3.1. Basic Chemical Composition
Our research findings regarding the basic chemical composition align well with the expected properties of ice cream. The average protein content seems similar across all samples, ranging from 3.00% to 3.03% (
Table 2). Similarly, the fat content also appears comparable between the control and other samples, ranging from 5.59% to 5.66%. The dry matter content shows a slightly wider range, with the control sample having the highest value (26.92%) and ics1–ics6 samples having the lowest (26.60%). However, the error margins are large enough that there may not be statistically significant differences.
The lactose content remains consistent across all samples, ranging from 26.71 to 28.36 mg/kg (
Table 3). On the other hand, sucrose content is notably higher, ranging from 110.26 to 116.97 mg/kg. Statistically significant differences were observed between the ics4 sample and the other samples (ics1–ics3, ics5, ics6 samples), indicating a potential sucrose decomposition during ice cream production or storage due to enzymatic activity from the microorganisms used (white bean homogenate fermented by
L. rhamnosus GG was utilized in the ics4 samples). Glucose, the third most prevalent sugar, also displays slight variations among samples (ranging from 4.99 to 5.26 mg/kg). These sugars’ content is generally low, averaging below 0.15 mg/kg. The lowest glucose content was found in the ics2 sample with the addition of white bean homogenate fermented by
L. acidophilus La-5. Carbohydrates such as raffinose, stachyose, and verbascose had low contents, with only stachyose showing statistically significant differences between samples fermented with different probiotics.
Dry matter, primarily composed of milk solids–not–fat and sugars, provides the solid structure of ice cream [
16], preventing it from becoming excessively icy or runny. A higher dry matter content results in denser and firmer ice cream. Additionally, it aids in trapping air bubbles incorporated during churning, creating the light and fluffy texture we all enjoy. Insufficient dry matter would lead to the disappearance of these air bubbles, resulting in a dense, potentially icy product. Although milk protein constitutes a smaller portion of ice cream compared to other ingredients, it plays a crucial role [
17]. Acting like tiny whisks, milk proteins evenly distribute fat droplets throughout the mixture. Like dry matter, protein also helps stabilize air bubbles for a delightful texture. Our research demonstrates the advantage of using white bean homogenate, as it does not significantly reduce protein content, contributing to an optimal recipe formulation. While the protein content in our ice cream formulations may not qualify them as a significant source of dietary protein, the inclusion of white beans introduces additional fiber and other potentially health-promoting compounds.
Milk fat is another key ingredient that influences both the taste and quality of ice cream [
18,
19]. When freezing occurs, milk fat crystallizes, forming a network that traps air bubbles and hinders the formation of large ice crystals. Studies have shown that higher fat content results in lower overrun (less air incorporated) [
20]. Additionally, fat content can contribute to satiety, but the type of fat must be considered. Our emphasis on using healthy fats, such as those found in milk and white beans, can offer certain health benefits.
While fat, sugar content, and bacterial count do not directly affect ice cream’s acidity, they can influence its pH and viscosity [
21]
. Our research confirms that yogurt bacteria and probiotic strains generally have minimal impact on the overall carbohydrate content of ice cream. Although they might utilize some sugars during fermentation, the amount is negligible compared to the total sugar content. However, probiotics may influence the activity of enzymes that break down carbohydrates (such as alpha-amylase and alpha-glucosidase) [
22]. The cold storage temperature of ice cream significantly hinders bacterial activity. In fact, some research suggests that optimizing sugar concentration might even improve probiotic survival [
23].
Although there is limited data on the direct effects of probiotics and yogurt bacteria on the carbohydrate content of ice cream, there is potential for the metabolic properties of probiotics to impact the composition and characteristics of dairy products enriched with these microorganisms. Further investigation is necessary to thoroughly understand these effects, especially within the low-temperature environment of ice cream. Changes in fermentative activity could result in reduced levels of unfavorable carbohydrates, such as oligosaccharides that cause flatulence, through breakdown processes. Probiotics, like
L. plantarum, might influence fermentation in white bean products used as ice cream ingredients, potentially altering carbohydrate content by converting simple and complex sugars [
11,
13]. Incorporating probiotics into white bean-based products for ice cream formulation could impact their nutritional value, including carbohydrate content, by improving digestibility and potentially stimulating the growth of desirable bacteria that ferment dietary fiber and other carbohydrates [
24]. This suggests potential health benefits associated with the consumption of these ice creams, warranting further investigation.
3.2. pH Value
In our experiments, the ice cream recipe incorporating fermented white bean homogenate (ics1–ics6 samples) showed a statistically significant decrease in pH compared to the control samples (ics0) containing nonfermented white bean homogenate (
Table 6). However, most ice cream samples maintained stable pH values over the 6-month storage period, except for the ics2 and ics3 samples which exhibited variations. These observed pH variations might influence the survival of probiotics and the antioxidant properties of the ice cream, topics that will be discussed in the following sections.
The pH value of milk ice cream varies depending on ingredients and production methods, typically ranging from 6.0 to 6.8 [
21,
25,
26,
27,
28,
29]. This range has a significant impact on various aspects of ice cream quality. However, limited information is available on how pH directly affects features such as bacterial survival, overrun, carbohydrate content, or antioxidant properties in ice cream. While some probiotic strains thrive in acidic environments, others struggle [
30]. The pH level of fermented ice cream significantly impacts probiotic survival, with variations depending on the bacterial strains used. For example, L. acidophilus thrives in slightly acidic environments (pH 5.5–6.0), whereas bifidobacteria prefer a more neutral range (pH 6.0–7.0). L. acidophilus demonstrates better tolerance to acidity compared to bifidobacteria, which experience significantly slowed growth below pH 5.5. Furthermore, even among Bifidobacterium species, tolerance to acidity can vary between strains. Therefore, precise pH management during ice cream fermentation is crucial. If the pH falls below 5.5, it can significantly reduce viable probiotic bacteria.
While pH does not directly affect overrun, it does influence the structure and stability of the ice cream’s protein matrix, impacting its ability to retain air during mixing and freezing. Balanced pH is important for achieving the desired ice cream texture. The direct effect of pH on carbohydrate content is limited. However, pH value can affect the stability of bioactive compounds in ice cream, including their antioxidant properties. Lower pH levels generally tend to increase the antioxidant activity of certain ingredients, but this effect varies depending on the specific compound. pH values can also affect the availability of phenols and other antioxidants in foods.
3.3. Ice Cream Overrun
Overrun, which refers to the amount of air incorporated during churning, plays a crucial role in determining ice cream texture, volume, and consumer acceptance. Higher overrun results in a lighter texture but can also strain probiotics. In our experiments, the overrun of ice cream was significantly affected by the type of white bean homogenate and the probiotic strain used for fermentation (
Table 5). Notably, ice cream produced with white bean homogenate fermented with
L. acidophilus La-5 (ics2) and
L. plantarum 299v (ics3) exhibited the highest overrun. This suggests that both the composition of the white bean homogenate and the bacterial strains employed in fermentation have a significant impact on ice cream overrun.
While there is limited data on overrun specifically for dairy and probiotic ice cream, some general observations can be made. Studies have shown that incorporating plant milk into probiotic ice cream without fermentation can improve sensory aspects and probiotic survival [
31]. Additionally, substituting skimmed milk with sweet potatoes in probiotic ice cream has been found to have no significant impact on overrun [
32]. Conversely, other research has found no impact of probiotic addition on overrun [
33,
34]. Further research is necessary to determine the exact mechanisms through which white bean homogenate and lactic acid bacteria influence ice cream overrun. Our findings suggest that overrun plays a crucial role in balancing the desired textural properties of probiotic ice cream with the viability of probiotic cultures, thereby impacting the potential health benefits for consumers. While higher overrun contributes to a lighter texture, it may also strain the probiotics, potentially reducing their survival during processing and storage [
35]. Previous research has highlighted the sensitivity of
Bifidobacterium bacteria to oxygen in dairy products [
36,
37,
38,
39]. While further research is required to determine the exact mechanisms, our research supports the idea that ice cream with lower overrun could provide a more conducive environment for probiotic survival [
40,
41]. Our study aligns with these observations by showing that ice cream with lower overrun levels tended to maintain a higher number of viable probiotic bacteria after processing and storage compared to those with high overrun. Future studies can investigate the interplay between overrun, probiotic strains, and ice cream formulation to optimize both sensory qualities and potential health benefits.
3.4. Survival of Yoghurt and Probiotic Bacteria
Probiotic ice cream offers a novel approach to delivering beneficial bacteria to the gut, potentially merging enjoyment with probiotic supplementation [
31,
42,
43]. Maintaining probiotic viability during processing and storage, while maintaining sensory and nutritional qualities, is crucial for these functional ice creams.
Table 4 shows the viability (colony forming units, CFU) of yogurt and probiotic bacteria strains in ice cream throughout the production process and during frozen storage. The data is expressed in logarithmic units (log CFU/mL) for easier comparison. The result confirms that the ice cream production process itself does not introduce any contaminating bacteria. All probiotic strains (ics1–ics6 samples) showed a statistically significant decrease in viability over the storage period. However, freezing the ice cream appears to have minimal impact on the initial bacterial count for most strains (when comparing values at 0 months). Overall, the results suggest that probiotic bacteria can survive in ice cream during production and frozen storage, although their viability declines over the 6-month storage period. The rate of decline varies among strains, as expected due to freezing and storage stress.
L. casei DN-114001 (ics5 samples) displayed the highest resilience, whereas
L. delbrueckii subsp.
bulgaricus (ics1 samples) showed the most significant decline. This data allows for comparing the survival of different probiotic strains (ics1–ics6 samples) in ice cream, which is valuable for selecting appropriate strains for ice cream products. Our findings regarding probiotic survival during storage, which generally exceeded the recommended minimum of 6 log CFU/g, suggest that this ice cream formulation holds promise for delivering viable probiotics to the gut, potentially enhancing health benefits. The observed differences in probiotic survival rates suggest that selecting strains with higher viability after storage (such as
L. plantarum 299v) could enhance the potential health benefits associated with consuming probiotic ice cream.
Previous research has shown that probiotics can survive in ice cream for up to 6 months when stored frozen at temperatures ranging from –18 to –28°C, with viable cell counts exceeding the recommended minimum of 6 log CFU/g [
23,
27,
44]. Factors influencing survival include the type of strain, production methods, storage temperature and duration, and the composition of the product (including bulking agents, sweeteners, and fat content) [
30]. Our findings align with Salem et al. [
41], who observed a decrease in live cell counts for various probiotic strains in ice cream stored at –26°C. Despite this decline, their ice cream remained a probiotic food with viable cell counts above the minimum threshold. Similarly, our study showed a decrease in live cell counts, albeit with different bacterial strains and ice cream formulations, highlighting the influence of both bacterial and ice cream composition on survival rates. Further research involving a wider range of formulations and storage conditions could provide additional insights into how these variables influence probiotic viability.
3.5. Antioxidant Capacity
DPPH: The DPPH assay measures a product’s ability to neutralize free radicals, which are harmful molecules linked to cellular damage and various diseases. Milk-based ice cream containing fermented white bean homogenate serves as a source of antioxidant compounds, including polyphenols and proteins derived from milk, white beans, and other ingredients. Through the DPPH assay, we can gauge the effectiveness of these compounds in scavenging free radicals. A higher level of antioxidant activity suggests that the ice cream might provide health benefits by protecting cells against oxidative stress. All ice cream samples with fermented white bean homogenate (ics1–ics6 samples) exhibited significantly higher DPPH activity compared to the control group (ics0 samples) at both time points (
Figure 1a), indicating that fermented white bean homogenate successfully enhances the ice cream’s antioxidant properties. Among the fermented white bean homogenate samples (ics1–ics6 samples), no statistically significant differences in DPPH values were observed after production or after 6 months of storage, except for the sample with
L. plantarum 299v (ics3 samples), where storage time had a significant effect. Our findings demonstrate that incorporating fermented white bean homogenate with probiotic bacteria into milk-based ice cream represents a promising approach for enhancing its antioxidant potential. This increase in antioxidant activity suggests potential health benefits, such as reducing cellular damage associated with oxidative stress. Further research could explore the specific contributions of different bean types, fermentation parameters, and storage conditions on the overall antioxidant profile of the ice cream.
ABTS: The DPPH and ABTS assays measure a product’s ability to neutralize free radicals, which can damage cells and contribute to various diseases. These assays help evaluate the efficacy of these compounds in scavenging free radicals. Higher antioxidant activity suggests potential health benefits by protecting cells from oxidative stress. In the DPPH assay, all ice cream samples with fermented white bean homogenate (ics1–ics6 samples) exhibited significantly higher ABTS values than the control (ics0 samples) both during production and after 6 months of storage (
Figure 1b). This affirms that fermented white bean homogenate effectively enhances the ice cream’s overall antioxidant capacity. Interestingly, the ABTS assay suggests some variation in the impact of different probiotic strains.
L. rhamnosus GG (ics4 samples) and
L. casei DN-114001 (ics5 samples) showed the highest values, while
L. plantarum 299v (ics3 samples) had a smaller increase. This suggests these strains might have a more substantial impact on the overall antioxidant profile, warranting further investigation into the specific mechanisms and compounds responsible. Our findings demonstrate that incorporating fermented white bean homogenate with probiotic bacteria is a promising strategy to improve the antioxidant potential of milk-based ice cream. This increase in antioxidant activity, as measured by both DPPH and ABTS assays, suggests potential health benefits by potentially reducing cellular damage caused by free radicals. Further research could explore the reasons for the observed strain-specific differences and identify the specific components contributing to the ice cream’s antioxidant properties.
FRAP: As observed in the DPPH and ABTS assays (sections 3.5.1 and 3.5.2), all ice cream samples containing fermented white bean homogenate (ics1–ics6 samples) exhibited significantly higher FRAP values compared to the control (ics0 samples) both at production and after 6 months of storage (
Figure 1c). This confirms that fermented white bean homogenate effectively enhances the ice cream’s overall antioxidant capacity, as measured by all three assays (DPPH, ABTS, and FRAP). Interestingly, the FRAP assay suggests some variability in the impact of different probiotic strains. Apart from ics0 and ics2, all samples showed increased FRAP values after storage, with
L. rhamnosus GG (ics4 samples) showing the most significant increase. This suggests that
L. rhamnosus GG may have a more significant impact on ferric reducing power, potentially by influencing the types of antioxidant compounds present in the ice cream. These findings, however, differ somewhat from the observations related to DPPH values, highlighting the potential for strain-specific effects on various aspects of antioxidant activity. Further investigation is warranted to identify the mechanisms driving these differences and to optimize probiotic selection for maximizing the antioxidant benefits of probiotic ice cream. The increased ferric reducing power observed with the FRAP assay suggests a potential for these ice creams to contribute to increased antioxidant activity in the body, potentially reducing cellular damage caused by free radicals.
TPC:
Figure 1d displays the TPC, expressed in milligrams of Gallic Acid Equivalents (mg GAE) per 100 mL of ice cream. All ice cream samples containing fermented white bean homogenate (ics1–ics6 samples) exhibited significantly higher TPC values compared to the control group (ics0 samples) both at production and after 6 months of storage. This indicates a greater abundance of phenolic compounds in the ice cream with added probiotic bacteria. The control group showed minimal changes in TPC, suggesting a low inherent phenolic content. These findings suggest that all yogurt and probiotic strains (ics1–ics6 samples) contribute to the ice cream’s overall phenolic content, potentially due to the presence of phenolics in the fermented white bean homogenate itself or those produced during fermentation. Interestingly, ics3 samples (
L. plantarum 299v fermentation) showed the most substantial and statistically significant increase in TPC at 6 months. This suggests that
L. plantarum 299v might have a more significant impact on the overall phenolic content, warranting further investigation into the specific types of phenolics produced and their contribution to antioxidant activity. The increased phenolic content, as measured by TPC, suggests a greater potential for antioxidant activity. Phenolic compounds can function as free radical scavengers, potentially conferring health benefits by reducing oxidative stress in the body. Further research could identify the specific types of phenolic compounds present in the ice cream and determine their individual contributions to the overall antioxidant profile.
The antioxidant capacity of ice cream is influenced by the presence and concentration of ingredients known for their antioxidant properties. Milk-based ice creams derive benefits from the inherent nutrients in milk, such as vitamins and proteins, while plant-based options often utilize fruits, nuts, and seeds that are rich in natural antioxidants, including vitamins and phenolics [
31,
45,
46,
47,
48]. Our study specifically focused on milk-based ice cream and investigated how incorporating fermented white bean homogenate with probiotic bacteria could enhance its antioxidant capacity compared to regular ice cream. Fermented white bean homogenate is likely to contribute natural antioxidants like polyphenols (e.g., flavonoids, phenolic acid) and proteins (e.g., albumins, globulins, and phaseolin) [
49,
50,
51,
52]. Processes like soaking, sprouting, and fermentation can influence the polyphenol content of white beans, with fermentation potentially converting complex polyphenols into simpler, more bioavailable forms [
9,
10]. Studies on other legumes, such as soybeans, suggest that fermentation can influence enzymatic activities and potentially affect polyphenol content [
53].
All assays (DPPH, ABTS, FRAP, TPC) demonstrated a significant increase in antioxidant activity in ice cream containing fermented white bean homogenate compared to the control. Fermentation can lead to the formation of bioactive compounds, including plant-derived polyphenols known for their antioxidant properties [
54,
55,
56]. White beans naturally contain polyphenols, and fermentation by lactic acid bacteria or bifidobacteria may release these compounds, potentially increasing the overall antioxidant capacity as observed in fermented soy milk [
57]. This suggests a greater ability to neutralize free radicals and potentially protect cells from oxidative stress.
It is important to consider factors influencing antioxidant capacity, such as the specific probiotic strain, fermentation conditions, and bean quality. L. rhamnosus GG (ics4 samples) consistently displayed high values across most assays and showed the greatest increase in FRAP over storage, suggesting a significant impact. Further research is necessary to identify the specific components responsible for the observed increase in antioxidant activity and how this process affects the health benefits of fermented white bean homogenate in ice cream. Storage time generally led to an increase in antioxidant activity as measured by ABTS and FRAP, with the exception of DPPH values. This suggests potential benefits from ongoing fermentation processes even during storage. The overall increase in antioxidant capacity observed in our study suggests potential health benefits for consumers, such as reduced cellular damage from free radicals.