The β-carotene and total carotenoid content for ripe and overripe A. triloba pulp from the 2022 harvest year is shown in Table 2. Overripe pulp contained significantly more total carotenoids than ripe pulp (p = 0.017), but no difference was observed in β-carotene between ripe and overripe pulp.

A.triloba pulp obtained from a composite of fruits of unknown cultivar during the 2023 harvest was used to explore carotenoid content (µg total carotenoids/100 g pulp) of pawpaw pulp for two main effects, ripeness level and refrigerated storage time. The main effect of ripeness revealed a significant difference in total carotenoids (p < 0.007) between ripe and overripe pulp, with overripe pulp containing 5-fold more carotenoids (1,143 µg total carotenoids/100 g pulp) than ripe pulp (230 µg total carotenoids/100 g pulp). The main effect of refrigerated storage time showed a significant decline in total carotenoids (p = 0.013), with pawpaw pulp stored for 0 and 7 days exhibiting significantly higher total carotenoids than pulp stored for 21 days. The two-way interactions (Table 3) show that these differences (p < 0.001) were driven solely by the decline of total carotenoids in the overripe pulp because total carotenoids in the fresh pulp that was stored refrigerated did not decline significantly during the 21-day storage period. Overripe pulp that was stored for 21 days contained significantly lower total carotenoids (23% to 35%) than overripe pulp that was stored refrigerated for 0, 7, or 14 days. Thus, it appears that more carotenoids are found in overripe pulp than in ripe pulp, but the levels in ripe pulp are stable during refrigerated storage, whereas the levels in the overripe pulp decline during refrigerated storage.

CIELAB L*, a*, b* values were measured for A. triloba harvested in 2022 (Table 2) and 2023 (Table 3). These values were used to calculate applied color attributes, of which color index, whiteness index, yellowness index, the ratio of a*/b*, and percent change in L* are reported for A. triloba for the first time. A statistical comparison between fresh ripe pulp for the two harvest years was undertaken and shows that pulp color is not homogenous. This observation is not surprising when considering that qualitative descriptions of pawpaw pulp from different cultivars almost always note a range of descriptors such as creamy white, bright yellow, or shades of orange [13]. The 2022 A. triloba pulp, obtained from fruit from a single tree, was statistically more yellow (higher b*, p < 0.001) and exhibited a correspondingly higher chroma (p < 0.001) and yellowness index (p < 0.001) than the 2023 pulp, which was a composite of different cultivars grown at a different location than the 2022 pulp. The ripe pulp from 2022 A. triloba was also more brown, as indicated by a higher browning index (p = 0.007) and a correspondingly lower whiteness index (p = 0.001) than the 2023 pulp.

There were many significant indicators that suggested that overripe pulp in A. triloba harvested in 2023 (Table 3) exhibited browning compared to ripe pulp, including lower L* value and whiteness and yellowness indices, and higher browning and total color indices between the overripe and ripe pulp. Browning index significantly increased in ripe pulp after day 0, but no further increase was observed during subsequent refrigerated storage. In overripe pulp, in which significant browning was already indicated at the onset of refrigerated storage, no further change in browning index was observed. Significantly lower L* value was also observed in overripe pulp compared to ripe pulp in A. triloba harvested in 2022 (Table 2).

Pearson correlation coefficients were computed from the 2023 A. triloba to assess the linear relationship between the applied color attributes and total carotenoids, ripeness level, and storage. There was a significant but moderately positive correlation between total carotenoids and both b* [r(22) = 0.515, p = 0.010] and chroma [r(22) = 0.482, p = 0.017], although a* was not significantly correlated with total carotenoids. Regardless, this result agrees with the literature that shows that positive correlations between carotenoids and color values, for example, in nectarines, where the association of red and yellow color was directly correlated with increased total carotenoid content that was independent of the initial white or yellow color of the pulp [14].

The correlations between ripeness and both b* and chroma, but not a*, mirror those for total carotenoids, with significant but moderately positive correlations for b* [r(22) = 0.527, p = 0.008] and chroma [r(22) = 0.510, p = 0.011]. The significant, strong, positive correlation between total carotenoids and ripeness [r(22) = 0.945, p < 0.001] echoes the results presented previously that indicate that total carotenoids increase as A. triloba becomes more ripe.

The Pearson correlation coefficients that were computed to assess the linear relationship between storage time and each of the 12 applied color attributes (Table 3) of A. triloba indicate that pulp color during storage is significantly correlated to a change in 9 of the 12 attributes, some of which are indicators of pulp browning. There were significant, positive correlations between storage time and color index [r(22) = 0.806, p < 0.001], percent L* reduction [r(22) = 0.795, p < 0.001], a*/b* ratio [r(22) = 0.779, p < 0.001], ΔE [r(22) = 0.706, p < 0.001], a* [r(22) = 0.625, p < 0.001], and browning index [r(22) = 0.498, p < 0.013]. There were significant, negative correlations between storage time and L* [r(22) = –0.795, p < 0.001], hue angle [r(22) = –0.779, p < 0.001], and whiteness index [r(22) = –0.667, p < 0.001].

The positive correlations between storage time and both the percent L* reduction and browning index, as well as the negative correlations between storage time and both L* and whiteness index, indicate that the color of the pulp becomes darker or more brown as the pulp is stored. Several factors can contribute to the darkening or browning of the pulp, but the major factor is probably enzymatic browning, where polyphenol oxidases catalyze the oxidation of phenolic compounds. It has been shown that polyphenol oxidase is active in pawpaw pulp [15], and the phenolic compounds that are substrates for polyphenol oxidase are present in the pulp [16].

Vitamin A is reported on most nutrition labels worldwide in retinol activity equivalents (RAE) or international units (IU). RAE conversion factors have been established for β-carotene (12 µg to 1 µg RAE) and total carotenoids (24 µg to 1 µg RAE), although the total carotenoid conversion factor relies on the assumption that the majority of total carotenoids are composed of dietary provitamin A carotenoids such as α-carotene and β-cryptoxanthin [17]. Using these conversion factors, A. triloba contains 36 µg RAE per 100 g edible pulp based on β-carotene and 23 µg RAE per 100 g edible pulp based on total carotenoids. The latter value probably should be used with caution because individual carotenoids of A. triloba have not been identified, so it is not known if this value actually reports the RAE solely of dietary provitamin A compounds. A. muricata, for example, contains carotenoids that are not classified as dietary provitamin A, such as lycopene and lutein. The same A. triloba pulp that was obtained from obviously overripe fruit during the 2022 harvest contained 389 µg β-carotene and 947 µg total carotenoids per 100 g edible pulp (Table 2), converting to 32 µg RAE and 39 µg RAE per 100 g edible pulp, respectively.

Using the 120 g serving size that has been established for A. triloba [18] and the seemingly more reliable conversion using β-carotene, ripe pulp provides 4.0% (males) and 5.1% (females) of the U.S. Recommended Dietary Allowance (USRDA) of vitamin A for individuals 14 years or older, and overripe A. triloba pulp provides 3.5% (males) and 4.6% (females) of the USRDA of vitamin A for this population. For comparison, a serving of mango provides 10% (males) and 13% (females), and a serving of banana provides less than 1% of the USRDA of vitamin A for these populations.

Annonaceae fruits are climacteric, and an increase in carotenoids in other climacteric fruits agrees with research that has shown that typical post-climacteric biochemical changes were not linked to post-climacteric carotenoid losses [19]. This research showed that carotenoids, especially lutein, were stable in the overripe stage [19] of nectarines [Prunus persica (L.) Batsch], hybrid persimmons (Diospyros virginiana L. × Diospyros kaki L.f.), and bananas (Musa acuminata Colla). Other research was similar. In the ‘red banana’ cultivar of bananas [20], yellow mombin fruit (Spondias mombin L.) of unknown cultivar [21], and mango (Mangifera indica L.) of unknown cultivar [22], higher carotenoid levels were found in fruits that were 4, 9, and 16 days post-climacteric, respectively.

A comparison of the carotenoid content of A. triloba and seven Anona spp. is shown in Table 4. The individual carotenoids in Annonaceae fruit have been well-characterized in A. muricata, A. reticulata, and A. squamosa, with 10, 8, and 8 individual carotenoids reported in the literature, respectively. The five other Annonaceae species, A. triloba, A. cherimola, A. coriacea, A. crassiflora, and A. purpurea, are not as thoroughly studied, with only 1–3 individual carotenoids reported in the literature for each. The provitamin A carotenoids (α-, β-, γ-carotene and β-cryptoxanthin) are nutritionally important because they can be converted into vitamin A in the body and have antioxidant properties [7]. β-Carotene has been quantified in A. triloba and six of the seven Annonaceae species, with values ranging from 0.19 to 1,970 µg per 100 g pulp (in A. muricata and A. crassiflora, respectively). The five species for which α-carotene has been quantified indicate that A. reticulata, A. squamosa, and A. crassiflora [23] have higher levels of α- compared to β-carotene [24], whereas A. cherimola [25] have higher levels of β- compared to α-carotene. The research on A. muricata is equivocal, with a study showing higher levels of β- compared to α-carotene [26] and another showing the reverse [24].

Using the conversion values for individual provitamin A carotenoids described previously [17] and assuming a 100 g serving size as a basis, the percentage per serving of the USRDA of vitamin A for individuals 14 years or older for the four most widely cultivated and commercially important Annona species ranges from: A. muricata (0.01–0.79% males, 0.01–1.01% females), A. cherimola (0.02% males, 0.03% females), A. squamosa (0.09% males, 0.12% females), and A. reticulata (0.10% males, 0.13% females). This suggests that Annona species do not provide significant dietary vitamin A on their own or compared to A. triloba. Annona species also are a source of xanthophylls, but do not contain high enough levels of lutein and zeaxanthin to meet the daily recommendation of these carotenoids for eye health [9, 10]. The presence of lycopene, which is neither a provitamin A carotenoid nor a xanthophyll, was reported only in A. muricata [24].

The C.I.E. color value data for different cultivars of A. triloba, A. cherimola, A. diversifolia, and A. muricata are shown in Table 5. The data were obtained from different studies and reinforce the finding that color values for A. triloba cultivars are not homogenous. However, this observation probably should not be universally applied to other Annonaceae fruits. The most obvious Annonaceae fruit that is non-homogenous in color is A. diversifolia, reported in the literature and on Table 5 as varieties labeled white, pink, and deep pink [32], which probably come from cultivars ‘Annona Blanca’, ‘Fairchild’ or ‘Rosendo Pérez’, and ‘Genova Red’, respectively. Not surprisingly, the color values are directional from white to deep pink with notably increasing redness (a*). A. muricata and A. cherimola redness (a*) and (b*) color values are relatively uniform, suggesting homogeneity, although there are some differences in lightness (L*).

As shown in Table 6, there are many nutritional similarities between the nutritional content of six Annonaceae fruits that can be found in research or a database. Detailed nutritional information is reported in the literature for A. triloba [18, 30] and A. macroprophyllata [32], and nutritional information for the four most widely cultivated Annona species, A. muricata, A. cherimola, A. squamosa, and A. reticulata, is reported in the U.S. FoodData Central database [25]. All species have a caloric content between 276–423 kJ (66–101 kcal) per 100 g edible fruit, ≤ 1% ash and lipid, and around 1% protein [18, 25, 30, 32]. There is some variation between species for total carbohydrates and moisture.

From the period spanning 1982–2018, A. triloba fruit pulp was mistakenly reported to be a complete protein, i.e., a food that contains all nine essential amino acids. This misinterpretation was due to the fact that the research that reported this finding in 1982 was conducted on A. triloba fruit pulp and the skin [38], which is generally considered inedible. Recent research (Table 6) now indicates that A. triloba fruit pulp is probably deficient in methionine and tryptophan [30]. No studies could be located in which a complete amino acid profile was conducted on any of the Annona species, so no conclusion can be made as to whether any of the Annona fruits are complete proteins.

Total carbohydrates (Table 6) ranged from 16.8% to 25.2% across the six species [18, 25, 30, 32]. In A. triloba, two nutritional studies reported carbohydrate contents of 18.6% [30] and 23.8% [18]. These two studies also reported higher dietary fiber (5.8% and 4.5%, respectively) than the 2% to 4.4% range reported for five Annona species [25, 32]. Annonaceae fruits have 13–16% total sugar, composed of sucrose, glucose, and fructose. However, the distribution of these three simple sugars varies between A. triloba and A. macroprophyllata, the only Annona species for which this data is reported. In A. triloba, the distribution of sucrose, glucose, and fructose is 70%, 17%, and 13%, respectively, whereas A. macroprophyllata has less sucrose, with a distribution of 53%, 20%, and 27%, respectively. It is not known if this result can be generalized to all Annona fruits until such a time as the sugar profile of other Annona fruits is reported.

Comparisons between the six Annona species can be made for vitamin C and five minerals (calcium, iron, magnesium, potassium, and sodium) because all six species have reported values for these nutrients. With respect to vitamin C, three tiers emerged. At the high end, A. squamosa contains vitamin C at an equivalent level to that of mango. A. muricata and A. reticulata contain about 45% less, and A. cherimola about 66% less. A. triloba and A. macroprophyllata have very little vitamin C. All Annonaceae species contained comparable amounts of calcium, iron, magnesium, potassium, and sodium, which were on par with a serving of mango.

With respect to carotenoids, higher levels were found in overripe A. triloba pulp than in ripe pulp, and the levels in ripe pulp were stable during refrigerated storage, whereas the levels in the overripe pulp declined during refrigerated storage. The significant correlation between total carotenoids and ripeness reinforces that total carotenoids increase as A. triloba becomes more ripe. The individual carotenoids in Annonaceae fruit have been well-characterized in A. muricata, A. reticulata, and A. squamosa, but not in other Annonaceae species. A. reticulata, A. crassiflora, and A. squamosa have higher levels of α- compared to β-carotene, whereas A. cherimola has higher levels of β- compared to α-carotene. The research on A. muricata is inconclusive.

An interesting finding from this study is the nutritional similarity of A. triloba, unique in the family Annonaceae because it is the only temperate genus, and the “normal” Annona species that are native to Latin America. Overall, Annonaceae fruits are nutrient-dense and provide a good amount of fiber and potassium, while being low in fat and protein. These fruits do not appear to be complete proteins, although studies are limited, for which a complete amino acid profile would provide confirmation. The carbohydrates from Annonaceae fruits come primarily from the sugars sucrose, glucose, and fructose, albeit with differing distributions. Fruits from some Annonaceae, such as A. squamosa, have a good amount of vitamin C, while others, such as A. triloba and A. macroprophyllata, have very little vitamin C. Fruits from all Annonaceae species contained comparable amounts of calcium, iron, magnesium, potassium, and sodium.

Promotion and marketing of the vitamins and other antioxidative compounds in Annonaceae fruit pulp would enrich their culinary scope and potential. Added benefits would include improving the shelf life and stability of products to which they are incorporated and a reduction in oxidative stress and the associated lower risk of chronic diseases in those who consume them. In addition to their health benefits, the unique flavor and texture of Annonaceae fruits add versatility for creative culinary applications that may encourage healthier eating habits and make delicious, nutritious foods more accessible.

This study has implications with respect to increased utilization of Annonaceae fruit. It would be beneficial if overripe fruits, often discarded, could be utilized in some fashion, but more research on the acceptability of products prepared from overripe pulp is needed. It would also be beneficial to know if other nutrients in addition to carotenoids are stable during refrigerated storage, because this research suggests that if the quality of the fruit can be maintained during refrigerated storage, the carotenoid content also will be maintained. Because the findings from this study apply only to A. triloba, further study would be required to determine whether these findings are universal for all Annonaceae fruits, which would add value to Annonaceae fruits.

The pawpaw research presented herein is preliminary in nature, mirroring the post-harvest research on Annonaceae, which is far behind in maturity than many other commercial fruit crops. Strict genetic control in research involving Annonaceae fruit is rarely undertaken, as was the case in this research. Because of this limitation, observed differences in the measured variables could be attributable to inherent genetic variations among individual pawpaw fruit rather than solely the experimental conditions, which could affect the generalizability of the findings. Future research that incorporates controlled experiments to specifically address the genetic background of Annonaceae samples would be important for more robust and generalizable conclusions.

Nonetheless, the results of this study underscore the importance of considering both ripeness and storage duration when evaluating the nutritional content in Annonaceae fruits. The stability of A. triloba carotenoids during refrigerated storage suggests that overripe pulp could be utilized in meal prep and food production, which offers practical solutions for both consumer health and sustainable practices in food manufacturing. However, the sensory attributes of overripe fruit pulp, i.e., color, texture, and flavor, will have to be taken into consideration before any generalizations can be made.

This study seeks to enhance the global understanding of the nutritional relevance of Annonaceae fruits, both temperate and tropical, especially in areas where these two climates exist in close proximity. For example, the diverse range of climates in Latin America suggests that A. triloba cultivars could be selected and introduced into areas near the native range of tropical Annonaceae, such as the Pampas region of Argentina or temperate regions of central Chile, Uruguay, and southern Brazil. Understanding Annonaceae fruits in this regional context may reveal previously hidden industrial potential and stimulate new awareness that contributes to enhancing local economies.