Bioprocessing of Jackfruit Seeds (Artocarpus heterophyllus Lam.) for Protein Enrichment in Semi-Solid State: Potential for Animal Feed Production

Abstract

Jackfruit residues represent 70% of the total by-products generated from the processing of the fruit. The seeds, which are composed of proteins, fibers, and starch, are widely used in human nutrition; however, its potential in animal nutrition should be further investigated. Thus, the objective was to study the protein enrichment of jackfruit seeds by semi-solid fermentation using Saccharomyces cerevisiae and applying an experimental design to verify the effects of yeast concentration (1, 3, and 5%) and process temperature (30, 35, and 40 °C) on the protein increase. Physical and chemical analysis of the substrate was performed at intervals of 0, 24, 48, 72, and 96 h. A decrease in water content and water activity was observed during the fermentation time. The total soluble solid content also declined due to the consumption of carbohydrates by yeast. After 96 h of the process, the crude protein content of the fermented substrate increased approximately 2.5 times, corresponding to a protein increase of 357%, with the use of 5% of yeast at 40 °C. Through semi-solid fermentation, the protein content and the concentration of mineral nutrients in the jackfruit seeds increased, making it an alternative product for animal feed with high added value.

1. Introduction

The search for new animal feed sources derived from agro-industrial waste rich in proteins and other nutrients is essential for making animal production more sustainable and efficient. The valorization of by-products such as discarded seeds, peels, and pulps reduces waste and minimizes the environmental impacts associated with improper disposal. Additionally, reusing these residues can decrease dependence on traditional ingredients such as soybean meal and corn, which have high costs and require large cultivation areas.

The global food industry faces the challenge of managing the high volume of waste generated throughout the supply chain. According to the Food and Agriculture Organization (FAO), approximately 1.3 billion tons of edible food are discarded annually, contributing to environmental pollution and the depletion of natural resources [1]. In agro-industrial fruit and vegetable processing, it is estimated that about 50% of waste is generated in the form of peels, seeds, and pulp, along with immature or damaged products [2,3]. Utilizing these residues in food and agro-industrial inputs can reduce costs and optimize resources, promoting a more sustainable and efficient production chain [4].

Jackfruit (Artocarpus heterophyllus Lam.), formerly known as the “poor man’s food”, has been recognized as a “superfood” due to its high nutritional value and pharmaceutical benefits [5]. Rich in functional compounds such as polysaccharides, flavonoids, sterols, and prenylated chromones, this fruit exhibits antibacterial, anti-inflammatory, antioxidant, antitumor, hypoglycemic, hypolipidemic, and immunomodulatory properties [6]. Its residual parts, mainly the peel, pericarp, and seeds, are often wasted despite their nutritional potential [7]. In some countries, mature jackfruit seeds are collected and sun-dried for further processing and storage; however, difficulties in this process lead to significant annual losses [8]. To extend the shelf life of jackfruit seeds and add value, they can be roasted and ground into flour for use in baking and confectionery due to their functional properties [9]. Additionally, fruit residues can be incorporated into animal feed, particularly in ruminant nutrition [10]. Jackfruit seeds are an excellent source of protein and carbohydrates and contain phytonutrients such as lignans, isoflavones, and saponins—compounds with antioxidant, anticancer, and anti-aging properties. Studies suggest that these compounds may help combat various health conditions, including cancer, hypertension, ulcers, nervous disorders, and asthma [11,12].

Semi-solid fermentation involves the growth of microorganisms on solid materials under controlled conditions, promoting changes in the chemical or physicochemical properties of the substrate. Recent research indicates that this process can enhance the nutritional properties of raw materials rich in plant protein [13]. This type of fermentation is widely used in industrial applications due to several advantages, including lower effluent generation, simple processing requirements with minimal technological support, reduced risks of environmental contamination, lower water consumption compared to submerged fermentation, and reduced capital and energy investment [14]. Given the scarcity of low-cost, high-nutritional-value products, the search for alternative protein sources has become essential to meet these demands. Among the microorganisms used in solid-state fermentation, Saccharomyces cerevisiae stands out as a promising candidate for protein production due to its safety profile, as it is generally considered free from allergenicity and toxicity concerns. Additionally, its proteins have a favorable nutritional profile, with high levels of essential amino acids [14,15]. The fermentation process using this yeast is particularly advantageous for animal feed applications, as it enhances the protein content of agro-industrial residues. In this context, utilizing fruit seeds in solid-state fermentation presents promising characteristics, offering a sustainable approach to converting fruit waste into nutritionally enriched feed ingredients.

Semi-solid fermentation, in turn, is a sustainable and low-cost alternative for adding value to agro-industrial waste, enabling its conversion into high-value-added products. Recent studies have demonstrated the feasibility of this approach for different applications. Kou et al. [16] produced butyric acid from the semi-solid fermentation of rapeseed straw using Clostridium beijerinckii, while Da Silva et al. [17] explored this method to obtain cellulose from passion fruit peels. Additionally, Khurshida et al. [18] found that inoculating Saccharomyces cerevisiae in cassava flour significantly improved the quality and texture of baked products. Compared to liquid fermentation, semi-solid fermentation consumes less water, requires less strict control of operational parameters, and enables better utilization of lignocellulosic substrates, making it a viable option for processes that use agro-industrial waste. Thus, semi-solid fermentation presents great technological and nutritional potential, being applicable to the use of jackfruit seeds as an animal food product with high protein content. This study aimed to investigate the protein enrichment of jackfruit residues through semi-solid fermentation using Saccharomyces cerevisiae. To achieve this, an experimental design associated with response surface methodology was employed to evaluate the effects of yeast concentration and fermentation temperature on protein increase.

2. Materials and Methods

2.1. Material

The experiments were carried out at the Laboratory of Storage and Processing of Agricultural Products situated at the Technology and Natural Resources Center and at the Food Biochemistry and Biotechnology Laboratory situated at the Education and Health Center, both at the Federal University of Campina Grande—PB, Brazil. The jackfruit seeds used were manually removed from fruits purchased at the Bujari farm, in the municipality of Cuité-PB. They were selected according to homogeneity in size and color intensity for further processing and crushing in an industrial blender at maximum speed, for a period of approximately 3 min, resulting in the substrate. In all experiments, 700 g of jackfruit seeds was used.

2.2. Fermentation Process

The fermentation process was carried out in a batch system, using rectangular plastic bioreactors, with dimensions of 10 × 27 × 9 cm, which were arranged in an air circulation oven, at temperatures of 30, 35, and 40 °C, defined according to the optimal temperature range for yeast growth [18], with a variation of +1 °C, for 96 h.

Pressed yeast S. cerevisiae, commercial biological yeast, with a water content of 70% (wet basis) and an average value of crude protein of 45% (dry basis), in concentrations corresponding to 1, 3, and 5% in relation to the substrate mass, was added to the substrate. The choice of these values was based on literature data referring to protein enrichment of several substrates. The choice of these values was based on the study developed by Sousa et al. [19].

Before, during, and after 96 h of fermentation, samples were collected to characterize the enriched residue for the analysis of water content, fixed mineral residue, total soluble solids, and crude protein, determined according to the methodology described by the Adolfo Lutz Institute [20]. To determine the water activity, the Aqualab 3TE equipment (Decagon Devices, Washington, DC, USA) was used. All analyses were performed in quadruplicate.

The calculation of the protein increase (PI) (%) of the jackfruit seeds was based on the protein value contained in the fresh substrate, being defined as the ratio between the difference in the protein value of the enriched substrate and the protein value of the fresh substrate, and the initial value of crude protein in natura (Equation (1)), as described by Sousa [19].

$$\mathrm{PI} \left(\%\right)={\displaystyle \frac{\left(\%\right) \mathrm{Crude} \mathrm{protein} \left(\mathrm{enriched}\right)-\left(\%\right) \mathrm{Crude} \mathrm{protein} \left(\mathrm{in} \mathrm{nature}\right) }{\left(\%\right) \mathrm{Crude} \mathrm{protein} \left(\mathrm{in} \mathrm{nature}\right)}}\times 100\%$$

(1)

2.3. Factorial Design

Factorial planning 3² plus one central point was applied to quantitatively evaluate the influence of the independent variables: the effect of the yeast concentration (%) and the fermentation temperature (°C) on the protein increase, as well as any possible interactions. The tests were performed in duplicate and in random order, to avoid systematic error, while varying the variables simultaneously. The matrix of factorial planning 3² is shown in

Table 1. Factorial planning matrix 3² +1.

Exp.	Yeast Concentration		Temperature
	Coded Value	Real Value (%)	Coded Value	Real Value (°C)
1	−1	1	−1	30
2	−1	1	0	35
3	−1	1	+1	40
4	0	3	−1	30
5	0	3	0	35
6	0	3	+1	40
7	+1	5	−1	30
8	+1	5	0	35
9	+1	5	+1	40
10	0	3	0	35

, along with the encodings and actual levels for each variable investigated (encodings: high level (+1), intermediate level (0), and low level (−1)).

2.4. Statistical Analysis

The mean data obtained were analyzed using Statistica software (StatSoft, Inc., Tulsa, OK, USA), which was used to generate and evaluate the factorial experimental design and regression analysis of the experimental data. Analysis of variance (ANOVA) was applied to estimate the significance of the model (p < 0.05) and individual response parameters.

3. Results and Discussions

3.1. Water Content and Water Activity

Table 2. Mean ± standard error values of water content (%) and water activity, at the initial timepoint and after 96 h of semi-solid fermentation of the jackfruit residues.

Experiments	Water Content (%)		Water Activity
	Initial	Final	Initial	Final
1	60.10 ± 0.40	42.34 ± 0.81	0.9883 ± 0.002	0.9149 ± 0.014
2	57.19 ± 0.25	33.11 ± 0.25	0.9883 ± 0.002	0.8110 ± 0.041
3	58.52 ± 0.25	39.72 ± 1.41	0.9881 ± 0.002	0.6429 ± 0.042
4	60.10 ± 0.40	43.33 ± 1.08	0.9883 ± 0.002	0.9023 ± 0.018
5	57.19 ± 0.25	33.70 ± 0.89	0.9881 ± 0.002	0.7606 ± 0.012
6	58.52 ± 0.25	33.51 ± 0.04	0.9858 ± 0.002	0.6352 ± 0.204
7	60.10 ± 0.40	44.22 ± 0.06	0.9883 ± 0.002	0.8975 ± 0.008
8	57.19 ± 0.25	30.76 ± 0.34	0.9883 ± 0.002	0.7553 ± 0.012
9	58.52 ± 0.25	32.19 ± 0.56	0.9881 ± 0.002	0.6071 ± 0.025
10	57.19 ± 0.25	32.08 ± 0.10	0.9881 ± 0.002	0.7202 ± 0.007

shows the results obtained regarding the water content and water activity at the initial and final timepoints, the latter being after 96 h of fermentation.

The differences in the reduction of water content and water activity between the experiments indicate that factors such as the type of substrate, the microbial strain used, and the conditions of the fermentation process directly influence moisture retention. In the experiments that presented higher final water activity values, such as 1, 4, and 7, water availability remained higher, possibly due to lower evaporation or greater water retention in the solid components. In the experiments with lower final water activity values, such as 6 and 9, moisture loss was more intense, which may be associated with greater water consumption by the microorganisms or the formation of compounds that alter the water retention capacity.

Water content can be defined as the total concentration of free water, which in some products may even be the majority component; however, this type of water can be easily removed by unit processes such as drying [21]. Water activity determines the availability of water in the food or substrate, directly influencing the growth and metabolism of the microorganisms responsible for fermentation. An adequate level of water activity is essential for yeasts and fermentative bacteria to perform their biochemical functions efficiently, promoting the production of desirable compounds and ensuring the quality, safety, and stability of the final product. Furthermore, controlling water activity helps to inhibit the development of undesirable microorganisms, preventing interference and contamination [22].

Reducing water activity plays a fundamental role in the microbiological stability of fermented material, as values below 0.7 hinder the growth of microorganisms responsible for spoilage. Thus, by controlling fermentation conditions, it is possible to adjust the final moisture content of the substrate, ensuring greater stability of the product or favoring the activity of certain microbial species. Thus, optimizing semi-solid fermentation parameters becomes essential to achieve the desired composition, whether to improve conservation, intensify biochemical transformations, or enhance the functional characteristics of fermented products [23].

3.2. Fixed Mineral Residues and Total Soluble Solids

Table 3. Mean ± standard error of the values of fixed mineral residues (%, dry basis) and total soluble solids (°Brix, dry basis) at the initial timepoint and after 96 h of semi-solid fermentation of the jackfruit residues.

Experiments	Fixed Mineral Waste (%)		Total Soluble Solids (°Brix)
	Initial	Final	Initial	Final
1	3.18 ± 0.06	7.44 ± 0.01	45.36 ± 0.44	8.27 ± 0.52
2	3.20 ± 0.02	6.31 ± 0.07	36.44 ± 0.42	6.26 ± 0.29
3	2.77 ± 0.14	7.23 ± 0.27	32.79 ± 0.42	5.54 ± 0.17
4	3.78 ± 0.26	7.51 ± 0.21	43.86 ± 0.15	7.77 ± 0.17
5	3.83 ± 0.37	6.54 ± 0.05	36.91 ± 0.28	5.49 ± 0.70
6	2.96 ± 0.09	7.47 ± 0.34	35.56 ± 0.21	5.01 ± 0.15
7	3.21 ± 0.03	7.44 ± 0.06	40.85 ± 0.24	6.09 ± 0.75
8	3.18 ± 0.09	6.54 ± 0.05	36.01 ± 0.64	4.74 ± 0.42
9	3.06 ± 0.03	7.47 ± 0.03	39.85 ± 0.61	4.22 ± 0.17
10	2.83 ± 0.06	6.54 ± 0.07	36.56 ± 0.92	7.71 ± 0.42

presents the average data on the fixed mineral residues and dry basis contents observed during the enrichment process of jackfruit seeds.

The data presented show the changes that occurred during a controlled fermentation process, with significant changes in the fixed mineral residues (%) and total soluble solids (°Brix). The fixed mineral residues increased from 2.77% to 3.83% at the beginning to 6.31% to 7.51% at the end, indicating a concentration of minerals, probably due to water loss or degradation of organic compounds. The mineral content varies depending on the biological matrix. Figueirôa et al. [24] obtained higher values of total minerals (20.423%) in Opuntia ficus-indica biomass. Total soluble solids decreased drastically, going from 32.79 °Brix to 45.36 °Brix to 4.22 °Brix to 8.27 °Brix, which suggests that sugars and other soluble compounds were consumed by microorganisms during fermentation.

The results show that the fermentation was efficient in consuming the soluble solids, reducing them to less than 25% of the initial values, while the fixed minerals increased by more than 100% in some cases. These changes are consistent with what is expected in controlled fermentations, where microorganisms metabolize substrates, transforming them into products such as alcohol, acids, or gases. The variability in the data may be related to differences in experimental conditions, such as temperature, pH, or aeration, which influence microbial activity. In summary, the data confirm the effectiveness of the fermentation process in transforming soluble compounds and concentrating minerals. The reduction of total soluble solids is directly related to the biological activity of the microorganisms involved in the fermentation process [25]. In a recent study, Douradinho et al. [26] investigated different strains of Saccharomyces cerevisiae regarding their fermentation efficiency and stress tolerance. The results demonstrated that strains with greater fermentation efficiency were able to promote a more significant reduction in total soluble solids during the process, highlighting the importance of selecting microorganisms to optimize fermentation [27].

3.3. Protein Content

Table 4. Mean ± standard error of the crude protein values, on a dry basis, of the jackfruit substrate and of the highest protein increase achieved during the 96 h of fermentation.

Exp.	YC (%)	Temp (°C)	Crude Protein (%)					Protein Increase (%)
			0 h	24 h	48 h	72 h	96 h
1	1	30	14.5 ± 0.6	16.9 ± 0.7	19.6 ± 0.6	21.4 ± 0.7	28.5 ± 0.2	115.0
2	1	35	13.9 ± 0.6	16.5 ± 0.2	20.6 ± 1.2	25.1 ± 1.2	27.4 ± 1.5	119.1
3	1	40	13.5 ± 0.4	15.6 ± 0.3	18.6 ± 0.6	25.1 ± 1.5	29.2 ± 1.4	125.3
4	3	30	14.6 ± 0.5	17.4 ± 0.3	19.7 ± 0.2	22.7 ± 1.1	28.6 ± 0.1	122.2
5	3	35	13.9 ± 0.6	15.8 ± 0.6	21.1 ± 0.7	25.1 ± 0.9	27.9 ± 0.4	115.3
6	3	40	13.8 ± 1.1	15.8 ± 0.4	19.7 ± 0.6	23.8 ± 0.6	30.5 ± 0.9	135.2
7	5	30	15.9 ± 0.4	17.5 ± 0.3	20.8 ± 0.7	23.5 ± 1.1	31.0 ± 1.3	138.2
8	5	35	14.2 ± 0.6	17.7 ± 0.7	22.0 ± 0.5	27.4 ± 0.6	30.4 ± 0.6	135.1
9	5	40	13.4 ± 1.0	20.1 ± 0.7	19.5 ± 0.3	24.4 ± 0.7	32.1 ± 0.7	146.9
10	3	35	14.2 ± 0.8	16.6 ± 1.3	21.9 ± 0.5	23.8 ± 1.0	28.6 ± 1.1	122.1

YC: Yeast concentration (%), Temp: Temperature (°C).

presents the crude protein content, on a dry basis, obtained in the different experiments with jackfruit seeds after the addition of yeast, following a 3² factorial design. It can be observed that the protein content in the fermented residues increased progressively as a function of fermentation time, temperature, and yeast concentration.

The data presented demonstrate that the fermentation process of jackfruit seeds, using yeast, promoted a significant increase in the crude protein content over time (0 h to 96 h). Similar results were observed by Tropea et al. [28], who investigated the fermentation of agro-industrial waste with Saccharomyces cerevisiae, in which the crude protein content increased over a 120 h fermentation period. In the present study, experiment 9 (5% yeast at 40 °C) stood out with an increase of 146.9%, going from 13.4% to 32.1% of crude protein. This result corroborates recent findings that indicate that higher temperatures and higher yeast concentrations accelerate microbial activity, favoring protein biosynthesis and carbohydrate degradation, which results in a fermented residue with greater nutritional value.

Other experiments also showed significant results, such as experiment 7 (5% yeast at 30 °C), which achieved a 138.2% increase in crude protein content, going from 15.9% to 31.0%. These findings are in line with a study by Sharma et al. [29] which demonstrated that, even at lower temperatures, a higher yeast concentration can be effective in increasing the protein content in fermented substrates. In addition, experiment 6 (3% yeast at 40 °C) also stood out, with an increase of 135.2%, indicating that the combination of moderately high temperature and intermediate yeast concentration can be a viable alternative for fermentation processes. The results indicate that fermentation is a promising strategy for adding value to agro-industrial waste, such as jackfruit seeds, transforming them into products with higher protein content. A recent study by Kychala et al. [30] highlighted the potential of microbial fermentation for the valorization of agricultural by-products, reducing waste and contributing to the circular economy.

Figure 1 graphically represents the variations in the protein increase of jackfruit substrates for 0, 24, 48, 72, and 96 h semi-solid fermentation times.

Based on Figure 1, it is possible to observe that the protein increase in jackfruit residues fermented with Saccharomyces cerevisiae varied according to the experimental conditions. In experiments Exp. 3, Exp. 5, and Exp. 9, the accumulated production was significantly higher, suggesting that under these conditions there was more efficient growth of S. cerevisiae, resulting in a more expressive protein enrichment. This indicates that the temperature and fermentation time in these experiments were more favorable for the conversion of residues into protein-rich microbial biomass. According to Bento et al. [31], semi-solid fermentation can modify the bioactive composition of these by-products, releasing phenolics and other bioactive compounds that are converted into beneficial metabolites during the final process.

On the other hand, in experiments Exp. 2, Exp. 6, and Exp. 10, a lower accumulated production was observed, which may be related to suboptimal conditions, such as inadequate temperatures or possible nutritional limitations in the substrate. This suggests that fermentation was less efficient, reducing protein enrichment. In the experiments Exp. 1, Exp. 4, Exp. 7, and Exp. 8, the accumulated production showed an intermediate behavior, indicating that there was some level of conversion of the residues into microbial biomass, but without reaching the peaks observed in the most productive experiments.

Thus, the results of the graph demonstrate that the semi-solid fermentation with S. cerevisiae was more efficient in certain experiments, especially Exp. 3, Exp. 5, and Exp. 9, where the conditions favored the assimilation of nutrients and protein synthesis. The analysis of these experiments allows us to identify the most appropriate parameters to maximize the use of jackfruit residues, contributing to the production of a by-product with greater nutritional value and potential application in animal and human food. Koutelidakis et al. [32] developed a high-protein Greek miso powder using industrially rejected third-grade chickpeas (25 g/L) combined with cereal milling by-products—mavragani (Triticum durum), wheat, and corn—in a 2:1:1 ratio (25 g/L). The optimized solid-state fermentation process used Aspergillus oryzae (0.05% w/w) for koji preparation and took place in two stages: the first at 4 °C for 10 days (20% moisture) and the second at 28 °C for 60 days (15% moisture). As a result, the final miso had a high protein content (17%), surpassing the average commercial miso (11.8 g/100 g), while also offering a low-cost, sustainable process that repurposes food industry by-products, adding nutritional value and reducing waste.

3.4. Optimization of the Fermentation

Thus, the optimization of the fermentation temperature and yeast concentration parameters was performed in a total of 20 experiments according to the factorial design 3² plus a central point, with repetition, for the protein enrichment of the jackfruit seeds during 96 h of semi-solid fermentation. The quantitative relationship between the protein increase and the different levels of these factors were explored to determine levels optimized by the response surface methodology. Thus,

Table 5. ANOVA results for jackfruit seed protein increase.

Fator	Quadratic Sum	Degree of Freedom	Quadratic Mean	F	p
YC (L)	616.107	1	616.01106	51.01106	0.000836
YC (Q)	111.04	1	111.044	0.48002	0.519270
T (L)	170.667	1	170.6667	14.13049	0.013170
T (Q)	105.190	1	105.1905	8.70933	0.031843
Erro	60.390	5	12.0779
Total SS	1045.604	9

Bold values indicate statistically significant differences (p < 0.05).

presents the results of the analysis of variance (ANOVA) for the protein increase that occurred during the fermentation process of the jackfruit seed residues.

Based on Table 5, we can conclude that there are significant variations between the different conditions for yeast concentration (YC (L)) and temperature (T(L) and T(Q)). The YC (L) factor, which represents the linear effect of yeast concentration, shows a p value of 0.000836, showing that yeast concentration has a significant impact on the fermentation process, although it does not have a critical point, since the quadratic effect is not significant. Temperature, on the other hand, presents both a significant linear effect (p = 0.013170) and a quadratic effect (p = 0.031843), demonstrating the significant existence of a point of maximum efficiency at which temperature reaches a peak and then decreases in efficiency. In statistics, a p value of less than 0.05 means that the probability of the results being random is low, allowing the null hypothesis to be rejected. This reinforces the validity of the model’s conclusions, indicating that the factors analyzed really influence the dependent variables [33]. Therefore, although yeast concentration can be optimized linearly, temperature has a critical point that should be investigated in more detail to maximize the efficiency of the fermentation process. Equation (2) and Figure 2 represent the second-order model obtained from the experimental data established for yeast concentration (YC) and fermentation temperature (T), with response to protein increase (PI).

PI = 118.7 + 8.0 YC + 8.4 YC² + 6.5 T + 10.0 T² − 0.4 YC.T + 3.2 YC.T² − 1.8 YC².T − 5.8 YC²·T² + 0

(2)

Figure 2 shows the response surface graph of the factorial design matrix.

The three-dimensional graph presented demonstrates the relationship between the protein increase (%) in the fermented jackfruit residues, the temperature (°C), and the yeast concentration (%). It can be seen that as the temperature and the concentration of Saccharomyces cerevisiae increase, the protein content also increases, reaching the highest values in the extreme conditions of both variables. Regions colored red indicate the points of greatest protein enrichment, while the green areas represent lower efficiency in the fermentation process. This suggests that higher temperatures and a higher concentration of yeast favor protein biosynthesis and microbial growth.

On the other hand, the lower part of the graph, where there are lower temperatures and low yeast concentration, shows a reduced protein increase, which indicates a lower use of the residues by the yeast. This trend may be associated with the lower enzymatic and metabolic activity of S. cerevisiae in less favorable conditions. Thus, the data confirm that optimizing these two factors—temperature and yeast concentration—is essential to maximize protein enrichment during semi-solid fermentation of jackfruit waste, making the process more efficient and sustainable for future applications.

Optimization through factorial design is essential in the development of semi-solid fermentation studies, as it allows the identification of the best combination of factors such as temperature, substrate concentration, humidity, pH, and inoculum concentration, aiming to maximize process efficiency. This systematic approach reduces the number of experiments required and allows an analysis of the interactions between factors, which is essential to understand the behavior of the fermentation system. In the case of protein enrichment of jackfruit seeds by S. cerevisiae, for example, the ideal combination of high temperature and high yeast concentration proved to be decisive in increasing protein production. Thus, factorial design becomes a crucial tool for achieving reproducible and efficient conditions in semi-solid fermentation processes. Shu et al. [34] analyzed the experimental conditions for flavor improvement of Apostichopus japonicus peptide using the response surface methodology during bacterial co-fermentation. Similarly, Jian et al. [35] investigated the optimization of fermentation conditions and dextran structure, evaluating their impact on the rheological and textural properties of a new enriched plant-based cheese. These studies demonstrate that the optimization approach through fermentation can be widely applied in different scientific contexts, contributing to the development of new products and the improvement of their functional characteristics.

4. Conclusions

The study demonstrated that the semi-solid fermentation of jackfruit seeds (Artocarpus heterophyllus Lam.) with Saccharomyces cerevisiae is an efficient technique for protein enrichment, resulting in an approximately 2.5-fold increase in crude protein content compared to the raw substrate. The combination of 5% yeast and a temperature of 40 °C, after 96 h of fermentation, provided the highest protein increase (146.9%), reaching 32% crude protein. These results highlight the potential of jackfruit seeds as a sustainable and low-cost alternative for producing protein-rich foods for animal nutrition. Furthermore, the semi-solid fermentation process proved viable for valorizing agro-industrial waste, contributing to waste reduction and the generation of high-value-added products. The use of protein-enriched jackfruit seeds can replace conventional protein sources, such as corn, in animal diets, offering an economically attractive and environmentally sustainable solution. Therefore, this approach represents a promising alternative for utilizing agricultural by-products and producing functional foods.