bacteria on cocoa bean fermentation \u0026mdash; LAB roles in acidification, competitive exclusion, and flavour\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eLAB strains (Lactobacillus, Leuconostoc)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.92%;\"\u003e\n \u003cp\u003eCocoa pulp simulation + bean fermentation (lab-scale)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.72%;\"\u003e\n \u003cp\u003eCocoa pulp/bean matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 27.68%;\"\u003e\n \u003cp\u003eLAB metabolic contributions, acidification kinetics, flavour impact\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations: LAB = lactic acid bacteria; AAB = acetic acid bacteria; SCOBY = symbiotic culture of bacteria and yeast. * Identified through hand search of reference lists.\u003c/em\u003e\u003c/p\u003e"},{"header":"7. Quorum Sensing as a Coordination Layer in Cocoa Pulp Juice ","content":"\u003cp\u003eFermentation\u003c/p\u003e\n\u003cp\u003eThe concept of biointelligent starter design rests on the hypothesis that microbial communication mechanisms\u0026mdash;particularly quorum sensing (QS)\u0026mdash;can be leveraged to coordinate community behaviour, synchronize metabolic transitions, and improve fermentation stability in complex, variable substrates such as cocoa pulp juice.\u003csup\u003e11,29\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e7.1 The AI-2/LuxS system in food-relevant LAB\u003c/p\u003e\n\u003cp\u003eThe most extensively characterized inter-species QS signal in food fermentation contexts is autoinducer-2 (AI-2), produced via the LuxS enzyme. AI-2 has been shown to regulate biofilm formation in \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e GG, with LuxS-deficient mutants showing substantially reduced biofilm and community stability,\u003csup\u003e13\u003c/sup\u003e and to modulate stress tolerance and adhesion capacity in \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e KLDS1.0391.\u003csup\u003e12\u003c/sup\u003e Johansen and Jespersen\u003csup\u003e29\u003c/sup\u003e provide a broader synthesis of QS effects across food fermentation ecosystems, documenting that AI-2 and acyl-homoserine lactone (AHL) signals can influence metabolite timing, succession dynamics, and competitive exclusion in LAB-dominated communities.\u003c/p\u003e\n\u003cp\u003e7.2 In silico evidence for QS in cocoa fermentation systems\u003c/p\u003e\n\u003cp\u003eAlmeida et al.\u003csup\u003e11\u003c/sup\u003e conducted an in silico investigation of QS potential across microbial communities involved in spontaneous cocoa bean fermentation, identifying LuxS/AI-2 pathway activity in several key fermentation-associated LAB species. This study provides the primary mechanistic rationale for hypothesizing QS-linked coordination in cocoa-associated fermentations, but it is important to note that this evidence is computational rather than experimental\u0026mdash;the in silico results indicate QS pathway presence and potential activity, not functional QS-driven fermentation control.\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e7.3 QS activity in benchmark food fermentation systems\u003c/p\u003e\n\u003cp\u003eDirect experimental evidence for functional AI-2 activity in food fermentations comes principally from kimchi. Park et al.\u003csup\u003e34\u003c/sup\u003e demonstrated that kimchi fermentation generates measurable AI-2 quorum-signalling activity associated with its LAB microbiota, with community-level AI-2 dynamics reflecting inter-species interactions among \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eWeissella\u003c/em\u003e, and \u003cem\u003eLeuconostoc\u003c/em\u003e taxa. This community-level QS activity is proposed to influence succession timing and competitive dynamics in the fermentation, supporting a role for inter-species communication in ecosystem stabilization.\u003csup\u003e34\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e7.4 Critical constraints on QS application in cocoa pulp juice\u003c/p\u003e\n\u003cp\u003eSeveral constraints limit the straightforward translation of QS-based coordination strategies to cocoa pulp juice fermentation. First, AI-2 signals are known to be pH-sensitive, with signal stability decreasing substantially under acidic conditions. Given that cocoa pulp juice fermentation typically acidifies from pH ~4.5\u0026ndash;5.0 to pH ~3.0\u0026ndash;3.5 over the fermentation course, the functional window for AI-2-mediated coordination may be narrow and largely confined to early fermentation phases.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSecond, the majority of QS mechanistic evidence comes from controlled single- or dual-species systems, simplified media, or in silico analyses. The functional relevance of QS in complex, multi-species, substrate-variable environments\u0026mdash;such as undiluted cocoa pulp juice with high indigenous microbial loads\u0026mdash;has not been validated. Extrapolating from controlled QS studies to real fermentation ecosystems requires substantial caution and targeted experimental work.\u003csup\u003e11,29\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThird, QS signal dynamics can be influenced by matrix components including polyphenols, which may quench or interfere with signalling molecules.\u003csup\u003e10\u003c/sup\u003e This interaction is potentially significant in cocoa pulp juice, where polyphenol concentrations are substantial.\u003c/p\u003e\n\u003cp\u003eFor these reasons, this review positions QS as a mechanistically plausible research target for improving coordination in modular starter systems, rather than as a validated feature of current cocoa pulp juice fermentation practice.\u003c/p\u003e"},{"header":"8. A Modular Biointelligent Blueprint for Cocoa Pulp Juice Fermentation","content":"\u003cp\u003eDrawing on the evidence synthesized in Sections 3\u0026ndash;7, a modular biointelligent starter culture framework for cocoa pulp juice fermentation can be conceptualized as a four-layer architecture designed for cooperative function under substrate stress (see Fig. 2 and Table 2).\u003c/p\u003e\n\u003ch2\u003e8.1 Layer 1 \u0026mdash; Anchor/Chassis module (early phase)\u003c/h2\u003e\n\u003cp\u003eThe chassis module drives rapid sugar conversion in the early phase of fermentation, establishes ecological dominance, and creates a predictable metabolic foundation for subsequent module transitions. Candidate chassis organisms are osmotolerant, thermotolerant yeasts with documented performance in cocoa pulp or pulp-mimicking media.\u003csup\u003e21,22,30\u003c/sup\u003e Selection criteria include: high fermentative capacity under osmotic stress; tolerance of polyphenol exposure; competitive dominance over undesirable indigenous taxa; and compatibility with downstream LAB colonization.\u003csup\u003e9,26\u003c/sup\u003e \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, \u003cem\u003ePichia kudriavzevii\u003c/em\u003e, \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e, and \u003cem\u003eTorulaspora delbrueckii\u003c/em\u003e are the most documented candidates in cocoa pulp contexts.\u003csup\u003e21,22,30\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003e8.2 Layer 2 \u0026mdash; Stability/Bioprotection module (mid phase)\u003c/h2\u003e\n\u003cp\u003eThe stability module controls acidification trajectory, suppresses spoilage organisms, and maintains process predictability through the mid-to-late fermentation phase. LAB are the primary members of this module, selected for: acid production kinetics compatible with the target product; polyphenol tolerance or detoxification capacity; competitive exclusion of undesirable taxa; and, where applicable, compatibility with inter-species communication dynamics.\u003csup\u003e12,13,14,32\u003c/sup\u003e Tannase-producing LAB strains\u0026mdash;capable of enzymatic polyphenol degradation\u0026mdash;are particularly valuable because they simultaneously contribute to detoxification and competitive stabilization.\u003csup\u003e10\u003c/sup\u003e The stability module should be designed to be communication-compatible with the chassis module to prevent antagonistic interactions during the module transition window.\u003csup\u003e29,34\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003e8.3 Layer 3 \u0026mdash; Optional oxidation/redox module (conditional late phase)\u003c/h2\u003e\n\u003cp\u003eAn oxidation module composed of AAB can be incorporated when the product intent requires targeted acetate generation (e.g., vinegar-type ingredients or certain functional beverages). This module should be activated deliberately through oxygen management rather than allowed to develop adventitiously, as uncontrolled AAB activity represents a common failure mode in pulp juice fermentations.\u003csup\u003e7,25\u003c/sup\u003e When a low-acidity or non-alcoholic beverage is the product target, this module should be excluded and oxygen minimized to prevent AAB colonization.\u003c/p\u003e\n\u003ch2\u003e8.4 Layer 4 \u0026mdash; Stress-guard mechanisms (cross-cutting)\u003c/h2\u003e\n\u003cp\u003eStress-guard mechanisms are not a separate organism group but a set of design features that improve consortium resilience across all phases. These include: functional redundancy (multiple strains with overlapping roles to buffer against individual strain failure); immobilization strategies (kefir grain analogs or carrier-based delivery to improve robustness and reuse potential); thermotolerance screening (selecting strains with demonstrated activity across the expected temperature range of 25\u0026ndash;40\u0026deg;C); and preparation methods that extend viability under field storage conditions without refrigeration.\u003csup\u003e5,26\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003e8.5 Inoculum strategy as a modular design variable\u003c/h2\u003e\n\u003cp\u003eConsistent with the sourdough process classification framework,\u003csup\u003e35\u003c/sup\u003e the inoculum regime should be treated as a deliberate design choice matched to the deployment context. In controlled production facilities, a defined multi-module starter can be applied as a single inoculation event. In decentralized smallholder settings, a hybrid approach\u0026mdash;seed inoculation followed by controlled backslopping using a fraction of the previous batch\u0026mdash;may offer better practical sustainability, at the cost of some precision in community composition over successive batches. The choice between these strategies represents a reproducibility\u0026ndash;deployability trade-off that must be explicitly addressed in modular starter system design.\u003csup\u003e31\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFig. 2 | Modular biointelligent starter culture framework for cocoa pulp juice (mucilage) fermentation. Panel A: substrate stress landscape (osmotic pressure from high sugars 10\u0026ndash;15% w/v, polyphenol inhibition 6\u0026ndash;8% w/v, and oxygen oscillations). Panel B: three functional modules \u0026mdash; Module 1 anchor/chassis (yeast-dominant, early phase), Module 2 stability/bioprotection (LAB-centred, mid phase), and Module 3 optional redox/oxidation (AAB, conditional late phase). Panel C: cross-cutting stress-guard layer. Panel D: biointelligent coordination layer (LuxS/AI-2 quorum sensing; mechanistically plausible, but validation in acidic pulp juice conditions required). Panel E: minimal viable monitoring and deployment variables. LAB = lactic acid bacteria; AAB = acetic acid bacteria; QS = quorum sensing; AI-2 = autoinducer-2.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 | Summary of the modular biointelligent starter culture blueprint for cocoa pulp juice fermentation. Each layer is described by its representative organisms, activation phase, key functions, and principal design requirements.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eModule / Layer\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eRepresentative Organisms\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKey Functions\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDesign Requirements\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLayer 1 \u0026mdash; Anchor/Chassis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSaccharomyces cerevisiae, Pichia kudriavzevii, Kluyveromyces marxianus, Torulaspora delbrueckii\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEarly (0\u0026ndash;48 h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRapid sugar conversion; ecological dominance; aroma-active metabolite formation; suppression of spoilage taxa\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOsmotolerance; thermotolerance (25\u0026ndash;40\u0026deg;C); polyphenol tolerance; competitive dominance; LAB-compatibility\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLayer 2 \u0026mdash; Stability/Bioprotection\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLactiplantibacillus plantarum, Leuconostoc mesenteroides, Lactobacillus fermentum, tannase-producing LAB strains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMid (48\u0026ndash;96 h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eControlled acidification; spoilage suppression; polyphenol detoxification; community stabilization via competitive exclusion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAcid production kinetics; polyphenol/tannase activity; inter-species QS compatibility (AI-2/LuxS); safety-relevant competitive exclusion; LAB role in flavour trajectory (Ho et al.32)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLayer 3 \u0026mdash; Oxidation/Redox (optional)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAcetobacter spp., Gluconobacter spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLate (\u0026gt;96 h, oxygen-dependent)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTargeted ethanol-to-acetate conversion; acidification for ingredient applications\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eActivated deliberately via oxygen management only; excluded for wine/kefir beverage targets to prevent volatile acidity off-notes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLayer 4 \u0026mdash; Stress-Guard (cross-cutting)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFunctional redundancy across all layers; immobilised consortia; carrier-based delivery systems\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAll phases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eResilience buffering; robustness under field temperature variability; extended inoculum shelf-life without cold chain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFunctional redundancy per module; immobilization strategy (kefir analog/carrier); thermotolerance screening; cold-chain-independent viability methods\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCoordination Layer (QS)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLuxS/AI-2-competent LAB strains (plausible but unvalidated in cocoa pulp juice context)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEarly\u0026ndash;mid transition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSynchronized metabolic transitions; succession timing; inter-species competitive signalling\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eValidation required under acidic pH trajectories (pH 4.5 to 3.0); validation in presence of cocoa polyphenols; experimental confirmation of functional AI-2 half-life in pulp juice\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations: LAB = lactic acid bacteria; AAB = acetic acid bacteria; QS = quorum sensing; AI-2 = autoinducer-2; LuxS = AI-2 synthase enzyme.\u003c/em\u003e\u003c/p\u003e"},{"header":"9. Validation Gaps Limiting Real-World Deployment","content":"\u003cp\u003eDespite the mechanistic plausibility and proof-of-concept evidence supporting modular starter design for cocoa pulp juice fermentation, a series of validation gaps must be addressed before these systems can be deployed reliably at scale.\u003c/p\u003e\n\u003ch2\u003e9.1 Comparative benchmarking under matched conditions\u003c/h2\u003e\n\u003cp\u003eThe most immediate gap in the existing literature is the absence of head-to-head comparison of different starter architectures (yeast-only, yeast+LAB, multi-kingdom/kefir-based, non-conventional fungal) under identical substrate, temperature, and oxygen conditions. Without such comparisons, it is not possible to make evidence-based recommendations about which starter configuration provides superior outcomes for a given product target.\u003csup\u003e4,5,6,21\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003e9.2 QS validation under acidic pulp conditions\u003c/h2\u003e\n\u003cp\u003eThe QS evidence base for cocoa-associated fermentations remains largely in silico\u003csup\u003e11\u003c/sup\u003e or derived from non-cocoa food systems.\u003csup\u003e29,34\u003c/sup\u003e Experimental validation of AI-2-mediated coordination in cocoa pulp juice—under realistic pH trajectories, temperature variation, and mixed indigenous microbial loads—is absent from the current literature. Specifically, the functional half-life of AI-2 signals along the acidification curve of cocoa pulp juice fermentation needs to be established, and the effect of pulp polyphenols on signal transmission and reception requires investigation.\u003c/p\u003e\n\u003ch2\u003e9.3 Tropical field-mimicking validation\u003c/h2\u003e\n\u003cp\u003eMost eligible studies were conducted under controlled laboratory conditions, often with diluted or pasteurized substrates, at temperatures that may not reflect tropical production environments.\u003csup\u003e9\u003c/sup\u003e Validation under field-mimicking conditions—including undiluted pulp juice, variable indigenous microbial loads, temperature fluctuations (25–40°C), and realistic hygiene constraints—is needed to determine whether laboratory-demonstrated starter performance translates to near-farm deployment.\u003csup\u003e9,28\u003c/sup\u003e\u003c/p\u003e\n\u003ch2\u003e9.4 Safety, stability, and reporting standardization\u003c/h2\u003e\n\u003cp\u003eAcross eligible studies, safety indicators (e.g., Enterobacteriaceae enumeration, mycotoxin screening) and shelf-life stability endpoints are rarely reported. For beverage applications, these endpoints are not optional—they are foundational requirements for any regulatory pathway. Standardized minimum reporting sets that include core metabolite profiles, microbial community endpoints, safety indicators, and sensory acceptance would substantially improve comparability across studies and accelerate the field toward evidence-based standardization.\u003csup\u003e8,9\u003c/sup\u003e\u003c/p\u003e"},{"header":"10. Conclusion","content":"\u003cp\u003eCocoa pulp juice (mucilage) is a credible, scalable substrate for applied fermentation, with demonstrated proof-of-concept across product classes including kefir-style functional beverages, wine-style alcoholic fermentations, and aroma-forward non-alcoholic prototypes.\u003csup\u003e2,4,5,6\u003c/sup\u003e However, the evidence mapped in this review shows that individual feasibility demonstrations have not yet translated into standardized, reproducible fermentation practice. The core barrier is that the cocoa pulp juice matrix imposes multi-dimensional stressors—osmotic pressure, polyphenol inhibition, oxygen instability, and temperature variability—that single-strain or single-trait starters cannot reliably navigate.\u003csup\u003e7,10\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe central implication of this scoping review is that cocoa pulp juice valorisation will be standardized most effectively through modular ecosystem design rather than single-strain optimization. The benchmark fermentation evidence from kimchi, sourdough, and tempeh collectively supports three convergent principles: anchor the early phase with a dominant stabilizer; build an active stability layer with community communication compatibility; and treat inoculum strategy as a deliberate design variable matched to the deployment context.\u003csup\u003e14,15,17,34,35\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eBiointelligent coordination via QS—particularly AI-2/LuxS signalling—represents a mechanistically plausible research target for improving succession stability and competitive exclusion in modular consortia, supported by in silico evidence from cocoa fermentation systems\u003csup\u003e11\u003c/sup\u003e and experimental evidence from food-relevant benchmark fermentations.\u003csup\u003e29,34\u003c/sup\u003e However, QS-mediated coordination cannot be considered a validated feature of cocoa pulp juice fermentation until experimental studies demonstrate functional signal activity under acidic pulp conditions, in the presence of polyphenols, and in the context of mixed indigenous microbial communities.\u003c/p\u003e\n\u003cp\u003eTranslation into smallholder-compatible deployment will require substrate stabilization strategies to reduce indigenous load variability;\u003csup\u003e37\u003c/sup\u003e affordable monitoring technologies for core state variables (pH, temperature, °Brix);\u003csup\u003e36\u003c/sup\u003e inoculum preparation methods that maintain viability without cold chain dependence;\u003csup\u003e26\u003c/sup\u003e and field-validated protocols that account for tropical variability and decentralized hygiene constraints.\u003csup\u003e9,28,31\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eThe field is at an inflection point. The substrate is proven. The starter framework is conceptually mature. The benchmark analogues provide transferable design principles. What is now needed are comparative, field-realistic experimental studies that benchmark modular starter architectures against matched controls, with standardized reporting of microbial, metabolite, safety, and sensory endpoints.\u003c/p\u003e\n\u003ch1\u003eFuture Perspectives\u003c/h1\u003e\n\u003cp\u003e\u003cstrong\u003eFrom demonstrations to benchmarks:\u0026nbsp;\u003c/strong\u003estandardization requires head-to-head comparisons of yeast-only, yeast+LAB, and multi-kingdom modular starters under identical cocoa pulp juice conditions, including matched substrate preparation, temperature, and oxygen management.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQS under real acidity:\u0026nbsp;\u003c/strong\u003eAI-2/LuxS performance must be experimentally tested along pulp-relevant pH trajectories and in mixed consortia to determine whether QS improves succession stability without unintended antagonism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmobilization as an enabling technology:\u0026nbsp;\u003c/strong\u003estructured consortia analogous to kefir grains or carrier-based systems may offer the most deployable route for smallholder adoption by improving reuse, robustness, and dosing simplicity without requiring refrigerated culture maintenance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMinimal viable monitoring:\u0026nbsp;\u003c/strong\u003etranslation into smallholder settings will depend on low-cost measurement of temperature, pH, and °Brix as operational state variables linked to starter-module transitions, supported by affordable sensor platforms.\u003csup\u003e36\u003c/sup\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAAB — Acetic Acid Bacteria; AHL — Acyl-Homoserine Lactone; AI-2 — Autoinducer-2; LAB — Lactic Acid Bacteria; PRISMA-ScR — Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews; QS — Quorum Sensing; SCOBY — Symbiotic Culture of Bacteria and Yeast.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eNot applicable. This is a scoping review article; no primary data are presented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions:\u0026nbsp;\u003c/strong\u003eAnthony Oppong Kyekyeku: Conceptualization, Methodology, Writing — original draft, Writing — review and editing, Visualization. Margaret Owusu: Supervision, Writing — review and editing. John Edem Kongor: Supervision, Writing — review and editing. Daniel Sitsofe Yabani: Writing — review and editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors acknowledge all who provided guidance and support during the development of the research programme underpinning this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUse of AI tools:\u0026nbsp;\u003c/strong\u003eThe authors used Anthropic's Claude AI assistant to support language polishing, structural review, and reference formatting during manuscript preparation. All AI-assisted outputs were critically reviewed, edited, and verified by the authors, who take full responsibility for the content and conclusions of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSoares TF, Oliveira MBPP (2022) Cocoa by-products: characterization of bioactive compounds and beneficial health effects. 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Future Foods 9:100330. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fufo.2024.100330\u003c/span\u003e\u003cspan address=\"10.1016/j.fufo.2024.100330\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"CSIR College of Science and Technology","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cocoa pulp juice, cocoa mucilage, starter culture, modular consortia, quorum sensing, AI-2, applied fermentation, scoping review","lastPublishedDoi":"10.21203/rs.3.rs-9587715/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9587715/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCocoa pulp juice (mucilage)—the fermentable liquid fraction released from the pulp surrounding Theobroma cacao beans—is an abundant yet underutilized substrate for applied fermentation. Unlike cocoa bean fermentation targeted at chocolate flavour development, cocoa pulp juice valorisation is substrate-centric and requires tailored microbial and process controls to deliver consistent beverage-quality outcomes. We conducted a scoping review to map the state of modular starter culture design and fermentation systems applied to cocoa pulp juice, with emphasis on microbial consortia, process configuration, reported metabolites, volatiles, microbial dynamics, and sensory outcomes. Eligible work was charted by starter architecture (yeast-dominated, multi-kingdom/immobilised, LAB-inclusive, non-conventional platforms), fermentation system (low-tech batch to controlled bioreactors), and outcome measures (ethanol, organic acids, aroma compounds, community composition, sensory acceptance). Evidence indicates that cocoa pulp juice supports diverse fermentation products, including wine-style beverages, kefir-like functional drinks, and aroma-forward non-alcoholic prototypes. However, comparative benchmarking of starter architectures under matched substrate conditions remains limited, and mechanistic links between microbial community transitions, quorum sensing (QS) regulation, and metabolite trajectories are insufficiently reported. QS-mediated regulation—particularly luxS/AI-2 signalling—is mechanistically plausible as a coordination layer in mixed fermentations, but its effectiveness in acidic cocoa pulp juice environments remains unvalidated. Key research priorities include comparative benchmarking of modular starter architectures, experimental validation of QS-mediated coordination in acidic pulp environments, and development of smallholder-accessible deployment and monitoring frameworks.\u003c/p\u003e","manuscriptTitle":"Modular Biointelligent Starter Cultures for Cocoa Pulp Juice (Mucilage) Fermentation: A Scoping Review","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 17:34:15","doi":"10.21203/rs.3.rs-9587715/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"997a5100-db4f-4edf-806d-1af740a02bf7","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":67382384,"name":"Food Science \u0026 Technology"},{"id":67382385,"name":"Applied \u0026 Industrial Microbiology"},{"id":67382386,"name":"General Microbiology"},{"id":67382387,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2026-05-04T17:34:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 17:34:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9587715","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9587715","identity":"rs-9587715","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}