Rx 5000

The kiwifruits (Actinidia deliciosa cv. Hayward) used to obtain the aqueous extracts were purchased at a local market in Catania, Italy. The fruits were preliminarily evaluated for their ripeness degree using a digital refractometer (Atago, RX-5000, Tokyo, Japan): unripe (9.5° Brix) and ripe (14° Brix). Cow’s milk was kindly provided by the La Cava dairy farm (Randazzo, Italy), and was used for cheesemaking at the laboratory scale.

Total soluble solids, pH, total acidity, tartaric acid and malic acid were determined in grapes at harvest. Harvest time was determined when the technological maturity was reached in grapes. Total soluble sugars were measured using an Abbé-type refractometer (Atago RX-5000, Tokyo, Japan) and pH and total acidity using and automatic titrator (Metrohm, Herisau, Switzerland) with 0.1 N NaOH. Tartaric and malic acid were measured using enzymatic kits from Boehringer 146 Mannheim GmbH (Mannhein, Germany). The methodology used to carry out these analyses is described in OIV (Organisation Internationale de la Vigne et du Vin) [32 ].

Berries for gene expression were collected at 24 h after treatment and the specific stages of sampled berries were pre-veraison, veraison and commercial maturity. The corresponding sampling dates were 12 May, 26 May, 12 July, 28 July and 28 August, in 2018 and 2019. Samples taken from the different stages were accordingly as follows: 12 May 2018, 26 May 2018, 12 July 2018, 28 July 2018 and 28 August 2018, and 12 May 2019, 26 May 2019, 12 July 2019, 28 July 2019 and 28 August 2019, respectively. The samples which were harvested on 28 August were also used for the determination of the average berry mass (ABM), total soluble solids (TSS), titratable acidity (TA) and the free or bound fraction of aroma compounds. Berries with different treatments were collected at different stages and instantly frozen in liquid nitrogen and stored at −80 °C until further use. Total soluble solids (TSS) were determined using an Abbé refractometer (type Rx-5000; Atago, Tokyo, Japan). Titratable acidity (TA), which was expressed as grams of tartaric acid per 100 g of fresh weight, was determined by titration with 0.1 N NaOH to a final pH of 8.1 using an automatic titration system [27 (link)].

The yield per vine, and berry weight were determined. Total soluble solids (°Brix) were analyzed using an Abbé-type refractrometer (Atago RX-5000) and titratable acidity and pH were measured using an automatic titrator (Metrohm, Herisau, Switzerland) with 0.1 N NaOH.

Collected milk was pooled and four subsamples of 150 mL per treatment were taken for subsequent physicochemical analysis by the following methods described by the AOAC [26 ]. Titratable acidity (% lactic acid) was measured by titration with 0.1 N sodium hydroxide. The pH value was determined using a digital potentiometer (HI-2210, HANNA^®, Ciudad de México, Mexico) by direct insertion of the electrode to the sample. Nonfat solids (NFS) were measured with a refractometer (ATAGO^®; RX-5000, Tokyo, Japan) and nitrogen content by the Kjeldahl method 991.20 [26 ], milk protein was calculated as N × 6.38 and the percentage of ash per incineration as 945.46 [26 ], and the concentration of peptides was determined by the modified ninhydrin colorimetric method [27 (link)]. All analyses were carried out in triplicate. The experimental yogurt samples were stored in a cold chamber at 6 ± 2 °C and the parameters mentioned above were evaluated on days 1, 7, and 14 of storage.

The traditional flesh parameters were measured: Total soluble solids (°Brix) using an Abbe-type refractometer (Atago RX-5000); titratable acidity using an automatic titrator (Metrohm, Herisau, Switzerland) with 0.1 N NaOH, and tartaric and malic acids using enzymatic kits from Boehringer 146 Mannheim GmbH (Mannheim, Germany).

The pH, the TSS and the total phenol content were monitored for all samples during fermentation. The pH was measured with a Mettler DL25 pH meter (Mettler–Toledo International Inc., Columbus, OH, USA) and the total soluble solids (TSS), expressed as °Brix, were measured using a refractometer (Atago, RX-5000, Milano, Italy). In addition, the total phenolic content was determined according to the Folin–Ciocalteu’s colorimetric method (FC). The tested samples were mixed with 5 mL of commercial FC reagent (Labochimica, Campodarsego, Italy) diluted with water (1:10 v/v) and added with 4 mL of a 7.5% sodium carbonate solution. Subsequently, samples were left in the dark at room temperature. After 2 h, the absorbance was measured spectrophotometrically at 765 nm (Cary 100 Scan UV-Visible, Agilent, CA, USA). The total phenolic content was expressed as mg gallic acid equivalent (GAE)/L of sample).