The Bioactivity of Byproducts from the Blackberry (Rubus fruticosus) Juice Industry ^†

Abstract

The blackberry (Rubus fruticosus) is a red fruit with great potential as a functional food thanks to its composition, which is rich in antioxidants. This work focused on the study of two byproducts of blackberry (skins and seeds) after juice production, with the aim of characterizing them and studying their bioactivity. The phenolic composition and antioxidant capacity of the products, determined by ABTS, as well as their colonic fermentation fractions, were analyzed. In addition, their genotoxicity and effect on the intestinal microbiota were evaluated after in vitro gastrointestinal digestion and fermentation. Blackberry byproducts, namely skin and seeds, are rich in phenolic compounds, especially the skin, which is rich in anthocyanins and presents an antioxidant capacity that makes it potentially usable as a functional ingredient. All the fermented samples present in vitro genoprotective activity and a modulation effect on the intestinal microbiota, promoting the growth of Bifidobacterium and Lactobacillus and reducing the abundance of the Clostridia XIVa cluster and Faecalibacterium prausnitzii. A similar effect was observed for the skin and seeds. The results provide insights into the digestive properties and health benefits of blackberry byproducts after consumption.

1. Introduction

Blackberries (Rubus, spp.) are fruits obtained from brambles, which are thorny shrubs of the Rosaceae family. More than 350 species of blackberry are known, with the most important being the American species (Rubus fruticosus) since most commercial varieties are derived from it [1].

Blackberry is a red fruit with great potential as a functional ingredient thanks to its composition, which is rich in antioxidants. Among these antioxidants, vitamin C, carotenoids, and phenolic compounds stand out.

The major phenolic compounds in the skin of blackberries are anthocyanins, which are pigments responsible for the characteristic coloration of this fruit, and they have been described to have a preventive effect against cardiovascular diseases [2]. Fruit seeds are also rich in polyphenols, with the most abundant being flavonols and procyanidins [3].

Blackberries are mainly consumed as fresh products, although they are also frozen and used to produce juices, liqueurs, and jams or syrups. The different processing mechanisms used generate a significant amount of waste, mainly composed of the remains of seeds and skin, which can account for between 20 and 30% of the weight of the processed blackberries. Blackberry seeds are the main component of the byproducts of blackberry processing, reaching 80% of the total byproducts [4]. The management of this waste increases production costs in addition to generating potential environmental risks.

Different studies have shown the positive effect of the phenolic compounds present in blackberries, describing anti-inflammatory, anticancer, neuroprotective, hypoglycemic, and cardioprotective properties [5]. Describing the potential of these byproducts for industrial applications as functional ingredients in food due to their potential health effects requires prior characterization studies and an analysis of their bioaccessibility and bioactivity.

The originality of this work, based on studying the application of blackberry juice byproducts as functional additives and the investigation of their bioactivity, lies in several innovative aspects. Firstly, this approach addresses the growing concern over sustainability in the food industry by exploring the potential of byproducts, which are generally considered waste. By investigating the bioactive properties of these byproducts, this study could reveal new applications in the food industry.

Furthermore, this work could provide valuable information about the transformations that occur in blackberry byproducts during the digestion and fermentation processes, thus contributing to scientific knowledge about their health benefits. The originality of this work is also reflected in the direct use of the byproducts efficiently and economically, which could have significant implications for industry and future research in this field.

This work aimed to characterize and study the bioactivity of two byproducts (skins and seeds) from the blackberry juice industry. To achieve this objective, in vitro gastrointestinal digestion and colonic fermentation were carried out on the byproducts. Their phenolic composition, antioxidant capacity, genotoxicity, and effect on the microbiota of the colonic fermentation fractions were analyzed.

2. Materials and Methods

2.1. Samples

The samples were byproducts generated during the production of blackberry juice (skin and seeds) harvested in 2021 and supplied by Agroberry S.L. (Zamora, Spain). Upon receipt, they were stored at −20 °C and ground and dried for use. For these byproducts, in vitro gastrointestinal digestion through a digestive simulation process was carried out following the methodology of Minekus et al., 2014 [6]. For the oral phase, α-amylase from the porcine pancreas (75 U/mL final concentration) was used; for the gastric phase, incubation was carried out with pepsin from porcine gastric mucosa (500 U/mL final concentration); and for the intestinal phase, pancreatin from the porcine pancreas (100 U/mL final concentration) and bile salts (10 mM) were added.

Colonic fermentation was carried out on the non-bioaccessible fractions of gastrointestinal digestion, following the methodology of Perez-Burillo et al. (2018) [7]. The solid residue obtained after in vitro digestion was fermented by the human gut microbiota from fecal samples obtained from three healthy adult donors (mean age range: 27 years; not taking antibiotics; no intestinal diseases; Body Mass Index range = 18.5–24.8), and this was approved by the Ethics Committee of the University of Burgos (IO27/2024). Fresh fecal samples were stored at 4 °C until inoculum was prepared and placed in sterile containers. Nitrogen was bubbled through the mixture to produce an anaerobic atmosphere, and the mixture was then incubated at 37 °C for 24 h under oscillation. After fermentation, the fractions obtained were the fermented bioaccessible (FB) and non-bioaccessible (FnB) fractions of skin and seeds, respectively.

2.2. Characterization of Byproducts and Fermented Fractions

2.2.1. Folin–Ciocalteu Assay (FC)

The total polyphenol content was determined [8], and the results were expressed as g gallic acid Equivalent/100 g.

2.2.2. Antioxidant Capacity (ABTS Assay)

The ABTS (2,2′-Azinobis 3-ethylbenzothiazoline-6-sulfonic acid) method [9] was carried out, and the results were expressed as mmol Trolox Equivalent/100 g. The samples were byproducts generated in the production of blackberry juice (skins and seeds).

2.3. Genotoxicity

The inhibition of oxidative damage to DNA was evaluated by inducing the generation of hydroxyl radicals (OH^·) and conducting subsequent agarose gel electrophoresis [10].

2.4. Microbiota Analysis

Total DNA was isolated from non-bioaccessible fermented fractions using the QIAamp Mini DNA kit (Qiagen, West Sussex, UK). Eluted DNA was treated with RNase, and the DNA concentration was measured spectrophotometrically by using a NanoDrop (BioTek, Winooski, VT, USA). Six groups of bacteria were analyzed by qPCR, and the results were expressed as log copy number per ng of DNA for Total Bacteria, Bacteroides, Bifidobacterium, Clostridia XIVa, Lactobacillus, and Faecalibacterium prausnitzii.

The 16S rDNA-specific primers used were the following:

• Total Bacteria: F-CGGTGAATACGTTCCCGG and R-TACGGCTACCTTGTTACGACTT;
• Bacteroides: F-GAGAGGAAGGTCCCCCAC and R-CGCACTTGGCTGGTTCAG;
• Bifidobacterium: F-GATTCTGGCTCAGGATGAACGC and R-CTGATAGGACGCGACCCCAT;
• Clostridia XIVa: F-CGGTACCTGACTAAGAAGC and R-AGTTTYATTCTTGCGAACG;
• F. prausnitzii: F-GGAGGAAGAAGGTCTTCGG and R-AATTCCCGCCTACCTCTGCACT.

3. Results and Discussion

Blackberry byproducts contain phenolic compounds that exhibit antioxidant activity. The results obtained in this work signified a higher phenolic content in the skin (4.69 g GAE/100 g) than in seeds (1.97 g GAE/100 g), mainly due to the anthocyanin content [11]. In addition, the antioxidant capacity of the byproducts was evaluated using the ABTS method, obtaining values of 11.12 mmol TE/100 g for the skin and 9.03 mmol TE/100 g for the seeds.

To evaluate the bioactivity of these compounds, it is not only important to quantify the proportion of antioxidants ingested but also the availability of them in the body. This concept is known as bioaccessibility [12]. Bioaccessibility is determined by simulating gastrointestinal digestion and, subsequently, colonic fermentation.

The transformations suffered after gastrointestinal digestion and fermentation imply an increase in the level of polyphenols (Figure 1) and antioxidant capacity (Figure 2), especially in non-bioaccessible fermented fractions. Non-bioaccessible polyphenols can be high-molecular-weight compounds such as proanthocyanidins or low-molecular-weight phenols associated with fiber [13]. This increase in the amount of polyphenols after digestion has been described in some studies related to the increase in the concentration of many phenolic acids, such as gallic, ferulic, p-coumaric, or vanillinic acid, derived from the catabolism of anthocyanins, flavonols, and tannins when undergoing these digestive processes [14]. In addition, studies on the intestinal microbiota [15] have described that the total polyphenols (TPs) increase after fermentation as a consequence of the metabolism of intestinal bacteria by certain phenolic compounds, such as p-cresol/p-cresyl sulfate or phenol. These post-fermentative metabolites could be responsible for the increase in TPs that appears in the analyzed samples of blackberry skin and seeds.

The protective effect of the bioaccesible and non-bioaccessible blackberry fermented samples on DNA was studied using, as an oxidizing agent, ascorbic acid and Cu⁺². In lanes 3, 4, and 7, the FnB skin samples show the greatest protection against the oxidative effect (Figure 3). In these lanes, they appear as higher-molecular-weight DNA fragments. The greater effect of non-bioaccessible fermented fractions correlates with the greater content of phenolic compounds in these fractions. These fractions are characterized by a high content of non-bioavailable polyphenols, either because they have a high molecular weight, such as proanthocyanins, or because they are low-molecular-weight polyphenols associated with fiber (proteins, hydrolyzable proteins) [16], which will exert their protective effect directly on intestinal cells.

The foods consumed in a diet will promote the growth and development of a series of intestinal bacteria, capable of synthesizing specific substances [17]. This modulation effect helps restore microbial communities beneficial to health.

The colonic fermentation of blackberry skin and seed samples modulated the gut microbiota (Figure 4), promoting the growth of the bacteria Bifidobacterium and Lactobacillus. These bacteria contribute to a beneficial microbiota due to their well-documented anti-inflammatory and immune-regulating effects, which help maintain the intestinal barrier integrity.

However, after fermentation, a significant decline in the abundance of Clostridia XIVa and Faecalibacterium prausnitzzi was observed. Similar results were obtained by other authors, who worked with whole blackberry fruits and observed a decrease in the Firmicutes group [18], which includes the Clostridium XIVa cluster and Faecalibacterium prausnitzii. These authors indicated that after fermentation with blackberry, the Firmicute level was decreased from 81.46% to 32.25%, while the Bacteroidetes level increased from 6.18% to 37.14%. The observed decline in the Firmicutes-to-Bacteroidetes (F/B) ratio may lead to a reduction in energy harvesting, which is conducive to lowering the risk of human obesity [19]. Therefore, these results suggest that blackberries could serve as a functional ingredient by modulating the composition of the bacterial microbiota. These findings on microbiota modulation do not differ between skin and seeds, as both have a similar effect.

Therefore, the skin and seed extracts show interesting results in maintaining healthy intestinal balance.

4. Conclusions

The byproducts of the blackberry industry contain phenolic compounds that exert antioxidant activity. After gastrointestinal digestion and colonic fermentation, no significant differences were observed between the skin and seeds, both exerting a similar genoprotective effect and modulation of the intestinal microbiota. These results contribute to adding value to these byproducts for their potential application as functional ingredients in food. However, future studies are necessary to clarify the mechanisms that regulate the possible effect of these products for their application in food.