Evaluation of the Effectiveness of Plant-Protein-Based Cleaning Agents in the Production of Industrial-Filtered Clarified Apple Juice

Abstract

Apple (Malus domestica Borkh.) juice is a globally popular beverage that is valued for its pleasing flavor, wide availability, and health benefits, including support for cardiovascular health and antioxidant properties. A critical element of the production process is the clarification procedure, which improves the product’s purity, visual appeal, and shelf stability by removing solids, colloids, and polyphenols. This study examines the efficacy of plant-based clarifiers, LittoFresh Liquid and FloaClair, in combination with three mineral agents—KlarSol30, GranuBent Pore-Tec, and Seporit Pore-Tec—on the quality of apple juice. The following analytical procedures were conducted: measurement of turbidity (NTU), color stability (ΔE*), transmittance at 440 nm, antioxidant capacity (FRAP), and total polyphenol content (TPC). The results showed that plant-based clarifiers were effective in reducing turbidity by up to 45% while improving transmittance levels by an average of 30% compared to untreated samples. Additionally, plant-based agents retained up to 20% more polyphenols and exhibited a 15% higher antioxidant capacity than traditional gelatin-based clarifiers.

1. Introduction

Apple juice is one of the most widely consumed fruit beverages worldwide. It is valued for its sensory attributes and potential health benefits [1]. Regular consumption has been associated with positive effects on cardiovascular health, oxidative stress reduction, and modulation of inflammatory markers due to its rich content of polyphenols and antioxidants [2,3]. However, the presence of suspended solids, pectin, starch, and proteins in pressed juice contributes to turbidity and viscosity, which can negatively impact its visual appeal and shelf stability in terms of the clarified, filtered apple juice [4]. To address consumer preferences and meet international food standards, apple juice undergoes a critical step in its processing: clarification [5].

The objective of clarification techniques is to remove suspended particles, enhance color stability, and improve organoleptic properties while maintaining the nutritional integrity of the juice. Conventional clarification methodologies encompass mechanical filtration, sedimentation, and centrifugation, frequently accompanied by enzymatic [6], chemical [7], or physical treatments [8]. Enzymatic clarification, particularly using pectinases, cellulases, and amylases, has become the preferred approach due to its efficiency in breaking down polysaccharides and reducing juice viscosity, thus improving yield and clarity [9,10].

Clarification is achieved through the combined use of negatively charged (silica, bentonite) and positively charged, highly surface-active, so-called clarifying agents [11]. These agents can be of mineral origin (e.g., bentonite), be of protein origin (e.g., casein, gelatin), or be synthetic materials (e.g., polymers, PVPP) [12].

The most prevalent of the positively charged agents is gelatin, a protein derived from collagen found in animal tissues, with other derivatives, such as chitosan, egg white, casein, and fish collagen, also being employed [11]. The number of consumers choosing vegan products has been increasing rapidly in recent years. The vegan food market was valued at USD 14.2 billion in 2018 and is predicted to reach USD 31.4 billion by 2026 [13,14]. This trend is prompting food companies to explore plant-based alternatives to traditional animal-based ingredients, such as gelatin, as demand for juices made exclusively with plant-based additives and processing aids is increasing [15]. In response to these demands, manufacturers are exploring strategies to meet both consumer and economic expectations.

Researchers are exploring alternative clarification methods, including the use of plant-based and mineral-fining agents. Vegan-friendly clarifiers, such as pea protein and other plant-derived proteins, have gained popularity as substitutes for traditional animal-based fining agents like gelatin or casein [16,17,18]. Additionally, mineral-based clarifiers, such as bentonite and silica sol, have been shown to be effective in removing colloidal haze and polyphenolic compounds [19]. The selection of a clarifier has been demonstrated to influence not only the clarity of the final product but also its color stability and sensory properties. Consequently, it is imperative to optimize treatment conditions to ensure the efficacy and consistency of the clarification process [20].

Moreover, individuals who follow a vegetarian or vegan diet, or those who abstain from animal-derived ingredients for cultural reasons, are also hesitant to choose products that contain animal-based additives. The increasing popularity of plant-based alternatives can be attributed to concerns regarding sustainability and ethical issues associated with animal proteins [21]. The efficacy of pea, potato, and wheat proteins is comparable, offering a vegan and environmentally friendly solution [22].

Several studies have been conducted on the utilization of plant proteins, with particular attention directed towards their application in the enhancement of quality in Moroccan red wines. Pea protein has been examined in this context, and the findings have indicated its potential as a substitute due to its ability to reduce bitterness, astringency, and oxidized tannins [23]. The use of vegetable proteins has been demonstrated to be effective in the clarification of muscatel juices [24] and the prevention of mad cow disease in sweet wines [25]. In the case of white wines, wheat gluten [26], grape seed flour [27], potato protein [28], and pea and soy proteins [29] have demonstrated comparable efficacy. Furthermore, cassava and rice starch have been shown to be effective in clarifying cashew juice [30]. In the purification of apple juice, red lentil protein isolates have exhibited a purification ability that is analogous to that of the conventional gelatin and bentonite pair. However, green lentils and pea protein have demonstrated a lower degree of efficiency [16]. These alternatives are in alignment with present-day consumer trends and have the potential to reduce the probability of allergic reactions or other health risks. Notably, these products are not subject to religious or cultural restrictions, a quality that enhances their adaptability in global markets. The development of advanced technologies, such as membrane filtration, high hydrostatic pressure, and pulsed electric fields, has led to the emergence of non-enzymatic methods for juice stabilization and clarification. These methods can be used in combination with enzymatic treatments to enhance product quality, reduce processing time, and minimize environmental impact [31].

A substantial proportion of the research is conducted within a laboratory setting, wherein the efficacy of the process and the characteristics of the product can be evaluated under controlled circumstances. To illustrate, parameters such as the turbidity (NTU), water-soluble solids (BRIX°), transmittance (T%), color (CIELab), antioxidant capacity, and polyphenol content of apple juice are subjected to analysis.

Nevertheless, it is not always evident that laboratory results accurately reflect the range of variability that may be encountered in an industrial setting. It is of considerable importance to simulate industrial conditions, particularly in the case of mixed or lower-quality raw materials, to identify the operational challenges and economic aspects of the manufacturing process. This guarantees that the final product will be stable and of high quality, even when manufactured using sub-optimal raw materials.

The objectives of this research are to evaluate the effectiveness of two types of plant-based clarification agents compared to gelatin in industrial apples and, furthermore, to study the effectiveness of positively charged plant proteins with negatively charged clarification agents. The results may not only provide a detailed insight into the efficiency of the clarifying processes and agents but may also contribute to the development of more sustainable and innovative technologies for the juice industry.

2. Materials and Methods

2.1. Apple and Apple Juice

The model of industrial conditions was created using enzyme-treated pressed juice, produced under industrial conditions, from mixed apple varieties, sourced from the AUSTRIA JUICE Hungary Kft. plant in Vásárosnamény, Hungary. The washed apples entered the plant through a floating channel, where, after sorting, the apples were chopped using a hammer crusher. The chopped apple was treated with Pectinex Ultra AFP (200 mL/1000 kg; Novozymes A/S, Bagsvaerd, Denmark) pectin-degrading enzymes for 1 h and, then, the juice was separated using a Flottweg-type belt press (Flottweg SE, Vilsbiburg, Germany). The pressed juice was used for the clarifying experiments. In the examination of the water-soluble solids (BRIX°), transmittance (T%), turbidity (NTU), antioxidant capacity (FRAP), and total polyphenol content (TPC), the unclarified sample was considered the control.

2.2. Clarification Agents

The clarification agents (

Table 1. Properties of the clarifying agents.

Code	Name of Agent	Properties	Surface Charge	Form	Recommended Dosage	Medium Dosage; mL/100 L (M)	High Dosage; mL/100 L (H)
K	KlarSol 30	alkaline, silica sol	−	Liquid	20–250 mL/100 L	135	250
G	GranuBent Pore-Tec	sodium bentonite, granules	−	Granules	35–75 g/100 L	55	75
S	Seporit Pore-Tec	calcium bentonite granules	−	Granules	100–200 g/100 L	150	200
E	ErbiGel Liquid	gelatine	+	Liquid	20–50 mL/100 L	35	50
L	LittoFresh Liquid	vegetable protein	+	Liquid	50–200 mL/100 L	125	200
F	FloaClair	pea protein isolate	+	Powder	20–60 g/100 L	40	60

) employed in the experiments, all provided by Erbslöh Geisenheim GmbH (Geisenheim, Germany) [32], consisted of a range of specialized products. The clarification agents included LittoFresh Liquid (L) and FloaClair (F), plant-protein-based agents; ErbiGel (E), gelatin-based clarifying agent; KlarSol 30 (K), 30% alkaline silica sol in liquid form; GranuBent Pore-Tec (G), sodium bentonite granule; and Seporit Pore-Tec (S), iron-free calcium bentonite granule. Additionally, Pectinex Ultra AFP, a liquid pectin-degrading enzyme (100 mL/1000 kg), was employed.

2.3. Spectrophotometer and Chemicals

The instrumental analysis of transmittance, antioxidant capacity, and total polyphenol content was conducted utilizing the Hitachi U-2900 spectrophotometer (Hitachi High Technologies Europe GmbH, Krefeld, Germany). All reagents were purchased in analytical grade from Sigma-Aldrich Chemical Co. (3050 Spruce Street, St. Louis, MO 63103, USA).

2.4. Methods

2.4.1. Volume of Sediment

The samples were prepared in 500 mL measuring cylinders. Half an hour after clarification, the sedimented volume on the side of the measuring cylinder was checked and converted into a percentage compared to the total 500 mL of the samples.

2.4.2. Water-Soluble Solids (Brix°)

Refraction was measured with a digital ATAGO DBX-55 refractometer (Saitama, Japan), which displayed the result in Brix°.

2.4.3. Transmittance (T%)

The transmittance percentage was measured with a spectrophotometer at 440 nm [33].

2.4.4. Color (ΔE*)

The color coordinates were determined in accordance with the C.I.E. LAB system, employing a digital colorimeter by Konica Minolta CR 410 (Konica Minolta Business Solutions Ltd., Mississauga, ON, Canada). The ΔE* value was calculated using the equation proposed by Lukács (1982) [34]:

$$\Delta E\ast =\sqrt{{{\left(\Delta L\ast \right)}^2\left(\Delta a\ast \right)}^2+{\left(\Delta b\ast \right)}^2}$$

(1)

The color difference serves to indicate the extent of the difference between two samples. In the case of a difference below 0.5, it is not noticeable. In samples with a difference between 0.5 and 1.5, the distinction is barely noticeable. In cases where the difference is between 1.5 and 3.0, it is noticeable. In samples with a range between 3.0 and 6.0, the difference is clearly visible. In samples with a difference above 6.0, the difference is a large one [34].

The ΔE* values were calculated to determine the color differences between apple juice samples treated with gelatin (E) and those treated with either LittoFresh Liquid (L) or FloaClair (F). These comparisons were made separately for each mineral-based clarification agent (KlarSol 30 (K), GranuBent Pore-Tec (G), and Seporit Pore-Tec (S)). This analysis offers insights into the impact of substituting gelatin with plant-based clarifiers on the coloration of apple juices.

2.4.5. Turbidity (NTU = Nephelometric Turbidity Unit)

To determine turbidity, I used a Hach 2100P Turbidimeter (HACH Company, Loveland, CO, USA), which displayed the Nephelometric Turbidity Unit (NTU) of the juice.

2.4.6. Antioxidant Capacity (FRAP)

The antioxidant capacity was determined using the ferric-reducing ability of plasma (FRAP) method, as described by Benzie and Strain (1996) [35]. The FRAP reagent was prepared by combining acetate buffer (pH 3.6), 2,4,6-tripyridyl-s-triazine (TPTZ), and FeCl₃ × 6H₂O in the appropriate proportions. The absorbance was measured at a wavelength of 593 nm after a five-minute incubation period. The results were expressed in ascorbic acid equivalent (mg AAE g⁻¹ L apple juice) using an ascorbic acid standard calibration curve. The calibration curve exhibited a range of 0.304 to 1.429, with an R² value of 0.998.

2.4.7. Total Polyphenol Content (TPC)

The total phenolic concentration was determined through the application of the Folin–Ciocalteu method, as originally described by Singleton and Rossi (1965) [36]. A volume of 1250 μL of Folin reagent (1:10 v/v Folin; distilled water) was added to the test tube, followed by the addition of 200 μL of methanol (4:1 v/v methanol; distilled water). Subsequently, 50 μL of the sample was added, followed by the addition of 1000 μL of sodium carbonate after one minute. Subsequently, the samples were maintained in a water bath at 50 °C for a period of five minutes. Lastly, the absorbance was measured at a wavelength of 760 nm. The results are expressed in gallic acid equivalents (mg GAE g⁻¹ L apple juice). The calibration curve exhibited a range of 0.112 to 0.588, with an R² value of 0.988.

2.4.8. Statistical Analysis

The data were analyzed statistically using IBM SPSS Statistics software, version 29.0.1.0 (IBM Corp., New York, NY, USA, 2023). Prior to conducting the analysis of variance (ANOVA), the conditions were verified through the implementation of two statistical tests: the Shapiro–Wilk test for the assessment of residual normality and Levene’s test for the evaluation of variance homogeneity. All statistical tests were conducted with a significance level of p = 0.05.

In instances where the ANOVA yielded statistically significant results, a series of pairwise comparisons was conducted. Tukey’s post hoc test was employed when the homogeneity of variance condition was met, whereas the Games–Howell post hoc test was utilized when the condition was not satisfied. These methods ensured the reliability of the comparisons and accounted for variations in data properties across factor levels.

To calculate the color difference (ΔE*) between apple juice samples, the CIELab color coordinates were employed and ΔE* was calculated using the 3D Pythagorean theorem. This approach permitted precise quantification of color differences, thus bypassing the necessity for statistical software analysis in this specific parameter.

The integration of these methodologies provided robust and comprehensive insights into the effects of clarification agents on apple juice quality attributes, including turbidity, clarity, antioxidant properties, and color stability.

3. Results and Discussion

3.1. Volume of Sediment

As demonstrated in Figure 1, the percentage of the sedimented fraction is shown to be highest when gelatine is used, regardless of the type of negatively charged clarification agent employed. However, within this category, the highest settled layer is obtained when silica (K) is combined with gelatine. Among the vegetable proteins, FloaClair (F) exhibited a significant difference in comparison to LittoFresh Liquid (L) only when used in combination with the mineral agent GranuBent Pore-Tec (G), with the higher amount of vegetable protein and the medium amount of bentonite (GF-MH).

3.2. Water-Soluble Solids (BRIX°)

As demonstrated in

Table 2. Water-soluble content of apple juices in Brix.

	E	L	F
	Klar Sol 30
MM	12.4 ± 0.1 a	12.2 ± 0.1 a	12.3 ± 0.0 a
HH	12.2 ± 0.0 a	12.2 ± 0.1 a	12.2 ± 0.1 a
MH	12.2 ± 0.0 a	12.2 ± 0.0 a	12.2 ± 0.0 a
HM	12.3 ± 0.1 a	12.3 ± 0.0 a	12.3 ± 0.1 a
	GranuBent Pore-Tec
MM	12.0 ± 0.0 b	11.8 ± 0.1 a	11.9 ± 0.1ab
HH	11.9 ± 0.0 a	11.8 ± 0.1 a	11.8 ± 0.1 a
MH	11.9 ± 0.1 a	11.9 ± 0.1 a	11.9 ± 0.1 a
HM	11.9 ± 0.0 a	11.9 ± 0.1 a	11.8 ± 0.1 a
	Seporit Pore-Tec
MM	11.9 ± 0.1 ab	11.8 ± 0.1 a	12.1 ± 0.1 b
HH	11.8 ± 0.1 a	11.9 ± 0.1 ab	12.0 ± 0.1 b
MH	12.0 ± 0.1 a	11.9 ± 0.1 a	12.1 ± 0.1 a
HM	11.9 ± 0.1 a	11.9 ± 0.1 a	12.0 ± 0.1 a

Each value is expressed as mean ± standard deviation. The presence of different letters indicates that the groups in question are significantly different. Lower-case letter: comparison of proteins at fixed mineral clarification agents and concentrations.

, the Brix values exhibited a narrow range, with a variation of less than 1 percentage point between 11.77% and 12.37%. The results of the statistical analysis confirmed a significant effect of the mineral-based clarification agent (F(2;101) = 250.692; p < 0.001), the protein-based clarification agent (F(2;101) = 8.586; p < 0.001), and the interaction between them as well (F(4;101) = 8.205; p < 0.001). In industrial practice, a Brix value of 13% is considered standard for raw materials. However, this standard was not met by the samples under investigation, likely due to the ripening stage of the apples at the time of harvest.

Within the Klar Sol and GranuBent groups, no significant differences were observed in the Brix values of the samples. However, differences were found between samples, where Seporit was used, in the case of SL-MM and SF-MM, as well as between SE-HH and SF-HH.

Statistically, equivalent results to the control were found in the case of the following samples: GE-MM, GE-HH, GE-MH, GE-MH, GL-HM, GF-MM, GF-MH, SE-MM, SE-MH, SE-HM, SL-HH, SF-MM, SF-HH, SF-MH, SF-HM. In comparison to the control samples, the following samples exhibited a lower water-soluble solid content: GL-MM, GL-HH, GL-MH, GF-HH, GF-HM, SE-HH, SL-MM, SL-MH, SL-HM.

3.3. Transmittance (T%)

The method for determining the intensity of the yellow color in apple juice is the transmittance percentage at 440 nm, the results of which are shown in

Table 3. Statistical analysis of T%.

	E	L	F
	Klar Sol 30
MM	7.6 ± 0.6 a	9.6 ± 0.6 b	11.2 ± 0.6 c
HH	7.9 ± 0.6 a	6.9 ± 1.0 a	8.9 ± 0.0 b
MH	10.1 ± 0.6 c	7.5 ± 0.6 b	5.7 ± 0.6 a
HM	6.8 ± 1.0 a	6.4 ± 1.0 a	11.4 ± 0.6 b
	GranuBent Pore-Tec
MM	29.9 ± 0.6 c	9.2 ± 0.0 a	24.5 ± 0.6 b
HH	21.5 ± 0.6 b	25.5 ± 0.6 c	7.9 ± 0.6 a
MH	18.4 ± 0.6 b	13.5 ± 0.6 a	13.7 ± 0.0 a
HM	16.8 ± 0.6 b	23.6 ± 0.0 c	9.3 ± 0.6 a
	Seporit Pore-Tec
MM	16.5 ± 0.6 c	6.7 ± 0.0 a	8.9 ± 0.0 b
HH	14.4 ± 0.0 a	11.6 ± 0.0 a	12.6 ± 0.6 a
MH	14.9 ± 0.6 c	12.0 ± 1.0 b	9.0 ± 1.0 a
HM	13.3 ± 1.0 a	16.5 ± 1.0 b	11.7 ± 1.0 a

Each value is expressed as mean ± standard deviation. The presence of different letters indicates that the groups in question are significantly different. Lower-case letter: comparison of proteins at fixed mineral clarification agents and concentrations.

.

It can be observed that all samples have higher T% values (p < 0.001) compared to the control sample (1.87 ± 0.1%). For the GranuBent (G) bentonite-based samples, exceptionally high (favorable) values were obtained. The results of the statistical analysis confirmed a significant effect of the mineral-based clarification agent (F(2;101) = 49.317; p < 0.001), the protein-based clarification agent (F(2;101) = 7.216; p < 0.01), and the interaction between them as well (F(4;101) = 3.878; p < 0.01). Statistical analysis also indicates that gelatine is no longer performing better since vegetable proteins frequently resulted in higher transmittance values at 440 nm.

The combination of bentonite and gelatin was found to be particularly effective in combination with ultrafiltration; the control sample value increased from 48.5 T% to 76.2 T%. With the bentonite–gelatin combination, they achieved a maximum of 60.1 T%. In their experiment, the standard was set at 40 T% based on the literature [37]. In the case of the McIntosh variety, after purification by electroflotation, only 43 T% could be achieved with a 30 s treatment at 20 mA/cm² without gelatin with the addition of 200 mg/L, 63.5 T% [38].

3.4. Color

The calculated ΔE* values, based on the C.I.E. LAB system, indicate notable differences in color between apple juice samples clarified with gelatin and samples clarified with either one of the plant-based agents.

The results showed that the color deviations were highly dependent on the type and concentration of the clarification agent used (

Table 4. ΔE* values calculated between samples with gelatin and plant-based proteins.

	Klar Sol 30		GranuBent Pore-Tec		Seporit Pore-Tec
	L	F	L	F	L	F
MM	2.95	3.11	9.63	9.11	9.28	8.57
HH	6.77	5.85	11.05	9.74	6.05	3.51
MH	11.38	10.11	10.95	7.46	10.08	7.41
HM	4.52	3.73	8.71	6.78	5.96	3.91

). For Klar Sol 30, the values ranged from 2.95 to 11.38, indicating a good to very good visible difference in most cases while, for GranuBent Pore-Tec, the values ranged from 6.78 to 11.05, falling into the very good visible category. The Seporit Pore-Tec treatments resulted in ΔE* values ranging from 3.51 to 10.08, also showing significant color differences. It can be seen that the difference between KL-MM and KE-MM (2.95) is noticeable while ΔE* values between 3 and 6 (e.g., KF-MM, KF-HH, KL-HM, SF-HH) showed already well-visible differences. Overall, the effect of the mineral derivative was found to be significant as it had a large effect on the color differences between the plant- and animal-protein-treated samples. Furthermore, it was observed that when greater amounts of protein-based agents were added, the differences also became larger. Thus, color is affected not only by the protein but also by the interaction of protein and mineral-based clarification agents.

In the study of the effectiveness of sepiolite, for which silicic acid, gelatin, and bentonite were used, it was found that the combination of sepiolite, gelatin, and silicic acid had the best effect on the clarity factor of apple juice and the worst value was obtained with the use of bentonite alone [39].

3.5. Turbidity (NTU)

The Nephelometric Turbidity Unit (NTU) values suggest that the application of bentonite-based agents resulted in superior outcomes (Figure 2). The findings indicate that turbidity reduction was most effective when both the mineral (negatively charged) and protein (positively charged) agents were used at medium concentrations (MM). When higher doses of protein (MH, HH) were applied alongside medium or high doses of minerals, turbidity reduction efficiency was less pronounced, likely due to oversaturation effects. The samples that demonstrated the most efficacious outcomes were KL-MM and GF-MM, which exhibited the lowest turbidity values.

It was observed that samples made with gelatine appeared to contain fewer unwanted components and were more translucent. When vegetable protein was used, many samples performed at the same low efficiency as the control (KL-MM, KF-MM, KF-HH, SL-HM, SF-HH; p = 1.000 in all cases) and, in some cases, the samples significantly exceeded the turbidity value of the control (KL-HM, KF-MH, GL-HH, GF-MM, GF-HH, SL-MM, SL-HH, SF-MH). This suggests that these combinations were not successful in terms of turbidity.

3.6. Antioxidant Capacity (FRAP)

The findings indicate that plant-based clarifying agents may offer a superior means of preserving polyphenols, as evidenced by elevated ferric-reducing antioxidant power (FRAP) values. Statistical analyses demonstrate significant differences (p < 0.05) among the treatments (Figure 3). The results suggest that, while medium doses of clarifiers (MM) achieved a balance between clarity and polyphenol retention, increasing protein doses (MH, HH) resulted in greater losses of antioxidant compounds due to stronger binding and precipitation. The samples that demonstrated the greatest efficacy were SL-MM and KF-HH, which exhibited the highest antioxidant capacities. In comparison with the control sample (47.4 mgAAE/L), the following samples did not demonstrate a statistically significant difference in antioxidant capacity: GL-HH (p = 0.572), GF-MH (p = 0.136), SE-MM (p = 1.000), and SL-HH (p = 0.97), SL-MH (p = 1.000), SL-HM (p = 1.000), SF-HM (p = 0.136). The remaining combinations exhibited a significant increase in antioxidant capacity compared to the control. Oszmianski et al. (2011) [40] examined the impact of enzyme treatment on the antioxidant capacity of apple juice (‘Shampion’ variety) during storage. In comparison with the control sample (150 mgAAE/L), the treated samples exhibited a decrease in concentration ranging from 136 to 158 mgAAEsl when subjected to various enzymes. Notably, the KE-MM sample yielded the highest value of 111.14 mgAAE/L, which is also lower than the lower limit of 136 mgAAE/L reported in the referenced source. These findings imply that the diverse enzymatic treatments exert a more pronounced effect on the antioxidant capacity than the clarification procedures employed.

3.7. Total Polyphenol Content (TPC)

The findings of this investigation indicate that negatively charged clarifiers have a greater impact on polyphenol retention compared to their positively charged counterparts. Statistical comparisons indicate significant differences (p < 0.05) in polyphenol preservation across treatments. The findings confirm that polyphenol retention was optimal when both clarifier types were applied at medium concentrations (MM), whereas increasing protein levels (MH, HH) resulted in excessive polyphenol removal, reducing the overall antioxidant benefits of the juice (Figure 4). The best-performing samples were GL-MM and KF-MM, which retained the highest polyphenol content. In comparison with the control sample, no significant differences in polyphenol content were observed in the following samples: KF-MM (p = 0.999), KF-HM (p = 0.998), GE-MM (p = 0.134), GL-HH (p = 1.000), GL-HM (0.072), GF-MM (0.337), SE-MM (0.967), SE-HH (0.110), SE-MH (1.000), SE-HM (0.134), SL-MH (p = 0.665), SL-HM (1.000), SF-MH (1.000). The remaining combinations all resulted in significantly higher polyphenol concentrations.

The efficacy of three plant-clarifying agents, red lentil, green lentil, and green pea protein, was investigated in the clarification of apple juice at concentrations ranging from 60 to 900 mg/L. Gelatin–bentonite treatment was used as a control at the same concentration. The results showed that the 300 mg/L concentration was the best treatment for all agents and, at this concentration, red lentil showed the highest clarification efficiency in terms of both flocculation and sedimentation. Regarding TPC content, the lowest value was for the juice treated with the red lentil agent (266 μgGAE/mL) while the values were higher for the green lentil and green pea agents (295 μg GAE/mL). The effect of red lentil protein was similar to that of the gelatin–bentonite-treated sample in terms of clarification efficiency and TPC. This study proposed red lentil protein as a promising alternative to commercial fining agents in the fruit juice industry [16].

In the case of pomegranate juice, compared to natural sedimentation (keeping the juice at 2 °C for 16 h), protein-based fining agents were found to be more effective in reducing total phenolic content and the number of hydrolyzable tannins that cause turbidity [41]. The process is based on the fact that proteins can form water-insoluble complexes with polyphenols through different mechanisms of action. The main mechanisms observed are electrostatic interactions between positively charged proteins and negatively charged phenolic compounds [42].

4. Conclusions

This study investigated the use of plant-based and traditional clarifying agents in the clarification of apple juice, with the objective of evaluating the impact of different treatments on the final product’s clarity, color, and antioxidant properties. This study’s findings indicated that gelatin-based combinations, particularly those containing silica, exhibited the most effective clarification results as they resulted in higher sedimentation volumes and enhanced turbidity reduction. Notably, plant-based proteins exhibited noteworthy outcomes, presenting a viable substitute for conventional animal-derived clarifiers. From a sustainability standpoint, the rising demand for plant-based solutions in food processing reinforces the necessity of the further exploration of these alternatives. While gelatin maintains its effectiveness, plant proteins could emerge as a competitive option if optimized formulations are developed.

The findings of the present study indicate that the most effective clarifying agent combination is plant-based (LittoFresh Liquid and FloaClair) in combined use with bentonite-based agents (GranuBent Pore-Tec or Seporit Pore-Tec). This combination has been shown to reduce turbidity, improve transmittance, and retain higher polyphenol and antioxidant levels in comparison with traditional gelatin-based clarifiers. The optimal action concentration was identified as the medium levels (MM: medium mineral and medium protein dosage), which exhibited the optimal balance between clarity and polyphenol retention. Higher doses of protein-based agents led to excessive polyphenol removal, thereby diminishing the antioxidant benefits of the juice.

Future research should focus on refining these alternatives, evaluating their sensory impacts, and scaling up their application in industrial fruit juice production. This study contributes to the ongoing efforts toward more sustainable and efficient juice clarification methods, aligning with consumer preferences and industry trends.