Effect of substrates on yield and physicochemical properties of Nata de Pina: Valorization strategy for low-grade fruits of pineapple (Ananas comosus (L.) Merr.) var. Mauritius

Introduction

Pineapple, belonging to bromeliaceae family is one of the widely cultivated fruit crops in tropics and sub tropics, sought out for its nutritional richness and excellent quality juice (Ancos et al., 2016) [1] . Pineapple fruits are generally consumed as such or processed into canned pineapple slices, wine, juice, jams, and other confectionary items further highlighting its market presence. Furthermore, worldwide production and cultivated area of pineapple fruit have shown an upward trend from 1994 to 2023, emphasizing its demand in food and beverage industries (FAO, 2024) [2] . Therefore, an escalation in waste generation is inexorable owing to the growing public health cognizance and elevated production levels. The non-edible parts such as crown, core, leaves, stems, and peels constituting about 80% of pineapple fruits are eliminated as waste during processing, transportation, and storage whereas waste generated after processing consisting of crown, peels, core, and pomace account for more than 60 % of the total fruit weight (Zainal Alam et al., 2020; Sarangi et al., 2022) [4,3] . Fruits that fail to meet established grading standards such as minimum total soluble solids content, uniformity in size and shape, and absence of defects etc. are often excluded from both international and domestic markets as well as processing chains, leading to significant underutilization and resource wastage in horticulture sector. Inadequate handling and disposal of these rich organic wastes lead to their decomposition, emitting potent greenhouse gases exacerbating climate change, and contributing to eutrophication of waterbodies endangering aquatic ecosystems, thus impacting the overall quality of environment (Rabiu et al., 2018; Sial et al., 2024) [5,6] . Multiple studies conducted on characterization of pineapple waste revealed higher quantities of sugars, carbohydrates, fibers, organic acids, and phenolic compounds making them valuable substrates for valorization processes (Khedkar et al., 2017; Zain et al., 2021) [7,8] . Leveraging this rich nutrient composition in processing residue, fermentation serves as an efficient and versatile pathway for valorization. Nata de pina or bacterial cellulose is a remarkable biopolymer and an acclaimed dessert synthesized and secreted by Acetobacter xylinum grown in a medium containing pineapple juice and necessary nutrients (Almeida et al., 2013) [9] . It exhibits distinct physico-chemical characteristics that render it a versatile ingredient for incorporation into a diverse array of food items, such as yogurt, salads, and pastries. Bacterial cellulose posses' unique properties including high purity, crystallinity and mechanical strength which extend its utility beyond desserts. Wang et al., (2017) [10] elucidated that the wide range of physical and mechanical attributes of bacterial cellulose makes it applicable across various fields such as tissue engineering, biomedicine, nano fluidics, wearable technology, functional foods, cosmetics, and bio composite materials. While the general principles of pineapple waste valorization and Nata de pina production are established, there remains a discernible gap in specific research focusing on the detailed utilization of off-grade fruits of pineapple variety Mauritius' for nata de pina synthesis and the comprehensive characterization of its physicochemical properties and yield. A similar study has been conducted by Rojsuntornkitti et al., (2020) [11] on the effect of various concentrations of ammonium sulphate on characteristics of nata produced form pineapple cultivar Pattavia. Beyond that, studies have been focused on effect of other variables such as fermentation time, nitrogen, and carbon source concentration etc. (Mulawi & Kristina, 2019; Syukri et al., 2025) [12,13] under different non-species-specific processing conditions (Sutanto, 2012; Khanagoudar & Narayanaswamy, 2016) [15,14] . Given that the changes in fermentation conditions will have significant effect on important nata characteristics, it becomes relevant to explore the effect of varying substrate type and concentration of pineapple variety Mauritius. Mauritius is a mid-season cultivar, commonly used for direct table consumption and holds great significance, by being predominantly grown in parts of India, such as Meghalaya and Kerala where Asia's largest pineapple market is situated. In this regard, the authors had conducted a study on physico-chemical characterization of processing waste of Mauritius pineapple revealed higher lignocellulosic and sugar content, making it ideal for nata de pina production (Jose et al., 2024) [16] This research aims to bridge the scientific gap by investigating the effect of processing residue of pineapple variety Mauritius on the yield of nata de pina, evaluating the physicochemical properties (e.g., thickness, weight, pH, acidity, total soluble solids) of nata de pina produced. Research on nata production conditions has significant industrial implications, primarily in optimizing production for increased efficiency and product quality. Understanding factors like substrate, temperature, pH, and nutrient levels allows for tailored processes that boost yield, reduce costs, and enhance product characteristics like texture and shelf life of nata de pina.

Materials and Methods materials required for fermentation

Off-grade (due to size and quality non-compliance) pineapple fruits of variety Mauritius, which are rejected and is unsuitable for processing, export, or local market sale were collected from Asia's largest pineapple market, Vazhakkulam. Various chemicals of pure grade such as ammonium sulphate, glacial acetic acid and sucrose were purchased from Sisco research laboratories pvt. Ltd. Polypropylene bottles (7 cm x 5.3 cm) used for fermentation were purchased from local markets.

Elemental analysis of pineapple peels

Peels of pineapple variety Mauritius was analysed for its elemental composition to determine C/N ratio using CHNS analyzer. 0.1 g of powdered and dried pineapple peels were used for this analysis.

Preparation of bacterial inoculum and nata starter

Bacterial inoculum of Acetobacter xylinum culture (NCIM 2526) was procured from the National Collection of Industrial Microorganisms (NCIM), Pune, Maharashtra. Freeze dried culture (Figure 1) was revived by emptying the content into 2-5 ml of Acetobacter suboxydans media prepared using the ingredients (Figure 2) (Table 1). One or two loopful of culture was streaked into Acetobacter suboxydans agar media and incubated at 28 °C for five days (Figure 3). It was then subcultured to obtain pure cultures, which were transferred to tomato agar slants for maintenance (Jagannath et al., 2011) [17] . For nata starter preparation, Acetobacter xylinum maintained on tomato agar slants were inoculated into sterilized Hestrin and Shramm media (Hestrin et al., 1954) [18] . The inoculated media was incubated statically at 30 °C for 7 days.

preparation of substrates for Nata production

The experiment was conducted in Completely Randomized Design (CRD) with each treatment carried out in five replicates. Nata de pina production was performed as per the procedure described by (Jose et al., 2024) [16] . Fruits were decrowned, cleaned and washed thoroughly with potable water after sanitizing with 120 ppm sodium hypochlorite solution for 15 minutes. The fruits were peeled and cut into pieces; juice was extracted using a fresh juice extractor (Sujatha Powermatic juicer). The peel was processed in an electric blender (Panasonic Mixer-Grinder MX AV425) for uniform blend. The juice/peel extract was diluted with distilled water in 1:2 and 1:3 ratio and used as the substrate for nata production. This media was mixed with ammonium sulphate (0.5 %) and sucrose (10%). The pH was adjusted to 3.5 and sterilized. The sterilized substrate was inoculated with starter culture (40 % of the concentration) under aseptic condi-tions, covered with muslin cloth and incubated at ambient conditions for 14 days. The treatments included were S 1 -Pineapple juice diluted with water in 1:2 ratio, S 2 -Pineapple peel diluted with water in 1:2 ratio, S 3 -Pineapple juice diluted with water in 1:3 ratio and S 4 -Pineapple peel diluted with water in 1:3 ratio.

Harvest and purification of Nata de pina

Upon completion of the fermentation period, the Nata de pina layer was carefully harvested. The harvested nata sheets were made to undergo a rigorous purification process involving repeated washing with distilled water to remove residual fermentation medium, unreacted sugars, and acetic acid etc. This purification step is crucial for obtaining pure nata de pina suitable for further characterization and application.

Physicochemical characterization of nata de pina

Physico-chemical characteristics of nata de pina such as thickness, weight, T.S.S, pH, acidity, yield, and T.S.S of the fermentation medium were analyzed. Thickness of nata sheet was measured at four different positions using a vernier caliper. Values were averaged and expressed as mm. Weight of nata (g) was measured using a weighing balance after draining off the water from nata for 5 minutes. Yield of nata was measured in g/ml and expressed in percentage. The harvested nata was washed, cut into pieces, and crushed using mortar and pestle until a fine paste was obtained. Total soluble solids were recorded using ERMA handheld refractometer (0-32 °B) and expressed in degree brix (°B). The pH of the nata was measured using an ECPHTUTOR-S CyberScan pH Tutor Meter (U.S.). Acidity of nata was estimated by acid-base titration using a phenolphthalein indicator (AOAC, 1998) [19] .

Statistical analysis

The collected data were analyzed using analysis of variance (ANOVA), Group means were compared and grouped based on Duncan's Multiple Range Test (DMRT), where significant effects were found.

Results and Discussion

Thickness of nata de pina

The thickness of nata de pina exhibited distinct trends influenced by both substrate type and concentration, indicating the varied metabolic responses of Acetobacter xylinum to different fermentation conditions (Figure 56789). Notably, initial nata formation differed significantly across treatments. While no discernible growth was detected in S 1 , S 2 , and S 3 during the first seven days, nata formation commenced on the fourth day in S 4 , achieving a thickness of 5.65 mm. Earlier initiation of nata formation in S 4 suggests a convenient and optimally balanced nutrient profile in the peel extract of pineapple variety Mauritius as outlined by (Jose et al., 2024) [16] for the initial proliferation of Acetobacter xylinum compared to other treatments. This is further supported by our elemental analysis (CHNS) of the pineapple peels, which revealed a high carbon-to-nitrogen (C/N) ratio of 84.72. Such a high C/N ratio typically indicates abundant carbon sources relative to nitrogen, which can strategically favor the carbon-intensive process of cellulose biosynthesis over rapid microbial biomass accumulation during the initial lag phase (Sari et al., 2020) [20] further elucidated that Acetobacter xylinum exhibits a higher substrate utilization rate in media with lower glucose content, whereas the rate of cellulose formation is enhanced in the presence of higher glucose concentrations. This kinetic behavior aligns with our observations. Despite a slower initial growth phase, the substrates diluted with twice amount of distilled water (S 1 and S 2 ), characterized by a higher immediate availability of fermentable glucose, and ultimately supported the maximum observed nata de pina thickness (9.48 mm and 9.27 mm respectively). The final thickness measurements on day 21 showed negligible differences from day 14 values across all four treatments (p < 0.05) (8.33 mm and 8.23 mm in S 3 and S 4 , respectively), suggesting an optimum fermentation time of approximately 14 days under these experimental conditions (Table 3). This finding partially aligns with (Juwita et al., 2020) [21] , who reported an optimum fermentation time of 21 days for nata de pina produced from peels, highlighting that optimal fermentation duration can vary depending on specific processing parameters. Beyond this optimal period, factors such as nutrient depletion, accumulation of inhibitory metabolic byproducts, or autolysis of bacterial cells may limit further cellulose synthesis (Ye et al., 2019) [22] . Figure 4. Effect of substrates on increase in thickness of nata de pina during incubation period Moreover, it was observed that the texture of nata in S 4 was significantly harder than all other treatments. This increased rigidity in peelderived nata can primarily be attributed to the inherent chemical composition of pineapple peels, which possess a higher content of lignocellulosic fibers and other complex carbohydrates (Roha et al., 2013) [23] . These macromolecular components likely facilitate the formation of a denser and more intricately interconnected bacterial cellulose network during A. xylinum biosynthesis, leading to a more robust final texture. While some strains of Acetobacter xylinum, such as BPR2001, have been reported to produce cellulase enzymes that can decrease the degree of polymerization and consequently the mechanical strength of bacterial cellulose, particularly under certain pH conditions (Tahara et al., 1997) [24] , our observed increase in hardness in S 4 suggests that any such cellulase activity in our specific strain and conditions was either minimal, or its impact was significantly overshadowed by the enhanced network formation facilitated by the peel substrate's composition (Figure 4). Conversely, the comparatively looser texture observed in the diluted juice treatment (S 3 ) might also be influenced by factors like lower initial substrate concentration leading to a less dense cellulose network, or potentially a relative increase in the impact of any endogenous cellulase activity relative to cellulose synthesis in that specific diluted environment.

Figure 5. CHNS composition of pineapple peels of variety Mauritius

Furthermore, the fermentation medium utilizing pineapple peels (S 2 , S 4 ) exhibited a deeper yellow hue. This is consistent with the natural pigmentation inherent to pineapple peels, and while Acetobacter xylinum is known for urease activity that can produce ammonia and influence pH affecting the color (Syafiqah et al., 2020) [25] , the predominant factor contributing to this deeper yellow in the final product is likely the direct transfer and stability of these natural pigments.

Weight and yield of nata de pina

The highest fresh weight and yield were observed in S 2 , while the lowest was recorded in S 3 . This observation aligns with Khanagoudar & Narayanaswamy (2016) [14] who reported a decrease in the fresh weight of nata with increasing dilution of pineapple juice. Our findings collectively suggest that S 2 provided an optimal balance of fermentable sugars and other nutrients, promoting maximal bacterial cellulose synthesis. In contrast, while dilution generally reduces yield due to lowered fermentable sugar availability (as clearly seen in the very low yield of S 3 ), the yields in S 1 and S 4 appear to be sub-optimal compared to S 2 , but for different underlying reasons. S 1 , as a less diluted juice medium, may have encountered conditions beyond the optimal range for Acetobacter xylinum activity, potentially due to factors such as substrate saturation or an elevated rate of gluconic acid production. While these are not necessarily inhibitory to the point of complete cessation, it could shift the pH rapidly away from the ideal range for peak cellulose synthesis, as noted by Chen et al. (2011) [26] . The yield of S 4 , which utilizes pineapple peels at a certain concentration, while being superior to S 3 , falls short of S 2 . This could be attributed to the inherent characteristics of the peel substrate (e.g., initial total sugar concentration, the rate of complex carbohydrate hydrolysis, or the presence of non-sugar components) that, at this specific concentration, did not perfectly match the highly optimized conditions found in S 2 . Thus, the similar yields of S 1 and S 4 represent convergence at a sub-optimal level, driven by distinct limitations related to their specific substrate types and concentrations. In addition to that, no significant statistical differences were observed between the weight and yield of nata layers obtained from S 1 and S 4 (p < 0.05). A significant finding of our study is the comparatively higher yield obtained from pineapple peels (S 2 , S 4 ) compared to pineapple juice (S 1 , S 3 ), underscoring the efficiency and potential for valorization of pineapple peels as a substrate for nata production. These results are consistent with Yovita et al. (2020) [27] , who also reported superior yields of nata de pina from pineapple fruit peel (12.02 %) compared to other parts of the fruit, including the core and juice. It can also be seen that yield of nata de pina from honey variety of pineapple was less than that of Mauritius variety for the same dilution. The enhanced yield from pineapple peels can be attributed to their distinct carbohydrate profile and complex matrix. While pineapple juice predominantly contains simple sugars, peels are rich in both fermentable sugars and complex carbohydrates. Preliminary experiments by Embuscado et al. (1994) [28] highlighted that sucrose, a predominant sugar in both pineapple peel and core extracts yielded significantly higher bacterial cellulose (5.25 g/L) compared to glucose (1 g/L). This suggests that the specific sugar composition of pineapple peels, potentially with a more favorable sucrose-to-glucose ratio, could contribute to better bacterial fermentation. Moreover, the higher cellulose content inherently present in pineapple peels can undergo partial acid hydrolysis during the prolonged fermentation process, forming glucose (Ifeanyi et al., 2024) [29] , which then becomes available for bacterial utilization. This continuous release of fermentable sugars from complex carbohydrates provides a sustained energy source, potentially leading to higher overall cellulose yields. Our study achieved remarkably high yields, averaging 12.5 g per 50 mL of medium (equivalent to 250 g/L). This significantly surpasses previously reported yields for bacterial cellulose production, particularly when considering the nature of the primary substrate. Sari et al. (2021) [20] reported yields of 1.5-1.4 g/L from a coconut water medium that was modified and optimized to maintain a carbon-to-nitrogen (C/N) ratio of 5.39. In contrast, our primary carbon source, the raw pineapple peel, exhibited an inherent C/N ratio of 84.72. While the actual C/N ratio of our complete fermentation medium (containing added nutrients like ammonium sulphate and sucrose) would differ, the dramatically higher C/N ratio of the foundational peel substrate itself strongly indicates its substantial carbon richness. This inherent abundance of carbon, readily available for metabolism and cellulose synthesis as discussed in Section 3.1, provides a compelling explanation for the substantially higher cellulose production observed in our peel-based system, despite the differences in substrate preparation and medium optimization strategies compared to other studies. The reduction in yield observed in S 3 and S 4 compared to S 1 and S 2 , is consistent with the findings of Sasithorn (2008) [30] , who reported that while an initial increase in dilution (e.g., from 1:1 to 1:2) could significantly increase yield, further increases in dilution beyond an optimum point led to a reduction. This indicates that while pineapple peels are an efficient substrate, an optimal concentration of fermentable sugars and other essential nutrients is critical for maximizing bacterial cellulose production, and excessive dilution can still limit overall yield, even with the benefits of the peel matrix. The abundant and potentially more complex nutrient matrix in the fermentation medium of treatment S 4 likely contributed to the development of a faster and harder-textured bacterial cellulose layer, as indicated by initial growth observations.

T.S.S, pH and acidity of Nata de pina

The total soluble solids (TSS), pH, and titratable acidity of the nata de pina samples varied notably across the different treatments, reflecting the dynamic metabolic activity of Acetobacter xylinum during fermentation.

International Journal of Horticulture and Food Science https://www.hortijournal.com while the lowest levels were recorded in S 3 and S 1 . Notably, TSS of S 3 and S 1 were significantly lower than in S 2 (Table 2). Interestingly, treatments using pineapple peel as a substrate generally maintained higher TSS values than those using pineapple juice as substrates, which could be attributed to the inherent differences in the initial physicochemical composition of the substrates. Higher residual TSS of the fermentation medium values in certain treatments, particularly S 4 , might suggest that not all fermentable solids were fully utilized by the bacteria during fermentation. Conversely, fermentation media with the lowest final TSS value, such as S 2 , demonstrated the highest yield, indicating more efficient conversion of available soluble solids into bacterial cellulose. This inverse relationship underscores the importance of optimizing substrate concentration for maximal substrate utilization and yield. Although, the treatments with sub-optimal yields (S 1 and S 4 ) did not exhibit this inverse relationship. Total soluble solids (TSS) of nata made from pineapple juice consistently measured 10 across both dilution levels, with the fermentation medium TSS also remaining stable at 18. In contrast, pineapple peel exhibited a different response when nata TSS increased from 11 at 1:2 dilution to 14 at 1:3 dilution, while its fermentation medium TSS similarly rose from 16 to 20. Simply stated, while pineapple juice nata and medium TSS remained constant when dilution increased from 1:2 to 1:3, pineapple peel nata and fermentation medium TSS both rose with the higher dilution. The breakdown of juice and peel matrix which is possibly catalyzed by endogenous peel enzymes, microbial enzymes (if present), or the acidic environment created by gluconic acid production (Verschuren et al., 2000) [31] , would release new soluble sugars and oligosaccharides into the medium. Consequently, the final TSS does not solely reflect unutilized initial fermentable sugars but also comprises newly solubilized components, making a direct correlation between TSS re-duction and cellulose yield more intricate than with simple sugar media. pH: The pH levels of the nata de pina varied significantly across treatments (Table 2). The highest pH was recorded in S 1 (2.84), while the lowest was observed in S 3 (2.67). There were no significant statistical differences in pH between S 2 , S 3 , and S 4 (p > 0.05). A notable trend observed was a decrease in the pH of nata de pina with increasing dilution when pineapple juice was used as the substrate. This could be explained by the reduced buffering capacity of more diluted juice, allowing the gluconic acid produced by Acetobacter xylinum to cause a more pronounced drop in pH. Conversely, an increase or stabilization in pH was observed when pineapple peels were used as the substrate. This contrasting behavior is likely due to the variation in the composition of both substrates. Pineapple peels possess a higher concentration of lignocellulosic and fiber components (Roha et al., 2013) [23] , which may provide a greater buffering capacity against the acidification caused by gluconic acid production, or be metabolized differently, resulting in a less drastic pH reduction. Comparing our results to other studies, nata de pina produced from Pattavia pineapple juice, which had a pH of ≈ 3.8, resulted in a product pH as low as 1.5 (Rojsuntornkitti et al., 2020) [11] . This difference from our Mauritius pineapple juice treatments (final pH ≈ 2.7-2.8) suggests potential varietal distinctions. The Mauritius pineapple, with its higher initial pH (5.43) might possess a greater buffering capacity or a different sugar profile that influences the rate or extent of gluconic acid accumulation, leading to a slightly less extreme final pH, despite robust acid production. The consistent final pH values across all our treatments (juice vs. peel, different dilutions) in the range of 2.6-2.8, despite varying initial pH values and substrate types, highlights the strong acid-producing capacity of the Acetobacter xylinum strain used and its tendency to drive the medium to a specific acidic equilibrium (Vandamme et al., 1998) [32] . Although these final pH values are slightly below the gen erally reported optimum range for A. xylinum growth, the high cellulose yields observed in our study (especially S 2 ) suggest that the strain is highly tolerant to such acidic conditions, or that the critical period for optimal activity occurs before the pH drops to these extreme levels. Furthermore, this low pH environment is crucial for inhibiting the growth of most contaminating microorganisms, contributing to the safety and stability of the nata de pina product. Optimal conditions for nata production involve temperatures ranging from 25 °C to 30 °C and a slightly acidic pH, favoring bacterial proliferation. (Talawar et al., 2015) [33] Acidity: S 1 and S 2 demonstrated similar titrable acidity levels (1.66%), as S 3 and S 4 (0.89%) (Table 2). No significant differences were observed between treatments using pineapple juice and those using pineapple peel as the primary substrate (p > 0.05). This suggests that the dilution ratio, rather than the specific type of substrate (juice vs. peel), played a primary role in determining the final acidity levels of nata de pina. This is corroborated by the findings of Thankappan & Anitha (2018) [34] who reported a titrable acidity of ~1.2 for nata produced from medium where pineapple juice was used as an acidulant. The concentration of fermentable sugars that are converted to gluconic acid by Acetobacter xylinum appears to be the dominant factor influencing the final titrable acidity. This implies that while peelbased substrates offer benefits in terms of yield and texture, the acidic profile can be modulated primarily through control of initial substrate concentration.

Conclusion

This study comprehensively evaluated the impact of different pineapple juice and peel concentrations on the yield, and physicochemical properties of nata de pina. Our findings strongly advocate for the valorization of peels and juice of off-grade pineapple fruits which are rejected due to non-compliance of quality characteristics as a highly efficient and sustainable substrate for bacterial cellulose production. This is primarily attributed to the complex carbohydrate matrix of peels, including beneficial sucrose levels and the potential for acid hydrolysis of lignocellulosic components, providing a sustained and diverse carbon source for Acetobacter xylinum. The distinct physicochemical properties of variety Mauritius demonstrate the tunability of nata de pina by manipulating substrate type and concentration. This has critical implications for developing diversified nata products with specific attributes for various food applications. The observed pH and acidity profiles, strongly influenced by dilution rather than solely substrate type, provide valuable insights for process control and ensuring product safety and stability. Future research should focus on a more detailed characterization of the precise sugar profiles and other nutrient compositions of both pineapple juice and peel extracts throughout the fermentation process to elucidate the exact metabolic pathways contributing to the observed differences in yield and texture. Further optimization of peel processing (e.g., enzyme pretreatment, different extraction methods) could potentially enhance fermentable sugar availability and further improve yields. This study provides a robust scientific basis for the sustainable production of high-quality nata de pina from pineapple variety Mauritius, contributing significantly to food waste reduction and the development of novel functional food ingredients.

Acknowledgement

AcknowledgementsWe thank Kerala Agricultural University for giving the facility and financial support for conducting for this research work. Declaration of interest statementNo competing interest was reported by the author(s)

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