Introduction

The production of fruit juices is often associated with the loss of many compounds with potential health-promoting properties contained in the starting material (fruits). This loss may occur in all processes applied during production, including washing, grinding, depectinization, pressing, clarification, filtration, pasteurization, and concentration [1, 2]. In these processes, the material is subjected to thermal treatment as well as the action of enzymes, including oxidizing and pectinolytic enzymes. As a result, the final product is poorer in valuable ingredients than the starting material [1, 3, 4]. Examples of such compounds are anthocyanins and ellagitannins, which are present in significant amounts in raspberry, blackberry, wild strawberry, and strawberry fruits [5,6,7,8]. These compounds are attributed properties that can prevent the occurrence of numerous diseases, including diabetes, cardiovascular diseases, and cancer [9,10,11,12].

According to literature data, in the production of raspberry or blackberry juice, ellagitannins are largely (60–70%) retained in pomace, and those that are transferred into the juice are low-molecular-weight ellagitannins [3, 13]. This indicates that these compounds are quite strongly bound to the fruit matrix, namely the seeds and skins, and technological processes based on thermal or enzymatic treatments may either reduce or increase their transfer to the juice.

In the processing of berries, thermal processes that may affect the content of ellagitannins and anthocyanins include, among others, the processes of depectinization and enzymatic clarification, as well as the pasteurization process. The use of enzymatic treatment in the processing of berries or cloudy juice usually takes place at moderately high temperatures of 40–50 °C, but it can take several hours, and thus often results in the degradation of certain polyphenols. The thermal loss of particular compounds may be partially compensated by the action of pectinolytic enzymes, which, by loosening the matrix, contribute to the release of polyphenolic compounds [14]. The stages of depectinization of the pulp or enzymatic clarification of cloudy juices are commonly used in processing, as they facilitate both the pressing of the juice and its filtration, thereby increasing the yield of the product. The facilitation of filtration results from, among other things, the degradation of hydrocolloid compounds presents in cloudy juices [15]. In the case of pasteurization, the temperatures used are in the range 60–95 °C, with the process time depending on the temperature. Currently, in industrial practice, to maintain microbiological safety and ensure the high quality of juices, rapid pasteurization lasting 20–120 s is used, and the finished juice is poured under aseptic conditions. Such a short pasteurization time usually contributes to the preservation of health-promoting compounds [16, 17]. According to the data provided by Azofeifa et al., a process of fast pasteurization of blackberry juice lasting 10–15 s, at temperatures of 75 and 92 °C, did not affect the content of ellagitannin compounds; however, it caused a decrease in the content of anthocyanins [18]. Similar results were obtained by Hager et al. who reported that pasteurization consisting in the heating of blackberry juice in a bottle to 90 °C and its spontaneous cooling did not affect the content of ellagitannins, but led to a 20% decrease in the content of anthocyanins [2, 3].

Another unit process that may affect the content of polyphenolic compounds in the juice is filtration, which follows the chemical treatment of the juice with fining agents (gelatin, bentonite, silica sol, diatomaceous earth) or enzymatic clarification. When filtration is used after these stages, the content of polyphenols in the juice can be reduced by more than 50% compared with the non-clarified juice [19]. Ellagitannins are particularly susceptible to removal in the filtration process: according to research conducted by Acosta et al., the use of appropriate filtration membranes leads to almost 100% retention of these compounds on the filtration barrier [20]. The factors favoring the retention of ellagitannins on the partitions include the relatively large size of ellagitannin molecules, as well as their ability to form complexes with proteins, hydrocolloids, and other components of fruit juice cell walls that are difficult to filter [15, 21].

The present study examined how individual unit processes, namely enzymatic clarification, filtration, and pasteurization, affect the content of the main ellagitannins (lambertianin C and sanguiin H-6) and the main anthocyanins (cyanidin-3-sophoroside and cyanidin-3-rutinoside) in raspberry juice. This knowledge is of particular importance for the production of raspberry preserves that retain the highest content of health-promoting ingredients.

Materials and methods

Plant material

The research material consisted of frozen red raspberry fruits (Rubus idaeus L.) purchased in a local market (Łódź, Poland). Before processing, the fruits were stored in commercial packaging at a temperature of –18 °C.

Juice production

The production of raspberry juice was carried out in accordance with the methodology described in an earlier publication by Sójka et al., with minor modifications [22]. Briefly, 1 kg of fruit, after thawing and grinding, was heated to 50 °C and subjected to enzyme treatment using Rohapect 10L (AB Enzymes, Darmstadt, Germany). In this process, which lasted 1 h, a dose of 0.2 mL of enzyme per 1 kg of fruit pulp was used. The pulp was then pressed using a laboratory hydraulic press (Lodz University of Technology, Poland) for 5 min at 100 bar. The pressed cloudy juice, in an amount of about 900 mL, was then analyzed to determine the impact of the clarification and filtration process. For analytical purposes, juice samples were taken before subsequent processes.

Clarification and filtration

The juice clarification process was carried out at a temperature of 45 °C for 1 h using an enzyme dose of 80 mg per liter of juice (Rohapect 10L, AB Enzymes, Darmstadt, Germany). The filtration process was carried out using a Hobrafilt type S40N cellulose filter screen with the following parameters: thickness 3.6 mm; retention 5 microns; flow capacity 420–540 L/m2/min (Hobra-Školník S.R.O., Broumov, Czech Republic). 300 mL of juice was filtered using a Büchner funnel (diameter 7 cm) at a pressure of 800 mbar. The juice obtained was then used for analytical purposes and to study the impact of pasteurization. To determine whether ellagitannins were retained on the filtration partition, the precipitate and the partition were washed with 100 mL of a solution containing acetone, water, and formic acid in a volume ratio of 70:30:0.05 (v/v/v), respectively.

Juice pasteurization

The effect of the pasteurization process on the stability of ellagitannins and anthocyanins was investigated in filtered juice (clear juice). Due to the low content of ellagitannins after filtration, and in particular the low content of lambertianin C, this juice was enriched with an ellagitannin preparation. The preparation used contained 60% ellagitannins, and was produced by the method described by Klewicka et al. [23]. 20 mg of the preparation was added to 100 mL of clarified juice, obtaining an average concentration of ellagitannins at a level of 150 mg/L. The maintenance of such a level of ellagitannins was critical from the analytical point of view, as the signal obtained, namely the height of the peaks corresponding to the main ellagitannins, was at a level ensuring proper repeatability, and any losses resulting from the pasteurization process did not reduce the content of ellagitannins to values below the LOQ.

The pasteurization process was carried out in four variants, using the following temperatures and pasteurization times: HTLT—high temperature, long time (85 °C, 60 s); HTST—high temperature, short time (85 °C, 20 s); MTLT—mild temperature, long time (65 °C, 60 s); MTST—mild temperature, short time (65 °C, 20 s) [17]. Pasteurization was carried out in a water bath at a temperature 3 °C higher than the defined pasteurization temperature. Before pasteurization, the juices were poured into glass vials with a volume of 3 mL, and these were tightly closed with a stopper and placed in the water bath. At the same time, a vial containing juice and equipped with a temperature sensor was placed in the bath. The pasteurization time was measured from the moment at which the defined temperature was reached. After pasteurization, that is, after 20 s or 60 s, the vials with juices were placed in a water bath at 20 °C for rapid cooling. Changes in juice temperature during the pasteurization process, depending on the variant used, are shown in Fig. 1. The pasteurized juices were immediately analyzed using HPLC.

Fig. 1
figure 1

Temperature changes in the pasteurization process HTLT (85 °C, 60 s), HTST (85 °C, 20 s), MTLT (65 °C, 60 s), MTST (85 °C, 20 s)

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Analysis of ellagitannins and anthocyanins

The determination of ellagitannins and anthocyanins was performed in accordance with the method described in an earlier publication and using the same apparatus [22]. Briefly, a Knauer Smartline chromatograph (Berlin, Germany) equipped with a degasser (Manager 500), a pump (P1000), an autosampler (3950), a thermostat and a PDA detector (2800) was used for the analysis. A Gemini C18 110 Å column of 250 mm × 4.6 mm i.d., 5 μm (Phenomenex, Torrance, CA) was used for the separation. Compounds were gradient eluted using phases: phase A (0.05% (v/v) phosphoric acid–water) and phase B (83:17 (v/v) acetonitrile–water with 0.05% phosphoric acid). Ellagitannins were detected at a wavelength of 250 nm, and anthocyanins at 520 nm. Before the analysis, the juice samples were diluted 1:1 (v/v) with acetone, sonicated for 10 min in a water bath, centrifuged for 5 min at 12,000 g, and transferred to chromatographic vials. In the case of the acetone solution obtained from washing the filtration barrier, it was diluted 1:1 (v/v) with phase A and centrifuged before analysis.

Statistical analysis

The results obtained were subjected to statistical analysis. The effect of the enzymatic clarification, filtration, and pasteurization processes on the content of ellagitannins and anthocyanins was examined using analysis of variance (ANOVA) and Duncan’s post hoc test at the significance level p ≤ 0.05. Statistical analysis was carried out using Statistica Version 12 software (StatSoft, Tulsa, USA).

Results and discussion

Impact of enzymatic treatment

The contents of lambertianin C, sanguiin H-6, three conjugates of ellagic acid, and ellagic acid were determined in the tested raspberry juices. Other ellagitannins, which were present only in trace amounts, were omitted from the calculations. According to previous studies, raspberry juice may contain derivatives of the two first-mentioned ellagitannins. The contents of two main anthocyanins, cyanidin-3-O-sophoroside and cyanidin-3-O-rutinoside, were also determined. As in the case of ellagitannins, other anthocyanins were omitted, due to their presence only in trace amounts and their poor chromatographic separation.

The clarification process used in this study consisted of two stages: enzymatic juice processing and filtration. The use of enzymatic treatment is important as it supports the filtration process by degrading the hydrocolloids [15]. Table 1 and Fig. 2 show the effect of enzymatic treatment on the content of the tested compounds. Based on the sums of the tested polyphenolic groups, the contents of ellagitannins and anthocyanins did not change as a result of this process (action of the pectolytic enzyme for one hour). This was also confirmed by statistical analysis. However, when individual compounds are considered, slight changes in the content of ellagic acid conjugates can be observed. The total content of both ellagic acid conjugates 1 and 2 decreased by 3.8 mg/L, while the content of conjugate 3 increased by 5.5 mg/L. These results suggest that these compounds may be interconverted during enzymatic treatment. In the case of the main ellagitannins (lambertianin C and sanguiin H-6) and anthocyanins (cyanidin-3-sophoroside and cyanidin-3-O-rutinoside), no significant effect of the enzymatic treatment process on their contents was observed. These results are similar to those obtained by Hager et al. [3], who found no decrease in the content of ellagitannins in blackberry pulp subjected to blanching and enzymatic treatment lasting 1 h at 40 °C. However, it should be noted that that study concerned the processing of pulp, not cloudy juice. Similar results were also observed in the case of enzymatic treatment of guava fruit, where the content of ellagitannin derivatives such as vescalagin, geraniin, pedunculagin, and castalagin did not decrease during the process [24]. In the case of anthocyanins, research by Hager et al. [2] showed a 34% decrease in anthocyanin content in blackberry pulp subjected to blanching and enzymatic treatment. In turn, research conducted by Dawarcı et al. on pomegranate juice showed that the process of depectinization and clarification with bentonite, kieselsol, and gelatine resulted in only a 7% decrease in anthocyanin content [25]. Our research clearly shows that the enzymatic treatment process before filtration did not significantly affect the content of ellagitannins and anthocyanins in cloudy raspberry juice. However, it should be noted here that the enzymatic process can have both a negative effect by degrading polyphenols, and a positive effect by increasing their extraction into the juice as a result of release from cell walls. According to research by Buchert et al., another extremely important factor is the profile of the enzyme preparations used, which, in addition to their activity against polygalacturonic acid, are also characterized by activity against compounds containing galactose, xylose, glucose, and other simple sugars [14].

Table 1 The influence of processes of enzymatic treatment of cloudy juice and filtration on the content of ellagitannins and anthocyanin content in raspberry juice
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Fig. 2
figure 2

The influence of the clarification and filtration process on the content of Ellagitannins (ET) and anthocyanins (ACY) in raspberry juice. The results above the bars marked by the same letter do not differ statistically at p < 0.05

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Impact of filtration

The study showed that the filtration process had a critical impact on the retention of the studied polyphenols in the juice. Table 1 and Fig. 2 also show a comparison of the content of ellagitannins in the juice before and after the filtration process. Based on analysis of the sum of the tested compounds, it is observed that the filtered (clear) juice retained only 18.2% of the ellagitannins that were present in the juice before filtration. Lambertianin C was completely retained on the filtration barrier, while sanguiin H-6 was transferred to the juice in an amount of 18.2%. In the case of ellagic acid and its conjugates, the amount transferred to the filtered juice was 49–87%. Retention on the filtration partition was confirmed by analysis of the acetone extract obtained from its washing. In this extract, 83% of lambertianin C and 57% of sanguiin H-6 were recovered, and total ellagitannin recovery was 60%. Summing the recovery of the tested compounds in the filtered juice and the washing extract, over 83% recovery of lambertianin C, 71% recovery of sanguiin H-6, and 95–101% recovery of ellagic acid and its conjugates were obtained. These results clearly indicate that during juice production, ellagitannin compounds may be largely removed during the filtration process. Comparing the two main ellagitannins, it was shown that lambertianin C, a trimeric ellagitannin with a high molecular weight, was subject to much higher retention than dimeric sanguiin H-6. It can be concluded from this that high-molecular-weight ellagitannins will be more susceptible to retention on filtration barriers. In the study by Hager et al., blackberry juice subjected to a clarification and filtration process had more than 60% lower content of ellagitannins than the juice before clarification, and the losses of lambertianin C and sanguiin H-6 were 59.5% and 73%, respectively [3]. The authors of that study did not specify how the juice was separated from the sediment formed during clarification. Numerous studies clearly show that the type of filtration, namely the use of conventional filtration (> 10 μm), micro-filtration (1–10 μm) or ultrafiltration (0.01–1 μm), has a significant impact on the content of certain groups of polyphenols in fruit juices [15, 16]. The tendency of ellagitannins to be retained on filtration membranes was used in research by Acosta et al., where lambertianin C and sanguiin.

H-6 were effectively isolated from blackberry juice and separated (to a degree of 90%) from anthocyanins [20]. From a technological point of view, the purpose of the clarification and filtration process is to remove undesirable turbidity and increase the color stability of the juice during storage. The formation of turbidity in juices is closely linked to the presence of polyphenols, particular those with a high molecular weight, which can form complexes with proteins and, thus, produce turbidity [16]. On the other hand, compounds with beneficial health properties are removed during this process [10].

The filtration process was not observed to have any significant effect on anthocyanins. The recovery of the tested anthocyanins in the filtered juice was 101%. Analysis of the acetone extract from the washing of the filtration partition showed the presence of an additional 10% of anthocyanins, which gives 111% total recovery of these compounds. The positive balance of anthocyanins is difficult to explain, but it may result from additional extraction of these compounds with a water–acetone solution from a filter cake consisting of water-insoluble pulp and skin fractions of raspberry fruit. Considering anthocyanins and ellagitannins as the most important groups of polyphenols in the tested juice, the total loss of these compounds as a result of enzymatic treatment and filtration was 35%. In a similar study of pomegranate juice by Farahmand et al. [26], it was shown that the process of enzymatic treatment and sedimentation caused a decrease in the polyphenol content by 25% in clarified juice, while the decrease in anthocyanin content was at a similar level. In turn, in a study by Fisher et al. [15], it was indicated that the process of enzymatic treatment and filtration did not significantly affect the content of polyphenols, including anthocyanins. However, those authors indicated that the application of the micro-filtration process, with the use of 0.2 µm ceramic membrane filters, can significantly reduce the content of these compounds.

Impact of pasteurization

Due to the low content of the main ellagitannins in the filtered juice, experiments on the effect of thermal processing of the juice on the content of ellagitannins were carried out on juices fortified with ellagitannin extract produced from pomace as described by Klewicka et al. [23]. The contents of lambertianin C and sanguiin H-6 in fortified unpasteurized juice were 68 and 65 mg/L, respectively, and the contents of conjugates and ellagic acid were 14.6 and 2.3 mg/L, respectively (Table 2). The content of anthocyanins was several times higher: the juice before pasteurization contained 421 mg/L of cyanidin-3-sophoroside and 97 mg/L of cyanidin-3-rutinoside.

Table 2 Influence of the pasteurization process on the stability of ellagitannins and anthocyanins in clarified, fortified with ellagitannins, raspberry juice
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Tests of the content of the analyzed compounds and statistical analysis of the results showed that the pasteurization conditions used in most variants did not have a significant effect on their content (Table 2, Fig. 3). Based on Duncan’s test, the only differences were observed for the two ellagic acid conjugates numbered 2 and 3: conjugate 2 showed a slight increase after pasteurization and conjugate 3 showed a slight decrease. However, it should be noted that the difference in content is small (1 mg/L) and may result from measurement error. As regards the content of the main ellagitannins—sanguiin H-6 and lambertianin C—a slight downward trend was observed for the high-temperature (HT) variants, regardless of process time. For both of these compounds, the average decrease for the HTLT and HTST variants was 5.5%. A downward trend in the case of high-temperature pasteurization was also observed for anthocyanins. In this case, the average decrease in the content of these compounds was 8%. However, the aforementioned downward trends were not confirmed by the statistical analysis; therefore, it can be concluded that the pasteurization process in the tested high-temperature conditions has only a minimal effect.

Fig. 3
figure 3

Influence of the pasteurization process on the stability of ellagitannins and anthocyanins in clarified, fortified with ellagitannins, raspberry juice. The results above the bars marked by the same letter do not differ statistically at p < 0.05

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The above results confirm the conclusions of Hager et al. [3], who found that ellagitannins in the environment of blackberry juice were thermostable compounds. In their study, pasteurization lasted a longer time, because it was carried out by steam-heating bottles filled with juice, with a volume of approximately 177 mL, to reach a temperature of 90 °C. The high stability of ellagitannins during juice pasteurization is also confirmed by research by Rojas-Garbanzo et al. [24], in which processes carried out on guava juice under the conditions T = 71 °C, t = 4 s and T = 60 °C, t = 8.2 min resulted in a 5% and 10% decrease in the content of ellagitannins, respectively, but the differences in content compared with unpasteurized juice were not statistically significant. In turn, research conducted by Muhacir-Güzel et al. on pomegranate juice showed increases in the content of selected ellagitannins (from 1 to 52%) after the pasteurization process (T = 85 °C, 10 min), but the authors did not indicate the factors responsible for this increase [27]. In a study by Mena et al., pasteurization of pomegranate juice using the HTST (90 °C, 5 s) and LTLT (65 °C, 30 s) variants resulted in decreases (between 10 and 28%) for ellagitannins such as punicalin and punicalagin-like, and in the case of the LTLT variant, a several-fold increase in punicalagin was observed, which the authors attribute to the degradation of other high-molecular-weight ellagitannins [28]. In the same study, the decrease in anthocyanin content in pomegranate juices during pasteurization in the HTST and LTLT variants was 7% and 5%, respectively, but these values were statistically insignificant [28]. According to those authors, the lack of degradation of anthocyanins results from the short pasteurization time, while greater losses, reaching almost 100%, occur during the storage of juices. In the case of anthocyanins, Hager et al. [2] indicated that the process of pasteurization of blackberry juice caused a decrease in content by 22%, the process being carried out in the same way as in the study on ellagitannins [3].

The results presented above clearly indicate that the enzymatic treatment of cloudy raspberry juice carried out for 1 h at 45 °C does not reduce the content of the main ellagitannins and anthocyanins. A critical part of the processing of cloudy raspberry juices is the filtration step, which can cause retention of the most important ellagitannins present in the juice. The present study indicates that filtration on a cellulose filter with a nominal retention of 5 μm particles can effectively retain lambertianin C (100%) and sanguiin H-6 (82%), and the resulting juice is characterized by a low content of both. The filtration process did not affect the content of anthocyanins; their content in the filtered juice was equal to that in the juice not subjected to this process. The research also showed that a rapid pasteurization process carried out for 20 or 60 s at temperatures of 65 and 85 °C did not significantly affect the content of ellagitannins and anthocyanins, with a slight downward trend observed in the higher-temperature conditions. The data obtained are particularly important for manufacturers interested in the production of juices with a high content of bioactive compounds, in particular ellagitannins.