Abstract
The utilization of sea buckthorn pomace (SBP) is attracting growing attention since it is valuable industrial waste. This pomace can find usage as a functional ingredient of food because it contains bioactive, health-promoting components, but to our knowledge, few scientists have so far studied utilization of the antimicrobial activity of fruit pomace. The study aims are to broaden our knowledge of antioxidant and antimicrobial status of SBP by utilizing pomace as a functional apple juice ingredient and by monitoring the antioxidant capacity, the total polyphenol content and microbial changes that occur during the storage of juice samples. Our results of this study highlight that the importance of the utilization of SBP because the results reported here provide further evidence that SBP can contribute to increasing the content of valuable components in apple juice samples and inhibiting the growth of microorganisms during storage.
1 Introduction
In the food industry, sea buckthorn (Hippophaë rhamnoides L.) is a widely recognized source of bioactive components due to its content of antioxidant molecules. Fruit juice is produced in the largest quantity from sea buckthorn, so its production results in a very large amount of pomace (it contains the seeds and peels of the berry) (Beveridge et al., 2002; Kant et al., 2012). Fruit pomace contains many valuable components that are often underutilized, although in the last decade the utilization of by-products has become a key issue in the food industry.
Several researchers have addressed the issue of quantitative and qualitative characterization of the by-product produced during the processing of fruit (Arora et al., 2012; Bánvölgyi et al., 2020; Cossuta et al., 2007; Galanakis, 2012; Gonelimali et al., 2021; Jian-Hua et al., 2021; Kant et al., 2012; Nour et al., 2021; Rösch et al., 2003).
SBP contains the beneficial components found in whole berries, including most of its antioxidant compounds (Richa, 2012). As noted by Rösch and Kroh (2003), in the juice, the flavonols are transferred in larger quantities than proanthocyanidins, which predominantly remain in the pomace thanks to the effective hydrogen bonds between the cellular compounds of the pomace.
In their 2004 study, Rösch et al. found that 75% of the total antioxidant capacity of SBP extract was attributed to oligomeric proanthocyanidins, and significant sitosterol, tocopherol and carotenoid contents were measured. While most studies (Kitryte et al., 2017; Korekar et al., 2011; Kreps et al., 2021) focus on the most effective method of extracting antioxidant compounds from pomace, this study presents one of the possibilities for utilizing SBP extract.
This paper discusses the case of adding SBP extract into the apple juice and monitored the antioxidant components content and microbiology status of juice samples during 8 weeks of storage.
2 Materials and methods
2.1 Materials
The pomace from ‘Ascora’ sea buckthorn (Hippophaë rhamnoides L.) is produced in Szolnok (47° 10′ 29″ N; 20° 11′ 47″ E) during juice extraction in accordance with the industrial practice.
The chemicals, solvents and materials of the measurements were provided by Reanal Laboratory Chemicals Ltd, Sigma-Aldricht Chemie GmbH, and Merck Chemicals Hungary and Merck Life Science Ltd. For HPLC measurement, the solvents were gradient grade.
2.2 Sample production
After drying (in LP-322 oven at 60 °C to moisture content of 5 (m/m) %), the dried pomace was ground to a fine powder. For the recovery of polyphenolic compounds present in dry pomace sample, the solid-liquid extractions using 40 (v/v)% acetone was carried out (for 1 h in ultrasound bath – 35 kHz) (Furulyas et al., 2018). The SBP extract was centrifuged (5,000 g, 10 min) and the total acetone was evaporated by drying in an oven at 60 °C. The sea buckthorn extract for the apple juice samples production was obtained by diluting the dried residue with water after evaporation.
The apple juices (AJ) were prepared from apple concentrate (70 Brix° dry matter content, provided by Sió-Eckes Ltd.), which was diluted to 11.2 Brix° in the case of the control sample with water (100% apple juice, in accordance with FVM decree 152/2009, XI. 12) and with two different concentrations of SBP extracts in the case of the fortified apple juices with sea buckthorn pomace (SPA). Table 1 shows the dilution ratios of samples.
Composition and code of the samples
Sample Name: | AJ | L-SPA | H-SPA |
Pomace concentration (ml 100 mL–1) | 0 | 15 | 30 |
Apple concentrate (ml 100 mL–1) | 14 | 14 | 14 |
SBPE (ml 100 mL–1) | 0 | 43 | 86 |
Water (ml 100 mL–1) | 86 | 43 | 0 |
AJ: 100% apple juice, L-SPA: Low fortified apple juice with sea buckthorn pomace extract, H-SPA: High fortified apple juice with sea buckthorn pomace extract, SBPE: Sea buckthorn pomace extract.
The apple juice samples were pasteurized using traditional heat treatment in 80 °C water bath with a 15-min heating and holding time, followed by rapid cooling. The samples were stored for 8 weeks at 10 °C.
2.3 Analytical methods
During the 8-week storage period, the apple juice was sampled every two weeks and stored in a −80 °C freezer until the start of the analytical and microbiological measurements.
2.3.1 Analysis of total polyphenol content and antioxidant activity
During the 8 weeks, 5 samplings were performed, the TPC and FRAP values of apple juice samples were determined using spectrophotographic method (UV–visible spectrophotometer –Hitachi U-2900).
We followed the method used by Singleton and Rossi (1965) for total polyphenol content (TPC), which was determined by using Folin Ciocalteau reagent and the absorbance was set at 760 nm after 5 min of reaction. The results were expressed in mg equivalents of gallic acid (GAE) per L of juice (mg L−1).
The antioxidant capacity (Ferric Reducing Ability Power-FRAP) was used to estimate the total antioxidant capacity. FRAP values were calculated by the method of Benzie and Strain (1966). The absorbance was measured at wavelength of 593 nm and the FRAP values were expressed in mg equivalents of ascorbic acid (AAE) per L (mg L−1).
2.3.2 RP-HPLC measurement of polyphenol components
The identification and measurement of polyphenol components were carried out on the fresh samples, and it was taken at the end of storage using a Shimadzu HPLC system (Shimadzu Corporation), comprised of 4.6 × 150 mm, 3 µm particle size, C18 column (Phenomenex, Torrance, California, USA) on the separation of components. The mobile phase consisted of high purity water with 1% formic acid (solvent A) and acetonitrile with 1% formic acid (solvent B). For the determination of phenolics the applied gradient elution included the following: 0–10 min: 95% A/5% B; 10–30 min: 75% A/25% B; 30–35 min: 100% A/0% B; 35–35.5-min: 95% A/5% B; 35.5–45 min: 95% A/5% B. Absorbance measures were recorded at 280 and 310 nm. The flow rate was 1.5 ml min−1 and the data analysis was performed using LC Solution Software.
For identification of phenolic compounds, the retention time of standard molecules was applied: catechin 15.654 min, chlorogenic acid 15.953 min, epicatechin 16.752 min, rutin 18.282 min, quercetin-3-glucoside 18.842 min. The quantitative evaluation was based on external five points calibration curves of standards. The polyphenol compounds were expressed in mg L−1 juice.
2.3.3 Microbiological tests
Microbiological tests were implemented to determine the total viable counts (TVC) and the yeast and molds count (YMC) on initial and final samples of the storage experiment.
The standard spread plate technique was employed according to the MSZ EN ISO 4833-1:2014 standard to TVC measurements. The plates were incubated at 30 ± 1 C for 72 h. The colonies appearing on the plates were then counted for the calculation of the final loads.
For molds and yeasts analysis, the selective agar (Malt agar) was used with the horizontal method in accordance with MSZ ISO 21527-1:2013 standard. Petri plates were incubated at 25 °C for 3–5 days. Negative and positive controls were also performed.
After incubation, the total number of colonies was determined by manual counting and the bacterial and fungal counts in the samples were expressed CFU 100 µL−1.
2.3.4 Statistical analysis
All analyses were performed in triplicate and the results were evaluated as means ± standard deviations. Statistical significance was determined using the SPSS Statistics V21 program. The significance difference between the means was analyzed using One-way analysis of variance (ANOVA) and Tukey multiple comparisons. To predict the evaluated relationship between the results, Pearson's correlation analysis was used. Significance levels were set at P > 0.05.
3 Results and discussion
The results of TPC and FRAP are presented in Table 2. Content of total phenolic in AJ was 85 ± 2 – 94 ± 31 mg L−1, and its antioxidant activity was between 77 ± 6 – 88 ± 4 mg L−1. Limited literature data exist about individual phenolic content or antioxidant activity of commercial apple juice. Similar results were obtained in the experiment by Gliszczynska-Swiglo and Tyrakowska (2003), Kahle et al. (2005) and Ribárszki et al. (2022).
Results of total polyphenol content and antioxidant capacity, MEAN ± SD, the statistical results are shown by letter, the different letter indicates a significant difference between the samples
Week of storage | AJ | L-SPA | H-SPA |
mg L−1 | mg L−1 | mg L−1 | |
Total polyphenol content | |||
0 | 87 ± 4 a | 616 ± 36 b | 636 ± 19 b |
2 | 86 ± 5 a | 889 ± 45 c | 933 ± 37 c |
4 | 85 ± 2 a | 958 ± 15 cd | 1,026 ± 43 de |
6 | 90 ± 4 a | 899 ± 56 c | 1,023 ± 38 de |
8 | 94 ± 3 a | 912 ± 40 c | 1,069 ± 64 e |
FRAP-Antioxidant activity | |||
0 | 88 ± 4 a | 1,132 ± 86 d | 1852 ± 60 e |
2 | 85 ± 3 a | 1,013 ± 50 cd | 1855 ± 62 e |
4 | 79 ± 13 a | 959 ± 77 cd | 1,654 ± 144 e |
6 | 77 ± 7 a | 814 ± 82 c | 1788 ± 219 e |
8 | 79 ± 16 a | 537 ± 59 b | 1709 ± 161 e |
AJ: 100% apple juice, L-SPA: Low fortified apple juice with sea buckthorn pomace extract, H-SPA: High fortified apple juice with sea buckthorn pomace extract.
The results show a significant difference between the control apple juice and the fortified apple juice samples containing SBP extracts both for the total polyphenol content and FRAP values. In the samples enriched with SBP extract, 7–7.3 times higher total polyphenol content was measured and 13–21 times higher antioxidant capacity was detected according to the results of the initial samples of storage. This increase indicates that sea buckthorn extracts can increase polyphenolic and antioxidant compounds in apple juice with a very high efficiency.
During the 8-week storage experiment, the apple juice was sampled every two weeks, a total of 5 times. From the data shown in Table 2, it is apparent that in the case of the ‘L-SPA’ sample, 44.4% increase in polyphenol content was measured in the 2nd week, after which no significant change was detectable until the end of storage. In the case of the ‘H-SPA’ sample, this increase was continuous; by the end of storage, the polyphenol content values were 68.1% higher than in the initial samples. This increase was likely caused by the increased release of phenols from potential polyphenol-matrix complexes and the formation of products from non-enzymatic browning reactions. These components can react with the Folin reagent to increase the TPC values (Klimczak et al., 2007; Moldovan and David, 2020; Sadilova et al., 2009; Spanos and Wrolstad, 1990).
In contrast, the results of the FRAP values followed a decreasing trend during the storage, at the end of storage, the antioxidant capacity value decreased by 52% in the case of the ‘L-SPA’ sample, significantly, while in the case of the ‘H-SPA’ sample, the 8-week storage did not cause a significant change in the FRAP value. The degree of increase in total polyphenol content (r = 0.86) and FRAP values (r = 0.98) correlates with the percentage of pomace extract in the fortified apple juice samples.
These trends are in line with those obtained by results of Rentsendavaa et al. (2021), juice containing more SBP caused the higher FRAP value and limited reduction of antioxidant activity was measured in the samples with more pomace content. One possible explanation for this decrease is the damage of phytochemicals, leading to reduction of antioxidant activity.
The initial and final results of storage experiments of polyphenol components are presented in Fig. 1 and are expressed in mg L−1.
Results of identification of polyphenolic components. Different letters over the columns show significant differences between the values AJ: 100% apple juice, L-SPA: Low fortified apple juice with sea buckthorn pomace extract, H-SPA: High fortified apple juice with sea buckthorn pomace extract.
Citation: Progress in Agricultural Engineering Sciences 20, 1; 10.1556/446.2024.00119
Five polyphenol components were identified in the samples, two of them were detectable with significantly higher values: catechin and epicatechin. In the case of 100% AJ samples, the catechin component was measured in the largest quantity, 0.8 mg L−1 the amount of catechin in fortified apple juices is ‘L-SPA’: 1.4 mg L−1 and ‘H-SPA’: 3.7 mg L−1. The amount of epicatechin was even higher, ‘L-SPA’: 2.5 mg L−1 and ‘H-SPA’: 5.0 mg L−1. Compared to the control apple juice, the amount of chlorogenic acid in the ‘L-SPA’ sample was increased 2 times, and in the ‘H-SPA’ sample by 5 times, following the addition of pomace extract. The identification of the polyphenolic compounds of berries has been investigated in many studies, but most of the research have overlooked the polyphenolic components that can be extracted from sea buckthorn pomace. Similarly to our results, in the work of CHU et al. (2003), catechin and epicatechin components were identified as the main polyphenolic compounds of sea buckthorn seeds.
All components remained stable during the 8-week storage, which is in agreement with the results of Rentsendavaa (2021), who investigated the stability of the amounts of quercetin (4.6–7.5 mg L−1) and rutin (0.7–3.1 mg L−1) in sea buckthorn juice during storage.
As shown in Table 3 an increase in total viable count (TVC) and mold and yeast count (YMC) was observed during storage. In samples containing SBP extract, the results of total colonies numbers showed lower rates of increase than in 100% apple juice (AJ). The lowest TVC and YMC were calculated for the ‘H-SPA’ sample. These results can be explained by the assumption that added SBP extracts presumably inhibited the growth of microorganisms in the apple juice during the storage time.
Results of microbiological measurement, the statistical results are shown by letter, the different letter indicates a significant difference between the samples
Weeks of storage | AJ | L-SPA | H-SPA |
Total viable count: CFU 100 µL−1 | |||
0-week | 0 a | 2 a | 2 a |
8-week | 48 c | 42 c | 3 a |
Yeast/mould count: CFU 100 µL−1 | |||
0-week | 0 a | 0 a | 0 a |
8-week | 6 b | 5 b | 1 a |
AJ: 100% apple juice, L-SPA: Low fortified apple juice with sea buckthorn pomace extract, H-SPA: High fortified apple juice with sea buckthorn pomace extract.
The plant extract can produce components that can have microbicidal and fungicide effects through a different type of action (Wang et al., 2016). The antibacterial effect of sea buckthorn has attracted considerable attention among research topics, several studies (Geetha et al., 2002; Tian et al., 2018; Sandulachi et al., 2022) have found sea buckthorn has good antibacterial properties thanks to the large amount of flavonoid components. These findings suggest a more thorough examination of the inhibitory effect of SBP for future research. Taken together, these results suggest a relationship between antioxidant activity and the antimicrobial effect.
4 Conlusions
Our study provides the foundation for a novel utilization method of by-products of sea buckthorn. The apple juice samples were prepared from apple concentrate which was diluted with different mixtures of water and sea buckthorn pomace extract, thus resulting in fortified apple juice samples with different SBP contents. The obtained products were subjected to chemical and microbiological analyses, determining the antioxidant properties and microbial stability of the enriched apple juice during the 8-week storage experiment.
We have obtained satisfactory results demonstrating that the extract of SBP can be integrated as functional ingredient into apple juice improving its microbial stability and enriching it with valuable compounds. In addition, the present findings might help to solve the problem of increasing production waste by utilizing waste from juice production as a valuable, useful by-product. Our results are encouraging but should be validated by a larger sample size. However, these works provide a significant step towards the development of a more natural method of preservation using bioactive compounds from by-products.
Acknowledgement
The research was supported by the Doctoral School of Food Science of the Hungarian University of Agricultural and Life Sciences ID: 232.
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