Black Chokeberry Fruit Polyphenols: A Valuable Addition to Reduce Lipid Oxidation of Muffins Containing Xylitol

The study aimed at assessing effects of black chokeberry polyphenol extract (ChPE) added (0.025–0.075%) to xylitol-containing muffins to reduce lipid oxidation, especially in preventing degradation of hydroperoxides throughout the storage period. Among polyphenolic compounds (3092 mg/100 g in total) in ChPE, polymeric procyanidins were the most abundant (1564 mg/100 g). ChPE addition resulted in a significantly increased capacity of scavenging free radicals and markedly inhibited hydroperoxides decomposition, as reflected by low anisidine values (AnV: 3.25–7.52) throughout the storage. On the other hand, sucrose-containing muffins had increased amounts of primary lipid oxidation products and differed significantly from other samples in conjugated diene hydroperoxides (CD values), which was in accordance with the decrease of C18:2 9c12c in those muffins after storage. In addition, sucrose-containing muffins were found to be those with the highest level of contamination with toxic carbonyl lipid oxidation products. Throughout the storage, no yeast or moulds contamination were found in higher enriched muffins. The incorporation of polyphenols to xylitol-containing muffins resulted in preventing decomposition of polyunsaturated fatty acids (PUFAs), and in reducing the content of some toxic aldehydes. ChPE could be regarded as a possible solution to xylitol-containing muffins to extend their shelf life. The results support the use of xylitol in muffin manufacture as being favourable in terms of suitability for diabetics.


Introduction
Consumers appreciate muffins, but those that contain sucrose, high-fructose syrup or invert sucrose have a high glycemic index and are not recommended for diabetics. Moreover, high-fructose syrups, containing 42-55% fructose, are considered more lipogenic than sucrose, thus more risky for non-alcoholic fatty liver disease and dyslipidemia [1]. Xylitol, used in this study instead of high-fructose syrups or sucrose is, like the majority of other polyols, only slowly absorbed from the It was also reported that lipid hydroperoxides were not harmless for human health, as they were elevated in patients suffering from e.g., hyperlipidemia, atherosclerosis, diabetes mellitus, stroke, and multiple sclerosis. The review of Ou et al. [21] suggested that the incorporation of polyphenols reduced the content of some toxic aldehydes in baked foods and scavenged toxic aldehydes in thermal-treated food. Previous results indicated about limitations in the content of chokeberry fruit extract Aronox (Agropharm,Tuszyn, Poland). Enrichment of cookies with 0.090% amount of this chokeberry extract revealed as having prooxidative properties and attributed to high intensity sensory astringent taste, acidic taste, off-taste and off-flavour [24]. We thus assumed that chokeberry fruit extract, rich in polyphenols, might be effective as an antioxidative agent for reducing lipid oxidation in muffins in lower than previously studied amounts. The aim of this study was thus to examine the effects of supplementing the xylitol-containing muffins with chokeberry fruit extract (in range of 0.025%, 0.05% and 0.075%) on reducing the lipid oxidation process, especially in preventing the degradation of hydroperoxides throughout storage, as well as on the acceptability by potential consumers.

Chokeberry Polyphenol Extract (ChPE)
Ripe black chokeberry fruits were manually collected at a plantation located on the slopes of the Eagle Mountains (southwestern Poland) in August 2017, at the fully ripened stage. The berries were washed with distilled water, surface-cleaned with cotton cloth and then dried in a laboratory dryer at 40 • C for 48 h. Dried berries were ground using an electric grinder to obtain particles measuring about 300 µm.
Polyphenol extract from dried chokeberry fruits powder was prepared by macerating berries in an Erlenmeyer flask (100 mL) on a shaker (Unimax 1010, Heidolph, Germany) at ambient temperature, and then extracted with 50% ethanol for 60 min, the solid-to-solvent ratio being 1:20. The flasks were covered with aluminum foil to avoid light exposure and ethanol evaporation. The extracts were separated by paper filtering (POCH, S.A., Poland). These extraction conditions were previously recommended by Cujić et al. [25] for the extraction of polyphenols from chokeberry. Next, the ethanol was evaporated in a rotary vacuum evaporator (RVO 400, INGOS s.r.o., Czech Republic) and the obtained extract was frozen (−80 • C, 60 min) and freeze-dried using Christ Alpha 1-2 LD plus lyophilizer (Martin Christ Gefriertrocknungsanlagen GmbH, Germany).

Muffin Preparation
To stabilize the extract in dough, ChPE, prior to adding, was dissolved in 8 mL of 1.59% citric acid solution. In addition to the four variants presented in Table 1, another one was prepared, in which the xylitol was replaced by sucrose and no ChPE was added. Muffins were prepared and baked as previously reported by Białek, Rutkowska, Adamska, and Bajdalow [26]. After cooling, 24 h from baking, muffins were packed in cellulose film, put into cardboard box without light access. Muffins were stored at 23 ± 1 • C until counts of moulds exceed 10 colony forming units accordingly PN-ISO 21527-2 (2009) [27].
Baked muffins were assayed as follows: immediately after production, after 2, 4, 6, and 8 weeks of storage (with regard to antioxidant properties, lipid oxidation products and fatty acid contents). Microbiological analysis was provided in nine intervals (0-8 weeks).

Antioxidative Properties of Muffins
To evaluate changes in antioxidant activity throughout the storage, ethanolic extracts of muffins were prepared by suspending 1 g of crushed sample in 10 mL of ethanol and left for 24 h without light access. Filter paper (POCH, S.A., Poland) was used to separate extracts.

Fatty Acid Composition of fat Extracted from Muffins
Lipids from muffins were extracted according to Folch, Less, and Sloane-Stanley [33], using a 2:1 chloroform/methanol (v/v) mixture. Fatty acid (FA) methyl esters (FAME) were prepared by transmethylation of fat samples using H 2 SO 4 (95%) and methanol accordingly American Oil Chemists' Society (AOCS, 2000) [34]. FAMEs were analysed by gas chromatography (GC) using an Agilent 6890N (USA) instrument equipped with flame ionisation detector (FID). FAMEs were separated on a capillary column Rtx 2330 with highly polar stationary phase (100 m × 0.25 mm I.D. × 0.1 µm thickness, Restek Corp., USA). The operating conditions and separation of FA methyl esters (FAME) were published in detail elsewhere [35]. The Supelco 37 standard (No. 47885-U, Sigma Aldrich) was applied to identify FAs. The FAs were quantified in relation to the internal standard, nonadecanoic FA (C19:0) (Sigma, Aldrich, USA) that was added before transesterification to lipid samples.

Determination of Lipid Oxidation Products in Muffins
Stability of the extracted lipid fraction of muffins was followed periodically at two-week intervals during storage. The content of primary lipid oxidation products expressed as hydroperoxide value (PV; mEq O/kg of fat) was determined according to the standard titration method [36]. The anisidine value (AnV) spectrophotometric assay reflected the content of secondary lipid oxidation products and was based on the reaction of carbonyl compounds with p-anisidine reagent and measurement of the yellow complex at 350 nm [37]. Additionally, the contents of conjugated dienes (CD) and conjugated trienes (CT) were determined spectrophotometrically according to Pegg [38] at 233 nm and 268 nm wavelength using ultraviolet-visible (UV-Vis) spectrophotometer (Specord 40, Analytic Jena AG, Germany) as reported [30].

Consumer Acceptance Evaluation of Muffins
The overall consumer's degree of liking muffins was estimated using a 9-point structured hedonic scale ranging from "like extremely -9" to "dislike extremely -1". The young consumer panel (faculty students aged 19-25 years) consisted of 50 male and 130 female subjects; they were trained in the testing procedure. The evaluation of muffins was performed 24 h from baking (fresh) and in two intervals during storage. Microbiological safety was a limitation for offering consumer panel stored muffins for evaluation. In all sessions, the same members tested all types of muffins. Muffins were delivered in coded plastic containers in a random order. Panellists were provided with spring water to cleanse the palate between tasted samples.

Microbiological Analysis of Muffins
The numbers of yeasts and moulds colonies were determined according to PN-ISO 21527-2 [27] presented in detail by Antoniewska et al. [30].

Data Analysis
All assays were run in triplicates, the results for each sample being expressed as mean ± standard deviation (SD). The effects of ChPE content and storage time on antioxidant activity and formation of lipid oxidation products in muffins and hedonic consumer acceptability were assessed by two-way ANOVA followed by Tukey's post-hoc test. Also, the effects of ChPE content and storage time on antioxidant activity of muffins were analyzed with regression analysis. In multiple linear regressions, the dependent variable (y) was ABTS + or DPPH, the independent ones were the time of storage and ChPE content. The results of fatty acid contents were subjected to one-way ANOVA followed by Tukey's post-hoc test. The level of p < 0.05 was considered significant. Statistical analyses were performed with GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA).

Polyphenol Composition of Chokeberry Extract (ChPE)
Application of the ultra-performance liquid chromatography coupled to photodiode array detection and mass spectrometry (UPLC/PDA/MS) technique enabled determining the contents of 21 phenolic compounds in ChPE belonging to 5 groups (total content 3092 mg/100g ChPE). Among polyphenolic compounds, polymeric procyanidins were most abundant (1564 mg/100g ChPE) compared with anthocyanins, phenolic acids, flavonols and flavonones (Table 2), as reported by Oszmiański and Lachowicz [13]. However, those authors found much higher content of polymeric procyanidins than in this study; this might have resulted from using sonication by Oszmiański and Lachowicz [13] during extraction procedure, whereas in this study maceration was used. As reported by Jovanović et al. [39], a much higher content of phenolic compounds was found when Thymus serpyllum herb was subjected to ultrasound-assisted fast extraction than when maceration was used. Those differences might have been also due to differences in chokeberry cultivation conditions and location, ripening stage, or in using different solvents [13,15].
As mentioned above, ChPE contained substantial amounts of polymeric procyanidins (about 50%) responsible for the pungent taste of chokeberry, and of anthocyanins, which made the dark blue colour of fruits (about 30%). Among anthocyanins, 7 compounds were separated, including the dominating cyanidin-3-O-galactoside, like reported by Oszmiański and Lachowicz [13]. However, in chokeberry cultivated in Slovenia, also small amounts of pelargonidines were detected [15].
Polymeric procyanidins, as well as anthocyanins, were reported to exhibit a variety of physiological activities, e.g., antioxidant properties, more effective than resveratrol or ascorbic acid in scavenging free radicals [40]. Their most impressive biological activities are: anti-inflammatory, antiplatelet, antimicrobial, hepatoprotective, gastroprotective, antiviral and others. Latest in vitro and ex vivo studies confirmed beneficial effect of procyanidins in cocoa bean and husk extract on vascular related dysfunction [41]. Anthocyanin-rich chokeberry extract is capable to protect endothelial progenitor cells against angiotensin II induced dysfunction [14].

Antioxidant Properties and Microbiological Safety of Muffins
Antioxidant properties of muffins significantly (p < 0.05) depended on both, ChPE content (x 2 ) and the time of storage (x 1 ), as reflected by multiple linear regressions (see Figure 1A,B). The enrichment of muffins with ChPE resulted in significantly (p < 0.05) increased capacity of scavenging free radicals as reflected by DPPH and ABTS + values ( Figure 1A,B). Other studies also revealed that bakery products enriched with ingredients derived from fruit (pomace, by-products) like berries or sour cherry, improved its antioxidant potential [7,16]. Addition of ChPE to muffins (0.025 to 0.075%) resulted in 5-25% increase of ABTS + and 5-10% of DPPH  , as compared with control samples. Higher level of incorporation of ChPE into muffin formulation resulted in improving the antioxidant activity of the final product, like when using sour cherry pomace extract, freeze-dried Japanese quince fruit [8,11]. It was due to a high Addition of ChPE to muffins (0.025 to 0.075%) resulted in 5-25% increase of ABTS + and 5-10% of DPPH, as compared with control samples. Higher level of incorporation of ChPE into muffin formulation resulted in improving the antioxidant activity of the final product, like when using sour cherry pomace extract, freeze-dried Japanese quince fruit [8,11]. It was due to a high amount of strongly antioxidant anthocyanins and procyanidins in chokeberry extract [42]. Although heat processing brought about losses of phenolic compounds, especially anthocyanins, due to their heat instability [20], the citric acid used to dissolve the extract in muffin preparation could have acted as a protective agent, retarding the losses of this compounds during baking [43,44]. Moreover, it was demonstrated that the degree of degradation depended on the type of phenolic compound, as well as on the thermal conditions applied. Górnaś et al. [19] observed that in muffins enriched with the pomace of various berries, baked at similar conditions as ours (180 • C/18 min), anthocyanins presented higher stability than when exposed to lower temperatures at longer time (140 • C/35 min). Also, substantial amount of phenolic acids in ChPE contributed to high antioxidant properties of muffins. As previously reported, in fat-rich bakery products, phenolic acids and their derivatives were less accessible and thus less degradable than in low-fat bread products [22]. Other authors reported also increased levels of phenolic acid in enriched muffins during baking. It ought to be remembered that phenolic acids are strongly linked to cell walls, so the baking process might release them [10].
Muffins enriched with chokeberry extract kept their high antioxidant potential throughout the 8 weeks of storage ( Figure 1B). As can be seen, sucrose addition significantly (p < 0.05) accelerated the storage time-related reduction of ABTS + and DPPH contents in muffins, as compared with xylitol addition ( Figure 1C). It was previously reported that over 50% of anthocyanins in blueberry puree were lost after 6-month storage. However, monomeric anthocyanins could have polymerized, these new polymeric compounds also revealed antioxidant activity [18]. The presence of xylitol in muffin formulation could be also a determinant factor. In food matrix rich in phenolic compounds, xylitol showed a protective effect on the polyphenol content, especially on anthocyanins and their antioxidant activity during storage, as previously reported by Nowicka and Wojdyło [45]. Moreover, the presence of xylitol seemed to enhance stability and to increase the intestinal bioavailability of phenolic compounds [3]. Probably because of that, the inclusion of ChPE into muffins resulted in much lower losses of scavenging capacity during storage than in control samples, as reflected by ABTS + (18-22% in enriched muffins, 41% in sucrose containing ones, and 38% xylitol control muffins) and DPPH levels (2-11%, 34% and 24%, respectively; Figure 1).
Throughout the 8 weeks of storage, no yeast or moulds with counts exceeding 10 colony forming units (CFU/g) were found in muffins containing 0.05% or 0.075% ChPE, and up to 6 weeks of storage-in muffins containing 0.025% ChPE. An increased level of moulds (4.5 × 10 4 CFU/g) was found in control muffins stored for 4 weeks, while muffins containing sucrose stored for only 2 weeks were markedly contaminated (8.5 × 10 4 CFU/g; Table 3). Our study also confirmed antimicrobial activities of black chokeberry polyphenols like reported by Denev et al. [12].

Oxidative Stability of Muffins during Storage
Oxidative stability of fat extracted from muffins, expressed as PV-measured hydroperoxides, was significantly (p < 0.05) related to the ChPE content and storage time ( Figure 2). As compared with control and sucrose-containing samples, the enriched muffins contained lower amounts of hydroperoxides, irrespectively of ChPE amount. This was due to the strong radical scavenging activity of polyphenolic compounds, mainly polymeric procyanidins and phenolic acids in ChPE, that are more stable than anthocyanins [10], as supported also by ABTS + assay of muffins ( Figure 1). Literature data indicated also that procyanidins were more effective than resveratrol or ascorbic acid in scavenging free radicals [40]. Our results also support the view that plant components rich in phenolic compounds, added to cookies, are efficient antioxidants in reducing lipid oxidation [11,30,46]. Antioxidants 2020, 9,394 10 of 17 hydroperoxides, irrespectively of ChPE amount. This was due to the strong radical scavenging activity of polyphenolic compounds, mainly polymeric procyanidins and phenolic acids in ChPE, that are more stable than anthocyanins [10], as supported also by ABTS + assay of muffins ( Figure  1). Literature data indicated also that procyanidins were more effective than resveratrol or ascorbic acid in scavenging free radicals [40]. Our results also support the view that plant components rich in phenolic compounds, added to cookies, are efficient antioxidants in reducing lipid oxidation [11,30,46]. Because of that, C18:2 9c12c was the dominating PUFA in fat extracted from muffins (Table 4), and the majority of primary lipid oxidation products were conjugated diene hydroperoxides. Only sucrose-containing muffins differed significantly (p < 0.05) from other samples in CD values, accompanying the decrease of C18:2 9c12c in those muffins after storage (Table 4, Figure 3). As compared with literature data, the levels of CD in enriched muffins (below 10.9 μmol/g) were much lower than in muffins partially substituted with buckwheat flakes and amaranth flour blend (˂24.30 μmol/g, [30]). It should be also noted that the addition of ChPE to muffins was very low (0.025-0.075%). The levels of CT in enriched muffins were lower than the control or sucrose-containing samples, and remained relatively constant throughout the storage, while in sucrose-containing samples sharply increased after 6 weeks of storage (Figure 3). This was due to the generation of primary lipid oxidation products and to a significant decomposition of α-linolenic acid C18:3 9c12c15c (Table 4, Figure 2). Our results support the view that polyphenols are effective antioxidants that prevent PUFAs decomposition. Aldehydes arising from the degradation of hydroperoxides, as lipid oxidation secondary products, were spectrophotometrically assayed in fat extracted from muffins using the AnV method. The results highlight the potential of ChPE in inhibiting the degradation of hydroperoxides and in forming secondary oxidation products, irrespectively of the ChPE level. AnVs of enriched muffins ranged from 3.25 to 7.52 (Figure 2) throughout the storage and did not exceed the recommended upper limit of secondary lipid oxidation products (AnV ˂ 8), except control muffins in which it was exceeded after six week of Because of that, C18:2 9c12c was the dominating PUFA in fat extracted from muffins (Table 4), and the majority of primary lipid oxidation products were conjugated diene hydroperoxides. Only sucrose-containing muffins differed significantly (p < 0.05) from other samples in CD values, accompanying the decrease of C18:2 9c12c in those muffins after storage (Table 4, Figure 3). As compared with literature data, the levels of CD in enriched muffins (below 10.9 µmol/g) were much lower than in muffins partially substituted with buckwheat flakes and amaranth flour blend (<24.30 µmol/g, [30]). It should be also noted that the addition of ChPE to muffins was very low (0.025-0.075%). The levels of CT in enriched muffins were lower than the control or sucrose-containing samples, and remained relatively constant throughout the storage, while in sucrose-containing samples sharply increased after 6 weeks of storage ( Figure 3). This was due to the generation of primary lipid oxidation products and to a significant decomposition of α-linolenic acid C18:3 9c12c15c (Table 4, Figure 2). Our results support the view that polyphenols are effective antioxidants that prevent PUFAs decomposition. Aldehydes arising from the degradation of hydroperoxides, as lipid oxidation secondary products, were spectrophotometrically assayed in fat extracted from muffins using the AnV method. The results highlight the potential of ChPE in inhibiting the degradation of hydroperoxides and in forming secondary oxidation products, irrespectively of the ChPE level. AnVs of enriched muffins ranged from 3.25 to 7.52 ( Figure 2) throughout the storage and did not exceed the recommended upper limit of secondary lipid oxidation products (AnV < 8), except control muffins in which it was exceeded after six week of storage, and except sucrose-containing muffins, the highest contaminated ones with toxic carbonyl lipid oxidation products (AnV ranging from 8.23 to 26.35). It was in agreement with the view that incorporation of polyphenols reduced the content of some toxic aldehydes in baked foods [21,47].  Using xylitol instead of simple sugars in muffins eliminated formation of other toxic carbonyl compounds, such as glyoxal or methylglyoxal [21]. That finding was confirmed by much lower levels of AnV in muffins containing xylitol, than in sucrose-containing samples ( Figure 2). It could be concluded that using both, ChPE and xylitol in muffin formulation, not only improved their antioxidant potential, but also enhanced their safety.

Consumer Evaluation of Muffins
Hedonic acceptability of muffins by young consumers was significantly (p < 0.05) related to ChPE content in the formulation and storage time (Table 5). Fresh muffins contained only 0.025% of ChPE were most appreciated in terms of overall acceptability, like the control samples. Hedonic acceptability sharply decreased at highest level of ChPE (0.075%), which resulted in the highest presence of polymeric procyanidins that probably brought about unacceptable taste of muffins. Polymeric procyanidins may impart a specific astringent and bitter taste in enriched products [13,24]. Substantial amounts of dark blue anthocyanins present in ChPE affected the colour of enriched muffins. Baking process significantly affected the anthocyanin stability in muffins and led to their degradation, as they were less stable than the colourless phenolic compounds [19]. However, higher scavenging capacity (ABTS + and DPPH) of enriched muffins compared with control samples (Figure 1A,B) indicated that the thermal degradation of anthocyanins was probably limited. Storage significantly (p < 0.05) influenced hedonic consumer acceptability of muffins except sucrose muffins, which were not evaluated during storage because of microbiological contamination after 2 weeks storage (Tables 3 and 5). Because of this, the microbiological safety is at a limitation for conducting consumer estimation of muffins after storage, for evaluation 3 weeks stored muffins were presented.
In all cases, hedonic acceptability of muffins stored for 3 weeks decreased as compared with fresh samples. In general, the consumer panel indicated a higher preference for stored muffins contained 0.025% and 0.05% ChPE. Also, in the case of enriched muffins with 0.025% and 0.05% ChPE, the decrease of acceptability after 3 weeks of storage was lower than in control muffins. As the hedonic acceptance of highest enriched muffins is limited, distribution of individual preferences during three intervals of storage (0, 3, and 6 weeks) is presented on Figure 4. Throughout storage increased number of consumers described their preferences for muffins with 0.075% ChPE as: from "dislike a little -4" to "dislike extremely -1".

Conclusions
Enrichment of muffins with black chokeberry polyphenols extract (ChPE) significantly improved their antioxidative potential (ABTS + and DPPH values), effectively inhibiting hydroperoxides decomposition (muffins containing 0.025% or 0.05% of ChPE had the lowest AnV) throughout the eight-week storage period. Thus, the richness of polyphenolic compounds, mainly proanthocyanidins and anthocyanins in ChPE, protected the lipid fraction of muffins with regard to the generation of toxic secondary lipid oxidation products (limitation AnV < 8). Moreover, the inclusion of ChPE in muffins might replace synthetic antioxidants used to prolong their shelf-life. Furthermore, the results represent an important approach to using xylitol and 0.025% or 0.05% ChPE in muffins in view of fulfilling the wishes of potential consumers and diabetics.

Funding:
The authors appreciate the funding support of Fundación Bancaria Ibercaja y Fundación CAI, Universidad de Zaragoza, Spain, within the fellowship CA 2/19 granted to Montserrat Martínez-Pineda.