# Designing the Quality Characteristics of Berry Processing Byproducts Using Fermentation
## 2 Materials and Methods
### Methods  
We assessed the microbiological quality, TPC, and antioxidant activity using the ABTS (2,2'-azinobis-(3-ethylbenzothazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydraz)), and FRAP (ferric reducing antioxidant power) methods. We also measured the vitamin C content.  
Pomace samples inoculated with microorganisms were microbiologically tested using the culture method, before and after fermentation. A volume of 1 mL of suspension was taken from the flasks, and a series of 10-fold dilutions were made in a sterile 0.9% sodium chloride physiological solution. The prepared dilutions were inoculated onto plates with the following media: MRS agar (number of lactic acid bacteria and _Saccharomyces cerevisiae_), SDA (number of fungi), and plate count agar (PCA; total number of microorganisms; Argenta, Poland). The cultures were incubated at 30 \({}^{\circ}\)C for 24-72 h.  
Fermentation was carried out using mixtures of reference strains of bacteria and yeast with a defined composition and count. To standardize the fermentation process, we utilized commercially available berry pomace (chokeberry, blackcurrant, raspberry, and strawberry) and the defined composition of reference strains of lactic acid bacteria and _Saccharomyces cerevisiae_ from the ATCC collection. Each strain was added to fermentation at a defined count of \(10^{8}\) CFU/mL. The number of viable microbial cells was controlled by measuring microbiological cultures for the control sample and the samples after fermentation.  
TPCs were measured using the colorimetric method [50], with gallic acid serving as the standard. Volumes of 5 mL of distilled water, 0.5 mL of Folin-Ciocalteu reagent, and 0.1 mL of the sample were added to volumetric flasks (10 mL). After 3 min of incubation at room temperature, 1.5 mL of 20% sodium carbonate solution was added to the flasks. Then, the flasks were filled with distilled water until reaching a designated mark. After incubating the infusions with the reagent for 2 h, the absorbance was measured at a wavelength of\(\lambda\) = 725 nm, and the phenolic compound content (in mg GA/L) was determined based on the standard curve.  
To assess the free radical scavenging ability, we employed the reaction of the examined samples with ABTS cation radicals. The determination was carried out following the method of [PERSON] et al. [51] with minor modification. Absorbance was measured at a wavelength of \(\lambda\) = 734 nm after 6 min of sample incubation at 37 \({}^{\circ}\)C. The antioxidant activity of the infusions was determined based on the standard curve and expressed in mmol Trolox/L.  
We evaluated the antioxidant properties of the extracts obtained from fruit tea polaces following the method of [PERSON] et al. [52], modified by using a DPPH radical solution with a concentration of 0.0025 g/100 mL in 96% ethyl alcohol. The DPPH radical (2,2-diphenyl-1-picrylhydrazyl) is stable under normal conditions. This method involves assessing the extent to which antioxidants present in the fruit pomaces reduce DPPH radicals. Absorbance measurements were taken every half minute for 10 min at a wavelength of \(\lambda\) = 515 nm. The antioxidant activity of the infusions was determined based on the standard curve and expressed in mmol Trolox/L.  
FRAP was measured using a technique previously described by [PERSON] et al. [53] with minor modification. The FRAP analysis was performed at a wavelength of \(\lambda\) = 593 nm. The antioxidant activity of the infusions was determined based on the standard curve and expressed in mmol FeSO\({}_{4}\)/L.  
The content of vitamin C, in the form of L-ascorbic acid, was determined using standard methods [54] and expressed in mg/100 g.  
The results represent the arithmetic mean of at least three replicates. Analysis of variance (ANOVA) was performed to compare the mean values. [PERSON]'s test was applied to verify the significance of differences between mean values, employing Statistica 13.3 software. A significance level of \(p<0.05\) was adopted for statistical estimation.